Petey W. Mumford

A Ketogenic Diet in Rodents Elicits Improved Mitochondrial Adaptations in Response to Resistance Exercise Training Compared to an Isocaloric Western Diet

Purpose: Ketogenic diets (KD) can facilitate weight loss, but their effects on skeletal muscle remain equivocal. In this experiment we investigated the effects of two diets on skeletal muscle mitochondrial coupling, mitochondrial complex activity, markers of oxidative stress, and gene expression in sedentary and resistance exercised rats. Methods: Male Sprague-Dawley rats (9–10 weeks of age, 300–325 g) were fed isocaloric amounts of either a KD (17 g/day, 5.2 kcal/g, 20.2% protein, 10.3% CHO, 69.5% fat, n = 16) or a Western diet (WD) (20 g/day, 4.5 kcal/g, 15.2% protein, 42.7% CHO, 42.0% fat, n = 16) for 6 weeks. During these 6 weeks animals were either sedentary (SED, n = 8 per diet group) or voluntarily exercised using resistance-loaded running wheels (EXE, n = 8 per diet group). Gastrocnemius was excised and used for mitochondrial isolation and biochemical analyses. Results: In the presence of a complex II substrate, the respiratory control ratio (RCR) of isolated gastrocnemius mitochondria was higher (p < 0.05) in animals fed the KD compared to animals fed the WD. Complex I and IV enzyme activity was higher (p < 0.05) in EXE animals regardless of diet. SOD2 protein levels and GLUT4 and PGC1α mRNA expression were higher (p < 0.05) in EXE animals regardless of diet. Conclusion: Our data indicate that skeletal muscle mitochondrial coupling of complex II substrates is more efficient in chronically resistance trained rodents fed a KD. These findings may provide merit for further investigation, perhaps on humans.

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.

Effects of testosterone treatment on markers of skeletal muscle ribosome biogenesis.

The effects of testosterone (TEST) treatment on markers of skeletal muscle ribosome biogenesis in vitro and in vivo were examined. C2C12 myotubes were treated with 100 nm TEST for short‐term (24‐h) and longer‐term (96‐h) treatments. Moreover, male 10‐month‐old Fischer 344 rats were housed for 4 weeks, and the following groups were included in this study: (i) Sham‐operated (Sham) rats, (ii) orchiectomised rats (ORX) and (iii) ORX+TEST‐treated rats (7.0 mg week−1). For in vitro data, TEST treatment increased c‐Myc mRNA expression by 38% (P = 0.004) after 96 h, but did not affect total RNA, 47S pre‐rRNA, Raptor mRNA, Nop56 mRNA, Bop1 mRNA, Ncl mRNA at 24 h or 96 h following the treatment. For in vivo data, ORX decreased levator ani/bulbocavernosus (LABC) myofibril protein versus Sham (P = 0.006), whereas ORX+TEST (P = 0.015) rescued this atrophic effect. ORX also decreased muscle ribosome content (total RNA) compared to Sham (P = 0.046), whereas ORX+TEST tended to rescue this effect (P = 0.057). However, other markers of ribosome biogenesis including c‐Myc mRNA, Nop56 mRNA, Bop1 mRNA, Ncl mRNA decreased with ORX independently of TEST treatments (P < 0.05). Finally, lower phospho‐(Ser235/236)‐to‐total rps6 protein and lower rpl5 protein levels existed in ORX+TEST rats versus other treatments, suggesting that chronic TEST treatment may lower translational capacity.

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-1β 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.

Testosterone inhibits expression of lipogenic genes in visceral fat by an estrogen-dependent mechanism

The influence of the aromatase enzyme on the chronic fat-sparing effects of testosterone requires further elucidation. Our purpose was to determine whether chronic anastrozole (AN, an aromatase inhibitor) treatment alters testosterone-mediated lipolytic/lipogenic gene expression in visceral fat. Ten-month-old Fischer 344 rats (n = 6/group) were subjected to sham surgery (SHAM), orchiectomy (ORX), ORX + treatment with testosterone enanthate (TEST, 7.0 mg/wk), or ORX + TEST + AN (0.5 mg/day), with drug treatment beginning 14 days postsurgery. At day 42, ORX animals exhibited nearly undetectable serum testosterone and 29% higher retroperitoneal fat mass than SHAM animals (P < 0.001). TEST produced a ∼380-415% higher serum testosterone than SHAM (P < 0.001) and completely prevented ORX-induced visceral fat gain (P < 0.001). Retroperitoneal fat was 21% and 16% lower in ORX + TEST than SHAM (P < 0.001) and ORX + TEST + AN (P = 0.007) animals, while serum estradiol (E2) was 62% (P = 0.024) and 87% (P = 0.010) higher, respectively. ORX stimulated lipogenic-related gene expression in visceral fat, demonstrated by ∼84-154% higher sterol regulatory element-binding protein-1 (SREBP-1, P = 0.023), fatty acid synthase (P = 0.01), and LPL (P < 0.001) mRNA than SHAM animals, effects that were completely prevented in ORX + TEST animals (P < 0.01 vs. ORX for all). Fatty acid synthase (P = 0.061, trend) and LPL (P = 0.043) mRNA levels were lower in ORX + TEST + AN than ORX animals and not different from SHAM animals but remained higher than in ORX + TEST animals (P < 0.05). In contrast, the ORX-induced elevation in SREBP-1 mRNA was not prevented by TEST + AN, with SREBP-1 expression remaining ∼117-171% higher than in SHAM and ORX + TEST animals (P < 0.01). Across groups, visceral fat mass and lipogenic-related gene expression were negatively associated with serum testosterone, but not E2 Aromatase inhibition constrains testosterone-induced visceral fat loss and the downregulation of key lipogenic genes at the mRNA level, indicating that E2 influences the visceral fat-sparing effects of testosterone.

The 1-Week and 8-Month Effects of a Ketogenic Diet or Ketone Salt Supplementation on Multi-Organ Markers of Oxidative Stress and Mitochondrial Function in Rats

We determined the short- and long-term effects of a ketogenic diet (KD) or ketone salt (KS) supplementation on multi-organ oxidative stress and mitochondrial markers. For short-term feedings, 4 month-old male rats were provided isocaloric amounts of KD (n = 10), standard chow (SC) (n = 10) or SC + KS (~1.2 g/day, n = 10). For long-term feedings, 4 month-old male rats were provided KD (n = 8), SC (n = 7) or SC + KS (n = 7) for 8 months and rotarod tested every 2 months. Blood, brain (whole cortex), liver and gastrocnemius muscle were harvested from all rats for biochemical analyses. Additionally, mitochondria from the brain, muscle and liver tissue of long-term-fed rats were analyzed for mitochondrial quantity (maximal citrate synthase activity), quality (state 3 and 4 respiration) and reactive oxygen species (ROS) assays. Liver antioxidant capacity trended higher in short-term KD- and SC + KS-fed versus SC-fed rats, and short-term KD-fed rats exhibited significantly greater serum ketones compared to SC + KS-fed rats indicating that the diet (not KS supplementation) induced ketonemia. In long term-fed rats: (a) serum ketones were significantly greater in KD- versus SC- and SC + KS-fed rats; (b) liver antioxidant capacity and glutathione peroxidase protein was significantly greater in KD- versus SC-fed rats, respectively, while liver protein carbonyls were lowest in KD-fed rats; and (c) gastrocnemius mitochondrial ROS production was significantly greater in KD-fed rats versus other groups, and this paralleled lower mitochondrial glutathione levels. Additionally, the gastrocnemius pyruvate-malate mitochondrial respiratory control ratio was significantly impaired in long-term KD-fed rats, and gastrocnemius mitochondrial quantity was lowest in these animals. Rotarod performance was greatest in KD-fed rats versus all other groups at 2, 4 and 8 months, although there was a significant age-related decline in performance existed in KD-fed rats which was not evident in the other two groups. In conclusion, short- and long-term KD improves select markers of liver oxidative stress compared to SC feeding, although long-term KD feeding may negatively affect skeletal muscle mitochondrial physiology.

Molecular, neuromuscular, and recovery responses to light versus heavy resistance exercise in young men

Recent evidence suggests that resistance training with light or heavy loads to failure results in similar adaptations. Herein, we compared how both training modalities affect the molecular, neuromuscular, and recovery responses following exercise. Resistance‐trained males (mean ± SE: 22 ± 2 years, 84.8 ± 9.0 kg, 1.79 ± 0.06 m; n = 15) performed a crossover design of four sets of leg extensor exercise at 30% (light RE) or 80% (heavy RE) one repetition maximum (1RM) to repetition failure, and heavy RE or light RE 1 week later. Surface electromyography (EMG) was monitored during exercise, and vastus lateralis muscle biopsies were collected at baseline (PRE), 15 min (15mPOST), and 90 min following RE (90mPOST) for examination of molecular targets and fiber typing. Isokinetic dynamometry was also performed before (PRE), immediately after (POST), and 48 h after (48hPOST) exercise. Dependent variables were analyzed using repeated measures ANOVAs and significance was set at P ≤ 0.05. Repetitions completed were greater during light RE (P < 0.01), while EMG amplitude was greater during heavy RE (P ≤ 0.01). POST isokinetic torque was reduced following light versus heavy RE (P < 0.05). Postexercise expression of mRNAs and phosphoproteins associated with muscle hypertrophy were similar between load conditions. Additionally, p70s6k (Thr389) phosphorylation and fast‐twitch fiber proportion exhibited a strong relationship after both light and heavy RE (r > 0.5). While similar mRNA and phosphoprotein responses to both modalities occurred, we posit that heavy RE is a more time‐efficient training method given the differences in total repetitions completed, lower EMG amplitude during light RE, and impaired recovery response after light RE.

Effect of caffeine on golf performance and fatigue during a competitive tournament.

PURPOSE This study aimed to determine the effect of a caffeine-containing supplement on golf-specific performance and fatigue during a 36-hole competitive golf tournament. METHODS Twelve male golfers (34.8 ± 13.9 yr, 175.9 ± 9.3 cm, 81.23 ± 13.14 kg) with a United States Golf Association handicap of 3-10 participated in a double-blind, placebo-controlled, crossover design in which they played an 18-hole round of golf on two consecutive days (36-hole tournament) and were randomly assigned to consume a caffeine-containing supplement (CAF) or placebo (PLA). CAF/PLA was consumed before and after nine holes during each 18-hole round. Total score, drive distance, fairways and greens in regulation, first putt distance, HR, breathing rate, peak trunk acceleration, and trunk posture while putting were recorded. Self-perceived ratings of energy, fatigue, alertness and concentration were also recorded. RESULTS Total score (76.9 ± 8.1 vs 79.4 ± 9.1, P = 0.039), greens in regulation (8.6 ± 3.3 vs 6.9 ± 4.6, P = 0.035), and drive distance (239.9 ± 33.8 vs 233.2 ± 32.4, P = 0.047) were statistically better during the CAF condition compared with those during PLA. Statistically significant main effects for condition (P < 0.05) and time (P < 0.001) occurred for perceived feelings of energy and fatigue. Compared with PLA, CAF reported more energy (P = 0.025) and less fatigue (P = 0.05) over the competitive round of golf. There were no substantial differences in HR or breathing rates, peak trunk acceleration, or putting posture between conditions or over the round (P > 0.05). CONCLUSIONS A moderate dose (1.9 ± 0.3 mg · kg(-1)) of caffeine consumed before and during a round of golf improves golf-specific measures of performance and reduces fatigue in skilled golfers.

Whey protein-derived exosomes increase protein synthesis and hypertrophy in C2­C12 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.

Aging in rats differentially affects markers of transcriptional and translational capacity in soleus and plantaris muscle

Alterations in transcriptional and translational mechanisms occur during skeletal muscle aging and such changes may contribute to age-related atrophy. Herein, we examined markers related to global transcriptional output (i.e., myonuclear number, total mRNA and RNA pol II levels), translational efficiency [i.e., eukaryotic initiation and elongation factor levels and muscle protein synthesis (MPS) levels] and translational capacity (ribosome density) in the slow-twitch soleus and fast-twitch plantaris muscles of male Fischer 344 rats aged 3, 6, 12, 18, and 24 months (n = 9–10 per group). We also examined alterations in markers of proteolysis and oxidative stress in these muscles (i.e., 20S proteasome activity, poly-ubiquinated protein levels and 4-HNE levels). Notable plantaris muscle observations included: (a) fiber cross sectional area (CSA) was 59% (p < 0.05) and 48% (p < 0.05) greater in 12 month vs. 3 month and 24 month rats, respectively, suggesting a peak lifetime value near 12 months and age-related atrophy by 24 months, (b) MPS levels were greatest in 18 month rats (p < 0.05) despite the onset of atrophy, (c) while regulators of ribosome biogenesis [c-Myc and upstream binding factor (UBF) protein levels] generally increased with age, ribosome density linearly decreased from 3 months of age and RNA polymerase (Pol) I protein levels were lowest in 24 month rats, and d) 20S proteasome activity was robustly up-regulated in 6 and 24 month rats (p < 0.05). Notable soleus muscle observations included: (a) fiber CSA was greatest in 6 month rats and was maintained in older age groups, and (b) 20S proteasome activity was modestly but significantly greater in 24 month vs. 3/12/18 month rats (p < 0.05), and (c) total mRNA levels (suggestive of transcriptional output) trended downward in older rats despite non-significant between-group differences in myonuclear number and/or RNA Pol II protein levels. Collectively, these findings suggest that plantaris, not soleus, atrophy occurs following 12 months of age in male Fisher rats and this may be due to translational deficits (i.e., changes in MPS and ribosome density) and/or increases in proteolysis rather than increased oxidative stress and/or alterations in global transcriptional mechanisms.