Phosphodiesterase-3B Signaling in the Hypothalamus and Obesity

描述(申请人提供)：肥胖是美国的主要健康危害之一。人们对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，并发挥中枢作用，特别是在下丘脑，以减少食物摄入量和体重(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。饮食诱导肥胖(DIO)的啮齿动物模型，即动物变得肥胖和高瘦素血症，似乎与人类肥胖相似。因此，了解啮齿动物DIO发展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，PI3K-PDE3B-cAMP通路与JAK2-STAT3通路相互作用，构成了下丘脑瘦素信号的关键组成部分。在慢性中枢注射瘦素的大鼠模型中，神经肽Y和前阿片黑素皮质素(POMC)神经元产生瘦素抵抗，但在下丘脑，STAT3途径仍然升高，而PI3K-PDE3B-cAMP途径受到影响。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，瘦素信号的PI3K-PDE3B-cAMP通路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一可能性。目的：验证下丘脑瘦素信号通路PDE3B-cAMP在DIO发生发展过程中受损的假说。目的2：验证脑或ObRb神经元特异性缺失PDE3B基因会导致肥胖的假说。目的：验证PDE3B基因POMC或AgRP神经元特异性缺失导致肥胖发生的假说。目的：验证成年小鼠ARC中PDE3B基因敲除将改变正常能量平衡的假说。将使用CRE-loxP技术删除PDE3B。PDE3B活性和cAMP水平将分别用酶分析法和EIA法测定。基因表达用定量聚合酶链式反应和原位杂交检测，蛋白质水平用免疫印迹法检测。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，因此将与肥胖症和饮食失调的治疗方法的发展相关。 与公共健康相关：瘦素主要由脂肪细胞产生，向下丘脑的关键调节中心发出营养状态信号，并已成为调节体重、体内平衡和能量平衡的重要信号。大多数肥胖者都有高瘦素血症，表明存在瘦素抵抗状态。因此，我们一直致力于研究，旨在了解下丘脑中瘦素信号的机制，以及它们在肥胖发展过程中的变化。在这方面，我们描述了下丘脑中一种新的瘦素信号通路，涉及PI3K和磷酸二酯酶3B(PDE3B)的激活以及cAMP水平的降低。我们对抑制剂的药理学研究表明，PDE3B信号在下丘脑中转导瘦素的厌食和减肥作用中起着重要作用。我们目前正致力于确定PDE3B-cAMP通路在下丘脑和肥胖发展过程中转导瘦素作用的生理作用，以便将这一通路作为治疗和/或预防肥胖症及相关疾病的靶点。由于饮食诱导肥胖(DIO)的啮齿动物模型似乎与人类肥胖更相似，我们将研究下丘脑中的PDE3B信号在小鼠DIO发生中的作用。我们将用几个转基因小鼠模型进行体内研究，以确定在低脂饮食或高脂饮食存在的情况下，大脑或神经元特异性PDE3B缺乏在DIO发生中的后果。总体而言，这些研究将在能量平衡的生理学和病理生理学的背景下，确定PDE3B信号在下丘脑中的关键作用。

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. PUBLIC HEALTH RELEVANCE: Leptin produced primarly by fat cells signals nutritional status to key regulatory centers in the hypothalamus and it has emerged as an important signal regulating body weight homeostasis and energy balance. Most obese individuals are hyperleptinimic suggesting a state of leptin resistance. We have thus been engaged in studies aimed at understanding the mechanisms of leptin signaling in the hypothalamus and how they change during the development of obesity. In this regard, we have described a novel leptin-signaling pathway in the hypothalamus involving activation of PI3K and phosphodiesterase 3B (PDE3B) and a decrease in cAMP levels. Our pharmacolgical studies with inhibitors have shown an important role of PDE3B signaling in transducing anorectic and body weight reducing actions of leptin in the hypothalamus. We are now working to establish the physiological role of the PDE3B-cAMP pathway in transducing leptin action in the hypothalamus and during the development of obesity so that this pathway could be targeted for the treatment and or prevention of the obesity and related disorders. Because rodent models of diet-induced obesity (DIO) appear to be more comparable to human obesity, we will examine the role of PDE3B signaling in the hypothalamus in the development of DIO in mice. We will perform in vivo studies with several transgenic mouse models to establish the consequences of brain- or neuron-specifc PDE3B deficiency in the development of DIO in the presence of a low-fat diet or a high-fat diet. Overall, these studies will define the critical role of PDE3B signaling in the hypothalamus in the context of the physiology and pathophysiology of energy homeostasis.