Mechanisms of Fatty-Acid Inhibition of the Insulin Gene

描述(由申请人提供)：这项建议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。基于我们先前的发现和初步数据，潜在的假说认为，糖代谢紊乱的早期阶段涉及两个互补的机制：1-抑制每Arnt Sim Kinase(PASK)的表达，导致转录因子胰腺-十二指肠同源盒-1(PDX-1)和禽类MAFA/L-MAF(MAFA)的哺乳动物同源物表达和活性降低，从而通过改变组蛋白密码和封闭的染色质构象导致胰岛素表达减少；以及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)激活FOXM1信号并导致功能障碍。在特定的目标1中，我们将确定抑制PASK表达如何在糖代谢紊乱中损害胰岛素基因的表达。我们的工作假设是，PASK使糖原合成酶激酶(GSK)3？磷酸化，从而使其失活，从而减轻PDX-1和MafA的蛋白酶体降解。利用啮齿动物遗传模型，我们建议进一步描述PASK和GSK3的功能关系及其在糖代谢紊乱条件下对PDX-1和MafA表达和功能的影响。在特定的目标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编码和DNA甲基化特征的改变。我们的工作假设是，糖代谢紊乱的PDX-1缺乏导致组蛋白甲基转移酶Set7/9的缺陷募集，以及胰岛素、PDX-1和MafA启动子的组蛋白甲基化特征的改变。利用体外和体内模型，我们建议确定导致糖代谢紊乱的表观遗传修饰。在具体目标3中，我们将确定胰岛素抵抗是如何在糖代谢障碍中诱导细胞增殖的。我们的工作假设是，6个月大鼠对营养过剩的胰岛素抵抗与循环因子的增加有关，FGF21可能是一个候选因素，它刺激FOXM1介导的细胞增殖。利用体内模型，我们建议确定胰岛素抵抗在营养过剩条件下促进细胞生长的机制。我们期望在本申请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.