Regulated Expression of Hypothalamic CPT1 Using Gutless Adenovirus

描述（由申请人提供）：肥胖和2型糖尿病共有几种代谢异常，包括细胞感知营养物质流量增加的能力受损。由于大脑在调节燃料代谢和体重的体内平衡中起着至关重要的作用，因此本提案的最终目标是了解营养感测的神经元机制。丙二酰辅酶A是葡萄糖代谢的产物，被认为是监测能量平衡的下丘脑神经元中燃料代谢的主要指标。据推测，在进食状态下，丙二酰辅酶A充当燃料丰度的传感器，因为其对酶肉毒碱棕榈酰转移酶1（CPT 1）的有效变构抑制，该酶控制脂肪酸进入线粒体，在线粒体中它们进行氧化。下丘脑中长链脂肪酰辅酶A（LCFA-CoA）水平的增加反过来导致食物摄入减少和内源性葡萄糖产生减少。为了测试下丘脑中丙二酰辅酶A水平是否通过调节脂肪酸氧化来调节摄食行为和代谢，我们建议在成年大鼠下丘脑中表达CPT 1基因（CPT 1AE 3A），该基因对丙二酰辅酶A的变构抑制不敏感，同时保留其全部催化活性。为了避免下丘脑CPT 1AE 3A表达的高组成性和超生理水平，我们在目标1中提出在单个颗粒中产生含有雷帕霉素类似物诱导型表达盒加上所有转录因子组分的无肠腺病毒（gAd）载体。在第二个目标中，我们将确定最佳的腺病毒载体和雷帕霉素剂量在体内下丘脑神经元中表达CPT 1AE 3A。最后，在第三个目标中，我们将评估下丘脑丙二酰辅酶A不敏感CPT 1的表达是否会改变摄食行为和葡萄糖代谢。在下丘脑神经元转导中使用诱导型gAd载体将是代谢研究的宝贵工具，因为迄今为止还没有开发出合适的基因表达系统，其允许以中等水平控制转基因的表达。此外，该技术应用于下丘脑中丙二酰辅酶A传感通路的研究将为将代谢营养传感与信号转导和神经传递耦合的神经元机制提供新的线索。 公共卫生相关声明：肥胖症在富裕社会的流行已达到流行病的程度，使人们更加关注与肥胖症有关的健康风险，包括2型糖尿病、高血压和动脉粥样硬化。随着人们对肥胖相关健康风险认识的提高，人们对肥胖发病机制的研究也越来越多。该提案的最终目标是描绘控制体重和代谢的新分子机制，并确定用于治疗肥胖和糖尿病的特定分子靶点。

DESCRIPTION (provided by applicant): Obesity and type 2 diabetes share several metabolic abnormalities, including an impaired ability of cells to sense increased nutrients flux. Since the brain plays a crucial role in regulating the homeostasis of fuel metabolism and body weight, the ultimate goal of this proposal is to understand the neuronal mechanisms of nutrient sensing. Malonyl CoA, a product of glucose metabolism, has been implicated as a major gauge of fuel metabolism in hypothalamic neurons that monitor energy balance. It is postulated that in the fed state malonyl CoA acts as a sensor of fuel abundance because of its potent allosteric inhibition of the enzyme carnitine palmitoyltransferase 1 (CPT1), which controls the entry of fatty acids into mitochondria where they undergo oxidation. Increased levels of long-chain fatty acyl-Co-enzyme A (LCFA-CoAs) in hypothalamus lead, in turn, to decreased food intake and decreased endogenous glucose production. To test whether the malonyl CoA levels in hypothalamus regulate feeding behavior and metabolism via modulation of fatty acid oxidation, we propose to express in hypothalamus of adult rats a CPT1 gene (CPT1AE3A) that is insensitive to the allosteric inhibition of malonyl CoA, while it retains its full catalytic activity. In order to avoid high constitutive, and therefore supraphysiologic levels of hypothalamic CPT1AE3A expression, we propose in aim 1 to generate gutless adenoviral (gAd) vectors containing a rapalog-inducible expression cassette plus all the transcription factor components, in a single particle. In the second aim we will determine the optimal adenoviral vector and rapalog doses to express CPT1AE3A in hypothalamic neurons in vivo. Finally, in the third aim we will assess whether hypothalamic expression of a malonyl CoA-insensitive CPT1 alters feeding behavior and glucose metabolism. The use of inducible gAd vectors in transduction of hypothalamic neurons will be an invaluable tool for the study of metabolism, since to date no suitable gene expression system has been developed that allows controlled expression of transgenes at moderate levels. Additionally, the application of this technology to the study of the malonyl CoA sensing pathway in the hypothalamus will shed new light in the neuronal mechanisms that couple metabolic nutrient sensing to signal transduction and neurotransmission. PUBLIC HEALTH RELEVANCE STATEMENT: The prevalence of obesity in affluent societies has reached epidemic proportions, heightening the concern for the health risks associated with obesity, which include type 2 diabetes, hypertension and atherosclerosis. The increased awareness of the health risks associated with obesity has increased the interest in understanding the pathogenesis of obesity. The ultimate goal of this proposal is to delineate novel molecular mechanisms controlling body weight and metabolism and to identify specific molecular targets for the treatment of obesity and diabetes.