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Exercise training exacerbates tourniquet ischemia-induced decreases in GLUT4 expression and muscle atrophy in rats
Life Sciences 78 (2006) 2953 – 2959 www.elsevier.com/locate/lifescie
Exercise training exacerbates tourniquet ischemia-induced decreases in GLUT4 expression and muscle atrophy in rats Ying-Lan Tsai c, Chien-Wen Hou a, Yi-Hung Liao a, Chung-Yu Chen a, Fang-Ching Lin a, Wen-Chih Lee b, Shih-Wei Chou d, Chia-Hua Kuo a,* a
c
Laboratory of Exercise Biochemistry, Taipei Physical Education College, 5 Dun-Hua N. Rd, Taipei 105, Taiwan, ROC b Committee of General Studies, Shih Hsin University, Taipei 116, Taiwan ROC Department of Athletic Training and Health, National College of Physical Education and Sports, Taoyuan 333, Taiwan, ROC d Department of Physical Medicine and Rehabilitation, Chang Gung Memorial Hospital, Taipei 333, Taiwan, ROC Received 29 August 2005; accepted 17 November 2005
Abstract The current study determined the interactive effects of ischemia and exercise training on glycogen storage and GLUT4 expression in skeletal muscle. For the first experiment, an acute 1-h tourniquet ischemia was applied to one hindlimb of both the 1-week exercise-trained and untrained rats. The contralateral hindlimb served as control. For the second experiment, 1-h ischemia was applied daily for 1 week to both trained (5 h postexercise) and untrained rats. GLUT4 mRNA was not affected by acute ischemia, but exercise training lowered GLUT4 mRNA in the acute ischemic muscle. GLUT4 protein levels were elevated by exercise training, but not in the acute ischemic muscle. Exercise training elevated muscle glycogen above untrained levels, but this increase was reversed by chronic ischemia. GLUT4 mRNA and protein levels were dramatically reduced by chronic ischemia, regardless of whether the animals were exercise-trained or not. Chronic ischemia significantly reduced plantaris muscle mass, with a greater decrease found in the exercise-trained rats. In conclusion, the exercise training effect on muscle GLUT4 protein expression was prevented by acute ischemia. Furthermore, chronic ischemia-induced muscle atrophy was exacerbated by exercise training. This result implicates that exercise training could be detrimental to skeletal muscle with severely impaired microcirculation. D 2005 Elsevier Inc. All rights reserved. Keywords: Hypoxia; Intermittent ischemia; Reperfusion; Glucose uptake; Glycogen; Rehabilitation; Muscle mass
Introduction To sustain normal cellular function under low arterial PO2, carbohydrate utilization increases in an effort to compensate for an energy deficit due to insufficient fat oxidation. This is demonstrated by the fact that ischemia significantly decreases muscle glycogen levels (Purshottam et al., 1977; Fraser et al., 1998; Carvalho et al., 1996; Katz, 1988); and treatments that enhances carbohydrate metabolism protects muscles from severe ischemic damage (Lopaschuk, 2000; Keller et al., 1998). GLUT4 protein, the main glucose transporter expressed in skeletal muscle (Olson and Pessin, 1996), is found to play a
* Corresponding author. Tel.: +886 2 25774624x831; fax: +886 2 25790526. E-mail address: [email protected] (C.-H. Kuo). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.11.021
key role in the regulation of muscle glycogen storage (Ren et al., 1993; Hansen et al., 1995; Tsao et al., 1996). Glycogen is the most abundant anaerobic fuel source in skeletal muscle. Exercise training, with sufficient recovery (5– 16 h in rats), will result in elevated muscle glycogen storage above pre-exercise levels (termed glycogen supercompensation) (Kuo et al., 1999). It is believed that the increase in glycogen storage, post-exercise, is due to the increased GLUT4 protein expression induced by exercise (Ivy and Kuo, 1998; Dohm, 2002). This adaptation is presumably beneficial in tolerating low PO2 in skeletal muscle. Similarly, it was found that reducing oxygen supply, by systemic hypoxia, increases GLUT4 protein expression in skeletal muscle (Dill et al., 2001; Xia et al., 1997). This evidence led us to speculate that low oxygen tension (or tissue ischemia) may be an important signal in the upregulation of GLUT4 protein in exercised skeletal muscle. In
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this study, we asked the question of whether muscle tissue ischemia can simulate the exercise training effect on GLUT4 protein expression and glycogen storage. The interactive effects of ischemia and exercise training were also investigated. For the first experiment, we determined the acute effect of 1-h ischemia on GLUT4 protein expression and glycogen storage in both the exercise-trained and untrained hindlimb muscles. For the second experiment, the chronic effects of ischemia (daily 1-h ischemia for 1 week) on the exercise-trained and untrained hindlimb muscles were determined. Materials and methods Animals Male Sprague –Dawley rats weighing 180– 190 g were housed in a room maintained on a 0700 to 1900 h light cycle and at a temperature of 21 -C. The rats were allowed free access to water and chow (PMI Nutrition International, Brentwood, Missouri, USA) except when indicated. All procedures were approved by the Animal Care and Use Committee of the Taipei Physical Education College and conformed to The Guideline for the Use of Laboratory Animal published by the Council of Agriculture, Executive Yuan, Taiwan ROC. Experimental design and procedures Two experiments were performed. For each experiment, rats were randomly assigned into two groups: exercise-trained (N = 8) and untrained (N = 8). For the first experiment, rats were exercised 3 h a day by swimming for 1 week (water maintained at 34 -C). Five hour after the last bout of exercise, tourniquet ischemia was applied for 1 h on one hindlimb followed by a 30-min recovery, whereas the contralateral hindlimb served as control. Ischemia was induced by placing a tourniquet around the upper hindlimb and proximal to the knee joint. To ensure total occlusion of blood flow to the hindlimb, a 350-mmHg pressure was employed. The animals were then anesthetized 30 min after completion of the ischemia for muscle sampling. For the second experiment, the same exercise training protocol was performed as in the acute ischemia study. However, ischemia was applied everyday, 5 h following the exercise bout. Rats were then anesthetized 5-h after completion of the exercise protocol for muscle sampling. All rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (65.0 mg/kg body wt). Following muscle sampling, rats were euthanized by a cardiac injection of pentobarbital sodium. Muscle sampling consisted of excising the red gastrocnemius of both legs. Plantaris muscle was used to determine the muscle mass. Muscles were rapidly separated over ice and clamped frozen with tongs cooled in liquid N2.
30 min. Dissolved homogenate was neutralized by glacial acetic acid and incubated overnight in acetate buffer (0.3 M sodium acetate, pH to 4.8) containing amyloglucosidase (Boehringer Mannheim, Indianapolis, IN). The reaction mixture was neutralized with 1 N NaOH. Samples were then analyzed by measuring glucosyl units by the Trinder reaction (Sigma, St. Louis, MO). Western blotting analysis for GLUT4 About 50 mg of RG was homogenized (1 : 20) in 20 mM ice-cold HEPES, 1 mM EDTA, and 250 mM sucrose buffer (HES buffer, pH 7.4) with a Polytron (Brinkmann Instrument, Littau, Switzerland). The protein concentration of the homogenate was determined using a BioRad protein assay reagent (Richmond, CA, USA), according to the manufacturer’s instructions. Sample homogenates and standards were diluted 1 : 1 with Laemmli sample buffer (125 mM Tris, 20% glycerol, 2% SDS, 0.008% bromophenol blue, pH 6.8). Fractionated protein from sample homogenates were transferred to a polyvinylidene fluoride (PVDF) membrane. GLUT4 antiserum directed against the carboxyl terminus was used for immunoblotting in a dilution of 5 1 : 5000 (Chemicon, Temecula, CA, USA). GLUT4 protein was visualized using an ECL Western Blot Detection Kit containing a secondary antibody against rabbit antibody (Amersham, Arlington Heights, IL, USA) on Kodak film according to the manufacturer’s instructions. Northern blotting analysis for GLUT4 mRNA The Northern blotting procedure was used to determine GLUT4 mRNA levels. For RNA extraction, RG was homogenized in guanidium isothiocyanate-beta-mercaptoethanol buffer with a Polytron. Total RNA was purified by a phenol – chloroform extraction method, and the RNA in the aqueous layer was then precipitated in 2 steps using isopropanol and ethanol. Total RNA (10 Ag) isolated from each tissue samples was denatured by heating at 60 -C for 10 min, then separated on a 1% agarose – formaldehyde gel, and subjected to vacuum transfer onto a nylon membrane. Hybridization with GLUT4 cRNA was performed according to the manufacturer’s instructions. A GLUT4 cDNA template was used to generate a fluorescent antisense GLUT4 probe using the Northern Start Kit (Roche Applied Science) for the hybridization procedure. 28 S rRNA on the striped blot of the ethidium bromide-stained gel was used for determining both the integrity and equal loading of total RNA on the gel. Autoradiographs of GLUT4 protein and GLUT4 mRNA were quantitated by a densitometric method with NIH imaging software (National Institutes of Health, Bethesda, MD, USA), according to the software instructions.
Glycogen assay
Statistical analysis
About 50 mg of skeletal muscle from the red portion of the gastrocnemius (RG) was dissolved in 1 N KOH at 70 -C for
A paired t test to distinguish mean difference between ischemia and non-ischemia muscle was performed on all
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0.45
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NIS IS
GLUT4 mRNA
0.4 28S rRNA
0.3 18S rRNA
0.25 0.2
1.6
0.15
1.4
0.1 0.05 0 Control
Exercise
Fig. 1. Effect of acute 1-h ischemia on the plantaris muscle mass in exercisetrained and untrained rats. IS: ischemia limb; NIS: non-IS limb.
variables. An independent Student t test was used to determine the mean difference between trained and untrained groups. A level of p < 0.05 was set for significance for all tests, and all values are expressed as means T SE.
RG GLUT4 mRNA (% Control)
Muscle weight (gram)
0.35
NIS IS
1.2 1 0.8 *#
0.6 0.4 0.2 0 Control
Results Acute ischemia effect An acute 1 –h ischemia did not significantly affect the muscle mass of plantaris of both trained and untrained rats (Fig. 1). Ischemia did not significantly change glycogen levels
Fig. 3. Effect of acute 1-h ischemia on GLUT4 mRNA of the red gastrocnemius muscle in exercise-trained and untrained rats. Representative autoradiograph of Northern blots is illustrated above the histogram. IS: ischemia limb; NIS: nonIS limb. *Significantly different from the IS level ( P < 0.05). #Significantly different from the control ( P < 0.05).
45
1.8
35
RG glycogen (µmol/g)
2
#
RG GLUT4 protein (% Control)
40
NIS IS
30 25 20 15
Exercise
# NIS IS
1.6 1.4 1.2 * 1 0.8 0.6 0.4
10 0.2 5
0 Control
Exercise
0 Control
Exercise
Fig. 2. Effect of acute 1-h ischemia on glycogen of the red gastrocnemius muscle in exercise-trained and untrained rats. IS: ischemia limb; NIS: non-IS limb. #Significantly different from the control ( P < 0.05).
Fig. 4. Effect of acute 1-h ischemia on GLUT4 protein of red gastrocnemius muscle in exercise-trained and untrained rats. Representative autoradiograph of Western blot is illustrated above the histogram. IS: ischemia limb; NIS: non-IS limb. *Significantly different from the IS level ( P < 0.05). #Significantly different from the control ( P < 0.05).
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0.4
NIS IS
Chronic ischemia effect
0.35
Muscle weight (gram)
0.3 * 0.25 0.2
*#
0.15 0.1 0.05 0 Control
Daily 1-h ischemia for 1 week (chronic ischemia) significantly lowered muscle mass in both trained and untrained rats (Fig. 5). Muscle mass in trained ischemic muscles were significantly lower than that in untrained ischemic muscles. Glycogen data are shown in Fig. 6. Exercise training significantly elevated muscle glycogen levels above untrained levels. Chronic ischemia did not affect glycogen levels in the untrained muscles. In the trained group, glycogen was significantly reduced by chronic ischemia. Chronic ischemia significantly lowered GLUT4 mRNA levels in both trained and untrained muscles (Fig. 7). GLUT4 protein data are shown in Fig. 8. Exercise training significantly elevated GLUT4 protein level in non-ischemic muscle when compared to the untrained group. Chronic ischemia dramatically lowered GLUT4 protein levels in both trained and untrained muscles.
Exercise
Fig. 5. Effect of 1-h ischemia repeated for 1 week on plantaris muscle mass in exercise-trained and untrained rats. IS: ischemia limb; NIS: non-IS limb. *Significantly different from the IS level ( P < 0.05). #Significantly different from the IS control ( P < 0.05).
Discussion The first novel finding of the study is that the normal increase in GLUT4 protein expression that is normally seen with exercise training was prevented by 1-h of ischemia.
in the trained or untrained muscle (Fig. 2). Exercise training significantly elevated muscle glycogen levels above untrained levels. Muscle GLUT4 mRNA data are shown in Fig. 3. Acute 1 –h ischemia did not affect GLUT4 mRNA level in untrained muscle. Whereas, in the trained muscle, GLUT4 mRNA of ischemic muscles were significantly lower than the nonischemic control. GLUT4 protein data are shown in Fig. 4. One-week of exercise training significantly increased GLUT4 protein in non-ischemic muscle, but this increase was completely abolished by 1-h ischemia.
40
NIS IS
RG glycogen (µmol/g)
18S rRNA
1.6
#
1.4
35 *
30
28S rRNA
25 20 15 10
RG GLUT4 mRNA (% Control)
45
GLUT4 mRNA
NIS IS
1.2 1 *
0.8
*
0.6 0.4 0.2
5 0
0 Control
Exercise
Fig. 6. Effect of acute 1-h ischemia repeated for 1 week on glycogen of red gastrocnemius muscle in exercise-trained and untrained rats. IS: ischemia limb; NIS: non-IS limb. *Significantly different from the IS level ( P < 0.05). # Significantly different from the control ( P < 0.05).
Control
Exercise
Fig. 7. Effect of acute 1-h ischemia repeated for 1 week on GLUT4 mRNA of red gastrocnemius muscle in exercise-trained and untrained rats. Representative autoradiograph of Northern blot is illustrated above the histogram. IS: ischemia limb; NIS: non-IS limb. *Significantly different from the IS level ( P < 0.05).
Y.-L. Tsai et al. / Life Sciences 78 (2006) 2953 – 2959
2
RG GLUT4 protein (% Control)
1.8
NIS IS
#
1.6 1.4 1.2 1 0.8 *
0.6 *
0.4 0.2 0 Control
Exercise
Fig. 8. Effect of acute 1-h ischemia repeated for 1 week on GLUT4 protein of red gastrocnemius muscle in exercise-trained and untrained rats. Representative autoradiograph of Western blots is illustrated above the histogram. IS: ischemia limb; NIS: non-IS limb. *Significantly different from the IS level ( P < 0.05). # Significantly different from the control ( P < 0.05).
GLUT4 mRNA in the exercise-trained group was also reduced by acute ischemia when compared to the untrained group, whereas this acute ischemia treatment did not significantly affect GLUT4 mRNA or protein levels in the untrained group. These results indicate that the acute ischemia strongly inhibited the exercise training effect on GLUT4 gene expression at the pretranslational level. The second novel finding of the study is that chronic ischemia (daily 1-h ischemia for a week) caused a remarkable reduction in muscle GLUT4 mRNA and protein regardless of whether the muscle was exercise-trained or not. In skeletal muscle, GLUT4 protein is the main glucose transporter protein and it plays the key role in insulin’s ability to stimulate glycogen storage (Tsao et al., 1996). Exercise training with a 5 h recovery is known to increase muscle glycogen storage above normal, which is associated with the increased expression of the GLUT4 protein (Ivy and Kuo, 1998). Thus, inhibition of GLUT4 protein expression by chronic ischemia suggests that glycogen storage capability of muscle could be blunted by ischemia, which may in turn explain the current observation that the exercise-induced increase in glycogen storage was eliminated by chronic ischemia. In the untrained muscle, glycogen levels in the acute ischemic muscle were not different from that of the nonischemic contralateral limb. This result is different from previous studies in that 1-h of tourniquet ischemia significantly reduced glycogen levels (Carvalho et al., 1996; Fraser et al., 1998). It could be due to the treatment protocol utilized, allowing a 30-min recovery following ischemia, which elicited a rapid hypoxia-induced increase in glucose transport. It has been frequently shown that hypoxic skeletal muscle can
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increase the rate of glucose transport rapidly (Cartee et al., 1991), by increasing the number of GLUT4 protein at plasma membrane (Ryder et al., 2000). Therefore, the discrepancy might be explained by an (post-ischemia recovery) increase in glucose influx due to increased intracellular GLUT4 protein translocation to the plasma membrane, which could lead to the fast recovery of glycogen storage. Our GLUT4 expression results indicate that the current tourniquet ischemia protocol is not comparable to systemic hypoxia and exercise training, which lowers tissue oxygen level. Previous studies by Dill et al. (2001) and Xia et al. (1997) show that GLUT4 protein in muscle is elevated by prolonged systemic hypoxia. Chiu et al. (2004) has further noted that chronic intermittent hypoxia significantly enhances the increase in muscle GLUT4 gene expression induced by exercise. Yet, the current study observed that ischemia to the hindlimb causes an opposite effect on GLUT4 expression in exercise-trained skeletal muscle. Ischemia treatment by tourniquet occlusion was done to create anoxia (total lack of oxygen) in skeletal muscle of the hindlimb, however, this may have been a more severe treatment when compared to systemic hypoxia and exercise. In addition, tourniquet ischemia could also impair peripheral nerve function, which in turn could decrease the normal exercise-training response in muscle GLUT4 gene expression. It has been reported that tourniquet ischemia application could slow down conduction velocity of the sciatic nerve (Rorabeck and Kennedy, 1980). This may explain the reason why ischemia prevented the exercise-training effect on GLUT4 gene expression. Exercise not only recruits muscle fibers, but also produces neurogenic factors regulating protein expression in recruited muscles (Gomez-Pinilla et al., 2002; Delbono, 2003). Elimination of neurogenic factors by denervation has been shown to inhibit muscle GLUT4 gene expression at the pretranslational level (Megeney et al., 1994; Paulsen et al., 2001), which is similar to the current result in GLUT4 mRNA and protein with chronic ischemia. Another interesting finding of the study is that ischemiainduced muscle atrophy was accelerated in the exercise-trained plantaris muscle. The observed muscle atrophy in the plantaris could be related to the impaired neuronal function by tourniquet ischemia. The importance of neuronal factor on muscle mass can be directly demonstrated by the evidence that sciatic nerve denervation results in rapid reduction in muscle mass and fiber diameters in muscle (Herbison et al., 1979). Exercise training has been suggested to prevent various conditions causing muscle atrophy (Kouidi et al., 1998; Kirby et al., 1992). In contrast, the current study clearly indicated that ischemia can be detrimental particularly to the exercisetrained skeletal muscle. The maintenance of appropriate muscle mass depends on a delicate balance between the rates of catabolic and anabolic activities in muscle. Catabolic signals are generally induced during exercise, which is normally counterbalanced by a protracted increase in anabolic activity during recovery. Apparently, tourniquet ischemia could be increasing catabolic stress beyond what typically occurs during exercise or decreasing the anabolic signal post-
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exercise (probably neurogenic factors), which can ultimately cause muscle atrophy. Impaired peripheral microcirculation (peripheral arterial disease) is a common complication of type 2 diabetes (Leibson et al., 2004). This pathogenic condition is characterized by cold feet and atrophy of peripheral tissue (Regensteiner et al., 1993). To some extent, this condition is similar to the current ischemia model. Exercise training is normally encouraged for diabetic patients based on the fact that exercise training is able to increase GLUT4 protein expression, capillary density, insulin sensitivity, glucose tolerance, and preventing muscle atrophy (Holloszy et al., 1986; Ivy et al., 1999). However, very few studies have investigated the effects of exercise training in diabetic subjects with seriously impaired peripheral microcirculation. Thus, whether exercise training is suitable for patients with seriously-impaired microcirculation warrants further evaluation. Conclusions In conclusion, previous studies have shown that hypoxia enhances the exercise-induced increases in GLUT4 protein and glycogen storage in skeletal muscle. However, in the current study, we found that the normal training effect on GLUT4 gene expression was completely eliminated by both acute and chronic ischemia at the pretranslational level. In addition, the chronic ischemia-induced muscle atrophy was more severe in the exercise-trained rats than in the untrained rats. This result suggests that for individuals with impaired microvascular conditions, exercise training might not be beneficial in maintaining muscle mass. Acknowledgements This study was partially supported by a research grant from the National Science Council, Republic of China, grant number NSC 94-2413-H-154-007. References Cartee, G.D., Douen, A.G., Ramlal, T., Klip, A., Holloszy, J.O., 1991. Stimulation of glucose transport in skeletal muscle by hypoxia. Journal of Applied Physiology 70 (4), 1593 – 1600. Carvalho, A.J., McKee, N.H., Green, H.J., 1996. Metabolic and contractile responses of fast- and slow-twitch rat skeletal muscles to ischemia. Canadian Journal of Physiology and Pharmacology 74 (12), 1333 – 1341. Chiu, L.L., Chou, S.W., Cho, Y.M., Ho, H.Y., Ivy, J.L., Hunt, D., Wang, P.S., Kuo, C.H., 2004. Effect of prolonged intermittent hypoxia and exercise training on glucose tolerance and muscle GLUT4 protein expression in rats. Journal of Biomedical Science 11 (6), 838 – 846. Delbono, O., 2003. Neural control of aging skeletal muscle. Aging Cell 2 (1), 21 – 29. Dill, R.P., Chadan, S.G., Li, C., Parkhouse, W.S., 2001. Aging and glucose transporter plasticity in response to hypobaric hypoxia. Mechanism of Ageing and Development 122 (6), 533 – 545. Dohm, G.L., 2002. Exercise effects on muscle insulin signaling and action: invited review: regulation of skeletal muscle GLUT-4 expression by exercise. Journal of Applied Physiology 93 (2), 782 – 787.
Fraser, H., Lopaschuk, G.D., Clanachan, A.S., 1998. Assessment of glycogen turnover in aerobic, ischemic, and reperfused working rat hearts. American Journal of Physiology 275 (5), H1533 – H1541. Gomez-Pinilla, F., Ying, Z., Roy, R.R., Molteni, R., Edgerton, V.R., 2002. Voluntary exercise induces a BDNF-mediated mechanism that promotes neuroplasticity. Journal of Neurophysiology 88 (5), 2187 – 2195. Hansen, P.A., Gulve, E.A., Marshall, B.A., Gao, J., Pessin, J.E., Holloszy, J.O., Mueckler, M., 1995. Skeletal muscle glucose transport and metabolism are enhanced in transgenic mice overexpressing the GLUT4 glucose transporter. Journal of Biological Chemistry 270 (5), 1679 – 1684. Herbison, G.J., Jaweed, M.M., Ditunno, J.F., 1979. Muscle atrophy in rats following denervation, casting, inflammation, and tenotomy. Archives of Physical Medicine and Rehabilitation 60 (9), 401 – 404. Holloszy, J.O., Schultz, J., Kusnierkiewicz, J., Hagberg, J.M., Ehsani, A.A., 1986. Effects of exercise on glucose tolerance and insulin resistance. Brief review and some preliminary results. Acta Medica Scandinavica. Supplementum 711, 55 – 65. Ivy, J.L., Kuo, C.H., 1998. Regulation of GLUT4 protein and glycogen synthase during muscle glycogen synthesis after exercise. Acta Physiologica Scandinavica 162 (3), 295 – 304. Ivy, J.L., Zderic, T.W., Fogt, D.L., 1999. Prevention and treatment of noninsulin-dependent diabetes mellitus. Exercise and Sport Sciences Reviews 27, 1 – 35. Katz, A., 1988. G-1,6-P2, glycolysis, and energy metabolism during circulatory occlusion in human skeletal muscle. American Journal of Physiology 255 (2), C140 – C144. Keller, V.A., Toporoff, B., Raziano, R.M., Pigott, J.D., Mills, N.L., 1998. Carnitine supplementation improves myocardial function in hearts from ischemic diabetic and euglycemic rats. Annals of Thoracic Surgery 66 (5), 1600 – 1603. Kirby, C.R., Ryan, M.J., Booth, F.W., 1992. Eccentric exercise training as a countermeasure to non-weight-bearing soleus muscle atrophy. Journal of Applied Physiology 73 (5), 1894 – 1899. Kouidi, E., Albani, M., Natsis, K., Megalopoulos, A., Gigis, P., GuibaTziampiri, O., Tourkantonis, A., Deligiannis, A., 1998. The effects of exercise training on muscle atrophy in haemodialysis patients. Nephrology Dialysis Transplantation 13 (3), 685 – 699. Kuo, C.H., Browning, K.S., Ivy, J.L., 1999. Regulation of GLUT4 protein expression and glycogen storage after prolonged exercise. Acta Physiologica Scandinavica 165 (2), 193 – 201. Leibson, C.L., Ransom, J.E., Olson, W., Zimmerman, B.R., O’Fallon, W.M., Palumbo, P.J., 2004. Peripheral arterial disease, diabetes, and mortality. Diabetes Care 27 (12), 2843 – 2849. Lopaschuk, G., 2000. Regulation of carbohydrate metabolism in ischemia and reperfusion. American Heart Journal 139 (2), S115 – S119. Megeney, L.A., Prasad, M.A., Tan, M.H., Bonen, A., 1994. Expression of the insulin-regulatable transporter GLUT-4 in muscle is influenced by neurogenic factors. American Journal of Physiology 266 (5), E813 – E816. Olson, A.L., Pessin, J.E., 1996. Structure, function, and regulation of the mammalian facilitative glucose transporter gene family. Annual Review of Nutrition 16, 235 – 256. Paulsen, S.R., Rubink, D.S., Winder, W.W., 2001. AMP-activated protein kinase activation prevents denervation-induced decline in gastrocnemius GLUT-4. Journal of Applied Physiology 91 (5), 2102 – 2108. Purshottam, T., Kaveeshwar, U., Brahmachari, H.D., 1977. Changes in tissue glycogen stores of rats under acute and chronic hypoxia and their relationship to hypoxia tolerance. Aviation Space & Environmental Medicine 48 (4), 351 – 355. Regensteiner, J.G., Wolfel, E.E., Brass, E.P., Carry, M.R., Ringel, S.P., Hargarten, M.E., Stamm, E.R., Hiatt, W.R., 1993. Chronic changes in skeletal muscle histology and function in peripheral arterial disease. Circulation 87 (2), 413 – 421. Ren, J.M., Marshall, B.A., Gulve, E.A., Gao, J., Johnson, D.W., Holloszy, J.O., Mueckler, M., 1993. Evidence from transgenic mice that glucose transport is rate-limiting for glycogen deposition and glycolysis in skeletal muscle. Journal of Biological Chemistry 268 (22), 16113 – 16115.
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Tsao, T.S., Burcelin, R., Katz, E.B., Huang, L., Charron, M.J., 1996. Enhanced insulin action due to targeted GLUT4 overexpression exclusively in muscle. Diabetes 45 (1), 28 – 36. Xia, Y., Warshaw, J.B., Haddad, G.G., 1997. Effect of chronic hypoxia on glucose transporters in heart and skeletal muscle of immature and adult rats. American Journal of Physiology 273 (5), R1734 – R1741.
Exercise training exacerbates tourniquet ischemia-induced decreases in GLUT4 expression and muscle atrophy in rats Ying-Lan Tsai c, Chien-Wen Hou a, Yi-Hung Liao a, Chung-Yu Chen a, Fang-Ching Lin a, Wen-Chih Lee b, Shih-Wei Chou d, Chia-Hua Kuo a,* a
c
Laboratory of Exercise Biochemistry, Taipei Physical Education College, 5 Dun-Hua N. Rd, Taipei 105, Taiwan, ROC b Committee of General Studies, Shih Hsin University, Taipei 116, Taiwan ROC Department of Athletic Training and Health, National College of Physical Education and Sports, Taoyuan 333, Taiwan, ROC d Department of Physical Medicine and Rehabilitation, Chang Gung Memorial Hospital, Taipei 333, Taiwan, ROC Received 29 August 2005; accepted 17 November 2005
Abstract The current study determined the interactive effects of ischemia and exercise training on glycogen storage and GLUT4 expression in skeletal muscle. For the first experiment, an acute 1-h tourniquet ischemia was applied to one hindlimb of both the 1-week exercise-trained and untrained rats. The contralateral hindlimb served as control. For the second experiment, 1-h ischemia was applied daily for 1 week to both trained (5 h postexercise) and untrained rats. GLUT4 mRNA was not affected by acute ischemia, but exercise training lowered GLUT4 mRNA in the acute ischemic muscle. GLUT4 protein levels were elevated by exercise training, but not in the acute ischemic muscle. Exercise training elevated muscle glycogen above untrained levels, but this increase was reversed by chronic ischemia. GLUT4 mRNA and protein levels were dramatically reduced by chronic ischemia, regardless of whether the animals were exercise-trained or not. Chronic ischemia significantly reduced plantaris muscle mass, with a greater decrease found in the exercise-trained rats. In conclusion, the exercise training effect on muscle GLUT4 protein expression was prevented by acute ischemia. Furthermore, chronic ischemia-induced muscle atrophy was exacerbated by exercise training. This result implicates that exercise training could be detrimental to skeletal muscle with severely impaired microcirculation. D 2005 Elsevier Inc. All rights reserved. Keywords: Hypoxia; Intermittent ischemia; Reperfusion; Glucose uptake; Glycogen; Rehabilitation; Muscle mass
Introduction To sustain normal cellular function under low arterial PO2, carbohydrate utilization increases in an effort to compensate for an energy deficit due to insufficient fat oxidation. This is demonstrated by the fact that ischemia significantly decreases muscle glycogen levels (Purshottam et al., 1977; Fraser et al., 1998; Carvalho et al., 1996; Katz, 1988); and treatments that enhances carbohydrate metabolism protects muscles from severe ischemic damage (Lopaschuk, 2000; Keller et al., 1998). GLUT4 protein, the main glucose transporter expressed in skeletal muscle (Olson and Pessin, 1996), is found to play a
* Corresponding author. Tel.: +886 2 25774624x831; fax: +886 2 25790526. E-mail address: [email protected] (C.-H. Kuo). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.11.021
key role in the regulation of muscle glycogen storage (Ren et al., 1993; Hansen et al., 1995; Tsao et al., 1996). Glycogen is the most abundant anaerobic fuel source in skeletal muscle. Exercise training, with sufficient recovery (5– 16 h in rats), will result in elevated muscle glycogen storage above pre-exercise levels (termed glycogen supercompensation) (Kuo et al., 1999). It is believed that the increase in glycogen storage, post-exercise, is due to the increased GLUT4 protein expression induced by exercise (Ivy and Kuo, 1998; Dohm, 2002). This adaptation is presumably beneficial in tolerating low PO2 in skeletal muscle. Similarly, it was found that reducing oxygen supply, by systemic hypoxia, increases GLUT4 protein expression in skeletal muscle (Dill et al., 2001; Xia et al., 1997). This evidence led us to speculate that low oxygen tension (or tissue ischemia) may be an important signal in the upregulation of GLUT4 protein in exercised skeletal muscle. In
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this study, we asked the question of whether muscle tissue ischemia can simulate the exercise training effect on GLUT4 protein expression and glycogen storage. The interactive effects of ischemia and exercise training were also investigated. For the first experiment, we determined the acute effect of 1-h ischemia on GLUT4 protein expression and glycogen storage in both the exercise-trained and untrained hindlimb muscles. For the second experiment, the chronic effects of ischemia (daily 1-h ischemia for 1 week) on the exercise-trained and untrained hindlimb muscles were determined. Materials and methods Animals Male Sprague –Dawley rats weighing 180– 190 g were housed in a room maintained on a 0700 to 1900 h light cycle and at a temperature of 21 -C. The rats were allowed free access to water and chow (PMI Nutrition International, Brentwood, Missouri, USA) except when indicated. All procedures were approved by the Animal Care and Use Committee of the Taipei Physical Education College and conformed to The Guideline for the Use of Laboratory Animal published by the Council of Agriculture, Executive Yuan, Taiwan ROC. Experimental design and procedures Two experiments were performed. For each experiment, rats were randomly assigned into two groups: exercise-trained (N = 8) and untrained (N = 8). For the first experiment, rats were exercised 3 h a day by swimming for 1 week (water maintained at 34 -C). Five hour after the last bout of exercise, tourniquet ischemia was applied for 1 h on one hindlimb followed by a 30-min recovery, whereas the contralateral hindlimb served as control. Ischemia was induced by placing a tourniquet around the upper hindlimb and proximal to the knee joint. To ensure total occlusion of blood flow to the hindlimb, a 350-mmHg pressure was employed. The animals were then anesthetized 30 min after completion of the ischemia for muscle sampling. For the second experiment, the same exercise training protocol was performed as in the acute ischemia study. However, ischemia was applied everyday, 5 h following the exercise bout. Rats were then anesthetized 5-h after completion of the exercise protocol for muscle sampling. All rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (65.0 mg/kg body wt). Following muscle sampling, rats were euthanized by a cardiac injection of pentobarbital sodium. Muscle sampling consisted of excising the red gastrocnemius of both legs. Plantaris muscle was used to determine the muscle mass. Muscles were rapidly separated over ice and clamped frozen with tongs cooled in liquid N2.
30 min. Dissolved homogenate was neutralized by glacial acetic acid and incubated overnight in acetate buffer (0.3 M sodium acetate, pH to 4.8) containing amyloglucosidase (Boehringer Mannheim, Indianapolis, IN). The reaction mixture was neutralized with 1 N NaOH. Samples were then analyzed by measuring glucosyl units by the Trinder reaction (Sigma, St. Louis, MO). Western blotting analysis for GLUT4 About 50 mg of RG was homogenized (1 : 20) in 20 mM ice-cold HEPES, 1 mM EDTA, and 250 mM sucrose buffer (HES buffer, pH 7.4) with a Polytron (Brinkmann Instrument, Littau, Switzerland). The protein concentration of the homogenate was determined using a BioRad protein assay reagent (Richmond, CA, USA), according to the manufacturer’s instructions. Sample homogenates and standards were diluted 1 : 1 with Laemmli sample buffer (125 mM Tris, 20% glycerol, 2% SDS, 0.008% bromophenol blue, pH 6.8). Fractionated protein from sample homogenates were transferred to a polyvinylidene fluoride (PVDF) membrane. GLUT4 antiserum directed against the carboxyl terminus was used for immunoblotting in a dilution of 5 1 : 5000 (Chemicon, Temecula, CA, USA). GLUT4 protein was visualized using an ECL Western Blot Detection Kit containing a secondary antibody against rabbit antibody (Amersham, Arlington Heights, IL, USA) on Kodak film according to the manufacturer’s instructions. Northern blotting analysis for GLUT4 mRNA The Northern blotting procedure was used to determine GLUT4 mRNA levels. For RNA extraction, RG was homogenized in guanidium isothiocyanate-beta-mercaptoethanol buffer with a Polytron. Total RNA was purified by a phenol – chloroform extraction method, and the RNA in the aqueous layer was then precipitated in 2 steps using isopropanol and ethanol. Total RNA (10 Ag) isolated from each tissue samples was denatured by heating at 60 -C for 10 min, then separated on a 1% agarose – formaldehyde gel, and subjected to vacuum transfer onto a nylon membrane. Hybridization with GLUT4 cRNA was performed according to the manufacturer’s instructions. A GLUT4 cDNA template was used to generate a fluorescent antisense GLUT4 probe using the Northern Start Kit (Roche Applied Science) for the hybridization procedure. 28 S rRNA on the striped blot of the ethidium bromide-stained gel was used for determining both the integrity and equal loading of total RNA on the gel. Autoradiographs of GLUT4 protein and GLUT4 mRNA were quantitated by a densitometric method with NIH imaging software (National Institutes of Health, Bethesda, MD, USA), according to the software instructions.
Glycogen assay
Statistical analysis
About 50 mg of skeletal muscle from the red portion of the gastrocnemius (RG) was dissolved in 1 N KOH at 70 -C for
A paired t test to distinguish mean difference between ischemia and non-ischemia muscle was performed on all
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0.45
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NIS IS
GLUT4 mRNA
0.4 28S rRNA
0.3 18S rRNA
0.25 0.2
1.6
0.15
1.4
0.1 0.05 0 Control
Exercise
Fig. 1. Effect of acute 1-h ischemia on the plantaris muscle mass in exercisetrained and untrained rats. IS: ischemia limb; NIS: non-IS limb.
variables. An independent Student t test was used to determine the mean difference between trained and untrained groups. A level of p < 0.05 was set for significance for all tests, and all values are expressed as means T SE.
RG GLUT4 mRNA (% Control)
Muscle weight (gram)
0.35
NIS IS
1.2 1 0.8 *#
0.6 0.4 0.2 0 Control
Results Acute ischemia effect An acute 1 –h ischemia did not significantly affect the muscle mass of plantaris of both trained and untrained rats (Fig. 1). Ischemia did not significantly change glycogen levels
Fig. 3. Effect of acute 1-h ischemia on GLUT4 mRNA of the red gastrocnemius muscle in exercise-trained and untrained rats. Representative autoradiograph of Northern blots is illustrated above the histogram. IS: ischemia limb; NIS: nonIS limb. *Significantly different from the IS level ( P < 0.05). #Significantly different from the control ( P < 0.05).
45
1.8
35
RG glycogen (µmol/g)
2
#
RG GLUT4 protein (% Control)
40
NIS IS
30 25 20 15
Exercise
# NIS IS
1.6 1.4 1.2 * 1 0.8 0.6 0.4
10 0.2 5
0 Control
Exercise
0 Control
Exercise
Fig. 2. Effect of acute 1-h ischemia on glycogen of the red gastrocnemius muscle in exercise-trained and untrained rats. IS: ischemia limb; NIS: non-IS limb. #Significantly different from the control ( P < 0.05).
Fig. 4. Effect of acute 1-h ischemia on GLUT4 protein of red gastrocnemius muscle in exercise-trained and untrained rats. Representative autoradiograph of Western blot is illustrated above the histogram. IS: ischemia limb; NIS: non-IS limb. *Significantly different from the IS level ( P < 0.05). #Significantly different from the control ( P < 0.05).
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0.4
NIS IS
Chronic ischemia effect
0.35
Muscle weight (gram)
0.3 * 0.25 0.2
*#
0.15 0.1 0.05 0 Control
Daily 1-h ischemia for 1 week (chronic ischemia) significantly lowered muscle mass in both trained and untrained rats (Fig. 5). Muscle mass in trained ischemic muscles were significantly lower than that in untrained ischemic muscles. Glycogen data are shown in Fig. 6. Exercise training significantly elevated muscle glycogen levels above untrained levels. Chronic ischemia did not affect glycogen levels in the untrained muscles. In the trained group, glycogen was significantly reduced by chronic ischemia. Chronic ischemia significantly lowered GLUT4 mRNA levels in both trained and untrained muscles (Fig. 7). GLUT4 protein data are shown in Fig. 8. Exercise training significantly elevated GLUT4 protein level in non-ischemic muscle when compared to the untrained group. Chronic ischemia dramatically lowered GLUT4 protein levels in both trained and untrained muscles.
Exercise
Fig. 5. Effect of 1-h ischemia repeated for 1 week on plantaris muscle mass in exercise-trained and untrained rats. IS: ischemia limb; NIS: non-IS limb. *Significantly different from the IS level ( P < 0.05). #Significantly different from the IS control ( P < 0.05).
Discussion The first novel finding of the study is that the normal increase in GLUT4 protein expression that is normally seen with exercise training was prevented by 1-h of ischemia.
in the trained or untrained muscle (Fig. 2). Exercise training significantly elevated muscle glycogen levels above untrained levels. Muscle GLUT4 mRNA data are shown in Fig. 3. Acute 1 –h ischemia did not affect GLUT4 mRNA level in untrained muscle. Whereas, in the trained muscle, GLUT4 mRNA of ischemic muscles were significantly lower than the nonischemic control. GLUT4 protein data are shown in Fig. 4. One-week of exercise training significantly increased GLUT4 protein in non-ischemic muscle, but this increase was completely abolished by 1-h ischemia.
40
NIS IS
RG glycogen (µmol/g)
18S rRNA
1.6
#
1.4
35 *
30
28S rRNA
25 20 15 10
RG GLUT4 mRNA (% Control)
45
GLUT4 mRNA
NIS IS
1.2 1 *
0.8
*
0.6 0.4 0.2
5 0
0 Control
Exercise
Fig. 6. Effect of acute 1-h ischemia repeated for 1 week on glycogen of red gastrocnemius muscle in exercise-trained and untrained rats. IS: ischemia limb; NIS: non-IS limb. *Significantly different from the IS level ( P < 0.05). # Significantly different from the control ( P < 0.05).
Control
Exercise
Fig. 7. Effect of acute 1-h ischemia repeated for 1 week on GLUT4 mRNA of red gastrocnemius muscle in exercise-trained and untrained rats. Representative autoradiograph of Northern blot is illustrated above the histogram. IS: ischemia limb; NIS: non-IS limb. *Significantly different from the IS level ( P < 0.05).
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2
RG GLUT4 protein (% Control)
1.8
NIS IS
#
1.6 1.4 1.2 1 0.8 *
0.6 *
0.4 0.2 0 Control
Exercise
Fig. 8. Effect of acute 1-h ischemia repeated for 1 week on GLUT4 protein of red gastrocnemius muscle in exercise-trained and untrained rats. Representative autoradiograph of Western blots is illustrated above the histogram. IS: ischemia limb; NIS: non-IS limb. *Significantly different from the IS level ( P < 0.05). # Significantly different from the control ( P < 0.05).
GLUT4 mRNA in the exercise-trained group was also reduced by acute ischemia when compared to the untrained group, whereas this acute ischemia treatment did not significantly affect GLUT4 mRNA or protein levels in the untrained group. These results indicate that the acute ischemia strongly inhibited the exercise training effect on GLUT4 gene expression at the pretranslational level. The second novel finding of the study is that chronic ischemia (daily 1-h ischemia for a week) caused a remarkable reduction in muscle GLUT4 mRNA and protein regardless of whether the muscle was exercise-trained or not. In skeletal muscle, GLUT4 protein is the main glucose transporter protein and it plays the key role in insulin’s ability to stimulate glycogen storage (Tsao et al., 1996). Exercise training with a 5 h recovery is known to increase muscle glycogen storage above normal, which is associated with the increased expression of the GLUT4 protein (Ivy and Kuo, 1998). Thus, inhibition of GLUT4 protein expression by chronic ischemia suggests that glycogen storage capability of muscle could be blunted by ischemia, which may in turn explain the current observation that the exercise-induced increase in glycogen storage was eliminated by chronic ischemia. In the untrained muscle, glycogen levels in the acute ischemic muscle were not different from that of the nonischemic contralateral limb. This result is different from previous studies in that 1-h of tourniquet ischemia significantly reduced glycogen levels (Carvalho et al., 1996; Fraser et al., 1998). It could be due to the treatment protocol utilized, allowing a 30-min recovery following ischemia, which elicited a rapid hypoxia-induced increase in glucose transport. It has been frequently shown that hypoxic skeletal muscle can
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increase the rate of glucose transport rapidly (Cartee et al., 1991), by increasing the number of GLUT4 protein at plasma membrane (Ryder et al., 2000). Therefore, the discrepancy might be explained by an (post-ischemia recovery) increase in glucose influx due to increased intracellular GLUT4 protein translocation to the plasma membrane, which could lead to the fast recovery of glycogen storage. Our GLUT4 expression results indicate that the current tourniquet ischemia protocol is not comparable to systemic hypoxia and exercise training, which lowers tissue oxygen level. Previous studies by Dill et al. (2001) and Xia et al. (1997) show that GLUT4 protein in muscle is elevated by prolonged systemic hypoxia. Chiu et al. (2004) has further noted that chronic intermittent hypoxia significantly enhances the increase in muscle GLUT4 gene expression induced by exercise. Yet, the current study observed that ischemia to the hindlimb causes an opposite effect on GLUT4 expression in exercise-trained skeletal muscle. Ischemia treatment by tourniquet occlusion was done to create anoxia (total lack of oxygen) in skeletal muscle of the hindlimb, however, this may have been a more severe treatment when compared to systemic hypoxia and exercise. In addition, tourniquet ischemia could also impair peripheral nerve function, which in turn could decrease the normal exercise-training response in muscle GLUT4 gene expression. It has been reported that tourniquet ischemia application could slow down conduction velocity of the sciatic nerve (Rorabeck and Kennedy, 1980). This may explain the reason why ischemia prevented the exercise-training effect on GLUT4 gene expression. Exercise not only recruits muscle fibers, but also produces neurogenic factors regulating protein expression in recruited muscles (Gomez-Pinilla et al., 2002; Delbono, 2003). Elimination of neurogenic factors by denervation has been shown to inhibit muscle GLUT4 gene expression at the pretranslational level (Megeney et al., 1994; Paulsen et al., 2001), which is similar to the current result in GLUT4 mRNA and protein with chronic ischemia. Another interesting finding of the study is that ischemiainduced muscle atrophy was accelerated in the exercise-trained plantaris muscle. The observed muscle atrophy in the plantaris could be related to the impaired neuronal function by tourniquet ischemia. The importance of neuronal factor on muscle mass can be directly demonstrated by the evidence that sciatic nerve denervation results in rapid reduction in muscle mass and fiber diameters in muscle (Herbison et al., 1979). Exercise training has been suggested to prevent various conditions causing muscle atrophy (Kouidi et al., 1998; Kirby et al., 1992). In contrast, the current study clearly indicated that ischemia can be detrimental particularly to the exercisetrained skeletal muscle. The maintenance of appropriate muscle mass depends on a delicate balance between the rates of catabolic and anabolic activities in muscle. Catabolic signals are generally induced during exercise, which is normally counterbalanced by a protracted increase in anabolic activity during recovery. Apparently, tourniquet ischemia could be increasing catabolic stress beyond what typically occurs during exercise or decreasing the anabolic signal post-
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exercise (probably neurogenic factors), which can ultimately cause muscle atrophy. Impaired peripheral microcirculation (peripheral arterial disease) is a common complication of type 2 diabetes (Leibson et al., 2004). This pathogenic condition is characterized by cold feet and atrophy of peripheral tissue (Regensteiner et al., 1993). To some extent, this condition is similar to the current ischemia model. Exercise training is normally encouraged for diabetic patients based on the fact that exercise training is able to increase GLUT4 protein expression, capillary density, insulin sensitivity, glucose tolerance, and preventing muscle atrophy (Holloszy et al., 1986; Ivy et al., 1999). However, very few studies have investigated the effects of exercise training in diabetic subjects with seriously impaired peripheral microcirculation. Thus, whether exercise training is suitable for patients with seriously-impaired microcirculation warrants further evaluation. Conclusions In conclusion, previous studies have shown that hypoxia enhances the exercise-induced increases in GLUT4 protein and glycogen storage in skeletal muscle. However, in the current study, we found that the normal training effect on GLUT4 gene expression was completely eliminated by both acute and chronic ischemia at the pretranslational level. In addition, the chronic ischemia-induced muscle atrophy was more severe in the exercise-trained rats than in the untrained rats. This result suggests that for individuals with impaired microvascular conditions, exercise training might not be beneficial in maintaining muscle mass. Acknowledgements This study was partially supported by a research grant from the National Science Council, Republic of China, grant number NSC 94-2413-H-154-007. References Cartee, G.D., Douen, A.G., Ramlal, T., Klip, A., Holloszy, J.O., 1991. Stimulation of glucose transport in skeletal muscle by hypoxia. Journal of Applied Physiology 70 (4), 1593 – 1600. Carvalho, A.J., McKee, N.H., Green, H.J., 1996. Metabolic and contractile responses of fast- and slow-twitch rat skeletal muscles to ischemia. Canadian Journal of Physiology and Pharmacology 74 (12), 1333 – 1341. Chiu, L.L., Chou, S.W., Cho, Y.M., Ho, H.Y., Ivy, J.L., Hunt, D., Wang, P.S., Kuo, C.H., 2004. Effect of prolonged intermittent hypoxia and exercise training on glucose tolerance and muscle GLUT4 protein expression in rats. Journal of Biomedical Science 11 (6), 838 – 846. Delbono, O., 2003. Neural control of aging skeletal muscle. Aging Cell 2 (1), 21 – 29. Dill, R.P., Chadan, S.G., Li, C., Parkhouse, W.S., 2001. Aging and glucose transporter plasticity in response to hypobaric hypoxia. Mechanism of Ageing and Development 122 (6), 533 – 545. Dohm, G.L., 2002. Exercise effects on muscle insulin signaling and action: invited review: regulation of skeletal muscle GLUT-4 expression by exercise. Journal of Applied Physiology 93 (2), 782 – 787.
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Tsao, T.S., Burcelin, R., Katz, E.B., Huang, L., Charron, M.J., 1996. Enhanced insulin action due to targeted GLUT4 overexpression exclusively in muscle. Diabetes 45 (1), 28 – 36. Xia, Y., Warshaw, J.B., Haddad, G.G., 1997. Effect of chronic hypoxia on glucose transporters in heart and skeletal muscle of immature and adult rats. American Journal of Physiology 273 (5), R1734 – R1741.