Pathogenesis of the Metabolic Syndrome

描述（由申请人提供）： 心脏代谢疾病的范围包括代谢综合征、糖尿病前期、2型糖尿病（T2 DM）和心血管疾病，并且这些疾病在退伍军人和美国人口中造成患者痛苦和医疗保健费用的巨大负担。骨骼肌水平的胰岛素抵抗是所有这些疾病表现的潜在发病机制的核心。关于胰岛素抵抗的机制，传统的假设是长链脂肪酸（LCFA）的氧化受损，而可用性增加，这导致肌内脂质（IMCL）和LCFA的代谢产物（如甘油二酯、LCFA-CoA和神经酰胺）的蓄积。这些后一种化合物激活各种丝氨酸激酶，如PKC或IKK 2（与NF-：B的激活相关），然后磷酸化胰岛素受体底物（IRS）分子，导致胰岛素信号转导受损。然而，我们的初步数据指向一个替代或补充的病理生理过程。第一条线索是IMCL是完全独立的非裔美国人的胰岛素敏感性，而与肌肉中的脂质过氧化有关的氧化应激是欧洲人和非洲裔美国人的胰岛素抵抗的预测。第二条线索来自对正常对照和胰岛素抵抗T2 DM患者血浆的代谢组学研究。T2 DM中的代谢组学特征与长链脂肪酸（LCFA）不完全氧化一致，如中链酰基肉毒碱蓄积所证明，而不是LCFA增加和IMCL甘油三酯蓄积，以及三羧酸（TCA）循环活性降低和回补减少。 鉴于这些观察结果，我们希望测试一个新的假设，即异常代谢组是固有的线粒体缺陷的结果，既损害FA氧化和产生活性氧（ROS）。丝氨酸激酶的激活然后直接由中链酰基肉毒碱和/或由激活炎性途径的线粒体ROS产生的过氧化脂质介导（例如，NG-：B）。在本提案中，我们将在人类中测试这些假设。血糖正常的胰岛素敏感性和胰岛素抵抗受试者以及T2 DM患者将通过高胰岛素钳夹、身体组成、IMCL、能量消耗和底物氧化率进行代谢特征分析，并将在改变胰岛素敏感性的扰动（包括短期极低热量饮食）前后、体重减轻10%后稳定后和胰岛素增敏性噻唑烷二酮治疗后进行研究。将评估血浆、尿液和活检肌肉组织中酰基肉毒碱、脂肪酸、有机酸、氨基酸和LCFA-CoA的代谢组学特征。此外，将使用高分辨率呼吸测定法、活性氧生成和单个呼吸复合物的活性对从肌肉中分离的线粒体的底物氧化能力进行功能研究。将通过羟基壬烯醛和蛋白质羰基来测量肌肉中的氧化应激。将检查氧化应激和/或代谢组学分析物激活肌肉中导致丝氨酸磷酸化和IRS和胰岛素信号转导脱敏的炎症途径的作用。 这些研究代表了新的代谢组学技术的最新应用，结合线粒体功能和胰岛素信号传导的分子研究，在人类肌肉中，并将测试有关异常脂质代谢和胰岛素抵抗之间联系的新假设。这些数据将可预见地确定胰岛素抵抗和T2 DM的新的诊断生物标志物，并推进我们对人类胰岛素抵抗的分子机制的理解。

DESCRIPTION (provided by applicant): The spectrum of cardiometabolic disease encompasses the Metabolic Syndrome, Pre-diabetes, Type 2 Diabetes (T2DM), and cardiovascular disease, and these diseases are responsible for a huge burden of patient suffering and health care costs among veterans and the US population in general. Insulin resistance at the level of skeletal muscle is central to the underlying pathogenesis of all these disease manifestations. Regarding the mechanism of insulin resistance, the conventional hypothesis is that oxidation of long-chain fatty acids (LCFA) is impaired while availability is increased, and this results in the accumulation of intramyocellular lipid (IMCL) and metabolites of LCFAs such as diacylglycerol, LCFA-CoAs, and ceramides. These latter compounds activate various serine kinases, such as PKC or IKK2 (in association with activation of NF-:B), which then phosphorylate insulin receptor substrate (IRS) molecules resulting in impaired insulin signal transduction. However, our preliminary data point to an alternative or complementary pathophysiological process. The first clue was that IMCL was completely independent of insulin sensitivity in African Americans, while oxidative stress related to lipid peroxidation in muscle was predictive of insulin resistance in both European- and African-Americans. The second clue was provided by metabolomic studies of plasma obtained from normal controls and insulin-resistant patients with T2DM. The metabolomic profile in T2DM was consistent with incomplete oxidation of long-chain fatty acids (LCFA) as evidenced by accumulation of medium chain acylcarnitines, rather than increased LCFAs and accumulation as IMCL triglyceride, together with decreased tri-carboxylic acid (TCA) cycle activity and diminished anaplerosis. Given these observations, we wish to test a novel hypothesis that the abnormal metabolome is the result of intrinsic mitochondrial defects that both impair FA oxidation and generate reactive oxygen species (ROS). Activation of serine kinases is then mediated either directly by medium-chain acylcarnitines and/or by peroxidized lipids generated by mitochondrial ROS that activate inflammatory pathways (e.g., NG-:B). In this proposal, we will test these hypotheses in humans. Normoglycemic insulin sensitive and insulin resistant subjects, and patients with T2DM will be metabolically characterized by hyperinsulinemic clamps, body composition, IMCL, energy expenditure and substrate oxidation rates, and will be studied before and after perturbations that alter insulin sensitivity including short-term very low calorie diet, after stabilization following 10% weight loss, and insulin-sensitizing thiazolidinedione treatment. Metabolomic profiles will be assessed for acylcarnitines, fatty acids, organic acids, amino acids, and LCFA-CoAs in plasma, urine, and in biopsied muscle tissue. In addition, mitochondria isolated from muscle will be functionally studied for substrate oxidation capacity using high resolution respirometry, reactive oxygen species generation, and activity of individual respiratory complexes. Oxidative stress will be measured in muscle by hydroxyl-nonenal and protein carbonyls. The effect of oxidative stress and/or metabolomic analytes to activate inflammatory pathways in muscle leading to serine phosphorylation and desensitization of IRS and insulin signaling will be examined. These studies represent a state-of-the-art application of new metabolomics technologies, combined with molecular studies of mitochondrial function and insulin signaling, in human muscle, and will test new hypotheses regarding the link between abnormal lipid metabolism and insulin resistance. These data will predictably identify new diagnostic biomarkers for insulin resistance and T2DM, and advance our understanding of molecular mechanisms underlying human insulin resistance.