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© 2002 by the American Diabetes Association, Inc.
Section 1: Insulin Release: Some Molecular Requisites |
Effect of the Insulin Mimetic L-783,281 on Intracellular [Ca2+] and Insulin Secretion From Pancreatic ß-Cells
1 Department of Chemistry, University of Florida, Gainesville, Florida
2 Department of Molecular Endocrinology, Merck Research Laboratories, R80W250, Rahway, New Jersey
3 Joslin Diabetes Center and Department of Medicine, Harvard Medical School, Boston, Massachusetts
L-783,281, an antidiabetic fungal metabolite that has previously been shown to activate insulin signaling in CHO cells, was tested for its effect on intracellular Ca2+ ([Ca2+]i) and insulin secretion in single mouse pancreatic ß-cells. Application of 10 µmol/l L-783,281 for 40 s to isolated ß-cells in the presence of 3 mmol/l glucose increased [Ca2+]i to 178 ± 10% of basal levels (n = 18) as measured by fluo-4 fluorescence. L-767,827, an inactive structural analog of the insulin mimetic, had no effect on ß-cell [Ca2+]i. The L-783,281-evoked [Ca2+]i increase was reduced by 82 ± 4% (n = 6, P < 0.001) in cells incubated with 1 µmol/l of the SERCA (sarco/endoplasmic reticulum calcium ATPase) pump inhibitor thapsigargin and reduced by 33 ± 6% (n = 6, P < 0.05) in cells incubated with 20 µmol/l of the L-type Ca2+-channel blocker nifedipine. L-783,281–stimulated [Ca2+]i increases were reduced to 31 ± 3% (n = 9, P < 0.05) and 48 ± 10% (n = 6, P < 0.05) of control values by the phosphatidylinositol 3-kinase (PI3-K) inhibitors LY294002 (25 µmol/l) and wortmannin (100 nmol/l), respectively. In ß-cells from IRS-1–/– mice, 10 µmol/l L-783,281 had no significant effect on [Ca2+]i (n = 5). L-783,281 also resulted in insulin secretion at single ß-cells. Application of 10 µmol/l L-783,281 for 40 s resulted in 12.2 ± 2.1 (n = 14) exocytotic events as measured by amperometry, whereas the inactive structural analog had no stimulatory effect on secretion. Virtually no secretion was evoked by L-783,281 in IRS-1–/– ß-cells. LY294002 (25 µmol/l) significantly reduced the effect of the insulin mimetic on ß-cell exocytosis. It is concluded that L-783,281 evokes [Ca2+]i increases and exocytosis in ß-cells via an IRS-1/PI3-K–dependent pathway and that the [Ca2+]i increase involves release of Ca2+ from intracellular stores.
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