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（申请人提供）：控制不良的糖尿病患者胃肠道功能常出现异常。临床研究表明，大多数这些异常可归因于迷走神经传入功能的缺陷。我们的初步研究表明，链脲佐菌素诱导的糖尿病大鼠（STZ- D）结节神经节（NG）神经元呈现超极化，导致兴奋性降低。这可能导致糖尿病状态下迷走神经功能异常。我们假设慢性高血糖激活“背景”TRESK钾通道，导致NG超极化和兴奋性降低。这是一个两步的过程。最初，糖尿病神经元细胞内钙的增加激活钙调磷酸酶。钙调磷酸酶与TRESK蛋白上的NFAT样对接位点结合，引起丝氨酸276的去磷酸化，导致通道激活并导致超极化。随着时间的推移，TRESK蛋白的上调不仅导致TRESK通道的打开频率增加，而且导致TRESK K+通道的数量增加。为了验证这一假设，我们有三个具体目标。目的1是证明STZ- D的高血糖改变了NG神经元的基本电生理特性。膜片钳记录将用来表征对照和STZ-D大鼠的NG神经节神经元的兴奋性。这些异常的生理意义将通过糖尿病大鼠NG的体内电生理记录来评估，并研究其对CCK、瘦素和分泌素刺激的反应性。目的2研究慢性糖尿病状态下NG神经元的超极化是否通过激活TRESK通道介导。NG中特定TESK钾通道的存在将通过电生理研究以及western blot和RT-PCR方法进行鉴定。TRESK通道的参与将通过使用基于病毒的siRNA递送系统来沉默NG中TRESK通道的表达来证明。为了评估TRESK在糖尿病中介导NG超极化中的功能重要性，我们将通过用TRESK siRNA电穿孔NG，在体内沉默TRESK通道表达后，研究电生理和GI异常的可逆性。目的3研究糖尿病中介导NG神经元膜修饰的信号转导级联。膜片钳记录和细胞内钙成像研究将与信使特异性激活剂或抑制剂一起进行，以确定特定细胞内级联元件对糖尿病动物NG兴奋性的作用。证明276丝氨酸在糖尿病中的去磷酸化将涉及到使用磷酸化蛋白质组方法。了解糖尿病状态下NG功能异常的细胞和分子机制将为慢性糖尿病患者GI功能异常的治疗提供重要的治疗靶点。