Ethical Approval and Participant Screening

Prior to initiating this study, the protocol was reviewed and approved by the Auburn University

Institutional Review Board (IRB), and was conducted in accordance with the Declaration of Helsinki (approved protocol #: 15–320 MR 1508; IRB contact: irbadmin@auburn.edu). Additionally, this trial was registered at ClinicalTrials.gov (Identifier: NCT03501628, date registered: April 18, 2018). Apparently healthy, college-aged males (i.e., 19–23 years old) naïve to resistance exercise training were recruited locally for this experiment. Participants provided both verbal and written informed consent to participate in the study and completed a medical history form before study initiation. Screening forms were administered to ensure participants had not engaged in regular resistance exercise for the previous six months and that participants were not consuming a high-protein diet (i.e., >2 g/kg/d). Additionally, participants verified no use of anabolic steroids, supplemental protein, creatine monohydrate, or prohormones. Baseline testing occurred one week following screening.

Experiment Overview

Readers are directed to our previous manuscript for a more detailed breakdown of the parent study design, procedures, and additional supplement information10. Figure 7 provides a visual representation of the timeline of the investigation, and the points at which biological samples analyzed herein were collected. Briefly, baseline (PRE) blood, body composition, and muscle biopsy samples were collected 12 weeks prior to the collection of post-intervention samples (POST). Between these two time points, subjects consumed either PLA, SPC, or WPC twice per day, while both subjects and researchers were blinded to subject grouping. Prior to PRE blood and tissue sample collections, subjects were instructed to refrain from rigorous physical activity for 4–5 days. Subjects were also instructed to report to the laboratory well-hydrated and at least 4 hours fasted on the day of testing. Subject hydration status was confirmed via urine specific gravity and DXA measurements were performed upon hydration confirmation. After DXA scans were complete, venipuncture and biopsies of the vastus lateralis muscle and subcutaneous fat in the gluteal region were performed. During the intervention, subjects completed resistance exercise three times per week, as previously described10, and were closely monitored by research staff for appropriate technical execution of the exercises. Resistance exercises included the barbell back squat, barbell bench press, trap-bar deadlift, and barbell bent row. Load magnitude for each exercise was progressively increased throughout the study, so long as subjects maintained proper lifting technique. The experimental procedures employed to process and analyze blood serum, body composition data, fat tissue, and muscle tissue samples are described in detail below. Additionally, LC-MS/MS methods for the measurement of supplement phytoestrogen contents are described below.

Figure 7 An overview of the experiment is provided. Subjects completed PRE testing consisting of dual-energy x-ray absorptiometry tests of body composition, a muscle biopsy of the vastus lateralis, a fat biopsy of subcutaneous fat in the gluteal region, and venipuncture. Participants then completed 12 weeks of resistance exercise training and consumed two servings of either PLA, SPC, or WPC per day. POST testing consisted of the same assessments as PRE, and both assessments were under a 4-hour fast around the same time of day (±2 hours). Additionally, PRE and POST tests were 72 hours following the last resistance exercise training bout along, and WPC/SPC/PLA supplementation occurred up to the day prior to POST assessments. Full size image

Supplement Phytoestrogen Analysis

We sought to accurately measure isoflavone contents of each supplement given their proposed role in androgen and estrogen signaling. For this analysis we randomly selected five separate packets of each supplement and used LC-MS/MS to measure the total daidzein and genistein concentrations in each packet. 2–3 mg of powder from each supplement was weighed and added to a 5 mL glass tube. Samples were sonicated in 1 mL methanol for 30 min to extract phytoestrogens. 500 μL of methanol extract (and standards) were collected and added to a 1.5 mL centrifuge tube. An aliquot of 100 µL of internal standard was added to sample tubes. Samples were centrifuged at 14,000 g for 15 min. 200 µL of supernatant was collected and transferred into an Agilent injection vial for LC-MS/MS measurement. The LC-MS/MS measurement of genistein, daidzein, and their internal standard (Quercetin), was performed on an Agilent 1290 UHPLC system coupled Agilent 6460 Triple Quad Mass Spectrometer. The mobile phase consists of 10 mM ammonium format and methanol. The samples were separated on ACQUITY UPLC® HSS T3 column (2.1 × 100 mm, 1.8 μm) using a gradient from 60% of methanol to 80% over 1.5 min, then ran for 0.5 min at 80% of methanol. The samples were introduced into the mass spectrometer with a flow rate of 0.4 mL/min using an Agilent Jet Stream Electrospray Ionization (AJ ESI) source. Nitrogen gas was used as the dry (10 L/min at 350 °C), nebulizer (45 psi), and collision gas. Mass spectra were acquired in negative-ion mode and capillary voltage was set at −3500 V. Mass transitions were monitored using multiple-reaction monitoring (MRM). The transitions for each analyte are listed in Table 4.

Table 4 LC-MS/MS Parameters. Full size table

Serum Hormone Analyses

Fasting blood samples were aseptically collected into 5 mL serum separator tubes (BD Vacutainer, Franklin Lakes, NJ, USA) from the antecubital vein before and after 12 weeks of supplementation and training during the same time of day (±2 hours). Notably, SPC, WPC or PLA supplementation occurred up until the day prior to blood and tissue extraction. After collection in serum tubes, blood was centrifuged at 4,000 g at 4 °C for 5 min. Serum was then pipetted into cryotubes and placed in a −80 °C freezer for batch-processing. 17β estradiol and total testosterone concentrations in serum were measured using commercially available enzyme linked immunosorbent assay kits (ELISAs, ALPCO Diagnostics, Salem, NH, USA).

Fat and Muscle Biopsies

Methodology for fat and muscle biopsies along with tissue processing procedures in our laboratory have been described in detail elsewhere10, however, a brief description follows. After the extraction of muscle tissue from the right vastus lateralis approximately midway between the patella and iliac crest using a 5 gauge Bergstöm needle with suction and sterile laboratory procedures, a fat tissue biopsy was also completed. A 20–40 mg portion of muscle tissue was placed in a cryomold and suspended in optimal cutting temperature media (OCT; Electron Microscopy Sciences, Hatfield, PA, USA) for histochemical analysis to measure the cross sectional area of muscle fibers. The approximate 100 mg of remaining muscle tissue was placed in foil and snap frozen for protein and RNA isolation batch processing. Approximately 100 mg of SQ fat was extracted from the gluteal aspect of the left hip. 20–40 mg of SQ fat was placed in 10% formalin and preserved for hematoxylin and eosin staining and histological analysis for adipocyte cross sectional area analysis. Approximately 50–75 mg of fat tissue was placed in foil and snap frozen in liquid nitrogen for protein and RNA isolation procedures.

Fat and Muscle mRNA Expression Analyses

At the conclusion of the study, ~25 mg of fat and muscle tissue placed in foil was removed and homogenized in 500 μL of Ribozol (Ameresco) depending on the amount of tissue available and stored at −80 °C for batch processing. Upon removal for batch processing, RNA isolation occurred according to manufacturer’s instructions. RNA concentrations were assessed using a NanoDrop Lite (Thermo Scientific, Waltham, MA, USA) prior to cDNA synthesis. cDNA was synthesized via reverse transcription from 1,000 ng of total RNA for real time PCR analyses employing a commercial cDNA synthesis kit (Quanta Biosciences, Gaithersburg, MD, USA). SYBR green-based methods with gene-specific primers designed using an online primer designer tool (Primer3Plus, Cambridge, MA, USA) were subsequently performed. Genes are denoted in Table 5. The comparative threshold cycle (CT) method (2−ΔΔCT) was used to calculate relative gene expression36. Melt curve analyses were performed on the first PCR plate for each gene to ensure that one PCR product was amplified per reaction.

Table 5 Primer Sequences for RT-PCR. Full size table

Fat and Muscle Protein Expression Analyses

Approximately 75 mg of fat and muscle tissue were placed in cryotubes containing 250–500 μL of cell lysis buffer, depending on the amount of tissue remaining after the previously mentioned tissue allocations. Insoluble proteins were removed by centrifugation at 500 g for 5 min at 4 °C and supernatants containing homogenates were stored at −80 °C. Tissue homogenates were batch-assayed for total protein content using a BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). After calculation of sample protein concentrations, homogenates were prepared for Western blotting using 4x Laemmli buffer. Fat tissue lysates were prepared at concentrations of 750 ng/μL and muscle tissue lysates were prepared at 1 μg/μL concentrations. For immunoblotting, 25 μL of samples were loaded onto 4–15% SDS-polyacrylamide gels (BioRad, Hercules, CA, USA) and subjected to electrophoresis (180 V for 60 min) using 1x SDS-PAGE running buffer (Ameresco). Proteins were subsequently transferred to polyvinyl difluoride membranes (BioRad), Ponceau stained, and imaged using a gel documentation system (UVP, Upland, CA, USA) to ensure equal protein loading between lanes. Additionally, Ponceau images served to provide a proxy of total sample loaded and protein expression values were normalized on a per-lane basis to account for any differences in sample loading. After this, membranes were blocked for 1 hour at room temperature with 5% nonfat milk powder. For muscle samples, membranes were incubated overnight with rabbit anti-human AR (1:1,000; Thermo Fisher Scientific) primary antibodies at 4 °C in 5% bovine serum albumin (BSA). For fat samples, membranes were incubated overnight with mouse ER-α (1:500; Hybridoma Bank) and separately with mouse ER-β (1:500; Hybridoma Bank) at 4 °C in 5% bovine serum albumin (BSA). The following day, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2000; Cell Signaling) and anti-mouse IgG (1:2000; Cell Signaling) secondary antibodies at room temperature for 1 h prior to membrane development. Membrane development was performed using an enhanced chemiluminescent reagent (Luminata Forte HRP substrate; Millipore, Billerica, MA, USA), and band densitometry was performed through the use of a gel documentation system and associated densitometry software (UVP). Densitometry values for all protein targets were normalized to Ponceau densities. POST protein expression values were subsequently normalized to PRE values in order to obtain fold-change values where: POST/PRE = fold-change.

Fat and Muscle Immunohistochemical Analyses

SQ aCSA and muscle fCSA analysis was performed as previously published by our laboratory10. SQ fat samples were removed from formalin and washed in cold running tap water, embedded, and stored in 70% alcohol. Dehydration was accomplished by gradually increasing percentages of ethyl alcohol to replace the water content in the tissue. Hemo-De was used thereafter to clear the tissue from the ethyl alcohol and allow infiltration with paraffin. The paraffin tissue blocks were sectioned into 6 µm slices and placed onto glass microscope slides. Paraffin was removed with xylene, mounted sections were stained with hematoxylin and eosin, and sample sections were enclosed with a coverslip and mounting media. Two 10x objective digital images per sample were obtained using bright-field imaging (Nikon Instruments), and CSAs were obtained from at least 100 adipocytes per image using ImageJ (National Institutes of Health, Bethesda, MD, USA). For muscle samples, sections from OCT‐preserved samples were cut at a thickness of 20 μm using a cryotome (Leica Biosystems; Buffalo Grove, IL, USA) and were adhered to positively-charged histology slides. Sections of muscle tissue were dried at room temperature for 30 min and incubated in a phosphate‐buffered saline (PBS) solution containing 0.5% Triton X‐100, and blocked with Pierce Super Blocker (Thermo Fisher Scientific). Sections were then rinsed in PBS and incubated with primary antibodies for 1 h. The primary antibodies used for muscle fiber-typing were rabbit anti-dystrophin IgG (Thermo Fisher Scientific; 10 µL antibody per 1 mL of blocking solution) and mouse anti-myosin II IgG (catalog #: SC71; Hybridoma Bank; 100 µL per 1 mL of blocking solution). Sections were then washed for 5 min in PBS and incubated in the dark for 60 min with a secondary antibody solution containing Texas Red-conjugated anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA), and Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Fisher Scientific) (10 µL of all secondary antibodies per 1 mL of blocking solution). Sections were then washed for 5 min in PBS, air-dried, and were mounted with fluorescent media containing 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories). Following mounting, slides were stored in the dark at 4 °C until immunofluorescent images were obtained. Digital images were captured using a fluorescence microscope (Nikon Instruments, Melville, NY, USA) and 20x objective. Approximate exposure times of 600 ms for red and green imaging were used while only 30 ms exposure occurred for blue imaging. For fiber typing, our staining method allowed the identification of cell membranes (detected by the Texas Red filter), type II fiber green cell bodies (detected by the FITC filter), type I fiber black cell bodies (unlabeled), and myonuclei (detected by the DAPI filter). Measurements of muscle fiber cross sectional area (CSA) were performed using the open-sourced software CellProfilerTM per modified methods previously described37, whereby the number of pixels counted within the border of each muscle fiber were converted to a total area in microns-squared (µm2). Based on data from Mackey et al.38 and Murach et al.39, at least 50 fibers per specimen were quantified in order to obtain accurate CSAs.

Statistical Analyses