Glucose/Secretagogue Metabolism in Pancreatic Islets

描述（由申请方提供）：广泛的长期目标是获得有关胰岛素分泌代谢信号的新信息。该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素，和所有其他可代谢的促分泌素，通过它们在线粒体中的代谢刺激胰岛素分泌。我们早期的工作表明，一半的葡萄糖衍生的丙酮酸通过丙酮酸羧化酶催化的羧化进入线粒体代谢，一半通过脱羧进入。从羧化产生的草酰乙酸和丙酮酸脱羧产生的乙酰辅酶A，可以合成任何柠檬酸循环中间体。最近的工作表明存在线粒体内和线粒体外的酶，包括琥珀酰- CoA：3-酮酸-CoA转移酶（SCOT），其催化线粒体产物的合成和利用。SCOT可从所有胰岛素促分泌素形成乙酰乙酸。因此，除了柠檬酸循环中间体之外，乙酰乙酸酯还可以将碳转移到胞质溶胶中，用于合成短链酰基辅酶A、脂质和其他因子。目的1A是研究线粒体中分子形成的途径，这些分子被输出到胞质溶胶，用于在胞质溶胶中合成支持或信号胰岛素分泌的化合物。目的1B研究这些化合物的线粒体外利用。具有敲低的丙酮酸羧化酶、SCOT、乙酰乙酰辅酶A合成酶、胞质苹果酸酶、脂肪酸合成酶和其他酶的细胞系;以及酶测定，包括线粒体苹果酸酶的测定，将促进这项工作。最近的研究表明，β细胞是一种脂肪生成组织。用气相色谱法测量细胞和亚细胞组分中的脂质。质谱法用于测量短链酰基辅酶A，并鉴定随促分泌素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。目标2A将进一步研究令人惊讶的观察结果，即在啮齿动物β细胞中非常活跃的回补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表明，丙酮酸羧化酶，回补的主要酶，是90%，低于正常人胰岛的啮齿类动物和克隆β细胞系的胰岛。因此，人辟田胞可以被编程为更大量地使用丙酮酸羧化酶的替代途径用于线粒体合成。将研究人类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是继续研究在啮齿动物和2型糖尿病患者胰岛中减少的线粒体内和线粒体外代谢酶以及可能调节其表达的因子。公共卫生相关性：了解促分泌素刺激的胰岛素分泌的正常代谢途径对于开发人工β细胞（例如通过改变正常的非胰岛素细胞，如肝细胞，或从干细胞开发胰岛素分泌细胞），对于保存用于治疗1型糖尿病的健康胰腺或胰岛，以及对于了解2型糖尿病中受损的胰岛素分泌是必不可少的。

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. PUBLIC HEALTH RELEVANCE: Understanding the normal metabolic pathways of secretagogue-stimulated insulin secretion is essential for developing artificial beta cells (e.g. by altering normal non-insulin cells, such as liver cells, or developing insulin-secreting cells from stem cells), for the preservation of healthy pancreas or islets for use in the treatment of type 1 diabetes and also for understanding the impaired insulin secretion in type 2 diabetes.