IFN GAMMA-TREATED PANCREATIC BETA-CELLS

与基因筛查和临床鉴定相结合的进展 糖尿病前期的标志物，对胰岛素依赖型未来风险的诊断 糖尿病(IDDM)更接近实际情况。严重失踪 是保存胰岛β细胞的有效治疗策略 一旦糖尿病前期患者被识别出来，它就会发挥作用。此应用程序是 基于对分子机制的阐明 在贝塔细胞破坏的早期阶段运作 对这类干预疗法的发展做出了巨大贡献。我们的 研究的重点是了解表型和功能的变化 在β细胞中对干扰素-伽马(IFNGamma)的反应，a 胰岛素样皮损中存在强大的T细胞衍生细胞因子，并需要 用于IDDM开发。证实了临床结果，我们有 证明了在干扰素-γ治疗的β-干扰素中发生了两个主要的变化。 细胞--葡萄糖反应性减弱和主要 组织相容性复合体(MHC)I类抗原处理途径。这个 本提案的总体目标是确定这些项目是否 两个事件通过互动机制联系在一起。我们的基础 假说是细胞内的胰岛素含量在 干扰素-γ处理的β细胞，因为前胰岛素原从 正常分泌途径作为MHC抗原肽的供体 I类程序集。实验的设计是为了在 分子水平、亚基组成和亚细胞定位 被认为在IFNGamma-中产生I类肽的蛋白水解物- 治疗后的β细胞。确定是否存在因果关系 在诱导这种低分子质量多肽(LMP)复合体和 β细胞功能减弱，葡萄糖刺激的胰岛素生物合成 将在LMP基因表达为 熄灭的或结构上升高的。此外，内源性的 与β细胞MHC I类分子结合的多肽将被分离并 测序以确定前胰岛素原是否是主要的多肽供体。都不是 胰岛素RNA表达和胰岛素分泌颗粒胞吐 由IFNGamma更改。干扰素-γ对β细胞的抑制作用 然而，功能确实需要基因转录。因此，IFNGamma的影响 必须由阻止翻译的诱导因素造成 胰岛素mRNA，增强胰岛素降解，或两者兼而有之。要解决这些问题 可能性，研究将区分IFNGamma 对胰岛素原生物合成、降解和转运的影响 葡萄糖刺激的β细胞。因为葡萄糖利用率的降低可能 选择性阻断胰岛素原前体的翻译、葡萄糖摄取和 我们还比较了对照组和干扰素-伽马处理的β细胞的代谢情况。 总的来说，这些研究将确定可能的分子机制 在糖尿病前期的β细胞中运行，从而提供信息 对于弥合IDDM诊断和治疗之间的差距至关重要。

With combined advances in genetic screening and identification of clinical markers of prediabetes, diagnosis for future risk of insulin-dependent diabetes mellitus (IDDM) is close to practical reality. Critically missing are effective therapeutic strategies to preserve pancreatic beta-cell function once prediabetic individuals are identified. This application is based on the contention that elucidation of the molecular mechanisms operative during the early stages of beta-cell destruction would contribute greatly to the development of such intervention therapies. Our research is focused on understanding the phenotypic and functional changes that occur in beta-cells in response to interferon-gamma (IFNgamma), a potent T cell-derived cytokine present in insulitic lesions and required for IDDM development. Corroborating clinical findings, we have demonstrated that two major alterations occur in IFN-gamma-treated beta- cells - - diminished glucose responsiveness and induction of the major histocompatibility complex (MHC) class I antigen - processing pathway. The overall objective of the present proposal is to determine whether these two events are linked through an interactive mechanism. Our underlying hypothesis is that intracellular insulin content is diminished in IFNgamma-treated beta-cells because pre-proinsulin is diverted from the normal secretory pathway and used as a donor of antigenic peptides for MHC class I assembly. Experiments are designed to characterize, at the molecular level, the subunit composition and subcellular location of the proteolytic complex thought to generate class I peptides in IFNgamma- treated beta-cells. To determine if a cause-effect relationship exists between induction of this low molecular mass polypeptide (LMP) complex and diminished beta-cell function, glucose-stimulated insulin biosynthesis will be measured in beta-cells in which LMP gene expression is either extinguished or constitutively elevated. In addition, the endogenous peptides bound to beta-cell MHC class I molecules will be isolated and sequenced to determine if pre-proinsulin is a major peptide donor. Neither insulin RNA expression nor insulin secretory granule exocytosis are altered by IFNgamma. The inhibitory effect of IFNgamma on beta-cell function does however require gene transcription. Thus, IFNgamma's effects must be contributed by induced factors that either block translation of insulin mRNA, enhance insulin degradation, or both. To resolve these possibilities, studies are included that will distinguish IFNgamma's effects on biosynthesis, degradation, and trafficking of pre-proinsulin in glucose-stimulated beta-cells. As a reduction in glucose utilization might selectively block pre-proinsulin translation, glucose uptake and metabolism are also compared in control and IFNgamma-treated beta-cells. Collectively, these studies will define molecular mechanisms likely operative in the prediabetic beta-cell, thereby providing information critical for bridging the gap between diagnosis and treatment of IDDM.