Véronique Lenoir

Increased Mitochondrial Fatty Acid Oxidation Is Sufficient to Protect Skeletal Muscle Cells from Palmitate-induced Apoptosis

The mechanisms underlying the protective effect of monounsaturated fatty acids (e.g. oleate) against the lipotoxic action of saturated fatty acids (e.g. palmitate) in skeletal muscle cells remain poorly understood. This study aimed to examine the role of mitochondrial long-chain fatty acid (LCFA) oxidation in mediating oleates protective effect against palmitate-induced lipotoxicity. CPT1 (carnitine palmitoyltransferase 1), which is the key regulatory enzyme of mitochondrial LCFA oxidation, is inhibited by malonyl-CoA, an intermediate of lipogenesis. We showed that expression of a mutant form of CPT1 (CPT1mt), which is active but insensitive to malonyl-CoA inhibition, in C2C12 myotubes led to increased LCFA oxidation flux even in the presence of high concentrations of glucose and insulin. Furthermore, similar to preincubation with oleate, CPT1mt expression protected muscle cells from palmitate-induced apoptosis and insulin resistance by decreasing the content of deleterious palmitate derivates (i.e. diacylglycerols and ceramides). Oleate preincubation exerted its protective effect by two mechanisms: (i) in contrast to CPT1mt expression, oleate preincubation increased the channeling of palmitate toward triglycerides, as a result of enhanced diacylglycerol acyltransferase 2 expression, and (ii) oleate preincubation promoted palmitate oxidation through increasing CPT1 expression and modulating the activities of acetyl-CoA carboxylase and AMP-activated protein kinase. In conclusion, we demonstrated that targeting mitochondrial LCFA oxidation via CPT1mt expression leads to the same protective effect as oleate preincubation, providing strong evidence that redirecting palmitate metabolism toward oxidation is sufficient to protect against palmitate-induced lipotoxicity.

Enhancing liver mitochondrial fatty acid oxidation capacity in obese mice improves insulin sensitivity independently of hepatic steatosis.

BACKGROUND & AIMS Despite major public health concern, therapy for non-alcoholic fatty liver, the liver manifestation of the metabolic syndrome often associated with insulin resistance (IR), remains elusive. Strategies aiming to decrease liver lipogenesis effectively corrected hepatic steatosis and IR in obese animals. However, they also indirectly increased mitochondrial long-chain fatty acid oxidation (mFAO) by decreasing malonyl-CoA, a lipogenic intermediate, which is the allosteric inhibitor of carnitine palmitoyltransferase 1 (CPT1A), the key enzyme of mFAO. We thus addressed whether enhancing hepatic mFAO capacity, through a direct modulation of liver CPT1A/malonyl-CoA partnership, can reverse an already established hepatic steatosis and IR in obese mice. METHODS Adenovirus-mediated liver expression of a malonyl-CoA-insensitive CPT1A (CPT1mt) in high-fat/high-sucrose (HF/HS) diet-induced or genetically (ob/ob) obese mice was followed by metabolic and physiological investigations. RESULTS In association with increased hepatic mFAO capacity, liver CPT1mt expression improved glucose tolerance and insulin response to a glucose load in HF/HS and ob/ob mice, showing increased insulin sensitivity, and corrected IR in ob/ob mice. Surprisingly, hepatic steatosis was not affected in CPT1mt-expressing obese mice, indicating a clear dissociation between hepatic steatosis and IR. Moreover, liver CPT1mt expression rescued HF/HS-induced impaired hepatic insulin signaling at the level of IRS-1, IRS-2, Akt, and GSK-3β, most likely through the observed decrease in the HF/HS-induced accumulation of lipotoxic lipids, oxidative stress, and JNK activation. CONCLUSIONS Enhancing hepatic mFAO capacity is sufficient to reverse a state of IR and glucose intolerance in obese mice independently of hepatic steatosis.

Modulation of the hepatic malonyl-CoA-carnitine palmitoyltransferase 1A partnership creates a metabolic switch allowing oxidation of de novo fatty acids.

Liver mitochondrial beta-oxidation of LCFAs (long-chain fatty acids) is tightly regulated through inhibition of CPT1A (carnitine palmitoyltransferase 1A) by malonyl-CoA, an intermediate of lipogenesis stimulated by glucose and insulin. Moreover, CPT1A sensitivity to malonyl-CoA inhibition varies markedly depending on the physiopathological state of the animal. In the present study, we asked whether an increase in CPT1A activity solely or in association with a decreased malonyl-CoA sensitivity could, even in the presence of high glucose and insulin concentrations, maintain a sustained LCFA beta-oxidation and/or protect from triacylglycerol (triglyceride) accumulation in hepatocytes. We have shown that adenovirus-mediated expression of rat CPT1wt (wild-type CPT1A) and malonyl-CoA-insensitive CPT1mt (CPT1AM593S mutant) in cultured fed rat hepatocytes counteracted the inhibition of oleate beta-oxidation induced by 20 mM glucose/10 nM insulin. Interestingly, the glucose/insulin-induced cellular triacylglycerol accumulation was prevented, both in the presence and absence of exogenous oleate. This resulted from the generation of a metabolic switch allowing beta-oxidation of de novo synthesized LCFAs, which occurred without alteration in glucose oxidation and glycogen synthesis. Moreover, CPT1mt expression was more effective than CPT1wt overexpression to counteract glucose/insulin effects, demonstrating that control of CPT1A activity by malonyl-CoA is an essential driving force for hepatic LCFA metabolic fate. In conclusion, the present study highlights that CPT1A is a prime target to increase hepatic LCFA beta-oxidation and that acting directly on the degree of its malonyl-CoA sensitivity may be a relevant strategy to prevent and/or correct hepatic steatosis.

Mitochondrial retrograde signaling mediated by UCP2 inhibits cancer cell proliferation and tumorigenesis

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

Oxidation of hydrogen sulfide by human liver mitochondria.

Hydrogen sulfide (H2S) is the third gasotransmitter discovered. Sulfide shares with the two others (NO and CO) the same inhibiting properties towards mitochondrial respiration. However, in contrast with NO or CO, sulfide at concentrations lower than the toxic (μM) level is an hydrogen donor and a substrate for mitochondrial respiration. This is due to the activity of a sulfide quinone reductase found in a large majority of mitochondria. An ongoing study of the metabolic state of liver in obese patients allowed us to evaluate the sulfide oxidation capacity with twelve preparations of human liver mitochondria. The results indicate relatively high rates of sulfide oxidation with a large variability between individuals. These observations made with isolated mitochondria appear in agreement with the main characteristics of sulfide oxidation as established before with the help of cellular models.

Increasing mitochondrial muscle fatty acid oxidation induces skeletal muscle remodeling toward an oxidative phenotype

Adult skeletal muscle is a dynamic, remarkably plastic tissue, which allows myofibers to switch from fast/glycolytic to slow/oxidative types and to increase mitochondrial fatty acid oxidation (mFAO) capacity and vascularization in response to exercise training. mFAO is the main muscle energy source during endurance exercise, with carnitine palmitoyltransferase 1 (CPT1) being the key regulatory enzyme. Whether increasing muscle mFAO affects skeletal muscle physiology in adulthood actually remains unknown. To investigate this, we used in vivo electrotransfer technology to express in mouse tibialis anterior (TA), a fast/glycolytic muscle, a mutated CPT1 form (CPT1mt) that is active but insensitive to malonyl‐CoA, its physiologic inhibitor. In young (2‐mo‐old) adult mice, muscle CPT1mt expression enhanced mFAO (+40%), but also increased the percentage of oxidative fibers (+28%), glycogen content, and capillary‐to‐fiber density (+45%). This CPT1mt‐induced muscle remodeling, which mimicked exercise‐induced oxidative phenotype, led to a greater resistance to muscle fatigue. In the context of aging, characterized by sarcopenia and reduced oxidative capacity, CPT1mt expression in TAs from aged (20‐mo‐old) mice partially reversed aging‐associated sarcopenia and fiber‐type transition, and increased muscle capillarity. These findings provide evidence that mFAO regulates muscle phenotype and may be a potential target to combat age‐related decline in muscle function.—Hénique, C., Mansouri, A., Vavrova, E., Lenoir, V., Ferry, A., Esnous, C., Ramond, E., Girard, J., Bouillaud, F., Prip‐Buus, C., Cohen, I. Increasing mitochondrial muscle fatty acid oxidation induces skeletal muscle remodeling toward an oxidative phenotype. FASEB J. 29, 2473‐2483 (2015). www.fasebj.org

Muscle expression of a malonyl-CoA-insensitive carnitine palmitoyltransferase-1 protects mice against high-fat/high-sucrose diet-induced insulin resistance

Impaired skeletal muscle mitochondrial fatty acid oxidation (mFAO) has been implicated in the etiology of insulin resistance. Carnitine palmitoyltransferase-1 (CPT1) is a key regulatory enzyme of mFAO whose activity is inhibited by malonyl-CoA, a lipogenic intermediate. Whereas increasing CPT1 activity in vitro has been shown to exert a protective effect against lipid-induced insulin resistance in skeletal muscle cells, only a few studies have addressed this issue in vivo. We thus examined whether a direct modulation of muscle CPT1/malonyl-CoA partnership is detrimental or beneficial for insulin sensitivity in the context of diet-induced obesity. By using a Cre-LoxP recombination approach, we generated mice with skeletal muscle-specific and inducible expression of a mutated CPT1 form (CPT1mt) that is active but insensitive to malonyl-CoA inhibition. When fed control chow, homozygous CPT1mt transgenic (dbTg) mice exhibited decreased CPT1 sensitivity to malonyl-CoA inhibition in isolated muscle mitochondria, which was sufficient to substantially increase ex vivo muscle mFAO capacity and whole body fatty acid utilization in vivo. Moreover, dbTg mice were less prone to high-fat/high-sucrose (HFHS) diet-induced insulin resistance and muscle lipotoxicity despite similar body weight gain, adiposity, and muscle malonyl-CoA content. Interestingly, these CPT1mt-protective effects in dbTg-HFHS mice were associated with preserved muscle insulin signaling, increased muscle glycogen content, and upregulation of key genes involved in muscle glucose metabolism. These beneficial effects of muscle CPT1mt expression suggest that a direct modulation of the malonyl-CoA/CPT1 partnership in skeletal muscle could represent a potential strategy to prevent obesity-induced insulin resistance.

Protective role of the ELOVL2/docosahexaenoic acid axis in glucolipotoxicity-induced apoptosis in rodent beta cells and human islets

Aims/hypothesisDietary n-3 polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), are known to influence glucose homeostasis. We recently showed that Elovl2 expression in beta cells, which regulates synthesis of endogenous DHA, was associated with glucose tolerance and played a key role in insulin secretion. The present study aimed to examine the role of the very long chain fatty acid elongase 2 (ELOVL2)/DHA axis on the adverse effects of palmitate with high glucose, a condition defined as glucolipotoxicity, on beta cells.MethodsWe detected ELOVL2 in INS-1 beta cells and mouse and human islets using quantitative PCR and western blotting. Downregulation and adenoviral overexpression of Elovl2 was carried out in beta cells. Ceramide and diacylglycerol levels were determined by radio-enzymatic assay and lipidomics. Apoptosis was quantified using caspase-3 assays and poly (ADP-ribose) polymerase cleavage. Palmitate oxidation and esterification were determined by [U-14C]palmitate labelling.ResultsWe found that glucolipotoxicity decreased ELOVL2 content in rodent and human beta cells. Downregulation of ELOVL2 drastically potentiated beta cell apoptosis induced by glucolipotoxicity, whereas adenoviral Elovl2 overexpression and supplementation with DHA partially inhibited glucolipotoxicity-induced cell death in rodent and human beta cells. Inhibition of beta cell apoptosis by the ELOVL2/DHA axis was associated with a decrease in ceramide accumulation. However, the ELOVL2/DHA axis was unable to directly alter ceramide synthesis or metabolism. By contrast, DHA increased palmitate oxidation but did not affect its esterification. Pharmacological inhibition of AMP-activated protein kinase and etomoxir, an inhibitor of carnitine palmitoyltransferase 1 (CPT1), the rate-limiting enzyme in fatty acid β-oxidation, attenuated the protective effect of the ELOVL2/DHA axis during glucolipotoxicity. Downregulation of CPT1 also counteracted the anti-apoptotic action of the ELOVL2/DHA axis. By contrast, a mutated active form of Cpt1 inhibited glucolipotoxicity-induced beta cell apoptosis when ELOVL2 was downregulated.Conclusions/interpretationOur results identify ELOVL2 as a critical pro-survival enzyme for preventing beta cell death and dysfunction induced by glucolipotoxicity, notably by favouring palmitate oxidation in mitochondria through a CPT1-dependent mechanism.

O61 La carnitine palmitoyltransférase 1 hépatique : une cible pour contrecarrer la stéatose hépatique et l’insulinorésistance ?

Introduction L’accumulation ectopique des lipides contribue a l’instauration d’une intolerance au glucose et d’une insulinoresistance. Le transport des acides gras a chaine longue (AGCL), catalyse par la carnitine palmitoyltransferase 1 hepatique (CPT1A), represente l’etape majeur du controle de l’oxydation mitochondriale des AGCL suite a l’inhibition de la CPT1A par le malonyl-CoA, un intermediaire de la lipogenese. Le but de l’etude est de determiner si une augmentation de l’oxydation hepatique des AGCL peut contrecarrer la steatose hepatique et l’insulinoresistance. Materiels et methodes Nous avons exprime par approche adenovirale une CPT1 constitutivement active (CPT1mt), i.e. insensible au malonyl-CoA, dans le foie de souris genetiquement obeses (ob/ob) ou rendues obeses via un regime riche en lipides et en saccharose (HFD). Apres 16 jours, les consequences physiologiques et metaboliques ont ete etudiees. Resultats Chez les souris mises sur regime standard (ST), l’expression hepatique de la CPT1mt entraine une augmentation respective de 1,4 et 3 du flux d’oxydation hepatique des AGCL et de la production hepatique de s-hydroxybutyrate. Une augmentation similaire est observee chez les souris HFD-CPT1mt. L’expression de la CPT1mt n’affecte pas la steatose hepatique des souris HFD et ob/ob mais diminue leur intolerance au glucose et leur insulino-resistance. Pour comprendre ces effets, nous avons etudie l’expression genique de plusieurs acteurs impliques dans le metabolisme glucido-lipidique du foie. Nos resultats indiquent que l’expression de la CPT1mt induit des enzymes cles impliquees dans la glycolyse, la lipogenese, l’esterification et l’export des lipoproteines a tres faible densite chez les souris ST et HFD. D’autres etudes sont en cours afin de determiner l’impact de l’expression hepatique de la CPT1mt sur la sensibilite du foie a l’insuline. Conclusion Nos resultats suggerent : 1/l’existence d’une dissociation entre steatose hepatique et insulinoresistance, et 2/que la CPT1A representerait une cible pour ameliorer la tolerance au glucose et la sensibilite a l’insuline chez des souris obeses.

P240 Caractérisation phénotypique d’un nouveau modèle de souris transgéniques exprimant dans le foie une carnitine palmitoyltransférase 1 constitutivement active

Introduction La carnitine palmitoyltransferase 1 (CPT1) represente le site majeur de controle de l’oxydation des acides gras a chaine longue (AGCL) du fait de sa capacite a etre inhibee par le malonyl-CoA, premier intermediaire de la lipogenese. Dans le foie d’animaux obeses ou diabetiques, l’existence d’une lipogenese accrue conduit a une augmentation du malonyl-CoA qui entraine une diminution de l’oxydation mitochondriale des AGCL. Materiels et methodes Nous avons developpe un nouveau modele de souris transgeniques (Tg-CPT1mt) exprimant specifiquement dans le foie (promoteur de la transthyretine) une CPT1 insensible au malonyl-CoA (CPT1mt). Resultats L’expression homozygote de la CPT1mt entraine une letalite embryonnaire. Chez les souris heterozygotes a l’etat nourri, l’expression de la CPT1mt entraine une diminution de la sensibilite de la CPT1 hepatique vis-a-vis du malonyl-CoA similaire a celle observee apres un jeune chez des souris temoins (WT). A l’âge de 3 mois, les souris Tg-CPT1mt presentent a l’etat nourri des concentrations circulantes de triglycerides et d’acides gras libres significativement plus faibles que chez les souris WT. Chez les souris Tg-CPT1mt âgees de 10 mois, seule la triglyceridemie est diminuee et ceci s’accompagne d’une diminution du poids corporel. Quel que soit leur âge, les souris Tg-CPT1mt ont a l’etat nourri une diminution du poids des tissus adipeux (epididymaire, visceral et sous cutane) et du contenu hepatique en glycogene. De plus, la glycemie des souris Tg-CPT1mt est plus faible a l’etat nourri comme a l’etat a jeun. Seules les souris Tg-CPT1mt âgees de 10 mois ont une tolerance au glucose et une sensibilite a l’insuline ameliorees. Chez ces souris, des experiences de clamp euglycemique-hyperinsulinemique suggerent que la diminution de la glycemie observee ne resulte pas d’une diminution de la production hepatique de glucose, mais d’une amelioration de l’utilisation basale de glucose. Conclusion Ces resultats suggerent que les souris Tg-CPT1mt presentent un phenotype evolutif et constituent un modele pertinent pour l’etude des consequences d’une augmentation de l’oxydation des AGCL dans le foie sur l’obesite, l’insulinoresistance et la steatose hepatique.