The effects of coenzyme Q10 on oxidative stress and heat shock proteins in rats subjected to acute and chronic exercise

Article information

J Exerc Nutrition Biochem. 2018;22(3):14-20

doi : https://doi.org/10.20463/jenb.2018.0019

Ragip Pala ¹, Fahrettin Beyaz ¹, Mehmet Tuzcu ², Besir Er ², Nurhan Sahin ³, Vedat Cinar ¹, Kazim Sahin ³^,

¹ Department of Movement and Training Science, Firat University, Elazig Turkey

² Department of Biology, Firat University, Elazig Turkey

³ Department of Animal Nutrition, Firat University, Elazig Turkey

^*Kazim Sahin, Ph.D. Department of Animal Nutrition, Faculty of Veterinary Medicine, Firat University, Elazig, Turkey. Tel: +90-424-237 3938 / Fax: +90-424-238 8173 E-mail: nsahinkm@yahoo.com

Received 2018 June 23; Revised 2018 July 28; Accepted 2018 August 24.

Abstract

[Purpose]

The aim of the study was to determine the effects of dietary CoQ10 on serum biochemical parameters, lipid peroxidation, and HSP expression in the liver and slow-twitch muscles (soleus and gastronemius deep portion) of exercise-trained rats.

[Methods]

A total of 42 Wistar albino rats were divided into six groups: 1) Control, 2) Coenzyme Q10 (CoQ10), 3) Chronic Exercise (CE), 4) CE + CoQ10, 5) Acute Exercise (AE), and 6) AE + CoQ10. The rats were subjected to the running test 5 days a week for 6 weeks after which CoQ10 was administered via the diet. AE (running on the treadmill until the rats were exhausted) was done on the last day

[Results]

The results showed no significant difference in serum glucose and liver functions in any of the groups. However, CoQ10 and exercise treatment were found to lower cholesterol and triglyceride levels. Serum and muscle malondialdehyde (MDA) levels were found to be lower in the CE and CE + CoQ10 groups compared to the control group. The highest levels of HSP60, HSP70, and HSP90 in liver and muscle were found in the AE group, and the lowest levels were found in CE + CoQ10 group. CoQ10 supplementation reduced HSP expression in both CE-and AE-trained rats (P < 0.05).

[Conclusion]

The results showed that CoQ10 supplementation could reduce MDA levels, protect against oxidative damage, and regulate HSP expression in CE-and AE-trained rats. CE and CoQ10 were shown to reduce oxidative stress synergistically.

Keywords: exercise; coenzyme Q10; oxidative stress; heat shock proteins

INTRODUCTION

The impairment of the antioxidant-mediated defense system during acute exercise (AE) training, which has been shown in muscles and the liver, results in the increase of reactive oxygen species (ROS) and inflammation markers¹. At the end of the maximal exercise, this condition of the muscle tissue causes free radicals to multiply, leading to lipid peroxidation of the membranes, and an increase in the abundance of macrophages and white blood cells². Exercise can increase the use of oxygen by over 200 times and increases the relaxation levels of working muscle fibers³. During exercise-training, muscle mitochondria mediates an increase in superoxide production with the increase in oxygen flow^3,4. Increased amounts of free radicals associated with excess oxygen consumption is counteracted by a defense system containing enzymatic and non-enzymatic antioxidants. Excessive exercise is manifested as muscle fatigue and muscle damage known as oxidative stress due to imbalance between ROS and antioxidants⁵. In a previous study, it was shown that regular exercise provides many benefits, while excessive exercise increases oxidative damage by increasing the ROS formation⁶. Studies have been conducted on both marathon runners and experimental animals in order to eliminate or mitigate such negative effects caused by exercise. These studies were mostly based on the removal of oxidative stress and were conducted with reinforcing substances which are thought to have strong antioxidant properties.

Heat shock proteins (HSPs) are molecular chaperones that help fold proteins into their original conformation, restore function, and contribute to the reduction of cellular damage^7-9. They have many functions in maintaining intracellular integrity via protection, repair, and even regulation of cell death signaling¹⁰. It is well documented that the expression of HSPs, particularly HSP70, has been found to be increased in the mammalian skeletal muscle under stress conditions caused by exercise^8,11,12. It has been reported that HSP70 expression is upregulated immediately after thermal stress in the soleus muscle¹³. The upregulation of HSPs may be responsible for cytoprotection via a mechanism capable of reestablishing protein homeostasis against numerous stressors, including exercise¹⁴. Chronic exercise (CE) may improve the overall health; however, a single bout of inadequate exercise may lead to oxidative stress and muscle damage^15,16. Taken together, these studies suggest that the expression of HSPs could be considered important for protection from and repair of skeletal muscle after exercise-induced stresses.

Coenzyme Q10 (CoQ10), naturally found in mitochondria, is a potent antioxidant that is endogenously synthesized and is soluble in fat¹⁷. Due to its antioxidant properties, it is able to effectively inhibit the oxidation of fat, protein, and DNA in the body¹⁸. CoQ10 suppresses lipid peroxidation, and thus, oxidative stress by suppressing the activity of enzymes involved in ROS production^19,20. One of the most important functions of CoQ10 is to serve as an electron carrier during oxidative phosphorylation by diffusing to the phospholipid layer of the cell membrane via a unique chain structure in mitochondria²¹. In addition, it plays an important role in energy production from carbohydrates and fatty acids in cells²². Besides, CoQ10 decreases serum cholesterol levels by suppressing cholesterol synthesis in the liver²³.

Oxidative stress, which occurs during extreme exercise, lowers CoQ10 levels in mitochondria²⁴. Thus, excessive free radicals are created that accumulate in the muscles, leading to fatigue and muscle damage in the body²⁵. It has been reported that CoQ10 supplementation is highly effective in transporting and using oxygen required at the cellular level²⁶ as well as in eliminating muscle damage and fatigue²⁷. Therefore, the aim of the present study was to determine the effects of dietary CoQ10 on serum biochemical parameters, lipid peroxidation, and the expression of heat shock proteins (HSP60, HSP70, and HSP90) in the liver and muscles of acutely and chronically exercised rats.

METHODS

Animals

This study was carried out by following the appropriate ethical rules for animal welfare and animal rights, after approval (No: 2014/01-07) from the Firat University Ethics Committee for Animal Experiments (Elazig, Turkey). A total of 42 male Wistar rats (8 weeks old, 180 ± 20 g weighted, 7 in each group) were housed in a controlled room with a 12/12 hour light/dark cycle at 22 ± 2 ° C and 55 ± 5% relative humidity in specially prepared and daily cleaned cages, and supplied with rat chow and water ad libitum.

The rats were divided randomly into the following 6 groups: (1) Control (C); rats received basal diet and were not exercised, (ii) CoQ10; rats received basal diet supplemented with CoQ10 and were not exercised, (iii) CE; rats received basal diet and were exercised (30 m/min, gradient 0 %, 30 min; exercise was performed for 5 days per week for 6 weeks), (iv) CE + CoQ10; rats received basal diet supplemented with CoQ10 and were subjected to CE (v) AE; rats received basal diet and were exercised (30 m/min, gradient 0 %, 30 min for 5 days per week for 6 weeks and exhaustion exercise was also performed); and (vi) AE+ CoQ10; rats received diet supplemented with CoQ10 and were subjected to AE. CoQ10 was administered daily for six weeks as an oral supplement by a gastric tube at a dosage of 300 mg/kg body weight. The selection of the dose (300 mg/kg b. wt.) was based on previous studies where this dosage elicited a significant antioxidant effect in rats²⁸. After exercise training of rats in the AE groups, exhaustion exercise (making the rats run rats until they are exhausted and collecting serum and tissue samples immediately) was performed.

Exercise protocol

The rats were subjected to a motor-driven rodent treadmill at a 0 % gradient (MAY-TME, Commat Limited, Ankara, Turkey). The treadmill was supplied with an electric shock grid on the rear barrier to provide exercise motivation to the rats. All exercise tests were done during the same time period of the day to diminish diurnal effects (11.30–13.30 hours). The rats in the CE groups were familiarized by treadmill exercise over a 5-d period such as: 1st day 10 m/min, 10 min; 2nd day 20 m/min, 10 min; 3rd day 25 m/min, 10 min; 4th day 25 m/min, 20 min; and 5th day 30 m/min, 0 % grade, 30 min at high intensity²⁹. Thereafter, animals were exercised at this level for 6 weeks, 5 days/week. Exhaustion was defined as the inability of a rat to right itself when being laid on its side. All animals were fasted for 12 h before death.

Sample collection

At the completion of the exercise program, rats were decapitated by cervical dislocation, and blood, slow-twitch muscles (soleus and gastronemius deep portion), and liver samples were taken. Blood samples were centrifuged at 5000 rpm at 4 °C for 10 minutes in a refrigerated centrifuge (Universal 320R, Hettich, Germany) with gliotic biochemical tubes (Standard plus & Medical Co., Ltd., Germany) to isolate serum. All the samples were stored at -80 °C (Hettich, Germany) for further analyses.

Laboratory analysis

Levels of serum glucose, cholesterol, triglyceride, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were analyzed with an autoanalyzer (Samsung LABGEO PT10, Samsung Electronics Co, Suwon, Korea). Repeatability and device/method precision of LABGEO PT10 was established according to the IVRPT06 guideline. The malondialdehyde (MDA) levels of serum, liver, and muscle were measured by high-performance liquid chromatography (HPLC; Shimadzu, Tokyo, Japan) using a Shimadzu UV–vis SPD-10 AVP detector and C18 ODS-3.5 μm, 4.6 mm × 250 mm column³⁰.

Analysis of gene expression

For total RNA extraction, 50 mg of tissue sample was homogenized using a Tissue Ruptor (Qiagen, Venlo, Netherland) homogenizer attached with a probe in 600 μL of 1:100 beta-mercaptoethanol in RLT buffer. The samples were then centrifuged for 3 min. The supernatant was collected and mixed with one volume of 70 % ethanol. The total RNA was isolated from 50 mg of liver and muscle samples using the RNeasy total RNA isolation Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. After isolation, amount and the quality of total RNA were determined using nanodrop spectrophotometry (Maestrogen, USA). Occurrence of any RNA degradation was checked with agarose gel electrophoresis. cDNA synthesis was carried out by using 500 ng of total RNA and oligo(dT)18 primer using a commercial First Strand cDNA Synthesis Kit (MBI Fermentas, USA) as per the manufacturer's instructions. Gene expression of desired proteins were determined with real time PCR by mixing 1 μl cDNA and 5 μl 2x SYBR Green Master-mix (Qiagen Fast Start Universal, SYBR Green Master Mix).

Real time PCR analysis

Predeveloped TaqMan primers and probes set for HSP60, HSP70, and HSP90 were designed at ABI based on gene sequence information obtained from GenBank (Qiagen Lot no: 20111214039, 20120813061, and 20110719024, respectively). The glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH), which has been widely used as a reference gene in rats³¹, was used as internal control (Qiagen Lot no: 20130917040). Then primer pairs (Table 1) were added at final concentrations of 0.5 mM to a final volume of 10 μl. The real time PCR program of the quantitative PCR (Light Cycler 480 II, Qiagen, Tokyo, Japan) was arranged as follows: initial denaturation at 95 °C for 10 minutes, denaturation at 95 °C for 15 s, annealing at 65 °C for 30 s, and extension at 72 °C for 15 s with 40 repeated thermal cycles measuring the green fluorescence at the end of each extension step. PCR reactions were carried out in triplicates and specificity of PCR products was verified by melt analysis. Also, negative controls lacking template were used in all reactions. The relative expression of genes with respect to GAPDH was calculated with efficiency corrected advance relative quantification tool provided by the software (http://www.qiagen.com/geneglobe Data Analysis Center). Gene expression profiles were indicated as ΔCT values. Gene expression fold changes were presented relative to the control and calculated using the 2 -ΔΔCT method. The differential gene expression was rated in pairs with a fold change cut off of 2 and significance value of P < 0.05³¹.

Table 1.

Effects of CoQ10 on biochemical parameters in rats subjected to AE and CE

Statistical analysis

Data was assessed using ANOVA in SPSS software program (Version 22.0; Chicago, IL, USA). Comparisons between groups were analyzed by the Tukey post hoc test. Data was expressed as group mean and standard error of the mean (SEM). P < 0.05 was considered to be significant.

RESULTS

Serum biochemical parameters

Serum glucose, cholesterol, triglyceride, AST, and ALT concentrations are shown in Table 1. Serum cholesterol and triglyceride levels in exercised or CoQ10 supplemented rats were significantly different from the control group rats (P < 0.0001, for both); however, differences in the serum glucose, AST, and ALT levels were not significant (P > 0.05). The serum cholesterol and triglyceride concentrations were decreased in the CE + CoQ10 group (P < 0.0001, for both; Table 1).

Serum and tissue MDA levels

As shown in Table 2, in general, serum, liver, and muscle MDA levels were found to be decreased by CE and CoQ10 supplementation in rats (P < 0.05). However, an increase in MDA level was detected with AE (P < 0.001). The lowest serum, liver, and muscle MDA levels were found in the CE + CoQ10 group, while the highest serum, liver, and muscle MDA levels were found in the AE group (P < 0.001, respectively; Table 2).

Table 2.

Effects of CoQ10 on oxidative stress in rats subjected to acute and chronic exercise training rats

Expression of heat shock proteins

Expression of HSP60, HSP70, and HSP90 genes in liver and muscles of rats in AE group were higher than those in the other groups (Fig 1A, B, C and 2A, B, C; P < 0.001). CoQ10 administration significantly decreased the expression of HSP60, HSP70, and HSP90 genes in liver and muscle (P < 0.05). The decrease in the expression of HSP60, HSP70, and HSP90 in the liver and muscle of the rats in the CE + CoQ10 group was more prominent than in the other groups (P < 0.05; Fig 1 and 2).

Fig. 1.

Effects of CoQ10 on the expression of liver heat shock proteins (HSPs) in rats subjected to CE and AE training. Control: rats fed a basal diet; CoQ10: rats fed a basal diet supplemented with CoQ10; CE: rats fed a basal diet and subjected to CE; CE + CoQ10: rats fed a basal diet supplemented with CoQ10 and subjected to CE; AE: rats fed a basal diet and subjected to AE; AE + CoQ10: rats fed a basal diet supplemented with CoQ10 and subjected to AE. Gene expression was analyzed by RT-PCR. Different superscripts (a–d) indicate group mean differences (P < 0.05).

Fig. 2.

Effects of CoQ10 on expression of muscle heat shock proteins (HSPs) in rats subjected to CE and AE training. Control: rats fed a basal diet; CoQ10: rats fed a basal diet supplemented with CoQ10; CE: rats fed a basal diet and subjected to CE; CE + CoQ10: rats fed a basal diet supplemented with CoQ10 and subjected to CE; AE: rats fed a basal diet and subjected to AE; AE + CoQ10: rats fed a basal diet supplemented with CoQ10 and subjected to AE. Gene expression was analyzed by RT-PCR. Different superscripts (a–d) indicate group mean differences (P < 0.05).

DISCUSSION

In the present study, the effects of CoQ10 supplementation on serum biochemical parameters, liver functions, lipid peroxidation, and expression of HSP60, HSP70, and HSP90 in the liver and muscle tissues of acutely and chronically exercised rats have been revealed. CoQ10 and AE/CE training were found to lower cholesterol and triglyceride levels (Table 1). These results show that during exercise training CoQ10 does not instantly mediate carbohydrate or lipid metabolism in the organism. CoQ10 is known as a redox molecule with intracellular antioxidant efficacy that is biochemically present in biological tissues both in reduced and oxidized form interacting with oxygen-derived radicals and singlet oxygen, thereby preventing the initiation of lipid peroxidation and damaging biomolecules^32,33. Also, CoQ10 is an ongoing antioxidant which plays a vital role in the regeneration of other antioxidants when exposed to plasma oxidants, although it is present in low concentrations compared to other antioxidants (e.g. α-tocopherol)^34,35. Furthermore, CoQ10 has been reported to have roles in membrane fixation, cell signaling, gene expression, cell growth, and supervision of apoptosis³⁶. Similar to our research, Pala et al. found that CoQ10 supplementation in exercised rats led to a decrease in cholesterol and triglyceride levels³⁷. Kim et al.³⁸ have previously reported that there was a significant difference in serum cholesterol and triglyceride levels, and Yoon and Park³⁹ have reported that there was a significant difference in serum triglyceride levels in exercised rats supplemented with antioxidant agents such as soy isoflavones, L-carnitine, and vitamin E. However, Díaz-Castro et al. found that there was no significant difference in cholesterol levels, but triglyceride levels were decreased in CoQ10 supplemented amateur male runners⁴⁰. It has been also been shown that during exercise, CoQ10 tend to improve skeletal muscle activity and triglyceride capacity that could be responsible for its ergogenic effects⁴¹.

Increased lipid peroxidation and tissue damage in overt exercises are associated with increased oxidative stress^42,43. MDA, the product of lipid peroxidation, is a dialdehyde that is the result of peroxidation of unsaturated fatty acids and is indicative of oxidative damage^44,45. In the present study, the control group exhibited decreased levels of serum, liver, and muscle MDA after CoQ10 supplementation and CE (Table 2). Díaz-Castro et al. stated that CoQ10 supplementation in amateur male runners reduced oxidative stress and inhibited muscle damage⁴⁰. In another study, Pala et al. reported that CoQ10, which acts as an antioxidant, suppresses oxidative stress in rats who have been practicing running exercises and consuming CoQ10³⁷. Gul et al. also reported that CoQ10 supplementation inhibited oxidative stress in 15 healthy and sedentary males who were subjected to Wingate tests⁴⁶. Kon et al. examined the effects of CoQ10 supplementation in rats subjected to exercise and found that CoQ10 is useful in reducing physical exercise-induced muscle damage²⁸. Ostman et al. have reported that CoQ10 may be effective on the alleviation of oxidative stress in moderately trained healthy men⁴⁷. In addition, Pala determined that end-of-match MDA levels of elite boxers were significantly decreased⁴⁸, and Pala et al. found that MDA levels caused due to oxidative stress decreased post-match in the coaches of Turkish boxing team⁴⁹.

In the present study, AE induced significant increases in the expression of HSP60, HSP70, and HSP90 in the liver and muscles. However, CE induced a decrease in the HSP60, HSP70, and HSP90 expression in the liver and muscles of rats (Fig. 1 and Fig. 2). We also demonstrated that CoQ10 supplementation decreased HSP60, HSP70, and HSP90 expression in the liver and muscle of acutely and chronically exercised rats. It was seen that the greatest reduction in expression of HSPs was in the chronic exercised and CoQ10-supplemented group. To the best of our knowledge, no previous studies related to investigative the effects of CoQ10 supplementation on the expression of HSPs in rats acutely and chronically exercised rats have been conducted. However, there have been studies on the effects of exercise on expression of HSPs^50-53. HSPs are known to act as cell defense mechanism, and they play an important role in the synthesis and repair of proteins⁵⁰. HSPs are expressed especially in oxidative stress conditions (e.g. exhaustive physical exercise)⁵¹, and mitochondria are effectively protected by members of the HSP family including HSP60, HSP70, and HSP90^52,53. Previously, the expression of the stress proteins were found to increase in rats subjected to running training^52,53. Moreover, exercise-induced HSP responses are dependent on exercise intensity and duration, reflecting a physiological response to heat shock and oxidative stress⁵⁴. Similar to our findings, it was reported that AE increased HSP60 mRNA, HSP90 mRNA and protein levels, and AE-induced oxidative stress (indicated by increased levels of HO-1 mRNA and protein), and HNE protein adducts⁵⁵. In addition, Starnes et al.⁵⁶ indicated that after three months of exercise for 5 days/week, HSP70 expression was not increased in the liver of aged rats, although it was significantly elevated in young rats⁵⁶. In a previous study, it was shown that administration of CoQ10 decreased the levels of colon tissue MDA, HSP70, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in an ulcerative colitis model of rats⁵⁷. There are also studies on the effects of antioxidants like Q10 on HSPs. For example, Oksala et al. reported that antioxidant supplementation effectively suppressed the heat shock protein expression in the liver of exercised rats⁵⁸. Wu et al. reported that lipoic acid significantly decreased the levels of HSP70 expression in different muscle types in heat-shock induced rats⁵⁹.

A decrease in lipid peroxidation and heat shock proteins in rats in the present study could have been due to positive effects of CoQ10 and/or CE, alleviating the negative effects of AE. In the present study, CE and dietary CoQ10 inclusions resulted in a synergic effect. More specifically, the combination of CoQ10 and CE provided the lowest levels of lipid peroxidation and HSP expression. It is apparent that a combination of dietary CoQ10 and CE offers a feasible way to reduce the increase in lipid peroxidation and HSP expression^29,37. CoQ plays a role in inhibiting lipid peroxidation by either scavenging ROS directly or in conjunction with α-tocopherol⁶⁰. Shimomura et al.⁶¹ have reported that intravenous CoQ10 supplementation suppressed the upregulated muscle damage markers (creatine kinase: CK, and lactate dehydrogenase: LDH) in rats following downhill running. The CoQ10 deficiency in skeletal muscle led to a spectrum of clinical manifestations and was it is suggested that this also leads to a secondary impairment of mitochondrial fatty acid oxidation⁶². In a previous study, it has been reported that CoQ10 supplementation increases CoQ concentration in muscle cell membranes and reduces AE-induced muscular injury by enhancing cell membrane stabilization⁶³.

CONCLUSION

In conclusion, these results showed that CoQ10 supplementation did not affect serum glucose levels and liver function, but reduced cholesterol and triglyceride concentrations in acutely and chronically exercised rats. In addition, CoQ10 showed protective effects against oxidative stress caused by AE by lowering MDA levels in acutely exercised rats. However, while AE increases oxidative stress, CE decreases oxidative stress by reducing lipid peroxidation. This effect has also been demonstrated by the regulation of the expression of heat-shock proteins in the liver and muscle tissues of CoQ10-fed rats. Meanwhile, CE and CoQ10 have been shown to reduce oxidative stress synergistically.

Acknowledgements

This project was supported by Firat University Scientific Research Projects Unit (FUBAP-BSY.14.03) and the Turkish Academy of Sciences (KS).

References

1. Goldfarb AH. Antioxidants: role of supplementation to prevent exercise-induced oxidative stress. Med Sci Sports Exerc 1993;25:232–6. 8450726.

2. Demopoulos HB, Santomier JP, Seligman ML, Pietronigro DD. Free radical pathology; rationale and toxicology of antioxidants and other supplements in sports medicine and exercise science In : In sport health and nutrition; the 1984 Olympic scientific congress proceedings Champaign; 1986. 2p. 139–90.

3. Pearson AM. Muscle growth and exercise. Crit Rev Food Sci Nutr 1990;29:167–96. 2222798.

4. Davies KJ, Quintanilha AT, Brooks GA, Packer L. Free radicals and tissue damage produced by exercise. Biochem Biophys Res Commun 1982;107:1198–205. 10.1016/s0006-291x(82)80124-1. 6291524.

5. Urso ML, Clarkson PM. Oxidative stress, exercise, and antioxidant supplementation. Toxicol 2003;189:41–54. 10.1016/s0300-483x(03)00151-3.

6. van Klaveren RJ, Nemery B. Role of reactive oxygen species in occupational and environmental obstructive pulmonary diseases. Curr Opin Pulm Med 1999;5:118–23. 10.1097/00063198-199903000-00007. 10813262.

7. Latchman DS. Heat shock proteins and cardiac protection. Cardiovasc Res 2001;51:637–46. 10.1016/s0008-6363(01)00354-6. 11530097.

8. Ogata T, Oishi Y, Higashida K, Higuchi M, Muraoka I. Prolonged exercise training induces long-term enhancement of HSP70 expression in rat plantaris muscle. Am J Physiol Regul Integr Comp Physiol 2009;296:R1557–63. 10.1152/ajpregu.90911.2008. 19244585.

9. Thakur SS, Swiderski K, Ryall JG, Lynch GS. Therapeutic potential of heat shock protein induction for muscular dystrophy and other muscle wasting conditions. Philos Trans R Soc Lond B Biol Sci 2018;19:373–373.

10. Kregel KC. Heat shock proteins: modifying factors in physiological stress responses and acquired thermotolerance. J Appl Physiol 2002;92:2177–86. 11960972.

11. Gonzalez B, Hernando R, Manso R. Stress proteins of 70 kDa in chronically exercised skeletal muscle. Pflugers Arch 2000;440:42–9. 10863996.

12. Archer AE, von Schulze AT, Geiger PC. Exercise, heat shock proteins and insulin resistance. Philos Trans R Soc Lond B Biol Sci 2018;373:19.

13. Oishi Y, Taniguchi K, Matsumoto H, Ishihara A, Ohira Y, Roy RR. Muscle type-specific response of HSP60, HSP72, and HSC73 during recovery after elevation of muscle temperature. J Appl Physiol 2002;92:1097–103. 10.1152/japplphysiol.00739.2001. 11842045.

14. Santoro MG. Heat shock factors and the control of the stress response. Biochem Pharmacol 2000;59:55–63. 10.1016/s0006-2952(99)00299-3. 10605935.

15. Cruzat VF, Krause M, Newsholme P. Amino acid supplementation and impact on immune function in the context of exercise. J Int Soc Sports Nutr 2014;11:61. 10.1186/s12970-014-0061-8. 25530736.

16. Moura CS, Lollo PCB, Morato PN, Risso EM, Amaya-Farfan J. Modulatory effects of arginine, glutamine and branched-chain amino acids on heat shock proteins, immunity and antioxidant response in exercised rats. Food Funct 2017;8:3228–38. 10.1039/c7fo00465f. 28812766.

17. Andree P, Dallner G, Ernster L. Ubiquinol: an endogenous lipid soluble antioxidant in animal tissues. In:free radicals, oxidative stress and antioxidant Plenum Press; 1998. p. 293–314.

18. Ernster L. Lipid peroxidation in biological membranes: mechanisms and implications. In active oxygens, lipid peroxides and antioxidants CRC Press; 1993. p. 1–38.

19. Stocker R, Frei B. Endogenous antioxidant defenses in human blood plasma, in oxidative stress: oxidants and antioxidants. in oxidative stress: oxidants and antioxidants H Sies ed. 1991. p. 213–43.

20. Bargossi AM, Battino M, Gaddi A, Fiorella PL, Grossi G, Barozzi G, Di Giulio R, Descovich G, Sassi S, Genova ML. Exogenous CoQ10 preserves plasma ubiquinone levels in patients treated with 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors. Int J Clin Lab Res 1994;24:171–6. 10.1007/bf02592449. 7819598.

21. Ernster L, Forsmark P, Nordenbrand K. The mode of action of lipid-soluble antioxidants in biological membranes: relationship between the effects of ubiquinol and vitamin E as inhibitors of lipid peroxidation in submitochondrial particles. J Nutr Sci Vitaminol 1992;548:1–51.

22. Ernsteer L, Dallner G. Biochemical, physiological and medical aspects of ubiquinone function. Biochim Biophys Acta 1995;1271:195–204. 7599208.

23. Modi K, Santani DD, Goyal RK, Bhatt PA. Effect of coenzyme Q10 on catalase activity and other antioxidant parameters in streptozotocin-induced diabetic rats. Biol Trace Elem Res 2006;109:25–34. 10.1385/bter:109:1:025. 16388100.

24. Mataix J, Manas M, Quies J, Battino M, Cassinello M, Lopez-Frias M, Huertas JR. Coenzyme Q content depends upon oxidative stress and dietary fat unsaturation. Mol Aspects Med 1997;18:129–35. 10.1016/s0098-2997(97)00019-8.

25. Gomez-Cabrera MC, Domenech E, Viña J. Moderate exercise is an antioxidant: upregulation of antioxidant genes by training. Free Radic Biol Med 2008;44:126–31. 10.1016/j.freeradbiomed.2007.02.001. 18191748.

26. Bhagavan HN, Chopra RK. Coenzyme Q10: absorption, tissue uptake, metabolism and pharmacokinetics. Free Radic Res 2006;40:445–53. 10.1080/10715760600617843. 16551570.

27. Linnane AW, Kios M, Vitetta L. Coenzyme Q (10) -its role as a prooxidant in the formation of superoxide anion/hydrogen peroxide and the regulation of the metabolome. Mitochondrion 2007;7:S51–61. 10.1016/j.mito.2007.03.005. 17482887.

28. Kon M, Kimura F, Akimoto T, Tanabe K, Murase Y, Ikemune S, Kono I. Effect of Coenzyme Q10 supplementation on exercise-induced muscular injury of rats. Exerc Immunol Rev 2007;13:76–88. 18198662.

29. Belviranlı M, Gökbel H, Okudan N, Basaralı K. Effects of grape seed extract supplementation on exercise-induced oxidaive stress in rats. Br J Nutr 2012;108:249–56. 10.1017/s0007114511005496. 22011589.

30. Karatepe M. Simultaneous determination of ascorbic acid and free malondialdehyde in human serum by HPLC–UV. Lc Gc North America 2004;22:362–5.

31. Nikzamir A, Palangi A, Kheirollaha A, Tabar H, Malakaskar A, Shahbazian H, Fathi M. Expression of glucose transporter 4 (GLUT4) is increased by cinnamaldehyde in C2C12 mouse muscle cells. Iran Red Cresecent Med J 2014;16:e13426. 10.5812/ircmj.13426.

32. Littarru GP, Tiano L. Bioenergetic and antioxidant properties of coenzyme Q10: recent developments. Mol Biotechnol 2007;37:31–7. 10.1007/s12033-007-0052-y. 17914161.

33. Bonakdar RA, Guarneri E. Coenzyme Q10. Am Fam Physician 2005;72:1065–70. 16190504.

34. Turunen M, Olsson J, Dallner G. Metabolism and function of coenzyme Q. Biochim Biophys Acta 2004;1660:171–99. 10.1016/j.bbamem.2003.11.012. 14757233.

35. Overvad K, Diamant B, Holm L, Holmer G, Mortensen SA, Stender S. Coenzyme Q10 in health and disease. Eur J Clin Nutr 1999;53:764–70. 10556981.

36. Crane FL. Biochemical functions of coenzyme Q10. J Am Coll Nutr 2001;20:591–8. 10.1080/07315724.2001.10719063. 11771674.

37. Pala R, Orhan C, Tuzcu M, Sahin N, Ali S, Cinar V, Atalay M, Sahin K. Coenzyme Q10 Supplementation Modulates NFκB and Nrf2 pathways in exercise training. J Sports Sci Med 2016;15:196–203. 26957943.

38. Kim E, Park H, Cha YS. Exercise training and supplementation with carnitine and antioxidants increases carnitine stores, triglyceride utilization, and endurance in exercising rats. J Nutr Sci Vitaminol 2004;50:335–43. 10.3177/jnsv.50.335. 15754494.

39. Yoon GA, Park S. Antioxidant action of soy isoflavones on oxidative stress and antioxidant enzyme activities in exercised rats. Nutr Res Pract 2014;8:618–24. 10.4162/nrp.2014.8.6.618. 25489400.

40. Díaz-Castro J, Guisado R, Kajarabille N, García C, Guisado IM, de Teresa C, Ochoa JJ. Coenzyme Q10 supplementation ameliorates inflammatory signaling and oxidative stress associated with strenuous exercise. Eur J Nutr 2012;51:791–9. 10.1007/s00394-011-0257-5. 21990004.

41. Hawley JA. Effect of increased fat availability on metabolism and exercise capacity. Med Sci Sports Exerc 2002;34:1485–91. 10.1097/00005768-200209000-00014. 12218743.

42. Fernstrom M, Bakkman L, Tonkonogi M, Shabalina IG, Rozhdestvenskaya Z, Mattsson CM, Enqvist JK, Ekblom B, Sahlin K. Reduced efficiency, but increased fat oxidation, in mitochondria from human skeletal muscle after 24-h ultraendurance exercise. J Appl Physiol 2007;102:1844–9. 17234801.

43. Mastaloudis A, Leonard SW, Traber MG. Oxidative stress in athletes during extreme endurance exercise. Free Radic Biol Med 2001;31:911–22. 10.1016/s0891-5849(01)00667-0. 11585710.

44. Cighetti G, Duca L, Bortone L, Sala S, Nava I, Fiorelli G, Cappellini MD. Oxidative status and malondialdehyde in beta-thalassaemia patients. Eur J Clin Invest 2002;32:55–60. 10.1046/j.1365-2362.2002.0320s1055.x. 11886433.

45. Goode HF, Cowley HC, Walker BE, Howdle PD, Webster NR. Decreased antioxidant status and increased lipid peroxidation in patients with septic shock and secondary organ dysfunction. Crit Care Med 1995;23:646–51. 10.1097/00003246-199504000-00011. 7712754.

46. Gul I, Gokbel H, Belviranli M, Okudan N, Buyukbas S, Basarali K. Oxidative stress and antioxidant defense in plasma after repeated bouts of supra maximal exercise: the effect of coenzyme Q10. J Sports Med Phys Fitness 2011;51:305–12. 21681167.

47. Ostman B, Sjödin A, Michaëlsson K, Byberg L. Coenzyme Q10 supplementation and exercise-induced oxidative stress in humans. Nutrition 2012;28:403–17. 22079391.

48. Pala R. Investigation of the Oxidative Stress Parameters of Elite Boxers. Aust J Basic Appl Sci 2013;7:692–6.

49. Pala R, Cinar V, Kilic Y, Alpay N, Orhan S, Bicer Y. Examining certain oxidative stress parameters of coaches of Turkish national boxing team before and after the matches. Euro J Exp Bio 2013;3:558–61.

50. Powers SK, Locke M, Demirel HA. Exercise, heat shock proteins, and myocardial protection from I-R. injury. Med Sci Sports Exerc 2001;33:386–92. 10.1097/00005768-200103000-00009. 11252064.

51. Snoeckx LH, Cornelussen RN, van Nieuwenhoven FA, Reneman RS, van Der Vusse GJ. Heat shock proteins and cardiovascular pathophysiology. Physiol Rev 2001;81:1461–97. 11581494.

52. Mattson JP, Ross CR, Kilgore JL, Musch TI. Induction of mitochondrial stress proteins following treadmill running. Med Sci Sports Exerc 2000;32:365–9. 10.1097/00005768-200002000-00016. 10694118.

53. Salo DC, Donovan CM, Davies KJ. HSP70 and other possible heat shock or oxidative stress proteins are induced in skeletal muscle, heart, and liver during exercise. Free Radic Biol Med 1991;11:239–46. 10.1016/0891-5849(91)90119-n. 1937141.

54. Liu Y, Lormes W, Baur C, Opitz-Gress A, Altenburg D, Lehmann M, Steinacker JM. Human skeletal muscle HSP70 response to physical training depends on exercise intensity. Int J Sports Med 2000;21:351–5. 10.1055/s-2000-3784. 10950444.

55. Lappalainen J, Oksala NKJ, Laaksonen DE, Khanna S, Kokkola T, Kaarniranta K, Sen CK, Atalay M. Suppressed heat shock protein response in the kidney of exercise-trained diabetic rats. Scand J Med Sci Sports 2018;28:1808–17. 10.1111/sms.13079. 29474750.

56. Starnes JW, Choilawala AM, Taylor RP, Nelson MJ, Delp MD. Myocardial heat shock protein 70 expression in young and old rats after identical exercise programs. J Gerontol A Biol Sci Med Sci 2005;60:963–9. 10.1093/gerona/60.8.963. 16127097.

57. El Morsy EM, Kamel R, Ahmed MA. Attenuating effects of coenzyme Q10 and amlodipine in ulcerative colitis model in rats. Immunopharmacol Immunotoxicol 2015;37:244–51. 10.3109/08923973.2015.1021357. 25753843.

58. Oksala NK, Laaksonen DE, Lappalainen J, Khanna S, Nakao C, Hänninen O, Sen CK, Atalay M. Heat shock protein 60 response to exercise in diabetes: effects of alpha-lipoic acid supplementation. J Diabetes Complications 2006;20:257–61. 16798478.

59. Wu PF, SC Luo, Chang LC. Heat-shock-induced glucose transporter 4 in the slow-twitch muscle of rats. Physiol Res 2015;64:523–30. 25470523.

60. Lass A, Sohal RS. Electron transport-linked ubiquinone-dependent recycling of alpha-tocopherol inhibits autooxidation of mitochondrial membranes. Arch Biochem Biophys 1998;352:229–36. 9587410.

61. Shimomura Y, Suzuki M, Sugiyama S, Hanaki Y, Ozawa T. Protective effect of coenzyme Q10 on exercise-induced muscular injury. Biochem Biophys Res Commun 1991;176:349–55. 10.1016/0006-291x(91)90931-v. 2018527.

62. Schaefer J, Navas P, Horvath Z, Jackson S. Myopathic coenzyme Q10 deficiency. Acta Myologica 2009;28:41.

63. Kon M, Kimura F, Akimoto T, Tanabe K, Murase Y, Ikemune S, Kono I. Effect of Coenzyme Q10 supplementation on exercise-induced muscular injury of rats. Exerc Immunol Rev 2007;13:76–88. 18198662.

Article information Continued

©2018 Ragip Pala et al.; License Journal of Exercise Nutrition and Biochemistry. This is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the orginal work is properly cited.

Funded by : Firat University Scientific Research Projects Unit

Award ID : FUBAP-BSY.14.03

Funded by : Turkish Academy of Sciences (KS)

Variables	Control	CoQ10	CE	CE + CoQ10	AE	AE + CoQ10
Glucose (mg/dL)	105.7 ± 5.68	107.2 ± 5.01	104.8 ± 5.91	107.3 ± 4.96	106.0 ± 3.30	105.3 ± 6.32
Cholesterol (mg/dL)	80.6 ± 3.12^a	71.1 ± 2.91^b	69.7 ± 0.85^b	68.4 ± 1.64^c	69.3 ± 0.62^b	68.7 ± 4.30^b
Triglyceride (mg/dL)	93.8 ± 2.85^a	83.8 ± 1.78^b	84.7 ± 2.58^b	72.0 ± 2.16^d	84.3 ± 2.42^b	74.3 ± 2.23^c
AST (U/L)	230.8 ± 19.44	221.5 ± 17.82	225.3 ± 26.34	221.9 ± 11.2	219.3 ± 15.42	222.0 ± 17.78
ALT (U/L)	92.0 ± 8.89	91.0 ± 6.26	85.7 ± 6.15	90.4 ± 7.89	92.3 ± 6.05	97.0 ± 7.02

Variables	Control	CoQ10	CE	CE + CoQ10	AE	AE + CoQ10
Serum MDA (nmol/mg protein)	0.88±0.04^c	0.61±0.03^d	0.55±0.02^d	0.43±0.03^e	1.37±0.05^a	0.91±0.03^b
Liver MDA (nmol/mg protein)	101.4±2.24^c	95.1±2.46^d	96.1±2.04^d	87.2±1.94^e	117.7±2.55^a	108.3±2.8^b
Muscle MDA (nmol/mg protein)	85.2±3.11^c	61.3±2.05^d	59.6±1.82^d	53.0±1.50^e	97.7±2.22^a	93.6±1.43^b