Effect of the insulin mimetic L-783,281 on intracellular Ca2+ and insulin secretion from pancreatic beta-cells.
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 Ca(2+) ([Ca(2+)](i)) and insulin secretion in single mouse pancreatic beta-cells. Application of 10 micromol/l L-783,281 for 40 s to isolated beta-cells in the presence of 3 mmol/l glucose increased [Ca(2+)](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 beta-cell [Ca(2+)](i). The L-783,281-evoked [Ca(2+)](i) increase was reduced by 82 +/- 4% (n = 6, P < 0.001) in cells incubated with 1 micromol/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 micromol/l of the L-type Ca(2+)-channel blocker nifedipine. L-783,281-stimulated [Ca(2+)](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 micromol/l) and wortmannin (100 nmol/l), respectively. In beta-cells from IRS-1-/- mice, 10 micromol/l L-783,281 had no significant effect on [Ca(2+)](i) (n = 5). L-783,281 also resulted in insulin secretion at single beta-cells. Application of 10 micromol/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-/- beta-cells. LY294002 (25 micromol/l) significantly reduced the effect of the insulin mimetic on beta-cell exocytosis. It is concluded that L-783,281 evokes [Ca(2+)](i) increases and exocytosis in beta-cells via an IRS-1/PI3-K-dependent pathway and that the [Ca(2+)](i) increase involves release of Ca(2+) from intracellular stores.
Roper
Michael G
MG
Department of Chemistry, University of Florida, Gainesville, FL, USA.
Qian
Wei-jun
WJ
Zhang
Bei B
BB
Kulkarni
Rohit N
RN
Kahn
C Ronald
CR
Kennedy
Robert T
RT
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003924:Diabetes Mellitus, Type 2 / Q000188:drug therapy / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology*; D007328:Insulin / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018716:Molecular Mimicry; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D019284:Thapsigargin / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007211:Indoles; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; C119066:L 783281; D010750:Phosphoproteins; D019284:Thapsigargin; D019869:Phosphatidylinositol 3-Kinases; D002118:Calcium