Increased adipocyte oxygen consumption by FFA-induced uncoupled respiration contributes to adipose tissue hypoxia in obesity. (A) Whole-mount immunohistochemistry analysis of eWAT from mice fed normal chow diet (NCD) or HFD for 3 days. Green, pimonidazole adduct; Red, perilipin; Blue, PECAM-1. (B) Western blot analysis of HIF-1α in eWAT. (C-D) Q-PCR analysis of VEGF expression (C) or lactate concentration (D) in eWAT from mice fed NCD or HFD. D1, 1 day HFD; D3, 3 day HFD; D7, 7 day HFD; W10, 10 week HFD. n=8 per group. (E) Relative oxygen consumption rate in the presence or absence of oligomycin in primary adipocytes from NCD/lean or HFD/obese mice. n=4 per group. (F) FFA level in eWAT of NCD or HFD mice. n=5-7 per group. *, P<0.05; **, P<0.01. (G) Oxygen consumption rate in 3T3-L1 adipocytes before and after BSA, KLA (Kdo2), or each of FFAs indicated (50 μM) in the presence or absence of an inhibitor of ANT, carboxyatractyloside (CAtr). *, P<0.05; **, P<0.01. (H) Palmitate-induced respiration is not inhibited by Etomoxir (Etom) or oligomycin (Oligo). (I) ATP production as calculated from Seahorse data as described previously (). (J) Palmitate induces hypoxia (pimonidazole adducts) in cultured 3T3-L1 adipocytes. Detailed protocols are described in the Extended Experimental Procedures. Blue, DAPI; Green, hypoxia probe. (K) Western blot analysis of HIF-1α protein in 3T3-L1 adipocytes with or without palmitate (100 μM) and/or CAtr (1 μM). (L) Acute injection of CAtr reduces adipocyte hypoxia in mice fed a HFD for 3 days. Green, hypoxia probe; Red, BODIPY. All data are mean+/-SEM. See also .