An unexpected role for the transcriptional coactivator isoform NT-PGC-1α in the regulation of mitochondrial respiration in brown adipocytes

Abstract

Brown adipose tissue dissipates energy as heat, a process that relies on a high abundance of mitochondria and high levels of electron transport chain (ETC) complexes within these mitochondria. Two regulators of mitochondrial respiration and heat production in brown adipocytes are the transcriptional coactivator PGC-1α and its splicing isoform NT-PGC-1α, which control mitochondrial gene expression in the nucleus. Surprisingly, we found that, in brown adipocytes, some NT-PGC-1α localizes to mitochondria, whereas PGC-1α resides in the nucleus. Here we sought to investigate the role of NT-PGC-1α in brown adipocyte mitochondria. Immunocytochemistry, immunotransmission electron microscopy, and biochemical analyses indicated that NT-PGC-1α was located in the mitochondrial matrix in brown adipocytes. NT-PGC-1α was specifically enriched at the D-loop region of the mtDNA, which contains the promoters for several essential ETC complex genes, and was associated with LRP130, an activator of mitochondrial transcription. Selective expression of NT-PGC-1α and PGC-1α in PGC-1α^-/- brown adipocytes similarly induced expression of nuclear DNA-encoded mitochondrial ETC genes, including the key mitochondrial transcription factor A (TFAM). Despite having comparable levels of TFAM expression, PGC-1α^-/- brown adipocytes expressing NT-PGC-1α had higher expression of mtDNA-encoded ETC genes than PGC-1α^-/- brown adipocytes expressing PGC-1α, suggesting a direct effect of NT-PGC-1α on mtDNA transcription. Moreover, this increase in mtDNA-encoded ETC gene expression was associated with enhanced respiration in NT-PGC-1α-expressing PGC-1α^-/- brown adipocytes. Our findings reveal a previously unappreciated and isoform-specific role for NT-PGC-1α in the regulation of mitochondrial transcription in brown adipocytes and provide new insight into the transcriptional control of mitochondrial respiration.

Keywords: adipocyte; adipose tissue metabolism; gene expression; mitochondrial respiratory chain complex; mtDNA; peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α).

Figure 1.

NT-PGC-1α is present in brown adipocyte mitochondria. A, schematic of subcellular fractionation. Brown adipose tissues were isolated from C57BL/6J mice exposed to 4 °C for 5 h and subjected to subcellular fractionation. B, presence of NT-PGC-1α in mitochondria. Top panel, schematic of the PGC-1α and NT-PGC-1α proteins. AD, transcription activation domain; LxxLL, nuclear receptor interaction motif; RS, arginine/serine-rich domain. Bottom left panel, PGC-1α-HA and NT-PGC-1α-HA were expressed in COS-1 cells and immunoblotted with a highly specific anti-PGC-1α monoclonal antibody (9). Bottom right panel, endogenous PGC-1α and NT-PGC-1α in BAT isolated from cold-exposed mice were immunoblotted with the same anti-PGC-1α antibody. The cytosolic (C), nuclear (N), and mitochondrial (M) markers were detected in their respective fractions. C, expression of NT-PGC-1α in PGC-1α^−/− brown adipocytes. WT brown adipocytes expressing an empty vector and PGC-1α^−/− brown adipocytes stably expressing NT-PGC-1α or an empty vector by retrovirus-mediated gene transfer were treated with 0.5 mm dibutyryl cAMP for 4 h. D, presence of NT-PGC-1α in brown adipocyte mitochondria. PGC-1α^−/− brown adipocytes stably expressing NT-PGC-1α were treated with dibutyryl cAMP and subjected to subcellular fractionation. E, cAMP-induced signaling does not regulate mitochondrial targeting of NT-PGC-1α. PGC-1α^−/− brown adipocytes stably expressing NT-PGC-1α were treated with vehicle or dibutyryl cAMP for 4 h. WGL, whole cell lysates. F, localization of NT-PGC-1α and PGC-1α in PGC-1α^−/− brown adipocytes. NT-PGC-1α-HA and PGC-1α-HA were expressed in PGC-1α^−/− brown adipocytes by adenovirus-mediated gene transfer and analyzed for cellular localization by indirect immunofluorescence with anti-HA antibody. Colocalized pixels were analyzed using an ImageJ analysis tool and are displayed in white on an RGB overlay image. Scale bars = 23 μm. G, correlation of the amounts of mitochondria and the protein levels of NT-PGC-1α. Mitochondrial enrichment of NT-PGC-1α was analyzed in increasing amounts of purified brown adipocyte mitochondria. H, mitochondrial NT-PGC-1α is protected from proteinase K digestion. Purified mitochondria (60 μg) were treated with increasing amounts of proteinase K.

Figure 2.

NT-PGC-1α localizes in the mitochondrial matrix. Immunotransmission electron microscopic analysis of NT-PGC-1α in brown adipocytes. Black dots represent immunogold particles reacted with PGC-1α antibody in PGC-1α^−/− brown adipocytes (KO) expressing NT-PGC-1α or an empty vector (pBABE). Mitochondrial localization of immunogold particles was examined in 6–8 grids/group (20–30 mitochondria/grid), and the relative number of immunogold particles localized in the mitochondria in each group is shown in the bottom panel. Data are presented as the mean ± S.E. One-way ANOVA was used to compare the difference between groups: **, p < 0.01.

Figure 3.

Mitochondrial NT-PGC-1α is enriched at the D-loop region of mitochondrial DNA. A, schematic of mtDNA. B, enrichment of mitochondrial NT-PGC-1α at the D-loop region of mtDNA in brown adipocytes. Mitochondria were isolated from wild-type brown adipocytes treated with 0.5 mm dibutyryl cAMP for 4 h and subjected to chromatin immunoprecipitation assays. The relative amounts of mtDNA immunoprecipitated with IgG, PGC-1α, and TFAM antibodies were analyzed by quantitative real-time PCR analysis (n = 6). Data represent mean ± S.E. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. C, mitochondrial NT-PGC-1α does not interact with TFAM but with LRP130. Mitochondria were isolated from PGC-1α^−/− brown adipocytes expressing NT-PGC-1α-HA or pBABE and subjected to immunoprecipitation (IP) using anti-HA antibody. D, enrichment of LRP130 at the mitochondrial D-loop region. Brown adipocyte mitochondria were isolated from wild-type brown adipocytes treated with 0.5 mm dibutyryl cAMP for 4 h, and mitochondrial ChIP was carried out with anti-LRP130 antibody. Data represent mean ± S.E. *, p < 0.05.

Figure 4.

NT-PGC-1α increases mitochondrial DNA-encoded gene expression. A, differential effects of NT-PGC-1α and PGC-1α on mtDNA-encoded gene expression. PGC-1α^−/− brown adipocytes stably expressing pBABE, NT-PGC-1α, and PGC-1α were assessed for nucDNA- and mtDNA-encoded gene expression (n = 6). Data represent mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001. B, Western blot analysis of TFAM in PGC-1α^−/− brown adipocytes stably expressing pBABE, NT-PGC-1α, and PGC-1α. Relative protein levels of TFAM normalized to β-actin were determined by densitometric analysis using ImageJ as described under “Experimental Procedures.” C, effects of NT-PGC-1α and PGC-1α on mitochondrial respiration in PGC-1α^−/− brown adipocytes. OCRs were measured at baseline and after stimulation with cAMP (n = 4) as described under “Experimental Procedures.” Representative results from three independent experiments are shown and presented as the mean ± S.E. Two-way ANOVA was used to compare the difference between groups: #, p < 0.0001.

Figure 5.

Increased mitochondrial targeting of NT-PGC-1α by addition of MLS. A, cellular localization of NT-PGC-1α and MLS-NT-PGC-1α. HeLa cells were transfected with NT-PGC-1α-HA and MLS-NT-PGC-1α-HA using FuGENE 6. Immunofluorescence was carried out using anti-HA antibody and MitoTracker Deep Red 633. Scale bars = 23 μm. B, subcellular distribution of NT-PGC-1α-HA and MLS-NT-PGC-1α-HA in HeLa cells. Relative protein levels of NT-PGC-1α-HA and MLS-NT-PGC-1α-HA in cytoplasmic (C), nuclear (N), and mitochondrial (M) fractions were determined by densitometric analysis using ImageJ as described under “Experimental Procedures.” C, effect of NT-PGC-1α and MLS-NT-PGC-1α on PPARγ/RXRα-mediated reporter gene expression in the nucleus. NT-PGC-1α or MLS-NT-PGC-1α was co-transfected with a luciferase reporter gene containing three copies of PPAR-binding sites, PPARγ, RXRα, and a Renilla luciferase reporter gene in HeLa cells. Luciferase activity was determined after 48-h transfection and normalized with Renilla luciferase activity. Data represent the mean ± S.E. of three independent experiments. One-way ANOVA was used to compare the difference between groups: ****, p < 0.0001. RLU, relative light units.

Figure 6.

MLS-NT-PGC-1α increases mitochondrial DNA-encoded gene expression and mitochondrial respiration. A, MLS-NT-PGC-1α increases mtDNA- and nucDNA-encoded gene expression. Quantitative real-time PCR was carried out in PGC-1α^−/− brown adipocytes expressing pBABE and MLS-NT-PGC-1α (n = 5). Data represent mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001; #, p < 0.0001. B, quantitative analysis of mitochondrial biogenesis. The ratio of mtDNA relative to nucDNA was analyzed by quantitative real-time PCR (n = 6/group). Data represent mean ± S.E. C, MLS-NT-PGC-1α enhances mitochondrial respiration in PGC-1α^−/− brown adipocytes. OCRs were measured at baseline and after injection of oligomycin and FCCP (n = 6) as described under “Experimental Procedures.” Representative results from four independent experiments are shown and presented as the mean ± S.E. *, p < 0.05; **, p < 0.01.

Figure 7.

A proposed scheme of the transcriptional control of mitochondrial respiratory function by NT-PGC-1α in brown adipocytes. Cold-induced PGC-1α and NT-PGC-1α transcriptionally regulate mitochondrial respiratory function in brown adipocytes by stimulating the expression of nucDNA-encoded ETC genes and other mitochondrial genes, including TFAM. TFAM is subsequently imported to mitochondria and regulates mitochondrial DNA replication and transcription, leading to an increase in mtDNA-encoded ETC gene expression. Our findings suggest an additional mechanism by which NT-PGC-1α regulates mtDNA transcription within brown adipocyte mitochondria. Simultaneous localization of NT-PGC-1α in the nucleus and mitochondria may contribute to the coordinated regulation of nucDNA- and mtDNA-encoded ETC gene expression in response to cold.