A truncated PPAR gamma 2 localizes to mitochondria and regulates mitochondrial respiration in brown adipocytes

Abstract

Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of brown adipocyte differentiation and thermogenesis. The PPARγ gene produces two isoforms, PPARγ1 and PPARγ2. PPARγ2 is identical to PPARγ1 except for additional 30 amino acids present in the N-terminus of PPARγ2. Here we report that the C-terminally truncated form of PPARγ2 is predominantly present in the mitochondrial matrix of brown adipocytes and that it binds to the D-loop region of mitochondrial DNA (mtDNA), which contains the promoter for mitochondrial electron transport chain (ETC) genes. Expression of mitochondrially targeted MLS-PPARγ2 in brown adipocytes increases mtDNA-encoded ETC gene expression concomitant with enhanced mitochondrial respiration. These results suggest that direct regulation of mitochondrially encoded ETC gene expression by mitochondrial PPARγ2, in part, underlies the isoform-specific role for PPARγ2 in brown adipocytes.

Fig 1. A C-terminally truncated form of PPARγ2 is enriched in brown adipose tissue mitochondria.

(A) Schematic of PPARγ1 and PPARγ2 proteins. AF-1, activation function 1; DBD, DNA binding domain; LBD, ligand binding domain; AF-2, activation function 2. Blue letters represent specific regions recognized by three different PPARγ antibodies. (B) Presence of a 52 kDa protein recognized by PPARγ2 antibody in the cytosolic and mitochondrial fractions. Brown adipose tissue was isolated from C57BL/6J mice and subjected to subcellular fractionation. Cytosolic (C), nuclear (N) and mitochondrial (M) markers were detected in their respective fractions. (C) Validation of three different PPARγ antibodies. PPARγ1 and PPARγ2 were expressed in HeLa cells and analyzed with three different PPARγ antibodies. *, a non-specific band at ~50kDa that is reacted with PPARγ (H100) antibody in HeLa cells. (D) Western blot analysis of brown adipose tissue extracts (WCE) and mitochondrial lysates (M) with three different PPARγ antibodies.

Fig 2. Localization of the full-length and truncated PPARγ2 during brown adipocyte differentiation.

(A) Analysis of PPARγ2 localization in brown adipocytes. Brown preadipocytes were differentiated and subjected to indirect immunofluorescence using anti-PPARγ2 antibody. (B, C) Western blot analysis of the full-length and truncated PPARγ2 in subcellular fractions of brown adipocytes during differentiation. Brown preadipocytes (day 0) were differentiated for 2, 4, and 7 days, homogenized and subjected to centrifugation at 1,000 × g (N, nuclear pellets; S, nuclei-free supernatant). The nuclei-free supernatant was further centrifuged at 10,000 × g to isolate mitochondria (M).

Fig 3. The truncated PPARγ2 localizes in the mitochondrial matrix.

(A) The truncated form of PPARγ2 in mitochondria is protected from proteinase K digestion. Purified brown adipose tissue mitochondria (60 μg) were treated with increasing amounts of proteinase K in the absence or presence of 1% Triton X-100. (B) Immuno-transmission electron microscopic analysis of the truncated PPARγ2 in brown adipocytes. Black dots indicated by arrow heads represent immunogold particles reacted with PPARγ (H100) antibody. Mitochondrial localization of immunogold particles was examined in 4–5 grids per group (20–30 mitochondria/grid), and the relative number of immunogold particles localized in the mitochondria was shown in the right panel. Data are presented as the mean ± SEM. Data represent mean ±SEM. ****P<0.0001.

Fig 4. The truncated PPARγ2 binds to the D-loop region of mitochondrial DNA.

(A) A schematic diagram of mitochondrial DNA (mtDNA). (B) Enrichment of the truncated PPARγ2 at the D-loop region of mtDNA in brown adipocyte mitochondria. Mitochondrial chromatin immunoprecipitation assay was carried out as described in Materials and Methods. The relative amounts of mtDNA immunoprecipitated with IgG or PPARγ (H100) antibody were analyzed by quantitative real-time PCR analysis (n = 4). Data represent mean ±SEM. **P<0.01.

Fig 5. MLS-PPARγ2 increases mitochondrial respiration by modulating mtDNA-encoded ETC gene expression.

(A) Expression of MLS-PPARγ2 in brown adipocytes increases mtDNA-encoded ETC gene expression. Quantitative real-time PCR was carried out in brown adipocytes expressing pBABE or MLS-PPARγ2 (n = 5). Data represent mean ±SEM. *P<0.05. (B) MLS-PPARγ2 enhances mitochondrial respiration in brown adipocytes. Cellular oxygen consumption rates (OCR) were measured at baseline and after injection of antimycin A (n = 6). The value of mitochondrial respiration was determined by subtracting antimycin A-independent non-mitochondrial respiration. Data represent mean ±SEM. *P<0.05.