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缺乏治疗肥胖症的有效干预策略表明需要更好地理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。产热控制外周组织，特别是棕色脂肪组织（BAT）的产热。BAT产热作用在成年人中也是有功能的，尽管关于BAT控制体重功能的重要性存在争议，但BAT大小与体重指数呈负相关，因此BAT产热作用的中枢调节剂是抗肥胖药物的潜在靶标。瘦素通过体温调节机制（包括BAT产热）部分调节体重。事实上，下丘脑瘦素反应神经元（例如在DMH/DHA）概括了已知的中央温度调节途径，我们假设介导食物摄入独立的体重调节。所提出的实验研究DMH/DHA瘦素作用在产热和能量稳态中的作用，强调生理功能、中枢和外周信号传导通路的调节以及DMH/DHA神经元的神经元连接（对DMH/DHA的抑制性/兴奋性LepRb输入）。小鼠瘦素受体（LepRb）神经元的拟议研究允许使用几种分子生物学最先进的工具（cre/loxP技术，神经元特异性示踪和报告基因表达），尚未用于体温调节研究领域。在目的1中，我们将通过使用DMH/DHA特异性瘦素或LepRb拮抗剂注射，或通过使用DREADD技术体内调节DMH/DHA LepRb神经元中的神经元活性，研究体温调节DMH/DHA瘦素作用对体重控制的生理重要性。通过温度变送器、VO 2/CO2交换、BAT功能的蛋白标记物监测野生型或瘦素缺陷型ob/ob小鼠中的体温调节瘦素作用，并研究瘦素诱导的变化（例如脂肪氧化、有丝分裂）对体重以及其它外周组织（例如肌肉和白色脂肪）的影响。在目标2中，将鉴定对DMH/DHA（例如来自POA）的抑制性/兴奋性（GABA-/GABA-）输入及其对瘦素的响应。此外，我们将研究LepRb DMH/DHA中谷氨酸缺失的小鼠，以测试体温调节和体重控制的生理后果。在目的3中，我们确定了LepRb DMH/DHA神经元的神经元连接。我们将使用位点和神经元特异性示踪技术（cre诱导的病毒示踪剂的立体定位注射）来可视化GABA-/神经元能投射（轴突EGFPf）及其二级靶神经元（跨突触示踪）。在具有LepRb驱动的cre/EGFP表达的瘦素缺陷型ob/ob小鼠中，研究了POA和DMH/DHA神经元中潜在的体温调节缺陷（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.