title: Inhibition of cholesterol biosynthesis impairs insulin secretion and voltage-gated calcium channel function in pancreatic beta-cells.
abstract: Insulin secretion from pancreatic beta-cells is mediated by the opening of voltage-gated Ca2+ channels (CaV) and exocytosis of insulin dense core vesicles facilitated by the secretory soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein machinery. We previously observed that beta-cell exocytosis is sensitive to the acute removal of membrane cholesterol. However, less is known about the chronic changes in endogenous cholesterol and its biosynthesis in regulating beta-cell stimulus-secretion coupling. We examined the effects of inhibiting endogenous beta-cell cholesterol biosynthesis by using the squalene epoxidase inhibitor, NB598. The expression of squalene epoxidase in primary and clonal beta-cells was confirmed by RT-PCR. Cholesterol reduction of 36-52% was observed in MIN6 cells, mouse and human pancreatic islets after a 48-h incubation with 10 mum NB598. A similar reduction in cholesterol was observed in the subcellular compartments of MIN6 cells. We found NB598 significantly inhibited both basal and glucose-stimulated insulin secretion from mouse pancreatic islets. CaV channels were markedly inhibited by NB598. Rapid photolytic release of intracellular caged Ca2+ and simultaneous measurements of the changes in membrane capacitance revealed that NB598 also inhibited exocytosis independently from CaV channels. These effects were reversed by cholesterol repletion. Our results indicate that endogenous cholesterol in pancreatic beta-cells plays a critical role in regulating insulin secretion. Moreover, chronic inhibition of cholesterol biosynthesis regulates the functional activity of CaV channels and insulin secretory granule mobilization and membrane fusion. Dysregulation of cellular cholesterol may cause impairment of beta-cell function, a possible pathogenesis leading to the development of type 2 diabetes.
authors.affiliation: Department of Medicine, University of Toronto, Toronto, Ontario, Canada M3J 1P3.
authors.forename: Fuzhen
authors.initials: F
authors.lastname: Xia
authors.forename: Li
authors.initials: L
authors.lastname: Xie
authors.forename: Anton
authors.initials: A
authors.lastname: Mihic
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Gao
authors.forename: Yi
authors.initials: Y
authors.lastname: Chen
authors.forename: Herbert Y
authors.initials: HY
authors.lastname: Gaisano
authors.forename: Robert G
authors.initials: RG
authors.lastname: Tsushima
mesh_terms: D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology*; D002460:Cell Line; D002784:Cholesterol / Q000096:biosynthesis*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure; D021962:Membrane Microdomains / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D008854:Microscopy, Electron; D015221:Potassium Channels / Q000502:physiology; D050600:SNARE Proteins / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism / Q000648:ultrastructure; D050603:Squalene Monooxygenase / Q000037:antagonists & inhibitors / Q000378:metabolism; D013876:Thiophenes / Q000494:pharmacology
chemical_list: D001596:Benzylamines; D015220:Calcium Channels; D004791:Enzyme Inhibitors; D007328:Insulin; D015221:Potassium Channels; D050600:SNARE Proteins; D013876:Thiophenes; C066059:NB 598; D002784:Cholesterol; D050603:Squalene Monooxygenase