The role of neurpopeptide Y in the arcuate nucleus of the hypothalamus in the regulation of energy expenditure and the involvment of its peripherally expressed receptor type-1 in the regulation of energy homeostasis in mice

目的1:NPY缺失在不同的弧神经元群体中是否影响能量平衡和细胞燃料选择？在9周龄以及研究结束时（16周龄），通过间接量热法检测条件KO小鼠和对照小鼠的身体组成（全身瘦体和脂肪量）、能量消耗和身体活动。研究完成后，将解剖血浆、棕色脂肪组织（BAT）和白色脂肪组织（WAT）、肝脏和肌肉，并通过实时PCR分析相关组织中参与线粒体功能和产热的各种关键调节因子的表达模式/活性水平，包括线粒体呼吸链复合物（IV）、柠檬酸合酶、过氧化物酶体增殖物激活受体ý共激活因子（PGC-1a）、解偶联蛋白-1 （UCP-1）。Western blot分析和/或酶活性测定。为了验证细胞燃料选择是否存在差异，我们将分析几个器官中涉及脂质氧化的调节因子，如肉碱棕榈酰基转移酶-1 （CPT-1）、β -羟酰基辅酶a脱氢酶（b-HAD）和中链酰基辅酶a脱氢酶（MCAD），如肝脏、WAT、肌肉。此外，还将收集血清，并对参与代谢调节的大量激素进行分析。脑弧不同神经元群的NPY缺失是否通过调节下丘脑肽影响能量消耗？为了观察能量消耗的变化是否可以归因于下丘脑肽表达模式和水平的变化，还将收集这些小鼠的大脑，并使用原位杂交技术评估不同肽的mRNA表达水平。我们将重点分析POMC和CART的mRNA表达水平，它们是与NPY和AGRP相反的神经递质。目标2。目的探讨外周Y1受体在能量稳态调节中的作用。外周缺失Y1受体是否影响能量平衡和细胞燃料选择？通过使用他莫昔芬诱导小鼠系，将在SD条件下分析条件Y1敲除小鼠，与Aim1中描述的类似。外周缺失Y1受体是否能预防饮食性肥胖？为了确定肥胖背景下组织特异性y1受体缺失的影响，第二组小鼠将以相同的方式进行分析，但在高脂肪喂养条件下。同样的参数将按照上面的描述进行分析。在这些条件下对这些动物进行测试也很重要，因为它将证明高热量和高脂肪摄入对哪个表达Y1的组织影响最大。

Aim 1: Does NPY deletion in different neuron populations of the arc effect energy balance and cellular fuel selection?Body composition (whole body lean and fat mass), energy expenditure and physical activity in the conditional KO and control mice will be examined by indirect calorimetry at 9 weeks of age as well as at the end of the study at 16 weeks of age. Upon completion of the study plasma, brown adipose tissue (BAT) and white adipose tissue (WAT), liver and muscle will be dissected and the expression patterns/activity levels of various key regulators involved in mitochondrial function and thermogenesis in relevant tissues, including mitochondrial respiratory chain complexes (IV), citrate synthase, peroxisome proliferator-activated receptor ý co-activator (PGC-1a), uncoupling protein-1 (UCP-1) will be analyzed by real time PCR, Western blot analysis and/or enzyme activity assay. To verify whether differences in cellular fuel selection are present, we will analyze regulators involved in lipid oxidation such as carnitine palmitoyltransferase-1 (CPT-1), beta-hydroxyacyl CoA dehydrogenase (b-HAD) and medium-chain acyl-CoA dehydrogenase (MCAD) in several organs, such as liver, WAT, muscle. Furthermore, serum will be collected and the analysis of an extensive panel of hormones involved in the regulation of metabolism will be carried out. Does NPY deletion in different neuron populations of the arc affect energy expenditure via regulation of hypothalamic peptides?In order to see if any changes in energy expenditure can be attributed to changes in expression patterns and levels of hypothalamic peptides, brains from these mice will also be collected and mRNA expression levels of different peptides will be evaluated by using in situ hybridization. Particular emphasis will be given to the analysis of the mRNA expression levels in the Arc of POMC and CART, the opposing neurotransmitters to NPY and AGRP. Aim 2. To determine the contribution of peripheral Y1 receptors to the regulation of energy homeostasis.Does peripherally deletion of Y1 receptor effect energy balance and cellular fuel selection? By using a tamoxifen-inducuble mouse line, conditional Y1 knockout mice will be analyzed on SD conditions similarly to what described in Aim1.Does peripherally deletion of Y1 receptor protects against diet-induced obesity? In order to determine the effect of tissue specific Y1-receptor deletion in an obese context, a second set of mice will be analyzed in the same way but under high fat feeding conditions. The same parameters will be analyzed as described above. It is important to test these animals also under these conditions, as it will demonstrate which Y1 expressing tissue is most affected by high calorie and high fat intake.