Adipose AMPK deficiency impaired cold tolerance and suppressed cold-induced thermogenesis specifically in inguinal white adipose tissue. (A) Western blot analysis of UCP1, p-AMPKα (T172) and AMPKα protein levels in the interscapular brown adipose tissue (BAT), inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) of 9-week-old male wild-type (WT) mice housed at RT or 4°C for 24 h. β-actin was used as a loading control. n = 4. (B) Western blot analysis of AMPKα1, α2, β1, β2, γ1, γ2 protein expression levels in the BAT, iWAT, eWAT and liver of 10-week-old male AKO mice and age-matched floxed littermates. β-actin was used as a loading control. n = 3. (C) Rectal temperature of 8-week-old chow-fed AKO and floxed mice at 4°C for 8 h. n = 9. (D–M) 8-week-old male chow-fed AKO mice and floxed mice were housed at 4°C for 48 h. Representative transmission electronic microscopy images of iWAT (D) and BAT (G), total number of mitochondria per micrograph in iWAT (E) and BAT (H) and the percentage of mitochondria with disrupted cristae over total mitochondria in iWAT (F) and BAT (I) from AKO mice and floxed mice were shown. n = 3. Scale bar: 2 μm in low magnification (×5,000, upper) and 0.5 μm in high magnification (×20,000, bottom). The relative mRNA levels of thermogenic genes and fatty acid oxidation (FAO)-related genes in the iWAT (J) and BAT (K) of AKO mice and floxed mice were analyzed by quantitative RT-PCR (normalized to 36b4). n = 8–9. The expression levels of AMPKα, p-AMPKα (T172), ACC, p-ACC (S79), UCP1 and PGC-1α in the iWAT (L) and BAT (M) of AKO mice and floxed mice were determined by western blot analysis. Data are presented as the means ± SEM. Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the indicated control group.