Metabolic and Mitochondrial Defects of Islet beta-cells of MODY-3

描述（由申请人提供）：首席研究员的长期目标是描述导致胰岛β细胞胰岛素分泌功能丧失的生化机制。利用多核核磁共振光谱技术和稳定同位素示踪剂方法，正在开发对胰岛β细胞代谢通量进行动态测量的方法。本研究旨在确定β细胞代谢与胰岛素分泌耦合所必需的几种代谢途径中的哪一种导致葡萄糖刺激胰岛素分泌受损，从而导致年轻3型糖尿病（MODY-3）的成熟发病。这项研究将加强正在进行的胰岛β细胞生理学的PI研究。MODY-3的特点是β细胞功能障碍，导致缺乏适当的胰岛素分泌反应和严重的高血糖。MODY-3是一种遗传性缺陷，与肝核因子1 α (hnf -1 α)转录因子的显性负突变有关。鉴定分子和功能靶点的尝试集中在HNF-1 α敲除小鼠模型和稳定的胰岛素瘤细胞系ns -1上，该细胞系转染了与MODY-3相关的最常见突变。这些模型表明糖酵解通量受损，线粒体改变！代谢和氧化磷酸化，或UCP-2表达上调可能导致胰岛素分泌减少。该研究将全面评估线粒体氧化和复变途径的调节，并通过核磁共振光谱测量改变的UCP-2活性和氧化磷酸化效率，以及这些MODY-3模型。现在人们认识到，如果没有大量的回凝通量进入克雷伯循环，线粒体ATP的产生本身就不足以分泌胰岛素。利用同位素分析方法对代谢途径进行了定量分析，结果表明胰岛素分泌与丙酮酸羧化酶（PC）的复排通量密切相关。PC通量与胰岛素分泌耦合的机制可能涉及依赖于丙酮酸循环的信号转导，或者可能是由于下游第二信使的产生。本研究将结合同位素分析和苹果酸酶（丙酮酸循环所必需的）的siRNA敲低来解决这个问题，并确定HNF-1 α在代谢与胰岛素分泌联系的途径中可能发挥的作用。

DESCRIPTION (provided by applicant): The Principal Investigator's long-term objectives are to characterize the biochemical mechanisms that lead to loss of functional viability of insulin secretion from pancreatic beta-cells. Methods are being developed to make dynamic measurements of metabolic flux in islet beta-cells by using multinuclear NMR spectroscopic techniques and stable isotopic tracer methodology. The present proposal seeks to identify which of the several metabolic pathways necessary for coupling beta-cell metabolism with insulin secretion cause the impaired glucose stimulated insulin secretion responsible for maturity-onset diabetes of the young type 3 (MODY-3). This research will augment on-going research of the PI into islet beta-cell physiology. MODY-3 is characterized by the onset of beta-cell dysfunction leading to absence of an appropriate insulin secretory response and severe hyperglycemia. MODY-3 is an inherited defect that has been linked to a dominant negative mutation of the hepatic nuclear factor 1 alpha (HNF-1alpha) transcription factor. Attempts to identify the molecular and functional targets have focused on the HNF-1 alpha knockout mouse model, and a stable insulinoma cell line, INS-1, transfected with the most common mutation associated with MODY-3. These models suggest that impaired glycolytic flux, altered mitochondria! metabolism and oxidative phosphorylation, or upregulation of UCP-2 expression may contribute to the reduction in insulin secretion. The proposed study will provide a comprehensive evaluation of the regulation of mitochondrial oxidative and anaplerotic pathways, complemented by NMR spectroscopic measurements of altered UCP-2 activity and oxidative phosphorytion efficiency, and in these MODY-3 models. It is now appreciated that without significant anaplerotic flux into the Kreb's cycle, mitochondrial ATP production per se is not sufficient for insulin secretion. Quantitative analysis of metabolic pathways, by isotopomer methods, indicates that insulin secretion correlates best with anaplerotic flux through pyruvate carboxylase (PC). The mechanism whereby PC flux couples with insulin secretion may involve transduction of a signal dependent on pyruvate cycling, or may be due to the generation of downstream second messenger. This study will combine isotopomer analysis with siRNA knockdown of malic enzyme (required for pyruvate cycling) to resolve this question and to determine the role that HNF-1 alpha may have on the pathways linking metabolism with insulin secretion.