Figure 7
Effects of inhibition of mitochondrial function on mitophagy, autophagy and mTORC1 activity. (A) Density of mitochondria determined as Mitotracker fluorescence after inhibition of mitochondrial function. Adipocytes from six nondiabetic subjects (age 46–71, average 60.8 years; BMI 21.5–31.6, average 26.7 kg/m2) were incubated with low concentration of dinitrophenol (50 µmol/L, DNP), rotenone (1 µmol/L), antimycin A (10 nmol/L), or vehicle (0.1 % ethanol, control) for 18 h, then mitochondrial density was determined by fluorescence microscopy. 20 cells per subject were analyzed. Results are given as mitochondria density in arbitrary fluorescence units, mean ± SE. (B) Number of lipofuscin particles per cell was determined after inhibition of mitochondrial function, as in (A), in adipocytes from five nondiabetic subjects (age 42–71, average 54.2 years; BMI 22.2–27.2, average 24.0 kg/m2). 20 cells per subject were analyzed. Results are given as number of lipofuscin particles per cell, mean ± SE. (C) Insulin-stimulated activation of mTORC1, determined as phosphorylation of S6K1, was assayed after incubating adipocytes from 11 nondiabetic subjects (age 35–82, average 56.0 years; BMI 22.2–31.6, average 26.2 kg/m2) with mitochondrial inhibitors, as in (A). Cells were then incubated without (−) or with (+) 10 nmol/L insulin for 10 min, when cells were subjected to SDS-PAGE and immunoblotting with antibodies against phospho-threonine(389)S6K1. Results are given as percent of without insulin, mean ± SE. The immunoblots of one experiment representing one subject are shown. (D) Insulin-stimulated phosphorylation of IRS1 at serine 307 (human sequence) was analyzed after incubating adipocytes from 12 nondiabetic subjects (age 35–82, average 53.9 years; BMI 22.2–31.6, average 26.5 kg/m2) with mitochondrial inhibitors, as in (A). Cells were then incubated without (−) or with (+) 10 nmol/L insulin for 10 min, when cells were subjected to SDS-PAGE and immunoblotting with antibodies against phospho-serine(307, human sequence)IRS1. Results are given as percent of without insulin, mean ± SE. The immunoblots of one experiment representing one subject are shown.