Cholesterol Regulates Insulin Secretion through Protein Prenylation and Membrane Arrangement in INS-1E Cells

Abstract

Cholesterol plays a key role in membrane structure and secretory function of pancreatic B cells. Prenyl molecules derived from the cholesterol biosynthesis pathway, such as geranylgeranyl pyrophosphate (GGPP), serve as substrates for post-translational modifications of small GTPases involved in insulin secretion. However, appropriate cholesterol content is required for the maintenance of the membrane physicochemical properties such as fluidity that directly impact insulin granule fusion. Since statins reduce both prenyl groups and cholesterol, our aim was to study the relative contribution of these lipid lowering compounds on glucose-stimulated insulin secretion (GSIS) in insulin secreting cells.INS-1E cells were cultured in the absence or presence of simvastatin (SIM, 1μM) or zaragozic acid (ZGA, inhibitor of cholesterol synthesis but not of prenyl groups, 20μM), in acute (2h) and chronic (24h) treatments; rat islets were acutely treated with SIM. To evaluate the role of protein prenylation, cells were treated for 24h with GGPP (20μM). Secreted insulin during 2h incubation at 2.8 and 16.7mM glucose was quantified by radioimmunoassay. Cellular cholesterol after treatment was measured by a colorimetric assay. Membrane lipid rafts after 2h SIM treatment were analyzed by immunofluorescence. Plasma membrane fluidity was measured by EPR using 5-DSA as probe.Acute SIM reduces GSIS, but does not affect total cholesterol content, nor membrane microdomain distribution or membrane fluidity. GSIS inhibition was not observed after acute ZGA treatment nor after acute SIM treatment on cells previously loaded with GGPP. Chronic SIM inhibits GSIS but enhances cholesterol reduction and increases plasma membrane fluidity. Our results show that protein prenylation contributes to GSIS and that plasma membrane cholesterol content may be a critical parameter for the maintenance of physicochemical properties that regulate cellular functions such as granule fusion and cargo exocytosis.