Glucagon secretion and islet neuropathy

描述(申请人提供)：胰岛素诱导的低血糖(IIH)的胰升糖素反应在1型糖尿病早期明显受损。我们之前已经证明，在非糖尿病动物和人类中，这种胰高血糖素反应主要是自主调节的。最近，我们在患有1型糖尿病的受试者及其动物模型中发现了一种早期交感胰岛神经病变(ESIN)，因此假设ESIN与这种高血糖素损伤有关。这项资助的目的是确定1)只有交感神经丢失，2)仅从胰岛丢失，3)仅在自身免疫性糖尿病中丢失的机制。解释这三种现象的机制是基于发育神经生物学中的新兴概念。我们假设，在自身免疫性糖尿病的发展过程中，由侵入胰岛的B淋巴细胞分泌的脑源性神经营养因子(BDNF)将胰岛神经营养因子的平衡从通过Trk受体的“轴突维持”转移到通过p75泛神经营养素受体(NTR)的“轴突修剪”，导致胰岛交感神经的选择性丢失，从而导致交感α细胞通路的功能障碍。因此，我们的第一个具体目标是证明BDNF治疗胰岛交感神经丢失的必要性。我们将通过转基因标记BDNF和免疫组织化学标记标记相结合的方法来定位糖尿病胰岛中的BDNF。我们将通过激光捕获这些胰岛并使用RT-PCR来定量检测这些胰岛中的BDNF mRNA。我们将通过敲除BDNF基因来证明BDNF的交感神经毒性，从而保护这些神经，防止交感神经激活时胰高血糖素反应的损害。在我们的第二个特定目标中，我们将通过敲除B淋巴细胞来证明它们的必要性，从而防止胰岛交感神经的丢失和对交感神经激活的胰升糖素反应的损害。在我们的第三个具体目标中，我们将通过敲除p75NTR来防止胰岛交感神经的丧失，再次保留对交感神经激活的胰高血糖素反应。意义：确定1型糖尿病患者胰岛交感神经丢失的机制将使我们能够预防或逆转这种丢失。由于交感神经-阿尔法细胞通路在IIH期间被激活，并参与了IIH对胰高血糖素的反应，我们希望这种预防可以改善1型糖尿病患者的这种胰高血糖素反应。使这种高血糖素反应正常化将减少胰岛素引起的低血糖的发生率和严重程度，使1型糖尿病患者能够得到更密集的治疗，从而减少导致这种疾病长期并发症的慢性高血糖。

DESCRIPTION (provided by applicant): The glucagon response to insulin-induced hypoglycemia (IIH) is markedly impaired early in type 1 diabetes. We have previously demonstrated that this glucagon response is largely autonomically mediated in nondiabetic animals and humans. More recently we discovered an early sympathetic islet neuropathy (eSIN) in human subjects with type 1 diabetes and animal models thereof and therefore hypothesized that eSIN contributes to this glucagon impairment. The purpose of this grant is to determine the mechanism by which 1) only sympathetic nerves are lost, 2) only from the islets and 3) only in autoimmune diabetes. The mechanism that accounts for all three is based on emerging concepts in developmental neurobiology. We hypothesize that brain derived neurotrophic factor (BDNF), secreted by B-lymphocytes invading the islet during the development of autoimmune diabetes, shifts the balance of islet neurotrophins away from "axonal maintenance" via Trk receptors towards "axonal pruning" via the p75 pan-neurotrophin receptor (NTR), causing a selective loss of islet sympathetic nerves and thereby dysfunction of the sympathetic alpha-cell pathway. Therefore our first Specific Aim is to demonstrate the necessity of BDNF for the loss of islet sympathetic nerves. We will localize BDNF in the diabetic islet by a combination of transgenically tagging BDNF and immunohistochemically staining the tag. We will quantify BDNF mRNA in these islets by laser capturing them and using RT-PCR. We will demonstrate the sympathetic neurotoxicity of BDNF by knocking out the BDNF gene and thereby sparing these nerves and preventing the impairment of the glucagon response to sympathetic nerve activation. In our second Specific Aim we will demonstrate the necessity of B-lymphocytes by knocking them out, thus preventing both the loss of islet sympathetic nerves and the impairment of the glucagon response to sympathetic activation. In our third Specific Aim we will prevent the loss of islet sympathetic nerves by knocking out p75NTR, again preserving the glucagon response to sympathetic nerve activation. Significance: Determining the mechanism for the loss of islet sympathetic nerves in type 1 diabetes will allow us to prevent or reverse that loss. Since the sympathetic-alpha cell pathway is activated during IIH and contributes to the glucagon response to IIH, we expect such prevention to improve this glucagon response in type 1 diabetes. Normalizing this glucagon response will reduce the incidence and severity of insulin induced hypoglycemia, allowing more intensive treatment of patients with type 1 diabetes, thereby reducing the chronic hyperglycemia that causes the long-term complications of this disease.