Amino Acids (2017) 49:1415–1426 DOI 10.1007/s00726-017-2443-0
ORIGINAL ARTICLE
Post‑resistance exercise ingestion of milk protein attenuates plasma TNFα and TNFr1 expression on monocyte subpopulations Adam J. Wells1 · Adam R. Jajtner2 · Alyssa N. Varanoske1 · David D. Church1 · Adam M. Gonzalez3 · Jeremy R. Townsend4 · Carleigh H. Boone1 · Kayla M. Baker1 · Kyle S. Beyer1 · Gerald T. Mangine5 · Leonardo P. Oliveira6 · David H. Fukuda1 · Jeffrey R. Stout1 · Jay R. Hoffman1
Received: 1 March 2017 / Accepted: 25 May 2017 / Published online: 29 May 2017 © Springer-Verlag Wien 2017
Abstract Attenuating TNFα/TNFr1 signaling in monocytes has been proposed as a means of mitigating inflammation. The purpose of this study was to examine the effects of a milk protein supplement on TNFα and monocyte TNFr1 expression. Ten resistance-trained men (24.7 ± 3.4 years; 90.1 ± 11.3 kg; 176.0 ± 4.9 cm) ingested supplement (SUPP) or placebo (PL) immediately post-exercise in a randomized, cross-over design. Blood samples were obtained at baseline (BL), immediately (IP), 30-min (30P), 1-h (1H), 2-h (2H), and 5-h (5H) post-exercise to assess plasma concentrations of myoglobin; tumor necrosis factor-alpha (TNFα); and expression of tumor necrosis factor receptor 1 (TNFr1) on classical, intermediate, and non-classical monocytes. Magnitude-based inferences were used to provide inferences on the true effects of SUPP compared to PL. Plasma TNFα concentrations were “likely attenuated” (91.6% likelihood effect) from BL to
30P in the SUPP group compared with PL (d = 0.87; mean effect: 2.3 ± 2.4 pg mL−1). TNFr1 expressions on classical (75.9% likelihood effect) and intermediate (93.0% likelihood effect) monocytes were “likely attenuated” from BL to 2H in the SUPP group compared with PL (d = 0.67; mean effect: 510 ± 670 RFU, and d = 1.05; mean effect: 2500 ± 2300 RFU, respectively). TNFr1 expression on non-classical monocytes was “likely attenuated” (77.6% likelihood effect) from BL to 1H in the SUPP group compared with PL (d = 0.69; mean effect: 330 ± 430 RFU). Ingestion of a milk protein supplement immediately postexercise appears to attenuate both plasma TNFα concentrations and TNFr1 expression on monocyte subpopulations in resistance-trained men.
Handling Editor: E. Rawson.
Keywords Inflammation · Tumor necrosis factor-alpha (TNFα) · Tumor necrosis factor receptor 1 (TNFr1) · Classical monocytes · Intermediate monocytes · Nonclassical monocytes
* Adam J. Wells
[email protected]
Introduction
1
Educational and Human Sciences, Institute of Exercise Physiology and Wellness, University of Central Florida, 12494 University Blvd, Orlando, FL 32816, USA
2
School of Health Sciences, Kent State University, Kent, OH 44242, USA
3
Department of Health Professions, Hofstra University, Hempstead, NY 11549, USA
4
Department of Kinesiology, Lipscomb University, Nashville, TN 37204, USA
5
Department of Exercise Science and Sport Management, Kennesaw State University, Kennesaw, GA 30144, USA
6
Department of Orthopaedic Surgery, University of Chicago Medical Center, Chicago, IL 60637, USA
The resolution of skeletal muscle homeostasis following damaging exercise is mediated to a large extent by cells of the innate immune system (Arnold et al. 2007; Ogle et al. 2016). The recruitment of circulating monocytes is generally regarded as a critical step in the post-injury inflammatory response (Arnold et al. 2007). These cells, which constitute approximately 10% of the total circulating leukocyte population in humans (Italiani and Boraschi 2014), give rise to both classically activated (M1) and alternatively activated (M2) macrophages, which present during specific stages of repair to promote debridement of the injury site, cell proliferation, angiogenesis, collagen deposition, and
13
1416
matrix remodeling (Novak and Koh 2013). In circulation, human monocytes exist in three functionally distinct subpopulations. These populations are characterized based upon their differential expression of antigenic markers, and are defined as classical (CD14++CD16−), non-classical (CD14+CD16++), and intermediate (CD14++CD16+) monocytes (Ziegler-Heitbrock et al. 2010). The early inflammatory response appears to be dominated by the selective recruitment of classical monocytes to injured tissue (Ingersoll et al. 2011; Wermuth and Jiminez 2015). These cells infiltrate at sites of damage/inflammation in response to damage-associated molecular patterns (DAMP’s), and differentiate into inflammatory M1 macrophages (Soehnlein and Lindbom 2010; Wermuth and Jiminez 2015). The subsequent fate of M1 macrophages, however, is a point of conjecture. Some evidence suggests that M2 macrophages (M2a, M2b, M2c) represent subsequent maturation stages in a common path of differentiation from the M1 phenotype (Arnold et al. 2007), while others suggest that M2 macrophages are independently derived from non-classical monocytes (Nahrendorf and Swirski 2013). While a potential role for intermediate monocytes has yet to be characterized, evidence suggests that classical and non-classical monocyte subpopulations may play independent facilitatory roles in skeletal tissue repair (Arnold et al. 2007; San Emeterio et al. 2017). Pursuant to muscle damage, the skeletal muscle microenvironment becomes rich with signals to recruit, position, and functionally instruct cells of the inflammatory system to program repair (Ogle et al. 2016). Tumor necrosis factoralpha (TNFα) is considered to be one of the “master regulators” of pro-inflammatory cytokine production (Maini et al. 1995; Parameswaran and Patial 2010), and is rapidly released following tissue trauma (Feldmann et al. 1994; Wells et al. 2016b). The effects of TNF alpha on monocytes appear to be dichotomous in nature. On the one hand, elevations in TNFα appear to promote the gene expression of adhesion molecules and chemokines resulting in increased recruitment and adhesion of monocytes to damaged tissue (Peterson et al. 2006). On the other hand, TNF has been reported to impair inflammatory monocyte development and function, resulting in premature egress, and a subsequent increase in systemic inflammation (Puchta et al. 2016). Furthermore, muscle catabolism has been attributed to TNFα in a number of inflammatory diseases (Moldawer and Sattler 1998; Anker and Rauchhaus 1999; Reid and Li 2001). The effects of TNFα are mediated via binding with its cognate membrane receptors TNFr1 (CD120a) and TNFr2 (CD120b) (Hijdra et al. 2012), and these receptors appear to mediate this respective dichotomy in cellular events (Grell et al. 1998; Wajant et al. 2003; Defer et al. 2007; Masli and Turpie 2009; Gane et al. 2016).
13
A. J. Wells et al.
Interestingly, monocytes are reported to direct their own behavior and fate via self-amplification of inflammatory responses through autocrine and paracrine signaling in a TNFα/TNFr1-dependent manner (Gane et al. 2016; San Emeterio et al. 2017). Indeed, monocytes are reported to be potent producers of TNFα, and all three subpopulations, albeit in differing relative proportions, express the TNFr1 receptor (Hijdra et al. 2012; Dimitrov et al. 2013). Accordingly, simultaneous elevations in plasma concentrations of TNFα and monocyte TNFr1 expression may lead to an amplified inflammatory response that could result in delayed recovery. Correspondingly, interventions targeting TNFα signaling in monocytes via TNFr1 may represent a viable means of attenuating the negative effects of TNFα signaling. This is consistent with the concept of selective in vivo TNFr1 blockade, which has recently been proposed as a means to promote TNFr2 ligation, and a subsequent anti-inflammatory response (Gane et al. 2016). Overwhelming evidence shows that the ingestion of protein/amino acids following damaging exercise is a potent means of enhancing recovery (Sharp and Pearson 2010; Duan et al. 2015). Furthermore, recent evidence suggests that amino acids, particularly branched chain amino acids, are important regulators of immune responses (Li et al. 2007). We have recently shown that orally administered amino acids maintain plasma MCP-1 concentrations and augment CCR2 expression on classical monocytes following damaging exercise, suggesting that exogenous amino acids may aid in classical monocyte recruitment (Wells et al. 2016a). Similarly, Townsend et al. (2013) have recently shown that a free-acid form of betahydroxy-beta-methylbutyrate (HMB) is able to attenuate both TNFα and TNFr1 expression on CD14+ monocytes immediately following resistance exercise. These authors postulate that the attenuation of TNFα/TNFr1 signaling in monocytes may reduce recovery time following intense exercise. However, to our knowledge, no study has investigated the effect of a protein supplement on plasma TNFα and TNFr1 expression on all three monocyte subpopulations following damaging exercise. Given that monocyte subpopulations may independently modulate skeletal muscle recovery processes, an examination of TNFr1 perturbations on all subpopulations following damaging exercise is warranted. Therefore, the purpose of this study was to examine the effect of a post-workout milk protein supplement on plasma TNFα and monocyte TNFr1 expression on monocyte subpopulations following an acute bout of highvolume, moderate intensity resistance exercise in experienced, resistance-trained men. We hypothesized that postworkout milk protein will result in an acute attenuation in TNFα concentration and TNFr1 expression on all monocyte subpopulations.
Post-resistance exercise ingestion of milk protein attenuates plasma TNFα and TNFr1…
Methods and materials Participants Ten resistance-trained men (24.7 ± 3.4 years; 90.1 ± 11.3 kg; 176.0 ± 4.9 cm; 14.1 ± 6.1% body fat) were recruited to participate in this randomized, counterbalanced, cross-over design research study. Using the procedures described by Beck (2013), a sample size of 10 participants per treatment produced a statistical power (1 − β) of 0.97. Power calculations were made using G*Power statistical analyses software (Version 3.1.9.2, Düsseldorf, Germany) and were based upon an effect size (dz) of 0.92 generated from changes in TNFr1 expression on CD14+ monocytes reported by Townsend et al. (2013) in response to lower body resistance exercise (mean difference ± SD from BL to 30P in placebo group). Strict recruitment criteria were implemented to increase homogeneity of the sample. Inclusion criteria required participants to be between the ages of 18 and 35 years, with a minimum of 1 year of resistance training experience, and the ability to squat a weight equivalent to their body mass (confirmed during 1-RM testing). Participants had 6.7 ± 4.6 years of resistance training experience with an average maximum barbell back squat of 172.7 ± 25.2 kg. All participants were free of any physical limitations that may have affected performance. In addition, all participants were free of any prescription or over the counter medications, performance enhancing drugs, and/or ergogenic aids including the use of creatine, beta-alanine, or any herbal/vitamin supplement, as determined by a health and activity questionnaire. Following an explanation of all procedures, risks, and benefits, each participant provided his written informed consent prior to participation in this study. The research protocol and the informed consent document were approved by the New England Institutional Review Board prior to participant enrollment (NEIRB# 14-272).
Maximal strength testing Prior to experimental trials, participants reported to the Human Performance Laboratory (HPL) to establish maximal strength (1RM) on all lifts involved in the exercise protocol. Participants performed a standardized warm-up consisting of 5 min on a cycle ergometer against a light resistance, ten body weight squats, ten body weight walking lunges, ten dynamic walking hamstring stretches, and ten dynamic walking quadricep stretches. Following the warm-up, 1RM testing for the barbell back squat and leg press exercises were performed. Briefly, each participant performed two warm-up sets using a resistance of
1417
approximately 40–60 and 60–80% of his perceived maximum, respectively. For each exercise, 3–4 subsequent trials were performed to determine the 1RM. A 3–5-min rest period was provided between each trial. Maximum strength testing for the back squat and leg press was administered by the same Certified Strength and Conditioning Specialist (CSCS) to ensure that each participant reached the parallel position for each repetition of the squat and that the exercise technique was consistent between sessions. For the leg extension, hamstring curl, and calf raise, 1RM was predicted using the Brzycki (1993) prediction equation. Trials were discarded where range of motion criteria for each exercise were not met, where repetitions performed for predicted 1RM were greater than 10, or where proper technique was not used, as determined by the CSCS.
Experimental trials On the morning of each trial, participants reported to the HPL after a 10-h overnight fast and having refrained from all forms of moderate-to-vigorous exercise for the previous 72 h. Participants were provided a standardized low protein, low carbohydrate breakfast bar (7-g protein, 3-g carbohydrate, and 13-g fat) following BL assessments. The exercise protocol was administered 20-min post-ingestion of the breakfast bar. Experimental trials were performed in a randomized counter-balanced order, and each experimental trial was separated by a minimum of 1 week to ensure adequate recovery. All participants performed the exercise protocol between the hours of 8–10 am on the day of each experimental trial, and each participant performed each trial at the same time of day to avoid diurnal variations. Participants provided a urine sample upon arrival to the HPL for analysis of urine-specific gravity (USG) by refractometry to ensure adequate hydration status (USG 0.05) was noted. However, a significant time effect for lactate was observed (F = 168.9.0; p ≤ 0.001; η2p = 0.95). With both trials combined, plasma lactate was significantly elevated above BL at all post-exercise time points (p’s
