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。特定代谢物的细胞内或细胞外浓度随着线粒体LCFA燃烧能力或效率的变化而变化，可能是影响胰岛素信号转导的生物活性分子。我们的母项目是利用多个代谢组学分析平台来发现与肌肉线粒体FA燃烧和T2 DM普遍相关的新代谢物，到目前为止，一个有趣的结果是发现了中链脂肪酰肉碱(MCFA-Carnitines[C6-C14-Carns]，反映不完全的LCFA氧化)，作为T2 DM血浆中增加的实体。原理验证研究表明，MCFA-肉毒碱激活了小鼠巨噬细胞系中与核因子？B相关的促炎通路。MCFA-肉毒碱的促炎作用可能是通过激活上游Toll样受体(TLRs)来实现的，该受体以前被包括C12：0和C14：0在内的饱和脂肪酸触发，但之前并未与酰卡尼汀联系在一起。像许多糖尿病前期患者和T2 DM患者中看到的那样，这种机制可能是与FA代谢紊乱相关的胰岛素抵抗的部分原因吗？我们新的初步结果指出，上游细胞表面受体Toll样受体2(TLR2)被MCFA-肉毒碱特异性激活，我们预计这反过来会在靶细胞中引起胰岛素抵抗表型。如果这是真的，这将是在理解T2 DM的病因以及确定脂肪酸代谢障碍和胰岛素抵抗之间的具体联系方面向前迈出的重要一步。这项拟议的项目是对我们的父项目的补充，它有两个主要目标，将明确回答酰卡尼汀是否通过TLR2(特别是TLR2-TLR1二聚化)引起炎症，以及酰卡尼汀是否可以通过TLR2依赖机制在体内和体外减弱胰岛素的作用。具体目标1--确认MCFA-肉毒碱特异性激活TLR2-TLR1，导致胰岛素信号转导受损。到目前为止，使用小鼠细胞系统和小鼠TLR基因构建物的研究表明，C12-CARN治疗通过激活TLR2-TLR1异构体而不是其他TLR系统来增加基于NF？B的促炎基因的表达。这一目标将通过：(A)评估敲除TLR2或其直接下游接头MyD88是否减少或消除MCFA-卡尼汀激活炎症通路；(B)测试TLR2显性-阴性突变体(但不包括其他TLR突变体)是否降低MCFA-卡尼汀的影响；(C)确定MCFA-卡尼汀是否诱导TLR2-TLR1异源二聚；以及(D)确定MCFA-卡尼汀是否可以通过TLR2介导的途径削弱胰岛素信号转导。特定目的2--确定MCFA-卡尼汀在体内是否通过TLR2依赖的机制激活促炎途径和诱导胰岛素抵抗。在体内，还没有研究考察酰卡尼汀对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(目标1和初步结果)将在体内得到概括，这将支持我们的工作假设，即T2 DM局部和/或全身乙酰肉碱的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)检查是否急性静脉注射。在小鼠体内注射C12-CARN触发 (A)研究TLR2/NF？B依赖的基因在靶组织(肌肉、肝脏、脂肪、血白细胞)中的促炎通路，并将确定这些结果在TLR2-KO小鼠中是否被取消或抑制，以及(B)测试长期暴露于高系统性C12-carn是否会增加组织炎症标志物和全身胰岛素抵抗，从而在TLR2-KO小鼠中消除或降低这种作用。 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。禁食肌肉的相对较差的脂肪燃烧通常与胰岛素抵抗有关，即使在糖尿病前期状态下也是如此，脂肪代谢的改变似乎也会削弱肝脏、脂肪组织和胰腺的胰岛素产生细胞的胰岛素作用。因此，我们研究的首要目标是确定与临床相关的脂肪代谢紊乱的代谢物生物标记物，并了解这些因素中是否至少有一些不仅标志着疾病易感性，而且还参与糖尿病的病因或加重。该项目的具体目标是跟踪初步结果，这些结果表明，自然产生的酰卡尼汀代谢物(不同链长的脂肪酸，与氨基酸类分子结合)在T2 DM血液中作为低效脂肪代谢的副产品而升高，通过特定的细胞表面受体Toll样受体2(TLR2)激活促炎/胰岛素抵抗途径。如果这是真的，这将对我们对糖尿病病理生理学的理解产生深远的影响，并可能为预防和治疗方式提供新的靶点。

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.