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

描述（由申请人提供）：组织内脂肪水平升高和长链脂肪酸（LCFA）氧化分解代谢能力降低/效率低下与肌肉、肝脏、脂肪和全身胰岛素抵抗高度相关。细胞内或细胞外浓度随线粒体LCFA燃烧能力或效率的变化而变化的特定代谢物可能作为影响胰岛素信号传导的生物活性分子。我们的母项目是利用多种代谢组学分析平台来发现与肌肉线粒体FA燃烧和T2DM相关的新代谢物，迄今为止一个有趣的结果是发现中链脂肪酰基肉碱（MCFA-carnitines [C6-C14-carns]，反映了LCFA不完全氧化）在T2DM血浆中的实体增加。原理验证研究表明mcfa -肉碱能激活NF？小鼠巨噬细胞系中与b相关的促炎通路。mcfa -肉毒碱促炎作用的机制，局部或全身作用，可能是通过激活上游toll样受体（TLRs），以前被证明是由饱和FAs触发的，包括C12:0和C14:0，但以前没有与酰基肉毒碱联系？在许多糖尿病前期和2型糖尿病患者中，是否存在与FA代谢功能失调相关的胰岛素抵抗的机制？我们新的初步结果表明，mcfa -肉碱可以特异性激活上游细胞表面受体toll样受体2 (TLR2)，我们预计这反过来会在靶细胞中引发胰岛素抵抗表型。如果这是真的，这将是了解2型糖尿病病因的重要一步，并在确定功能失调的脂肪酸代谢和胰岛素抵抗之间的具体联系。该项目是对我们的母项目的补充，有两个主要目的，即明确回答酰基肉碱是否通过TLR2（特别是TLR2- tlr1二聚化）引起炎症，以及酰基肉碱是否可以通过TLR2依赖机制在体内和体外减弱胰岛素作用。特异性目标1-确认mcfa -肉毒碱特异性激活TLR2-TLR1导致胰岛素信号受损。迄今为止使用小鼠细胞系统和小鼠TLR基因构建的研究表明，c12 - can治疗可增加NF？通过激活TLR2-TLR1异源二聚化而非其他TLR系统来表达基于b的促炎基因。这一目标将通过以下方式确认和扩大这些成果：(a)评估TLR2或其直接下游接头MyD88的下调是否会减少或消除mcfa -肉碱对炎症通路的激活，(b)测试TLR2显性阴性突变体（但不包括其他TLR突变体）是否会降低mcfa -肉碱的作用，(c)确定mcfa -肉碱是否会诱导TLR2- tlr1异二聚体化，(d)确定mcfa -酰基肉碱是否会通过TLR2介导的途径损害胰岛素信号传导。特定目标2-确定mcfa -肉碱给药是否激活促炎途径并诱导体内胰岛素抵抗，通过tlr2依赖机制介导。没有研究检验酰基肉碱对体内炎症和胰岛素作用的影响。我们预测，在培养细胞中观察到的对c12 - can反应的炎症表型（Aim 1和初步结果）将在体内重现，这将支持我们的工作假设，即T2DM中酰基肉碱的局部和/或系统性积累会加剧慢性炎症，从而在胰岛素抵抗表型中发挥作用。这些概念验证研究将：(a)检查小鼠急性静脉注射c12 - can是否会触发

DESCRIPTION (provided by applicant): Elevated fat levels within tissues and reduced capacity/inefficiencies in long-chain fatty acid (LCFA) oxidative catabolism are highly correlated with muscle, liver, adipose, and whole-body insulin resistance. Specific metabolites whose intra- or extra-cellular concentrations shift in response to changes in capacity or efficiency of mitochondrial LCFA combustion may act as bioactive molecules that impact insulin signaling. Our parent project is leveraging multiple metabolomics analysis platforms to discover new metabolites that correlate with muscle mitochondrial FA combustion and T2DM generally, and one interesting outcome to date has been the identification of medium-chain fatty acylcarnitines (MCFA-carnitines [C6-C14-carns], reflective of incomplete LCFA ¿-oxidation) as entities increased in T2DM plasma. Proof-of-principle studies demonstrated activation by MCFA-carnitines of NF?B-related pro-inflammatory pathways in a murine macrophage cell line. Might the mechanism underlying the pro-inflammatory effect of MCFA-carnitines, acting locally or systemically, be via activation of upstream Toll-like receptors (TLRs), previously shown to be triggered by saturated FAs including C12:0 and C14:0 but not previously linked to acylcarnitines? Could such a mechanism underlie part of the insulin resistance associated with dysfunctional FA metabolism, as seen in many pre-diabetics and T2DM patients? Our novel preliminary results point to specific activation of the upstream cell-surface receptor Toll-like receptor 2 (TLR2) by MCFA-carnitines, and we anticipate that this in turn elicits insulin resistance phenotypes in target cells. If true, this would be a major step forward in understanding the etiology of T2DM, and in identifying the specific links between dysfunctional fatty acid metabolism and insulin resistance. The proposed project, which complements our parent project, has two primary Aims that will definitively answer if acylcarnitines evoke inflammation via TLR2 (and specifically, TLR2-TLR1 dimerization), and whether acylcarnitines can attenuate insulin action in vivo and in vitro through TLR2-dependent mechanisms. Specific Aim 1--Confirm that MCFA-Carnitines Specifically Activate TLR2-TLR1 Leading to Impaired Insulin Signaling. Studies to date using murine cell systems and murine TLR gene constructs suggest that C12-carn treatment increases NF?B-based pro-inflammatory gene expression through activation of TLR2-TLR1 heterodimerization but not other TLR systems. This aim will confirm and extend those results by: (a) evaluating if knockdown of TLR2 or its immediate downstream adaptor MyD88 reduces or abolishes the activation of inflammatory pathways by MCFA-carnitine, (b) testing if a TLR2 dominant-negative mutant (but not other TLR mutants) reduces the effects of MCFA-carnitine, (c) determining whether MCFA-carnitine induces TLR2-TLR1 heterodimerization, and (d) determining if MCFA-acylcarnitine can impair insulin signaling through TLR2-mediated pathways. Specific Aim 2--Determine if MCFA-Carnitine Administration Activates Pro-Inflammatory Pathways and Induces Insulin Resistance in vivo, Mediated Through TLR2-Dependent Mechanisms. No studies have examined effects of acylcarnitines on inflammation and insulin action in vivo. We predict that the inflammatory phenotype observed in cultured cells in response to C12-carn (Aim 1 & Preliminary Results) will be recapitulated in vivo, which would support our working hypothesis that local and/or systemic accumulation of acylcarnitines in T2DM exacerbates chronic inflammation and hence plays a role in the insulin resistance phenotype. These proof-of-concept studies will: (a) examine if acute i.v. injection of C12-carn in mice triggers TLR2/NF?B-dependent gene pro-inflammatory pathways in target tissues (muscle, liver, fat, blood leukocytes), and will determine if these outcomes are abolished or dampened in TLR2-KO mice, and (b) test if longer-term exposure to high systemic C12-carn increases tissue inflammatory markers and whole-body insulin resistance, with effects abolished or reduced in TLR2-KO mice. 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). Relatively poor fat combustion by fasting muscle is often correlated with insulin resistance, even in the pre-diabetic state, and altered fat metabolism also appears to diminish insulin action in liver, fat tissue, and the insulin-producing cells of the pancreas. Thus, the overarching aim of our research is to identify clinically-relevant metabolite biomarkers of dysfunctional fat metabolism, and to understand if at least some of these factors not only mark disease susceptibility but also participate in diabetes causation or exacerbation. Specific to this project is the goal to follow-up on initial results indicating that naturally-occurring acylcarnitine metabolites (fatty acids of differing chain-lengths, bound to an amino-acid-like molecule), which are elevated in the T2DM blood as byproducts of inefficient fat metabolism, activate pro-inflammatory/insulin-resistance pathways through the specific cell-surface receptor Toll-like receptor 2 (TLR2). If true, this will have a profound influence on our understanding of diabetes pathophysiology and may provide new targets for preventive and therapeutic modalities.