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

脂肪酸β-氧化的肌肉特异性生物标志物的鉴定

• 批准号: 7809146
• 负责人: Sean Harrison Adams
• 金额: $ 58.27万
• 依托单位: U.S. AGRICULTURAL RESEARCH SERVICE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-20 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7809146
• 关键词: Acute; Address; Adipocytes; Adipose tissue; Affect; Amino Acids; Animals; Attenuated; Automobile Driving; Binding; Biological Markers; Blood; Blood Glucose; Carnitine; Catabolism; Cell Line; Cell Surface Receptors; Cell surface; Cells; Chronic; Complement; Cultured Cells; Development; Diabetes Mellitus; Dimerization; Disease susceptibility; Dominant-Negative Mutation; Etiology; Event; Exposure to; Fasting; Fatty Acids; Fatty acid glycerol esters; Functional disorder; Gene Expression; Genes; Goals; Hepatic; Heterodimerization; Hypertrophy; In Vitro; Inflammation; Inflammatory; Injection of therapeutic agent; Insulin; Insulin Resistance; Insulin Resistance Pathway; Knock-out; Length; Leukocytes; Link; Lipids; Liver; Mediating; Metabolic; Metabolism; Mitochondria; Modality; Molecular; Mus; Muscle; Muscle Fibers; Non-Insulin-Dependent Diabetes Mellitus; Outcome; Pancreas; Pancreatic Hormones; Pathway interactions; Patients; Phenotype; Phosphotransferases; Plasma; Play; Preventive; Receptor Gene; Research; Role; System; TLR1 gene; Testing; Therapeutic; Tissues; Toll-Like Receptor 2; Toll-like receptors; Work; acylcarnitine; attenuation; base; clinically relevant; diabetic; fatty acid metabolism; follow-up; genetic manipulation; in vivo; inflammatory marker; insulin sensitivity; insulin signaling; interest; lipid metabolism; long chain fatty acid; macrophage; metabolomics; mutant; novel; oxidation; parent project; prevent; public health relevance; research study; response; uptake

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.

描述（由适用提供）：长链脂肪酸（LCFA）氧化物分解代谢的脂肪水平升高，降低了能力/效率低下，与肌肉，肝脏，脂肪和全身胰岛素耐药性高度相关。特定的代谢产物，其内或细胞外浓度随着线粒体LCFA组合的容量或效率的变化而转移的特定代谢产物可能是影响胰岛素信号传导的生物活性分子。 Our parent project is leveraging multiple metabolomics analysis platforms to discover new metabolites that correlate with muscle mitochondrial FA combination 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等离子体。原始研究证明，在鼠巨噬细胞系中，NF？b？b相关的促炎途径的MCFA - 肉碱激活。 MCFA - 肉碱促炎作用的基础机制是否可以通过在本地或系统上作用，是通过激活上游收费受体（TLR）的激活，以前被证明是由饱和FAS触发的，包括C12：0和C14和C14和C14：0，但没有链接到酰基碳氮胺？这种机制能否构成与功能失调的FA代谢相关的胰岛素抵抗的部分，如许多糖尿病前和T2DM患者所见吗？我们新的初步结果表明，MCFA - 肉碱素对上游细胞表面受体Toll样受体2（TLR2）的特异性激活，我们预计这反过来又引起了靶细胞中胰岛素抵抗表型。如果是真的，这将是理解T2DM病因的主要一步，并确定功能障碍脂肪酸代谢和胰岛素抵抗之间的特定联系。提出的符合我们父母项目的拟议项目具有两个主要目的，可以确定地回答是否通过TLR2引起注射（特别是TLR2-TLR1二聚体化），以及酰基碳氮碱是否可以减弱体内和通过TLR2依赖性机制在体内和体外的胰岛素胰岛素作用。具体目标1-确认MCFA - 肉碱特异性激活TLR2-TLR1导致胰岛素信号受损。迄今为止使用鼠类细胞系统和鼠TLR基因构建体的研究表明，C12-CARN处理通过激活TLR2-TLR1异二聚化而不是其他TLR系统来增加基于NF？B的促炎基因表达。此目标将通过：（a）评估TLR2或其直接下游适配器MyD88是否会减少或废除MCFA - 肉碱的激活或废除炎症途径的激活，（b）测试TLR2是否占主导地位的突变体（但不是其他TLR突变体）是否会重新效应。 MCFA - 肉碱诱导TLR2-TLR1异二聚化，（D）确定MCFA-酰基碱是否可以通过TLR2介导的途径损害胰岛素信号传导。具体目标2-确定性如果MCFA - 肉碱施用会激活促炎途径并诱导通过TLR2依赖性机制介导的体内胰岛素抵抗。尚无研究检查酰基肉碱对体内炎症和胰岛素作用的影响。我们预测，将在培养细胞中观察到的炎症表型对C12-CARN（AIM 1＆初步结果）将在体内概括，这将支持我们的工作假设，即T2DM在T2DM中的局部和/或全身积累在T2DM中促进了慢性感染，因此在持续性中具有persipty蛋白的作用。这些概念验证研究将：（a）检查急性静脉注射。在小鼠中注射C12-CARN触发 TLR2/NF？b依赖性基因促疾病（肌肉，肝脏，脂肪，血液白细胞）中的促炎途径，并将确定这些结果是否在TLR2-KO小鼠中取消或减弱，并且（B）在较高的系统中抗抗体均可抑制较高的系统和整个组织的反溶液，并且（b）测试是否会导致较高的抗溶液和整个抗溶液，并固定整个杂物或整个杂物。 TLR2-KO小鼠。 公共卫生相关性：胰腺胰岛素触发组织吸收血糖的能力降低是在2型糖尿病（T2DM）发展过程中的早期事件。禁食肌肉相对较差的脂肪组合通常与胰岛素抵抗相关，即使在糖尿病前，也会改变脂肪代谢的改变也会减少肝脏，脂肪组织和胰腺产生胰岛素的细胞中的胰岛素作用。这就是我们研究的总体目的是确定功能失调的脂肪代谢的临床代谢物生物标志物，并了解至少其中一些因素不仅标记了疾病易感性，而且还参与糖尿病原因或恶化。 Specific to this project is the goal to follow-up on initial results indicating that naturally-occurring acylcarnitine metabolites (fatty acids of different chain-lengths, bound to an amino-acid-like molecule), which are elevated in the T2DM blood as byproducts of ineffective fat metabolism, activate pro-inflammatory/insulin-resistance pathways through the specific cell-surface receptor Toll-like受体2（TLR2）。如果是真的，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标。