TRPV1-Dependent Autonomic Control in Diabetes

描述(申请人提供)：全身性葡萄糖稳态在很大程度上受中枢自主神经回路的调节，如果存在自主神经功能障碍，则发展为2型糖尿病的风险很高。因此，如果不完全了解自主神经前神经元的调控机制，就会阻碍对血糖状态控制的理解。这项建议的总体长期目标是阐明中枢自主神经控制和血糖稳态之间的基本关系。下丘脑室旁核(PVN)是控制自主神经流出的重要指挥中心，从而影响血糖和能量平衡。由于糖尿病患者糖平衡受损涉及控制自主神经输出的中枢回路，本研究的直接目标是利用体内和体外方法确定瞬时受体电位香草样物质1型(TRPV1)依赖的机制，参与调节自主神经前PVN神经元的血糖状态。TRPV1在糖尿病中的作用已得到证实，最近我实验室的研究表明，TRPV1通过增加兴奋性神经递质的释放，是自主神经前PVN神经元的重要控制器。此外，这种依赖于TRPV1的肝脏相关PVN神经元的兴奋在糖尿病小鼠模型中是不存在的。我们的初步观察表明，PVN中TRPV1的激活可能通过增加肌肉对葡萄糖的摄取和减少肝脏的糖异生来降低对照组小鼠的全身血糖水平。这些观察结果导致了一个中心假设，即PVN前自主神经接收到TRPV1表达的输入，这些输入被整合到一个协调的自主输出信号中，以产生适当的血糖反应。拟议的活体研究将确定PVN回路中TRPV1激活对自主神经终点的影响。TRPV1在PVN中的激活对葡萄糖稳态的影响将通过清醒小鼠的高胰岛素-正常血糖钳夹研究来确定，并将通过遥测来确定对血压和心率的影响。利用光遗传学进行的系统水平研究将利用TRPV1cre小鼠确定选择性刺激和抑制TRPV1输入对葡萄糖稳态的影响。全细胞膜片钳研究将揭示TRPV1的激活和抑制对PVN神经元的细胞影响，我们将确定由TRPV1表达的投射调节的PVN中的自主神经前神经元群。此外，将利用光刺激在TRPV1cre小鼠身上确定TRPV1激活对神经传递的影响。这些研究将确定中枢TRPV1通过自主神经前PVN神经元对全身血糖稳态的作用，并将区分TRPV1突触前和突触后位置在该系统中的重要性。拟议研究的结果有望开启创新的临床策略和药物靶点，通过自主神经控制改善糖尿病患者的血糖状况。

DESCRIPTION (provided by applicant): Systemic glucose homeostasis is substantially regulated by central autonomic circuits and there is a high risk of developing type 2 diabetes if autonomic dysfunction is present. Therefore, without a full understanding of the mechanisms governing the preautonomic neurons, there is a barrier to the understanding of the control of glycemic status. The overall long term goal of this proposal is to elucidate the fundamental relationship between central autonomic control and glucose homeostasis. The paraventricular nucleus (PVN) of the hypothalamus is a critical command center controlling autonomic outflow and, thereby, influencing glucose and energy homeostasis. Since impaired glucose homeostasis in diabetic patients involves central circuits controlling autonomic output, the immediate objective of this proposal is to identify transient receptor potential vanilloid type 1 (TRPV1)-dependent mechanisms involved in the regulation of preautonomic PVN neurons in the control of glycemic status using in vivo and in vitro approaches. The involvement of TRPV1 in diabetes has been established and recent studies from my laboratory have indicated that TRPV1 is a vital controller of preautonomic PVN neurons through increasing excitatory neurotransmitter release. Furthermore, this TRPV1- dependent excitation of liver-related PVN neurons was absent in a diabetic mouse model. Our preliminary observations demonstrate that activation of TRPV1 in the PVN lowers systemic blood glucose levels in control mice likely through increasing glucose uptake of the muscle and decreasing gluconeogenesis by the liver. These observations lead to the central hypothesis that preautonomic PVN neurons receive TRPV1-expressing inputs and these inputs are integrated into a coordinated autonomic output signal to generate an appropriate glycemic response. The proposed in vivo studies will determine the effect of TRPV1 activation in PVN circuits on autonomic endpoints. The effect of TRPV1 activation in PVN on glucose homeostasis will be determined using hyperinsulinemic-euglycemic clamp studies in conscious mice, and on blood pressure and heart rate using telemetry. Systems level studies using optogenetics will determine the effect of selective stimulation and inhibition of TRPV1 inputs on glucose homeostasis using TRPV1cre mice. Whole-cell, patch-clamp studies will reveal the cellular effects of activation and inhibition of TRPV1 inputs on PVN neurons, and we will identify preautonomic neuronal populations in the PVN regulated by TRPV1-expressing projections. Furthermore, the effect of TRPV1 activation on neurotransmission will be determined using photostimulation in TRPV1cre mice. These studies will define the role of central TRPV1 action on whole body glucose homeostasis through preautonomic PVN neurons, and will differentiate the importance of pre- and postsynaptic locations of TRPV1 on this system. The outcomes of the proposed studies hold the promise of opening innovative clinical strategies and drug targets for the improvement of glycemic status in diabetic patients via autonomic control.