Role of Sterols and Insulin in Cardiac Autonomic Response

描述（由申请人提供）：糖尿病自主神经病变是糖尿病的严重并发症。超过50%有10年糖尿病史的患者表现出心脏对副交感神经刺激的反应受损，并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副交感神经反应性的降低有关。心脏的副交感神经刺激涉及乙酰胆碱与M2毒蕈碱受体的结合，以及G蛋白激活的内向整流K+通道（GIRK 1）2/（GIRK 4）2的激活，其负责IKACh，即引起心脏膜超极化和心率降低的超极化K+电流。固醇调节元件结合蛋白（Sterol regulatory element binding proteins，SREBPs）是调节脂肪酸和胆固醇合成相关基因的转录因子。糖原合成酶激酶3 β（GSK 3 β）是胰岛素信号通路的关键调节组分，具有组成性活性，并受Akt介导的磷酸化作用抑制。研究表明，GSK 3 β在SREBP的泛素化和降解中起作用。胰岛素已被证明可以增加SREBP-1水平。我们以前证明，秋田糖尿病小鼠，它有一个点突变的胰岛素原基因（ins 2），表现出显着降低的反应，副交感神经刺激的心脏。使用这种小鼠，我们先前表明G1 i2和GIRK 1被SREBP-1上调，并且糖尿病小鼠中的低胰岛素血症导致GIRK 1表达减少和IKACh减少。SREPBP-1的腺病毒表达逆转了秋田小鼠心房肌细胞中IKACh的这种损伤。Akt/GSK 3 β是胰岛素代谢作用的重要介质。在本申请中，将测试4个主要假设：1）糖尿病心脏中的GSK 3 β活性增加，并且这与SREBP、GIRK 1和GIRK 4水平降低相关。2)GSK 3通过影响SRBP-1的周转在转录水平上调节GIRK 1和GIRK 4的表达，3）GSK 3的化学抑制剂和DN-1的过表达，GSK 3逆转了秋田小鼠心房肌细胞中IKACh的损伤，并刺激了秋田小鼠心房肌细胞中GIRK 1和GIRK 4的表达，用GSK 3抑制剂治疗小鼠逆转了IKACh的损伤。这些小鼠中的副交感神经功能障碍和4）将具有GSK 3的条件性心脏特异性KO的小鼠与秋田I型糖尿病小鼠杂交保护小鼠免于发展副交感神经功能障碍，并且这种作用被这些小鼠心房中DA-GSK 3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确定新的途径和潜在的治疗靶点，而且应该测试GSK 3抑制剂在治疗和预防糖尿病这种毁灭性并发症中的疗效。 公共卫生相关性：大脑无法调节心脏跳动的速率和力量是糖尿病的主要并发症，这与糖尿病人群的猝死有关。糖原合成酶激酶（GSK 3）是一种通常由胰岛素控制的分子;在这里，我们将确定I型糖尿病小鼠中的胰岛素缺乏是否导致GSK 3不受控制，以及GSK 3的增加是否导致大脑控制心跳的能力丧失。我们将测试逆转糖尿病心脏中增加的GSK 3的药物，以恢复心脏对大脑信号的反应;这些研究有可能开发新的治疗靶点，用于治疗和预防糖尿病的这种衰弱并发症。

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.