Fig. 2

Mipep depletion alters MIPEP substrate levels and decreases its activity in the WAT and BAT of adipocyte-specific Mipep knockout (aMKO) mice. The effects of Mipep knockout in the mitochondria of gWAT (A–F) and BAT (G–I) from 22-week-old control and aMKO mice. (A) Protein expression levels of MIPEP substrates (SIRT3, COX4, and MDH2), AcSOD2, and total SOD2 in gWAT were obtained by immunoblotting. (B and C) Quantitative values of MIPEP substrates (B) and the ratio of AcSOD2/total SOD2 (C). (D) COX activity in gWAT from 22-week-old control and aMKO mice. (E) MDH2 activity in gWAT from 27-week-old control and aMKO mice. (F) mRNA expression levels of substrates were obtained by real-time PCR in gWAT from 27-week-old control and aMKO mice. (G) Protein expression levels of MIPEP substrates (SIRT3, COX4, and MDH2), AcSOD2, and total SOD2 in BAT were obtained by immunoblotting. (H and I) Quantitative values of MIPEP substrates (H) and the ratio of AcSOD2/total SOD2 (I). (J) COX activity of BAT from 22-week-old control and aMKO mice. (K) MDH2 activity of BAT from 27-week-old control and aMKO mice. (L) mRNA expression levels of substrates were obtained by real-time PCR in BAT from 27-week-old control and aMKO mice. Ponceau S stain was used as a loading control for immunoblotting. Each dot represents one mouse. Values are mean ± SD. Differences between values were analyzed by Student’s t-test. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control. AcSOD2, AcK122 acetylated-SOD2; BAT, brown adipose tissue; COX4, cytochrome c oxidase 4; gWAT, gonadal white adipose tissue; MDH2, malate dehydrogenase 2; MIPEP, mitochondrial intermediate peptidase; SIRT3, sirtuin 3.