Mechanisms of Fatty-Acid Inhibition of the Insulin Gene

描述（由申请人提供）：本提案的总体目标是阐明葡萄糖和脂肪酸水平升高对胰腺细胞产生不利影响的分子机制，这种现象被称为糖脂功能障碍。基于我们之前的发现和初步数据，潜在的假设提出，早期阶段的糖脂功能障碍涉及两个互补的机制：1-抑制Per Arnt Sim kinase （PASK）的表达，导致转录因子胰-十二指肠同源盒-1 （Pdx-1）和禽类MafA/l-Maf （MafA）的哺乳动物同源物的表达和活性降低，通过组蛋白编码的改变和封闭的染色质构象导致胰岛素表达降低；2-营养诱导的细胞增殖，由循环中激活FoxM1信号的成纤维细胞生长因子21 （FGF21）水平升高驱动，并导致功能失调的细胞团。在特定的目标1中，我们将确定抑制PASK表达如何损害糖脂功能障碍中胰岛素基因的表达。我们的工作假设是PASK磷酸化，从而使糖原合成酶激酶（GSK） 3¿失活，从而减轻蛋白酶体对Pdx-1和MafA的降解。利用啮齿类动物遗传模型，我们建议进一步描述PASK和GSK3¿之间的功能关系及其在糖脂功能障碍条件下对Pdx-1和MafA表达和功能的影响。在特定的Aim 2中，我们将描述与糖脂功能障碍相关的Pdx-1、MafA和胰岛素启动子的组蛋白代码和DNA甲基化谱的修饰。我们的工作假设是，糖脂功能障碍中Pdx-1的缺乏导致组蛋白甲基转移酶Set7/9的募集缺陷，以及胰岛素、Pdx-1和MafA启动子处组蛋白甲基化谱的改变。使用离体和体内模型，我们建议确定负责启动糖脂功能障碍的表观遗传修饰。在特定的Aim 3中，我们将确定胰岛素抵抗如何诱导糖脂功能障碍患者的细胞增殖。我们的工作假设是，6岁大鼠对营养过剩的胰岛素抵抗与循环因子的增加有关，FGF21是一个可能的候选者，它刺激FoxM1介导的细胞增殖。使用体内模型，我们建议确定在营养过剩条件下胰岛素抵抗促进细胞生长的机制。我们期望在本应用中描述的研究将揭示胰腺细胞中糖脂功能障碍的分子特征。我们预计这些发现将作为设计新的治疗方法的基础，以防止T2D中细胞功能的恶化。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to elucidate the molecular mechanisms by which elevated levels of glucose and fatty acids adversely affect the pancreatic ¿-cell, a phenomenon referred to as glucolipodysfunction. The underlying hypothesis, based on our previous findings and preliminary data, proposes that early stages of glucolipodysfunction involve two complementary mechanisms: 1- Inhibition of expression of the enzyme Per Arnt Sim kinase (PASK) which results in diminished expression and activity of the transcription factors pancreatic-duodenal homeobox-1 (Pdx-1) and mammalian homologue of avian MafA/l-Maf (MafA), leading to decreased insulin expression via alterations of the histone code and a closed chromatin conformation; and 2- Nutrient-induced ¿-cell proliferation driven by elevated circulating levels of fibroblast growth factor 21 (FGF21) activating FoxM1 signaling and leading to a dysfunctional ¿-cell mass. In specific Aim 1 we will determine how inhibition of PASK expression impairs insulin gene expression in glucolipodysfunction. Our working hypothesis is that PASK phosphorylates, and thereby inactivates, glycogen synthase kinase (GSK) 3¿ which alleviates proteasomal degradation of Pdx-1 and MafA. Using rodent genetic models we propose to further delineate the functional relationship between PASK and GSK3 ¿ and its consequences on Pdx-1 and MafA expression and function under conditions of glucolipodysfunction. In specific Aim 2 we will characterize the modifications of the histone code and DNA methylation profile at the Pdx-1, MafA, and insulin promoters associated with glucolipodysfunction. Our working hypothesis is that Pdx-1 deficiency in glucolipodysfunction results in defective recruitment of the histone methyltransferase Set7/9 and alterations of the histone methylation profile at the insulin, Pdx-1, and MafA promoters. Using ex vivo and in vivo models we propose to identify the epigenetic modifications responsible for the initiation of glucolipodysfunction. In specific Aim 3 we will ascertain how insulin resistance induces ¿ -cell proliferation in glucolipodysfunction. Our working hypothesis is that insulin resistance in response to nutrient excess in 6-mo-old rats is associated with a rise in circulating factors, FGF21 being a likely candidate, which stimulate FoxM1- mediated ¿-cell proliferation. Using in vivo models we propose to identify the mechanisms whereby insulin resistance promotes ¿-cell growth under conditions of nutrient excess. We expect that the studies described in this application will reveal the molecular signature of glucolipodysfunction in the pancreatic ¿-cell. We anticipate that these findings will serve as a basis to design novel therapeutic approaches to prevent the deterioration of ¿-cell function in T2D. PUBLIC HEALTH RELEVANCE: Pancreatic ¿-cell function inexorably declines during the course of type 2 diabetes mellitus. This deterioration is due, at least in part, to the metabolic perturbations associated with diabetes, chiefly chronic hyperglycemia and dyslipidemia, which themselves have detrimental effects on ¿-cell function. By discovering the molecular mechanisms by which chronic excess of nutrients impair insulin production from the ¿-cell we hope to identify potential therapeutic targets to curtail the degradation of glucose homeostasis in type 2 diabetes.