Molecular Mechanisms Mediating Resistance to Leptin Signaling During Fat Gain

描述（由申请人提供）：肥胖与2型糖尿病和心血管疾病发展的风险增加有关。肥胖治疗干预的新前沿在于认识到人类体重增加的初始阶段所涉及的新机制。瘦素是一种外周循环饱腹感因子，在平衡能量消耗和防止脂肪增加方面起着关键作用，人们对它的作用越来越感兴趣。然而，肥胖人群的瘦素水平高于非肥胖人群。在常见的人类肥胖形式中，高瘦素不能引起体重减轻，这表明瘦素作用的衰减（瘦素抵抗）。为此，我们提出了一种新的假设，即对瘦素信号传导的抗性是小泡膜微域分子和结构变化的结果。为了验证我们的假设，我们提出了一系列研究，旨在调查体重增加过程中瘦素信号中断的分子机制，使用从过度喂食方案中获得体脂的健康人类受试者的腹部脂肪组织。我们还建议研究这些变化在减脂过程中的可逆性。在我们的初步研究中，我们首先观察到，在体重增加期间，循环瘦素水平的增加伴随着内皮介导的血管舒张功能的减少和脂肪细胞脂肪积累的增加。其次，在体重增加期间，小窝蛋白-1和内皮型一氧化氮合酶（eNOS）表达增加，而在体重减轻期间，这些变化发生逆转。第三，体外瘦素依赖性caveolin-1表达增加。第四，caveolin-1体外抑制瘦素信号传导的能力。这些观察结果表明，在高瘦素水平下，小洞蛋白-1和小洞微结构域作为一种反馈机制，可能在瘦素信号的破坏中起作用。在本提案中，我们将结合组织学和分子生物学方法来研究与体重增加和体重减轻相关的腔泡膜微域的形态和组成变化（AIM#1），以及脂肪细胞和脂肪组织微血管在体重增加和体重减轻中的作用（AIM#2）。我们假设，在体重增加期间，循环瘦素增加导致脂肪组织中小洞蛋白-1表达增加，这反过来又导致小洞蛋白-1和Ob-R在小洞微域中的相互作用增加。这种增加的相互作用可能导致下游Ob-R信号的破坏，从而导致瘦素抵抗。瘦素信号的衰减将导致脂质积累增加和eNOS活性降低。本提案的独特性和翻译强度在于在分子水平上研究人类受试者的动态重量变化，以及我们能够在脂肪细胞和脂肪组织微血管中进行差异研究以及体外研究。该建议的长期意义在于理解瘦素信号在人类体重增加过程中的衰减机制及其在体重减轻过程中的可逆性。这种理解对于瘦素抵抗和肥胖相关疗法的发展至关重要。公共卫生相关性：拟议的研究将对体重增加期间瘦素信号衰减的分子机制及其在体重减轻期间的可逆性具有明确和重要的意义。此外，我们提出的研究结果可能对瘦素抵抗和肥胖相关治疗的发展至关重要。

DESCRIPTION (provided by applicant): Obesity is associated with increased risk for the development of type 2 diabetes mellitus and cardiovascular disorders. New frontiers in therapeutic intervention against obesity lie in the recognition of novel mechanisms involved during the initial stages of weight gain in humans. There has been increasing interest in the role leptin, a peripheral circulating satiety factor, which plays a key role in balancing energy expenditure and preventing fat gain. However, leptin is found at higher levels in obese humans than in non-obese humans. The failure of elevated leptin to elicit weight loss in common forms of human obesity suggests the attenuation of leptin action (leptin resistance). To this end, we propose a novel hypothesis that resistance to leptin signaling is a result of molecular and structural changes in caveolar membrane microdomains. To test our hypothesis we propose a series of studies directed at investigating the molecular mechanisms involved in disruption of leptin signaling during weight gain, using abdominal adipose tissue obtained from healthy human subjects who gain body fat in response to an overfeeding protocol. We also propose to investigate the reversibility of these changes during fat loss. In our preliminary studies we have observed first, increases in circulating leptin levels during weight gain accompanied by decreased endothelium mediated vasodilation and increased adipocyte fat accumulation. Second, increases in caveolin-1 and endothelial nitric oxide synthase (eNOS) expression during weight gain and reversal of these changes during weight loss. Third, increases in leptin-dependent caveolin-1 expression in vitro. Fourth, the ability of caveolin-1 to inhibit leptin signaling in-vitro. These observations point to a probable role of caveolin-1 and caveolar microdomains in disruption of leptin signaling as a feedback mechanism in response to high leptin levels. In this proposal we will combine histological and molecular biologic approaches to study the morphological and compositional changes in caveolar membrane microdomains associated with weight gain and weight loss (AIM#1), and the role of caveolin-1 during weight gain and weight loss in adipocytes and adipose tissue microvasculature (AIM#2). We hypothesize that during weight gain, increased circulating leptin leads to increased caveolin-1 expression in adipose tissue, which in turn leads to increased caveolin-1 and Ob-R interaction in the caveolar microdomains. This increased interaction may result in disruption of downstream Ob-R signaling which would then lead to leptin resistance. Attenuation of leptin signaling will result in increased lipid accumulation and decreased eNOS activity. The uniqueness and translational strength of this proposal lies in studying at the molecular level dynamic weight changes in human subjects, and in our ability to differentially study them in adipocytes and adipose tissue microvessels along with in-vitro studies. The long term significance of the proposal will be in understanding the mechanisms involved in attenuation of leptin signaling during weight gain in human subjects and its reversibility during weight loss. This understanding would be pivotal in development of therapeutics related to leptin resistance and obesity. PUBLIC HEALTH RELEVANCE: The proposed studies will have clear and important implications regarding the molecular mechanisms involved in attenuation of leptin signaling during weight gain and its reversibility during weight loss. In addition the results of our proposed studies may be critical in development of therapeutics relating to leptin resistance and obesity.