, Roche PhosSTOP phosphatase inhibitor cocktail). SDS page and Western blotting was performed using the NuPAGE system and iBlot (Invitrogen). Membranes were probed using antibodies against pAKT-S473 (4060, RIPA buffer containing protease inhibitors (Roche Complete) and phosphatase inhibitors

, Measurement of fasting glucose and insulin. For QUICKI calculation (1/[log(I0) + log(G0)], where I0 is fasting insulin (µU/ml) and G0 is fasting glucose (mg/dl)), fasting plasma glucose and insulin were determined respectively with a Glucotrend Accu-Chek Performa (Roche SAS) and by ELISA (Mercodia)

, For glucose and insulin tolerance tests, 1 mg/g body weight of D-(+) glucose (Sigma) and 0.6mU/g body weight of insulin (Lilly) were administered by intraperitoneal route to overnight and 6h fasted mice, respectively. Glycemia was checked on blood from tail vein 30 min and 15 min before glucose injection and then at various times after glucose administration with a Glucotrend Accu-Chek Performa (Roche SAS). For insulin bolus experiments, Glucose and insulin tolerance tests and insulin bolus injection

, Radiolabelled 3 H-glucose was also perfused at the same time (bolus of 30µCi/mice followed by a 30µCi/min/kg perfusion). Euglycemia was maintained at 100mg/dl by adjusting the glucose perfusion, 5U/kg of insulin or PBS control. Mice were culled 10 minutes after insulin injection. Euglycemic-hyperinsulinemic clamp. A catheter was inserted into the femoral vein on anaesthetized animals by isoflurane

, For hyperglycemic hyperinsulinemic clamp in young men, total RNA from abdominal subcutaneous adipose tissue was obtained using RNeasy mini kit (Qiagen). 500ng of total RNA were reverse transcribed using Superscript II (Thermo Fischer Scientific). mRNA levels were evaluated using SYBR qPCR Premix Ex Taq (Tli RNaseH Plus) (Ozyme) with RotorGene 6000 (Qiagen). TBP (forward, TGGTGTGCACAGGAGCCAAG; reverse, TTCACATCACAGCTCCCCAC) was used as control to normalize gene expression. Other primer sequences are mentioned above. For morbidly obese subjects undergoing bariatric surgery, subcutaneous fat biopsy specimen was obtained by needle aspiration under local anesthesia from the mid-abdominal area. It was rapidly washed in saline and immediately thereafter frozen at -70 o C. All samples (before/after surgery) were simultaneously extracted for RNA and subsequent micro-array analyses as described in Dahlman, Gene expression analysis. For women with differing obese and metabolic status, procedures were similar as for human adipocytes described above

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