All chemical reagents were purchased from Sigma–Aldrich Sweden AB, if not otherwise indicated. To determine intrinsic differences in mRNA expression between myotubes derived from T2D patients and NGT subjects, the mRNA level of several metabolic genes was determined by qPCR. Genes of interest include insulin receptor [INSR], insulin-like growth factor I receptor [IGF1R], glucose transporter 4 (GLUT4) [SLC2A4], Akt1 [AKT1] and Akt2 [AKT2], as well as muscle specific markers desmin [DES] and myogenin [MYF4]. RNA was extracted with RNeasy Mini Kit (Qiagen), and the cDNA was synthesized using SuperScript First-Strand Etoposide Synthesis System for RT-PCR (Invitrogen). The primers and FAM

probes for all genes were purchased from ABI (Applied Biosystem, Stockholm, Sweden). Using the CT comparative method, the relative abundance of the target transcript was calculated from duplicate samples after normalization against a housekeeping gene. Three housekeeping genes were tested (18s, GAPDH, and beta-actin) to standardize expression from myoblasts and myotubes and beta-actin

find more was chosen in this study specifically as the most stably expressed reference gene for normalization to ensure reliable results and highest accuracy of analysis. Myotubes were initially studied using several assays that characterize possible inherent differences in substrate metabolism. Glucose incorporation into glycogen was determined from duplicate samples, as previously described [29]. Myotubes were incubated with or without insulin (120 nM) AZD9291 price for 30 min before adding 1 μCi/ml d-[U-14C] glucose (PerkinElmer CA, USA) for the final 90 min. Cells were harvested and [14C]-labeled glycogen was purified and counted in a liquid scintillation counter (Win-Spectral 1414 liquid scintillation counter; Wallac, Turku, Finland). Lactate measurement was performed using the colorimetric l-Lactate Assay Kit according to manufacturer’s instructions (Biomedical Research Center, Buffalo, NY, catalog no. A-108). Lactate production from duplicate samples was determined after 6 h of incubation with or without insulin (120 nM) in cell culture media. Free fatty acid oxidation assessment was performed from duplicate samples

as previously described [30], with modifications including the use of a non-radioactive lipid (palmitate) for the measurement of the specific activity (tracer–tracee ratio). Myotubes were incubated with or without insulin (120 nM) for 6 h in serum-free DMEM supplemented with 0.2% fatty acid-free albumin, 50 μM of cold palmitate and 0.5 μCi palmitic acid [9,10(n)-3H] (PerkinElmer, CA, USA). The non-metabolized free palmitate was removed by charcoal treatment and the metabolic product [3H] H2O from the supernatant phase was determined in a liquid scintillation counter. Myotubes grown in 6 well plates, were incubated with or without insulin (120 nM) for 6 h in serum-free DMEM media, supplemented with [14C] phenylalanine (1 μCi/ml) at 37 °C.