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Browsing by Author "Penque, Brent A."
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Item Chromium Enhances Insulin Responsiveness via AMPK(Elsevier, 2014-05) Hoffman, Nolan J.; Penque, Brent A.; Habegger, Kirk M.; Sealls, Whitney; Tackett, Lixuan; Elmendorf, Jeffrey S.; Department of Cellular & Integrative Physiology, IU School of MedicineTrivalent 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.Item Effect of Corncob bedding on feed conversion efficiency in a high-fat diet-induced prediabetic model in C57Bl/6J mice(American Association for Laboratory Animal Science, 2014-09) Ambery, Ashley G.; Tackett, Lixuan; Penque, Brent A.; Hickman, Debra I.; Elmendorf, Jeffrey S.; Department of Cellular and Integrative Physiology, IU School of MedicineLaboratory 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.Item 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(Wiley, 2017-08-15) Ambery, Ashley G.; Tackett, Lixuan; Penque, Brent A.; Brozinick, Joseph T.; Elmendorf, Jeffrey S.; Cellular and Integrative Physiology, School of MedicineInsulin 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.Item Hexosamines Provoke Membrane Cholesterol Accrual, Filamentous Actin Loss, and GLUT4 Dysregulation in Adipocytes through Transcriptional Activation of Specificity Protein 1(Poster session presented at IUPUI Research Day 2012, Indianapolis, Indiana., 2012-04-13) Penque, Brent A.; Elmendorf, Jeffrey S.The 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.Item Mechanisms of hexosamine-induced cholesterol accumulation and therapeutic actions of chromium(2014-01-03) Penque, Brent A.; Elmendorf, Jeffrey S.; Atkinson, Simon; Considine, Robert V.; Evans-Molina, Carmella; Kempson, Stephen A.Excess caloric intake and/or obesity currently remain the largest predisposing risk factors for the development of type 2 diabetes. Discerning the cellular and molecular mechanisms responsible and amendable to therapy represents a growing challenge in medicine. At a cellular level, increased activity of the hexosamine biosynthesis pathway (HBP), a sensor of excess energy status, has been suggested to promote the exacerbation of insulin resistance through increasing adipose tissue and skeletal muscle membrane cholesterol content. This in turn compromises cortical filamentous actin structure necessary for proper incorporation of the insulin-sensitive glucose transporter GLUT4 into the plasma membrane. The current studies attempted to elucidate the mechanism by which hexosamines provoke membrane cholesterol toxicity and insulin resistance. In 3T3-L1 adipocytes cultured with pathophysiologic hyperinsulinemia to induce insulin resistance, increased HBP flux was observed. This occurred concomitant with gains in the mRNA and protein levels of HMG-CoA reductase (HMGR), the rate limiting enzyme in cholesterol synthesis. Mechanistically, immunoprecipitation demonstrated increased HBP-induced N-acetylglucosamine (O-GlcNAc) modification of specificity protein 1 (Sp1), a regulator of HMGR synthesis. This was associated with increased affinity toward and activity of Hmgcr, the gene encoding HMGR. Global HBP inhibition or Sp1 binding to DNA prevented membrane cholesterol accrual, filamentous actin loss, and glucose transport dysfunction. Furthermore, hyperinsulinemia and HBP activation impaired cholesterol efflux in adipocytes, exacerbating cholesterol toxicity and potentially contributing to cardiovascular disease. In this regard, chromium picolinate (CrPic), known to have beneficial effects on glucose and lipoprotein metabolism, improved cholesterol efflux and restored membrane cholesterol content. To test the role of membrane cholesterol accumulation in vivo, studies were conducted on C57Bl/6J mice fed a low or high fat diet. High fat feeding promoted increased HBP activity, membrane cholesterol accumulation, and insulin resistance. Supplementation of mice with CrPic in their drinking water (8µg/kg/day) countered these derangements and improved insulin sensitivity. Together, these data provide mechanistic insight for the role of membrane cholesterol stress in the development of insulin resistance, as well as cardiovascular disease, and highlight a novel therapeutic action of chromium entailing inhibition of the HBP pathway.