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

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Post by Cr6 on Fri Nov 23, 2018 8:14 pm

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

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Post by Cr6 on Fri Nov 23, 2018 8:32 pm

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 ( Cool, 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


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Post by Cr6 on Fri Nov 23, 2018 8:35 pm

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.

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Post by Cr6 on Fri Nov 23, 2018 8:40 pm

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
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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.

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Post by Cr6 on Sat Dec 22, 2018 1:35 am

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/

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Post by Cr6 on Sat Dec 22, 2018 1:44 am


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–Cool. 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

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Post by Cr6 on Sat Dec 22, 2018 1:50 am

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

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Cancer and ATP: The Photon Energy Pathway (DCA as anti-tumor) - Page 3 Empty Re: Cancer and ATP: The Photon Energy Pathway (DCA as anti-tumor)

Post by Cr6 on Sat Dec 22, 2018 1:55 am

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(Cool–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

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Post by Cr6 on Sat Dec 22, 2018 2:01 am

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

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Post by Cr6 on Sat Dec 22, 2018 3:31 am

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

--------------------------

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.
Cancer and ATP: The Photon Energy Pathway (DCA as anti-tumor) - Page 3 Nihms864914f4

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/


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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 (Cool, 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
--------------------------

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/

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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/

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Post by Jared Magneson on Sat Jan 05, 2019 4:00 am

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.

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Post by Cr6 on Tue Jan 08, 2019 11:32 pm

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.

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Cancer and ATP: The Photon Energy Pathway (DCA as anti-tumor) - Page 3 Empty Re: Cancer and ATP: The Photon Energy Pathway (DCA as anti-tumor)

Post by Cr6 on Mon Jan 14, 2019 1:24 am

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.
An external file that holds a picture, illustration, etc. Object name is gr1.jpg
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.

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Cancer and ATP: The Photon Energy Pathway (DCA as anti-tumor) - Page 3 Empty Re: Cancer and ATP: The Photon Energy Pathway (DCA as anti-tumor)

Post by Cr6 on Mon Jan 14, 2019 1:34 am

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*
Author information Article notes Copyright and License information Disclaimer
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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.
An external file that holds a picture, illustration, etc. Object name is OXIMED2012-740849.001.jpg
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).
An external file that holds a picture, illustration, etc. Object name is OXIMED2012-740849.002.jpg
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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

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Cancer and ATP: The Photon Energy Pathway (DCA as anti-tumor) - Page 3 Empty Re: Cancer and ATP: The Photon Energy Pathway (DCA as anti-tumor)

Post by Cr6 on Mon Jan 14, 2019 1:46 am

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, Cool. 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 (Cool, 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

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Post by Cr6 on Mon Jan 14, 2019 2:04 am

Flavins and cross linked proteins with Genipin:
------------
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.Cool 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/

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Post by Cr6 on Sat Sep 14, 2019 11:24 pm

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.  cheers

-------------------------

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).
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https://www.nature.com/articles/d41586-019-02638-w

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