a, Mito-Tracker Green fluorescence in adipocytes and electron microscopy in WAT mitochondria (Mito-EM). KO adipocytes exhibit significantly elevated citrate synthase activity (b) and ATP content (c). d, Representative oxygen consumption by mitochondria isolated from WAT in the presence of pyruvate/malate, ADP and the inhibitors oligomycin and actractyloside. The oxygen consumption rate (nmol/min/mg prot) is shown below the trace after the addition of the mentioned substrates and inhibitors. e, KO primary adipocytes exhibit significantly increased oxygen consumption. The fold differences in oxygen consumption are derived from the calculated slope of oxygen utilization. f, Respiratory control ratio (State3/State4; RCR) of isolated WAT mitochondria demonstrating increased mitochondrial uncoupling, as evident by a lower RCR, in KO tissue (n=4 separate mitochondrial preparations per genotype). g, Oxygen consumption rate (OCR) is increased in intact ShSmad3 lentivirus infected 3T3-L1 cells in basal conditions and further enhanced by the addition of 1 μM norepinephrine (NE). NE does not affect OCR in control cells. The addition of complex III inhibitor, antimycin A (AA), inhibits respiration in both cells. For basal OCR versus OCR after NE addition, Control versus ShSmad3 p<0.001 for all measurements. h, i, Resting oxygen consumption at 20°C (h) and respiratory exchange ratio (i) in 3-month old female Smad3+/+ (WT) and Smad3−/− (KO) mice fed chow diet. *p < 0.05; **, p< 0.005; ***, p<0.001.