Phosphodiesterase-3B Signaling in the Hypothalamus and Obesity

描述（由申请人提供）：肥胖是美国主要的健康危害之一。肥胖背后的潜在机制知之甚少。瘦素是肥胖基因的产物，主要由脂肪细胞分泌，并在中枢，特别是下丘脑中起作用，以减少食物摄入和体重（wt）。由于大多数肥胖个体是高瘦素血症，瘦素抵抗状态似乎是人类和啮齿动物肥胖的主要原因。啮齿类动物的饮食诱导的肥胖（DIO）模型，其中动物变得肥胖和hyperleptinimic与高脂肪喂养，似乎是人类肥胖。因此，了解啮齿类动物DIO发展背后的机制可能与人类肥胖的神经生物学直接相关。虽然DIO与中枢瘦素抵抗有关，但这一现象背后的机制尚不清楚。我们的研究表明，PI 3 K-PDE 3B-cAMP通路与JAK 2-STAT 3通路相互作用构成了下丘脑瘦素信号传导的关键组成部分。在慢性中枢瘦素输注的大鼠模型中，其中神经肽Y和阿黑皮素原（POMC）神经元产生瘦素抗性，下丘脑中的STAT 3通路保持升高，但PI 3 K-PDE 3B-cAMP通路受损。最近，我们发现DIO小鼠下丘脑中瘦素信号的PI 3 K通路受损。因此，瘦素信号传导的PI 3 K-PDE 3B-cAMP途径中的缺陷可能是中枢瘦素抵抗和DIO发展的基础。四个具体目标将检验这一可能性。目的1：验证下丘脑PDE 3B-cAMP瘦素信号通路在DIO发病过程中受损的假说。目的2：验证脑或ObRb神经元特异性PDE 3B缺失导致肥胖的假设。目的3：验证POMC或AgRP神经元特异性PDE 3B缺失导致肥胖的假设。目的4：检验成年小鼠ARC中PDE 3B的敲低将改变正常能量稳态的假设。将使用Cre-LoxP技术删除PDE 3B。将分别通过酶测定和EIA测量PDE 3B活性和cAMP水平。将通过qPCR和ISH测量基因表达，并通过Western印迹测量蛋白质水平。这些研究将进一步加深我们对中枢瘦素抵抗和肥胖发生机制的理解，因此将与肥胖和进食障碍治疗方法的发展相关。

DESCRIPTION (provided by applicant): Obesity is one of the major health hazards in the United States. The underlying mechanism behind obesity is poorly understood. Leptin, a product of the obese gene, is secreted primarily by fat cells and acts centrally, particularly in the hypothalamus, to reduce food intake and body weight (wt). Since most obese individuals are hyperleptinemic, a state of leptin resistance appears to be the main cause of obesity in humans and rodents. Rodent models of diet-induced obesity (DIO), in which animals become obese and hyperleptinimic with high-fat feeding, appear to be comparable to human obesity. Thus understanding the mechanisms behind the development of DIO in rodents may have direct relevance to the neurobiology of human obesity. Although DIO is associated with central leptin resistance, the mechanisms behind this phenomenon are not clearly understood. Our study suggests that a PI3K-PDE3B-cAMP pathway interacting with the JAK2-STAT3 pathway constitutes a critical component of leptin signaling in the hypothalamus. In a rat model of chronic central leptin infusion in which neuropeptide Y and proopiomelanocortin (POMC) neurons develop leptin resistance, the STAT3 pathway remains elevated but the PI3K-PDE3B-cAMP pathway is compromised in the hypothalamus. Recently, we have shown an impaired PI3K pathway of leptin signaling in the hypothalamus of DIO mice. Thus, a defect in the PI3K-PDE3B-cAMP pathway of leptin signaling could underlie the development of central leptin resistance and DIO. Four specific aims will test this possiblity. Aim 1: To test the hypothesis that the hypothalamic PDE3B-cAMP pathway of leptin signaling is impaired during the development of DIO. Aim 2: To test the hypothesis that brain or ObRb neuron-specific deletion of PDE3B will result in the developemt of obesity. Aim 3: To test the hypothesis that POMC or AgRP neuron-specific deletion of PDE3B will result in the developement of obesity. Aim 4: To test the hypothesis that knockdown of PDE3B in the ARC of adult mice will alter normal energy homeostasis. PDE3B will be deleted using Cre-LoxP technology. PDE3B activity and cAMP levels will be measured by enzyme assay and EIA, respectively. Gene expression will be measured by qPCR and ISH, and protein levels by Western blot. These studies will further our understanding on the mechanisms underlying the development of central leptin resistance and obesity, and therefore will be relevant to the development of therapeutic approaches to obesity and eating disorders.