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 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.

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

Ceramides 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.

Sphingosine-1-Phosphate Metabolism in the Regulation of Obesity/Type 2 Diabetes.

Obesity is a pathophysiological condition where excess free fatty acids (FFA) target and promote the dysfunctioning of insulin sensitive tissues and of pancreatic ß cells. This leads to the dysregulation of glucose homeostasis, which culminates in the onset of type 2 diabetes (T2D). FFA, which accumulate in these tissues, are metabolized as lipid derivatives such as ceramide, and the ectopic accumulation of the latter has been shown to lead to lipotoxicity. Ceramide is an active lipid that inhibits the insulin signaling pathway as well as inducing pancreatic ß cell death. In mammals, ceramide is a key lipid intermediate for sphingolipid metabolism as is sphingosine-1-phosphate (S1P). S1P levels have also been associated with the development of obesity and T2D. In this review, the current knowledge on S1P metabolism in regulating insulin signaling in pancreatic ß cell fate and in the regulation of feeding by the hypothalamus in the context of obesity and T2D is summarized. It demonstrates that S1P can display opposite effects on insulin sensitive tissues and pancreatic ß cells, which depends on its origin or its degradation pathway.

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.

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

One main mechanism of insulin resistance (IR), a key feature of type 2 diabetes, is the accumulation of saturated fatty acids (FAs) in the muscles of obese patients with type 2 diabetes. Understanding the mechanism that underlies lipid-induced IR is an important challenge. Saturated FAs are metabolized into lipid derivatives called ceramides, and their accumulation plays a central role in the development of muscle IR. Ceramides are produced in the endoplasmic reticulum (ER) and transported to the Golgi apparatus through a transporter called CERT, where they are converted into various sphingolipid species. We show that CERT protein expression is reduced in all IR models studied because of a caspase-dependent cleavage. Inhibiting CERT activity in vitro potentiates the deleterious action of lipotoxicity on insulin signaling, whereas overexpression of CERT in vitro or in vivo decreases muscle ceramide content and improves insulin signaling. In addition, inhibition of caspase activity prevents ceramide-induced insulin signaling defects in C2C12 muscle cells. Altogether, these results demonstrate the importance of physiological ER-to-Golgi ceramide traffic to preserve muscle cell insulin signaling and identify CERT as a major actor in this process.

Inhibition of central de novo ceramide synthesis restores insulin signaling in hypothalamus and enhances ß-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 ß-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 ß-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.