Gluco-lipotoxicity inhibits ceramide transport between endoplasmic reticulum and Golgi apparatus in pancreatic beta cells

Background and aims: In type 2 diabetes (TD2) the chronic adverse effects of fatty acids (FA) on beta cell function and viability have been correlated to hyperglycaemia. Gluco-lipotoxicity refers to the combined, deleterious effects of elevated glucose and fatty acid levels on pancreatic beta-cell function and survival. Gluco-lipotoxicity caused beta-cell apoptosis and may thus contribute to progressive beta-cell loss in TD2. The molecular pathways and regulators involved in the detrimental effects of chronic exposure to FA, in particular palmitate, include the de novo synthesis of ceramide (Cer) in the endoplasmic reticulum (ER). However, increasing Cer levels in ER can also be due to a reduction of Cer utilization for complex sphingolipids synthesis. Therefore, we studied the effect of gluco-lipotoxicity on Cer metabolism in beta cells and its impact on beta-cell apoptosis. Materials and methods: INS-1 cells were cultured with 0.4 mM palmitate and 5 or 30 mM of glucose. INS-1 cell apoptosis was determined by caspase 3/7 activity assay. Sphingolipid metabolites were analyzed by liquid chromatography mass spectrometry. Metabolism of Cer was measured in INS-1 beta cells with [3H]sphingosine, a precursor of sphingolipid biosynthesis. Transport of C5-Bodipy-ceramides between ER and Golgi apparatus was analysed by fluorescence confocal microscopy. Down-regulation of Cer transporter CERT was made by specific siRNA. Results: Both nutrients taken separately did not induce INS-1 cell death, whereas the combined treatment with palmitate and glucose resulted in an extensive beta cell apoptosis and this was associated to a significant increase of Cer levels. The presence of fumonisin-B1, an inhibitor of Cer biosynthesis, partially reversed the apoptosis induced by the combined treatment with palmitate and glucose. Metabolic studies using [3H]sphingosine as precursor of sphingolipid biosynthesis show that treatment with palmitate results in a small but significant increase of [3H]Cer associated to a decrease of [3H] sphingomyelin (SM) at 5 mM glucose. Inhibition of Cer utilization for SM biosynthesis was significantly potentiated by the presence of 30 mM glucose. Lipidomic analysis showed that gluco-lipotoxicity inhibited biosynthesis of complex sphingolipids. Fluorescence microscopy studies using C5-Bodipy- Cer show that at high gluco-lipotoxicity reduces the fluorescence accumulation in the perinuclear region representative of the Golgi apparatus. Interestingly, this was associated with an inhibition of the Cer transporter CERT function. Inhibition of CERT was mediated by a decrease of its expression and an increase of its phosphorylation status. Finally, selective silencing of CERT expression increased INS-1 cell apoptosis induced by palmitate. Conclusion: Altogether these data suggest that gluco-lipotoxicity increased Cer accumulation in the ER through also through a decreased utilization of newly synthesized Cer for SM biosynthesis. Moreover, these results support a role of Cer transport between ER and Golgi apparatus in the regulation of beta cell death induced by gluco-lipotoxicity.