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. 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. Insulin resistance plays a major role in the pathogenesis of type 2 diabetes (T2D). Skeletal muscles are quantitatively the largest glucose users in response to insulin and are considered as the main effectors for the development of insulin resistance (1Katz L.D. Glickman M.G. Rapoport S. Ferrannini E. DeFronzo R.A. Splanchnic and peripheral disposal of oral glucose in man.Diabetes. 1983; 32: 675-679Google Scholar). It is now clear that circulating free fatty acids (FAs), highly increased in T2D, are key players for the development of muscle insulin resistance, and high plasma concentrations of FAs are generally associated with an increased risk of developing diabetes (2Charles M.A. Eschwege E. Thibult N. Claude J.R. Warnet J.M. Rosselin G.E. et al.The role of non-esterified fatty acids in the deterioration of glucose tolerance in caucasian subjects: results of the paris prospective study.Diabetologia. 1997; 40: 1101-1106Google Scholar). In healthy individuals, free FAs are stored as lipid droplets in adipocytes. In situations like obesity and T2D, FAs coming from both lipolysis and food intake are in excess and eventually accumulate in insulin tissues (liver, skeletal muscles). Ectopic fat accumulation is associated with insulin resistance, a phenomenon called lipotoxicity. However, FA themselves are not directly involved but rather their metabolic derivatives such as ceramides or diacylglycerols (DAGs) (3Hage Hassan R. Bourron O. Hajduch E. Defect of insulin signal in peripheral tissues: important role of ceramide.World J. Diabetes. 2014; 5: 244-257Google Scholar, 4Bandet C.L. Tan-Chen S. Bourron O. Le Stunff H. Hajduch E. Sphingolipid metabolism: new insight into ceramide-induced lipotoxicity in muscle cells.Int. J. Mol. Sci. 2019; 20: E479Google Scholar). In the context of obesity-associated FA overload, ceramides are mainly produced de novo from saturated FAs (SFAs) and particularly palmitate. This synthesis occurs in the endoplasmic reticulum (ER) and begins with the condensation of L-serine with palmitoyl-CoA by serine palmitoyltransferase (SPT), generating 3-keto-dihydrosphingosine, reduced to dihydrosphingosine by 3-keto-dihydrosphingosine reductase. Dihydrosphingosine is then acylated by ceramide synthase (CerS) isoforms to form dihydroceramide species (3Hage Hassan R. Bourron O. Hajduch E. Defect of insulin signal in peripheral tissues: important role of ceramide.World J. Diabetes. 2014; 5: 244-257Google Scholar). The 4-5 position of the sphingoid base backbone is finally desaturated by dihydroceramide desaturase-1 to form different ceramide species (5Zheng W. Kollmeyer J. Symolon H. Momin A. Munter E. Wang E. et al.Ceramides and other bioactive sphingolipid backbones in health and disease: lipidomic analysis, metabolism and roles in membrane structure, dynamics, signaling and autophagy.Biochim. Biophys. Acta. 2006; 1758: 1864-1884Google Scholar). Interestingly, ceramide can also be synthetized through a salvage pathway which involves the acylation of sphingosine by CerS isoforms (6Kitatani K. Idkowiak-Baldys J. Hannun Y.A. The sphingolipid salvage pathway in ceramide metabolism and signaling.Cell. Signal. 2008; 20: 1010-1018Google Scholar). Sphingosine is only generated from the degradation of complex sphingolipids such as glycosphingolipids or deacetylation of ceramide in lysosomes by ceramidases. Then, sphingosine can be re-acylated by CerS into ceramide (6Kitatani K. Idkowiak-Baldys J. Hannun Y.A. The sphingolipid salvage pathway in ceramide metabolism and signaling.Cell. Signal. 2008; 20: 1010-1018Google Scholar). Thus, through this salvage pathway, sphingolipids can be recycled to give new molecules of ceramide. Interestingly, this pathway was shown to contribute from 50 to 90% of sphingolipid biosynthesis (6Kitatani K. Idkowiak-Baldys J. Hannun Y.A. The sphingolipid salvage pathway in ceramide metabolism and signaling.Cell. Signal. 2008; 20: 1010-1018Google Scholar), and a growing body of evidence is also starting to point toward roles of this pathway in many biological responses (6Kitatani K. Idkowiak-Baldys J. Hannun Y.A. The sphingolipid salvage pathway in ceramide metabolism and signaling.Cell. Signal. 2008; 20: 1010-1018Google Scholar, 7Mullen T.D. Spassieva S. Jenkins R.W. Kitatani K. Bielawski J. Hannun Y.A. et al.Selective knockdown of ceramide synthases reveals complex interregulation of sphingolipid metabolism.J. Lipid Res. 2011; 52: 68-77Google Scholar). Many studies including ours showed that ceramides play a crucial role in lipotoxicity-induced insulin resistance (3Hage Hassan R. Bourron O. Hajduch E. Defect of insulin signal in peripheral tissues: important role of ceramide.World J. Diabetes. 2014; 5: 244-257Google Scholar) and demonstrated that downregulating de novo ceramide biosynthesis prevents their deleterious action on insulin signaling in peripheral tissues (3Hage Hassan R. Bourron O. Hajduch E. Defect of insulin signal in peripheral tissues: important role of ceramide.World J. Diabetes. 2014; 5: 244-257Google Scholar, 8Bellini L. Campana M. Mahfouz R. Carlier A. Veret J. Magnan C. et al.Targeting sphingolipid metabolism in the treatment of obesity/type 2 diabetes.Expert Opin. Ther. Targets. 2015; 19: 1037-1050Google Scholar, 9Bandet C.L. Mahfouz R. Veret J. Sotiropoulos A. Poirier M. Giussani P. et al.Ceramide transporter CERT is involved in muscle insulin signaling defects under lipotoxic conditions.Diabetes. 2018; 67: 1258-1271Google Scholar). In contrast, only few studies showed that ceramides supplied from the salvage pathway are implied as potential actors of lipotoxicity in peripheral tissues (10Verma M.K. Yateesh A.N. Neelima K. Pawar N. Sandhya K. Poornima J. et al.Inhibition of neutral sphingomyelinases in skeletal muscle attenuates fatty-acid induced defects in metabolism and stress.Springerplus. 2014; 3: 255Google Scholar, 11Manukyan L. Ubhayasekera S.J. Bergquist J. Sargsyan E. Bergsten P. Palmitate-induced impairments of beta-cell function are linked with generation of specific ceramide species via acylation of sphingosine.Endocrinology. 2015; 156: 802-812Google Scholar, 12Choi S. Snider A.J. Sphingolipids in high fat diet and obesity-related diseases.Mediators Inflamm. 2015; 2015520618Google Scholar). Ceramide species are involved in many processes such as regulation of apoptosis, cell differentiation, and insulin signaling (3Hage Hassan R. Bourron O. Hajduch E. Defect of insulin signal in peripheral tissues: important role of ceramide.World J. Diabetes. 2014; 5: 244-257Google Scholar). In the liver, C16-ceramide species are harmful for insulin sensitivity (13Turpin S.M. Nicholls H.T. Willmes D.M. Mourier A. Brodesser S. Wunderlich C.M. et al.Obesity-induced CerS6-dependent C16:0 ceramide production promotes weight gain and glucose Intolerance.Cell Metab. 2014; 20: 678-686Google Scholar). Conversely, very-long chain ceramide species (C22/C24/C24:1) appear to exert protective functions in hepatocytes (14Zigdon H. Kogot-Levin A. Park J.W. Goldschmidt R. Kelly S. Merrill A.H. et al.Ablation of ceramide synthase 2 causes chronic oxidative stress due to disruption of the mitochondrial respiratory chain.J. Biol. Chem. 2013; 288: 4947-4956Google Scholar, 15Raichur S. Wang S.T. Chan P.W. Li Y. Ching J. Chaurasia B. et al.CerS2 Haploinsufficiency inhibits beta-oxidation and confers susceptibility to diet-induced steatohepatitis and insulin resistance.Cell Metab. 2014; 20: 687-695Google Scholar). In muscle cells, most of ceramide species are increased in response to lipotoxicity (3Hage Hassan R. Bourron O. Hajduch E. Defect of insulin signal in peripheral tissues: important role of ceramide.World J. Diabetes. 2014; 5: 244-257Google Scholar, 4Bandet C.L. Tan-Chen S. Bourron O. Le Stunff H. Hajduch E. Sphingolipid metabolism: new insight into ceramide-induced lipotoxicity in muscle cells.Int. J. Mol. Sci. 2019; 20: E479Google Scholar, 16Chavez J.A. Summers S.A. A ceramide-centric view of insulin resistance.Cell Metab. 2012; 15: 585-594Google Scholar, 17Mahfouz R. Khoury R. Blachnio-Zabielska A. Turban S. Loiseau N. Lipina C. et al.Characterising the inhibitory actions of ceramide upon insulin signaling in different skeletal muscle cell models: a mechanistic insight.Plos One. 2014; 9e101865Google Scholar), but the species that mediates insulin resistance is still not clear, even if C18-ceramide is often associated to insulin resistance in this tissue (9Bandet C.L. Mahfouz R. Veret J. Sotiropoulos A. Poirier M. Giussani P. et al.Ceramide transporter CERT is involved in muscle insulin signaling defects under lipotoxic conditions.Diabetes. 2018; 67: 1258-1271Google Scholar, 18Bergman B.C. Brozinick J.T. Strauss A. Bacon S. Kerege A. Bui H.H. et al.Muscle sphingolipids during rest and exercise: a C18:0 signature for insulin resistance in humans.Diabetologia. 2016; 59: 785-798Google Scholar, 19Turpin-Nolan S.M. Hammerschmidt P. Chen W. Jais A. Timper k Awazawa M. et al.CerS1-derived C18:0 ceramide in skeletal muscle promotes obesity-induced insulin resistance.Cell Rep. 2019; 26: 1-10.e7Google Scholar, 20Tan-Chen S. Guitton J. Bourron O. Le Stunff H. Hajduch E. Sphingolipid metabolism and signaling in skeletal muscle: from physiology to physiopathology.Front. Endocrinol. (Lausanne). 2020; 11: 491Google Scholar). Mechanism by which ceramides act negatively on the insulin signaling pathway has been well characterized in muscle cells (4Bandet C.L. Tan-Chen S. Bourron O. Le Stunff H. Hajduch E. Sphingolipid metabolism: new insight into ceramide-induced lipotoxicity in muscle cells.Int. J. Mol. Sci. 2019; 20: E479Google Scholar). To summarize, ceramides rapidly target and inhibit Akt through the activation of either the phosphatase PP2A or the PKC ζ (16Chavez J.A. Summers S.A. A ceramide-centric view of insulin resistance.Cell Metab. 2012; 15: 585-594Google Scholar, 21Litherland G.J. Hajduch E. Hundal H.S. Intracellular signalling mechanisms regulating glucose transport in insulin-sensitive tissues (review).Mol. Membr. Biol. 2001; 18: 195-204Google Scholar, 22Hajduch E. Balendran A. Batty I. Litherland G.J. Blair A.S. Downes C.P. et al.Ceramide impairs the insulin-dependent membrane recruitment of protein kinase B leading to a loss in downstream signalling in L6 skeletal muscle cells.Diabetologia. 2001; 44: 173-183Google Scholar, 23Turban S. Hajduch E. Protein kinase C isoforms: mediators of reactive lipid metabolites in the development of insulin resistance.FEBS Lett. 2011; 585: 269-274Google Scholar), and in longer term, they target and inhibit insulin-induced insulin receptor substrate-1 activity in a PKR/JNK– and/or Prep1-p160–dependent manner (24Cimmino I. Lorenzo V. Fiory F. Doti N. Ricci S. Cabaro S. et al.A peptide antagonist of Prep1-p160 interaction improves ceramide-induced insulin resistance in skeletal muscle cells.Oncotarget. 2017; 8: 71845-71858Google Scholar, 25Hage Hassan R. Pacheco de Sousa A.C. Mahfouz R. Hainault I. Blachnio-Zabielska A. Bourron O. et al.Sustained action of ceramide on the insulin signaling pathway in muscle cells: implication of the double-STRANDED RNA-activated protein kinase.J. Biol. Chem. 2016; 291: 3019-3029Google Scholar). Most of the studies investigating ceramide effects used biologically active, cell-permeable, short-chain ceramide analogs such as C2- or C6-ceramide species (17Mahfouz R. Khoury R. Blachnio-Zabielska A. Turban S. Loiseau N. Lipina C. et al.Characterising the inhibitory actions of ceramide upon insulin signaling in different skeletal muscle cell models: a mechanistic insight.Plos One. 2014; 9e101865Google Scholar, 22Hajduch E. Balendran A. Batty I. Litherland G.J. Blair A.S. Downes C.P. et al.Ceramide impairs the insulin-dependent membrane recruitment of protein kinase B leading to a loss in downstream signalling in L6 skeletal muscle cells.Diabetologia. 2001; 44: 173-183Google Scholar, 25Hage Hassan R. Pacheco de Sousa A.C. Mahfouz R. Hainault I. Blachnio-Zabielska A. Bourron O. et al.Sustained action of ceramide on the insulin signaling pathway in muscle cells: implication of the double-STRANDED RNA-activated protein kinase.J. Biol. Chem. 2016; 291: 3019-3029Google Scholar, 26Summers S.A. Garza L.A. Zhou H. Birnbaum M.J. Regulation of insulin-stimulated glucose transporter GLUT4 translocation and Akt kinase activity by ceramide.Mol. Cell. 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Even if the presence of C2-ceramide (C2-cer) was established in mouse brain and liver, its concentration is approximately 5000-fold less than long-chain ceramide species (31Van O.H. Denizot Y. Baes M. Van Veldhoven P.P. On the presence of C2-ceramide in mammalian tissues: possible relationship to etherphospholipids and phosphorylation by ceramide kinase.Biol. Chem. 2007; 388: 315-324Google Scholar) usually used to induced lipotoxic responses (17Mahfouz R. Khoury R. Blachnio-Zabielska A. Turban S. Loiseau N. Lipina C. et al.Characterising the inhibitory actions of ceramide upon insulin signaling in different skeletal muscle cell models: a mechanistic insight.Plos One. 2014; 9e101865Google Scholar, 32Amati F. Dube J.J. Carnero E.A. Edreira M.M. Chomentowski P. Coen P.M. et al.Skeletal-muscle triglycerides, diacylglycerols, and ceramides in insulin resistance: another paradox in endurance-trained athletes?.Diabetes. 2011; 60: 2588-2597Google Scholar, 33Coen P.M. Dube J.J. Amati F. Stefanovic-Racic M. Ferrell R.E. Toledo F.G. et al.Insulin resistance is associated with higher intramyocellular triglycerides in type I but not type II myocytes concomitant with higher ceramide content.Diabetes. 2010; 59: 80-88Google Scholar, 34Bajpeyi S. Myrland C.K. Covington J.D. Obanda D. Cefalu W.T. Smith S.R. et al.Lipid in skeletal muscle myotubes is associated to the donors’ insulin sensitivity and physical activity phenotypes.Obes. (Silver. Spring). 2013; 22: 426-434Google Scholar). Since C18-ceramide is the predominant ceramide species in skeletal muscle and since skeletal muscle CerS1-derived C18-ceramide promotes insulin resistance (19Turpin-Nolan S.M. Hammerschmidt P. Chen W. Jais A. Timper k Awazawa M. et al.CerS1-derived C18:0 ceramide in skeletal muscle promotes obesity-induced insulin resistance.Cell Rep. 2019; 26: 1-10.e7Google Scholar), how can C2-cer reproduce the deleterious action of endogenous C18-ceramides in muscle cells? The goal of the present study was to evaluate whether C2-cer needs to be converted into longer chain ceramide species in muscle cells using the sphingolipid salvage pathway in order to induce insulin resistance. When incubated with palmitate, C2C12 myotubes become rapidly insulin resistant, as shown by the inhibition of insulin-induced phosphorylation of Akt (Fig. 1A). Preincubation of cells with myriocin, a potent and specific inhibitor of SPT, the rate limiting enzyme of de novo ceramide synthesis (17Mahfouz R. Khoury R. Blachnio-Zabielska A. Turban S. Loiseau N. Lipina C. et al.Characterising the inhibitory actions of ceramide upon insulin signaling in different skeletal muscle cell models: a mechanistic insight.Plos One. 2014; 9e101865Google Scholar, 32Amati F. Dube J.J. Carnero E.A. Edreira M.M. Chomentowski P. Coen P.M. et al.Skeletal-muscle triglycerides, diacylglycerols, and ceramides in insulin resistance: another paradox in endurance-trained athletes?.Diabetes. 2011; 60: 2588-2597Google Scholar), completely prevented the inhibitory action of palmitate, demonstrating that palmitate inhibits insulin signaling through de novo production of ceramide. Short-chain C2-cer mimicked the effect of palmitate on C2C12 myotubes. Indeed, C2-cer incubation strongly reduced insulin-induced Akt phosphorylation (Fig. 1B). However, pretreatment of cells with myriocin did not prevent C2-cer–induced inhibition of Akt phosphorylation by insulin (Fig. 1B). In order to analyze whether C2-cer was used as a backbone to generate endogenous ceramide species, we incubated C2C12 myotubes with C2-cer up to 2 h and endogenous ceramide species were quantified. First, we detected ceramide intracellular content by immunofluorescence using an anticeramide antibody that specifically detects both C16- and C24-ceramide species (35Nganga R. Oleinik N. Kim J. Selvam S.P. De Palma R. Johnson K.A. et al.Receptor-interacting Ser/Thr kinase 1 (RIPK1) and myosin IIA-dependent ceramidosomes form membrane pores that mediate blebbing and necroptosis.J. Biol. Chem. 2019; 294: 502-519Google Scholar). Figure 1C shows that, in the basal state, very low content of both ceramide species was detected in muscle cells. Addition of C2-cer induced a rapid and robust increase in the concentration of both C16- and C24-ceramide species in C2C12 myotubes (Fig. 1C). Then, we refined these results by assessing intracellular ceramide species concentrations using ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC/MS/MS). Figure 1D shows that incubation of C2-cer for 2 h of C2C12 myotubes increased several endogenous long chain ceramide species such as C14:0, C16:0, C18:1, C18:0, C24:0, and C24:1-ceramide. Overall, C2-cer increased the level of endogenous ceramides by around 2-fold (Fig. 1D). This is in line with what was observed previously in C2C12 myotubes treated 16 h with palmitate (9Bandet C.L. Mahfouz R. Veret J. Sotiropoulos A. Poirier M. Giussani P. et al.Ceramide transporter CERT is involved in muscle insulin signaling defects under lipotoxic conditions.Diabetes. 2018; 67: 1258-1271Google Scholar). In parallel, we tested in our cell model the action of another short-chain ceramide species (C6-ceramide, C6-cer), first ceramide species shown to be remodeled into longer-chain endogenous ceramides in cells (36Ogretmen B. Pettus B.J. Rossi M.J. Wood R. Usta J. Szulc Z. et al.Biochemical mechanisms of the generation of endogenous long chain ceramide in response to exogenous short chain ceramide in the A549 human lung adenocarcinoma cell line. Role for endogenous ceramide in mediating the action of exogenous ceramide.J. Biol. Chem. 2002; 277: 12960-12969Google Scholar). First, Fig. S1A shows that C6-cer inhibited the insulin response in a dose-dependent manner. Furthermore, and as observed with C2-cer (Fig. 1D), C6-cer was remodeled into several longer-chain ceramide species (Fig. S1B). This result shows that this intracellular recycling mechanism is not limited to one ceramide analog. Interestingly, and in contrast with palmitate, no increase in DAG content was observed in response to C2-cer treatment (Fig. 1E), confirming that the deleterious action of exogenous C2-cer on insulin signaling remains a ceramide-driven process in muscle cells. Generation of endogenous long-chain ceramide species from C2-cer could happen through a C2-cer deacylation/re-acylation process that has already been shown to occur on short-chain ceramide in a human lung adenocarcinoma cell line (36Ogretmen B. Pettus B.J. Rossi M.J. Wood R. Usta J. Szulc Z. et al.Biochemical mechanisms of the generation of endogenous long chain ceramide in response to exogenous short chain ceramide in the A549 human lung adenocarcinoma cell line. Role for endogenous ceramide in mediating the action of exogenous ceramide.J. Biol. Chem. 2002; 277: 12960-12969Google Scholar). We first tested whether the expression of enzymes involved in this mechanism could be modulated in response to C2-cer. Fig. S2 shows that neither the expression of the six CerS isoforms nor that of the three ceramidase isoforms are significantly modified after 2 h treatment with C2-cer. Then, we treated C2C12 myotubes with ceranib-2, a nonlipid ceramidase inhibitor (37Draper J.M. Xia Z. Smith R.A. Zhuang Y. Wang W. Smith C.D. Discovery and evaluation of inhibitors of human ceramidase.Mol. Cancer Ther. 2011; 10: 2052-2061Google Scholar), and after 2 h treatment, we assessed ceramide content in cells. Ceranib-2 alone induced a 50% accumulation in basal total endogenous ceramide concentrations (Fig. 2A). It is interesting to note that, like C2-cer, ceranib-2 increased most ceramide species (except C14:0 and C20:0, Fig. 2A). Unexpectedly, ceranib-2–induced ceramide content within cells did not cause any default in insulin response, as insulin-induced Akt phosphorylation remained identical with or without the ceramidase inhibitor (Fig. 2B). Then, we checked whether preventing C2-cer deacylation into sphingosine would stop C2-cer–induced endogenous ceramide synthesis and thus its inhibitory action on insulin signaling. Figure 3A shows that both sphingosine and sphingosine-1-phosphate (S1P) concentrations were increased following C2-cer addition, demonstrating that C2-cer was converted into both sphingolipids in muscle cells. Ceranib-2 significantly reduced C2-cer–induced sphingosine and S1P content (Fig. 3A). In addition, C2-cer treatment alone induced a 60% increase in total ceramide content in muscle cells (Fig. 3B), but in this case, and in opposite to what was observed in response to ceranib-2 alone (Fig. 2), a sharp inhibition of the insulin signal was observed in the presence of C2-cer (Fig. 3D). Interestingly, inhibition of ceramidase activity with ceranib-2 reduced C2-cer–induced endogenous ceramide content (residual 25% increase, Fig. 3C), confirming that endogenous ceramide species synthetized from C2-cer occurred to a large extent through a salvage ceramide pathway in C2C12 myotubes. In this situation, the ceranib-2 inhibitor restores the insulin response in a dose-dependent manner (Figs. 3D and S3A), suggesting that newly ceramide species synthetized from C2-cer were responsible for the loss of insulin signal. To confirm a sphingosine re-acylation process in the insulin resistance mediated by C2-cer, we treated muscle cells with fumonisin B1 (FB1), an inhibitor of CerS, enzymes that would add different chain length fatty acyl-CoAs to the short chain ceramide-derived sphingosine backbone to generate newly synthetized ceramide species (36Ogretmen B. Pettus B.J. Rossi M.J. Wood R. Usta J. Szulc Z. et al.Biochemical mechanisms of the generation of endogenous long chain ceramide in response to exogenous short chain ceramide in the A549 human lung adenocarcinoma cell line. Role for endogenous ceramide in mediating the action of exogenous ceramide.J. Biol. Chem. 2002; 277: 12960-12969Google Scholar). Figure 4A shows that FB1 prevented the generation of total endogenous ceramide in response to C2-cer, suggesting that sphingosine derived from C2-cer is undeniably used as backbone to produce other ceramide species in cells. In contrast, and as a negative control, inhibition of SPT with myriocin did not affect C2-cer–induced endogenous ceramide synthesis (Fig. 4B). We checked whether FB1 would prevent C2-cer–inhibited insulin signaling in C2C12 myotubes. We treated C2C12 myotubes with C2-cer in the presence or not in the presence of FB1 for 2 h and then with insulin for the last 10 min of the incubation process, and we assessed Akt phosphorylation status. We observed the usual loss in the insulin response with C2-cer (Fig. 4C). Interestingly, pretreatment of cells with FB1 prevented the negative action of C2-cer and restored the ability of insulin to induce Akt phosphorylation in a dose-dependent manner (Figs. 4C and S3A). Altogether, these data confirm that long chain ceramide species generated from C2-cer through a deacylation/re-acylation process are responsible for the loss in insulin signal observed after C2-cer treatment. Then, we evaluated whether preventing C2-cer–induced generation of long chain ceramide species would have consequences on glucose metabolism downstream of insulin signaling. Figure 4D shows that, as previously described in muscle cells (38Alvim R.O. Cheuhen M.R. Machado S.R. Sousa A.G. Santos P.C. General aspects of muscle glucose uptake.An. Acad. Bras. Cienc. 2015; 87: 351-368Google Scholar), insulin induces the translocation of the insulin-regulated glucose transporter (GLUT4) to the plasma membrane where it facilitates glucose uptake into the cell. However, in the presence of C2-cer, GLUT4 could not be recruited anymore to the plasma membrane (Fig. 4D). As expected, inhibition of endogenous long chain ceramide generation from C2-cer by FB1 prevented completely the inhibitory effect of C2-cer on insulin-induced GLUT4 translocation to the plasma membrane (Fig. 4D). Finally, we assessed the effect of inhibiting CerS activity on insulin-induced glucose uptake. As shown in Figure 4E, inhibition of insulin-induced 2-deoxy-glucose (2-DG) uptake by C2-cer was prevented when CerS activity was inhibited by FB1. T2D is a prevalent metabolic disorder characterized by hyperglycemia (glucotoxicity) and hyperlipidemia (lipotoxicity). High circulating glucose concentrations were shown to potentiate the effect of lipotoxicity through ceramide synthesis (8Bellini L. Campana M. Mahfouz R. Carlier A. Veret J. Magnan C. et al.Targeting sphingolipid metabolism in the treatment of obesity/type 2 diabetes.Expert Opin. Ther. Targets. 2015; 19: 1037-1050Google Scholar). Hyperglycaemia is also responsible for FA synthesis through the lipogenic pathway in several tissues, including muscle (39Aas V. Kase E.T. Solberg R. Jensen J. Rustan A.C. Chronic hyperglycaemia promotes lipogenesis and triacylglycerol accumulation in human skeletal muscle cells.Diabetologia. 2004; 47: 1452-1461Google Scholar). Thus, the FA-synthetized de novo could serve as precursors for endogenous ceramide formation from sphingosine. To test this hypothesis, we incubated muscle cells with 25 mM or 5 mM glucose 16 h before to add C2-cer and to check the insulin response. At 25 mM glucose, C2-cer prevented completely insulin-induced Akt phosphorylation (Figure 5A). However, at 5 mM glucose, C2-cer action was unable to alter the insulin signal (Fig. 5A). Then, we inhibited lipogenesis by using an inhibitor of acetyl-CoA carboxylase, 5-tetradecyloxy-2-furoic acid (TOFA) (40Loft