IFN GAMMA-TREATED PANCREATIC BETA-CELLS

随着遗传筛查和临床鉴定的综合进展， 糖尿病前期标志物，胰岛素依赖性未来风险的诊断 糖尿病（IDDM）是接近现实的。严重失踪 是保护胰腺β细胞的有效治疗策略， 功能一旦前驱糖尿病个体被识别。 本申请是 基于对分子机制的阐明 在β细胞破坏的早期阶段起作用， 这将极大地促进此类干预疗法的发展。 我们 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-γ（IFN-γ）的反应中发生， 在胰岛病变中存在的有效T细胞衍生的细胞因子， 对于IDDM的发展。根据临床结果，我们有 表明IFN-γ处理的β- 细胞-减少葡萄糖反应和诱导的主要 组织相容性复合体（MHC）I类抗原加工途径。的 本提案的总体目标是确定这些 两项活动通过互动机制联系在一起。我们的基本 假设细胞内胰岛素含量减少， IFN γ处理的β-细胞，因为前胰岛素原从 正常分泌途径，并用作MHC抗原肽的供体 class I assembly.实验旨在表征，在 分子水平上，亚基组成和亚细胞定位的 蛋白水解复合物被认为在IFN γ中产生I类肽， 处理的β细胞。确定是否存在因果关系 在诱导这种低分子量多肽（LMP）复合物和 β细胞功能减弱，葡萄糖刺激的胰岛素生物合成 将在β细胞中测量，其中LMP基因表达是 熄灭或者本质上升高。此外，内源性 将分离与β-细胞MHC I类分子结合的肽， 测序以确定前胰岛素原是否是主要的肽供体。既不 胰岛素RNA表达和胰岛素分泌颗粒胞吐作用 被IFN γ改变IFN γ对β细胞的抑制作用 然而，功能需要基因转录。因此，IFN γ的作用 必须由诱导因素促成，这些因素要么阻止翻译， 胰岛素mRNA、增强胰岛素降解或两者。解决这些 可能性，研究包括将区分IFN γ的 对前胰岛素原生物合成、降解和运输的影响 葡萄糖刺激的β细胞。由于葡萄糖利用率的降低可能 选择性阻断前胰岛素原翻译、葡萄糖摄取和 还比较了对照和IFN γ处理的β细胞中的代谢。 总的来说，这些研究将确定分子机制可能 在糖尿病前期β细胞中起作用，从而提供信息 这对于弥合胰岛素依赖型糖尿病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.