Contraction of skeletal muscle generates pressure stimuli to intramuscular tissues. However, the effects of pressure stimuli, other than those created by electricity or nerve impulse, on physiological and biochemical responses in skeletal muscles are unknown. The purpose of this study is to examine the effects of a pure pressure stimulus on metabolic responses in a skeletal muscle cell line. Atmospheric pressure was applied to L6 myoblasts using an original apparatus. Succinate dehydrogenase (SDH) activity was evaluated by colorimetric assay using tetrazolium monosodium salt. The amounts of 2-deoxy-[3H]glucose uptake and lactate release were measured. SDH activity was 2.6- to 2.9-fold higher in pressurized L6 cells than in nonpressurized L6 cells (P < 0.01), and 2-deoxy-[3H]glucose uptake was 2.2-fold higher (P < 0.001). In addition, the amount of released lactate decreased from 6.8 to 3.7 μmol/dish when pressure was applied (P < 0.001). In contrast, the intracellular lactate contents of the pressurized cells were higher than those of nonpressurized cells (P < 0.01). However, the total amount of released lactate and intracellular lactate was lower in the pressurized cells than in nonpressurized cells. These findings demonstrate that a pure pressure stimulus enhances aerobic metabolism in L6 skeletal muscle cells and raise the possibility that elevated intramuscular pressure during muscle activity may be an important factor in stimulating oxidative metabolic responses in skeletal muscles.
- mechanical pressure
- aerobic metabolism
- glucose uptake
when a muscle contracts, it not only receives electrical stimulation but is also subjected to mechanical forces. The mechanical forces to skeletal muscles are classified into two components: stretching and intramuscular pressure. The former stimulus is developed during elongation of muscle length by external-strain force. On the other hand, intramuscular pressure is generated by contraction (i.e., force production) of the pressure-applied muscle itself, independent of alterations in muscle length (1). The effects of stretching stimuli on metabolism, intracellular signal events, and gene expression in skeletal muscle tissues or cells have been shown (3, 6, 9, 15, 16). In those studies, passive stretching (i.e., unrelated to innervation) caused glucose uptake in isolated muscles and cultured muscle cells (6, 9, 16). On the basis of those reports, it appears that skeletal muscle is sensitive to mechanical forces independent of electrical stimuli and humoral factors.
With regard to intramuscular pressure, Ballard et al. (1) demonstrated that the intramuscular pressures at concentric contraction during walking and running were ∼180 and 270 mmHg, respectively. However, very little is known about the effects of a pressure stimulus as a component of mechanical forces during contraction on the physiological and biochemical responses of skeletal muscles. On the basis of the intensity of the intramuscular pressure reported by Ballard et al. and the fact that a pressure stimulus is produced in more active phase during skeletal muscle activity than stretch stimuli, a pressure stimulus during muscle contraction might have some effects on metabolic or on adaptive responses to the activity of skeletal muscles.
The purpose of the present study was to examine whether applying artificial pressure affected aerobic metabolism in skeletal muscle cells. To load homogeneous pressure efficiently in the absence of cell distortions induced by electricity or stretch stimuli, we used atmospheric pressure and cultured myocytes in the present study.
L6 myoblast cells were provided by the Cell Bank of the Japanese Collection of Research Bioresources. 2-Deoxy-d-[3H]glucose (2-[3H]DG) was from Amersham Pharmacia Biotech (Buckinghamshire, UK). 2-Deoxy-d-glucose, lactate, succinate, 1-methoxy-5-methylphenazinium methylsulfate (mPMS), and 2-(4-iodophenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) were purchased from Wako Pure Chemical (Osaka, Japan). Phloridzin and penicillin-streptomycin solutions were obtained from ICN Biomedicals (Aurora, OH). Gadolinium (III) chloride hexahydrate and cycloheximide were purchased from Sigma (St. Louis, MO). All other chemicals were purchased from Sigma unless otherwise noted.
Rat L6 myoblasts were cultured in 10-cm dishes with high-glucose DMEM (4,500 mg glucose/l) containing 5% FBS and 1% penicillin-streptomycin solution. The cells were incubated at 37°C in a humidified 5% CO2 atmosphere. Beginning 3 days before the experiments, L6 cells were cultured in 12-well plates, except for the measurements of lactate contents and the immunoblot analysis. On the day before each experiment, the cells were rendered quiescent in normal-glucose DMEM (1,000 mg glucose/l) containing 0.1% FBS for at least 24 h.
During culture, L6 cells differentiate into multinucleated fibers (i.e., myotubes) that become cross-striated and express sensitivity to both insulin and passive stretching (13, 16, 21, 27). We knew, however, that the cells would not become well-differentiated myotubes during the culture period between seeding and pressure loading. Therefore, we used L6 cells in the myoblastic state, in which they were insensitive to insulin and passive stretching (16).
An original pressure loading apparatus based on previous reported systems (7, 29) was used, as described in detail previously (10, 17). The plates were set up in the chamber of the apparatus in fresh normal-glucose DMEM containing 20 mM HEPES (pH 7.4) with 10 mM lactate. To raise internal atmospheric pressure of the chamber, room air was pumped in until the pressure reached 160 mmHg. The L6 cells were maintained at this pressure for 3 h at 37°C. The control cells were incubated with the same medium under normal pressure for 3 h at 37°C. The intensity of the pressure was based on the value of intramuscular pressure during walking reported by Ballard et al. (1). In our previous report (17), we verified that the pH of the medium under both conditions was similar (pH 7.41 ± 0.02). Although it was not possible to monitor the actual morphology of pressurized cells in the chamber, we did not find any changes in cell size or morphology after pressurization in light microscopic investigations. In our pilot study, the L6 cells released lactate constantly to culture medium, and, after incubation for 24 h in our standard conditions, the lactate concentration reached ∼20 mM in a 10-cm dish. Because lactate was decreased after pressure treatment, 10 mM lactate was added to the medium during the experiments.
Succinate dehydrogenase activity.
To evaluate aerobic metabolic activity, we measured succinate dehydrogenase (SDH) activity, since enzyme activity is considered an indicator of tricarboxylic acid (TCA) cycle activity. To measure SDH activity, we used the methods described by Levine et al. (14) and Yang et al. (28), with slight modifications. In brief, SDH activity was evaluated by a colorimetric assay based on redox reaction with the use of a highly water-soluble formazan former, WST-1, instead of nitro blue tetrazolium, which was used by Levine et al. and Yang et al. Immediately after incubation for 3 h in the presence or absence of pressure stimulation, sodium azide was added to each well (final concentration 10 mM) to inhibit the electron transfer system, which is a main site of redox reactions, and then incubated for 5 min. The medium was replaced by normal-glucose DMEM containing 100 μM mPMS, 500 μM WST-1, 20 mM HEPES, 10 mM NaN3, and 93 mM succinate. After the cells were incubated for 20 min at 37°C, the degree of color of the medium was measured at a wavelength of 450 nm. Nonspecific coloring was determined in parallel samples in the absence of succinate and was subtracted from all measurements reported.
To investigate whether substrates were utilized, we first measured 2-[3H]DG uptake into L6 cells. After treatments with pressure stimulation or normal pressure incubation, the cells were washed three times with HEPES-buffered Krebs-Ringer solution [123.2 mM NaCl, 4.98 mM KCl, 1 mM CaCl2, 2.5 mM MgSO4, and 22 mM HEPES (pH 7.4)] with 0.1% bovine serum albumin (buffer A). The 10 μM 2-[3H]DG (0.5 μCi/ml) uptake was measured for 10 min under linear uptake conditions. To terminate the transport reaction, phloridzin and HgCl2 were added to each well (final concentration 0.2 and 1 mM, respectively). Nonspecific uptake was determined in the presence of 0.2 mM phloridzin and 1 mM HgCl2 and was subtracted from the total uptake. Then, the radioactive incubation medium was rapidly aspirated off, and the cells were rinsed three times with buffer A. After the cells were disrupted with 0.5% SDS solution, radioactivity inside the cells was measured by liquid scintillation counting. All experiments were performed at least three times, and, within each experiment, triplicate determination was used.
Lactate release and intracellular lactate concentration.
Concentrations of lactate, an anaerobic metabolic end product, were measured to determine whether the pressure stimulus had caused any changes in metabolic products. Using a lactate analyzer (YSI 1500 SPORT Lactate Analyzer; Yellow Springs Instruments, Yellow Springs, OH), we measured the lactate contents in fresh medium and then in the medium after pressure loading; the difference was the lactate release. To evaluate intracellular lactate concentration, the cells treated with pressure stimulation or with normal pressure incubation were harvested in a constant amount (500 μl) of medium. All samples were sonicated and then boiled for 3 min to inactivate enzymes. The lactate content in a sample aliquot was measured, and the value was corrected according to the original amount of the medium in the well.
Total RNA was extracted from L6 cells after treatment with pressure stimulation or normal pressure incubation by use of an RNeasy Protect Mini Kit (Qiagen, Valencia, CA), following the procedures recommended by the manufacturer. A Perkin-Elmer DNA thermal cycler (Norwalk, CT) was utilized for the assay with a OneStep RT-PCR kit (Qiagen). Total RNA (8 ng) was reverse transcribed and PCR amplified in a 50-μl volume with the use of an equal amount of the RNA sample, 5× Qiagen OneStep RT-PCR buffer, each primer at 2 μM, each dNTP at 400 μM, and 2 μl of Qiagen OneStep RT-PCR Enzyme Mix in the thermal cycler. Samples were heated for 30 min at 50°C for reverse transcription and further heated for 15 min at 95°C for activation of HotStarTaq DNA polymerase and inactivation of reverse transcriptases, and then the following conditions were applied: 35 cycles of 45-s denaturation at 94°C, 50-s annealing at 55°C, and 1-min extension at 72°C. Samples were then kept for 10 min at 72°C and cooled at 4°C. The following oligonucleotide primers were used: inducible nitrate oxide synthase (iNOS; Ref. 5) forward (sense), 5′-TGG-ATC-AAT-AAC-CTG-AAG-CCC-GAA-3′, and reverse (antisense), 5′-TTG-CCC-TTT-TTC-ACT-CCA-TA-3′; and α-skeletal actin (22) forward (sense), 5′-CGT-CAC-CAG-GGT-GTC-ATG-G-3′, and reverse (antisense), 5′-TGT-AGA-AGG-TGT-GGT-GCC-AGA-T-3′. Amplified samples (15 μl) were electrophoresed on 1.5% agarose gel in Tris-acetate-EDTA buffer containing 10 μg of ethidium bromide. As a positive control, we used lipopolysaccharide (LPS)-treated L6 cells, which have been reported to express iNOS mRNA (2).
After treatment with pressure stimulation or normal pressure incubation, the cells were washed twice with ice-cold PBS and harvested in PBS containing protease inhibitors (100 μM benzamidine, 2 μM leupeptin, 0.15 μM aprotinin, 1.5 μM pepstatin A, and 100 μM phenylmethylsulfonyl fluoride), and 1% Phosphatase Inhibitor Cocktail I and II (Sigma). The cells were subjected to three freeze-thaw cycles at −150°C. Samples were then centrifuged for 20 min at 10,000 g to obtain cell lysates. The protein content of each sample was determined using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). Twenty-five micrograms of protein per lane were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). Membranes were blocked for 1 h at room temperature with 4% nonfat dry milk in PBS containing 0.2% Tween 20. Hypoxia-inducible factor-1α (HIF-1α) was detected by use of rabbit anti-HIF-1α (Santa Cruz Biotechnology, Santa Cruz, CA) as the primary antibody and peroxidase-conjugated donkey anti-rabbit IgG (Amersham) as the secondary antibody, in concentrations recommended by the manufacturer. Antibody-bound protein was detected by chemiluminescence detection system (Amersham) and light emission exposed to X-ray film (model RX-U; Fujifilm, Kanagawa, Japan) for 3 min to 2 h. To verify the reactivity of the anti-HIF-1α antibody, a fusion protein corresponding to amino acids 575–780 of HIF-1α (Santa Cruz Biotechnology) was applied at the same time.
All results are expressed as means ± SD. Statistical significance was tested with the Student's two-tailed t-test or one-way ANOVA; in the latter case, the test was combined with Scheffé's test for post hoc analysis. Values of P < 0.05 were considered statistically significant.
Pressure stimulus enhanced SDH activity and glucose uptake.
In nonpressurized L6 cells, only subtle color differences were apparent between presence and absence of succinate. In contrast, the pressurized cells showed significantly larger differences in the degree of coloring (2.6-fold increase; Fig. 1). As shown in Fig. 2, the amount of 2-[3H]DG was greater in pressurized than in nonpressurized L6 cells (2.2-fold increase). These findings suggest that the pressure stimulus enhanced SDH activity and resulted in enhancement of glucose uptake in L6 skeletal muscle cells.
Effects of Gd3+ and cycloheximide on pressure-induced SDH activation.
We tested whether stretching stimuli were involved in pressure-induced SDH activation. It has been reported that stretch-activated channels are critically involved in stretch-induced cellular responses and that Gd3+ is a potent blocker of stretch-activated channels (11). When pressure was applied to the cells in the presence of 30 μM Gd3+, SDH activity was enhanced to a similar degree as when pressure was given alone (pressurized cells, 2.9-fold increase; Gd3+ treatment with pressure, 3.1-fold increase; Fig. 3A). This suggested that stretch stimuli were not involved in the pressure-induced SDH activation.
To examine the effects of protein synthesis on SDH activation by pressure stimulus, we determined whether cycloheximide, a protein synthesis inhibitor, suppressed the pressure-induced SDH activation. At the concentration of 20 μg/ml, used by Hatfaludy et al. (6), absorption indicating SDH activity was reduced under both the nonpressurized and the pressurized condition compared with that in untreated controls (data not shown). Because this concentration of cycloheximide may cause a degree of damage to the cells, we used the inhibitor at a concentration of 10 μg/ml in the medium. As shown in Fig. 3B, although pressure stimulus significantly enhanced SDH activity in the presence of cycloheximide (1.6-fold increase compared with that in nonpressurized controls), the degree of the pressure-induced activation of SDH was reduced when compared with the pressurized cells in the absence of cycloheximide. These results suggested that protein synthesis was at least partly involved in the pressure-induced SDH activation.
Pressure reduced lactate release.
Under nonpressure conditions, 6.8 ± 0.7 μmol/dish lactate were released, whereas the amount of released lactate in the pressure-applied cells was 3.7 ± 0.5 μmol/dish (Fig. 4A). On this point, we investigated whether enhanced lactate utilization caused the reduction of lactate in the pressure-applied cells. The contents of intracellular lactate in nonpressurized cells and pressurized cells were 0.23 and 0.32 μmol/dish, respectively (Fig. 4B). Although significantly more intracellular lactate was found in the pressurized cells, the amounts and difference in intracellular lactate between pressurized and nonpressurized cells were an order of magnitude smaller than that of released lactate. Consequently, the total amount of released lactate and intracellular lactate was significantly lower in the pressurized cells than in the nonpressurized cells (P < 0.001).
Effects of oxygen content on L6 cells.
We tested whether both pressurized and nonpressurized cells were exposed to hypoxic, normoxic, or hyperoxic conditions, respectively. It has been reported that iNOS and HIF-1α are detected under hypoxic conditions (12, 18, 19, 26). As shown in Fig. 5A, iNOS mRNA of L6 cells, in contrast to that of LPS-treated cells, was weakly exhibited in both pressurized and nonpressurized conditions. In immunoblot analysis, HIF-1α protein was hardly detected in either group (Fig. 5B).
Intramuscular pressure is elevated in the contraction phase of active muscles during physical activity. However, the effects of elevated intramuscular pressure per se on the physiological and biochemical responses of skeletal muscles are unknown. In this study, we have attempted to approach these questions by investigating the effects of an artificial pressure stimulus on metabolic responses in cultured skeletal muscle cells. The present study demonstrates for the first time that a pressure stimulus activated SDH activity and glucose uptake and reduced lactate release in L6 myoblasts. It is thought that enhancement of SDH, which is an indicator of TCA cycle activity, increases the capacity to degrade substrates (e.g., lactate and glucose). Thus the pressurization of L6 cells stimulated lactate utilization and glucose uptake. These findings suggest that mechanical pressure enhanced aerobic metabolism in skeletal muscle cells and may provide valuable clues toward elucidating the nervous system-independent mechanism(s) for metabolic activation and/or adaptation by skeletal muscle contraction.
The mechanisms by which exercise and/or contractile activity stimulates skeletal muscle metabolism are of great interest and importance. Until now, adaptive and metabolic responses to contractile activity of skeletal muscle have been investigated only by electrical stimulation and stretch stimuli in ex vivo studies. The effects of pressure stimuli, which are produced by muscle contraction, on adaptation and metabolic activity of skeletal muscle are not yet well recognized. In the present study, we examined the contribution of stretch stimuli in pressure-activated aerobic metabolism by treatment with a stretch-activated channel inhibitor, Gd3+. Although Caldwell et al. (4) demonstrated a possibility that treatment with Gd3+ to identify the involvement of stretch-activated channels could lead to false-negative conclusions, the present experiments did not use the phosphate, carbonate, and sulfate buffers that were shown to react with Gd3+ in the study (4). In addition, a decrease of lactate release induced by pressure stimulus in the present study was opposite to the result of stretch-applied cells reported by Hatfaludy et al. (6). Thus we could exclude electricity and stretching as factors in demonstrating the enhancing effects of a pressure stimulus on aerobic metabolism of L6 skeletal muscle cells. However, we do not yet understand the mechanism(s) underlying the pressure-activated aerobic metabolism in L6 cells.
On the basis of our results and the fact that both SDH activity and TCA cycle activity are regulated mainly by the ADP-to-ATP ratio, we could consider the following explanations as possibly responsible for pressure-activated metabolism. The activation of SDH was at least partially attributable to enhancement of protein synthesis, because cycloheximide partly inhibited the pressure-activated metabolic shift. Protein synthesis is a metabolically costly process requiring a large amount of ATP, possibly driving an increase in SDH activity. The suppression of the pressure-induced SDH activation by cycloheximide could be the result of an inhibition of the synthesis of enzymes of mitochondrial metabolism and/or an inhibition of general protein synthesis. Also, L6 cells can differentiate into myofibers from myotubes and myoblasts (27). This process requires many proteins as well as ATP. Therefore, the pressure stimulus may accelerate protein synthesis, cell growth, and/or the process of differentiation, resulting in an increase in aerobic metabolism. As for the protein synthesis-independent effects of the pressure on aerobic metabolism, one possible explanation is an increase in active ion transport. Transport of ions (e.g., Ca2+ uptake into sarcoplasmic reticulum and Ca2+ influx) is carried out by consumption of ATP. Thus there is a possibility that enhancement of ion shuttling induced by pressure stimulus promoted some degree of the activation of the aerobic metabolism. Although further studies are needed to elucidate the functional implications and the mechanism(s) of pressure-activated aerobic metabolism, the results of the present study may provide a new perspective in the study of the mechanisms by which muscle contraction induces metabolic activation and/or adaptations.
Another interesting finding of the present study was that glucose and lactate may be used as a substrate in the pressure-activated aerobic metabolism of L6 cells. For a long time, lactate was thought to be a metabolic end product of glycolysis. Recently, however, lactate was shown to be incorporated into skeletal muscles and was utilized during skeletal muscle contraction and/or exercise (23–25). In the present study, we found that the pressure stimulus reduced lactate release. Considering that the pressure stimulus enhanced the aerobic metabolic capacity and glucose uptake, the reduction of the release of lactate from the L6 cells raises the following two possibilities: a decrease in lactate production and enhancement of lactate utilization. The first possibility (i.e., less conversion of pyruvate to lactate) is consistent with enhanced aerobic metabolism. We observed, however, small but significant increases of intracellular lactate in the pressurized cells in the presence of enhanced glucose uptake and aerobic metabolism (i.e., no decrease in lactate production). Thus we considered that the latter possibility was largely responsible for the reduction in released lactate in the cells.
During pressure loading experiments, increased oxygen partial pressure (Po2) may occur in the culture medium because of the use of room air (theoretically 21% O2; normobaric condition, 150 mmHg Po2; hyperbaric condition, 180 mmHg Po2). It may be possible for increased Po2 to affect metabolic responses in insufficient Po2 conditions. However, our pressure loading experiments were performed in an ambient Po2 condition, which is much higher than tissue Po2 (10–20 mmHg Po2; Ref. 8). In fact, HIF-1α and iNOS, which are induced by hypoxia (12, 18, 19, 26), were not expressed or altered under our pressurized and nonpressurized conditions, indicating that the cells were not exposed to an insufficient Po2 condition. Additionally, with respect to effects of increasing Po2, an in vitro study showed that oxygen concentration had no influence on enzyme activity and metabolite production involved in TCA cycle turnover and glycolysis (20). Taken together, these previous findings and our results support our view that atmospheric pressure-increased Po2 did not affect the activation of aerobic metabolism by the pressure stimulus.
In conclusion, we indicate for the first time that a pressure stimulus induced the activation of SDH and increased glucose transport and lactate utilization in L6 myoblasts, and we suggest that elevated intramuscular pressure during muscle contraction may be an important stimuli inducing metabolic adaptive responses in skeletal muscles.
This research was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Health, Labor and Welfare of Japan (no. H14-trans-013).
We thank Fuyuko Kanda for technical assistance.
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- Copyright © 2004 by American Physiological Society