Role of Sterols and Insulin in Cardiac Autonomic Response

描述(申请人提供：糖尿病自主神经病变是糖尿病的严重并发症。有10年糖尿病病史的患者中，超过50%的人表现出心脏对副交感神经刺激的反应受损，并由此导致交感迷走神经失衡。此外，糖尿病患者猝死的发生率显著增加，这可能至少部分与心脏的副交感反应性降低有关。对心脏的副交感刺激涉及乙酰胆碱与M2 M受体的结合，以及G蛋白激活的内向整流钾通道(GIRK1)2/(GIRK4)2的激活，这是导致IKACh的超极化K+电流，导致心肌膜超极化和心率减慢。固醇调节元件结合蛋白(SREBPs)是调节脂肪酸和胆固醇合成相关基因的转录因子。糖原合成酶3β(GSK3β)被认为是胰岛素信号通路的关键调节成分，具有结构性活性，并被Akt介导的胰岛素反应的磷酸化所抑制。有人认为GSK3β在SREBPs的泛素化和降解中起作用。胰岛素已被证明可以增加SREBP-1的水平。我们先前证明，在原胰岛素基因(Ins2)有点突变的秋田糖尿病小鼠，对心脏副交感神经刺激的反应显著降低。在这只小鼠身上，我们以前发现SREBP-1上调了G1i2和GIRK1的表达，糖尿病小鼠的低血糖导致GIRK1表达减少和IKACh减少。腺病毒表达SREPBP-1可逆转IKACh在秋田小鼠心房肌细胞中的这种损伤。AKT/GSK3β是胰岛素代谢效应的重要调节因子。在本申请中，将检验4个主要假设：1)糖尿病心脏中GSK3β活性增加，这与SREBP、GIRK1和GIRK4水平下降有关。2)GSK3通过影响SRBP-1的周转在转录水平调节GIRK1和GIRK4的表达；3)GSK3的化学抑制剂和dN-GSK3的过表达逆转了秋田小鼠心房肌细胞IKACh的损伤，刺激了秋田小鼠心房肌细胞GIRK1和GIRK4的表达，GSK3抑制剂治疗小鼠可逆转这些小鼠的副交感神经功能障碍；4)将GSK3的有条件心脏特异性KO与Akita I型糖尿病小鼠杂交可保护小鼠免受副交感神经功能障碍的发展，这种作用可由这些小鼠的心房中DA-GSK3的表达逆转。这些研究不仅应该为糖尿病自主神经病变的发病机制和治疗寻找新的途径和潜在的治疗靶点，而且还应该测试GSK3抑制剂在治疗和预防糖尿病这一毁灭性并发症方面的疗效。 公共卫生相关性：大脑无法调节心脏跳动的速度和力量是糖尿病的一个主要并发症，它与糖尿病人群中的猝死有关。糖原合成酶激酶(GSK3)是一种通常由胰岛素控制的分子；在这里，我们将确定I型糖尿病小鼠的胰岛素缺乏是否会导致GSK3失控，以及GSK3的增加是否会导致大脑失去控制心跳的能力。我们将测试逆转糖尿病心脏中增加的GSK3的药物，以了解它们恢复心脏对来自大脑的信号的反应的能力；这些研究有可能开发一种新的治疗目标，用于治疗和预防这种使人衰弱的糖尿病并发症。

DESCRIPTION (provided by applicant: Diabetic Autonomic Neuropathy is a severe complication of diabetes. More than 50% of patients with a 10-year history of diabetes demonstrate an impaired response of the heart to parasympathetic stimulation and a resulting sympathovagal imbalance. Furthermore, there is a marked increase in the incidence of sudden death in diabetics which may be associated, at least in part, with a decrease in parasympathetic responsiveness of the heart. Parasympathetic stimulation of the heart involves acetylcholine binding to M2 muscarinic receptor, and activation of the G protein-activated inward rectifier K+ channel, (GIRK1)2/(GIRK4)2 which is responsible for IKACh, the hyperpolarizing K+ current that causes a hyperpolarization of the cardiac membrane and a decrease in heart rate. Sterol regulatory element binding proteins (SREBPs) are the transcription factors that regulate genes involved in fatty acid and cholesterol synthesis. Glycogen synthase kinase 3beta (GSK3beta), which was found to be a key regulatory component of the insulin-signaling pathway, is constitutively active and is inhibited by Akt-mediated phosphorylation in response to insulin. It has been suggested that GSK3beta plays a role in the ubiquitination and degradation of SREBPs. Insulin has been shown to increase SREBP-1 levels. We previously demonstrated that the Akita diabetic mouse, which has a point mutation in the pro-insulin gene (ins2), demonstrates a markedly decreased response to parasympathetic stimulation of the heart. Using this mouse we previously showed that G1i2 and GIRK1 are up-regulated by SREBP-1 and that the hypoinsulinemia in the diabetic mouse resulted in decreased expression of GIRK1 and a decrease in IKACh. Adenoviral expression of SREPBP-1 reversed this impairment of IKACh in atrial myocytes from the Akita mouse. Akt/GSK3beta are important mediators of the metabolic effects of insulin. In this application will test 4 major hypotheses: 1) that GSK3beta activity in the diabetic heart is increased and that this is associated with decreased levels of SREBP, GIRK1 and GIRK4. 2) that GSK3 regulates the expression of GIRK1 and GIRK4 at the level of transcription via an effect on the turnover of SRBP-1, 3) that chemical inhibitors of GSK3 and overexpression of a DN-GSK3 reverse the impairment of IKACh in atrial myocytes from Akita mice and stimulates the expression of GIRK1 and GIRK4 in atrial myocytes form Akita mice and that treatment of mice with an inhibitor of GSK3 reverses parasympathetic dysfunction in these mice and 4) that crossing a mouse with a conditional cardiac specific KO of GSK3 with the Akita type I diabetic mouse protects the mouse from developing parasympathetic dysfunction and that this effect is reversed by the expression of a DA-GSK3 in the atria of these mice. These studies should not only identify a new pathway and potential therapeutic target for the pathogenesis and treatment of diabetic autonomic neuropathy, but test the efficacy of GSK3 inhibitors in the treatment and prevention of this devastating complication of diabetes. PUBLIC HEALTH RELEVANCE: The inability of the brain to regulate the rate and force of beating of the heart is a major complication of diabetes which has been associated with sudden death in the diabetic population. Glycogen Synthase Kinase (GSK3) is a molecule that is normally controlled by insulin; here we will determine whether the insulin deficiency in the Type I diabetic mice results in uncontrolled GSK3 and whether this increase in GSK3 results in the loss of the ability of the brain to control the heart beat. We will test drugs that reverse the increased GSK3 in the diabetic heart for their ability to restore the response of the heart to signals from the brain; these studies have the potential of developing a new therapeutic target for the treatment and prevention of this debilitating complication of diabetes.