Free fatty acids (FFAs), in addition to glucose, have been shown to stimulate insulin release through the G protein-coupled receptor (GPCR)40 receptor in pancreatic beta-cells. Intracellular free calcium concentration ([Ca(2+)](i)) in beta-cells is elevated by FFAs, although the mechanism underlying the [Ca(2+)](i) increase is still unknown. In this study, we investigated the action of linoleic acid on voltage-gated K(+) currents. Nystatin-perforated recordings were performed on identified rat beta-cells. In the presence of nifedipine, tetrodotoxin, and tolbutamide, voltage-gated K(+) currents were observed. The transient current represents less than 5%, whereas the delayed rectifier current comprises more than 95%, of the total K(+) currents. A long-chain unsaturated FFA, linoleic acid (10 microm), reversibly decreased the amplitude of K(+) currents (to less than 10%). This reduction was abolished by the cAMP/protein kinase A system inhibitors H89 (1 microm) and Rp-cAMP (10 microm) but was not affected by protein kinase C inhibitor. In addition, forskolin and 8'-bromo-cAMP induced a similar reduction in the K(+) current as that evoked by linoleic acid. Insulin secretion and cAMP accumulation in beta-cells were also increased by linoleic acid. Methyl linoleate, which has a similar structure to linoleic acid but no binding affinity to GPR40, did not change K(+) currents. Treatment of cultured cells with GPR40-specific small interfering RNA significantly reduced the decrease in K(+) current induced by linoleic acid, whereas the cAMP-induced reduction of K(+) current was not affected. We conclude that linoleic acid reduces the voltage-gated K(+) current in rat beta-cells through GPR40 and the cAMP-protein kinase A system, leading to an increase in [Ca(2+)](i) and insulin secretion.
Extracellular adenosine triphosphate (ATP) has distinct effects on insulin secretion from pancreatic beta-cells between rats and mice. Using a confocal microscope, we compared changes between rats and mice in cytosolic free calcium concentration ([Ca2+]c) in pancreatic beta-cells stimulated by extracellular ATP. Extracellular ATP (50 microM) induced calcium release from intracellular calcium stores by activating P2Y receptors in both rat and mouse beta-cells. The intracellular calcium release stimulated by extracellular ATP is significantly smaller in amplitude and longer in duration in rat beta-cells than in mouse. In response to extracellular ATP, rat beta-cells activate store-operated calcium entry following intracellular calcium release. This response is lacking in mouse beta-cells. Rat and mouse beta-cells both responded to 9 mM glucose by increasing [Ca2+]c. This increase, however, was pronounced only in the rat beta-cells. In 9 mM glucose, extracellular ATP induced a pronounced calcium release above the increased level of [Ca2+]c in rat beta-cells. In mouse beta-cells, however, extracellular ATP did not exhibit calcium release on top of the increased level of [Ca2+]c in 9 mM glucose. These results demonstrate distinct responses between rat and mouse beta-cells to extracellular ATP under the condition of low and high glucose. Considering that extracellular ATP inhibits insulin secretion from mouse beta-cells but stimulates insulin secretion from rat beta-cells, we suggest that store-operated Ca2+ entry may be related to exocytosis in pancreatic rat beta-cells.
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