Ethical Approval and Participant Screening
Prior to any data collection, this study was approved by the Auburn University Institutional Review Board (IRB) (Protocol # 19–249 MR 1907), conformed to standards set by the latest revision of the Declaration of Helsinki, and was registered as a clinical trial (NCT04015479). Men and women aged 50–80 years with minimal RT experience, defined here as not having performed structured RT for at least three months prior, were recruited for this study. Participants were recruited via flyer, email inquiry and newspaper advertisement. Interested participants were informed of the study and testing procedures either over the phone or face-to-face at the Auburn University School of Kinesiology. Eligibility criteria indicated that potential participants had to: 1) be between the ages of 50–80 years old, 2) not actively be participating in structured RT for at least 3 months prior, 3) be free of metal implants, and 4) possess blood pressure readings within normal ranges, with or without medication (i.e. <140/90 SBP/DBP). Exclusion criteria included: 1) individuals having a known peanut allergy, 2) individuals having a body mass index ≥ 35 kg/m2, 3) individuals being exposed to medically-necessary radiation in the last 6 months, or 4) individuals having a medical condition contradicting participation in a RT program, giving blood or donating a skeletal muscle biopsy (i.e. blood clotting disorders or taking blood thinning medications). Participants deemed eligible based on the aforementioned criteria provided written and verbal consent to participate. A medical history questionnaire was obtained at the time of consenting and participants were scheduled to return to the Auburn University School of Kinesiology to complete study procedures described below.
Study Design
Our original intent was to recruit two separate ten-week cohorts. Due to the SARS-CoV-2 pandemic, we voluntarily decided to end the second cohort after only six weeks of training. As such, the primary difference between cohorts was the length of the intervention. The study design for the 10-week and 6-week cohorts is presented in Fig. 1 below.
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Briefly, participants in the 10-week cohort reported to the Auburn University School of Kinesiology on 24 separate occasions, whereas participants in the 6-week cohort reported on 16 separate occasions. Visit one (V1) included screening to determine eligibility, gathering consent and obtaining a health history. Visit two (V2; PRE) occurred at least three days prior to visit 3 (V3) and included a battery of assessments comprised of urine specific gravity (USG), height and body mass, ultrasound of the right leg vastus lateralis (VL), full body dual energy x-ray absorptiometry (DXA), peripheral quantitative computed tomography (pQCT) scan at the mid-thigh of the right leg and right leg strength assessment using an isokinetic dynamometer. Following the battery of assessments, participants were provided with deuterium oxide (D2O)-enriched water, a three-day food log, and three separate salivettes to measure D2O enrichment. The food log was returned prior to V3 at each participant’s convenience.
V3 included the participant’s first muscle tissue sample collection, randomization to either the peanut protein supplement group (PP) or wait-list control (CTL), the participant’s first resistance exercise bout, and immediate post-exercise PP supplementation or no supplementation. A complete nutritional breakdown for the PP supplement is presented in Table 1. V4 included the participant’s second muscle tissue sample collection and salivette return. Visit five (V5) through visit twenty-three (V23) for the 10-week cohort and V5 through visit fifteen (V15) for the 6-week cohort included a single RT session. During V23 for the 10-week cohort and V15 for the 6-week cohort participants were provided with their second set of food logs. Visit twenty-four (V24; POST) for the 10-week cohort and visit sixteen (V16; POST) for the 6-week cohort occurred roughly 72 hours following V23 and V15, respectively, and included a repeat of the V2 testing battery. Specific testing methodologies are detailed below.
Table 1
Baseline Participant Characteristics
Variable (units) | Mean ± SD | p-value |
Gender | PP CTL | 12 M / 8 F 10 M / 9 F | N/A |
Age (years) | PP CTL | 60 ± 9 58 ± 7 | p = 0.61 |
Height (cm) | PP CTL | 171.7 ± 8.3 172.0 ± 9.3 | p = 0.41 |
Weight (kg) | PP CTL | 84.9 ± 17.6 88.8 ± 20.5 | p = 0.52 |
BMI (kg/m2) | PP CTL | 27.8 ± 5.5 29.7 ± 6.2 | p = 0.23 |
DXA % body fat (%) | PP CTL | 36.0 ± 7.1 36.2 ± 7.6 | p = 0.92 |
Legend: Baseline participant characteristics are presented as means ± standard deviation values. Abbreviations: PP, peanut protein supplemented participants (n = 20); CTL, non-supplemented participants (n = 19); DXA, dual x-ray absorptiometry; BMI, body mass index; cm, centimeters; kg, kilograms; kg/m2, kilograms per meter squared. |
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Pre- and Post-intervention Testing Battery
The testing sessions described below occurred during morning hours (05:00–09:00) following an overnight fast for all but 7 participants who reported to the laboratory after working hours at 17:00–18:30 following a ~ 4–5 hour fast.
Body Composition Assessments. During V2 and V24 (10-week participants) or V2 and V16 (6-week participants), participants reported to the Auburn University School of Kinesiology wearing casual sports attire (i.e. athletic shirt and shorts, tennis shoes). Participants submitted a urine sample (~ 5 mL) to assess USG levels using a handheld refractometer (ATAGO; Bellevue, WA, USA). Notably, all participants possessed USG values less than 1.020 indicating that they were well hydrated. Height and body mass were assessed using a digital column scale (Seca 769; Hanover, MD, USA) with mass and height being collected to the nearest 0.1 kg and 0.5 cm, respectively. Thereafter, right leg VL images were captured in the transverse plane using real-time B-mode ultrasonography (LOGIQ S7 Expert, GE Healthcare, USA) utilizing a multi-frequency linear-array transducer (3–12 MHz, GE Healthcare, USA) and subsequently analyzed for VL thickness. Participants were instructed to stand and displace bodyweight to the left leg to ensure the right leg was relaxed. Measurements were standardized by placing the transducer at the midway point between the inguinal crease and proximal border of the patella. All images were captured and analyzed by the same investigator (S.C.O.) with a 24-hr test-retest reliability using intraclass correlation coefficient (ICC3,1), standard error of the measure (SEM), and minimal difference (MD) to be considered real of 0.991, 0.06, and 0.16 cm, respectively. Participants then underwent a full body dual-energy x-ray absorptiometry (DXA) scan (Lunar Prodigy; GE Corporation, Fairfield, CT, USA) for determination of total lean soft tissue mass (LSTM) and fat mass (FM). Quality assurance testing and calibration were performed the morning of data-collection days to ensure the scanner was operating to manufacturer specification. Scans were analyzed by the same technician using the manufacturer’s standardized software. Test-retest reliability using ICC3,1, SEM, and MD were previously determined for LSTM (0.99, 0.36, and 0.99 kg, respectively) and FM (0.99. 0.43, and 1.19 kg). Following the DXA scan, a cross-sectional image of the right thigh at 50% of the femur length was acquired using a pQCT scanner (Stratec XCT 3000, Stratec Medical, Pforzheim, Germany). Scans were acquired using a single 2.4 mm slice thickness, a voxel size of 0.4 mm and scanning speed of 20 mm/sec. All images were analyzed for total muscle cross-sectional area (mCSA, cm2) and density (mg/cm3) using the pQCT BoneJ plugin freely available through ImageJ analysis software (NIH, Bethesda, MD). All scans were performed and analyzed by the same investigator (K.C.Y.). Test-retest reliability using ICC3,1, SEM, and MD was previously determined for mCSA (0.99, 0.84, and 2.32 cm2, respectively).
Right Leg Isokinetic Strength Assessment. Participants performed maximal isokinetic right leg extensions on an isokinetic dynamometer (System 4 Pro, BioDex Medical Systems, Shirley, NY, USA). Participants were fastened to the dynamometer so that the right knee was aligned with the axis of the dynamometer. Seat height was adjusted to ensure the hip angle was approximately 90°. Prior to peak torque assessment, each participant performed a warmup consisting of submaximal to maximal isokinetic knee extensions. Participants then completed five maximal voluntary isokinetic knee extension actions at 60°/sec and 120°/sec. Sets were separated by 60 sec of rest. Participants were provided verbal encouragement during each set. The isokinetic extension resulting in the greatest peak torque value was used for analyses. Right leg extensor peak torque testing occurred ~ 1–3 days prior to the muscle biopsy at the PRE (V2) time point in both the 10-week and 6-week cohorts, whereas this test occurred approximately 10 minutes following the biopsy at the POST time point for the 10-week cohort only (V24). This difference in methodology between time points was due to logistical constraints. However, we have unpublished data suggesting peak torque values are not affected by muscle biopsies when isokinetic testing occurs within a 10-minute post-biopsy window (20).
Supplement Randomization and Resistance Training
During V3, immediately following collection of the first muscle sample, participants were randomized to either consume PP during the intervention (n = 20) or after the intervention (n = 19). The PP supplement (PBfit; BetterBody Foods, Lindon, UT, USA) provided the following per daily serving: 315 kcal, 35 g protein, 10.7 g essential amino acids (where 2.44 g was L-leucine), 9.0 g fat and 22.5 g carbohydrate (with 14.8 g fiber and 7.7 g sugars).
Randomization was stratified by gender in blocks of four, hence the slight differences in allocation to study arms. Afterwards, participants were escorted to the Auburn University School of Kinesiology Fitness and Performance Optimization Laboratory for their first resistance exercise session. Participants were provided detailed instructions on proper posture, technique, range-of-motion, body positioning and breathing to ensure safety. Participants completed supervised RT twice weekly for either ten weeks or six weeks. All RT sessions were separated by at least 48 hours to allow for a period of recovery. Each RT session consisted of five exercises including seated leg press, leg extensions, lying leg curls, barbell bench press and cable pull-downs. For each exercise, participants performed three sets of 10–12 repetitions with 1 minute of rest between sets. At the end of each set, participants were asked to rate the level of difficulty where 0 = easy, 5 = moderate difficulty and 10 = hard. If values were below 7, weight was modestly added to increase exertion on the subsequent set. If values were 10, or the participant could not complete the set, weight was removed prior to the next set. Participants were encouraged to be as truthful as possible when assessing difficulty and were provided verbal encouragement and feedback during and following each set. The intent of this training method was to consistently challenge participants so that perceived exertion after each set of 10–12 repetitions was at a 7–9 rating. Training data for each participant were logged, allowing us to ensure that training effort was maximized within each training session, and participants were successfully implementing progressive overload in an individualized fashion.
Notably, study personnel supervised all training throughout the study. Participants in the PP group were instructed to consume one daily serving of the PP supplement. On workout days, PP supplements were provided to participants in the PP group immediately following exercise, and supplementation compliance was supervised. On non-workout days, participants were instructed to consume one serving between meals. Product bottles were returned to the study coordinator to ensure compliance to the supplementation protocol.
Muscle Sample Collection and Integrated Myofibrillar Protein Synthesis Rate Determination using Deuterium Oxide
MyoPS rates were determined after the first bout of training with or without PP supplementation using the integrated D2O technique. Briefly, participants consumed a total 4.5 mL∙kg− 1 of lean body mass (LBM) of D2O-enriched water (70 atom percent; Sigma-Aldrich, St. Louis, MO) over the course of four separate days beginning 2 days prior to V2 through V3. Participants were provided with six individual servings of D2O. Three of these servings contained 1 mL∙kg− 1 LBM D2O and were consumed in a single day as a loading phase, and three of these servings contained 0.5 mL∙kg− 1 LBM D2O and were consumed over the next three consecutive days.
Skeletal muscle biopsies at V3 and V4 were obtained from the right thigh (i.e VL; in the same plane as ultrasound and pQCT assessments) midway between the patella and iliac crest using a 5-gauge needle with suction and sterile laboratory procedures. Briefly, upon arrival to the laboratory, participants were instructed to lie in a supine position on an athletic training table. Approximately 5 minutes afterwards, 1.5 mL of 1% lidocaine was injected subcutaneously above the skeletal muscle fascia a small pilot incision was made for needle insertion using a sterile Surgical Blade No. 11 (AD Surgical; Sunnyvale, CA, USA). After 5 minutes of allowing the anesthetic to take effect, the biopsy needle was inserted into the pilot incision just beyond the fascia and approximately 50–100 mg of skeletal muscle was removed using a double chop method and applied suction (21). Following biopsies, tissue was rapidly teased of blood and connective tissue and subsequently stored at -80 °C until the isolation of myofibrils. The day of myofibril isolation, all samples were batch-processed using the recently published MIST method; for further details refer to our recent publication (22). Thereafter, isolated myofibrils and saliva (collected from salivettes) were shipped on dry ice to Metabolic Solutions (Nashua, NH, USA) for tracer analyses.
MyoPS rates over the 24-hour period following the first training bout were calculated similar to Bell et al. (23) (see equation below).
Participants were instructed to self-report their habitual food intake for three consecutive days and return these food logs at V3 and V24 or V16 (10- and 6-week cohort, respectively). Participants were asked not to change their diet in any way, with the exception of PP participants who were instructed to consume the supplement as described above. Study staff entered each food log into the Automated Self-Administered 24-Hour Dietary Assessment tool (ASA24), which uses the United States Department of Agriculture Food and Nutrient Database for Dietary Studies to provide values for 195 nutrients, nutrient ratios and other food components (