1 Department of Medicine, Washington University School of Medicine, St. Louis 63110; and 2 Departments of Physiology and Internal Medicine and Diabetes and Cardiovascular Biology Program, School of Medicine, University of Missouri, Columbia, Missouri 65212
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ABSTRACT |
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Muscle contractions cause numerous disturbances in intracellular homeostasis. This makes it impossible to use contracting muscle to identify which of the many signals generated by contractions are responsible for stimulating mitochondrial biogenesis. One purpose of this study was to evaluate the usefulness of L6 myotubes, which do not contract, for studying mitochondrial biogenesis. A second purpose was to evaluate further the possibility that increases in cytosolic Ca2+ can stimulate mitochondrial biogenesis. Continuous exposure to 1 µM ionomycin, a Ca2+ ionophore, for 5 days induced an increase in mitochondrial enzymes but also caused a loss of myotubes, as reflected in an ~40% decrease in protein per dish. However, intermittent (5 h/day) exposure to ionomycin, or to caffeine or W7, which release Ca2+ from the sarcoplasmic reticulum, did not cause a decrease in protein per dish. Raising cytosolic Ca2+ intermittently with these agents induced significant increases in mitochondrial enzymes. EGTA blocked most of this effect of ionomycin, whereas dantrolene, which blocks Ca2+ release from the sarcoplasmic reticulum, largely prevented the increases in mitochondrial enzymes induced by W7 and caffeine. These findings provide evidence that intermittently raising cytosolic Ca2+ stimulates mitochondrial biogenesis in muscle cells.
caffeine; exercise; gene expression; ionomycin; L6 myotubes
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INTRODUCTION |
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THIRTY-FIVE YEARS HAVE ELAPSED since the discovery that endurance exercise induces an increase in muscle respiratory capacity, mediated by an increase in mitochondrial enzymes (11). Although progress has been made in explaining how the increase in muscle mitochondria contributes to enhancement of exercise capacity and endurance, relatively little is known regarding the mechanisms responsible for this adaptation (2, 4, 5). One of the reasons for this slow progress is that muscle contractions cause numerous disturbances in intracellular homeostasis. This makes it impossible to use contracting muscle for elucidating which of the many signals generated by exercise is/are responsible for stimulation of mitochondrial biogenesis. It is, therefore, necessary to use another model in which it is possible to study these potential signaling mechanisms individually. In this context, one purpose of this study was to evaluate the usefulness of L6 myotubes in culture for studying the regulation of mitochondrial biogenesis.
One of the signals by which contractile activity stimulates mitochondrial biogenesis could be the increase in cytosolic Ca2+ that is triggered by each wave of sarcolemmal depolarization. In support of this possibility, Lawrence and Salsgiver (16) reported that exposure of primary cultures of fetal rat muscle cells to 3 µM A-23187, a Ca2+ ionophore, induced increases in mitochondrial enzymes. Similarly, Freyssenet et al. (6) found that continuous exposure of L6E9 myoblasts to A-23187 induced an increase in cytochrome c mRNA. In a preliminary experiment in our laboratory, using primary cultures of fetal rat skeletal muscle, we confirmed that continuous exposure to 3 µM A-23187 induces an increase in mitochondrial enzymes; however, we also found that continuous exposure to 3 µM A-23187 for 4 days has a deleterious effect on cell viability, with a decrease in total protein content, a decrease in number of cells, and atrophy of some cells (E. A. Gulve and J. O. Holloszy, unpublished findings). Enzyme activities vary markedly between individual myotubes in primary cultures of rat skeletal muscle (21). So, this finding of a decrease in myotubes raised the possibility that the sustained increase in cytosolic Ca2+ may have selectively killed myotubes with a low mitochondrial content rather than stimulated mitochondrial biogenesis. Therefore, a second aim of this study was to evaluate further the possibility that intermittent increases in cytosolic Ca2+ can stimulate mitochondrial biogenesis in muscle cells. L6 myotubes do not contract in response to an increase in cytosolic Ca2+, thus avoiding the generation of other potential signals induced by breakdown of high-energy phosphates (~P), increases in glycolytic intermediates, changes in redox state and pH, and so forth, in contracting muscle.
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MATERIALS AND METHODS |
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Materials.
Reagents for SDS-PAGE were purchased from Bio-Rad. Reagents for
enhanced chemiluminescence (ECL) were obtained from Amersham Pharmacia
Biotech. L6 myocytes were purchased from American Type Culture
Collection (ATCC, Manassas, VA). [1-14C]oleic acid was
from American Radiolabeled Chemicals (St. Louis, MO). Rabbit polyclonal
antibodies (directed) against the 19 COOH-terminal amino acids of
-aminolevulinate synthase (ALAS) and the 20 COOH-terminal amino
acids of citrate synthase were generated by Alpha Diagnostic International (San Antonio, TX). A mouse anti-human cytochrome oxidase
(COX) subunit I monoclonal antibody was purchased from Molecular Probes
(Eugene, OR). A mouse anti-cytochrome c monoclonal antibody
was purchased from Pharmingen International (San Diego, CA). A rabbit
polyclonal phospho-AMP kinase-
antibody (Thr172) was
obtained from Cell Signaling Technology. Horseradish
peroxidase-conjugated secondary antibodies were from the Jackson
Laboratory.
5-Aminoimidazole-4-carboxamide-
-D-ribofuranoside (AICAR)
was obtained from Toronto Research Chemicals (North York, Ontario,
Canada). All other reagents were purchased from Sigma.
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, 5% horse serum, and 10% 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 with the use of 0.25% trypsin. Differentiation was induced by switching to medium containing 2% heat-inactivated horse serum when the myoblasts were ~80% confluent. The 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 5% horse serum and 10% FBS. Treatment of the myotubes with 5 mM caffeine, 1 µM ionomycin, or 50 µM W7 with or without 10 µM dantrolene or 1 mM EGTA was for 5 h/day for 5 days. To remove these agents, the myotubes were washed twice with PBS.
Measurement of cytosolic Ca2+ levels. Cytosolic Ca2+ was determined with the use of fura-2 epifluorescence digital microscopy as described previously (10, 23).
Western blotting.
Myotubes were homogenized in 250 mM sucrose containing 10 mM HEPES and
1 mM EDTA, pH 7.4. Homogenate protein concentration was measured, 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 COOH-terminus of ALAS, a rabbit polyclonal antibody against
the COOH-terminus of citrate synthase, a monoclonal antibody against
COX-I, a monoclonal antibody against cytochrome c, or a
rabbit polyclonal antibody against a phosphopeptide corresponding to
residues surrounding Thr172 of AMP kinase-. The blots
were then incubated with the appropriate horseradish
peroxidase-conjugated anti-IgG antibody. Antibody-bound protein was
detected using ECL.
Oleate oxidation. Myotubes were scraped from the culture plates and homogenized in 175 mM KCl containing 0.1 mM EDTA, using a glass Potter-Elvehjem homogenizer immersed in ice water. The capacity of whole homogenates of myotubes to oxidize [14C]oleate was assessed by measuring the rate of 14CO2 production as described previously (1, 20). The reaction mixture, contained in a final volume of 2 ml, consisted of homogenate equivalent to 2 mg protein, 5 mM MgCl2, 87.5 mM KCl, 40 mM potassium phosphate buffer, 2 mM EDTA, 2 mM ADP, 10 mM Tris · HCl, 0.078 mM cytochrome c, 0.15 mM fatty acid-free albumin, and 0.75 mM oleate containing 0.25 µCi [1-14C]oleate (per flask). The oleate was solubilized in 1 mM fatty acid-free albumin. The reaction mixtures were placed in flasks fitted with serum caps and hanging center wells containing 0.4 ml hyamine hydrozide. The flasks were incubated in Dubnoff shaking incubators. The 14CO2 produced was trapped, and radioactivity was measured using a scintillation counter. Results are expressed per milligram protein.
ATP measurement.
Cells were scraped from plates and homogenized in 0.6 M perchloric acid
(PCA) at 10°C. PCA was extracted from the homogenates by
use of 1:4 (vol/vol) trioctylamine:Freon. ATP concentration was
determined with the use of high-pressure liquid chromatography as
described by Scott et al. (27).
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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AICAR induces increases in ALAS and cytochrome c in L6 myotubes.
AICAR is converted to the AMP analog
5-aminoimidazole-4-carboxamide-1--D-ribofuranosyl
5'-monophosphate (ZMP) after it is transported into cells and
thus results in activation of AMP kinase (AMPK). It has been shown that
administration of AICAR to rats induces an increase in mitochondrial
enzymes in skeletal muscle (32). To validate L6 myocytes
as an appropriate model for studying mitochondrial biogenesis, we
therefore examined the effect of AICAR. As shown in Fig.
1, exposure of myotubes to 1 mM
AICAR for 5 days resulted in significant increases in ALAS and
cytochrome c, which were used as mitochondrial marker
proteins. Exposure of L6 myotubes to AICAR also resulted in a
significant increase in AMPK phosphorylation (Fig.
2), providing evidence for activation of
AMPK kinase and AMPK.
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Continuous vs. intermittent exposure of L6 myotubes to ionomycin.
We used ionomycin instead of A-23187, because it is a more effective
Ca2+ ionophore (18). Continuous exposure to 1 µM ionomycin for 5 days induced large increases in the protein
concentrations of COX-I and ALAS, which were used as mitochondrial
marker enzymes (Fig. 3). On microscopic
examination, there were fewer myotubes in the culture dishes treated
with ionomycin than in the untreated controls. This was reflected in a
significant decrease in total protein per dish: 3.5 ± 0.3 mg/plate for ionomycin treatment vs. 5.8 ± 0.4 mg/plate for
control, P < 0.05. This finding, which confirmed our
pilot study findings in primary muscle cell cultures, led us to
investigate the effect of intermittent exposure to ionomycin. Exposure
of myotubes to 1 µM ionomycin for 5 h/day for 5 days resulted in
slightly smaller, but still highly significant increases in COX-I and
ALAS (Fig. 3) but no significant decrease in protein: 5.8 ± 0.4 mg/plate for controls and 5.5 ± 0.4 mg/plate for myotubes treated
for 5 h with ionomycin.
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Effects of ionomycin, W7, and caffeine on cytosolic
Ca2+ concentrations.
Before conducting further studies of the effect of raising cytosolic
Ca2+, we measured the effects of ionomycin, which acts as a
Ca2+ ionophore and facilitates Ca2+ entry from
the medium (18), and of two agents, W7 and caffeine, that
release Ca2+ from the sarcoplasmic reticulum (SR)
(25) on cytosolic Ca2+ concentration. As for
ionomycin, the intermittent exposure of myotubes to caffeine or W7 did
not result in a decrease in protein. As shown in Fig.
4, all three agents caused increases in
cytosolic Ca2+ in L6 myotubes. [Fig. 4C,
showing the effect of caffeine, has been published previously
(23) and is included here for comparison with W7 and
ionomycin.]
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ATP concentrations in L6 myotubes exposed to ionomycin or W7. Although L6 myotubes do not contract in response to an increase in cytosolic Ca2+, it still seemed possible that the increase in Ca2+ might result in a decrease in ATP. However, exposure of myotubes to 1 µM ionomycin or 50 µM W7 for 5 h had no significant effect on ATP concentrations, which averaged 5.01 ± 0.23 µmol/100 mg protein for untreated control myotubes, 4.92 ± 0.29 µmol/100 mg protein for myotubes exposed to ionomycin, and 4.80 ± 0.24 µmol/100 mg protein for myotubes exposed to W7. L6 myotubes contain negligible amounts of phosphocreatine, which were too low to measure accurately.
Increases in mitochondrial enzymes induced by agents that raise
cytosolic Ca2+.
As shown in Fig. 5, exposure of L6
myotubes to either 50 µM W7, 1 µM ionomycin, or 5 mM caffeine for 5 h/day for 5 days induced significant increases in ALAS, COX-I, and
cytochrome c or citrate synthase, which were used as
mitochondrial marker proteins. The increases in these enzymes induced
by W7 and caffeine were, like the increase in cytosolic
Ca2+, partially blocked by inclusion of 10 µM dantrolene
in the medium containing these agents. Dantrolene is an inhibitor of
Ca2+ release from the SR (29). Similarly, the
Ca2+ chelator EGTA partially inhibited the increases in
enzyme protein induced by ionomycin. As shown in Fig. 2, neither
caffeine, ionomycin, W7, nor dantrolene treatment resulted in an
increase in AMPK phosphorylation.
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Rates of oxidation of oleate.
To determine whether the increases in mitochondrial marker enzymes
reflect an increase in functional mitochondria, we measured the
capacity of myotube homogenates to oxidize [14C]oleate to
14CO2 under conditions in which availability of
ADP and Pi are not limiting. As shown in Fig.
6, oleate oxidation was increased
significantly in response to 5 days of caffeine treatment.
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DISCUSSION |
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One aim of this study was to evaluate the usefulness of L6 myotubes as a model for studying the regulation of mitochondrial biogenesis. One reason for choosing L6 myotubes instead of primary cultures of rat skeletal muscle is that L6 myotubes have the advantage that they do not contract. Our results show that L6 myotubes can respond to adaptive stimuli with rapid increases in the concentrations of mitochondrial enzymes and in the capacity to oxidize fat and carbohydrate. The latter finding provides good evidence for an increase in functional mitochondria. From these findings, we conclude that L6 myotubes are a suitable model for studying mitochondrial biogenesis.
The second purpose of this study was to reevaluate the effect of raising cytosolic Ca2+ on mitochondrial biogenesis in muscle cells. The evidence that Ca2+ stimulates mitochondrial biogenesis has come from two studies in which continuous exposure of primary cultures of rat myotubes (16) or L6E9 myotubes (6) to A-23187 induced increases in a number of mitochondrial enzymes. One reason for reevaluating the role of Ca2+ was the evidence that activation of AMPK stimulates mitochondrial biogenesis (32) and the possibility that the effect of A-23187 on mitochondrial biogenesis was mediated by a decrease in ATP, with an increase in AMP, resulting in activation of AMPK, rather than by the increase in Ca2+ per se. Another reason was our finding in a preliminary experiment that continuous exposure of primary cultures of rat myotubes to 3 µM A-23187 had a deleterious effect on cell viability, with an ~50% decrease in total protein because of atrophy and loss of cells (E. A. Gulve and J. O. Holloszy, unpublished results). Because enzyme activities can vary up to eightfold between individual myotubes in primary cultures of rat myotubes (21), this finding raised the possibility that the increases in mitochondrial enzymes might be due to selective killing by A-23187 of myotubes with the lowest mitochondrial content. A third reason was that during contractile activity, Ca2+ is released from and increases in the region of the SR, whereas A-23187 mediates Ca2+ entry through the sarcolemma. We therefore also wanted to evaluate the effects of agents that release Ca2+ from the SR.
Our results provide strong evidence that increases in cytosolic Ca2+, mediated either by intermittent release of Ca2+ from the SR or Ca2+ entry through the sarcolemma, stimulate mitochondrial biogenesis. They also show that this effect is mediated by Ca2+, not by a decrease in ~P. In support of the role of Ca2+, we found that dantrolene, at a concentration that inhibited most of the increase in cytosolic Ca2+ induced by caffeine or W7, also largely inhibited the increases in mitochondrial proteins. Similarly, removal of Ca2+ from the medium with EGTA attenuated both the increase in cytosolic Ca2+ and the stimulation of mitochondrial biogenesis by ionomycin.
These findings, which implicate increases in cytosolic Ca2+
in stimulating mitochondrial biogenesis, raise a question regarding the
role of decreases in phosphocreatine and ATP (~P) in mediating the
increase in mitochondria induced by endurance training. The evidence
that a decrease in ~P stimulates mitochondrial biogenesis, although
not conclusive, is also strong. This evidence includes the finding that
a number of other stimuli that lower ~P also induce increases in
mitochondrial proteins. These include thyrotoxicosis (30),
cold exposure (14), severe iron deficiency
(22), feeding of -guanidinopropionate (
-GPA)
(26, 28, 33), and mitochondrial uncoupling (17,
19). Although severe iron deficiency results in a decrease in
the iron-containing mitochondrial constituents, it can result in large
increases in non-iron-containing mitochondrial enzymes
(22). A mutation that results in partial mitochondrial uncoupling is associated with a remarkable increase in muscle mitochondria (19). In HeLa cells containing plasmids
allowing doxycycline-inducible expression of uncoupling protein 1 (UCP-1), induction of UCP-1 expression resulted in a decrease
in phosphorylation potential, as evidenced by an increase in oxygen
consumption and rapid increases in nuclear respiratory factor (NRF)-1
and ALAS expression (17). Feeding rats the creatine analog
-GPA, which competitively inhibits creatine uptake and markedly
lowers ~P concentrations, induces increases in mitochondrial enzymes
(26, 28, 33) and in GLUT4 (26), the
expression of which is regulated in parallel with mitochondrial
biogenesis (3, 7, 12, 26, 32).
Recent studies have shown that many of the metabolic effects induced by a decrease in ~P are mediated by activation of AMPK (31). Inhibition of this enzyme is released by decreases in phosphocreatine and ATP, and activation is induced by increases in AMP (31). Much of the information regarding the roles of AMPK has come from the use of AICAR, which is taken up by muscle and converted to the AMP analog ZMP, which activates AMPK (31). Activation of AMPK appears to be the mechanism by which muscle contractions stimulate glucose transport (8, 9, 15). AICAR also induces increases in mitochondrial enzymes and GLUT4 in skeletal muscle (13, 24, 32, 34) and L6 myotubes (23, Fig. 1). These findings suggest that activation of AMPK may mediate the effects of a decrease in ~P not only on glucose transport but also on mitochondrial biogenesis and GLUT4 expression.
In view of this evidence, it is our tentative conclusion that both increases in cytosolic Ca2+ and decreases in ~P stimulate signaling pathways that lead to increases in mitochondrial biogenesis and GLUT4 expression. Mitochondria are necessary for survival, so the existence of more than one mechanism for the regulation of mitochondrial biogenesis is not surprising. Although there is now evidence for only two independent signaling pathways, one activated by increases in Ca2+, the other by decreases in ~P, for stimulating mitochondrial biogenesis, there may well be additional mechanisms still to be discovered.
In conclusion, the present results show that L6 myocytes are a useful model for investigation of mitochondrial biogenesis. Using this model, we have shown that intermittently raising cytosolic Ca2+, either by release from the SR or entry across the sarcolemma, stimulates mitochondrial biogenesis in muscle cells, with a rapid increase in mitochondria.
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ACKNOWLEDGEMENTS |
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We are grateful to Victoria Reckamp for expert assistance with preparation of the manuscript.
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FOOTNOTES |
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This research was supported by National Institutes of Health Grants AG-00425 to J. O. Holloszy and HL-62552 to M. Sturek. E. O. Ojuka and T. E. Jones were supported by National Institute on Aging Institutional National Research Service Award AG-00078, and Brian Wamhoff was supported by an American Heart Association doctoral fellowship.
Address for reprint requests and other correspondence: J. O. Holloszy, Washington Univ. School of Medicine, Dept. of Internal Medicine, Campus Box 8113, 4566 Scott Ave., St. Louis, MO 63110 (E-mail: jhollosz{at}im.wustl.edu).
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.
July 24, 2002;10.1152/ajpendo.00242.2002
Received 4 June 2002; accepted in final form 17 July 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Baldwin, KM,
Klinkerfuss GH,
Terjung RL,
Molé PA,
and
Holloszy JO.
Respiratory capacity of white, red, and intermediate muscle: adaptive response to exercise.
Am J Physiol
222:
373-378,
1972
2.
Booth, FW,
and
Baldwin KM.
Muscle plasticity: energy demand and supply processes.
In: Handbook of Physiology. Exercise: Regulation and Integration of Multiple Systems. Bethesda, MD: Am. Physiol. Soc, 1996, sec. 12, chapt. 24, p. 1075-1123.
3.
Casla, A,
Rovira A,
Wells JA,
and
Dohm GL.
Increased glucose transporter (GLUT4) protein expression in hyperthyroidism.
Biochem Biophys Res Commun
171:
182-188,
1990[ISI][Medline].
4.
Constable, SH,
Favier RJ,
McLane JA,
Fell RD,
Chen M,
and
Holloszy JO.
Energy metabolism in contracting rat skeletal muscle: adaptation to exercise training.
Am J Physiol Cell Physiol
253:
C316-C322,
1987
5.
Dudley, GA,
Tullson PC,
and
Terjung RL.
Influence of mitochondrial content on the sensitivity of respiratory control.
J Biol Chem
262:
9109-9114,
1987
6.
Freyssenet, D,
DiCarlo M,
and
Hood DA.
Calcium-dependent regulation of cytochrome c gene expression in skeletal muscle cells.
J Biol Chem
274:
9305-9311,
1999
7.
Friedman, JE,
Sherman WM,
Reed MJ,
Elton CW,
and
Dohm GL.
Exercise-training increases glucose transporter protein GLUT4 in skeletal muscle of obese Zucker (fa/fa) rats.
FEBS Lett
268:
13-16,
1990[ISI][Medline].
8.
Hayashi, T,
Hirshman MF,
Fujii N,
Habinowski SA,
Witters LA,
and
Goodyear LJ.
Metabolic stress and altered glucose transport. Activation of AMP-activated protein kinase as a unifying coupling mechanism.
Diabetes
48:
537-531,
2000.
9.
Hayashi, T,
Hirshman MF,
Kurth EJ,
Winder WW,
and
Goodyear LJ.
Evidence for 5'AMP-activated protein kinase mediation of the effect of muscle contraction on glucose transport.
Diabetes
47:
1369-1373,
1998[Abstract].
10.
Hill, BJF,
Katwa LC,
Wamhoff BR,
and
Sturek M.
Enhanced endothelium(A) receptor-mediated calcium mobilization and contraction in organ cultured coronary arteries.
J Pharmacol Exp Ther
295:
484-491,
2000
11.
Holloszy, JO.
Biochemical adaptations in muscle. Effects of exercise on mitochondrial O2 uptake and respiratory enzyme activity in skeletal muscle.
J Biol Chem
242:
2278-2282,
1967
12.
Holloszy, JO,
and
Hansen PA.
Regulation of glucose transport into skeletal muscle.
In: Reviews of Physiology, Biochemistry and Pharmacology, edited by Blaustein MP,
Grunicke H,
Habermann E,
Pette D,
Schultz G,
and Schweiger M.. Berlin: Springer-Verlag, 1996, p. 99-193.
13.
Holmes, BF,
Kurth-Kraczek EJ,
and
Winder WW.
Chronic activation of 5'-AMP-activated protein kinase increases GLUT-4, hexokinase, and glycogen in muscle.
J Appl Physiol
87:
1990-1995,
1999
14.
Klingenspor, M,
Ivemeyer M,
Wiesinger H,
Haas K,
Heldmaier G,
and
Wiesner RJ.
Biogenesis of thermogenic mitochondria in brown adipose tissue of Djungarian hamsters during cold adaptation.
Biochem J
316:
607-613,
1996[ISI][Medline].
15.
Kurth-Kraczek, EJ,
Hirshman MF,
Goodyear LJ,
and
Winder WW.
5' AMP-activated protein kinase activation causes GLUT4 translocation in skeletal muscle.
Diabetes
48:
1667-1671,
1999[Abstract].
16.
Lawrence, JC, Jr,
and
Salsgiver WJ.
Levels of enzymes of energy metabolism are controlled by activity of cultured rat myotubes.
Am J Physiol Cell Physiol
244:
C348-C355,
1983[Abstract].
17.
Li, B,
Holloszy JO,
and
Semenkovich CF.
Respiratory uncoupling induces -aminolevulinate synthase expression through a nuclear respiratory factor-1-dependent mechanism in HeLa cells.
J Biol Chem
274:
17534-17540,
1999
18.
Liu, C,
and
Hermann TE.
Characterization of ionomycin as a calcium ionophore.
J Biol Chem
253:
5892-5894,
1978[Abstract].
19.
Luft, R,
Ikkos D,
Palmieri G,
Ernster L,
and
Afzelius B.
A case of severe hypermetabolism of nonthyroid origin, with a defect in the maintenance of mitochondrial respiratory control. A Correlated clinical biochemical and morphological study.
J Clin Invest
41:
1776-1804,
1962[ISI].
20.
Molé, PA,
Oscai LB,
and
Holloszy JO.
Adaptation of muscle to exercise. Increase in levels of palmityl CoA synthetase, and in the capacity to oxidize fatty acids.
J Clin Invest
50:
2323-2330,
1971[ISI][Medline].
21.
Nemeth, PM,
Solanki L,
and
Lawrence JC, Jr.
Control of enzyme activities in individual myotubes cultured without nerve.
Am J Physiol Cell Physiol
249:
C313-C317,
1985[Abstract].
22.
Ohira, Y,
Cartier LJ,
Chen M,
and
Holloszy JO.
Induction of an increase in mitochondrial matrix enzymes in muscle of iron-deficient rats.
Am J Physiol Cell Physiol
253:
C639-C644,
1987
23.
Ojuka, EO,
Jones TE,
Nolte LA,
Chen M,
Wamhoff BR,
Sturek M,
and
Holloszy JO.
Regulation of GLUT4 biogenesis in muscle: evidence for involvement of AMPK and Ca2+.
Am J Physiol Endocrinol Metab
282:
E1008-E1013,
2002
24.
Ojuka, EO,
Nolte LA,
and
Holloszy JO.
Increased expression of GLUT-4 and hexokinase in rat epitrochlearis muscles exposed to AICAR in vitro.
J Appl Physiol
88:
1072-1075,
2000
25.
Palade, P.
Drug induced Ca2+ release from isolated sarcoplasmic reticulum. II. Releases involving a Ca2+ induced Ca2+ release channel.
J Biol Chem
262:
6142-6148,
1987
26.
Ren, JM,
Semenkovich CF,
and
Holloszy JO.
Adaptation of muscle to creatine depletion: effect on GLUT-4 glucose transporter expression.
Am J Physiol Cell Physiol
264:
C146-C150,
1993
27.
Scott, MD,
Baudendistel LJ,
and
Dahms TE.
Rapid separation of creatine, phosphocreatine and adenosine metabolites by ion-pair reversed-phase high performance liquid chromatography in plasma and cardiac tissue.
J Chromatogr A
576:
149-154,
1992[ISI].
28.
Shoubridge, EA,
Challis AJ,
Hayes DJ,
and
Radda GK.
Biochemical adaptation in the skeletal muscle of rats depleted of creatine with the substrate analogue beta-guanidinopropionic acid.
Biochem J
232:
125-131,
1985[ISI][Medline].
29.
Van Winkle, WB.
Calcium release from skeletal muscle sarcoplasmic reticulum: site of action of dantrolene sodium.
Science
193:
1130-1131,
1976[ISI][Medline].
30.
Winder, WW,
Fitts R,
Holloszy JO,
Kaiser K,
and
Brooke M.
Effects of thyroid hormones on different types of skeletal muscle.
In: Plasticity of Muscle, edited by Pette D.. Berlin: Walter de Gruyter, 1980, p. 583-591.
31.
Winder, WW,
and
Hardie DG.
AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes.
Am J Physiol Endocrinol Metab
277:
E1-E10,
1999
32.
Winder, WW,
Holmes BF,
Rudink DS,
Jensen EB,
Chen M,
and
Holloszy JO.
Activation of AMP-activated protein kinase increases mitochondrial enzymes in skeletal muscle.
J Appl Physiol
88:
2219-2226,
2000
33.
Yaspelkis, BB, III,
Castle AL,
Ding Z,
and
Ivy JL.
Attenuating the decline in ATP arrests the exercise training-induced increases in muscle GLUT4 protein and citrate synthase activity.
Acta Physiol Scand
165:
71-79,
1999[ISI][Medline].
34.
Zheng, D,
MacLean PS,
Pohnert SC,
Knight JB,
Olson AL,
Winder WW,
and
Dohm GL.
Regulation of muscle GLUT-4 transcription by AMP-activated protein kinase.
J Appl Physiol
91:
1073-1083,
2001