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Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA
1Correspondence: Washington University School of Medicine Section of Applied Physiology, Campus Box 8113, 4566 Scott Ave., St. Louis, MO 63110, USA. E-mail: jhollosz{at}im.wustl.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: PPAR
coactivator 1 • mitochondrial transcription factor A • NRF-1 • NRF-2
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. These transcription factors activate nuclear genes that encode some mitochondrial respiratory chain proteins (1
, 2)
as well as 
-aminolevulinate (ALA) synthase, which limits the rate of heme synthesis (4)
, and mitochondrial transcription factor A (mtTFA), which activates mitochondrial DNA transcription and replication (3)
.
More recent studies by Spiegelman’s group (5
, 6)
have provided new insights regarding how the signals generated by adaptive stimuli result in a coordinated response of the transcriptional factors responsible for mitochondrial biogenesis. They discovered a coactivator of nuclear receptors, induced by cold in brown adipose tissue, that they termed PPAR coactivator-1 (PGC-1) because of its interaction with PPAR (5)
. It was subsequently found that in addition to coactivating PPAR, PGC-1 also coactivates PPAR
and NRF-1 and induces increased expression of NRF-1 and NRF-2 (6)
. Overexpression of PGC-1 greatly stimulates mitochondrial biogenesis in cultured myocytes and adipocytes (5
, 6)
, and in heart muscle of transgenic mice (7)
.
Skeletal muscle adapts to exercise, such as prolonged running or swimming, with an increase in mitochondria (8
9
10)
. This adaptation results in an enhanced capacity to generate ATP via oxidative phosphorylation (9
, 11
, 12)
. A single bout of swimming induces increases in PGC-1 mRNA (13
, 14)
, and protein (15)
, NRF-1 (15
, 16)
, and NRF-2 (15)
. These findings suggest that PGC-1, NRF-1 and NRF-2 are involved in the stimulation of mitochondrial biogenesis by exercise.
Contractile activity causes numerous perturbations of intracellular homeostasis. These include increases in cytosolic Ca2+, decreases in high-energy phosphates, increases in glycolytic intermediates and NADH, a decrease in pH, changes in redox state, and increased free radical production. This makes it difficult to identify the signal(s) responsible for mediating the exercise-induced increase in mitochondria. Cultured muscle cells have therefore been used to investigate the involvement of individual potential signals in inducing an increase in mitochondria (17
, 18)
. Using this approach, we found that intermittent increases in cytosolic Ca2+ induced by exposing L6 myotubes to caffeine or ionomycin for 5 h per day results in an increase in mitochondria (19)
.
A question raised by this finding is whether the increase in mitochondria induced by intermittently raising cytosolic Ca2+ in L6 myotubes is mediated by the same mechanism as the stimulation of mitochondrial biogenesis in skeletal muscle by exercise. If the same mechanism is involved, the L6 myotube model could be used to investigate the signaling pathway that mediates the exercise-induced increase in mitochondria. One purpose of this study was to determine whether, like exercise, intermittently raising cytosolic Ca2+ induces increased expression of PGC-1, NRF-1, and NRF-2 and whether an increase in mtTFA is involved in the adaptive response. A second purpose was to obtain preliminary information regarding whether Ca2+-calmodulin kinase (CAMK) is involved in mediating the increase in mitochondrial biogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-32P] dCTP was purchased from NEN Life Science Products (Boston, MA, USA). Rabbit polyclonal antibodies directed against the 19 carboxyl-terminal amino acids of ALA synthase and against the 20 carboxyl-terminal amino acids of citrate synthase were generated by Alpha Diagnostic International (Los Angles, CA, USA). A mouse anti-human cytochrome oxidase subunit (COXI) monoclonal antibody was purchased from Molecular Probes (Eugene, OR, USA). A mouse anti-cytochrome c monoclonal antibody was purchased from PharMingen International (San Diego, CA, USA). A polyclonal antibody against a peptide mapping near the carboxy terminus of PGC-1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase-conjugated secondary antibodies were from The Jackson Laboratory (West Grove, PA, USA). The bicinchoninic acid (BCA) protein assay kit was purchased from Pierce Chemical (Rockford, IL, USA). All other reagents were purchased from Sigma (St. Louis, MO, USA).
Cell culture
L6 myocytes were maintained at 37°C on 100 mm collagen-coated plastic dishes in 5% CO2/95% humidified air. The culture medium consisted of low glucose (5 mM) DMEM supplemented with 0.5 mM oleic acid, 1% BSA, 1 mM L-carnitine, 100 µU/mL penicillin, 100 µU/mL streptomycin, 0.25 µg/mL fungizone, 10% horse serum, and 5% fetal bovine serum (FBS). The oleate was solubilized in 1 mM fatty acid-free albumin. Media were sterilized by filtration through a 0.2 µm filter. Cells were maintained in continuous passage by trypsinization of subconfluent cultures using 0.25% trypsin. Differentiation was induced by switching to medium containing 2% heat-inactivated horse serum when the myoblasts were 
80% confluent. Experimental treatments were started after 7–9 days, by which time nearly all of the myoblasts had fused to form myotubes. At this time, we switched back to the medium containing 10% horse serum and 5% FBS. Treatment of the myotubes with 5 mM caffeine with or without 10 µM dantrolene or 10 µM KN93 was for 5 h/day for 5 days. To remove these agents, myotubes were washed twice with warm PBS.
Western blot
Myotubes were homogenized in 250 mM sucrose containing 10 mM HEPES and 1 mM EDTA, pH 7.4. Homogenate protein concentration was measured using a BCA protein assay kit, and the homogenate volumes were adjusted to give the same protein concentration in homogenates of cells from the different culture dishes. Aliquots of homogenate were solubilized in Laemmli sample buffer, subjected to SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were blocked overnight at 4°C with 5% nonfat dry milk in PBS containing 0.1% Tween. The blots were probed with the following primary antibodies: a rabbit polyclonal antibody against the carboxyl terminus of ALAS; a rabbit polyclonal antibody against the carboxyl terminus of citrate synthase; a monoclonal antibody against cytochrome oxidase subunit I; a monoclonal antibody against cytochrome c; a polyclonal antibody against the carboxyl terminus of PGC-1; or a polyclonal antibody against mtTFA, kindly given to us by Dr. David A. Clayton (Stanford University). The blots were then incubated with the appropriate horseradish peroxidase-conjugated anti-IgG antibody. Antibody-bound protein was detected using enhanced chemiluminescence.
Electrophoretic mobility shift assay
Oligonucleotides A and B containing a functional NRF-1 binding site in the ALA synthase promoter (4)
were synthesized as follows (the recognition sequence for NRF-1 is underlined): Oligo A, 5' G GCC GCT GCG CAT GCG C TGT G 3'; Oligo B, 3-GG CGA CGC GTA CGC GAC ACC C 5'. Oligonucleotides C and D, containing a functional NRF-2 binding site in the cytochrome oxidase subunit IV promoter were synthesized as follows (the recognition sequence is underlined): Oligo C, 5' GATC CGG GAC CC G CTC TTC CGG T CG CGA A 3'; Oligo D, 3' GCC CTG GG C GAG AAG GCC A GC GCT TTC GA 5'. Probes were labeled using Klenow in reaction mixtures containing [-32P]dCTP.
Nuclear extracts were prepared by the method of Dignam et al. (20)
as modified by Towler et al. (21)
. Binding assays were performed for 30 min at 20°C in a total volume of 20 µL containing 50,000 cpm 32P-labeled oligos, 10 µg of acetylated BSA, and 0.01 µg/µL poly-dI:dC. Preliminary experiments were performed to determine the range of input nuclear protein that generated a linear response for the gel shift signal on autoradiographs. Quantitative binding experiments were performed under conditions of probe excess. For supershifting, reactions included 1 µL of undiluted goat anti-NRF-1 antiserum, anti-NRF-2 antiserum (kindly given to us by Richard Scarpulla, Northwestern University), or 1 µL of undiluted control antiserum (rabbit anti-ALA synthase antiserum). To compete away gel-shifted bands, an excess of unlabeled oligonucleotide was included in the reaction mix. After 20 min at room temperature, binding reactions were loaded on pre-run 7% native gels. DNA binding was quantified using imaging densitometry.
Statistics
Values are expressed as means ± SE. Statistically significant differences were determined using unpaired Student’s t tests or ANOVA, as appropriate. When ANOVA showed significant differences, post hoc analysis was performed using Fisher’s least significant differences post hoc test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 22)
and that intermittently treating L6 myotubes with caffeine for 5 days induces significant increases in mitochondrial enzyme proteins and in substrate oxidative capacity (19)
. We also showed that the adaptive response to caffeine is blocked by 10 µM dantrolene (19
, 22)
, which inhibits Ca2+ release from the sarcoplasmic reticulum (19
, 22
, 23)
. In the present study, our first step was to confirm the finding that exposure to caffeine induces an increase in mitochondrial proteins. As before, significant increases in four mitochondrial marker proteins occurred during 5 days of intermittent exposure to caffeine (Fig. 1
). In addition, we examined the response of L6 myotubes to exposure to caffeine for 5 h/day for 2 days. ALA synthase has a short half-life, as evidenced by significant increases in ALAS protein in rat skeletal muscle in response to one bout of exercise (24)
. ALA synthase and COXI, which apparently also has a short half-life, were increased by 30% and 20% respectively, 18 h after the second exposure to caffeine (Fig. 1)
. Cytochrome c and citrate synthase, which have half-lives of 7 days in rat skeletal muscle (25)
were not measurably increased after 2 days of exposure to caffeine but were significantly increased after 5 days. These results confirm our previous finding of significant increase in mitochondrial enzyme proteins in L6 myotubes treated intermittently for 5 days with caffeine to raise cytosolic Ca2+ (19)
.
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Raising cytosolic Ca2+ induces an increase in PGC-1 expression
Overexpression of PGC-1 has been shown to cause large increases in the mitochondrial content of adipocytes and muscle cells in culture (5
, 6)
and in myocardial muscle of transgenic mice (7)
. It therefore seemed possible that an increase in PGC-1 might be involved in the increase in mitochondria induced by increases in cytosolic Ca2+. To investigate this possibility, we determined the effect of 5 h of exposure to caffeine on PGC-1 expression in homogenates of L6 myotubes harvested 16 h after caffeine treatment. As shown in Fig. 2
, there was a significant increase in PGC-1 protein in L6 myotubes exposed to caffeine. This response was examined after the second and fifth exposures to caffeine; as the increases in PGC-1 were similar, they were combined to give the average values shown in Fig. 2
. The increase in PGC-1 was prevented by a low concentration of dantrolene, which blocks Ca2+ release from the sarcoplasmic reticulum (SR) (Fig. 2)
.
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Increases in cytosolic calcium induce increases in NRF-1 and NRF-2
The transcription factors NRF-1 and NRF-2 are involved in regulating expression of nuclear genes encoding some mitochondrial enzymes and factors that regulate mitochondrial DNA transcription and replication (2)
. We evaluated the effect of raising cytosolic calcium on NRF-1 and NRF-2 by electrophoretic mobility shift assay, using an oligonucleotide containing an NRF-1 binding site from the ALA synthase promoter and an oligonucleotide containing the NRF-2 protein binding site from the cytochrome oxidase subunit IV promoter. There was an 90% increase in binding of NRF-1 (Fig. 3
A, C, E) and an 65% increase in binding of NRF-2 (Fig. 3B, D, E
) 16 h after exposure to caffeine. Increases in NRF-1 and in NRF-2 binding were evident 12 h after exposure to caffeine (Fig. 3A, B
). Approximately 50% of the increase in NRF protein binding was lost by 24 h after caffeine exposure (Fig. 3A, B
). Dantrolene prevented the increases in NRF binding (Fig. 3C-E
).
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Mitochondrial transcription factor A expression increases in response to caffeine exposure
Functional NRF-1 and NRF-2 binding sites have been identified in the promoter of the nuclear gene that encodes mtTFA, a transcription factor that regulates mitochondrial DNA transcription and replication (3)
. We evaluated the effect of raising cytosolic Ca2+ on mtTFA expression by means of Western blot of homogenates of L6 myotubes harvested 18 h after a 5 h exposure to caffeine. As shown in Fig. 4
, there was a significant increase in mtTFA protein, which was prevented by the presence of dantrolene during the caffeine exposure. The response of mtTFA to caffeine was measured 18 h after the second or fifth exposure to caffeine; as the increases in mtTFA were similar, the 2 and 5 days data were combined in the average shown in Fig. 4
.
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KN93, an inhibitor of CAMK, blocks the Ca2+-induced increase in mitochondrial proteins
As a preliminary step in the investigation of the pathway by which increases in cytosolic Ca2+ stimulate mitochondrial biogenesis, we used KN93, an inhibitor of CAMKs (26)
, to evaluate the possibility that the first step in the Ca2+ signaling pathway involves activation of a CAMK. Inclusion of 10 µM KN93 with caffeine in the culture medium completely blocked the Ca2+-induced increases in some mitochondrial marker proteins (Fig. 5
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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18
19)
. A previous study demonstrated that intermittently raising cytosolic Ca2+ in L6 myotubes by exposing them to caffeine or ionomycin induces increases in mitochondrial proteins and functional mitochondria as evidenced by an enhanced capacity for substrate oxidation (19)
. The present results show that this adaptation involves induction of PGC-1, mtTFA, NRF-1, and NRF-2 by Ca2+. This response induced by raising cytosolic Ca2+ mimics that induced in skeletal muscle by endurance exercise, which also involves increases in PGC-1, mtTFA, NRF-1, NRF-2, and mitochondria (9
, 13
14
15
16
, 35)
. Caffeine causes skeletal muscle to contract by releasing Ca2+ from the SR (36
, 37)
. Unlike skeletal muscle, L6 myotubes do not contract in response to increases in cytosolic Ca2+. As a result, raising cytosolic Ca2+ in L6 myotubes does not cause a decrease in high-energy phosphates (P) and the associated other potential signals generated by muscle contractions (19)
. It therefore seems probable that increases in cytosolic Ca2+ per se provide the signal that initiates the events leading to stimulation of mitochondrial biogenesis in L6 myotubes induced by caffeine and other agents that raise cytosolic Ca2+. This interpretation is supported by the finding that dantrolene, which blocks the increase in Ca2+ induced by caffeine in L6 myotubes (19
, 22)
, prevents this increase in mitochondrial proteins (19)
, PGC-1, NRF-1, NRF-2, and mtTFA (Figs. 2
3
4)
.
It also seems probable, in light of these findings, that the increase in cytosolic Ca2+ in contracting muscle is one of the signals that mediate the stimulation of mitochondrial biogenesis in skeletal muscle. We say "one of the signals," because there is evidence that activation of AMP-activated protein kinase (AMPK) also leads to increased mitochondrial biogenesis in both skeletal muscle and L6 myotubes (19
, 27)
. It is interesting that a low concentration of the CAMK inhibitor, KN93, completely blocks the stimulation of mitochondrial biogenesis (Fig. 5)
induced by increases in cytosolic Ca2+. AMPK and the CAMKs are closely related enzymes that belong to the same protein kinase subfamily and recognize the same amino acid consensus sequence (38)
. Thus, it seems possible that either CAMK or AMPK may activate the next step in the enzymatic pathway leading to the stimulation of mitochondrial biogenesis. In support of this possibility, Wu et al. (39)
reported that expression of a constitutively active form of CAMK IV in skeletal muscle in transgenic mice resulted in an increase in mitochondria.
It is now our working hypothesis that the adaptive increase in muscle mitochondria induced by exercise is mediated by both the increase in cytosolic Ca2+, possibly by activating CAMK, and the decrease in high energy phosphates (P), resulting in activation of AMPK. If this hypothesis is correct, it seems reasonable that during the prolonged steady-state exercise that results in modest perturbations in P concentrations and minimal activation of AMPK, the primary adaptive stimulus is the increase in cytosolic Ca2+. On the other hand, intense training that involves periods of very strenuous exercise such as are used in interval training would, by activating both CAMK and AMPK, result in a more powerful stimulus to mitochondrial biogenesis. This would explain the greater effectiveness of high intensity interval training compared with steady-state, moderate intensity exercise in inducing the adaptive response (40)
. Although it has not been conclusively proven that increases in cytosolic Ca2+ and decreases in P are responsible for the stimulation of mitochondrial biogenesis in skeletal muscle, there is now considerable evidence supporting this hypothesis (17
18
19
, 22
, 27
28
29
30
31
32
33
34)
. In view of the importance of rapid adaptive increases in skeletal muscle mitochondria for survival under conditions requiring prolonged vigorous exercise, it is not surprising that redundant mechanisms evolved for stimulating mitochondrial biogenesis and GLUT4 expression.
There is strong evidence that PGC-1 is the key regulator of mitochondrial biogenesis and GLUT4 expression. It appears to be responsible for mediating the responses to adaptive stimuli that induce increases in the capacity for glucose transport and generation of ATP via respiration (5
6
7
, 41
, 42)
. PGC-1 induces mitochondrial biogenesis by interacting with and activating transcription factors that regulate expression of mitochondrial proteins, including NRF-1 and PPAR, as well by inducing increased expression of NRF-1 and NRF-2 (6
, 41
, 42)
.
Although it appears to be established that PGC-1 is responsible for stimulating/coordinating mitochondrial biogenesis, this effect of PGC-1 can be enhanced by either activation of existing PGC-1 or increased expression of PGC-1 (5
6
7
, 43)
. It is not clear whether the increase in PGC-1 protein induced in skeletal muscle by exercise (15)
and in L6 myotubes by increases in cytosolic Ca2+ (Fig. 2)
is responsible for the initial activation of mitochondrial biogenesis. The alternative possibility is that stimulation of these processes is initiated by activation of PGC-1 and that the subsequent increase in PGC-1 expression sustains the adaptive response. Thus the increase in PGC-1 protein could either initiate or be a component of the adaptation. In the next phase of this research, we are therefore addressing the question: does PGC-1 expression increase sufficiently rapidly to account for the stimulation of mitochondrial biogenesis? To this end, we are comparing the time course of the increase in PGC-1 protein with the time course of the increases in the NRFs, and mtTFA in rat skeletal muscle in response to a bout of exercise.
In conclusion, it appears from our present and previous results that L6 myotubes are a suitable model for investigating the mechanisms by which increases in cytosolic Ca2+ and/or activation of AMPK induce mitochondrial biogenesis and GLUT4 expression. The results of this study show that increases in PGC-1, NRF-1, NRF-2, and mtTFA are involved in the adaptive response to increases in cytosolic Ca2+ that leads to an increase in mitochondria in L6 myotubes. This response mimics that seen in skeletal muscle in response to a bout of exercise. These findings support our hypothesis that increases in cytosolic Ca2+ provide one of the signals that mediate the exercise-induced increase in skeletal muscle mitochondria.
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ACKNOWLEDGMENTS |
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Received for publication October 14, 2002. Accepted for publication December 23, 2002.
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