Insulin Action on GLUT4 and FOXO1 in the Brain

描述（由申请人提供）：胰岛素刺激的葡萄糖摄取受损是发生2型糖尿病的主要风险因素。胰岛素通过促进葡萄糖转运蛋白4（GLUT 4）的转位促进葡萄糖摄取。GLUT 4定位于主要的胰岛素响应组织、骨骼肌和脂肪，但也定位于不需要胰岛素来摄取葡萄糖的器官和细胞类型，如CNS神经元。在表达GLUT4的小鼠组织中缺失胰岛素受体（GIRKO）会导致胰岛素抵抗性糖尿病和肝脏中的实质性胰岛素抵抗。该项目研究是否相同的机制，允许GLUT4从细胞质转运到质膜在脂肪组织和肌肉有助于葡萄糖的摄取在大脑中。将使用单细胞显微镜和荧光葡萄糖类似物来可视化GLUT 4表达的质膜位置处的葡萄糖摄取，并确定葡萄糖摄取是否发生在神经元的特定区域（例如突触与索马）。这些实验将在小鼠下丘脑神经元的原代培养物中进行。一种高分辨率显微镜技术，全内反射显微镜（TIRF），将可视化病毒含有标记GLUT 4在原代神经元培养。可以在胰岛素刺激之前和之后对质膜上标记的GLUT 4进行定量，以确定这些表达GLUT 4的细胞的胰岛素反应性。在该提案的第二部分，下丘脑胰岛素作用的功能丧失或获得 通过在表达Nkx2.1的细胞中分别删除胰岛素受体或Foxo1来产生小鼠模型。这些模型有望有助于研究下丘脑胰岛素作用及其对胰腺胰岛素分泌和肝脏葡萄糖产生的影响。还将确定来自这些功能丧失和获得模型的原代神经元中的GLUT 4定位。这些研究的目的是确定神经元GLUT4是否易位到质膜以及在什么条件下，以及下丘脑中胰岛素敏感性的丧失或获得如何影响表达GLUT4的神经元的功能以及对外周的影响。 公共卫生相关性：在1型和2型糖尿病中，面对低血糖时采取适当的反调节反应以及维持足够的葡萄糖摄入的能力对于避免危险的高血糖和低血糖水平至关重要。该项目研究大脑中表达葡萄糖转运蛋白GLUT 4和基因转录因子FOXO 1的神经元的功能，以及这些神经元中的胰岛素作用如何维持外周葡萄糖稳态。

DESCRIPTION (provided by applicant): Impairment in insulin-stimulated glucose uptake is a major risk factor for developing Type 2 Diabetes. Insulin promotes glucose uptake through translocation of the facilitative glucose transporter 4 (GLUT4). GLUT4 is localized to the main insulin-responsive tissues, skeletal muscle and fat, but also to organs and cell types that do not require insulin for glucose uptake, such as CNS neurons. Deletion of insulin receptors in GLUT4-expressing tissues of mice (GIRKO) causes insulin-resistant diabetes and substantial insulin resistance in liver. This project studies whether the same mechanisms allowing GLUT4 translocation from the cytoplasm to the plasma membrane in the adipose tissue and muscle contribute to glucose uptake in the brain. Single cell microscopy and a fluorescent glucose analog will be used to visualize the uptake of glucose at the plasma membrane location where GLUT4 is expressed and determine whether glucose uptake occurs in a specific region of the neuron (e.g. synapses vs. soma). These experiments will be performed in primary cultures of neurons from the mouse hypothalamus. A high-resolution microscopy technique, total internal reflection microscopy (TIRF), will visualize a virus containing labeled GLUT4 in primary neuronal culture. Quantitation of labeled GLUT4 at the plasma membrane can be performed before and after insulin stimulation to determine the insulin responsiveness of these GLUT4 expressing cells. In the second part of the proposal, loss- or gain-of-function of hypothalamic insulin action mouse models will be generated by deleting the insulin receptor or Foxo1, respectively, in cells that express Nkx2.1. These models are expected to aid in the study of hypothalamic insulin action and its effects on insulin secretion from the pancreas and hepatic glucose production. GLUT4 localization in the primary neurons from these loss- and gain-of-function models will also be determined. The goals of these studies are to determine whether neuronal GLUT4 translocates to the plasma membrane and under what conditions, and how the loss or gain of insulin sensitivity in the hypothalamus affects the function of GLUT4-expressing neurons and the resulting effects on the periphery. PUBLIC HEALTH RELEVANCE: In Type 1 and Type 2 Diabetes, the ability to mount a proper counterregulatory response in the face of hypoglycemia as well as maintain sufficient glucose uptake is critical in avoiding dangerous high and low glucose levels. This project studies the function of neurons in the brain that express the glucose transporter GLUT4 and gene transcription factor FOXO1 and how insulin action in those neurons maintains peripheral glucose homeostasis.