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重要组成部分。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。 公共卫生相关性：瘦素主要由脂肪细胞产生，将营养状态信号传递到下丘脑的关键调节中心，并且已经成为调节体重稳态和能量平衡的重要信号。大多数肥胖个体是高瘦素血症，表明瘦素抵抗状态。因此，我们一直在从事研究，旨在了解瘦素信号在下丘脑的机制，以及它们如何在肥胖的发展过程中变化。在这方面，我们已经描述了一种新的瘦素信号通路，在下丘脑涉及激活PI 3 K和磷酸二酯酶3B（PDE 3B）和降低cAMP水平。我们对抑制剂的药理学研究表明，PDE 3B信号转导在下丘脑中瘦素的厌食和体重减轻作用中具有重要作用。我们现在正在努力确定PDE 3B-cAMP途径在下丘脑和肥胖发展过程中转导瘦素作用的生理作用，以便该途径可以作为治疗和/或预防肥胖和相关疾病的靶点。由于饮食诱导的肥胖（DIO）的啮齿动物模型似乎更接近人类肥胖，我们将研究小鼠下丘脑中PDE 3B信号转导在DIO发展中的作用。我们将用几种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下，脑或神经元特异性PDE 3B缺乏在DIO发展中的后果。总体而言，这些研究将定义PDE 3B信号传导在能量稳态的生理学和病理生理学背景下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.