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CELLULAR METABOLISM

Pyruvate induces mitochondrial biogenesis by a PGC-1 -independent mechanism

Diabetes and Metabolism Disease Area, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts

Submitted 10 August 2006 ; accepted in final form 19 December 2006

	ABSTRACT

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Oxidative cells increase mitochondrial mass in response to stimuli such as changes in energy demand or cellular differentiation. This plasticity enables the cell to adapt dynamically to achieve the necessary oxidative capacity. However, the pathways involved in triggering mitochondrial biogenesis are poorly defined. The present study examines the impact of altering energy provision on mitochondrial biogenesis in muscle cells. C2C12 myoblasts were chronically treated with supraphysiological levels of sodium pyruvate for 72 h. Treated cells exhibited increased mitochondrial protein expression, basal respiratory rate, and maximal oxidative capacity. The increase in mitochondrial biogenesis was independent of increases in peroxisomal proliferator activator receptor- coactivator-1 (PGC-1) and PGC-1 mRNA expression. To further assess whether PGC-1 expression was necessary for pyruvate action, cells were infected with adenovirus containing shRNA for PGC-1 before treatment with pyruvate. Despite a 70% reduction in PGC-1 mRNA, the effect of pyruvate was preserved. Furthermore, pyruvate induced mitochondrial biogenesis in primary myoblasts from PGC-1 null mice. These data suggest that regulation of mitochondrial biogenesis by pyruvate in myoblasts is independent of PGC-1, suggesting the existence of a novel energy-sensing pathway regulating oxidative capacity.

oxidative metabolism; peroxisomal proliferator activator receptor- coactivator-1, mitochondria; muscle

Despite a number of stimuli being known, the signaling pathways controlling mitochondrial biogenesis in skeletal muscle are not clearly defined. An emerging regulator of mitochondrial replication is the peroxisomal proliferator activator receptor- coactivator-1 (PGC-1), which acts through coactivation of a number of transcription factors (20, 28). For example, PGC-1 coactivates the peroxisome proliferator-activated receptors (PPARs) to upregulate fatty acid oxidation while it induces the transcription of oxidative phosphorylation genes by coactivating nuclear respiratory factors (NRF)-1 and -2. A close homolog of PGC-1, PGC-1 may share a similar role in mitochondrial metabolism (12, 26).

In the present study, we explore pyruvate-induced mitochondrial biogenesis in muscle myoblasts and the role of PGC-1 in mediating this effect. We report that supraphysiological concentrations of pyruvate increase mitochondrial mass and functionality as determined by a comprehensive array of mitochondrial measures. Furthermore, we establish that this action does not increase PGC-1 or - mRNA expression and, in fact, is not dependent on PGC-1.

	MATERIALS AND METHODS

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Cell culture. C2C12 cells (American Type Culture Collection, Manassas, VA) were seeded in either basal media or test media. Basal media was DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 1% antibiotics, and 4 mM L-glutamine. Pyruvate test media were DMEM with no sodium pyruvate (Invitrogen) supplemented with 10% FBS, 1% antibiotics, 4 mM L-glutamine, and 50 mM sodium pyruvate (Sigma, St. Louis, MO). Sodium chloride or sodium bicarbonate test medium was basal medium supplemented with either an additional 50 mM sodium chloride or sodium bicarbonate, respectively. Cells were incubated for 72 h before manipulation. Sodium chloride and pyruvate-free DMEM were obtained as a customized reagent (Invitrogen). All studies were performed on myoblasts, i.e., undifferentiated proliferating muscle progenitors.

Flow cytometry. Cells were trypsinized, centrifuged at 1,000 rpm for 5 min, resuspended, and counted. Cells were incubated with MitoTracker Green FM (Molecular Probes, Eugene, OR) at a final concentration of 100 nM for 30 min at 37°C. After incubation, cells were centrifuged at 1,000 rpm for 5 min. Media were removed, and cells were resuspended in PBS at a concentration of 1 x 10⁶ cells/ml. The forward scatter vs. side scatter area was used to analyze a homogeneous population of live cells after doublet exclusion using a FacsAria Flow Cytometer (BD Biosciences, San Diego, CA).

Confocal microscopy. Following treatment, media were removed, and cells were incubated with 100 nM MitoTracker Green FM for 30 min at 37°C, then with 2 µg/ml Hoechst 33342 dye for an additional 10 min. Phenol red-free DMEM (Invitrogen) was added, and the cells were visualized on a Zeiss 510 Meta Confocal Microscope at x63 magnification (Carl Zeiss, Thornwood, NY).

Protein expression studies. Cytochrome c protein level was measured using a commercially available enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. For Western blot analyses, cells were washed three times with PBS, then solubilized with PBS containing 0.5% Triton X-100 and protease inhibitors for 10 min at 4°C. Extracts were briefly sonicated, and the protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Pierce). Ten micrograms of cell lysate were mixed with LDS sample buffer and reducing reagent (Invitrogen), vortexed, and heated at 70°C for 10 min. Samples were loaded and resolved on a 4–12% Bis-Tris mini gel (Invitrogen). Following transfer, the nitrocellulose membranes were incubated with indicated primary antibodies. Immunoreactive bands were detected using ECL Western blotting detection reagents (Amersham Biosource). Anti-mitochondrial protein antibodies were purchased from MitoSciences (Eugene, OR). Anti-silent information regulator-2 (anti-Sir2) antibodies were purchased from Upstate (Lake Placid, NY).

Cellular respiration assay. Cells were washed with PBS, trypsinized, and resuspended in respiration medium (PBS without Ca⁺/Mg²⁺, 25 mM HEPES, pH 7.0, 5.55 mM glucose, 1% BSA, fatty acid free); 300,000 cells in 2 ml were transferred to a Clark-type oxygen electrode chamber that was connected to a circulating water bath at 37°C. Basal respiration was measured, and then nonphosphorylating (uncoupled) respiration was determined in the presence of the ATP synthase inhibitor oligomycin (2 µg/ml). Carbonylcyanide-3-chlorophenylhydrazone (CCCP), a chemical uncoupler, was titrated into the cell solution to determine oxidative capacity (maximal respiration). Data obtained from a background (blank) measurement of oxygen flux using heavy gas titration were subtracted from sample data to control for instrumental influence.

Adenoviral studies. A recombinant adenoviral vector expressing a short hairpin RNA (shRNA) targeted to mouse PGC-1 was kindly provided by Dr. Marc Montminy, Salk Institute for Biological Studies, La Jolla, CA, and was generated as described by Koo et al. (9). C2C12 myoblasts were plated on six-well plates in basal media at a density of 3 x 10⁴ cells per well (control) or 8 x 10⁴ cells per well (treated). Cells were infected 6 h after seeding with either Ad-sh-PGC-1 (3.0 x 10¹² particle/ml) or Ad-sh-scrambled (3.0 x 10¹² particle/ml) at 1.25 x 10⁵ particle/cell in basal media. The media were changed 24 h later to either basal media or test media. Virus at 1 x 10⁵ particle/cell was added to each well 24 h after the media were changed, and cells were harvested 48 h after the second infection.

Real-time quantitative PCR analysis. Following the isolation of total RNA and preparation of cDNA, the expression profiles of PGC-1 and PGC-1 genes (accession nos. NM_008904 and NM_133249, respectively) were measured using real-time PCR. Quantitative RT-PCR was performed on an ABI Prism 7900HT Sequence Detection System and analyzed using SDS 2.0 software (Applied Biosystems, Foster City, CA). The expression of each target gene was normalized by the endogenous control 18S rRNA (Applied Biosystems). An Assay-on-Demand 20x mix containing primers and probe specific for each target gene (ID nos. PGC-1/Mm00447183_m1 and PGC-1/Mm00504720_m1) was obtained from Applied Biosystems. Data were determined in duplicate from three wells per treatment and expressed as means ± SE. Differences were considered statistically significant at P < 0.05 by one-way ANOVA with Dunnett's multiple-comparison test.

Gene chip analysis. The GeneChip experiment was conducted in the Genomics Factory, Novartis PHARMA, Basel, Switzerland, on a Gene Chip Mouse Genome 430 2.0 Expression Array (Affymetrix, Santa Clara, CA). The oligonucleotide probes were 25mers, and 11 probe pairs per sequence were used. The data discussed in this publication have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) and are accessible through GEO series accession number GSE5497 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE5497). Gene set enrichment analysis was performed as previously described (15).

PGC-1 null myoblast studies. Myoblasts derived from PGC-1 null mice and wild-type littermates were seeded in Ham's F-10 medium containing 20% FBS, 1% penicillin-streptomycin (Invitrogen), and 2.5 ng/ml basic fibroblast growth factor (13). Once the cells attached, the medium was removed, and cells were incubated in either fresh basal medium or pyruvate (50 mM) test medium for 72 h at 37°C.

Statistical analyses. Experiments were repeated on at least two independent days, and representative figures are provided. Within one experiment, n = 3 refers to three different culture plates. Statistical analyses were performed on the data presented using one-way ANOVA and Tukey's multiple comparison test, except where noted. P < 0.05 was considered significant.

	RESULTS

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C2C12 myoblasts were incubated in either basal media containing 1 mM sodium pyruvate or test media supplemented with 50 mM sodium pyruvate (SP) for 72 h. The cells were then stained with MitoTracker Green FM, a mitochondrial-specific fluorescent dye, and the mitochondrial mass was assessed using fluorescence-activated cell sorting (FACS) (Fig. 1A). FACS analyses revealed significantly greater mitochondrial staining in SP-treated cells than in control cells. The increase in MitoTracker fluorescence was not a result of increased cell size as determined by size-gating analysis (Fig. 1B). Further evidence of increased mitochondrial content was demonstrated using confocal microscopy and MitoTracker Green FM staining (Fig. 1C).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Mitochondrial mass in muscle cells is increased with pyruvate treatment. C2C12 myoblasts were incubated in either basal or sodium pyruvate (SP) test media for 72 h. Cells were trypsinized and stained with 100 nM MitoTracker Green FM. Fluorescence-activated cell sorting (FACS) analysis of the total cell population (A) and size-gated populations (B; Green-A indicates MitoTracker staining) was performed (n = 3 ± SE). A: RFU, relative fluorescence units. *P < 0.05. B: cells were gated according to size as follows: blue, smaller cells; gray, medium-sized cells; red, larger cells. Fluorescence is represented on the x-axis and cell no. on the y-axis. A right shift in distribution indicates an increase in fluorescence. C: stained cells were visualized using confocal microscopy. Nuclei were stained with 2 µg/ml Hoechst stain.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Pyruvate stimulates mitochondrial protein expression and cellular respiration in C2C12 myoblasts. A: cytochrome c protein levels in total cellular lysates from cells incubated in basal or test media were measured by an ELISA. Test media were basal media supplemented with either an additional 50 mM SP, 50 mM NaCl, or 50 mM NaHCO3 (n = 6 ± SE). B: extracts from basal or SP-treated cells were subjected to Western blot analysis. Resulting blots were probed for F1 (ATP synthase), core 2 (complex III), 30-kDa Ip subunit (complex II), and 20-kDa subunit (complex I). C: basal, uncoupled, and maximum respiration of cells was determined in cells incubated in basal or SP-treated media (n = 3 ± SE). CCCP, carbonylcyanide-3-chlorophenylhydrazone. *P < 0.05.

We also measured cellular respiration to evaluate whether functional changes accompanied the apparent increase in mitochondrial mass (Fig. 2C). Using a Clark-type oxygen electrode, we measured oxygen consumption in intact cells. SP-treated cells had a significantly higher basal respiration rate than control cells. Maximal oxidative capacity, in the presence of CCCP, was approximately twice that of control cells, whereas no significant difference in uncoupling was apparent. These data suggest that pyruvate treatment results in increased oxidative capacity and not increased uncoupling.

PGC-1 has been implicated as a key regulator of mitochondrial biogenesis; however, it is expressed at very low levels in C2C12 myoblasts (26, 28). Using quantitative RT-PCR, we analyzed PGC-1 mRNA expression levels in control and SP-treated C2C12 cells and found that pyruvate treatment did not upregulate PGC-1 expression (Fig. 3A). Interestingly, PGC-1 mRNA levels were significantly reduced following pyruvate treatment (Fig. 3B). Despite low PGC-1 expression in C2C12 myoblasts, we employed shRNA technology to further knock down PGC-1 in these cells. Cells were infected with either PGC-1 or scrambled shRNA adenoviruses and then incubated in either basal or SP test media for 72 h. Cytochrome c protein expression was increased in SP-treated cells compared with control cells despite a further 70% decrease in PGC-1 mRNA expression (Fig. 3C). Cells treated with control virus were not significantly different from untreated cells (data not shown). Also, protein levels of sirtuin 1 (SIRT1), a regulator of PGC-1 activity, were unchanged over a 24-h period of pyruvate treatment (Fig. 3D).

View larger version (11K): [in this window] [in a new window]   Fig. 3. Pyruvate-induced mitochondrial biogenesis is not dependent on peroxisomal proliferator activator receptor- coactivator-1 (PGC-1). C2C12 myoblasts were incubated in basal media or SP test media following infection with either PGC-1 or scrambled short hairpin (sh)RNA. Total RNA was extracted for quantitative real-time PCR analysis of PGC-1 (A) and PGC-1 (B) expression levels (n = 3 ± SE). C: cells were also extracted for cytochrome c protein determination (n = 9 ± SE). B and C: *P < 0.05. D: total cellular lysates were made from C2C12 myoblasts incubated in basal or SP test media at indicated time points. SIRT1 protein expression was analyzed by Western blot analysis and normalized against actin (n = 6 ± SE).

View larger version (13K): [in this window] [in a new window]   Fig. 4. Mitochondrial biogenesis is induced by pyruvate in myoblasts from PGC-1 null mice. Primary myoblasts from PGC-1 null mice and wildtype (WT) littermates were incubated in either basal or SP test media for 48 h. A: cells were incubated with 100 nM MitoTracker Green FM for 30 min, and the mitochondrial mass was assessed using FACS analysis (n = 6 ± SE). B: cytochrome c protein levels in total cellular lysates were measured using an ELISA (n = 4 ± SE). *P < 0.05.

View larger version (11K): [in this window] [in a new window]   Fig. 5. Gene set enrichment analysis (GSEA) of pyruvate-treated C2C12 myoblasts. Cells were incubated in either basal or 50 mM pyruvate [high sodium pyruvate (hsp)] test media for 72 h. Total RNA extracted from cells was subjected to GeneChip array analysis. Relative gene expression in the 2 conditions was plotted in scatter. GSEA was performed to assess pathway gene expression levels. Two representative probe sets are shown by black dots (n = 3 ± SE).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A previous study examined the action of high pyruvate concentrations in L6 myoblasts. The authors reported increased mitochondrial content as indicated by nonyl acridine orange (NAO) staining and an increase in mitochondrial membrane potential with pyruvate treatment (2). Some controversy exists over the use of NAO in estimating mitochondrial content, particularly when there are changes in mitochondrial membrane potential (7, 8). Therefore, we sought to corroborate these findings with a comprehensive panel of mitochondrial markers and then examine the mechanistic basis for pyruvate action.

In agreement with the previous study, treatment of myoblasts with high pyruvate concentrations induced mitochondrial biogenesis, as determined by cytochrome c expression, mitochondrial staining [using MitoTracker Green FM, a mitochondrial dye insensitive to changes in membrane potential (19)], expression of complexes I and III and basal and uncoupled respiration. It is somewhat surprising that pyruvate did not increase all electron transport chain proteins; however, it is conceivable that, in this setting of increased mitochondrial biogenesis, these proteins are not rate limiting. Importantly, these effects were not a result of changes in osmolarity, a parameter that was not controlled in the previous study.

Differentiation of myoblast precursor cells to more metabolically active myotubes results in an increased mitochondrial mass. Therefore, it is important to establish whether agents that increase mitochondrial biogenesis are doing so by inducing cellular differentiation. Pyruvate treatment did not induce myoblast fusion or genes associated with muscle differentiation. In fact, with the use of gene set enrichment analysis, it was apparent that the gene program associated with myocellular determination was downregulated. Interestingly, by use of the same analysis, genes associated with free radical scavenging, a key auxiliary requirement to mitochondrial activity, were upregulated. Increased production of reactive oxygen species following pyruvate treatment has been observed in a number of cellular systems (18, 29) and likely stimulates upregulation of anti-oxidative machinery. This provides some support of the observation that pyruvate has anti-oxidative properties (1). In addition, it is possible that increased free radical production could in some way induce mitochondrial replication.

The nuclear receptor coactivator PGC-1 is a key regulator of mitochondrial biogenesis and gluconeogenesis in muscle and liver, respectively. Ectopic expression of PGC-1, or a close homolog, PGC-1, results in increases in mitochondrial mass (10, 26, 28). The extent to which these factors act independently and/or redundantly in controlling oxidative capacity is seemingly dependent on tissue type and stimulus. For example, unlike PGC-1, PGC-1 is not upregulated during exercise or cold stress (14). Moreover, PGC-1 null mice have reduced exercise tolerance and other metabolic derangements associated with diminished mitochondrial capacity (11).

PGC-1 mRNA was expressed at low levels in C2C12 myoblasts under basal conditions and did not increase with pyruvate treatment. However, we could not exclude the possibility that posttranslational modification of existing PGC-1 protein could lead to increased activity, independently of upregulation of the gene. Therefore, we treated cells with shRNA targeted to PGC-1, resulting in a further reduction in target mRNA levels. Even with this reduction, pyruvate induction of the mitochondrial marker cytochrome c was unaffected. Interestingly, PGC-1 expression was decreased with pyruvate treatment and unaffected by PGC-1 knockdown. Importantly, primary myoblasts from the PGC-1 null mouse (13) also responded to pyruvate treatment.

One potential fate of acetyl-CoA generated from pyruvate entry into the mitochondria is the formation of acetyl-carnitine. In addition to liberating mitochondrial CoA, acetyl-carnitine can pass out of the mitochondria and cell, providing an overflow mechanism for excess energy supply when the cell is energy replete. Intriguingly, a number of reports have demonstrated that supplementing diets with acetyl-carnitine can help maintain or improve mitochondrial function in rats (4, 5). It is also noteworthy that administration of pyruvate reduced the reaccumulation of weight gain following diet-induced weight loss in humans (24, 25). The present data imply that pyruvate-induced mitochondrial biogenesis and increased oxidative capacity may have contributed to this phenotype.

A second fate of excess pyruvate is the production of lactate with the concomitant oxidation of NADH to NAD⁺. The NAD⁺-dependent histone deacetylase SIRT1 interacts with PGC-1 (17) and regulates PGC-1-dependent gene expression in a nutrient-sensitive manner in hepatocytes (21). Intriguingly, increased SIRT1 activity also has been linked to downregulation of muscle cell differentiation (3), a function that is similarly affected by pyruvate treatment in the present study. We were unable to detect increases in Sir2 protein levels with pyruvate treatment; however, we cannot exclude the possibility that SIRT1 activity levels are increased and can function independently of PGC-1. An alternate possibility is that the decrease in myogenicity is a direct result of increased mitochondrial content, as suggested by Seyer et al. (23).

Combined, our results indicate that cell autonomous regulation of mitochondrial mass and function is sensitive to three-carbon fuel availability. However, pyruvate sensing does not invoke the participation of the transcriptional coactivators PGC-1 and PGC-1. Given that lower organisms (e.g., yeast, fruit flies) lack PGC-1, our data support the view that additional control elements governing mitochondrial replication may play a role in tissues exposed to high flux of oxidative substrates, including pyruvate.

	ACKNOWLEDGMENTS

 
We thank Bruce Spiegelman for the kind gift of primary PGC-1 null and wildtype myoblasts, Mark Montminy for the shRNA PGC-1 adenovirus, Deborah Ahern-Ridlon and Akos Szilvasi for technical assistance with the confocal microscopy and FACS analyses, Nanguneri Nirmala and Joseph Szustakowski for the GeneChip data analysis, Daniel Kemp for the myogenic gene set, and Thomas Hughes for critical review of this manuscript.

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. Halse, Novartis Institutes for BioMedical Research, 100 Technology Square, Cambridge, MA 02139 (e-mail: reza.halse{at}novartis.com)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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This Article Abstract Full Text (PDF) All Versions of this Article: 292/5/C1599    most recent 00428.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wilson, L. Articles by Halse, R. Search for Related Content PubMed PubMed Citation Articles by Wilson, L. Articles by Halse, R.