Research Division, Joslin Diabetes Center, and Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts
Submitted 22 December 2004 ; accepted in final form 7 May 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
exercise; p38; ERK; AMPK signaling
Despite these advances in understanding the molecular regulation of contracting muscle fibers, it has become increasingly apparent that these signaling events do not explain all of the acute metabolic changes and long-term adaptations that occur in response to exercise. This fact, along with the complex nature of the contraction stimulus, suggests that contractile activity may stimulate additional signaling cascades in skeletal muscle. Nuclear factor-B (NF-
B) has been studied most extensively for its central role in the regulation of innate immunity, inflammation, and apoptosis. In contrast, its physiological and pathological roles in cells outside of the immune system are only beginning to be understood. As a universal mechanism, NF-
B dimers are sequestered basally in the cytoplasm by inhibitor of nuclear factor-
B
(I
B
). When activated, the IKK complex, consisting of two catalytic subunits (IKK
and IKK
) and a regulatory IKK
subunit, phosphorylates serine residues of I
B
and targets it for ubiquitination and proteosomal degradation (49). Liberation of the NF-
B dimers (predominantly p50 and p65) promotes their nuclear translocation and NF-
B-mediated gene transcription (34).
Several lines of evidence have led us to hypothesize that NF-B is regulated by exercise in skeletal muscle. Studies using cell systems or cardiac muscle have proposed that ERK, p38, and AMPK can regulate IKK/NF-
B signaling (11, 33, 50). NF-
B has also been shown to be regulated by elevations in intracellular calcium (23, 24) and reactive oxygen species (28, 35, 37, 46), both of which occur as a consequence of skeletal muscle contraction (4, 14, 31, 42). In fact, while this article was in preparation, Ji et al. (25) showed that exhaustive exercise resulted in increases in NF-
B binding in rat skeletal muscle. In contrast to this finding, NF-
B activity has also been reported to decrease in response to fatiguing skeletal muscle contraction (16). These data are intriguing due to recent findings, which have highlighted an important role for skeletal muscle NF-
B in cachexia and other muscle wasting syndromes (7). Because sustained activation of NF-
B leads to muscle wasting, we reasoned that discontinuous or episodic activation, as might occur during periods of exercise, could play an important role in normal muscle biology. In line with this hypothesis, acute exercise has been shown to induce apoptosis in skeletal muscle, an event involved in skeletal muscle plasticity, repair, and regeneration (40, 41, 44, 45).
In the present study we tested the hypothesis that submaximal exercise and contraction of isolated muscles activate IKK and NF-B. We demonstrate that both treadmill exercise in vivo and contraction in vitro activate IKK signaling in adult rat skeletal muscle and that this results in transient increases in NF-
B activity postexercise. Data from inhibitor studies suggest that exercise-induced activation of IKK is influenced by both ERK and p38 MAPK.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals and housing. All protocols for animal use were approved by the Institutional Animal Care and Use Committee of the Joslin Diabetes Center and were in accordance with National Institutes of Health guidelines. Male Sprague-Dawley rats (6080 g for muscle incubation studies; 175200 g for in vivo studies) were obtained from Taconic (Germantown, NY), provided standard rodent chow and water ad libitum, and were housed under standard laboratory conditions (12:12 h light-dark cycle). Animals were fasted 4 h before exercise and in vitro muscle contraction experiments described below.
In vivo treadmill exercise. Rats were familiarized with the treadmill (Quinton model 42) by running 510 min on the 2 days before the onset of experimentation. Animals were then subjected to a treadmill running protocol consisting of 5, 15, 30, or 60 min of moderate-intensity steady-state exercise (0.8 mph, 12% incline). Other animals were exercised for 60 min and euthanized 1, 3, or 5 h later. The rats were euthanized at the various times indicated in the figures by stunning and cervical dislocation. Soleus, red, and white gastrocnemius muscles were quickly separated, removed, and snap frozen in liquid nitrogen until subsequent processing and analyses.
Contraction of isolated muscles. Contraction experiments were performed as previously described (43). Briefly, fasted rats were euthanized by cervical dislocation and extensor digitorum longus (EDL) and soleus muscles were rapidly dissected. Both tendons were tied with a silk suture, mounted on an incubation apparatus to maintain resting length, and preincubated for 20 min in 37°C oxygenated Krebs-Ringer bicarbonate buffer (117 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, and 24.6 mM NaHCO3, pH 7.5) containing 2.0 mM pyruvic acid as an exogenous carbon source. Muscles were preincubated in U-0126 or SB-203580 for 20 min in concentrations indicated in the figures. After preincubation, muscles were transferred to a tissue support apparatus with stimulating electrodes (Harvard Apparatus, Holliston, MA) and incubated for an additional 10 min before the onset of contractions. Muscles were then stimulated for 10 min using the following protocol: train rate 2/min; train duration, 10 s; pulse rate, 100 Hz; pulse duration, 0.1 ms at 100 V. Force production was monitored during the entire contraction protocol with an isometric force transducer (Kent Scientific, Litchfield, CT) and a chart recorder (Kipp and Zonen, Delft, Holland). Immediately after contractions, muscles were removed from the buffer, dismounted, and snap-frozen in liquid nitrogen.
5-Aminomidazole-4-carboxamide-1--D-ribofurosanide treatment.
EDL muscles from fasted rats were isolated as described above. Muscles incubated in the presence or absence of 5-aminomidazole-4-carboxamide-1-
-D-ribofurosanide (AICAR; 2 mM) for 40 min were dismounted and snap-frozen in liquid nitrogen. For in vivo studies, fasted rats were treated with a maximal dose of AICAR (1.0 mg/g body wt) by intraperitoneal injection. A group of control rats were injected with saline. Sixty minutes after the injections, the rats were euthanized by stunning and cervical dislocation and mixed gastrocnemius muscles were quickly removed and snap-frozen in liquid nitrogen until subsequent processing and analyses.
Preparation of skeletal muscle lysates. Frozen muscles were pulverized and Polytron homogenized (Brinkman Instruments) in ice-cold lysis buffer (20 mM Tris, 5 mM EDTA, 10 mM Na3PO4, 100 mM NaF, 2 mM Na3VO4, 1% Nonidet P-40, 10 µM leupeptin, 3 mM benzamidine, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.5). Muscle homogenates were rotated end-over-end for 60 min at 4°C and centrifuged at 14,000 g for 10 min at 4°C. Protein concentrations of lysates were determined by the Bradford method with bovine serum albumin as a standard using a kit from Bio-Rad Laboratories (Hercules, CA). Muscle lysates were frozen in liquid nitrogen and stored at 80°C until used for immunoblotting.
Immunoblotting. Lysate proteins (40100 µg) were separated by 8% SDS-PAGE, transferred to nitrocellulose membranes, and membranes were blocked for 60 min in Tris-buffered saline with 0.050.1% Tween 20 (TBST), and either 5% nonfat milk or bovine serum albumin. Proteins of interest were probed by incubating membranes in TBST containing 35% nonfat milk or bovine serum albumin and corresponding primary antibodies overnight on a rocker at 4°C. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000) in TBST with 5% nonfat milk for 60 min at room temperature. Bands were visualized on film by enhanced chemiluminescence (Perkin-Elmer Life Sciences, Boston, MA), scanned, and quantitated by densitometry.
NF-B activity assays.
Gastrocnemius muscle protein was extracted through homogenization in passive lysis buffer (Promega, Madison, WI). Protein concentration was determined using the Bio-Rad Bradford protein assay. Electrophoretic mobility shift assays (EMSA) were performed using a Gel Shift Assay Systems Kit (Promega). Double-stranded consensus oligonucleotides (1.75 pmol/µl) for the NF-
B binding site (5-AGTTGAGGGGACTTTCCCAGG-3) were labeled with T4 polynucleotide kinase (510 U/µl) and [
-32P]ATP (3000Ci/mM) (Perkin-Elmer Life Sciences). End-labeled probe was purified through Sephadex G-25 spin column (Roche, Indianapolis, IN). Equal amounts of muscle protein (100 µg) were mixed with the labeled probe in the presence of 50 µg/ml poly(dI-dC) in 40 µl of binding buffer composed of 10 mM Tris·HCl, pH 7.5, 50 mM NaCl, 0.5 mM dithiothreitol, 1 mM MgCl2, and 10% glycerol. A 100-fold excess of cold probe was added into negative competitive control to ensure the specificity. After 30 min of incubation at room temperature, protein-DNA complexes were separated in a 3% polyacrylamide gel under nondenaturing conditions. Gels were subsequently dried, autoradiographied with a Storm phosphorimager (Molecular Dynamics), and analyzed using ImageQuant 5.1 software.
Statistical analyses. Data are expressed as means ± SE. Significance was determined a priori at P < 0.05. Student's t-test and ANOVA with Tukey's post hoc methods were used for group comparisons.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Contraction of isolated skeletal muscle activates IKK.
IKK/
and NF-
B are activated by circulating cytokines, such as TNF-
and IL-1
, as well as intracellular mediators such as reactive oxygen species. To determine whether exercise-mediated IKK
/
phosphorylation is regulated systemically or locally, we incubated and contracted isolated rat EDL and soleus muscles. Figure 2A shows that IKK
/
are phosphorylated by 10 min of contraction in EDL muscles, similar to treadmill exercise in vivo. Similar to exercise in vivo, total amounts of IKK
, IKK
, and I
B
protein were unchanged by contraction. The increase in IKK
/
phosphorylation was accompanied by a significant increase in the phosphorylation of I
B
(Fig. 2B).
|
|
AICAR does not alter IKK phosphorylation or NF-B activity.
AMPK is another kinase that is activated in muscle in response to exercise and has been shown to mediate oxidative stress-induced NF-
B signaling in neuroblastoma cells (27). We determined whether AMPK activation by AICAR results in IKK phosphorylation in skeletal muscle. Rats were injected intraperitoneally with a maximal dose of AICAR and muscles were examined 60 min later. As shown in Fig. 4, compared with saline-injected controls, AMPK phosphorylation in skeletal muscle was significantly increased in response to AICAR treatment in vivo. Despite robust increases in AMPK phosphorylation, skeletal muscle IKK
/
phosphorylation (Fig. 4, left) and NF-
B activity (data not shown) did not change after 60 min of AICAR treatment. Shorter exposures to AICAR for 15 or 30 min also had no effect on IKK phosphorylation (data not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In cells of the immune system, NF-B orchestrates the expression of over 150 target genes, including those encoding cytokines and chemokines, cytokine, chemokine, and immune receptors, stress response genes, cell adhesion molecules, regulators of apoptosis, and acute phase proteins (39). These proteins play pivotal roles in regulating innate immunity, inflammation, and apoptosis. The biology of NF-
B in tissues outside of the immune system is less well understood. In muscle, we now know that even high-level activation of NF-
B does not increase the expression of genes typically thought of as regulating immunity, inflammation, and apoptosis (7). Instead, sustained activation of NF-
B in muscle leads to increased expression of MuRF1, a muscle-specific E3 ligase (5, 17), and the C2 and C9 subunits of the proteasome. The net effect of chronic, unopposed activation of NF-
B in muscle is profound muscle wasting, much like that seen in cachexia, and muscle wasting syndromes associated with cancer, AIDS, bacterial sepsis, and immobilization (7). The opposite turns out to be true as well, that either genetic or pharmacological inhibition of NF-
B prevents or reduces muscle wasting, for example in cancer and denervation, confirming that NF-
B acts as a pathological mediator.
These findings raise intriguing questions about what effect episodic activation of NF-B, as occurs during intermittent exercise, might have on muscle physiology. Whereas sustained activation of this pathway causes muscle wasting, episodic activation might activate the same processes but with distinct end results. In fact, acute exercise has been shown to induce apoptosis in skeletal muscle (40, 41, 44, 45). Consistent with the possible involvement of NF-
B in muscle damage, a previous report (25) showed prolonged increases in NF-
B binding activity in deep vastus lateralis muscles after exhaustive exercise. Interestingly, our data demonstrate relatively rapid increases in IKK activation and transient postexercise increases in NF-
B activity in red gastrocnemius muscles in response to a nonexhaustive, submaximal exercise protocol. Differences in the relative intensity of the exercise protocols likely explain the transient vs. prolonged increases in NF-
B activation. The exhaustive nature of the exercise bout used in the previous study also might explain why they observed decreases in I
B
protein levels, whereas basal levels of I
B
were not altered by the moderate intensity exercise protocol used in the current study. Collectively, exercise-mediated NF-
B activation is likely to play multiple roles, mediating cellular events in response to low- and moderate-intensity exercise, and regulating normal repair and regeneration after high-intensity exercise-induced muscle damage.
Physical exercise has been shown previously to induce the expression of a variety of potential NF-B target genes in skeletal muscle, including IL-1
(8, 36), IL-6 (26, 38), IL-8 (9, 36), IL-10 (36), TNF-
(36), and MnSOD (22). In particular, skeletal muscle IL-6 production increases >100-fold during intense exercise (29, 47). Because persistent NF-
B activation alone is not sufficient to induce the expression of these genes in skeletal muscle (7), it appears that exercise-mediated increases in these putative NF-
B targets (e.g., IL-6) require additional signaling inputs. In cultured cells, IL-6 expression has also been shown to be p38 dependent (3, 12, 13, 30, 32, 50). Furthermore, p38-mediated IL-6 expression in cardiac myocytes is dependent on NF-
B because a point mutation that abolishes the
B site in the IL-6 promoter also abrogates p38-inducible IL-6 reporter activity (12). Our present findings implicate p38, as well as ERK, as kinases that influence the activities of IKK and NF-
B in contracting skeletal muscle. This possibility is underscored by the fact that like NF-
B, p38 is involved in the expression of numerous cytokines (33), and the degree of p38 activation is highly correlated with exercise-mediated skeletal muscle IL-6 expression (10). However, our data do not distinguish two possible mechanisms, one with p38 and ERK in linear pathways leading to IKK and NF-
B activation and the second with p38 and ERK in parallel pathways. It is possible, in the second case, that cells respond to decreased p38 or ERK activity by dampening IKK/NF-
B activity, and vice versa. Additional experiments will be needed to distinguish these possibilities.
In summary, we show that exercise in vivo and muscle contractile activity in vitro regulate NF-B signaling in adult skeletal muscle. Our data show that the IKK activation in response to exercise in vivo is a local event, triggered by either paracrine molecules and/or signals intrinsic to the muscle. We also provide evidence that p38 and ERK are likely involved in the coordinated activation of contraction-induced IKK activity. Furthermore, whereas AMPK has been implicated in the activation of NF-
B, our data suggest that AMPK is not upstream in skeletal muscle IKK or NF-
B activation. IKK and NF-
B are novel exercise-induced signals in skeletal muscle that represent candidate regulators of gene expression during and following exercise.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Aronson D, Violan MA, Dufresne SD, Zangen D, Fielding RA, and Goodyear LJ. Exercise stimulates the mitogen-activated protein kinase pathway in human skeletal muscle. J Clin Invest 99: 12511257, 1997.
3. Baeza-Raja B and Munoz-Canoves P. p38 MAPK-induced nuclear factor-B activity is required for skeletal muscle differentiation: role of interleukin-6. Mol Biol Cell 15: 20132026, 2004.
4. Bejma J and Ji LL. Aging and acute exercise enhance free radical generation in rat skeletal muscle. J Appl Physiol 87: 465470, 1999.
5. Bodine SC, Latres E, Baumhueter S, Lai VK, Nunez L, Clarke BA, Poueymirou WT, Panaro FJ, Na E, Dharmarajan K, Pan ZQ, Valenzuela DM, DeChiara TM, Stitt TN, Yancopoulos GD, and Glass DJ. Identification of ubiquitin ligases required for skeletal muscle atrophy. Science 294: 17041708, 2001.
6. Boppart MD, Asp S, Wojtaszewski JF, Fielding RA, Mohr T, and Goodyear LJ. Marathon running transiently increases c-Jun NH2-terminal kinase and p38 activities in human skeletal muscle. J Physiol 526: 663669, 2000.
7. Cai D, Frantz JD, Tawa NE Jr, Melendez PA, Oh BC, Lidov HG, Hasselgren PO, Frontera WR, Lee J, Glass DJ, and Shoelson SE. IKK/NF-
B activation causes severe muscle wasting in mice. Cell 119: 285298, 2004.[CrossRef][ISI][Medline]
8. Cannon JG, Fielding RA, Fiatarone MA, Orencole SF, Dinarello CA, and Evans WJ. Increased interleukin-1 in human skeletal muscle after exercise. Am J Physiol Regul Integr Comp Physiol 257: R451R455, 1989.
9. Chan MH, Carey AL, Watt MJ, and Febbraio MA. Cytokine gene expression in human skeletal muscle during concentric contraction: evidence that IL-8, like IL-6, is influenced by glycogen availability. Am J Physiol Regul Integr Comp Physiol 287: R322R327, 2004.
10. Chan MH, McGee SL, Watt MJ, Hargreaves M, and Febbraio MA. Altering dietary nutrient intake that reduces glycogen content leads to phosphorylation of nuclear p38 MAP kinase in human skeletal muscle: association with IL-6 gene transcription during contraction. FASEB J 18: 17851787, 2004.
11. Chen BC and Lin WW. PKC- and ERK-dependent activation of IB kinase by lipopolysaccharide in macrophages: enhancement by P2Y receptor-mediated CaMK activation. Br J Pharmacol 134: 10551065, 2001.[CrossRef][ISI][Medline]
12. Craig R, Larkin A, Mingo AM, Thuerauf DJ, Andrews C, McDonough PM, and Glembotski CC. p38 MAPK and NF-B collaborate to induce interleukin-6 gene expression and release. Evidence for a cytoprotective autocrine signaling pathway in a cardiac myocyte model system. J Biol Chem 275: 2381423824, 2000.
13. Craig R, Larkin A, Mingo AM, Thuerauf DJ, Andrews C, McDonough PM, and Glembotski CC. p38 MAPK and NF-B collaborate to induce interleukin-6 gene expression and release. Evidence for a cytoprotective autocrine signaling pathway in a cardiac myocyte model system. J Biol Chem 275: 2381423824, 2000.
14. Davies KJ, Quintanilha AT, Brooks GA, and Packer L. Free radicals and tissue damage produced by exercise. Biochem Biophys Res Commun 107: 11981205, 1982.[CrossRef][ISI][Medline]
15. De Alvaro C, Teruel T, Hernandez R, and Lorenzo M. Tumor necrosis factor- produces insulin resistance in skeletal muscle by activation of inhibitor
B kinase in a p38 MAPK-dependent manner. J Biol Chem 279: 1707017078, 2004.
16. Durham WJ, Li YP, Gerken E, Farid M, Arbogast S, Wolfe RR, and Reid MB. Fatiguing exercise reduces DNA binding activity of NF-B in skeletal muscle nuclei. J Appl Physiol 97: 17401745, 2004.
17. Glass DJ. Signalling pathways that mediate skeletal muscle hypertrophy and atrophy. Nat Cell Biol 5: 8790, 2003.[CrossRef][ISI][Medline]
18. Gomez DA, Martinez-Martinez S, Maldonado JL, Ortega-Perez I, and Redondo JM. A role for the p38 MAP kinase pathway in the nuclear shuttling of NFATp. J Biol Chem 275: 1387213878, 2000.
19. Goodyear LJ, Chung PY, Sherwood D, Dufresne SD, and Moller DE. Effects of exercise and insulin on mitogen-activated protein kinase signaling pathways in rat skeletal muscle. Am J Physiol Endocrinol Metab 271: E403E408, 1996.
20. Han J, Jiang Y, Li Z, Kravchenko VV, and Ulevitch RJ. Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation. Nature 386: 296299, 1997.[CrossRef][ISI][Medline]
21. Hayashi T, Hirshman MF, Dufresne SD, and Goodyear LJ. Skeletal muscle contractile activity in vitro stimulates mitogen-activated protein kinase signaling. Am J Physiol Cell Physiol 277: C701C707, 1999.
22. Hollander J, Fiebig R, Gore M, Ookawara T, Ohno H, and Ji LL. Superoxide dismutase gene expression is activated by a single bout of exercise in rat skeletal muscle. Pflügers Arch 442: 426434, 2001.[CrossRef][ISI][Medline]
23. Hughes K, Antonsson A, and Grundstrom T. Calmodulin dependence of NF-B activation. FEBS Lett 441: 132136, 1998.[CrossRef][ISI][Medline]
24. Hughes K, Edin S, Antonsson A, and Grundstrom T. Calmodulin-dependent kinase II mediates T cell receptor/CD3- and phorbol ester-induced activation of IB kinase. J Biol Chem 276: 3600836013, 2001.
25. Ji LL, Gomez-Cabrera MC, Steinhafel N, and Vina J. Acute exercise activates nuclear factor (NF)-B signaling pathway in rat skeletal muscle. FASEB J 18: 14991506, 2004.
26. Jonsdottir IH, Schjerling P, Ostrowski K, Asp S, Richter EA, and Pedersen BK. Muscle contractions induce interleukin-6 mRNA production in rat skeletal muscles. J Physiol 528: 157163, 2000.
27. Jung JE, Lee J, Ha J, Kim SS, Cho YH, Baik HH, and Kang I. 5-Aminoimidazole-4-carboxamide-ribonucleoside enhances oxidative stress-induced apoptosis through activation of nuclear factor-B in mouse neuro 2a neuroblastoma cells. Neurosci Lett 354: 197200, 2004.[CrossRef][ISI][Medline]
28. Kamata H, Manabe T, Oka S, Kamata K, and Hirata H. Hydrogen peroxide activates IB kinases through phosphorylation of serine residues in the activation loops. FEBS Lett 519: 231237, 2002.[CrossRef][ISI][Medline]
29. Keller C, Steensberg A, Pilegaard H, Osada T, Saltin B, Pedersen BK, and Neufer PD. Transcriptional activation of the IL-6 gene in human contracting skeletal muscle: influence of muscle glycogen content. FASEB J 15: 27482750, 2001.
30. Kosmidou I, Vassilakopoulos T, Xagorari A, Zakynthinos S, Papapetropoulos A, and Roussos C. Production of interleukin-6 by skeletal myotubes: role of reactive oxygen species. Am J Respir Cell Mol Biol 26: 587593, 2002.
31. Kumar CT, Reddy VK, Prasad M, Thyagaraju K, and Reddanna P. Dietary supplementation of vitamin E protects heart tissue from exercise-induced oxidant stress. Mol Cell Biochem 111: 109115, 1992.[ISI][Medline]
32. Luo G, Hershko DD, Robb BW, Wray CJ, and Hasselgren PO. IL-1 stimulates IL-6 production in cultured skeletal muscle cells through activation of MAP kinase signaling pathway and NF-
B. Am J Physiol Regul Integr Comp Physiol 284: R1249R1254, 2003.
33. Maulik N, Sato M, Price BD, and Das DK. An essential role of NFB in tyrosine kinase signaling of p38 MAP kinase regulation of myocardial adaptation to ischemia. FEBS Lett 429: 365369, 1998.[CrossRef][ISI][Medline]
34. Muller JM, Krauss B, Kaltschmidt C, Baeuerle PA, and Rupec RA. Hypoxia induces c-fos transcription via a mitogen-activated protein kinase-dependent pathway. J Biol Chem 272: 2343523439, 1997.
35. Muller JM, Rupec RA, and Baeuerle PA. Study of gene regulation by NF-B and AP-1 in response to reactive oxygen intermediates. Methods 11: 301312, 1997.[CrossRef][ISI][Medline]
36. Nieman DC, Davis JM, Henson DA, Walberg-Rankin J, Shute M, Dumke CL, Utter AC, Vinci DM, Carson JA, Brown A, Lee WJ, McAnulty SR, and McAnulty LS. Carbohydrate ingestion influences skeletal muscle cytokine mRNA and plasma cytokine levels after a 3-h run. J Appl Physiol 94: 19171925, 2003.
37. Oka S, Kamata H, Kamata K, Yagisawa H, and Hirata H. N-Acetylcysteine suppresses TNF-induced NF-B activation through inhibition of I
B kinases. FEBS Lett 472: 196202, 2000.[CrossRef][ISI][Medline]
38. Ostrowski K, Rohde T, Zacho M, Asp S, and Pedersen BK. Evidence that interleukin-6 is produced in human skeletal muscle during prolonged running. J Physiol 508: 949953, 1998.
39. Pahl HL. Activators and target genes of Rel/NF-B transcription factors. Oncogene 18: 68536866, 1999.[CrossRef][ISI][Medline]
40. Podhorska-Okolow M, Krajewska B, Carraro U, and Zabel M. Apoptosis in mouse skeletal muscles after physical exercise. Folia Histochem Cytobiol 37: 127128, 1999.[ISI][Medline]
41. Podhorska-Okolow M, Sandri M, Zampieri S, Brun B, Rossini K, and Carraro U. Apoptosis of myofibres and satellite cells: exercise-induced damage in skeletal muscle of the mouse. Neuropathol Appl Neurobiol 24: 518531, 1998.[CrossRef][ISI][Medline]
42. Rose AJ and Hargreaves M. Exercise increases Ca2+-calmodulin-dependent protein kinase II activity in human skeletal muscle. J Physiol 553: 303309, 2003.
43. Sakamoto K, Hirshman MF, Aschenbach WG, and Goodyear LJ. Contraction regulation of Akt in rat skeletal muscle. J Biol Chem 277: 1191011917, 2002.
44. Sandri M, Carraro U, Podhorska-Okolov M, Rizzi C, Arslan P, Monti D, and Franceschi C. Apoptosis, DNA damage and ubiquitin expression in normal and Mdx muscle fibers after exercise. FEBS Lett 373: 291295, 1995.[CrossRef][ISI][Medline]
45. Sandri M, Podhorska-Okolow M, Geromel V, Rizzi C, Arslan P, Franceschi C, and Carraro U. Exercise induces myonuclear ubiquitination and apoptosis in dystrophin-deficient muscle of mice. J Neuropathol Exp Neurol 56: 4557, 1997.[ISI][Medline]
46. Schreck R, Rieber P, and Baeuerle PA. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-B transcription factor and HIV-1. EMBO J 10: 22472258, 1991.[Abstract]
47. Steensberg A, Van Hall G, Osada T, Sacchetti M, Saltin B, and Klarlund PB. Production of interleukin-6 in contracting human skeletal muscles can account for the exercise-induced increase in plasma interleukin-6. J Physiol 529: 237242, 2000.
48. Widegren U, Ryder JW, and Zierath JR. Mitogen-activated protein kinase signal transduction in skeletal muscle: effects of exercise and muscle contraction. Acta Physiol Scand 172: 227238, 2001.[CrossRef][ISI][Medline]
49. Zandi E, Rothwarf DM, Delhase M, Hayakawa M, and Karin M. The IB kinase complex (IKK) contains two kinase subunits, IKK
and IKK
, necessary for I
B phosphorylation and NF-
B activation. Cell 91: 243252, 1997.[CrossRef][ISI][Medline]
50. Zechner D, Craig R, Hanford DS, McDonough PM, Sabbadini RA, and Glembotski CC. MKK6 activates myocardial cell NF-B and inhibits apoptosis in a p38 mitogen-activated protein kinase-dependent manner. J Biol Chem 273: 82328239, 1998.