Resistance training can increase strength and skeletal muscle mass, but the adaptive responses can be enhanced through an increased protein intake, and in particular an increased intake of whey protein (Cribb et al., 2006; Miller et al., 2014). Whey protein is a rich source of amino acids including branched-chain amino acids (BCAA) that are critical for increasing muscle protein synthesis (Ha and Zemel, 2003) and also contains small amounts of growth factors which may contribute to increases in muscle size and strength (Walzem et al., 2002). Other dairy foods that are rich in growth factors have also been shown to improve muscular adaptations to resistance training, such as bovine colostrum. Bovine colostrum is the first milk produced by cows after calving and contains high concentrations of growth factors (Francis et al., 1988). Bovine colostrum has been shown to increase lean tissue mass (Antonio et al., 2001) and muscular power (Buckley et al., 2003) when consumed during resistance exercise training. However, the availability of colostrum is limited relative to that of normal cow's milk. This has led to the development of chromatography technologies to extract growth factors from milk and/or whey (Francis et al., 1995) to produce growth factor-rich products to potentially increase muscular adaptations to exercise training.
Whey Growth Factor Extract (WGFE) is a concentrated protein source consisting primarily of Lactoperoxidase (62%) and Lactoferrin (16%) proteins, but is also enriched in other growth factors such as insulin-like growth factors (Collier et al., 1991) that are naturally present in milk at low concentrations. A preliminary study using untrained volunteers indicated that WGFE supplementation (2 g/day) during 12 weeks of resistance training resulted in ~35% greater increase in leg strength (Carey et al., 2006; Crittenden et al., 2009). However, whether WGFE enhances strength increases in individuals who are already resistance trained is unclear.
Muscle hypertrophy in response to resistance training requires activation of the AKT/mTOR pathway (Philp et al., 2011), which can be activated by IGF-1 (Rommel et al., 2001). This pathway activates the ribosomal complex and initiates translation downstream of the kinase [P70.sup.s6k], increasing the rate of new protein synthesis. Simultaneously intense exercise may stimulate the ubiquitin proteasome pathway and the induction of muscle-specific E3-ubiquitin ligases, atrogin-1 and muscle RING finger-1 (MuRF1) which promote muscle turnover (Rahbek et al., 2015; Stefanetti et al., 2014). This is achieved by the E3-ubiquitin ligases regulating transcription of the Forkhead Box (FOXO) proteins, including the FOXO3 isoform (Okamoto et al., 2011). There is evidence that the ingestion of dairy protein can influence the mTOR pathway (Mitchell et al., 2015), possibly due to its IGF-1 content (Rommel et al., 2001), but not the FOXO pathway (Stefanetti et al., 2014). Studies are yet to address the actions of WGFE on these pathways.
The aim of this study was to examine the effect of supplementation with WGFE on muscle strength, body composition and molecular pathways controlling skeletal muscle hypertrophy in men with a history of resistance exercise training. We hypothesised that WGFE would promote activation of the AKT/mTOR pathway and thus increase muscle anabolism enabling greater increases in lean tissue mass and strength.
This study used a randomised, double-blind, placebo-controlled parallel design. All participants undertook a 12-week progressive resistance exercise training program and were allocated to concurrent daily consumption of 20 g of whey protein isolate together with 1.6 g of cellulose (CONT n = 24) or 1.6 g of WGFE (n = 22). A dose of 1.6 g/day, rather than the 2 g/day used in the preliminary study (Carey et al., 2006; Crittenden et al., 2009), was chosen to improve the commercial viability of the product as a supplement by reducing cost per effective dose. Ten participants from each group underwent vastus lateralis muscle biopsies pre- and post-training at baseline and after the 12-week training program. To account for potential confounding effects of differences in baseline muscle strength participants were allocated to treatment via minimisation (Altman and Bland, 2005) based upon peak isometric knee extension torque data collected during a pre-intervention familiarisation session. Participants were further stratified on the basis of training experience and age. Assessments at baseline and after the 12-week training program were performed at the same time of day to control for circadian variation. A diagram of the study protocol is provided in Figure 1. This study was approved by the Human Research Ethics Committee of the University of South Australia and conformed to the standards set by the Declaration of Helsinki. All participants provided written informed consent prior to participating.
Adult males aged 18-30 years who had been participating in regular ([greater than or equal to]2 sessions per week) resistance exercise training for at least six months immediately prior to the study were recruited via public advertisement. All participants reported being free from current or prior musculoskeletal injury which would prevent them from undertaking the training required for the study. Prospective participants were excluded if they were: (a) smokers or had recently (within the previous 6 months) quit smoking; (b) engaged in other athletic training that might confound the outcomes of the present study; (c) consumed prescription medication; (d) were allergic to/sensitive to/intolerant of dairy proteins or lactose; or (e) had recently (within the past 6 months) taken any form of supplement intended to increase physical performance or enhance recovery. All potential participants were administered the Sports Medicine Australia pre-exercise screening questionnaire (Sports Medicine Australia, 2005), with only those classified as low risk accepted into the study.
All study supplements were commercially available products (Murray Goulburn Co-Operative Co Ltd, Melbourne, Australia). The protein and growth factor composition of the WGFE supplement is provided in Table 1.
Both WGFE and CONT groups consumed 20 g of whey protein isolate powder daily, mixed with 250 mL of water. Additionally both groups consumed four capsules each morning. Those randomised to the WGFE group consumed capsules each containing 400 mg of whey growth factor extract (i.e. 1.6 g dose; Catalyst, Murray Goulburn, Parkville, Australia), whilst each CONT capsule contained 400 mg of cellulose (i.e. 1.6 g dose). The capsules were identical in appearance. Supplements were consumed immediately upon rising on non-training days and immediately after training on training days. Compliance with supplementation was determined by capsule counting.
Anthropometry and body composition measures
At baseline height was measured using a stadiometer (SECA, Hamburg). Body mass was measured using digital scales (Tanita Ultimate Scale, Tokyo) at baseline and after 6 and 12 weeks and mid-thigh girth was measured using a tape (Lufkin, Apex Tool Group, Maryland) following International Society for the Advancement of Kinanthropometry (ISAK) protocols (Marfell-Jones et al., 2007). Body composition was assessed at Weeks 0 and 12 using dual-energy x-ray absorptiometry (DXA, Lunar Prodigy, General Electric, Madison, WI, USA, using enCORE 2003 software version 7.52.002), with whole body and regional (right thigh) non-bone lean and fat tissue mass determined. The thigh segment assessed in the regional analysis was defined as the area bordered distally by a line passing through the medial and lateral joint spaces of the knee, parallel to the tibial plateau and proximally by a line passing immediately distal to the most inferior point of the ischial tuberosity and immediately proximal to the superior border of the greater trochanter. Medial and lateral borders included all lower limb soft tissue falling between the proximal and distal boundaries. Test-retest reliability for data extraction was assessed on baseline DXA scans from all 46 subjects. Region of interest data were extracted from the same scan on separate days, and reliability of extraction was excellent for both lean tissue mass (ICC [+ or -] 95%CI: 1.000 [+ or -] 0.001) and fat tissue mass (ICC [+ or -] 95%CI: 1.000 [+ or -] 0.001).
Maximal isometric torque of the right knee extensors was assessed using an isokinetic dynamometer (Biodex System 4, Biodex...