Leptin and Central Control of Thermoregulation

描述（由申请人提供）：肥胖的持续急剧上升和缺乏有效的干预策略来治疗肥胖表明需要更好地了解维持能量稳态的机制。产热是维持体温所必需的，但也影响能量消耗和体重。生热作用控制外周组织，特别是棕色脂肪组织（BAT）的产热。BAT产热在成年人中也有功能，尽管关于BAT功能控制体重的重要性存在争议，但BAT的大小与体重指数呈负相关，因此BAT产热的中枢调节因子是抗肥胖药物的潜在靶点。瘦素部分通过包括BAT产热在内的体温调节机制调节体重。事实上，下丘脑瘦素反应神经元（例如DMH/DHA）概括了已知的中枢体温调节途径，我们假设它介导食物摄入独立的体重调节。本实验研究DMH/DHA瘦素在产热和能量稳态中的作用，强调DMH/DHA神经元的生理功能、中枢和外周信号通路的调节以及神经元连接（抑制/兴奋LepRb输入DMH/DHA）。提出的小鼠瘦素受体（LepRb）神经元的研究允许使用几种分子生物学最先进的工具（cre/loxP技术，神经元特异性追踪和报告基因表达），这些工具尚未在体温调节研究领域使用。在Aim 1中，我们将通过注射DMH/DHA特异性瘦素或LepRb拮抗剂，或使用DREADD技术在体内调节DMH/DHA LepRb神经元的神经元活性，研究体温调节DMH/DHA瘦素对体重控制的生理重要性。通过温度传递器监测野生型或瘦素缺乏ob/ob小鼠的热调节瘦素作用，VO2/CO2交换，BAT功能的蛋白质标记物以及对体重和其他外周组织（如肌肉和白色脂肪）的影响，研究瘦素诱导的变化（如脂肪氧化，有丝分裂）。目的2将确定DMH/DHA的抑制性/兴奋性（GABA-/谷氨酸能）输入（例如来自POA）及其对瘦素的反应。此外，我们将研究LepRb DMH/DHA中谷氨酸缺失的小鼠，以测试其对体温调节和体重控制的生理影响。在Aim 3中，我们鉴定了LepRb DMH/DHA神经元的神经元连通性。我们将使用位点和神经元特异性示踪技术（立体定向注射可诱导病毒示踪剂）来可视化GABA-/谷氨酸能投射（轴突EGFPf）及其二级目标神经元（跨突触示踪）。在LepRb驱动cre/EGFP表达的瘦素缺陷ob/ob小鼠中，研究了POA和DMH/DHA神经元中潜在的热调节缺陷（例如LepRb表达，神经元投射）。

DESCRIPTION (provided by applicant): The ongoing dramatic rise in obesity and the lack of efficient intervention strategies to treat obesity demonstrates the need to better understand mechanisms to maintain energy homeostasis. Thermogenesis is necessary to maintain body temperature, but also affects energy expenditure and body weight. Thermogenesis controls heat production in peripheral tissues, particularly the brown adipose tissue (BAT). BAT thermogenesis is also functional in adult humans and despite controversial opinions on the importance of BAT function to control body weight, BAT size correlates negatively with body mass index and thus central regulators of BAT thermogenesis are potential targets for anti-obesity drugs. Leptin regulates body weight in part via thermoregulatory mechanisms including BAT heat production. Indeed, hypothalamic leptin responsive neurons (e.g. in the DMH/DHA) recapitulate known central thermoregulatory pathways, that we hypothesize to mediate food-intake independent body weight regulation. The proposed experiments investigate the role of DMH/DHA leptin action in thermogenesis and energy homeostasis, emphasizing physiological function, regulation of central and peripheral signaling pathways and neuronal connectivity of DMH/DHA neurons (inhibitory/excitatory LepRb inputs to the DMH/DHA). The proposed study of leptin receptor (LepRb) neurons in mice allows the use of several molecular biological state-of-the-art tools (cre/loxP technology, neuron specific tracing and reporter expression), that have not been used in the thermoregulation research field, yet. In Aim 1 we will study the physiological importance of thermoregulatory DMH/DHA leptin action on body weight control by using DMH/DHA specific leptin or LepRb antagonist injections, or by in vivo modulation of neuronal activity in DMH/DHA LepRb neurons using DREADD technology. Thermoregulatory leptin action in wildtype or leptin deficient ob/ob mice is monitored by temperature transmitters, VO2/CO2 exchange, protein markers of BAT function and effects on body weight as well as other peripheral tissues (e.g. muscle and white fat) are investigated for leptin induced changes (e.g fat oxidation, mitogenesis). In Aim 2 will identify inhibitory/excitatory (GABA-/glutamatergic inputs to the DMH/DHA (e.g. from the POA) and their response to leptin. Furthermore, we will study mice with loss of glutamate in LepRb DMH/DHA to test the physiological consequence for thermoregulation and body weight control. In Aim 3 we identify the neuronal connectivity of LepRb DMH/DHA neurons. We will use site and neuron- specific tracing techniques (stereotaxic injection of cre-inducible viral tracers) to visualize GABA-/glutamatergic projections (axonal EGFPf) and their 2nd order target neurons (transsynaptic tracing). In leptin deficient ob/ob mice with LepRb-driven cre/EGFP expression potential thermoregulatory defects (e.g. LepRb expression, neuronal projections) in POA & DMH/DHA neurons are investigated. PUBLIC HEALTH RELEVANCE: Current obesity intervention strategies (e.g. drugs and life style modification) are not satisfactory, demonstrated by the ongoing dramatic rise in obesity; and indicates the need for a better understanding of the mechanisms controlling body weight. In humans and other mammals, the brown adipose tissue (BAT) is able to produce heat and maintain body temperature, which requires BAT to burn body fat stores. In this proposal, we will investigate the importance and mechanisms of leptin -a key regulator of body weight- to regulate BAT heat production via the central nervous system, which would have an important impact as potential target to treat obesity.