Cholesterol Toxicity as a Promising Target for Diabetes Prevention

毋庸置疑，过量葡萄糖通量通过己糖胺生物合成途径（HBP）可引起胰岛素抵抗。临床研究结果支持这样一种观点，即葡萄糖诱导的胰岛素抵抗可能在2型糖尿病发病前几年就开始了，甚至在糖尿病前期被发现之前。虽然机制尚不清楚，但体外数据表明，HBP活性的增加增加了Sp1的o -连接n -乙酰氨基葡萄糖修饰，导致HMG-CoA还原酶的转录激活，HMG-CoA还原酶是胆固醇合成中的限速酶。这种hbp诱导的反应增加了质膜（PM）胆固醇，从而损害胰岛素刺激的葡萄糖转运蛋白glut4介导的葡萄糖转运。抑制HBP活性或阻断o - glcnac修饰的Sp1与DNA的结合可防止PM胆固醇积累和GLUT4/葡萄糖转运失调。这些细胞培养数据支持了一个新的假设，即胰岛素抵抗中葡萄糖稳态的破坏是继发于hbp介导的胆固醇生物合成增加。体内观察到过量PM胆固醇的事实表明，在胰岛素抵抗脂肪/肌肉中，防止细胞胆固醇积累/毒性的调节机制可能存在缺陷。为了支持这种可能性，hbp -胆固醇生成反应也会损害atp结合盒转运蛋白A1 （ABCA1）介导的胰岛素抵抗型3T3-L1脂肪细胞的胆固醇外排。总的来说，这些数据与最近的基因表达研究一致，表明胆固醇代谢基因网络的改变与T2D风险相关。来自细胞和组织的数据表明，PM胆固醇积累减少了对GLUT4调节重要的皮质丝状肌动蛋白（f -肌动蛋白）。尽管f -肌动蛋白缺失，但对胰岛素抵抗的3T3-L1脂肪细胞的初步机制研究表明，GLUT4储存囊泡（GSVs）被胰岛素动员到胆固醇含量高的PM下方的位置，但随后无法整合和运输葡萄糖。数据表明，这种损伤是由磷脂酶D1 （PLD1）介导的磷脂酸（PA）产生缺陷引起的，磷脂酸可以促进GSV/PM融合。该项目将确定体内胰岛素抵抗脂肪/肌肉中PM胆固醇的增加是否由于hbp驱动的Sp1转录事件，以及ABCA1和/或abcg1介导的PM胆固醇积累保护缺陷是否会加剧胰岛素抵抗（Aim 1）。由于f -肌动蛋白聚合和PLD1激活的调控都发生在富含胆固醇的小泡PM微域，该项目还将确定这些细胞骨架/膜glut4调控步骤中过量的PM胆固醇驱动缺陷是否与胰岛素抵抗有因果关系（目的2）。该应用的一个关键假设是体内葡萄糖耐受不良的发展涉及hbp诱导的胆固醇生成反应，该反应损害了GLUT4调节的一个或多个远端膜机制。这一认识的进步将重塑我们对胰岛素抵抗发展的理解，并为其预防和/或治疗确定新的治疗靶点。

There is little doubt that excess glucose flux through the hexosamine biosynthesis pathway (HBP) can cause insulin resistance. Clinical findings support the contention that glucose-induced insulin resistance likely starts years before the onset of type 2 diabetes, even before prediabetes is recognized. Although a mechanism is not known, in vitro data suggest that increased HBP activity increases O-linked N-acetylglucosamine modification of Sp1, leading to transcriptional activation of HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. This HBP-induced response increases plasma membrane (PM) cholesterol that impairs insulin-stimulated glucose transporter GLUT4-mediated glucose transport. Inhibition of HBP activity or blockade of O-GlcNAc-modified Sp1 binding to DNA prevents PM cholesterol accumulation and GLUT4/glucose transport dysregulation. These cell culture data support a novel hypothesis that the breakdown of glucose homeostasis in insulin resistance is secondary to increased HBP-mediated cholesterol biosynthesis. The fact excess PM cholesterol is seen in vivo suggests that regulatory mechanisms that protect against cellular cholesterol accumulation/toxicity may be defective in insulin-resistant fat/muscle. In support of this possibility, the HBP-cholesterolgenic response also impairs ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux from insulin-resistant 3T3-L1 adipocytes. Collectively, these data are in accord with recent gene expression studies showing that alterations in a network of cholesterol metabolism genes are associated with T2D risk. Data from cells and tissues suggest PM cholesterol accumulation diminishes cortical filamentous actin (F-actin) important for GLUT4 regulation. Despite this loss of F-actin, preliminary mechanistic studies in insulin-resistant 3T3-L1 adipocytes show GLUT4 storage vesicles (GSVs) are mobilized by insulin to a position just beneath the cholesterol-laden PM but then fail to incorporate and transport glucose. Data suggest that this impairment results from defective phospholipase D1 (PLD1)-mediated production of phosphatidic acid (PA), which is known to promote GSV/PM fusion. This project will determine whether the in vivo increase in PM cholesterol in insulin-resistant fat/muscle is due to HBP-driven Sp1 transcriptional events, and if defective ABCA1 and/or ABCG1-mediated protection against PM cholesterol accumulation occurs exacerbating insulin resistance (Aim 1). With both the regulation of F-actin polymerization and PLD1 activation occurring at cholesterol-enriched caveolae PM microdomains, this project will also determine if excess PM cholesterol-driven defects in these cytoskeletal/membrane GLUT4-regulatory steps are causally linked to insulin resistance (Aim 2). A key postulate of this application is that the development of glucose intolerance in vivo involves a HBP-induced cholesterolgenic response that impairs one or more distal membrane-based mechanisms of GLUT4 regulation. Advancement of this understanding will reshape our understanding of insulin resistance development and identify new therapeutic targets for its prevention and/or treatment.