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Plasma Apelin Concentration in Exercised Horses: Preliminary Study
Journal of Equine Veterinary Science 80 (2019) 16e19
Contents lists available at ScienceDirect
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Original Research
Plasma Apelin Concentration in Exercised Horses: Preliminary Study ska b, Tatiana Wawak a, Iwona Janczarek c, Izabela Wilk c, Witold Ke˛ dzierski a, Anna Cywin d, * Sylwester Kowalik a
Department of Biochemistry, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Lublin, Poland Department of Pathology and Veterinary Diagnostic, Faculty of Veterinary Medicine, Warsaw University of Life Sciences - SGGW, Warszawa, Poland c Department of Horse Breeding and Use, Faculty of Biology, Animal Sciences and Bioeconomy, University of Life Sciences in Lublin, Lublin, Poland d Department of Animal Physiology, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Lublin, Poland b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 April 2019 Received in revised form 17 June 2019 Accepted 17 June 2019 Available online 3 July 2019
Physical effort is one of the key aspects of keeping horses in good condition. The condition of the animal is reflected by multiple blood parameters. The newly discovered cytokineeapelin can pose one of them, however, so far, has not been studied in the horse population. Apelin is produced by adipocytes and myocytes and plays an important role in the energy metabolism of the body through the influence, for example, on the process of adipogenesis and lipolysis. The aim of this study was to investigate if physical effort of various intensity affects the plasma concentration of apelin in horses. The study involved 20 purebred Arabian horses divided into two groups. The first group included 10 race horses, aged 3 years, and second group included 10 horses aged from 6 to 12 years, used in endurance rides. Blood samples were collected from each horse at rest and after exercise. The concentrations of apelin, lactic acid, cortisol, uric acid (UA), triacylglycerols, total plasma protein, and glucose were determined in plasma samples. Race training sessions induced significant decreases in plasma apelin concentrations (P < .05). In endurance horses, significant correlations were found only between exercise-induced changes of apelin and cortisol (r ¼ 0.55) and apelin and UA (r ¼ 0.67) concentrations. In conclusion, the concentration of apelin in equine plasma decreases in response to short-time exercise unlike endurance exercise. Blood plasma apelin concentration determined at rest is lower in long-time regularly trained horses than in the ones at the beginning of their training process. © 2019 Elsevier Inc. All rights reserved.
Keywords: Apelin Horses Exercise Training
1. Introduction Apelin is a novel cytokine produced mainly by adipocytes [1,2] and myocytes [3,4]. The name apelin includes a group of peptides composed of 12, 13, 17, and 36 amino acids [5,6]. Apelin has a multifunctional and beneficial influence on adipose tissue and other organs. It has been shown to influence lipid metabolism [7,8] and food intake [9], increase insulin sensitivity [10e12] and glucose
Animal welfare/ethical statement: The study was conducted in accordance with the Guidelines for Animal Care and Treatment of the European Union and were approved by the Local Ethic Review Committee for Animal Experiments (reference number 45/2017). Conflicts of interest statement: The authors declare no conflict of interest. * Corresponding author at: Sylwester Kowalik, Department of Animal Physiology, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Akademicka 12, 20-033 Lublin, Poland. E-mail address: [email protected] (S. Kowalik). https://doi.org/10.1016/j.jevs.2019.06.012 0737-0806/© 2019 Elsevier Inc. All rights reserved.
uptake [13], reduce arterial blood pressure [14] and arterial stiffness [15], and enhance antioxidant defense [16e18]. Plasma apelin concentration is higher in overweight and/or insulin-resistant subjects than in slim and healthy individuals [19e22]. It is well known that regular exercise promotes body weight (BW) control and glucose tolerance. In humans, there are a general consent that single bout of exercise leads to an increase in plasma apelin concentration [23,24]. However, studies regarding the influence of regular training on the level of apelin brought contradictory results. Some studies indicated the increase in this adipokine plasma level with training [4,15,25e28], whereas others noted significant decreases in this parameter [19,20,23,29,30]. To the authors’ knowledge, there is no scientific reports regarding apelin regulation in horses. These animals are used primarily for physical work, including regular sport training. Athletic horses are routinely trained and submitted to extremally intensive exercise tests. Purebred Arabian horses are special in this regard, as they are trained on race tracks and used for races when they are aged 3
W. Ke˛ dzierski et al. / Journal of Equine Veterinary Science 80 (2019) 16e19
years, and then, as 6 years or later, they participate in endurance rides. On the other hand, some nonexercised horses may suffer from overweight, insulin resistance, and other metabolic disorders [31,32]. Studies on other adipokines in trained horses, such as leptin, visfatin, and adropin, have shown some fluctuation depending on the intensity of exercise or training [33e35]. Taking into account the role of apelin in the regulation of lipid and glucose metabolism, changes in the release of this cytokine in exercised horses can be expected. Thus, the aim of the study was to investigate if training process and exercise of various intensity influence plasma apelin concentration in horses. 2. Materials and Methods 2.1. Horses Two groups of purebred Arabian horses were involved in the study. First group was composed of 10 race horses, aged 3 years, five stallions, and five mares (mean body condition score [BCS] 5.0), and the second group included 10 older horses, aged from 6 to 12 years, trained for endurance rides (four mares, three stallions, and three geldings, mean BCS 4.7). Young horses were trained and competed in official races on the Sluzewiec Horse Race Track in Warsaw (Poland). They began regular training in late March and started to participate in official races at least once a month starting from the middle of May. Throughout the season, all race horses were under the care of one trainer. They were carried and fed in the same manner recommended for race horses. The stallions and mares were kept in separate stables in the boxes 4 4 m. Each horse received an individually calculated ration of hay, oats, and concentrate according to its nutritional requirements arising from maintenance and workload. This diet included an average of digestible energy 0.27 MJ/kg BW per day and digestible protein 2.5 g/kg BW per day, distributed over three feedings per day. A mineral salt block and fresh water were constantly available. During the training season, the horses were routinely exercised five times per week. Each training session included about 10 minutes of walking as a warm up, 10 minutes of trot or canter, and then gallop over the distance of 1,200 m, at a speed of 6e12 m/s, according to horse performance and the trainer's decision. After that, the horses were cooled down on an automatic horse walker for 30 minutes. Horses from this group were investigated three times during routine training sessions performed in the middle of May, July, and October, which coincided with the beginning (BEG), middle (MID), and the end (END) of race season. In all time points, blood samples were collected from each horse at rest (early morning), immediately after exercise, and after the recovery (30 minutes later). Details regarding the exercise protocols are given in Table 1. Endurance horses were investigated during official competitions at 120 km, which took place in July. The horses were trained individually by their trainers or owners in different training centers and were brought to the place of the competitions at least a day before the start. All horses passed veterinary examination and were
Table 1 Body condition scores (BCSs) of the studied horses and technical conditions of the exercise they performed. Horses
BCS
n
Length
Velocity
Duration
Race horses at BEG Race horses in MID Race horses at END Endurance horses
5.0 4.8 4.5 4.7
10 10 8 10
1,200 m 2,000 m 1,200 m 120 km
10 m/s 6 m/s 9 m/s 3.3 m/s
2.0 min 5.5 min 2.2 min 10.5 hr
BEG, MID, END: the beginning, middle, and the end of race season.
17
allowed to participate in the competitions. Only horses which completed the full distance were involved in the study. Blood samples were taken at rest and immediately after finishing the competitions. For each horse, the BCS was evaluated by the same, experienced veterinarian who used the 9-point rating system devised by Henneke et al. [36]. 2.2. Blood Sampling and Analysis From each horse, samples were collected by jugular venipuncture to the tubes containing di-potassium ethylenediaminetetraacetic acid and immediately put into a water bath at a temperature of 4 C and then centrifuged to obtain plasma for further investigations. Blood lactic acid (LA) concentrations in the samples collected at rest and after exercise were determined immediately using the enzymatic method (Cormay, Warsaw, Poland) and a Dr Lange portable analyzer (Dr Lange Laboratory System LP450, Germany). The remaining plasma was frozen and stored at 74 C until analyzing other biochemical parameters: apelin, cortisol, uric acid (UA), triacylglycerols together with free glycerol (TG), total plasma protein (TPP), and glucose. Plasma apelin concentrations were determined in duplicate in the samples taken at rest and 30 minutes after exercise using a commercial test based on enzyme-immunoassay method (Apelin12 EIA-kit; Phoenix Pharmaceuticals, Inc, Burlingame), with a sensitivity 0.07 ng/mL, and the linear range from 0.07 to 1.33 ng/ mL. Manufacturer indicated the specificity of this kit as 100% for humans, rat, mouse, and bovine apelin-12. The kit shows crossreactivity with biologically active isoforms of apelin-36 and 13. Absorbance was measured at 450 nm using a Multiskan reader (Labsystem, Helsinki, Finland) supported by GENESIS V 3.00 software (VWR International Ltd, Lutterworth, UK). The intra-assay variations for the used kit were <10%, and interassay <15%. Horse plasma diluted 1:2, 1:4, and 1:6 demonstrated linearity and parallelism when these dilutions were plotted against the used curve of standard apelin serial dilution. Two samples of equine plasma were added to 0.1 and 1.0 ng/mL of apelin standards resulted in 115% and 90% recovery of the used standards, respectively. Results are expressed as human equivalents of immunereactive total apelin, including apelin-12, 13, and 36. For plasma cortisol determination, the CORTISOL ELISA kit (DRG International Inc, Mountainside, NJ) was used. The absorbance was measured by the same equipment. The intra- and inter-assay coefficient of variation for plasma cortisol concentration determined in the laboratory reached 6% and 8%, respectively. The values are expressed as nanogram/milliliter. Other biochemical parameters were determined using enzymatic kits (Cormay, Warsaw, Poland) and Dr Lange analyzer. 2.3. Statistical Analysis The results are presented as means ± standard deviation. Statistical analyses were performed by two-way repeated measures analysis of variance using the statistical software package GraphPad Prism (Graph Pad Software, La Jolla, CA). Data were tested for normality of distribution using ShapiroeWilk method. The normal distribution was confirmed. Post hoc comparisons between the results obtained at rest and after exercise in studied groups and periods of the study were made by the Tukey's test. For each horse, the differences between results obtained after the end of exercise and basal values were calculated. The coefficient correlations between these differences for apelin, cortisol, LA, UA, TG, and glucose values were assessed by the Pearson test. The statistical significance was accepted at P < .05.
W. Ke˛ dzierski et al. / Journal of Equine Veterinary Science 80 (2019) 16e19
18
3. Results Mean values of BCS in endurance horses amounted to 4.7, and in race horses were BEG 5.0, MID 4.8, END 4.5, and did not differ significantly (Table 1). Plasma LA concentrations at rest did not exceed 1 mmol/L and did not significantly change in the studied period and between groups of horses. Plasma apelin, cortisol, and postexercise LA concentrations are presented in Table 2. Plasma apelin concentration determined at rest was significantly lower in endurance horses than in race horses examined at BEG and MID. Cortisol concentration at rest was higher in endurance horses than in race horses. All race training sessions induced significant decreases in plasma apelin concentrations. A drop in the value of this parameter in endurance horses was not significant. The highest increase in LA concentration was observed during race training session performed at BEG and less pronounced, but still significant postexercise increases occurred in race horses at END and in endurance horses. Significant increase in plasma cortisol concentration was stated in race horses at BEG and END only. In endurance horses after the end of exercise, cortisol levels dropped in some horses, but increased few times in others; thus, the mean value was unchanged compared with the value at rest and ranged from 112 to 1,074 ng/mL. Among other studied parameters, plasma UA concentration increased significantly after race exercise at BEG and in endurance horses (P < .05). Triacylglycerols together with free glycerol increased in race horses at BEG and END, glucose increased in race horses at BEG and END and decreased in endurance horses, and TPP did not significantly change (P < .05). Significant correlations were found in endurance horses only between exerciseinduced changes of apelin and cortisol (r ¼ 0.55) and apelin and UA (r ¼ 0.67) concentrations. 4. Discussion Plasma apelin concentration determined at rest was lower in older, long-time trained horses than in 3-year-olds, which began the race training process. The influence of training on plasma level of this peptide in humans and laboratory animals is not clearly defined. Training-induced decrease in plasma apelin level was described in obese humans [19,20,30], and diabetic rats [29]. On the other hand, increase in plasma apelin level was observed in intensively trained older, hypertensive people [27], obese men [25], healthy adults [15], and diabetic rats [26,28]. Some other researchers did not find any relation between plasma apelin and training [3,37,38]. It is difficult to find the reason for these differences. However, apelin is synthetized in adipose tissue as well as in muscles. Training, leading to reduce body fat mass, was frequently associated with the decrease in plasma apelin concentration [19,30], but lack of such changes was also noted [20]. Other studies showed that training increases apelin messenger RNA expression in muscles [3,4]. Thus, in obese subjects, a reduction in body fat mass can in turn reduce the source of apelin,
whereas increases are in line with the hypothesis of trained muscles as a source of circulating apelin. Probably, a balance between adipose and muscle tissuesedelivered apelin influenced its plasma level. However, this hypothesis did not explain the results obtained in the horses investigated in the study because their BCS remained unchanged. Other possible reason of observed decrease in plasma apelin value in studied horses can be of hormonal origin. It is known that insulin stimulates apelin synthesis, whereas cortisol and other corticoids play an inhibitory role [39,40]. Moreover, cortisol can upregulate expression of apelin receptors [41]. Heavy exercise induces a decrease in insulin level and increase in the concentrations of circulating corticosteroids; thus, constantly repeated exercise sessions can lead to decrease in apelin synthesis and secretion. Decreases in plasma apelin concentration after each training session in race horses seem to confirm the thesis mentioned previously. However, the studies in humans showed an exerciseinduced increase in plasma apelin level [23,24]. Moreover, the results obtained in endurance horses indicated that changes in plasma apelin and cortisol or UA after long-lasting exercise were positively correlated. It means that the exercise-induced significant increase in cortisol or UA values in horses seems to be accompanied by a tendency to increase in plasma apelin concentration. UA increases after strong effort as a result of adenosine 5'-triphosphate degradation and can be used as a marker of relative exercise intensity [42,43]. Horses seem less sensitive to exercise load than humans, and only an extreme, exhaustive effort is able to induce an increase in plasma apelin level. However, an influence of some other, so far undefined factors on plasma apelin concentration cannot be excluded. Despite the influence of insulin and cortisol on apelin synthesis and release, there must be other factors which lead to an increase in plasma apelin values during exercise. For example, hypoxia and inflammation can induce apelin expression via appropriate peptide factors [44]. Other substances inducing apelin expression are polyunsaturated fatty acids [45]; however, their role is not related to exercise. In general, this is the first study focused on apelin in horses. Although, the present study reports exercised-induced changes in plasma apelin concentration in race horses, its design is not without limitations. First, the samples were collected in field conditions precisely control some sources of possible mistakes. Moreover, there might be some relevant factors, which additionally influence on the level of circulating apelin in horses, for example, horse's breed, type of use, and maintaining routine. And finally, there is the lack of similar studies in a horse; thus, it was not possible to discuss our results with others. Thus, further research is needed to explain the regulation of apelin release and its role in exercised horses. In conclusion, the concentration of apelin in equine plasma decreases in response to short-time exercise and is lower in longtime regularly trained horses than in the ones at the beginning of their training process.
Table 2 Plasma adropin, lactic acid (LA), and cortisol concentrations in the studied horses (means ± standard deviation). Horses
Apelin (ng/mL)
Race horses at BEG Race horses in MID Race horses at END Endurance horses
0.59 0.62 0.55 0.43
At Rest
x, y, z
± ± ± ±
0.14x 0.15x 0.11xy 0.09y
30 min After Exercise 0.46 0.46 0.45 0.38
± ± ± ±
0.07a 0.09a 0.08a 0.08
Means in columns with different letters differ significantly. BEG, MID, END: the beginning, middle, and the end of race season. a Mean significantly different in comparison to the value obtained at rest.
LA (mmol/L)
Cortisol (ng/mL)
After Exercise
At Rest
11.1 0.96 2.56 3.83
± ± ± ±
4.04xa 0.14y 2.22za 0.66za
110 109 123 213
± ± ± ±
31.1x 25.9x 18.9x 59.2y
After Exercise 175 121 167 334
± ± ± ±
55.3a 24.4 37.9a 262
W. Ke˛ dzierski et al. / Journal of Equine Veterinary Science 80 (2019) 16e19
Acknowledgments This work was supported by the funds of the University of Life Sciences in Lublin (WKB-DS-4).
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[22] Karbek B, Bozkurt NC, Topaloglu O, Aslan MS, Gungunes A, Cakal E, et al. Relationship of vaspin and apelin levels with insulin resistance and atherosclerosis in metabolic syndrome. Minerva Endocrinol 2014;39:99e105. [23] Fortunato AK, Pontes WM, De Souza DMS, Prazeres JSF, Marcucci-Barbosa LS, Santos JMM, et al. Strength training session induces important changes on physiological, immunological, and inflammatory biomarkers. J Immunol Res 2018;26:9675216. [24] Dundar A, Kocahan S, Arslan C. Effects of different loading exercises on apelin levels and physical and hematologic parameters of swimmers. Horm Mol Biol Clin Investig 2019;32. in press. [25] Nikseresht M, Hafezi Ahmadi MR, Hedayati M. Detraining-induced alterations in adipokines and cardiometabolic risk factors after nonlinear periodized resistance and aerobic interval training in obese men. Appl Physiol Nutr Metabol 2016;41:1018e25. [26] Son JS, Kim HJ, Son Y, Lee H, Chae SA, Seong JK, et al. Effects of exerciseinduced apelin levels on skeletal muscle and their capillarization in type 2 diabetic rats. Muscle Nerve 2017;56:1155e63. [27] Izadi MR, Ghardashi Afousi A, Asvadi Fard M, Babae Bigi MA. High-intensity interval training lowers blood pressure and improves apelin and NOx plasma levels in older treated hypertensive individuals. J Physiol Biochem 2018;74: 47e55. [28] Kazemi F, Zahediasl S. Effects of exercise training on adipose tissue apelin expression in streptozotocin-nicotinamide induced diabetic rats. Gene 2018;662:97e102. [29] Delavar R, Heidarianpour A. The effect of aerobic exercise training on plasma apelin levels and Pain Threshold in T1DM rats. Iran Red Crescent Med J 2016;18:e31737. [30] Jang SH, Paik IY, Ryu JH, Lee TH, Kim DE. Effects of aerobic and resistance exercises on circulating apelin-12 and apelin-36 concentrations in obese middle-aged women: a randomized controlled trial. BMC Womens Health 2019;19:23. [31] Frank N, Elliott SB, Brandt LE, Keisler DH. Physical characteristics, blood hormone concentrations, and plasma lipid concentrations in obese horses with insulin resistance. J Am Vet Med Assoc 2006;228:1383e90. [32] Durham AE, Frank N, McGowan CM, Menzies-Gow NJ, Roelfsema E, Vervuert I, et al. EIMS consensus statement on equine metabolic syndrome. J Vet Intern Med 2019;33:335e49. [33] Ke˛ dzierski W. The effects of exercise on plasma leptin concentrations in horses. J Equine Vet Sci 2016;47:36e41. [34] Ke˛ dzierski W, Janczarek I, Wilk I, Staniszewska M, Kowalik S. Plasma visfatin response to the intensity of exercise and training in race-horses. Pferdeheilkunde 2018;34:525e30. [35] Kowalik S, Ke˛ dzierski W, Janczarek I, Wilk I. Plasma adropin in exercised horses: a pilot study. Pferdeheilkunde 2017;33:585e90. [36] Henneke DR, Potter GD, Kreider JL, Yeates BF. Relationship between condition score, physical measurements and body fat percentage in mares. Equine Vet J 1983;15:371e2. [37] Sanchis-Gomar F, Alis R, Rampinini E, Bosio A, Ferioli D, La Torre A, et al. Adropin and apelin fluctuations throughout a season in professional soccer players: are they related with performance? Peptides 2015;70:32e6. [38] Kolahdouzi S, Baghadam M, Kani-Golzar FA, Saeidi A, Jabbour G, Ayadi A, et al. Progressive circuit resistance training improves inflammatory biomarkers and insulin resistance in obese men. Physiol Behav 2018;205:15e21. [39] Wei L, Hou X, Tatemoto K. Regulation of apelin mRNA expression by insulin and glucocorticoids in mouse 3T3-L1 adipocytes. Regul Pept 2005;132:27e32. [40] Jiang H, Ye XP, Yang ZY, Zhan M, Wang HN, Cao HM, et al. Aldosterone directly affects apelin expression and secretion in adipocytes. J Mol Endocrinol 2013;51:37e48. [41] O'Carroll AM, Don AL, Lolait SJ. APJ receptor mRNA expression in the rat hypothalamic paraventricular nucleus: regulation by stress and glucocorticoids. J Neuroendocrinol 2003;15:1095e101. [42] Escribano BM, Molina A, Valera M, Tovar P, Agüera EI, Santisteban R, et al. Genetic analysis of haematological and plasma biochemical parameters in the Spanish purebred horse exercised on a treadmill. Animal 2013;7:1414e22. [43] Ke˛ dzierski W. Changes in plasma leptin concentration during different types of exercises performed by horses. Animal 2014;8:1456e61. [44] Han S, Wang G, Qi X, Lee HM, Englander EW, Greeley Jr GH. A possible role for hypoxia-induced apelin expression in enteric cell proliferation. Am J Physiol Regul Integr Comp Physiol 2008;294:R1832e9. [45] Mortazavi A, Nematipoor E, Djalali M, Keshavarz SA, Samavat S, Zarei M, et al. The effect of omega-3 fatty acids on serum apelin levels in cardiovascular disease: a randomized, double-blind, placebo-controlled trial. Rep Biochem Mol Biol 2018;7:59e66.
Contents lists available at ScienceDirect
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Original Research
Plasma Apelin Concentration in Exercised Horses: Preliminary Study ska b, Tatiana Wawak a, Iwona Janczarek c, Izabela Wilk c, Witold Ke˛ dzierski a, Anna Cywin d, * Sylwester Kowalik a
Department of Biochemistry, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Lublin, Poland Department of Pathology and Veterinary Diagnostic, Faculty of Veterinary Medicine, Warsaw University of Life Sciences - SGGW, Warszawa, Poland c Department of Horse Breeding and Use, Faculty of Biology, Animal Sciences and Bioeconomy, University of Life Sciences in Lublin, Lublin, Poland d Department of Animal Physiology, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Lublin, Poland b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 April 2019 Received in revised form 17 June 2019 Accepted 17 June 2019 Available online 3 July 2019
Physical effort is one of the key aspects of keeping horses in good condition. The condition of the animal is reflected by multiple blood parameters. The newly discovered cytokineeapelin can pose one of them, however, so far, has not been studied in the horse population. Apelin is produced by adipocytes and myocytes and plays an important role in the energy metabolism of the body through the influence, for example, on the process of adipogenesis and lipolysis. The aim of this study was to investigate if physical effort of various intensity affects the plasma concentration of apelin in horses. The study involved 20 purebred Arabian horses divided into two groups. The first group included 10 race horses, aged 3 years, and second group included 10 horses aged from 6 to 12 years, used in endurance rides. Blood samples were collected from each horse at rest and after exercise. The concentrations of apelin, lactic acid, cortisol, uric acid (UA), triacylglycerols, total plasma protein, and glucose were determined in plasma samples. Race training sessions induced significant decreases in plasma apelin concentrations (P < .05). In endurance horses, significant correlations were found only between exercise-induced changes of apelin and cortisol (r ¼ 0.55) and apelin and UA (r ¼ 0.67) concentrations. In conclusion, the concentration of apelin in equine plasma decreases in response to short-time exercise unlike endurance exercise. Blood plasma apelin concentration determined at rest is lower in long-time regularly trained horses than in the ones at the beginning of their training process. © 2019 Elsevier Inc. All rights reserved.
Keywords: Apelin Horses Exercise Training
1. Introduction Apelin is a novel cytokine produced mainly by adipocytes [1,2] and myocytes [3,4]. The name apelin includes a group of peptides composed of 12, 13, 17, and 36 amino acids [5,6]. Apelin has a multifunctional and beneficial influence on adipose tissue and other organs. It has been shown to influence lipid metabolism [7,8] and food intake [9], increase insulin sensitivity [10e12] and glucose
Animal welfare/ethical statement: The study was conducted in accordance with the Guidelines for Animal Care and Treatment of the European Union and were approved by the Local Ethic Review Committee for Animal Experiments (reference number 45/2017). Conflicts of interest statement: The authors declare no conflict of interest. * Corresponding author at: Sylwester Kowalik, Department of Animal Physiology, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Akademicka 12, 20-033 Lublin, Poland. E-mail address: [email protected] (S. Kowalik). https://doi.org/10.1016/j.jevs.2019.06.012 0737-0806/© 2019 Elsevier Inc. All rights reserved.
uptake [13], reduce arterial blood pressure [14] and arterial stiffness [15], and enhance antioxidant defense [16e18]. Plasma apelin concentration is higher in overweight and/or insulin-resistant subjects than in slim and healthy individuals [19e22]. It is well known that regular exercise promotes body weight (BW) control and glucose tolerance. In humans, there are a general consent that single bout of exercise leads to an increase in plasma apelin concentration [23,24]. However, studies regarding the influence of regular training on the level of apelin brought contradictory results. Some studies indicated the increase in this adipokine plasma level with training [4,15,25e28], whereas others noted significant decreases in this parameter [19,20,23,29,30]. To the authors’ knowledge, there is no scientific reports regarding apelin regulation in horses. These animals are used primarily for physical work, including regular sport training. Athletic horses are routinely trained and submitted to extremally intensive exercise tests. Purebred Arabian horses are special in this regard, as they are trained on race tracks and used for races when they are aged 3
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years, and then, as 6 years or later, they participate in endurance rides. On the other hand, some nonexercised horses may suffer from overweight, insulin resistance, and other metabolic disorders [31,32]. Studies on other adipokines in trained horses, such as leptin, visfatin, and adropin, have shown some fluctuation depending on the intensity of exercise or training [33e35]. Taking into account the role of apelin in the regulation of lipid and glucose metabolism, changes in the release of this cytokine in exercised horses can be expected. Thus, the aim of the study was to investigate if training process and exercise of various intensity influence plasma apelin concentration in horses. 2. Materials and Methods 2.1. Horses Two groups of purebred Arabian horses were involved in the study. First group was composed of 10 race horses, aged 3 years, five stallions, and five mares (mean body condition score [BCS] 5.0), and the second group included 10 older horses, aged from 6 to 12 years, trained for endurance rides (four mares, three stallions, and three geldings, mean BCS 4.7). Young horses were trained and competed in official races on the Sluzewiec Horse Race Track in Warsaw (Poland). They began regular training in late March and started to participate in official races at least once a month starting from the middle of May. Throughout the season, all race horses were under the care of one trainer. They were carried and fed in the same manner recommended for race horses. The stallions and mares were kept in separate stables in the boxes 4 4 m. Each horse received an individually calculated ration of hay, oats, and concentrate according to its nutritional requirements arising from maintenance and workload. This diet included an average of digestible energy 0.27 MJ/kg BW per day and digestible protein 2.5 g/kg BW per day, distributed over three feedings per day. A mineral salt block and fresh water were constantly available. During the training season, the horses were routinely exercised five times per week. Each training session included about 10 minutes of walking as a warm up, 10 minutes of trot or canter, and then gallop over the distance of 1,200 m, at a speed of 6e12 m/s, according to horse performance and the trainer's decision. After that, the horses were cooled down on an automatic horse walker for 30 minutes. Horses from this group were investigated three times during routine training sessions performed in the middle of May, July, and October, which coincided with the beginning (BEG), middle (MID), and the end (END) of race season. In all time points, blood samples were collected from each horse at rest (early morning), immediately after exercise, and after the recovery (30 minutes later). Details regarding the exercise protocols are given in Table 1. Endurance horses were investigated during official competitions at 120 km, which took place in July. The horses were trained individually by their trainers or owners in different training centers and were brought to the place of the competitions at least a day before the start. All horses passed veterinary examination and were
Table 1 Body condition scores (BCSs) of the studied horses and technical conditions of the exercise they performed. Horses
BCS
n
Length
Velocity
Duration
Race horses at BEG Race horses in MID Race horses at END Endurance horses
5.0 4.8 4.5 4.7
10 10 8 10
1,200 m 2,000 m 1,200 m 120 km
10 m/s 6 m/s 9 m/s 3.3 m/s
2.0 min 5.5 min 2.2 min 10.5 hr
BEG, MID, END: the beginning, middle, and the end of race season.
17
allowed to participate in the competitions. Only horses which completed the full distance were involved in the study. Blood samples were taken at rest and immediately after finishing the competitions. For each horse, the BCS was evaluated by the same, experienced veterinarian who used the 9-point rating system devised by Henneke et al. [36]. 2.2. Blood Sampling and Analysis From each horse, samples were collected by jugular venipuncture to the tubes containing di-potassium ethylenediaminetetraacetic acid and immediately put into a water bath at a temperature of 4 C and then centrifuged to obtain plasma for further investigations. Blood lactic acid (LA) concentrations in the samples collected at rest and after exercise were determined immediately using the enzymatic method (Cormay, Warsaw, Poland) and a Dr Lange portable analyzer (Dr Lange Laboratory System LP450, Germany). The remaining plasma was frozen and stored at 74 C until analyzing other biochemical parameters: apelin, cortisol, uric acid (UA), triacylglycerols together with free glycerol (TG), total plasma protein (TPP), and glucose. Plasma apelin concentrations were determined in duplicate in the samples taken at rest and 30 minutes after exercise using a commercial test based on enzyme-immunoassay method (Apelin12 EIA-kit; Phoenix Pharmaceuticals, Inc, Burlingame), with a sensitivity 0.07 ng/mL, and the linear range from 0.07 to 1.33 ng/ mL. Manufacturer indicated the specificity of this kit as 100% for humans, rat, mouse, and bovine apelin-12. The kit shows crossreactivity with biologically active isoforms of apelin-36 and 13. Absorbance was measured at 450 nm using a Multiskan reader (Labsystem, Helsinki, Finland) supported by GENESIS V 3.00 software (VWR International Ltd, Lutterworth, UK). The intra-assay variations for the used kit were <10%, and interassay <15%. Horse plasma diluted 1:2, 1:4, and 1:6 demonstrated linearity and parallelism when these dilutions were plotted against the used curve of standard apelin serial dilution. Two samples of equine plasma were added to 0.1 and 1.0 ng/mL of apelin standards resulted in 115% and 90% recovery of the used standards, respectively. Results are expressed as human equivalents of immunereactive total apelin, including apelin-12, 13, and 36. For plasma cortisol determination, the CORTISOL ELISA kit (DRG International Inc, Mountainside, NJ) was used. The absorbance was measured by the same equipment. The intra- and inter-assay coefficient of variation for plasma cortisol concentration determined in the laboratory reached 6% and 8%, respectively. The values are expressed as nanogram/milliliter. Other biochemical parameters were determined using enzymatic kits (Cormay, Warsaw, Poland) and Dr Lange analyzer. 2.3. Statistical Analysis The results are presented as means ± standard deviation. Statistical analyses were performed by two-way repeated measures analysis of variance using the statistical software package GraphPad Prism (Graph Pad Software, La Jolla, CA). Data were tested for normality of distribution using ShapiroeWilk method. The normal distribution was confirmed. Post hoc comparisons between the results obtained at rest and after exercise in studied groups and periods of the study were made by the Tukey's test. For each horse, the differences between results obtained after the end of exercise and basal values were calculated. The coefficient correlations between these differences for apelin, cortisol, LA, UA, TG, and glucose values were assessed by the Pearson test. The statistical significance was accepted at P < .05.
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3. Results Mean values of BCS in endurance horses amounted to 4.7, and in race horses were BEG 5.0, MID 4.8, END 4.5, and did not differ significantly (Table 1). Plasma LA concentrations at rest did not exceed 1 mmol/L and did not significantly change in the studied period and between groups of horses. Plasma apelin, cortisol, and postexercise LA concentrations are presented in Table 2. Plasma apelin concentration determined at rest was significantly lower in endurance horses than in race horses examined at BEG and MID. Cortisol concentration at rest was higher in endurance horses than in race horses. All race training sessions induced significant decreases in plasma apelin concentrations. A drop in the value of this parameter in endurance horses was not significant. The highest increase in LA concentration was observed during race training session performed at BEG and less pronounced, but still significant postexercise increases occurred in race horses at END and in endurance horses. Significant increase in plasma cortisol concentration was stated in race horses at BEG and END only. In endurance horses after the end of exercise, cortisol levels dropped in some horses, but increased few times in others; thus, the mean value was unchanged compared with the value at rest and ranged from 112 to 1,074 ng/mL. Among other studied parameters, plasma UA concentration increased significantly after race exercise at BEG and in endurance horses (P < .05). Triacylglycerols together with free glycerol increased in race horses at BEG and END, glucose increased in race horses at BEG and END and decreased in endurance horses, and TPP did not significantly change (P < .05). Significant correlations were found in endurance horses only between exerciseinduced changes of apelin and cortisol (r ¼ 0.55) and apelin and UA (r ¼ 0.67) concentrations. 4. Discussion Plasma apelin concentration determined at rest was lower in older, long-time trained horses than in 3-year-olds, which began the race training process. The influence of training on plasma level of this peptide in humans and laboratory animals is not clearly defined. Training-induced decrease in plasma apelin level was described in obese humans [19,20,30], and diabetic rats [29]. On the other hand, increase in plasma apelin level was observed in intensively trained older, hypertensive people [27], obese men [25], healthy adults [15], and diabetic rats [26,28]. Some other researchers did not find any relation between plasma apelin and training [3,37,38]. It is difficult to find the reason for these differences. However, apelin is synthetized in adipose tissue as well as in muscles. Training, leading to reduce body fat mass, was frequently associated with the decrease in plasma apelin concentration [19,30], but lack of such changes was also noted [20]. Other studies showed that training increases apelin messenger RNA expression in muscles [3,4]. Thus, in obese subjects, a reduction in body fat mass can in turn reduce the source of apelin,
whereas increases are in line with the hypothesis of trained muscles as a source of circulating apelin. Probably, a balance between adipose and muscle tissuesedelivered apelin influenced its plasma level. However, this hypothesis did not explain the results obtained in the horses investigated in the study because their BCS remained unchanged. Other possible reason of observed decrease in plasma apelin value in studied horses can be of hormonal origin. It is known that insulin stimulates apelin synthesis, whereas cortisol and other corticoids play an inhibitory role [39,40]. Moreover, cortisol can upregulate expression of apelin receptors [41]. Heavy exercise induces a decrease in insulin level and increase in the concentrations of circulating corticosteroids; thus, constantly repeated exercise sessions can lead to decrease in apelin synthesis and secretion. Decreases in plasma apelin concentration after each training session in race horses seem to confirm the thesis mentioned previously. However, the studies in humans showed an exerciseinduced increase in plasma apelin level [23,24]. Moreover, the results obtained in endurance horses indicated that changes in plasma apelin and cortisol or UA after long-lasting exercise were positively correlated. It means that the exercise-induced significant increase in cortisol or UA values in horses seems to be accompanied by a tendency to increase in plasma apelin concentration. UA increases after strong effort as a result of adenosine 5'-triphosphate degradation and can be used as a marker of relative exercise intensity [42,43]. Horses seem less sensitive to exercise load than humans, and only an extreme, exhaustive effort is able to induce an increase in plasma apelin level. However, an influence of some other, so far undefined factors on plasma apelin concentration cannot be excluded. Despite the influence of insulin and cortisol on apelin synthesis and release, there must be other factors which lead to an increase in plasma apelin values during exercise. For example, hypoxia and inflammation can induce apelin expression via appropriate peptide factors [44]. Other substances inducing apelin expression are polyunsaturated fatty acids [45]; however, their role is not related to exercise. In general, this is the first study focused on apelin in horses. Although, the present study reports exercised-induced changes in plasma apelin concentration in race horses, its design is not without limitations. First, the samples were collected in field conditions precisely control some sources of possible mistakes. Moreover, there might be some relevant factors, which additionally influence on the level of circulating apelin in horses, for example, horse's breed, type of use, and maintaining routine. And finally, there is the lack of similar studies in a horse; thus, it was not possible to discuss our results with others. Thus, further research is needed to explain the regulation of apelin release and its role in exercised horses. In conclusion, the concentration of apelin in equine plasma decreases in response to short-time exercise and is lower in longtime regularly trained horses than in the ones at the beginning of their training process.
Table 2 Plasma adropin, lactic acid (LA), and cortisol concentrations in the studied horses (means ± standard deviation). Horses
Apelin (ng/mL)
Race horses at BEG Race horses in MID Race horses at END Endurance horses
0.59 0.62 0.55 0.43
At Rest
x, y, z
± ± ± ±
0.14x 0.15x 0.11xy 0.09y
30 min After Exercise 0.46 0.46 0.45 0.38
± ± ± ±
0.07a 0.09a 0.08a 0.08
Means in columns with different letters differ significantly. BEG, MID, END: the beginning, middle, and the end of race season. a Mean significantly different in comparison to the value obtained at rest.
LA (mmol/L)
Cortisol (ng/mL)
After Exercise
At Rest
11.1 0.96 2.56 3.83
± ± ± ±
4.04xa 0.14y 2.22za 0.66za
110 109 123 213
± ± ± ±
31.1x 25.9x 18.9x 59.2y
After Exercise 175 121 167 334
± ± ± ±
55.3a 24.4 37.9a 262
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Acknowledgments This work was supported by the funds of the University of Life Sciences in Lublin (WKB-DS-4).
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