Ceramide flow from the endoplasmic reticulum to the Golgi apparatus is impaired in glucolipotoxic conditions in INS-1 beta cells

Ceramide flow from the Endoplasmic Reticulum to the Golgi apparatus is impaired in Glucolipotoxic conditions in INS-1  cells Giussani Paola1, Gjoni Enida1, Cinque Alessandra1, Le Stunff Hervé2, Riboni Laura1, Viani Paola1 1Department of Medical Biotechnology and Translational Medicine, University of Milano, LITA Segrate, Via Fratelli Cervi, 93, 20090 Segrate (MI) Italy 2Unité Biologie Fonctionnelle et Adaptative - EAC CNRS 4413, Laboratoire de Biologie et Pathologie du Pancréas Endocrine, Université PARIS- DIDEROT (7),4, rue Marie-Andrée Lagroua Weill-Halle, 75205 PARIS Cedex 13 France In type 2 diabetes the detrimental effects of chronic exposure to NEFA, in particular palmitate, on -cell function and viability have been associated to hyperglycaemia. ER stress is among the molecular pathways and regulators involved in these negative effects. Moreover, it is known that accumulation of ceramide due to glucolipotoxicity can be associated to the induction of ER stress. The increased ER ceramide levels can be due to an enhanced ceramide biosynthesis and a decrease in ceramide utilization as well. In the control of ER ceramide levels it is crucial ceramide trafficking between ER and Golgi, which represents the site of complex sphingolipid biosynthesis. In order to gain insight into the molecular mechanism(s) of glucolipotoxicity we studied the effect of glucolipotoxic conditions on ceramide traffic in INS-1 cells. Metabolic studies show that treatment with palmitate results in the inhibition of ceramide utilization for SM and glycosphingolipid biosynthesis. Fluorescence microscopy studies suggest a reduced ER to Golgi flow of ceramide. To evaluate if ceramide accumulation could be due to a defect in the vesicular and/or CERT-mediated transport of ceramide we studied CERT expression and ceramide metabolism in INS-1 cells silenced for CERT incubated with glucose in the presence or not of palmitate. Glucolipotoxicity decreased the total amount of CERT and, by inducing CERT phosphorylation, prevented its localization to the Golgi of pancreatic -cells. Moreover in CERT-downregulated cells palmitate still inhibited ceramide utilization for SM biosynthesis. These results demonstrate that both the vesicular-mediated and CERT-mediated transport are involved in the reduced ER to Golgi flow of ceramide in glucolipotoxic conditions. Altogether these data suggest that the accumulation of ceramide can be due to a decrease in utilization of newly synthesized ceramide for SM biosynthesis thus supporting a role of ER-associated ceramide in the regulation of -cell death induced by gluco-lipotoxicity.