Academic literature on the topic 'Citrate shuttle'

The activation of the Ras signaling pathway is a crucial process in hepatocarcinogenesis. Till now, no reports have scrutinized the role of dynamic metabolic changes in Ras oncogene-induced transition of the normal and precancerous liver cells to hepatocellular carcinoma in vivo. In the current study, we attempted a comprehensive investigation of Hras12V transgenic mice (Ras-Tg) by concatenating nontargeted metabolomics, transcriptomics analysis, and targeted-metabolomics incorporating [U-13C] glucose. A total of 631 peaks were detected, out of which 555 metabolites were screened. Besides, a total of 122 differently expressed metabolites (DEMs) were identified, and they were categorized and subtyped with the help of variation tendency analysis of the normal (W), precancerous (P), and hepatocellular carcinoma (T) liver tissues. Thus, the positive or negative association between metabolites and the hepatocellular carcinoma and Ras oncogene were identified. The bioinformatics analysis elucidated the hepatocarcinogenesis-associated significant metabolic pathways: glycolysis, mitochondrial citrate-malate shuttle, lipid biosynthesis, pentose phosphate pathway (PPP), cholesterol and bile acid biosynthesis, and glutathione metabolism. The key metabolites and enzymes identified in this analysis were further validated. Moreover, we confirmed the PPP, glycolysis, and conversion of pyruvate to cytosol acetyl-CoA by mitochondrial citrate-malate shuttle, in vivo, by incorporating [U-13C] glucose. In summary, the current study presented the comprehensive bioinformatics analysis, depicting the Ras oncogene-induced dynamic metabolite variations in hepatocarcinogenesis. A significant finding of our study was that the mitochondrial citrate-malate shuttle plays a crucial role in detoxification of lactic acid, maintenance of mitochondrial integrity, and enhancement of lipid biosynthesis, which, in turn, promotes hepatocarcinogenesis.

The main aim of this study was to investigate whether enzyme levels of the malate-aspartate and alpha-glycerophosphate shuttles and of cytochrome b5 reductase in human skeletal muscle are affected by strength training. Muscle biopsy samples from the deltoid muscle of the nondominant arm in untrained (n = 12) and strength-trained (n = 12) subjects were compared. The strength-trained muscles were characterized by a tendency to a higher percentage of type I fibers (67 vs. 59%), a lower percentage of type IIb fibers (12 vs. 18%), 34% larger mean fiber areas, and 19% more capillaries per fiber (P less than 0.1). No difference was noted in levels of enzymes representing the citric acid cycle, fatty acid oxidation, and glycolysis, nor in the number of capillaries per square millimeter. Neither did the levels of malate-aspartate and alpha-glycerophosphate shuttle enzymes nor cytochrome b5 reductase differ. Levels of cytochrome b5 reductase correlated (r = 0.59, P less than 0.01) with levels of the mitochondrial marker enzyme citrate synthase. It is concluded that strength training does not appear to result in increased levels of NADH shuttle enzymes and cytochrome b5 reductase.

ABSTRACT Acetyl coenzyme A (acetyl-CoA) is a central metabolite in carbon and energy metabolism. Because of its amphiphilic nature and bulkiness, acetyl-CoA cannot readily traverse biological membranes. In fungi, two systems for acetyl unit transport have been identified: a shuttle dependent on the carrier carnitine and a (peroxisomal) citrate synthase-dependent pathway. In the carnitine-dependent pathway, carnitine acetyltransferases exchange the CoA group of acetyl-CoA for carnitine, thereby forming acetyl-carnitine, which can be transported between subcellular compartments. Citrate synthase catalyzes the condensation of oxaloacetate and acetyl-CoA to form citrate that can be transported over the membrane. Since essential metabolic pathways such as fatty acid β-oxidation, the tricarboxylic acid (TCA) cycle, and the glyoxylate cycle are physically separated into different organelles, shuttling of acetyl units is essential for growth of fungal species on various carbon sources such as fatty acids, ethanol, acetate, or citrate. In this review we summarize the current knowledge on the different systems of acetyl transport that are operational during alternative carbon metabolism, with special focus on two fungal species: Saccharomyces cerevisiae and Candida albicans.

ABSTRACT The potential role of outer membrane proteins in electron transfer to insoluble Fe(III) oxides by Geobacter sulfurreducens was investigated because this organism is closely related to the Fe(III) oxide-reducing organisms that are predominant in many Fe(III)-reducing environments. Two of the most abundant proteins that were easily sheared from the outer surfaces of intact cells were c-type cytochromes. One, designated OmcS, has a molecular mass of ca. 50 kDa and is predicted to be an outer membrane hexaheme c-type cytochrome. Transcripts for omcS could be detected during growth on Fe(III) oxide, but not on soluble Fe(III) citrate. The omcS mRNA consisted primarily of a monocistronic transcript, and to a lesser extent, a longer transcript that also contained the downstream gene omcT, which is predicted to encode a second hexaheme outer membrane cytochrome with 62.6% amino acid sequence identity to OmcS. The other abundant c-type cytochrome sheared from the outer surface of G. sulfurreducens, designated OmcE, has a molecular mass of ca. 30 kDa and is predicted to be an outer membrane tetraheme c-type cytochrome. When either omcS or omcE was deleted, G. sulfurreducens could no longer reduce Fe(III) oxide but could still reduce soluble electron acceptors, including Fe(III) citrate. The mutants could reduce Fe(III) in Fe(III) oxide medium only if the Fe(III) chelator, nitrilotriacetic acid, or the electron shuttle, anthraquinone 2,6-disulfonate, was added. Expressing omcS or omcE in trans restored the capacity for Fe(III) oxide reduction. OmcT was not detected among the sheared proteins, and genetic studies indicated that G. sulfurreducens could not reduce Fe(III) oxide when omcT was expressed but OmcS was absent. In contrast, Fe(III) oxide was reduced when omcS was expressed in the absence of OmcT. These results suggest that OmcS and OmcE are involved in electron transfer to Fe(III) oxides in G. sulfurreducens. They also emphasize the importance of evaluating mechanisms for Fe(III) reduction with environmentally relevant Fe(III) oxide, rather than the more commonly utilized Fe(III) citrate, because additional electron transfer components are required for Fe(III) oxide reduction that are not required for Fe(III) citrate reduction.

To investigate the effects of microgravity on murine skeletal muscle fiber size, muscle contractile protein, and enzymatic activity, female C57BL/6J mice, aged 64 days, were divided into animal enclosure module (AEM) ground control and spaceflight (SF) treatment groups. SF animals were flown on the space shuttle Endeavour (STS-108/UF-1) and subjected to ∼11 days and 19 h of microgravity. Immunohistochemical analysis of muscle fiber cross-sectional area revealed that, in each of the muscles analyzed, mean muscle fiber cross-sectional area was significantly reduced ( P < 0.0001) for all fiber types for SF vs. AEM control. In the soleus, immunohistochemical analysis of myosin heavy chain (MHC) isoform expression revealed a significant increase in the percentage of muscle fibers expressing MHC IIx and MHC IIb ( P < 0.05). For the gastrocnemius and plantaris, no significant changes in MHC isoform expression were observed. For the muscles analyzed, no alterations in MHC I or MHC IIa protein expression were observed. Enzymatic analysis of the gastrocnemius revealed a significant decrease in citrate synthase activity in SF vs. AEM control.

ABSTRACT The hyperthermophilic archaeon Pyrobaculum aerophilum used 20 mM Fe(III) citrate, 100 mM poorly crystalline Fe(III) oxide, and 10 mM KNO3 as terminal electron acceptors. The two forms of iron were reduced at different rates but with equal growth yields. The insoluble iron was reduced when segregated spatially by dialysis tubing, indicating that direct contact with the iron was not necessary for growth. When partitioned, there was no detectable Fe(III) or Fe(II) outside of the tubing after growth, suggesting that an electron shuttle, not a chelator, may be used as an extracellular mediator of iron reduction. The addition of 25 and 50% (vol vol−1) cell-free spent insoluble iron media to fresh media led to growth without a lag phase. Liquid chromatography analysis of spent media showed that cultures grown in iron, especially insoluble iron, produced soluble extracellular compounds that were absent or less abundant in spent nitrate medium. NADH-dependent ferric reductase activity increased approximately 100-fold, while nitrate reductase activity decreased 10-fold in whole-cell extracts from iron-grown cells relative to those from nitrate-grown cells, suggesting that dissimilatory iron reduction was regulated. A novel 2,6-anthrahydroquinone disulfonate oxidase activity was more than 580-fold higher in iron-grown cells than in nitrate-grown cells. The activity was primarily (>95%) associated with the membrane cellular fraction, but its physiological function is unknown. Nitrate-grown cultures produced two membrane-bound, c-type cytochromes that are predicted to be monoheme and part of nitrite reductase and a bc 1 complex using genome analyses. Only one cytochrome was present in cells grown on Fe(III) citrate whose relative abundance was unchanged.

To evaluate the effects of endurance training on the expression of monocarboxylate transporters (MCT) in human vastus lateralis muscle, we compared the amounts of MCT1 and MCT4 in total muscle preparations (MU) and sarcolemma-enriched (SL) and mitochondria-enriched (MI) fractions before and after training. To determine if changes in muscle lactate release and oxidation were associated with training-induced changes in MCT expression, we correlated band densities in Western blots to lactate kinetics determined in vivo. Nine weeks of leg cycle endurance training [75% peak oxygen consumption (V˙o 2 peak)] increased muscle citrate synthase activity (+75%, P < 0.05) and percentage of type I myosin heavy chain (+50%, P < 0.05); percentage of MU lactate dehydrogenase-5 (M4) isozyme decreased (−12%, P < 0.05). MCT1 was detected in SL and MI fractions, and MCT4 was localized to the SL. Muscle MCT1 contents were consistent among subjects both before and after training; in contrast, MCT4 contents showed large interindividual variations. MCT1 amounts significantly increased in MU, SL, and MI after training (+90%, +60%, and +78%, respectively), whereas SL but not MU MCT4 content increased after training (+47%, P < 0.05). Mitochondrial MCT1 content was negatively correlated to net leg lactate release at rest ( r = −0.85, P < 0.02). Sarcolemmal MCT1 and MCT4 contents correlated positively to net leg lactate release at 5 min of exercise at 65%V˙o 2 peak ( r = 0.76, P < 0.03 and r = 0.86, P < 0.01, respectively). Results support the conclusions that 1) endurance training increases expression of MCT1 in muscle because of insertion of MCT1 into both sarcolemmal and mitochondrial membranes, 2) training has variable effects on sarcolemmal MCT4, and 3) both MCT1 and MCT4 participate in the cell-cell lactate shuttle, whereas MCT1 facilitates operation of the intracellular lactate shuttle.

Le diabète est une maladie métabolique qui se caractérise par une résistance à l’insuline des tissus périphériques et par une incapacité des cellules β pancréatiques à sécréter les niveaux d’insuline appropriés afin de compenser pour cette résistance. Pour mieux comprendre les mécanismes déficients dans les cellules β des patients diabétiques, il est nécessaire de comprendre et de définir les mécanismes impliqués dans le contrôle de la sécrétion d’insuline en réponse au glucose. Dans les cellules β pancréatiques, le métabolisme du glucose conduit à la production de facteurs de couplage métabolique, comme l’ATP, nécessaires à la régulation de l’exocytose des vésicules d’insuline. Le mécanisme par lequel la production de l’ATP par le métabolisme oxydatif du glucose déclenche l’exocytose des vésicules d’insuline est bien décrit dans la littérature. Cependant, il ne peut à lui seul réguler adéquatement la sécrétion d’insuline. Le malonyl-CoA et le NADPH sont deux autres facteurs de couplage métaboliques qui ont été suggérés afin de relier le métabolisme du glucose à la régulation de la sécrétion d’insuline. Les mécanismes impliqués demeurent cependant à être caractérisés. Le but de la présente thèse était de déterminer l’implication des navettes du pyruvate, découlant du métabolisme mitochondrial du glucose, dans la régulation de la sécrétion d’insuline. Dans les cellules β, les navettes du pyruvate découlent de la combinaison des processus d’anaplérose et de cataplérose et permettent la transduction des signaux métaboliques provenant du métabolisme du glucose. Dans une première étude, nous nous sommes intéressés au rôle de la navette pyruvate/citrate dans la régulation de la sécrétion d’insuline en réponse au glucose, puisque cette navette conduit à la production dans le cytoplasme de deux facteurs de couplage métabolique, soit le malonyl-CoA et le NADPH. De plus, la navette pyruvate/citrate favorise le flux métabolique à travers la glycolyse en réoxydation le NADH. Une étude effectuée précédemment dans notre laboratoire avait suggéré la présence de cette navette dans les cellules β pancréatique. Afin de tester notre hypothèse, nous avons ciblé trois étapes de cette navette dans la lignée cellulaire β pancréatique INS 832/13, soit la sortie du citrate de la mitochondrie et l’activité de l’ATP-citrate lyase (ACL) et l’enzyme malique (MEc), deux enzymes clés de la navette pyruvate/citrate. L’inhibition de chacune de ces étapes par l’utilisation d’un inhibiteur pharmacologique ou de la technologie des ARN interférant a corrélé avec une réduction significative de la sécrétion d’insuline en réponse au glucose. Les résultats obtenus suggèrent que la navette pyruvate/citrate joue un rôle critique dans la régulation de la sécrétion d’insuline en réponse au glucose. Parallèlement à notre étude, deux autres groupes de recherche ont suggéré que les navettes pyruvate/malate et pyruvate/isocitrate/α-cétoglutarate étaient aussi importantes pour la sécrétion d’insuline en réponse au glucose. Ainsi, trois navettes découlant du métabolisme mitochondrial du glucose pourraient être impliquées dans le contrôle de la sécrétion d’insuline. Le point commun de ces trois navettes est la production dans le cytoplasme du NADPH, un facteur de couplage métabolique possiblement très important pour la sécrétion d’insuline. Dans les navettes pyruvate/malate et pyruvate/citrate, le NADPH est formé par MEc, alors que l’isocitrate déshydrogénase (IDHc) est responsable de la production du NADPH dans la navette pyruvate/isocitrate/α-cétoglutarate. Dans notre première étude, nous avions démontré l’importance de l’expression de ME pour la sécrétion adéquate d’insuline en réponse au glucose. Dans notre deuxième étude, nous avons testé l’implication de IDHc dans les mécanismes de régulation de la sécrétion d’insuline en réponse au glucose. La diminution de l’expression de IDHc dans les INS 832/13 a stimulé la sécrétion d’insuline en réponse au glucose par un mécanisme indépendant de la production de l’ATP par le métabolisme oxydatif du glucose. Ce résultat a ensuite été confirmé dans les cellules dispersées des îlots pancréatiques de rat. Nous avons aussi observé dans notre modèle que l’incorporation du glucose en acides gras était augmentée, suggérant que la diminution de l’activité de IDHc favorise la redirection du métabolisme de l’isocitrate à travers la navette pyruvate/citrate. Un mécanisme de compensation à travers la navette pyruvate/citrate pourrait ainsi expliquer la stimulation de la sécrétion d’insuline observée en réponse à la diminution de l’expression de IDHc. Les travaux effectués dans cette deuxième étude remettent en question l’implication de l’activité de IDHc, et de la navette pyruvate/isocitrate/α-cétoglutarate, dans la transduction des signaux métaboliques reliant le métabolisme du glucose à la sécrétion d’insuline. La navette pyruvate/citrate est la seule des navettes du pyruvate à conduire à la production du malonyl-CoA dans le cytoplasme des cellules β. Le malonyl-CoA régule le métabolisme des acides gras en inhibant la carnitine palmitoyl transférase 1, l’enzyme limitante dans l’oxydation des acides gras. Ainsi, l’élévation des niveaux de malonyl-CoA en réponse au glucose entraîne une redirection du métabolisme des acides gras vers les processus d’estérification puis de lipolyse. Plus précisément, les acides gras sont métabolisés à travers le cycle des triglycérides/acides gras libres (qui combinent les voies métaboliques d’estérification et de lipolyse), afin de produire des molécules lipidiques signalétiques nécessaires à la modulation de la sécrétion d’insuline. Des études effectuées précédemment dans notre laboratoire ont démontré que l’activité lipolytique de HSL (de l’anglais hormone-sensitive lipase) était importante, mais non suffisante, pour la régulation de la sécrétion d’insuline. Dans une étude complémentaire, nous nous sommes intéressés au rôle d’une autre lipase, soit ATGL (de l’anglais adipose triglyceride lipase), dans la régulation de la sécrétion d’insuline en réponse au glucose et aux acides gras. Nous avons démontré que ATGL est exprimé dans les cellules β pancréatiques et que son activité contribue significativement à la lipolyse. Une réduction de son expression dans les cellules INS 832/13 par RNA interférant ou son absence dans les îlots pancréatiques de souris déficientes en ATGL a conduit à une réduction de la sécrétion d’insuline en réponse au glucose en présence ou en absence d’acides gras. Ces résultats appuient l’hypothèse que la lipolyse est une composante importante de la régulation de la sécrétion d’insuline dans les cellules β pancréatiques. En conclusion, les résultats obtenus dans cette thèse suggèrent que la navette pyruvate/citrate est importante pour la régulation de la sécrétion d’insuline en réponse au glucose. Ce mécanisme impliquerait la production du NADPH et du malonyl-CoA dans le cytoplasme en fonction du métabolisme du glucose. Cependant, nos travaux remettent en question l’implication de la navette pyruvate/isocitrate/α-cétoglutarate dans la régulation de la sécrétion d’insuline. Le rôle exact de IDHc dans ce processus demeure cependant à être déterminé. Finalement, nos travaux ont aussi démontré un rôle pour ATGL et la lipolyse dans les mécanismes de couplage métabolique régulant la sécrétion d’insuline.
Diabetes is a metabolic disorder characterized by a combination of insulin resistance in peripheral tissues with an inappropriate amount of insulin secreted by the pancreatic β-cells to overcome this insulin resistance. In order to help find a cure for diabetic patients, we need to elucidate the mechanisms underlying the proper control of insulin secretion in response to glucose. In pancreatic β-cells, glucose metabolism leads to the production of metabolic coupling factors, like ATP, implicated in the regulation of insulin vesicle exocytosis. The mechanism linking ATP production by the oxidative metabolism of glucose to the triggering of insulin release that involves Ca2+ and metabolically sensitive K+ channels is relatively well known. Other mechanisms are also involved in the regulation of insulin secretion in response to glucose and other nutrients, such as fatty acids and some amino acids. Malonyl-CoA and NADPH are two metabolic coupling factors that have been suggested to be implicated in the transduction of metabolic signaling coming from glucose metabolism to control the release of insulin granules. However, the mechanisms implicated remained to be defined. The goal of the present thesis was to further our understanding of the role of the pyruvate shuttles, derived from mitochondrial glucose metabolism, in the regulation of insulin secretion. In pancreatic β-cells, pyruvate shuttles are produced by the combination of anaplerosis and cataplerosis processes and are thought to link glucose metabolism to the regulation of insulin secretion by the production metabolic coupling factors. In our first study, we wished to determine the role of the pyruvate/citrate shuttle in the regulation of glucose-induced insulin secretion. The pyruvate/citrate shuttle leads to the production in the cytoplasm of both malonyl-CoA and NADPH and also stimulates the metabolic flux through the glycolysis by re-oxidating NADH. A previous study done in the group of Dr Prentki has suggested the feasibility of the pyruvate/citrate shuttle in pancreatic β-cells. To investigate our hypothesis, we inhibited three different steps of this shuttle in INS 832/13 cells, a pancreatic β-cell line. Specifically, we repressed, using pharmacological inhibitors or RNA interference technology, the mitochondrial citrate export to the cytoplasm and the expression of malic enzyme (MEc) and ATP-citrate lyase (ACL), two key enzymes implicated in the pyruvate/citrate shuttle. The inhibition of each of those steps resulted in a reduction of glucose-induced insulin secretion. Our results underscore the importance of the pyruvate/citrate shuttle in the pancreatic β-cell signaling and the regulation of insulin secretion in response to glucose. Other research groups are also interested in studying the implication of pyruvate cycling processes in the regulation of insulin exocytosis. They suggested a role for the pyruvate/malate and the pyruvate/isocitrate/α-ketoglutarate shuttles. Therefore, three different shuttles derived from the mitochondrial glucose metabolism could be implicated in the regulation of glucose-induced insulin release. All those three shuttles can produce NADPH in the cytoplasm. In the pyruvate/malate and the pyruvate/citrate shuttles, the NADPH is formed by cytosolic malic enzyme (MEc), whereas in the pyruvate/isocitrate/α-ketoglutarate, NADPH is produced by cytosolic isocitrate dehydrogenease (IDHc). In our first study, we established the importance of MEc expression in the regulation of insulin secretion. In our second study, we wanted to investigate the importance of IDHc expression in glucose-induced insulin secretion. The reduction of IDHc expression in INS 832/13 cells stimulated insulin release in response to glucose by a mechanism independent of ATP production coming from glucose oxidative metabolism. This stimulation was also observed in isolated rat pancreatic cells. IDHc knockdown cells showed elevated glucose incorporation into fatty acids, suggesting that isocitrate metabolism could be redirected into the pyruvate/citrate shuttle in these cells. Taken together, these results suggest that IDHc is not essential for glucose-induced insulin secretion and that a compensatory mechanism, probably involving the pyruvate/citrate shuttle, explains the enhanced insulin secretion in IDHc knockdown cells . The pyruvate/citrate shuttle is the only pyruvate shuttle that is linked to the production of malonyl-CoA. Malonyl-CoA is a known inhibitor of carnitine palmitoyl transferase 1, the rate-limiting step in fatty acid oxidation. Therefore, the raising level of malonyl-CoA in response to glucose redirects the metabolism of fatty acids into the triglycerides/free fatty acids cycle which combine esterification and lipolysis processes. Previous studies done in the laboratory of Dr Prentki supported the concept that lipolysis of endogenous lipid stores is an important process for the appropriate regulation of insulin secretion. A first lipase, hormone-sensitive lipase (HSL), has been identified in pancreatic β-cells. HSL expression is important, but not sufficient, for the β-cell lipolysis activity. In a complementary study, we have investigated the role of another lipase, adipose triglyceride lipase (ATGL), in the regulation of insulin secretion in response to glucose and to fatty acids. We first demonstrated the expression and the activity of ATGL in pancreatic β-cells. Reducing ATGL expression using shRNA in INS 832/13 cells caused a reduction in insulin secretion in response to glucose and to fatty acids. Pancreatic islets from ATGL null mice also showed defect in insulin release in response to glucose and to fatty acids. The results demonstrate the importance of ATGL and intracellular lipid signaling in the regulation of insulin secretion. In conclusion, the work presented in this thesis suggests a role for the pyruvate/citrate shuttle in the regulation of insulin secretion in response to glucose. This mechanism possibly implicates the production of NADPH and malonyl-CoA in the cytoplasm. The results also points to a re-evaluation of the role of IDHc in glucose-induced insulin secretion. The precise role of IDHc in pancreatic β-cells needs to be determined. Finally, the data have also documented a role of lipolysis and ATGL in the coupling mechanisms of insulin secretion in response to both fuel and non-fuel stimuli.