Mechanisms of Fatty-Acid Inhibition of the Insulin Gene

描述(申请人提供)：本提案的目的是确定长期暴露于高脂肪酸影响胰岛素原基因转录的信号和分子机制，这是一种导致2型糖尿病患者β细胞功能不可避免地恶化的现象。此前，我们已经证明棕榈酸酯通过神经酰胺合成在转录水平上抑制胰岛素基因的表达。具体目的1：确定棕榈酸酯抑制胰岛和胰岛素分泌细胞胰岛素基因转录的信号通路。我们将通过测量葡萄糖和棕榈酸酯的反应中的激酶活性和磷酸化状态来评估MAPK和PI3激酶通路的参与。我们将尝试通过使用药物抑制剂和腺病毒介导的激酶亚型过表达来调节棕榈酸抑制转录因子活性和胰岛素基因转录。特定目的2：确定棕榈酸抑制分离的胰岛和胰岛素分泌细胞中MafA表达和PDX-1的核定位的机制。我们将通过测量mRNA稳定性和使用启动子-报告结构来确定棕榈酸酯是否在转录或转录后水平影响MafA的表达；通过免疫组织化学方法确定棕榈酸酯是否影响PX-1核定位；通过染色质免疫沉淀试验检测MafA和PDX-1与内源性胰岛素基因启动子的结合。具体目的3：确定合并高脂血症和高血糖是否影响长期输注的大鼠和高脂喂养的小鼠胰岛的胰岛素基因转录。我们将确定PDX-1和MafA的表达和结合活性，胰岛素基因启动子的活性，以及胰岛素mRNA水平是否受到影响 1)Wistar大鼠单独或联合输注葡萄糖和脂肪酸24小时和72小时； 2)高脂饲料喂养C57BI/6J小鼠。该项目有可能揭示过量脂肪酸对胰岛β细胞功能产生不利影响的细胞和分子机制，从而导致2型糖尿病过程中葡萄糖稳态的恶化。它将提供新的治疗目标，旨在保护2型糖尿病患者的胰岛素分泌。2型糖尿病是一种毁灭性的疾病，影响着1800多万美国人。

DESCRIPTION (provided by applicant): The objective of this proposal is to identify the signaling and molecular mechanisms whereby prolonged exposure to elevated fatty acids affects preproinsulin gene transcription, a phenomenon that contributes to the inexorable deterioration of beta-cell function in type 2 diabetes. Previously, we have demonstrated that palmitate inhibits insulin gene expression at the transcriptional level via ceramide synthesis in isolated islets. Specific Aim 1: To identify the signaling pathways mediating palmitate inhibition of insulin gene transcription in isolated islets and insulin-secreting cells. We will assess the involvement of the MAPK and PI3 kinase pathways by measuring kinase activity and phosphorylation state in response to glucose and palmitate. We will attempt to modulate palmitate-inhibition of transcription factor activity and insulin gene transcription by using pharmacological inhibitors and adenovirus-mediated overexpression of kinase isoforms. Specific Aim 2: To determine the mechanisms whereby palmitate inhibits expression of MafA and nuclear localization of PDX-1 in isolated islets and insulin-secreting cells. We will ascertain whether palmitate affects MafA expression at the transcriptional or post-transcriptional level by measuring mRNA stability and using promoter-reporter constructs; determine whether palmitate affects PX-1 nuclear localization by immunohistochemistry; and examine binding of MafA and PDX-1 to the endogenous insulin gene promoter by chromatin immunoprecipitation assays. Specific Aim 3: To ascertain whether combined hyperlipidemia and hyperglycemia affect insulin gene transcription in islets from chronically infused rats and high-fat fed mice. We will ascertain whether expression and binding activity of PDX-1 and MafA; activity of the insulin gene promoter; and insulin mRNA levels are affected in islets isolated from 1) Wistar rats following a 24- and 72-h infusion of glucose and fatty acids, alone or in combination; and 2) high-fat fed C57BI/6J mice. This project has the potential to uncover the cellular and molecular mechanisms by which excessive levels of fatty acids adversely affect pancreatic beta-cell function and thereby contribute to the deterioration of glucose homeostasis during the course of type 2 dabetes. It will provide new therapeutic targets aimed at preserving insulin secretion in type 2 diabetes, a devastating disease that affects more than 18 million Americans.