Regulated Expression of Hypothalamic CPT1 Using Gutless Adenovirus

利用无肠腺病毒调节下丘脑 CPT1 的表达

• 批准号: 7386180
• 负责人: SILVANA OBICI
• 金额: $ 24.78万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7386180
• 关键词: Acyl Coenzyme A; Adenovirus Vector; Adenoviruses; Adult; Affect; Alanine; Amino Acids; Animals; Atherosclerosis; Awareness; Body Weight; Brain; Carnitine; Carnitine O-Palmitoyltransferase; Cells; Characteristics; Diabetes Mellitus; Dose; Eating; Energy Metabolism; Enzyme Inhibition; Enzymes; Epidemic; Equilibrium; Failure; Fatty Acids; Feeding behaviors; Gene Expression; Generations; Genes; Glucose; Glutamates; Goals; Health; Hepatic; Homeostasis; Hyperphagia; Hypertension; Hypothalamic structure; Inflammatory Response; Lead; Light; Liver; Malonyl Coenzyme A; Metabolic; Metabolic Pathway; Metabolism; Mitochondria; Molecular; Molecular Target; Monitor; Mutation; Neurons; Non-Insulin-Dependent Diabetes Mellitus; Nutrient; Obesity; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Phenotype; Play; Positioning Attribute; Prevalence; Property; Protein Isoforms; Rattus; Regulation; Risk; Role; Satiation; Secondary to; Signal Transduction; Sirolimus; Societies; Specific qualifier value; System; Technology; Testing; Trans-Activators; Transferase; Viral; Viral Vector; base; brain cell; design; detection of nutrient; energy balance; fatty acid oxidation; feeding; glucose metabolism; glucose production; gutless adenoviral vector; immunogenicity; in vitro testing; in vivo; interest; metabolic abnormality assessment; mutant; neurotransmission; novel; obesity treatment; oxidation; particle; promoter; public health relevance; sensor; small molecule; tool; transcription factor; transduction efficiency; transgene expression; vector

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.

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动态平衡方面起着至关重要的作用，因此这一提议的最终目标是了解营养感知的神经机制。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燃料代谢的主要指标。据推测，在进食状态下，丙二酰辅酶A是燃料丰度的传感器，因为它对肉碱棕榈酰转移酶1(CPT1)的变构抑制作用很强，CPT1控制脂肪酸进入线粒体，在那里它们经历氧化。下丘脑中长链脂肪酰辅酶A(LCFA-COAS)水平的增加反过来会导致食物摄入量减少和内源性葡萄糖产生减少。为了测试下丘脑丙二酰辅酶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表达一个CPT1基因(CPT1AE3A)，该基因对丙二酰辅酶A的变构抑制不敏感，但仍保持其完全的催化活性。为了避免下丘脑CPT1AE3A的高组成和超生理水平的表达，我们在目标1中建议在单个颗粒中产生无胆型腺病毒(Gad)载体，该载体包含Rapalog可诱导的表达盒和所有转录因子成分。在第二个目的中，我们将确定在体内表达CPT1AE3A的最佳腺病毒载体和Rapalog剂量。最后，在第三个目标中，我们将评估下丘脑对丙二酰辅酶A不敏感的CPT1的表达是否改变了摄食行为和葡萄糖代谢。在下丘脑神经元的转导中使用可诱导的Gad载体将是研究新陈代谢的宝贵工具，因为到目前为止还没有开发出合适的基因表达系统来控制中等水平的转基因表达。此外，将该技术应用于下丘脑丙二酰辅酶A传感通路的研究，将为新陈代谢营养传感与信号转导和神经传递相结合的神经机制提供新的线索。 公共卫生相关声明：肥胖症在富裕社会的流行程度已经达到流行病的程度，这加剧了人们对与肥胖症相关的健康风险的关注，这些风险包括2型糖尿病、高血压和动脉粥样硬化。人们对肥胖带来的健康风险的认识增加了人们对了解肥胖发病机制的兴趣。这项提议的最终目标是描绘控制体重和新陈代谢的新分子机制，并确定治疗肥胖症和糖尿病的特定分子靶点。