Carnitine Acetyltransferase in Defending Mitochondrial and Metabolic Function

描述(申请人提供)：本项目旨在阐明肉碱乙酰转移酶(CrAT)作为线粒体功能和糖耐量的重要调节因子的作用，以及L肉碱疗法抗糖尿病作用的主要中介。肉碱乙酰转移酶(CrAT)是一种线粒体基质酶，在乙酰肉碱等短链肉碱偶联物的合成和外排过程中起着关键作用。这种酶在肉碱含量丰富的组织中含量最高，如骨骼肌和心脏，但其确切的代谢功能在很大程度上仍未被探索。CRAT活性主要受L肉碱可获得性的调节，这是一种条件性必需营养素，以其在允许线粒体摄取和氧化长链脂肪酸方面的强制性作用而闻名。除了在脂肪氧化中的必要作用外，肉碱还允许在线粒体内形成酰基肉碱结合物，从而促进线粒体外流多余的碳燃料。我们实验室最近的研究表明，衰老和/或营养过剩导致的肉碱不足通过影响CrAT的活性来损害燃料代谢和胰岛素的作用。值得注意的是，膳食肉碱补充改善了这些模型中的代谢结果，并与血浆和尿乙酰肉碱水平的强劲上升有关。乙酰肉碱的产生和外排之间的生理相关性还知之甚少，而且令人惊讶的是研究不足。我们试图了解CrAT作为肉碱效应器在维持代谢动态平衡方面的具体作用。我们将解决两个中心假设：1)CrAT在调节线粒体底物在葡萄糖和脂肪酸燃料之间的转换中起关键作用，2)CrAT衍生的酰肉碱的线粒体外流提供保护，防止慢性营养过剩引起的肌肉胰岛素抵抗和氧化应激。这些假说将在原代人类骨骼肌细胞和基因敲除小鼠模型中使用功能获得和功能丧失的基因工程方法进行验证。主要结果测量将包括间接量热法、胰岛素作用和代谢流量的多种测量，以及最先进的基于质谱学的代谢图谱。 公共卫生相关性：本项目研究肉碱缺乏的作用，肉碱缺乏是胰岛素抵抗和2型糖尿病等代谢性疾病发展和进展的一个促成因素。L肉碱是一种条件性必需氨基酸衍生物，经常被宣传为增强能量的营养素。我们实验室最近的动物研究表明，衰老和肥胖会破坏全身肉碱的动态平衡，而补充肉碱则可以改善代谢调节和糖耐量。肉碱疗法的有益效果与一种名为肉碱乙酰转移酶(CrAT)的线粒体酶的活性增加有关。本项目的主要目标是阐明补充L肉碱改善糖耐量的机制，并确定CrAT酶在调节能量代谢中的确切作用。拟议研究的结果可能会对L-卡尼汀的治疗特性产生新的见解，同时也促进了对线粒体功能和胰岛素作用之间相互作用的理解。这些都是与临床相关的话题，引起了强烈的科学兴趣和争议。

DESCRIPTION (provided by applicant): This project seeks to elucidate the role of carnitine acetyltransferase (CrAT) as an important regulator of mitochondrial function and glucose tolerance, and the principal mediator of the antidiabetic actions of L-carnitine therapy. Carnitine acetyltransferase (CrAT) is a mitochondrial matrix enzyme that plays a key role in the synthesis and efflux of short chain carnitine conjugates, such as acetyl-carnitine. This enzyme is most abundant in carnitine-rich tissues such as skeletal muscle and heart, but its precise metabolic function remains largely unexplored. CrAT activity is mainly regulated by availability of L-carnitine, a conditionally essential nutrient that is best known for its obligatory role in permitting mitochondrial uptake and oxidation of long chain fatty acids. In addition to its requisite role in fat oxidation, carnitine also permits the intramitochondrial formation of acylcarnitine conjugates, thereby facilitating mitochondrial efflux of excess carbon fuels. Recent studies by our laboratory suggest that carnitine insufficiency caused by aging and/or overnutrition impairs fuel metabolism and insulin action by compromising CrAT activity. Remarkably, dietary carnitine supplementation improved metabolic outcomes in these models in association with robust increases in plasma and urinary acetyl-carnitine levels. The physiological relevance of acetyl-carnitine production and efflux is poorly understood and surprisingly understudied. We seek to understand the specific role of CrAT as a carnitine effector that defends metabolic homeostasis. We will address two central hypotheses: 1) CrAT plays a key role in regulating mitochondrial substrate switching between glucose and fatty acid fuels, and 2) mitochondrial efflux of CrAT-derived acylcarnitines affords protection against muscle insulin resistance and oxidative stress caused by chronic overnutrition. These hypotheses will be tested using gain- and loss-of-function genetic engineering approaches in primary human skeletal myocytes as well as knockout mouse models. Primary outcome measures will include indirect calorimetry, multiple measures of insulin action and metabolic flux, along with state-of-the-art mass spectrometry-based metabolic profiling. PUBLIC HEALTH RELEVANCE: This project examines the role of carnitine insufficiency a contributing factor to the development and progression of metabolic disorders such as insulin resistance and type 2 diabetes. L- carnitine is a conditionally essential amino acid derivative that is often advertised as an energy enhancing nutrient. Recent animal studies by our laboratory have shown that systemic carnitine homeostasis is compromised by aging and obesity, whereas carnitine supplementation improved metabolic regulation and glucose tolerance. The salutary effects of carnitine therapy were linked to increased activity of a mitochondrial enzyme known as carnitine acetyltransferase (CrAT). The overarching goal of this project is to elucidate mechanisms through which supplemental L-carnitine improves glucose tolerance and to determine the precise role of the CrAT enzyme in regulating energy metabolism. Results from the proposed studies are likely to yield new insights regarding the therapeutic properties of L-carnitine while also advancing understanding of the interplay between mitochondrial function and insulin action. These are clinically relevant topics of intense scientific interest and controversy.