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尚不清楚。瘦素是肥胖基因的产物，主要由脂肪细胞分泌，并集中起作用，特别是在下丘脑，以减少食物摄入和体重（wt）。由于大多数肥胖者患有高瘦素血症，瘦素抵抗状态似乎是人类和啮齿动物肥胖的主要原因。啮齿类动物的饮食性肥胖（DIO）模型，即动物在高脂肪喂养下变得肥胖和高瘦素血症，似乎与人类肥胖相似。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与中枢性瘦素抵抗有关，但这一现象背后的机制尚不清楚。我们的研究表明，PI3K-PDE3B-cAMP途径与JAK2-STAT3途径相互作用，构成了下丘脑瘦素信号传导的关键组成部分。在慢性中枢性瘦素输注的大鼠模型中，神经肽Y和proopiomelanocortin （POMC）神经元产生瘦素抵抗，下丘脑的STAT3通路仍然升高，但PI3K-PDE3B-cAMP通路受损。最近，我们发现DIO小鼠下丘脑中瘦素信号的PI3K通路受损。因此，瘦素信号通路PI3K-PDE3B-cAMP的缺陷可能是中枢性瘦素抵抗和DIO发生的基础。四个具体目标将检验这种可能性。目的1：验证下丘脑PDE3B-cAMP瘦素信号通路在DIO发展过程中受损的假说。目的2：验证PDE3B脑或ObRb神经元特异性缺失会导致肥胖发生的假说。目的3：验证POMC或AgRP神经元特异性PDE3B缺失会导致肥胖发生的假设。目的4：验证成年小鼠ARC中PDE3B基因敲低会改变正常能量稳态的假设。使用Cre-LoxP技术删除PDE3B。PDE3B活性和cAMP水平将分别通过酶测定和EIA测定。qPCR和ISH检测基因表达，Western blot检测蛋白水平。这些研究将进一步加深我们对中枢瘦素抵抗和肥胖发展的机制的理解，因此将与肥胖和饮食失调的治疗方法的发展有关。

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.