MITOCHONDRIAL FUNCTION AND INSULIN RESISTANCE

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 高血糖和高胰岛素血症的共存表明肝脏和骨骼肌的葡萄糖稳态功能严重失调。该项目(由Newgard博士领导的项目4)的动机是希望了解支链氨基酸(BCAA)与人体两个器官中中间代谢的相互作用。如本申请的概述部分和项目1所述，支链氨基酸可能会与其他大量营养素竞争骨骼肌中的氧化，肌肉和肝脏中的降脂通量可能都是异常的。在高脂肪饮食存在的情况下，预期的次要影响包括肌肉中酰肉碱和细胞内脂肪的积累，这两种物质都是骨骼胰岛素抵抗的标志。当然，特定的氨基酸对胰岛素分泌、肉碱代谢等生化方面也有复杂的影响，目前还不清楚支链氨基酸代谢异常是否会显著影响这两个器官的线粒体功能。我们在计划项目1、2和3中的同事将在分离的细胞和动物模型中测试这些想法，但仍需要测量相关途径的流量，并测量相关人体受试者肝脏和骨骼肌中的特定代谢物。尽管13C示踪方法在研究骨骼肌代谢方面具有潜在的力量，但除了少数实验室外，这项技术很少被使用，原因很简单：还没有找到在柠檬酸循环中有效竞争氧化的合适的富含13C的底物。一个重点是开发更好的方法，在骨骼肌和肝脏中同时提供高13C浓缩的柠檬酸循环中间体。虽然目前的目标是在血浆支链氨基酸升高的情况下评估生物化学，但这些新方法将广泛适用于人体新陈代谢研究，并与该计划项目将技术开发与假说驱动的糖尿病研究联系起来的历史主题保持一致。该项目的具体目标如下： 1)开发一种方案，在清醒大鼠的骨骼肌和肝脏中标记13C到高水平(&gt；20%)的柠檬酸循环中间体。在单独的实验中，富含13C的醋酸盐、羟丁酸盐加丙酸盐、辛酸、乳酸或丙酮酸将被静脉输注，并将在稳态下测定骨骼肌谷氨酸、肝脏谷氨酸和血糖中的13C富集量。能够在骨骼肌中产生高13C丰度和血糖的最佳组合的示踪剂组合(S)将被选择用于进一步的人类研究。 2)将在动物体内开发的~(13)C输注方案翻译到健康人身上，并改进~(31)P磁化转移方法。在骨骼肌中实现稳定同位素标记所需的输注条件将通过7T下的系列1H去耦合13C核磁共振谱来确定。将监测血浆葡萄糖中的13C标记模式，以确保葡萄糖同位素异构体的稳定状态，并确定由肝脏产生的富含13C的葡萄糖是否对血糖有显著贡献。为此，我们还将比较两种31P核磁共振方法，倒置转移和饱和转移，以测量静息骨骼肌的ATP合成率。 3)检验BCAA升高的超重受试者骨骼肌线粒体功能改变和肝脏糖异生改变的基本假设。患者的选择将通过本计划的核心B分析支链氨基酸和相关代谢物来实现。具体地说，我们预计这些个体的骨骼肌线粒体功能(31P核磁共振的ATP周转率)可能是正常的，但这些个体的中间骨骼肌代谢将发生改变，反映为IMCL过多、酰肉碱过多和失调症增加。我们还将测试这些受试者中葡萄糖产生途径的网络是否异常：糖原分解、甘油糖异生和柠檬酸循环中的糖异生。 总而言之，我们的目标是将新的高场核磁共振技术与已有的主要用于了解人类肝脏和骨骼肌中线粒体功能的技术相结合，从而成为一个将项目1、2和3的生物学/机械发现转化为人类研究的项目。将高度优先地制定一项将被患者很好地接受的方案。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Coexistence of hyperglycemia and hyperinsulinemia indicates profound disruption of the glucose homeostasis functions of liver and skeletal muscle. This Project (Project 4 of a Program Project headed by Dr. Newgard) is motivated by the desire to understand the interaction of branched chain amino acids (BCAAs) with intermediary metabolism in both organs in human subjects. As outlined in the Overview section and Project 1 of this application, BCAAs may compete with other macro-nutrients for oxidation in skeletal muscle, and anaplerotic fluxes in both muscle and liver may be abnormal. In the presence of a high fat diet, anticipated secondary effects include accumulation of acylcarnitines and intra-cellular lipids in muscle, both markers of skeletal insulin resistance. Of course, specific amino acids also have complex effects on insulin secretion, carnitine metabolism and other aspects of biochemistry, and at this point it is not clear if abnormal BCAA metabolism significantly impacts mitochondrial function in either organ. Our colleagues in Projects 1, 2 and 3 of the Program Project will test these ideas in isolated cells and in animal models, but there will still be a need to measure fluxes in relevant pathways and measure specific metabolites in both the liver and skeletal muscle in relevant human subjects. In spite of the potential power of 13C tracer methods for studying skeletal muscle metabolism, this technology is little used, outside of a handful of labs, for one simple reason: suitable 13C enriched substrates that compete effectively for oxidation in the citric acid cycle have not been identified. A focus is to develop better methods to deliver high 13C enrichment of citric acid cycle intermediates in skeletal muscle and liver simultaneously. Although the immediate target is to assess biochemistry in the setting of elevated plasma BCAAs, these new methods will be widely applicable to studies of human metabolism and are in keeping with the historical theme of this program project of linking technology development with hypothesis-driven diabetes investigation. The specific aims of the project are as follows: 1) To develop a protocol that labels citric acid cycle intermediates with 13C to a high level (>20%) in both skeletal muscle and liver in conscious rats. In separate experiments, 13C-enriched acetate, ¿ hydroxybutyrate plus propionate, octanoate, and lactate or pyruvate will be infused intravenously, and 13C enrichment in skeletal muscle glutamate, hepatic glutamate and blood glucose will be determined at steady-state. The combination of tracer(s) that yield the best combination of high 13C enrichment in skeletal muscle and plasma glucose will be selected for further studies in humans. 2) To translate the 13C infusion protocol developed in animals to healthy human subjects and refine 31P magnetization transfer methods. The infusion conditions necessary to achieve steady-state isotope labeling in skeletal muscle will be determined by serial 1H decoupled 13C NMR spectra at 7T. The 13C labeling pattern in plasma glucose will be monitored to assure steady-state in glucose isotopomers and to determine whether 13C enriched glucose, generated by the liver, contributes significantly to plasma glucose. In this aim, we will also compare two 31P NMR methods, inversion transfer and saturation transfer, for measurement of ATP synthesis rates in resting skeletal muscle. 3) To test the primary hypothesis that overweight subjects with elevated BCAAs have altered mitochondrial function in skeletal muscle plus altered liver gluconeogenesis. Selection of patients will be achieved by analysis of BCAA and related metabolites by Core B of this program. Specifically, we anticipate that skeletal muscle mitochondrial function (ATP turnover by 31P NMR) may be normal in these individuals but that intermediary skeletal muscle metabolism in these individuals will be altered as reflected by excess IMCL, excess acylcarnitines and an increase in anaplerosis. We will also test whether the network of glucose production pathways  glycogenolysis, gluconeogenesis from glycerol and gluconeogenesis from the citric acid cycle  in liver is abnormal among these subjects. In summary, our goal is to integrate new high field NMR technologies with those already largely in hand for understanding mitochondrial function in liver and skeletal muscle of humans, thereby serving as a project that translates the biological / mechanistic findings of Projects 1, 2, and 3 to human studies. A high priority will be placed on developing a protocol that will be well-accepted by patients.