Xia Fuzhen, Xie Li, Mihic Anton, Gao Xiaodong, Chen Yi, Gaisano Herbert Y, Tsushima Robert G
Department of Medicine, University of Toronto, Toronto, Ontario, Canada M3J 1P3.
Endocrinology. 2008 Oct;149(10):5136-45. doi: 10.1210/en.2008-0161. Epub 2008 Jul 3.
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.
胰腺β细胞的胰岛素分泌是由电压门控Ca2+通道（CaV）的开放介导的，并且分泌性可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体蛋白机制促进胰岛素致密核心囊泡的胞吐作用。我们之前观察到β细胞胞吐作用对膜胆固醇的急性去除敏感。然而，关于内源性胆固醇的慢性变化及其生物合成在调节β细胞刺激-分泌偶联中的作用知之甚少。我们使用角鲨烯环氧酶抑制剂NB598研究了抑制内源性β细胞胆固醇生物合成的作用。通过RT-PCR证实了角鲨烯环氧酶在原代和克隆β细胞中的表达。用10 μM NB598孵育48小时后，在MIN6细胞、小鼠和人胰岛中观察到胆固醇降低了36 - 52%。在MIN6细胞的亚细胞区室中也观察到类似的胆固醇降低。我们发现NB598显著抑制小鼠胰岛的基础和葡萄糖刺激的胰岛素分泌。CaV通道被NB598显著抑制。细胞内笼锁Ca2+的快速光解释放以及膜电容变化的同步测量表明，NB598也独立于CaV通道抑制胞吐作用。这些作用通过补充胆固醇得以逆转。我们的结果表明，胰腺β细胞中的内源性胆固醇在调节胰岛素分泌中起关键作用。此外，胆固醇生物合成的慢性抑制调节CaV通道的功能活性以及胰岛素分泌颗粒的动员和膜融合。细胞胆固醇的失调可能导致β细胞功能受损，这是导致2型糖尿病发生的一种可能的发病机制。
