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（细胞内脂质）与肌肉和全身胰岛素抵抗高度相关，在肥胖中更为普遍。驱动这种关联的分子联系和代谢变化仍有争议，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具有巨大的糖尿病预后和诊断价值。此外，表征与肌肉线粒体脂肪代谢相关的代谢物应该揭示将FA β氧化与胰岛素信号传导联系起来的候选信号因子。我们建议首次使用多种代谢组学分析平台来鉴定肌肉FA β -氧化的特定生物标志物，以比较来自不同肌肉脂肪燃烧的生物系统样品中的代谢物谱，包括：分离的线粒体细胞器和肌肉细胞以不同的速率分解FA， UCP3转基因动物模型，以及具有UCP3截断多态性的人类受试者（目的1和2）。Aim 3中的试点验证研究将测试是否通过旨在改善肌肉健康和FA燃烧的饮食-运动方案，在肥胖、胰岛素抵抗的受试者中，血浆代谢物和/或追踪肌肉FA β氧化的代谢物特征（如Aims 1和2中所确定的）可以实验性地增加。目标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，这些模型显示脂肪酸β -氧化显著改变。我们将确定在肌肉β -氧化增加的模型（解偶联蛋白3过表达的肌肉细胞系和肌肉ucp3转基因小鼠）中代谢物谱是如何变化的，并假设ucp3过表达系统中的谱将反映FA β -氧化增加。补充研究将确定线粒体在体外棕榈酸分解代谢过程中产生的组织特异性代谢物，并将肌肉与肝脏和肾脏制剂进行比较。目的2-鉴定人类肌肉脂肪燃烧的代谢物生物标志物，其中包含UCP3错义多态性等位基因。我们预测，与没有这种多态性的受试者相比，具有这种多态性的受试者（产生截断的UCP3和减少50%的全身脂肪燃烧）将显示出独特的血浆代谢物谱，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代谢健康的变化。我们假设，在一组久坐不动的肥胖受试者中，反映正常到增加的肌肉β -氧化的生物标志物会增加，而表明肌肉脂肪燃烧不良的标志物会减少，这些受试者接受4个月的饮食-运动方案，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（T2DM）发展过程中的一个早期事件，肌肉床是许多人出现这一现象的重要部位。禁食肌肉相对较差的脂肪燃烧通常与胰岛素抵抗有关，即使在糖尿病前期也是如此。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、体育活动和药物干预阻止2型糖尿病的发展至关重要。

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.