Impact of Neprilysin on Islet Function

描述（由申请人提供）：2型糖尿病以胰岛细胞衰竭为特征，由高血糖和高脂血症引起。NEP （NEP）是一种广泛表达的质膜肽酶，我们已经证明，随着长期暴露于葡萄糖和脂肪升高，胰岛中NEP的表达会增加。与二肽基肽酶-4 （DPP-4）一样，NEP可以降解并使胰高血糖素样肽-1 （GLP-1）失活，GLP-1是一种由肠L细胞和胰岛分泌的葡萄糖依赖的促胰岛素肽。细胞。我们的初步数据表明，在饮食脂肪增加的情况下，小鼠NEP消融可提高活性GLP-1水平，从而增加葡萄糖刺激胰岛素分泌（GSIS）并改善葡萄糖耐量。因此，在本提案中，我们将验证NEP活性增加限制GLP-1对胰岛素分泌和葡萄糖稳态有益作用的假设。为了解决这一假设，确定了以下具体目标：1)确定胰岛NEP通过其降解活性GLP-1的能力对降低GSIS的贡献。首先，将利用体外培养模型来测量在棕榈酸盐或高葡萄糖培养或不培养的人和小鼠胰岛中活性GLP-1水平，以确定GLP-1水平是否在与NEP活性增加相关的条件下降低。对NEP进行药物抑制或基因消融的胰岛将用于比较。其次，将利用体内模型，将野生型或NEP-/-胰岛移植到链脲佐菌素糖尿病野生型或NEP-/-同基因受体中，以评估胰岛NEP对移植后维持活性GLP-1水平和正常细胞功能的贡献。2)通过肠道NEP降解活性GLP- 1的能力，确定肠道NEP对降低GSIS的贡献。L细胞释放的GLP-1直接通过体循环或间接通过神经信号促进GSIS

DESCRIPTION (provided by applicant): Type 2 diabetes is characterized by islet �-cell failure, contributed to by hyperglycemia and hyperlipidemia. Neprilysin (NEP) is a widely expressed plasma membrane peptidase that we have shown is increased in islets with prolonged exposure to elevated glucose and fat. Like dipeptidyl peptidase-4 (DPP-4), NEP can degrade and inactivate glucagon-like peptide-1 (GLP-1), a glucose-dependent insulinotropic peptide secreted by intestinal L cells and islet ? cells. Our preliminary data show that under conditions of increased dietary fat, NEP ablation in mice enhances active GLP-1 levels thereby contributing to increased glucose-stimulated insulin secretion (GSIS) and improved glucose tolerance. Thus in this proposal, we will test the hypothesis that increased NEP activity limits the beneficial effect of GLP-1 on insulin secretion and glucose homeostasis. To address this hypothesis, the following specific aims have been identified: 1) To determine the contribution of islet NEP to reduced GSIS via its ability to degrade active GLP-1. First, an in vitro culture model will be utilized to measure active GLP-1 levels in human and mouse islets cultured with or without palmitate or high glucose to determine whether GLP-1 levels are reduced in conditions associated with increased NEP activity. Islets with pharmacological inhibition or genetic ablation of NEP will be used for comparison. Second, an in vivo model will be utilized in which wild-type or NEP-/- islets will be transplanted into streptozocin-diabetic wild-type or NEP-/- syngeneic recipients to evaluate the contribution of islet NEP to the maintenance of active GLP-1 levels and normal �-cell function following transplantation. 2) To determine the contribution of intestinal NEP to reduced GSIS via its ability to degrade active GLP- 1. GLP-1 released from L cells facilitates GSIS directly via the systemic circulation and indirectly via neural signals from vagal afferents. We will assess whether selective reduction of NEP activity in mouse intestine using pharmacologic and genetic (NEPflox ? Villin-Cre) approaches increases portal and peripheral active GLP- 1 levels and thereby enhances GSIS. The contribution of neural signaling to enhanced GSIS will be determined using selective hepatic vagotomies or capsaicin to block activation of pancreatic vagal efferents. 3) To compare the efficacy of NEP versus DPP-4 inhibition in enhancing active GLP-1 levels and improving fat-induced insulin secretory dysfunction in vivo. NEP, DPP-4 or both will be pharmacologically inhibited in wild-type control mice fed a low or high fat diet for 18 weeks. Active GLP-1 levels and GSIS will be measured to determine the relative contributions of each peptidase to preservation of �-cell function in mice fed a high fat diet. Dual peptidase inhibition will test whether any beneficial effect observed wit NEP inhibition can be augmented with concurrent DPP-4 inhibition. These studies will delineate a novel role for islet and intestinal NEP in modulating �-cell function, and will have significant implications for development of therapeutics to treat �-cell dysfunction in diabetes.