Identification of Muscle-Specific Biomarkers of Fatty Acid beta-Oxidation

描述（由申请人提供）：骨骼肌细胞内脂肪水平升高（肌细胞内脂质）与肌肉和全身胰岛素抵抗高度相关，在肥胖症中更为普遍。驱动这种关联的分子联系和代谢变化仍然存在争议，但值得注意的是，肌肉线粒体脂肪酸（FA）β-氧化减少在胰岛素抵抗/糖尿病患者中更为普遍。因此，发现反映个体肌肉FA β-氧化活性或能力状态的生物标志物将在糖尿病方面具有巨大的预后和诊断价值。此外，与肌肉线粒体脂肪代谢相关的代谢物的表征应该揭示将FA β-氧化与胰岛素信号传导联系在一起的候选信号传导因子。我们建议首次使用多个代谢组学分析平台来识别肌肉FA β氧化的特定生物标志物，以比较来自显示不同肌肉脂肪燃烧的生物系统的样品中的代谢物谱，包括：分离的线粒体细胞器和以不同速率分解代谢FA的肌肉细胞，UCP 3转基因动物模型，和具有UCP 3截短多态性的人类受试者（目的1和2）。目标3中的初步验证研究将测试是否可以通过旨在改善肌肉健康和FA燃烧的饮食-运动方案，在肥胖、胰岛素抵抗受试者中通过实验增加追踪肌肉FA β氧化的血浆代谢物和/或代谢物特征（如目标1和2中所确定的）。目的1--在细胞器、细胞和动物模型中识别肌肉脂肪燃烧的代谢物生物标志物，这些生物标志物显著改变脂肪酸β-氧化。我们将确定代谢物谱如何在显示增加的肌肉β-氧化的模型中变化（解偶联蛋白3过表达的肌肉细胞系和肌肉UCP 3转基因小鼠），并假设UCP 3过表达系统中的谱将反映增加的FA β-氧化。补充研究将确定组织特异性代谢物产生的线粒体在过程中的棕榈酸酯catalysts在体外，比较肌肉，肝脏和肾脏的准备。目的2--鉴定携带UCP 3错义多态等位基因的人类肌肉脂肪燃烧的代谢物生物标志物。我们预测，与没有多态性的受试者相比，具有这种多态性的受试者（产生截短的UCP 3和50%的全身脂肪燃烧减少）将显示出独特的血浆代谢产物谱，表明肌肉FA氧化减少。目的3--确定反映肌肉脂肪燃烧的代谢组学特征是否能预测肥胖受试者饮食和运动干预后的代谢健康变化。我们假设，在经历4个月饮食-运动方案的久坐肥胖受试者队列中，反映正常到增加的肌肉β-氧化的生物标志物将增加，并且指示不良肌肉脂肪燃烧的标志物减少，这将增加肌肉健康并改善胰岛素作用。 公共卫生相关性：胰腺激素胰岛素触发组织摄取血糖的能力降低是2型糖尿病（T2 DM）发展过程中的早期事件，并且肌肉床是许多人中这种现象的重要部位。即使在糖尿病前期状态下，禁食肌肉相对较差的脂肪燃烧通常与胰岛素抵抗相关。因此，我们研究的总体目标--确定肌肉脂肪代谢的临床相关生物标志物--对于帮助识别高危人群、确定疾病病因并最终通过营养、体力活动和药物干预阻止T2 DM的发展至关重要。

DESCRIPTION (provided by applicant): Elevated fat levels within skeletal muscle cells (intramyocellular lipids) are highly correlated with muscle and whole-body insulin resistance, and more prevalent in obesity. The molecular links and metabolic shifts driving this association remain open to debate, but notably, reduced muscle mitochondrial fatty acid (FA) beta-oxidation is more prevalent among insulin-resistant/diabetic persons. Therefore, discovery of biomarkers reflective of the status of an individual's muscle FA beta-oxidation activity or capacity would have tremendous prognostic and diagnostic value in terms of diabetes. Furthermore, characterization of metabolites associated with muscle mitochondrial fat metabolism should uncover candidate signaling factors which tie FA beta-oxidation to insulin signaling. We propose to identify, for the first time, specific biomarkers of muscle FA beta- oxidation using multiple metabolomic analytical platforms to compare metabolite profiles in samples derived from biological systems displaying disparate muscle fat combustion, including: isolated mitochondrial organelles and muscle cells catabolizing FA at different rates, a UCP3 transgenic animal model, and human subjects harboring a UCP3 truncation polymorphism (Aims 1 & 2). Pilot validation studies in Aim 3 will test whether plasma metabolites and/or metabolite signatures that track muscular FA beta-oxidation (as identified in Aims 1 and 2) can be experimentally increased in obese, insulin-resistant subjects via a diet-exercise regimen designed to improve muscle fitness and FA combustion. Aim 1--Identify Metabolite Biomarkers of Muscle Fat Combustion in Organelle, Cell, and Animal Models Displaying Significantly Altered Fatty Acid beta-Oxidation. We will determine how metabolite profiles shift in models displaying increased muscle beta-oxidation (uncoupling protein 3-overexpressing muscle cell line and muscle UCP3-transgenic mice), and hypothesize that profiles in UCP3-overexpressing systems will reflect increased FA beta-oxidation. Complementary studies will identify tissue-specific metabolites generated by mitochondria in the course of palmitate catabolism in vitro, comparing muscle to liver and kidney preparations. Aim 2--Identify Metabolite Biomarkers of Muscle Fat Combustion in Humans Harboring a UCP3 Missense Polymorphic Allele. We predict that subjects with this polymorphism (which yields a truncated UCP3 and 50% decreased whole-body fat combustion) will display a distinctive plasma metabolite profile indicative of reduced muscle FA oxidation, when compared to subjects without the polymorphism. Aim 3--Determine Whether Metabolomic Profiles Reflective of Muscle Fat Combustion Predict Metabolic Health Changes Following Diet & Exercise Intervention in Obese Subjects. We hypothesize that biomarkers reflective of normal to increased muscle beta-oxidation will be increased, and markers indicative of poor muscle fat combustion reduced, in a cohort of sedentary obese subjects undergoing a 4 month diet- exercise protocol which will increase muscle fitness and improve insulin action. PUBLIC HEALTH RELEVANCE: A reduced ability of the pancreatic hormone insulin to trigger tissue uptake of blood sugar is an early event in the course of development of type 2 diabetes mellitus (T2DM), and the muscle beds are important sites for this phenomenon in many people. Relatively poor fat combustion by fasting muscle is often correlated with insulin resistance, even in the pre-diabetic state. Thus, the overarching aim of our research-- identification of clinically-relevant biomarkers of muscle fat metabolism--is critical to help identify at-risk persons, determine etiology of disease, and ultimately thwart development of T2DM through nutritional, physical activity, and pharmacological interventions.