1751-P: SERCA2 Regulates ER Lipid Composition and Proinsulin Processing

Abstract

Endoplasmic reticulum calcium (ER Ca2+) levels are primarily maintained through the sarco-ER Ca2+ ATPase (SERCA) pump. We have demonstrated reduced SERCA2 expression in islets from human cadaveric donors with Type 2 Diabetes (T2D) compared to normoglycemic controls and linked β-cell specific deletion of SERCA2 with impaired proinsulin processing. To test mechanisms of how reduced ER Ca2+ and SERCA2 deficiency regulate proinsulin processing, a SNAP-tagged human proinsulin adenovirus (SNAP-proinsulin) was overexpressed in rat insulinoma β-cells where SERCA2 had been deleted (S2KO INS-1) using CRISPR/Cas9 and in islets from C57BL/6 mice with SERCA2 haploinsufficiency fed a high fat diet or treated ex vivo with glucolipotoxicity (GLT). Immunoblotting SNAP-tagged cleavage products showed reduced proinsulin processing in SERCA2 deficient models compared to wildtype (WT), these defects were exacerbated by GLT treatment. To determine whether reduced processing could be related to impaired trafficking of proinsulin within the β-cell secretory pathway, SNAP-proinsulin was fluorescently labelled in C57BL/6 mouse islets treated with GLT. The colocalization of SNAP-proinsulin with Golgi immuno-marker, Giantin, suggested an accumulation in this compartment. The ER is the main site of lipid synthesis and lipid remodeling is known to regulate β-cell function. Preliminary mass spectrometry data revealed that loss of SERCA2 increases ER levels of phosphatidylcholine and decreases ceramide and sphingomyelin in S2KO INS-1 cells compared to WT cells. Taken together, these results demonstrate that ER Ca2+ deficiency changes ER lipid composition, which may affect proinsulin processing and trafficking. Characterizing this novel mechanism could provide valuable insight into β-cell dysfunction in T2D. Disclosure M.Hartley: Employee; Eli Lilly and Company. M.J.Pearson: None. F.Syed: None. H.Bui: None. T.Kono: None. K.D.Roth: Employee; Eli Lilly and Company. C.Evans-molina: Advisory Panel; Provention Bio, Inc., DiogenX, Avotres Inc., Neurodon, MaiCell Therapeutics, Other Relationship; Isla Technology, Bristol-Myers Squibb Company, Nimbus Therapeutics, Research Support; Lilly, Astellas Pharma Inc. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK093954, R01DK127308, R01DK127236)