Figure 4
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.