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. 2010 Jun 18;285(25):19460-71.
doi: 10.1074/jbc.M110.113092. Epub 2010 Apr 19.

Peroxisome proliferator-activated receptor gamma coactivator 1alpha or 1beta overexpression inhibits muscle protein degradation, induction of ubiquitin ligases, and disuse atrophy

Jeffrey J Brault  1 , Jakob G Jespersen, Alfred L Goldberg
Affiliations

Peroxisome proliferator-activated receptor gamma coactivator 1alpha or 1beta overexpression inhibits muscle protein degradation, induction of ubiquitin ligases, and disuse atrophy

Jeffrey J Brault et al. J Biol Chem. .

Abstract

Overexpression of the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha), like exercise, increases mitochondrial content and inhibits muscle atrophy. To understand these actions, we tested whether PGC-1alpha or its close homolog, PGC-1beta, influences muscle protein turnover. In myotubes, overexpression of either coactivator increased protein content by decreasing overall protein degradation without altering protein synthesis rates. Elevated PGC-1alpha or PGC-1beta also prevented the acceleration of proteolysis induced by starvation or FoxO transcription factors and prevented the induction of autophagy and atrophy-specific ubiquitin ligases by a constitutively active FoxO3. In mouse muscles, overexpression of PGC-1beta (like PGC-1alpha) inhibited denervation atrophy, ubiquitin ligase induction, and transcription by NFkappaB. However, increasing muscle PGC-1alpha levels pharmacologically by treatment of mice with 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside failed to block loss of muscle mass or induction of ubiquitin ligases upon denervation atrophy, although it prevented loss of mitochondria. This capacity of PGC-1alpha and PGC-1beta to inhibit FoxO3 and NFkappaB actions and proteolysis helps explain how exercise prevents muscle atrophy.

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Figures

FIGURE 1.
FIGURE 1.
Overexpression of PGC-1α or PGC-1β in C2C12 myotubes increases mitochondrial content and total protein content without altering protein synthetic rate. A, fully differentiated myotubes were infected at various times with adenoviruses expressing PGC-1α, PGC-1β, or GFP before all being collected at the same time after differentiation. Activity of citrate synthase, a mitochondrial enzyme, was determined spectrophotometrically from cell homogenates. *, p < 0.001 versus GFP. B, myotubes were infected with PGC-1α, PGC-1β, or GFP adenoviruses. Total protein per well of a 6-well plate was measured 48 h later. *, p < 0.001 versus GFP. C, protein synthetic rate was determined by measuring the incorporation of [3H]tyrosine for 2 h at the indicated times after adenoviral infection. No statistically significant differences were found.
FIGURE 2.
FIGURE 2.
PGC-1α or PGC-1β overexpression inhibits overall protein degradation by slowing both proteasomal and lysosomal degradation. A, after labeling with [3H]tyrosine, C2C12 myotubes were infected with adenoviruses expressing PGC-1α, PGC-1β, or GFP (1st Virus). 24 h later, the myotubes were given fresh media containing a second adenovirus (2nd Virus). *, p < 0.05 versus GFP-GFP; **, p < 0.05 versus GFP-caFoxO3. B, myotubes were infected with PGC-1α and caFoxO3 adenoviruses as in A. Protein levels were determined by Western blot. C, myotubes were incubated with [3H]tyrosine and 1st Virus as in A. 24 h later, fresh medium was added either with (control) or without serum. *, p < 0.05 versus GFP-control; **, p < 0.05 versus GFP-No serum. In D: Top panel, myotube proteins were labeled with [3H]tyrosine for 24 h and then infected with GFP or PGC-1α adenoviruses for 48 h. Fresh DMEM (control) or HBSS (starved) medium, both containing 2% horse serum, was then added with or without the proteasome (bortezomib = Bortz) or lysosomal (concanamycin A = ConcA) inhibitors. Rates of proteolysis were determined 1 h later as described previously. Middle panel, the amount of proteolysis sensitive to each inhibitor represented the amount of proteasome or lysosome-mediated degradation and was calculated from data in the top panel by subtracting the rates of proteolysis in cells treated with the inhibitors from those of untreated cells. Bottom panel, the PGC-1α-induced decrease in proteolysis was calculated by subtracting the amount of lysosomal or proteasomal protein degradation rate (i.e. the inhibitor-sensitive component) in the PGC-1α-overproducing cells from that in the controls (GFP-expressing).
FIGURE 3.
FIGURE 3.
PGC-1α or PGC-1β inhibits caFoxO3-induced Atrogin1 or MuRF1 promoter activity in myotubes. A, myoblasts were transfected with pRL-TK and an Atrogin1 promoter luciferase reporter plasmids and allowed to differentiate for 3 days. Myotubes were then infected with adenoviruses (1st) expressing GFP, PGC-1α, or PGC-1β. 24 h later, the myotubes were infected with a second adenovirus (2nd) expressing either GFP or caFoxO3. Extracts were collected 24 h later and analyzed for luciferase activity. *, p < 0.05 versus GFP-GFP; **, p < 0.05 versus GFP-caFoxO3. B, cells were prepared and data analyzed precisely as in A except that myoblasts were transfected with a MuRF1-promoter reporter in place of the Atrogin1 reporter.
FIGURE 4.
FIGURE 4.
PGC-1α and mitochondrial content decrease in TA muscle 10 days after denervation. A, weight of the TA decreased 10 days after cutting the sciatic nerve (denervated). *, p < 0.001, n = 6. B, PGC-1α and mitochondrial proteins were decreased in denervated TA. Protein levels were quantified by Western blot. Equal amounts of total protein were loaded per lane. *, p < 0.05. C, the activity of the mitochondrial enzymes, citrate synthase and succinate dehydrogenase, decreased in the denervated TA. *, p < 0.001.
FIGURE 5.
FIGURE 5.
Electroporation of PGC-1β in mouse muscle inhibits fiber atrophy, and both PGC-1α and PGC-1β decrease Atrogin1 promoter activity and NFκB activity. Frequency histograms (right) showing the distribution of cross-sectional areas of innervated (A) or 10-day denervated (B) muscle fibers of the TA. Muscles were electroporated with PGC-1β-IRES-GFP plasmids at the same time as denervation. Transfected fibers were identified in transverse sections by GFP expression (left). Scale bar represents 30 μm. Tibialis anterior (TA) or soleus muscles of adult mice were co-electroporated with the pRL-TK and Atrogin1 promoter (C) or NFκB binding (D) luciferase reporter plasmids together with GFP (control), PGC-1α, or PGC-1β plasmids. Five or ten days later, muscles were collected and luciferase activity was measured. *, p < 0.05 versus innervated GFP-transfected fibers; **, p < 0.05 versus denervated GFP-transfected fibers.
FIGURE 6.
FIGURE 6.
AICAR treatment for 4 weeks increases PGC-1α and mitochondrial content but has no effect on muscle mass or expression of ubiquitin ligases. A, levels in the gastrocnemius muscle of PGC-1α protein and the mitochondrial proteins, cytochrome c, and succinate dehydrogenase (SDH), increase with AICAR treatment, and decrease with denervation. *, p < 0.05 versus innervated vehicle-treated muscle, n = 6. B, 2 weeks after denervation, the weights of several muscles decreased similarly with or without AICAR treatment. *, p < 0.01 versus innervated vehicle-treated muscle. C, the levels of the ubiquitin ligases Atrogin1 and MuRF1 proteins in the gastrocnemius muscle increased after denervation similarly with or without AICAR treatment. *, p < 0.05 versus innervated vehicle-treated muscle. D, the content of NRF-1 protein decreased with AICAR treatment and with denervation. *, p < 0.01 versus innervated vehicle-treated muscle, in contrast to the induction of NRF-1 following PGC-1 overexpression (cf. Fig. 2B).
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