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.
Highlights
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
No change in mitochondrial content of NT-PGC-1␣ was observed after treatment of brown adipocytes with cAMP, indicating that mitochondrial targeting of NT-PGC-1␣ is independent of cAMP signaling (Fig. 1E)
We provide the first evidence showing that NT-PGC-1␣ is present in brown adipocyte mitochondria, where it is enriched at the D-loop region of mtDNA, associated with LRP130, and involved in mtDNA transcription
Summary
We have demonstrated that NT-PGC-1␣ is predominantly cytoplasmic and that its nuclear accumulation is increased by cold/-adrenergic receptor/cAMP/PKA-mediated phosphorylation [21]. To test whether mitochondrial NT-PGC-1␣ is recruited to the D-loop region of mtDNA, we isolated mitochondria from wild-type brown adipocytes treated with cAMP for 4 h and performed mitochondrial ChIP (mtChIP) assays using an anti-PGC-1␣ polyclonal antibody that has been confirmed for its specificity to immunoprecipitate NT-PGC-1␣ [9, 10, 22]. A transcriptional co-activation assay using a luciferase reporter containing the PPAR response element (PPRE) elements showed that co-expression of NT-PGC-1␣ with PPAR␥ and retinoid X receptor ␣ (RXR␣) increased reporter gene expression by 10.3-fold compared with control cells, whereas MLSNT-PGC-1␣ only led to a 3.7-fold increase in reporter gene expression (Fig. 5C) This result may suggest that the nuclear content of MLS-NT-PGC-1␣ is in part reduced because of increased mitochondrial targeting. OCRs were measured at baseline and after injection of oligomycin and FCCP (n ϭ 6) as described under “Experimental Procedures.” Representative results from four independent experiments are shown and presented as the mean Ϯ S.E. *, p Ͻ 0.05; **, p Ͻ 0.01
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