Phase 1 (cell culture tests)
C2C12 myoblasts (ATCC; Manassas, VA, USA) were plated at approximately 30% confluence and grown for 24 hours in 10% FBS High Glucose DMEM with antibiotics (100 μg/ml streptomycin and 100 U/ml penicillin; Sigma-Aldrich; St. Louis, MO, USA). At 16 hours prior to the experiment, myoblasts cells were switched to serum free high glucose DMEM (no antibiotics) and were approximately 70% confluent at the time of the experiment. All stimulants were dissolved in chloroform to yield a concentration of 10 mg/mL, with the exception of DAG which was dissolved at 2 mg/mL and G3P which was dissolved at 6 mg/mL. Each stimulant was then dried with a stream of nitrogen gas and resuspended in PBS to obtain either 20 or 60 nmol/100 μL, such that 100 μL added to 2 mL of media resulted in 10 or 30 μM respectively. Accordingly, cells were stimulated for 20 minutes with vehicle (Control; 100 μL of PBS) 10 or 30 μM of soy-derived (S) phosphatidylserine (S-PS, SerinAid®, Chemi Nutra, White Bear Lake, MN, USA), phosphatidylinositol (S-PI), phosphatidyl-ethanolamine (S-PE), phosphatidylcholine (S-PC), PA (S-PA, Mediator®, Chemi Nutra, White Bear Lake, MN, USA), lysophosphatidic acid (S-LPA), diacylglycerol (DAG), glycerol-3-phosphate (G3P), or egg-derived PA (E-PA). Cells were then harvested in lysis buffer (40 mM Tris, pH 7.5; 1 mM EDTA; 5 mM EGTA; 0.5% Triton X-100; 25 mM β-glycerophosphate; 25 mM NaF; 1 mM Na3VO4; 10 μg/mL leupeptin; and 1 mM PMSF) and subjected to immunoblotting with anti-phospho-p70 S6 Kinase (Thr389; Cell signaling #9234; 1:1000; Danvers, MA, USA) as previously described [19]. Once the appropriate image was captured, membranes were stripped for 30 minutes in stripping buffer (100 mM β-Mercaptoethanol, 2% SDS, 62.5 mM Tris HCL pH 6.8) maintained at 50°C. Membranes were washed with TBST, blocked with 5% powdered milk in TBST for 1 h, and then immunoblotted with anti-p70 S6 Kinase (cell signaling #2708, 1:2000; Danvers, MA, USA). Once the appropriate image was captured, the membranes were stained with Coomassie Blue to verify equal loading in all lanes. Densitometric measurements were performed by determining the density of each band using the public domain ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/nih-image/). The ratio of P-p70-389 to total p70 was used as readout for mTOR signaling. C2C12 myoblasts were chosen, as it has previously been established that changes in P-p70-389 phosphorylation are a valid marker of PA induced changes in mTOR signaling [20].
Phase 2 (human efficacy study)
Thirty-four males were recruited from the University of Tampa to participate in this study. Twenty eight males (21 ± 3 years, 77 ± 7 kg, 176 ± 9 cm) were used for data analysis. Three subjects elected to discontinue their participation prior to beginning the intervention, citing a more demanding schedule than they had anticipated; two subjects ceased participation, claiming the resistance training protocol was too onerous for them to complete; and one subject was removed from data analysis due to a failure to comply with the prescribed diet. Subjects were equally divided into the PA (n = 14, 78 ± 9 kg, 177 ± 7 cm) and PLA (n = 14, 76 ± 6 kg, 175 ± 11 cm) groups. All participants were required to abstain from consuming any muscle-building supplements (e.g. creatine) for 1 month prior to pretest measures, be non-smokers, have RT experience of no less than one year, and have participated in RT at least three days per week for the past six months to be included in this study. Participants were allowed, although not provided with, multivitamin and protein powder supplementation during the month prior to the initiation of the study. Participants were carefully matched according to their lean body mass (LBM), rectus femoris cross sectional area (CSA), and leg press 1-repetition-maximum (1RM), and they were then equally divided into either the PA or placebo (PLA) groups. Measures of leg press and bench press 1RM, LBM, fat mass, total mass, and CSA were taken prior to, and following, the RT protocol.
RT occurred three days per week with 48–72 hours between RT sessions. Each body part was trained 1–2 times per week following a daily undulating periodized scheme. Each participant performed a 5 RM for each exercise prior to the first four weeks with the exception of the bench press and leg press, in which true 1RM values were determined. 5 RM testing was repeated at the end of week 4 for the new exercises. The 1RM testing protocol consisted of 1 set of 10–12 repetitions at approximately 50% 1RM followed by 1 set of 2–3 repetitions at approximate intensities of 75% and 85% 1RM. After the final warm up set, weight was increased in 5-20 lb increments until 1RM was attained. 5RM determination followed an identical pattern; however, intensities were relative to 5RM instead of 1RM. These RM values were used to calculate the load used for each exercise for each participant. These exercises were also altered at week 5 to introduce a more novel stimulus. All participants were required to perform the prescribed number of repetitions with their prescribed weight. In the event that a subject reached muscular failure, a laboratory researcher assisted with the completion of the exercise. A comprehensive outline of the workouts can be found in Table 1.
Strength was assessed via 1-RM testing of the leg press and bench press. Total strength was calculated as the sum of both leg press and bench press. Body composition (lean body mass, fat mass, and total mass) was determined on a Lunar Prodigy DXA apparatus (software version, enCORE 2008, Madison, Wisconsin, U.S.A.). Skeletal muscle hypertrophy was determined via changes in CSA of the rectus femoris at 50% femur length while the participant rested supine with a portable ultrasound device (Logiq e, General Electric Medical Systems, Milwaukee, WI, USA) in B mode with a wide-band linear array transducer (12 L-RS, General Electric Medical Systems, Milwaukee, WI, USA) sampling at 12 MHz. Ultrasound has previously been verified as a valid measurement of CSA [21]. However in the present study, a simpler, conventional method was used as opposed to the panoramic method used by Ahtiainen et al. Using the conventional method, a single image is captured for subsequent measurement instead of combing several images along a path, as with panoramic. Femur length was defined as the distance between the anterior superior iliac spine and the superior aspect of the patella. Probe placement was traced with permanent marker, and each subject was instructed to reapply the mark daily. Additionally, researchers affirmed and reapplied the mark during each laboratory and resistance training visit. Upon retesting, 50% femur length was remeasured to assure the mark had not deviated. The average of the greatest two out of three measurements was used to CSA value. Power was assessed during a maximal cycling ergometry test. During the cycling test, the volunteer was instructed to cycle against a predetermined resistance (7.5% of body weight) as fast as possible for 10 seconds [22]. The saddle height was adjusted to the individual’s height to produce a 5–10° knee flexion while the foot was in the low position of the central void. A standardized verbal stimulus was provided to the subjects. Power output was recorded in real time by a computer connected to the Monark standard cycle ergometer (Monark model 894e, Vansbro, Sweden) during the 10-second sprint test. Peak power (PP) was recorded using Monark Anaerobic test software (Monark Anaerobic Wingate Software, Version 1.0, Monark, Vansbro, Sweden). From completion of Wingate tests performed over several days, interclass correlation coefficient for peak power was 0.96.
Two weeks prior to and throughout the study, subjects were placed on a diet consisting of 25% protein, 50% carbohydrates, and 25% fat by a registered dietician who specialized in sport nutrition. Subjects met as a group with the dietitian, and they were given individual meal plans at the beginning of the study. Daily total of calories were determined by the Harris Benedict equation and tracked by weekly logs to ensure compliance. The PA group received 750 mg of soy-derived PA (Mediator®, Chemi Nutra, White Bear Lake, MN) per day, while the PLA group received 750 mg of rice flour, each delivered in 5 visually identical capsules. On RT days, participants consumed 450 mg of their respective supplement 30 minutes prior to RT and 300 mg immediately following RT with 24 g of hydrolyzed collagen protein powder from beef skin (Peptiplus XB agglomerated, Gelita AG, Eberbach, Germany) mixed with 500 ml water. The protein supplement was provided in order to ensure control for post-exercise meals between groups and hydrolyzed collagen was chosen as an incomplete protein source low in leucine (3.2 weight%). On non-RT days, participants consumed 450 mg of their respective supplement with breakfast and the remaining 300 mg with dinner. Participants were required to return to the laboratory with their empty bags to ensure compliance. Post-study analysis of the subjects’ diet revealed that it consisted of 25% protein, 51% carbohydrates, and 24% fat, with no differences between groups. The PA group consumed 2,865 ± 271 Calories per day (79.5 ± 6.7 g fat; 358.1 ± 28.6 g carbohydrate; 179.0 ± 15.0 g protein), and the PLA group consumed 2,795 ± 236 Calories per day (74.5 ± 5.4 g fat; 356.4 ± 32.1 g carbohydrate; 174.6 ± 12.9 g protein). Since the dietician had weekly interaction with the subjects, all subjects remained compliant throughout the study. Product formulations were blinded to both the investigators and the volunteers and coded so that neither knew which formulation was consumed during each trial.
All participants were informed of all risks associated with the study, and each participant signed an informed consent prior to beginning the study. This investigation was approved by the University’s Institutional Review Board.
Statistics
Phase 1 data was analyzed using a one way analysis of variance (ANOVA) for all data. A Tukey’s Multiple Comparison Test was used to determine significant differences between treatments. Significance was set at P < 0.05. Statistical analyses were performed on SigmaStat software (San Jose, CA, USA). A repeated measures ANOVA model was used to measure group, time, and group by time interactions in phase 2. If any main effects were observed, a Tukey post-hoc was employed to locate where differences occurred. Data are presented as mean ± standard deviation. All phase 2 statistics were run using Statistica software (Statsoft, Tulsa, OK, USA).