Brain Insulin Action Regulates Hypothalamic Glucose Sensing

描述（由申请人提供）：严重低血糖发作（hypoglycemia）是1型糖尿病患者的主要问题。正常情况下，大脑会感觉到低血糖，并触发“战斗或逃跑”的应激反应，以迅速提高血糖，防止癫痫发作或昏迷。不幸的是，1型糖尿病患者不能像非糖尿病患者那样检测到低血糖，他们抵御低血糖的能力严重受损。因此，他们经历严重低血糖发作的风险更大。低血糖是糖尿病治疗的限制步骤，排除了与强化血糖控制相关的益处。由于这些后果，NIDDK的一项主要研究计划是预防或减少1型糖尿病患者的低血糖，特别是通过定义脑葡萄糖感知的“机制和调节剂”。本研究旨在研究胰岛素调节脑葡萄糖感知的作用和机制。关键证据表明，胰岛素在脑内调节葡萄糖稳态、中枢神经系统葡萄糖感知和低血糖反调节反应（CRR），但中枢神经系统胰岛素作用的部位和方法尚不清楚。本研究将1)研究胰岛素是否作用于下丘脑神经元调节脑葡萄糖感知；2)通过评估胰岛素对关键葡萄糖传感器和中枢神经系统葡萄糖摄取的影响，确定胰岛素如何调节葡萄糖感知。首先，利用缺乏CNS胰岛素作用的遗传小鼠模型（NIRKO小鼠），本研究将评估CNS胰岛素作用是否调节大脑检测和应对低血糖的能力。通过低血糖钳夹研究和c-fos免疫染色来测量低血糖反应中的CRR和神经元激活。此外，为了阐明中枢神经系统胰岛素作用的机制，本研究还将评估胰岛素是否调节关键的葡萄糖传感器和/或中枢神经系统的葡萄糖摄取。葡萄糖传感蛋白，包括葡萄糖转运蛋白（GLUTs）和葡萄糖激酶（GK），将通过标准免疫组织化学技术检测在NIRKO小鼠中的表达变化。同样，为了检测脑葡萄糖摄取的变化，将使用放射性同位素测定法评估区域脑葡萄糖利用情况。总之，本研究将为胰岛素在调节中枢神经系统葡萄糖感知和低血糖反调节反应中的作用提供新的见解。由于强化胰岛素治疗增加了严重低血糖（低血糖发作）的风险，低血糖阻碍了糖尿病患者严格的血糖管理。因此，了解大脑如何调节对低血糖的应激反应对于设计治疗低血糖的方法至关重要。

DESCRIPTION (provided by applicant): Episodes of severe low blood sugar (hypoglycemia) are a major problem for Type 1 diabetic patients. Normally, the brain senses hypoglycemia and triggers a "fight-or-flight" stress response to rapidly increase blood sugar and prevent a seizure or coma. Unfortunately, people with Type 1 diabetes do not detect low blood glucose as well as non-diabetic individuals and their ability to defend against it is severely impaired. Therefore, they are at a greater risk of experiencing severe hypoglycemic episodes. Hypoglycemia is the limiting step in the treatment of diabetes and precludes against the benefits linked to intensive glucose control. Due to these consequences, a major research initiative of the NIDDK is to prevent or reduce hypoglycemia in Type 1 diabetics, specifically by defining the "mechanisms and modulators" of brain glucose sensing. This proposal is designed to study the role and mechanism by which insulin regulates brain glucose sensing. Key evidence suggests that insulin acts in the brain to regulate glucose homeostasis, CNS glucose sensing, and the counterregulatory response (CRR) to hypoglycemia, but the site and method of CNS insulin action are still unknown. This study will 1) investigate whether insulin acts on hypothalamic neurons to regulate brain glucose sensing and 2) determine how insulin regulates glucose sensing by evaluating its effects on key glucose sensors and CNS glucose uptake. First, taking advantage of a genetic mouse model that lacks CNS insulin action (NIRKO mouse), this study will assess whether CNS insulin action regulates the brain's ability to detect and respond to hypoglycemia. The CRR and neuronal activation in response to hypoglycemia will be measured via hypoglycemic clamp studies and c-fos immunostaining. Further, to clarify a mechanism of CNS insulin action, this study will also assess whether insulin regulates key glucose sensors and/or CNS glucose uptake. Glucose sensing proteins, including glucose transporters (GLUTs) and glucokinase (GK), will be assayed for altered expression in the NIRKO mouse via standard immunohistochemical techniques. Similarly, to detect changes in brain glucose uptake, regional brain glucose utilization will be evaluated using a radioisotope assay. Overall, this study will provide new insights into insulin's role in regulating CNS glucose sensing and the hypoglycemic counterregulatory response. Because intensive insulin therapy increases the risk of severe hypoglycemia (low blood glucose episode), hypoglycemia impedes tight glucose management in diabetic patients. Therefore, understanding how the brain regulates the stress response to hypoglycemia is critical to devise therapies to combat hypoglycemia.