Acute effects of commercial energy drink consumption on exercise performance and cardiovascular safety: a randomized, double-blind, placebo-controlled, crossover trial.

The aim of this study was to examine the acute effects of a non-caloric energy drink (C4E) compared to a traditional sugar-containing energy drink (MED) and non-caloric placebo (PLA) on exercise performance and cardiovascular safety. Thirty healthy, physically active males (25 ± 4 y) completed three experimental visits under semi-fasted conditions (5-10 h) and in randomized order, during which they consumed C4E, MED, or PLA matched for volume, appearance, taste, and mouthfeel. One hour after drink consumption, participants completed a maximal, graded exercise test (GXT) with measurement of pulmonary gases, an isometric leg extension fatigue test (ISOFTG), and had their cardiac electrical activity (ECG), leg blood flow (LBF), and blood pressure (BP) measured throughout the visit. Neither MED nor C4E had an ergogenic effect on maximal oxygen consumption, time to exhaustion, or peak power during the GXT (p > 0.05). Compared to PLA, MED reduced fat oxidation (respiratory exchange ratio (RER) +0.030 ± 0.01; p = 0.026) during the GXT and did not influence ISOFTG performance. Compared to PLA, C4E did not alter RER (p = 0.94) and improved impulse during the ISOFTG (+0.658 ± 0.25 V·s; p = 0.032). Relative to MED, C4E did not significantly improve gas exchange threshold (p = 0.05-0.07). Both MED and C4E increased systolic BP at rest (+7.1 ± 1.2 mmHg; p < 0.001 and + 5.7 ± 1.0 mmHg; p < 0.001, respectively), C4E increased SBP post-GXT (+13.3 ± 3.8 mmHg; p < 0.001), and MED increased SBP during recovery (+3.2 ± 1.1 mmHg; p < 0.001). Neither MED nor C4E influenced ECG measures (p ≥ 0.08) or LBF (p = 0.37) compared to PLA. C4E may be more efficacious for improving performance in resistance-type tasks without altering fat oxidation under semi-fasted conditions during fatiguing exercise bouts, but promotes similar changes in BP and HR to MED.

Novel Energy Drink Improves Cognitive Function and Mood, without Influencing Myocardial Oxygen Demand or Ventricular Repolarization in Adult Gamers: A Randomized, Double-Blind, Placebo-Controlled, Crossover Trial.

OBJECTIVES: To examine the efficacy of acute consumption of a novel energy drink (C4S) versus placebo for improving cognitive and gaming performance and mood. Secondarily, we examined the cardiovascular safety profile of acute C4S consumption. METHODS: Forty-five healthy, young adult video gamers completed two experimental visits in randomized order where they consumed either C4S or a placebo and then completed a validated battery of neurocognitive tests, played five video games, and completed a mood state survey. Blood pressure (BP), heart rate (HR), oxygen saturation, and electrocardiogram measurements were taken at baseline and repeated throughout each visit. RESULTS: Acute consumption of C4S improved cognitive flexibility (absolute mean or median difference [95% CI] = +4.3 [2.2-6.4]; p < 0.001; d = 0.63), executive function (+4.3 [2.3-6.3]; p < 0.001; d = 0.63), sustained attention (+2.1 [0.6-3.6]; p = 0.01; d = 0.44), motor speed (+2.9 [0.8-4.9]; p < 0.001; d = 0.44), psychomotor speed (+3.9 [0.1-7.7]; p = 0.04; d = 0.32) working memory (+1.0 [0.1-1.9]; p = 0.02; d = 0.35), and performance in the two-dimensional visuospatial game Tetris (+463 [-419-2,065] pts; p = 0.049; d = 0.30) compared to placebo. C4S also improved Fatigue-Inertia (-1 [-3-0]; p = 0.004; d = 0.45), Vigor-Activity (+2.4 [1.3-3.6]; p < 0.001; d = 0.64), Friendliness (+0 [0-1]; p = 0.04; d = 0.32), and Total Mood Disturbance (-3 [-6-0]; p = 0.002; d = 0.44). BP increased slightly in C4S versus placebo, while HR decreased from baseline to post-drink in the C4S condition. Rate-pressure-product was higher in C4S versus placebo independent of time but did not increase from baseline. There was no effect on corrected QT interval. CONCLUSION: Acute consumption of C4S was efficacious for cognitive performance, visuospatial gaming performance, and mood enhancement, and had no effect on myocardial oxygen demand or ventricular repolarization, despite being associated with increases in BP.

The effects of a sugar-free amino acid-containing electrolyte beverage on 5-kilometer performance, blood electrolytes, and post-exercise cramping versus a conventional carbohydrate-electrolyte sports beverage and water.

OBJECTIVE: The purpose of this study was to examine the acute effects of a multi-ingredient, low calorie dietary supplement (MIDS, XTEND® Healthy Hydration) on 5-kilometer (5-km) time trial performance and blood electrolyte concentrations compared to a carbohydrate-electrolyte beverage (CE, GATORADE® Thirst Quencher) and distilled water (W). METHODS: During visit 1 (V1), participants (10 men and 10 women, 20-35 years old, BMI ≤ 29 kg/m2, recreationally active) reported to the laboratory whereby the following tests were performed: i) height and weight measurements, ii) body composition analysis, iii) treadmill testing to measure maximal aerobic capacity, and iv) 5-km time trial familiarization. The second visit (V2) was one week after V1 in the morning (0600 - 0900) and participants arrived 12-14 h fasted (no food or drink). The first battery of assessments (V2-T1) included nude body mass, urine specific gravity (USG), a profile of mood states (POMS) questionnaire, and the completion of a visual analogue scale (VAS) questionnaire to quantify cramping. Then heart rate (HR), blood pressure (BP), total body hydration (via bioelectrical impedance spectroscopy [BIS]) were examined. Finally, a measurement of blood markers via finger stick was performed. Participants consumed a randomized beverage (16 fl. oz. of MIDS, 16 fl. oz. of W, or 16 fl. oz. of CE) within 3 min followed by a 45-min rest. Following the rest period, a second battery (V2-T2) was performed whereby participants' USG was assessed and they completed the POMS and VAS questionnaires, and HR, BP, and blood markers were measured. The participants then performed a 5-km treadmill time trial. Immediately following the 5-km time trial, participants completed a third testing battery (V2-T3) that began with blood markers, HR and BP assessments, followed by nude body weight assessment, and the POMS and VAS questionnaires. After 60 min, a fourth battery (V2-T4) was performed that included HR, BP, and blood markers. After sitting quietly for another 60 min a fifth battery assessment was performed (V2-T5) that included participants' USG, POMS and VAS questionnaires, HR, BP, blood markers, and total body hydration. Visits 3 (V3) and 4 (V4) followed the same protocol except a different randomized drink (16 oz. of CE, MIDS, or W) was consumed; all of which were separated by approximately one week. RESULTS: No differences occurred between conditions for 5-km time trial completion, indirect calorimetry outcomes during 5-km time trials, USG, or nude mass measurements (p > 0.05 for all relevant statistical tests). However, blood potassium and the sodium/potassium ratio displayed significant interactions (p < 0.05), and post hoc testing indicated these values were better maintained in the MIDS versus other conditions. Post-exercise cramp prevalence was greater in the CE (p < 0.05) and trended higher with W (p = 0.083) compared to the MIDS condition. Post-exercise cramp severity was also elevated with the W and CE beverages (p < 0.05) but not the MIDS (p = 0.211). CONCLUSIONS: The MIDS did not affect 5-km time trial performance but exhibited favorable effects on blood electrolyte and post-exercise self-reporting cramp outcomes compared to the CE and W drinks.

Impact of Protein and Carbohydrate Supplementation on Musculoskeletal Injuries in Army Initial Entry Training Soldiers.

This project investigated whey protein and/or carbohydrate supplementation effects on musculoskeletal injury (MSI) outcomes. Four groups of Initial Entry Training soldiers consumed either: (1) one protein (38.6 g, 293 kcal); (2) one carbohydrate (63.4 g, 291 kcal); (3) two protein (77.2 g, 586 kcal); or (4) two carbohydrate servings/day (126.8 g, 582 kcal) after physical training and before bed, or before bed only. Odds Ratio, Chi-square and Wilcoxon ranked-sum test compared supplementation/no supplementation, number of servings, and protein/carbohydrate for MSI and limited/missed duty rates and limited/missed training days. Non-matched pairs group averages were compared to 2015/2016 historical data. Non-supplemented soldiers were approximately 5× more likely to sustain a MSI (χ2 = 58.48, p < 0.001) and 4× more likely to miss training (χ2 = 9.73, p = 0.003) compared to two servings. Non-supplemented soldiers missed five additional training days compared to two servings (W = 6059.5, p = 0.02). Soldiers consuming one serving were approximately 3× more likely to sustain a MSI than two servings (χ2 = 9.55, p = 0.002). There was no difference in limited/missed duty rates or limited/missed training days between consuming one or two servings. There was no difference between consuming one serving versus no supplementation or protein versus carbohydrate supplementation for any outcome variable. Soldiers consuming 2 servings/day of protein or carbohydrate had lower MSI rates, limited/missed duty rates, and limited/ missed training days compared to non-supplemented soldiers.

Effect of Whey Protein Supplementation on Physical Performance and Body Composition in Army Initial Entry Training Soldiers.

We investigated the effects of whey protein (WP) supplementation on body composition and physical performance in soldiers participating in Army Initial Entry Training (IET). Sixty-nine, male United States Army soldiers volunteered for supplementation with either twice daily whey protein (WP, 77 g/day protein, ~580 kcal/day; n = 34, age = 19 ± 1 year, height = 173 ± 6 cm, weight = 73.4 ± 12.7 kg) or energy-matched carbohydrate (CHO) drinks (CHO, 127 g/day carbohydrate, ~580 kcal/day; n = 35, age = 19 ± 1 year, height = 173 ± 5 cm, weight = 72.3 ± 10.9 kg) for eight weeks during IET. Physical performance was evaluated using the Army Physical Fitness Test during weeks two and eight. Body composition was assessed using 7-site skinfold assessment during weeks one and nine. Post-testing push-up performance averaged 7 repetitions higher in the WP compared to the CHO group (F = 10.1, p < 0.001) when controlling for baseline. There was a significant decrease in fat mass at post-training (F = 4.63, p = 0.04), but no significant change in run performance (F = 3.50, p = 0.065) or fat-free mass (F = 0.70, p = 0.41). Effect sizes for fat-free mass gains were large for both the WP (Cohen's d = 0.44) and CHO (Cohen's d = 0.42) groups. WP had a large effect on fat mass (FM) loss (Cohen's d = -0.67), while CHO had a medium effect (Cohen's d = -0.40). Twice daily supplementation with WP improved push-up performance and potentiated reductions in fat mass during IET training in comparison to CHO supplementation.

Author Correction: Soy protein supplementation is not androgenic or estrogenic in college-aged men when combined with resistance exercise training.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Skeletal muscle mitochondrial volume and myozenin-1 protein differences exist between high versus low anabolic responders to resistance training.

BACKGROUND: We sought to examine how 12 weeks of resistance exercise training (RET) affected skeletal muscle myofibrillar and sarcoplasmic protein levels along with markers of mitochondrial physiology in high versus low anabolic responders. METHODS: Untrained college-aged males were classified as anabolic responders in the top 25th percentile (high-response cluster (HI); n = 13, dual x-ray absorptiometry total body muscle mass change (Δ) = +3.1 ± 0.3 kg, Δ vastus lateralis (VL) thickness = +0.59 ± 0.05 cm, Δ muscle fiber cross sectional area = +1,426 ± 253 µm2) and bottom 25th percentile (low-response cluster (LO); n = 12, +1.1 ± 0.2 kg, +0.24 ± 0.07 cm, +5 ± 209 µm2; p < 0.001 for all Δ scores compared to HI). VL muscle prior to (PRE) and following RET (POST) was assayed for myofibrillar and sarcoplasmic protein concentrations, myosin and actin protein content, and markers of mitochondrial volume. Proteins related to myofibril formation, as well as whole lysate PGC1-α protein levels were assessed. RESULTS: Main effects of cluster (HI > LO, p = 0.018, Cohen's d = 0.737) and time (PRE > POST, p = 0.037, Cohen's d = -0.589) were observed for citrate synthase activity, although no significant interaction existed (LO PRE = 1.35 ± 0.07 mM/min/mg protein, LO POST = 1.12 ± 0.06, HI PRE = 1.53 ± 0.11, HI POST = 1.39 ± 0.10). POST myofibrillar myozenin-1 protein levels were up-regulated in the LO cluster (LO PRE = 0.96 ± 0.13 relative expression units, LO POST = 1.25 ± 0.16, HI PRE = 1.00 ± 0.11, HI POST = 0.85 ± 0.12; within-group LO increase p = 0.025, Cohen's d = 0.691). No interactions or main effects existed for other assayed markers. DISCUSSION: Our data suggest myofibrillar or sarcoplasmic protein concentrations do not differ between HI versus LO anabolic responders prior to or following a 12-week RET program. Greater mitochondrial volume in HI responders may have facilitated greater anabolism, and myofibril myozenin-1 protein levels may represent a biomarker that differentiates anabolic responses to RET. However, mechanistic research validating these hypotheses is needed.

Soy protein supplementation is not androgenic or estrogenic in college-aged men when combined with resistance exercise training.

It is currently unclear as to whether sex hormones are significantly affected by soy or whey protein consumption. Additionally, estrogenic signaling may be potentiated via soy protein supplementation due to the presence of phytoestrogenic isoflavones. Limited also evidence suggests that whey protein supplementation may increase androgenic signaling. Therefore, the purpose of this study was to examine the effects of soy protein concentrate (SPC), whey protein concentrate (WPC), or placebo (PLA) supplementation on serum sex hormones, androgen signaling markers in muscle tissue, and estrogen signaling markers in subcutaneous (SQ) adipose tissue of previously untrained, college-aged men (n = 47, 20 ± 1 yrs) that resistance trained for 12 weeks. Fasting serum total testosterone increased pre- to post-training, but more so in subjects consuming WPC (p < 0.05), whereas serum 17ß-estradiol remained unaltered. SQ estrogen receptor alpha (ERα) protein expression and hormone-sensitive lipase mRNA increased with training regardless of supplementation. Muscle androgen receptor (AR) mRNA increased while ornithine decarboxylase mRNA (a gene target indicative of androgen signaling) decreased with training regardless of supplementation (p < 0.05). No significant interactions of supplement and time were observed for adipose tissue ERα/ß protein levels, muscle tissue AR protein levels, or mRNAs in either tissue indicative of altered estrogenic or androgenic activity. Interestingly, WPC had the largest effect on increasing type II muscle fiber cross sectional area values (Cohen's d = 1.30), whereas SPC had the largest effect on increasing this metric in type I fibers (Cohen's d = 0.84). These data suggest that, while isoflavones were detected in SPC, chronic WPC or SPC supplementation did not appreciably affect biomarkers related to muscle androgenic signaling or SQ estrogenic signaling. The noted fiber type-specific responses to WPC and SPC supplementation warrant future research.

Biomarkers associated with low, moderate, and high vastus lateralis muscle hypertrophy following 12 weeks of resistance training.

We sought to identify biomarkers which delineated individual hypertrophic responses to resistance training. Untrained, college-aged males engaged in full-body resistance training (3 d/wk) for 12 weeks. Body composition via dual x-ray absorptiometry (DXA), vastus lateralis (VL) thickness via ultrasound, blood, VL muscle biopsies, and three-repetition maximum (3-RM) squat strength were obtained prior to (PRE) and following (POST) 12 weeks of training. K-means cluster analysis based on VL thickness changes identified LOW [n = 17; change (mean±SD) = +0.11±0.14 cm], modest (MOD; n = 29, +0.40±0.06 cm), and high (HI; n = 21, +0.69±0.14 cm) responders. Biomarkers related to histology, ribosome biogenesis, proteolysis, inflammation, and androgen signaling were analyzed between clusters. There were main effects of time (POST>PRE, p<0.05) but no cluster×time interactions for increases in DXA lean body mass, type I and II muscle fiber cross sectional area and myonuclear number, satellite cell number, and macronutrients consumed. Interestingly, PRE VL thickness was ~12% greater in LOW versus HI (p = 0.021), despite POST values being ~12% greater in HI versus LOW (p = 0.006). However there was only a weak correlation between PRE VL thickness scores and change in VL thickness (r2 = 0.114, p = 0.005). Forced post hoc analysis indicated that muscle total RNA levels (i.e., ribosome density) did not significantly increase in the LOW cluster (351±70 ng/mg to 380±62, p = 0.253), but increased in the MOD (369±115 to 429±92, p = 0.009) and HI clusters (356±77 to 470±134, p<0.001; POST HI>POST LOW, p = 0.013). Nonetheless, there was only a weak association between change in muscle total RNA and VL thickness (r2 = 0.079, p = 0.026). IL-1ß mRNA levels decreased in the MOD and HI clusters following training (p<0.05), although associations between this marker and VL thickness changes were not significant (r2 = 0.0002, p = 0.919). In conclusion, individuals with lower pre-training VL thickness values and greater increases muscle total RNA levels following 12 weeks of resistance training experienced greater VL muscle growth, although these biomarkers individually explained only ~8-11% of the variance in hypertrophy.

Acute and chronic resistance training downregulates select LINE-1 retrotransposon activity markers in human skeletal muscle.

Herein, we examined if acute or chronic resistance exercise affected markers of skeletal muscle long interspersed nuclear element-1 (LINE-1) retrotransposon activity. In study 1, 10 resistance-trained college-aged men performed three consecutive daily back squat sessions, and vastus lateralis biopsies were taken before (Pre), 2 h following session 1 (Post1), and 3 days following session 3 (Post2). In study 2, 13 untrained college-aged men performed a full-body resistance training program (3 days/wk), and vastus lateralis biopsies were taken before ( week 0) and ~72 h following training cessation ( week 12). In study 1, LINE-1 mRNA decreased 42-48% at Post1 and 2 ( P < 0.05), and reverse transcriptase (RT) activity trended downward at Post2 (-37%, P = 0.067). In study 2, LINE-1 mRNA trended downward at week 12 (-17%, P = 0.056) while LINE-1 promoter methylation increased (+142%, P = 0.041). Open reading frame (ORF)2p protein expression (-24%, P = 0.059) and RT activity (-26%, P = 0.063) also trended downward by week 12. Additionally, changes in RT activity versus satellite cell number were inversely associated ( r = -0.725, P = 0.008). Follow-up in vitro experiments demonstrated that 48-h treatments with lower doses (1 µM and 10 µM) of efavirenz and nevirapine (non-nucleoside RT inhibitors) increased myoblast proliferation ( P < 0.05). However, we observed a paradoxical decrease in myoblast proliferation with higher doses (50 µM) of efavirenz and delavirdine. This is the first report suggesting that resistance exercise downregulates markers of skeletal muscle LINE-1 activity. Given our discordant in vitro findings, future research is needed to thoroughly assess whether LINE-1-mediated RT activity enhances or blunts myoblast, or primary satellite cell, proliferative capacity.

Effects of Whey, Soy or Leucine Supplementation with 12 Weeks of Resistance Training on Strength, Body Composition, and Skeletal Muscle and Adipose Tissue Histological Attributes in College-Aged Males.

We sought to determine the effects of L-leucine (LEU) or different protein supplements standardized to LEU (~3.0 g/serving) on changes in body composition, strength, and histological attributes in skeletal muscle and adipose tissue. Seventy-five untrained, college-aged males (mean ± standard error of the mean (SE); age = 21 ± 1 years, body mass = 79.2 ± 0.3 kg) were randomly assigned to an isocaloric, lipid-, and organoleptically-matched maltodextrin placebo (PLA, n = 15), LEU (n = 14), whey protein concentrate (WPC, n = 17), whey protein hydrolysate (WPH, n = 14), or soy protein concentrate (SPC, n = 15) group. Participants performed whole-body resistance training three days per week for 12 weeks while consuming supplements twice daily. Skeletal muscle and subcutaneous (SQ) fat biopsies were obtained at baseline (T1) and ~72 h following the last day of training (T39). Tissue samples were analyzed for changes in type I and II fiber cross sectional area (CSA), non-fiber specific satellite cell count, and SQ adipocyte CSA. On average, all supplement groups including PLA exhibited similar training volumes and experienced statistically similar increases in total body skeletal muscle mass determined by dual X-ray absorptiometry (+2.2 kg; time p = 0.024) and type I and II fiber CSA increases (+394 µm² and +927 µm²; time p < 0.001 and 0.024, respectively). Notably, all groups reported increasing Calorie intakes ~600-800 kcal/day from T1 to T39 (time p < 0.001), and all groups consumed at least 1.1 g/kg/day of protein at T1 and 1.3 g/kg/day at T39. There was a training, but no supplementation, effect regarding the reduction in SQ adipocyte CSA (-210 µm²; time p = 0.001). Interestingly, satellite cell counts within the WPC (p < 0.05) and WPH (p < 0.05) groups were greater at T39 relative to T1. In summary, LEU or protein supplementation (standardized to LEU content) does not provide added benefit in increasing whole-body skeletal muscle mass or strength above PLA following 3 months of training in previously untrained college-aged males that increase Calorie intakes with resistance training and consume above the recommended daily intake of protein throughout training. However, whey protein supplementation increases skeletal muscle satellite cell number in this population, and this phenomena may promote more favorable training adaptations over more prolonged periods.

Whey protein-derived exosomes increase protein synthesis and hypertrophy in C2-C12 myotubes.

We sought to examine potential amino acid independent mechanisms whereby hydrolyzed whey protein (WP) affects muscle protein synthesis (MPS) and anabolism in vitro. Specifically, we tested (1) whether 3-h and 6-h treatments of WP, essential amino acids, or l-leucine (Leu) affected MPS, and whether 6-h treatments with low-, medium-, or high doses of WP versus Leu affected MPS; (2) whether knockdown of the primary Leu transporter affected WP- and Leu-mediated changes in MPS, mammalian target of rapamycin (mTOR) signaling responses, or both, following 6-h treatments; (3) whether exosomes isolated from WP (WP-EXO) affected MPS, mTOR signaling responses, or both, compared with untreated (control) myotubes, following 6-h, 12-h, and 24-h treatments, and whether they affected myotube diameter following 24-h and 48-h treatments. For all treatments, 7-d post-differentiated C2C12 myotubes were examined. In experiment 1, 6-h WP treatments increased MPS compared with control (+46%), Leu (+24%), and essential amino acids (+25%). Moreover, the 6-h low-, medium-, and high WP treatments increased MPS by approximately 40 to 50% more than corresponding Leu treatments. In experiment 2 (LAT short hairpin RNA-transfected myotubes), 6-h WP treatments increased MPS compared with control (+18%) and Leu (+19%). In experiment 3, WP-EXO treatments increased MPS over controls at 12h (+18%) and 24h (+45%), and myotube diameters increased with 24-h (+24%) and 48-h (+40%) WP-EXO treatments compared with controls. The WP-EXO treatments did not appear to operate through mTOR signaling; instead, they increased mRNA and protein levels o eukaryotic initiation factor 4A. Bovine-specific microRNA following 24-h WP-EXO treatments were enriched in myotubes (chiefly miR-149-3p, miR-2881), but were not related to hypertrophic gene targets. To summarize, hydrolyzed WP-EXO increased skeletal MPS and anabolism in vitro, and this may be related to an unknown mechanism that increases translation initiation factors rather than enhancing mTOR signaling or the involvement of bovine-specific microRNA.

Effects of Hydrolyzed Whey versus Other Whey Protein Supplements on the Physiological Response to 8 Weeks of Resistance Exercise in College-Aged Males.

OBJECTIVE: The objective of this study was to compare the chronic effects of different whey protein forms on body composition and performance when supplemented with resistance training. METHODS: Resistance-trained men (N = 56, 21.4 ± 0.4 years, 79.5 ± 1.0 kg) participated in an 8-week resistance training regimen (2 upper-body sessions and 2 lower-body sessions per week) and received one of 4 double-blinded treatments: 30 g/serving carbohydrate placebo (PLA) or 30 g/serving protein from either (a) 80% whey protein concentrate (WPC), (b) high-lactoferrin-containing WPC (WPC-L), or (c) extensively hydrolyzed WPC (WPH). All subjects consumed 2 servings of treatment per day; specifically, once immediately before and after training and between meals on nontraining days. Blood collection, one repetition maximum (1RM) testing for bench press and hack squat, and body composition assessment using dual-energy x-ray absorptiometry (DXA) occurred prior to training and 48 hours following the last training session. RESULTS: Total body skeletal muscle mass increased in all groups (p < 0.0125). There were similar between-group increases in upper-body (4%-7%, analysis of covariance [ANCOVA] interaction p = 0.73) and lower-body (24%-35%, ANCOVA interaction p = 0.85) 1RM strength following the intervention. Remarkably, WPH reduced fat mass (-6%), which was significantly different from PLA (+4.4%, p < 0.0125). No time or between-group differences were present for serum markers of health, metabolism, or muscle damage, with the exception of blood urea nitrogen being significantly lower for WPH than WPC (p < 0.05) following the intervention. CONCLUSIONS: WPH may augment fat loss but did not provide any other advantages when used in combination with resistance training. More mechanistic research is needed to examine how WPH affects adipose tissue physiology.

Western diet-induced hepatic steatosis and alterations in the liver transcriptome in adult Brown-Norway rats.

BACKGROUND: The purpose of this study was to investigate the effects of sub-chronic high fat, high sucrose diet (also termed 'Westernized diet' or WD) feeding on the liver transcriptome during early nonalcoholic fatty liver disease (NAFLD) development. METHODS: Brown Norway male rats (9 months of age) were randomly assigned to receive ad libitum access to a control (CTL; 14 % kcal fat, 1.2 % sucrose by weight) diet or WD (42 % kcal from fat, 34 % sucrose by weight) for 6 weeks. RESULTS: Six weeks of WD feeding caused hepatic steatosis development as evidenced by the 2.25-fold increase in liver triacylglycerol content, but did not induce advanced liver disease (i.e., no overt inflammation or fibrosis) in adult Brown Norway rats. RNA deep sequencing (RNA-seq) revealed that 94 transcripts were altered in liver by WD feeding (46 up-, 48 down-regulated, FDR < 0.05). Specifically, the top differentially regulated gene network by WD feeding was 'Lipid metabolism, small molecular biochemistry, vitamin and mineral metabolism' (Ingenuity Pathway Analysis (IPA) score 61). The top-regulated canonical signaling pathway in WD-fed rats was the 'Superpathway of cholesterol biosynthesis' (10/29 genes regulated, p = 1.68E-17), which coincides with a tendency for serum cholesterol levels to increase in WD-fed rats (p = 0.09). Remarkably, liver stearoyl-CoA desaturase (Scd) mRNA expression was by far the most highly-induced transcript in WD-fed rats (approximately 30-fold, FDR = 0.01) which supports previous literature underscoring this gene as a crucial target during NAFLD development. CONCLUSIONS: In summary, sub-chronic WD feeding appears to increase hepatic steatosis development over a 6-week period but only induces select inflammation-related liver transcripts, mostly acute phase response genes. These findings continue to outline the early stages of NAFLD development prior to overt liver inflammation and advanced liver disease.

Effects of oral phosphatidic acid feeding with or without whey protein on muscle protein synthesis and anabolic signaling in rodent skeletal muscle.

BACKGROUND: Phosphatidic acid (PA) is a diacyl-glycerophospholipid that acts as a signaling molecule in numerous cellular processes. Recently, PA has been proposed to stimulate skeletal muscle protein accretion, but mechanistic studies are lacking. Furthermore, it is unknown whether co-ingesting PA with other leucine-containing ingredients can enhance intramuscular anabolic signaling mechanisms. Thus, the purpose of this study was to examine if oral PA feeding acutely increases anabolic signaling markers and muscle protein synthesis (MPS) in gastrocnemius with and without whey protein concentrate (WPC). METHODS: Overnight fasted male Wistar rats (~250 g) were randomly assigned to four groups: control (CON, n = 6-13), PA (29 mg; n = 8), WPC (197 mg; n = 8), or PA + WPC (n = 8). Three hours post-feeding, gastrocnemius muscle was removed for markers of Akt-mTOR signaling, gene expression patterns related to skeletal muscle mass regulation and metabolism, and MPS analysis via the SUnSET method. RESULTS: Compared to CON rats, PA, WPC and PA + WPC resulted in a significant elevation in the phosphorylation of mTOR (Ser2481) and rps6 (Ser235/236) (p < 0.05) in the gastrocnemius though there were no differences between the supplemented groups. MPS levels in the gastrocnemius were significantly (p < 0.05) elevated in WPC versus CON rats, and tended to be elevated in PA versus CON rats (p = 0.08), though MPS was less in PA + WPC versus WPC rats (p < 0.05) in spite of robust increases in mTOR pathway activity markers in the former group. C2C12 myoblast data agreed with the in vivo data herein showing that PA increased MPS levels 51% (p < 0.001) phosphorylated p70s6k (Thr389) levels 67% (p < 0.001). CONCLUSIONS: Our results are the first in vivo evidence to demonstrate that PA tends to increases MPS 3 h post-feeding, though PA may delay WPC-mediated MPS kinetics within a 3 h post-feeding window.

Effects of protein type and composition on postprandial markers of skeletal muscle anabolism, adipose tissue lipolysis, and hypothalamic gene expression.

BACKGROUND: We examined the acute effects of different dietary protein sources (0.19 g, dissolved in 1 ml of water) on skeletal muscle, adipose tissue and hypothalamic satiety-related markers in fasted, male Wistar rats (~250 g). METHODS: Oral gavage treatments included: a) whey protein concentrate (WPC, n = 15); b) 70:30 hydrolyzed whey-to-hydrolyzed egg albumin (70 W/30E, n = 15); c) 50 W/50E (n = 15); d) 30 W/70E (n = 15); and e) 1 ml of water with no protein as a fasting control (CTL, n = 14). RESULTS: Skeletal muscle analyses revealed that compared to CTL: a) phosphorylated (p) markers of mTOR signaling [p-mTOR (Ser2481) and p-rps6 (Ser235/236)] were elevated 2-4-fold in all protein groups 90 min post-treatment (p < 0.05); b) WPC and 70 W/30E increased muscle protein synthesis (MPS) 104% and 74% 180 min post-treatment, respectively (p < 0.05); and c) 70 W/30E increased p-AMPKα (Thr172) 90 and 180-min post-treatment as well as PGC-1α mRNA 90 min post-treatment. Subcutaneous (SQ) and omental fat (OMAT) analyses revealed: a) 70 W/30 W increased SQ fat phosphorylated hormone-sensitive lipase [p-HSL (Ser563)] 3.1-fold versus CTL and a 1.9-4.4-fold change versus all other test proteins 180 min post-treatment (p < 0.05); and b) WPC, 70 W/30E and 50 W/50E increased OMAT p-HSL 3.8-6.5-fold 180 min post-treatment versus CTL (p < 0.05). 70 W/30E and 30 W/70E increased hypothalamic POMC mRNA 90 min post-treatment versus CTL rats suggesting a satiety-related response may have occurred in the former groups. However, there was a compensatory increase in orexigenic AGRP mRNA in the 70 W/30E group 90 min post-treatment versus CTL rats, and there was a compensatory increase in orexigenic NPY mRNA in the 30 W/70E group 90 min post-treatment versus CTL rats. CONCLUSIONS: Higher amounts of whey versus egg protein stimulate the greatest post-treatment anabolic skeletal muscle response, though test proteins with higher amounts of WPH more favorably affected post-treatment markers related to adipose tissue lipolysis.

Oral adenosine-5'-triphosphate (ATP) administration increases blood flow following exercise in animals and humans.

INTRODUCTION: Extracellular adenosine triphosphate (ATP) stimulates vasodilation by binding to endothelial ATP-selective P2Y2 receptors; a phenomenon, which is posited to be accelerated during exercise. Herein, we used a rat model to examine how different dosages of acute oral ATP administration affected the femoral blood flow response prior to, during, and after an exercise bout. In addition, we performed a single dose chronic administration pilot study in resistance trained athletes. ANIMAL STUDY: Male Wistar rats were gavage-fed the body surface area, species adjusted human equivalent dose (HED) of either 100 mg (n=4), 400 mg (n=4), 1,000 mg (n=5) or 1,600 mg (n=5) of oral ATP as a disodium salt (Peak ATP®, TSI, Missoula, MT). Rats that were not gavage-fed were used as controls (CTL, n=5). Blood flow was monitored continuously: a) 60 min prior to, b) during and c) 90 min following an electrically-evoked leg-kicking exercise. Human Study: In a pilot study, 12 college-aged resistance-trained subjects were given 400 mg of ATP (Peak ATP®, TSI, Missoula, MT) daily for 12 weeks, and prior to an acute arm exercise bout at weeks 1, 4, 8, and 12. Ultrasonography-determined volumetric blood flow and vessel dilation in the brachial artery was measured at rest, at rest 30 minutes after supplementation, and then at 0, 3, and 6 minutes after the exercise. ANIMAL STUDY: Rats fed 1,000 mg HED demonstrated significantly greater recovery blood flow (p < 0.01) and total blood flow AUC values (p < 0.05) compared to CTL rats. Specifically, blood flow was elevated in rats fed 1,000 mg HED versus CTL rats at 20 to 90 min post exercise when examining 10-min blood flow intervals (p < 0.05). When examining within-group differences relative to baseline values, rats fed the 1,000 mg and 1,600 mg HED exhibited the most robust increases in blood flow during exercise and into the recovery period. Human study: At weeks 1, 8, and 12, ATP supplementation significantly increased blood flow, along with significant elevations in brachial dilation. CONCLUSIONS: Oral ATP administration can increase post-exercise blood flow, and may be particularly effective during exercise recovery.

Herbal adaptogens combined with protein fractions from bovine colostrum and hen egg yolk reduce liver TNF-α expression and protein carbonylation in Western diet feeding in rats.

BACKGROUND: We examined if a purported anti-inflammatory supplement (AF) abrogated Western-diet (WD)-induced liver pathology in rats. AF contained: 1) protein concentrates from bovine colostrum and avian egg yolk; 2) herbal adaptogens and antioxidants; and 3) acetyl-L-carnitine. METHODS: Nine month-old male Brown Norway rats were allowed ad libitum access to WD for 41-43 days and randomly assigned to WD + AF feeding twice daily for the last 31-33 days (n = 8), or WD and water-placebo feeding twice daily for the last 31-33 days (n = 8). Rats fed a low-fat/low-sucrose diet (CTL, n = 6) for 41-43 days and administered a water-placebo twice daily for the last 31-33 days were also studied. Twenty-four hours following the last gavage-feed, liver samples were analyzed for: a) select mRNAs (via RT-PCR) as well as genome-wide mRNA expression patterns (via RNA-seq); b) lipid deposition; and, c) protein carbonyl and total antioxidant capacity (TAC). Serum was also examined for TAC, 8-isoprostane and clinical chemistry markers. RESULTS: WD + AF rats experienced a reduction in liver Tnf-α mRNA (-2.8-fold, p < 0.01). Serum and liver TAC was lower in WD + AF versus WD and CTL rats (p < 0.05), likely due to exogenous antioxidant ingredients provided through AF as evidenced by a tendency for mitochondrial SOD2 mRNA to increase in WD + AF versus CTL rats (p = 0.07). Liver fat deposition nor liver protein carbonyl content differed between WD + AF versus WD rats, although liver protein carbonyls tended to be lower in WD + AF versus CTL rats (p = 0.08). RNA-seq revealed that 19 liver mRNAs differed between WD + AF versus WD when both groups were compared with CTL rats (+/- 1.5-fold, p < 0.01). Bioinformatics suggest that AF prevented WD-induced alterations in select genes related to the transport and metabolism of carbohydrates in favor of select genes related to lipid transport and metabolism. Finally, serum clinical safety markers and liver pathology (via lesion counting) suggests that chronic consumption of AF was well tolerated. CONCLUSIONS: AF supplementation elicits select metabolic, anti-inflammatory, and anti-oxidant properties which was in spite of WD feeding and persisted up to 24 hours after receiving a final dose.

Comparing serum responses to acute feedings of an extensively hydrolyzed whey protein concentrate versus a native whey protein concentrate in rats: a metabolomics approach.

We examined how gavage feeding extensively hydrolyzed whey protein (WPH) versus a native whey protein concentrate (WPC) transiently affected serum biochemical profiles in rodents. Male Wistar rats (250-300 g) were 8 h fasted and subsequently fed isonitrogenous amounts of WPH or WPC, or remained unfed (control). Animals were sacrificed 15 min, 30 min, and 60 min post-gavage for serum extraction, and serum was analyzed using untargeted global metabolic profiling via gas chromatography/mass spectrometry (MS) and liquid chromatography/MS/MS platforms. We detected 333 serum metabolites amongst the experimental and control groups. Both WPH and WPC generally increased amino acids (1.2-2.8-fold), branched-chain amino acids (1.2-1.7-fold), and serum di- and oligo-peptides (1.1-2.7-fold) over the 60 min time course compared with control (q < 0.05). However, WPH increased lysine (false discovery rate using a q-value <0.05) and tended to increase isoleucine and valine 15 min post-feeding (q < 0.10) as well as aspartylleucine 30 min post-feeding compared with WPC (q < 0.05). While both protein sources led to a dramatic increase in free fatty acids compared with control (up to 6-fold increases, q < 0.05), WPH also uniquely resulted in a 30 min post-feeding elevation in free fatty acids compared with WPC (q < 0.05), an effect which may be due to the robust 30 min postprandial increase in epinephrine in the WPH cohort. These data provide a unique postprandial time-course perspective on how WPH versus WPC feedings affect circulating biochemicals and will guide future research comparing these 2 protein sources.

Effects of oral adenosine-5'-triphosphate supplementation on athletic performance, skeletal muscle hypertrophy and recovery in resistance-trained men.

BACKGROUND: Currently, there is a lack of studies examining the effects of adenosine-5'-triphosphate (ATP) supplementation utilizing a long-term, periodized resistance-training program (RT) in resistance-trained populations. Therefore, we investigated the effects of 12 weeks of 400 mg per day of oral ATP on muscular adaptations in trained individuals. We also sought to determine the effects of ATP on muscle protein breakdown, cortisol, and performance during an overreaching cycle. METHODS: The study was a 3-phase randomized, double-blind, and placebo- and diet-controlled intervention. Phase 1 was a periodized resistance-training program. Phase 2 consisted of a two week overreaching cycle in which volume and frequency were increased followed by a 2-week taper (Phase 3). Muscle mass, strength, and power were examined at weeks 0, 4, 8, and 12 to assess the chronic effects of ATP; assessment performance variables also occurred at the end of weeks 9 and 10, corresponding to the mid and endpoints of the overreaching cycle. RESULTS: There were time (p<0.001), and group x time effects for increased total body strength (+55.3 ± 6.0 kg ATP vs. + 22.4 ± 7.1 kg placebo, p<0.001); increased vertical jump power (+ 796 ± 75 ATP vs. 614 ± 52 watts placebo, p<0.001); and greater ultrasound determined muscle thickness (+4.9 ± 1.0 ATP vs. (2.5 ± 0.6 mm placebo, p<0.02) with ATP supplementation. During the overreaching cycle, there were group x time effects for strength and power, which decreased to a greater extent in the placebo group. Protein breakdown was also lower in the ATP group. CONCLUSIONS: Our results suggest oral ATP supplementation may enhance muscular adaptations following 12-weeks of resistance training, and prevent decrements in performance following overreaching. No statistically or clinically significant changes in blood chemistry or hematology were observed. TRIAL REGISTRATION: ClinicalTrials.gov NCT01508338.