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的风险增加有关。肥胖治疗干预的新前沿在于认识到在人类体重增加的初始阶段涉及的新机制。瘦素是一种外周循环饱足因子，在平衡能量消耗和防止脂肪增加方面发挥着关键作用，人们对瘦素的作用越来越感兴趣。然而，肥胖者体内的瘦素水平高于非肥胖者。在常见的人类肥胖症中，升高的瘦素未能导致体重减轻，这表明瘦素的作用减弱(瘦素抵抗)。为此，我们提出了一个新的假设，即对瘦素信号的抵抗是由于腔泡膜微域的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，旨在研究体重增加期间瘦素信号中断的分子机制，使用从健康受试者身上获得的腹部脂肪组织，这些受试者因过量进食而增加体脂。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，在体重增加过程中，循环中的瘦素水平增加，同时内皮介导的血管扩张功能减弱，脂肪细胞脂肪堆积增加。第二，在体重增加期间小窝蛋白-1和内皮型一氧化氮合酶(ENOS)的表达增加，并在体重减轻期间逆转这些变化。第三，瘦素依赖的小窝蛋白-1在体外表达增加。第四，小窝蛋白-1在体外抑制瘦素信号的能力。这些观察结果表明，小窝蛋白-1和小窝微区可能在干扰瘦素信号转导中起作用，作为对高水平瘦素的反应的反馈机制。在这项提案中，我们将结合组织学和分子生物学的方法来研究与体重增加和体重下降相关的腔泡膜微区的形态和组成变化(AIM#1)，以及小窝蛋白-1在脂肪细胞和脂肪组织微血管系统(AIM#2)体重增加和体重下降过程中的作用。我们假设，在体重增加期间，循环中的瘦素增加会导致脂肪组织中小窝蛋白-1表达增加，进而导致小窝微域中小窝蛋白-1和Ob-R相互作用的增加。这种增加的相互作用可能导致下游Ob-R信号的中断，从而导致瘦素抵抗。瘦素信号的减弱会导致脂质堆积增加，eNOS活性降低。这一建议的独特性和翻译强度在于在分子水平上研究受试者的动态重量变化，以及我们在体外研究的同时，在脂肪细胞和脂肪组织微血管中区别研究它们的能力。该提案的长期意义将在于了解受试者在体重增加过程中瘦素信号减弱的机制，以及在体重减轻过程中瘦素信号的可逆性。这一认识将对开发与瘦素抵抗和肥胖相关的治疗方法至关重要。与公共健康相关：拟议的研究将对体重增加期间瘦素信号减弱的分子机制以及体重减轻期间瘦素信号的可逆性具有明确而重要的影响。此外，我们建议的研究结果可能对开发与瘦素抵抗和肥胖相关的治疗方法至关重要。

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.