CerS6-dependent ceramide synthesis in hypothalamic neurons promotes ER/mitochondrial stress and impairs glucose homeostasis in obese mice

Dysregulation of hypothalamic ceramides has been associated with disrupted neuronal pathways in control of energy and glucose homeostasis. However, the specific ceramide species promoting neuronal lipotoxicity in obesity have remained obscure. Here, we find increased expression of the C$_{16:0}$ ceramide-producing ceramide synthase (CerS)6 in cultured hypothalamic neurons exposed to palmitate in vitro and in the hypothalamus of obese mice. Conditional deletion of CerS6 in hypothalamic neurons attenuates high-fat diet (HFD)-dependent weight gain and improves glucose metabolism. Specifically, CerS6 deficiency in neurons expressing pro-opiomelanocortin (POMC) or steroidogenic factor 1 (SF-1) alters feeding behavior and alleviates the adverse metabolic effects of HFD feeding on insulin sensitivity and glucose tolerance. POMC-expressing cell-selective deletion of CerS6 prevents the diet-induced alterations of mitochondrial morphology and improves cellular leptin sensitivity. Our experiments reveal functions of CerS6-derived ceramides in hypothalamic lipotoxicity, altered mitochondrial dynamics, and ER/mitochondrial stress in the deregulation of food intake and glucose metabolism in obesity

Insulinorésistance musculaire : rôle du métabolisme des sphingolipides

The prevalence of obesity and type 2 diabetes are increasing at the same rate. These two pathologies are linked to a sedentary lifestyle and a diet rich in lipids. When the storage capacity of adipose tissue is exceeded, excess saturated fatty acids accumulate in peripheral tissues, such as skeletal muscle. These ectopic lipid deposits lead to the formation of ceramides (Cer) in muscle cells. Cer are known to inhibit insulin signalling by targeting two major proteins of this pathway, Akt and IRS-1. The deleterious role of Cer has been demonstrated by the use short-chain Cer (C2-Cer) analogous. Endogenous ceramides are produced in the endoplasmic reticulum (ER) and are transported by the CERT transporter to be metabolized into sphingomyelin (SM) in the trans-Golgi network. My thesis project was (i) to understand the role of the CERT transporter in the onset of muscle insulin resistance and (ii) to demonstrate how C2-Cer can mimic the effect of endogenous long-chains ceramides. We observed that the expression of CERT is decreased in lipotoxic conditions and we demonstrated the importance of the transport of CER from the ER to the Golgi for the maintenance of a good insulin sensitivity of cells, by inhibiting or overexpressing CERT in vitro and in vivo. In my second study, we showed that C2-Cer were metabolized to endogenous long-chain Cer via the recycling pathway, and that these endogenous Cer were responsible for the loss of cell insulin sensitivity. Our study therefore validates C2-Cer as a good model for studying the effects of long-chain endogenous Cer.Les prévalences de l’obésité et du diabète de type 2 augmentent parallèlement. Ces deux pathologies sont liées à la sédentarité et à une alimentation riche en lipides. Lorsque la capacité de stockage du tissu adipeux est dépassée, les acides gras saturés en excès s’accumulent au niveau des tissus périphériques, comme les muscles squelettiques. Ces dépôts ectopiques de lipidiques conduisent à la formation de céramides (Cer) dans les cellules musculaires. Les Cer sont connus pour inhiber la signalisation insulinique en ciblant deux protéines majeures de cette voie, Akt et IRS-1. Le rôle délétère des Cer a été mis en évidence grâce à des analogues des Cer à chaînes courtes (C2-Cer). Les céramides endogènes sont produits au niveau du réticulum endoplasmique (RE) puis sont transportés par le transporteur CERT pour être métabolisés en sphingomyéline (SM) au niveau du Golgi. Mon projet de thèse s’est articulé en deux partie : (i) comprendre le rôle du transporteur CERT dans l’installation de l’insulinorésistance musculaire et (ii) comprendre comment les C2-Cer permettent de mimer l’effet de céramides endogènes à chaînes longues. Premièrement, nous avons observé que l’expression de CERT était diminuée en conditions lipotoxiques et nous avons montré l’importance du transport des Cer du RE vers le Golgi pour le maintien d’une bonne sensibilité à l’insuline des cellules en inhibant ou en surexprimant CERT in vitro et in vivo. Dans la seconde étude, nous avons montré que les C2-Cer étaient métabolisés en Cer endogènes à chaînes plus longues via la voie de recyclage, et que ces Cer endogènes étaient responsable de la perte de sensibilité à l’insuline des cellules

Muscle insulin resistance : role of sphingolipid metabolism

Les prévalences de l’obésité et du diabète de type 2 augmentent parallèlement. Ces deux pathologies sont liées à la sédentarité et à une alimentation riche en lipides. Lorsque la capacité de stockage du tissu adipeux est dépassée, les acides gras saturés en excès s’accumulent au niveau des tissus périphériques, comme les muscles squelettiques. Ces dépôts ectopiques de lipidiques conduisent à la formation de céramides (Cer) dans les cellules musculaires. Les Cer sont connus pour inhiber la signalisation insulinique en ciblant deux protéines majeures de cette voie, Akt et IRS-1. Le rôle délétère des Cer a été mis en évidence grâce à des analogues des Cer à chaînes courtes (C2-Cer). Les céramides endogènes sont produits au niveau du réticulum endoplasmique (RE) puis sont transportés par le transporteur CERT pour être métabolisés en sphingomyéline (SM) au niveau du Golgi. Mon projet de thèse s’est articulé en deux partie : (i) comprendre le rôle du transporteur CERT dans l’installation de l’insulinorésistance musculaire et (ii) comprendre comment les C2-Cer permettent de mimer l’effet de céramides endogènes à chaînes longues. Premièrement, nous avons observé que l’expression de CERT était diminuée en conditions lipotoxiques et nous avons montré l’importance du transport des Cer du RE vers le Golgi pour le maintien d’une bonne sensibilité à l’insuline des cellules en inhibant ou en surexprimant CERT in vitro et in vivo. Dans la seconde étude, nous avons montré que les C2-Cer étaient métabolisés en Cer endogènes à chaînes plus longues via la voie de recyclage, et que ces Cer endogènes étaient responsable de la perte de sensibilité à l’insuline des cellules.The prevalence of obesity and type 2 diabetes are increasing at the same rate. These two pathologies are linked to a sedentary lifestyle and a diet rich in lipids. When the storage capacity of adipose tissue is exceeded, excess saturated fatty acids accumulate in peripheral tissues, such as skeletal muscle. These ectopic lipid deposits lead to the formation of ceramides (Cer) in muscle cells. Cer are known to inhibit insulin signalling by targeting two major proteins of this pathway, Akt and IRS-1. The deleterious role of Cer has been demonstrated by the use short-chain Cer (C2-Cer) analogous. Endogenous ceramides are produced in the endoplasmic reticulum (ER) and are transported by the CERT transporter to be metabolized into sphingomyelin (SM) in the trans-Golgi network. My thesis project was (i) to understand the role of the CERT transporter in the onset of muscle insulin resistance and (ii) to demonstrate how C2-Cer can mimic the effect of endogenous long-chains ceramides. We observed that the expression of CERT is decreased in lipotoxic conditions and we demonstrated the importance of the transport of CER from the ER to the Golgi for the maintenance of a good insulin sensitivity of cells, by inhibiting or overexpressing CERT in vitro and in vivo. In my second study, we showed that C2-Cer were metabolized to endogenous long-chain Cer via the recycling pathway, and that these endogenous Cer were responsible for the loss of cell insulin sensitivity. Our study therefore validates C2-Cer as a good model for studying the effects of long-chain endogenous Cer

Sphingolipid Metabolism: New Insight into Ceramide-Induced Lipotoxicity in Muscle Cells

Insulin-resistance is a characteristic feature of type 2 diabetes (T2D) and plays a major role in the pathogenesis of this disease. Skeletal muscles are quantitatively the biggest glucose users in response to insulin and are considered as main targets in development of insulin-resistance. It is now clear that circulating fatty acids (FA), which are highly increased in T2D, play a major role in the development of muscle insulin-resistance. In healthy individuals, excess FA are stored as lipid droplets in adipocytes. In situations like obesity and T2D, FA from lipolysis and food are in excess and eventually accumulate in peripheral tissues. High plasma concentrations of FA are generally associated with increased risk of developing diabetes. Indeed, ectopic fat accumulation is associated with insulin-resistance; this is called lipotoxicity. However, FA themselves are not involved in insulin-resistance, but rather some of their metabolic derivatives, such as ceramides. Ceramides, which are synthetized de novo from saturated FA like palmitate, have been demonstrated to play a critical role in the deterioration of insulin sensitivity in muscle cells. This review describes the latest progress involving ceramides as major players in the development of muscle insulin-resistance through the targeting of selective actors of the insulin signaling pathway

Compte rendu de congrès : les journées 2016 de l’école doctorale « physiologie, physiopathologie et thérapeutique » de l’Université Pierre et Marie Curie

Les doctorants organisateurs de ces journées proposent un résumé des principaux événements scientifiques qui se sont déroulés les 25 et 26 mai 2016 au centre de recherche des Cordeliers (CRC) lors des journées de l’école « physiologie, physiopathologie et thérapeutique » (ED 394). Outre la présentation de leurs travaux scientifiques lors des sessions « communications orales » et « posters », les participants ont entendu une conférence plénière du Professeur Pierre Corvol sur l’intégrité scientifique et participé à une table ronde consacrée à la médiation scientifique. Ces deux conférences ont permis d’échanger les points de vue sur ces problématiques actuelles

Ceramide analog C2-cer induces a loss in insulin sensitivity in muscle cells through the salvage/recycling pathway

International audienceCeramides have been shown to play a major role in the onset of skeletal muscle insulin resistance and therefore in the prevalence of type 2 diabetes. However, many of the studies involved in the discovery of deleterious ceramide actions used a nonphysiological, cell-permeable, short-chain ceramide analog, the C2-ceramide (C2-cer). In the present study, we determined how C2-cer promotes insulin resistance in muscle cells. We demonstrate that C2-cer enters the salvage/recycling pathway and becomes deacylated, yielding sphingosine, re-acylation of which depends on the availability of long chain fatty acids provided by the lipogenesis pathway in muscle cells. Importantly, we show these salvaged ceramides are actually responsible for the inhibition of insulin signaling induced by C2-cer. Interestingly, we also show that the exogenous and endogenous monounsaturated fatty acid oleate prevents C2-cer to be recycled into endogenous ceramide species in a diacylglycerol O-acyltransferase 1-dependent mechanism, which forces free fatty acid metabolism towards triacylglyceride production. Altogether, the study highlights for the first time that C2-cer induces a loss in insulin sensitivity through the salvage/recycling pathway in muscle cells. This study also validates C2-cer as a convenient tool to decipher mechanisms by which long-chain ceramides mediate insulin resistance in muscle cells and suggests that in addition to the de novo ceramide synthesis, recycling of ceramide could contribute to muscle insulin resistance observed in obesity and type 2 diabetes

Inhibition of central de novo ceramide synthesis restores insulin signaling in hypothalamus and enhances β-cell function of obese Zucker rats

Objectives: Hypothalamic lipotoxicity has been shown to induce central insulin resistance and dysregulation of glucose homeostasis; nevertheless, elucidation of the regulatory mechanisms remains incomplete. Here, we aimed to determine the role of de novo ceramide synthesis in hypothalamus on the onset of central insulin resistance and the dysregulation of glucose homeostasis induced by obesity. Methods: Hypothalamic GT1-7 neuronal cells were treated with palmitate. De novo ceramide synthesis was inhibited either by pharmacological (myriocin) or molecular (si-Serine Palmitoyl Transferase 2, siSPT2) approaches. Obese Zucker rats (OZR) were intracerebroventricularly infused with myriocin to inhibit de novo ceramide synthesis. Insulin resistance was determined by quantification of Akt phosphorylation. Ceramide levels were quantified either by a radioactive kinase assay or by mass spectrometry analysis. Glucose homeostasis were evaluated in myriocin-treated OZR. Basal and glucose-stimulated parasympathetic tonus was recorded in OZR. Insulin secretion from islets and β-cell mass was also determined. Results: We show that palmitate impaired insulin signaling and increased ceramide levels in hypothalamic neuronal GT1-7 cells. In addition, the use of deuterated palmitic acid demonstrated that palmitate activated several enzymes of the de novo ceramide synthesis pathway in hypothalamic cells. Importantly, myriocin and siSPT2 treatment restored insulin signaling in palmitate-treated GT1-7 cells. Protein kinase C (PKC) inhibitor or a dominant-negative PKCζ also counteracted palmitate-induced insulin resistance. Interestingly, attenuating the increase in levels of hypothalamic ceramides with intracerebroventricular infusion of myriocin in OZR improved their hypothalamic insulin-sensitivity. Importantly, central myriocin treatment partially restored glucose tolerance in OZR. This latter effect is related to the restoration of glucose-stimulated insulin secretion and an increase in β-cell mass of OZR. Electrophysiological recordings also showed an improvement of glucose-stimulated parasympathetic nerve activity in OZR centrally treated with myriocin. Conclusion: Our results highlight a key role of hypothalamic de novo ceramide synthesis in central insulin resistance installation and glucose homeostasis dysregulation associated with obesity. Keywords: Ceramide, Lipotoxicity, Hypotalamus, Insulin resistance, Insulin secretio

Ceramide Transporter CERT Is Involved in Muscle Insulin Signaling Defects Under Lipotoxic Conditions

International audienc

CerS6-dependent ceramide synthesis in hypothalamic neurons promotes ER/mitochondrial stress and impairs glucose homeostasis in obese mice

Abstract Dysregulation of hypothalamic ceramides has been associated with disrupted neuronal pathways in control of energy and glucose homeostasis. However, the specific ceramide species promoting neuronal lipotoxicity in obesity have remained obscure. Here, we find increased expression of the C16:0 ceramide-producing ceramide synthase (CerS)6 in cultured hypothalamic neurons exposed to palmitate in vitro and in the hypothalamus of obese mice. Conditional deletion of CerS6 in hypothalamic neurons attenuates high-fat diet (HFD)-dependent weight gain and improves glucose metabolism. Specifically, CerS6 deficiency in neurons expressing pro-opiomelanocortin (POMC) or steroidogenic factor 1 (SF-1) alters feeding behavior and alleviates the adverse metabolic effects of HFD feeding on insulin sensitivity and glucose tolerance. POMC-expressing cell-selective deletion of CerS6 prevents the diet-induced alterations of mitochondrial morphology and improves cellular leptin sensitivity. Our experiments reveal functions of CerS6-derived ceramides in hypothalamic lipotoxicity, altered mitochondrial dynamics, and ER/mitochondrial stress in the deregulation of food intake and glucose metabolism in obesity