CELL BIOLOGICAL STUDIES ON DIABETES AND METABOLISM

本项目的超微结构和生物化学研究将 增加我们对调节聚集的机制的了解， 胰岛素受体的内化和细胞内加工 复杂，并将决定这些进程，特别是核 胰岛素的转运及胰岛素对大分子摄取的影响 与胰岛素的调节有关 细胞新陈代谢和生长的关键 未来五年的具体目标 年是：1）使用免疫电镜技术，以文件 组织、分配、横向流动、内部化途径 以及肝脏和骨骼上胰岛素受体的细胞内加工 肌肉. 这些组织，除了脂肪细胞， 被研究的是主要的胰岛素敏感组织。 三 组织对胰岛素的反应不同。 组织特异 胰岛素受体复合物加工的差异，这可能是 观察，应该是有价值的，在理解之间的关系， 胰岛素-受体复合物加工和胰岛素作用。 2)使用高 分辨率超微结构技术和细胞表达正常 或突变的人胰岛素受体，以确定哪些结构域的胰岛素受体， 胰岛素受体负责正常组织， 分布、横向移动、内化和细胞内 胰岛素-受体复合物的加工。 这些研究将提供 超微结构分析，以补充有关 胰岛素受体的分子结构之间的关系 以及聚集、内化和适当的细胞内 靶向胰岛素受体复合物，导致正常胰岛素 行动上 类似的研究将使用表达正常细胞的细胞进行。 或突变的人IGF I受体。 3)利用超微结构和 生物化学技术来表征细胞内途径 负责胰岛素的移位和核积累。 通过确定这种细胞内途径，我们应该更多地了解 胰岛素调节基因转录和细胞的机制 增长 在一些研究中， 胰岛素的积累将用于强调 胞内易位路线。 4)以确定是否 胰岛素对细胞增殖或基因表达影响 随着胰岛素的核积累和/或胰岛素对 大分子进入细胞核和从细胞核排出。 我们的假设， 胰岛素在细胞核中的积累及其对 大分子易位具有生理学意义， 可能与细胞生长和/或基因表达相关， 通过评估细胞类型中的这些过程，包括那些表达 突变的人胰岛素受体，具有不同的生长相关反应 到胰岛素。 超微结构，细胞和分子技术， 包括原位电镜杂交、北方印迹 分析等等，将用于这些研究。 我们将确定 胰岛素的积累和胰岛素刺激的大分子 核摄取是细胞周期依赖性的。 所得到的资料 该项目的研究将提供对正常机制的见解， 胰岛素作用及胰岛素抵抗和糖尿病的潜在原因 糖尿病。 这些见解可能会提供可能的替代方案， 干预或治疗。

The ultrastructural and biochemical studies in this project will increase our knowledge of the mechanisms that regulate the aggregation, internalization and intracellular processing of the insulin-receptor complex and will determine whether those processes, especially nuclear translocation of insulin and insulin's effects on macromolecular uptake into and efflux from the nucleus, are involved in insulin's regulation of cellular metabolism and growth. The specific aims for the next five years are: 1) To use immunoelectron microscopic techniques to document the organization, distribution, lateral mobility, internalization routes and intracellular processing of insulin receptors on liver and skeletal muscle. These tissues, in addition to the adipocyte that has already been studied, are the primary insulin-sensitive tissues. The three tissues differ in their responses to insulin. Tissue-specific differences in insulin-receptor complex processing, which may be observed, should be valuable in understanding the relationship between hormone-receptor complex processing and insulin action. 2) To use high resolution ultrastructural techniques and cells expressing either normal or mutated human insulin receptors to determine which domains of the insulin receptor are responsible for the normal organization, distribution, lateral mobility, internalization and intracellular processing of the insulin-receptor complex. These studies will provide ultrastructural analysis to complement biochemical data concerning the relationships between the molecular structure of the insulin receptor and the aggregation, internalization and appropriate intracellular targeting of the insulin-receptor complex leading to normal insulin action. Similar studies will be performed using cells expressing normal or mutated human IGF I receptors. 3) To use ultrastructural and biochemical techniques to characterize the intracellular route responsible for the translocation and nuclear accumulation of insulin. By determining this intracellular pathway, we should learn more about the mechanism by which insulin regulates gene transcription and cell growth. In some of these studies agents that affect the nuclear accumulation of insulin will be used to accentuate key components of the intracellular translocation itinerary. 4) To determine whether or not insulin's effects on cell proliferation or gene expression correlate with the nuclear accumulation of insulin and/or insulin's effects on macromolecular uptake into and efflux from the nucleus. Our hypothesis, that the nuclear accumulation of insulin and its effects on macromolecular translocation are physiologically significant and possibly related to cell growth and/or gene expression, will be tested by evaluating these processes in cell types, including those expressing mutated human insulin receptors, with different growth-related responses to insulin. Ultrastructural, cellular and molecular techniques, including in situ electron microscopic hybridization, Northern blot analysis, etc., will be used for these studies. We will determine whether insulin accumulation and insulin-stimulated macromolecular nuclear uptake is cell-cycle dependent. The information gained from studies in this project will provide insights into normal mechanisms of insulin action and potential causes of insulin-resistance and diabetes mellitus. These insights may then provide possible alternatives for intervention or therapy.