Cancer and ATP: The Photon Energy Pathway (DCA as anti-tumor)

Public Release: 6-Jun-2006
Gardenia fruit compound starting point for diabetes therapy

Cell Press

A Gardenia fruit extract traditionally used in Chinese medicine to treat the symptoms of type 2 diabetes does indeed contain a chemical that reverses some of the pancreatic dysfunctions that underlie the disease, researchers report in the June 7, 2006, Cell Metabolism. The chemical therefore represents a useful starting point for new diabetes therapies, they said.

Such a drug could offer a big advance, the group added, as no currently available therapy for diabetes actually targets the underlying causes of disease in insulin-producing pancreatic beta cells. Insulin controls blood levels of glucose, the body's main energy source. In those with diabetes, insulin deficiency or insulin resistance causes blood sugar concentrations to rise.

The team discovered that Gardenia extract contains the chemical "genipin." Previously known for its ability to cross-link proteins, they now find that the chemical also blocks the function of the enzyme called uncoupling protein 2 (UCP2) through another mechanism. In both animals and humans, high concentrations of UCP2 appear to inhibit insulin secretion from the pancreas and increase the risk of type 2 diabetes.

"We think the increase in UCP2 activity is an important component of the pathogenesis of diabetes," said Bradford Lowell of Beth Israel Deaconess Medical Center and Harvard Medical School. "Our goal therefore was to discover a UCP2 inhibitor capable of working in intact cells, as such an inhibitor could theoretically represent a lead compound for agents aimed at improving beta cell function in type 2 diabetes."

Study coauthor Chen-Yu Zhang's familiarity with traditional Chinese medicine led the team to consider the extract of Gardenia jasminoides Ellis fruits. Pancreas cells taken from normal mice secreted insulin when treated with the extract, they found, whereas the cells of mice lacking UCP2 did not. The results suggested that the extract worked through its effects on the UCP2 enzyme.

"When I first saw the results, I was in disbelief," Lowell said. "I didn't think we could ever be that lucky." However, blinded repetition of the initial experiments confirmed the results every time, he said.

Through a series of chemical analyses, the researchers then zeroed in on genipin as the active compound. Genipin, like the extract, stimulated insulin secretion in control but not UCP2-deficient pancreas cells.

They further found that acute addition of genipin to isolated pancreatic tissue reversed high glucose- and obesity-induced dysfunction of insulin-producing beta cells. A derivative of genipin that lacked the chemical's cross-linking activity continued to inhibit UCP2, they reported.

That's a good sign for the therapeutic potential of genipin-related compounds, according to Lowell, as such indiscriminate cross-linking would likely have adverse effects. However, further work will need to examine whether inhibition of UCP2 itself might also have some negative consequences.

In addition to the possibility of new drugs, the findings might also prove a boon to the use of Gardenia extract itself for the treatment of disease, particularly in eastern Asia, Zhang said.

Irrespective of genipin's potential for clinical applications, its benefits within the scientific community are already clear, Lowell added.

"Genipin represents an extremely useful investigational tool for studying a number of aspects of UCP2 biology," Lowell added. UCP2 plays a role in the process by which food is converted into energy storage molecules by cellular powerhouses called mitochondria in cells throughout the body.

https://www.eurekalert.org/pub_releases/2006-06/cp-gfc053006.php

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Cell Metab. 2006 Jun;3(6):417-27.
Genipin inhibits UCP2-mediated proton leak and acutely reverses obesity- and high glucose-induced beta cell dysfunction in isolated pancreatic islets.
Zhang CY1, Parton LE, Ye CP, Krauss S, Shen R, Lin CT, Porco JA Jr, Lowell BB.
Author information

Abstract

Uncoupling protein 2 (UCP2) negatively regulates insulin secretion. UCP2 deficiency (by means of gene knockout) improves obesity- and high glucose-induced beta cell dysfunction and consequently improves type 2 diabetes in mice. In the present study, we have discovered that the small molecule, genipin, rapidly inhibits UCP2-mediated proton leak. In isolated mitochondria, genipin inhibits UCP2-mediated proton leak. In pancreatic islet cells, genipin increases mitochondrial membrane potential, increases ATP levels, closes K(ATP) channels, and stimulates insulin secretion. These actions of genipin occur in a UCP2-dependent manner. Importantly, acute addition of genipin to isolated islets reverses high glucose- and obesity-induced beta cell dysfunction. Thus, genipin and/or chemically modified variants of genipin are useful research tools for studying biological processes thought to be controlled by UCP2. In addition, these agents represent lead compounds that comprise a starting point for the development of therapies aimed at treating beta cell dysfunction.

https://www.ncbi.nlm.nih.gov/pubmed/16753577

Also on UCP2
https://www.ncbi.nlm.nih.gov/pubmed/11440717

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Research paper on Genipin:
https://www.wou.edu/las/physci/ch350/Projects_2006/Vaandering/Genipin.htm

Mitochondrial Uncoupling and the Warburg Effect: Molecular Basis for the Reprogramming of Cancer Cell Metabolism
Ismael Samudio, Michael Fiegl and Michael Andreeff
DOI: 10.1158/0008-5472.CAN-08-3722 Published March 2009


Abstract

The precise mitochondrial alterations that underlie the increased dependence of cancer cells on aerobic glycolysis for energy generation have remained a mystery. Recent evidence suggests that mitochondrial uncoupling—the abrogation of ATP synthesis in response to mitochondrial membrane potential—promotes the Warburg effect in leukemia cells, and may contribute to chemoresistance. Intriguingly, leukemia cells cultured on bone marrow–derived stromal feeder layers are more resistant to chemotherapy, increase the expression of uncoupling protein 2, and decrease the entry of pyruvate into the Krebs cycle—without compromising the consumption of oxygen, suggesting a shift to the oxidation of nonglucose carbon sources to maintain mitochondrial integrity and function. Because fatty acid oxidation has been linked to chemoresistance and mitochondrial uncoupling, it is tempting to speculate that Warburg's observations may indeed be the result of the preferential oxidation of fatty acids by cancer cell mitochondria. Therefore, targeting fatty acid oxidation or anaplerotic pathways that support fatty acid oxidation may provide additional therapeutic tools for the treatment of hematopoietic malignancies. [Cancer Res 2009;69(6)–6]
The Warburg Effect and Mitochondrial Uncoupling

More than half a century ago, Otto Warburg ( 1) proposed that cancer cells originated from non-neoplastic cells acquired a permanent respiratory defect that bypassed the Pasteur effect, i.e., the inhibition of fermentation by oxygen. This hypothesis was based on results of extensive characterization of the fermentation and oxygen consumption capacity of normal and malignant tissues—including mouse ascites and Earle's cells of different malignancies but same genetic origin—that conclusively showed a higher ratio of fermentation to respiration in the neoplastic cells. Moreover, the data indicated that the more malignant Earle's cancer cells displayed a higher ratio of fermentation to respiration than their less malignant counterparts, suggesting to Warburg and his colleagues that a gradual and cumulative decrease in mitochondrial activity was associated with malignant transformation. Interestingly, the precise nature of these gradual and cumulative changes has eluded investigators for nearly 80 years, albeit Warburg's observations of an increased rate of aerobic glycolysis in cancer cells have been reproduced countless times—not to mention the wealth of positron emission tomography images that support an increased uptake of radiolabeled glucose in tumor tissues.

It is noteworthy that although Warburg's hypothesis remains a topic of discussion among cancer biologists, Otto Warburg himself had alluded to an alternative hypothesis put forth by Feodor Lynen—one which did not necessitate permanent or transmissible alterations to the oxidative capacity of mitochondria—that suggested the possibility that the increased dependence of cancer cells on glycolysis stemmed not from their inability to reduce oxygen, but rather from their inability to synthesize ATP in response to the mitochondrial proton gradient (ΔΨM; ref. 1). Lynen's hypothesis was partly based on his work ( 2) and the previous work of Ronzoni and Ehrenfest ( 3) using the prototypical protonophore 2,4-dinitrophenol, which causes a “short circuit” in the electrochemical gradient that abolishes the mitochondrial synthesis of ATP, and decreases the entry of pyruvate into the Krebs cycle. Subsequent work showed that mitochondrial uncoupling (i.e., the abrogation of ATP synthesis in response to ΔΨM) results in a metabolic shift to the use of nonglucose carbon sources to maintain mitochondrial function ( 4, 5). Given the elusiveness of permanent transmissible alterations to the oxidative capacity of cancer cells that could broadly support Warburg's hypothesis, could Lynen's hypothesis better explain the dependence of cancer cells on glycolysis for ATP generation?

Over the past several decades, it has become increasingly clear that mitochondrial uncoupling occurs under physiologic conditions, such as during cold acclimation in mammals, and is mediated, at least in part, by uncoupling proteins (UCP; ref. 6, 7). UCP1 was the first UCP identified, and was shown to play a role in energy dissipation as heat in mammalian brown fat ( 6). During cold acclimation, UCP1 accumulates in the inner mitochondrial membrane and short circuits ΔΨM (created by the mitochondrial respiratory chain) by sustaining an inducible proton conductance ( 7). Other UCPs have been identified in humans (UCP2-4), although their functions may be unrelated to the maintenance of core body temperature, and instead involved in the reprogramming of metabolic pathways. For instance, recent work shows that UCP2 is necessary for efficient oxidation of glutamine ( , and may promote the metabolic shift from glucose oxidation to fatty acid oxidation ( 4). Likewise, UCP3 has also been shown to promote fatty acid oxidation in muscle tissue via, in part, an increased flux of fatty acid anions ( 9); however, such as for UCP2, the nature of its proton conductance remains controversial (reviewed in ref. 10). More interesting, perhaps, are recent observations that UCP2 is overexpressed in various chemoresistant cancer cell lines and primary human colon cancer, and that overexpression of this UCP leads to an increased apoptotic threshold ( 11), suggesting that in addition to metabolic reprogramming, UCPs may ipso facto provide a prosurvival advantage to malignant cells.

It is important to point out that physiologic fatty acid oxidation has been shown to be associated with an uncoupling and/or thermogenic phenotype in various cell types (reviewed in ref. 12). In addition, it is also significant that glycolysis-derived pyruvate, as well as α-ketoglutarate derived from glutaminolysis, may be necessary to provide anaplerotic substrates (i.e., those that replenish intermediates in metabolic cycles) for efficient Krebs cycle use of fatty acid-derived acetyl CoA ( 13), suggesting the possibility that in certain cell types, high rates of aerobic glycolysis may be necessary for efficient mitochondrial oxidation of fatty acids (“fats burn in the fire of carbohydrates”). The above support the concept—and indirectly, Lynen's hypothesis—that the Warburg effect may, in fact, be the result of fatty acid and/or glutamine oxidation in favor of pyruvate use.

http://cancerres.aacrjournals.org/content/69/6/2163

BTW, excess Glucagon can feed a cancer:
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Mitochondrial Protein UCP2 Controls Pancreas Development

Benjamin Broche1,2,3, Selma Ben Fradj1,2,3, Esther Aguilar1,2,3, Tiphaine Sancerni1,2,3,4, Matthieu Bénard1,2,3, Fatna Makaci1,2,3, Claire Berthault1,2,3, Raphaël Scharfmann1,2,3, Marie-Clotilde Alves-Guerra1,2,3⇑ and Bertrand Duvillié1,2,3⇑

Corresponding authors: Bertrand Duvillié, bertrand.duvillie{at}curie.fr, and Marie-Clotilde Alves-Guerra, clotilde.alves-guerra{at}inserm.fr.

Diabetes 2018 Jan; 67(1): 78-84. https://doi.org/10.2337/db17-0118



Abstract

The mitochondrial carrier uncoupling protein (UCP) 2 belongs to the family of the UCPs. Despite its name, it is now accepted that UCP2 is rather a metabolite transporter than a UCP. UCP2 can regulate oxidative stress and/or energetic metabolism. In rodents, UCP2 is involved in the control of α- and β-cell mass as well as insulin and glucagon secretion. Our aim was to determine whether the effects of UCP2 observed on β-cell mass have an embryonic origin. Thus, we used Ucp2 knockout mice. We found an increased size of the pancreas in Ucp2−/− fetuses at embryonic day 16.5, associated with a higher number of α- and β-cells. This phenotype was caused by an increase of PDX1+ progenitor cells. Perinatally, an increase in the proliferation of endocrine cells also participates in their expansion. Next, we analyzed the oxidative stress in the pancreata. We quantified an increased nuclear translocation of nuclear factor erythroid 2–related factor 2 (NRF2) in the mutant, suggesting an increased production of reactive oxygen species (ROS). Phosphorylation of AKT, an ROS target, was also activated in the Ucp2−/− pancreata. Finally, administration of the antioxidant N-acetyl-l-cysteine to Ucp2−/− pregnant mice alleviated the effect of knocking out UCP2 on pancreas development. Together, these data demonstrate that UCP2 controls pancreas development through the ROS-AKT signaling pathway.

Genipin-Induced Inhibition of Uncoupling Protein-2 Sensitizes Drug-Resistant Cancer Cells to Cytotoxic Agents

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2953501/

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https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024792


Research Article
Cellular Model of Warburg Effect Identifies Tumor Promoting Function of UCP2 in Breast Cancer and Its Suppression by Genipin

   Vanniarajan Ayyasamy ,
   Kjerstin M. Owens ,
   Mohamed Mokhtar Desouki,
   Ping Liang,
   Andrei Bakin,
   Kumarasamy Thangaraj,
   Donald J. Buchsbaum,
   Albert F. LoBuglio,
   Keshav K. Singh

PLOS

   Published: September 15, 2011
   https://doi.org/10.1371/journal.pone.0024792


Abstract

The Warburg Effect is characterized by an irreversible injury to mitochondrial oxidative phosphorylation (OXPHOS) and an increased rate of aerobic glycolysis. In this study, we utilized a breast epithelial cell line lacking mitochondrial DNA (rho0) that exhibits the Warburg Effect associated with breast cancer. We developed a MitoExpress array for rapid analysis of all known nuclear genes encoding the mitochondrial proteome. The gene-expression pattern was compared among a normal breast epithelial cell line, its rho0 derivative, breast cancer cell lines and primary breast tumors. Among several genes, our study revealed that over-expression of mitochondrial uncoupling protein UCP2 in rho0 breast epithelial cells reflects gene expression changes in breast cancer cell lines and in primary breast tumors. Furthermore, over-expression of UCP2 was also found in leukemia, ovarian, bladder, esophagus, testicular, colorectal, kidney, pancreatic, lung and prostate tumors. Ectopic expression of UCP2 in MCF7 breast cancer cells led to a decreased mitochondrial membrane potential and increased tumorigenic properties as measured by cell migration, in vitro invasion and anchorage independent growth. Consistent with in vitro studies, we demonstrate that UCP2 over-expression leads to development of tumors in vivo in an orthotopic model of breast cancer. Genipin, a plant derived small molecule, suppressed the UCP2 led tumorigenic properties, which were mediated by decreased reactive oxygen species and down-regulation of UCP2. However, UCP1, 3, 4 and 5 gene expression was unaffected. UCP2 transcription was controlled by SMAD4. Together, these studies suggest a tumor-promoting function of UCP2 in breast cancer. In summary, our studies demonstrate that i) the Warburg Effect is mediated by UCP2; ii) UCP2 is over-expressed in breast and many other cancers; iii) UCP2 promotes tumorigenic properties in vitro and in vivo and iv) genipin suppresses the tumor promoting function of UCP2.
Figures
Figure 4
Figure 5
Figure 6
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 1
Figure 2
Figure 3
 

Citation: Ayyasamy V, Owens KM, Desouki MM, Liang P, Bakin A, Thangaraj K, et al. (2011) Cellular Model of Warburg Effect Identifies Tumor Promoting Function of UCP2 in Breast Cancer and Its Suppression by Genipin. PLoS ONE 6(9): e24792. https://doi.org/10.1371/journal.pone.0024792

Editor: Alfred Lewin, University of Florida, United States of America

Received: May 17, 2011; Accepted: August 18, 2011; Published: September 15, 2011

Copyright: 2011 Ayyasamy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This study was supported by grants from the National Institutes of Health (R01 121904 and 116430) and DST-STIO to KKS, and Breast Cancer SPORE Pilot project to KKS and DJB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.
Introduction

Mitochondria play a central role in the cell growth, metabolism and cell death. Mitochondria produce energy by oxidative phosphorylation (OXPHOS) and are involved in the metabolism of fatty acids, nucleotides, amino acids and carbohydrates; synthesis of heme, Fe-S, ubiquinone and cofactors; DNA replication, repair and methylation; and antibacterial defense [1], [2], [3], [4]. Since mitochondria perform multiple cellular functions, defective mitochondria contribute to a vast number of human diseases [5], [6], [7], [8].

Otto Warburg in 1956 proposed that cancer was caused by defects in mitochondria, forcing cells to shift to energy production through glycolysis despite aerobic conditions [9]. This characteristic of cancers is described as the “Warburg Effect.” The Warburg Effect plays an important role in tumor development by remodeling the metabolic profile, which allows tumor cell survival under adverse conditions [10], [11], [12], [13], [14]. Warburg stated that cancer cells originate in two phases: i) “The first phase is the irreversible injury to respiration (OXPHOS).” ii) “The irreversible injury to respiration (OXPHOS) is followed, by a long struggle for existence by the injured cells to maintain their structure, in which a part of the cells perish (apoptosis) for lack of energy, while another part succeed in replacing the lost respiration energy by aerobic glycolysis” [9]. Our studies and those conducted by others suggest that the underlying cause of “irreversible injury” to OXPHOS includes reduced mtDNA content and mutations in mtDNA and in nuclear genes affecting OXPHOS [15], [16], [17], [18]. Our previous studies have also revealed that defects in OXPHOS induce a “mitocheckpoint” response involving epigenetic and genetic changes in the nuclear genome [3], [19], [20], [21]. We reported an undetectable level of mtDNA-encoded cytochrome c-oxidase subunit II in more than 40% of breast and ovarian tumors, suggesting a significant depletion of mtDNA in primary tumors [22], [23]. Other laboratories have also described a decrease in mtDNA content in breast [24], [25], renal [26], hepatocellular [27], gastric [28] and prostate tumors [16]. Depletion of mtDNA is also proportional to a decrease in OXPHOS levels in renal tumors [29]. A reduced mtDNA copy number is also associated with resistance to apoptosis and increased metastasis [30], [31], [32].

We recently developed a breast epithelial cell line devoid of mitochondrial DNA (rho0) that recapitulates the Warburg Effect [33] and mimics depletion of mtDNA in the variety of cancers described above. The rho0 cells lack mtDNA and thus lack the critical subunits of the respiratory chain, causing irreversible injury to respiration and forcing the cells to utilize aerobic glycolysis for ATP production [33], [34], [35]. The rho0 cells exhibiting the Warburg Effect serve as a valuable tool for identifying genomic and epigenomic changes associated with tumorigenesis [33], [36]. In this paper, we determined whether the gene expression changes associated with rho0 state in epithelial cells reflect changes in cancer cell lines and in primary tumors. Among many genes, we confirmed that UCP2 was over-expressed in rho0 epithelial cells, breast cancer cell lines and primary breast tumors. UCP2 is a member of the family of uncoupling proteins located in the inner mitochondrial membrane [37]. UCP2 function is linked to obesity and diabetes [38]. The role of UCP2 in cancers is not well understood. This paper describes the tumor promoting properties of UCP2 in vitro and in vivo in a mouse xenograft model. We also describe that genipin, a small molecule extracted from the gardenia plant, reduces the tumor promoting properties induced by over-expression of UCP2.

Keep in mind the Glucagon (released by the liver) is seen to interrupt cancer.
http://cancerres.aacrjournals.org/content/canres/19/5/512.full.pdf
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The absence of an effect of glucagon on either
the growth of the L cells in tissue culture or the
uptake of glucose by the tumor tissue in vitro, together
with the rapid resumption of growth of the
lymphoma in vivo following cessation of glucagon
injections in the DBA mice, suggests that the effect
of glucagon on tumor growth in vivo is due to
some reversible systemic effect on the tumor-bearing
host.
The hyperglycemia evoked in the intact animal
in response to glucagon is attributed in part to
liver glycogenolysis (4, 7) and in part to gluconeogenesis;
the latter is reflected in a greatly increased
nitrogen excretion (5). Attention is drawn to the
fact that, while the reduction in food intake in the
glucagon-treated animals did not differ materially
from that of the control animals, the former
showed a much greater weight loss. Such evidence
as is available therefore suggests that the enhancement
in protein catabolism and gluconeogenesis
which occurs during the administration of glucagon
may interfere with tumor growth by limiting
the supply of nitrogenous materials required for
this process.

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UCP2 Regulates the Glucagon Response to Fasting and Starvation

https://dash.harvard.edu/bitstream/handle/1/12407032/3636632.pdf?sequence=1&isAllowed=y
Author
Allister, Emma M.
Robson-Doucette, Christine A.
Prentice, Kacey J.
Hardy, Alexandre B.
Sultan, Sobia
Gaisano, Herbert Y.
Kong, DongHARVARD
Gilon, Patrick
Herrera, Pedro L.
Lowell, Bradford B.HARVARD
Wheeler, Michael B.
Note: Order does not necessarily reflect citation order of authors.
Published Version
https://doi.org/10.2337/db12-0981

Allister, E. M., C. A. Robson-Doucette, K. J. Prentice, A. B. Hardy, S. Sultan, H. Y. Gaisano, D. Kong, et al. 2013. “UCP2 Regulates the Glucagon Response to Fasting and Starvation.” Diabetes 62 (5): 1623-1633. doi:10.2337/db12-0981. http://dx.doi.org/10.2337/db12-0981.

Abstract
Glucagon is important for maintaining euglycemia during fasting/starvation, and abnormal glucagon secretion is associated with type 1 and type 2 diabetes; however, the mechanisms of hypoglycemia-induced glucagon secretion are poorly understood. We previously demonstrated that global deletion of mitochondrial uncoupling protein 2 (UCP2−/−) in mice impaired glucagon secretion from isolated islets. Therefore, UCP2 may contribute to the regulation of hypoglycemia-induced glucagon secretion, which is supported by our current finding that UCP2 expression is increased in nutrient-deprived murine and human islets. Further to this, we created α-cell–specific UCP2 knockout (UCP2AKO) mice, which we used to demonstrate that blood glucose recovery in response to hypoglycemia is impaired owing to attenuated glucagon secretion. UCP2-deleted α-cells have higher levels of intracellular reactive oxygen species (ROS) due to enhanced mitochondrial coupling, which translated into defective stimulus/secretion coupling. The effects of UCP2 deletion were mimicked by the UCP2 inhibitor genipin on both murine and human islets and also by application of exogenous ROS, confirming that changes in oxidative status and electrical activity directly reduce glucagon secretion. Therefore, α-cell UCP2 deletion perturbs the fasting/hypoglycemic glucagon response and shows that UCP2 is necessary for normal α-cell glucose sensing and the maintenance of euglycemia.

Other Sources
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3636632/pdf/

UCP2 is highly expressed in pancreatic α-cells and influences secretion and survival

Jingyu Diao, Emma M. Allister, Vasilij Koshkin, Simon C. Lee, Alpana Bhattacharjee, Christine Tang, Adria Giacca, Catherine B. Chan, and Michael B. Wheeler
PNAS August 19, 2008 105 (33) 12057-12062; https://doi.org/10.1073/pnas.0710434105

Edited by Donald F. Steiner, University of Chicago, Chicago, IL, and approved May 21, 2008

↵*J.D. and E.M.A. contributed equally to this work. (received for review November 6, 2007)

Abstract

In pancreatic β-cells, uncoupling protein 2 (UCP2) influences mitochondrial oxidative phosphorylation and insulin secretion. Here, we show that α-cells express significantly higher levels of UCP2 than do β-cells. Greater mitochondrial UCP2-related uncoupling was observed in α-cells compared with β-cells and was accompanied by a lower oxidative phosphorylation efficiency (ATP/O). Conversely, reducing UCP2 activity in α-cells was associated with higher mitochondrial membrane potential generated by glucose oxidation and with increased ATP synthesis, indicating more efficient metabolic coupling. In vitro, the suppression of UCP2 activity led to reduced glucagon secretion in response to low glucose; however, in vivo, fasting glucagon levels were normal in UCP2−/− mice. In addition to its effects on secretion, UCP2 played a cytoprotective role in islets, with UCP2−/− α-cells being more sensitive to specific death stimuli. In summary, we demonstrate a direct role for UCP2 in maintaining α-cell function at the level of glucose metabolism, glucagon secretion, and cytoprotection.

Blood-glucose levels are tightly regulated by the islet hormones insulin and glucagon. Insulin is secreted from β-cells when glucose levels are high to increase glucose utilization, whereas glucagon is secreted from α-cells when glucose levels are low to elevate blood glucose. It is well established that β-cell dysfunction, resulting in a lack of insulin secretion, is a key event in the development of hyperglycemia that is associated with both type 1 and 2 diabetes (1, 2). In type 2 diabetes, β-cell dysfunction can in part be explained by the loss of proper glucose sensing, leading to abnormal insulin secretion. However, in both forms of diabetes, glucagon secretion can be dysregulated during hyper- and hypoglycemia (3, 4), suggesting that glucose sensing by the α-cell is also impaired. For this reason, it is important to understand mechanistically how glucagon is regulated by glucose in normal and diseased states.

High plasma levels of glucose inhibit glucagon secretion; however, it is still unclear whether this in vivo response is mediated directly via glucose sensing or indirectly by neuronal modulation and/or paracrine/endocrine effects (5–. Pancreatic α-cells, like β-cells, possess ATP-dependent K+ (KATP) channels; however, the metabolism/oxidation of glucose resulting in closure of the KATP channels causes inhibition of glucagon secretion (9, 10). It is suggested that N-type Ca2+ channels modulate this alternate excitability downstream of KATP-channel closure (10). Glucose metabolism in α-cells generates a proton-motive force (pmf) in the inner mitochondria that drives the synthesis of ATP via ATP synthase. Uncoupling proteins (UCPs) are mitochondrial carrier proteins that can dissipate the proton gradient to prevent the pmf from becoming excessive when there is nutrient overload, which can reduce reactive oxygen species (ROS) produced by electron transport (11). There are five mitochondrial UCP homologues in mammals (12). The closely related UCPs are UCP1–3. UCP1 is mainly expressed in brown adipose tissue and UCP3 in muscle and adipose tissue, whereas UCP2 has been found in liver, brain, pancreas, and adipose tissue and immune cells (13, 14). Specifically, UCP2 is expressed in pancreatic islets where its β-cell overexpression increases mitochondrial uncoupling, decreases mitochondrial membrane potential (ΔΨm), reduces mitochondrial ROS production and cytoplasmic ATP content, and therefore attenuates glucose stimulated insulin secretion (GSIS) by antagonizing the KATP-channel pathway (15–17). Uncoupling processes have not been studied in α-cells where they could regulate ATP production and glucagon secretion. UCP2 may be cytoprotective in some cell types, such as macrophages, cardiomyocytes, and neurons (18, 19), and thus expression of UCP2 in α-cells may modulate susceptibility to stress stimuli and influence cell survival (20). This study aims to identify whether UCP2 is expressed in α-cells, and if so, to characterize the role it plays in regulating glucagon secretion and cell survival.

https://www.pnas.org/content/105/33/12057

PPARα suppresses insulin secretion and induces UCP2 in insulinoma cells

   Karen Tordjman 1 , Kara N. Standley, Carlos Bernal-Mizrachi, Teresa C. Leone, Trey Coleman, Daniel P. Kelly and Clay F. Semenkovich 2

   Departments of Medicine, Cell Biology and Physiology, Molecular Biology and Pharmacology, and the Center for Cardiovascular Research, Washington University School of Medicine, St. Louis, MO

   2To whom correspondence should be addressed at the Division of Atherosclerosis, Nutrition, and Lipid Research, Washington University School of Medicine, Campus Box 8046, 660 South Euclid Avenue, St. Louis, MO 63110. e-mail: semenkov{at}im.wustl.edu

Abstract

Fatty acids may promote type 2 diabetes by altering insulin secretion from pancreatic β cells, a process known as lipotoxicity. The underlying mechanisms are poorly understood. To test the hypothesis that peroxisome proliferator-activated receptor α (PPARα) has a direct effect on islet function, we treated INS-1 cells, an insulinoma cell line, with a PPARα adenovirus (AdPPARα) as well as the PPARα agonist clofibric acid. AdPPARα-infected INS-1 cells showed PPARα agonist- and fatty acid-dependent transactivation of a PPARα reporter gene. Treatment with either AdPPARα or clofibric acid increased both catalase activity (a marker of peroxisomal proliferation) and palmitate oxidation. AdPPARα induced carnitine-palmitoyl transferase-I (CPT-I) mRNA, but had no effect on insulin gene expression. AdPPARα treatment increased cellular triglyceride content but clofibric acid did not. Both AdPPARα and clofibric acid decreased basal and glucose-stimulated insulin secretion. Despite increasing fatty acid oxidation, AdPPARα did not increase cellular ATP content suggesting the stimulation of uncoupled respiration. Consistent with these observations, UCP2 expression doubled in PPARα-treated cells. Clofibric acid-induced suppression of glucose-simulated insulin secretion was prevented by the CPT-I inhibitor etomoxir.

These data suggest that PPARα-stimulated fatty acid oxidation can impair β cell function

http://www.jlr.org/content/43/6/936.full

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Ketone body β-hydroxybutyrate blocks the NLRP3 inflammasome-mediated inflammatory disease

Natural Medicine
Author(s)

Yun-Hee Youm, Kim Y. Nguyen, Ryan W. Grant, Emily L. Goldberg, Monica Bodogai, Dongin Kim, Dominic D’Agostino, Noah Planavsky, Christopher Lupfer, Thirumala D. Kanneganti, Seokwon Kang, Tamas L. Horvath, Tarek M. Fahmy, Peter A. Crawford, Arya Biragyn, Emad Alnemri, and Vishwa Deep Dixit
Abstract

Ketone bodies , β-hydroxybutyrate (BHB) and acetoacetate support mammalian survival during states of energy deficit by serving as alternative source of ATP1. BHB levels are elevated during starvation, high-intensity exercise or by the low carbohydrate ketogenic diet2. Prolonged caloric restriction or fasting reduces inflammation as immune system adapts to low glucose supply and energy metabolism switches towards mitochondrial fatty acid oxidation, ketogenesis and ketolysis2-6. However, role of ketones bodies in regulation of innate immune response is unknown. We report that BHB, but neither acetoacetate nor structurally-related short chain fatty acids, butyrate and acetate, suppresses activation of the NLRP3 inflammasome in response to several structurally unrelated NLRP3 activators, without impacting NLRC4, AIM2 or non- canonical caspase-11 inflammasome activation. Mechanistically, BHB inhibits NLRP3 inflammasome by preventing K+ efflux and reducing ASC oligomerization and speck formation. The inhibitory effects of BHB on NLRP3 were not dependent on chirality or classical starvation regulated mechanisms like AMPK, reactive oxygen species (ROS), autophagy or glycolytic inhibition. BHB blocked NLRP3 inflammasome without undergoing oxidation in TCA cycle, independently of uncoupling protein-2 (UCP2), Sirt2, receptor Gpr109a and inhibition of NLRP3 did not correlate with magnitude of histone acetylation in macrophages. BHB reduced the NLRP3 inflammasome mediated IL-1β and IL-18 production in human monocytes. In vivo, BHB attenuates caspase-1 activation and IL-1β secretion in mouse models of NLRP3-mediated diseases like Muckle-Wells Syndrome (MWS), Familial Cold Autoinflammatory syndrome (FCAS) and urate crystal induce body cavity inflammation. Taken together, these findings suggest that the anti- inflammatory effects of caloric restriction or ketogenic diets may be mechanistically linked to BHB-mediated inhibition of the NLRP3 inflammasome, and point to the potential use of interventions that elevate circulating BHB against NLRP3-mediated pro-inflammatory diseases.
Date

March 21, 2015

https://www.drperlmutter.com/study/ketone-body-%ce%b2-hydroxybutyrate-blocks-nlrp3-inflammasome-mediated-inflammatory-disease/

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Sirt1 Regulates Insulin Secretion by Repressing UCP2 in Pancreatic β Cells

Laura Bordone,
Maria Carla Motta,
Frederic Picard,
Ashley Robinson,
Ulupi S. Jhala,
Javier Apfeld,
Thomas McDonagh,
Madeleine Lemieux,
Michael McBurney,
Akos Szilvasi,
Erin J. Easlon,
Su-Ju Lin,
Leonard Guarente

PLOS

Published: December 29, 2015
https://doi.org/10.1371/journal.pbio.1002346

Fig 5. Sirt1 Binds at the UCP2 Promoter and Represses the Gene.

(A) In vitro CAT assay. 293T cells were transfected with a CAT reporter driven by the UCP2 promoter. Cells were also co-transfected with Sirt1 or not and with PPARγ or not, as indicated. CAT activity was determined (n = 3 experiments done in triplicate, *p < 0.05 in the no Sirt1 transfection experiment, ANOVA). (B) Schematic representation of the primer sets (arrows) in the UCP2 promoter (shown schematically and with excerpted DNA sequence). (C) Chromatin-immunoprecipitation (IP) was carried out on INS-1 control cells (lanes 1–3) or Sirt1 knockdown cells (columns 4–6) using Sirt1 antibody or a Gal4 control antibody, as indicated. PCR was carried out with the indicated primers. INPUT (columns 7–10) refers to PCR carried out on samples prepared prior to immunoprecipitation. Negative controls for the PCR (minus DNA) are also indicated (columns 11 and 12). In preparing the original panel for publication, the shadow/midtone/highlight input levels in the gray channel were adjusted uniformly to approximately 35/1.00/85 units. Vertical lines indicate where the original gel image was spliced together.

Fig 6. Knockdown of UCP2 in Sirt1 Knockdown Cells Restores Glucose-Induced Insulin Secretion.

(A) Northern blot for UCP2 RNA in control INS-1 cells, and cells knocked down for Sirt1 (SiRNA Sirt1), UCP2 (SiRNA UCP2), or both Sirt1 and UCP2 (SiRNA Sirt1-SiRNA UCP2). (B) Insulin secretion in INS-1 control cells and cells with knockdown levels of Sirt1, UCP2, or both Sirt1 and UCP2 after treatment with 16.7 mM glucose (+) or 4mM glucose (−) for 1 h (n = 3 experiments done in triplicate, *p < 0.05 compared to no glucose, ANOVA).

https://doi.org/10.1371/journal.pbio.1002346.g003

https://doi.org/10.1371/journal.pbio.1002346.g002

The Mitochondrial Uncoupling Protein-2 Promotes Chemoresistance in Cancer Cells

Zoltan Derdak, Nicholas M. Mark, Guido Beldi, Simon C. Robson, Jack R. Wands and György Baffy


DOI: 10.1158/0008-5472.CAN-08-0053 Published April 2008

Abstract

Cancer cells acquire drug resistance as a result of selection pressure dictated by unfavorable microenvironments. This survival process is facilitated through efficient control of oxidative stress originating from mitochondria that typically initiates programmed cell death. We show this critical adaptive response in cancer cells to be linked to uncoupling protein-2 (UCP2), a mitochondrial suppressor of reactive oxygen species (ROS). UCP2 is present in drug-resistant lines of various cancer cells and in human colon cancer. Overexpression of UCP2 in HCT116 human colon cancer cells inhibits ROS accumulation and apoptosis after exposure to chemotherapeutic agents. Tumor xenografts of UCP2-overexpressing HCT116 cells retain growth in nude mice receiving chemotherapy. Augmented cancer cell survival is accompanied by altered NH2-terminal phosphorylation of the pivotal tumor suppressor p53 and induction of the glycolytic phenotype (Warburg effect). These findings link UCP2 with molecular mechanisms of chemoresistance. Targeting UCP2 may be considered a novel treatment strategy for cancer. [Cancer Res 2008;68(–9]

Introduction

Cancers are often exposed to adverse conditions such as nutrient limitation, ischemia, hypoxia, host defense mechanisms, and anticancer therapy. Cancer cells typically respond to these stimuli by increased abundance of reactive oxygen species (ROS) resulting in oxidative stress ( 1). In this complex interplay, ROS promote further genomic instability and stimulate signaling pathways of cellular growth and proliferation. Paradoxically, ROS may also initiate cell death pathways, if present in excessive amounts ( 2, 3). The ability of cancer cells to regulate ROS levels greatly contributes to autonomous growth, evasion of apoptosis, and other hallmarks of adaptation associated with chemoresistance ( 4, 5). A better understanding of how oxidative stress is controlled by cancer cells is therefore essential to identifying new molecular targets for the treatment of cancer.

Mitochondria are the primary source of metabolically derived ROS ( 6). Substrate oxidation by mitochondrial respiration generates a proton gradient across the mitochondrial inner membrane that establishes the electrochemical potential (Δψm). The energy contained within Δψm can be either used for ATP synthesis (oxidative phosphorylation) or dissipated as heat that is mediated via proton leak in a process termed uncoupling ( 7). Elevated Δψm levels impede rapid flow of electrons along the respiratory chain, facilitating escape of more electrons and formation of superoxide, the primary mitochondrial ROS ( 6). Because proton leak decreases Δψm and the rate of superoxide production, mitochondrial uncoupling is a principal mechanism in the regulation of oxidative stress ( 8, 9). Accordingly, uncoupling protein-2 (UCP2), a widely distributed member of the anion carrier protein superfamily located in the mitochondrial inner membrane, is the major regulator of mitochondrial ROS ( 9, 10).

UCP2 expression correlates with neoplastic changes in human colon cancer ( 11), and drug-resistant sublines of various cancer cells also exhibit increased levels of UCP2, lower Δψm, and reduced susceptibility to oxidative damage ( 12). Overexpression of UCP2 in HepG2 human hepatoma cells lowers intracellular ROS levels and attenuates apoptosis induced by various challenges ( 13). Thus, whereas UCP2 is a marker of chemoresistance, expression of this mitochondrial protein may facilitate cancer cell adaptation to oxidative stress. However, the precise molecular mechanisms by which increased UCP2 expression may promote cancer cell survival are not known and have been examined here.
....
Results and Discussion

UCP2 overexpression protects cancer cells from apoptosis and oxidative stress. To examine the functional importance of UCP2 in cancer cells, we have overexpressed the plasmid-encoded cDNA of human UCP2 in HCT116, a human colon cancer cell line with low endogenous UCP2 levels ( Fig. 1A and B ). Recombinant UCP2 was synthesized at high levels and targeted successfully to the mitochondrial inner membrane of HCT116 cells ( Fig. 1C). Consistent with increased uncoupling ( 9), UCP2-overexpressing HCT116 cells displayed diminished baseline Δψm ( Fig. 1D, left) and increased oxygen consumption ( Fig. 1D, middle), whereas their intracellular ATP levels remained unchanged ( Fig. 1D, right). Because UCP2 has no apparent effect on net proton conductance unless activated by superoxide or ROS-derived alkenals ( 9), these findings affirm that baseline oxidant levels are sufficiently high to activate plasmid-encoded UCP2 in HCT116 cells.

Uncoupling mimics the effect of UCP2 in cancer cells. A, at the doses indicated, HCT116 cells were treated with the protonophore FCCP 30 min before the addition of CPT for 24 h. Apoptosis was assessed by Annexin V staining (left), DNA ladder formation (right top), caspase-3 cleavage (right middle), and disappearance of Bcl-XL (right bottom). For additional details, please see Fig. 1. Note the increased number of cells staining for both Annexin V and propidium iodide in response to 5 μmol/L FCCP (and at higher doses; data not shown) in the top right quadrants, indicating concomitant increase in necrotic cell death. Results are each from at least two independent experiments. B, columns, mean intracellular ROS levels (expressed as the percentage of levels measured by DCF in untreated cells) at baseline and in response to treatment with 2.5 μmol/L CPT for 30 min; bars, SE. ROS levels in cells treated with the antioxidant N-acetylcysteine (NAC; 2.5 mmol/L) are shown for comparison. C, intracellular ATP content (pmol/103 cells ± SE) measured by luciferin-luciferase assay shows dose-dependent decrease following treatment with FCCP, but FCCP has no further effect on markedly decreased ATP levels in cells exposed to 2.5 μg CPT for 24 h. *, P < 0.05, between cells with or without treatment with CPT; ‡, P < 0.05, between cells with or without treatment with FCCP.

UCP2 overexpression promotes the glycolytic phenotype in cancer cells. p53 seems to be involved in the regulation of energy metabolism, potentially via interactions with UCP2. As recently reported, p53 stimulates mitochondrial oxygen consumption by inducing the expression of SCO2, a subunit of the cytochrome c oxidase complex that is embedded in the respiratory chain, revealing a further novel mechanism for tumor suppression ( 30). Furthermore, the product of another p53-inducible gene, TP53-induced glycolysis and apoptosis regulator (TIGAR), lowers the intracellular levels of fructose-2,6-bisphosphate, a key substrate in glycolysis ( 31). Thus, p53 may compromise the Warburg effect, a metabolic hallmark of many cancer cells ( 32) by increasing oxidative phosphorylation, inhibiting glycolysis, and preserving the balance between these two differing ATP-generating pathways. Indeed, there is decreased oxygen consumption and increased lactate production in p53-deficient cells ( 30). In line with these observations, we found that HCT116 cells that stably overexpress UCP2 produce progressively more lactate compared with empty vector–transfected control cells in culture ( Fig. 5C). Moreover, treatment of UCP2-overexpressing HCT116 cells with the glucose analogue 2-deoxyglucose, a potent inhibitor of glycolysis, results in suppression of cell growth, consistent with increasing dependence on glycolytic ATP production ( Fig. 5D).
Conclusions

Our findings indicate that UCP2 modulates the cellular adaptive response of cancer cells. Increased expression of UCP2 may provide a marker of chemoresistance in p53-mutant cell lines ( Fig. 1A) and in the setting of neoplasia ( 11) of the human colon. We also show that UCP2 seems to have an active role in promoting cancer cell survival that is linked to mitochondrial suppression of ROS production. Moreover, the antiapoptotic effects of UCP2 via ROS involve modulation of the p53 pathway, a pivotal tumor suppression mechanism. Finally, this interaction also affects the balance of cellular energy production because UCP2 overexpression preferentially induces the glycolytic phenotype in cancer cells. Altogether, the data identify UCP2 as a potential molecular target of novel treatment strategies in cancer.


http://cancerres.aacrjournals.org/content/68/8/2813

Sirt1 Regulates Insulin Secretion by Repressing UCP2 in Pancreatic β Cells

Abstract

Sir2 and insulin/IGF-1 are the major pathways that impinge upon aging in lower organisms. In Caenorhabditis elegans a possible genetic link between Sirt1 and the insulin/IGF-1 pathway has been reported. Here we investigate such a link in mammals. We show that Sirt1 positively regulates insulin secretion in pancreatic β cells. Sirt1 represses the uncoupling protein (UCP) gene UCP2 by binding directly to the UCP2 promoter. In β cell lines in which Sirt1 is reduced by SiRNA, UCP2 levels are elevated and insulin secretion is blunted. The up-regulation of UCP2 is associated with a failure of cells to increase ATP levels after glucose stimulation. Knockdown of UCP2 restores the ability to secrete insulin in cells with reduced Sirt1, showing that UCP2 causes the defect in glucose-stimulated insulin secretion. Food deprivation induces UCP2 in mouse pancreas, which may occur via a reduction in NAD (a derivative of niacin) levels in the pancreas and down-regulation of Sirt1. Sirt1 knockout mice display constitutively high UCP2 expression. Our findings show that Sirt1 regulates UCP2 in β cells to affect insulin secretion.==Abstract== Sirt1 is shown to regulate the expression of the metabolic decoupling gene UCP2 in pancreatic β cells, highlighting a possible role for Sirt1 in coordinating insulin release in response to changing dietary conditions.

Introduction

Glucose homeostasis is maintained, in part, by pancreatic β cells, which secrete insulin in a highly regulated sequence of dependent events[1]. β cells metabolize glucose, resulting in an increase in the ATP/ADP ratio, the closing of the ATP-dependent K+ channel, the activation of the voltage-gated Ca+ channel and Ca+ influx, and the fusion of secretory vesicles to the plasma membrane to release insulin. Insulin is part of an organismal physiological axis in which it stimulates glucose uptake in metabolic tissues, such as muscle, and stores energy in the form of fat in white adipose tissue (WAT). Short-term food limitation (i.e., overnight [O/N] fasting) will therefore elicit the mobilization of glycogen stores and then fat from WAT for metabolism, and the lower level of blood glucose during fasting will result in low levels of insulin production by β cells.

Long-term calorie restriction (CR) has been known for 70 years to extend the life span of mammals dramatically[2], and it can also work in a variety of organisms, including yeast, flies, and rodents [3]–[4], although the mechanism of this effect has remained obscure. In mammals a characteristic set of physiological changes takes place during long-term CR, which overlaps the rapid physiological adaptations to short-term food limitation. One such change is the use of dietary fat or fat mobilized from WAT for energy [5]. Another is a large reduction in blood insulin levels accompanied by an increase in insulin sensitivity, i.e., the ability of insulin to promote glucose utilization [5]. In addition, gluconeogenesis is activated in the liver. These changes keep glucose available for the brain, and are closely associated with the longevity elicited by CR. The paucity of fat in WAT appears to be sufficient per se to promote a degree of longevity, since mice engineered for leanness—for example, a WAT-specific knockout (KO) of the insulin receptor—live longer [6],[7].

Findings in model organisms suggest a mechanism for the longevity engendered by CR that implicates the silent mating type information regulation 2 gene (Sir2). This gene regulates the life span in yeast[8] and Caenorhabditis elegans[9] as a longevity determinant. In yeast, CR works by up-regulating the activity of Sir2 [10],[11], a NAD-dependent deacetylase [12]–[13] (NAD is a derivative of niacin), by increasing respiration, and by increasing the NAD/NADH ratio [14] (NADH is the reduced form of NAD). CR is also reported to activate the NAD salvage pathway, which would deplete a Sir2 inhibitor, nicotinamide [3],[10]. The Drosophila melanogaster Sir2 gene was also shown to mediate life extension in response to dietary restriction[15],[16].

Since Sir2 appears to mediate the effects of CR on life span in simple model organisms, it seemed possible that Sir2 proteins also regulate the effects of food limitation and CR in mammals. The homolog of the yeast silencing information regulator2 (Sirt1) has also been implicated in several aspects of food limitation and CR in mammals. In WAT, Sirt1 represses the key regulatory protein peroxisome proliferator-activated receptor gamma (PPARγ), resulting in fat mobilization in response to food limitation[17]. In addition, Sirt1 regulates the FOXO (forkhead Box O) set of forkhead transcription factors [18],[19], providing another link to metabolism and diet. Also, gluconeogenesis in the liver is regulated by Sirt1 [18], which works in concert with the transcriptional co-activator, peroxisome proliferator-activated receptor coactivator, PGC-1α [20]. Finally, Sirt1 may play a role in the observed stress resistance of CR animals, since it down-regulates several pro-apoptotic factors, such as p53, FOXO, and Bax [18],[19],[21]–[22].

In addition to the classical paradigm for insulin regulation by glucose outlined above, reports suggest a role of an uncoupling protein (UCP) in insulin secretion. UCPs belong to a family of mitochondrial inner membrane proteins. They function to uncouple oxygen consumption during respiration from the production of ATP by allowing proton leakage down an electrochemical gradient from the cytoplasm to the mitochondria[23]–[24]. Several mammalian UCP homologues have been identified and characterized [25]. UCP2 has been shown to promote proton leakage across the mitochondrial membrane [26]–[27], and has been proposed to play a role in lipid metabolism, insulin resistance, glucose utilization, regulation of reactive oxygen species, and macrophage-mediated immunity [25],[28],[29]. A role for UCP2 in insulin secretion has been demonstrated, since UCP2 KO mice show higher ATP levels in islets and increased insulin secretion upon glucose stimulation [30]. Conversely, UCP2 overexpression in cultured β cell lines reduces ATP levels and glucose-stimulated insulin secretion [31],[32].

In this study, we show that Sirt1 functions as a positive regulator of insulin secretion in response to glucose by directly repressing the UCP2 gene. Our findings suggest that UCP2 is thus used in β cells to modulate the insulin response pathway as a function of diet. The fact that Sirt1 is a positive regulator of insulin may seem surprising, in light of the fact that sir-2.1 in C. elegans appears to be a negative regulator of the insulin-like signaling pathway[9]. We discuss how this difference makes sense in light of the physiological adaptations that take place in mammals in response to dietary changes.

Results
Sirt1 Is Preferentially Expressed in Pancreatic β Cells

To determine whether Sirt1 could potentially play a role in β cells, immunofluorescence of whole murine pancreas with anti-Sirt1 antibody was carried out, and 20 islets were examined by fluorescence microscopy. A representative islet is shown in Figure 1A (see lower left panel for hematoxylin-eosin staining). All pancreatic sections examined showed intense staining concentrated in islets using Sirt1 antibodies (Figure 1A, top right) but not with secondary antibody alone (Figure 1A, lower right), and a lower level of expression in the surrounding exocrine cells. DAPI bright spots (Figure 1A, top left) mark nuclei and their corresponding cells in the islets and surrounding tissue. This enrichment of Sirt1 in the islets and not exocrine cells is noteworthy, in light of the ubiquitous expression of this sirtuin in most somatic and germ tissues[33],[34].

Sirt1 KO Mice Have Low Levels of Blood Insulin

Because Sirt1 is highly expressed in β cells, we investigated whether this sirtuin plays a functional role in insulin production. First, we determined whether Sirt1 KO mice[33],[34] showed any defects in the pancreatic β cells or the islets. Pancreases from wild-type, Sirt1+/− heterozygotes, or Sirt1−/− KO mice (4−5 each) were sectioned and stained with antibodies against insulin (blue), glucagons (red), and somatostatin (green). These stains mark islets for β cells, α cells, and δ cells, respectively. As shown in a typical section (Figure 1B), no differences were observed in the staining pattern of these three markers between wild-type and mutant islets. We measured pancreatic islet areas using Image-Pro 4.1 Plus software, and expressed the islet areas as a percentage of the total pancreatic area (Figure 1C). There were no significant differences in islet areas comparing wild-type, heterozygous, and KO mice. The absolute islet size was also not appreciably different in the mutant mice.

Next, we determined whether insulin production was altered in Sirt1 KO mice. Blood insulin was measured in males 2–4 mo old fed ad libitum or after 18 h of starvation (Figure 2A). Sirt1 KO mice (black bars) had much lower blood levels of insulin compared with littermate control animals (open bars) when the samples were collected under ad libitum conditions. This difference was also observed when the animals were starved O/N, in which case insulin levels were very low in both wild-type and KO mice. To assess more precisely the ability of animals to produce insulin, mice were given an injection of glucose after O/N starvation and insulin was measured after 2, 10, and 20 min. Whereas insulin induction was clearly evident in the wild-type mice at 10 min (open bars), induction was not observed in Sirt1 KO animals (black bars in Figure 2B, n = 4 or 5 for each measurement). A similar trend was noted at 20 min.

To further investigate the defect in insulin production, islets from four wild-type or four Sirt1 KO animals were isolated and incubated in vitro with or without glucose for 1 h, and insulin secretion was determined. As shown in Figure 2C, the basal level of insulin secreted into the media from islets of Sirt1 KO mice (black bars) was significantly lower than wild-type controls (open bars). Moreover, the islets isolated from Sirt1 KO mice were not induced to secrete insulin by glucose, while control islets were inducible, as expected.

Levels of blood glucose were then determined and, surprisingly, were lower in Sirt1 KO mice (Figure 2D). Further, these mice appeared to be hypermetabolic, since they ate more food per body weight than the wild-type (unpublished data). These findings suggested that the KO mice had a better ability to use the lower levels of insulin for glucose uptake, i.e., were more insulin sensitive. To address this possibility, we performed glucose tolerance tests in both wild-type and KO mice by injecting glucose intraperitoneally and measuring the kinetics of glucose clearance from the blood. The KO mice cleared the glucose significantly faster than the wild-type (Figure 2E). This increased glucose tolerance explains why the KO mice maintained lower levels of blood glucose, even with reduced levels of insulin. The surprising effect of knocking out Sirt1 on glucose tolerance likely derives from tissues other than the pancreas and is currently being investigated in greater detail. In summary, we do not know whether the reduction in insulin in Sirt1 KO mice is due to a β cell defect or is an indirect consequence of increased glucose tolerance in these animals.
Sirt1 Drives Glucose-Induced Insulin Secretion in Cultured Cells

To address whether Sirt1 played a positive role in insulin production specifically in β-cells, the pancreatic β-cell lines, INS-1 from rat, and MIN6 from mouse were employed. INS-1 cells were grown in culture, and nuclear expression of Sirt1 was evident by immunofluorescence (Figure 3) and Western blot (Figure 3C and 3E). INS-1 or MIN6 cells were treated with 10 mM nicotinamide, an inhibitor of Sirt1[35], for 48 h, and the levels of insulin secreted into the media were measured without and with induction by glucose (Figure 3B and 3F, respectively). Whereas control cells showed robust induction of insulin secretion by glucose, nicotinamide treatment blocked this induction in both cell lines.

To test if the effects of nicotinamide were due specifically to inhibition of Sirt1, levels of this sirtuin were lowered by RNA interference. INS-1 and MIN6 cells were infected with a retrovirus carrying a Sirt1-SiRNA construct[17] or a control vector (pSUPER-SiRNA GFP), and stable cell lines were created as puromycin-resistant infected pools. These cells displayed Sirt1 protein levels that were knocked down compared with drug-resistant control pools generated from the vector (Figure 3C [INS-1] and 3E [MIN]). When these stable Sirt1 knockdown cells (Figure 3D, 3F, and 3G) were treated with glucose, induction of insulin secretion was eliminated, whereas contemporaneous assays of vector control cells (open bars) showed normal induction (Figure 3D and 3F).

We then tested if the defect in glucose-induced insulin secretion was due to a different glucose uptake between control and knockdown cells by measuring transport of fluorescence analog 2-NBDG. Glucose uptake[36],[37] was not reduced and was perhaps slightly elevated in this assay in the knockdown cells (Figure 3G). Further, no decrease in the glucose transporter Glut 2 was observed (unpublished data). Finally, cell growth measurements revealed no difference in the growth rate of INS-1 knockdown cells compared with controls (Figure S1). These results show that knockdown of Sirt1 suppresses glucose-stimulated insulin secretion in two different β cell lines.
ATP/ADP Ratio Is Lower in Sirt1 Knockdown Cells

We next sought to investigate the mechanism by which Sirt1 regulates insulin secretion in β cells. First, we assayed the RNA level of the INS1 gene, encoding insulin, by Northern blot and found no difference in control versus Sirt1 knockdown β cells (Figure 4A). Similarly, we found no difference by Western blot in expression level of c/EBPβ, a transcription factor that regulates INS1 (Figure 4B). Because the insulin receptor is part of an autocrine induction loop for insulin[38], we also determined by Western blot the levels of this receptor (α and β subunits) and observed no difference between control and knockdown cells (Figure 4B). Next, we investigated whether Sirt1 affected the levels of the K+ channel by Western blot, and again found no effect (Figure 4C). While this analysis suggests that Sirt1 does not function by altering levels of these factors, it does not rule out the possibility that Sirt1 regulates their activity.

Finally, because of the central role of ATP in insulin secretion, we surmised that Sirt1 could play a role in the energetics of glucose utilization in β cells. We thus measured the ATP/ADP ratio in control and Sirt1 knockdown INS-1 cells after glucose induction. Control cells (Figure 4D, open bars) responded to glucose by increasing the ratio of ATP to ADP, as expected (Figure 4D). In contrast, in cells with the knockdown levels of Sirt1 (Figure 4D, black bars), the ATP/ADP ratio did not increase upon glucose stimulation. These findings show that knocking down Sirt1 results in a defect in ATP production in response to glucose. Basal ATP levels are not significantly altered in knockdown cells, consistent with the fact that they grow as well as control cells.

UCP2 Levels Are Increased in Sirt1 KO Mice and Knockdown Cells

One possibility for the failure of the Sirt1 knockdown cells to make ATP is that respiration is more uncoupled than in control cells, which would square well with the known link between the UCP2 and insulin production in β cells (36). Thus, we determined by Western blot the levels of UCP2 in control and Sirt1 knockdown cells. Strikingly, there was a significant increase in the UCP2 protein level in the knockdown cells (Figure 4E). This increase in protein level was mirrored by an increase in the mRNA in the same cells (Figure 4F), indicating that Sirt1 regulates UCP2 transcription. It was previously shown that UCP2 expression reduced NADH levels[39]. We therefore determined NADH levels by autofluorescence [40] and found a significantly lower level of NADH in the knockdown cells (Figure 4G).

Western blot for UCP2 in Sirt1 KO mice showed a similar effect. We observed an increase in UCP2 protein in the whole pancreas (unpublished data), which is a measure of the islets, since UCP2 is expressed in only the endocrine cells of the pancreas[32]. Moreover, UCP2 protein was also up-regulated in isolated islets of Sirt1 KO mice (Figure 4H). In summary, our expression studies suggest that Sirt1 is a repressor of UCP2 transcription in β cells, and by repressing this UCP, this sirtuin may allow cells to secrete insulin in response to glucose.

Sirt1 Binds to the UCP2 Promoter

To study further the repression of UCP2 by Sirt1, we carried out reporter assays in 293T cells transfected with a chloramphenicol acetyl-transferase (CAT) gene, whose expression is driven by the UCP2 promoter. Cells were also transfected with a control vector or with an expression vector for PPARγ, which is known to bind to and activate the UCP2 promoter[41]. In a control experiment, PPARγ activated the reporter in this assay (Figure 5A, left bars). In a parallel experiment, cells were also co-transfected with a Sirt1 expression vector. Sirt1 clearly repressed the activation of the UCP2 promoter (Figure 5A, right bars). Repression of this reporter by endogenous or expressed Sirt1 was alleviated by nicotinamide, a known inhibitor of Sirt1 (unpublished data).

To determine whether repression of UCP2 was due to the direct binding of Sirt1 at the promoter, INS-1 cells were subjected to chromatin-immunoprecipitation, using Sirt1 or control antibodies. Primers specifically designed to span a known regulatory region of the UCP2 promoter (43) (Figure 5B, primer set 5) were used to probe by PCR the DNA in the immunoprecipitate. Sirt1 bound to this region of the UCP2 promoter in the control cells (Figure 5C, column 3) and to a significantly lesser extent in the Sirt1 knockdown cells (Figure 5C, column 6). Control primers designed in a different region of UCP2 upstream DNA (Figure 5B, primer set 4) showed no amplification (Figure 5C, columns 2 and 5). An anti-gal4 antibody control using primer set 5 also showed no binding (Figure 5C, columns 1 and 4). These findings indicate that Sirt1 represses UCP2 transcription by binding directly at the UCP2 promoter.

Reduction of UCP2 Restores Insulin Secretion in Sirt1 Knockdown β cells

The above findings show that Sirt1 represses UCP2, and alleviation of this repression correlated with blunted insulin secretion in response to glucose. To address whether the increase in UCP2 in Sirt1 knockdown cells caused the failure to secrete insulin, UCP2 was also knocked down in INS-1 cells with reduced Sirt1. Stable cell lines with UCP2 knocked down by SiRNA were derived from two different lines in which Sirt1 was already knocked down, as well as from control INS-1 cells. Several different UCP2 sequences were inserted into a pSUPER hairpin vector with the neomycin-resistance drug marker. These constructs were transfected into the SiRNA Sirt1 puromycin resistant cells or control cells, and stable populations of NeoR cells were assayed for UCP2 RNA and protein by Northern and Western blots. In the case of two different UCP2 SiRNA constructs, the levels of UCP2 RNA (Figure 6A) and protein (unpublished data) were clearly reduced in transfected cells. The levels of Sirt1 remained low in the double knockdown cells (unpublished data).

Next, glucose-stimulated insulin secretion was assayed in Sirt1 or UCP2 knockdown cells and in cells with both Sirt1 and UCP2 knocked down. The Sirt1 knockdown cells were again defective in induction of insulin secretion, as expected. However, the double knockdown cells or cells with only the UCP2 SiRNA construct displayed insulin secretion in response to glucose (Figure 6B). A similar effect was observed in the second Sirt1 knockdown line in which UCP2 was also knocked down (unpublished data). This result demonstrates that the failure of Sirt1 knockdown cells to secrete insulin is due to the elevated levels of UCP2 in these cells. We conclude that Sirt1 acts as a positive regulator of insulin secretion in wild-type cells by repressing UCP2, thereby allowing coupling of glucose metabolism to ATP synthesis.

https://en.wikisource.org/wiki/Wikisource:WikiProject_Open_Access/Programmatic_import_from_PubMed_Central/Sirt1_Regulates_Insulin_Secretion_by_Repressing_UCP2_in_Pancreatic_%CE%B2_Cells

Intravenous Mycobacterium Bovis Bacillus Calmette-Guérin Ameliorates Nonalcoholic Fatty Liver Disease in Obese, Diabetic ob/ob Mice

   Masashi Inafuku ,
   Goro Matsuzaki,
   Hirosuke Oku

PLOS

   Published: June 3, 2015
   https://doi.org/10.1371/journal.pone.0128676


Abstract

Inflammation and immune response profoundly influence metabolic syndrome and fatty acid metabolism. To analyze influence of systemic inflammatory response to metabolic syndrome, we inoculated an attenuated vaccine strain of Mycobacterium bovis Bacillus Calmette–Guérin (BCG) into leptin-deficient ob/ob mice. BCG administration significantly decreased epididymal white adipose tissue weight, serum insulin levels, and a homeostasis model assessment of insulin resistance. Serum high molecular weight (HMW) adiponectin level and HMW/total adiponectin ratio of the BCG treated mice were significantly higher than those of control mice. Hepatic triglyceride accumulation and macrovesicular steatosis were markedly alleviated, and the enzymatic activities and mRNA levels of lipogenic-related genes in liver were significantly decreased in the BCG injected mice. We also exposed human hepatocellular carcinoma HepG2 cells to high levels of palmitate, which enhanced endoplasmic reticulum stress-related gene expression and impaired insulin-stimulated Akt phosphorylation (Ser473). BCG treatment ameliorated both of these detrimental events. The present study therefore suggested that BCG administration suppressed development of nonalcoholic fatty liver disease, at least partly, by alleviating fatty acid-induced insulin resistance in the liver.
Figures

Citation: Inafuku M, Matsuzaki G, Oku H (2015) Intravenous Mycobacterium Bovis Bacillus Calmette-Guérin Ameliorates Nonalcoholic Fatty Liver Disease in Obese, Diabetic ob/ob Mice. PLoS ONE 10(6): e0128676. https://doi.org/10.1371/journal.pone.0128676

Academic Editor: Makoto Makishima, Nihon University School of Medicine, JAPAN

Received: January 26, 2015; Accepted: April 29, 2015; Published: June 3, 2015

Copyright: 2015 Inafuku et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported by the JSPS KAKENHI, Grant Number 2400755(Identify author, MI), (http://www.jsps.go.jp/english/e-grants). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.
Introduction

Obesity, especially visceral obesity, contributes to the pathogenesis of the metabolic syndrome, a cluster of metabolic abnormalities that includes hyperlipidemia, type 2 diabetes mellitus (T2DM), and hypertension [1]. The metabolic syndrome is a widespread and an increasingly prevalent disease in both developed and developing countries, and contributes to an increase in the rates of morbidity and mortality by cardiovascular disorders [1]. Nonalcoholic fatty liver disease (NAFLD) is also associated with metabolic syndrome. It encompasses a wide spectrum of disease from simple hepatic steatosis to steatohepatitis, advanced fibrosis, and cirrhosis [2]. It has recently been reported that various immune cells play key roles in the development of obesity-related metabolic abnormalities [3]. Obesity-related insulin resistance (IR) is also associated with elevated cytokine levels, including tumor necrosis factor (TNF)-α and interleukin (IL)-6, and elevated serum free fatty acid levels [4, 5].

Recent studies have demonstrated altered immune function in obese human compared with that in healthy patients compared with healthy individuals, suggesting that obesity may result in altered immune surveillance and in an impaired host defense [6]. Decreased resistance against viral,bacterial, and fungal infections has been shown in genetic- and diet-induced obesity model animals [7–9]. Many animal studies have shown that both type 1 diabetes mellitus (T1DM) and T2DM lead to increased susceptibility to infection with Mycobacterium tuberculosis (Mtb), which is responsible for human tuberculosis (TB) [10, 11].

One-third of the world’s population is infected with Mtb, with over 9 million new cases and 1.5 million deaths estimated from TB in 2013 [12]. An attenuated strain of M. bovis Bacillus Calmette–Guérin (BCG) is used worldwide as a vaccine against TB. Not only have cohort studies demonstrated that diabetes is a moderate-to-strong risk factor for the development of active TB [13], but the severity of diabetes has also been suggested to increase the risk of TB. Conversely, obesity is associated with a lower risk of active pulmonary TB, although that finding did not extend to extra-pulmonary TB [14]. Human T1DM is caused by an absolute deficiency of insulin production by pancreatic β–cells due to autoimmune T cell-induced destruction. This T-cell–dependent autoimmunity against islets β-cells plays an equally central role in the pathogenesis of nonobese diabetic (NOD) mouse models, which are used as an animal model for human T1DM.

It has been reported that BCG administration can prevent insulitis and T1DM in NOD mice [15, 16], and recent clinical trial data has suggested that BCG treatment can ameliorate human T1DM by stimulating the host innate immune response [17]. Interferon (IFN)-γ produced from mycobacterial antigen-specific T cells are considered important in the protective immunity against Mtb infection [18], being released as a first-line host defense mechanism after BCG vaccination [19]. TNF-α is another important cytokine in protective immunity against Mtb infection [20], and BCG administration is well known to induce TNF-α production [19]. TNF-α treatment prevents some autoimmune diseases including T1DM because autoreactive T cells were more susceptible to TNF-α-induced apoptosis than healthy T cells [21]. BCG administration may prevent T1DM by the TNF-α-induced apoptosis of diabetogenic T cells [17, 22]. In contrast, TNF-α also appears to be important in the progression of metabolic disorders such as IR and T2DM [4].

These results led us to propose that BCG treatment likely modulates a state of metabolic syndrome. However, no study has reported the effect of mycobacteria on the pathogenesis of obesity-related metabolic disorders. Although one study did report the effect of intranasal Mtb infection in leptin-deficient ob/ob mice, it only examined the immune response to TB [23]. Leptin is well known as a key mediator of energy metabolism; in addition, it is now recognized to play a role in the immune system. Wieland et al. suggested that leptin plays a role in early immune response to pulmonary TB [23]. Therefore, in this study, we aimed to examine the effects of intravenous (i.v.) BCG administration on the development of metabolic syndrome in leptin-deficient ob/ob mice.

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0128676

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Uncoupling Protein 2 and Metabolic Diseases
Annapoorna Sreedhar and Yunfeng Zhao*
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Abstract

Mitochondria are fascinating organelles involved in various cellular-metabolic activities that are integral for mammalian development. Although they perform diverse, yet interconnected functions, mitochondria are remarkably regulated by complex signaling networks. Therefore, it is not surprising that mitochondrial dysfunction is involved in plethora of diseases, including neurodegenerative and metabolic disorders. One of the many factors that lead to mitochondrial-associated metabolic diseases is the uncoupling protein-2, a family of mitochondrial anion proteins present in the inner mitochondrial membrane. Since their discovery, uncoupling proteins have attracted considerable attention due to their involvement in mitochondrial-mediated oxidative stress and energy metabolism.

This review attempts to provide a summary of recent developments in the field of uncoupling protein 2 relating to mitochondrial associated metabolic diseases.
Keywords: Uncoupling proteins, mitochondrial dysfunction, metabolic disorder, cancer, obesity, diabetes

1. Introduction

The increase in the prevalence of metabolic syndrome among the US population constitutes a serious threat to public health. Metabolic syndrome is not a ‘disease’ per se. Instead, metabolic syndrome is a group of metabolic abnormalities which are associated with high blood sugar, elevated blood pressure, excess body fat, and abnormal cholesterol levels, which can ultimately lead to plethora of diseases, such as diabetes, obesity, cardiovascular disease and cancer. Moreover, since these diseases are associated with increased mortality and morbidity rates, metabolic syndrome is a consequential life-threatening condition [1–6]. One of the many factors that lead to this condition is the alteration of uncoupling protein-2 (UCP2) [7–9].

Uncoupling proteins (UCPs) are a family of mitochondrial proteins present in the inner mitochondrial membrane whose physiological role is to transport the protons back into the mitochondrial matrix [8–11]. In recent times, there has been immense interest in defining the role of uncoupling proteins; UCP2 is often dysregulated in various metabolic conditions. Mutations in UCP2 show association with congenital hyperinsulinism and obesity, while studies from our lab and the labs of other medical institutions have shown that UCP2 is overexpressed in many aggressive cancers [12–17]. Since a growing body of evidence supports the emerging functions of UCP2 in metabolic diseases, the aim of this integrative review is to collate the evidence and summarize the role of UCP2 in metabolic syndromes, mainly, diabetes, obesity and cancer.

2. Uncoupling Proteins: What are they?

UCPs are proteins encoded by nuclear DNA and located in the inner mitochondrial membrane. Mitochondrial respiration results in the extrusion of the protons (H+) out of the mitochondria and into the intermembrane space, establishing the mitochondrial membrane potential that drives the ATP synthase. UCPs pump the protons from the intermembrane space into the mitochondrial matrix and thereby dissipate the proton gradient, reduce the ATP production and diminish superoxide production. Hence, UCPs appear to play important roles in redox regulation, mitochondrial and metabolic processes [18–20].

2.1 The family of UCPs

Among the family of UCPs, UCP1 is well-characterized with known functions and is almost exclusively present in the mitochondria of brown adipocytes [21,22]. It accounts for up to 8% of total mitochondrial protein. UCP1 was first discovered in 1997 and has since been characterized as a mitochondrial membrane transporter essential to nonshivering thermogenesis. Cold, thyroid hormone, norepinephrine, adrenergic stimulation, and cyclic adenosine monophosphate (cAMP) can increase UCP1 gene expression. In addition, UCP1 gene expression is enhanced by fatty acids and inhibited by purine nucleotides (GDP, ATP, ADP). It plays important roles in regulation of energy expenditure, thermogenesis, mitochondrial membrane potential, and ROS. Since these mechanisms are associated with diabetes, obesity, UCP1 is an excellent candidate gene for the pathogenesis of diabetes and obesity.

Next are the UCP2 and UCP3, homologues of UCP1 [23,24]. UCP2 has 59% identity with UCP1 and is ubiquitously expressed. UCP2 is widely present in the mitochondria of adipose tissue, skeletal muscle, spleen, liver, lung, and macrophages. Whereas, UCP3 is specifically expressed in skeletal muscle [25]. Interestingly, UCP2 and UCP3 have 73% identity with each other [24]. UCP3 is 57% identical with UCP1 and after its discovery in 1997, UCP3 was thought to be the skeletal muscle analogue of UCP1. Conversely, UCP4 and UCP5 are very recently discovered, mainly expressed in the neurons of the central nervous system (CNS) and its functions are largely unknown [26,27]. More interestingly, Sukolova and colleagues have identified an invertebrate UCP homologue similar to UCP2 and 3, termed UCP6 [28]. Given the physiological roles of UCPs and their association with various pathophysiology conditions, there exists an exciting potential for investigation and potential therapeutic applications.

2.2. UCP2: What is special?

An enormous interest has been created since the discovery of UCP2 in 1997. Unlike the other UCPs, the major difference is that UCP2 mRNA is present in many tissues and cell types – adipose tissue, heart, lung, spleen, kidney, thymus, lymphocytes and macrophages [Fleury C et al.]. In mammals, they reduce mitochondrial membrane potential, attenuate mitochondrial ROS production and protect against oxidative damage. Thus, the primary physiological function of UCP2 is redox regulation, ROS handling and immunity [29]. In addition, UCP2 has a role in lipid and fatty acid metabolism, glucose metabolism and transportation of TCA cycle metabolites [30–32]. UCP2 is unique. It is regulated at both transcriptional and translational levels [33,34].

Various studies have demonstrated that free fatty acids induce transcription of UCP2 [35]. As well as increased production of ROS, particularly superoxide can activate UCP2 even in the absence of FFA (Figure 3) [36]. However, the precise pathway through which superoxide activates UCP2 is unknown. Nevertheless, the superoxide-UCP2 pathway is involved in pathogenesis of hyperglycemia, hyperlipidemia and β-cell dysfunction [10]. Several physiological states and pathological conditions (like high-fat diet, stress, exercise, obesity, and diabetes) are known to regulate UCP2 expression and activity [37–39]. Hence, substantial efforts are being made to understand how UCP2 is regulated. In addition, UCP2 is very unstable and has an unusually short half-life (30min) making it a novel protein to explore [40].
An external file that holds a picture, illustration, etc. Object name is nihms864914f3.jpg
Figure 3

Schematic diagram of uncoupling protein 2 activation
2.3. Characteristics of UCP2 gene

The UCP2 gene is located on chromosome 7 of mice and chromosome 11 of humans [10]. Ironically, chromosome 11 is one of the disease-rich chromosomes in humans. Both human and mouse UCP2 genes are located 7–10 kilobases (kb) downstream of UCP3 stop codon and particularly present in exon 2 of several ATG-translational initiation codons. UCP2 coding sequence begins in the exon 3. Furthermore, UCP2 promoter region does not contain a TATA box, a DNA sequence (5′-TATAAA-3′) which is typically present within the core promoter region on the DNA. Rather, it contains potential binding motifs for several transcription factors such as specificity protein 1 (Sp1), activator proteins (AP-1, AP-2) and the cyclic AMP response element binding protein (CREB) [9].

UCP2, member of the family of mitochondrial uncoupling proteins have the widest tissue distribution. Since its discovery, UCP2 has been shown to be involved in various cellular and physiological processes. Numerous speculations on the possible role of UCP2 in tissue-specific functions are growing. Furthermore, UCP2 expression is generally increased in response to oxidative stress, which is implicated in several metabolic diseases. Alteration in expression and activity of UCP2 are associated with metabolic diseases (Figure 4).

5. UCP2 in cancer

Cancer has been one of the biggest challenges of modern medicine. Cancer is not a single disease, rather a name given to a collection of related diseases, where the cells divide abnormally and uncontrollably and spread into the surrounding tissues. Cancer is the second leading cause of mortality and morbidity worldwide. In 1971, cancer was thought to be primarily a genetic disease caused by mutation in DNA and a ‘war on cancer’ was declared by then US President Richard Nixon [61–62]. Since cancer is a disease in DNA, it was thought that mutated genes resulted in cancer. However, no single mutation or combination of mutations was identified as required for initiating the disease. Scientists concluded that cancer is more of a mutational complexity and that cancer is not a genetic disease alone. Later, cancer was thought to be resulting from defective metabolism. With the advent of science and technology, cancer is considered as genetic, epigenetic and a metabolic disease. In addition, aging, lifestyle (smoking, diet, obesity), environmental factors (chemicals, radiations), infectious agents (human papillomavirus, Epstein-Barr virus) are risk factors for cancer [63,65]. In recent times, cancer research has seen remarkable progress. Breakthroughs in cancer treatments like Immunotherapy and personalized medicine as a treatment for cancer are fueling new hope [66–69]. Our lab has been doing extensive research supporting the role of UCP2 in cancer [12,17,69]. In this paper, we review a few of the most recent works from our lab and the labs of other institutions in UCP2 upregulation and the metabolic reprogramming associated with cancer.

Uncoupling protein 2 is often upregulated in various pathological conditions. It is no surprise that tumor cells have high oxidative stress, and increase in ROS levels in cancer cells play an important role in tumor promotion, proliferation and differentiation. Since the physiological proteins of UCPs are involved in energy-dissipation, it has been speculated to be involved in tumor promotion [70]. Mitochondria, one of the most important organelles, is often dysregulated in cancers. Furthermore, mitochondrial dysregulation is associated with tumor survival, proliferation and differentiation [71–73]. Since mitochondria are the leading source of ROS production, there is a strong correlation between mitochondrial dysfunction and oxidative stress [74–76]. Thus, the higher the mitochondrial membrane potential, the higher is the ROS production. Since, the uncoupling activity induced by UCP2 expression can inhibit the mitochondrial membrane potential, and ROS production as well, they act as natural antioxidants. Consistent with this proposal, upregulation of UCP2 in cancer is known to decrease ROS production leading to chemo-resistance [77–78]. Z. Derdak in 2008 demonstrated that overexpression of UCP2 promoted chemo-resistance [77]. A more recent study demonstrated that knockout of UCP2 sensitized breast cancer cells to chemotherapeutic agents by increasing ROS, thus suggesting an inter-talk between UCP2 expression levels and oxidative stress [79]. This suggests that UCP2 could be a marker of chemo-resistance. We and others’ have shown that UCP2 is upregulated in many aggressive human cancers. Most of the studies in humans point to the upregulation of UCP2 in breast, prostate, skin, head and neck and colon cancers [17,80]. However, the exact role of UCP2 upregulation in cancer remains unclear. Paradoxically, superoxide is thought to induce the expression of UCP2, and elevated UCP2 in turn decreases ROS production. It is hard to explain whether UCP2 upregulation is the cause or the effect of oxidative stress and/or cancer. To determine the exact role of UCP2 upregulation in skin tumorigenesis, we performed a chemically-induced skin tumorigenesis study using wild type and UCP2 knockout mice [79]. Our results demonstrated that knockout of UCP2 suppressed skin formation in the animal model in vivo, suggesting UCP2 might serve as a tumor promoter and that increased UCP2 expression confer pro-survival advantage for cancer cells. One possible growth advantage of UCP2 overexpression in cancer is the UCP2-induced chemo-resistance. Another growing speculation is existence of a link between UCP2 expression and cancer cell metabolism [81,82,31]. Since, UCP2 can ‘uncouple’ or disengage electron transport chain (ETC) from ATP synthesis, and the fact that UCP2 is upregulated in aggressive cancers, it is hypothesized that UCP2 overexpression could affect energy metabolism in cancer cells. Interestingly, it has been demonstrated that UCP2 exports TCA cycle metabolites out of the mitochondria [31], thus preventing mitochondrial glucose oxidation and favoring aerobic glycolysis. Consistent with this explanation, we have found that UCP2 overexpressed JB6 cells show enhanced glycolysis leading to lactic acid production. Thus, UCP2 overexpression may sustain the Warburg effect in cancer cells. Furthermore, fatty acids are shown to activate UCP2 expression and cancer cells often exhibit enhanced fatty acid oxidation, leading to speculation on the association between UCP2 expression and fatty acid oxidation in cancer [83] These studies demonstrate a critical role of UCP2 in cancer cell energy metabolism. C. Pecquer et al. showed that UCP2 controls proliferation by promoting fatty acid oxidation and limiting glycolysis-derived pyruvate utilization [84]. Therefore, UCP2 expression may promote a metabolic switch thus regulating fatty acid oxidation and glucose metabolism in favor of tumorigenesis. In conclusion, tumor promotion, metabolic reprogramming, chemo-resistance, and redox regulation are some of the advantages of UCP2 overexpression in cancers. Overall, various studies demonstrate that UCP2 overexpression promotes cancer cell survival and adaption and targeting UCP2 could serve as a potential therapeutic approach for cancer prevention and/or therapy.


https://www.deepdyve.com/lp/elsevier/metabolic-features-of-macrophages-in-inflammatory-diseases-and-cancer-yfHTpVQEpi

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477468/

https://puredca.com/papers/ijo.2015.2953_AOP_PDF.pdf

Dichloroacetate stimulates changes in the mitochondrial network morphology via partial mitophagy in human SH-SY5Y neuroblastoma cells

DAVID PAJUELO-REGUERA, LUKÁŠ ALÁN, TOMÁŠ OLEJÁR and PETR JEŽEK
Department of Membrane Transport Biophysics, No. 75, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Received January 29, 2015; Accepted March 11, 2015
DOI: 10.3892/ijo.2015.2953

Introduction

Dichloroacetate (DCA) is a metabolic modulator that has been used in humans for decades for the treatment of lactic acidosis and inherited mitochondrial diseases (1). DCA is a pyruvate dehydrogenase kinase (PDK) inhibitor, which activates pyruvate dehydrogenase (PDH), increasing glucose oxidation by promoting an influx of pyruvate into the tricarboxylic acid cycle (2).

DCA affects multiple pathways of intermediary metabolism. It stimulates peripheral glucose utilization and inhibits gluconeogenesis, thereby reducing hyperglycemia in animals and humans with diabetes mellitus. It inhibits lipogenesis and cholesterolgenesis, thereby decreasing circulating lipid and lipoprotein levels in short-term studies of patients with acquired or hereditary disorders of lipoprotein metabolism. By stimulating the activity of pyruvate dehydrogenase, DCA facilitates oxidation of lactate and decreases morbidity in acquired and congenital forms of lactic acidosis (3). It has been shown that DCA reverses the metabolic-electrical remodeling in several cancer lines, increases ROS production, produces hyperpolarized mitochondria, activates NFAT1, induces apoptosis and decreases tumor growth (1). DCA has been found to have antitumor properties in pulmonary epithelial cells (1), breast tumor cells (4), colorectal cancer cells (5) and prostate tumors (6) without affecting normal cells. It has also been shown that DCA has an increased effect when combined with other drugs, such as with arsenic trioxide in breast cancer (7), sulindac in lung cancer (, bortezomib in multiple myeloma (9) or in combination with radiation in prostate cancer (6). Currently, clinical trials are being conducted with DCA to prove its effectiveness (http://clinicaltrial.gov/show/NCT01111097).

All organisms need energy not only to survive but also to prosper and proliferate. Accordingly, properly functioning mitochondria are essential to any cell, including cancer cells (10-12). Metabolic activities of normal cells rely predominately on mitochondrial oxidative phosphorylation (OXPHOS) for energy generation in the form of ATP. On the contrary, cancer cells predominately rely on glycolysis rather than on oxidative phosphorylation (13). There is growing evidence linking cancer with diseases or mutations affecting mitochondrial function and their metabolic pathways (14). Although mitochondrial function and intact mtDNA are essential for cancer cell growth and tumorigenesis, mtDNA mutations and/or reductions in mtDNA copy number that alter the OXPHOS physiology are common features of cancer (15). When mtDNA has a high mutation rate, de novo mtDNA mutations create a mixture of mutant and normal mtDNAs in cells, a state known as heteroplasmy. As the proportion of mutant mtDNAs increases, the energy output capacity of the cell declines until there is insufficient energy to sustain cellular function, termed the bioenergetic threshold. Mitochondria form a reticular network that is constantly undergoing fusion and fission, which is necessary for the maintenance of organelle fidelity (16).

The quality of a mitochondrial population is maintained through mitophagy, a form of specific autophagy in which defective mitochondria are selectively degraded (17). Some antitumor therapies, such as PI3K/mTOR inhibitors, are known to induce autophagy in cancer cells (18). To evaluate autophagy, LC3b is a commonly used marker because it is an essential component of autophagosomes. The level of the phosphatidylethanolamine-conjugated LC3b (LC3b-II) inserted into the autophagosome membrane and the ratio of LC3b-II/LC3b-I determine the rate of autophagy. However, autophagy itself is a very fast process leading to an immediate fusion of autophagosomes with lysosomes and subsequent degradation of LC3b-II. Blocking the autophagy process in a late phase just prior to lysosome degradation is needed to observe the LC3b-II/LC3b-I ratio. For this reason, NH4Cl and chloroquine (CHLQ) have been used to block the completion of autophagy (19).

Due to the importance of mitophagy in mitochondrial quality control and because of the known changes that DCA produces in energy metabolism, we hypothesized that DCA could cause a decrease in mitochondrial density in SH-SY5Y neuroblastoma cells through the activation of mitophagy. As a model, we chose SH-SY5Y cells, originally derived from the SK-N-SH cell line (20), for our study because they are well known and frequently studied. Moreover, neuroblastoma is the most common type of extracranial childhood solid tumor, accounting for 15% of pediatric cancer-related deaths (21). We used undifferentiated SH-SY5Y cells in this study.

We demonstrate that treatment with DCA ≤60 mM stimulates the reorganization of the mitochondrial network in SH-SY5Y cells, leading to shorter and more fragmented mitochondrial filaments. This change in the mitochondrial network was related to an imbalance in the expression of proteins.
...
DCA induces morphology changes in the mitochondrial network. Confocal microscopy showed that no significant change in the cell size was observed in the DCA-treated groups compared with controls. The control group tended to have a mitochondrial structure that was long and contained interconnected filaments. As the DCA concentration increased, the mitochondrial network contained shorter and more fragmented filaments. At higher DCA concentrations, there were clustered mitochondria filaments more frequently present, causing the filaments of the mitochondrial network not to be distributed homogeneously within the cell cytosol (Fig. 2).
....

We suggest that DCA induces partial mitophagy preserving nucleoids. It is for this reason we could not detect a decrease in the number of mtDNA copies nor TFAM protein levels.

In conclusion, in this study, we show that DCA causes cell death in SH-SY5Y neuroblastoma cells. The surviving cells after DCA treatment exhibit an altered mitochondrial network morphology that was made up of shorter and more fragmented filaments. We related this mitochondrial network restructuring to changes in FIS1, PINK1, Parkin and LC3b protein levels. Although DCA treatment did not change the number of mtDNA copies and TFAM levels, we suggest that DCA causes partial mitophagy preserving the nucleoids.

https://puredca.com/papers/ijo.2015.2953_AOP_PDF.pdf
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Cancer Cell Int. 2009; 9: 14.
Published online 2009 May 29. doi: 10.1186/1475-2867-9-14
PMCID: PMC2694762
PMID: 19480693

Acetoacetate reduces growth and ATP concentration in cancer cell lines which over-express uncoupling protein 2

Eugene J Fine,corresponding author1,2 Anna Miller,3 Edward V Quadros,2,3 Jeffrey M Sequeira,2,3 and Richard D Feinman3

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Abstract
Background

Recent evidence suggests that several human cancers are capable of uncoupling of mitochondrial ATP generation in the presence of intact tricarboxylic acid (TCA) enzymes. The goal of the current study was to test the hypothesis that ketone bodies can inhibit cell growth in aggressive cancers and that expression of uncoupling protein 2 is a contributing factor. The proposed mechanism involves inhibition of glycolytic ATP production via a Randle-like cycle while increased uncoupling renders cancers unable to produce compensatory ATP from respiration.
Methods

Seven aggressive human cancer cell lines, and three control fibroblast lines were grown in vitro in either 10 mM glucose medium (GM), or in glucose plus 10 mM acetoacetate [G+AcA]. The cells were assayed for cell growth, ATP production and expression of UCP2.
Results

There was a high correlation of cell growth with ATP concentration (r = 0.948) in a continuum across all cell lines. Controls demonstrated normal cell growth and ATP with the lowest density of mitochondrial UCP2 staining while all cancer lines demonstrated proportionally inhibited growth and ATP, and over-expression of UCP2 (p < 0.05).

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2694762/

Cell. 2001 Jun 15;105(6):745-55.
Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes.

Zhang CY1, Baffy G, Perret P, Krauss S, Peroni O, Grujic D, Hagen T, Vidal-Puig AJ, Boss O, Kim YB, Zheng XX, Wheeler MB, Shulman GI, Chan CB, Lowell BB.
Author information
Abstract

beta cells sense glucose through its metabolism and the resulting increase in ATP, which subsequently stimulates insulin secretion. Uncoupling protein-2 (UCP2) mediates mitochondrial proton leak, decreasing ATP production. In the present study, we assessed UCP2's role in regulating insulin secretion. UCP2-deficient mice had higher islet ATP levels and increased glucose-stimulated insulin secretion, establishing that UCP2 negatively regulates insulin secretion. Of pathophysiologic significance, UCP2 was markedly upregulated in islets of ob/ob mice, a model of obesity-induced diabetes. Importantly, ob/ob mice lacking UCP2 had restored first-phase insulin secretion, increased serum insulin levels, and greatly decreased levels of glycemia. These results establish UCP2 as a key component of beta cell glucose sensing, and as a critical link between obesity, beta cell dysfunction, and type 2 diabetes.
Comment in

The pancreatic beta cell heats up: UCP2 and insulin secretion in diabetes. [Cell. 2001]

https://www.ncbi.nlm.nih.gov/pubmed/11440717/

So much information here... I'm kind of shocked I hadn't seen this one lately! Thanks for sharing all this, now to spend a few weeks parsing it all and finding commonalities between all the theories. The cells are so much more complex than the atomic structures we've been dealing with, generally! It's rather daunting.

Jared Magneson wrote:So much information here... I'm kind of shocked I hadn't seen this one lately! Thanks for sharing all this, now to spend a few weeks parsing it all and finding commonalities between all the theories. The cells are so much more complex than the atomic structures we've been dealing with, generally! It's rather daunting.

No problem Jared. At the end of the day, I think Miles (team here) could win big with a CF description of chronic disease formation. UCP2/Glycosis/ROS/OXPHOS/etc. play in the formation of diseases that cost globally 2+ trillion USD. Fixing this, with attributes to Miles and team could create an endless revenue stream. There is a clear relationship between diabetes (1,2)/Tuberculosis/Starvation effect/myco-bacterium/mitochondria energy creation that could hopefully be resolve through the C.F. and our work on atomic structures. All living things are fueled by the Charge Field... it is now specifying how charge interacts to produce "health" or "aging/sickness" that needs to be resolved. This may be the key for the full on revolution needed IMHO.

A good article on the mechanics of Cancer/ROS at the molecular level...of course using traditional models instead of the charge field.

(more at link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4444525/ )
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Int J Biochem Cell Biol. 2015 Jun; 63: 16–20.
doi: 10.1016/j.biocel.2015.01.021
PMCID: PMC4444525
PMID: 25666559

Mitochondria: Much ado about nothing? How dangerous is reactive oxygen species production?
☆

Eliška Holzerováa,b and Holger Prokischa,b,⁎
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Abstract

For more than 50 years, reactive oxygen species have been considered as harmful agents, which can attack proteins, lipids or nucleic acids. In order to deal with reactive oxygen species, there is a sophisticated system developed in mitochondria to prevent possible damage. Indeed, increased reactive oxygen species levels contribute to pathomechanisms in several human diseases, either by its impaired defense system or increased production of reactive oxygen species. However, in the last two decades, the importance of reactive oxygen species in many cellular signaling pathways has been unraveled. Homeostatic levels were shown to be necessary for correct differentiation during embryonic expansion of stem cells. Although the mechanism is still not fully understood, we cannot only regard reactive oxygen species as a toxic by-product of mitochondrial respiration anymore.

This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.
Abbreviations: I, complex I; II, complex II; III, complex III; IV, complex IV; α-KGDH, α-ketoglutarate dehydrogenase; AO, alternative oxidase; cyt, cytochrome; DHODH, dihydroorotate dehydrogenase; ETF, electron transfer flavoprotein; GLRX, glutaredoxin; GPx, glutathione peroxidase; GSH GSSG, glutathione; GSR, glutathione reductase; mGPDH, mitochondrial glycerophosphate dehydrogenase; MAO, monoamine oxidase; NADH DH, external NADH dehydrogenase; NOS, nitric oxide synthase; OXPHOS, oxidative phophorylation; PDH, pyruvate dehydrogenase; PRXIII, peroxiredoxin III; ROS, reactive oxygen species; TXN2, thioredoxin 2; TXNRD2, thioredoxin reductase
Keywords: Reactive oxygen species, ROS scavenging, ROS signalization
Key facts

   • Reactive oxygen species are produced in various cell compartments.
   • Previously thought of as harmful agents only, they are now considered as important signaling molecules with potential therapeutic effect.

Organelle facts

   • Mitochondria produce vital energy in the form of ATP via oxidative phosphorylation.
   • Mitochondria have their own genome, called mitochondrial DNA.
   • Mitochondria are responsible for most of the reactive oxygen species via oxidative phosphorylation.
   • Mutations in nuclear encoded genes of mitochondrial proteins potentially result in inherited diseases, with an incidence of 1 in 10,000, most of them causing neuropathies or myopathies.
   • Mitochondrial diseases of oxidative phosphorylation can be connected with increased ROS
   • Mitochondria have their own ROS defense system.

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1. Introduction

The discussion about reactive oxygen species (ROS) started around the year 1956 (Harman, 1956) with the finding that 2% of the oxygen which is used up by the respiratory chain in mitochondria can be released and transformed into a superoxide radical anion O2•− by consuming a single electron coming from the respiratory chain. Traditionally, most of the ROS production is believed to originate from the electron transport chain in mitochondria, especially from complexes I and III. Later on, many other proteins were described as potential ROS producers, but the exact contribution from different sites is not yet fully understood. Many ROS producers arise with disruption of cell homeostasis, but, in contrast, several proteins produce ROS to restore this homeostasis. Here, we summarize sites of reactive oxygen species production and mitochondrial defense mechanisms and focus on described roles of ROS in cell signalization as a beneficial, yet often overlooked effect of ROS in cell metabolism.
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2. Organelle function: mitochondrial sites of ROS production

Mitochondria play a key role in aerobic cellular metabolism. The incomplete oxidation of oxygen to water results in superoxide production, virtually ROS. Even though it is still unclear if ROS are only harmful or beneficial, many ROS producing sites were described (Fig. 1). However, an exact contribution of each enzyme is not yet known. Complexes of the respiratory chain in mitochondria are considered as main producers, especially complex I in several sites of the enzyme (Koopman et al., 2010), complex III in subunits interacting with coenzyme Q (Raha et al., 2000; Turrens et al., 1985) and complex II under low substrate conditions (Quinlan et al., 2012) as well.
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Fig. 1

Sites of ROS production. Many different sites of ROS production exist within a cell. Most of them are located in the mitochondrial environment such as the complexes of the respiratory chain: complex I (I), complex II (II), complex III (III), or mitochondrial glycerophosphate dehydrogenase (mGPDH) next to α-ketoglutharate dehydrogenases (α-KGDH), electron transfer flavoprotein (ETF) and ETF ubiquinone oxidoreductase, pyruvate dehydrogenase (PDH), aconitase, alternative oxidase (AO), complex IV (IV), dihydroorotate dehydrogenase (DHODH), external NADH dehydrogenase (NADH DH), protein p66Shc, cytochrome (cyt) b5 reductase, monoamine oxidase (MAO) and nitric oxide synthase (NOS). Other proteins or organelles can also contribute to ROS production. Respiratory chain complexes are displayed in blue, other ROS contributors in green, organelles in violet. (For interpretation of the references to color in figure legend, the reader is referred to the web version of the article.)

Within mitochondria, minor ROS producers can also be found. First of all, one protein, which is able to transfer electrons to the coenzyme Q pool as well as to contribute to the ROS formation, is the mitochondrial glycerolphosphate dehydrogenase (Drahota et al., 2002). It is located in the inner membrane facing the intermembrane space. Another significant contribution to ROS production occurs during fatty acid oxidation due to electron transfer flavoprotein (ETF) that accepts electrons from different dehydrogenases and transfers them through its membrane partner ETF ubiquinone oxidoreductase to the coenzyme Q pool in the inner membrane (Ruzicka and Beinert, 1977). Next, there is a multisubunit pyruvate dehydrogenase complex (Starkov et al., 2004) and a structurally similar membrane bound enzyme complex of α-ketoglutarate dehydrogenase (α-KGDH) which has been proposed as a source of superoxide and hydrogen peroxide under low availability of NAD+, the natural electron acceptor of α-KGDH (Starkov et al., 2004). Many of the aforementioned proteins contain flavin in their active site, which is directly interacting with electrons and plays a possible role in the electron leakage.

Aconitase, an enzyme in the mitochondrial matrix, is able to transform hydrogen peroxide into hydroxyl radicals during a Fenton reaction with its iron–sulphur cluster. Aconitase, though, is easily inhibited by the presence of superoxide (Vasquez-Vivar et al., 2000). The function of many proteins is changed upon oxidative stress in cells. Redox disbalance and subsequent oxidation of, for example, protein p66Shc, which plays an important role in the regulation of apoptosis, translocates it into the intermembrane space to produce H2O2 (Pelicci, 2005).

There are several other proteins contributing to mitochondrial ROS production either as a superoxide or as a hydrogen peroxide, most of them non-mammalian: external NADH dehydrogenase (Fang and Beattie, 2003b) or alternative oxidase (Fang and Beattie, 2003a), proline dehydrogenase (White et al., 2007), cytochrome b5 reductase (Whatley et al., 1998), monoamine oxidase (Koopman et al., 2010) and dihydroorotate dehydrogenase (Forman and Kennedy, 1976) as well as potentially complex IV (Koopman et al., 2010).

Next to the mitochondrial ROS production, numerous contributing proteins were identified in the cytoplasm as well. Most important is the family of NADPH oxidases, proteins crucial for host defense and killing of microorganisms during phagocytosis due to increased ROS production (Hampton et al., 1998). Special conditions lead to increased ROS production in other organells too: upon stress, such as the unfolded protein response in the endoplasmatic reticulum and as part of the long-chain fatty acid oxidation in peroxisomes (Holmstrom and Finkel, 2014). In the cytoplasm or within organelles we find producers like lipoxygenase, xanthine oxidase, cyclooxygenase, cytochrome P450 monoxygenase as well as d-amino acid oxidase (Holmstrom and Finkel, 2014). Nitric oxide synthase is a protein responsible for the formation of reactive nitrogen species (Koopman et al., 2010) in the cell cytoplasm. An overview of proteins so far reported as possible ROS producers within or outside mitochondria is displayed in Fig. 1.
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3. Cell physiology: ROS scavenging and signalization

Thiol groups within redox-sensitive proteins are most important not only for ROS scavenging but also for ROS signaling. Cysteine residues, especially with low pKa and a thiolate anion (—S−) at a physiological pH, are the most prominent responders to redox changes, but methionines, tryptophans and tyrosines are prone as well. Besides, iron–sulphur clusters containing proteins provide this sulphur from their cysteine residues (e.g. aconitase). An attack of the peroxide bond leads to the formation of reversible sulphenic acid (—SOH), which is reactive and can easily be transformed into a disulfide bridge (Fig. 2A). These changes in thiol groups are reverted by glutathione or thioredoxin systems (Fig. 2B) with the help of the peroxiredoxin family of enzymes. However, upon ongoing exposure to hydrogen peroxide in the microenvironment, sulphenic groups can be further oxidized into irreversible sulphinic (—SO2H) or sulphonic (—SO3H) acids (Koopman et al., 2010).
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Fig. 2

Principle of ROS signalization and ROS scavenging mechanism. (A) Redox signalization causes reversible or irreversible modification in redox sensitive proteins in thiol molecules of cysteine residues. (B) ROS are produced within oxidative phophorylation (OXPHOS) or by other proteins. Reversible redox modifications are restored due to proteins or molecules of the ROS scavenging systems of thioredoxin and glutathione. Within mitochondria, hydrogen peroxide is sensed by peroxiredoxin III (PRXIII) and oxidation of PRXIII is reduced by thioredoxin 2 (TXN2) with help of thioredoxin reductase 2 (TXNRD2). In the glutathione pathway, glutathione peroxidase (GPx) reduces H2O2 and it is subsequently sensed by glutathione (GSH) molecule, which forms dimers (GSSG). The GSH dimers are reduced by the glutathione reductase (GSR) or by a molecule of glutaredoxin (GLRX).

The ROS scavenging mechanism occurs mainly in mitochondria, but analogous proteins exist in the cytosol. Specifically in peroxisomes, organelles producing ROS for their functioning, we find catalase, an enzyme capable of decomposing hydrogen peroxide to water and oxygen, whereas mitochondria have very low levels of this enzyme. The same removal of hydrogen peroxide is also used in mitochondria by peroxiredoxin and glutathione peroxidase (Fig. 2B), which are oxidized in return. Proteins of the peroxiredoxin family form dimers upon oxidation, which are oxidized by thioredoxins. Oxidized thioredoxin is then reduced by thioredoxin reductase in the presence of NADPH. In the other part of the pathway, oxidized glutathione peroxidase is reduced by a molecule of glutathione which forms dimers that are restored by the glutathione reductase. Glutathione is part of the cellular nonenzymatic antioxidant system also including vitamines C and E, carotenoids and flavonoids (Koopman et al., 2010). Reduction of glutathione peroxidase can also be performed by glutaredoxin, a molecule providing a possible crosstalk with the thioredoxin pathway, specifically with peroxiredoxin and thioredoxin (Hanschmann et al., 2010). On the other hand, thioredoxin reductase can directly affect peroxiredoxin as well as glutaredoxin and glutathione is able to reduce thioredoxin directly (Casagrande et al., 2002).

Due to indirect inhibition of proteins by oxidation of their thiol group, some of the signaling pathways become unblocked and therefore active. This is specifically true for the inactivation of protein phosphatases by H2O2, thereby increasing the level of protein phosphorylation (Meng et al., 2002). In contrast, redox dependent inactivation of protein tyrosine phosphatases may be specific and reversible (Denu and Tanner, 1998). ROS can also directly affect kinase signaling, for example a receptor tyrosine kinase (Truong and Carroll, 2013). A second example for the direct role of ROS in signal transduction was observed with increased tyrosine phosporylation occurring after growth factor stimulation preceded by a burst of ROS generation (Bae et al., 1997; Sundaresan et al., 1995). As previously mentioned, ROS attack DNA, but their signalization affects transcription factors, e.g. bacterial OxyR, as well. This redox sensitive protein regulates the antioxidant stress response. Oxidation of cytosine residues in OxyR creates an intramolecular disulphide bond locking the transcription factor in its active configuration (Lee et al., 2004; Xanthoudakis and Curran, 1992). Many other pathways and proteins such as actin polymerization, activity of the calcium/calmodulin-dependent kinase, DNA binding of transcription factors activator protein 1 or forkhead box protein O, pathways of Kelch-like ECHS-associated protein 1 and nuclear factor erythroid 2-related factor 2, essential autophagy protein ATG4 are all connected with ROS production (Holmstrom and Finkel, 2014). Additionally, various roles were described in circadian rhythms, innate immunity, metabolic regulations gut homeostasis, stem cell biology, the pathogenesis of cancer and why and how we are aging (Holmstrom and Finkel, 2014). In many of these mechanisms, mitophagy caused by increased ROS production serves as a quality control system. Based on several observations, a principle of redox signaling reveals:

   Mutation in gene/Specific conditions → ↑ ROS → ↑ ROS signaling → higher compensation → (PARTIAL) recovery of normal status

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4. Organelle pathology: impairment of ROS scavenging system

A number of mutations has been described in genes coding for respiratory chain complexes, some of them increasing ROS production, some of them having no effect. To date, no significant positive effects of antioxidant treatments were observed within properly performed clinical trials (Pfeffer et al., 2013). On one hand decreasing ROS amount in patients had no effect on disease improvement. On the other hand no increased ROS production was observed in the mutator mouse model, which is accumulating mutations in mitochondrial DNA over time (Bratic and Larsson, 2013), nor in mouse model with mutations in NDUFS4 subunit of complex I (Chouchani et al., 2014). The subunits of the respiratory chain complexes are impaired by the mutations, yet in these cases ROS are not part of the pathomechanism. In case of natural ROS increase, reactive radicals are removed as previously described by compensatory mechanisms. Nevertheless, several pathologies concerning the scavenging mechanism have been revealed in the past few years. Recently, Familial glucocorticoid deficiency caused by mutations in the mitochondrial antioxidant thioredoxin reductase (TXNRD2) has been described (Prasad et al., 2014). Several living patients were reported in this study. However, in vitro experiments demonstrated that the glutathione system is unable to fully compensate for the TXNRD2 deficiency leading to increased mitochondrial superoxide production. Moreover, a mouse knock-out model showed to be embryonic lethal (Conrad et al., 2004). This is also the case for a thioredoxin 2 mouse model, where only heterozygous mice are surviving (Nonn et al., 2003). A frameshift mutation in human glutaredoxin 2 causing hearing loss was also reported, yet unfortunately, impaired ROS production was not investigated (Imtiaz et al., 2014). A state characterized by low level of catalase in the cell environment is called acatalasemia. This is not directly causing a disease, but it can contribute to increased risk of developing other disorders. Understanding the whole mechanism of ROS scavenging can help to clarify specific problems and potential therapies in such condition. Concerning all so far reported mutations, we can conclude that this system is quite robust and even with some missing pieces, it is able to compensate or just skip lacking part via a functioning crosstalk.
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5. Future outlook

Although redox signalization seems to be less specific than other types of signalization, mechanisms like controlled production, cellular localization, time and exposure of cysteine residues play an important role. Also, rapid and reversible changes of protein function create this extraordinary signalization. Special attention should be applied to the specific level of ROS occurring in the cell environment, making it one of its greatest aspects. Principles can be similar in many cases, revealing potential selective therapeutic directions as well. Therefore, it is essential to identify definite targets and their function. It can be expected that genome sequencing of patients will discover more mutations in the defense system. This will contribute to our understanding of the system. Particular potential lies in H2O2 which exists naturally in 10 nm intracellular concentration and, as it is not charged, is not as reactive as other radicals and can pass through membranes.

Antioxidant and Regulatory Role of Mitochondrial Uncoupling Protein UCP2 in Pancreatic β-cells

P. JEŽEK1, T. OLEJÁR1, K. SMOLKOVÁ1, J. JEŽEK1, A. DLASKOVÁ1,
L. PLECITÁ-HLAVATÁ1, J. ZELENKA1, T. ŠPAČEK1, H. ENGSTOVÁ1,
D. PAJUELO REGUERA1, M. JABŮREK1
1Department of Membrane Transport Biophysics, Institute of Physiology Academy of Sciences of
the Czech Republic, Prague, Czech Republic

http://www.biomed.cas.cz/physiolres/pdf/63%20Suppl%201/63_S73.pdf
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Oxid Med Cell Longev. 2012; 2012: 740849.
Published online 2012 Sep 16. doi: 10.1155/2012/740849
PMCID: PMC3458419
PMID: 23029600

Mitochondrial Hormesis in Pancreatic β Cells: Does Uncoupling Protein 2 Play a Role?

Ning Li,* Suzana Stojanovski, and Pierre Maechler*
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Abstract

In pancreatic β cells, mitochondrial metabolism translates glucose sensing into signals regulating insulin secretion. Chronic exposure of β cells to excessive nutrients, namely, glucolipotoxicity, impairs β-cell function. This is associated with elevated ROS production from overstimulated mitochondria. Mitochondria are not only the major source of cellular ROS, they are also the primary target of ROS attacks. The mitochondrial uncoupling protein UCP2, even though its uncoupling properties are debated, has been associated with protective functions against ROS toxicity. Hormesis, an adaptive response to cellular stresses, might contribute to the protection against β-cell death, possibly limiting the development of type 2 diabetes. Mitochondrial hormesis, or mitohormesis, is a defense mechanism observed in ROS-induced stress-responses by mitochondria. In β cells, mitochondrial damages induced by sublethal exogenous H2O2 can induce secondary repair and defense mechanisms. In this context, UCP2 is a marker of mitohormesis, being upregulated following stress conditions. When overexpressed in nonstressed naïve cells, UCP2 confers resistance to oxidative stress. Whether treatment with mitohormetic inducers is sufficient to restore or ameliorate secretory function of β cells remains to be determined.

1. Introduction

Type 2 diabetes (T2D) is characterized by insufficient insulin release from pancreatic β  cells that should compensate for peripheral insulin resistance [1]. Pancreatic β  cell dysfunction and, eventually, death are considered to occur in response to metabolic stresses, which trigger mitochondrial oxidative damages, consequently interfering with glucose metabolism responsible for induction of insulin exocytosis. Accordingly, β  cell should be equipped with efficient defense and adaptive mechanisms against chronic over stimulation of mitochondria, counteracting the adverse effects of oxidative stress. Emerging evidence indicate a hormetic nature of mitochondrial defensive response, that is, a cellular defense adaptation promoted by ROS-triggered signaling. Here, we discuss the putative mitohormetic role of mitochondrial uncoupling protein 2 (UCP2) in β  cells and protection from mitochondrial oxidative damages.

2. Mitochondrial Metabolism, Glucose Sensing, and the Secretory Response

Pancreatic β  cells function as glucose sensors to adjust insulin secretion to blood glucose levels, thereby maintaining glucose homeostasis. Translating nutrient signals into regulated insulin exocytosis relies on optimally tuned mitochondrial function [4]. Although glucose is the chief secretagogue for the β  cell, metabolic profile of mitochondria is modulated by the relative contribution of glucose and lipid products for oxidative catabolism [5]. In the mitochondrion, substrates derived from glucose and fatty acids are oxidized and converted to ATP by the mitochondrial electron transport chain located in the inner mitochondrial membrane. Synthesized ATP is subsequently translocated to the cytosol, triggering insulin exocytosis thanks to calcium elevation secondary to ATP-mediated plasma membrane depolarization [6]. One byproduct of mitochondrial electron transportation is the generation of reactive oxygen species (ROS) [5].

3. Impact of Mitochondrial ROS on β Cell Function

Physiological levels of glucose and fatty acids are essential to normal β  cell function. However, continuous overstimulation of β  cells by these nutrients may be deleterious to β  cell function, a phenomenon referred to as glucolipotoxicity. Accordingly, pancreatic β  cells chronically exposed to hyperglycemic and hyperlipidemic conditions steadily undergo deterioration and ultimately failure of insulin secreting capacity [7]. This loss of β  cell function has been attributed to a variety of mechanisms, most of which having in common the formation of ROS [8–10]. Elevated ROS affect the function and survival of β  cells through a direct oxidation of cellular macromolecules [11, 12] and activation of cellular stress-sensitive signaling pathway [13]. Glucose infusion in rats for 48 h to achieve chronic hyperglycaemia increases mitochondrial islet superoxide and reduces glucose-stimulated insulin secretion [14]. However, it should be noticed that physiological glucose stimulation prevents formation and accumulation of ROS [15, 16]. Increasing glucose usage by pharmacological activation of glucokinase reduces ROS toxicity in insulin-secreting cells [17].

4. Mitochondria Generate ROS

ROS refer to a diverse range of species, such as superoxide (O2∙−), hydrogen peroxide (H2O2), and hydroxyl radical. The biological consequences of ROS rely on the specific species being involved and the physiological or pathological context. Superoxide can be converted to less reactive H2O2 by superoxide dismutase (SOD) and then to O2 and H2O by catalase, glutathione peroxidase (GPX), and peroxiredoxin, which constitute antioxidant defenses [18].

Mitochondrial electron transport chain is a potent producer of O2∙− within cell. Electrons from sugar, fatty acids, and amino acid catabolism accumulate in the electron carriers of the respiratory chain. O2∙− formation is coupled to this electron transportation as a byproduct of normal mitochondrial respiration through one electron reduction of molecular O2∙−. Complexes I and III of the respiratory chain are the major sites for O2∙− generation [19]. In complex I, electrons carried by NADH are accepted by flavin mononucleotide (FMN) and transferred to mobile electron carrier Coenzyme Q (CoQ), with O2∙− formation at FMN. This formation of O2∙− requires FMN in a fully reduced form, which is determined by the NADH/NAD+ ratio [20]. Accumulation of NADH and enhanced ROS formation are favored by slowdown of mitochondrial respiration caused by complex I inhibition (e.g., rotenone effect), secondary to damages of respiratory chain, or because of low cellular ATP demand [21, 22].

Reverse electron transport (RET) from complex II to complex I also generates large amounts of O2∙−. Under conditions of substantial proton motive force, RET occurs when electron supply induces CoQ reduction driving electrons back to complex I, thereby reducing NAD+ to NADH at the FMN site [23]. Succinate and fatty acid oxidation promote high proton motive force along with electron supply to CoQ, giving rise to O2∙− formation under RET. Generation of O2∙− by complex I, in particular through RET, is very sensitive to mitochondrial uncoupling because of the required proton motive force [24]. When inhibited by antimycin, complex III can produce significant amounts of O2∙−, although its production under physiological conditions is only marginal compared to that of complex I [22].

5. Mitochondrial ROS, Friends or Foes?

ROS impact differently on cell function depending on specific reactive oxygen species, their concentrations, and effectiveness of detoxifying systems; thereby, defining signaling or toxic effects of ROS [25]. In insulin-secreting cells, low concentrations of H2O2 have been reported to contribute to the stimulation of insulin secretion [26]. However, exposure to robust concentrations of H2O2 impairs β  cell function [27]. When β  cells are continuously overstimulated by nutrients, accumulation of ROS can overwhelm detoxification systems and induce deleterious effects [8]. This is particularly relevant for pancreatic islets because of the shifted redox balance favored by high metabolic rate, in particular under glucolipotoxic conditions, and relatively weak detoxifying systems [28]. Being a major source of ROS, mitochondria are in the eye of the storm. Specifically, H2O2 exposure to insulin-secreting cells inactivates mitochondria, thereby interrupting mitochondrial signals normally linking glucose metabolism to insulin exocytosis [27]. One single oxidative stress applied for just 10 minutes induces β  cell dysfunction lasting over days, explained by persistent damages in mitochondrial components and accompanied by subsequent generation of endogenous ROS of mitochondrial origin [29]. In the close vicinity of free radical production, mitochondrial inner membrane components are particularly prone to oxidative injuries, such as subunits of electron transport chain complexes and the adenine nucleotide translocase (ANT) [29, 30]. Moreover, some iron-sulfur centers of mitochondrial matrix proteins, among them aconitase, are susceptible to damages induced by direct reaction with O2∙− [31] or nitric oxide [32], leading to impaired mitochondria and β  cell dysfunction.

Because of relatively low antioxidant-enzyme activities in pancreatic islets, enhancing expression of corresponding genes in insulin-secreting cells has been foreseen as potential protective intervention. However, contradictory findings were reported in studies testing this promising approach. The concept is favored by some studies, among them: (i) β  cell-specific overexpression of cytosolic SOD (SOD1) enhances mouse resistance to alloxan-induced diabetogenesis [33]; (ii) adenovirus-mediated overexpression of mitochondrial SOD (SOD2) in isolated islets extends islet function following transplantation into streptozotocin (STZ)-treated nonobese diabetic (NOD) mice [34]; (iii) β  cell-specific overexpression of SOD2 and catalase protects islets from STZ-induced oxidative stress [35]; (iv) β  cell-specific overexpression of glutathione peroxidase in db/db mice improves β  cell volume and granulation [36]. Tempering these promising results, overexpression of antioxidant enzymes, such as catalase and metallothionein, specifically in β  cells of NOD mice increases β  cell death and sensitizes islets to cytokine-induced injuries [37]. The latter results suggest that a mild dominance of host ROS over detoxifying systems might exhibit beneficial effects.

6. Mitochondrial Protection against ROS: Role of UCP2

Mitochondrial uncoupling refers to the dissociation of electron-dependent oxygen consumption to ATP generation on the respiratory chain. The most efficient way to induce mitochondrial uncoupling is to allow protons to circulate freely across the inner mitochondrial membrane, in other words to create a proton leak. In this regard, UCP1 is a professional mitochondrial uncoupler by inducing proton leakage. As a result, the energy contributed by electron flow is dissipated as heat instead of ATP generation. UCP1 expression in brown adipose tissue confers to these fat depots highly thermogenic properties [38]. UCP2 was named after its loose homology (59%) with UCP1 and exhibits heterogeneous tissue expression, including in pancreatic islets [39]. Similarly to UCP1, UCP2 was proposed to induce proton leakage and to dissipate proton motive force [40], consequently limiting ATP production and glucose-stimulated insulin secretion in pancreatic β  cells [41, 42].

Due to tight dependency of complex I on proton motive force for ROS formation, putative uncoupling effects of UCP2 were suggested to compromise mitochondrial ROS generation and associated cell damages [43, 44]. In insulin-secreting cells, a series of in vitro studies have shown that increasing UCP2 expression attenuates ATP synthesis and insulin secretion in response to glucose [41, 42, 45, 46]. Conversely, UCP2 deficiency enhances glucose-stimulated insulin secretion, as shown in islets isolated from both global [47, 48] and β  cell-specific [49] UCP2 knockout mice. In diet-induced T2D mouse model, the lack of UCP2 improves blood glucose levels and insulin secretory capacity [47]. Chronic exposure of INS-1 β  cells to fatty acid decreases the secretory response to glucose, along with UCP2 gene induction and partial mitochondrial uncoupling [50].

Over the last decade, UCP2 effects in β  cells have been tightly correlated with its presumed uncoupling properties and consequences on ATP synthesis from oxidative phosphorylation [41, 42, 45–48]. However, in β  cell-specific UCP2-null mice, the potentiated glucose-stimulated insulin secretion correlates with higher intracellular ROS levels, without any changes on mitochondrial coupling and ATP generation [49]. Moreover, pancreatic islets form global UCP2 knockout mice studied on three congenic backgrounds, as opposed to mixed genetic background [48], exhibit impaired glucose response accompanied by increased ROS production and persistent oxidative stress [51]. Finally, cytokine-induced ROS production is reduced in insulin-secreting cells overexpressing UCP2, independently of uncoupling effects [3]. Collectively, these observations are contradictory regarding putative uncoupling properties of UCP2 and its effects on the secretory function of β  cells. Instead, they suggest a role in defense mechanisms against oxidative stress [52], as shown by induction of UCP2 that prevents cytokine-induced β  cell death through suppression of ROS production [3, 53].

Because UCP2 might play a protective role against ROS, one can hypothesize cooperation and feedback mechanisms with dedicated antioxidant enzymes. In β  cells overexpressing UCP2, the associated sheltering effect against oxidative injuries is not associated with changes in antioxidant enzymes (personal communication from Françoise Assimacopoulos-Jeannet, University of Geneva). Ablation of UCP2 in β  cells favors both ROS formation and induction of H2O2-scavenging GPX, but not of superoxide scavenger SODs [49]. In islets lacking SOD1 or GPX1, UCP2 is upregulated as a protective response against excessive cellular ROS [54]. Conversely, UCP2 is downregulated upon induction of GPX1 in mouse islets [55]. Collectively, these data indicate crosstalk between UCP2 and antioxidant enzymes through unidentified mechanisms.

7. Hormesis, a Stress-Induced Protective Response

Theophrastus Bombastus von Hohenheim, a Swiss pharmacist born in 1493 also named Paracelsus, developed this revolutionary idea at the Renaissance period: “Alle Ding' sind Gift, und nichts ohn' Gift; allein die Dosis macht, daß ein Ding kein Gift ist” freely translated to “the dose makes the poison.” Five centuries later, this notion has been extended to the so-called hormesis. Hormesis is a phenomenon whereby exposure of cells or organs to low levels of a given toxin confers resistance to subsequent contacts to higher concentrations [56]. Accordingly, hormesis describes an adaptive response to continuous cellular stresses. Hormesis is well illustrated by ischemic preconditioning, a situation where short ischemic episodes protect brain and heart from prolonged lack of oxygen and nutrients [57, 58]. Regarding pancreatic β  cells, emerging concepts suggest that efficiency of hormetic responses to detrimental lifestyle factors might set the level of protection, impacting on the progression of T2D [59].

8. Adaptation and Hormesis in β cells

Obesity is a strong risk factor for T2D, appearing in subjects developing β  cell dysfunction and death in response to metabolic and inflammatory stresses [60, 61]. However, about half of obese individuals do not develop diabetes, due to efficient long-term adaptation to insulin resistance by increasing β  cell mass and insulin secretion. In these resistant individuals, β  cells may develop adaptive stress responses to prevent their loss, at least transiently. Peroxisome proliferator-activated receptor alpha (PPARα) is a transcription factor controlling lipid and glucose homeostasis. PPARα-deficient mice on an obese (ob/ob) background develop β  cell dysfunction characterized by reduced islet area and glucose response [62]. Human islets treated with PPARα agonist are protected against fatty acid-induced impairment of glucose-induced insulin secretion and apoptosis [62]. This indicates that PPARα could be an adaptive candidate in β  cells under pathological conditions, such as lipid-induced dysfunction [63].

Converging evidence suggest that stresses can induce specific responses rendering β  cells more resistant to the stress-molecule, or even to other toxins. Pre-exposure to low dose IL-1β renders β  cells less susceptible to toxin-induced cell necrosis and to radical-induced damages, though with a loss of normal phenotype [64]. Moreover, islets from pancreatectomized hyperglycemic rats exhibit reduced sensitivity to STZ, an effect associated with induction of protective antioxidant and antiapoptotic genes during chronic hyperglycemia [65]. Finally, islets from GK/Par rat (non-obese model of T2D) also show strong resistance to toxic effects of exogenous ROS, secondary to an adaptive response to the diabetic milieu [66]. Thus, β  cells possess hormetic mechanisms in response to inflammatory and metabolic stresses. Stressors are not merely toxic; they can also prime the stressed cell to future pathogenic challenges by rendering them more resistant.


9. Mitochondrial Adaptation and Hormesis, or Mitohormesis, in β Cells

Mitochondrial adaptation and hormesis, or mitohormesis, originally referred to the hypothetical model of cell preservation in response to ROS-induced stresses originating from mitochondria [67]. The concept was substantiated by findings in C. elegans revealing that glucose restriction activates mitochondria and ROS formation, promoting hormetic extension of life span [68]. In conflict with Harman's free radical theory of aging [69], protective effects depend on mitochondrial ROS formation inducing an adaptive response, in turn conferring increased stress-resistance. This might ultimately give rise to long-term cell preservation. In agreement with this model, calorie restriction extend life span in different organisms by increasing mitochondrial ROS production [70]. In pancreatic β  cells, mitohormetic response is suggested by adaptation to dietary fat-induced insulin resistance attributed to increased mitochondrial function [71], an effect correlating with elevated ROS levels secondary to fatty acid treatment of insulin-secreting cells [10].

The nature of mitohormesis in insulin-secreting cells can be studied when cells recover from a single transient exposure to sublethal H2O2. We previously reported that INS-1E β  cells and rat islets subjected to a 10 min H2O2 exposure exhibit impaired secretory response associated with interrupted mitochondrial signals measured right after stress [27]. Loss of mitochondrial function occurs within the first minutes of oxidative stress [27], as revealed by collapse of mitochondrial membrane potential (Figure 1(a)). Monitored concomitantly on the same cells, gradual discontinuous mitochondrial network is observed, eventually exhibiting some globular patterns 60 min after stress (Figure 1(b)). These phenomena were not observed in control nonstressed cells (Figures 1(c) and 1(d)). Then, the question is whether such oxidative stress results in prolonged mitochondrial damages, recovery of cell function, or improved resistance to stress. After a 3-day recovery period following the 10 min stress, we observed increased mitochondrial H2O2 formation and persistence of mitochondrial dysfunction altering metabolism-secretion coupling [29]. The ROS-induced endogenous H2O2 generation contributes to prolongation of oxidative attacks days after exposure to exogenous H2O2. This is accompanied by increased expression of genes participating to recovery of mitochondrial function, detoxification, and cell survival; such as subunits of mitochondrial electron transport chain complexes and antioxidant enzymes [29]. Mitochondrial defects induced by acute 10 min oxidant exposure are carried on to daughter cells. These cells ultimately achieve gradual turnover of mitochondrial components enabling recovery of their function in the following weeks of culture period [29]. Three weeks after transient oxidant exposure, those insulin-secreting cells respond normally to physiological stimuli. Remarkably, the recovered cells are more resistant than naïve cells to a new exogenous oxidative stress. This beneficial “memory” of mitochondrial oxidative injury represents mitohormetic property and is associated with a higher UCP2 gene expression 3 weeks post-stress [29], suggesting a protective role for UCP2.
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Figure 1

Simultaneous monitoring of mitochondrial membrane potential and morphology in INS-1E β  cell under transient oxidative stress. Real-time imaging of INS-1E cells by simultaneous fluorescence recordings of mitochondrial potential (ΔΨm) by TMRE (a and c) and mitochondrial morphology by ΔΨm-independent mito-eYFP (b and d) as described [2]. (a), Signals recorded before oxidant exposure (before-stress), during the 10 min 200 μM H2O2 exposure (stress), and after neutralization of extracellular H2O2 by the addition of 100 U/mL catalase (after-stress). (b), Corresponding mitochondrial morphology monitored simultaneously with ΔΨm shown in (a). (c) and (d) show control nonstressed cells.
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10. Does UCP2 Participate to Mitohormesis in β cell?

As described above, some studies have highlighted UCP2 as a protective element under stress conditions [3, 29, 49, 52], possibly implicated in β  cell mitohormetic response. To address this question, INS-1 β  cells with doxycycline-inducible overexpression of human UCP2 [3] were challenged with an oxidative stress by exposure to 200 μM H2O2 for 10 min as described [27, 29]. Consistent with previous report [3], increased expression of UCP2 (Figure 2(a)) did not alter mitochondrial coupling (Figures 2(b) and 2(c)). Indeed, INS-1 cells with induced UCP2 overexpression exhibited similar respiration upon glucose stimulation compared to non induced cells (Figure 2(c)). Moreover, state 3 respiration measured on isolated mitochondria stimulated with succinate plus ADP was even slightly higher versus controls (Figure 2(b)). INS-1E cells with basal UCP2 expression are highly sensitive to oxidative stress regarding mitochondrial respiration, exhibiting marked reduction of state 3 (−59% versus control nonstressed cells) 3 days after oxidative stress [29]. On the contrary, cells overexpressing UCP2 did not show any impairment of oxygen consumption at day 3 after stress, as shown both on isolated mitochondria and intact cells (Figures 2(b) and 2(c), resp.). Acute oxidant exposure did not further elevate UCP2 protein levels 3 days after stress in UCP2-induced cells (Figure 2(a)). Collectively, these observations support the concept that UCP2 upregulation observed previously as a mitohormetic response [29] can serve as defense mechanism against mitochondrial oxidative damages (Figure 2).
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Figure 2

Effects of UCP2 overexpression and oxidative stress on mitochondrial respiration in INS-1 cells. UCP2 was induced in INS-1 cells (hUCP2 INS-1-r9, [3]) by 250 ng/mL doxycycline (+Dox) 2 days before oxidative stress and during stress period. (a) Immunoblotting showing UCP2 protein levels in noninduced (−Dox) versus induced (+Dox) INS-1 cells and nonstressed (−stress) versus stressed cells (+stress, 200 μM H2O2 for 10 min 3 days before analysis). Cytochrome oxidase (COX IV) is shown as control for inner mitochondrial protein. (b) O2 consumption measured on mitochondria isolated from INS-1 cells 3 days after-stress. Respiration was induced by 5 mM succinate (Succinate) followed by addition of 150 μM ADP (Succinate + ADP). (c) O2 consumption measured on intact INS-1 cells stimulated by 15 mM glucose (Glc), compared to basal respiration at 2.5 mM glucose (Basal). Data are means ± SE of 3 independent experiments expressed as nmol O2/min per 100 μg mitochondrial protein (b) or nmol O2/min per 106 cells (c) normalized to basal respiration of controls (no Dox, no stress). *P < 0.05 versus basal O2 consumption of corresponding condition.

11. Conclusions

In pancreatic β  cells, stress-response hormesis can develop under different metabolic insults, such as lipotoxicity, cytokines, or ROS. In particular, oxidative stress induces mitohormesis, rendering mitochondria more resistant to oxidative attacks. Various studies in this field reported conflicting results. However, converging evidence points to UCP2 as a marker of mitohormesis, this protein being upregulated following stress conditions. Moreover, overexpression of UCP2 in naïve cells lacking hormesis adaptation is sufficient to confer resistance to oxidative stress (Figure 2). The exact function of UCP2 is still unknown, although its partial homology with uncoupling UCP1 protein suggests a functional link with the electron transport chain. Whether treatment with UCP2 inducers, such as glutamine [72], could promote mitohormesis and protect β  cells under metabolic stress remains to be determined.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3458419/

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November 2016, Volume 54, Issue 2, pp 276–283 | Cite as
A hypothetical model to solve the controversy over the involvement of UCP2 in palmitate-induced β-cell dysfunction

   Authors

   Alaa Shaheen
       1Email author
   Ahmad M. A. Aljebali
       2

   1.Kafr El-Sharakwa Medical Center, Kafr El-SharakwaAgaEgypt
   2.Department of Zoology, Faculty of ScienceOmar Al Mukhtar UniversityBaydaLibya

Review
First Online: 04 August 2016

Abstract

The aim of this article is to solve an existing controversy over the involvement of uncoupling protein-2 in the impairment of glucose-stimulated insulin secretion induced by chronic exposure of β-cells to palmitate. We analyzed and compared the results of studies that support and that deny the involvement of uncoupling protein-2 in this impairment. We observed that this impairment could occur in multiple stages. We provide a model in which palmitate-induced impairment of glucose-stimulated insulin secretion is proposed to occur in two stages, early stage and late stage, depending on the integrity of electron supply (glycolysis and Krebs cycle) and transport system through electron transport chain after palmitate treatment. Prolonged exposure of β-cells to palmitate can impair this system. Early-stage impairment occurs due to uncoupling by uncoupling protein-2 when this system is still intact. When this system becomes impaired, late-stage impairment occurs mainly due to reduced glucose-stimulated adenosine triphosphate production independent of uncoupling by uncoupling protein-2. The change in glucose-stimulated oxygen uptake after palmitate treatment reflects the integrity of this system and can be used to differentiate between the two stages. Some β-cells lines and islets appear to be more resistant to palmitate-induced impairment of electron supply and transport system than others, and therefore early stage is prominent in the more resistant cell lines and less prominent or absent in the less resistant cell lines. This may help to resolve the pathogenesis of diabetes and to monitor the progression of palmitate-induced β-cell dysfunction.

https://link.springer.com/article/10.1007/s12020-016-1051-1

More on UCP2
http://www.biomed.cas.cz/physiolres/pdf/63%20Suppl%201/63_S73.pdf

Flavins:
https://en.wikipedia.org/wiki/Flavin_adenine_dinucleotide

Function

Flavoproteins utilize the unique and versatile structure of flavin moieties to catalyze difficult redox reactions. Since flavins have multiple redox states they can participate in processes that involve the transfer of either one or two electrons, hydrogen atoms, or hydronium ions. The N5 and C4a of the fully oxidized flavin ring are also susceptible to nucleophilic attack.[14] This wide variety of ionization and modification of the flavin moiety can be attributed to the isoalloxazine ring system and the ability of flavoproteins to drastically perturb the kinetic parameters of flavins upon binding, including flavin adenine dinucleotide (FAD).

The number of flavin-dependent protein encoded genes in the genome (the flavoproteome) is species dependent and can range from 0.1% - 3.5%, with humans having 90 flavoprotein encoded genes.[15] FAD is the more complex and abundant form of flavin and is reported to bind to 75% of the total flavoproteome[15] and 84% of human encoded flavoproteins.[16] Cellular concentrations of free or non-covalently bound flavins in a variety of cultured mammalian cell lines were reported for FAD (2.2-17.0 amol/cell) and FMN (0.46-3.4 amol/cell).[17]

FAD has a more positive reduction potential than NAD+ and is a very strong oxidizing agent. The cell utilizes this in many energetically difficult oxidation reactions such as dehydrogenation of a C-C bond to an alkene. FAD-dependent proteins function in a large variety of metabolic pathways including electron transport, DNA repair, nucleotide biosynthesis, beta-oxidation of fatty acids, amino acid catabolism, as well as synthesis of other cofactors such as CoA, CoQ and heme groups. One well-known reaction is part of the citric acid cycle (also known as the TCA or Krebs cycle); succinate dehydrogenase (complex II in the electron transport chain) requires covalently bound FAD to catalyze the oxidation of succinate to fumarate by coupling it with the reduction of ubiquinone to ubiquinol.[11] The high-energy electrons from this oxidation are stored momentarily by reducing FAD to FADH2. FADH2 then reverts to FAD, sending its two high-energy electrons through the electron transport chain; the energy in FADH2 is enough to produce 1.5 equivalents of ATP[18] by oxidative phosphorylation. There are also redox flavoproteins that non-covalently bind to FAD like Acetyl-CoA-dehydrogenases which are involved in beta-oxidation of fatty acids and catabolism of amino acids like leucine (isovaleryl-CoA dehydrogenase), isoleucine, (short/branched-chain acyl-CoA dehydrogenase), valine (isobutyryl-CoA dehydrogenase), and lysine (glutaryl-CoA dehydrogenase).[19] Additional examples of FAD-dependent enzymes that regulate metabolism are glycerol-3-phosphate dehydrogenase (triglyceride synthesis) and xanthine oxidase involved in purine nucleotide catabolism.[20] There are other noncatalytic roles that FAD can play in flavoproteins such as structural roles, or involved in blue-sensitive light photoreceptors that regulate biological clocks and development, generation of light in bioluminescent bacteria.[19]
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A radical explanation for glucose-induced β cell dysfunction
Michael Brownlee

First published December 15, 2003 - More info

   Abstract

   The development of type 2 diabetes requires impaired β cell function. Hyperglycemia itself causes further decreases in glucose-stimulated insulin secretion. A new study demonstrates that hyperglycemia-induced mitochondrial superoxide production activates uncoupling protein 2, which decreases the ATP/ADP ratio and thus reduces the insulin-secretory response. These data suggest that pharmacologic inhibition of mitochondrial superoxide overproduction in β cells exposed to hyperglycemia could prevent a positive feed-forward loop of glucotoxicity that drives impaired glucose tolerance toward frank type 2 diabete

   The diabetes epidemic and its consequences

   Diabetes and impaired glucose tolerance currently affect an estimated 29 million people in the US (1). For those born in 2000, the estimated lifetime risk of developing diabetes is 36% (2). People with diabetes have large reductions in life expectancy and in quality of life (2), due to diabetes-specific microvascular complications in the retina, renal glomerulus, and peripheral nerve, and to extensive atherothrombotic macrovascular disease affecting arteries that supply the heart, brain, and lower extremities. It has been estimated that up to 70% of patients with acute myocardial infarction have either diabetes or impaired glucose tolerance (3).

   As a consequence of its microvascular pathology, diabetes is the leading cause of blindness, end-stage renal disease, and a variety of debilitating neuropathies. Diabetics are the fastest-growing group of renal dialysis and transplant recipients, and in the US, their 5-year survival rate is only 21 percent, worse overall than that for all forms of cancer combined. Over 60% of diabetic patients suffer from neuropathy, which accounts for 50% of all nontraumatic amputations in the US (4).

   Insulin resistance, β cell function, and the natural history of type 2 diabetes

   Both genetic and environmental factors (mainly obesity) contribute to insulin resistance. Recent work with tissue-conditional knockouts of both Glut4 and the insulin receptor in mice have shown that adipose tissue plays a central role in the pathogenesis of insulin resistance, and that there is significant cross-talk among insulin target tissues (5).

   Insulin resistance induces a compensatory increase in β cell mass, which in many people results in normal glucose levels. In other people, intrinsic defects in this compensatory β cell response prevent adequate compensation, and impaired glucose tolerance or type 2 diabetes occurs. Impaired glucose tolerance leads to type 2 diabetes in a significant number of people, and type 2 diabetes, in turn, becomes progressively unresponsive to oral antidiabetic agents, until treatment with insulin is necessary.

   Glucose toxicity and the decline of β cell function

   Hyperglycemia is widely recognized as the causal link between diabetes and diabetic complications (6). More recently, adverse effects of hyperglycemia on insulin target tissues and on pancreatic β cells have also been recognized, and this phenomenon has been termed “glucotoxicity.” Chronic hyperglycemia has been shown to induce multiple defects in β cells, including early decreases in glucose-stimulated insulin secretion, and late irreversible changes in insulin-gene transcription and β cell mass (7, . In patients with impaired glucose tolerance, lowering of glucose levels dramatically reduces the progression to type 2 diabetes (9), suggesting that glucotoxicity plays a major role in this transition.

   The central role of mitochondria in glucose-stimulated insulin secretion

   Pancreatic β cells sense the ambient plasma glucose concentration because (a) the high-Km glucose transporter GLUT2 facilitates rapid equilibration across the cell membrane, and (b) the high-Km hexokinase isoform glucokinase allows the generation of a proportionate signal through glycolytic and mitochondrial metabolism of glucose (10). This results in an increased ATP/ADP ratio, which closes an ATP-sensitive potassium channel in the cell membrane, thereby depolarizing the cell membrane and activating a voltage-gated calcium channel. The resultant influx of calcium triggers secretion of insulin granules (Figure 1).
   Model of glucose-stimulated insulin secretion in the pancreatic β cell. FolFigure 1

   Model of glucose-stimulated insulin secretion in the pancreatic β cell. Following phosphorylation by glucokinase (GK), glucose is converted to pyruvate by glycolysis. Pyruvate enters the mitochondria and fuels the TCA cycle, resulting in the transfer of reducing equivalents to the respiratory chain, hyperpolarization of the mitochondrial membrane, and ATP generation. Subsequent closure of KATP channels depolarizes the cell membrane, which opens voltage-gated calcium channels, increasing the concentration of cytosolic calcium ([Ca2+]c). This influx of calcium triggers insulin release from the cell. Figure modified with permission from Nature (17). Pyr, pyruvate; Δψm, mitochondrial membrane potential; Δψc, cell membrane potential.

   The glucose-sensitive increase in the ATP/ADP ratio is caused by greater electron flux through the mitochondrial electron-transport system (Figure 2). Pyruvate derived from glycolysis is transported into the mitochondria, where it is oxidized by the tricarboxylic acid (TCA) cycle to produce NADH and reduced flavin adenine dinucleotide (FADH2). Mitochondrial NADH and FADH2 provide energy for ATP production via oxidative phosphorylation by the electron-transport chain.
   Effect of hyperglycemia on mitochondrial electron-transport chain functionFigure 2

   Effect of hyperglycemia on mitochondrial electron-transport chain function in the pancreatic β cell. Hyperglycemia increases production of electron donors from the tricarboxylic acid (TCA) cycle (NADH and FADH2). This increases the membrane potential (ΔμH+), because protons are pumped across the mitochondrial inner membrane in proportion to electron flux through the electron-transport chain. Inhibition of electron transport at Complex III by increased ΔμH+ increases the half-life of free radical intermediates of coenzyme Q, which reduce O2 to superoxide. Krauss and colleagues (13) have demonstrated that hyperglycemia-induced mitochondrial superoxide activates UCP2-mediated proton leak, thus lowering ATP levels and impairing glucose-stimulated insulin secretion. Figure modified with permission from Nature (15). Pi, inorganic phosphorus.

   Electron flow through the mitochondrial electron-transport chain is carried out by four inner membrane–associated enzyme complexes, plus cytochrome c and the mobile carrier coenzyme Q. NADH derived from the TCA cycle donates electrons to Complex I. Complex I ultimately transfers its electrons to coenzyme Q. Coenzyme Q is also reduced by electrons donated from several FADH2-containing dehydrogenases, such as the TCA cycle succinate:ubiquinone oxidoreductase (Complex II). Electrons from reduced coenzyme Q are then transferred to Complex III. Electron transport then proceeds through cytochrome c, Complex IV, and, finally, molecular oxygen.

   Electron transfer through Complexes I, III, and IV generates a proton (voltage) gradient. Much of the energy of this voltage gradient (ΔμH+) is used to generate ATP, as the collapse of the proton gradient through ATP synthase (Complex V) drives the ATP synthetic machinery. This energy can also be dissipated as heat through the mediation of uncoupling proteins (UCPs). When the electrochemical potential difference generated by this proton gradient is high, electron transport in Complex III is partially inhibited, resulting in a backup of electrons to coenzyme Q and their donation to molecular oxygen, leading to increased generation of the free radical superoxide.

   Excess activation of UCP2 by superoxide causes β cell dysfunction

   How does hyperglycemia cause β cell dysfunction? The answer to this question has not been clear. Although elegant studies by Zhang and coworkers (11) have suggested that the level of UCP2 gene expression in β cells is an important determinant, conflicting data in the literature suggested that changes in expression alone are not the whole story (12).

   In this issue of the JCI, Krauss et al. (13) now show that the key missing element in hyperglycemia-induced β cell dysfunction is activation of UCP2. This UCP2 activation is accomplished by hyperglycemia-induced superoxide formation by the mitochondrial electron-transport chain. In vitro studies have suggested that superoxide could activate UCPs (14), but these data have been somewhat controversial. Now, Krauss et al. demonstrate that under physiologic conditions, endogenous superoxide generated by hyperglycemia activates UCP2. This activation diverts energy away from ATP synthesis (Figure 2), thereby decreasing the ATP/ADP ratio. This results in impaired glucose-stimulated insulin secretion (13).

   Therapeutic implications and future directions

   The data reported by Krauss et al. (13) have enormous clinical implications. They suggest that pharmacologic inhibition of mitochondrial superoxide overproduction in β cells exposed to hyperglycemia could prevent the positive feed-forward loop of glucotoxicity that pushes impaired glucose tolerance into frank type 2 diabetes. Since the incidence and rate of progression of diabetic complications increase in proportion to the level of hyperglycemia (6), prevention or even significant delay of the transition from impaired glucose tolerance to type 2 diabetes would have a major positive impact on diabetes-associated morbidity and mortality. Interestingly, the process of hyperglycemia-induced overproduction of superoxide by the mitochondrial electron transport chain also appears to be the central mechanism underlying all the major molecular mechanisms implicated in glucose-mediated vascular damage (15, 16). Thus, a common unifying mechanism may underlie hyperglycemic damage in β cells, endothelial cells, and other targets of glucotoxicity.

   An important aspect of β cell glucose toxicity that remains to be clarified is the possible role of hyperglycemia-induced superoxide production by the mitochondria and subsequent activation of UCP2 in long-term loss of β cell mass. Reactive oxygen leads to irreversible decreases in the level of the transcription factor PDX-1 (, which is critical for insulin-gene expression, and also for β cell neogenesis.

   Future studies on the role of altered mitochondrial production of both superoxide and ATP, based on the work of Krauss et al. (13), may also lead to a better understanding of the mechanisms underlying the inexorable decline of β cell insulin production and β cell mass with increased duration of type 2 diabetes.

   Acknowledgments

   Michael Brownlee is supported by grants from the NIH, the American Diabetes Association, and the Juvenile Diabetes Research Foundation.

https://www.jci.org/articles/view/20501?utm_campaign=cover-page&utm_content=short_url&utm_medium=pdf&utm_source=content

Flavins and cross linked proteins with Genipin:
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Novel Biomedical Applications of Crosslinked Collagen
The biomedical potential of natural collagen is limited by its poor mechanical strength, thermal stability, and enzyme resistance, but exogenous chemical, physical, or biological crosslinks have been used to modify the molecular structure of collagen to minimize degradation and enhance mechanical stability.

Crosslinked collagen-based scaffolds have been extensively studied for tissue engineering to promote tissue regeneration or repair. Nanoparticles act as crosslinking agents for collagen stabilization as well as functionalized carriers for crosslinking to collagen scaffolds for novel biomolecular applications.

Genipin cross-linked type II collagen/chondroitin sulfate composite hydrogel-like cell delivery system induces differentiation of adipose-derived stem cells and regenerates degenerated nucleus pulposus.

https://www.cell.com/trends/biotechnology/fulltext/S0167-7799(18)30303-2
http://rsif.royalsocietypublishing.org/content/royinterface/14/129/20170014.full.pdf

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Mar Drugs. 2015 Dec; 13(12): 7314–7338.
Published online 2015 Dec 11. doi: 10.3390/md13127068
PMCID: PMC4699241
PMID: 26690453

Genipin-Crosslinked Chitosan Gels and Scaffolds for Tissue Engineering and Regeneration of Cartilage and Bone
(more at link:   https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4699241/ )
Riccardo A. A. Muzzarelli,1,* Mohamad El Mehtedi,2 Carlo Bottegoni,3 Alberto Aquili,3 and Antonio Gigante3
Hitoshi Sashiwa, Academic Editor
Author information Article notes Copyright and License information Disclaimer
PMC

Abstract

The present review article intends to direct attention to the technological advances made since 2009 in the area of genipin-crosslinked chitosan (GEN-chitosan) hydrogels. After a concise introduction on the well recognized characteristics of medical grade chitosan and food grade genipin, the properties of GEN-chitosan obtained with a safe, spontaneous and irreversible chemical reaction, and the quality assessment of the gels are reviewed. The antibacterial activity of GEN-chitosan has been well assessed in the treatment of gastric infections supported by Helicobacter pylori. Therapies based on chitosan alginate crosslinked with genipin include stem cell transplantation, and development of contraction free biomaterials suitable for cartilage engineering. Collagen, gelatin and other proteins have been associated to said hydrogels in view of the regeneration of the cartilage. Viability and proliferation of fibroblasts were impressively enhanced upon addition of poly-l-lysine. The modulation of the osteocytes has been achieved in various ways by applying advanced technologies such as 3D-plotting and electrospinning of biomimetic scaffolds, with optional addition of nano hydroxyapatite to the formulations. A wealth of biotechnological advances and know-how has permitted reaching outstanding results in crucial areas such as cranio-facial surgery, orthopedics and dentistry. It is mandatory to use scaffolds fully characterized in terms of porosity, pore size, swelling, wettability, compressive strength, and degree of acetylation, if the osteogenic differentiation of human mesenchymal stem cells is sought: in fact, the novel characteristics imparted by GEN-chitosan must be simultaneously of physico-chemical and cytological nature. Owing to their high standard, the scientific publications dated 2010–2015 have met the expectations of an interdisciplinary audience.
Keywords: chitosan, genipin, tissue engineering, biomedical uses, biochemical properties

1. Introduction and Scope

The most important applications of genipin in conjunction with chitosan are the preparation of elastic cartilage substitutes, the manufacture of carriers for the controlled release of drugs, the encapsulation of biological products and living cells, the biofabrication of tissues such as muscle and arterial walls, and the dressing of wounds in animals and humans. Genipin has definitely replaced glutaraldehyde and other crosslinkers mainly owing to the expanded biochemical significance of the genipin-crosslinked hydrogels (GEN-chitosan), but also owing to the advantages of stability, biocompatibility, well defined chemistry and general safety of the products whose manipulation, handling and quality assessment are currently done with advanced techniques and clearly defined protocols that guarantee absence of cytotoxicity.

1.1. Characteristic Properties of Genipin

The first review article on genipin was published in 2009 [1], but two early papers [2,3] on the isolation and structure of genipin deserve to be cited here because they are valid examples of exhaustive research and scientific soundness obtained with advanced equipment. Working in the early 1950s with Syntex S.A. in Mexico City, Carl Djerassi first synthesized 19-nor-17α-ethynyltestosterone (norethisterone). This steroid, derived from inedible yams of a wild plant Dioscorea, proved to be the most effective orally administered progestational agent discovered at that time. This was the start of a very fortunate research program that led to hundreds and hundreds of journal articles and patents. Syntex could boast of possessing the most advanced equipment such infrared and NMR spectrometers, at a time when neither the pharmaceutical industries, as Djerassi wrote, “nor my Alma Mater, the University of Wisconsin, had such equipment which proved to be enormously useful for steroid research” [4]. The work done paved the way to the first synthesis of a steroid contraceptive in 1953, “the Pill” that changed the habits of mankind [4]. In the frame of said research program, several other plants were investigated and several extracts were described scientifically with avalanches of data, thus starting the evolution of the empirical medicaments of the traditional medicine into scientifically assessed plant extracts, as it was the case of Genipa americana and Gardenia jasminoides Ellis that yielded commercial genipin. A more recent example is the food supplement from Serenoa repens (Permixon Pierre Fabre, Giem, France). Thus genipin is a part of the cultural legacy from Carl Djerassi.

Because it is recognized that genipin, rather than geniposide, is the main compound that exerts pharmacological activities [5], there is interest in its isolation and purification for use in therapy and in the manufacture of food commodities [6]. Genipin is choleretic; anti-depressant; antidiabetic; anticancer; antithrombotic; anti-inflammatory; antibacterial; gastro-, hepato-, and neuro-protective [7]; it prevents lipid peroxidation; and it protects the hippocampal neurons against the Alzheimer’s amyloid beta protein [8].

The biochemical significance of genipin emerges in fact from a number of research projects in the areas of the therapies of vascular diseases, diabetes, hepatic dysfunctions, as well as biofabrication, dentistry, ophthalmology, wound healing and regeneration of nerve, tendon and other tissues, just to mention a few [9,10,11,12,13,14,15,16,17,18,19,20].

The main specifications of genipin (CAS 6902-77. are the following: white crystalline powder soluble in water, methanol, ethanol and acetone; chemical formula C11H14O5; molar mass 226.226 g/mol; melting point 120–121 °C; UV (CH3OH) λmax 240 nm.

Although a minor molar ratio of genipin to chitosan is necessary for crosslinking the latter or other aminated polymers, genipin is expensive because during its preparation a large quantity is wasted owing to homopolymerization. Therefore Fusarium solani was screened as an efficient source of β-glucosidase for genipin preparation from geniposide by extraction with a 10-L ethyl acetate-water biphasic system. HPLC data indicated that immediately after hydrolysis genipin was extracted from the aqueous phase into ethyl acetate thus escaping homopolymerization that would have been unavoidable in the aqueous phase. With Fusarium solani ACCC 36223, genipin in the ethyl acetate phase was 15.7 g/L, corresponding to yields of 0.65 g·L−1·h−1. Efficient substrate conversion and side reactions elimination were the key aspects of the advances made; moreover genipin was easily purified via the sole recrystallization. These most recent conceptual and technical approaches will certainly permit a more convenient production at lower price [21]. The available methods for recovery of genipin and geniposide were described, as well as the methods for genipin and geniposide identification and quantification based on instrumental analyses. Analytical methods for genipin were implemented in view of effective recovery protocols [22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37].

1.2. Characteristic Properties of Chitosans

Chitins and chitosans of various origins along with some of their derivatives are today protagonists in the scenario of wound healing, tissue engineering, gene therapy, and other advanced biomedical areas, owing to their unique properties. Basic information on these polysaccharides, relevant to the title topic, can be found in books and review articles [38,39,40,41,42,43,44,45,46,47,48,49].

Being biocompatible, non-toxic, stable, sterilizable and biodegradable, chitosan exhibits most appreciated properties that enhance its versatility in the biomedical and biotechnological fields, such as immunostimulation, activation of macrophages, mucoadhesion, antimicrobial activity, and well assessed chemistry [50]. Moreover, chitosan can also be prepared in a variety of forms, namely hydrogels and xerogels, powders, beads, films, tablets, capsules, microspheres, microparticles, nanofibrils, textile fibers, and inorganic composites. Chitosan is today a protagonist in advanced fields, for example it is a high performing non-viral vector for DNA and gene delivery.

1.3. Genipin-Crosslinked Chitosan Hydrogels

Genipin reacts promptly with chitosan, as well as with proteins or amines in general [51], as a bi-functional crosslinking compound, thus producing blue-colored fluorescent hydrogels. The reaction between chitosan and genipin is well understood for a variety of experimental conditions and yields composites and complexes with no cytotoxicity for human and animal cells (Figure 1).

An external file that holds a picture, illustration, etc. Object name is marinedrugs-13-07068-g001.jpg
Figure 1

Genipin crosslinks chitosan spontaneously at a quite small molar ratio. On the right, two chitosan chains (represented by their structural units) react covalently with one mole of genipin to yield two newly formed chemical functions, namely the monosubstituted amide and the tertiary amine.

Chitosan nanoparticles crosslinked with genipin were prepared by reverse microemulsion that allowed obtaining highly monodisperse nanogels. Whilst 13C·NMR provides evidence of the reaction as shown in Figure 2, the incorporation of genipin into chitosan was also confirmed and quantitatively evaluated by 1H·NMR [52,53]. The hydrodynamic diameter of the genipin-chitosan nanogels ranged from 270 to 390 nm and no difference was found when the crosslinking degree was varied. The hydrodynamic diameters of the nanoparticles increased slightly at acidic pH. TEM data indicated that the nanoparticles had average diameters of from 3 to 20 nm and that they are spherical, have nearly uniform particle size distribution, and are not affected by particle agglomeration; these being interesting qualities for drug delivery. The progressive protonation of the amino groups as pH decreases was confirmed by measuring the electrokinetic potential of the nanogels. The variation of water solubility of chitosan due to the crosslinking with genipin is a compromise between the decrease of crystallinity and the elastic force within the generated network. There was an insignificant variation of the average hydrodynamic diameter of the nanoparticles with pH, but a large progressive variation of zeta potential (from +30 to −7 mV) in the pH interval 4–9, indicative of the fact that these hydrogels are pH-sensitive [53].

An external file that holds a picture, illustration, etc. Object name is marinedrugs-13-07068-g002.jpg
Figure 2

13C NMR spectrum of chitosan film crosslinked with genipin 0.10%. At 23.0 ppm the resonance signal of alkyl groups in the crosslinked chitosan was attributed to the chitosan + genipin linkage. The signal at 170.5 ppm, assigned to the ester group of plain genipin, disappeared as a consequence of the reaction, thus the resonance at 181.3 ppm is assigned to the amide generated by the reaction between the amino group of chitosan and the ester group of genipin.

Biodegradable polymers such as chitosan need to be crosslinked in order to modulate their general properties and to last long enough for delivering drugs over a desired period of time. Certain chemicals have been used for crosslinking chitosan such as glutaraldehyde, tripolyphosphate, ethylene glycol, diglycidyl ether and diisocyanate. However, the synthetic crosslinking reagents are all more or less cytotoxic and may impair the biocompatibility of a chitosan delivery system. Hence, efforts were made to provide crosslinking reagents that have low cytotoxicity and that form stable and biocompatible crosslinked products, for example tyrosinase was used to mediate quinone tanning of chitosans [54].

Chitosan can be used as a scaffold for tissue regeneration in porous or film form. However, as a porous scaffold it exhibits mechanical weakness: for example, when mouse fibroblasts are cultured on a porous chitosan scaffold, the narrow site of attachment and general weakness drastically depress the adhesion, and the cells tend to become round thus losing their prerogatives. On the other hand, when the cells are anchored to a surface endowed with stiffness, the cellular growth and differentiation rates are better, migration and aggregation become evident, and the cellular shapes favored by the support are those associated with proliferation, differentiation, and apoptosis.

A number of research teams are interested in using genipin to obtain stable and biocompatible chitosan hydrogels. Yao et al. indicated that the fibroblasts adhering to the GEN-chitosan scaffolds were 2.29 times more numerous compared to the fibroblasts on the pristine scaffold surface, the characteristic modulus of a genipin-crosslinked chitosan surface, ≈2.3 GPa, being nearly the double of the control [55]. A genipin crosslinked scaffold retains its own chemical composition while having significantly larger Young’s modulus and hardness. Thus, the mechanical properties of a porous chitosan scaffold in film form are enhanced by genipin. In turn the enhanced general properties induce cell adhesion and proliferation in the modified porous scaffold. Interestingly, the pore size and mechanical properties of chitosan can be tuned for specific tissue regeneration.

Moreover, survival and proliferation of L929 fibroblasts were up-regulated after crosslinking with genipin, especially 0.5% genipin solutions. Analogous data were presented by Bao et al. for carboxymethylchitosan crosslinked with genipin in an article devoted to the mechanical properties of that class of hydrogels and their biocompatibility [56].

GEN-Chitosan hydrogels were prepared by incubation of solutions containing mixtures of genipin and chitosan in different ratios. They turned dark blue and became opaque, owing to exaggerated quantity of genipin. Upon lyophilization they yielded macroporous sponge-like scaffolds [57]. The in vitro cytocompatibility of hydrogels was demonstrated with L929 fibroblasts by the MTT method, in agreement with other authors [58]. The macroporous structure of the chitosan hydrogels could be tailored so that they enhanced their storage modulus, and also altered their hydrophilicity and swelling properties. The crosslinked hydrogels did not induce cytotoxic effects. Flow cytometry showed that fibroblasts possessed good viability on the surface of crosslinked gels (88.4%–90.9%) close to that on blank plates (93.7%) and chitosan films (92.8%). There was no quantitative difference in apoptotic or dead cells, thus crosslinking had little influence on viability, but the stiffness was the most important parameter influencing cell growth and made it possible to switch the cells either toward round or spreading shapes upon modulation of the hydrogel stiffness.

Safety of use was amply confirmed, thus chitosan composites have been taken into consideration in view of the production of biomaterials with desirable physicochemical and biological properties for tissue engineering. It is worth emphasizing that the safety of genipin has been demonstrated by a number of approaches, for example although the blue pigments derived from genipin and aminoacids have been used as value-added colorants for foods over the last 20 years in Eastern Asia, their biochemical significance has been explored as recently as in 2012 by Wang, Q.S. et al. who demonstrated that blue pigments did not only inhibit iNOS and COX-2 gene expression induced by LPS and subsequent production of NO and PGE2, but reduced the production of cytokines (TNF-α, IL-6) induced by LPS in macrophages by the inhibition of signaling cascades leading to the activation of NF-κB [33]. Therefore, the results of recent studies provide strong scientific evidence for blue pigments to be developed as nutraceuticals for prevention and treatment of chronic inflammatory diseases. Nitric oxide is recognized as a mediator and regulator of inflammatory responses being produced in high amounts by iNOS in activated inflammatory cells. Blue pigments were found to inhibit LPS-induced NO production (Figure 3). Also the mRNA expression of iNOS was decreased by blue pigments, confirming the inhibitory effect of blue pigments on the NO production. That work also showed that blue pigments inhibited the expression of iNOS mRNA in LPS-stimulated macrophages. The effect of blue pigments on LPS-induced iNOS expression might result from the transcriptional inhibition of the iNOS gene. Further, the anti-inflammatory effect of blue pigments might be attributed to their inhibitory effect on PGE2 production through blocking COX-2 gene and protein expression. Therefore, besides being safe, genipin is also beneficial owing to its positive action when present in functional foods.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4699241/

Interesting article below.

You can do this yourself with:
- Berberine (works like Metformin)
- IGF-1 Deer Antler Velvet (Growth hormone similar to HGH)
- DHEA

All three can be bought together on Amazon for under $100 USD.  

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First hint that body’s ‘biological age’ can be reversed

Alison Abbott 05 September 2019

In a small trial, drugs seemed to rejuvenate the body’s ‘epigenetic clock’, which tracks a person’s biological age.

A small clinical study in California has suggested for the first time that it might be possible to reverse the body’s epigenetic clock, which measures a person’s biological age.

For one year, nine healthy volunteers took a cocktail of three common drugs — growth hormone and two diabetes medications — and on average shed 2.5 years of their biological ages, measured by analysing marks on a person’s genomes. The participants’ immune systems also showed signs of rejuvenation.

The results were a surprise even to the trial organizers — but researchers caution that the findings are preliminary because the trial was small and did not include a control arm.

“I’d expected to see slowing down of the clock, but not a reversal,” says geneticist Steve Horvath at the University of California, Los Angeles, who conducted the epigenetic analysis. “That felt kind of futuristic.” The findings were published on 5 September in Aging Cell1.

“It may be that there is an effect,” says cell biologist Wolfgang Wagner at the University of Aachen in Germany. “But the results are not rock solid because the study is very small and not well controlled.”

The latest trial was designed mainly to test whether growth hormone could be used safely in humans to restore tissue in the thymus gland. The gland, which is in the chest between the lungs and the breastbone, is crucial for efficient immune function. White blood cells are produced in bone marrow and then mature inside the thymus, where they become specialized T cells that help the body to fight infections and cancers. But the gland starts to shrink after puberty and increasingly becomes clogged with fat.

Evidence from animal and some human studies shows that growth hormone stimulates regeneration of the thymus. But this hormone can also promote diabetes, so the trial included two widely used anti-diabetic drugs, dehydroepiandrosterone (DHEA) and metformin, in the treatment cocktail.

Epigenetics: The sins of the father

The Thymus Regeneration, Immunorestoration and Insulin Mitigation (TRIIM) trial tested 9 white men between 51 and 65 years of age. It was led by immunologist Gregory Fahy, the chief scientific officer and co-founder of Intervene Immune in Los Angeles, and was approved by the US Food and Drug Administration in May 2015. It began a few months later at Stanford Medical Center in Palo Alto, California.

Fahy’s fascination with the thymus goes back to 1986, when he read a study in which scientists transplanted growth-hormone-secreting cells into rats, apparently rejuvenating their immune systems. He was surprised that no one seemed to have followed up on the result with a clinical trial. A decade later, at age 46, he treated himself for a month with growth hormone and DHEA, and found some regeneration of his own thymus.

In the TRIIM trial, the scientists took blood samples from participants during the treatment period. Tests showed that blood-cell count was rejuvenated in each of the participants. The researchers also used magnetic resonance imaging (MRI) to determine the composition of the thymus at the start and end of the study. They found that in seven participants, accumulated fat had been replaced with regenerated thymus tissue.
....

Cancer immunologist Sam Palmer at the Heriot-Watt University in Edinburgh says that it is exciting to see the expansion of immune cells in the blood. This “has huge implications not just for infectious disease but also for cancer and ageing in general”.

Nature 573, 173 (2019)
doi: 10.1038/d41586-019-02638-w
References

1.

Fahy, G. M. et al. Aging Cell https://doi.org/10.1111/acel.13028 (2019).
Article
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(more at link...)
https://www.nature.com/articles/d41586-019-02638-w

Chinese research on Zhi-Zhi (Genipin) and Diabetes - Cancer.
(Note: I take Genipin for UCP-2 down regulation enhancement. Warning that it will put you to sleep if you take it during the day-time. Also taking Yohimbine for effects.)
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Review Article

Chemistry and bioactivity of Gardenia jasminoides
Author links open overlay panelWenpingXiaoabShimingLiaSiyuWangcChi-TangHoc
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https://doi.org/10.1016/j.jfda.2016.11.005Get rights and content
Under a Creative Commons licenseopen access
Highlights
• Complete list of phytochemicals in Gardenia jasminoides.
• Thorough review of isolation methods of compounds in G. jasminoides.
• Comprehensive review of biological property of G. jasminoides compounds.
• Major review of genipin, geniposide, gardenoside, and crocin in their chemistry and biology.


Abstract

Gardenia jasminoides, grown in multiple regions in China, was commonly used as a natural yellow dye but has been one of the popular traditional Chinese medicines since the discovery of its biological property a few decades ago. It has been reported that G. jasminoides possess multiple biological activities, such as antioxidant properties, hypoglycemic effect, inhibition of inflammation, antidepression activity, and improved sleeping quality. In this review, our aim was to have a comprehensive summary of its phytochemistry including the extraction, isolation, and characterization of volatiles and bioactive molecules in G. jasminoides, focusing on the two major phytochemicals, genipin and crocin, which possess potent medicinal properties. Furthermore, this study attempted to establish a structure–activity relationship between the two major series of molecules with two pharmcophores and their biological activities, which would serve further exploration of the properties of phytocompounds in G. jasminoides as potential functional foods and medicines.

Graphical abstract

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Keywords
crocinGardenia jasminoidesgeniposidegenipiniridiod
1. Introduction
Gardenia jasminoides, an evergreen tree that belongs to the Rubiaceae family, is cultivated in multiple areas in China, with a Chinese name of Zhi Zi. It grows in many temperate regions and has fragrant white flowers [1]. It is not only used as natural yellow dyes for many years [2], [3], but also has various biological activities, such as antidiabetic [4], anti-inflammatory [5], antidepression [6], and antioxidant properties [7], and improvement of the quality of sleep [8]. It is commonly used in traditional Chinese medicine. The chemical analysis of G. jasminoides has been mainly focused on extraction technologies in recent years. Obtained extracts have exerted certain biological activities both in vitro and in vivo. Recent research showed that the oil extract from the G. jasminoides had antidepressant activity [6], and other new techniques to extract the oil and the complex biological activity have also been discussed. Herein, we reviewed the chemical components and biological activities of G. jasminoides as well as new techniques to extract and isolate the natural compounds from G. jasminoides.

2. Chemistry
A number of chemical components of G. jasminoides have been isolated and characterized, including iridoids, iridoid glucosides, triterpenoids, organic acids, and volatile compounds. Geniposide, genipin, gardenoside, crocin, and iridiod are the major bioactive compounds found in G. jasminoides. For instance, the yield of geniposide reached 10.9% under certain extraction conditions [9].

2.1. Volatiles in G. jasminoides
The major volatile compounds in essential oil of G. jasminoides are aliphatic acids, ketones, aldehydes, esters, alcohols, and aromatic derivatives [6], [10]. Because of the different temperature and duration of processing, the essential oil from G. jasminoides contains varied contents and proportions of volatile compounds. In addition, unstable components such as iridoids may be partially converted to volatile components during high temperature processing [6].

Gas chromatography–mass spectrometry (GC/MS) was the major technique used to identify volatile components from G. jasminoides [6]. He et al [11] reported that the oil of the fruits of G. jasminoides was extracted by supercritical fluid CO2, in which 16 major components of the oil extract were revealed by GC/MS. Myristic acid (15.3%) had the highest relative content, whereas the lowest one was caproic acid (0.24%) [12]. Because of the pharmacological activities exerted by G. jasminoides oil and the availability of modern extraction techniques, many efforts were invested in the extraction of the G. jasminoides, in order to find the optimal extraction method. The extraction methods of volatile oil from G. jasminoides are listed in Table 1.

Table 1. Extraction method of volatile oil from Gardenia jasminoides.

Extraction method Parameters of extraction Results (%) Refs
Supercritical fluid extraction (SFE) Extraction pressure: 36.8 MPa
Temperature: 65°C
CO2 flow rate: 15 kg/h Linoleic acid, 44; palmitic acid, 26.4;
oleic acid, 24.6 [12]
Temperature: 49.94°C
Pressure: 29.89 MPa
Time: 93.82 min 16 major components of the oil extract were characterized [6]
Pressure: 30 MPa
Temperature: 55°C
CO2 flow rate: 15 kg/h Linoleic acid, 44.38; oleic acid, 24.96; palmitic acid, 24.83; stearic acid, 2.55; linolenic acid, 1.31 [13]
Pressure: 12 MPa or 25 MPa
Temperature: 45°C Oil yield, 12 [13]
Subcritical fluid extraction Subcritical butane Fatty acids, 77.6 [14]
Ultrasound-assisted extraction 28 kHz & 100 W
Material/solvent ratio: 1:10 (g/mL) Oil yield, 16.49 [15]
Steam distillation Distilled water Oil yield, 0.12 [14]
2.2. Iridoids and iridoid glycoside
Iridoids and iridoid glycoside are rich in G. jasminoides. The iridoids and iridiod glycosides include genipin, geniposide, and gardenoside. Many researchers have found multiple positive health effect of geniposide, including anti-inflammation [16], antidepression [16], antidiabetic properties [4], antithrombotic activities [18], as well as protection against lipopolysaccharide (LPS)-induced apoptotic liver damage [19]. The content of iridoid glycosides may vary from different regions at about 5–6% [20]. A study quantified the content of geniposide, gardenoside, geniposidic acid, and chlorogenic acid as 56.37 ± 26.24 μg/mg, 49.57 ± 18.78 μg/mg, 3.15 ± 3.27 μg/mg, and 0.69 ± 0.39 μg/mg, respectively, measured in 68 samples from different regions in China and Korea [21].

2.2.1. Extraction of iridoids
Because of the pharmacological effects of G. jasminoides and the application of modern extraction technology, many efforts have been invested in the preparation of different G. jasminoides extracts, in an attempt to find a potent ingredient that can have significant effects on diseases, such as high blood pressure, hyperglycemia, cancer, hyperlipidemia, and Alzheimer’s disease (AD) [22]. Certain positive results have been obtained with specified extracts, but in the future, further fractionation and evaluation of the active components of this plant should be a better direction. With the emergence of several new extraction methods, several minor components with potent biological activity may be found in G. jasminoides. Extraction methods such as solvent extraction, as well ultrasound- and microwave-assisted extraction (MAE) have been used to extract iridoids.

https://www.sciencedirect.com/science/article/pii/S1021949816301740

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Wikipedia on Iridoids:

Iridoid

Chemical structure of iridomyrmecin
Iridoids are a type of monoterpenoids in the general form of cyclopentanopyran, found in a wide variety of plants and some animals. They are biosynthetically derived from 8-oxogeranial.[1] Iridoids are typically found in plants as glycosides, most often bound to glucose.

The chemical structure is exemplified by iridomyrmecin, a defensive chemical produced by the genus Iridomyrmex, for which iridoids are named. Structurally, they are bicyclic cis-fused cyclopentane-pyrans. Cleavage of a bond in the cyclopentane ring gives rise to a subclass known as secoiridoids, such as oleuropein and amarogentin.

Aucubin and catalpol are two of the most common iridoids in the plant kingdom.

The iridoids produced by plants act primarily as a defense against herbivores or against infection by microorganisms.[citation needed] The variable checkerspot butterfly also contains iridoids obtained through its diet which act as a defense against avian predators.[2] To humans and other mammals, iridoids are often characterized by a deterrent bitter taste.

Aucubin and catalpol are two of the most common iridoids in the plant kingdom.[citation needed] Iridoids are prevalent in the plant subclass Asteridae, such as Ericaceae, Loganiaceae, Gentianaceae, Rubiaceae, Verbenaceae, Lamiaceae, Oleaceae, Plantaginaceae, Scrophulariaceae, Valerianaceae, and Menyanthaceae.[3]

Iridoids have been the subject of research into their potential biological activities.[3][4]

Biosynthesis

The iridoid ring scaffold is synthesized, in plants, by the enzyme iridoid synthase.[5] In contrast with other monoterpene cyclases, iridoid synthase uses 8-oxogeranial as a substrate. The enzyme uses a two-step mechanism, with an initial NADPH-dependent reduction step followed by a cyclization step that occurs through either a Diels-Alder reaction or an intramolecular Michael addition.[5]

Loganic acid is an iridoid substrate converted to strictosidine which reacts with tryptamine, eventually leading to the indole alkaloids which include many biologically active compounds such as strychnine, yohimbine, vinca alkaloids, and ellipticine.

https://en.wikipedia.org/wiki/Iridoid

UCP2 and NAFLD (Non alcoholic Fatty Liver Disease) - Related to Cancer and T2D:

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Published online 2016 Jun 14. doi: 10.1186/s12967-016-0936-3
PMCID: PMC4908770
PMID: 27301474

Major components of metabolic syndrome and nutritional intakes in different genotype of UCP2 −866G/A gene polymorphisms in patients with NAFLD

Mahdieh Abbasalizad Farhangi,corresponding author Fatemeh Mohseni, Safar Farajnia, and Mohammad-Asghari Jafarabadi

Background

Nonalcoholic fatty liver disease (NAFLD) is defined as abnormal lipid deposition in the hepatocytes in the absence of significant amount of alcohol intake. NAFLD includes nonalcoholic steatohepatitis (NASH) associated with markedly increased risk of cardiovascular and liver-related mortality which finally progress to more severe forms of liver disease such as advanced fibrosis, cirrhosis and even hepatocellular carcinoma [1, 2].

In developed countries, the prevalence of the NAFLD is up to 30 % in the general population, 50 % in patients with type 2 diabetes mellitus (T2DM), 76 % in obese people and almost 100 % in patients with morbid obesity [3, 4]. According to data from the US National Health and Nutrition Examination Survey (NHANES) report, the prevalence of NAFLD has increased from 47 to 75 % between 1988 and 2008 [5, 6]. In a large population-based study in southern regions of Iran in 2011, 21.5 % the prevalence of NAFLD in the adult general population was reported [5].

In addition to genetic predisposition, change in lifestyles and dietary habits increases the prevalence of obesity, diabetes mellitus, metabolic syndrome, cardiovascular disease and their consequences such as NAFLD throughout the world [7–9]. The long-term excessive food intake and dietary composition in food groups, macronutrients and micronutrients is associated with progression of NAFLD mostly recognized by abnormal ultrasonography (US) findings or elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations as markers of liver injury [10, 11]. In general, lower antioxidant consumption, higher intake of calorie, carbohydrate, protein and high dietary cholesterol stimulate hepatic lipid accumulation leading to development of fatty liver disease [12–14]. In addition, inadequate intake of micronutrients, copper and iron may also be involved in pathogenesis of NAFLD [15]. NAFLD symptoms manifested by abnormalities in serum and hepatic stores of copper and iron in rodent models [16, 17] and low hepatic copper levels is known to be correlated with NAFLD progression and components of metabolic syndrome [16, 17]. However no report of the association between dietary intakes of these micronutrients and different genotypes of UCP2 in NAFLD is available. Additionally, oxidative stress has been involved in the pathogenesis of NAFLD; vitamin C and E are well-known antioxidants capable in blocking distribution of radical reactions [18]. These antioxidants play important roles in histological improvement of inflammation in NAFLD [18, 19].

Dietary recommendation should be appropriate according to an individual status and even genetic background [20]. Several studies mostly in animal models introduced diet as a potent modifier of NAFLD-related genes expression [10]; for example enhanced ω-6/ω-3 poly unsaturated fatty acids (PUFAs) ratio interacts with PNPLA3 rs738409 gene in the GG homozygote and enhances ALT concentrations and hepatic fat accumulation in human [21]. Other studies also revealed the role of ω-3 fatty acids as regulators of hepatic gene expression by mainly aiming the transcription factors sterol regulatory element binding transcription factor 1 (SREBP-1c) and down-regulating inflammatory genes [22].

Three common polymorphisms in uncoupling protein 2 (UCP2) are −866G>A (rs659366), 55 Ala/Val (rs660339) and 3-UTR ins > del [23]. The relationship of −866G>A gene polymorphism of UCP2 (rs659366) with obesity and type 2 diabetes has been reported previously [23]. UCP2 is located in the inner membrane of mitochondria, acts as a mediator of proton leak and ultimately leads to decreased ATP production and energy release [24]. The wide tissue distribution of UCP2 shows its potent role in several pathologic events on specific tissue or organs [25, 26]. Enhanced UCP2 expression is able to respond oxidative stress by controlling production of mitochondrial superoxide [26]; therefore, it may be a therapeutic target for management of oxidative damage and metabolic imbalance in NAFLD [25].

Considering the lack of knowledge about the interaction between UCP2 gene polymorphism and nutrient intakes in NAFLD patients we aimed to investigate the interaction between energy and nutrients intake and −866G>A gene polymorphism of uncoupling protein 2 (UCP2) in patients with NAFLD.

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UCP2 inhibition induces ROS/Akt/mTOR axis: Role of GAPDH nuclear translocation in genipin/everolimus anticancer synergism

IlariaDandoaRaffaellaPacchianaaElisa DallaPozzaaIvanaCataldobStefanoBrunocPaolaContidMarcoCordanieAnnaGrimaldifGiovannaButeraaMicheleCaragliafAldoScarpabMartaPalmieriaMassimoDonadellia
https://doi.org/10.1016/j.freeradbiomed.2017.09.022
Get rights and content

Abstract

Several studies indicate that mitochondrial uncoupling protein 2 (UCP2) plays a pivotal role in cancer development by decreasing reactive oxygen species (ROS) produced by mitochondrial metabolism and by sustaining chemoresistance to a plethora of anticancer drugs. Here, we demonstrate that inhibition of UCP2 triggers Akt/mTOR pathway in a ROS-dependent mechanism in pancreatic adenocarcinoma cells. This event reduces the antiproliferative outcome of UCP2 inhibition by genipin, creating the conditions for the synergistic counteraction of cancer cell growth with the mTOR inhibitor everolimus. Inhibition of pancreatic adenocarcinoma cell growth and induction of apoptosis by genipin and everolimus treatment are functionally related to nuclear translocation of the cytosolic glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The synthetic compound (S)-benzyl-2-amino-2-(S)-3-bromo-4,5-dihydroisoxazol-5-yl-acetate (AXP3009), which binds GAPDH at its redox-sensitive Cys152, restores cell viability affected by the combined treatment with genipin and everolimus, suggesting a role for ROS production in the nuclear translocation of GAPDH. Caspase-mediated apoptosis by genipin and everolimus is further potentiated by the autophagy inhibitor 3-methyladenine revealing a protective role for Beclin1-mediated autophagy induced by the treatment. Mice xenograft of pancreatic adenocarcinoma further confirmed the antiproliferative outcome of drug combination without toxic effects for animals. Tumor masses from mice injected with UCP2 and mTOR inhibitors revealed a strong reduction in tumor volume and number of mitosis associated with a marked GAPDH nuclear positivity. Altogether, these results reveal novel mechanisms through which UCP2 promotes cancer cell proliferation and support the combined inhibition of UCP2 and of Akt/mTOR pathway as a novel therapeutic strategy in the treatment of pancreatic adenocarcinoma.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4908770/

Dorit Koren, Andrew Palladino, in Genetic Diagnosis of Endocrine Disorders (Second Edition), 2016

Hyperinsulinism Resulting from Mutations in UCP2 (Uncoupling Protein 2)
UCP2 encodes a mitochondrial uncoupling protein that facilitates anion transfer from the inner to the outer mitochondrial membrane in exchange for protons. It is part of a larger family of uncoupling proteins that separate (“uncouple”) oxidative phosphorylation from ATP synthesis; the resulting energy is dissipated as heat. UCP2 is widely expressed in many tissues, including pancreatic islets.73 UCP2 acts as a negative regulator of insulin secretion in pancreatic β-cells by decreasing ATP content and thus inhibiting glucose-stimulated insulin secretion.

(More at link: https://www.sciencedirect.com/science/article/pii/B9780128008928000038 )

Mitochondrial Retrograde Signaling Mediated by UCP2 Inhibits Cancer Cell Proliferation and Tumorigenesis

Pauline Esteves, Claire Pecqueur, Céline Ransy, Catherine Esnous, Véronique Lenoir, Frédéric Bouillaud, Anne-Laure Bulteau, Anne Lombès, Carina Prip-Buus, Daniel Ricquier and Marie-Clotilde Alves-Guerra
DOI: 10.1158/0008-5472.CAN-13-3383 Published July 2014
ArticleFigures & DataInfo & Metrics PDF

Abstract

Cancer cells tilt their energy production away from oxidative phosphorylation (OXPHOS) toward glycolysis during malignant progression, even when aerobic metabolism is available. Reversing this phenomenon, known as the Warburg effect, may offer a generalized anticancer strategy. In this study, we show that overexpression of the mitochondrial membrane transport protein UCP2 in cancer cells is sufficient to restore a balance toward oxidative phosphorylation and to repress malignant phenotypes. Altered expression of glycolytic and oxidative enzymes mediated the effects of this metabolic shift. Notably, UCP2 overexpression increased signaling from the master energy-regulating kinase, adenosine monophosphate-activated protein kinase, while downregulating expression of hypoxia-induced factor. In support of recent new evidence about UCP2 function, we found that UCP2 did not function in this setting as a membrane potential uncoupling protein, but instead acted to control routing of mitochondria substrates. Taken together, our results define a strategy to reorient mitochondrial function in cancer cells toward OXPHOS that restricts their malignant phenotype.

Cancer Res; 74(14); 3971–82. 2014 AACR.

Introduction

Cancer is a multistep process involving gene modifications and chromosomal rearrangements in the tumor cells that promote unchecked proliferation, abrogate cell death, and reprogram metabolism (1, 2). Indeed, tumor cell proliferation requires rapid synthesis of macromolecules, including lipids, proteins, and nucleotides. Dysregulation of cellular metabolism has been associated with malignant transformation and may be triggered directly through mutations in oncogenes or through signaling pathways involved in metabolism (2, 3). Many cancer cells exhibit rapid glucose consumption, with most of the glucose-derived carbon being secreted as lactate despite abundant oxygen availability (Warburg effect). Glycolysis is also important for generating precursors and reducing equivalents needed for cellular biogenesis and antioxidant defense (4). Moreover, the bioenergetic reprogramming of tumoral cells from oxidative phosphorylation (OXPHOS) to the use of glycolysis for ATP production allows cells to be metabolically less oxygen dependent, thus favoring invasion processes.

Mitochondria have been directly involved in tumor development (5). Indeed, cancer-associated mutations have been identified in several metabolic enzymes genes such as the ones coding succinate dehydrogenase (SDH), fumarate hydratase (FH), and isocitrate dehydrogenase (IDH), all enzymes of the tricarboxylic acid cycle (TCA; refs. 6–. In addition, in tight relation to their bioenergetic status, mitochondria play a crucial role in the control of apoptosis, are known to release reactive oxygen species (ROS), and contain potent antioxidant defense. Acting on metabolism, and more particularly on mitochondria, thus represents a therapeutic perspective for cancer therapy (9).

The uncoupling protein 2 (UCP2) is the second member identified in the UCP family (10), a subfamily of the mitochondrial carriers. The first member, UCP1, acts as a passive proton transporter in the mitochondrial inner membrane in brown adipose tissue (BAT). In this tissue, upon cold exposure, the mitochondrial respiration is uncoupled, meaning that the electron transport chain is no longer coupled to ATP synthesis. UCP2 exhibits singular features that distinguish it from the other uncoupling proteins. UCP2 mRNA is found in many tissues, whereas UCP1 and UCP3 are respectively restricted to BAT and muscle. UCP2 is regulated at both the transcriptional and translational levels (11, 12). The inhibition of UCP2 translation can be relieved in vitro by addition of glutamine and in vivo by fasting or an inflammatory state. Furthermore, the half-life of the protein is very short (13), suggesting that UCP2 is a suitable candidate for regulating rapid biologic responses. At first, sequence similarities between UCP proteins led to the conclusion that they act in a similar way but with different intensities due to the lower UCP2 and UCP3 expression level compared with UCP1 (11). The generation of Ucp2−/− mice allowed us to show that UCP2 mitigates immunity through a reduction in ROS production (11, 14) and plays a role in inflammation (11, 15–17). Although partial uncoupling would decrease mitochondrial ROS release, our present point of view is that activities other than uncoupling have to be considered for UCP2. Previously, we showed that loss of UCP2 is associated with increased proliferation in primary fibroblasts (18). Interestingly, although no difference in the mitochondrial respiration and the ATP/ADP ratio was recorded, we demonstrated that Ucp2−/− fibroblasts were more dependent on glucose and oxidized less long-chain fatty acids. The correlation between higher rate of division and increased dependency to glucose in the absence of UCP2 recalls the Warburg hypothesis on metabolic alteration in cancer. Its present reformulation takes into account that complete oxidation leads to the loss of carbon skeleton. Consequently, dividing cells with an intense biosynthesis rate depend on a shift toward a larger availability of precursors favoring aerobic glycolysis. Therefore, UCP2 is a good candidate to understand the crosstalk between metabolic alteration and promotion of cancer initiation, progression, and invasion.

Our goal was to determine whether UCP2 is able to control metabolic reprogramming in cancer cells and to modify their proliferation. In this article, we show that cells overexpressing UCP2 shift their metabolism from glycolysis toward oxidative phosphorylation and become poorly tumorigenic. UCP2 overexpression generates a mitochondrial retrograde signaling that modifies expression of glycolytic and oxidative enzymes, leading to enhanced oxidative phosphorylation. Moreover, UCP2 overexpression is associated with an activation of adenosine monophosphate-activated protein kinase (AMPK) signaling together with a downregulation of hypoxia-induced factor (HIF) expression. Finally, UCP2 overexpression can amplify apoptosis induced by chemotherapeutic drugs, such as staurosporine. The crucial role of UCP2 in tumor metabolism makes UCP2 a promising target for tumor therapy.

https://cancerres.aacrjournals.org/content/74/14/3971

From an accidental discovery at Cardiff University:  

https://www.telegraph.co.uk/science/2020/01/20/immune-cell-kills-cancers-discovered-accident-british-scientists/

Immune cell that kills most cancers discovered by accident by British scientists

Immune cell that kills most cancers discovered by accident by British scientists .

More at link...

A new type of immune cell which kills most cancers has been discovered by accident by British scientists, in a finding which could herald a major breakthrough in treatment.

Researchers at Cardiff University were analysing blood from a bank in Wales, looking for immune cells that could fight bacteria, when they found an entirely new type of T-cell.

That new immune cell carries a never-before-seen receptor which acts like a grappling hook, latching on to most human cancers, while ignoring healthy cells.

New T-cell discovery at the University of Cardiff.

https://www.cardiff.ac.uk/news/view/1749599-discovery-of-new-t-cell-raises-prospect-of-universal-cancer-therapy
...

Discovery of new T-cell raises prospect of ‘universal’ cancer therapy
20 January 2020

Professor Andrew Sewell with Research Fellow Garry Dolton in a lab

Researchers at Cardiff University have discovered a new type of killer T-cell that offers hope of a “one-size-fits-all” cancer therapy.

T-cell therapies for cancer - where immune cells are removed, modified and returned to the patient’s blood to seek and destroy cancer cells - are the latest paradigm in cancer treatments.

The most widely-used therapy, known as CAR-T, is personalised to each patient but targets only a few types of cancers and has not been successful for solid tumours, which make up the vast majority of cancers.

Cardiff researchers have now discovered T-cells equipped with a new type of T-cell receptor (TCR) which recognises and kills most human cancer types, while ignoring healthy cells.

This TCR recognises a molecule present on the surface of a wide range of cancer cells as well as in many of the body’s normal cells but, remarkably, is able to distinguish between healthy cells and cancerous ones, killing only the latter.

The researchers said this meant it offered “exciting opportunities for pan-cancer, pan-population” immunotherapies not previously thought possible.

How does this new TCR work?
Conventional T-cells scan the surface of other cells to find anomalies and eliminate cancerous cells - which express abnormal proteins - but ignore cells that contain only “normal” proteins.

The scanning system recognises small parts of cellular proteins that are bound to cell-surface molecules called human leukocyte antigen (HLA), allowing killer T-cells to see what’s occurring inside cells by scanning their surface.

HLA varies widely between individuals, which has previously prevented scientists from creating a single T-cell-based treatment that targets most cancers in all people.

But the Cardiff study, published today in Nature Immunology, describes a unique TCR that can recognise many types of cancer via a single HLA-like molecule called MR1.

Unlike HLA, MR1 does not vary in the human population - meaning it is a hugely attractive new target for immunotherapies.

What did the researchers show?
T-cells equipped with the new TCR were shown, in the lab, to kill lung, skin, blood, colon, breast, bone, prostate, ovarian, kidney and cervical cancer cells, while ignoring healthy cells.

To test the therapeutic potential of these cells in vivo, the researchers injected T-cells able to recognise MR1 into mice bearing human cancer and with a human immune system.

This showed “encouraging” cancer-clearing results which the researchers said was comparable to the now NHS-approved CAR-T therapy in a similar animal model. More at link...

Genipin attenuates sepsis-induced immunosuppression through inhibition of T lymphocyte apoptosis

Author links open overlay panelJoon-SungKim1Sun-MeeLee

https://doi.org/10.1016/j.intimp.2015.04.034

• Genipin attenuates sepsis-induced immunosuppression.
• Genipin inhibits T lymphocyte apoptosis in the late phase of sepsis.
• Genipin suppresses Th1/Th2 cytokine shift in the late p ase of sepsis.


Abstract

Sepsis, a systemic inflammatory response to infection, initiates a complex immune response consisting of an early hyperinflammatory response and a subsequent hypoinflammatory response that impairs the removal of infectious organisms. The importance of sepsis-induced immunosuppression and its contribution to mortality has recently emerged. Apoptotic depletion of T lymphocytes is a critical cause of immunosuppression in the late phase of sepsis. Genipin is a major active compound of gardenia fruit that has anti-apoptotic and anti-microbial properties. This study investigated the mechanisms of action of genipin on immunosuppression in the late phase of sepsis. Mice received genipin (1, 2.5 and 5 mg/kg, i.v.) at 0 (immediately) and 24 h after cecal ligation and puncture (CLP). Twenty-six hours after CLP, the spleen and blood were collected. Genipin improved the survival rate compared to controls. CLP increased the levels of FADD, caspase-8 and caspase-3 protein expression, which were attenuated by genipin. Genipin increased the level of anti-apoptotic B-cell lymphoma-2 protein expression, while it decreased the level of pro-apoptotic phosphorylated-Bim protein expression in CLP. CLP decreased the CD4+ and CD8+ T cell population, while it increased the regulatory T cell (Treg) population and the level of cytotoxic T lymphocyte-associated antigen 4 protein expression on Treg. These changes were attenuated by genipin. The splenic levels of interferon-γ and interleukin (IL)-2 were reduced, while the levels of IL-4 and IL-10 increased after CLP. Genipin attenuated these alterations. These findings suggest that genipin reduces immunosuppression by inhibiting T lymphocyte apoptosis in the late phase of sepsis.

More on Genipin....

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Molecular Hats

Genipin is a natural product found in the fruits of flowering plants such as Gardenia jasminoides and Gardenia americana. Interestingly, genipin is a natural cross-linking agent, meaning it can react with chemical groups on different biochemical molecules and effectively link them together (1). In addition, genipin is found in traditional Chinese medicine formulations used for a variety of diseases, ranging from reducing inflammation to cancer treatment (2). Given the extensive versatility of this compound, scientists are interested in understanding more about its mechanism of action.

In the recent article, “Molecular Mechanisms Responsible for Pharmacological Effects of Genipin on Mitochondrial Proteins,” published in Biophysical Journal, a group of researchers used extensive biochemical methods to better characterize the specific effects of genipin on mammalian cells (3). While previous work suggested genipin targets a specific protein found in the membrane of mitochondria called UCP2 (uncoupling protein 2) (4), it was unclear if other proteins could be affected by this molecule. UCP2 is part of a family of proteins in the mitochondrial membrane that dissipate the proton gradient that accumulates during oxidative phosphorylation by transporting protons into the mitochondrial inner membrane (5).

To directly probe the role of genipin on UCP1, UCP2, and UCP3, the authors of this work reconstituted each purified protein into a lipid bilayer and demonstrated that the addition of genipin caused inhibition proton transport for all three proteins. This proves genipin can act on more than just UCP2. While this experiment showed genipin could inhibit these proteins, it did not demonstrate the molecular mechanism of action, which the authors sought to determine.

Interestingly, it appeared that when reconstituted UCP1 was treated with high concentrations of genipin (1 mM), membrane conductance showed an initial decrease and then subsequent increase. The authors concluded that the UCP1-specific effects occurred at low concentration (<200 µM), followed by non-specific ion transport at higher concentrations which leads to an increase in membrane conductance. To understand this specific inhibition, the authors speculated the genipin molecules may be reacting with atoms on the protein as a result of the cross-linking behavior of this unique molecule. Using mass spectrometry, they identified two specific residues of UCP1 that become labeled by genipin, suggesting this modification could be the source of UCP inhibition via genipin.

To confirm this hypothesis, the authors performed an experiment in which they chemically blocked the residues of UCP1 labeled with genipin and investigated whether this blockage prevented inhibition. The authors expected that blocking these residues would cause UCP1 to no longer be inhibited by genipin— but to their surprise, this was not the case. In fact, blocking these residues had no impact on genipin inhibition, suggesting that this cross-linking modification by genipin was not the source of inhibition.

Previous work has shown that UCP1 is inhibited by purine nucleotides and that there are key arginine residues required for this inhibition (6). To conclude whether the mechanism of UCP1 inhibition by genipin is similar to that of purine inhibition, the authors mutated these arginine residues and in fact confirmed that genipin can no longer inhibit the protein in the absence of these critical residues. This suggests the mechanism by which purine nucleotides inhibit the proton transport functions of UCP molecules is the same for the plant-derived natural product, genipin and requires critical arginine residues situated in the UCP1 pocket.


More at link:  https://www.biophysics.org/blog/genipin-a-versatile-chemical-that-wears-many-different-molecular-hats

More research on UCPs:
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Arch Biochem Biophys
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. 2018 Nov 1;657:41-55.
doi: 10.1016/j.abb.2018.09.006. Epub 2018 Sep 11.

Uncoupling proteins: Martin Klingenberg's contributions for 40 years

Karim S Echtay  1 , Martin Bienengraeber  2 , Peter Mayinger  3 , Simone Heimpel  4 , Edith Winkler  5 , Doerthe Druhmann  5 , Karina Frischmuth  5 , Frits Kamp  5 , Shu-Gui Huang  6
Affiliations

   PMID: 30217511 DOI: 10.1016/j.abb.2018.09.006

Abstract

The uncoupling protein (UCP1) is a proton (H+) transporter in the mitochondrial inner membrane. By dissipating the electrochemical H+ gradient, UCP1 uncouples respiration from ATP synthesis, which drives an increase in substrate oxidation via the TCA cycle flux that generates more heat. The mitochondrial uncoupling-mediated non-shivering thermogenesis in brown adipose tissue is vital primarily to mammals, such as rodents and new-born humans, but more recently additional functions in adult humans have been described. UCP1 is regulated by β-adrenergic receptors through the sympathetic nervous system and at the molecular activity level by nucleotides and fatty acid to meet thermogenesis needs. The discovery of novel UCP homologs has greatly contributed to the understanding of human diseases, such as obesity and diabetes. In this article, we review the progress made towards the molecular mechanism and function of the UCPs, in particular focusing on the influential contributions from Martin Klingenberg's laboratory. Because all members of the UCP family are potentially promising drug targets, we also present and discuss possible approaches and methods for UCP-related drug discovery.

Keywords: Brown adipose tissue; Drug discovery; Mitochondria; Non-shivering thermogenesis; Obesity; Solute transport; Uncoupling protein.

Copyright 2018 Elsevier Inc. All rights reserved.

https://pubmed.ncbi.nlm.nih.gov/30217511/
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Cancers (Basel)

. 2010 Apr 16;2(2):567-91.
doi: 10.3390/cancers2020567.

Role of uncoupling proteins in cancer

Adamo Valle  1 , Jordi Oliver, Pilar Roca
Affiliations

   PMID: 24281083 PMCID: PMC3835092 DOI: 10.3390/cancers2020567

Uncoupling proteins (UCPs) are a family of inner mitochondrial membrane proteins whose function is to allow the re-entry of protons to the mitochondrial matrix, by dissipating the proton gradient and, subsequently, decreasing membrane potential and production of reactive oxygen species (ROS). Due to their pivotal role in the intersection between energy efficiency and oxidative stress, UCPs are being investigated for a potential role in cancer. In this review we compile the latest evidence showing a link between uncoupling and the carcinogenic process, paying special attention to their involvement in cancer initiation, progression and drug chemoresistance.
https://pubmed.ncbi.nlm.nih.gov/24281083/

..................

Cancers (Basel)

. 2010 Apr 16;2(2):567-91.
doi: 10.3390/cancers2020567.
Role of uncoupling proteins in cancer

Adamo Valle  1 , Jordi Oliver, Pilar Roca

https://pubmed.ncbi.nlm.nih.gov/24281083/

............


Mitochondrion
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. 2010 Apr;10(3):243-52.
doi: 10.1016/j.mito.2009.12.143. Epub 2009 Dec 21.

Uncoupling protein-2 and cancer

Gyorgy Baffy  1
Affiliations

   PMID: 20005987 DOI: 10.1016/j.mito.2009.12.143

Abstract

Cancer cells respond to unfavorable microenvironments such as nutrient limitation, hypoxia, oxidative stress, and host defense by comprehensive metabolic reprogramming. Mitochondria are linked to this complex adaptive response and emerging evidence indicates that uncoupling protein-2 (UCP2), a mitochondrial inner membrane anion carrier, may contribute to this process. Effects of UCP2 on mitochondrial bioenergetics, redox homeostasis, and oxidant production in cancer cells may modulate molecular pathways of macromolecular biosynthesis, antioxidant defense, apoptosis, cell growth and proliferation, enhancing robustness and promoting chemoresistance. Elucidation of these interactions may identify novel anti-cancer strategies.
https://pubmed.ncbi.nlm.nih.gov/20005987/

.............


Inflammopharmacology
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. 2015 Dec;23(6):365-9.
doi: 10.1007/s10787-015-0250-3. Epub 2015 Nov 5.

Overexpression of uncoupling protein-2 in cancer: metabolic and heat changes, inhibition and effects on drug resistance

Michael A Pitt  1
Affiliations

   PMID: 26542482 DOI: 10.1007/s10787-015-0250-3

Abstract

This paper deals with the role of uncoupling protein-2 (UCP2) in cancer. UCP2 is overexpressed in cancer. This overexpression results in uncoupling of mitochondrial oxidative phosphorylation and a shift in production of ATP from mitochondrial oxidative phosphorylation to cytosolic aerobic glycolysis. UCP2 overexpression results in the following changes. Mitochondrial membrane potential (Δψ(m)) is decreased and lactate accumulates. There is a diminished production of reactive oxygen species and apoptosis is inhibited post-exposure to chemotherapeutic agents. There is an increase in heat and entropy production and a departure from the stationary state of non-cancerous tissue. Uncoupling of oxidative phosphorylation may also be caused by protonophores and non-steroidal anti-inflammatory drugs. UCP2 requires activation by superoxide and lipid peroxidation derivatives. As vitamin E inhibits lipid peroxidation, it might be expected that vitamin E would act as a chemotherapeutic agent against cancer. A recent study has shown that vitamin E and another anti-oxidant accelerate cancer progression. UCP2 is inhibited by genipin, chromane compounds and short interfering RNAs (siRNA). Genipin, chromanes and siRNA are taken up by both cancer and non-cancerous cells. Targeting the uptake of these agents by cancer cells by the enhanced permeability and retention effect is considered. Inhibition of UCP2 enhances the action of several anti-cancer agents.

Keywords: Cancer; Non-steroidal anti-inflammatory drugs; Uncoupling protein-2 inhibition; Uncoupling protein-2 inhibition effect on chemotherapeutic agents; Uncoupling protein-2 overexpression; Vitamin E.

https://pubmed.ncbi.nlm.nih.gov/26542482/

Published: 24 July 2011

Mitochondrial uncoupling protein 2 structure determined by NMR molecular fragment searching

Marcelo J. Berardi, William M. Shih, Stephen C. Harrison & James J. Chou

Nature volume 476, pages 109–113 (2011)Cite this article

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310 Citations

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Abstract

Mitochondrial uncoupling protein 2 (UCP2) is an integral membrane protein in the mitochondrial anion carrier protein family, the members of which facilitate the transport of small molecules across the mitochondrial inner membrane1,2. When the mitochondrial respiratory complex pumps protons from the mitochondrial matrix to the intermembrane space, it builds up an electrochemical potential2. A fraction of this electrochemical potential is dissipated as heat, in a process involving leakage of protons back to the matrix2. This leakage, or ‘uncoupling’ of the proton electrochemical potential, is mediated primarily by uncoupling proteins2. However, the mechanism of UCP-mediated proton translocation across the lipid bilayer is unknown. Here we describe a solution-NMR method for structural characterization of UCP2. The method, which overcomes some of the challenges associated with membrane-protein structure determination3, combines orientation restraints derived from NMR residual dipolar couplings (RDCs) and semiquantitative distance restraints from paramagnetic relaxation enhancement (PRE) measurements. The local and secondary structures of the protein were determined by piecing together molecular fragments from the Protein Data Bank that best fit experimental RDCs from samples weakly aligned in a DNA nanotube liquid crystal. The RDCs also determine the relative orientation of the secondary structural segments, and the PRE restraints provide their spatial arrangement in the tertiary fold. UCP2 closely resembles the bovine ADP/ATP carrier (the only carrier protein of known structure4), but the relative orientations of the helical segments are different, resulting in a wider opening on the matrix side of the inner membrane. Moreover, the nitroxide-labelled GDP binds inside the channel and seems to be closer to transmembrane helices 1–4. We believe that this biophysical approach can be applied to other membrane proteins and, in particular, to other mitochondrial carriers, not only for structure determination but also to characterize various conformational states of these proteins linked to substrate transport.

https://www.nature.com/articles/nature10257
https://genomebiology.biomedcentral.com/articles/10.1186/gb-2002-3-12-reviews3015
https://pubmed.ncbi.nlm.nih.gov/10605819/

Int J Biochem Cell Biol

. 1999 Nov;31(11):1261-78.
doi: 10.1016/s1357-2725(99)00049-7.
The mitochondrial uncoupling protein-2: current status
C Fleury 1 , D Sanchis
Affiliations

PMID: 10605819 DOI: 10.1016/s1357-2725(99)00049-7

Abstract

In eukaryotic cells ATP is generated by oxidative phosphorylation, an energetic coupling at the mitochondrial level. The oxidative reactions occurring in the respiratory chain generate an electrochemical proton gradient on both sides of the inner membrane. This gradient is used by the ATPsynthase to phosphorylate ADP into ATP. The coupling between respiration and ADP phosphorylation is only partial in brown adipose tissue (BAT) mitochondria, where the uncoupling protein UCP1 causes a reentry of protons into the matrix and abolishes the electrochemical proton gradient. The liberated energy is then dissipated as heat and ATP synthesis is reduced. This property was for a long time considered as an exception and specific to the non-shivering thermogenesis found in BAT. The recent cloning of new UCPs expressed in other tissues revealed the importance of this kind of regulation of respiratory control in metabolism and energy expenditure. The newly characterised UCPs are potential targets for obesity treatment drugs which could favour energy expenditure and diminish the metabolic efficiency. In 1997, we cloned UCP2 and proposed a role for this new uncoupling protein in diet-induced thermogenesis, obesity, hyperinsulinemia, fever and resting metabolic rate. Currently, an abundant literature deals with UCP2, but its biochemical and physiological functions and regulation remain unclear. The present review reports the status of our knowledge of this mitochondrial carrier in terms of sequence, activity, tissue distribution and regulation of expression. The putative physiological roles of UCP2 will be introduced and discussed.


https://pubmed.ncbi.nlm.nih.gov/10605819/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477468/
https://www.ncbi.nlm.nih.gov/Structure/pdb/2C3E

..........
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research
The transport mechanism of the mitochondrial ADP/ATP carrier

https://www.sciencedirect.com/science/article/pii/S0167488916300684
https://www.sciencedirect.com/science/article/pii/S0167488916300684/pdfft?md5=150d33d9c4d850b34a8a877577fee173&pid=1-s2.0-S0167488916300684-mainext.pdf

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The mitochondrial ADP/ATP carrier: structural, physiological and pathological aspects
C Fiore 1 , V Trézéguet, A Le Saux, P Roux, C Schwimmer, A C Dianoux, F Noel, G J Lauquin, G Brandolin, P V Vignais
Affiliations

PMID: 9587671 DOI: 10.1016/s0300-9084(98)80020-5

Abstract

Under the conditions of oxidative phosphorylation, the mitochondrial ADP/ATP carrier catalyses the one to one exchange of cytosolic ADP against matrix ATP across the inner mitochondrial membrane. The ADP/ATP transport system can be blocked very specifically by two families of inhibitors: atractyloside (ATR) and carboxyatractyloside (CATR) on one hand, and bongkrekic acid (BA) and isobongkrekic acid (isoBA) on the other hand. It is well established that these inhibitors recognise two different conformations of the carrier protein, the CATR- and BA-conformations, which exhibit different chemical, immunochemical and enzymatic reactivities. The reversible transition of the ADP/ATP carrier between the two conformations was studied by fluorometric techniques. This transconversion, which is only triggered by transportable nucleotides, is probably the same as that which occurs during the functioning of ADP/ATP transport system. The fluorometric approach, using the tryptophanyl residues of the yeast carrier as intrinsic fluorescence probes, was combined to a mutagenesis approach to elucidate the ADP/ATP transport mechanism at the molecular level. Finally, recent reports that myopathies might result from defect in ADP/ATP transport led us to develop a method to quantify the carrier protein in muscular biopsies.

https://pubmed.ncbi.nlm.nih.gov/9587671/

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January 7, 2019
How do carrier proteins transport ADP and ATP in and out of mitochondria?

by MRC Mitochondrial Biology Unit

Scientists at the MRC-MBU in Cambridge, U.K., have discovered how a key transport protein, called the mitochondrial ADP/ATP carrier, transports adenosine triphosphate (ATP), the chemical fuel of the cell. This process is vital to keep us alive, every second of our lives, for all of our lives. This work will help us understand how mutations can affect the function of these proteins, resulting in a range of neuromuscular, metabolic and developmental diseases.

Cellular structures, called mitochondria, are the powerhouses of our cells. Every day, we humans need our own body weight in ATP to fuel all of the cellular activities. Nerve impulses, muscle contraction, DNA replication and protein synthesis are just some examples of essential processes that depend upon a supply of ATP. Since we only have a small amount of ATP in our body, we need to remake it from the spent product ADP (adenosine diphosphate) and phosphate using an enzyme complex, called ATP synthase, which is located in mitochondria. In this way, every molecule of ATP is recycled roughly 1300 times a day. For ADP to reach the enzyme, and for the product ATP to refuel the cell, each molecule has to cross an impermeable lipid membrane that surrounds the mitochondria. The mitochondrial ADP/ATP carrier is involved in the transport of ADP in and ATP out of mitochondria.

The carrier cycles between two states; in one state, the central binding site is accessible for binding of ADP, called the cytoplasmic-open state, and in another, the binding site is accessible for binding newly synthesized ATP, called the matrix-open state. A key question has been how the protein is able to convert between these two states, changing its shape to transport ADP and ATP specifically, without letting other small molecules or ions leak across the membrane.

The paper, "The molecular mechanism of transport by the mitochondrial ADP/ATP carrier," published in Cell, describes how scientists have solved the structure of the carrier trapped in the matrix-open state. The carrier was trapped in this state by using a compound called bongkrekic acid, a lethal toxin that binds to the protein and stops it from working. The researchers could also rely on Nanobody technology. Nanobodies are fragments of llama antibodies, which bind specifically to the matrix-open state, and the structure of carrier-nanobody complex with bound bongkrekic acid was determined by X-ray crystallography. Together with earlier structures of the cytoplasmic-open state, this discovery reveals how the carrier works at the atomic scale. The carrier is incredibly dynamic, using six moving elements to transport ADP or ATP across the membrane in a unique and carefully orchestrated way.

The ADP/ATP carrier is just one member of a large family of related transport proteins that bring different compounds in and out of mitochondria, and based on this discovery, the scientists believe that this mechanism is likely to work in a similar way for the whole family. There are many diseases associated with dysfunction of these carriers and for the first time we understand how mutations affect their molecular function.

More information: Jonathan J. Ruprecht et al. The Molecular Mechanism of Transport by the Mitochondrial ADP/ATP Carrier, Cell (2019). DOI: 10.1016/j.cell.2018.11.025

https://phys.org/news/2019-01-carrier-proteins-adp-atp-mitochondria.html

Vine Tea (Dihydroquercetin (DHQ)) apparently works as an anti-inflammatory and anti-cancer drink in Asia:
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Exploring the genes involved in biosynthesis of dihydroquercetin and dihydromyricetin in Ampelopsis grossedentata

Zheng-Wen Yu, Ni Zhang, Chun-Yan Jiang, Shao-Xiong Wu, Xia-Yu Feng & Xiao-Ying Feng
Scientific Reports volume 11, Article number: 15596 (2021) Cite this article

1019 Accesses

6 Citations

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Abstract
Dihydroquercetin (DHQ), an extremely low content compound (less than 3%) in plants, is an important component of dietary supplements and used as functional food for its antioxidant activity. Moreover, as downstream metabolites of DHQ, an extremely high content of dihydromyricetin (DHM) is up to 38.5% in Ampelopsis grossedentata. However, the mechanisms involved in the biosynthesis and regulation from DHQ to DHM in A. grossedentata remain unclear. In this study, a comparative transcriptome analysis of A. grossedentata containing extreme amounts of DHM was performed on the Illumina HiSeq 2000 sequencing platform. A total of 167,415,597 high-quality clean reads were obtained and assembled into 100,584 unigenes having an N50 value of 1489. Among these contigs, 57,016 (56.68%) were successfully annotated in seven public protein databases. From the differentially expressed gene (DEG) analysis, 926 DEGs were identified between the B group (low DHM: 210.31 mg/g) and D group (high DHM: 359.12 mg/g) libraries, including 446 up-regulated genes and 480 down-regulated genes (B vs. D). Flavonoids (DHQ, DHM)-related DEGs of ten structural enzyme genes, three myeloblastosis transcription factors (MYB TFs), one basic helix–loop–helix (bHLH) TF, and one WD40 domain-containing protein were obtained. The enzyme genes comprised three PALs, two CLs, two CHSs, one F3’H, one F3’5’H (directly converts DHQ to DHM), and one ANS. The expression profiles of randomly selected genes were consistent with the RNA-seq results. Our findings thus provide comprehensive gene expression resources for revealing the molecular mechanism from DHQ to DHM in A. grossedentata. Importantly, this work will spur further genetic studies about A. grossedentata and may eventually lead to genetic improvements of the DHQ content in this plant.

Introduction
Dihydroquercetin (DHQ), also known as 3,5,7,3,4-pentahydroxy flavanone or commonly as taxifolin, is a kind of bioactive flavonoid. Because the molecular structure of DHQ contains five phenolic hydroxyl groups, it is considered to be one of the best and rarest natural powerful antioxidants in the world1. The safety of DHQ also has been investigated since it is a key component of dietary supplements2. Additionally, DHQ has a wide range of pharmacological activities, including anti-inflammatory3, anti-microbial4 anti-cancer5, anti-Alzheimer6, anti-toxoplasmosis effects7, health-promoting effects on hepatoprotective and cardiovascular systems8, 9. Due to its excellent pharmacological activity, DHQ is routinely used in pharmaceuticals, health products, foods and agriculture.

DHQ is most prevalent in larch, Douglas fir bark, French maritime pine bark, milk thistle, and onions2, 10,11,12,13. Among them, larch is the plant with the highest content (about 3%) of DHQ2, 14, but its distribution is sporadic, limited in range to a few countries, namely China, Japan, and Russia. Recently, DHQ has been obtained with low yield from plant, thereby limiting its widespread applications. It is less than annual output of 20 tons in global. Among them, China currently only produces around 5 tons. Unfortunately, many plants not only have a low content of DHM, but also upstream compounds (eriodictyol, dihydrokaempfrol) and downstream compounds (dihydromyricetin, quercetin, leucocyanidin) involved in the biosynthesis of DHQ likewise occur in low amounts15, 16. Consequently, it is very difficult to enhance the content of DHQ in these plants via conventional genetic techniques for improvement.

Ampelopsis grossedentata, a member of the Vitaceae plant family, grows widely in mountainous areas of southern China, and contains a high content of dihydromyricetin (DHM) in immature leaves: over 20% in dry leaves of most individuals and up to 38.5% in a few less common ones17,18,19. Interestingly, the plant contains a low content of DHQ, which is a direct precursor of DHM’s biosynthesis20, 21. DHM shares a similar molecular structure with DHQ, the former converted from DHQ by adding a phenolic hydroxyl under the catalysis of special enzymes. Therefore, genetic manipulation of such related metabolic pathways is one useful strategy to improve the yield of DHQ. However, the candidate genes involved in flavonoid (DHQ and DHM) biosynthesis and regulation in A. grossedentata remain unclear.

The enzymes and related genes involved in flavonoid biosynthesis and regulation have been reported in many plants, such as Phyllanthus emblica (L.)22, Ginkgo biloba23, Semen Trigonellae24, Meconopsis25, Arabidopsis thaliana26, Mangifera indica27, Salvia miltiorrhiza28and A. grossedentata29. Several enzymes in the biosynthetic pathway of flavonoids have been studied, including phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumaroyl-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI), which are known to catalyze naringenin synthesis from L-phenylalanine (L-Phe). Subsequently, flavanone 3-hydroxylase (F3H), flavonoid 3’-hydroxylase (F3’H), flavonoid-3’,5’-hydroxylase (F3’5’H), dihydroflavonol-4-reducatse (DFR), flavonol synthase (FLS), and anthocyanidin synthase (ANS) are responsible for the later steps in the synthesis pathway30. Flavonoid biosynthesis is regulated by myeloblastosis transcription factors (MYB TFs), basic helix–loop–helix (bHLH) TFs, and WD40 proteins31. Among them, AtMYB12 has been identified as the main regulator of phenylpropanoid biosynthesis that up-regulates the expression of CHS, CHI, F3H, and FLS32.

In order to illustrate the physiological, biochemical and environmental factors for accumulation of dihydromyricetin, the effects of PAL and CHI on the metabolism of dihydromyricetin under different soil conditions were studied and analyzed in our research33, 34. RNA sequencing (RNA-Seq) has been applied in transcriptome studies of A. grossedentata in different tissues and leaf stages, the study just showed the expression patterns and differential distribution of genes related to DHM bisosynthesis in A. grossedentata29. However, there have been no studies investigating and discussing the molecular mechanisms of flavonoid (DHQ and DHM) formation and accumulation in A. grossedentata with same developmental period, different genetic backgrounds. In this study, A. grossedentata containing an extreme content of DHM were chosen as the experimental material. The candidate genes involved in the flavonoid (DHQ and DHM) biosynthesis and regulation pathways in A. grossedentata was analyzed and investigated by the comparative transcriptome, especially the transformation relationship between DHQ and DHM. The obtained transcriptome data will thus serve as reference sequences for genetic studies of A. grossedentata in the future. Additionally, this study provides useful resources for further study of the transformation from DHQ to DHM, and plays crucial roles for revealing the DHQ’s formation mechanism in A. grossedentata.

Materials and methods
Plant materials
A. grossedentata was collected from Dayu of Jiangxi Province (No. D1–D3, three independent individuals) and Jiangkou of Guizhou Province (No. B1–B3, three independent individuals) in 2012. The collected scions were preserved in the form of sapling and planted in the A. grossedentata germplasm resource repository of Guizhou Normal University (26°26′18.23″ N, 106°39′45.32″ E, at 1100.5 m a.s.l.) (Guiyang, Guizhou Province, China). The plant was identified by Associate Prof. Chao Zhang of School of Life Sciences, Guizhou Normal University. The voucher specimens (accession number: GZNUYZW202002001) was deposited in the herbarium of School of Life Sciences, Guizhou Normal University. For comparative transcriptome analysis, same-aged individuals of B1, B2, B3, D1, D2 and D3 were cultivated closely and under the same management practices (consistent light, soil, and moisture conditions) (Fig. 1A). In May 2018, their young leaf samples were frozen in liquid nitrogen and stored at –80°C until later use.

https://www.nature.com/articles/s41598-021-95071-x

More on Curcumin mentioned earlier.  It is a natural PBM (Photon-Bio-Modulation) note the article immediately below is a bit speculative like there isn't (Zero Point Energy):
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May 23, 2007

Cancer and ATP: The Photon Energy Pathway

Could cancer be a functional deviation of the cellular energy production mechanism that is open to correction by relatively simple means instead of a genetic mutation that is passed down through cell division? Heinrich Kremer, MD, says that this is indeed the case. His hypothesis of a photon-mediated cellular energy pathway may turn out to substantially add to our understanding of what cancer is, and why such natural substances as curcumin may be effective cancer fighting agents.


Turmeric (Curcuma longa) - a curative spice and coloring agent -


According to the World Health Organization, cancer accounted for 13 per cent of deaths world wide in 2005. The trend is rising, which means we have not found the real cause of the disease. Our efforts to treat cancer are centered on symptoms, rather than the factors that cause the disease.

The WHO's cancer information page also says that cancer is the result of a genetic failure of cells:

Cancer arises from one single cell. The transformation from a normal cell into a tumour cell is a multistage process, typically a progression from a pre-cancerous lesion to malignant tumours...
Cancer researchers however have long known that cancer cells are distinguished from their 'normal' cousins by an alteration of the cells' energy metabolism. The mitochondria inside the cells become inactive and the cells switch to a secondary mode of energy production that is based on the fermentation of sugars. This metabolic change has recently been confirmed by a study at Johns Hopkins.

The Hopkins scientists report that the loss of a single gene in kidney cancer cells causes them to stop making mitochondria, the tiny powerhouses of the cell that consume oxygen to generate energy.

Instead, the cancer cells use the less efficient process of fermentation, which generates less energy but does not require oxygen. As a result, the cancer cells must take in large amounts of glucose.

Researchers at the University of Alberta recently discovered a relatively simple way to re-activate the mitochondria and make them re-start normal energy production, using a commonly available substance, dichloroacetate or DCA:

Dr. Evangelos Michelakis, a professor at the U of A Department of Medicine, has shown that dichloroacetate (DCA) causes regression in several cancers, including lung, breast, and brain tumors.

Michelakis and his colleagues, including post-doctoral fellow Dr. Sebastian Bonnet, have published the results of their research in the journal Cancer Cell.

Scientists and doctors have used DCA for decades to treat children with inborn errors of metabolism due to mitochondrial diseases. Mitochondria, the energy producing units in cells, have been connected with cancer since the 1930s, when researchers first noticed that these organelles dysfunction when cancer is present.

According to Wikipedia, dichloroacetate decreases lactate production by shifting the metabolism of pyruvate from glycolysis towards oxidation in the mitochondria. That is the reason the substance has been used to treat lactic acidosis. Its use in cancer has only recently been pioneered and the University of Alberta researchers caution that more trials are needed, before DCA can be recommended as an anti-tumor agent.

It is against this background that we should see the discovery of Heinrich Kremer, MD, a German medical doctor who is perhaps best known for his unconventional views on AIDS. Kremer says that the current view, according to which the mitochondria's normal energy production pathway is based on chemical oxidation does not go deep enough to allow an understanding of the underlying mechanisms of cancer.

Kremer's discovery is described in his book The Silent Revolution in Cancer and AIDS, which is available here in English: http://aliveandwellsf.org/kremer/book.html and here in Italian.

The new view on cancer is explained in detail in an article titled The Secret of Cancer: Short-circuit in the Photon Switch, due for publication shortly. I will link it here as soon as it becomes available. Meanwhile, here is a sneak preview.

- - -

Cancer and ATP: The Photon Energy Pathway

(for the full article, we must await publication in the July issue of Townsend Letter for Doctors)

Although the mutation theory of oncogenesis is generally accepted today, it does not explain how cancer cells seemingly are able to evade all the body's normal mechanisms that prevent and correct such mutations, and how they can invade and metastasize in different tissues from those that are primarily concerned.

Consequently, our standard therapies, which are based on the assumption that the deviated cells must be destroyed and which attempt to do so by a slash, burn and poison approach operated by the surgeon, the radiologist and the oncologist, are of little use in prolonging the patient's life or effecting real cures.


Evolution

To understand the new concept of oncogenesis, we must take a look at the evolution of cells and organisms. Cells as present in today's organisms are the result of a fusion, in prehistoric times, of two different types of unicellular life forms into a unique symbiotic combination. A type of cell of the archaea family and another type of the bacteria family entered into symbiosis and formed what is now known as a protist. The cells of mammals including humans today contain genes from both original families. The bacterial symbionts have evolved into the mitochondria which are delegated to take care of energy production.

ATP Energy Pathways

In cancer, the bacterial symbionts go on strike - they refuse to produce any more of the ATP energy molecules they are normally busy churning out all day. The cells thus have to revert to an alternate mode of energy production (glycolysis) which involves fermentation of sugars. This is very much more inefficient than the normal cellular energy mechanism.

But more importantly, and here comes Kremer's very interesting discovery, the normal mode of energy production is not a pure chemical energy pathway. ATP (adenosine triphosphate) is made up of three molecule groups. A base adenine ring that absorbs light quanta in the near ultraviolet band of 270 nanometer wavelength, one sugar molecule and a molecular string with three phosphate groups.

The currently accepted view is that energy production and storage in ATP is by means of chemical energy, stored in the phosphate bonds. The bond energy is then released by hydrolysis in the cytoplasm, where it is used to drive energetic and metabolic processes. Not so, says Kremer. Hydrolysis only yields heat energy, which is not sufficient to drive all the various cell processes. The secret lies in the adenine groups of ATP which absorb photons, but the role of adenine is not adequately explained in the prevailing hypothesis.

The essential components of mitochondrial cell respiration are light absorbing molecules that react to frequencies from the near ultraviolet band down to the yellow/orange spectral range of visible light. Yet, the source of energy for these cellular power plants is not sunlight, as one might easily be led to assume. The flow of para-magnetically aligned electrons in the respiratory organelles gives rise to a low frequency pulsating electromagnetic field which, enormously accelerated through catalytic processes activated by enzymes, in turn activates a spin-mediated information and energy transfer from the physical vacuum, the zero point field (Cr6--probably better explained by Miles' Charge Field tbh), to the biological entity. Consequently, the human organism isn't governed by heat transfer but by a light frequency modulated energy transformation from space background or physical vacuum to the living organism.

Cancer is a result of the disturbance of the enzyme mediated transformation of that energy. The affected cells lose their ability to communicate with other cells around them and they change not only their way of making energy but they become - for all practical purposes - separate unicellular entities that must divide and form a colony to survive. That colony is what we then see as the tumor, the visible manifestation of cancer.

The exact mechanism of that transformation and how the disturbance, once active, feeds back to cause these changes in the cancer cells, is explained - it is a rather technical subject - in Kremer's paper. We will have to wait for its publication to get the whole story.


Curcumin

In the meantime, however, we can say that curcumin, a natural substance in the family of polyphenols contained in turmeric root or curcuma longa and used as a natural coloring agent and a spice, has been found to be beneficial to cancer patients in research at the Anderson Cancer Research Center. See Can a Common Spice Be Used to Treat Cancer?.

Kremer explains that the anti-cancer properties of curcumin are a consequence of its ability to absorb photons in the violet spectral range of visible light at a wavelength of 415 nanometers. This particular property of the healthy spice is what enables it to bridge the broken pathway of photonic energy production and information transfer, thus bringing the affected cells back into the fold, to make them once more function as parts of the organism.

While, admittedly, more research is needed, the pioneering efforts of Kremer will go a long way to point us in the right direction. I hope I was able to stimulate some interest in that new discovery and that you, my readers, are as eagerly awaiting publication of Kremer's research article and book in English as I am.

Watch here for links as soon as they become available.


Meanwhile, there is news that microwaves - as used in mobile telephony - drastically increase the risk of certain cancers.

"By screening for aneuploidy, you could detect the cancer early and also see what possible drugs to use and whether drugs would even help," Duesberg noted. "Then, you wouldn't have to give a cocktail of drugs that includes all the best poisons, but you could leave out those you could tell wouldn't work. If you could cut chemotherapy drug toxicity in half or two-thirds, and direct it better at cancer, that is some progress. But it is not a cure."

...

Turmeric, Spice of Health

Turmeric comes from the root of the Curcuma longa plant and has a tough brown skin and a deep orange flesh. The volatile oil fraction of turmeric has been demonstrated significant anti-inflammatory activity in a variety of experimental models. Even more potent than its volatile oil is the yellow or orange pigment of turmeric, which is called curcumin. Curcumin is thought to be the primary pharmacological agent in turmeric. In numerous studies, curcumin's anti-inflammatory effects have been shown to be comparable to the potent drugs hydrocortisone and phenylbutazone as well as over-the-counter anti-inflammatory agents such as Motrin. Unlike the drugs, which are associated with significant toxic effects (ulcer formation, decreased white blood cell count, intestinal bleeding), curcumin produces no toxicity.

posted by Sepp Hasslberger on Wednesday May 23 2007
updated on Tuesday December 7 2010

http://www.newmediaexplorer.org/sepp/2007/05/23/cancer_and_atp_the_photon_energy_pathway.htm

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Photobiomodulation and nitric oxide signaling
Satoshi Kashiwagi 1, Atsuyo Morita 2, Shinya Yokomizo 3, Emiyu Ogawa 4, Eri Komai 2, Paul L Huang 2, Denis E Bragin 5, Dmitriy N Atochin 6
Affiliations expand
PMID: 36462596 PMCID: PMC9808891 (available on 2024-01-01) DOI: 10.1016/j.niox.2022.11.005

Abstract
Nitric oxide (NO) is a well-known gaseous mediator that maintains vascular homeostasis. Extensive evidence supports that a hallmark of endothelial dysfunction, which leads to cardiovascular diseases, is endothelial NO deficiency. Thus, restoring endothelial NO represents a promising approach to treating cardiovascular complications. Despite many therapeutic agents having been shown to augment NO bioavailability under various pathological conditions, success in resulting clinical trials has remained elusive. There is solid evidence of diverse beneficial effects of the treatment with low-power near-infrared (NIR) light, defined as photobiomodulation (PBM). Although the precise mechanisms of action of PBM are still elusive, recent studies consistently report that PBM improves endothelial dysfunction via increasing bioavailable NO in a dose-dependent manner and open a feasible path to the use of PBM for treating cardiovascular diseases via augmenting NO bioavailability. In particular, the use of NIR light in the NIR-II window (1000-1700 nm) for PBM, which has reduced scattering and minimal tissue absorption with the largest penetration depth, is emerging as a promising therapy. In this review, we update recent findings on PBM and NO.

Keywords: Cardiovascular diseases; Endothelial cells; Near-infrared light; Nitric oxide; Photobiomodulation.

Copyright 2022 Elsevier Inc. All rights reserved.

https://pubmed.ncbi.nlm.nih.gov/36462596/

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J Biomed Opt
. 2020 Aug;25(:1-20. doi: 10.1117/1.JBO.25.8.085001.

Wavelength- and irradiance-dependent changes in intracellular nitric oxide level

Nathaniel J Pope 1, Samantha M Powell 2, Jeffrey C Wigle 3, Michael L Denton 3
Affiliations expand
PMID: 32790251 PMCID: PMC7423318 DOI: 10.1117/1.JBO.25.8.085001
Free PMC article

Abstract

Significance: Photobiomodulation (PBM) refers to the beneficial effects of low-energy light absorption. Although there is a large body of literature describing downstream physiological benefits of PBM, there is a limited understanding of the molecular mechanisms underlying these effects. At present, the most popular hypothesis is that light absorption induces release of nitric oxide (NO) from the active site of cytochrome c oxidase (COX), allowing it to bind O2 instead. This is believed to increase mitochondrial respiration, and result in greater overall health of the cell due to increased adenosine triphosphate production.

Aim: Although NO itself is a powerful signaling molecule involved in a host of biological responses, less attention has been devoted to NO mechanisms in the context of PBM. The purpose of our work is to investigate wavelength-specific effects on intracellular NO release in living cells.

Approach: We have conducted in-depth dosimetry analyses of NO production and function in an in vitro retinal model in response to low-energy exposure to one or more wavelengths of laser light.

Results: We found statistically significant wavelength-dependent elevations (10% to 30%) in intracellular NO levels following laser exposures at 447, 532, 635, or 808 nm. Sequential or simultaneous exposures to light at two different wavelengths enhanced the NO modulation up to 50% of unexposed controls. Additionally, the immediate increases in cellular NO levels were independent of the function of NO synthase, depended greatly on the substrate source of electrons entering the electron transport chain, and did not result in increased levels of cyclic guanosine monophosphate.

Conclusions: Our study concludes the simple model of light-mediated release of NO from COX is unlikely to explain the wide variety of PBM effects reported in the literature. Our multiwavelength method provides a novel tool for studying immediate and early mechanisms of PBM as well as exploring intracellular NO signaling networks.

Keywords: fluorescence; low-level laser; low-level light; nitric oxide; photobiomodulation; retinal pigment epithelium.

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(Interesting they mention "Quantum")

Quantum biology in low level light therapy: death of a dogma

Andrei P Sommer 1, Peter Schemmer 2, Attila E Pavláth 3, Horst-Dieter Försterling 4, Ádám R Mester 5, Mario A Trelles 6
Affiliations expand
PMID: 32395484 PMCID: PMC7210155 DOI: 10.21037/atm.2020.03.159
Free PMC article

Abstract
Background: It is shown that despite exponential increase in the number of clinically exciting results in low level light therapy (LLLT), scientific progress in the field is retarded by a wrong fundamental model employed to explain the photon-cell interaction as well as by an inadequate terminology. This is reflected by a methodological stagnation in LLLT, persisting since 1985. The choice of the topics is, by necessity, somewhat arbitrary. Obviously, we are writing more about the fields we know more about. In some cases, there are obvious objective reasons for the choice. Progress in LLLT is currently realized by a trial and error process, as opposed to a systematic approach based on a valid photon-cell interaction model.

Methods: The strategy to overcome the current problem consists in a comprehensive analysis of the theoretical foundation of LLLT, and if necessary, by introducing new interaction models and checking their validity on the basis of the two pillars of scientific advance (I) agreement with experiment and (II) predictive capability. The list of references used in this work, does contain a representative part of what has been done in the photon-cell interaction theory in recent years, considered as ascertained by the scientific community.

Results: Despite the immense literature on the involvement of cytochrome c oxidase (COX) in LLLT, the assumption that COX is the main mitochondrial photoacceptor for R-NIR photons no longer can be counted as part of the theoretical framework proper, at least not after we have addressed the misleading points in the literature. Here, we report the discovery of a coupled system in mitochondria whose working principle corresponds to that of field-effect transistor (FET). The functional interplay of cytochrome c (emitter) and COX (drain) with a nanoscopic interfacial water layer (gate) between the two enzymes forms a biological FET in which the gate is controlled by R-NIR photons. By reducing the viscosity of the nanoscopic interfacial water layers within and around the mitochondrial rotary motor in oxidatively stressed cells R-NIR light promotes the synthesis of extra adenosine triphosphate (ATP).

Conclusions: Based on the results of our own work and a review of the published literature, we present the effect of R-NIR photons on nanoscopic interfacial water layers in mitochondria and cells as a novel understanding of the biomedical effects R-NIR light. The novel paradigm is in radical contrast to the theory that COX is the main absorber for R-NIR photons and responsible for the increase in ATP synthesis, a dogma propagated for more than 20 years.

Keywords: Low level light therapy (LLLT); adenosine triphosphate (ATP); biological field-effect transistor (FET); biostimulation; cytochrome c oxidase (COX); interfacial water; mitochondria; quantum biology.

2020 Annals of Translational Medicine. All rights reserved.

https://pubmed.ncbi.nlm.nih.gov/32395484/

More on PBM:
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Systemic glucose levels are modulated by specific wavelengths in the solar light spectrum that shift mitochondrial metabolism

Michael B Powner 1, Glen Jeffery 2
Affiliations expand
PMID: 36327250 PMCID: PMC9632789 DOI: 10.1371/journal.pone.0276937
Free PMC article

Abstract

Systemic glucose levels can be modulated with specific solar wavelengths that influence mitochondrial metabolism. Mitochondrial respiration can be modulated using light that shifts ATP production with exceptional conservation of effect across species, from insects to humans. Known wavelengths have opposing effects of photobiomodulation, with longer wavelengths (660-900 nm red/infrared) increasing ATP production, and 420 nm (blue) light suppressing metabolism. Increasing mitochondrial respiration should result in a greater demand for glucose, and a decrease should result in a reduced demand for glucose. Here we have tested the hypothesis that these wavelengths alter circulating glucose concentration. We first established an oral glucose tolerance test curve in a bumblebee model, which showed sustained increase in systemic glucose beyond that seen in mammals, with a gradual normalisation over eight hours. This extended period of increased systemic glucose provided a stable model for glucose manipulation. Bees were starved overnight and given a glucose load in the morning. In the first group glucose levels were examined at hourly intervals. In the second group, bees were additionally exposed to either 670 nm or 420 nm light and their blood glucose examined. Increasing mitochondrial activity with 670 nm light at the peak of circulating glucose, resulted in a significant 50% reduction in concentration measured. Exposure to 420nm light that retards mitochondrial respiration elevated systemic glucose levels by over 50%. The impact of 670 nm and 420 nm on mitochondria is highly conserved. Hence, different wavelengths of visible light may be used to modulate systemic metabolism bidirectionally and may prove an effective agent in mammals.

https://pubmed.ncbi.nlm.nih.gov/36327250/

Looks like there is a relationship between pathways for ATP creation with mental illness (Schizophrenia, Autism, et. al) and cancer. The same dynamics look correlated to a partial degree:
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Interesting paper below. Has some ties with the Cancer thread here.
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https://bmcpsychiatry.biomedcentral.com/articles/10.1186/s12888-023-04784-y
Research
Open Access
Published: 19 April 2023
Alterations in inflammasome-related immunometabolites in individuals with severe psychiatric disorders
Ulrika Hylén, Eva Särndahl, Susanne Bejerot, Mats B Humble, Tuulia Hyötyläinen, Samira Salihovic & Daniel Eklund
BMC Psychiatry volume 23, Article number: 268 (2023) Cite this article

918 Accesses

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Abstract

Introduction

Psychiatric disorders are common and significantly impact the quality of life. Inflammatory processes are proposed to contribute to the emergence of psychiatric disorders. In addition to inflammation, disturbances in metabolic pathways have been observed in individuals with different psychiatric disorders. A suggested key player in the interaction between inflammation and metabolism is the Nod-like receptor 3 (NLRP3) inflammasome, and NLRP3 is known to react to a number of specific metabolites. However, little is known about the interplay between these immunometabolites and the NLRP3 inflammasome in mental health disorders.

Aim
To assess the interplay between immunometabolites and inflammasome function in a transdiagnostic cohort of individuals with severe mental disorders.

Methods
Mass spectrometry-based analysis of selected immunometabolites, previously known to affect inflammasome function, were performed in plasma from low-functioning individuals with severe mental disorders (n = 39) and sex and aged-matched healthy controls (n = 39) using a transdiagnostic approach. Mann Whitney U test was used to test differences in immunometabolites between psychiatric patients and controls. To assess the relationship between inflammasome parameters, disease severity, and the immunometabolites, Spearman’s rank-order correlation test was used. Conditional logistic regression was used to control for potential confounding variables. Principal component analysis was performed to explore immunometabolic patterns.

Results
Among the selected immunometabolites (n = 9), serine, glutamine, and lactic acid were significantly higher in the patient group compared to the controls. After adjusting for confounders, the differences remained significant for all three immunometabolites. No significant correlations were found between immunometabolites and disease severity.

Conclusion
Previous research on metabolic changes in mental disorders has not been conclusive. This study shows that severely ill patients have common metabolic perturbations. The changes in serine, glutamine, and lactic acid could constitute a direct contribution to the low-grade inflammation observed in severe psychiatric disorders.

Peer Review reports
Introduction
Psychiatric disorders affect millions of people each year and are today one of the leading causes of disability in the world. The mortality risk is up to 60% higher in individuals with severe psychiatric disorders, and their life expectancy is reduced by 10–20 years [1].

Psychiatric disorders are diagnosed using interviews and questionnaires and are solely based on clinical symptoms [2]. The disorders often overlap, and within the different diagnoses there is often heterogeneity regarding symptoms [3]. The comorbidity can be extensive, especially among the more severely ill individuals [4]. The etiology of psychiatric disorders is still largely unknown but is thought to be multifactorial [5], and several proposed causal mechanisms have been reported across psychiatric disorders [6]. This had led to the idea that psychiatric disorders share common denominators, thus transdiagnostic studies are now put into focus, which take into account that the same mechanisms can be present across several symptoms [7]. Such common mechanisms include inflammatory processes, which have been proposed to contribute to the emergence of psychiatric disorders, such as depression, schizophrenia spectrum disorder (SSD), autism spectrum disorder (ASD), and obsessive-compulsive disorder (OCD) [8].

In addition to inflammation, metabolic shifts toward anabolism and increased synthesis of lactic acid, called the Warburg effect, have been observed in individuals with SSD, ASD, bipolar disorder, and anxiety [9]. Previous studies have found increased levels of lactic acid in individuals with bipolar disorder [10], ASD [11], and SSD [12]. Other examples of metabolic perturbations in mental disorders include mitochondrial dysfunction, oxidative stress [13], disturbances in amino acid metabolism [12], and changes in circulating lipids [14]. These metabolic changes have in recent years been shown to have a direct effect on inflammatory pathways in immune cells [15].

Metabolites in both glucose and fatty acid metabolism, as well as intermediates in the Krebs cycle, have been found to act as positive or negative regulators of inflammation. These immunologically active metabolites are referred to as immunometabolites [16]. For example, Krebs cycle intermediates such as succinate and citrate have been found to accumulate upon activation of myeloid cells, an event that promotes increased gene expression levels proinflammatroy genes of [17, 18], while accumulation of itaconate has suppressive effects inflammatory gene expression [19]. Increases in the glycolytic end-product lactic acid, through the Warburg effect, is a hallmark of activated immune cells that in turn regulates inflammatory responses by affecting myeloid cell differentiation, NFkB signaling and inflammasome activation [20, 21]. Other immunometabolites, such as ketone metabolite β-hydroxybutyrate, have been highlighted due to their specific effects on inflammasome activity [22, 23]. Moreover, amino acids, such as serine and glutamine, are known to promote inflammation through increased gene expression of proinflammatory mediators like IL-1β [24, 25], while tryptophan metabolites and arginine mainly suppress IL-1β and IL-18 [26, 27].

We have previously observed increased expression and activity of the inflammasome in a transdiagnostic cohort of severely ill individuals with psychiatric disorders [28]. In light of this, the current study aims to investigate if immunometabolites known as regulators of the inflammasome/IL-1-family cytokine axis also participate in the altered inflammatory response of these patients with severe psychiatric disorders.

Materials and methods

Participants

In this exploratory, cross-sectional study, 39 individuals with severe mental disorder and 39 age and sex-matched healthy controls were included. The inclusion criteria for participants with mental disorder were ages between 16 and 47 years and being diagnosed with SSD, ASD, OCD, and/or nonsuicidal self-injury disorder (NSSID) prior to enrolment by a board-certified psychiatrist. The Clinical Global Impression – Severity scale (CGI-S) was used. The exclusion criteria included neurological autoimmune disorder and/or an ongoing infection at the time of blood sampling. Participants with mental disorders were recruited from psychiatric clinics across Örebro County and surrounding counties. Interviews with participants were held at Örebro University hospital or in the participants´ homes.

Inclusion criteria for the healthy controls were based on age and sex, matched to the participants with psychiatric disorders. Exclusion criteria included being diagnosed with any psychiatric or medical disorder, however, three of the controls had been diagnosed with a psychical condition (psoriasis, asthma, nut allergy) but presented no current symptoms. However, none of the controls received medical treatment for their diseases at the time of the study, and they were regarded as being in clinical remission. Diagnostic interviews were performed with the controls to exclude individuals with current or previous psychiatric disorder (see below). Healthy controls were recruited through flyers or word of mouth. None of the participants was related to one another.

All participants with severe psychiatric disorder were recruited between November 2016 and June 2018, and the controls were recruited between January 2017 and June 2018.


Discussion

In this study, immunometabolites with known regulatory effects on the NLRP3 inflammasome and the production of IL-1 family cytokines were investigated in markedly ill individuals with severe psychiatric disorders and in age and sex-matched healthy controls. The results showed that plasma levels of three positive regulators of inflammasome function, serine, glutamine, and lactic acid, were significantly elevated in the patients compared to the healthy controls.

Lactic acid is a metabolite known to have immunomodulatory effects and to regulate the activation of immune cells [30]. During immune activation, up-regulation of glycolysis is observed in immune cells, leading to increased fermentation of glucose to lactic acid. This process is induced despite the availability of oxygen, and is known as the Warburg effect, and is a process in which the cell favors rapid mobilization of energy via aerobic glycolysis over the Krebs cycle and cellular respiration [31]. Lactic acid, produced during immune activation, has both proinflammatory and immunosuppressive effects [32]. In a previous in vitro study, increased fermentation of glucose to lactic acid was found to increase activation of the NLRP3 inflammasome and the release of IL-1β [33]. Increased levels of lactic acid are also known to be associated with dysfunction in the mitochondria due to oxidative stress, which promotes inflammation [34]. Interestingly, in control individuals, significant correlations was observed between a number of immunometabolites and lactic acid, while in patients the significant increase in lactic acid did not correlate to the altered levels of other immunometabolites (Fig. 1); data that might reflect an increased Warburg effect associated with increased immune activity in patients, or more evident mitochondrial dysfunction.

Considering previous clinical findings, studies has reported differences in energy metabolism and a change towards production of lactic acid in several psychiatric illnesses [9]. However, the findings are contradictory; in some studies, plasma levels of lactic acid were found to be up-regulated [35, 36], whereas others report down-regulation of lactic acid in patients compared to controls [12, 37]. In some studies, increased levels of lactic acid have also been found in both plasma and in some areas of the brain in patients compared to controls [11, 38]. It should be noted that most studies investigating lactic acid have been performed in patients with SSD and ASD, while studies on individuals with OCD are sparse, and no studies, to our knowledge, has been performed on individuals with NSSID.

Serine is a non-essential amino acid that plays an important role in energy metabolism. Most of the serine in the body is synthesized from 3-phosphoglycerate, an intermediate in glycolysis [39]. Recent research has found that serine regulates inflammatory responses, but the results are still insufficient. An in vitro study supports a positive effect of serine on inflammasome activity by regulating IL-1β production in macrophages, and deprivation of serine reduces gene expression and plasma levels of IL-1β [24]. The increase of serine observed in patients in the current study is in line with previous research on individuals with SSD [40]. However, concerning ASD, results from previous research are contradictory [41, 42], showing both increased and decreased plasma levels of serine in these patients. No studies have, to our knowledge, explored serine levels in patients with OCD and NSSID, respectively.

Glutamine is a versatile amino acid that acts as an important source for TCA intermediates, as well as to serve as a precursor for nucleotides and lipid synthesis. From an immunological point of view, glutamine is a key metabolite in most immune cells, where it promotes cell proliferation and cytokine production [43]. Glutamine drives the production of IL-1β through its conversion into succinate, which acts by increasing the gene expression of IL1B [25]. In individuals with psychiatric disorders, glutamine levels have been found to be affected but the results are inconsistent. Parksepp et al. (2020) found higher levels of glutamine in sera of patients with schizophrenia, five years after introduction of anti-psychotic medication, compared to controls [44], data that agrees with the findings of increased plasma levels of glutamine in the patients of our study. Contrarily, decreased levels of glutamine has been found in individuals with chronic schizophrenia and in first-episode schizophrenia patients compared to controls [45, 46]. Finally, Loureiro et al. (2020) found no differences in glutamine levels between patients with first-episode psychosis, unaffected siblings, and healthy controls [47].

In our previous study, investigating the same patients as in the current study, we detected increased levels of inflammasome components and IL-1 cytokines in the patients compared to controls [28]. The increased levels of lactic acid, serine, and glutamine in the current study (previously suggested to positively affect the activity of the NLRP3 inflammasome) supports the idea of an immunometabolic contribution to this our previous observations of increased inflammasome activity. However, this should be interpreted with caution since previous studies on the effects of the immunometabolites on inflammation and inflammasome activity have primarily been performed using in vitro or animal models, and these results do therefore not necessarily reflect the immunological properties of these metabolites in humans in vivo. In addition, there were no significant correlations between the immunometabolites and disease severity or between the metabolites and any of the diagnosis-specific rating scales. This is in line with previous studies showing no correlation between symptom severity and serine or glutamine [45, 48]. Contrarily, a negative correlation between glutamine and severity scores was detected in patients with first episode psychosis [40]. During subgroup analyses, lactic acid remained significant across all diagnostic groups, except for OCD, while serine and glutamine showed no significant difference between patients and controls, when the patients were divided into small diagnostic subgroups. This result indicates a larger difference between patients and controls for lactic acid than for serine and glutamine. Nevertheless, these same findings identified across different diagnostic groups, taken together with the fact that the data failed to cluster diagnoses in the PCA, indicate that the metabolic perturbations is not inherent to a single diagnostic group but instead is a transdiagnostic phenomenon.

An increased knowledge about immunometabolism within the field of psychiatry can help us to identify an immunometabolic phenotype, which is a transdiagnostic rather than a single diagnosis approach. This phenotype is associated with atypical symptoms connected to disturbances in the energy metabolism, such as anhedonia and fatigue [49, 50]. Other atypical symptoms could be hypersomnia and suicidal ideation [50]. Stratification into an immunometabolic phenotype can create new opportunities for development of different pharmacological treatments where patients are more likely to respond.

Limitations in this study include a small sample size, which makes comparisons between the diagnostic groups difficult and induces a risk for type II error. Another limitation is the design of the study involving only one measuring point of the immunometabolites, and thereby lacks the possibility to follow changes in the levels of metabolites over time. However, great care was taken to minimize diurnal variations of metabolites by taking fasting morning samples. Lifestyle factors, such as diet and physical activity, may affect the levels of immunometabolites. It is well-known that individuals with severe psychiatric disorders tend to have more unhealthy dietary habits, and to be less physical active than healthy controls. To adjust for these potential impacts are by design difficult but the identified difference between the patients and controls in regard to BMI was adjusted for in the analyses. Treatment with antipsychotic medication may increase the risk for metabolic disturbances, and to lead to increased levels of immunometabolites [37]. The correlation analysis (Fig. 1) showed a clear correlation between lactate and BMI in the patient group. While the study was designed with matched controls to adjust for these potential impacts, there might be an effect on immunometabolite levels, including lactate, due to BMI. However, this was adjusted for in the regression analyses and when removing SSD patients (who displayed the highest mean BMI) and their matched controls, lactate, serine and glutamine remained significantly different between patients and controls (data not shown). In addition, most of these differences remained significantly different in the subgroup analyses, and stratification on antipsychotic medication did not affect the significant observations, showing that medication does not influence the results of the current study in a major way.

Taken together, the study reveals that among immunometabolites with known effect on inflammation and in particular the NLRP3 inflammasome and the IL-1 family cytokines, lactic acid, serine, and glutamine are increased in individuals with psychiatric disorders irrespective of primary diagnosis. The increase of these immunometabolites could constitute a direct contribution to the observed low-grade inflammation observed by us and other in severe psychiatric disorders. However, further studies should address the interplay between the inflammasome and immunometabolites in detail by studying immune cells from larger groups of individuals with severe psychiatric disorders.

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Also:
Editorial: Antidepressant Prescriptions in Children and Adolescents
https://www.frontiersin.org/articles/10.3389/fpsyt.2020.600283/full


Published: 17 January 2022
Ketogenic diet for human diseases: the underlying mechanisms and potential for clinical implementations
Huiyuan Zhu, Dexi Bi, Youhua Zhang, Cheng Kong, Jiahao Du, Xiawei Wu, Qing Wei & Huanlong Qin
Signal Transduction and Targeted Therapy volume 7, Article number: 11 (2022) Cite this article

Abstract

The ketogenic diet (KD) is a high-fat, adequate-protein, and very-low-carbohydrate diet regimen that mimics the metabolism of the fasting state to induce the production of ketone bodies. The KD has long been established as a remarkably successful dietary approach for the treatment of intractable epilepsy and has increasingly garnered research attention rapidly in the past decade, subject to emerging evidence of the promising therapeutic potential of the KD for various diseases, besides epilepsy, from obesity to malignancies. In this review, we summarize the experimental and/or clinical evidence of the efficacy and safety of the KD in different diseases, and discuss the possible mechanisms of action based on recent advances in understanding the influence of the KD at the cellular and molecular levels. We emphasize that the KD may function through multiple mechanisms, which remain to be further elucidated. The challenges and future directions for the clinical implementation of the KD in the treatment of a spectrum of diseases have been discussed. We suggest that, with encouraging evidence of therapeutic effects and increasing insights into the mechanisms of action, randomized controlled trials should be conducted to elucidate a foundation for the clinical use of the KD.

Introduction

“Diseases enter by the mouth”, the literal meaning of the Chinese idiom “” (bìng cóng kǒu rù), which was first recorded in the Tsin/Jin () Dynasty, was initially assumed to convey the concept of dietetic hygiene; however, at present, the idiom aptly emphasizes the fact that dietary factors are closely associated with many diseases.1 Dietary planning is increasingly popular, not only as an intervention to maintain health but also as an important non-pharmaceutical option for fighting disease. Choosing a proper diet can have profound implications for health and may induce therapeutic effects. At present, there are numerous types of diets, including low-carbohydrate diets (LCDs; e.g., ketogenic diet [KD]), paleo-type diets, plant-forward diets, intermittent fasting, clean eating, traditional regional diets (e.g., Mediterranean diet), and other specifically designed diets (e.g., dietary approaches to stop hypertension diet, Mayo Clinic diet), that diversify food patterns or fulfill specific purposes. With the explosion of experimental and clinical research on microbiota in the past decade, the important roles of microbiota in health and disease have become well-known.2 Recently, microbiota-directed food invention, which was developed to exert therapeutic effects through the manipulation of gut microbiota components, was found to be an effective dietary supplementation strategy for undernourished children.3,4 One of the diets on the burgeoning list of diets, the KD, has a long history of clinical use and has recently gained considerable interest owing to its promising potential effects on a wide spectrum of diseases.

https://www.nature.com/articles/s41392-021-00831-w

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Free access | 10.1172/JCI120843


Altered mitochondrial function in insulin-deficient and insulin-resistant states
Gregory N. Ruegsegger, Ana L. Creo, Tiffany M. Cortes, Surendra Dasari, and K. Sreekumaran Nair
Published August 31, 2018 - More info

https://www.jci.org/articles/view/120843
https://www.jci.org/articles/view/120843/pdf

View PDF

Abstract

Diabetes profoundly alters fuel metabolism; both insulin deficiency and insulin resistance are characterized by inefficient mitochondrial coupling and excessive production of reactive oxygen species (ROS) despite their association with normal to high oxygen consumption. Altered mitochondrial function in diabetes can be traced to insulin’s pivotal role in maintaining mitochondrial proteome abundance and quality by enhancing mitochondrial biogenesis and preventing proteome damage and degradation, respectively. Although insulin enhances gene transcription, it also induces decreases in amino acids. Thus, if amino acid depletion is not corrected, increased transcription will not result in enhanced translation of transcripts to proteins. Mitochondrial biology varies among tissues, and although most studies in humans are performed in skeletal muscle, abnormalities have been reported in multiple organs in preclinical models of diabetes. Nutrient excess, especially fat excess, alters mitochondrial physiology by driving excess ROS emission that impairs insulin action. Excessive ROS irreversibly damages DNA and proteome with adverse effects on cellular functions. In insulin-resistant people, aerobic exercise stimulates both mitochondrial biogenesis and efficiency concurrent with enhancement of insulin action. This Review discusses the association between both insulin-deficient and insulin-resistant diabetes and alterations in mitochondrial proteome homeostasis and function that adversely affect cellular functions, likely contributing to many diabetic complications.

Introduction

The worldwide prevalence of diabetes, especially type 2 diabetes (T2D), and the associated global economic burden are substantial and rapidly growing, with major adverse impacts on health and social security systems (1). Although T2D, characterized by insulin resistance and insufficient insulin secretion, and type 1 diabetes mellitus (T1D), characterized by absolute insulin deficiency, have different etiologies (2), both profoundly derange fuel metabolism, including metabolism of glucose, fat, and amino acids (AAs), the main energy sources for humans. Moreover, well-documented upregulation of whole-body oxygen consumption in T1D and T2D indicates higher resting energy expenditure in untreated individuals (3–6). Emerging evidence clearly indicates that these alterations in energy metabolism can be traced to mitochondria, organelles that produce the majority of a cell’s ATP from macronutrients through cellular respiration and oxidative phosphorylation (OXPHOS).

Mitochondria’s chief role, cellular respiration, is in turn associated with various functions, including reactive oxygen species (ROS) production and detoxification; β-oxidation and processing of acetyl-CoA, generated from fatty acids (FAs), glucose, and AA oxidation through the tricarboxylic acid (TCA) cycle; and mitochondrial electron transport chain (ETC) complex activities (7, . Mitochondria possess approximately 1,500 predominantly nuclear-encoded proteins in addition to 13 proteins encoded by their own DNA (mtDNA). In humans, mtDNA contains 37 genes, which also encode 22 transfer RNAs and two RNA subunits, and all are critical to maintaining mitochondrial function (9). mtDNA replication, mitochondrial biogenesis (involving transcription, translation, and proteome homeostasis), and mitochondrial function are tightly regulated and highly complex. As almost all cellular processes require ATP, it is unsurprising that both physiological and pathological alterations in energy demand and fuel supply influence mitochondrial functions, including mitochondrial dynamics (fusion and fission), biogenesis, and mitophagy (10). Insulin, the predominant hormone involved in fuel metabolism, is involved in many cellular functions, including protein turnover, a highly ATP-dependent process; moreover, insulin impairment in diabetes is associated with altered energy metabolism.

Here, we synthesize results showing insulin’s key role in mitochondrial function and in regulation of mitochondrial proteome homeostasis, including biogenesis, at the transcriptional, translational, and posttranslational levels, as well as in mitophagy. Our primary aim is to broadly highlight how insulin deficiency in T1D and insulin resistance in T2D (and other conditions such as polycystic ovarian syndrome) have detrimental effects on mitochondrial function, primarily in skeletal muscle. We also provide data on mitochondrial function in heart, liver, kidney, adipose tissue (AT), and brain. Detailed review of mitochondrial functions in other aspects of metabolism and in specific tissues is beyond the scope of this Review, but readers are referred to other publications (11–15). Additionally, we discuss how altered mitochondrial function can adversely affect insulin’s actions. We propose that impaired insulin secretion and action may explain many of the alterations in mitochondrial function observed in T1D and T2D, respectively, and in turn explain how altered mitochondrial function contributes to many pathologies in diabetes.

Insulin regulates mitochondrial biogenesis, degradation, and function

In canonical insulin signaling, insulin binds to insulin receptor (IR), inducing IR autophosphorylation, recruitment of insulin receptor substrate (IRS) adaptor proteins, and AKT pathway stimulation (16). The insulin/IRS/AKT pathway is central to glucose homeostasis and regulates multiple ATP-dependent processes, including stimulating glucose uptake in muscle and AT, inhibiting glycogenosis and glucose release in the liver, promoting lipid synthesis and storage in liver and AT, and regulating protein synthesis and degradation. The insulin/IRS/AKT pathway’s regulation of mTOR activity and FOXO transcription factors has key implications for mitochondrial function, as mTOR (17, 18) and FOXO1 (19, 20) control mitochondrial oxidative function via regulation of PPARγ coactivator 1α (PGC1A). PGC1A is a master regulator of mitochondrial biogenesis via the transcription factors nuclear respiratory factor 1 (NRF1) and NRF2, which control nuclear-encoded and mitochondrial-encoded genes via transcription factor A, mitochondrial (TFAM) (ref. 21 and Figure 1). All nuclear-encoded mitochondrial proteins are synthesized in cytosolic ribosomes and imported into mitochondria, whereas mitochondrial ribosomes synthesize 13 mitochondrial proteins (22). Mitochondrial complexes are formed with both nuclear-encoded imported proteins and mitochondrial-encoded proteins. Insulin’s role in these mitochondrial outer and inner membrane transporters and complex formation remains unclear.

The mitochondrial proteome in the presence and absence of insulin signaling Figure 1

The mitochondrial proteome in the presence and absence of insulin signaling. (A) Insulin binding to the insulin receptor (IR) initiates a downstream signaling cascade leading to the phosphorylation of insulin receptor substrates (IRS1/2) and activation of AKT, the major node of insulin signaling. Of the many downstream effectors of AKT, the inhibition of FOXO1 influences the expression and activity of PPARγ coactivator 1α (PGC1A), which activates transcription factors (e.g., myocyte enhancer factor 2 [MEF2], nuclear respiratory factor 1 [NRF1], NRF2) that control nuclear-encoded mitochondrial gene expression. PGC1A also enhances the transcription of mitochondrial genes through transcription factor A, mitochondrial (TFAM). In the presence of appropriate amino acid concentrations, activation of mTOR and its downstream targets increases the synthesis of nuclear-encoded mitochondrial proteins in the cytoplasm, which are imported into the mitochondria to form TCA cycle, β-oxidation, and ETC complex proteins. Mitochondrial-encoded genes are synthesized in the mitochondria and form ETC complex proteins. Collectively, these increase the mitochondrial proteome and ATP production. (B) Insulin deficiency decreases the transcription of nuclear-encoded and mitochondrial-encoded mitochondrial genes and the synthesis of mitochondrial proteins, and increases oxidative stress and gene expression central to the proteasome pathway (e.g., the E3 ubiquitin ligase TRIM63) and autophagy pathway (e.g., Beclin), leading to increased degradation of mitochondrial proteins. Collectively, these responses decrease the mitochondrial proteome and ATP production. Additional abbreviations: 4EBP1, eukaryotic translation initiation factor 4E–binding protein 1; p70S6K, 70-kDa ribosomal S6 kinase.

Mitochondrial protein synthesis and turnover are critical for maintaining protein quality and function (23–25). Insulin’s effects on protein synthesis vary considerably between different proteins and tissues (26). Stump et al. (27) demonstrated that high physiological-level insulin infusion in healthy humans increased maximal ATP production in freshly isolated mitochondria from human skeletal muscle. Similarly, insulin infusion increased mRNA levels of mitochondrial genes (e.g., NADH dehydrogenase subunit IV [MT-ND4]) and nuclear genes encoding mitochondrial proteins (e.g., cytochrome c oxidase subunit IV [COX4])) increased mitochondrial protein synthesis, and increased activities of the key metabolic enzymes COX and citrate synthase (CS) in muscle. In healthy humans, high insulin concentrations stimulate mitochondrial protein synthesis in muscle, but only when coinfused with AAs (28), as acute insulin infusion lowers plasma AA concentrations by suppressing protein degradation (29, 30).

Like mitochondrial protein synthesis, increases in mitochondrial enzyme activities and ATP production also depend on AA availability. In healthy adults, insulin infusion without AA supplementation upregulated the mRNA expression of COX3, PGC1A, and NRF1; however, insulin alone failed to increase muscle mitochondrial protein synthesis, COX and CS activity, ATP production, and phosphorylation of the protein synthesis activators mTOR, 4EBP1, and p70S6K (31). Similarly, Ling et al. (32) associated increased muscle PGC1A mRNA with increased glucose oxidation following insulin infusion but noted that PGC1A protein level was unaltered. These observations (27, 28, 31, 33) indicate that insulin stimulates transcription of specific genes with potential effects on mitochondrial function, but only enhances mitochondrial protein expression and function when appropriate AA concentrations are present. Insulin inhibits not only degradation of endogenous proteins but also fat storage. Thus, when carbohydrate alone is ingested, insulin signaling promotes carbohydrate utilization for energy needs, preserving protein and fat stores. Since both AAs and insulin are needed for protein synthesis, carbohydrate or fat alone is unlikely to promote protein synthesis. Not surprisingly, protein ingestion is associated with higher thermogenesis than carbohydrate and fat ingestion, likely because of protein synthesis’s higher ATP demand (34). It remains undetermined whether carbohydrate-induced increases in insulin signaling fail to stimulate protein synthesis in muscle (especially mitochondria) in order to reserve AAs for synthesis of essential proteins, such as clotting factors in liver, when AA availability is limited.

Gene manipulation studies in mice have provided molecular links between insulin signaling and mitochondrial function. IR deletion in muscle, heart, and brain decreases mitochondrial respiration and increases oxidative stress (35–37). It also impairs ATP production and FA oxidation (FAO) in the heart (35). Adipose-specific IR knockout decreases mitochondrial content and oxygen consumption in brown AT (BAT), while depleting white AT (WAT) mass by over 90% (38). Similarly, ATP production is decreased in β cell–specific IR-knockout mice despite increased oxygen consumption, and restoring IR expression in β cells restored deficits in mitochondrial ATP production, suggesting that insulin regulates β cell mitochondrial function (39). The cardiomyocyte-specific deletion of Irs1 and Irs2 decreased ADP-stimulated mitochondrial oxygen consumption and ATP production concomitant with downregulation of OXPHOS and FAO genes (40). Similar impairments in mitochondrial FAO and mitochondrial morphology in the heart were observed following double knockout (DKO) of IR and IGF1 receptor (Igf1r), whose signaling cascades converge at IRS proteins (41).

Studies in Irs1/2–DKO mice provided mechanistic links between hepatic insulin action and mitochondrial function. Blocking hepatic insulin signaling in Irs1/2–DKO mice led to insulin resistance and FOXO1 hyperactivation, which disrupted ETC complexes and reduced NAD+ concentration (42). Consequently, the NAD+-dependent protein deacetylase SIRT1 reduces its deacetylation of PGC1A, decreasing mitochondrial biogenesis and drastically impairing mitochondrial coupling and ATP production (19). Thus, insulin may enhance mitochondrial function through redox regulation of PGC1A.

Recent studies show that insulin stimulates mitochondrial respiration in isolated cortical neurons from healthy mice but not Alzheimer’s disease–prone (AD-prone) apolipoprotein E ε4–knockin mice (43). Given that 80% of AD patients display impaired glucose tolerance or T2D (44), and the AD brain displays insulin resistance and impaired mitochondrial function (45, 46), strategies that enhance insulin sensitivity in diabetes may also have therapeutic value in AD. Collectively, these data demonstrate insulin’s importance for normal mitochondrial function in multiple tissues.

Effects of insulin deficiency on mitochondrial function
T1D results from insulin deficiency and produces long-term macrovascular and microvascular complications. Alterations in cellular oxidation and ATP production likely contribute to the pathogenesis of diabetes-related complications. Benedict and Joslin’s seminal findings before the discovery of insulin showed that whole-body oxygen consumption is increased in untreated T1D (47), but this observation was criticized because the comparison was with similarly wasted nondiabetic controls who had lower whole-body oxygen consumption (48). More recent observations show that transient withdrawal from insulin in insulin-treated T1D increased within-individual whole-body oxygen consumption and energy expenditure; these were also elevated in comparison with predicted energy values for non-T1D people of similar age and body composition (4, 49). Moreover, glucagon, whose effects on blood glucose counteract insulin’s, has a thermogenic effect (50). Further studies have shown that high glucagon concentrations during insulin deficiency may explain increased oxygen consumption (49), suggesting that the liver, which is rich in glucagon receptors, mediates this observed increase in oxygen consumption. Glucagon promotes gluconeogenesis (4) and increased protein turnover (34, 51), both ATP-dependent processes. However, many other insulin-dependent cellular processes involving energy storage, including glycogen synthesis and lipogenesis, are reduced during insulin deficiency and thus reduce ATP demand (4), prompting a hypothesis that insulin deficiency causes uncoupling of mitochondrial respiration pathways and thereby increases energy waste.

In support of the uncoupled mitochondrial respiration hypothesis, it has been shown that despite increasing whole-body oxygen consumption, transient insulin deficiency in T1D patients decreased maximal ATP production in mitochondria isolated from muscle and downregulated expression of TCA and OXPHOS mRNA (52). These results were concurrent with increased inflammation, cytoskeleton signaling, and mRNA expression of integrin signaling proteins, suggesting potential interaction between these important pathways, mitochondrial function, and associated metabolic derangement; however, these observations may be secondary to hyperglycemia and increased metabolism of FAs, AAs, and ketones that occur following insulin deficiency (52). Insulin treatment maintains mitochondrial function in T1D patients, as resting or postexercise mitochondrial capacity and maximal oxidative ATP synthesis were unchanged in in vivo measurements from the calf muscle of treated T1D patients compared with nondiabetic controls (53). However, among the diabetic patients, elevated hemoglobin A1c, a blood-based marker of glycemic control, negatively correlated with mitochondrial capacity (53), suggesting that reduced insulin action and/or hyperglycemia adversely affects mitochondrial capacity.

Insulin deprivation impairs mitochondrial function in muscle. Recent proteomic and lipidomic studies have provided mechanistic insight into altered muscle mitochondrial function during insulin deficiency. Zabielski et al. compared mice with streptozotocin-induced diabetes (STZ mice) continuously treated with insulin implants and mice in which insulin implants were removed after the reestablishment of healthy glycemic control (54). Insulin deprivation did not significantly change ex vivo maximal mitochondrial oxygen consumption but led to significant declines in coupling efficiency and ATP production in isolated muscle mitochondria, concurrent with marked increases in ROS emission that caused oxidative damage to proteins. The above changes were explained on the basis of substantial downregulation of mitochondrial proteins involved in OXPHOS, TCA cycle, and β-oxidation in comparison with nondiabetic controls. However, insulin treatment prevented deficits in mitochondrial function, supporting the hypothesis that insulin is critical to maintaining mitochondrial proteome and functional homeostasis.

Insulin deprivation in STZ mice also substantially increased mitochondrial protein degradation and markers of mitophagy (Beclin) and proteasome degradation (E3 ubiquitin ligase TRIM63), primary protein degradation pathways (55). Mitophagy, the specific autophagic targeting of mitochondria, is the primary pathway for degrading mitochondrial proteins (56). Thus, mitophagic signaling is likely upregulated during insulin deficiency to remove damaged mitochondria. Both STZ mice and T1D humans showed that insulin deficiency selectively increased the peptides in muscle that represent in vivo degradation of proteins involving mitochondrial function (e.g., ATP synthase subunit-γ and COX6) and protein translation (55). These studies suggest that insulin deficiency in T1D increases muscle mitochondrial protein degradation, leading to reduced mitochondrial protein expression and impaired mitochondrial function.

Paradoxically, proteins involved in myocellular uptake of FAs, muscular glycogen breakdown, and glycolysis were upregulated by insulin deprivation, while mitochondrial proteins involved in β-oxidation were downregulated, leading to the accumulation of short-chain acyl-carnitines, ceramides, and incomplete FA β-oxidation products (54, 57). Similarly, treating mouse myotubes with the FA palmitate in the presence of a β-oxidation inhibitor reduced mitochondrial coupling and increased ROS emission, suggesting that incomplete β-oxidation and increased accumulation of intramuscular lipids and lipid metabolites impair mitochondrial function during insulin deprivation (54).

Mitochondrial dysfunction in diabetic cardiomyopathy. Altered mitochondrial function in T1D is central to the progression of diabetic cardiomyopathy (DCM), a leading cause of heart failure and death among diabetic patients. In the Akita T1D mouse model, which develops diabetes as a consequence of a single–base pair substitution in Ins2, maximal ADP-stimulated mitochondrial respiration and ATP synthesis decreased despite unchanged ROS emission in cardiac tissue (58). Similarly, mRNA levels of transcriptional regulators of mitochondrial mass and function (e.g., Pgc1a, Pgc1b, Tfam) were downregulated in Akita mouse hearts (59). Proteomic analysis of OVE26 mice, a model of early-onset T1D in which transgenic overexpression of calmodulin in β cells leads to deficient insulin production, revealed that mitochondrial proteins were upregulated in DCM, corresponding with increased mitochondria number and area, although mitochondria had severely damaged morphology and decreased respiration (60). Similarly, mitochondria in coronary epithelial cells of STZ mice and mitochondria from human coronary epithelial cells exposed to high glucose are fragmented, suggesting that altered mitochondrial dynamics influence endothelial function in T1D, an effect that is secondary to increased oxidative stress (60). Proteomic and microarray analyses in the hearts of rats with STZ-induced diabetes also suggest increased cardiac FAO, in addition to modest reductions in ETC proteins that may impair ATP synthesis (60, 61). These studies suggest that increased oxidative stress likely diminishes cardiac mitochondrial function in T1D, which may promote protein damage and coincident compensatory mitochondrial biogenesis, depending on the model organism.

Diabetes-induced mitochondrial impairments in the kidney. Diabetes is the leading cause of kidney failure and kidney transplantation, and roughly 25% diabetic adults have kidney disease (62). Various rodent models of T1D display impairments in kidney mitochondrial functions, such as increased basal membrane potential, oxidized glutathione level, and H2O2 production; alterations in mitochondrial respiration; and decreased ETC complex protein activities (63–66). Insulin treatment appeared ineffective at correcting alterations in kidney mitochondrial respiration in T1D (64). Studies from Akita T1D mice suggest that morphology and function of renal mitochondria are minimally affected (59). Studies on kidney mitochondrial function in T1D patients are limited, although the development of T1D renal disease is associated with specific methylation patterns of genes affecting mitochondrial function (67).

Influence of T1D on brain mitochondrial function. How T1D affects mitochondrial function in the CNS is unclear. Studies in rodents have concluded that the brain may be spared from alterations to mitochondrial morphology and function in T1D (59, 68, 69). However, increased NRF2 protein levels and mtDNA copy number were observed in the cortex during insulin deficiency, suggesting that compensatory mechanisms protect the CNS from the effects of impaired mitochondrial function (69). It is important to study specific areas of the brain that are rich in IRs (e.g., hypothalamus, hippocampus, and cortex) to clearly evaluate whether insulin deficiency or resistance affects the brain’s mitochondrial homeostasis. Additionally, human studies testing the effect of insulin withdrawal on in vivo brain mitochondrial functions are lacking and require measurement of mitochondrial function by noninvasive approaches such as NMR spectroscopy. As insulin (70) and mitochondrial function (71, 72) are implicated in multiple brain-related diseases (e.g., neurodegeneration, mood disorders), there is a critical need to investigate how insulin and T1D influence brain mitochondrial function.

In summary, insulin deficiency with and without concurrent hyperglycemia alters mitochondrial function in multiple organs. The impact of insulin deficiency and resulting hyperglycemia on mitochondrial function is tissue-specific, although it affects mostly tissues with high glucose uptake, including muscle, heart, kidney, and brain (73, 74).

Insulin resistance and T2D mutually influence mitochondrial function

Alterations in mitochondrial function that implicate insulin resistance mechanisms have stimulated substantial interest (75, 76). T2D is much more prevalent than T1D, and etiologically, insulin resistance is the primary factor leading to insufficient β cell responses to increasing blood glucose levels. Moreover, insulin resistance in muscle occurs in a variety of conditions, including obesity, polycystic ovarian syndrome (PCOS), and hypertension. Various features of mitochondrial function are impaired in insulin-resistant muscle; however, the causal relationship between impaired mitochondrial oxidative capacity and insulin resistance is highly debated.

Mitochondrial function in insulin-resistant muscle. Kelley and colleagues (75, 77) reported decreased mitochondrial enzymatic activity, content, and FAO in muscle from adults with obesity and/or T2D during insulin resistance. Additional studies confirmed that mitochondrial content is decreased and many factors regulating mitochondria are altered in obese, insulin-resistant adults as well as the offspring of parents with T2D (33, 78–82). These findings formed the basis for the notion that decreased mitochondrial oxidative capacity leads to accumulation of intramuscular lipids, which in turn promote the development of insulin resistance. This hypothesis is supported by in vivo reports that the incorporation of 13C-acetate into glutamate and mitochondrial phosphorylation was approximately 30% lower in muscle of insulin-resistant versus lean individuals (81, 83). However, muscle’s resting energy demand is very low compared with its maximal oxygen uptake, which can increase approximately 150-fold during maximal exercise in well-trained individuals (84), and it is argued that a 30% reduction in mitochondrial content or decrease in energetics would not influence FAO at rest (85).

Insulin resistance is not always associated with reduced mitochondrial respiration, as reported in rodents on a high-fat diet (HFD) (86–89) or in humans (90). Weight loss by caloric restriction in obese people enhanced insulin sensitivity but did not improve maximal mitochondrial oxidative capacity in isolated mitochondria or mitochondrial content (91, 92). Moreover, high-intensity interval training enhanced maximal mitochondrial function in isolated mitochondria in offspring of individuals with T2D, but failed to improve insulin sensitivity, in contrast to its effects in the offspring of nondiabetic mothers (93). Animals with severely impaired mitochondrial function and increased myocellular fat also demonstrated dissociation between insulin-stimulated glucose uptake in muscle and mitochondrial deficiency (94, 95). However, HFD has been shown to increase muscle mitochondrial ROS emission that adversely affects insulin sensitivity (96) despite increasing mitochondrial respiration (88). Additionally, overexpressing the antioxidant enzyme catalase in muscle mitochondria completely preserved insulin sensitivity despite HFD (96), supporting the notion that insulin resistance results from oxidative stress. Similarly, maximal ex vivo mitochondrial respiration and content did not differ in young, lean people versus obese, insulin-resistant people despite increased H2O2 emission in the obese group, suggesting that reductions in mitochondrial respiratory capacity and content are not required for the initial manifestation of insulin resistance (97). Rather, excessive ROS emission may lead to the serine phosphorylation of IRS proteins, thus inhibiting insulin signaling (98). Collectively, these studies support a hypothesis that insulin sensitivity and mitochondrial function mutually influence each other, possibly promoting a cycle whereby insulin resistance further impairs mitochondrial function, and vice versa (Figure 2).

Proposed interactions between decreased insulin sensitivity, insulin deficiency Figure 2

Proposed interactions between decreased insulin sensitivity, insulin deficiency, and impaired mitochondrial function in skeletal muscle and in the pathophysiology of diabetes and its complications. Excess nutrient overload and high-fat diet increase leak respiration and ROS emission, which contributes to insulin resistance and protein and DNA damage. Conversely, insulin resistance enhances leak respiration and ROS emission and damages protein and DNA. Similarly, insulin deficiency increases leak respiration and ROS emission, and decreases mitochondrial protein synthesis and increases degradation of mitochondrial proteins. Damage to protein and DNA also contributes to decreased mitochondrial protein synthesis and increased mitochondrial protein degradation and damage, as well as decreases mtDNA and protein content. Lower mitochondrial content and its quality also impair mitochondrial respiration, ATP production, and ATP-dependent processes. Impairments in mitochondrial ATP production and ATP-dependent processes (e.g., protein turnover) lead to declines in many cellular functions, including those in mitochondria. Reduced ATP availability and ROS-induced damage to proteins and DNA likely contribute to age-related sarcopenia and frailty that, in combination with sedentariness, contribute to increased fat accumulation, decreased energy expenditure, and type 2 diabetes and its complications. CVD, cardiovascular disease.

Women with PCOS are insulin-resistant, but their muscle has similar maximal mitochondrial oxygen flux to insulin-sensitive lean women’s, despite displaying lower mitochondrial coupling and phosphorylation efficiencies and higher ROS emission (99). Moreover, 12 weeks of aerobic exercise training corrected mitochondrial abnormalities besides improving insulin sensitivity in women with PCOS (99). These results, together with the observation that high physiological insulin induced enhancement of mitochondrial oxidative capacity in nondiabetic but not in insulin-resistant T2D people (27), suggest that interactions between insulin resistance and mitochondrial abnormalities are more complex than the previous notion that reduced muscle mitochondrial respiration may cause insulin resistance. The reduced mitochondrial response to insulin also is consistent with reduced thermic response to meals and hyperinsulinemic-euglycemic clamp reported in T2D (5, 6). It also is important to consider the implication of observations that T2D individuals require lower rates of glucose infusion to maintain similar glucose levels than nondiabetic controls during high-dose insulin infusion. Most of the increased oxygen consumption following glucose/insulin administration is used for disposing glucose, and insulin itself has only a small effect (100), thus supporting a notion that the lack of increased ATP production in T2D individuals during high-dose insulin infusion may arise from lower amounts of glucose infusion needed to maintain euglycemia, linking insulin resistance directly to reduced mitochondrial response. It is clear, however, that both insulin deficiency and resistance adversely affect mitochondrial coupling efficiency and increase oxidative stress.

As in T1D, decreased expression of OXPHOS genes is a potential molecular basis for altered mitochondrial function in T2D. Initial gene array analysis of muscle from T2D patients following a 2-week withdrawal of insulin treatment for hyperglycemia identified 85 altered transcripts in comparison with nondiabetic controls; moreover, decreased expression of mRNA central to mitochondrial maintenance (e.g., mitochondrial superoxide dismutase [SOD2]) and mitochondrial OXHPOS was prominent in T2D (82). In the same study, insulin treatment reduced the difference between diabetic and nondiabetic individuals in all but 11 mRNA transcripts, and transcripts pertaining to mitochondrial maintenance and energy production were almost completely normalized, suggesting that loss of insulin action contributes to altered mitochondrial gene expression and function in T2D (82). Insulin resistance and T2D are also associated with reduced mRNA expression of PGC1A and multiple NRF1-dependent genes encoding enzymes that are key to OXPHOS and mitochondrial function (80).

Mitochondrial dynamics during insulin resistance. Mitophagy’s influence on mitochondrial quality and insulin sensitivity is an emerging research area. Studies in mice support mitophagy’s beneficial effect in maintaining mitochondrial quality and insulin sensitivity (101, 102). Similarly, mitophagy mediators such as HSP72 (103, 104) and PTEN-induced putative kinase 1 (PINK1) (105) were reported as decreased in obesity and T2D, while another report showed no change in mitophagic muscle in T2D (106). Interestingly, transgenic mice with muscle deletion of autophagy-related 7 (Atg7), a manipulation that impairs basal autophagy, have poorly functioning mitochondria but are protected against developing insulin resistance (107). Similarly, muscle-specific deletion of FUN14 domain–containing 1 (Fundc1), which mediates mitophagy, also impairs mitochondrial function but alleviates HFD-induced obesity and insulin resistance (108). Thus, the relationship between mitophagy, mitochondrial function, and insulin resistance requires additional research. Other reviews discuss the relationship between mitophagy and insulin resistance in more detail (10, 56, 109).

Mitochondrial fission and fusion also play a key role in maintaining the mitochondrial network. Obesity and T2D downregulate mitofusin 2 (MFN2) mRNA, suggesting an imbalance between mitochondrial fusion and fission (110, 111). Additionally, muscle MFN2 mRNA positively associates with insulin-mediated glucose oxidation, and increased MFN2 mRNA level may explain increased glucose oxidation following bariatric surgery (111).

Mitochondrial function in the insulin-resistant heart. T2D is also associated with myocardial insulin resistance and increased myocellular lipid accumulation. 31P-NMR studies show decreased phosphocreatine/ATP ratios in cardiac tissue of obese and T2D subjects (112–114). Data for right atrium heart biopsies in T2D patients demonstrated decreased maximal mitochondrial respiratory capacity for glutamate and FA substrates, and increases in myocardial triglycerides, mitochondrial ROS emission, propensity for mitochondrial permeability transition pore opening, and caspase-9 activity, leading to persistent oxidative stress and apoptosis (115, 116). Animal studies also support a role for T2D in altering myocardial mitochondrial function, such as decreased maximal mitochondrial ATP production despite increased mitochondrial biogenesis, altered palmitate and glucose oxidation, increased ROS emission, and increased mitochondrial uncoupling (117, 118). Decrements in myocardial mitochondrial function appear more severe with advanced T2D progression. High-resolution respirometry of human right atrial tissue in lean, overweight, obese nondiabetic, and T2D individuals found decreases in mitochondrial respiration and mitochondrial coupling efficiency and increased ROS emission in the T2D individuals, while similar observations were absent in nondiabetic individuals (119). Impaired mitochondrial function in the T2D heart associated with increased mitochondrial fragmentation and approximately 5-fold decreased MFN1 protein levels irrespective of BMI (119). These observations show that T2D associates with decreased myocardial mitochondrial respiration, increased oxidative stress, and mitochondrial fragmentation independent of BMI, while deficits in mitochondrial oxidative capacity and redox balance are less pronounced in obesity.

Mitochondrial function in insulin-resistant AT. Mitochondrial activity in adipocytes crucially regulates the release of free FAs into the circulation, where they can promote insulin resistance and T2D (120). Several studies report reduced mitochondrial content and OXPHOS, PGC1A, and NRF1 mRNA in subcutaneous AT (SAT) in T2D and obesity (121–124). Maximal mitochondrial respiration did not differ in SAT of obese and T2D subjects, suggesting that altered AT mitochondrial function may be secondary to increased BMI rather than T2D (125). The influence of obesity and T2D on AT mitochondrial function also appears to be depot-specific. In contrast to SAT, obesity increased mitochondrial content in visceral AT, although maximal respiration decreased when normalized to mitochondrial content, indicating impaired mitochondrial function in obesity (126).

Mitochondrial function in the insulin-resistant liver. Insulin resistance appears to have tissue-specific effects on mitochondrial characteristics that likely correlate with the vastly different metabolic demands of divergent insulin-sensitive tissues. For example, initial gene expression studies revealed altered metabolic pathways in T2D patient livers, specifically the upregulation of mRNAs central to OXPHOS, ROS generation, and gluconeogenesis (127, 128), which contrasts with the downregulation of mitochondrial mRNAs in muscle. Additionally, increased hepatic OXPHOS mRNA expression was predictive of fasting blood glucose independent of age, BMI, or fasting insulin levels (127). The presence of insulin-dependent and insulin-independent glucose uptake in myocytes and hepatocytes, respectively, may influence how a tissue’s energy status regulates its mitochondrial function (129). However, as in muscle, 31P-NMR measurements of hepatic mitochondrial ATP production reveal 23%–26% decreased ATP production in T2D individuals compared with age- and weight-matched nondiabetic controls (130, 131). In these studies, hepatic ATP production more strongly associated with hepatic than peripheral insulin sensitivity (130, 131). Further, postprandial increases in hepatic ATP concentration were increased 6-fold in obese, insulin-resistant subjects compared with lean controls, suggesting augmented postprandial hepatic metabolism during insulin resistance (132). Likewise, high fructose consumption in obese, T2D individuals depleted hepatic ATP concentrations and impaired recovery from ATP depletion (133). Hepatic ATP content also inversely correlated with BMI (134). These data suggest that despite increasing mitochondrial mRNA expression, T2D impairs hepatic ATP production. The unknown mechanism underlying intriguing differences between hepatic and muscle tissue in response to a meal indicates the need for additional measures of mitochondrial function in T2D patients.

Potential therapies to rescue mitochondrial defects with insulin resistance
Exercise. Aerobic exercise is a powerful stimulant of mitochondrial respiration and ATP production (135, 136) and improves insulin sensitivity (137). As mentioned above, in obese, insulin-resistant women with PCOS, 12 weeks of aerobic exercise increased mitochondrial respiration and coupling efficiency while decreasing ROS emission and irreversible DNA damage toward those of lean, insulin-sensitive individuals (99). Additionally, improvements in mitochondrial energetics paralleled improvements in insulin sensitivity, which were likely due to improved cellular redox status (99). Mouse experiments also demonstrated that Irs1 and Irs2 were indispensable for increased PGC1A protein content, mitochondrial respiration and ATP production, and mtDNA copy number following exercise training, suggesting that exercise modulates mitochondrial function though insulin signaling (138). Aerobic exercise also prevented age-related declines in mitochondrial oxidative capacity (93, 136, 139, 140) concurrent with improvement in insulin sensitivity. Recent findings indicate that high-intensity interval training, rather than moderate aerobic exercise, has the greatest impact toward increasing mitochondrial oxidative capacity and mitochondrial protein synthesis and content while concurrently improving insulin sensitivity in older adults (141). Thus, sedentary lifestyles exacerbate mitochondrial deficits associated with insulin resistance and T2D, which may be at least partially rescued by moderate aerobic or high-intensity exercise.

Mitochondrial uncoupling. OXPHOS, oxidative stress, and heat production are intimately interconnected, and perturbation in one component affects the other two. Mitochondrial uncoupling is the process by which the ETC’s proton-motive force uncouples from OXPHOS by proton leak or by uncoupling proteins (UCPs) in the inner mitochondrial membrane, thus generating heat rather than ATP. Under conditions of high dietary fat and glucose, modest mitochondrial uncoupling and heat production can function as a sink for excessive substrates to alleviate oxidative stress, in turn modulating the development of obesity and insulin resistance (142). Muscle-specific Ucp3 overexpression rendered mice resistant to diet-induced obesity despite hyperphagia (143). Similarly, UCP3 mRNA was decreased in muscle from obese women who were resistant to weight loss following caloric restriction (144), and UCP3 positively correlated with resting metabolic rate in Pima Indians, a T2D-susceptible population (145). No clear evidence directly links UCPs with physiological regulation of metabolic rate and energy balance in humans. However, recent reports revealing significant amounts of metabolically active BAT in adults (146, 147) indicated a potential role of this mitochondria-rich fat in energy regulation. Preclinical studies indicate BAT’s potential role in glucose homeostasis (148). Furthermore, exercise-induced “browning” of WAT via many molecular factors has been proposed as a potential approach to influencing energy balance (149).

Oral ingestion of niclosamide ethanolamine salt, which induces mild mitochondrial uncoupling, by diabetic db/db mice increased energy expenditure, prevented HFD-induced insulin resistance, and improved glycemic control (150). Thus, mild mitochondrial uncoupling may offer therapeutic value for the treatment of T2D. However, it should be noted that uncontrolled mitochondrial uncoupling could lead to a life-threatening increase in body temperature and ATP depletion (151).

Concluding remarks
Recently our understanding of the dynamic relationship between mitochondrial function and diabetes has advanced tremendously. This interactive relationship is not surprising given that altered fuel metabolism characterizes both insulin-deficient and insulin-resistant diabetes. Research has identified insulin as a critical regulator of mitochondrial biogenesis and function in numerous tissues. Both insulin deficiency and resistance result in oxidative stress and consequent oxidative damage to DNA and proteins that likely contribute to diabetic complications. Insulin not only regulates oxidative metabolism and ROS emission, but ROS can affect insulin actions, as occurs following HFD. Although development of insulin resistance does not require impaired mitochondrial function, pathways promoting insulin resistance may impair mitochondrial function and further increase ROS production, resulting in a detrimental feedback loop. Aerobic exercise and caloric restriction disrupt this vicious loop potentially by preventing accumulation of damaged mitochondrial proteome with substantial improvement of insulin sensitivity. Although therapies targeted to enhance β-oxidation, as occurs following exercise, may improve insulin sensitivity by reducing adiposity, it remains to be determined whether increased fuel oxidation may increase oxidative stress.

Mitochondria are highly adaptable and dynamic within different environments, including various cell types and tissues. Consideration of this adaptability is critically important in planning and interpreting studies using variable experimental conditions (e.g., in vivo versus in vitro, different tissue/cell types, temperature differences, etc.). These aspects are reviewed elsewhere (e.g., refs. 152, 153).

Although mitochondrial respiration in resting muscle is unlikely to have any major impact on energy balance or fuel metabolism, maximal mitochondrial capacity to produce ATP is a key determinant of maximal aerobic capacity (154), which in turn determines exercise capacity with substantial impact on reducing mortality in various chronic diseases, including T2D (155). However, in many other organs, including brain and liver, methodologies to measure resting-state mitochondrial respiration are critically important to understanding the role of mitochondria in normal physiology and pathological states in diabetes. Given the epidemic levels of T2D and high incidence of comorbid dementia, understanding links between diabetes and mitochondrial impairment in cognitive decline is of great interest. However, unlike muscle, different anatomical regions of brain have distinct functions and variable distribution of mitochondria and IRs, necessitating studies with brain region–specific focus. Greater understanding of the molecular regulatory points altered by insulin resistance and deficiency offers opportunities to target therapeutic molecules to mitochondria with the potential to prevent T2D and diabetic complications.

Acknowledgments
This work was supported by grants from the NIH (R01DK101402, U24DK100469, and U24KD112326 to KSN and T32AG49672 to GNR) and by the Murdock-Dole Professorship (to KSN).

Small molecule targeting of transcription-replication conflict for selective chemotherapy

Long Gu
Min Li
Caroline M. Li
Robert J. Hickey
J. Jefferson P. Perry
Linda H. Malkas 12
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Show footnotesOpen AccessPublished:August 01, 2023DOI:https://doi.org/10.1016/j.chembiol.2023.07.001

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Introduction

Proliferating cell nuclear antigen (PCNA) is an evolutionarily conserved multifaceted protein found in all eukaryotic cells, and it plays a critical role in DNA synthesis and in DNA repair. PCNA forms a homo-trimeric ring structure encircling DNA1 and it acts as a central “hub” of the replisome, to provide an anchorage for the many proteins involved in the replication and repair pathways. The cellular functions of PCNA can be modulated through post-translational modifications on the surface of the protein, altering partner interactions2,3 that occur predominantly through the outer hydrophobic surface of PCNA, adjacent to its inter-domain connector loop (IDCL).4,5 Historically, PCNA has been widely used as a tumor progression marker and more recent studies have demonstrated that PCNA can play a mitogenic role, to distantly rejuvenate senescent cells via extracellular vesicles.6
DNA replication stress is a hallmark of cancer cells.7,8 It is used as a major anti-cancer therapeutic strategy by exploiting this cancer-associated feature, through introduction of further DNA damage resulting in catastrophic damage to the cancer cell. Due to its central role in DNA replication and repair, PCNA is a potential target for this anti-cancer strategy. Moreover, the identification of a distinct isoform of PCNA associated with cancer cells has potentially opened a novel avenue for the development of new chemotherapeutics. Early effects in targeting PCNA have identified several molecules of interest, both small molecule and peptide-based, which have indicated that directly targeting PCNA for cancer therapy may be a viable approach.9,10,11,12,13,14,15 More recent studies identified PCNA ligands, which show synergy with existing chemotherapeutic agents.16,17,18,19 We previously described a compound, AOH1160,17,20 functioning as a potential inhibitor hit compound of the cancer-associated PCNA isoform (caPCNA), but this compound lacked suitable metabolic properties to proceed further into preclinical/clinical studies.

Here, we describe both the identification and detailed molecular characterization of AOH1996, an analog of AOH1160 that exhibits remarkable therapeutic properties: It is orally administrable in a formulation compatible with its clinical use, and in animal studies it almost completely inhibits the growth of xenograft tumors and sensitizes them to topoisomerase inhibition. In studies that follow the good laboratory practice (GLP) guidelines of the US Food and Drug Administration (FDA), AOH1996 causes no discernible toxicity at 6 or more times the effective dose in mice and dogs

Looks like research with DCA is advancing:
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https://pubmed.ncbi.nlm.nih.gov/31827705/'


Oxid Med Cell Longev

. 2019 Nov 14:2019:8201079.
doi: 10.1155/2019/8201079. eCollection 2019.
Dichloroacetate (DCA) and Cancer: An Overview towards Clinical Applications
Tiziana Tataranni 1 , Claudia Piccoli 1 2
Affiliations

PMID: 31827705 PMCID: PMC6885244 DOI: 10.1155/2019/8201079

Free PMC article
Abstract

An extensive body of literature describes anticancer property of dichloroacetate (DCA), but its effective clinical administration in cancer therapy is still limited to clinical trials. The occurrence of side effects such as neurotoxicity as well as the suspicion of DCA carcinogenicity still restricts the clinical use of DCA. However, in the last years, the number of reports supporting DCA employment against cancer increased also because of the great interest in targeting metabolism of tumour cells. Dissecting DCA mechanism of action helped to understand the bases of its selective efficacy against cancer cells. A successful coadministration of DCA with conventional chemotherapy, radiotherapy, other drugs, or natural compounds has been tested in several cancer models. New drug delivery systems and multiaction compounds containing DCA and other drugs seem to ameliorate bioavailability and appear more efficient thanks to a synergistic action of multiple agents. The spread of reports supporting the efficiency of DCA in cancer therapy has prompted additional studies that let to find other potential molecular targets of DCA. Interestingly, DCA could significantly affect cancer stem cell fraction and contribute to cancer eradication. Collectively, these findings provide a strong rationale towards novel clinical translational studies of DCA in cancer therapy.

Copyright 2019 Tiziana Tataranni and Claudia Piccoli.
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Unraveling the therapeutic mechanisms of dichloroacetic acid in lung cancer through integrated multi-omics approaches: metabolomics and transcriptomics
Malong Feng 1 2 , Ji Wang 3 , Jianying Zhou 1
Affiliations

PMID: 37359381 PMCID: PMC10285292 DOI: 10.3389/fgene.2023.1199566

Free PMC article
Abstract

Objective: The aim of this study was to investigate the molecular mechanisms underlying the therapeutic effects of dichloroacetic acid (DCA) in lung cancer by integrating multi-omics approaches, as the current understanding of DCA's role in cancer treatment remains insufficiently elucidated. Methods: We conducted a comprehensive analysis of publicly available RNA-seq and metabolomic datasets and established a subcutaneous xenograft model of lung cancer in BALB/c nude mice (n = 5 per group) treated with DCA (50 mg/kg, administered via intraperitoneal injection). Metabolomic profiling, gene expression analysis, and metabolite-gene interaction pathway analysis were employed to identify key pathways and molecular players involved in the response to DCA treatment. In vivo evaluation of DCA treatment on tumor growth and MIF gene expression was performed in the xenograft model. Results: Metabolomic profiling and gene expression analysis revealed significant alterations in metabolic pathways, including the Warburg effect and citric acid cycle, and identified the MIF gene as a potential therapeutic target in lung cancer. Our analysis indicated that DCA treatment led to a decrease in MIF gene expression and an increase in citric acid levels in the treatment group. Furthermore, we observed a potential interaction between citric acid and the MIF gene, suggesting a novel mechanism underlying the therapeutic effects of DCA in lung cancer. Conclusion: This study underscores the importance of integrated omics approaches in deciphering the complex molecular mechanisms of DCA treatment in lung cancer. The identification of key metabolic pathways and the novel finding of citric acid elevation, together with its interaction with the MIF gene, provide promising directions for the development of targeted therapeutic strategies and improving clinical outcomes for lung cancer patients.

Keywords: dichloroacetic acid (DCA); drug mechanism; gene expression; lung cancer; metabolomics; molecular mechanisms; multi-omics; therapeutic target.

Copyright 2023 Feng, Wang and Zhou.

https://pubmed.ncbi.nlm.nih.gov/37359381/

More on Harmine/Syrian Rue treating Neuroblastomas. More at link:
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Published: 07 June 2018

Harmine, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) inhibitor induces caspase-mediated apoptosis in neuroblastoma

   Katie L. Uhl, Chad R. Schultz, Dirk Geerts & André S. Bachmann
Cancer Cell International volume 18, Article number: 82 (2018) Cite this article

Abstract

Background

Neuroblastoma (NB) is an early childhood malignancy that arises from the developing sympathetic nervous system. Harmine is a tricyclic β-carboline alkaloid isolated from the harmal plant that exhibits both cytostatic and cytotoxic effects. Harmine is capable of blocking the activities of dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family proteins and mitogen-activated protein kinase. These kinases promote proliferation and inhibit apoptosis.

Methods

Four human NB cell lines were used to study the effects of harmine treatment: SKNBE and KELLY (MYCN-amplified) as well as SKNAS and SKNFI (MYCN non-amplified). The anti-cancer properties of harmine were examined by RealTime-Glo MT cell viability assays, caspase activity assays, PARP cleavage using Western blot analysis, and flow cytometry-based Annexin V detection. A molecular interaction model of harmine bound to the DYRK2 family kinase was generated by computational docking using X-ray structures. NB tumors from human patients were profiled for DYRK mRNA expression patterns and clinical correlations using the R2 platform.

Results

The IC50 values for harmine after 72 h treatment were 169.6, 170.8, and 791.7 μM for SKNBE, KELLY, and SKNFI, respectively. Exposure of these NB cell lines to 100 μM of harmine resulted in caspase-3/7 and caspase-9 activation as well as caspase-mediated PARP cleavage and Annexin V-positive stained cells, as early as 24 h after treatment, clearly suggesting apoptosis induction, especially in MYCN-amplified cell lines. Elevated DYRK2 mRNA levels correlated with poor prognosis in a large cohort of NB tumors.
Conclusion

Harmine is a known inhibitor of DYRK family kinases. It can induce apoptosis in NB cell lines, which led us to investigate the clinical correlations of DYRK family gene expression in NB tumors. The patient results support our hypothesis that DYRK inhibition by harmine and the subsequent triggering of caspase-mediated apoptosis might present a novel approach to NB therapy.
Background

Neuroblastoma (NB) is an early childhood malignancy that arises from the developing sympathetic nervous system, resulting in aggressive tumor formation in the sympathetic ganglia and/or the adrenal glands [1]. Amplification of the MYCN gene, leading to over-expression of the MYCN protein, is the most prevalent NB genetic aberration. It is found in ~ 20% of NB, predominantly in high-stage tumors and has been linked to high risk disease, and poor patient prognosis [1, 2]. Of those high risk patients that respond initially to chemotherapy, the majority will succumb to the disease after a relapse into a chemotherapy-resistant state [2]. In addition to MYCN gene amplifications, mutations in genes encoding the mitogen-activated protein kinase (MAPK) pathway, and in the ALK gene have been identified as main drivers in the majority of NB [1].

The harmal plant (Peganum harmala L., family Zygophyllaceae), also called Syrian rue, is a perennial shrub native to the eastern Mediterranean region. Various parts of the plant have long been used in traditional folk medicine [3]. Harmine is a tricyclic β-carboline alkaloid isolated from harmal seeds and acts as a monoamine oxidase A (MAO-A) inhibitor [3,4,5]. Intake of alkaloids from the harmal plant can have anti-depressive, analgesic, and anti-bacterial pharmacological effects [6,7,8].

Harmine has been shown to induce apoptosis and inhibit cell proliferation, migration, and invasion in a dose-dependent manner in various human cancer cell lines including C334, CCD18LU, HeLa, HL-60, K562, SW480, BGC-823, and SGC-790 [9]. While lower concentrations of harmine typically induced cytostasis, higher concentrations are associated with cytotoxicity [10].

The dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family proteins are related to the MAPK family. However, the activating tyrosine phosphorylation of DYRK family kinases is not catalyzed by upstream kinases but occurs through autophosphorylation [10]. There is growing interest in the role of DYRK family kinases in cancer, as they can act as regulators of protein stability during the cell cycle and regulate the activity of the proteasome [11, 12]. Remarkably, harmine inhibits all DYRK family members (DYRK1A, DYRK1B, DYRK2, and DYRK4), with the highest affinity for DYRK1A [13, 14].

In this study, we identified that harmine induces apoptotic cell death in NB cells, generated a molecular interaction model for harmine bound to DYRK2, and showed that DYRK2 mRNA expression patterns in a large cohort of human NB tumors suggest the involvement of DYRK2 in NB tumorigenesis. Together, our results offer a potential new route of NB therapy.

Methods

Chemicals

Harmine (Fig. 1), LDN-192960, and INDY were purchased from Cayman Chemical. The compound solids were stored at − 20 °C. Stock solutions were prepared by dissolving harmine (100 mM), LDN-192960 (40 mM) and INDY (40 mM) into sterile DMSO (VWR). Stock concentrations were filtered prior to being added to the cell cultures.

Chemical structure of harmine. Harmine is a β-carboline alkaloid present in Peganum harmala plant, and several other Mediterranean plant species. Harmine has a number of pharmaceutical characteristics including irreversible inhibition of monoamine oxidase A (MAO-A), and demonstrates cytotoxicity in various cancer cell lines. The molecular weight of harmine is 212.25 g/mol

Cell culture

The human NB cell lines KELLY (also known as N206; #92110411 from Sigma), SK-N-AS (called SKNAS in this study; CRL-213 from ATCC), SK-N-BE (clone SKNBE(2)C, called SKNBE in this study; CRL-2268 from ATCC), and SK-N-FI (called SKNFI in this study; from the Children’s Oncology Group) were cultured in RPMI 1640 (VWR), supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), Penicillin (100 IU/mL) and Streptomycin (100 μg/mL) (30-002-CI, Corning). All cells were purchased from their respective suppliers within the last 2 years. The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2.
Cell viability assay

Cell viability and IC50 was determined using the RealTime-Glo MT cell viability assay (G9712, Promega). This reagent allows continuous measurement of cell viability in the same well. Cells were plated at a density of 4000 cells/well into white-walled, opaque assay plates. After the plated cells had been given 24 h to adhere, they were treated with harmine concentrations ranging from 0 to 1 mM. The MT Cell Viability Substrate and NanoLuc Enzyme were equilibrated to 37 °C, 2× RealTime-Glo reagent was prepared, and an equal volume was added to each well. For time zero measurements, cells were incubated with reagent for 20 min at 37 °C, and luminescence was measured on a Biotek Synergy microplate reader. Luminescence was measured at 24, 48, and 72 h after addition of harmine.
Cytotoxicity assay

The colorimetric SRB assay was used to measure cytotoxicity as previously described, following the treatment with DYRK2 inhibitor LDN-192960 or DYRK1A/B inhibitor INDY [15,16,17]. Briefly, NB cells were plated in transparent flat 96-well plates and allowed to attach overnight. At the initiation of each experiment (t = 0) and after drug treatments, cells were fixed with 10% TCA at 4 °C for 1 h, washed with deionized water, and dried at room temperature. Cells were then stained with 100 μl of 0.4% SRB in 1% acetic acid for 20 min at room temperature, rinsed five times with 1% acetic acid and allowed to dry at room temperature. One hundred µl of 10 mM Tris–HCl pH 7.0 was added to each well, shaken for 10 min at room temperature and read at 540 nm using a Molecular Devices Flexstation 3 microplate reader.
Caspase activity assays

The quantification of the relative caspase activity in harmine-treated cells was carried out using the Caspase-Glo 3/7 and Caspase-Glo 9 Assay kits (G8091 and G8211, respectively, from Promega). Cell lines were plated at a density of 16,000 cells/well into white-walled, opaque 96-well plates. Twenty-four hours after plating, cells were treated with 0, 25, 50 and 100 µM harmine for 24 h. Caspase Glo reagents were added to the cells and incubated at room temperature. Luminescence was measured using a Biotek Synergy microplate reader every 20 min for 3 h.
Western blot analysis

Whole cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer, (20 mM Tris–HCl [pH 7.5], 135 mM NaCl, 2 mM EDTA, 0.1% (w/v) sodium lauryl sulfate, 10% (v/v) glycerol, 0.5% (w/v) sodium deoxycholate, and 1% (v/v) Triton X-100). The RIPA buffer was supplemented with cOmplete Protease Inhibitor Cocktail (Roche), and 0.27 mM Na3VO4 and 20 mM NaF as phosphatase inhibitors. Protein concentration was determined using the Bradford dye reagent protein assay (Bio-Rad). Equal amounts of protein were resolved using 12% SDS-PAGE, and transferred to 0.45 µM polyvinylidene difluoride Immobilon-P membrane (Millipore). The transferred proteins were incubated with the following primary antibodies: PARP rabbit polyclonal antibody (#9542) from Cell Signaling Technologies at 1:1000 dilution and GAPDH mouse monoclonal antibody (#SC-47724) from Santa Cruz Biotechnology at 1:1000 dilution. After incubating the blots with the primary antibody at 4 °C for at least 8 h, they were washed and incubated with secondary antibodies at 1:10,000 dilution for 1 h at room temperature. The secondary antibodies used were LiCor IRDye 680RD Goat anti-Mouse IgG, (925-68070), and LiCor IRDye 680RD Goat anti-Rabbit IgG, (925-68710).
Annexin V detection

To quantify the percentage (%) of apoptosis induction in harmine-treated NB cells, the Annexin V Detection Kit APC (88-8007, Invitrogen) assay was used. Samples were prepared according to the manufacturer’s protocol and analyzed by flow cytometry. In brief, the cells were plated overnight and then treated with 0 μM or 100 μM harmine for 24 h at 37 °C. Cells were collected by centrifugation and stained with Annexin V APC/DAPI staining solution at room temperature for 20 min in the dark. Next, cells were collected by centrifugation suspended in PBS (pH 7.4) and analyzed immediately using flow cytometry Excitation/emission for Annexin APC and DAPI were 633/700 and 350/450 nm respectively.
Molecular docking of DYRK2 and harmine

The molecular docking model for DYRK2 in complex with harmine was constructed by identifying the target structures from the Protein Data Bank (PDB) [18]. The crystal structure of human DYRK1A (PDB ID: 3ANR) in complex with harmine was previously published [13, 18]. To predict if DYRK2 also forms a complex with harmine, we generated a molecular docking model with coordinates from an existing crystal structure of DYRK2 in complex with Leucettine (PDB:4AZF) using the online docking web server SwissDock (http://www.swissdock.ch/) [18,19,20,21]. Chain A of 4AZF was isolated from the original crystal structure, and the Leucettine L41 ligand was removed. The modified structure was then entered into SwissDock, along with the chemical structure of harmine (ZINC ID: 27646846) taken from the ZINC molecule database (http://zinc.docking.org/) [19, 20]. The conformational ΔG was calculated and deemed best fit by the parameters set by SwissDock. The resulting molecular interaction model was visualized using visual molecular dynamics (VMD) [22].

NCBI BLAST sequence alignment

Alignment of the DYRK family sequences was carried out using the Blastp tool provided by the National Center for Biotechnology Information (NCBI) [23, 24]. The amino acid sequences for human DYRK1A and human DYRK2 used for the alignment were taken from the Basic Local Alignment Search Tool (BLAST) database, (Accession No. Q13627 and AAH06375, respectively) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) [18, 24]. The query cover reported herein was given as part of the output of the Blastp program, as was the percentage of identity and the corresponding E-value.
NB public mRNA expression dataset analysis

For analysis of DYRK gene expression in human NB patients, the largest public NB cohort for which genome-wide tumor RNA-sequencing has been performed, SEQC-498, (n = 498; GSE62564) was analyzed using the R2 genomics analysis and visualization platform developed in the Department of Oncogenomics at the Academic Medical Center—University of Amsterdam (http://r2.amc.nl). Expression data for the datasets were retrieved from the public Gene Expression Omnibus (GEO) dataset on the NCBI website (http://www.ncbi.nlm.nih.gov/geo/) and analyzed as previously described [25].
Statistical analyses

The statistical significance for the cell viability measurements (Fig. 2b) was calculated using the GraphPad Prism 7 package (https://www.graphpad.com/) and an unpaired student’s t-test assuming the null hypothesis was performed. The change in caspase activity was quantified by calculating the average fold change, after normalizing experimental samples to their respective controls. The significance of both caspase activation and Annexin V apoptotic measurements (Figs. 3, 4) was calculated using an unpaired student’s t-test, assuming the null hypothesis. DYRK gene NB tumor mRNA expression correlation with survival probability (Fig. 7) was evaluated by Kaplan–Meier analysis using the log-rank test as described [26]. To determine the optimal value of gene expression to set as cutoff value, all tumor samples were first sorted according to gene mRNA expression and subsequently divided into two groups. Analyses were performed on groups separated by median or average tumor mRNA expression values. DYRK mRNA expression correlation with tumor MYCN gene amplification was determined using the non-parametric, rank-based Kruskal–Wallis test. For all tests, a P-value < 0.05 was considered to be statistically significant.

Fig. 2
figure 2

Harmine induces NB cell death. a NB cell lines (SKNBE, KELLY, SKNAS, and SKNFI) were treated with 100 μM harmine or left untreated for 24–72 h. Representative micrographs show the effects of 100 μM harmine on cell morphology after 72 h (see Additional file 1: Fig. S1 for micrographs representative of harmine treatments after 24 and 48 h). b IC50 curves representing cell viability measurements using Real-Time Glo (Promega). NB cell lines were continuously exposed to a range of harmine concentrations (0–1 mM) for a total of 72 h, with measurements after 24, 48, and 72 h. Values were normalized to control and represent the average of three independent experiments ± S.D. (n = 3)
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Fig. 3
figure 3

Harmine activates caspase-3/7 and caspase-9 in NB cells. NB cell lines (SKNBE, KELLY, SKNAS, and SKNFI) were treated with harmine (0, 50, 100 μM) for 24 h and caspase activities measured. a Caspase-3/7 and b caspase-9 are significantly activated in the presence of harmine (100 μM). Results were obtained using the Caspase-Glo Assay Systems as stated in the “Methods” section. The results were normalized to the control value of each cell line; the fold change for each cell line is shown. Data represent the average of three independent experiments, each performed in duplicate ± S.D. (n = 6). The change in activation was calculated using an unpaired student’s t-test, assuming the null hypothesis. Asterisk denotes statistically significant changes in caspase activity compared to control (P < 0.05)
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Fig. 4
figure 4

Harmine induces PARP cleavage in NB cells. NB cell lines (SKNBE, KELLY, SKNAS, and SKNFI) were exposed to increasing concentrations of harmine, (0–100 μM) for 24 h and probed for PARP cleavage, an indicator of progressive apoptosis. Whole cell lysates were collected using RIPA lysis buffer and analyzed for PARP cleavage using Western blot. Data are representative of three independent experiments (n = 3)
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Results
Harmine induces dose- and time-dependent NB cell death

To study the effect of harmine on the morphology of NB cells, four NB cell lines were exposed to 100 μM harmine. As early as 24 h after treatment, the plated cells began to show signs of morphological changes associated with apoptosis (Additional file 1: Fig. S1). In comparison to control cells, treated cells were smaller in size, rounded in shape, and more detached from the plate surface. After 72 h, the majority of treated cells had become spherical in shape and had detached (Fig. 2a). To determine if harmine induces dose- and time-dependent cell death, the four NB cell lines were treated with increasing drug concentrations (0, 6, 12.5, 25, 50, and 100 μM) and cell viability was measured after 24, 48 or 72 h of treatment (Fig. 2b). Increased treatment length with harmine caused decreased IC50 in all four cell lines. The IC50 values for SKNBE, KELLY, and SKNFI after a 72 h treatment of harmine were 169.6 ± 0.10, 170.8 ± 0.10, and 791.7 ± 0.77 μM, respectively. The IC50 value of SKNAS after 72 h could not be calculated (for all IC50 values including 24 and 48 h time points; see Additional file 1: Table S1). The IC50 decrease was consistent with the morphological changes observed in the treated cells (Fig. 2a). Moreover, the IC50 values suggest that harmine is more toxic to MYCN-amplified NB cell lines (SKNBE and KELLY), than to NB cell lines with a normal MYCN gene copy number (SKNAS and SKNFI).
Harmine activates caspase-3/7 and caspase-9 in NB cells

The dose- and time-dependent cell death and associated morphological changes in response to harmine treatment prompted us to determine if harmine induces caspases, known to be activated during apoptosis. NB cells were treated with 0, 50, and 100 μM of harmine for 24 h, after which caspase activity was measured using the Promega Caspase-Glo 3/7 and Caspase-Glo 9 Assay kits. Caspase-3/7 activity increased with increasing harmine concentration (Fig. 3a) and this was significant for all four cell lines at the highest concentration (100 μM). The average fold changes for the caspase-3/7 activity were: SKNBE = 4.32 (P < 1.0 × 10−4), KELLY = 8.26 (P < 1.0 × 10−4), SKNAS = 6.98 (P < 1.0 × 10−4), and SKNFI = 6.77 (P < 1.0 × 10−4). Under identical cell treatment conditions, a significant increase in caspase-9 activation was observed in all four cell lines (Fig. 3b). The average fold changes for caspase-9 activity were: SKNBE = 2.51 (P < 1.0 × 10−4), KELLY = 2.96 (P < 1.0 × 10−4), SKNAS = 4.18 (P < 1.0 × 10−4), and SKNFI = 3.86 (P < 1.0 × 10−4). The results suggest that harmine triggers apoptotic cell death in NB cells.
Harmine induces progressive apoptosis in NB cells

To confirm that caspase activation leads to progressive apoptosis, harmine-treated NB cells were analyzed for caspase-mediated PARP cleavage. As shown in Fig. 4, cleaved PARP appeared in whole cell lysates of harmine-treated SKNBE and KELLY cells, but not SKNAS or SKNFI cells. These results confirm that harmine induces apoptosis, with a profound effect on MYCN-amplified NB cells (SKNBE and KELLY); both cell lines representing the most aggressive sub-types of NB tumors.

To further confirm actual apoptosis in the harmine-treated NB cells, the amount of apoptotic cells in four NB cell lines was quantified using Annexin V staining and flow cytometry (Fig. 5a). The total amount of apoptotic cells in the SKNBE cells significantly increased from 12.5% ± 0.60 (control) to 36.8% ± 14.5 in cells that had been treated with harmine (100 μM) (Fig. 5b). Similarly, harmine significantly increased the total amount of apoptotic cells in KELLY cells from 11.3% ± 2.31 (control) to 45.3% ± 6.96 in the presence of harmine (100 μM). The change in apoptotic cells in SKNAS cells was less dramatic and increased from 6.20% ± 2.24 (control) to 16.6% ± 5.31 (100 μM) and there was no change in the percentage of apoptotic cells in SKNFI cells (Fig. 5b). The results further confirm our observation that harmine appears more toxic to MYCN-amplified NB cells (SKNBE, KELLY) than to NB cells with a normal MYCN gene copy number (SKNAS, SKNFI).
Fig. 5
figure 5

Harmine induces apoptosis, marked by increase in Annexin V-positive NB cells. a NB cell lines (SKNBE, KELLY, SKNAS, and SKNFI) were incubated with 100 μM harmine for 24 h, after which Annexin V presence was analyzed by flow cytometry using the Annexin V Apoptosis Detection Kit APC. The numbers in each quadrant represent the average number of events of three experiments (n = 3). b Bar graph representation showing an increase in the percentage of total apoptotic cells with harmine treatment. Data represent the average of three experiments ± S.D. (n = 3). Asterisk denotes a statistically significant increase in the percentage of apoptotic cells compared to control. The p-values are as follows: SKNBE P = 4.4 × 10−2; KELLY P = 1.3 × 10−3; and SKNAS P = 3.6 × 10−2. The p-value of SKNFI cells was not significant
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Molecular docking model predicts interaction of DYRK2 with harmine

Based on literature supporting the potential interaction between harmine and DYRK2, we performed a docking simulation using SwissDock [19, 20]. To accomplish this, we utilized the crystal structures of human DYRK1A in complex with harmine (PDB ID: 3ANR) (Fig. 6a) and human DYRK2 in complex with Leucettine (PDB:4AZF) [13, 21]. Chain A of human DYRK2 was isolated and was entered into the SwissDock portal with harmine as the ligand, after removing Leucettine. The structure of harmine was provided by the ZINC molecular database (ZINC ID: 27646846). The most favorable conformation of the DYRK2/harmine complex had a ΔG value of − 6.89 kJ mol−1 (Fig. 6a). The figure displays the highest affinity binding between human DYRK2 and harmine. The crystal structure of chain A of DYRK1A in complex with harmine is provided for comparison (Fig. 6b). The two proteins were also compared by performing a sequence alignment between human DYRK1A (Accession No. Q13627) and human DYRK2 (Accession No. AAH06375) using the NCBI Blastp online tool (Fig. 6c) [24]. The query identity was 41%, with an E-value of 4 × 10−92.
Fig. 6
figure 6

Molecular interaction of harmine with DYRK2. a The crystal structure of human DYRK1A chain A bound to harmine (PDB ID: 3ANR), as previously published [13]. Chain A is colored blue, and the structure of harmine is colored red. b Crystal structure of human DYRK2 chain A (PDB ID: 4AZF, ligand removed), as previously published [21] and docked with harmine. Chain A is shown in blue, and harmine is colored red. The figure was created using SwissDock to dock the structure of harmine (ZINC ID: 27646846), as detailed on the ZINC molecule database, to the published structure of chain A of the human DYRK2 protein. The resulting conformation had a ΔG value of − 6.89 kJ mol−1 and was deemed best fit by the parameters set by SwissDock. c BLAST sequence alignment of DYRK1A (Accession No. Q13627) and DYRK2 (Accession No. AAH06375) using the NCBI Blastp online tool [24]. The query cover was 51%, with an identity of 41% and an E-value of 4 × 10−92. Identical amino acids between the two sequences have been highlighted
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DYRK family genes are expressed in NB tumors and DYRK1B/2/3 predict poor patient outcome

Our in vitro data in NB cell lines were supported by crystal structure-derived computational docking models and suggest that targeting DYRK family kinases through harmine treatment may be a potential new route of therapy for patients with NB. This notion prompted us to investigate DYRK family kinases in human NB tumors. To accomplish this, we analyzed the mRNA expression of the DYRK gene family in SEQC-498, the largest RNASeq dataset on human NB samples in the public domain. Analyses were performed using the R2 website (see Materials and Methods). We first determined whether DYRK genes are expressed in human NB tumors (Fig. 7a). All five DYRK genes show robust expression, especially DYRK1A and DYRK2.
Fig. 7
figure 7

DYRK gene NB tumor mRNA expression and prognostic significance. The mRNA expression of the DYRK gene family was analyzed in SEQC-498, the largest genome-wide RNA sequencing dataset on human NB tumors in the public domain. a Bar plot of DYRK gene tumor mRNA expression in the SEQC-498 patient cohort. The Y-axis represents expression in RPM (reads per million), vertical bars represent mean expression ± standard deviation. All five DYRK genes show significant expression, with DYRK1A and DYRK2 as the most highly expressed mRNAs. b–f Kaplan–Meier graphs representing the overall survival prognosis of NB patients in the SEQC-498 cohort based on grouping of the patients according to median DYRK gene tumor mRNA expression. c–e High-level tumor mRNA expression of DYRK1B, DYRK2, and DYRK3 is significantly predictive of poor patient outcome. Significant correlations are also found when patients are grouped according to, e.g. average DYRK mRNA tumor expression, or when event-free survival is predicted (results not shown), suggesting that DYRK mRNA expression is a robust predictor for NB patient outcome
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We next determined whether high DYRK gene expression is beneficial to NB tumorigenesis and translates into poor patient outcome (Fig. 7b–f). Kaplan–Meier graphs representing the overall survival prognosis of NB patients in the SEQC-498 cohort based on grouping of the patients according to median DYRK gene tumor mRNA expression showed that high-level tumor mRNA expression of DYRK1B, DYRK2, and DYRK3, but not of DYRK1A or DYRK4, is significantly predictive of poor patient outcome (Fig. 7b–f). Although DYRK1A and DYRK2 are the most highly expressed, only DYRK2 is prognostic of survival outcome, suggesting that in NB the deleterious effect of harmine treatment acts mostly through DYRK2. Harmine toxicity tests and apoptosis analyses on NB cell lines suggest that MYCN-amplified cell lines are more sensitive to harmine than NB cell lines with two MYCN gene copies. We were interested whether this was reflected in the correlation between DYRK gene mRNA expression and MYCN tumor amplification in human NB tumors. We found that DYRK2 and DYRK3 mRNA expression in NB tumors was significantly higher in samples with MYCN gene amplification (Fig. . In contrast, DYRK1B and DYRK4 tumor expression was not correlated to MYCN amplification status, and DYRK1A was actually lower in tumors with MYCN amplification. These results might indicate that the most aggressive, MYCN-amplified NB tumors that have the highest DYRK2 and DYRK3 expression could be more sensitive to harmine treatment, similar to the results we found in NB cell lines. These expression profiles along with the harmine-DYRK2 interaction model would suggest that the cytotoxic effect of harmine in NB is primarily through DYRK2 inhibition.
Fig. 8
figure 8

DYRK gene NB tumor expression correlated to MYCN gene amplification. The mRNA expression of the DYRK gene family was analyzed in SEQC-498 and correlated to MYCN amplification by separating the tumors into samples with normal MYCN gene copy number (n = 401) and with MYCN amplification (n = 92) and comparing the groups using the non-parametric Kruskal–Wallis test. The Y-axis represents DYRK gene mRNA expres​sion(rank-based). The results show that DYRK1A mRNA expression is highest in tumors without MYCN amplification, but that both DYRK2 and DYRK3 are most highly expressed in tumors with MYCN amplification. DYRK1B and DYRK4 expression are not correlated to MYCN gene copy number
Full size image
Discussion

NB is a rare but lethal childhood tumor. Patients often present with advanced disease with survival chances below 50%, in spite of aggressive, multimodal therapy [9]. Almost all survivors of high-stage NB suffer from late therapy effects that severely impact quality of life. Current treatment might have reached a therapeutic plateau, clarifying an urgent need for more specific, more effective, novel therapies. The use of gene-targeting treatments is still very rare, and no current therapies target MYCN, the most important NB oncogene, that is almost invariantly over-expressed in advanced NB.

We investigated the use of harmine, a tricyclic β-carboline alkaloid with known, dose-dependent in vitro cytotoxicity for cell lines from different cancer types [9,10,11]. Harmine has been shown to inhibit MAO-A activity, increase BDNF levels, and inhibit human topoisomerase I [6, 27]. It is also a potent inhibitor of the DYRK family that interferes with neurite formation and DYRK1A plays a role in neurodegenerative disorders [14]. Although harmine displayed the highest in vitro potency against recombinant DYRK1A, it also inhibited DYRK1B and DYRK2, and, at much lower potency, DYRK4 [14]. Recently, harmine was found to synergize with the anthracycline doxorubicin in the breast cancer cell line MCF-7 [28] and harmine-mediated inhibition of DYRK1A destabilized epidermal growth factor receptor (EGFR) in aggressive glioblastomas and reduced EGFR-dependent glioblastoma growth [29]. Although harmine clearly binds to and inhibits DYRK proteins, it cannot be entirely ruled out that its anticancer effects at least in part were influenced by the inhibition of other potential harmine targets such as MAO-A or topoisomerase.

We observed strong cell death in harmine-treated NB cell lines that acting in part through the activation of caspase-3/7 and caspase-9. PARP cleavage and Annexin V staining analyses showed that apoptosis progressed into late stages. Further increasing the dose range of harmine resulted in similar effects (Additional file 1: Fig. S2). Excitingly, late apoptosis was significantly more prevalent in SKNBE and KELLY, two NB cell lines with MYCN amplification and concomitant MYCN oncoprotein expression that represent high-stage NB, than in two NB cell lines, SKNAS and SKNFI, with normal MYCN copy number and without MYCN over-expression. It has been previously shown that MYCN has the ability to promote apoptosis and specific targeted therapy combinations have been tested to exploit the apoptosis-primed state of MYCN-amplified NB cells. Moreover, caspases have been shown to contribute to cell cycle regulation independent of apoptosis, and caspase inhibitors prevented cell proliferation through induction of cell cycle arrest at mitotic phase [30, 31]. Our observations in vitro were further supported by mRNA expression data we generated in human NB tumors. We found that DYRK2 and DYRK3, both harmine targets, are most highly expressed in NB patients with poor prognosis, and show MYCN-correlated expression. Although the expression of DYRK1A was also high in NB, it was not of prognostic value. These results strongly support our in vitro findings.

To verify the importance of DYRK2 in NB, we treated four NB cell lines SKNBE, KELLY, SKNAS, and SKNFI with LDN-192960, a DYRK2/haspin inhibitor and INDY, a DYRK1A/B inhibitor [13, 32]. Strikingly, inhibition of DYRK2 with LDN-192960 induced significant cytotoxic effects whereas inhibition of DYRK1A/B with INDY had no effect under identical conditions (Fig. 9), thus underlining the importance of DYRK2 in NB.

https://cancerci.biomedcentral.com/articles/10.1186/s12935-018-0574-3

Chinese researchers on Harmine:
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Antibacterial, Antifungal, Antiviral, and Antiparasitic Activities of Peganum harmala and Its Ingredients: A Review

by Zihao Zhu, Shujuan Zhao and Changhong Wang

The MOE Key Laboratory for Standardization of Chinese Medicines, Shanghai Key Laboratory of Compound Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

Author to whom correspondence should be addressed.
Molecules 2022, 27(13), 4161;
https://doi.org/10.3390/molecules27134161
Received: 10 June 2022 / Revised: 25 June 2022 / Accepted: 27 June 2022 / Published: 29 June 2022


Abstract

Infectious diseases have always been the number one enemy threatening health and well-being. With increasing numbers of infectious diseases, growing resistance of pathogens, and declining roles of antibiotics in the treatment of infectious diseases, it is becoming increasingly difficult to treat new infectious diseases, and there is an urgent need to develop new antibiotics to change the situation. Natural products tend to exhibit many special biological properties. The genus Peganum (Zygophyllaceae) has been used, for a long time, to treat cough, asthma, lumbago, hypertension, diabetes, and Alzheimer’s disease. Over the past two decades, a growing number of studies have shown that components from Peganum harmala Linn and its derivatives can inhibit a variety of microorganisms by inducing the accumulation of ROS in microorganisms, damaging cell membranes, thickening cell walls, disturbing cytoplasm, and interfering with DNA synthesis. In this paper, we provide a review on the antibacterial, antifungal, antiviral, and antiparasitic activities of P. harmala, with a view to contribute to research on utilizing P. harmala for medicinal applicaitons and to provide a reference in the field of antimicrobial and a basis for the development of natural antimicrobial agents for the treatment of infectious diseases.

Keywords:
antimicrobial; antibacterial; antifungal; antiviral; genus Peganum; Peganum harmala; review

1. Introduction

Infectious diseases pose significant harm to the health of humans, animals, and plants, and they occur all over the world because of the diversity and strong infectivity of their pathogens. The effective prevention and treatment of infectious diseases are major medical problems. For example, the spread of COVID-19 to more than 200 countries and regions around the world in 2020 caused profound influences on human healthy life and survival [1,2] and, as of 28 July 2021, the World Health Organization (WHO) data showed that the number of confirmed cases of COVID-19 in the world reached 195 million, with a cumulative death toll of 4.18 million, representing an infectious disease with the largest death toll since SARS in 2002 and the greatest impact on human health [3,4].

Antimicrobial drugs play an important role in the prevention and treatment of infectious diseases [1,2]. According to statistics, in the past seven decades since the discovery, mass production, and clinical use of antibiotics, billions of patients have been saved worldwide [5]. Despite continuous developments and progress in medical science, many pathogens have evolved drug resistance to antimicrobial drugs due to genetic changes, reducing the efficacy of antibiotics and significantly limiting clinical applications. In addition, the development of antibiotics has almost stagnated in the past decade due to long R&D cycles of antibiotics, high R&D costs, and low commercial returns [6,7]. Therefore, there is an urgent need for new antimicrobial drugs to address diseases caused by drug-resistant microorganisms.
In recent years, numerous studies have shown that more and more natural products of plants have special biological activities as natural antibiotics that play important roles in prevention, treatment, and reducing disease prevalence [8,9,10,11], such as the artemisinin extracted from herb Artemisia annua L. with antimalarial effect [12].

Peganum harmala L., a perennial herb that belongs to the Zygophyllaceae family, is distributed in the Mediterranean region of Europe, Central Asia, and southern South America. It is commonly used as winter feed for cattle, sheep, camels, and other livestock and also as a traditional Chinese medicine for treating a variety of human diseases [13]. Phytochemical studies have discovered that the main chemical components in the plant are alkaloids, flavonoids, volatile oils, and trace elements [14]. P. harmala has many clinical pharmacological effects, such as antibacterial, anti-inflammatory, antibacterial, leukemia resisting, psoriasis resisting, and memory enhancement, and has been clinically used for treating cough, hypertension, diabetes, jaundice, colic, lumbago, malaria, Alzheimer’s disease, and other human diseases [13]. Reports on the antibacterial, antifungal, antiviral, and antiparasitic activities of P. harmala have been increasing each year. However, to the best of our knowledge, the antimicrobial effects and mechanisms of P. harmala have not been reported in detail, and therefore, it has been difficult for researchers to understand the antimicrobial effects of P. harmala and to develop its R&D and application potential in the antimicrobial field. In this paper, we review the antibacterial, antifungal, antiviral, and antiparasitic activities and mechanisms of P. harmala extracts and its ingredients by retrieving the relevant literature in globally recognized databases such as Web of Science, PubMed, Google Scholar, Elsevier, and Chinese National Knowledge Infrastructure (CNKI). This literature review should provide a reference for research on the utilization of P. harmala for medicinal purposes and the basis for the development of natural antimicrobial drugs.

2. Biological Characteristics of P. harmala

P. harmala, is also known as “smelly ancient flower” in the Xinjiang Uyghur Autonomous Region, China; it is a glabrous perennial herb plant that is 30–70 cm high. It has numerous roots that are up to 2 cm thick and stems that are erect or spreading with numerous branched from the base. Its leaves are alternate, ovate, and divided into 3–5 lanceolate-striate lobes that are 1–3.5 cm long and 1.5–3 mm wide [15]. The morphology of P. harmala is shown in Figure 1. Molecules 27 04161 g001 550

Figure 1. P. harmala (A) plant; (B) flower; (C) ripe fruits; (D) seeds.

3. Main Components of P. harmala

As early as 1900, a researcher studied the inhibitory effects of total alkaloids from P. harmala on retinoblastoma cells. People have studied the pharmacological, chemical, and biological activities of P. harmala for more than 100 years. There are many different compounds in P. harmala [14,16], which were reported in detail in 2017 [13]. More than 308 compounds have been isolated from P. harmala, including 97 alkaloids, 24 flavonoids, 10 triterpenoids, 3 anthraquinones, 2 phenylpropanoids, 18 carbohydrates, 17 amino acids, 99 volatile oils, 26 fatty acids, 3 sterols, 1 vitamin, 1 protein, 1 carotene, and 6 other trace elements. Among these compounds, the highest content is β-carboline alkaloids (βCs). The alkaloid content is up to 10% in seeds, followed by roots, and the least amount in leaves. They main compounds include harmine, harmaline, harmalol, harmane, and harmol; a few new compounds have been reported in recent years.

In 2017, Wang et al. [17] conducted a study on the chemical composition of P. harmala seeds. The seeds of P. harmala were extracted by ethanol reflux, and then the extract was concentrated, the pH adjusted, and separated by silica gel column to obtain nine compounds. Among them, (−)-peharmaline A (1) (Figure 2) and (+)-peharmaline A (2) (Figure 2) were rare carboline-vasicinone hybrid alkaloid enantiomers with an unknown hybrid dimer system.

Molecules 27 04161 g002 550
Figure 2. Structures of the twenty new compounds from P. harmala.
Wang et al. [18] extracted the seeds of P. harmala with ethanol reflux, adjusted the pH of the extract, extracted it, and passed it through the silica gel column. Six new βCs were identified, i.e., pegaharmines F–K (3– (Figure 2).

In a study by Li et al. [19], two pairs of new alkaloid glycoside dimorphisms (9–12) (Figure 2) and a new enantiomer (13) (Figure 2) were identified in the ethanol extracts of P. harmala seeds. The names of the compounds 9–12 are (S)- and (R)-1-(2-aminobenzyl)-3-hydroxypyrrolidin-2-one β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (9,10), (S)- and (R)-vasicinone β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside (11,12).

In 2019, Fang et al. [20] extracted seeds of P. harmala with 95% ethanol and concentrated them. The concentrate was re-extracted with n-hexane and ethyl acetate. The extracts were subjected to silica gel column chromatography, ODS column chromatography, MCI resin, and other technologies, and a new compound was found and named N-[3-(2-amino-4-methoxyphenyl)-3-oxopropyl] acetamide (14) (Figure 2).
In 2020, Wu et al. [21] isolated six new compounds against HSV-2 virus from crude alkaloids of P. harmala seeds and identified the structures of six new βCs based on HR-ESI-MS data, named pegaharines A–F (15–20) (Figure 2).

4. Antimicrobial Activity

4.1. Antibacterial Activity
The antibacterial effects of P. harmala have been demonstrated in many Gram-positive and Gram-negative bacteria.

Ahmad et al. [22] determined the antibacterial activity of harmine, harmaline, and their derivatives using the broth incorporation method with tryptone soya broth (Oxoid) and recorded the minimum inhibitory concentrations (MICs). They found that the MICs of harmine against Bacillus pumilus, B. subtilis, Corynebacterium hofmannii, Sarcina lutea, Staphylococcus citreus, S. lactis, Aeromonas hydrophila, and Salmonella paratyphi A were 100 μg/mL, respectively. This was the same activity as harmaline against C. hofmannii and S. lutea.

Abutbul et al. [23] studied the antibacterial (A. hydrophila, Streptococcus iniae, and Vibrio alginolyticus) activity of water extracts from desert plant extracts (including P. harmala) on fish pathogenic bacteria in vitro using the disk diffusion method. It was found that P. harmala seeds extract had the highest inhibition effects on A. hydrophila and V. alginolyticus; the inhibition area ranged from 18.0 to 20.5 mm.
Muhaisen et al. [24] observed the in vitro antibacterial (Bacillus cereus ATCC 11778, B. subtilis ATCC 6633, Enterococcus faecalis ATCC 29212, E. coli ATCC 25922, P. aeruginosa ATCC 27853, S. aureus ATCC 25923, and S. epidermidis ATCC 12228) activities of ethanol, hexane, chloroform, and methanol extracts from eight medicinal plants (including P. harmala leaves) using broth microdilution. It was found that there was inhibitory activity of P. harmala against Gram-negative bacteria (MIC range of 4–8 mg/mL) and the chloroform extract from P. harmala leaves. The results showed the strongest inhibition of extract from P. harmala leaves against P. aeruginosa with MICs ranging from 0.25 to 1.0 mg/mL.

Arshad et al. [25] tested the activities of methanol, ethanol, and water extracts of 12 medicinal plants (including P. harmala) against 20 bacteria (Acinetobacter sp. 7987, Clostridium sp. 1729, E. coli-SN-11, E coli-SN-07, E. coli-5457, E. coli-6058, E. coli-5706, E. coli-1253, E. coli-1303, E. coli-3931, Pasteurella multocida 1294, Staphylococci sp. 772, Streptococci sp. 1959, Proteus sp. 6433, Salmonella sp. 2853, Salmonella sp. 3102, Salmonella sp. 4922, Salmonella sp. 4377, and Salmonella sp. 3402) by performing disk and agar diffusion experiments. The extract of P. harmala seeds was found to inhibit the growth of all bacteria at concentrations of 0.38~1.65 mg/mL. The antimicrobial activities of crude extracts and ingredients from P. harmala were further compared using the microdilution method and the order of antimicrobial activities was harmane, harmaline, harmalol, and harmine. It was suggested that P. harmala or the ingredients could play a significant role in drug development for controlling bacteria.

Darabpour et al. [26] examined the antibacterial activity of methanol extracts from different parts of P. harmala (root, stem, leaf, flower, and seed) against 11 bacteria (Gram-positive bacterial species of Bacillus anthracis, B. cereus, B. pumilus, S. aureus, S. epidermidis, Listeria monocytogenes, and S. pyogenes, and Gram-negative bacterial species of P. aeruginosa, Brucella melitensis, P. mirabilis, S. typhi, E. coli, and K. pneumoniae) using disk diffusion. It was found that root extract had better antibacterial activity against Gram-positive bacteria than seed extract. The antibacterial activity of the leaf was medium, while those of the stem and flower were weak. The MIC and MBC of the root and seed extracts against MRSA were 0.625 mg/mL, which was similar to the seed extract against E. coli and S. typhi. It was also found that seed and root extracts showed synergistic effects when combined with neomycin, colistin, and carbenicillin. In conclusion, harmine could be used in the development of drugs for the treatment of infectious diseases caused by MRSA.

Omar et al. [27] investigated the anti-Cronobacter sakazakii (ATCC 29004) activity of methanol and petroleum ether extracts from six plants (including P. harmala). It was found that the methanol extract of P. harmala effectively inhibited the growth of the strain, with an MIC of 0.00375 mg/mL. The presence of 2% reconstituted infant milk formula could reduce the inhibitory effect of plants on the growth of C. sakazakii, and the mixture of nisin and disodium ethylenediamine tetraacetate had a slight synergistic effect on the inhibition of C. sakazakii.

Fazal et al. [28] studied the antibacterial (S. aureus 6538, P. aeruginosa 9721, E. coli 25922, K. pneumoniae, Salmonella typhi, B. subtilis, and B. cereus) activities of 11 medicinal plants including P. harmala, using the disk diffusion method. It was found that the ethanol and hexane extracts of P. harmala showed the optimal inhibitory activity against K. pneumoniae (the inhibition zone was 25.8 mm), while the inhibition zone against E. coli was 10.8 mm. The chloroform extract was the most effective against S. aureus (inhibition zone of 24.5 mm). It was indicated that plants, such as P. harmala, could be used in the development of antibiotics for the treatment of diseases caused by K. pneumoniae.

Soltani et al. [29] studied the activity of essential oil and aqueous extracts of 24 medicinal plants (including P. harmala) against Xanthomonas arboretum pv juglandis. Most of the plant extracts were observed to possess antibacterial activity using an agar disk diffusion test. The aqueous extracts of six plants had significant antibacterial activity with antibacterial area of 6.0 mm. It was concluded that the aqueous extracts of these plants had broad possibilities in plant disease control and could be used as natural biological control agents in walnut orchards.

Irshaid et al. [30] identified the activities of methanol extracts from aerial parts of four medicinal plants (including P. harmala) against five bacteria (S. aureus, E. coli, P. aeruginosa, E. cloacae, P. mirabilis) using broth dilution and a disk diffusion test. The results showed that P. harmala extracts inhibited the growth of these bacteria with MICs of 0.8, 1.2, 0.9, 1.0, and 0.9 mg/mL, respectively.

Apostolico et al. [31] performed an agar diffusion test to evaluate the antibacterial activities of essential oils from P. harmala seeds from Algeria, Egypt, Libya, Morocco, and Tunisia, on Bacillus cereus 4313, B. cereus 4384, E. coli 857, Pseudomonas aeruginosa 50071, and S. aureus 25693. The results showed that all samples inhibited the growth of the bacteria. E. coli showed the highest sensitivity to these oils, especially the oil of P. harmala from Egypt at a concentration of 15 μg/mL with an inhibition zone of 10.0 mm, which was higher than that of the control group (tetracycline, inhibition zone of 10.0 mm). It was speculated that this may be due to the differences in the growth environment of P. harmala in different countries, resulting in different percentages of secondary metabolites of plants such as oxygen-containing monoterpenes, sesquiterpenes, and oxygen-containing sesquiterpenes. These findings were considered to be important in an era of serious problems with antimicrobial resistance.

The ethanol extract of P. harmala seeds showed an inhibitory effect (MIC of 4 μg/mL) on E. faecalis, but the inhibitory effect was not significantly different from that of 0.5% NaOH [32]. It is expected to become a safe therapeutic agent. Khalid et al. [33] assessed the anti-S. aureus and anti-P. aeruginosa activities of low-molecular-weight peptides in seeds and leaves of 20 plant species (including P. harmala) using a dish diffusion assay. P. harmala peptides (PhAMP) isolated from P. harmala had a maximum zone of inhibition against the two laboratory bacterial strains. Then, the authors studied the antibacterial potential of PhAMP against pathogens in burn wounds (S. aureus, P. aeruginosa, and K. pneumoniae) and surgical wounds (P. aeruginosa and K. pneumoniae). It was found that PhAMP was effective at disrupting biofilm formation of all pathogens after 36 h of treatment. These data indicate that P. harmala has the potential to be developed into a natural antibiotic drug.

Ait Abderrahimet al. [34] measured the antibacterial activities of the methanol extracts from P. harmala and Ziziphi spinosae against four pathogenic microorganisms (S. aureus, E. coli, Candida albicans, and P. aeruginosa) and used an agar dilution method to determine the MICs. It was found that the extracts of P. harmala seeds showed growth inhibition against all the test strains, with MIC values of 0.5, 1.0, and 6.0 mg/mL, respectively. The antibacterial effect of methanol extracts from P. harmala was better than that of Z. jujuba. It was demonstrated that P. harmala was an effective antibacterial agent. In the same year, Iranshahy et al. [35] assessed the antibacterial activity of the chloroform extracts of the fruits and flowers of P. harmala against five microorganisms including M. luteus by using the disk diffusion method. They found that the total alkaloids had a strong specificity for M. luteus and a low sensitivity to Gram-negative bacteria, especially P. aeruginosa.

Nenaah et al. [36] evaluated the antibacterial activities of four βCs (harmane, harmine, harmaline, and harmalol) from P. harmala seeds using the disc diffusion method with high potency biodisc. In separate alkaloid resistance tests, Escherichia coli, P. vulgaris, S. aureus, and Bacillus subitilis were proven to be the most susceptible to inhibition by harmane, harmine, harmaline, and harmalol, with inhibition zones of 17.9, 24.7, 14.7, and 21.1 mm and MICs of 0.50, 0.83, 1.00, and 0.75 mg/mL, respectively. Harmane had the highest activity against E. coli and harmalol had moderate antibacterial activity. When used with the binary mixture of harmane and harmaline, the inhibitor was the most potent against P. vulgaris (inhibitory zone of 28.9 mm and MIC of 0.41 mg/mL) and against B. subitilis (inhibitory zone of 26.1 mm and MIC of 0.33 mg/mL). In addition, it was concluded that the selection of compounds that act synergistically with P. harmala alkaloids had great potential in the treatment of diseases caused by P. vulgaris. Shaheen et al. [37] evaluated the inhibitory activity of P. harmala seed alkaloid extracts against four plant pathogens (R. solanacearum Physiotype II, Erwinia Amylovora, Pectobacterium Carotovorum subsp., and Burkholderia gladioli) in vitro using the agar diffusion method. R. solanacearum phylotype II was the most sensitive to the extract (MBC of 150 μg/mL), followed by B. gladioli (MBC of 200 μg/mL). When the concentration was reached between 4 and 300 μg/mL, the extract showed a significant inhibitory effect against R. solanacearum. Transmission electron microscopy revealed that bacterial wilt cells were severely damaged, with coagulated genome, thickened cytoplasm, cell wall, and disorganized structure. In vivo studies indicated that extracts of 300 μg/mL reduced plant brown spot symptoms. It was concluded that total alkaloid extract of P. harmala could be an alternative to chemical antimicrobial agents in the treatment of these bacterial diseases. Siddique et al. [38] also tested the in vitro inhibitory effect of seven plant extracts including P. harmala on bacteria using a paper disc diffusion method and in vivo inhibitory effect using pot experiments. Oxytetracycline treatment was a positive control. The extracts (100%, 75%, and 50% concentrations) of P. harmala, M. piperita, A. sativum, W. somnifera, M. azedarach, C. processra, and N. oleander could inhibit the growth of C. michiganensis. The undiluted water extract of P. harmala had the highest antibacterial activity in these plants, and the inhibition zone was 14.40 mm. The positive control oxytetracycline (200 ppm) showed the highest antibacterial activity inhibition zone, which was 24.70 mm. In vivo experiments showed that after 56 days of treatment, the plant disease degree of P. harmala dry powder treatment was significantly reduced, and the plant height was 10.33% higher than that of the plant treated with clarithromycin. It was suggested that dry powder or extract from these plants could be used to control canker of tomato.

The inhibitory activity of the n-butanol extract of P. harmala seeds against three species of P. aeruginosa was superior to that of cefazolin and vaamox [39]. When incubated with three P. aeruginosa at 500 μg/mL for 4 h, the sterilization rate reached 100%, and the compound further isolated is harmaline.
P. harmala seeds smoke can act as an air disinfectant to reduce the concentration of bacteria in air [40]. The removal rate of bacteria in air after 5 g of seeds in residential areas produced smoke for 5 min reached a maximum of 71.4%. In an educational setting, the bacterial removal rate in the air after 10 min of smoke from 10 g of seeds reached 92.8%.

Therefore, so far, there is an inhibitory effect of P. harmala extract on a variety of pathogenic bacteria that cause disease in animals and plants, with a wide antibacterial spectrum. The potential of its resistance to plant pathogens needs further exploration. The extracts of seeds and roots have good antibacterial effect as compared with some antimicrobials, which provides clues for future studies on synergistic antibacterial with other drugs.

4.2. Antifungal Activity

The antifungal effects of P. harmala have been demonstrated in various pathogenic fungi.
Ahmad et al. [22] tested 16 fungi with harmine, harmaline, and their derivatives at a concentration of 50–500 μg/mL on Sabouraud dextrose agar (Oxoid) slants, and recorded inhibition zones to compare with the MICs. It was found that harmine of 100 μg/mL was effective against all eight dermatophytes used (Epidermophyton floccosum, Microsporum canis, T. longjifisis, T. mentagrophytes, T. rubrum, T. simii, T. tonsurans, and T. violaceum), while harmaline inhibited M. canis, T. longjifisis, T. rubrum, and T. tonsurans at 500 μg/mL. Other compounds reduced the growth of some fungi at higher concentrations as compared with the control group (terbinafine of 0.01–25 μg/mL).

Shahverdi et al. [41] mixed different amounts (0.156, 0.312, 0.612, 1.25, 2.5, and 5 mg) of dichloromethane and n-hexane extracts of smoke from P. harmala smoldering seeds with Mueller–Hinton agar, and determined the inhibition zone of fungi (Aspergillus niger PIM, C. albicans ATCC 14053, and Cryptococcus neoformans kf 33) using a conventional disk-diffusion method. It was found that there was no antibacterial activity of the dichloromethane extract at an amount of 0.156 mg, but higher contents showed good antifungal activities against S. epidermidis and C. neoformans.

Sarpeleh et al. [42] studied the inhibition of aqueous, alcoholic, and methanolic extracts of leaves, floral tissues, and seeds of P. harmala to 13 fungal (Alternaria sp., Botrytis cinera, Cladosporium cucumerinum, Corynespora cassiicola, Fusarium oxysporum f.sp melonis, Macrophomina phaseolina, Monosporascus annovballus, Phytophthora drechsleri, Rhizoctonia solani, Sclerotinia sclerotiorum, Trichoderma harzianum, Ulocladium sp., and Verticillium dahliae) species using mycelial growth inhibition assay. It was found that the mycelium growth rate of most fungi decreased when treated with water extract and methanol extract, and the seed extract had the highest activity. The water-soluble seed extract could inhibit the spore germination of F. oxysporum. The authors suggested that extracts of P. harmala could be used as a substitute for antifungal agents. Another study also illustrated this point [43].

Nenaah et al. [36] determined the activities of four alkaloids (harman, harmine, harmaline, and harmalol) from P. harmala seeds against two fungi (A. niger and C. albicans) using the disk diffusion method. It was found that harmaline had the best inhibitory effect on C. albicans, with inhibitory zones between 21.2 and 24.7 mm. When used in the form of total alkaloid mixture, the inhibition zone reached 31.5 mm. Its binary mixture was recommended for use as a novel antifungal agent.

Diba et al. [44] studied the activities of alcohol extract of P. harmala seeds against C. glabrata and C. tropicalis using serial dilutions in tubes and serial dilutions in agar media. It was confirmed that the MIC of P. harmala extract against C. glabrata was 0.312 mg/mL and the MFC was 0.62 mg/mL. The MFC against C. tropicalis was 0.125 mg/mL. It was suggested that there was antifungal activity of P. harmala seed extract against opportunistic yeast and Candida spp.

The conidial suspension of two fungi (Penicillium digitatum and B. cinerea) was prepared in buffers of pH 5 and pH 9, and treated with 1 mM harmol for 24 h to evaluate the number of pathogens. As compared with the control, the counts of pathogens decreased by two times at pH 5, and the spore viability was completely lost [45].

Hajji et al. [46] mixed oil of P. harmala seeds at concentrations of 50%, 25%, 12.5%, 6.25%, 3.125%, 1.562%, 0.781%, 0.39%, and 0.195% with melted agar and measured the hyphae to study the inhibitory effect of different concentrations of oil to 10 fungi (R. solani, M. phaseolina, Pythium sp. 1, Pythium sp. 2, Alternaria sp., Colletotrichum sp., M. cannonballus, F. solani f. sp. cucurbitae, F. oxysporum f. sp. melonis, and F. oxysporum f. sp. niveum). The results of mycelial measurements revealed that the oil of P. harmala seeds at a concentration of 50% had good activity against Pythium sp. with inhibition rates of mycelial growth ranging from 56% to 82%, followed by F. solani f. sp. cucurbitae with inhibition rates ranging from 15% to 55.7%. Seed oil had a moderate inhibitory effect on Colletotrichum sp. with the inhibition from 5.19% to 42%. It was concluded that P. harmala was expected to be a future drug for controlling some microorganisms.

Izadi et al. [47] extracted the dry leaves and fruits of P. harmala with 70% ethanol. After removing organic solvents, chitosan nanoparticles were used to encapsulate the extracts and forsythia essential oil (NCE), and the activity of encapsulated nanoparticles against Alternaria spp. was evaluated in vitro and in vivo. The results showed that the antifungal activity of P. harmala extract at 100 ppm was similar to that at 1000 ppm, and the fungus was completely inhibited at 750 ppm. Encapsulated with forsythia essential oil, the total inhibitory concentration decreased to 200 ppm. It was speculated that chitosan attached to the cell membrane near the release of content and enhanced antifungal activity. P. harmala seed smoke reduced the number of fungi in the air. After 30 min of exposure to air from the smoke generated by 10 g seeds, the fungal removal rate reached 94.7% [40].

From the current studies, it can be seen that there are many antifungal studies on P. harmala, which have revealed that there is good antifungal activity of P. harmala. The active part is mostly the crude extract of seeds. However, studies on screening antifungal compounds and structure optimization of high antibacterial compounds are rare. It is an urgent need to determine the optimization of active compounds and lead compounds in future studies.

4.3. Antiviral Activity

The inhibitory effects of P. harmala on a variety of animal viruses and plant viruses have been reported.
Ma et al. [48] prepared P. harmala seed crude protein extract (PHP) with 50–80% ammonium sulfate and tested the anti-HIV-1 RT activity of PHP according to the instructions of the HIV-1 RT kit, which showed a maximum inhibition rate of 69.1% at a PHP concentration of 3.75 μM and estimated an IC50 value of 1.26 μM.

Song et al. [49] tested the anti-TMV activities of β-carboline, dihydro-β-carboline, and tetrahydro-β-carboline alkaloids and their derivatives using the Ishida’s method. It was found that all alkaloids and some of their derivatives exhibited higher anti-TMV activities than ribavirin in vitro and in vivo. In particular, the in vitro and in vivo activities of harmalan (60.3%) and tetradydroarmane (59.5%) at 500 μg/mL were much higher than that of ribavirin (38.5%).

Moradi et al. [50,51] evaluated the effects of ethanol extract from P. harmala seeds and its total alkaloids on MDCK cells infected with influenza A virus (A/Puerto Rico/8/34 (H1N1, PR8) virus), and the mechanism of RNA polymerase blocking was studied. The results showed that there were good inhibition effects of the crude extract and total alkaloids on the virus with the CC50 values of 122.9 and 133.9 µg/mL, respectively. The extract reduced viral mRNA expression and inhibited viral protein synthesis in a dose-dependent manner, with no effect on hemagglutination inhibition and virucidal activity.

Wu et al. [21] compared the anti-HSV-2 activities of pegaharine A–F from P. harmala seeds with two representative β-carboline alkaloids (harmine and harmaline). Pegaharine D showed the strongest antibacterial activity of these compounds with an IC50 value of 2.12 ± 0.14 μM.

Edziri et al. [52] evaluated the antiviral activities of extracts of P. harmala leaves against the HCMV strain AD-169 and Coxsackie B virus type 3 (CoxB-3). Except for the petroleum ether extract of P. harmala leaves without resistance, there was anti-HCMV activities of all the other extracts. The methanol extract had the strongest activity. Antiviral activities varied from 80% to 95% at concentrations from 25 to 100 mg/mL. In the same year, Chen et al. [53] reported that harmine from 502 natural products was the most effective agent for EV71 virus treatment in vitro with CC50 values between 400.0 and 500.0 μM. Harmine at concentrations of 10, 30, and 100 μM was observed under an inverted microscope to inhibit viral activity, and to downregulate the RNA and protein levels of EV71. It reduced the cytopathogenic effect in a dose-dependent manner. DCFH-DA probe measurements showed that harmine inhibited ROS production in a dose-dependent manner, which was related to the downregulation of NF-κβ activation.

A complex of harmala has been shown to have highly selective anti-H1N1 activity without affecting host cells. The complex of 2-hydroxypropyl-β-cyclodextrin and harmala loaded into PLGA nanoparticles showed highly selective antiviral activity against H1N1 (IC50 of 2.7 μg/mL), with low toxicity to host cells [54].
The current studies have reported the antiviral activity of different components of P. harmala. In most of these studies, only the antiviral extracts of P. harmala have been reported, but research on screening antiviral compounds in the extracts is rare, and there is less research on the antiviral mechanism of P. harmala. Therefore, there is still more research needed on these aspects.

4.4. Antiparasitic Activity

P. harmala has been proved to have significant anti-parasitic activity. Lasa et al. [55] studied the antileishmanial activities of different forms of harmine through a hamster model. It was found that free, liposome, vesicle, and nano-articular forms of harmine reduced the parasitic load of the spleen by about 40%, 60%, 70%, and 80%, respectively. A cell cycle analysis study using flow cytometry showed that harmine interfered with the cell division stage and confocal microscopy showed that cell death was caused by non-specific membrane damage.

Mirzaei et al. [56] treated 50 cattle naturally infected with Theileria annulata with extract of the aerial parts of P. harmala at a dose of 5 mg/kg for 5 days. After about 15 ± 3 days, the symptoms and parasites of 39 cattles had disappeared. The recovery rate was 78%. Schizonts of T. annulata were not observed in lymph node biopsy smears.

Astulla et al. [57] tested the anti-P. falciparum activities of four alkaloids (harmine, harmaline, vasicinone, and deoxyvasicinone) from P. harmala seed extract and found that there was a moderate inhibition of harmine (IC50 value of 8.0 μg/mL) and harmaline (IC50 value of 25.1 μg/mL) on P. falciparumin in in vitro. Vasicinone and deoxyvasicinone did not display inhibitory effects on the protozoa (IC50 > 10 μg/mL). In human monocyte toxicity studies, it was found that harman and tetrahydroharman reduced THP1 cells in the S phase of the cell cycle, presumably inhibiting total protein synthesis [57].

Derakhshanfar et al. [58] infected 5–6-month-old lambs with T. hirci, and treated them with the extracts of the aerial parts of P. harmala at a dose of 5 mg/kg. Microscopic observation showed that the parasites disappeared in 9 days and the rectal temperature returned to normal in the lymph nodes and peripheral blood smears of lambs in the treatment group. These results proved that there were effects of P. harmala extracts in treating sheep tissue damage induced by T. hirci.

After 48 h of treatment with 1 mg/mL methanol extract of P. harmala seeds, miracidial formation reduced to 0.5%. The methanol extract of P. harmala seeds at a dose of 3 mg/mL could inhibit the formation of miracidial [59].

Shoaib et al. [60] incubated A. castellanii (ATCC 50492) isolated from a keratitis patient in 24-well plates with three plant extracts, including P. harmala seeds, and determined the number and activity of A. castellanii using cytometer counting and trypan blue exclusion. The in vitro results showed that P. harmala extracts of 1.5 mg/mL had a significant amoebicidal effect on Acanthamoeba in a concentration-dependent manner.

Tanweer et al. [61] treated coccidiosis chicken with the methanol extract of P. harmala seeds at concentrations of 200 mg/L (PH-200), 250 mg/L (PH-250), and 300 mg/L (PH-300) in drinking water. On the 14th day, chicken were infected with the coccidiosis larvae at a standard dose until the age of 35 days, and the weekly weight gain, feed intake, feed-to-meat ratio, cecal section of the birds were experimentally analyzed. It was found that weight gain, total body weight, and feed-to-meat ratio increased linearly with an increased dose of P. harmala. Histopathological observations revealed that the cecal injury and leukocyte infiltration were significantly reduced in broilers of group PH-300. It was indicated that P. harmala extract had good anticoccidial effects in broiler chicks.

Shafiq et al. [62] studied the acaricidal activities of methanol extracts of three plants (rhizome of Curcuma longa, fruit of Citrullus colocynthis, and seed of P. harmala) against R. microplus using a modified larval immersion method (syringe method). It was found that the activities of all three extracts reached peak values when the dose was 50 mg/mL for 6 days. The lowest acaricidal efficacy was observed at 24 h with P. harmala at 3.125 mg/mL alone. Based on these observations, it was suggested that this inexpensive and readily available plant combination formulation could be used on farms.

Tabari et al. [63] collected Trichomonas gallinae from pigeons using the wet mount method, cultured it in trypsin/yeast extract/maltose (TYM) medium on multiwell plates with P. harmala alkaloid extract, metronidazole, harmine, and harmaline at concentrations of 5, 10, 15, 20, 30, 50, and 100 μg/mL, to determine the MICs. The metronidazole of 50 mg/kg body weight or alkaloids of 25 mg/kg body weight were administered to 60 pigeons experimentally infected with T. gallinae. The MICs of P. harmala alkaloid extract, metronidazole, harmine, and harmaline were 15, 50, 30, and 100 µg/mL, respectively. Infected pigeons were completely recovered after 3 days of treatment with alkaloids but they were not completely recovered with metronidazole.

At present, P. harmala seed extract has shown good inhibitory effects on pathogens and inhibition of some protozoa (T. pigeon), which was superior to a positive control drug in some cases. However, there are few studies on its mechanism of action, and the material basis of its efficacy is not clear enough. Therefore, further studies are needed using modern molecular biology and other means.

5. Antimicrobial Mechanisms of P. harmala

A growing number of studies have shown superior inhibition of P. harmala against a wide range of microorganisms, and the antimicrobial mechanisms have also been identified. The antimicrobial mechanisms include: increasing ROS content; destroying the microorganism’s cell membrane and cell wall, causing cytoplasmic disorder; downregulating the expression of bacterial flagellum motility genes and upregulating the expression of capsular polysaccharide synthesis genes and intercellular adhesion genes; inhibiting the generation of microorganisms’ violacein peptide; inhibiting the replication of pathogenic nucleic acids and protein synthesis (Table 1).

Table 1. The antimicrobial mechanism of P. harmala.
Table

5.1. Effects on ROS and Cell Membrane

Under normal circumstances, active oxygen in organisms is in dynamic equilibrium, and metabolism imbalance leads to cell membrane damage. The conidial suspension of plant pathogenic fungus P. digtiatum was treated with 1 mM harmol. After 24 h, the cell membrane of the conidia was destroyed, the cell membrane was ruptured, and the cytoplasm was disordered. When harmol-treated P. digtiatum conidia were exposed to UVA, a significant increase in intracellular ROS accumulation was detected by H2DCFDA probe [45].

In another experiment using the MTT assay to determine the inhibition of MDR E. coli by harmine and its derivatives, a number of cell ruptures and cell debris were observed in the treated E. coli. The lucigenin CL assay showed a dose-dependent increase in ROS in treated E. coli [64].

PhAMP has good destructive effects on the biofilm formed by pathogenic bacteria. The inhibitory effects of PhAMP on burn and surgical wound pathogens (S. aureus, P. aeruginosa, K. pneumoniae, P. aeruginosa, and K. pneumoniae) were studied using the disc diffusion method, and then the effect of PhAMP on pathogens isolated from burn-wound biofilm was tested on a 24-well plate. After 36 h of treatment, the formation of crystal violet-stained biofilm decreased and the biofilm was destroyed, as observed under an optical microscope [33]. In another experiment, to evaluate the effect of P. harmala extracts on the inhibition of Acinetobacter baumannii biofilm, a decrease in biofilm was also observed with scanning electron microscopy [65].

5.2. Effects on Nucleic Acid

Nucleic acid is the main information molecule of cells, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In one study, PhAMP was incubated with P. aeruginosa and S. aureus under shaking for 16 h, and then the expression levels of biofilm-related genes were detected by real-time PCR. It was found that the expression levels of flagellum gene (flgK), fimbriae protein gene (pilA), and fimbriae gene (cupA1) in P. aeruginosa were significantly downregulated. The expressions of the capsular polysaccharide synthesis gene (CPS5) and the intercellular adhesion gene (icaA) were upregulated in S. aureus [69].
The total alkaloid extract from seeds of P. harmala was cultured with inocula of R. solanacearum in broth medium. In addition to the thickening of cell wall, disorder of cytoplasm, and serious damage of cells, genome condensation was also observed under transmission electron microscope [37].
In another study, eight plant extracts were mixed with yeast extract sucrose (YES), and the expression levels of aflatoxin B1 synthesis genes were analyzed by RT-PCR. It was found that the reduction in aflatoxin B1 biosynthesis was related to a reduction in (or blocking of) the expressions of aflR, aflM, and aflP by plant extracts such as P. harmala [66].

It can also affect virus replication. MDBK cells infected with Bohv-1 were treated with harmine at different stages. It was found that the viral production was significantly reduced, and harmine affected viral replication at early and later stages [67].

5.3. Effects on Reverse Transcriptase (RT) Activity

Reverse transcriptase plays a catalytic role in viral reverse transcription. Studies have shown that it can inhibit the proliferation of tumor cells and reduce the activity of HIV-1 RT. A new antifungal protein was isolated from the seeds of P. harmala and its inhibition rate on tumor cells was studied according to the cytotoxicity curve. The HIV-1 RT kit was used to test the inhibition effect on HVI-1 RT and it was found that the new antifungal protein could inhibit the proliferation of esophageal cancer, cervical cancer, gastric cancer, and melanoma cells, and the activity of HIV-1 RT was decreased [48].

6. Conclusions

P. harmala is rich in resources, widely distributed in the world, and its medicinal history is more than 2000 years. Its traditional use in disinfectants and mosquito control has contributed to its successful application in the treatment and prevention of human, animal, and plant diseases. In recent years, epidemics have become the greatest threat to human health and plant quality. The emergence and variation of new pathogens and the emergence of drug resistance pose a huge threat to humans, and therefore, there is an urgent need for effective drugs to change this situation. P. harmala extract, harmine, and other βCs have broad-spectrum inhibitory effects on many microorganisms. Therefore, P. harmala is a potential source of safe and natural antimicrobial drugs, especially at a time when viruses have become the greatest enemy threatening the safety of human lives.

The antimicrobial activity and antimicrobial mechanism of βCs, such as harmine, were studied to provide a theoretical basis for the development of potential antibacterial drugs. The inhibitory activity of P. harmala on broad-spectrum microorganisms may be the reason for its therapeutic effect on many pathogen-related diseases such as wound treatment, skin inflammation, hemorrhoids, and cough. P. harmala is a potential resource for the prevention or treatment of plant infectious diseases, such as rust and wilt, due to its insecticidal effect and inhibition of the activity of various plant pathogens.

A rich variety of compounds is the material basis for its broad-spectrum antimicrobial activity. There have been at least 308 chemical constituents isolated and identified in P. harmala, including alkaloids, flavonoids, triterpenoids, anthraquinones, phenylpropanes, carbohydrates, amino acids, volatile oils, sterols, vitamins, proteins, carotene, and trace elements, of which alkaloids have better antimicrobial activity tests. However, due to the diversity of plant secondary metabolites and the advancement of technology, new compounds have been discovered from P. harmala extracts. The antimicrobial activities of these new compounds need to be further studied. A very meaningful research focus would be to develop efficient broad-spectrum antimicrobial drugs using the compounds in P. harmala as antimicrobial scaffolds. In addition, the current research has revealed the biological activities of P. harmala, but the application of these biological activities in the treatment of human, animal, and plant diseases is limited, and the application of biological activities in medicine should be further studied in the future.

In conclusion, P. harmala inhibits a variety of pathogenic microorganisms and has great potential in the treatment of infectious diseases and air sterilization. Current studies have shown that it contains hundreds of compounds and has a variety of biological activities. These results herald the bright prospect of P. harmala as a natural source of next-generation medicines, and also lay the foundation for further elucidation of its therapeutic mechanisms, thus, revealing the relationships among the clinical uses, chemical components, and biological activities of P. harmala.

Author Contributions
Conceptualization, methodology, and data analysis, Z.Z., S.Z. and C.W.; writing—original draft preparation, Z.Z.; writing—review and editing, C.W. and Z.Z.; project administration, C.W. All authors have read and agreed to the published version of the manuscript.

This work was supported by the Technology Cooperation Projects of Science in Shanghai, China (no. 20015800100), the National Nature Science Foundation of China (no. 82173885), the National Natural Science Foundation of China and Xinjiang Uyghur Autonomous Region of China (no. U1130303), and the Graduate Student Innovation Ability Project of Shanghai University of Traditional Chinese Medicine (Y2021004) awarded to Professor Changhong Wang for financial support of this study.

https://www.mdpi.com/1420-3049/27/13/4161