Effects of β-Hydroxy-β-Methylbutyrate Supplementation on IL-4 , IL-10 and TGF-β 1 during Resistance Exercise in Athletes

A The aim of this study was to investigate the effect of β-Hydroxy-β-methylbutyrate (HMB) supplementation on anti-inflammatory cytokines including IL-4, IL-10 and TGF-β during an acute bout of resistance exercise (RE) in young resistance trained men. Ten resistance-trained men in a randomized, double-blind, placebo-controlled and crossover study, were administered a 7-day HMB supplementation (3×1 g.d-1 of HMB) and placebo (3×1 g.d-1 of Maltodextrin) with a 7 days washout period. After supplementation periods, subjects performed three sets of bench press, lat pull down, leg extension, leg curl, biceps curl, triceps curl and shoulder press to failure with 85% of one repetition to maximum (1RM). Blood samples were obtained before(Pre), immediately post(IP) and 1 hour-post RE (1h P) to assess serum concentrations of IL-4, IL-10 and TGF-β1. The data were analyzed using 2 (treatment: HMB and PL) × 3 (time points: Pre, IP and 1hP) repeated measures analysis of variance (ANOVA) followed by the Bonferroni post hoc test with a significant level of p<0.05. Serum IL-4 was significantly higher at IP resistance exercise in HMB compared to placebo. Circulating IL-4 and TGF-β1 were significantly raised at IP compared to Pre in both HMB and placebo treatments. No significant differences between treatments were observed for IL-10 and TGF-β1at any time points. In conclusion, HMB supplementation increased the circulating level of IL-4 during RE in resistance-trained men, which may attenuate inflammation and facilitate adaptation to RE.


Introduction
The It is generally accepted that chronic resistance exercise (RE) is associated with reduced risk of low grade systemic inflammation in atherosclerosis, obesity and insulin resistance [1], paradoxically, a single bout of RE appears to promote inflammation [2,3].Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1 and C reactive protein (CRP) are considered as low grade systemic inflammatory cytokines [4].Studies examining the acute effect of RE on inflammatory markers revealed significant elevations [5,6] and no changes [2,7] in inflammatory cytokines.The inflammatory responses to a single bout of RE may be due to RE-induced muscle damage [8].In addition to muscle damage, RE may trigger cytokine production by other physiological factors such as neuroendocrinological factors (i.e.adrenaline, noradrenaline, growth hormone and cortisol), ROS production, acidosis and the rise in muscle temperature [9][10][11].
Recently, the concept of anti-inflammatory effects of exercise has attracted particular attention in sport science community [12].The anti-inflammatory cytokines including IL-4, IL-10 and TGF-β1 are able to attenuate inflammation by limiting inflammatory cytokine production, up-regulating their soluble antagonist binding proteins, and suppressing inflammatory cell activity [13,14].IL-4 and IL-10 are produced largely by the TH2 subset of lymphocyte and TGF-β1 is produced mainly by regulatory T cells [14].The anti-inflammatory cytokines (Il-4 and IL-10) responses to acute RE are equivocal [3].However, there are no studies reporting TGF-β1 response to an acute RE in athletes.Recently, it was demonstrated TGF-β1 increased immediately after acute RE at low intensity (50% of 1 RM; 2x18 rps) in Coronary Artery Disease patients [15].In addition to the effect of exercise on the immune system, previous studies have shown that diet and nutritional supplements can reinforce the immune system in athletes [6,[16][17][18].
β-Hydroxy-β-methylbutyrate (HMB) is a metabolite of the amino acid leucine that has previously been shown to increase protein synthesis, strength and power, and decrease circulating markers of muscle damage [19].To date, three studies only have been evaluated the effect of HMB on immune function during RE in humans [6,17,18].Recently, it was proposed that HMB may exerts immunoregulatory effects including increased the anti-inflammatory cytokine IL-10 and decreasing pro-inflammatory cytokine IFN-γ and IL-1β after resistance training [17].In addition, it was reported that HMB could reduced TNF-α and monocyte TNF-α receptor 1 expression after strenuous RE [6].In a vitro study demonstrated decrease in inflammatory cytokine IFN-γ in T-lymphocytes was incubated with varying concentrations of HMB [20].Due to the lack of information regarding the effect of HMB supplementation on anti-inflammatory cytokines during RE in athletes, the purpose of this study was to investigate the effects of HMB on IL-4, IL-10 and TGF-β1 during intense RE in athletes.

Participants
Ten resistance-trained men voluntarily participated in this randomized, double-blind, placebo-controlled and crossover study (Table 1).The study protocol was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board for the human participants.All procedures, risks and benefits of this study were explained to participants, and then participants provided written informed consent.Participants had at least 2 years of resistance-training experience.Exclusion criteria were included smoking, taking supplements (i.e., creatine, HMB, lucine, or other amino acids), anabolic and catabolic hormones, and anti-inflammatory agents.The participants were asked to refrain from engaging in any exercise for 48 h before each resistance exercise bout as well as to complete 2-day food diary before the first RE bout and repeat it before the subsequent RE bout.

Study design
To investigate the effect of one week HMB supplementation on anti-inflammatory cytokines during heavy RE, participants ingested HMB (3 capsules of 1 g per day) or placebo (3 capsules of 1 g Maltodextrin per day) three times a day for one week prior to intense resistance exercise in a randomized, double-blind, placebo-controlled and crossover study.Participants were called to the Exercise Physiology Laboratory on three different sessions.In the first session, maximal strength tests based on one repetition maximum for bench press, lat pull down, leg extension, leg curl, biceps curl, triceps curl and shoulder press were calculated using methods described previously (Table 2) [21].During the second and third sessions, participants performed intense resistance exercise that included of three sets of bench press, lat pull down, leg extension, leg curl, biceps curl, triceps curl and shoulder press to failure with 85% of 1RM and 2 min of rest between sets and exercises.During two supplementation periods, participants in a randomized, double-blind, placebo-controlled and crossover design received one week HMB (Olimp, Poland ) and placebo that separated by a one week washout period.Both HMB and PL capsules were identical in shape, size and color.At the end of each one week supplementation periods, the participants performed heavy RE with 85% of 1RM.Prior to resistance exercise protocols, all participants performed warm-up, which consisted of 3 min running, 5-10 repetitions at 50% of perceived maximum and stretching.
Table 2. Baseline 1RM in resistance exercises

Blood sampling
Blood samples were obtained from a forearm vein at Pre, IP and 1h P into serum vacutainer tubes that allowed clotting at room temperature for 30 min and afterward centrifuged at 3000 g for 15 min at 4 º C. Serum tubes were stored at -80 °C for later analysis.IL-4, IL-10 and TGF-β1 were analyzed using enzyme-linked immunosorbant assays (ELISA kits with Catalogue No.: abx050119; abx050094 and abx252257, respectively), with sensitivities of 1.5 pg/mL, 0.5 pg/mL and 18.75 pg/mL.

Statistical analysis
Statistical analysis of the data was performed using IBM SPSS Statistics 20.0 software for windows (SPSS Inc., Chicago, Illinois).P value less than 0.05 were considered statistically significant.The Shapiro-Wilk test confirmed the normal distribution of the data.The data were analyzed using 2 (treatment: HMB and PL) × 3 (time points: Pre, IP and 1hP) repeated measures analysis of variance (ANOVA).A Bonferroni post hoc test was used for pairwise comparisons when the F-statistic revealed a significant p-value.Data are reported as mean ± standard deviations (SD) for all values.

Discussion
The primary aim of the present study was to evaluate the effect of HMB supplementation in a randomized, double-blind, placebo-controlled and crossover design in resistance-trained men on anti-inflammatory cytokines during intense RE.The main finding of the present study was that short-term HMB supplementation (3 g.d -1 for 7 days) significantly raised circulating IL-4 at immediately post RE in resistance-trained men.IL-4 is an important anti-inflammatory cytokine which leads to polarization of T helper (Th) cell differentiation toward Th2-like cells [14].These cells are able to secretes anti-inflammatory cytokines of IL-4 and IL-10 that cause to suppression of Th1 responses as well as block the secretion of inflammatory cytokines including IL-1, TNF-α, IL-6 and IL-8 produced by monocytes [14,22].Furthermore, it has been demonstrated that IL-4 can directly stimulate myogenesis, which is due to enhance myoblast fusion [23].
HMB supplementation increased circulating IL-4 by 19.75% immediately after heavy RE in resistance-trained men, which may be attributed to anti-inflammatory effect of HMB that supported by previous studies [6,24,25].Consistent with our findings, previous study has shown that HMB exerts anti-inflammatory effect by attenuating elevations in circulating TNF-α immediately after acute bout of heavy RE and as well as reducing TNFR1 expression on human monocytes at 30 min post RE [6].In another study, a single dose of 3 g.d - prior to RE attenuated C-reactive protein (CRP) 24 h post-RE in resistance-trained men [24].In addition, HMB-free acid administration can attenuate complement receptor type 3 (CR3) at 30 min post heavy RE with 70-80% of 1RM in resistance-trained men [25].Inflammatory cytokine responses to 23 days of HMB supplementation during heavy military training with sleep deprivation revealed decreases in circulating IL-6, IL-8, IL-10, IL-1ra, CX3CL1 and G-CSF [26].In contrast, 7 weeks of HMB supplementation in elite volleyball players had no significant effect on inflammatory markers of IL-6 and IL-1 after the first 7 weeks of the volleyball season [27].
IL-10 known as an important anti-inflammatory cytokine which is a potent inhibitor of Th1cytokines, including IL-2 and IFN-γ [14] as well as inhibits nuclear factor ҠB (NF-ҠB), cell surface expression of major histocompatibility complex class II molecules, B7 accessory molecules and signal molecule CD14 [14,28].In addition, it appears that the anti-inflammatory effects of exercise is mediated by elevations in circulating IL-10 and to be beneficial for recovery [4].Thus, it has been shown that circulating IL-10 can attenuate adaptive immune response and reduced the damage to tissue caused by inflammation in response to exercise [29].In the present study greater increase (4.5%) in IL-10 immediately post RE was observed after HMB supplementation compared to placebo but this change did not reach statistically significant.Few studies have evaluated HMB supplementation on IL-10 and their results are conflicting [24,26].Similar to our results, HMB supplementation in resistance-trained men did not alter IL-10 post lower body RE [24].In contrast, HMB supplementation was shown to attenuate circulating IL-10 response to military training with sleep deprivation [26].Also, regarding other nutritional supplements have been demonstrated that carbohydrate beverage intake reduces exercise-induced increases in circulating IL-10 compared to placebo [30][31][32].In addition, carbohydrate supplementation during hypoxia condition didn't change serum IL-10 concentration in healthy male who exercised for 60 min at an intensity of 50% VO2peak [33].Quercetin ingestion (1000 mg/day for 24 days) in trained male cyclists significantly reduced blood leukocyte IL-10 mRNA expression but without effect on plasma IL-10 after prolonged cycling [31].However, it was demonstrated that caffeine ingestion (6 mg.kg -1 body weight) lead to higher increases in circulating IL-10 after 15-km run competition.
TGF-β1 like IL-4 and IL-10 is an important anti-inflammatory cytokine which suppresses the proliferation and differentiation of T and B lymphocyte cells and reduces IL-2, IFN-γ and TNF-α secretion [14,37].Higher circulating levels of TGF-β1 after exercise contribute to mediating anti-inflammatory effects of exercise.Our findings show that circulating TGF-β1 significantly increased in both HMB (14.25%) and placebo (11.94%) condition at immediately post RE in resistance trained men, with no differences between conditions.We observed a trend towards a greater increase in TGF-β1 (2.31%) following HMB intake compared to placebo at immediately post RE in resistance trained men.To our knowledge there are no data on TGF-β1 response to HMB supplementation during RE in athletes.In addition, little research has examined the effect of acute RE on TGF-β1in humans [38].Our findings are in line with a previous study showing an increase in serum TGF-β1 following an acute bout of RE with 50% of 1RM in coronary artery dieses patients [38].It has been reported a rise in circulating TGF-β1 level after chronic resistance training in healthy adults [39] and in patients with type 2 diabetes [40,41].However, other studies reported no change in TGF-β1 level following resistance training in the elderly [42] and in older adults [43].Our results indicate that intense RE with 85% of 1RM lead to a rise in serum TGF-β1 in resistance-trained men, which may indicate a protective effect leading to prevention or reduction of atherosclerosis progression [38,44].

Conclusions
In conclusion, to the best of our knowledge this is the first study to investigate the effect of HMB supplementation on anti-inflammatory cytokines during strenuous RE in resistance-trained men in a randomized, double-blind, placebo-controlled and crossover design.Our findings revealed that short-term HMB supplementation lead to significant increment in IL-4 level after acute strenuous RE in resistance-trained men, which can directly stimulate myogenesis [23].Moreover, a trend towards greater increase in circulating IL-10 (4.5%) and TGF-β1(2.31%)were observed in HMB supplementation compared to placebo at immediately after RE in resistance-trained men.Taken together, our results show that short-term HMB supplementation during RE can amplify anti-inflammatory effects of exercise as shown by significant increase in IL-4 and non-significant increase in TGF-β1and IL-10.Additional study is needed to directly investigate how HMB exert anti-inflammatory effects during acute strenuous RE in resistance-trained men.

Figure 1 .
Figure 1.Effects of HMB supplmentation on serum concentration of IL-4 at pre-, immediately post-and 1h post-resistance exercise in resistance-trained men.* siginficant difference (P≤0.05) from corresponding pre-RE value.† siginficant difference (P≤0.05) between HMB and placebo at corresponding time point.

Figure 2 .
Figure 2. Effects of HMB supplmentation on serum concentration of IL-10 at pre-, immediately post-and 1h post-resistance exercise in resistance-trained men.

Figure 3 .
Figure 3. Effects of HMB supplmentation on serum concentration of TGF-β1 at pre-, immediately post-and 1h post-resistance exercise in resistance-trained men.* siginficant difference (P≤0.05) from corresponding pre-RE value.

Table 1 .
Baseline anthropometrics of subjects 1.98, P=0.0001, ƞ 2 =0.29).Serum IL-4 significantly increased at immediately post RE in HMB supplementation compared to placebo; with no significant difference at 1h post RE between both conditions.