CONTROL OF INSULIN BIOSYNTHESIS

描述(由申请人提供)：胰岛素受体底物2缺陷小鼠(Irs2-/-)出生后严重的细胞丢失和糖尿病证实了在胰岛素抵抗的背景下维持？细胞胰岛素信号以适应扩张和生存的必要性。之前在几种细胞类型和一些细胞中的研究已经确立了FoxO和GSK3蛋白的抗增殖和促凋亡作用，这两种蛋白受胰岛素信号的负面调节。研究表明，FoxO1缺乏可以部分恢复IRS2-/-小鼠的细胞团。我们在Irs2-/-小鼠身上的初步研究表明，GSK3缺乏显著增加了细胞的增殖和存活，维持了细胞的质量，还预防了糖尿病。然而，关于这方面的信息仍然很少。 GSK3的细胞特异性效应，或细胞中GSK3或FoxO蛋白缺失的功能后果。为了解决这一问题，我们建立了三种特定细胞的小鼠模型：1)过表达具有构成活性的GSK3？(RIP-GSK3？CA)，2)条件删除GSK3？基因(？-GSK3？KO)，以及3)表达功能缺失的FoxO1突变体(RIP-FoxO？256)。在这项应用中，我们描述了利用这些模型和体外细胞系统来测试假设，即减少细胞中的胰岛素信号(即抵抗)会导致FoxO1和GSK3活性增加，这两种抗增殖和促凋亡蛋白协同工作，促进细胞质量的减少。第一个具体目的是通过鉴定细胞特异性GSK3？缺陷(？-GSK3？KO)小鼠与Irs2/-胰岛素抵抗小鼠杂交，确定GSK3？在细胞扩张和存活中的作用，并结合体外和体内GSK3活性改变的联合模型来确定GSK3？下游的分子机制。另一个目的是通过研究细胞特异性功能丧失的FoxO1小鼠(RIP-FoxO？256)，通过检测IRS2-/-小鼠与RIP-FoxO？256小鼠的杂交，来研究FoxO蛋白在细胞扩张和存活中的作用，并通过体外细胞胰岛素抵抗模型来评估FoxO下游的分子机制。在最后一个目标中，我们将评估单个细胞GSK3和FoxO蛋白的单独和联合缺陷对细胞群适应饮食诱导的肥胖的扩张的影响。在这些模型中将解决几个问题：1)GSK3和FoxO蛋白在 -细胞代谢应激？2)抑制这些蛋白能预防某些形式的糖尿病吗？这些影响是相加的吗？3)下游的效应是什么，其中一些是GSK3和FoxO蛋白所共有的吗？这些研究的预期结果可能有益于避免肥胖胰岛素抵抗患者的糖尿病发病。

DESCRIPTION (provided by applicant): The severe postnatal loss of ¿-cells and diabetes in insulin receptor substrate 2 deficient mice (Irs2-/-) has confirmed the necessity of maintaining ¿-cell insulin signaling for adaptive expansion and survival in the context of insulin resistance. Previous work in several cell types and some in ¿-cells has established the anti-proliferative and pro- apoptotic effects of FoxO and Gsk3¿ proteins that are negatively regulated by insulin signaling. Haplo-insufficiency of FoxO1 has been shown to partially restore ¿-cell mass in Irs2-/- mice. Our preliminary studies in Irs2-/- mice now show that haplo-insufficiency of Gsk3¿ markedly increases ¿-cell proliferation and survival, maintaining ¿-cell mass and also preventing diabetes. However, there is still little information about ¿-cell specific effects of Gsk3¿, or about functional consequences of a deficiency of either Gsk3¿ or FoxO proteins in ¿-cells. To address this we have generated three ¿-cell specific mouse models with: 1) over-expression of a constitutively active Gsk3¿ (RIP-Gsk3¿CA), 2) conditional deletion of the Gsk3¿ gene (¿-Gsk3¿KO), and 3) expression of a loss-of-function mutant of FoxO1 (RIP-FoxO?256). In this application we describe experiments that utilize these models and in vitro cell systems to test the hypothesis that diminished insulin signaling (i.e. resistance) in the ¿-cell leads to increased activity of FoxO1 and GSK3¿, two anti-proliferative and pro-apoptotic proteins that work in concert to promote loss of ¿-cell mass. The first Specific Aim is to determine the role of Gsk3¿ in ¿-cell expansion and survival by characterizing ¿-cell specific Gsk3¿ deficient (¿-Gsk3¿KO) mice, in ¿-Gsk3¿KO mice crossed with Irs2-/- insulin resistant mice, and to define Gsk3¿ downstream molecular mechanisms with combined in vitro and in vivo models of altered Gsk3¿ activity. Another aim is to examine the role of FoxO proteins in ¿-cell expansion and survival by characterizing ¿-cell specific loss-of-function FoxO1 mice (RIP-FoxO?256), by examining the Irs2-/- mouse crossed with the RIP-FoxO?256 mouse, and to assess FoxO downstream molecular mechanisms with an in vitro model of ¿-cell insulin resistance. In the last aim the effects of individual and combined deficiencies of ¿-cell Gsk3¿ and FoxO proteins on the adaptive expansion of ¿-cell mass to diet induced obesity will be assessed. Several questions will be addressed in these models: 1) what are the roles of Gsk3¿ and FoxO proteins in the response of ¿-cells to metabolic stress? 2) Will suppression of these proteins prevent some forms of diabetes and are the effects additive? 3) What are the downstream effectors and are some of them shared by Gsk3¿ and FoxO proteins? The anticipated results of these studies may be beneficial in averting diabetes onset in obese insulin resistant individuals.