Glucose/Secretagogue Metabolism in Pancreatic Islets

描述(由申请人提供)：广泛的长期目标是获得有关胰岛素分泌的代谢信号的新信息。这个项目的直接目的是更好地了解线粒体生物合成途径在支持胰岛素分泌中的作用。葡萄糖，最有效的胰岛素促分泌剂，和所有其他可代谢的促分泌剂，通过它们在线粒体中的新陈代谢来刺激胰岛素的分泌。我们早期的工作表明，葡萄糖衍生的丙酮酸有一半是通过丙酮酸羧基酶催化的羧化作用进入线粒体代谢的，另一半是通过脱羧基作用进入线粒体的。由丙酮酸脱羧得到的草酰乙酸酯和乙酰辅酶A合成的柠檬酸循环中间体可以合成任何柠檬酸循环中间体。最近的研究表明，线粒体内和线粒体外的酶的存在，包括琥珀酰辅酶A：3-酮酸-辅酶A转移酶(SCOT)，催化线粒体产物的合成和利用。SCOT可以从所有的胰岛素促分泌剂中生成乙酰乙酸酯。因此，乙酰乙酸酯除了是柠檬酸循环的中间体外，还可以将碳转移到胞浆中，用于合成短链酰基COAs、脂类等因子。目标1A是研究线粒体中分子的形成途径，这些分子被输出到胞浆中，以合成胞浆中支持或信号胰岛素分泌的化合物。目的1B研究这些化合物对线粒体外的利用。具有击倒的丙酮酸羧基酶、SCOT、乙酰乙酰-CoA合成酶、胞质苹果酸酶、脂肪酸合成酶和其他酶的细胞系；以及酶分析，包括线粒体苹果酸酶的分析，将有助于这项工作。最近的研究表明，β细胞是一种生脂组织。用气相色谱测定细胞和亚细胞组分中的脂类。质谱学被用来测量短链酰基-COA，并识别随着促分泌剂刺激而变化的小分子和脂质。目的2是辨别线粒体生物合成与人类正常和异常胰岛素分泌的相关性。Aim 2A将进一步研究这一令人惊讶的观察结果，即在啮齿动物β细胞中非常活跃的逆转途径在人类胰岛中的活性要低得多。我们已经积累了确凿的证据，丙酮酸羧基酶是复苏的主要酶，在正常人的胰岛中比在啮齿类动物的胰岛和克隆性β细胞系中低90%。因此，人类β细胞可能被编程为更多地使用替代途径来丙酮酸羧化酶来合成线粒体。将研究人胰岛中丙酮酸羧化酶表达和丙酮酸羧化速率的调节。目的2B是继续研究啮齿类动物和人类2型糖尿病患者胰岛线粒体内和线粒体外代谢酶的减少，以及可能调节它们表达的因素。

DESCRIPTION (provided by applicant): The broad long-term objective is to obtain new information about the metabolic signals for insulin secretion. The immediate purpose of this project is to gain a better understanding of the role of mitochondrial biosynthetic pathways in supporting insulin secretion. Glucose, the most potent insulin secretagogue, and all other metabolizable secretagogues, stimulate insulin secretion via their metabolism in mitochondria. Our earlier work showed that one-half of glucose-derived pyruvate enters mitochondrial metabolism via carboxylation catalyzed by pyruvate carboxylase and one-half enters via decarboxylation. From the resulting oxaloacetate from carboxylation and acetyl-CoA from decarboxylation of pyruvate, any citric acid cycle intermediate can be synthesized. Recent work showed the presence of intra- and extra-mitochondrial enzymes, including succinyl- CoA: 3-ketoacid-CoA transferase (SCOT), that catalyze the synthesis and utilization of mitochondrial products. SCOT can form acetoacetate from all insulin secretagogues. Thus acetoacetate, in addition to citric acid cycle intermediates, can transfer carbon to the cytosol for the synthesis of short chain acyl-CoAs, lipids and other factors. Aim 1A is to study the pathways in mitochondria of formation of molecules that are exported to the cytosol for the synthesis of the compounds in the cytosol that support or signal insulin secretion. Aim 1B is to study the extramitochondrial utilization of these compounds. Cell lines with knocked down pyruvate carboxylase, SCOT, acetoacetyl-CoA synthetase, cytosolic malic enzyme, fatty acid synthase and other enzymes; as well as enzyme assays, including an assay for mitochondrial malic enzyme, will facilitate this work. Recent work has shown the beta cell is a lipogenic tissue. Lipids in cells and subcellular fractions are measured with gas chromatography. Mass spectrometry is used to measure short chain acyl-CoAs and to identify small molecules and lipids that change with secretagogue stimulation. Aim 2 is to discern the relevance of mitochondrial biosynthesis to normal and abnormal insulin secretion in humans. Aim 2A will further study the surprising observation that pathways of anaplerosis that are very active in rodent beta cells are far less active in human islets. We have accumulated conclusive evidence that pyruvate carboxylase, the major enzyme of anaplerosis, is 90% lower in normal human islets than in islets of rodents and clonal beta cell lines. Thus, the human beta cell may be programmed to more heavily use alternative pathways to pyruvate carboxylase for mitochondrial synthesis. The regulation of pyruvate carboxylase expression and rate of pyruvate carboxylation in human islets will be studied. Aim 2B is to continue to study intra- and extra- mitochondrial metabolic enzymes that are decreased in islets of rodents and humans with type 2 diabetes and factors that might regulate their expression.