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

描述（由申请方提供）：主要研究者的长期目标是表征导致胰腺β细胞胰岛素分泌功能活力丧失的生化机制。目前正在开发利用多核核磁共振波谱技术和稳定同位素示踪法动态测量胰岛β细胞代谢通量的方法。本提案旨在确定β细胞代谢与胰岛素分泌偶联所需的几种代谢途径中的哪一种导致葡萄糖刺激的胰岛素分泌受损，这是年轻3型成熟型糖尿病（MODY-3）的原因。这项研究将加强PI对胰岛β细胞生理学的持续研究。MODY-3的特征在于β细胞功能障碍的发作，导致缺乏适当的胰岛素分泌反应和严重的高血糖症。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INS-1上，INS-1转染了与MODY-3相关的最常见突变。这些模型表明，受损的糖酵解通量，改变线粒体！代谢和氧化磷酸化或UCP-2表达的上调可能有助于胰岛素分泌的减少。拟议的研究将提供线粒体氧化和回补途径的调节的全面评估，通过改变UCP-2活性和氧化磷酸化效率的NMR光谱测量，并在这些MODY-3模型中进行补充。现在认识到，如果没有显著的回补通量进入Kreb循环，线粒体ATP产生本身不足以用于胰岛素分泌。通过同位素方法对代谢途径的定量分析表明，胰岛素分泌与通过丙酮酸羧化酶（PC）的回补通量相关性最好。PC通量与胰岛素分泌偶联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。本研究将结合联合收割机同位素分析与苹果酸酶（丙酮酸循环所需的）的siRNA敲低来解决这个问题，并确定HNF-1 α可能在连接代谢与胰岛素分泌的途径中的作用。

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.