Adaptive Medicine 4(1): 33-39, 2012 DOI: 10.4247/AM.2012.

ABB019

33

Attenuation of Age-Dependent Lipid Profile by Treadmill Running in Different Skeletal Muscle Fibers of Old Rats
Korivi Mallikarjuna 1, 2, Reddy T. Bhaskar 1, Kondeti R. Shanmugam 1, and Reddy K. Sathyavelu 1
1

Division of Molecular Biology and Exercise Physiology, Department of Zoology, Sri Venkateswara University, Tirupati - 517 502, A. P, India 2 Laboratory of Exercise Biochemistry, Department of Sports Sciences, Taipei Physical Education College, Taipei 11153, Taiwan, Republic of China

The goal of this study was to distinguish the impact of regular treadmill running on age-induced altered lipid profiles in different skeletal muscle fiber types of rats. Wistar strain male albino rats of two age groups, young (3 months, n = 12) and aged/old (18 months, n = 12) were divided into two groups such as, control (Con) and exercise trained (ExT; 23 m/min, 30 min/day, 5 days/ week for 12-week). After completion of the last training session, lipid metabolic profiles, including total cholesterol (TC), triglycerides (TG), phospholipids and malondialdehyde (MDA, lipid peroxidation marker) levels were monitored in soleus (SOL), red gastrocnemius (RG) and white gastrocnemius (WG) muscle fibers of rat. The TC, TG and MDA contents were significantly (P < 0.05) increased with advancement of age, while, phospholipid content was decreased with age in all muscle fiber types. However, here we found significantly (P < 0.05) decreased TC and TG levels, and increased phospholipid contents after treadmill exercise in all muscle fiber types of aged rats. The decreased TC content was more in young muscles (WG 31%), and the increased phospholipid content was more in old muscles fibers after treadmill running compared to their respective counter age group rats. However, increased MDA levels in old muscle fibers (SOL-132.13 2, RG-129 2 and WG-147 2.5 moles/g) were not attenuated by exercise training (SOL151 2.4, RG-148 2.7 and WG-157 3 moles/g) in this study. These results demonstrated that decreased TC and TG, and increased phospholipid contents by regular treadmill running might be beneficial to counteract the age-associated malfunctions in different muscle fiber types and also avoid the musculoskeletal disorders in aged rats. Key Words: cholesterol, triglycerides, lipid peroxidation, muscle fibers, aging, exercise

Introduction
Aging is accompanied by a progressive loss of skeletal

mass and strength, leading to loss of functional capacity and an increased risk of developing chronic metabolic diseases. Age related increased reactive oxygen species (ROS) production may alter the structure and function of lipids, increased lipid peroxidation and cause cell death (13, 14, 22). Lipid peroxidation is a complex process initiated by high amount of ROS, and indexed by elevated levels of malondialdehyde (MDA) at old age. Peroxidation of membranes certainly causes loss of fluidity and permeability of the cells (24). Lipid profiles serves as sensitive markers of the metabolic status of tissues and its metabolism is a complex process that is intimately related to carbohydrate and protein metabolism and hormonal balance. It was found that lipids serve as a good substrate for peroxidation reaction. Changes in membrane lipids have been reported in several pathological conditions and during aging (26). Selected lipid profiles such as cholesterol, triglycerides (TG) and phospholipids are considered as clinical and chemical variables that indicate the health status. The difference in lipid profiles directly involved with changes in tissue oxidative capacity, compensating mechanisms, associated with oxidative stress and represents a metabolic perturbation (9). Skeletal muscle is a heterogeneous tissue composed of two main distinct fiber categories, type-I and type-II, plays an crucial role in whole body energy metabolism (1). Type I or slow twitch fibers (soleus) are predominantly oxidative and contain more mitochondria than do type II or fast twitch, glycolytic fibers (red and white portions of gastrocnemius) (3). The fiber type distribution, TG storage, and the fatty acid composition of membrane phospholipids in skeletal muscle have been linked to several diseases (1). Literature revealed that regular physical exercise is an effective intervention in augmenting life span

Corresponding author: Dr. Reddy Kesireddy Sathyavelu, Professor of Zoology, Division of Molecular Biology & Exercise Physiology, Sri Venkateswara University, Tirupati-517502, A.P, India. Tel: +91 9849023339, Fax: +91 877-2261801, E-mail: ksreddy2008@hotmail.com/ sathyakreddy@hotmail.com Received: October 21, 2011; Revised: January 19, 2012; Accepted: February 22, 2012. 2012 by The Society of Adaptive Science in Taiwan and Airiti Press Inc. ISSN : 2076-944X. http://www.sast.org.tw

34

Mallikarjuna, Bhaskar, Shanmugam and Sathyavelu

and delaying degenerative diseases in rodents through maintaining the stable lipid profiles (21). Although information is available on lipid metabolic profiles with exercise in different tissues, little is known about the impact of regular treadmill running on lipid profiles in oxidative and glycolytic muscle fibers of old age rats. In this study, we investigated the impact of treadmill running on soleus, red and white gastrocnemius muscles cholesterol, triglyceride, phospholipids and MDA levels in old rats.

hind limb of rats by using dissecting microscope. Respective muscle fibers (SOL, RG and WG) were identified based on their structure and appearance as described by Delp and Duan (3). Special care has been taken to distinguish the different fibers and avoid the mixing of one fiber type with another. The excised muscle fibers were rapidly dissected free of fat, tendon, blotted and immediately frozen them in liquid nitrogen. Tissues were stored at -80C until biochemical analysis. The total time for each muscle excision, dissection and freezing was ~5 min. Biochemical Assays

Materials and Methods

Animals Care and Maintenance Pathogen free Wistar strain male albino rats (n = 24) of two different age groups i.e., young (3 months, weighing 170 10 g) and moderately aged/old (18 months, weighing 240 10 g) were used in the present study. The rats were housed in hygienic polypropylene cages, six rats per cage and maintained under temperature controlled room (25 2C) with a photoperiod of 12 h light and 12 h dark cycle. The rats were fed with a standard laboratory chow (Hindustan lever Ltd., Mumbai, India) and water ad libitum. This study was approved by the Institutional Animal Ethics Committee (Regd. No.438/01/a/CP CSEA/dt.17.07. 2001). Grouping of Animals Both age groups rats i.e., young (n = 12) and moderately aged/old (n = 12) were equally divided into control and exercise groups as mentioned below. Control (Con): Young (n = 6) and aged rats (n = 6) in this group were placed on a six channel motor driven treadmill and given 2 m/min exercise for 5 min for equivalent handling. Exercise Trained (ExT): Rats from both ages, young (n = 6) and old (n = 6) were performed exercise training on a six channel motor driven treadmill for 5 days/ week for a period of 12 weeks and given 23 m/min exercise for 30 min. All animals in both age groups completed exercise training protocol. The running program was scheduled between 6.00 and 8.00 a.m. Treadmill was custom-built at University Scientific Instrumentation Center (USIC), Sri Venkateswara University Campus. Muscle Sampling Procedure All the rats were sacrificed after 24 h of the last training session along with sedentary controls by cervical dislocation and selected muscle fibers, including soleus (SOL), red gastrocnemius (RG) and white gastrocnemius (WG) were quickly removed from the In this study, total cholesterol (TC) content in the muscle samples was estimated by using Lieberman Burchard reaction as described by Natelson (18). Skeletal muscle TG content was determined by the method of Natelson (19) with slight modifications. The phospholipids in different fiber types were monitored by the method of Zilversmidth and Davis (29). Oxidative damage to lipids known as lipid peroxidation was assayed by monitoring the levels malondialdehyde (MDA) in all muscle fiber types as described by Hiroshi et al. (8). Statistical Analysis Two factor analysis of variance (ANOVA) was carried out by using the SPSS and the data have been analyzed for the significance of the main effects i.e., age and exercise along with their interaction. Tukeys multiple comparison post-hoc test was performed to determine the significant levels. Results were reported as the means SD of six observations, with the level of significance set at P < 0.05.

Results
Fig. 1 depicts the TC content, which was increased with advancement of age in all muscle fibers types. In this study, we found decreased TC with exercise training in old rats as well as in young rats compared to their respective unexercised rats. The decreased TC content with exercise training was more pronounced in WG (31%, Fig. 1C) of young and RG (20%, Fig. 1B) of moderately aged/old rats. The TG content was increased in all muscle fibers of old rats when compared to young rats. A significant (P < 0.05) decline in TG content was observed after 12 weeks treadmill running in both age groups. The decline in the TG content with exercise training was as follows; in young rats: WG (35%) > SOL (32%) > RG (23%), and in old rats: WG (43%) > RG (35%) > SOL (27%) (Fig. 2).

Exercise Attenuates Lipid Profiles in Aged Rat Skeletal Muscles

35

A. Soleus (SOL)
60

A. Soleus (SOL)
Con ExT
Triglyceride (mg/g Tissue)

0.5

Total Cholesterol (mg/g Tissue)

50 40 30 20 10 0

Con ExT

0.4

0.3

* *

0.2

0.1

Young

Old

Young

Old

B. Red Gastrocnemius (RG)

60

B. Red Gastrocnemius (RG)

Con ExT
Triglyceride (mg/g Tissue)

0.4 0.3

Total Cholesterol (mg/g Tissue)

50

Con ExT

*
40 30 20 10 0

0.2

0.1

Young

Old

Young

Old

C. White Gastrocnemius (WG)

60

C. White Gastrocnemius (WG)

Con ExT
0.4
Triglyceride (mg/g Tissue)

Total Cholesterol (mg/g Tissue)

Con ExT

50 40 30 20 10 0

0.3

0.2

0.1

Young

Old

0 Young Old

Fig. 1. Effect of treadmill running on total cholesterol content (mg/g) in soleus (A), red gastrocnemius (B) and white gastrocnemius (C) muscle fibers of young and old rats. Values are significant compared to control (Con, *P < 0.05), and compared to young group (P < 0.05).

Fig. 2. Effect of treadmill running on triglyceride content (mg/g) in soleus (A), red gastrocnemius (B) and white gastrocnemius (C) muscle fibers of young and old rats. Values are significant compared to control (Con, *P < 0.05), and compared to young group (P < 0.05).

Changes in phospholipid content in different skeletal muscle fibers with exercise training and aging was represented in Fig. 3, A, B and C. The drop in phospholipid content as a function of age was clearly observed in all types of muscle fibers. However,

phospholipid content was significantly (P < 0.05) increased by treadmill running in all muscle fiber types in aged rats, and this elevation was more in all respective muscle fibers of old rats compared to their young rats.

36

Mallikarjuna, Bhaskar, Shanmugam and Sathyavelu

A. Soleus (SOL)
10

A. Soleus (SOL)
Con ExT 180

*
Phospholipids (mg/g Tissue)

*
150

Con ExT

MDA (moles/g Tissue)

120 90 60 30 0 Young

0 Young Old

Old

B. Red Gastrocnemius (RG)

12
Phospholipids (mg/g Tissue)

B. Red Gastrocnemius (RG)

Con ExT 180

*
10

*
150

Con ExT

*
8 6 4 2 0 Young Old

MDA (moles/g Tissue)

120 90 60 30 0 Young

Old

C. White Gastrocnemius (WG)

10
Phospholipids (mg/g Tissue)

C. White Gastrocnemius (WG)

Con ExT 180

* *

*
MDA (moles/g Tissue)
150 120 90 60 30 0 Young Old

8 6 4 2 0 Young Old

Con ExT

Fig. 3. Effect of treadmill running on phospholipid content (mg/g) in soleus (A), red gastrocnemius (B) and white gastrocnemius (C) muscle fibers of young and old rats. Values are significant compared to control (Con, *P < 0.05), and compared young group (P < 0.05).

Fig. 4. Effect of treadmill running on malondialdehyde (MDA) levels (moles/g) in soleus (A), red gastrocnemius (B) and white gastrocnemius (C) muscle fibers of young and old rats. Values are significant compared to control (Con, *P < 0.05), and compared to young group (P < 0.05).

Fig. 4 shows the effect of 12 weeks exercise running on MDA levels in both age groups of rats. Aged unexercised rats exhibited higher MDA levels in all muscle fibers than that of young unexercised

rats. Among the fiber types, WG fibers have been reported with higher MDA levels than other fiber types. In our study, treadmill running was unable to attenuate the elevated MDA levels in aged rats.

Exercise Attenuates Lipid Profiles in Aged Rat Skeletal Muscles

37

Discussion
In the present investigation, we emphasize the beneficial effects of treadmill exercise training against age-induced fluctuations in lipid metabolic profiles in different skeletal muscle fibers types. We used 18month aged rats as old rats to compare the age effect on lipid metabolic profiles. We found that increased TC and TG contents in aged skeletal muscle fibers were possibly decreased after 12 weeks treadmill running. Age dependent decreased phospholipids in all muscle fibers were significantly increased above to baseline by exercise training even in old rats. Different muscle fibers composed with different fiber types, like type I or type II (oxidative or glycolytic) fibers, which were progress to lose with advancing of age. Furthermore, recruitment of muscle fibers during treadmill running is also varied. Therefore, in our study lipid profiles exhibited slightly different results in response to exercise in different muscle fiber types of young and old age rats. The striking characteristic feature of aging was evidenced by increased TC content in all old muscle fibers of old rat. The increased TC may be due to the decreased exchange of cholesterol between the blood and muscle in older animals, which indicates aged rats are more susceptible to heart diseases. Similar to our findings William and Smith (27) reported 20% higher muscle cholesterol levels in aging rats. Timiras (25) reported a significant increase in the plasma cholesterol and LDL (low density lipoprotein) cholesterol of humans with advancement of age. However, in our study we found significantly decreased cholesterol levels in all muscle fibers of aged rats with treadmill running, particularly decreased cholesterol was pronounced in RG muscle. The decrease in TC content in exercised muscle fibers might be due to mobilization of cholesterol from the muscles to plasma, liver and/or adipose tissue. Drop in cholesterol content might also attribute its involvement in the synthesis of steroid hormones. Previous studies demonstrated the effect of exercise training in reducing the cardiovascular diseases by reducing cholesterol and/or raising high density lipoprotein cholesterol (4, 20). The TG content was drastically elevated in SOL, RG and WG muscle fibers of aged rats compare to their young rats. Among three muscle fiber types, slow twitch oxidative muscle (SOL) have high amount of TG than the fast twitch and glycolytic fibers. An increased TG with age has been reported in an earlier study (25), which were associated with a higher incidence of coronary heart disease. The augmentation of TG content might be due to increased output of TG from liver and intra muscular TG pool and/or due to the decreased lipoprotein lipase (LPL)

activity in liver and intramuscular cells. Barakat et al. (2) found that decrease in LPL activity with age in adipose tissue may be responsible, at least in part for the elevation of the TG levels. Nevertheless, endurance exercise training significantly decreased the TG content in all muscle fiber types of old age rats. Halle et al. (5) reported decreased serum TG after 6 months bicycle exercise training in men and women. According to Novelli et al. (21) studies, long-term exercise has been shown to decrease muscle TG level about 50% in aged rats. Decrease TG content in fast twitch oxidative glycolytic fibers suggesting that intracellular TG is providing fuel for muscle contraction during exercise. Mengelkoch et al. (15) reported that the prevalence of coronary heart disease risk factor remained low and generally stable in older athletes, who have maintained habitual exercise training. The decreased TG content during exercise training may be attributed to the increased metabolic utilization of TG for the fuel and also due to the increased LPL activity. Phospholipids are the major structural components of biological membranes and, as such, have significant influence on their physical properties. The decreased phospholipid content with age in this study serves as metabolic alarm to the animal in the sense that the membrane integrity is lost. Under such circumstances, animal needs to make such necessary preparatory mechanisms either to repair or resist to these changes in the membrane integrity. This is vital function for maintaining the structural integrity of intracellular components like mitochondria (12). Age related reduction in phospholipid content is considered as a result of alteration in enzyme activities, reduction of bulk lipid and change in permeability properties of the membranes. The decreased phospholipid content may be due to reduced synthesis of phospholipids and/or elevated degradation for energy purposes, since they are actively degradable among the lipids (17). Schmucker et al. (23) found decreased total phospholipid content in rat liver microsomes progressively from 3 to 22 months of age. However, in this study, we found decreased phospholipids were attenuated by 12-week treadmill running in the all muscle fiber types. Elevated phospholipids in exercised SOL, RG and WG muscles may be due to increased synthesis or reduced deregulation of phospholipids with exercise training (2). Previously, Mitchell et al. (16) also demonstrated a remodeling of phospholipid profile in glycolytic and oxidative muscle fibers of rats after exercise training. Helge et al. (6) showed modulation of membrane phospholipid profile by regular exercise training in soleus, red and white quadriceps of rats. The cell membranes enriched with polyunsaturated fatty acids (PUFAs) are more prone to free

38

Mallikarjuna, Bhaskar, Shanmugam and Sathyavelu

radical mediated lipid peroxidation. MDA, a byproduct of lipid peroxides is most frequently considered as a marker of oxidative stress in various tissues (12, 24). Irrespective of fiber types, we observed increased MDA content in all muscle fiber types with age. Lawler and Demaree (10) also reported increased hydroperoxides in old rats compared to young rats. Age-induced decreased antioxidant enzyme status and increased ROS may responsible for the elevated MDA levels in various tissues (14). On the other hand, high-intensity exercise has been shown to ameliorate the insulin-mediate vasorelaxation by reduced production of ROS, in particular superoxide in rats (28). However, in this study, our exercise training protocol was unable to reduce the elevated MDA levels in aged muscle fibers. Similar to our studies, Lawler et al. (11) observed increased MDA content in RG muscles of young exercised rats, which means exercise also partly increase the MDA content in rats. The increased MDA content with treadmill running was more pronounced in the fast twitch fibers than slow twitch fibers. This study suggests that old animals may need low intensity exercise to overcome from elevated lipid peroxidation. The differences in MDA content among fiber types may be due to the result of muscle fiber recruitment, differences in antioxidant capacity and/or the amount of free radicals generated in specific fiber types (7, 11). From these findings it was concluded that 12week treadmill running could be beneficial to manage the age-induced alterations in lipid profiles of oxidative (soleus) and glycolytic muscle fibers (red and white gastrocnemius). Decreased cholesterol and TG levels and increased phospholipids in aged muscle fibers by treadmill running might be helpful to prevent the various lipids mediated diseases in aged animals. Therefore, regular physical activity is highly recommended to older subject to avoid possible ageassociated musculoskeletal disorders and to maintain the good quality of life. Although we found altered lipid profiles were ameliorated by regular treadmill running in old animals, increased MDA levels were unable to control by exercise training. This might be associated with involvement of free radicals, which production increased with age. Future studies are necessary to overcome this limitation by providing the external antioxidant supplements and/or by low intensity exercise training to old animals.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

References
19. 1. Andersson, A., Sjodin, A. and Hedman, A. Fatty acid profile of skeletal muscle phospholipids in trained and untrained young men. Am. J. Physiol. Endocrinol. Metab. 279: E744-E752, 2000. 2. Barakat, H.A., Dohm, G.L., Shukla, N., Marks, R.H.L., Kern, M., Carpenter, J.W. and Mazzeo, R.S. Influence of age and exercise

20.

training on lipid metabolism in Fisher-344 rats. J. Appl. Physiol. 67: 1638-1642, 1989. Delp, M.D. and Duan, C. Composition and size of type I, IIA, IID/X and IIB fibers and citrate synthase activity of rat muscle. J. Appl. Physiol. 80: 261-270, 1996. DeSouza, C.A., Shapiro, L.F., Clevenger, C.M., Dinenno, F.A., Monahan, K.D., Tanaka, H. and Seals, D.R. Regular aerobic exercise prevents and restores age-related declines in endothelium dependent vasodilation in healthy men. Circulation 102: 13511357, 2000. Halle, M., Berg, A., Konig, D., Keul, J. and Baumstark, M.W. Differences in the concentration and composition of low density lipoprotein sub-fraction particles between sedentary and trained hyper cholesterolemic man. Meta. Clin. Exp. 2: 186-191, 1997. Helge, J.W., Ayre, K.J., Hulbert, A.J., Kiens, B. and Storlien, L.H. Regular exercise modulates muscle membrane phospholipid profile in rats. J. Nutr. 129: 1636-1642, 1999. Heunks, L.M.A. and Dekhuijzen, P.N.R. Respiratory muscle function and free radicals: From cell to COPD. Thorax 55: 704-716, 2000. Hiroshi, O., Ohishi, N. and Yagi, K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95: 351-358. 1979. Kitts, D.D., Yuan, Y.V. and Godin, D.V. Plasma and lipoprotein lipid composition and hepatic antioxidant status in spontaneously hypertensive and normotensive rats. Can. J. Physiol. Pharmacol. 72: 202-209, 1998. Lawler, J.M. and Demaree, S.R. Relationship between NADPspecific isocitrate dehydrogenase and glutathione peroxidase in aging rat skeletal muscle. Mech. Aging Develop. 122: 291-304, 2001. Lawler, J.M., Powers, S.K., Visser, T., Dijk, V.H., Kordus, M.J. and Ji, L.L. Acute exercise training and skeletal muscle antioxidant and metabolic enzymes: effects of fiber type and age. Am. J. Physiol. 265: R1644-R1650, 1993. Mallikarjuna, K. Alcohol effects and exercise benefits: Hepatic antioxidant enzyme homeostasis in two different age groups of male albino rats. Ph.D. Thesis, Sri Venkateswara University, Tirupati, India. 2005. Mallikarjuna, K., Hou, C.W., Chen, C.Y., Lee, J.P., Sathyavelu Reddy, K. and Kuo, C.H. Angiogenesis: Role of exercise training and aging. Adapt. Med. 2: 29-41, 2010. Mallikarjuna, K., Nishanth, K., Hou, C.W., Kuo, C.H. and Sathyavelu Reddy, K. Effect of exercise training on ethanol-induced oxidative damage in aged rats. Alcohol 43: 59-64, 2009. Mengelkoch, L.J., Pollock, M.L., Limacher, M.L., Graves, J.E., Shereman, R.B., Riley, W.J., Lowenthal, W.T. and Lean, A.S. Effect of age physical training and physical fitness on coronary heart disease risk factor in older track athletics of twenty-year follow-up. J. Am. Geriatr. Soc. 45: 1446-1453, 1997. Mitchell, T.W., Turner, N., Hulbert, A.J., Else, P.L., Hawley, J.A., Lee, J.S., Bruce, C.R. and Blanksby, S.J. Exercise alters the profile of phospholipid molecular species in rat skeletal muscle. J. Appl. Physiol. 97: 1823-1829, 2004. Murray, R.K., Granner, D.K., Mayes, P.A. and Rodwell, V.W. In: Harpers Biochemistry, Lang Medical Book, 25th edition. Appleton and Lange, Prentice Hall Publ. New Jersey, USA, 2000. Natelson. Total cholesterol procedure Liberman Burchand reagent. In: Techniques in Clinical Chemistry, 3rd edition, edited by Charles, C. Thomas Publications, Springfield, Illinois (USA). 1971a, pp. 263-267. Natelson. Triglycerides. In: Techniques in Clinical Chemistry, 3rd edition, edited Charles, C. Thomas Publications, Springfield, Illinois (USA), 1971b, pp. 720-723. Nicklas, B.J., Katzel, L.I., Busbywhitehead, J. and Goldberg, A.P. Increase in high density lipoprotein cholesterol with endurance exercise training are blunted in obese compared with lean man.

Exercise Attenuates Lipid Profiles in Aged Rat Skeletal Muscles

39

Meta. Clin. Exp. 46: 556-591, 1997. 21. Novelli, M., Pocai, A., Skalicky, M., Viidik, A., Bergmini, E. and Masiello, P. Effects of life-long exercise on circulating free fatty acids and muscle triglyceride content in ageing rats. Exp. Gerontol. 39: 1333-1340, 2004. 22. Powers, S.K., Kavazis, A.N. and DeRuisseau, K.C. Mechanisms of disuse muscle atrophy: role of oxidative stress. Am. J. Physiol. Reg. Integr. Comp. Physiol. 288: 337-344, 2005 23. Schmucker, D.L., Vessey, D.A., Wang, R.K., James, J.L. and Maloney, A. Age dependent alterations in the physicochemical properties of rat liver microsomes. Mech. Ageing Dev. 27: 207-217, 1984. 24. Starnes, J.W., Cantu, G., Farrar, R.P. and Kehrer, J.P. Skeletal muscle lipid peroxidation in exercised and food-restricted rats during aging. J. Appl. Physiol. 67: 69-75, 1989. 25. Timiras, P.S. Disuse and aging: same problem, different outcomes.

J. Gravit. Physiol. 1: 5-7, 1994. 26. Tollet-Egnell, P., Parini, P., Sthlberg, N., Lnnstedt, I., Lee, N.H., Rudling, M., Flores-Morales, A. and Norstedt, G. Growth hormone-mediated alteration of fuel metabolism in the aged rat as determined from transcript profiles. Physiol. Genomics 16: 261267, 2004. 27. Williams, K.D. and Smith, D.O. Cholesterol conservation in skeletal muscle associated with age- and denervation-related atrophy. Brain Res. 493: 14-22, 1989. 28. Yang, A.L., Lo, C.W., Lee, J.T. and Su, C.T. Enhancement of vasorelaxation in hypertention following high-intensity exercise. Chinese J. Physiol. 54: 87-95, 2011. 29. Zilversmidth, D.B. and Davis, B.S. Micro determination of plasma phospholipids by trichloroacetic acid precipitation. J. Lab. Clin. Med. 35: 155-166, 1950.