β-Guanadinopropionic acid (β-GPA) feeding leads to reductions in skeletal muscle phosphagen concentrations and has been used as a tool with which to study the effects of energy charge on skeletal muscle metabolism. Supplementing standard rodent diets with β-GPA leads to increases in mitochondrial enzyme content in fast but not slow-twitch muscles from male rats. Given this apparent discrepancy between muscle types we used β-GPA feeding as a model to study signaling pathways involved in mitochondrial biogenesis. We hypothesized that β-GPA feeding would result in a preferential activation of p38 MAPK and AMPK signaling and reductions in RIP140 protein content in triceps but not soleus muscle. Despite similar reductions in high-energy phosphate concentrations, 6 wk of β-GPA feeding led to increases in mitochondrial proteins in triceps but not soleus muscles. Differences in the response of mitochondrial proteins to β-GPA feeding did not seem to be related to a differential activation of p38 MAPK and AMPK signaling pathways or discrepancies in the induction of PPARγ coactivator (PGC)-1α and -1β. The protein content and expression of the nuclear corepressor RIP140 was reduced in triceps but not soleus muscle. Collectively our results indicate that chronic reductions in high-energy phosphates lead to the activation of p38 MAPK and AMPK signaling and increases in the expression of PGC-1α and -1β in both soleus and triceps muscles. The lack of an effect of β-GPA feeding on mitochondrial proteins in the soleus muscles could be related to a fiber type-specific effect of β-GPA on RIP140 protein content.
- β-guanadinopropionic acid
- peroxisome proliferator-activated receptor-γ coactivator-1α
- skeletal muscle
an increase in skeletal muscle mitochondrial content, i.e., mitochondrial biogenesis, is a well-documented adaptation to endurance exercise training. It is thought that the repeated and transient reductions in high-energy phosphates that occur within contracting skeletal muscle serve as an initial signal in the induction of this process. β-Guanadinopropionic acid (β-GPA) is a creatine analog that has been used as a tool with which to study the effects of “energy charge” on skeletal muscle mitochondrial content (3, 8, 21, 22, 34). When supplemented in standard rodent diets, β-GPA leads to marked reductions in intramuscular phosphagen levels and increases in mitochondrial enzyme protein content and activity. Interestingly, some (6, 8, 24), but not all (18), investigations have reported fiber type-specific differences in the response to β-GPA feeding, with fast-twitch muscles displaying a more pronounced increase in mitochondrial enzymes compared with slow-twitch oxidative muscle.
The pathway through which decreases in high-energy phosphates leads to the induction of mitochondrial biogenesis would appear to involve the activation of the energy-sensing enzyme 5′-AMP-activated protein kinase (AMPK). This enzyme is robustly activated by perturbations in high-energy phosphate levels, as occurs with β-GPA feeding (3, 22, 34). Similarly, it has been reported that β-GPA-induced mitochondrial biogenesis is absent in fast-twitch muscle from AMPK-dominant-negative mice (34). AMPK likely mediates increases in mitochondrial content through PPARγ coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis (11). Although AMPK has been shown to have a direct effect on PGC-1α expression, some have argued that AMPK can also activate p38 MAPK (12, 13), a signaling pathway that has been implicated in the control of PGC-1α and mitochondrial biogenesis (1, 19, 32). Given the fact that mitochondrial enzyme content appears to increase to a much larger extent in fast- compared with slow-twitch skeletal muscle (6, 8, 24) following β-GPA feeding, it would seem reasonable to speculate that a differential activation of p38 MAPK signaling could in part explain these differences. To date, this premise has not been explored.
PGC-1α gain- and loss-of-function studies demonstrate the importance of this molecule in the regulation of skeletal muscle mitochondrial biogenesis (14, 30). However, increasing evidence points toward the involvement of additional factors in the control of this intricate process. For instance, RIP140, a corepressor of nuclear receptors, has been shown to suppress genes involved in the TCA cycle and fatty acid oxidation (23). The deletion of RIP140 results in mitochondrial biogenesis in skeletal muscle, whereas the overexpression of RIP140 leads to reductions in skeletal muscle mitochondrial enzyme expression (23). At this juncture, it is not known whether decreases in high-energy phosphates regulate RIP140 content in skeletal muscle. However, it is interesting to note that RIP140 deletion increases mitochondrial content in fast- but not slow-twitch skeletal muscle, a phenotype similar to what has been reported in skeletal muscle following β-GPA feeding (7).
The purpose of the present study was to explore in further detail the signaling pathways that are activated in rat triceps and soleus muscle following long-term reductions in high-energy phosphates. We sought to exploit the apparent divergence of fiber type responses to reductions in phosphagens as a tool to further elucidate the mechanisms underlying mitochondrial biogenesis. Specifically, we hypothesized that β-GPA feeding would lead to increases in markers of mitochondrial biogenesis in triceps but not soleus muscle and that these changes would be associated with a differential regulation of AMPK, p38 MAPK and RIP140 in fast- vs. slow-twitch muscle.
Reagents, molecular weight marker and nitrocellulose membranes for SDS-PAGE were purchased from Bio-Rad (Mississauga, ON, Canada). ECL Plus was a product of Amersham Pharmacia Biotech (Arlington Heights, IL). Antibodies against COX IV (cytochrome c oxidase IV; cat. no. A6403), COX I (cat. no. A6403), and CORE I (ubiquinol-cytochrome c reductase complex core protein-1; cat. no. A21362) were purchased from Molecular Probes (Eugene, OR). δ-Aminolevulinate synthase (ALAS) antibodies were a kind gift from Dr. John Holloszy (Washington University School of Medicine). Antibodies against p38 MAPK (cat. no. 9212), phospho-p38 MAPK (cat. no. 9211), ACC (cat. no. 3662), phospho-ACC (acetyl-CoA carboxylase; cat. no. 3661), phospho-ATF-2 (activating transcription factor 2; cat. no. 9221), AMPKα (cat. no. 2532), and p-AMPK (cat. no. 2531) were purchased from Cell Signaling (Danvers, MA). Medium-chain acyl-CoA dehydrogenase (MCAD) antibodies (cat. no. 50587) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-α-actin antibodies (cat. no. 2172) were a product of Sigma (St. Louis, MO). An antibody against RIP140 (cat. no. ab3425) was a product of Abcam (Cambridge, MA). PGC-1 antibodies were obtained from Chemicon (cat. no. ab3242). Horseradish peroxidase-conjugated donkey anti-rabbit and goat anti-mouse IgG secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). RNeasy extraction kits were purchased from Qiagen (Mississauga, ON, Canada). SuperScript II Reverse Transcriptase was a product of Invitrogen (Burlington, ON, Canada). Taqman Gene Expression Assays for β-actin, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), MCAD, and ALAS were from Applied Biosytems (Foster City, CA). Forward and reverse primers for PGC-1α, PGC-1β, RIP140, and COX I were from Integrated DNA technologies (Coraville, IA), while probes for these genes were from Applied Biosytems. Citrate synthase assay kits were purchased from Sigma.
Treatment of animals.
All protocols followed Canadian Council on Animal Care (CCAC) guidelines and were approved by the Animal Use and Welfare Committee at the University of Alberta. Male Wistar rats (Charles River, Wilmington, MA) weighing ∼180 g were housed two per cage with a 12:12-h light-dark cycle. Rats were fed a powdered, semipurified diet (15% fat wt/wt, 27% wt/wt protein, 43% wt/wt carbohydrate) supplemented with or without 1% wt/wt β-GPA for 6 wk. The control diet was supplemented with an additional 1% (wt/wt) cellulose to maintain equivalent nutrient density. The nutrient composition of the basal diet has been previously described in detail (29), the only difference being that in the present study the source of fat in the diet was flaxseed oil, soy tallow, and sunflower oil, providing a P:S ratio of 0.5. In preliminary experiments, we found that food intake was reduced in rats fed a β-GPA-supplemented diet. Consequently, control animals were pair fed to minimize differences in body weight. Food was weighed every day in the morning between 9:00 and 11:00 AM. Control-fed rats were given the same amount of food that the β-GPA-fed rats consumed the day prior. Average food intake was calculated as the average of the daily food intake during the duration of the 6-wk feeding study, i.e., the average amount of food consumed as recorded on 41 consecutive days. Between ∼9:00 and 11:00 AM, animals were anesthesized with pentabaritol sodium (5 mg/100 g body wt). Triceps and soleus muscles were carefully dissected, washed in sterile saline to remove any blood, and clamp-frozen in aluminum tongs cooled to the temperature of liquid nitrogen. Samples were stored at −80°C until analysis.
Clamp-frozen muscle samples were homogenized in 15 vol of ice-cold cell lysis buffer supplemented with Protease Inhibitor Cocktail (Sigma) and phenylmethylsulfonyl fluoride. Homogenized samples were sonicated for 5 s and centrifuged for 15 min at 2,500 g at 4°C. The supernatant was collected and protein concentration determined using the BCA method. There were no differences in protein concentrations between control and β-GPA-fed rats in either the soleus (control 10.52 ± 0.40, β-GPA 10.52 ± 0.34 μg/μl), or triceps (control 5.84 ± 0.54, β-GPA 6.58 ± 0.23 μg/μl). The protein content and/or phosphorylation status of CORE 1, COX IV, COX I, MCAD, ALAS, p38, ACC, AMPK, ATF-2, and RIP140 were determined by Western blot analysis as described previously (32). Briefly, equal amounts of protein were separated on 6.25% (RIP140, ACC, PGC-1), 10% (CORE 1, p38, ATF-2, AMPK, COX I, MCAD, ALAS) or 15% (COX IV) gels. Proteins were wet-transferred to nitrocellulose membranes at 200 mA/tank. Membranes were blocked in tris-buffered saline-0.01% Tween 20 (TBST) supplemented with 5% nonfat dry milk for 1 h at room temperature with gentle agitation. Membranes were incubated in TBST-5% nonfat dry milk supplemented with appropriate primary antibodies overnight at 4°C with gentle agitation. The following morning, blots were briefly washed in TBST and then incubated in TBST-1% nonfat dry milk supplemented with HRP-conjugated secondary antibodies for 1 h at room temperature. Bands were visualized using ECL Plus and captured using a Typhoon Imaging system (GE Health Care). Imagequant software was used to quantify relative band intensities. α-Actin was used as an internal control, as we found that it was not altered by β-GPA feeding.
RNA was isolated from skeletal muscle by use of a Fibrous RNeasy kit according to the manufacturer's instructions. One microgram of RNA was used for the synthesis of complementary DNA (cDNA) using SuperScript II reverse transcriptase, oligo(dT), and dNTP. Real-time PCR was performed using a 7900HT Fast Real-Time PCR system (Applied Biosytems). Taqman gene expression assays were used to determine the expression of GAPDH, β-actin, MCAD, and ALAS. Primers and probes for PGC-1α and -1β, COX I, and RIP140 were designed using Primer Express 3.0 software (sequences are available upon request). Samples were run in duplicate in a 96-well-plate format. For gene expression determined using Taqman expression assays, each well (20 μl total volume) contained 1 μl of gene expression assay, 1 μl of cDNA template, 10 μl of Taqman Fast Universal PCR Master Mix, and 8 μl of RNase-free water. For PGC-1α and -1β, each 20-μl reaction contained 12.5 μl of PCR Master Mix, 0.225 μl each of forward and reverse primers, 0.05 μl of probe, and 3.0 μl of RNase-free water.
In soleus muscle, β-actin was used as an endogenous control, as the expression of this gene did not change following β-GPA feeding. In the triceps, we used GAPDH as our control gene, as β-actin expression was decreased with β-GPA feeding (data not shown). Relative differences in gene expression between control and β-GPA fat-fed rats were determined using the 2 method (15). Standard-curve assays were performed for GAPDH/β-actin and the genes of interest. The amplification efficiencies of the gene of interest and GAPDH/β-actin were equivalent as determined using the equation 10(−1/slope) −1. Likewise, when plotting log cDNA dilution vs. (ΔCT, CT gene of interest − CT β-actin), the slope of this relationship was <0.1, indicating that the genes of interest were amplified with equal efficiency.
Mitochondrial DNA copy number.
Relative mitochondrial DNA (mtDNA) copy number was determined as described previously (25). Briefly, relative mtDNA copy number was measured by determining the ratio of a mtDNA target sequence (mitochondrial D-loop) to the expression of a nuclear target sequence (β-actin for soleus and GAPDH for triceps) by real-time PCR. These target genes have previously been used to determine relative amounts of mtDNA in rat tissue (4, 16, 25). In preliminary experiments, we found that there was a trend toward an increase in β-actin expression in rat triceps muscle from β-GPA-fed rats. Therefore, GAPDH was used as the nuclear encoded gene in the triceps. Primers and probes for β-actin and the mitochondrial D-loop were designed using Primer Express 1.5 software (sequences are available upon request), whereas GAPDH was measured using a Taqman Gene Expression Assay. Samples were run in duplicate using a 96-well-plate format using a 7900HT Fast Real-Time PCR system (Applied Biosytems). Relative differences in mtDNA copy number in skeletal muscle between control and β-GPA-fed rats was determined using 2 as described previously (25).
Citrate synthase activity.
Samples were prepared as described above for Western blotting. Citrate synthase activity was determined using a commercially available kit from Sigma. The formation of 5-thio-2-nitrobenzoic acid was measured spectrophotometrically at 412 nm in 96-well format. The CV of this assay in our laboratory is <10%.
Determination of ATP and phosphocreatine concentrations.
Data are presented as means ± SE. Comparisons between the means of control and β-GPA-fed groups were made using an unpaired Student's t-test. Statistical significance was set at P < 0.05.
Differences in body weight following β-GPA feeding.
There were no differences in body weight between control and β-GPA-fed rats at the beginning of the study. Following 6 wk of feeding, rats in the β-GPA group weighed significantly less than control rats (Table 1). The average daily food intake during the course of the study did not differ between control (24.3 ± 0.6 g/day) and β-GPA-fed rats (23.8 ± 0.6 g/day).
β-GPA feeding leads to reductions in high-energy phosphate concentrations.
Following 6 wk of dietary β-GPA supplementation, there were marked reductions in ATP, PCr, creatine (Cr), and calculated total Cr in both soleus and triceps muscles (Table 2).
β-GPA feeding increases mitochondrial proteins in triceps but not soleus muscle.
Six weeks of β-GPA feeding led to increases in mitochondrial proteins in rat triceps such as COX IV, COX I, CORE 1, MCAD, and ALAS (Fig. 1). Similarly, citrate synthase activity was higher in triceps from β-GPA-fed rats compared with controls, expressed relative to either tissue (control 19.9 ± 1.8, β-GPA 24.9 ± 1.2 μmol·min−1·g tissue−1, P < 0.05) or protein (control 95.1 ± 3.4, β-GPA 132.1. ± 5.8 μmol·min−1·g protein−1, P < 0.05). In contrast to the triceps, the protein content of mitochondrial marker proteins and the activity of citrate synthase (control 56.4 ± 1.4, β-GPA 59.4 ± 2.7 μmol·min−1·g tissue−1, control 194.2 ± 5.8, β-GPA 205.9 ± 9.5 μmol·min−1·g protein−1) in soleus muscle was not increased following β-GPA feeding.
To further assess the effects of β-GPA feeding on mitochondrial biogenesis, we assessed changes in the mRNA expression of mitochondrial enzymes and relative mtDNA copy number. As seen in Fig. 2, 6 wk of β-GPA feeding resulted in significant increases in ALAS and MCAD in triceps but not soleus muscles. There were no differences in relative mtDNA copy number in either muscle type following β-GPA feeding (soleus 0.85 ± 0.15-fold vs. control, triceps 0.99 ± 0.24-fold vs. control).
β-GPA feeding increases PGC-1 expression in skeletal muscle.
β-GPA feeding led to increases in the mRNA expression of PGC-1α and the related transcriptional coactivator PGC-1β in both rat triceps and soleus muscle (Fig. 3). Likewise, the protein content of PGC-1 was increased in both muscles from β-GPA fed-rats.
β-GPA feeding increases AMPK signaling.
β-GPA feeding led to increases in the phosphorylation of AMPK and its downstream substrate ACC in triceps and soleus muscles from β-GPA-fed rats (Fig. 4). ACC has been shown to be a sensitive marker of AMPK activation (9, 17), and changes in ACC phosphorylation have been used as a marker of AMPK activity following long-term treatment with AMPK agonists such as AICAR (2, 20). There were no differences in the total amount of ACC or AMPK following β-GPA feeding in either muscle.
β-GPA feeding increases p38 MAPK signaling.
Following 6 wk of β-GPA feeding, the phosphorylation of p38 MAPK and its downstream substrate ATF-2 were increased in both triceps and soleus muscles (Fig. 5).
β-GPA feeding reduces RIP140 mRNA expression and protein content in triceps but not soleus muscles.
The mRNA expression of RIP140 was decreased ∼40% in triceps muscles from β-GPA-fed rats. Although reduced, RIP140 mRNA expression in the triceps was still greater than in the soleus by ∼50% (data not shown). Similar to mRNA expression, β-GPA feeding led to an ∼40% reduction in RIP140 protein content. Conversely, neither the expression nor the protein content of RIP140 protein content was reduced in soleus muscles following β-GPA feeding (Fig. 6).
Supplementing rodent diets with the Cr analog β-GPA is a widely used model with which to study the effects of reduced phosphagen levels on skeletal muscle metabolism and gene expression. It has been demonstrated in skeletal muscle from male rats that β-GPA feeding leads to increases in mitochondrial enzymes in primarily fast- but not slow-twitch muscles (6, 8, 24). In an attempt to explain these apparent discrepancies between fiber types, we examined the activation of reputed mediators of mitochondrial biogenesis in rat triceps and soleus muscles, common examples of fast- and slow-twitch skeletal muscle, respectively.
Consistent with previous findings (6, 8, 24) we found that dietary supplementation of β-GPA led to increases in mitochondrial enzymes in triceps but not soleus muscle. These findings were not a result of differences in the degree to which our diet manipulation perturbed phosphagen levels, since ATP, PCr, and Cr levels were reduced to a similar extent in both muscle types. Previous work from Shulman's laboratory has clearly demonstrated that β-GPA feeding increases AMPK activation (3, 22, 34). Moreover, the overexpression of a dominant negative AMPK mutant blocks the effects of β-GPA on mitochondrial biogenesis in fast-twitch mouse skeletal muscle (34). Consistent with these findings, we found that the phosphorylation of ACC was increased in triceps muscles from β-GPA-fed rats. In soleus muscle, despite the fact that mitochondrial enzyme content was not augmented with β-GPA feeding, we saw increases in AMPK and ACC phosphorylation. These results are in line with a recent report demonstrating that the chronic activation of AMPK with AICAR did not lead to increases in mitochondrial enzyme activity in rat soleus muscle (2). Moreover, several groups have demonstrated a similar disconnect between AMPK activation and glucose transport in rat soleus muscle (7, 33).
In addition to AMPK, mounting evidence has suggested that the p38 MAPK signaling pathway is a central regulator of skeletal muscle mitochondrial biogenesis. For instance, Akimoto et al. (1), have reported that the overexpression of MAPK kinase-3 (MKK-3), an upstream activator of p38 MAPK, leads to increases in PGC-1α expression in skeletal muscle cells. This effect was prevented by the expression of a dominant negative mutant of ATF-2, a transcription factor that is a direct substrate of p38 MAPK and that binds to the PGC-1α promoter. In contrast to a previous study that analyzed p38 MAPK phosphorylation in skeletal muscles from creatine kinase-deficient mice (28), we found that β-GPA feeding led to increases in p38 MAPK and ATF-2 phosphorylation in both triceps and soleus. Although we do not have any evidence showing a direct effect of AMPK on the activation of the p38 MAPK signaling pathway, our results are consistent with others’ (10, 11) who have demonstrated cross talk between these pathways. Regardless of the mechanism through which p38 MAPK is activated following β-GPA feeding, differences in the activation of this enzyme do not appear to account for the differential effects of chronic reductions in high-energy phosphates on mitochondrial proteins between the triceps and soleus muscles. Moreover, it would appear that the activation of the p38 MAPK-ATF-2 signaling pathway, at least in soleus muscle, is not sufficient to cause increases in mitochondrial enzymes.
The activations of both AMPK (11, 26) and p38 MAPK (1, 19) have been shown to increase the expression of PGC-1α. As would be expected with the chronic activation of these signaling pathways by the dietary administration of β-GPA, we found that PGC-1α and the related transcriptional coactivator PGC-1β were increased to a similar extent in both soleus and triceps muscles. Taken in context with our signaling data, it would seem that many of the proximal signals and molecules that serve as positive effectors of mitochondrial biogenesis respond in a similar fashion to chronic reductions in high-energy phosphates regardless of the fiber type composition of the muscle examined. Within this framework it seems reasonable to assume that differences in the protein content and/or expression of negative regulators of mitochondrial biogenesis could potentially explain, at least in part, the fiber type-specific differences of β-GPA on skeletal muscle mitochondrial biogenesis. One likely candidate could be RIP140, a corepressor of nuclear receptors. The deletion of RIP140 has been shown to lead to increases in mitochondrial enzymes in fast-twitch mouse muscle that occur independently of increases in PGC-1α expression (23) whereas the overexpression of RIP140 results in reductions in mitochondrial content in slow-twitch skeletal muscle (23). It is thought that RIP140 exerts a negative effect on mitochondrial enzyme gene expression by acting as a scaffold protein between nuclear receptors and chromatin remodeling enzymes involved in transcriptional repression (31). Furthermore, it has been shown that RIP140 binds to the promoter regions of nuclear receptor target genes and can also interact with, and inhibit the activity of, PGC-1α (10).
In the present study, we found that β-GPA feeding led to marked reductions in both the protein content and mRNA expression of RIP140 in fast-twitch rat triceps muscles. To the best of our knowledge, this is the first investigation demonstrating that chronic reductions in skeletal muscle high-energy phosphate levels leads to decreases in RIP140 content. Given the similar effects of β-GPA feeding and exercise training on skeletal muscle mitochondrial biogenesis, it would seem likely that exercise could reduce skeletal muscle RIP140 content and that these changes could be involved in the mechanisms through which exercise induces mitochondrial biogenesis. However, arguing against this premise, it has recently been reported that the overexpression of RIP140 does not blunt the exercise-induced increase in skeletal muscle oxidative capacity (23). Clearly, the effects of exercise on RIP140 content, and the role of this protein in exercise-induced skeletal muscle mitochondrial biogenesis needs to be explored in further detail.
In contrast to the triceps, we did not see any differences in RIP140 protein content or mRNA expression in soleus muscle from control and β-GPA-fed rats. It has been suggested that RIP140 and PGC-1α have mutually antagonizing roles in the regulation of skeletal muscle mitochondrial gene expression (23). It has further been suggested that this antagonism may be mediated by the expression of RIP140 relative to PGC-1α. Thus, increases in PGC-1α, independent of reductions in RIP140, may not necessarily lead to increases in mitochondrial enzymes. The findings that certain markers of mitochondrial biogenesis did not increase in the soleus muscle despite a marked induction of PGC-1α and -1β would be consistent with this view.
In summary, this is the first investigation to examine fiber type-specific differences in the regulation of AMPK, p38 MAPK, and RIP140 by β-GPA feeding. We found that chronic reductions in phosphagens led to the activation of p38 MAPK and AMPK signaling and reductions in RIP140 in the triceps and that these changes were associated with increases in mitochondrial enzyme content. On the other hand, the activation of p38 MAPK and AMPK and the induction of PGC-1 mRNA did not appear to be sufficient to lead to increases in mitochondrial enzymes in the soleus. Although clearly a complex process involving multiple signaling pathways and transcriptional regulators, our results suggest that the differences in the response to long-term reductions in phosphagens between fast-twitch muscles like the triceps and slow-twitch muscles like the soleus, may be accounted for, at least in part, by a differential effect of β-GPA feeding on RIP140.
D. C. Wright is an Alberta Heritage Foundation for Medical Research Scholar, Canadian Diabetes Association Scholar, and Canadian Institutes of Health Research New Investigator. L. N. Sutherland was supported by an Alberta Heritage Foundation for Medical Research Studentship and a Natural Sciences and Engineering Research Council of Canada Graduate Student Scholarship. M. R. Bomhof and S. A. U. Basaraba were supported by Natural Sciences and Engineering Research Council of Canada summer studentships. This research was supported by NSERC Discovery Grants to C. J. Field, D. J. Dyck, and D. C. Wright.
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