Figure 4. Mitochondrial energetics, mtDNA copy numbers and eNOS expression. OXPHOS activity in the WHT of mice was spectrophotometrically assessed through analysis of the activity of individual OXPHOS complexes I (A), II (B), and IV (C). Complex enzymatic activity was determined according to the consumption of 1 nmol of NADH (complex I) or reduced cytochrome C (complex III, IV) in 1 g of tissue/min in the reaction system. For each value expressed as %, the 3.1CG value was set to 100, and the compared samples were normalized to this level. The enzymatic activity of CKMT2 in WHT was analyzed with a Mouse CKMT2 ELISA Kit (D). The values were spectrophotometrically obtained and converted to ng/ml by normalization to standard values (D, lower-right graph). MtDNA copy numbers (E) were analyzed with a Detroit-Mouse-mt-DNA analysis kit. For each group, the value is expressed as a percentage (%); the 3.1CG value was set to 0, and the rest of the groups were normalized to this level for comparison. For eNOS (F) and 18S rRNA (reference gene) expression, each primer was analyzed with SYBR Green fluorescence detection, and the transcript levels of eNOS were normalized to those of the endogenous control 18S rRNA. (G) eNOS immunoblot results and relative protein values were obtained by Western blot analysis and quantified with Image Lab 6.1 software. The values were normalized to GAPDH expression; the 3.1CG expression was set as 1.0. The data are the mean ± SD. *P < 0.05; **P < 0.01 vs. the 3.1CG group, unless otherwise specified.