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J Appl Physiol 102: 314-320, 2007. First published August 17, 2006; doi:10.1152/japplphysiol.00549.2006
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Effect of nitric oxide synthase inhibition on mitochondrial biogenesis in rat skeletal muscle

G. D. Wadley and G. K. McConell

Department of Physiology, The University of Melbourne, Parkville, Victoria, Australia

Submitted 15 May 2006 ; accepted in final form 2 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The purpose of this study was to determine whether nitric oxide synthase (NOS) inhibition decreased basal and exercise-induced skeletal muscle mitochondrial biogenesis. Male Sprague-Dawley rats were assigned to one of four treatment groups: NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME, ingested for 2 days in drinking water, 1 mg/ml) followed by acute exercise, no L-NAME ingestion and acute exercise, rest plus L-NAME, and rest without L-NAME. The exercised rats ran on a treadmill for 53 ± 2 min and were then killed 4 h later. NOS inhibition significantly (P < 0.05; main effect) decreased basal peroxisome proliferator-activated receptor-{gamma} coactivator 1beta (PGC-1beta) mRNA levels and tended (P = 0.08) to decrease mtTFA mRNA levels in the soleus, but not the extensor digitorum longus (EDL) muscle. This coincided with significantly reduced basal levels of cytochrome c oxidase (COX) I and COX IV mRNA, COX IV protein and COX enzyme activity following NOS inhibition in the soleus, but not the EDL muscle. NOS inhibition had no effect on citrate synthase or beta-hydroxyacyl CoA dehydrogenase activity, or cytochrome c protein abundance in the soleus or EDL. NOS inhibition did not reduce the exercise-induced increase in peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha} (PGC-1{alpha}) mRNA in the soleus or EDL. In conclusion, inhibition of NOS appears to decrease some aspects of the mitochondrial respiratory chain in the soleus under basal conditions, but does not attenuate exercise-induced mitochondrial biogenesis in the soleus or in the EDL.

contraction; metabolic regulation; peroxisome proliferator-activated receptor-{gamma} coactivator 1

THERE IS NOW CLEAR EVIDENCE for a role of nitric oxide (NO) in mitochondrial biogenesis. NO donors increase the protein and mRNA expression of peroxisome proliferator-activated receptor coactivator 1 (PGC-1), nuclear respiratory factor 1 (NRF-1), and mitochondrial transcription factor A (mtTFA) in muscle cells and adipocytes of rodents (25, 26), which are involved in coordinating mitochondrial biogenesis (40). This process is dependent on the downstream messenger of NO, cGMP, via the activation of soluble guanylate cyclase (sGC) (25, 26). Furthermore, blocking the activation of sGC also blocks mitochondrial biogenesis induced by NO donors (26). NO is formed in the conversion of L-arginine to L-citrulline by NO synthase (NOS) (23).

NOS is constitutively active at rest in rodent skeletal muscle (4, 29, 32), and there is evidence from knockout mice that the endothelial (eNOS) and neuronal (nNOS) isoforms may be differentially involved in the regulation of mitochondrial biogenesis in skeletal muscle. In mice deficient in eNOS (eNOS–/–), the gastrocnemius muscle has decreased PGC-1{alpha} mRNA levels and reduced mitochondrial density and mitochondrial DNA levels (26). The gastrocnemius of mice deficient in nNOS (nNOS–/–) display increased citrate synthase activity (but not protein abundance), and decreases in some but not all complexes of the electron transport chain, despite similar mitochondrial density (35). Unfortunately, it was not examined whether the differences in mitochondrial biogenesis observed in eNOS–/– (26) and nNOS–/– (35) mice occur in the red (oxidative) or white (glycolytic) portions of the gastrocnemius, or both. The oxidative characteristics of the muscle(s) being studied are important because eNOS is more abundant in oxidative and nNOS is more abundant in glycolytic rodent skeletal muscle, respectively (12, 16, 18). Therefore, one could anticipate that a lack of eNOS would impact mitochondrial biogenesis more in oxidative muscle, whereas a lack of nNOS would impact more in glycolytic muscles. Consistent with this, in eNOS–/– mice, oxidative skeletal muscle (soleus) has decreases in various aspects of respiratory chain function, including decreased mitochondrial respiration, citrate synthase, and cytochrome c oxidase (COX) activity, whereas glycolytic (white gastrocnemius) skeletal muscle does not (21, 22). However, despite the novel insights provided by these NOS knockout studies, it is difficult to determine whether any of these effects could be due to compensatory mechanisms brought about by the long-term lack of either eNOS or nNOS. An alternative method of investigating the role of NOS on mitochondrial biogenesis in skeletal muscle is to inhibit all NOS isoforms pharmacologically. This avoids the problem of NOS isoform compensatory mechanisms that may arise from long-term gene ablation. No study has examined the effect of ingesting a nonspecific NOS inhibitor such as NG-nitro-L-arginine methyl ester (L-NAME), which greatly reduces basal skeletal muscle NOS activity in rodents (29), on skeletal muscle mitochondrial biogenesis. Furthermore, no study has examined the effects of NOS inhibition (or a lack of eNOS or nNOS) on mitochondrial biogenesis markers (such as expression of PGC-1, NRF-1, or mtTFA) in skeletal muscle with differing oxidative characteristics, such as the soleus (oxidative) and extensor digitorum longus (EDL; glycolytic).

Endurance exercise potently stimulates increases in skeletal muscle mitochondrial volume (9, 11), and the increased mitochondrial biogenesis following exercise training (2, 9, 24, 28) is thought to be largely attributed to the cumulative effects of each acute bout of exercise (10, 28). The mRNA levels of key components of the mitochondrial biogenesis pathway such as PGC-1{alpha}, NRF1 and mtTFA are increased several hours following an acute exercise bout in skeletal muscle and are involved in coordinating this response (2, 24, 27, 28, 38). NOS activity also increases during contraction in rodent skeletal muscle (18, 29) and may therefore be involved in the upregulation of mitochondrial biogenesis by exercise. To date, no study has examined the effects of either NOS inhibition, or eNOS or nNOS deficiency, on mitochondrial biogenesis markers following an acute exercise bout. However, eNOS–/– mice have decreased COX activity in the soleus, but not the white (glycolytic) gastrocnemius following voluntary chronic physical activity (22), suggestive of reduced mitochondrial biogenesis. A confounding factor with eNOS–/– mice is that NO levels might still increase during exercise, because cGMP levels increase in skeletal muscle of these mice (presumably from nNOS) following electrical stimulation (18). To avoid such confounding factors involved with gene ablation, the present study sought to use an alternative model involving 2 days of L-NAME ingestion followed by acute exercise.

Based on the findings in skeletal muscle of eNOS–/– mice (21, 22), we hypothesized that 1) NOS inhibition in rats would attenuate several markers of basal mitochondrial biogenesis in the soleus (oxidative) but not the EDL (glycolytic) muscle and 2) that NOS inhibition in rats would attenuate the increases in mRNA levels of PGC-1{alpha}, NRF-1, and mtTFA 4 h following exercise in the soleus but not the EDL muscle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animal Care and Dietary Treatment

Male Sprague-Dawley rats, aged 6 wk old and weighing 246 ± 3 g, were obtained from The University of Melbourne Pharmacology and Physiology Animal House. Animals were housed in an environmentally controlled laboratory (temperature 22°C) with a reverse 12:12-h light-dark cycle (light 1900–0700). Animals were familiarized to treadmill running 1 wk before experimentation on 2 separate days for 20 min each day. The University of Melbourne Animal Experimentation Ethics Committee approved all experimental procedures.

Animals were assigned to one of four groups on the basis of 1) whether they were ad libitum fed L-NAME (1 mg/ml; Sigma, St. Louis, MO) in the drinking water for 48 h and rested (–Ex/+L-NAME; n = 8), 2) were fed L-NAME (1 mg/ml) in the drinking water for 48 h and acutely exercised (+Ex/+L-NAME; n = 7), 3) remained rested with ad libitum access to water (–Ex/–L-NAME; n = 8), or 4) ad libitum access to water for 48 h and then acutely exercised (+Ex/–L-NAME; n = 8). There is strong evidence that pharmacological NOS inhibition is effective at blocking NOS activity and downstream signaling mediators such as cGMP levels following contraction in rodent skeletal muscle (18, 29). The protocol of 2 days of L-NAME ingestion used in the present study has previously been shown to inhibit NOS activity (>95%) in rat skeletal muscle at rest and immediately after treadmill running (29). Furthermore, NOS inhibition also prevents the increase in cGMP levels following electrical stimulation of isolated mouse muscle (18).

Rested controls.   On the experimental day, two groups of rats (–Ex/–L-NAME and –Ex/+L-NAME) rested in their cages with ad libitum access to water or L-NAME. Food was withdrawn for 5 h, and the rats were then anesthetized with an intraperitoneal injection of xylazine (30 mg/kg) and ketamine (225 mg/kg) followed by cervical dislocation. Soleus and EDL muscles were rapidly excised and frozen and stored in liquid N2.

Exercised rats.   On the experimental day, two groups of rats (+Ex/–L-NAME and +Ex/+L-NAME) ran on a motor-driven treadmill at 25 m/min on a 5% incline for a maximum of 60 min or until exhaustion. The exercise duration was matched between both groups so that mean ± SE duration of running was 53 ± 2 min. Rats were then placed back in their cage with ad libitum access to water or L-NAME, and food was withdrawn. Four hours after exercise rats were killed (at the same time of day as the rested rats). This exercise protocol adequately recruits the soleus and EDL muscle groups during treadmill running. Preliminary investigations (8 rats for rested vs. exercise groups) in our laboratory have found that this exercise stimulus significantly reduces muscle glycogen levels immediately after exercise in the soleus by ~35% (P < 0.05; 27 ± 1 vs. 18 ± 2 mmol/kg wet wt, rested vs. exercise, respectively) and in the EDL by ~25% (P < 0.05, 34 ± 1 vs. 26 ± 2 mmol/kg wet wt, rested vs. exercise, respectively).

When investigating changes in gene expression and subsequent protein abundance after exercise, it is often difficult to sample at time point(s) that will adequately reflect changes in all of the genes (and proteins) of interest (41), and the present study is no exception. Previous studies have found the mRNA levels of PGC-1{alpha} increase ~10-fold following acute exercise, with peak mRNA levels occurring ~1–4 h following exercise, depending on the intensity and duration of the preceding exercise bout (28, 33). The mRNA levels of NRF1 and mtTFA typically increase to peak levels of 1.5- to 3.0-fold by 6 h postexercise (24, 28). Based on this information, examination of muscle 4 h following exercise was chosen because this time point was expected to best represent the response of PGC-1{alpha}, NRF-1, and mtTFA mRNA to an acute bout of exercise.

Preparation of Rat Tissue

RNA was isolated from frozen rat soleus and EDL using Trizol (Invitrogen, Melbourne, Australia). For immunoblotting and enzyme activity, frozen muscle (10 µl of buffer/mg of muscle) was homogenized in freshly prepared ice-cold buffer [50 mM Tris at pH 7.5 containing 1 mM EDTA, 10% vol/vol glycerol, 1% vol/vol Triton X-100, 50 mM NaF, 5 mM Na4P2O7, 1 mM DTT, 1 mM PMSF and 5 µl/ml Protease Inhibitor Cocktail (P8340, Sigma, St. Louis, MO)]. Tissue lysates were incubated on ice for 20 min and then spun at 10,000 g for 20 min at 4°C. Protein concentration was determined using a bicinchoninic protein assay (Pierce, Rockford, IL) with BSA as the standard.

Gene Expression

RNA integrity was verified and the concentration determined on the Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA). First strand cDNA was generated from 0.5 µg RNA using AMV Reverse Transcriptase (Promega, Madison, WI) as previously described (39). Primers were designed using the Primer Express software package version 1.0 (Perkin-Elmer, Norwalk, CT) from gene sequences obtained from GenBank (PGC-1{alpha}: AY237127 [GenBank] , PGC-1beta: AY188951 [GenBank] , mtTFA: AB014089 [GenBank] , COX III: AF504920 [GenBank] , COX IV: J05425 [GenBank] ). The primer sequences were validated using BLAST (1) to ensure that each primer was homologous with the desired mRNA of rat skeletal muscle. The primer sequences are shown in Table 1. Real-time PCR using SYBRgreen chemistry was performed in triplicate using the Rotor-gene 3000 system (Corbett Research, Sydney, Australia). Because SYBRgreen indiscriminately binds to double-stranded DNA and other products such as primer dimers, the samples were subjected to a heat-dissociation protocol after the final cycle of PCR to ensure that only one product was detected. Heat dissociation of oligonucleotides detects differences in melting temperature and produces a single dissociation peak for each nucleotide within a 2°C difference in melting temperature. NRF1 (catalog no. Rn01455954_m1) and beta2-microglobulin (catalog no. Rn00560865_m1) were assessed using predesigned/prevalidated 5'-6-carboxyfluorescein (FAM)-labeled Assays-on-Demand from Applied Biosystems (Applied Biosystems, Foster City, CA). All genes were normalized to beta2-microglobulin using the cycle threshold (2Formula) method. beta2-Microglobulin mRNA levels have been reported not to change in response to an acute bout of endurance exercise in skeletal muscle (19). Furthermore, using one-way analysis of variance, preliminary investigations in the present study found there to be no difference in beta2-microglobulin mRNA levels measured from the 2Formula values between all four groups of rats (n = 8 rats per group) for either the soleus or EDL muscles (data not shown).


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  		Table 1. Gene primer sequences

 
Immunoblotting

Total lysates for determination of PGC-1{alpha}, COX IV, and cytochrome c were solubilized in Laemmli sample buffer. Bound proteins were separated by SDS-PAGE and electrotransfer of proteins from the gel to PVDF membranes (25 mmol/l Tris, pH 8.3, 192 mmol/l glycine, and 20% vol/vol methanol) was performed for 90 min at 95 V (constant). Blots were probed with anti-PGC-1{alpha} rabbit polyclonal (Chemicon, Temecula, CA), anti-COX IV mouse monoclonal (Molecular Probes, Eugene, OR) and anti-cytochrome c mouse monoclonal (BD Biosciences Pharmingen, San Diego, CA) antibodies. Binding was detected with IRDye 800-conjugated anti-rabbit IgG (Rockland, Gilbertsville, PA) or IRDye 680-conjugated anti-mouse IgG (Molecular Probes) secondary antibodies. All data were analyzed in duplicate and expressed as integrated intensity following infrared detection (Odyssey Imaging system, LI-COR Biosciences, Lincoln, NE).

Enzyme Activities

All enzyme activities were measured spectrophotometrically at room temperature using the muscle homogenates and expressed in micromoles per minute per gram of total protein. The total activity of COX (electron transport chain) was assayed by measuring the decrease in absorbance at 550 nm corresponding to the oxidation of ferrocytochrome c by COX using a commercially available kit (Sigma). beta-Hydroxyacyl CoA dehydrogenase (beta-HAD; beta-oxidation of fatty acids) activity was measured at 340 nm by following the disappearance of NADH. Citrate synthase (Krebs cycle) activity was measured by following the increase in 5,5'-dithiobis-2-nitrobenzoate at 412 nm (36).

Statistical Analyses

Results were analyzed using two-factor analysis of variance (with/without L-NAME; with/without exercise). If this analysis revealed a significant interaction, specific differences between mean values were located using the Fisher's least significance difference test. All data are presented as means ± SE. The level of significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Only two of the seven rats in the +Ex/+L-NAME group were able to complete 60 min of treadmill running without becoming exhausted before 60 min. The running time for the +Ex/+L-NAME group was 53 ± 2 min and the running times for the +Ex/–L-NAME running group was subsequently matched, with none of the rats in the +Ex/–L-NAME group being exhausted at the end of exercise.

Effect of L-NAME on Basal Levels of Mitochondrial Biogenesis

In the soleus, the basal mRNA levels of both the mitochondrial encoded (COX III) and nuclear encoded (COX IV) subunits of COX, along with COX activity and the protein abundance of the COX IV subunit were all ~15–20% lower (P < 0.05, main effect for L-NAME; Fig. 1, A–D, respectively) following 2 days of L-NAME ingestion. Also in the soleus, the mRNA levels of PGC-1beta was ~27% lower (P < 0.05; main effect for L-NAME, Fig. 2A) following L-NAME ingestion. There was also a tendency for mRNA levels of mtTFA to be lower in the L-NAME groups (P = 0.08 for + L-NAME vs. –L-NAME, Fig. 2B). Two days of L-NAME ingestion did not alter the basal levels of any other measures of mitochondrial biogenesis in the soleus such as the mRNA level of NRF-1 (Fig. 2C), protein abundance of cytochrome c and PGC-1{alpha}, and the enzyme activities of citrate synthase and beta-HAD (Table 2).


Figure 1
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  		Fig. 1. Cytochrome c oxidase (COX) III mRNA (A), COX IV mRNA (B), COX activity (C), and COX IV protein abundance (D) in the soleus of rats following 2 days of ingestion of water [without NG-nitro-L-arginine methyl ester (–L-NAME)] or a nitric oxide synthase inhibitor (+L-NAME) and 4 h after 53 ± 2 min of treadmill running (+Ex) or rest (–Ex). Values are means ± SE; n = 8 for all groups except n = 7 for +Ex/+L-NAME group and for COX activity n = 7 for –Ex/+L-NAME. Western blots are representative from 1 rat from each treatment group. + P < 0.05 main effect for L-NAME.

 

Figure 2
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  		Fig. 2. Peroxisome proliferator-activated receptor-{gamma} coactivator 1beta (PGC-1beta) mRNA (A), mitochondrial transcription factor A (mtTFA) mRNA (B), and nuclear respiratory factor 1 (NRF-1) mRNA (C) in the soleus of rats following 2 days of ingestion of water (–L-NAME) or a nitric oxide synthase inhibitor (+L-NAME) and 4 h after 53 ± 2 min of treadmill running (+Ex) or rest (–Ex). Values are means ± SE; n = 8 for all groups except n = 7 for +Ex/+L-NAME group. + P < 0.05 main effect for L-NAME.

 

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  		Table 2. Effect of L-NAME ingestion on basal (–Ex) levels of mitochondrial biogenesis markers in the soleus

 
In the EDL, 2 days of L-NAME ingestion did not alter the basal levels of any measure of mitochondrial biogenesis (Table 3, see Fig. 4).


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  		Table 3. Effect of L-NAME ingestion on basal (–Ex) levels of mitochondrial biogenesis markers in the EDL

 

Figure 4
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  		Fig. 4. PGC-1{alpha} mRNA (A) and mtTFA mRNA (B) in the EDL of rats following 2 days of ingestion of water (–L-NAME) or a NOS inhibitor (+L-NAME) and 4 h after 53 ± 2 min of treadmill running (+Ex) or rest (–Ex). Values are means ± SE; n = 8 for all groups except n = 7 for +Ex/+L-NAME group. *P < 0.05 main effect for exercise. {dagger}P < 0.05 vs. –Ex/+L-NAME. {ddagger}P < 0.05 vs. +Ex/–L-NAME and vs. –Ex/–L-NAME.

 
Effect of L-NAME ingestion on mitochondrial biogenesis following exercise.   The acute exercise bout resulted in ~8-to 11-fold higher PGC-1{alpha} mRNA levels (P < 0.05, main effect for exercise; Fig. 3) 4 h following exercise in the soleus. Acute exercise also increased soleus COX activity by ~14% compared with the nonexercised groups (P < 0.05, main effect for exercise; 382 ± 16 vs. 436 ± 26 µmol·min–1·g protein–1, respectively). Acute exercise had no effect on the soleus mRNA levels of COX III, COX IV, PGC-1beta, mtTFA, and NRF-1 (P > 0.05; Fig. 1, A and B, Fig. 2, A–C, respectively) or the protein abundance (data not shown) of PGC-1{alpha}, COX IV, cytochrome c, and enzyme activities of citrate synthase and beta-HAD 4 h following exercise. There was no significant effect of L-NAME on any of the exercise-induced responses for any of these measured variables in the soleus (Fig. 1, A and B; Fig. 2, A–C; Fig. 3; and data not shown).


Figure 3
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  		Fig. 3. Peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha} (PGC-1{alpha}) mRNA in the soleus of rats following 2 days of ingestion of water (–L-NAME) or a nitric oxide synthase inhibitor (+L-NAME) and 4 h after 53 ± 2 min of treadmill running (+Ex) or rest (–Ex). Values are means ± SE; n = 8 for all groups except n = 7 for +Ex/+L-NAME group. *P < 0.05 main effect for exercise.

 
Acute exercise significantly increased PGC-1{alpha} mRNA in the EDL muscle (P < 0.05, main effect for exercise; Fig. 4A) 4 h following exercise, especially in the +Ex/+L-NAME group (P < 0.05 vs. all other groups; Fig. 4A). Exercise also increased mtTFA mRNA by ~20% (P < 0.05, main effect for exercise; Fig. 4B) in the EDL. There was no effect of exercise or L-NAME on the mRNA level, protein abundance or enzyme activity of the other variables measured in the EDL (P > 0.05; Table 3 and data not shown for +Ex/–L-NAME and +Ex/+L-NAME groups for all the variables measured in Table 3).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
A major finding of this study was that ingestion of the NOS inhibitor L-NAME resulted in reduced basal COX activity, COX III and COX IV mRNA, and COX IV protein expression in the soleus of rats. Given that cytochrome c protein, however, was not decreased with NOS inhibition in the soleus, these findings suggest a role for NO in basal regulation of some (but not all) aspects of the electron transport chain in oxidative muscle. The mechanism for this decreased mitochondrial biogenesis could be via reduced PGC-1beta, because L-NAME ingestion also significantly decreased basal PGC-1beta mRNA in the soleus. The second major finding, contrary to our original hypothesis, was that NOS inhibition did not attenuate the exercise-induced increase in skeletal muscle PGC-1{alpha} mRNA levels 4 h after acute exercise. These results suggest that NO may play a role in basal mitochondrial biogenesis in skeletal muscle but that it is not involved in the increase in mitochondrial biogenesis following exercise.

Our finding of decreased basal COX activity with NOS inhibition in soleus muscle is consistent with findings in the soleus of eNOS–/– mice (22) and we extend these findings to decreased COX mRNA and protein abundance. A possible mechanism for the reduced COX in the soleus following NOS inhibition could be due to the downregulation of PGC-1beta. L-NAME ingestion was observed to decrease PGC-1beta mRNA and tended to decrease mtTFA mRNA levels (Fig. 2) in the soleus, concomitant with decreased COX mRNA levels, protein abundance and enzyme activity (Fig. 1). Both PGC-1{alpha} and PGC-1beta appear to play important, but perhaps differing roles, in the regulation of mitochondrial biogenesis. Overexpression of either PGC-1{alpha} or PGC-1beta in L6 myoblasts increases mitochondrial number and basal oxygen consumption (20), and mice that overexpress PGC-1beta have elevated basal energy expenditure and beta-oxidation, consistent with increased mitochondrial biogenesis (13). Although the role of PGC-1beta is unclear, it has been hypothesized that PGC-1beta plays more of a role in the basal regulation of mitochondrial biogenesis, whereas PGC-1{alpha} may be more involved in mitochondrial biogenesis following metabolic perturbations such as exercise, cold exposure, fasting, or denervation (17, 20, 40). Indeed, similar to previous findings (20), the present study found PGC-1{alpha}, but not PGC-1beta, mRNA levels increased following an acute bout of exercise in skeletal muscle. No other study had previously examined the effect of NOS inhibition (or lack of eNOS or nNOS) on basal PGC-1beta mRNA expression. NO donors increase PGC-1{alpha} expression in adipocytes but have no effect on PGC-1beta (25). Therefore, the mechanism whereby decreased NOS activity may also decrease PGC-1beta mRNA and subsequently downregulate COX is an important question that remains to be answered.

A potential alternative explanation for the observed reduction in COX mRNA expression, protein abundance and activity in the soleus following L-NAME treatment could be related to the well-known inhibition of COX, and mitochondrial respiration, by NO. In competition with oxygen, NO binds to COX to inhibit COX activity in a reversible reaction (6, 23, 34). Although, based on this a NOS inhibitor would be expected to increase COX activity, it is possible that mitochondria actually respond to the inhibition of COX via NO by increasing mitochondrial biogenesis in an attempt to maintain oxygen consumption. Indeed, NO donors increase COX expression (25, 26). Therefore, when the inhibition of COX by NO is removed by NOS inhibition, it is possible that the response is a downregulation of COX mRNA, protein, and activity, as observed in the present study.

It is difficult to reconcile the different responses at rest in mitochondrial biogenesis observed between the soleus and EDL following L-NAME treatment in the present study. Some of the differences could possibly be explained by the distribution (and perhaps function) of eNOS in oxidative and glycolytic skeletal muscle. The eNOS isoform is strongly correlated with mitochondrial content (16) and is accordingly more highly expressed in the soleus compared with the EDL (18) and gastrocnemius (14). Therefore, if eNOS has a role in the basal regulation of mitochondrial biogenesis in skeletal muscle, then inhibition of eNOS would only be expected to alter basal mitochondrial biogenesis in the soleus and not the EDL, consistent with the findings of the present study. Indeed, eNOS–/– mice have decreased basal levels of mitochondrial respiration, citrate synthase and COX activity in the soleus (oxidative) muscle but not the white gastrocnemius (glycolytic) (21, 22).

A surprising finding from the present study was the significantly greater increase in EDL PGC-1{alpha} mRNA levels 4 h following exercise in the NOS inhibition group compared with the control exercise group (Fig. 4A). The reasons for this are unclear. Although speculative, NOS inhibition may (7, 8, 31) have attenuated the increase in glucose uptake during exercise (3, 5, 15, 30), which then caused a greater muscle energy imbalance. The greater energy imbalance may then have activated AMP-activated protein kinase, which is a known stimulator of PGC-1{alpha} in skeletal muscle (37, 38, 42).

In the present study, there was no increase in NRF-1 mRNA 4 h following exercise in the soleus or EDL muscle groups and only a small increase in mtTFA mRNA levels in the EDL. This would suggest that 4 h postexercise is not an adequate time point to observe exercise-induced changes in mRNA levels of NRF-1 or mtTFA and longer time points such as 6 h are required as observed by others (24, 28). Nevertheless, 4 h postexercise was sufficient to show that exercise increases mRNA levels of PGC-1{alpha} in the soleus and EDL and, in contrast to our hypothesis, that ingestion of the NOS inhibitor, L-NAME, does not attenuate this response.

The present study has demonstrated that L-NAME treatment does not attenuate the exercise-induced increase in PGC-1{alpha} mRNA following acute exercise. However, the findings of the present study do not completely rule out the involvement of NO in the regulation of exercise-induced mitochondrial biogenesis. Future studies should examine whether exercise training in other models of NOS inhibition (i.e., eNOS–/– or nNOS–/– mice) results in normal increases in mitochondrial biogenesis markers.

In conclusion, the present study demonstrated decreased basal levels of PGC-1beta mRNA and a trend for decreased mtTFA mRNA in the soleus muscle of rats following L-NAME treatment. This result may explain the decreased basal COX III and COX IV mRNA levels, COX IV protein abundance, and COX enzyme activity in rat soleus muscle following L-NAME treatment, and it suggests a role for NOS in the basal regulation of mitochondrial biogenesis in oxidative skeletal muscle. However, contrary to what we originally hypothesized, NOS inhibition did not attenuate the exercise-induced increase in the mRNA levels of PGC-1{alpha} in rat muscle. These results indicate that NOS inhibition in skeletal muscle via L-NAME treatment does not attenuate the increases in mitochondrial biogenesis following acute exercise.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was supported by grants from the National Health and Medical Research Council of Australia and Diabetes Australia.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors thank Dr. Robert Lee-Young, Associate Professor David Cameron-Smith, and Zheng Ruan for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Wadley, Dept. of Physiology, The Univ. of Melbourne, Parkville 3010, Australia (e-mail: gdwadley{at}unimelb.edu.au)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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