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.
New evidence suggests that the breakdown of glucose homeostasis, characteristic of insulin resistance in obesity and diabetes, may be secondary to plasma membrane cholesterol accumulation in fat and muscle cells. Strikingly, study has demonstrated that returning the excess plasma membrane cholesterol to normal levels in these cells restores insulin sensitivity. Study of this cause of insulin resistance in animals, along with further investigation of the cholesterol-induced mechanism of glucose transport dysregulation in these cells will significantly impact therapeutic strategies to promote glycemic health.
