Effect of the Insulin Mimetic L-783,281 on Intracellular [Ca²⁺] and Insulin Secretion From Pancreatic β-Cells

Abstract

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 β-cells. Application of 10 μmol/l L-783,281 for 40 s to isolated β-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 β-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 β-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 β-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^–/– β-cells. LY294002 (25 μmol/l) significantly reduced the effect of the insulin mimetic on β-cell exocytosis. It is concluded that L-783,281 evokes [Ca²⁺]_i increases and exocytosis in β-cells via an IRS-1/PI3-K–dependent pathway and that the [Ca²⁺]_i increase involves release of Ca²⁺ from intracellular stores.