otential to counteract an eventual age-dependent decline in insulin sensitivity. Exercise Prevents Aging-Induced Insulin Resistance Thus, we isolated satellite cells from young healthy males or middle-aged males who were either lifelong sedentary or extremely active, differentiated them into myotubes and measured the effects of physical activity level and aging on the insulin signaling pathway. Materials and Methods Materials F10 nutrient mixture, Dulbecco’s modified Eagle’s medium, fetal bovine serum, horse serum, penicillin/streptomycin and Fungizone antimycotic were obtained from Invitrogen. Insulin was from Novo Nordisk. Complete mini protein phosphatase inhibitor tablets were purchased from Boehringer-Roche Diagnostics, and protein protease inhibitor I and II were from Sigma-Aldrich. 2-deoxy-D–glucose was from 22837009 Perkin Elmer Life Sciences. Phospho-Akt/Akt, phospho-GSK3a/b, anti-Akt anti-a-actinin, anti-b-tubulin and p21 Waf1/Cip1 antibodies were from Cell Signaling Technology. Anti-Myosin heavy chain was from Developmental studies Hybridoma Bank, Alexa Fluor 488-conjugated goat anti-rabbit IgG antibodies and Alexa Fluor 647-conjugated wheat germ agglutinin were obtained from Molecular Probes, Inc. TRIzol was from Invitrogen. connective tissue the muscle biopsy was minced into small pieces and digested in buffer containing 0.05% typsin-EDTA, 1 mg/ml collagenase IV and 10 mg/ml bovine serum albumin for 5 min at 37uC. Subsequently, digestion solution containing liberated muscle precursor cells were transferred to cold FBS to inactivate trypsin activity. The solution was centrifuged at 800 g for 7 min. The supernatant was removed and washed with F10/ HAM. To minimize fibroblast contamination, the cell suspension was pre-plated in a culture plate for 3 hours in growth media containing 20% FBS, 1% PS and 1% FZ in F10/HAM. The unattached cells were seeded onto Matrigel coated culture flask and cultured for 4 days in growth media in a humidified incubator with 5% O2 and 5% CO2 at 37uC. After 4 days of incubation, the cell culture medium was changed and then every second day thereafter. At 100% confluence, cells were transferred to intermediate medium. After 2 days, medium was changed into differentiation media in order to Cyanidin 3-O-glucoside chloride manufacturer induce differentiation into myotubes. All experiments were performed on fully differentiated myocytes at 7 days of differentiation at passage 4 to 6. For experiments myocytes were serum starved in DMEM containing 1 g/L glucose for 2 hours. Glucose Uptake Assay Cells were treated with or without insulin during the penultimate 30 minutes of serum starvation. Cells were washed twice with Hepes buffered saline. Cells were then incubated with Hepes buffered saline containing 10 mM 2-deoxy-D- glucose for 10 min. Nonspecific glucose uptake was determined in the presence of 10 mM cytochalasin B and subtracted from total uptake to get specific glucose uptake. The 11358331 2-deoxy-D- glucose uptake was terminated by removing uptake buffer and washing cells twice with 0.9% ice-cold NaCl. Cells were then solubilized with 50 mM NaOH for 30 min and radioactivity was measured using scintillation counter. Protein concentration was determined using the Bradford reagent. Glucose uptake experiments were carried out in cells from n = 5 individuals per group in triplicate. Subjects Skeletal muscle biopsies from 
vastus lateralis were obtained from either: 1) young, healthy, recreationally active, 2) middle-aged sedentary or 3) middle-aged active ) males
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