Phosphatidic acid enhances mTOR signaling and resistance exercise induced hypertrophy

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.

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.

Cell culture tests

As shown in Figure 1, S-PI, S-PE, S-PC, DAG, and G3P elicited no increase in the ratio of P-p70-389 to total p70 compared to vehicle stimulated cells. In contrast, elevated mTOR signaling was observed at all tested concentrations of S-PS (529, and 558%), E-PA (206, and 221%), S-LPA (638, and 694%), and S-PA (658, and 636%; p < 0.05). In addition, S-LPA and S-PA increased mTOR signaling to a greater degree than did E-PA at all concentrations (p < 0.05).

Body composition

No differences existed between groups at baseline for any measure. There was a significant group x time effect (p = 0.02) for CSA (Figure 2a), in which the PA group increased (pre 4.5 ± 1.1 cm², post 5.5 ± 1.3 cm², Effect Size (ES) = 0.92) to a greater extent than the PLA group (pre 4.5 ± 1.1 cm², post 5.1 ± 1.2 cm², ES = 0.52). There was a significant group × time effect (p = 0.01) for LBM (Figure 2b), in which the PA group increased to a greater extent (pre 59.7 ± 6.0 kg, post 62.1 ± 5.5 kg, ES = 0.42) than the PLA group (pre 59.5 ± 4.7 kg, post 60.7 ± 4.7 kg, ES = 0.26). There was a significant time effect (p = 0.02) for Total Body Mass (TBM) in which the PA group increased from 78.1 ± 8.7 to 78.7 ± 7.9 kg and the PLA group increased from 75.7 ± 5.8 to 76.5 ± 6.1 kg, but no differences existed between groups (p = 0.71). There was a significant time effect (p < 0.01) for fat mass (Figure 2c), in which there was a trend (p = 0.068) for fat mass to decrease to a greater extent in the PA group (pre 15.1 ± 4.8 kg, post 13.8 ± 4.2 kg, ES = −0.28) than the PLA group (pre 13.0 ± 6.5 kg, post 12.5 ± 6.9 kg, ES = −0.07).

Strength and power

There was a significant group x time effect (p < 0.05) for leg press 1RM, in which the PA group increased to a greater extent (pre 228.7 ± 49.5 kg, post 280.6 ± 36.2 kg, ES = 1.2) than the PLA group (pre 226.3 ± 47.2 kg, post 258.7 ± 36.1 kg, ES = 0.78). There was a significant time effect (p < 0.01) for bench press 1RM, in which both the PA (pre 98.0 ± 13.5 kg, post 105.0 ± 12.4 kg, ES = 0.5) and PLA (pre 91.4 ± 19.1 kg, post 96.1 ± 17.0 kg, ES = 0.25) increased; however, no differences were present between groups (p = 0.11). There was a significant group x time effect (p < 0.05) for total strength, in which the PA group increased to a greater extent (pre 327.4 ± 59.9 kg, post 386.4 ± 45.8 kg, ES = 1.1) than the PLA group (pre 318.3 ± 60.3, post 355.5 ± 48.4, ES = 0.68). Change values for strength can be found in Figure 3. There was a significant time effect (p < 0.01) for Wingate peak power, which increased in the PA group from 760.5 ± 166.0 W to 822.8 ± 217.3 W and from 733.5 ± 105.8 to 797.3 ± 122.3 W in the PLA group; however, there were no differences between groups (p = 0.97).

Effects of PA supplementation on CSA and LBM

For decades, it has been well documented that RT leads to increases in muscle mass [29]. However, the mechanism for muscle hypertrophy due to a mechanical stimulus has only just begun to be elucidated. Recent research indicates that the lipid second messenger, PA, could be at least partially responsible for translating the mechanical stimulus of RT into the chemical signal for skeletal muscle hypertrophy [23]. For example, research conducted by O’Neil et al. [20] has demonstrated that the PA content of the cell is increased following eccentric contractions and this effect is associated with a robust activation of mTOR signaling.

It is interesting to note that the endogenously produced PA can directly bind to, and activate, mTOR signaling which, in-turn, can promote an increase in protein synthesis. These effects appear to be independent of the PI3K and ERK signaling pathways [14, 20]. However, exogenous PA can also be hydrolyzed to LPA, which binds to the LPA family of G-protein-coupled receptors. The binding of LPA to these receptors results in a cascade of events which stimulate the ERK signaling pathway and also increase PLD activity within the cell [15]. The result is a dual mediated increase in mTOR signaling via increased PA content within the cell as well as via the activation of the ERK signaling pathway [15]. Thus through the activation of multiple pathways, supplementing with PA could result in greater rates of MPS than RT alone and might explain why we observed a greater increase in skeletal muscle accretion when supplementing with PA.

While a pilot study [18] found that changes in LBM were likely enhanced by PA supplementation, they did not find as robust of an effect as the present study in either the control or experimental conditions. For example, their results found no significant change in LBM in the placebo group (+0.1 kg LBM), and they found smaller effects in the experimental group (+1.7 kg LBM) compared to the present study. These findings may indicate that the training stimulus was inadequate to enhance LBM by itself, and it may indicate that changes in LBM were primarily driven by the supplement itself. It is also possible that these findings are the result of unsupervised training as research demonstrates greater increases in neuromuscular adaptations in supervised, as compared to unsupervised, training [30]. In contrast, the present study found increases in both LBM and hypertrophy in both the placebo and PA conditions. These findings may indicate that skeletal muscle hypertrophy was driven by both endogenous (training) and exogenous (PA supplementation) mechanisms. Therefore, future research may be interested in examining the effects of PA on muscle hypertrophy in the absence of a RT stimulus. Additionally, subjects were administered a collagen protein supplement in order to standardize for post-exercise nutrition. It should be noted, however, that there is a low leucine content in collagen protein. Therefore, we posit that collagen supplementation likely had minimal effects in stimulating training-induced responses in strength and body composition, as leucine is primarily responsible for protein’s effects on muscle protein synthesis [31]. We chose a collagen protein supplement to minimize possible effects of protein supplementation. Moreover while mTOR and P-p70-389 influence MPS, MPS was not directly measured in this study, and this mechanism for muscle hypertrophy must be validated in future research.

It is interesting to note that there was a trend (p = 0.068) for PA to decrease body fat (ES = 0.28). Past research strongly suggests that RT alone does not provide a strong stimulus for fat loss [32]. However since muscle mass is a very metabolically active tissue [33], the fat loss may be explained by the increased LBM. While we presently can only speculate as to why or if this may be the case, future research may be interested in exploring the possibility of PA as a fat loss agent, as PA is known to interact with many complexes [34].

Strength and power

Strength is one of the most critical attributes underlying success in sport [35, 36]. The collective results of the present study, as well as those from Hoffman [18], suggest that changes in strength following supervised and non-supervised RT are enhanced by PA supplementation. The observed increases in skeletal muscle CSA certainly contribute to the increases observed in strength, and as PA is not expected to directly increase strength, we believe the increases in strength are due to the increases in CSA, which has been well documented [29]. Additionally, we believe no differences were observed for power as the participants did not train with a power-oriented stimulus. Rather they trained for hypertrophy twice per week, and strength once per week. Thus, further research will be needed to determine if PA can enhance improvements in power.