Peroxisomes are induced in brown fat during differentiation and in cold exposure, along with mitochondria. mRNA expression of peroxisomal (A) and mitochondrial (B) genes in undifferentiated and differentiated brown adipocytes. All values were normalized using 36B4. (C and D) mRNA expression of peroxisomal genes in brown fat tissue of 7-wk-old C57BL/6J female mice maintained at 29 °C or after cold exposure at 4 °C for 7.5 h (n = 3). All values were normalized using 18S. (E) Immunohistochemistry of brown fat tissue obtained from 9-wk-old C57BL/6J female mice kept at 29 °C or cold exposed at 4 °C for 7.5 h, using antibodies against PMP70 and Alexa Fluor 568 goat anti-rabbit (red). The samples were counterstained with the nuclear dye DAPI (blue) (magnification 100×). (F) Quantification of peroxisomal abundance in brown fat tissue at 29 °C or after cold exposure (at 4 °C). Results are shown as relative peroxisomal number per cell ± SEM (n = 3). Statistical significance was determined by unpaired two-tailed Student's test. ^#P < 0.01, ^##P < 0.001, *P < 0.05, **P < 0.01, ***P < 0.001.

PGC-1α is sufficient to induce peroxisomal gene expression, protein, and number in vitro. mRNA expression of peroxisomal genes in brown fat (A), 10T1/2 (B), and FAO (C) cells infected with control or PGC-1α adenovirus for 72 h. Values were normalized using 36B4 (A) or 18S (B and C). Results are shown as the mean of three independent experiments ± SEM. (D) Western blot analysis of whole-cell lysates from 10T1/2 and FAO cells infected with control or PGC-1α adenovirus for 72 h. (E) mRNA expression of peroxisomal genes in U2OS cells infected with control or PGC-1α adenovirus for 48 h. All values were normalized using 36B4. Results are shown as the mean of three independent experiments ± SEM. (F) Differences in fluorescent peroxisomes in U2OS cells stably expressing RFP–SKL (RFP–SKL–U2OS) after 72-h infection with GFP or PGC-1α adenovirus, costained with DAPI (magnification 63×). (G) Peroxisome quantification in RFP–SKL–U2OS cells infected with GFP or PGC-1α adenovirus for 48 or 72 h. Results are shown as relative peroxisomal number per cell ± SEM. Statistical significance was determined by unpaired two-tailed Student's test. *P < 0.05, **P < 0.01, ***P < 0.001.

PGC-1α is able to induce the peroxisomal program in vivo. (A) Western blot analysis of whole-cell lysates of liver samples obtained from mice injected with GFP or PGC-1α adenovirus for 5 d. (B) Immunohistochemistry of liver cryosections of 8-wk-old male C57BL/6J mice injected with GFP or PGC-1α adenovirus for 5 d using the PMP70 and the Alexa Fluor 568 goat anti-rabbit antibodies (red) and counterstained with the nuclear dye DAPI (blue) (magnification 63×). (C) Quantification of peroxisome abundance in mouse livers injected with GFP and PGC-1α adenovirus for 5 d. Results are shown as relative number of peroxisomes per cell ± SEM. Statistical significance was determined by unpaired two-tailed Student's test. *P < 0.05.

PGC-1α is required for the induction of peroxisomal genes. (A) mRNA expression of peroxisomal genes in preadipocytes or in fully differentiated WT and PGC-1α KO brown adipocytes. Results are presented as average of three independent experiments ± SEM. Values were normalized using 36B4. (B) mRNA expression of peroxisomal genes in differentiated WT and PGC-1α KO brown adipocytes treated with vehicle or with cAMP for 4 h. Data are shown as average of three independent experiments ± SEM. Values were normalized using 36B4. (C) mRNA expression of peroxisomal genes in brown fat and liver tissues from 10-wk-old male C57BL/6J mice fed with regular chow in the presence or absence of 0.2% fenofibrate for 2 wk (n = 3). Values were normalized using 36B4. (D) mRNA expression of peroxisomal genes in brown fat tissue obtained from 8-wk-old male C57BL/6J WT and PPARα KO mice kept at 29 °C or in the cold (4 °C) for 5 h (n = 3). Values were normalized using 36B4. Results are shown as mean ± SEM. (E) Confocal imaging of liver cryosections from 8-wk-old male C57BL/6J WT and PPARα KO mice injected with GFP or PGC-1α adenovirus for 5 d stained with PMP70 and the Alexa Fluor 594 goat anti-rabbit antibodies (red) and counterstained with DAPI (blue) (magnification 63×). (F) MetaMorph quantification of peroxisomes in mouse livers from WT and PPARα KO mice injected with GFP (open bar) and PGC-1α (filled bar) adenovirus for 5 d. Results are shown as relative number of peroxisomes per cell ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ^#P < 0.05, ^##P < 0.01. Statistical significance was determined by two-way ANOVA followed by Bonferroni posttest in A, B, D, and F and by unpaired two-tailed Student's test in C.