Cholesterol Toxicity as a Promising Target for Diabetes Prevention

毫无疑问，通过己糖胺生物合成途径(HBP)过量的葡萄糖流量会导致胰岛素抵抗。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很可能在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就开始了。虽然机制尚不清楚，但体外数据表明，HBP活性增加增加了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，从而损害了胰岛素刺激的葡萄糖转运体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一种新的假设，即胰岛素抵抗中葡萄糖稳态的破坏是HBP介导的胆固醇生物合成增加的次要原因。在体内观察到过量PM胆固醇的事实表明，保护细胞内胆固醇积累/毒性的调节机制可能在胰岛素抵抗脂肪/肌肉中存在缺陷。为了支持这种可能性，HBP的胆固醇生成反应也损害了ATP结合盒转运体A1(ABCA1)介导的胰岛素抵抗3T3-L1脂肪细胞的胆固醇外流。总的来说，这些数据与最近的基因表达研究是一致的，这些研究表明，胆固醇代谢基因网络的变化与T2D风险有关。来自细胞和组织的数据表明，PM胆固醇的积累减少了对GLUT4调节重要的皮质细丝肌动蛋白(F-肌动蛋白)。尽管F-肌动蛋白的丢失，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(GLUT4)可被胰岛素动员到恰好位于胆固醇含量较高的PM下方的位置，但随后无法结合和运输葡萄糖。数据表明，这种损伤是由于磷脂酶D1(PLD1)缺陷介导的磷脂酸(PA)的产生，这是已知的促进GSV/PM融合的因素。该项目将确定胰岛素抵抗脂肪/肌肉中PM胆固醇的体内增加是否是由于HBP驱动的Sp1转录事件，以及ABCA1和/或Abcg1介导的缺陷对PM胆固醇积累的保护是否发生加剧胰岛素抵抗(目标1)。随着F-肌动蛋白聚合和PLD1激活的调节都发生在胆固醇丰富的小窝PM微域中，该项目还将确定这些细胞骨架/膜GLUT4调节步骤中过量的PM胆固醇驱动的缺陷是否与胰岛素抵抗有因果联系(目标2)。这一应用的一个关键假设是，体内葡萄糖耐量异常的发展涉及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基于膜的机制。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.