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Browsing Cellular & Integrative Physiology Department Theses and Dissertations by Subject "Actin"
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Item ADF/Cofilin Activation Regulates Actin Polymerization and Tension Development in Canine Tracheal Smooth Muscle(2009-09-03T15:28:09Z) Zhao, Rong; Gunst, Susan J.; Atkinson, Simon J.; Elmendorf, Jeffrey S.; Sturek, Michael S.The contractile activation of airway smooth muscle tissues stimulates actin polymerization and the inhibition of actin polymerization inhibits tension development. Actin depolymerizing factor (ADF) and cofilin are members of a family of actin–binding proteins that mediate the severing of F–actin when activated by dephosphorylation at serine 3. The role of ADF/cofilin activation in the regulation of actin dynamics and tension development during the contractile activation of airway smooth was evaluated in intact canine tracheal smooth muscle tissues. Two–dimensional gel electrophoresis revealed that ADF and cofilin exist in similar proportions in the muscle tissues and that approximately 40% of the total ADF/cofilin in unstimulated tissues is phosphorylated (inactivated). Phospho–ADF/cofilin decreased concurrently with tension development in response to stimulation with acetylcholine (ACh) or potassium depolarization indicating the activation of ADF/cofilin. Expression of an inactive phospho–cofilin mimetic (cofilin S3E), but not WT cofilin in the smooth muscle tissues inhibited endogenous ADF/cofilin dephosphorylation and ACh–induced actin polymerization. Expression of cofilin S3E in the tissues depressed tension development in response to ACh, but it did not affect myosin light chain phosphorylation. The ACh–induced dephosphorylation of ADF/cofilin required the Ca2+–dependent activation of calcineurin (PP2B). Expression of Slingshot (SSH) inactive phosphatase (C393S) decreased force development and cofilin dephosphorylation. Activation of ADF/cofilin was also required for the relaxation of tracheal muscle tissues induced by forskolin and isoproterenol. Cofilin activation in response to forskolin was not Ca2+–dependent and was not inhibited by calcineurin inhibitors, suggesting it was regulated by a different mechanism. Cofilin activation is required for actin dynamics and tension development in response to the contractile stimulation of tracheal smooth muscle and is regulated by both contractile and relaxing stimuli. These concepts are critical to understanding the mechanisms of smooth muscle contraction and relaxation, which may provide novel targets for therapeutic intervention in the treatment of abnormal airway responsiveness.Item The Effects of Chromium on Skeletal Muscle Membrane/Cytoskeletal Parameters and Insulin Sensitivity(2012-07-03) Hoffman, Nolan John; Elmendorf, Jeffrey S.; Considine, Robert V.; Morral, Nuria; Pavalko, Fredrick M.A recent review of randomized controlled trials found that trivalent chromium (Cr3+) supplementation significantly improved glycemia among patients with diabetes, consistent with a long-standing appreciation that this micronutrient optimizes carbohydrate metabolism. Nevertheless, a clear limitation in the current evidence is a lack of understanding of Cr3+ action. We tested if increased AMP-activated protein kinase (AMPK) activity, previously observed in Cr3+-treated cells or tissues from Cr3+-supplemented animals, mediates improved glucose transport regulation under insulin-resistant hyperinsulinemic conditions. In L6 myotubes stably expressing the glucose transporter GLUT4 carrying an exofacial myc-epitope tag, acute insulin stimulation increased GLUT4myc translocation by 69% and glucose uptake by 97%. In contrast, the hyperinsulinemic state impaired insulin stimulation of these processes. Consistent with Cr3+’s beneficial effect on glycemic status, chromium picolinate (CrPic) restored insulin’s ability to fully regulate GLUT4myc translocation and glucose transport. Insulin-resistant myotubes did not display impaired insulin signaling, nor did CrPic amplify insulin signaling. However, CrPic normalized elevated membrane cholesterol that impaired cortical filamentous actin (F-actin) structure. Mechanistically, data support that CrPic lowered membrane cholesterol via AMPK. Consistent with this data, siRNA-mediated AMPK silencing blocked CrPic’s beneficial effects on GLUT4 and glucose transport regulation. Furthermore, the AMPK agonist 5-aminoimidazole-4-carboxamide-1-ß-D-ribonucleoside (AICAR) protected against hyperinsulinemia-induced membrane/cytoskeletal defects and GLUT4 dysregulation. To next test Cr3+ action in vivo, we utilized obesity-prone C57Bl/6J mice fed a low fat (LF) or high fat (HF) diet for eight weeks without or with CrPic supplementation administered in the drinking water (8 µg/kg/day). HF feeding increased body weight beginning four weeks after diet intervention regardless of CrPic supplementation and was independent of changes in food consumption. Early CrPic supplementation during a five week acclimation period protected against glucose intolerance induced by the subsequent eight weeks of HF feeding. As observed in other insulin-resistant animal models, skeletal muscle from HF-fed mice displayed membrane cholesterol accrual and loss of F-actin. Skeletal muscle from CrPic-supplemented HF-fed mice showed increased AMPK activity and protection against membrane cholesterol accrual and F-actin loss. Together these data suggest a mechanism by which Cr3+ may positively impact glycemic status, thereby stressing a plausible beneficial action of Cr3+ in glucose homeostasis.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.