Mechanisms of Fatty-Acid Inhibition of the Insulin Gene

描述（由申请人提供）：本提案的目的是确定长期暴露于升高的脂肪酸影响胰岛素前原基因转录的信号和分子机制，这种现象导致2型糖尿病β细胞功能不可避免地恶化。以前，我们已经证明棕榈酸盐在转录水平上通过神经酰胺合成抑制胰岛素基因表达。特异性目的1：在离体胰岛和胰岛素分泌细胞中鉴定介导棕榈酸盐抑制胰岛素基因转录的信号通路。我们将通过测量对葡萄糖和棕榈酸盐反应的激酶活性和磷酸化状态来评估MAPK和PI3激酶途径的参与。我们将尝试通过使用药理学抑制剂和腺病毒介导的激酶亚型的过表达来调节棕榈酸盐对转录因子活性和胰岛素基因转录的抑制。特异性目的2：确定棕榈酸酯在离体胰岛和胰岛素分泌细胞中抑制MafA表达和PDX-1核定位的机制。我们将通过测量mRNA稳定性和使用启动子-报告子结构来确定棕榈酸盐是否在转录或转录后水平影响MafA的表达；免疫组化检测棕榈酸盐是否影响PX-1核定位；并通过染色质免疫沉淀法检测MafA和PDX-1与内源性胰岛素基因启动子的结合。目的3：探讨高脂血症和高血糖症是否影响长期输注大鼠和高脂喂养小鼠胰岛胰岛素基因转录。我们将确定PDX-1和MafA的表达和结合活性；胰岛素基因启动子活性；胰岛中胰岛素mRNA水平受到影响

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.