Polygenic mechanisms underpinning the response to exercise-induced muscle damage in humans: In vivo and in vitro evidence.

We investigated whether 20 candidate single nucleotide polymorphisms (SNPs) were associated with in vivo exercise-induced muscle damage (EIMD), and with an in vitro skeletal muscle stem cell wound healing assay. Sixty-five young, untrained Caucasian adults performed 120 maximal eccentric knee-extensions on an isokinetic dynamometer to induce EIMD. Maximal voluntary isometric/isokinetic knee-extensor torque, knee joint range of motion (ROM), muscle soreness, serum creatine kinase activity and interleukin-6 concentration were assessed before, directly after and 48 h after EIMD. Muscle stem cells were cultured from vastus lateralis biopsies from a separate cohort (n = 12), and markers of repair were measured in vitro. Participants were genotyped for all 20 SNPs using real-time PCR. Seven SNPs were associated with the response to EIMD, and these were used to calculate a total genotype score, which enabled participants to be segregated into three polygenic groups: 'preferential' (more 'protective' alleles), 'moderate', and 'non-preferential'. The non-preferential group was consistently weaker than the preferential group (1.93 ± 0.81 vs. 2.73 ± 0.59 N ∙ m/kg; P = 9.51 × 10-4 ) and demonstrated more muscle soreness (p = 0.011) and a larger decrease in knee joint ROM (p = 0.006) following EIMD. Two TTN-AS1 SNPs in linkage disequilibrium were associated with in vivo EIMD (rs3731749, p ≤ 0.005) and accelerated muscle stem cell migration into the artificial wound in vitro (rs1001238, p ≤ 0.006). Thus, we have identified a polygenic profile, linked with both muscle weakness and poorer recovery following EIMD. Moreover, we provide evidence for a novel TTN gene-cell-skeletal muscle mechanism that may help explain some of the interindividual variability in the response to EIMD.

Influence of sex and fiber type on the satellite cell pool in human skeletal muscle.

The repair, remodeling, and regeneration of myofibers are dependent on satellite cells (SCs), although, the distribution of SCs in different fiber types of human muscle remains inconclusive. There is also a paucity of research comparing muscle fiber characteristics in a sex-specific manner. Therefore, the aim of this study was to investigate fiber type-specific SC content in men and women. Muscle biopsies from vastus lateralis were collected from 64 young (mean age 27 ± 5), moderately trained men (n = 34) and women (n = 30). SCs were identified by Pax7-staining together with immunofluorescent analyses of fiber type composition, fiber size, and myonuclei content. In a mixed population, comparable number of SCs was associated to type I and type II fibers (0.07 ± 0.02 vs 0.07 ± 0.02 SCs per fiber, respectively). However, unlike men, women displayed a fiber type-specific distribution, with SC content being lower in type II than type I fibers (P = .041). Sex-based differences were found specifically for type II fibers, where women displayed lower SC content compared to men (P < .001). In addition, positive correlations (r-values between 0.36-0.56) were found between SC content and type I and type II fiber size in men (P = .03 and P < .01, respectively), whereas similar relationships could not be detected in women. Sex-based differences were also noted for fiber type composition and fiber size, but not for myonuclei content. We hereby provide evidence for sex-based differences present at the myocellular level, which may have important implications when studying exercise- and training-induced myogenic responses in skeletal muscle.

Modeling statin myopathy in a human skeletal muscle microphysiological system.

Statins are used to lower cholesterol and prevent cardiovascular disease. Musculoskeletal side effects known as statin associated musculoskeletal symptoms (SAMS), are reported in up to 10% of statin users, necessitating statin therapy interruption and increasing cardiovascular disease risk. We tested the hypothesis that, when exposed to statins ex vivo, engineered human skeletal myobundles derived from individuals with (n = 10) or without (n = 14) SAMS and elevated creatine-kinase levels exhibit statin-dependent muscle defects. Myoblasts were derived from muscle biopsies of individuals (median age range of 62-64) with hyperlipidemia with (n = 10) or without (n = 14) SAMS. Myobundles formed from myoblasts were cultured with growth media for 4 days, low amino acid differentiation media for 4 days, then dosed with 0 and 5µM of statins for 5 days. Tetanus forces were subsequently measured. To model the change of tetanus forces among clinical covariates, a mixed effect model with fixed effects being donor type, statin concentration, statin type and their two way interactions (donor type*statin concentration and donor type* statin type) and the random effect being subject ID was applied. The results indicate that statin exposure significantly contributed to decrease in force (P<0.001) and the variability in data (R2C [R square conditional] = 0.62). We found no significant differences in force between myobundles from patients with/without SAMS, many of whom had chronic diseases. Immunofluorescence quantification revealed a positive correlation between the number of straited muscle fibers and tetanus force (R2 = 0.81,P = 0.015) and negative correlation between number of fragmented muscle fibers and tetanus force (R2 = 0.482,P = 0.051) with no differences between donors with or without SAMS. There is also a correlation between statin exposure and presence of striated fibers (R2 = 0.833, P = 0.047). In patient-derived myobundles, statin exposure results in myotoxicity disrupting SAA organization and reducing force. We were unable to identify differences in ex vivo statin myotoxicity in this system. The results suggest that it is unlikely that there is inherent susceptibility to or persistent effects of statin myopathy using patient-derived myobundles.

Super-relaxed state of myosin in human skeletal muscle is fiber-type dependent.

The myosin super-relaxed state (SRX) in skeletal muscle is hypothesized to play an important role in regulating muscle contractility and thermogenesis in humans but has only been examined in model organisms. Here we report the first human skeletal muscle SRX measurements, using quantitative epifluorescence microscopy of fluorescent 2'/3'-O-(N-methylanthraniloyl) ATP (mantATP) single-nucleotide turnover. Myosin heavy chain (MHC) isoform expression was determined using gel electrophoresis for each permeabilized vastus lateralis fiber, to allow for novel comparisons of SRX between fiber types. We find that the fraction of myosin in SRX is less in MHC IIA fibers than in MHC I and IIAX fibers (P = 0.008). ATP turnover of SRX is faster in MHC IIAX fibers compared with MHC I and IIA fibers (P = 0.001). We conclude that SRX biochemistry is measurable in human skeletal muscle, and our data indicate that SRX depends on fiber type as classified by MHC isoform. Extension from this preliminary work would provide further understanding regarding the role of SRX in human muscle physiology.

Deconstructing sarcomeric structure-function relations in titin-BioID knock-in mice.

Proximity proteomics has greatly advanced the analysis of native protein complexes and subcellular structures in culture, but has not been amenable to study development and disease in vivo. Here, we have generated a knock-in mouse with the biotin ligase (BioID) inserted at titin's Z-disc region to identify protein networks that connect the sarcomere to signal transduction and metabolism. Our census of the sarcomeric proteome from neonatal to adult heart and quadriceps reveals how perinatal signaling, protein homeostasis and the shift to adult energy metabolism shape the properties of striated muscle cells. Mapping biotinylation sites to sarcomere structures refines our understanding of myofilament dynamics and supports the hypothesis that myosin filaments penetrate Z-discs to dampen contraction. Extending this proof of concept study to BioID fusion proteins generated with Crispr/CAS9 in animal models recapitulating human pathology will facilitate the future analysis of molecular machines and signaling hubs in physiological, pharmacological, and disease context.

Age-related changes to the satellite cell niche are associated with reduced activation following exercise.

Skeletal muscle satellite cell (SC) function and responsiveness is regulated, in part, through interactions within the niche, in which they reside. Evidence suggests that structural changes occur in the SC niche as a function of aging. In the present study, we investigated the impact of aging on SC niche properties. Muscle biopsies were obtained from the vastus lateralis of healthy young (YM; 21 ± 1 yr; n = 10) and older men (OM; 68 ± 1 yr; n = 16) at rest. A separate group of OM performed a single bout of resistance exercise and additional muscle biopsies were taken 24 and 48 hours post-exercise; this was performed before and following 12 wks of combined exercise training (OM-Ex; 73 ± 1; n = 24). Muscle SC niche measurements were assessed using high resolution immunofluorescent confocal microscopy. Type II SC niche laminin thickness was greater in OM (1.86 ± 0.06 µm) as compared to YM (1.55 ± 0.09 µm, P < .05). The percentage of type II-associated SC that were completely surrounded by laminin was greater in OM (13.6%±4.2%) as compared to YM (3.5%±1.5%; P < .05). In non-surrounded SC, the proportion of active MyoD+ /Pax7+ SC were higher compared to surrounded SC (P < .05) following a single bout of exercise. This "incarceration" of the SC niche by laminin appears with aging and may inhibit SC activation in response to exercise.

Preserved capacity for satellite cell proliferation, regeneration, and hypertrophy in the skeletal muscle of healthy elderly men.

Blunted muscle hypertrophy and impaired regeneration with aging have been partly attributed to satellite cell (SC) dysfunction. However, true muscle regeneration has not yet been studied in elderly individuals. To investigate this, muscle injury was induced by 200 electrically stimulated (ES) eccentric contractions of the vastus lateralis (VL) of one leg in seven young (20-31 years) and 19 elderly men (60-73 years). This was followed by 13 weeks of resistance training (RT) for both legs to investigate the capacity for hypertrophy. Muscle biopsies were collected Pre- and Post-RT, and 9 days after ES, for immunohistochemistry and RT-PCR. Hypertrophy was assessed by MRI, DEXA, and immunohistochemistry. Overall, surprisingly comparable responses were observed between the young and elderly. Nine days after ES, Pax7+ SC number had doubled (P < .05), alongside necrosis and substantial changes in expression of genes related to matrix, myogenesis, and innervation (P < .05). Post-RT, VL cross-sectional area had increased in both legs (~15%, P < .05) and SCs/type II fiber had increased ~2-4 times more with ES+RT vs RT alone (P < .001). Together these novel findings demonstrate "youthful" regeneration and hypertrophy responses in human elderly muscle. Furthermore, boosting SC availability in healthy elderly men does not enhance the subsequent muscle hypertrophy response to RT.

Single-cell transcriptional profiles in human skeletal muscle.

Skeletal muscle is a heterogeneous tissue comprised of muscle fiber and mononuclear cell types that, in addition to movement, influences immunity, metabolism and cognition. We investigated the gene expression patterns of skeletal muscle cells using RNA-seq of subtype-pooled single human muscle fibers and single cell RNA-seq of mononuclear cells from human vastus lateralis, mouse quadriceps, and mouse diaphragm. We identified 11 human skeletal muscle mononuclear cell types, including two fibro-adipogenic progenitor (FAP) cell subtypes. The human FBN1+ FAP cell subtype is novel and a corresponding FBN1+ FAP cell type was also found in single cell RNA-seq analysis in mouse. Transcriptome exercise studies using bulk tissue analysis do not resolve changes in individual cell-type proportion or gene expression. The cell-type gene signatures provide the means to use computational methods to identify cell-type level changes in bulk studies. As an example, we analyzed public transcriptome data from an exercise training study and revealed significant changes in specific mononuclear cell-type proportions related to age, sex, acute exercise and training. Our single-cell expression map of skeletal muscle cell types will further the understanding of the diverse effects of exercise and the pathophysiology of muscle disease.

Correlation of Fiber-Type Composition and Sprint Performance in Youth Soccer Players.

Metaxas, T, Mandroukas, A, Michailidis, Y, Koutlianos, N, Christoulas, K, and Ekblom, B. Correlation of fiber-type composition and sprint performance in youth soccer players. J Strength Cond Res 33(10): 2629-2634, 2019-The aim of this study was to examine the correlation between muscle fiber type and sprint performance in elite young soccer players of different age groups of the same team. Twenty-eight young players participated in this study (group U15, n = 8; group U13, n = 9; and group U11, n = 11). Anthropometric assessments, acceleration (10 m), and Bangsbo modified sprint test (30 m) were performed. Muscle biopsies were obtained from the vastus lateralis, and after that, fiber-type composition was determined by immunohistochemistry. No significant correlations were found between the sprint test and muscle fiber distribution for the groups U13 and U11 (p > 0.05). Also, no correlations were found between cross-sectional areas in the types of fibers with the sprint test in all groups (p > 0.05). A positive correlation was found between type I fibers and the performance in the acceleration test (10 m) (r = 0.77, p < 0.05) was found only in group U15 and a negative correlation between type IIA fibers and the performance in the acceleration test (10 m) (r = -0.89, p < 0.05). The correlations were observed only in group U15, which may indicate that the duration and the intensity of the soccer systematic training can affect the plasticity of the muscle fibers. Specific soccer training in youth is one of the factors that can affect fiber-type plasticity. The specific training programs and status of U15 are more intensive, and the exercises are oriented more to improve physical fitness.

A Combination of Intrathecal and Intramuscular Application of Human Mesenchymal Stem Cells Partly Reduces the Activation of Necroptosis in the Spinal Cord of SOD1G93A Rats.

An increasing number of studies have demonstrated the beneficial effects of human mesenchymal stem cells (hMSC) in the treatment of amyotrophic lateral sclerosis (ALS). We compared the effect of repeated intrathecal applications of hMSC or their conditioned medium (CondM) using lumbar puncture or injection into the muscle (quadriceps femoris), or a combination of both applications in symptomatic SOD1G93A rats. We further assessed the effect of the treatment on three major cell death pathways (necroptosis, apoptosis, and autophagy) in the spinal cord tissue. All the animals were behaviorally tested (grip strength test, Basso Beattie Bresnahan (BBB) test, and rotarod), and the tissue was analyzed immunohistochemically, by qPCR and Western blot. All symptomatic SOD1 rats treated with hMSC had a significantly increased lifespan, improved motor activity and reduced number of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells. Moreover, a combined hMSC delivery increased motor neuron survival, maintained neuromuscular junctions in quadriceps femoris and substantially reduced the levels of proteins involved in necroptosis (Rip1, mixed lineage kinase-like protein, cl-casp8), apoptosis (cl-casp 9) and autophagy (beclin 1). Furthermore, astrogliosis and elevated levels of Connexin 43 were decreased after combined hMSC treatment. The repeated application of CondM, or intramuscular injections alone, improved motor activity; however, this improvement was not supported by changes at the molecular level. Our results provide new evidence that a combination of repeated intrathecal and intramuscular hMSC applications protects motor neurons and neuromuscular junctions, not only through a reduction of apoptosis and autophagy but also through the necroptosis pathway, which is significantly involved in cell death in rodent SOD1G93A model of ALS. Stem Cells Translational Medicine 2019;8:535-547.

Normative data of muscle fiber diameter of vastus lateralis during childhood: a field test.

INTRODUCTION: Currently, our knowledge of standard data for muscle morphology in children is largely limited to the 1969 article by Brooke and Engel (BE). In 2016, we reported normal muscle morphology from vastus lateralis biopsies in ambulant children with cerebral palsy (CP). This report compares our normal biopsy results against BE standard value criteria. METHODS: Single-blind prospective cross-sectional study design. RESULTS: Results of biopsies taken in ambulant children with CP were normal according to morphometry and light and electron microscopy; however, only 5 of 10 fulfilled the BE standard value criteria. DISCUSSION: This short report highlights the requirement for contemporary age-specific normative data from a larger number of biopsies, including typically developing children. Review of the literature suggests that biopsy material may be available from typically developing children who were control patients in research trials. This morphometric data could contribute to expanding the normative data set. Muscle Nerve 59:590-590, 2019.

Determination of a Critical Size Threshold for Volumetric Muscle Loss in the Mouse Quadriceps.

IMPACT STATEMENT: The goal of this study was to determine the threshold for a critically sized, nonhealing muscle defect by characterizing key components in the balance between fibrosis and regeneration as a function of injury size in the mouse quadriceps. There is currently limited understanding of what leads to a critically sized muscle defect and which muscle regenerative components are functionally impaired. With the substantial increase in preclinical VML models as testbeds for tissue engineering therapeutics, defining the critical threshold for VML injuries will be instrumental in characterizing therapeutic efficacy and potential for subsequent translation.

Fiber Type Composition and Rate of Force Development in Endurance- and Resistance-Trained Individuals.

Methenitis, S, Spengos, K, Zaras, N, Stasinaki, A-N, Papadimas, G, Karampatsos, G, Arnaoutis, G, and Terzis, G. Fiber type composition and rate of force development in endurance- and resistance-trained individuals. J Strength Cond Res 33(9): 2388-2397, 2019-The purpose of the study was to investigate the relationship between muscle fiber composition and the rate of force development (RFD) in well-trained individuals with different training background. Thirty-eight young men with different training background participated: 9 endurance runners, 10 power-trained individuals, 9 strength-trained individuals, and 10 sedentary individuals. They performed maximal isometric leg press for the measurement of RFD. Body composition (dual x-ray absorptiometry) and vastus lateralis fiber type composition were also evaluated. When all participants were examined as a group, moderate correlations were found between the percent of type II muscle fibers and RFD between 100 and 600 milliseconds (r = 0.321-0.497; p ≤ 0.05). The correlation coefficients were higher for the cross-sectional area (CSA) and the %CSA of type II and IIx muscle fibers (r = 0.599-0.847; p < 0.001). For the power group, RFD up to 250 milliseconds highly correlated with % type IIx muscle fibers and type IIx fiber CSA (r = 0.670-0.826; p ≤ 0.05), as well as with %CSA of type IIx fibers (r = 0.714-0.975; p ≤ 0.05). Significant correlations were found between the relative RFD (·kg lower extremities lean mass) and CSA-%CSA of type II and IIx fibers for the power group (r = 0.676-0.903; p ≤ 0.05). No significant correlations were found between muscle morphology and RFD for the other groups. In conclusion, the present data suggest that there is a strong link between the type IIx muscle fibers and early RFD and relative RFD in power-trained participants. Type II fibers seem to be moderately linked with RFD in non-power-trained individuals.

A double-blind, randomized, placebo-controlled clinical trial evaluating the safety and efficacy of autologous muscle derived cells in female subjects with stress urinary incontinence.

PURPOSE: The purpose of the study was to assess safety and efficacy of autologous muscle derived cells for urinary sphincter repair (AMDC-USR) in female subjects with predominant stress urinary incontinence. METHODS: A randomized, double-blind, multicenter trial examined intra-sphincteric injection of 150 × 106 AMDC-USR versus placebo in female subjects with stress or stress predominant, mixed urinary incontinence. AMDC-USR products were generated from vastus lateralis needle biopsies. Subjects were randomized 2:1 to receive AMDC-USR or placebo and 1:1 to receive 1 or 2 treatments (6 months after the first). Primary outcome was composite of ≥ 50% reduction in stress incontinence episode frequency (IEF), 24-h or in-office pad weight tests at 12 months. Other outcome data included validated subject-recorded questionnaires. Subjects randomized to placebo could elect to receive open-label AMDC-USR treatment after 12 months. Subject follow-up was up to 2 years. RESULTS: AMDC-USR was safe and well-tolerated with no product-related serious adverse events or discontinuations due to adverse events. Interim analysis revealed an unexpectedly high placebo response rate (90%) using the composite primary outcome which prevented assessment of treatment effect as designed and thus enrollment was halted at 61% of planned subjects. Post hoc analyses suggested that more stringent endpoints lowered placebo response rates and revealed a possible treatment effect. CONCLUSIONS: Although the primary efficacy finding was inconclusive, these results inform future trial design of AMDC-USR to identify clinically meaningful efficacy endpoints based on IEF reduction, understanding of placebo response rate, and refinement of subject selection criteria to more appropriately align with AMDC-USR's proposed mechanism of action.

Age-related reduction in single muscle fiber calcium sensitivity is associated with decreased muscle power in men and women.

Age-related declines in human skeletal muscle performance may be caused, in part, by decreased responsivity of muscle fibers to calcium (Ca2+). This study examined the contractile properties of single vastus lateralis muscle fibers with various myosin heavy chain (MHC) isoforms (I, I/IIA, IIA and IIAX) across a range of Ca2+ concentrations in 11 young (24.1±1.1years) and 10 older (68.8±0.8years) men and women. The normalized pCa-force curve shifted rightward with age, leading to decreased activation threshold (pCa10) and/or Ca2+ sensitivity (pCa50) for all MHC isoforms examined. In older adults, the slope of the pCa-force curve was unchanged in MHC I-containing fibers (I, I/IIA), but was steeper in MHC II-containing fibers (IIA, IIAX), indicating greater cooperativity compared to young adults. At sub-maximal [Ca2+], specific force was reduced in MHC I-containing fibers, but was minimally decreased in MHC IIA fibers as older adults produced greater specific forces at high [Ca2+] in these fibers. Lessor pCa50 in MHC I fibers independently predicted reduced isokinetic knee extensor power across a range of contractile velocities, suggesting that the Ca2+ response of slow-twitch fibers contributes to whole muscle dysfunction. Our findings show that aging attenuates Ca2+ responsiveness across fiber types and that these cellular alterations may lead to age-related reductions in whole muscle power output.

Intraoperative and biomechanical studies of human vastus lateralis and vastus medialis sarcomere length operating range.

The vast majority of musculoskeletal models are not validated against primary experimental data. Conversely, most human experimental measurements are not explained theoretically using models to provide a mechanistic understanding of experimental results. Here we present a study with both primary human data and primary modeling data. Intraoperative sarcomere length was measured on the human vastus lateralis (VL) and vastus medialis (VM) muscles (n = 8) by laser diffraction. These data were compared to a biomechanical model based on muscle architecture and moment arms obtained independently from cadaveric specimens (n = 9). Measured VL sarcomere length ranged from about 3.2 µm with the knee flexed to 45° to 3.8 µm with the knee flexed to 90°. These values were remarkably close to theoretical predictions. Measured VM sarcomere length ranged from 3.6 µm with the knee flexed to 45° to 4.1 µm with the knee flexed to 90°. These values were dramatically longer than theoretical predictions. Our measured sarcomere length values suggest that human vasti may have differing functions with regard to knee extension and patellar stabilization. This report underscores the importance of validating experimental data to theoretical models and vice versa.

Characterisation of L-Type Amino Acid Transporter 1 (LAT1) Expression in Human Skeletal Muscle by Immunofluorescent Microscopy.

The branch chain amino acid leucine is a potent stimulator of protein synthesis in skeletal muscle. Leucine rapidly enters the cell via the L-Type Amino Acid Transporter 1 (LAT1); however, little is known regarding the localisation and distribution of this transporter in human skeletal muscle. Therefore, we applied immunofluorescence staining approaches to visualise LAT1 in wild type (WT) and LAT1 muscle-specific knockout (mKO) mice, in addition to basal human skeletal muscle samples. LAT1 positive staining was visually greater in WT muscles compared to mKO muscle. In human skeletal muscle, positive LAT1 staining was noted close to the sarcolemmal membrane (dystrophin positive staining), with a greater staining intensity for LAT1 observed in the sarcoplasmic regions of type II fibres (those not stained positively for myosin heavy-chain 1, Type II-25.07 ± 5.93, Type I-13.71 ± 1.98, p < 0.01), suggesting a greater abundance of this protein in these fibres. Finally, we observed association with LAT1 and endothelial nitric oxide synthase (eNOS), suggesting LAT1 association close to the microvasculature. This is the first study to visualise the distribution and localisation of LAT1 in human skeletal muscle. As such, this approach provides a validated experimental platform to study the role and regulation of LAT1 in human skeletal muscle in response to various physiological and pathophysiological models.

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.

Protein kinase N2 regulates AMP kinase signaling and insulin responsiveness of glucose metabolism in skeletal muscle.

Insulin resistance is central to the development of type 2 diabetes and related metabolic disorders. Because skeletal muscle is responsible for the majority of whole body insulin-stimulated glucose uptake, regulation of glucose metabolism in this tissue is of particular importance. Although Rho GTPases and many of their affecters influence skeletal muscle metabolism, there is a paucity of information on the protein kinase N (PKN) family of serine/threonine protein kinases. We investigated the impact of PKN2 on insulin signaling and glucose metabolism in primary human skeletal muscle cells in vitro and mouse tibialis anterior muscle in vivo. PKN2 knockdown in vitro decreased insulin-stimulated glucose uptake, incorporation into glycogen, and oxidation. PKN2 siRNA increased 5'-adenosine monophosphate-activated protein kinase (AMPK) signaling while stimulating fatty acid oxidation and incorporation into triglycerides and decreasing protein synthesis. At the transcriptional level, PKN2 knockdown increased expression of PGC-1α and SREBP-1c and their target genes. In mature skeletal muscle, in vivo PKN2 knockdown decreased glucose uptake and increased AMPK phosphorylation. Thus, PKN2 alters key signaling pathways and transcriptional networks to regulate glucose and lipid metabolism. Identification of PKN2 as a novel regulator of insulin and AMPK signaling may provide an avenue for manipulation of skeletal muscle metabolism.

Overexpression of PGC-1α increases peroxisomal activity and mitochondrial fatty acid oxidation in human primary myotubes.

Peroxisomes are indispensable organelles for lipid metabolism in humans, and their biogenesis has been assumed to be under regulation by peroxisome proliferator-activated receptors (PPARs). However, recent studies in hepatocytes suggest that the mitochondrial proliferator PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1α) also acts as an upstream transcriptional regulator for enhancing peroxisomal abundance and associated activity. It is unknown whether the regulatory mechanism(s) for enhancing peroxisomal function is through the same node as mitochondrial biogenesis in human skeletal muscle (HSkM) and whether fatty acid oxidation (FAO) is affected. Primary myotubes from vastus lateralis biopsies from lean donors (BMI = 24.0 ± 0.6 kg/m2; n = 6) were exposed to adenovirus encoding human PGC-1α or GFP control. Peroxisomal biogenesis proteins (peroxins) and genes (PEXs) responsible for proliferation and functions were assessed by Western blotting and real-time qRT-PCR, respectively. [1-14C]palmitic acid and [1-14C]lignoceric acid (exclusive peroxisomal-specific substrate) were used to assess mitochondrial oxidation of peroxisomal-derived metabolites. After overexpression of PGC-1α, 1) peroxisomal membrane protein 70 kDa (PMP70), PEX19, and mitochondrial citrate synthetase protein content were significantly elevated (P < 0.05), 2) PGC-1α, PMP70, key PEXs, and peroxisomal ß-oxidation mRNA expression levels were significantly upregulated (P < 0.05), and 3) a concomitant increase in lignoceric acid oxidation by both peroxisomal and mitochondrial activity was observed (P < 0.05). These novel findings demonstrate that, in addition to the proliferative effect on mitochondria, PGC-1α can induce peroxisomal activity and accompanying elevations in long-chain and very-long-chain fatty acid oxidation by a peroxisomal-mitochondrial functional cooperation, as observed in HSkM cells.