Fatty acid required for glucose-induced change in beta cell plasma membrane potential leading to insulin secretion
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Abstract
The pancreatic β−cell secretes insulin through a combination of triggering and amplification pathways in response to glucose. Glucose metabolism is known to increase intracellular calcium and trigger insulin secretion from the β−cell while fatty acid (FA), an essential component of glucose stimulated insulin secretion (GSIS), is known to amplify secretion through varied mechanisms. Orlistat, a lipase inhibitor, blocks GSIS although the mechanism has not been clearly elucidated. We show that orlistat can also inhibit basal insulin secretion. FA can prevent/reverse orlistat-induced inhibition of secretion but fails to amplify GSIS after orlistat treatment. Here we test the hypothesis that FA is required to maintain normal plasma membrane potential and calcium influx in the β−cell. Clonal pancreatic β−cells (INS-1 832/13) were cultured in RPMI media containing 11 mM glucose and 10% FBS. Insulin secretion was measured over 2 hours with and without FA and orlistat (200 µM) using homogeneous time resolved fluorescence insulin assay (HTRF, Cisbio) and reported as ng/million cells. Intracellular calcium was measured in single cells using fura-2 AM. Single cell membrane potential was measured with virally expressed ARC Lite protein (Montana molecular). High glucose (8 mM) stimulated insulin secretion at least 3-fold over basal glucose (2 mM) and addition of FA enhanced GSIS. Orlistat almost completely abolished GSIS. This inhibition was mostly prevented in the presence of FA. Orlistat blocked calcium influx required for triggering insulin release while addition of FA recovered normal calcium homeostasis. The monoacylglycerol lipase inhibitor JZL 184 reduced both calcium influx and the increase in plasma membrane potential induced by KCl. FA recovered plasma membrane potential inhibited by JZL 184. Our results suggest that reducing β−cell intracellular FA availability by lipase inhibition blocks GSIS by preventing the glucose-induced rise in plasma membrane potential required to induce β−cell calcium influx through voltage dependent calcium channels (VDCC). FA is thus required to maintain normal nutrient metabolic coupling to insulin secretion. Whether the effect of FA to modulate plasma membrane potential plays a role in β−cell insulin hypersecretion resulting from excess nutrients requires further investigation.