Leptin and Central Control of Thermoregulation

描述(由申请人提供)：肥胖症的持续急剧上升和治疗肥胖症的有效干预策略的缺乏表明，需要更好地了解维持能量平衡的机制。生热是维持体温所必需的，但也会影响能量消耗和体重。生热作用控制周围组织，特别是棕色脂肪组织(BAT)的产热。蝙蝠的产热作用在成人中也有作用，尽管对蝙蝠功能控制体重的重要性存在争议，但蝙蝠的大小与体重指数呈负相关，因此蝙蝠产热的中枢调节因子是抗肥胖药物的潜在靶点。瘦素部分通过体温调节机制调节体重，包括蝙蝠产热。事实上，下丘脑瘦素反应神经元(例如，在DMH/DHA中)概括了已知的中枢体温调节通路，我们假设这些通路是为了调节摄食独立的体重调节。本实验研究DMH/DHA瘦素在生热和能量平衡中的作用，重点研究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的连接(DMH/DHA的抑制性/兴奋性LepRb输入)。对小鼠瘦素受体(LepRb)神经元的拟议研究允许使用几种最先进的分子生物学工具(cre/loxP技术、神经元特异性示踪和报告表达)，这些工具尚未用于体温调节研究领域。在目标1中，我们将通过注射DMH/DHA特异性瘦素或LepRb拮抗剂，或通过DREADD技术在体内调节DMH/DHA LepRb神经元的活动，来研究体温调节DMH/DHA瘦素在体重控制中的生理意义。通过温度传递监测野生型或瘦素缺乏ob/ob小鼠的体温调节瘦素作用，研究VO2/CO2交换、BAT功能的蛋白质标志物以及瘦素对体重和其他外周组织(如肌肉和白色脂肪)的影响，以了解瘦素诱导的变化(如脂肪氧化、有丝分裂)。在Aim 2中，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中谷氨酸丢失的小鼠，以测试其对体温调节和体重控制的生理后果。在目标3中，我们确定了LepRb DMH/DHA神经元的连通性。我们将使用部位和神经元特异性示踪技术(立体定向注射可诱导的病毒示踪剂)来可视化GABA/谷氨酸能投射(轴突EGFPf)及其二级靶神经元(跨突触示踪)。在瘦素缺乏的ob/ob小鼠中，研究了LepRb驱动的cre/EGFP表达的POA和DMH/DHA神经元潜在的温度调节缺陷(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.