(A) Western blot of mitochondrial proteins from whole tissue lysates (Total), crude mitochondria purified by differential centrifugation (Crude), and mitochondria purified on a sucrose gradient (Pure). 129SVE mice were cold-exposed (4°C) for one week. Beige-fat mitochondria (Bg), and brown-fat mitochondria (Br).

(B) Western blot of cytosolic (GAPDH) and endoplasmic reticulum (ER) (CLNX and KDEL) proteins from whole tissue lysates (Total), crude mitochondria purified by differential centrifugation (Crude), and mitochondria purified on a sucrose gradient (Pure). Mice were cold-exposed (4°C) for one week.

(C) Yield of beige and brown adipose mitochondria from 129SVE mice acclimated to thermoneutral (30°C) or cold (4°C) temperature. ∗p < 0.05 and ∗∗∗p < 0.01, comparison of temperature. #p < 0.05 and ###p < 0.01, comparison of tissue.

(D) Oxygen consumption rate (OCR) of beige- and brown-fat mitochondria isolated from 129SVE mice, respiring on pyruvate/malate. Mitochondria (5 μg) were incubated in buffer without GDP (-GDP) or with GDP (+GDP), followed by the addition of the following compounds: 1 mM GDP (G), 1 mM ADP (A), 14 μM Oligomycin (O), 10 μM FCCP (F), and 4 μM each of rotenone/antimycin (RA).

(E) Western blot of beige- and brown-fat-enriched mitochondrial proteins from whole-tissue lysates (Total), crude mitochondria purified by differential centrifugation (Crude), and mitochondria purified on a sucrose gradient (Pure).

(F) Quantification of western blots, by densitometry, of mitochondrial proteins (ATP5A1, PGAM5, HMGCS2) that were identified as beige selective by proteomics. The signal intensity from beige- and brown-fat mitochondria from C57BL/6 and 129SVE mice was plotted as a percentage of the total sum (set to 100%) of all the intensities combined.

(G) Quantification of western blots, by densitometry, of mitochondrial proteins (UCP1, COX5B, ACO2) that were identified as brown-selective by proteomics. The signal intensity from beige- and brown-fat mitochondria from C57BL/6 and 129SVE mice was plotted as a percentage of the total sum (set to 100%) of all the intensities combined.

(H) CK activity of beige- and brown-fat mitochondria from cold-exposed (4°C) C57BL/6 and 129SVE mice.

(I) Creatine concentration in iWAT and BAT from mice (129SVE) housed at 30°C or seven days at 4°C; n = 6 mice per group.

(J) Phosphocreatine (PCr) concentration in iWAT and BAT from mice (129SVE) housed at 30°C or seven days at 4°C; n = 6 mice per group.

(K) Creatine concentration in soleus and extensor digitorum longus (EDL) from mice (129SVE) housed at 30°C or seven days at 4°C; n = 6 mice per group.

Data are presented as means ± SEM. ∗p < 0.05, ∗∗∗p < 0.01.