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.

Enhanced autophagy in T2D. (A) Autophagosome-related structures visualized in thin sections of adipocytes by transmission electron microscopy; ap, autophagosome; cLD, central lipid droplet; LD, cytosolic lipid droplet; pm, plasma membrane; cav, caveolae. (B) Number of autophagosome-related structures in adipocytes from five nondiabetic (open bar; age 49–76, average 63.6 years; BMI 26.7–34.9, average 29.1 kg/m²) and five diabetic (filled bar; age 32–63, average 46.6 years; BMI 37.2–45.4, average 40.9 kg/m²) subjects determined by transmission electron microscopy of thin sections of five different whole cells from each subject. Results are given as number of autophagosome-related structures per whole-cell thin-section, mean ± SE. (C) Immunofluorescence confocal microscopy of nondiabetic (left) and diabetic (right) adipocytes that were incubated with 50 nmol/L rapamycin and 5 μmol/L chloroquine for 18 h, when LC3 was detected. (D) Autophagic activity, amount and turnover of particulate LC3. Adipocytes were isolated from seven nondiabetic subjects (open bars; age 28–85, average 53.3 years; BMI 19–35, average 26.1 kg/m²) and seven patients with T2D (filled bars; age 32–69, average 48.4 years; BMI 29–45.4, average 39.5 kg/m²) and incubated, with or without rapamycin and chloroquine, as in (C), when LC3 was determined by immunofluorescence microscopy. We analyzed 20 randomly selected cells per subject and condition. Results are given as fluorescence per imaged area of the cells, mean ± SE.

Reduced number of lipofuscin particles in T2D. (A) Visualization of autofluorescent lipofuscin particles in adipocytes from a nondiabetic subject (left), after treatment with 5 mmol/L CuSO₄, 50 min (middle), and from a diabetic subject (right), using confocal fluorescence microscopy. Cell nuclei were stained with TO-PRO3 (blue). (B) Quantification of lipofuscin particles in adipocytes from 10 nondiabetic subjects (age 34–80, average 60.7 years; BMI 21–35, average 24.9 kg/m²). Cells were incubated without additions (control, open bar), with 50 nmol/L rapamycin (filled bar) or with 50 nmol/L rapamycin and 5 μmol/L chloroquine (hatched bar) for 18 h and then the number of lipofuscin particles was determined by fluorescence microscopy. Microscopic image of one cell after the respective treatment is shown above the corresponding graph bar. In each condition 20 cells were analyzed per subject. Results are given as number of lipofuscin particles per cell as a percentage of that in controls, for each subject, mean ± SE. (C) Quantification of lipofuscin particles in adipocytes from 17 nondiabetic subjects (open bar; age 34–80, average 56.7 years; BMI 21–38.3, average 26.9 kg/m²) and 10 diabetic patients (filled bar; age 46–76, average 60.9 years; BMI 27–66.7, average 39.7 kg/m²); 20 cells were analyzed per patient. Results are given as number of lipofuscin particles per cell, mean ± SE.

Mitochondria in T2D. (A) Relative abundance of PGC1α 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 PGC1α mRNA was determined by RT-PCR. Results are given as amount of PGC1α mRNA in relation to GAPDH mRNA, mean ± SE. (B) Mitochondria constitute a tubular network in human adipocytes. Fluorescence microscopic image of an adipocyte incubated with Mitotracker Green. (C) Density of mitochondria determined as Mitotracker Green fluorescence. Adipocytes from eight nondiabetic (open bars; age 27–71, average 47 years; BMI 18.7–32.9, average 25.7 kg/m²) and eight diabetic subjects (filled bars; age 32–63, average 46.5 years; BMI 33.2–47.9, average 41.3 kg/m²) were incubated without additions (control), with 5 μmol/L chloroquine, with 50 nmol/L rapamycin, or with 50 nmol/L rapamycin and 5 μmol/L chloroquine for 18 h, and then total mitochondrial volume was determined by fluorescence microscopy. Twenty cells per subject and condition were analyzed. Results are given as fluorescence per imaged area of the cells, mean ± SE. (D) Mitochondria (M) in the cytosolic space between plasma membrane (pm) and central lipid droplet (cLD) visualized by transmission electron microscopy; cav, caveolae. (E) Morphometric analyses of number or volume fraction of mitochondria with developed cristae in adipocytes from five non-diabetic (open bars; age 49–76, average 63.6 years; BMI 26.7–34.9, average 29.1 kg/m²) and five diabetic (filled bars; age 32–63, average 46.6 years; BMI 37.2–45.4, average 40.9 kg/m²), determined by transmission electron microscopy of thin sections of five different whole cells from each subject. Results are given as number of mitochondria per whole-cell thin section, mean + SE; or as mitochondrial volume fraction (that is, area per whole-cell thin section) in arbitrary units, mean ± SE. (F) Amount of oxidative phosphorylation protein in adipocytes from seven nondiabetic (open bar; age 27–71, average 51.4 years; BMI 18.7–32.9, average 32.6 kg/m²) and seven diabetic (filled bar; age 32–80, average 51.9 years; BMI 27.5–47.9, average 38.8 kg/m²) subjects. Cells were subjected to SDS-PAGE and immunoblotting with antibodies against the electron transport chain protein ubiquinol-cytochrome c oxidoreductase (complex III, core protein 2, UQCRC2). The amount of UQCRC2 protein was normalized to the amount of actin in each sample. Results are given as arbitrary densitometric units, mean ± SE.

Increased number of lipid droplets in T2D. (A) Lipid droplets (LD) visualized by transmission electron microscopy. Left, parts of three cells are seen; in the leftmost cell there are small lipid droplets in the cytosol separate from the central lipid droplet (cLD). LD-patch, patch of cytosolic lipid droplets; pm, plasma membrane; ecm, extracellular matrix. (B) Amount of cytosolic lipid droplets (>100 nm) in adipocytes from five nondiabetic subjects (open bar; age 49–76, average 63.6 years; BMI 26.7–34.9, average 29.1 kg/m²) and five diabetic patients (filled bar; age 32–63, average 46.6 years; BMI 37.2–45.4, average 40.9 kg/m²) determined by transmission electron microscopy of thin sections of five different whole cells from each subject, as described in Experimental Procedures. Results are given as number of cytosolic droplets per whole-cell thin section, mean ± SE. (C) Autophagy of lipid droplets visualized by immunofluorescence confocal microscopy. Adipocytes were incubated with 50 nmol/L rapamycin and 5 μmol/L chloroquine for 18 h, when cells were incubated with antibodies against perilipin (red) and LC3 (green). Confocal section close to the plasma membrane shown. LD-patch, patch of cytosolic lipid droplets. (D) Autophagy of lipid droplets visualized by transmission electron microscopy; ap, autophagosome with lipid droplets; LD, lipid droplets; cLD, central lipid droplet; pm, plasma membrane.

Correlation of insulin-stimulated mTORC1 activity or glucose transport to whole body insulin sensitivity (QUICKI). Adipocytes from 26 nondiabetic subjects (Supplemental Table S1) were examined. (A) Glucose transport into isolated adipocytes was determined, in triplicate, as uptake of 2-deoxy-_D-glucose with (filled circles) or without (open circles) preincubation with 10 nmol/L insulin for 15 min. (B–D) The effect of insulin on the phosphorylation of S6K1 (B), S6 (C), and IRS1 at serine-307 (D) were determined in isolated adipocytes after incubation 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 (B), phosphoserine(235/236)S6 (C), or phosphoserine(307, human sequence)IRS1 (D). Results are given as fold over of controls without insulin, mean of triplicates.

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 phosphoserine(307, human sequence)IRS1. Results are given as percent of without insulin, mean ± SE. The immunoblots of one experiment representing one subject are shown.

Schematic overview of mTOR involvement in insulin signaling and dysregulation in T2D.