Long-term voluntary wheel running effects on markers of long interspersed nuclear element-1 in skeletal muscle, liver, and brain tissue of female rats.

We sought to determine the effects of long-term voluntary wheel running on markers of long interspersed nuclear element-1 (L1) in skeletal muscle, liver, and the hippocampus of female rats. In addition, markers of the cGAS-STING DNA-sensing pathway that results in inflammation were interrogated. Female Lewis rats (n = 34) were separated into one of three groups including a 6-mo-old group to serve as a young comparator group (CTL, n = 10), a group that had access to a running wheel for voluntary wheel running (EX, n = 12), and an age-matched group that did not (SED, n = 12). Both SED and EX groups were carried out from 6 mo to 15 mo of age. There were no significant differences in L1 mRNA expression for any of the tissues between groups. Methylation of the L1 promoter in the soleus and hippocampus was significantly higher in SED and EX than in CTL group (P < 0.05). ORF1p expression was higher in older SED and EX rats than in CTL rats for every tissue (P < 0.05). There were no differences between groups for L1 mRNA or cGAS-STING pathway markers. Our results suggest there is an increased ORF1 protein expression across tissues with aging that is not mitigated by voluntary wheel running. In addition, although previous data imply that L1 methylation changes may play a role in acute exercise for L1 RNA expression, this does not seem to occur during extended periods of voluntary wheel running.

An intron variant of the GLI family zinc finger 3 (GLI3) gene differentiates resistance training-induced muscle fiber hypertrophy in younger men.

We examined the association between genotype and resistance training-induced changes (12 wk) in dual x-ray energy absorptiometry (DXA)-derived lean soft tissue mass (LSTM) as well as muscle fiber cross-sectional area (fCSA; vastus lateralis; n = 109; age = 22 ± 2 y, BMI = 24.7 ± 3.1 kg/m2 ). Over 315 000 genetic polymorphisms were interrogated from muscle using DNA microarrays. First, a targeted investigation was performed where single nucleotide polymorphisms (SNP) identified from a systematic literature review were related to changes in LSTM and fCSA. Next, genome-wide association (GWA) studies were performed to reveal associations between novel SNP targets with pre- to post-training change scores in mean fCSA and LSTM. Our targeted investigation revealed no genotype-by-time interactions for 12 common polymorphisms regarding the change in mean fCSA or change in LSTM. Our first GWA study indicated no SNP were associated with the change in LSTM. However, the second GWA study indicated two SNP exceeded the significance level with the change in mean fCSA (P = 6.9 × 10-7 for rs4675569, 1.7 × 10-6 for rs10263647). While the former target is not annotated (chr2:205936846 (GRCh38.p12)), the latter target (chr7:41971865 (GRCh38.p12)) is an intron variant of the GLI Family Zinc Finger 3 (GLI3) gene. Follow-up analyses indicated fCSA increases were greater in the T/C and C/C GLI3 genotypes than the T/T GLI3 genotype (P < .05). Data from the Auburn cohort also revealed participants with the T/C and C/C genotypes exhibited increases in satellite cell number with training (P < .05), whereas T/T participants did not. Additionally, those with the T/C and C/C genotypes achieved myonuclear addition in response to training (P < .05), whereas the T/T participants did not. In summary, this is the first GWA study to examine how polymorphisms associate with the change in hypertrophy measures following resistance training. Future studies are needed to determine if the GLI3 variant differentiates hypertrophic responses to resistance training given the potential link between this gene and satellite cell physiology.

Synergist ablation-induced hypertrophy occurs more rapidly in the plantaris than soleus muscle in rats due to different molecular mechanisms.

We examined molecular mechanisms that were altered during rapid soleus (type I fiber-dominant) and plantaris (type II fiber-dominant) hypertrophy in rats. Twelve Wistar rats (3.5 mo old; 6 female, 6 male) were subjected to surgical right-leg soleus and plantaris dual overload [synergist ablation (SA)], and sham surgeries were performed on left legs (CTL). At 14 days after surgery, the muscles were dissected. Plantaris mass was 27% greater in the SA than CTL leg (P < 0.001), soleus mass was 13% greater in the SA than CTL leg (P < 0.001), and plantaris mass was higher than soleus mass in the SA leg (P = 0.001). Plantaris total RNA concentrations and estimated total RNA levels (suggestive of ribosome density) were 19% and 47% greater in the SA than CTL leg (P < 0.05), protein synthesis levels were 64% greater in the SA than CTL leg (P = 0.038), and satellite cell number per fiber was 60% greater in the SA than CTL leg (P = 0.003); no differences in these metrics were observed between soleus SA and CTL legs. Plantaris, as well as soleus, 20S proteasome activity was lower in the SA than CTL leg (P < 0.05), although the degree of downregulation was greater in the plantaris than soleus muscle (-63% vs. -20%, P = 0.001). These data suggest that early-phase plantaris hypertrophy occurs more rapidly than soleus hypertrophy, which coincided with greater increases in ribosome biogenesis, protein synthesis, and satellite cell density, as well as greater decrements in 20S proteasome activity, in the plantaris muscle.

Skeletal muscle LINE-1 retrotransposon activity is upregulated in older versus younger rats.

Long interspersed element-1 (LINE-1) is a retrotransposon capable of replicating and inserting LINE-1 copies into the genome. Others have reported skeletal muscle LINE-1 markers are higher in older versus younger mice, but data are lacking in other species. Herein, gastrocnemius muscle from male Fischer 344 rats that were 3, 12, and 24 mo old (n = 9 per group) were analyzed for LINE-1 mRNA, DNA, promoter methylation and DNA accessibility. qPCR primers were designed for active (L1.3) and inactive (L1.Tot) LINE-1 elements as well as part of the ORF1 sequence. L1.3, L1.Tot, and ORF1 mRNAs were higher (P < 0.05) in 12/24 versus 3-mo-old rats. L1.3 DNA was higher in the 24-mo-old rats versus other groups, and ORF1 DNA was greater in 12/24 versus 3-mo-old rats. ORF1 protein was higher in 12/24 versus 3-mo-old rats. RNA-sequencing indicated mRNAs related to DNA methylation (Tet1) and histone acetylation (Hdac2) were lower in 24 versus 3-mo-old rats. L1.3 DNA accessibility was higher in 24-mo-old versus 3-mo-old rats. No age-related differences in nuclear histone deacetylase (HDAC) activity existed, although nuclear DNA methyltransferase (DNMT) activity was lower in 12/24 versus 3-mo-old rats (P < 0.05). In summary, markers of skeletal muscle LINE-1 activity increase across the age spectrum of rats, and this may be related to deficits in DNMT activity and/or increased LINE-1 DNA accessibility.

Concomitant external pneumatic compression treatment with consecutive days of high intensity interval training reduces markers of proteolysis.

PURPOSE: To compare the effects of external pneumatic compression (EPC) and sham when used concurrently with high intensity interval training (HIIT) on performance-related outcomes and recovery-related molecular measures. METHODS: Eighteen recreationally endurance-trained male participants (age: 21.6 ± 2.4 years, BMI: 25.7 ± 0.5 kg/m2, VO2peak: 51.3 ± 0.9 mL/kg/min) were randomized to balanced sham and EPC treatment groups. Three consecutive days of HIIT followed by EPC/sham treatment (Days 2-4) and 3 consecutive days of recovery (Days 5-7) with EPC/sham only on Days 5-6 were employed. Venipuncture, flexibility and pressure-to-pain threshold (PPT) measurements were made throughout. Vastus lateralis muscle was biopsied at PRE (i.e., Day 1), 1-h post-EPC/sham treatment on Day 2 (POST1), and 24-h post-EPC/sham treatment on Day 7 (POST2). 6-km run time trial performance was tested at PRE and POST2. RESULTS: No group × time interaction was observed for flexibility, PPT, or serum measures of creatine kinase (CK), hsCRP, and 8-isoprostane. However, there was a main effect of time for serum CK (p = 0.005). Change from PRE in 6-km run times at POST2 were not significantly different between groups. Significant between-groups differences existed for change from PRE in atrogin-1 mRNA (p = 0.018) at the POST1 time point (EPC: - 19.7 ± 8.1%, sham: + 7.7 ± 5.9%) and atrogin-1 protein concentration (p = 0.013) at the POST2 time point (EPC: - 31.8 ± 7.5%, sham: + 96.0 ± 34.7%). In addition, change from PRE in poly-Ub proteins was significantly different between groups at both the POST1 (EPC: - 26.0 ± 10.3%, sham: + 34.8 ± 28.5%; p = 0.046) and POST2 (EPC: - 33.7 ± 17.2%, sham: + 21.4 ± 14.9%; p = 0.037) time points. CONCLUSIONS: EPC when used concurrently with HIIT and in subsequent recovery days reduces skeletal muscle markers of proteolysis.

Differential vascular reactivity responses acutely following ingestion of a nitrate rich red spinach extract.

INTRODUCTION: Inorganic nitrate ingestion has been posited to affect arterial blood pressure and vascular function. PURPOSE: We sought to determine the acute effect of a red spinach extract (RSE) high in inorganic nitrate on vascular reactivity 1-h after ingestion in peripheral conduit and resistance arteries. METHODS: Fifteen (n = 15; males 8, females 7) apparently healthy subjects (aged 23.1 ± 3.3 years; BMI 27.2 ± 3.7 kg/m2) participated in this crossover design, double-blinded study. Subjects reported to the lab ≥2-h post-prandial and consumed RSE (1000 mg dose; ~90 mg nitrate) or placebo (PBO). Venipuncture was performed on three occasions: baseline, 30-min post-ingestion and between 65 to 75-min post-ingestion. Baseline vascular measurements [i.e., calf venous occlusion plethysmography, brachial artery flow-mediated dilation (FMD)], 30-min of continuous blood pressure (BP) and heart rate (HR) analysis, and follow-up vascular measurements beginning at 40-min post-ingestion were also performed. RESULTS: Humoral nitrate following RSE ingestion was significantly higher at 30- (+54 %; P = 0.039) and 65 to 75-min post-ingestion compared to baseline (+255 %, P < 0.001) and PBO at the same time points (P < 0.05). No significant changes in BP or HR occurred in either condition. Peak reactive hyperemia (RH) calf blood flow increased significantly (+13.7 %; P = 0.016) following RSE ingestion, whereas it decreased (-14.0 %; P = 0.008) following PBO ingestion. No significant differential FMD responses were detected (P > 0.05), though RH was decreased following the baseline measure in both conditions. CONCLUSIONS: RSE significantly increased plasma nitrate 30-min post-ingestion, but acute microvascular (i.e., resistance vasculature) reactivity increases were isolated to the lower limb and no appreciable change in brachial artery FMD was observed.

Skeletal Muscle Nuclei in Mice are not Post-mitotic.

The skeletal muscle research field generally accepts that nuclei in skeletal muscle fibers (ie, myonuclei) are post-mitotic and unable to proliferate. Because our deuterium oxide (D2O) labeling studies showed DNA synthesis in skeletal muscle tissue, we hypothesized that resident myonuclei can replicate in vivo. To test this hypothesis, we used a mouse model that temporally labeled myonuclei with GFP followed by D2O labeling during normal cage activity, functional overload, and with satellite cell ablation. During normal cage activity, we observed deuterium enrichment into myonuclear DNA in 7 out of 7 plantaris (PLA), 6 out of 6 tibialis anterior (TA), 5 out of 7 gastrocnemius (GAST), and 7 out of 7 quadriceps (QUAD). The average fractional synthesis rates (FSR) of DNA in myonuclei were: 0.0202 ± 0.0093 in PLA, 0.0239 ± 0.0040 in TA, 0.0076 ± 0. 0058 in GAST, and 0.0138 ± 0.0039 in QUAD, while there was no replication in myonuclei from EDL. These FSR values were largely reproduced in the overload and satellite cell ablation conditions, although there were higher synthesis rates in the overloaded PLA muscle. We further provided evidence that myonuclear replication is through endoreplication, which results in polyploidy. These novel findings contradict the dogma that skeletal muscle nuclei are post-mitotic and open potential avenues to harness the intrinsic replicative ability of myonuclei for muscle maintenance and growth.

Skeletal Muscle DNA Methylation and mRNA Responses to a Bout of Higher versus Lower Load Resistance Exercise in Previously Trained Men.

We sought to determine the skeletal muscle genome-wide DNA methylation and mRNA responses to one bout of lower load (LL) versus higher load (HL) resistance exercise. Trained college-aged males (n = 11, 23 ± 4 years old, 4 ± 3 years self-reported training) performed LL or HL bouts to failure separated by one week. The HL bout (i.e., 80 Fail) consisted of four sets of back squats and four sets of leg extensions to failure using 80% of participants estimated one-repetition maximum (i.e., est. 1-RM). The LL bout (i.e., 30 Fail) implemented the same paradigm with 30% of est. 1-RM. Vastus lateralis muscle biopsies were collected before, 3 h, and 6 h after each bout. Muscle DNA and RNA were batch-isolated and analyzed using the 850k Illumina MethylationEPIC array and Clariom S mRNA microarray, respectively. Performed repetitions were significantly greater during the 30 Fail versus 80 Fail (p < 0.001), although total training volume (sets × reps × load) was not significantly different between bouts (p = 0.571). Regardless of bout, more CpG site methylation changes were observed at 3 h versus 6 h post exercise (239,951 versus 12,419, respectively; p < 0.01), and nuclear global ten-eleven translocation (TET) activity, but not global DNA methyltransferase activity, increased 3 h and 6 h following exercise regardless of bout. The percentage of genes significantly altered at the mRNA level that demonstrated opposite DNA methylation patterns was greater 3 h versus 6 h following exercise (~75% versus ~15%, respectively). Moreover, high percentages of genes that were up- or downregulated 6 h following exercise also demonstrated significantly inversed DNA methylation patterns across one or more CpG sites 3 h following exercise (65% and 82%, respectively). While 30 Fail decreased DNA methylation across various promoter regions versus 80 Fail, transcriptome-wide mRNA and bioinformatics indicated that gene expression signatures were largely similar between bouts. Bioinformatics overlay of DNA methylation and mRNA expression data indicated that genes related to "Focal adhesion," "MAPK signaling," and "PI3K-Akt signaling" were significantly affected at the 3 h and 6 h time points, and again this was regardless of bout. In conclusion, extensive molecular profiling suggests that post-exercise alterations in the skeletal muscle DNA methylome and mRNA transcriptome elicited by LL and HL training bouts to failure are largely similar, and this could be related to equal volumes performed between bouts.

Effects of Peanut Protein Supplementation on Resistance Training Adaptations in Younger Adults.

Protein supplementation is a commonly employed strategy to enhance resistance training adaptations. However, little research to date has examined if peanut protein supplementation is effective in this regard. Thus, we sought to determine if peanut protein supplementation (PP; 75 total g/d of powder providing 30 g/d protein, >9.2 g/d essential amino acids, ~315 kcal/d) affected resistance training adaptations in college-aged adults. Forty-seven college-aged adults (n = 34 females, n = 13 males) with minimal prior training experience were randomly assigned to a PP group (n = 18 females, n = 5 males) or a non-supplement group (CTL; n = 16 females, n = 8 males) (ClinicalTrials.gov trial registration NCT04707963; registered 13 January 2021). Body composition and strength variables were obtained prior to the intervention (PRE). Participants then completed 10 weeks of full-body resistance training (twice weekly) and PP participants consumed their supplement daily. POST measures were obtained 72 h following the last training bout and were identical to PRE testing measures. Muscle biopsies were also obtained at PRE, 24 h following the first exercise bout, and at POST. The first two biopsy time points were used to determine myofibrillar protein synthesis (MyoPS) rates in response to a naïve training bout with or without PP, and the PRE and POST biopsies were used to determine muscle fiber adaptations in females only. Dependent variables were analyzed in males and females separately using two-way (supplement × time) repeated measures ANOVAs, unless otherwise stated. The 24-h integrated MyoPS response to the first naïve training bout was similar between PP and CTL participants (dependent samples t-test p = 0.759 for females, p = 0.912 for males). For males, the only significant supplement × time interactions were for DXA-derived fat mass (interaction p = 0.034) and knee extensor peak torque (interaction p = 0.010); these variables significantly increased in the CTL group (p < 0.05), but not the PP group. For females, no significant supplement × time interactions existed, although interactions for whole body lean tissue mass (p = 0.088) and vastus lateralis thickness (p = 0.099) approached significance and magnitude increases in these characteristics favored the PP versus CTL group. In summary, this is the second study to determine the effects of PP supplementation on resistance training adaptations. While PP supplementation did not significantly enhance training adaptations, the aforementioned trends in females, the limited n-size in males, and this being the second PP supplementation study warrant more research to determine if different PP dosing strategies are more effective than the current approach.

A novel tetracycline-responsive transgenic mouse strain for skeletal muscle-specific gene expression.

BACKGROUND: The tetracycline-responsive system (Tet-ON/OFF) has proven to be a valuable tool for manipulating gene expression in an inducible, temporal, and tissue-specific manner. The purpose of this study was to create and characterize a new transgenic mouse strain utilizing the human skeletal muscle α-actin (HSA) promoter to drive skeletal muscle-specific expression of the reverse tetracycline transactivator (rtTA) gene which we have designated as the HSA-rtTA mouse. METHODS: To confirm the HSA-rtTA mouse was capable of driving skeletal muscle-specific expression, we crossed the HSA-rtTA mouse with the tetracycline-responsive histone H2B-green fluorescent protein (H2B-GFP) transgenic mouse in order to label myonuclei. RESULTS: Reverse transcription-PCR confirmed skeletal muscle-specific expression of rtTA mRNA, while single-fiber analysis showed highly effective GFP labeling of myonuclei in both fast- and slow-twitch skeletal muscles. Pax7 immunohistochemistry of skeletal muscle cross-sections revealed no appreciable GFP expression in satellite cells. CONCLUSIONS: The HSA-rtTA transgenic mouse allows for robust, specific, and inducible gene expression across muscles of different fiber types. The HSA-rtTA mouse provides a powerful tool to manipulate gene expression in skeletal muscle.

Physiological Differences Between Low Versus High Skeletal Muscle Hypertrophic Responders to Resistance Exercise Training: Current Perspectives and Future Research Directions.

Numerous reports suggest there are low and high skeletal muscle hypertrophic responders following weeks to months of structured resistance exercise training (referred to as low and high responders herein). Specifically, divergent alterations in muscle fiber cross sectional area (fCSA), vastus lateralis thickness, and whole body lean tissue mass have been shown to occur in high versus low responders. Differential responses in ribosome biogenesis and subsequent protein synthetic rates during training seemingly explain some of this individual variation in humans, and mechanistic in vitro and rodent studies provide further evidence that ribosome biogenesis is critical for muscle hypertrophy. High responders may experience a greater increase in satellite cell proliferation during training versus low responders. This phenomenon could serve to maintain an adequate myonuclear domain size or assist in extracellular remodeling to support myofiber growth. High responders may also express a muscle microRNA profile during training that enhances insulin-like growth factor-1 (IGF-1) mRNA expression, although more studies are needed to better validate this mechanism. Higher intramuscular androgen receptor protein content has been reported in high versus low responders following training, and this mechanism may enhance the hypertrophic effects of testosterone during training. While high responders likely possess "good genetics," such evidence has been confined to single gene candidates which typically share marginal variance with hypertrophic outcomes following training (e.g., different myostatin and IGF-1 alleles). Limited evidence also suggests pre-training muscle fiber type composition and self-reported dietary habits (e.g., calorie and protein intake) do not differ between high versus low responders. Only a handful of studies have examined muscle biomarkers that are differentially expressed between low versus high responders. Thus, other molecular and physiological variables which could potentially affect the skeletal muscle hypertrophic response to resistance exercise training are also discussed including rDNA copy number, extracellular matrix and connective tissue properties, the inflammatory response to training, and mitochondrial as well as vascular characteristics.

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.

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.

The Three-Month Effects of a Ketogenic Diet on Body Composition, Blood Parameters, and Performance Metrics in CrossFit Trainees: A Pilot Study.

Adopting low carbohydrate, ketogenic diets remains a controversial issue for individuals who resistance train given that this form of dieting has been speculated to reduce skeletal muscle glycogen levels and stifle muscle anabolism. We sought to characterize the effects of a 12-week ketogenic diet (KD) on body composition, metabolic, and performance parameters in participants who trained recreationally at a local CrossFit facility. Twelve participants (nine males and three females, 31 ± 2 years of age, 80.3 ± 5.1 kg body mass, 22.9 ± 2.3% body fat, 1.37 back squat: body mass ratio) were divided into a control group (CTL; n = 5) and a KD group (n = 7). KD participants were given dietary guidelines to follow over 12 weeks while CTL participants were instructed to continue their normal diet throughout the study, and all participants continued their CrossFit training routine for 12 weeks. Pre, 2.5-week, and 12-week anaerobic performance tests were conducted, and pre- and 12-week tests were performed for body composition using dual X-ray absorptiometry (DXA) and ultrasound, resting energy expenditure (REE), blood-serum health markers, and aerobic capacity. Additionally, blood beta hydroxybutyrate (BHB) levels were measured weekly. Blood BHB levels were 2.8- to 9.5-fold higher in KD versus CTL throughout confirming a state of nutritional ketosis. DXA fat mass decreased by 12.4% in KD (p = 0.053). DXA total lean body mass changes were not different between groups, although DXA dual-leg lean mass decreased in the KD group by 1.4% (p = 0.068), and vastus lateralis thickness values decreased in the KD group by ~8% (p = 0.065). Changes in fasting glucose, HDL cholesterol, and triglycerides were similar between groups, although LDL cholesterol increased ~35% in KD (p = 0.048). Between-group changes in REE, one-repetition maximum (1-RM) back squat, 400 m run times, and VO2peak were similar between groups. While our n-sizes were limited, these preliminary data suggest that adopting a ketogenic diet causes marked reductions in whole-body adiposity while not impacting performance measures in recreationally-trained CrossFit trainees. Whether decrements in dual-leg muscle mass and vastus lateralis thickness in KD participants were due to fluid shifts remain unresolved, and increased LDL-C in these individuals warrants further investigation.

Effects of Graded Whey Supplementation During Extreme-Volume Resistance Training.

We examined hypertrophic outcomes of weekly graded whey protein dosing (GWP) vs. whey protein (WP) or maltodextrin (MALTO) dosed once daily during 6 weeks of high-volume resistance training (RT). College-aged resistance-trained males (training age = 5 ± 3 years; mean ± SD) performed 6 weeks of RT wherein frequency was 3 d/week and each session involved 2 upper- and 2 lower-body exercises (10 repetitions/set). Volume increased from 10 sets/exercise (week 1) to 32 sets/exercise (week 6), which is the highest volume investigated in this timeframe. Participants were assigned to WP (25 g/d; n = 10), MALTO (30 g/d; n = 10), or GWP (25-150 g/d from weeks 1-6; n = 11), and supplementation occurred throughout training. Dual-energy x-ray absorptiometry (DXA), vastus lateralis (VL), and biceps brachii ultrasounds for muscle thicknesses, and bioelectrical impedance spectroscopy (BIS) were performed prior to training (PRE) and after weeks 3 (MID) and 6 (POST). VL biopsies were also collected for immunohistochemical staining. The GWP group experienced the greatest PRE to POST reduction in DXA fat mass (FM) (-1.00 kg, p < 0.05), and a robust increase in DXA fat- and bone-free mass [termed lean body mass (LBM) throughout] (+2.93 kg, p < 0.05). However, the MALTO group also experienced a PRE to POST increase in DXA LBM (+2.35 kg, p < 0.05), and the GWP and MALTO groups experienced similar PRE to POST increases in type II muscle fiber cross-sectional area (~+300 µm2). When examining the effects of training on LBM increases (ΔLBM) in all participants combined, PRE to MID (+1.34 kg, p < 0.001) and MID to POST (+0.85 kg, p < 0.001) increases were observed. However, when adjusting ΔLBM for extracellular water (ECW) changes, intending to remove the confounder of edema, a significant increase was observed from PRE to MID (+1.18 kg, p < 0.001) but not MID to POST (+0.25 kg; p = 0.131). Based upon DXA data, GWP supplementation may be a viable strategy to improve body composition during high-volume RT. However, large LBM increases observed in the MALTO group preclude us from suggesting that GWP supplementation is clearly superior in facilitating skeletal muscle hypertrophy. With regard to the implemented RT program, ECW-corrected ΔLBM gains were largely dampened, but still positive, in resistance-trained participants when RT exceeded ~20 sets/exercise/wk.

Red Spinach Extract Increases Ventilatory Threshold during Graded Exercise Testing.

Background: We examined the acute effect of a red spinach extract (RSE) (1000 mg dose; ~90 mg nitrate (NO 3 - )) on performance markers during graded exercise testing (GXT). Methods: For this randomized, double-blind, placebo (PBO)-controlled, crossover study, 15 recreationally-active participants (aged 23.1 ± 3.3 years; BMI: 27.2 ± 3.7 kg/m²) reported >2 h post-prandial and performed GXT 65⁻75 min post-RSE or PBO ingestion. Blood samples were collected at baseline (BL), pre-GXT (65⁻75 min post-ingestion; PRE), and immediately post-GXT (POST). GXT commenced with continuous analysis of expired gases. Results: Plasma concentrations of NO 3 - increased PRE (+447 ± 294%; p < 0.001) and POST (+378 ± 179%; p < 0.001) GXT with RSE, but not with PBO (+3 ± 26%, -8 ± 24%, respectively; p > 0.05). No effect on circulating nitrite (NO 2 - ) was observed with RSE (+3.3 ± 7.5%, +7.7 ± 11.8% PRE and POST, respectively; p > 0.05) or PBO (-0.5 ± 7.9%, -0.2 ± 8.1% PRE and POST, respectively; p > 0.05). When compared to PBO, there was a moderate effect of RSE on plasma NO 2 - at PRE (g = 0.50 [-0.26, 1.24] and POST g = 0.71 [-0.05, 1.48]). During GXT, VO2 at the ventilatory threshold was significantly higher with RSE compared to PBO (+6.1 ± 7.3%; p < 0.05), though time-to-exhaustion (-4.0 ± 7.7%; p > 0.05) and maximal aerobic power (i.e., VO2 peak; -0.8 ± 5.6%; p > 0.05) were non-significantly lower with RSE. Conclusions: RSE as a nutritional supplement may elicit an ergogenic response by delaying the ventilatory threshold.

Does external pneumatic compression treatment between bouts of overreaching resistance training sessions exert differential effects on molecular signaling and performance-related variables compared to passive recovery? An exploratory study.

PURPOSE: We sought to compare the effects of external pneumatic compression (EPC) and sham when used concurrently with resistance training on performance-related outcomes and molecular measures related to recovery. METHODS: Twenty (N = 20) resistance-trained male participants (aged 21.6±2.4 years) were randomized to balanced sham or EPC intervention groups. The protocol consisted of 3 consecutive days of heavy, voluminous back squat exercise followed by EPC/sham treatment (Days2-4) and 3 consecutive days of recovery (Days5-7) with EPC/sham only on Days5-6. On Day1 (PRE), and Days3-7, venipuncture, flexibility and pressure-to-pain threshold (PPT) measures were performed. Vastsus lateralis muscle tissue was biopsied at PRE, 1-h post-EPC/sham treatment on Day2 (POST1) and 24-h post-EPC/sham treatment on Day7 (POST2). Isokinetic peak torque was assessed at PRE and POST2. RESULTS: Peak isokinetic strength did not change from PRE to POST2 in either group. The PPT was significantly lower on Days3-6 with sham, indicating greater muscle soreness, though this was largely abolished in the EPC group. A significant decrease in flexibility with sham was observed on Day3 (+16.2±4.6% knee joint angle; P<0.01) whereas there was no change with EPC (+2.8±3.8%; P>0.01). Vastus lateralis poly-ubiquitinated proteins significantly increased at the POST2 time point relative to PRE with sham (+66.6±24.6%; P<0.025) and were significantly greater (P<0.025) than those observed with EPC at the same time point (-18.6±8.5%). 4-hydroxynonenal values were significantly lower at POST2 relative to PRE with EPC (-16.2±5.6%; P<0.025) and were significantly lower (P<0.025) than those observed with sham at the same time point (+11.8±5.9%). CONCLUSION: EPC mitigated a reduction in flexibility and PPT that occurred with sham. Moreover, EPC reduced select skeletal muscle oxidative stress and proteolysis markers during recovery from heavy resistance exercise.

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.

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.