Impaired mTOR activation in T2D. (A) Outline of major mTOR functions. (B) Phosphorylation of S6K1 in response to insulin stimulation of adipocytes from seven diabetic (d, filled bars; age 36–76, average 62.6 years; BMI 29.0–48.0, average 38.0 kg/m2) and seven nondiabetic subjects (n-d, open bars; age 46–61, average 52.7 years; BMI 20.8–32, average 25.4 kg/m2). Adipocytes were incubated with (+) or without (−) 10 nmol/L insulin for 10 min, when cells were subjected to SDS-PAGE and immunoblotting with antibodies against phospho-threonine(389)S6K1. Phosphorylation was normalized to the amount of S6K1 protein. Results are given as % of control without insulin, for each subject, mean ± SE. Immunoblots show the state of phosphorylation of S6K1 (T389P) and amount of S6K1 protein. (C,D) Abundance of mTOR (C) or S6K1 (D) protein in adipocytes from seven diabetic (filled bars; age 32–80, average 51.9 years; BMI 27.5–47.9, average 38.8 kg/m2) and seven nondiabetic subjects (open bars; age 27–71, average 51.4 years; BMI 18.7–32.9, average 32.6 kg/m2). Adipocytes were subjected to SDS-PAGE and immunoblotting with antibodies against mTOR or S6K1. The amount of mTOR or S6K1 protein was normalized to the amount of actin in each sample. Results are given as arbitrary densitometric units, mean ± SE. (E,F) Abundance of mTOR (E) or S6K1 (F) mRNA. Total RNA was extracted from isolated adipocytes from eight nondiabetic subjects (open bars; age 22–73, average 48.9 years; BMI 20–32.9, average 25.3 kg/m2) and eight patients with T2D (filled bars; age 32–63, average 48.1 years; BMI 29–45.4, average 40.0 kg/m2). The amount of mTOR or S6K1 mRNA was determined by RT-PCR. Results are given as amount of indicated mRNA in relation to GAPDH mRNA, mean ± SE.