Hexosamines Provoke Membrane Cholesterol Accrual, Filamentous Actin Loss, and GLUT4 Dysregulation in Adipocytes through Transcriptional Activation of Specificity Protein 1

poster abstractThe hexosamine biosynthesis pathway (HBP) serves as a sensor of excess nutrient bioavailability and has been implicated in the pathogenesis of type 2 diabetes. Previous study observed that hyperinsulinemic culturing conditions akin to those seen clinically activate the HBP provoking gains in plasma membrane (PM) cholesterol content in L6 myotubes and 3T3-L1 adipocytes. This, in turn, compromised the cortical filamentous actin (F-actin) structure necessary for the proper incorporation of the insulin sensitive glucose transporter GLUT4 into the membrane. The mechanism(s), however, by which HBP activation provokes PM cholesterol accrual, remains unclear. Here, the hypothesis that HBP engages a cholesterolgenic transcriptional response resulting in PM cholesterol accrual/toxicity was tested. In 3T3-L1 adipocytes, pathophysiologically relevant doses of hyperinsulinemia (0.25, 0.5, and 5 nM) resulted in a dose-dependent gain in PM cholesterol as well as mRNA and protein levels of HMG-CoA reductase (HMGR), the rate limiting enzyme in cholesterol synthesis. Immunoprecipitation experiments demonstrated that hyperinsulinemia induced elevations in O-linked N-acetylglucosamine post-translational modification of the cholesterolgenic transcription factor specificity protein 1 (Sp1). This modification was prevented in cells in which the HBP was inhibited. Chromatin immunoprecipitation demonstrated that hyperinsulinemia induced a ~4 fold increase in the affinity of Sp1 to the promoter region of HMGR, which was lost with HBP inhibition. Luciferase assays confirmed that this altered binding resulted in a ~50% increase in promoter activity of this cholesterolgenic gene. Hyperinsulinemia also augmented Sp1 binding to the promoter of the sterol response element binding protein gene, resulting in increased total and nuclear content of this factor. To further delineate the role of Sp1 in this process, a specific inhibitor, mithramycin (MTR), of Sp1 binding to DNA was employed. This inhibitor prevented against hyperinsulinemia-induced gains in HMGR and PM cholesterol as well as F-actin loss. Importantly, this treatment corrected the impaired insulin-stimulated GLUT4 translocation and glucose transport induced by hyperinsulinemia. These data suggest hyperinsulinemia-induced HBP activity provokes cholesterol synthesis and PM cholesterol accrual/F-actin loss that compromises GLUT4/glucose transport regulation by insulin

New aspects of cellular cholesterol regulation on blood glucose control- review and perspective on the impact of statin medications on metabolic health

Cholesterol is an essential component of cell membranes, and during the past several years, diabetes researchers have found that membrane cholesterol levels in adipocytes, skeletal muscle fibers and pancreatic beta cells influence insulin action and insulin secretion. Consequently, it is thought that dysregulated cell cholesterol homeostasis could represent a determinant of type 2 diabetes (T2D). Recent clinical findings compellingly add to this notion by finding increased T2D susceptibility in individuals with alterations in a variety of cholesterol metabolism genes. While it remains imperfectly understood how statins influence glucose metabolism, the fact that they display an influence on blood glucose levels and diabetes susceptibility seems to intensify the emerging importance of understanding cellular cholesterol in glucose metabolism. Taking this into account, this review first presents cell system and animal model findings that demonstrate the negative impact of cellular cholesterol accumulation or diminution on insulin action and insulin secretion. With this framework, a description of how changes in cholesterol metabolism genes are associated with T2D susceptibility will be presented. In addition, the connection between statins and T2D risk will be reviewed with expanded information on pitavastatin, a newer statin medication that displays actions favoring metabolic healt

Evidence that Hyperinsulinemia, known to Accelerate Diabetes Progression, may also Contribute to Dyslipidemia via Impairing ApoA1/ABCA1-Mediated Cholesterol Efflux

poster abstractLow levels of plasma high-density lipoprotein cholesterol (HDL-C) are associated with insulin resistance and type 2 diabetes (T2D). As it is well appreciated that hyperinsulinemia contributes to the progression/worsening of insulin resistance, we tested here if this key metabolic derangement impaired cellular mechanisms of HDL-C generation. An initial event in this process is the binding of apolipoprotein A1 (ApoA1) to the plasma membrane (PM)-localized ATPbinding cassette cholesterol transporter protein ABCA1. Subcellular fractionation analyses revealed that 3T3-L1 adipocytes exposed to chronic insulin (12h, 5nM) displayed a 25% decrease (P<0.05) in PM ABCA1 content and a reciprocal increase in endosomal ABCA1 content. These insulin-induced changes in cellular ABCA1 distribution occurred concomitantly with a decrease in ApoA1-mediated cellular cholesterol efflux. Consistent with endosomal/cytosolic cycling of the small molecular GTPase Rab8 playing a functional role in ABCA1 vesicle trafficking, we found a 50% increase (P<0.05) in endosomal Rab8 content and a 30% decrease (P<0.05) in cytosolic Rab8 content. New data shows that increased HBP activity increases cholesterol biosynthesis and increased endosomal cholesterol content inhibits the functional cycling of Rab proteins. In line with these observations, we found that cells treated with the cholesterol-lowering agent methyl-β-cyclodextrin were protected against insulininduced defects in ABCA1/Rab8 vesicle trafficking and PM cholesterol accrual. These data are consistent with the concept that the coexistence of low plasma HDL-C with insulin resistance and T2D may reflect a negative influence of hyperinsulinemia on Rab8-mediated trafficking of ABCA1 to the PM for ApoA1-mediated cholesterol efflux

Identification of an Actin-Based Antidiabetic Action of Chromium in Skeletal Muscle

poster abstractWe recently demonstrated that cortical filamentous actin (F-actin) loss contributes to cellular insulin resistance induced by hyperinsulinemia. New animal and human analyses suggest a similar loss of F-actin is present in insulin-resistant skeletal muscle and results from cellular cholesterol accrual. Interestingly, we found that chromium picolinate (CrPic), a dietary supplement recognized to improve insulin action, lowers plasma membrane cholesterol in cultured adipocytes. Understanding whether CrPic can improve F-actin structure in insulinresistant skeletal muscle via lowering membrane cholesterol is not known, yet significant, as skeletal muscle is responsible for a large majority of insulin-stimulated glucose transport. In L6 myotubes stably expressing the insulin-responsive glucose transporter GLUT4 carrying an exofacial myc-epitope tag, acute insulin stimulation (20 min, 100 nM) increased myc-epitope labeling at the surface of intact cells by ~2-fold (P<0.05). In contrast, the ability of insulin to stimulate this process was inhibited 25% (P<0.05) by sustained exposure of L6 myotubes to insulin (12 h, 5 nM). Defects in insulin signaling did not readily account for the observed disruption. However, we found that insulin-induced insulin-resistant myotubes displayed a 28% elevation (P<0.05) in membrane cholesterol with a reciprocal 14% loss (P<0.05) in F-actin. This cholesterol/actin imbalance and insulin/GLUT4 dysfunction was corrected by the cholesterollowering action of CrPic. Mechanistically, CrPic increased the activity of the AMP-activated protein kinase (AMPK). Tests also revealed that other well-recognized activators of AMPK (e.g., AICAR, DNP) lowered membrane cholesterol and that, in a fashion similar to that witnessed for CrPic, improved regulation of GLUT4 in insulin-induced insulin-resistant myotubes. These data, as well as findings from ongoing siRNA-mediated AMPK knockdown experiments, are consistent with AMPK mediating its antidiabetic action by lowering cellular cholesterol. We predict that chromium, via AMPK activation, protects against cholesterol accrual that induces skeletal muscle F-actin loss and insulin resistance

Effect of Corncob bedding on feed conversion efficiency in a high-fat diet-induced prediabetic model in C57Bl/6J mice

Laboratory facilities use many varieties of contact bedding, including wood chips, paper products, and corncob, each with its own advantages and disadvantages. Corncob bedding, for example, is often used because of its high absorbency, ability to minimize detectable ammonia, and low cost. However, observations that mice eat the corncob lead to concerns that its use can interfere with dietary studies. We evaluated the effect of corncob bedding on feed conversion (change in body weight relative to the apparent number of kcal consumed over 7 d) in mice. Four groups of mice (6 to 12 per group) were housed in an individually ventilated caging system: (1) low-fat diet housed on recycled paper bedding, (2) low-fat diet housed on corncob bedding, (3) high-fat diet housed on recycled paper bedding, and (4) high-fat diet housed on corncob bedding. After 4 wk of the high-fat diet, feed conversion and percentage body weight change both were lower in corncob-bedded mice compared with paper-bedded mice. Low-fat-fed mice on corncob bedding versus paper bedding did not show statistically significant differences in feed conversion or change in percentage body weight. Average apparent daily feed consumption did not differ among the 4 groups. In conclusion, these data suggest that corncob bedding reduces the efficiency of feed conversion in mice fed a high-fat diet and that other bedding choices should be favored in these models

Exercise training prevents skeletal muscle plasma membrane cholesterol accumulation, cortical actin filament loss, and insulin resistance in C57BL/6J mice fed a western‐style high‐fat diet

Insulin action and glucose disposal are enhanced by exercise, yet the mechanisms involved remain imperfectly understood. While the causes of skeletal muscle insulin resistance also remain poorly understood, new evidence suggest excess plasma membrane (PM) cholesterol may contribute by damaging the cortical filamentous actin (F‐actin) structure essential for GLUT4 glucose transporter redistribution to the PM upon insulin stimulation. Here, we investigated whether PM cholesterol toxicity was mitigated by exercise. Male C57BL/6J mice were placed on low‐fat (LF, 10% kCal) or high‐fat (HF, 45% kCal) diets for a total of 8 weeks. During the last 3 weeks of this LF/HF diet intervention, all mice were familiarized with a treadmill for 1 week and then either sham‐exercised (0 m/min, 10% grade, 50 min) or exercised (13.5 m/min, 10% grade, 50 min) daily for 2 weeks. HF‐feeding induced a significant gain in body mass by 3 weeks. Sham or chronic exercise did not affect food consumption, water intake, or body mass gain. Prior to sham and chronic exercise, “pre‐intervention” glucose tolerance tests were performed on all animals and demonstrated that HF‐fed mice were glucose intolerant. While sham exercise did not affect glucose tolerance in the LF or HF mice, exercised mice showed an improvement in glucose tolerance. Muscle from sham‐exercised HF‐fed mice showed a significant increase in PM cholesterol, loss of cortical F‐actin, and decrease in insulin‐stimulated glucose transport compared to sham‐exercised LF‐fed mice. These HF‐fed skeletal muscle membrane/cytoskeletal abnormalities and insulin resistance were improved in exercised mice. These data reveal a new therapeutic aspect of exercise being regulation of skeletal muscle PM cholesterol homeostasis. Further studies on this mechanism of insulin resistance and the benefits of exercise on its prevention are needed

Signaling of the p21-activated kinase (PAK1) coordinates insulin-stimulated actin remodeling and glucose uptake in skeletal muscle cells

Skeletal muscle accounts for ~80% of postprandial glucose clearance, and skeletal muscle glucose clearance is crucial for maintaining insulin sensitivity and euglycemia. Insulin-stimulated glucose clearance/uptake entails recruitment of glucose transporter 4 (GLUT4) to the plasma membrane (PM) in a process that requires cortical F-actin remodeling; this process is dysregulated in Type 2 Diabetes. Recent studies have implicated PAK1 as a required element in GLUT4 recruitment in mouse skeletal muscle in vivo, although its underlying mechanism of action and requirement in glucose uptake remains undetermined. Toward this, we have employed the PAK1 inhibitor, IPA3, in studies using L6-GLUT4-myc muscle cells. IPA3 fully ablated insulin-stimulated GLUT4 translocation to the PM, corroborating the observation of ablated insulin-stimulated GLUT4 accumulation in the PM of skeletal muscle from PAK1−/− knockout mice. IPA3-treatment also abolished insulin-stimulated glucose uptake into skeletal myotubes. Mechanistically, live-cell imaging of myoblasts expressing the F-actin biosensor LifeAct-GFP treated with IPA3 showed blunting of the normal insulin-induced cortical actin remodeling. This blunting was underpinned by a loss of normal insulin-stimulated cofilin dephosphorylation in IPA3-treated myoblasts. These findings expand upon the existing model of actin remodeling in glucose uptake, by placing insulin-stimulated PAK1 signaling as a required upstream step to facilitate actin remodeling and subsequent cofilin dephosphorylation. Active, dephosphorylated cofilin then provides the G-actin substrate for continued F-actin remodeling to facilitate GLUT4 vesicle translocation for glucose uptake into the skeletal muscle cell

Chromium Enhances Insulin Responsiveness via AMPK

Trivalent chromium (Cr3+) is known to improve glucose homeostasis. Cr3+ has been shown to improve plasma membrane-based aspects of glucose transporter GLUT4 regulation and increase activity of the cellular energy sensor 5′ AMP-activated protein kinase (AMPK). However, the mechanism(s) by which Cr3+ improves insulin responsiveness and whether AMPK mediates this action is not known. In this study we tested if Cr3+ protected against physiological hyperinsulinemia-induced plasma membrane cholesterol accumulation, cortical filamentous actin (F-actin) loss and insulin resistance in L6 skeletal muscle myotubes. In addition, we performed mechanistic studies to test our hypothesis that AMPK mediates the effects of Cr3+ on GLUT4 and glucose transport regulation. Hyperinsulinemia-induced insulin-resistant L6 myotubes displayed excess membrane cholesterol and diminished cortical F-actin essential for effective glucose transport regulation. These membrane and cytoskeletal abnormalities were associated with defects in insulin-stimulated GLUT4 translocation and glucose transport. Supplementing the culture medium with pharmacologically relevant doses of Cr3+ in the picolinate form (CrPic) protected against membrane cholesterol accumulation, F-actin loss, GLUT4 dysregulation and glucose transport dysfunction. Insulin signaling was neither impaired by hyperinsulinemic conditions nor enhanced by CrPic, whereas CrPic increased AMPK signaling. Mechanistically, siRNA-mediated depletion of AMPK abolished the protective effects of CrPic against GLUT4 and glucose transport dysregulation. Together these findings suggest that the micronutrient Cr3+, via increasing AMPK activity, positively impacts skeletal muscle cell insulin sensitivity and glucose transport regulation

The actin-related p41ARC subunit contributes to p21-activated kinase-1 (PAK1)-mediated glucose uptake into skeletal muscle cells

Defects in translocation of the glucose transporter GLUT4 are associated with peripheral insulin resistance, preclinical diabetes, and progression to type 2 diabetes. GLUT4 recruitment to the plasma membrane of skeletal muscle cells requires F-actin remodeling. Insulin signaling in muscle requires p21-activated kinase-1 (PAK1), whose downstream signaling triggers actin remodeling, which promotes GLUT4 vesicle translocation and glucose uptake into skeletal muscle cells. Actin remodeling is a cyclic process, and although PAK1 is known to initiate changes to the cortical actin-binding protein cofilin to stimulate the depolymerizing arm of the cycle, how PAK1 might trigger the polymerizing arm of the cycle remains unresolved. Toward this, we investigated whether PAK1 contributes to the mechanisms involving the actin-binding and -polymerizing proteins neural Wiskott-Aldrich syndrome protein (N-WASP), cortactin, and ARP2/3 subunits. We found that the actin-polymerizing ARP2/3 subunit p41ARC is a PAK1 substrate in skeletal muscle cells. Moreover, co-immunoprecipitation experiments revealed that insulin stimulates p41ARC phosphorylation and increases its association with N-WASP coordinately with the associations of N-WASP with cortactin and actin. Importantly, all of these associations were ablated by the PAK inhibitor IPA3, suggesting that PAK1 activation lies upstream of these actin-polymerizing complexes. Using the N-WASP inhibitor wiskostatin, we further demonstrated that N-WASP is required for localized F-actin polymerization, GLUT4 vesicle translocation, and glucose uptake. These results expand the model of insulin-stimulated glucose uptake in skeletal muscle cells by implicating p41ARC as a new component of the insulin-signaling cascade and connecting PAK1 signaling to N-WASP-cortactin-mediated actin polymerization and GLUT4 vesicle translocation