Academic literature on the topic 'Pyruvate/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.

To gain insight into the regulation of pancreatic β-cell mitochondrial metabolism, the direct effects on respiration of different mitochondrial substrates, variations in the ATP/ADP ratio and free Ca2+ were examined using isolated mitochondria and permeabilized clonal pancreatic β-cells (HIT). Respiration from pyruvate was high and not influenced by Ca2+ in State 3 or under various redox states and fixed values of the ATP/ADP ratio; nevertheless, high Ca2+ elevated pyridine nucleotide fluorescence, indicating activation of pyruvate dehydrogenase by Ca2+. Furthermore, in the presence of pyruvate, elevated Ca2+ stimulated CO2 production from pyruvate, increased citrate production and efflux from the mitochondria and inhibited CO2 production from palmitate. The latter observation suggests that β-cell fatty acid oxidation is not regulated exclusively by malonyl-CoA but also by the mitochondrial redox state. α-Glycerophosphate (α-GP) oxidation was Ca2+-dependent with a half-maximal rate observed at around 300 nM Ca2+. We have recently demonstrated that increases in respiration precede increases in Ca2+ in glucose-stimulated clonal pancreatic β-cells (HIT), indicating that Ca2+ is not responsible for the initial stimulation of respiration [Civelek, Deeney, Kubik, Schultz, Tornheim and Corkey (1996) Biochem. J. 315, 1015–1019]. It is suggested that respiration is stimulated by increased substrate (α-GP and pyruvate) supply together with oscillatory increases in ADP [Nilsson, Schultz, Berggren, Corkey and Tornheim (1996) Biochem. J. 314, 91–94]. The rise in Ca2+, which in itself may not significantly increase net respiration, could have the important functions of (1) activating the α-GP shuttle, to maintain an oxidized cytosol and high glycolytic flux; (2) activating pyruvate dehydrogenase, and indirectly pyruvate carboxylase, to sustain production of citrate and hence the putative signal coupling factors, malonyl-CoA and acyl-CoA; and (3) increasing mitochondrial redox state to implement the switch from fatty acid to pyruvate oxidation.

Substrate preferences of isolated mitochondria and maximal enzyme activities were used to assess the oxidative capacities of red muscle (RM) and white muscle (WM) of carp (Cyprinus carpio). A 14-fold higher activity of citrate synthase (CS) in RM reflects the higher mitochondrial density in this tissue. RM mitochondria oxidize pyruvate and fatty acyl carnitines (8:O, 12:O, 16:O) at similarly high rates. WM mitochondria oxidize these fatty acyl carnitines at 35–70% the rate of pyruvate, depending on chain length. WM has only half the carnitine palmitoyl transferase/CS ratio of RM, but similar ratios of beta-hydroxyacyl CoA dehydrogenase/CS. Ketone bodies are poor substrates for mitochondria from both tissues. In both tissues mitochondrial alpha-glycerophosphate oxidation was minimal, and alpha-glycerophosphate dehydrogenase was present at low activities, suggesting the alpha-glycerophosphate shuttle is of minor significance in maintaining cytosolic redox balance in either tissue. The mitochondrial oxidation rates of other substrates relative to pyruvate are as follows: alpha-ketoglutarate 90% (RM and WM); glutamate 45% (WM) and 70% (RM); proline 20% (WM) and 45% (RM). Oxidation of neutral amino acids (serine, glycine, alanine, beta-alanine) was not consistently detectable. These data suggest that RM and WM differ in mitochondrial properties as well as mitochondrial abundance. Whereas RM mitochondria appear to be able to utilize a wide range of metabolic fuels (fatty acids, pyruvate, amino acids but not ketone bodies), WM mitochondria appear to be specialized to use pyruvate.

Ethanol oxidation causes redox effects. The coupling of this oxidation via NADH to intermediary metabolism was studied in order to reveal the underlying mechanisms. Isolated rat hepatocytes were incubated with [1,1-2H2]-, (1R)-[1-2H]- and (1S)-[1-2H]-ethanol and the 2H incorporation was measured in lactate, beta-hydroxybutyrate, fumarate, malate, succinate, alpha-oxoglutarate and citrate. The results differed in the following ways from results obtained in intact rats. Lactate became labelled to an increasing extent, and in more than one position, indicating labelling of pyruvate. A small and constant fraction of malate and fumarate was formed without access to [2H]coenzyme. Addition of aspartate increased this fraction, which was concluded to be formed in the mitochondria. Citrate was essentially unlabelled. The 2H from (1R)-[1-2H]ethanol contributed to malate to a larger extent and to beta-hydroxybutyrate to a smaller extent, and 2H from (1S)-[1-2H]ethanol contributed to lactate to a smaller extent. These results indicate that the exchange via shuttle system was less efficient in isolated hepatocytes than in intact rats. The 2H incorporation was independent of concentration of [1,1-2H2]ethanol when this was above 5mM. Additions known to increase ethanol elimination, and cyanamide, which decreases it, had no marked effect on the 2H incorporation. This indicates equilibration of the NADH bound to alcohol dehydrogenase with free NADH. Disulfiram and cyanamide caused a decrease in the relative incorporation from (1S)-[1-2H]ethanol into malate in liver of intact rats. Addition of cyanamide to incubations with hepatocytes resulted in a decrease of the contribution of 2H from (1S)-[1-2H]ethanol in lactate, beta-hydroxybutyrate and malate. This indicates that acetaldehyde was only oxidized in the mitochondrial compartment.

Hepatocellular carcinoma (HCC) is a common malignancy. Despite progress in treatment, HCC is still one of the most lethal cancers. Therefore, deepening molecular mechanisms underlying HCC pathogenesis and development is required to uncover new therapeutic strategies. Metabolic reprogramming is emerging as a critical player in promoting tumor survival and proliferation to sustain increased metabolic needs of cancer cells. Among the metabolic pathways, the tricarboxylic acid (TCA) cycle is a primary route for bioenergetic, biosynthetic, and redox balance requirements of cells. In recent years, a large amount of evidence has highlighted the relevance of the TCA cycle rewiring in a variety of cancers. Indeed, aberrant gene expression of several key enzymes and changes in levels of critical metabolites have been observed in many solid human tumors. In this review, we summarize the role of the TCA cycle rewiring in HCC by reporting gene expression and activity dysregulation of enzymes relating not only to the TCA cycle but also to glutamine metabolism, malate/aspartate, and citrate/pyruvate shuttles. Regarding the transcriptional regulation, we focus on the link between NF-κB-HIF1 transcriptional factors and TCA cycle reprogramming. Finally, the potential of metabolic targets for new HCC treatments has been explored.

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.