Functioning of Nodose Ganglia in Diabetes

结状神经节在糖尿病中的功能

• 批准号: 8257173
• 负责人: CHUNG OWYANG
• 金额: $ 31.94万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8257173
• 关键词: Action Potentials; Afferent Neurons; Afferent Pathways; Animals; Appetite Regulation; Autonomic nervous system; Binding; Calcineurin; Calcium; Cells; Cholecystokinin; Chronic; Clinical Trials; Cyclic AMP; Data; Diabetes Mellitus; Docking; Eating Behavior; Electroporation; Elements; Frequencies; Fura-2; Ganglia; Gene Expression; Human; Hyperglycemia; Image; Insulin; Label; Lead; Leptin; Link; Mediating; Mediation; Membrane; Membrane Potentials; Methods; Modification; Molecular; Motor; Nervous system structure; Neurons; Nodose Ganglion; Pancreas; Pathway interactions; Patients; Physiological; Play; Potassium Channel; Process; Property; Protein Dephosphorylation; Proteins; Rattus; Reflex action; Resistance; Rest; Reverse Transcriptase Polymerase Chain Reaction; Role; Secondary to; Secretin; Sensory; Serine; Signal Transduction; Signaling Molecule; Site; Small Interfering RNA; Stomach; Streptozocin; Subfamily lentivirinae; System; Testing; Time; Transfection; Up-Regulation; Virus; Western Blotting; base; cell motility; diabetes control; diabetic; diabetic patient; diabetic rat; gastrointestinal function; improved; in vivo; inhibitor/antagonist; neuronal excitability; pancreatic juice; patch clamp; promoter; protein expression; public health relevance; response; stomach motility; therapeutic target; voltage

DESCRIPTION (provided by applicant): Gastrointestinal function is frequently abnormal in patients with poorly controlled diabetes. Clinical investigations indicate that most of these abnormalities can be attributed to defective vagal afferent functioning. Our preliminary studies indicate that nodose ganglia (NG) neurons in streptozotocin induced diabetic rats (STZ- D) display hyperpolarization leading to decreased excitability. This may contribute to abnormal vagal function in the diabetic state. We hypothesize that chronic hyperglycemia activates "background" TRESK potassium channels, leading to hyperpolarization of the NG and decreased excitability. This is a two-step process. Initially increase in intracellular calcium in diabetic neurons activates calcineurin. Calcineurin binds to a NFAT- like docking site on the TRESK protein and causes dephosphorylation of serine 276, resulting in activation of the channel and leading to hyperpolarization. Over time upregulation of the TRESK protein occurs resulting in not only increased frequency of the opening of TRESK channel but an increase number of TRESK K+ channels. To test this hypothesis we have 3 specific aims. Aim 1 is to demonstrate that hyperglycemia in STZ- D modifies basic electrophysiological properties of NG neurons. Patch clamp recordings will be performed to characterize the excitability of NG ganglia neurons from control and STZ-D rats. Physiological implications of these abnormalities will be evaluated by in vivo electrophysiological recording of NG in diabetic rats and study its responsiveness to CCK, leptin and secretin stimulation. Aim 2 examines whether hyperpolarization of NG neurons in the chronic diabetic state is mediated by activation of TRESK channels. The presence of specific TESK potassium channels in NG will be identified using electrophysiological studies as well as western blot and RT-PCR methods. The participation of the TRESK channel will be demonstrated by the use of a virus based system for delivery of siRNA to silence the expression of the TRESK channel in NG. To evaluate the functional importance of TRESK in the mediation of hyperpolarization of NG in diabetes, we will examine reversibility of the electrophysiological and GI abnormalities following silencing TRESK channel expression in vivo through electroporation of the NG with TRESK siRNA. Aim 3 investigates the signal transduction cascades that mediate the membrane modifications of NG neurons in diabetes. Patch clamp recordings and intracellular calcium imaging studies will be performed with messenger specific activators or inhibitors to determine the role of specific intracellular cascade elements on NG excitability in diabetic animals. The demonstration of desphosphorylation of serine 276 in diabetes will involve the use of phosphoproteome method. Understanding the cellular and molecular mechanism responsible for abnormal functioning of the NG in the diabetic state will provide important therapeutic targets for the management of abnormal GI function in chronic diabetes. PUBLIC HEALTH RELEVANCE: Gastrointestinal functions are frequently abnormal in patients with poorly controlled diabetes, including abnormal stomach motility, diminished pancreatic and stomach secretions and abnormal eating behavior. Many of these functions are controlled through a vagus nervous system which is frequently abnormal in diabetic patients. The purpose of this study is to understand the molecular and cellular mechanisms responsible for the abnormalities of this group of sensory neurons. This could provide important therapeutic targets and lead to improved management of diabetic patients with GI complications.

描述（由申请人提供）：控制不良的糖尿病患者经常异常胃肠功能。临床研究表明，这些异常大多数可能归因于迷走神经传入功能有缺陷。我们的初步研究表明，链蛋白酶诱导的糖尿病大鼠（STZ-D）中的淋巴结神经元（NG）神经元显示出超极化导致兴奋性降低。这可能导致糖尿病状态的迷走神经功能异常。我们假设慢性高血糖会激活“背景” TRESK钾通道，从而导致NG的超极化并降低兴奋性。这是一个两步的过程。糖尿病神经元中细胞内钙的最初增加会激活钙调神经蛋白酶。钙调神经蛋白与TRESK蛋白上的NFAT类似的对接位点结合，并引起丝氨酸276的去磷酸化，从而导致通道激活并导致超极化。随着时间的流逝，TRESK蛋白的上调不仅会增加Tresk通道的开放频率，而且增加了Tresk K+通道的数量。为了检验这一假设，我们有3个具体目标。目的1是证明STZ-D中的高血糖修饰了NG神经元的基本电生理特性。将执行斑块夹记录，以表征对控制和STZ-D大鼠NG神经元神经元的兴奋性。这些异常的生理含义将通过体内电生理记录在糖尿病大鼠中的体内电生理记录来评估，并研究其对CCK，瘦素和秘密蛋白刺激的反应性。 AIM 2检查了在慢性糖尿病状态下NG神经元的超极化是否是由TRESK通道的激活介导的。使用电生理学研究以及蛋白质印迹和RT-PCR方法，将鉴定出NG中特斯克钾通道的存在。 TRESK通道的参与将通过使用基于病毒的系统传递siRNA来证明TRESK通道在NG中的表达。为了评估TRESK在糖尿病中NG超极化介导的功能重要性，我们将检查通过用TRESK SIRNA的NG电穿孔在体内沉默的TRESK通道表达后电生理和GI异常的可逆性。 AIM 3研究了介导糖尿病中NG神经元的膜修饰的信号转导级联反应。贴片夹记录和细胞内钙成像研究将使用信使特异性激活剂或抑制剂进行，以确定特定细胞内级联元素对糖尿病动物NG兴奋性的作用。糖尿病中丝氨酸276的去磷酸化的证明将涉及使用磷酸蛋白酶法。了解负责NG在糖尿病状态下功能异常功能的细胞和分子机制将为慢性糖尿病异常GI功能的管理提供重要的治疗靶点。 公共卫生相关性：控制不良的糖尿病患者（包括异常的胃运动，胰腺和胃分泌降低以及异常饮食行为）的胃肠道功能经常异常。这些功能中的许多功能都是通过迷走神经系统来控制的，迷走神经系统在糖尿病患者中经常异常。这项研究的目的是了解导致这组感觉神经元异常的分子和细胞机制。这可以提供重要的治疗靶标，并导致改善胃肠道并发症的糖尿病患者的治疗。