Evidence of MyomiR Regulation of the Pentose Phosphate Pathway during Mechanical Load-Induced Hypertrophy

Many of the molecular and cellular mechanisms discovered to regulate skeletal muscle hypertrophy were first identified using the rodent synergist ablation model. This model reveals the intrinsic capability and necessary pathways of skeletal muscle growth in response to mechanical overload (MOV). Reminiscent of the rapid cellular growth observed with cancer, we hypothesized that in response to MOV, skeletal muscle would undergo metabolic programming to sustain increased demands to support hypertrophy. To test this hypothesis, we analyzed the gene expression of specific metabolic pathways taken from transcriptomic microarray data of a MOV time course. We found an upregulation of genes involved in the oxidative branch of the pentose phosphate pathways (PPP) and mitochondrial branch of the folate cycle suggesting an increase in the production of NADPH. In addition, we sought to determine the potential role of skeletal muscle-enriched microRNA (myomiRs) and satellite cells in the regulation of the metabolic pathways that changed during MOV. We observed an inverse pattern in gene expression between muscle-enriched myomiR-1 and its known target gene glucose-6-phosphate dehydrogenase, G6pdx, suggesting myomiR regulation of PPP activation in response to MOV. Satellite cell fusion had a significant but modest impact on PPP gene expression. These transcriptomic findings suggest the robust muscle hypertrophy induced by MOV requires enhanced redox metabolism via PPP production of NADPH which is potentially regulated by a myomiR network

LAT1 Protein Content Increases Following 12 Weeks of Resistance Exercise Training in Human Skeletal Muscle

Introduction: Amino acid transporters are essential for cellular amino acid transport and promoting protein synthesis. While previous literature has demonstrated the association of amino acid transporters and protein synthesis following acute resistance exercise and amino acid supplementation, the chronic effect of resistance exercise and supplementation on amino acid transporters is unknown. The purpose herein was to determine if amino acid transporters and amino acid metabolic enzymes were related to skeletal muscle hypertrophy following resistance exercise training with different nutritional supplementation strategies. Methods: 43 college-aged males were separated into a maltodextrin placebo (PLA, n = 12), leucine (LEU, n = 14), or whey protein concentrate (WPC, n = 17) group and underwent 12 weeks of total-body resistance exercise training. Each group\u27s supplement was standardized for total energy and fat, and LEU and WPC supplements were standardized for total leucine (6 g/d). Skeletal muscle biopsies were obtained prior to training and ~72 h following each subject\u27s last training session. Results: All groups increased type I and II fiber cross-sectional area (fCSA) following training (p \u3c 0.050). LAT1 protein increased following training (p \u3c 0.001) and increased more in PLA than LEU and WPC (p \u3c 0.050). BCKDHα protein increased and ATF4 protein decreased following training (p \u3c 0.001). Immunohistochemistry indicated total LAT1/fiber, but not membrane LAT1/fiber, increased with training (p = 0.003). Utilizing all groups, the change in ATF4 protein, but no other marker, trended to correlate with the change in fCSA (r = 0.314; p = 0.055); however, when regression analysis was used to delineate groups, the change in ATF4 protein best predicted the change in fCSA only in LEU (r2 = 0.322; p = 0.043). In C2C12 myoblasts, LAT1 protein overexpression caused a paradoxical decrease in protein synthesis levels (p = 0.002) and decrease in BCKDHα protein (p = 0.001). Conclusions: Amino acid transporters and metabolic enzymes are affected by resistance exercise training, but do not appear to dictate muscle fiber hypertrophy. In fact, overexpression of LAT1 in vitro decreased protein synthesis

Bovine Milk Extracellular Vesicles (EVs) Modification Elicits Skeletal Muscle Growth in Rats

The current study investigated how bovine milk extracellular vesicles (EVs) affected rotarod performance and biomarkers of skeletal muscle physiology in young, growing rats. Twenty-eight-day Fisher 344 rats were provided an AIN-93G-based diet for 4 weeks that either remained unadulterated [EVs and RNA-sufficient (ERS; n = 12)] or was sonicated [EVs and RNA-depleted (ERD; n = 12)]. Prior to (PRE) and on the last day of the intervention (POST), animals were tested for maximal rotarod performance. Following the feeding period, the gastrocnemius muscle was analyzed at the histological, biochemical, and molecular levels and was also used to measure mitochondrial function and reactive oxygen species (ROS) emission. A main effect of time was observed for rotarod time (PRE > POST, p = 0.001). Terminal gastrocnemius mass was unaffected by diet, although gastrocnemius muscle fiber cross sectional area was 11% greater (p = 0.018) and total RNA (a surrogate of ribosome density) was 24% greater (p = 0.001) in ERD. Transcriptomic analysis of the gastrocnemius indicated that 22 mRNAs were significantly greater in ERS versus ERD (p < 0.01), whereas 55 mRNAs were greater in ERD versus ERS (p < 0.01). There were no differences in gastrocnemius citrate synthase activity or mitochondrial coupling (respiratory control ratio), although mitochondrial ROS production was lower in ERD gastrocnemius (p = 0.016), which may be explained by an increase in glutathione peroxidase protein levels (p = 0.020) in ERD gastrocnemius. Dietary EVs profiling confirmed that sonication in the ERD diet reduced EVs content by ∼60%. Our findings demonstrate that bovine milk EVs depletion through sonication elicits anabolic and transcriptomic effects in the gastrocnemius muscle of rapidly maturing rats. While this did not translate into a functional outcome between diets (i.e., rotarod performance), longer feeding periods may be needed to observe such functional effects

RESEARCH Open Access

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

Bovine Milk Extracellular Vesicles (EVs) Modification Elicits Skeletal Muscle Growth in Rats

The current study investigated how bovine milk extracellular vesicles (EVs) affected rotarod performance and biomarkers of skeletal muscle physiology in young, growing rats. Twenty-eight-day Fisher 344 rats were provided an AIN-93G-based diet for 4 weeks that either remained unadulterated [EVs and RNA-sufficient (ERS; n = 12)] or was sonicated [EVs and RNA-depleted (ERD; n = 12)]. Prior to (PRE) and on the last day of the intervention (POST), animals were tested for maximal rotarod performance. Following the feeding period, the gastrocnemius muscle was analyzed at the histological, biochemical, and molecular levels and was also used to measure mitochondrial function and reactive oxygen species (ROS) emission. A main effect of time was observed for rotarod time (PRE \u3e POST, p = 0.001). Terminal gastrocnemius mass was unaffected by diet, although gastrocnemius muscle fiber cross sectional area was 11% greater (p = 0.018) and total RNA (a surrogate of ribosome density) was 24% greater (p = 0.001) in ERD. Transcriptomic analysis of the gastrocnemius indicated that 22 mRNAs were significantly greater in ERS versus ERD (p \u3c 0.01), whereas 55 mRNAs were greater in ERD versus ERS (p \u3c 0.01). There were no differences in gastrocnemius citrate synthase activity or mitochondrial coupling (respiratory control ratio), although mitochondrial ROS production was lower in ERD gastrocnemius (p = 0.016), which may be explained by an increase in glutathione peroxidase protein levels (p = 0.020) in ERD gastrocnemius. Dietary EVs profiling confirmed that sonication in the ERD diet reduced EVs content by ∼60%. Our findings demonstrate that bovine milk EVs depletion through sonication elicits anabolic and transcriptomic effects in the gastrocnemius muscle of rapidly maturing rats. While this did not translate into a functional outcome between diets (i.e., rotarod performance), longer feeding periods may be needed to observe such functional effects

Resistance training in humans and mechanical overload in rodents do not elevate muscle protein lactylation

Although several reports have hypothesized that exercise may increase skeletal muscle protein lactylation, empirical evidence in humans is lacking. Thus, we adopted a multifaceted approach to examine if acute and subchronic resistance training (RT) altered skeletal muscle protein lactylation levels. In mice, we also sought to examine if surgical ablation-induced plantaris hypertrophy coincided with increases in muscle protein lactylation. To examine acute responses, participants’ blood lactate concentrations were assessed before, during, and after eight sets of an exhaustive lower body RT bout (n = 10 trained college-aged men). Vastus lateralis biopsies were also taken before, 3-h post, and 6-h post-exercise to assess muscle protein lactylation. To identify training responses, another cohort of trained college-aged men (n = 14) partook in 6 weeks of lower-body RT (3x/week) and biopsies were obtained before and following the intervention. Five-month-old C57BL/6 mice were subjected to 10 days of plantaris overload (OV, n = 8) or served as age-matched sham surgery controls (Sham, n = 8). Although acute resistance training significantly increased blood lactate responses ~7.2- fold (p \u3c 0.001), cytoplasmic and nuclear protein lactylation levels were not significantly altered at the post-exercise time points, and no putative lactylation-dependent mRNA was altered following exercise. Six weeks of RT did not alter cytoplasmic protein lactylation (p = 0.800) despite significantly increasing VL muscle size (+3.5%, p=0.037), and again, no putative lactylation-dependent mRNA was significantly affected by training. Plantaris muscles were larger in OV versus Sham mice (+43.7%, p \u3c 0.001). However, cytoplasmic protein lactylation was similar between groups (p = 0.369), and nuclear protein lactylation was significantly lower in OV versus Sham mice (p \u3c 0.001). The current null findings, along with other recent null findings in the literature, challenge the thesis that lactate has an appreciable role in promoting skeletal muscle hypertrophy

Pre-training Skeletal Muscle Fiber Size and Predominant Fiber Type Best Predict Hypertrophic Responses to 6 Weeks of Resistance Training in Previously Trained Young Men

Limited evidence exists regarding differentially expressed biomarkers between previously-trained low versus high hypertrophic responders in response to resistance training. Herein, 30 college-aged males (training age 5 ± 3 years; mean ± SD) partook in 6 weeks of high-volume resistance training. Body composition, right leg vastus lateralis (VL) biopsies, and blood were obtained prior to training (PRE) and at the 3-week (W3) and 6-week time points (W6). The 10 lowest (LOW) and 10 highest (HIGH) hypertrophic responders were clustered based upon a composite hypertrophy score of PRE-to-W6 changes in right leg VL mean muscle fiber cross-sectional area (fCSA), VL thickness assessed via ultrasound, upper right leg lean soft tissue mass assessed via dual x-ray absorptiometry (DXA), and mid-thigh circumference. Two-way ANOVAs were used to compare biomarker differences between the LOW and HIGH clusters over time, and stepwise linear regression was performed to elucidate biomarkers that explained significant variation in the composite hypertrophy score from PRE to W3, W3 to W6, and PRE to W6 in all 30 participants. PRE-to-W6 HIGH and LOW responders exhibited a composite hypertrophy change of +10.7 ± 3.2 and -2.1 ± 1.6%, respectively (p < 0.001). Compared to HIGH responders, LOW responders exhibited greater PRE type II fCSA (+18%, p = 0.022). Time effects (p < 0.05) existed for total RNA/mg muscle (W6 > W3 > PRE), phospho (p)-4EBP1 (PRE > W3&W6), pan-mTOR (PRE > W3 < W6), p-mTOR (PRE > W3 < W6), pan-AMPKα (PRE > W3 < W6), pan-p70s6k (PRE > W3), muscle ubiquitin-labeled proteins (PRE > W6), mechano growth factor mRNA (W6 > W3&PRE), 45S rRNA (PRE > W6), and muscle citrate synthase activity (PRE > W3&W6). No interactions existed for the aforementioned biomarkers and/or other assayed targets (muscle 20S proteasome activity, serum total testosterone, muscle androgen receptor protein levels, muscle glycogen, or serum creatine kinase). Regression analysis indicated PRE type II fiber percentage (R2 = 0.152, β = 0.390, p = 0.033) and PRE type II fCSA (R2 = 0.207, β = -0.455, p = 0.019) best predicted the PRE-to-W6 change in the composite hypertrophy score. While our sample size is limited, these data suggest: (a) HIGH responders may exhibit more growth potential given that they possessed lower PRE type II fCSA values and (b) possessing a greater type II fiber percentage as a trained individual may be advantageous for hypertrophy in response to resistance training

Resistance training in humans and mechanical overload in rodents do not elevate muscle protein lactylation

Although several reports have hypothesized that exercise may increase skeletal muscle protein lactylation, empirical evidence in humans is lacking. Thus, we adopted a multi-faceted approach to examine if acute and subchronic resistance training (RT) altered skeletal muscle protein lactylation levels. In mice, we also sought to examine if surgical ablation-induced plantaris hypertrophy coincided with increases in muscle protein lactylation. To examine acute responses, participants’ blood lactate concentrations were assessed before, during, and after eight sets of an exhaustive lower body RT bout (n = 10 trained college-aged men). Vastus lateralis biopsies were also taken before, 3-h post, and 6-h post-exercise to assess muscle protein lactylation. To identify training responses, another cohort of trained college-aged men (n = 14) partook in 6 weeks of lower-body RT (3x/week) and biopsies were obtained before and following the intervention. Five-month-old C57BL/6 mice were subjected to 10 days of plantaris overload (OV, n = 8) or served as age-matched sham surgery controls (Sham, n = 8). Although acute resistance training significantly increased blood lactate responses ∼7.2-fold (p < 0.001), cytoplasmic and nuclear protein lactylation levels were not significantly altered at the post-exercise time points, and no putative lactylation-dependent mRNA was altered following exercise. Six weeks of RT did not alter cytoplasmic protein lactylation (p = 0.800) despite significantly increasing VL muscle size (+3.5%, p = 0.037), and again, no putative lactylation-dependent mRNA was significantly affected by training. Plantaris muscles were larger in OV versus Sham mice (+43.7%, p < 0.001). However, cytoplasmic protein lactylation was similar between groups (p = 0.369), and nuclear protein lactylation was significantly lower in OV versus Sham mice (p < 0.001). The current null findings, along with other recent null findings in the literature, challenge the thesis that lactate has an appreciable role in promoting skeletal muscle hypertrophy

BarA-UvrY Two-Component System Regulates Virulence of Uropathogenic E. coli CFT073

Uropathogenic Escherichia coli (UPEC), a member of extraintestinal pathogenic E. coli, cause ∼80% of community-acquired urinary tract infections (UTI) in humans. UPEC initiates its colonization in epithelial cells lining the urinary tract with a complicated life cycle, replicating and persisting in intracellular and extracellular niches. Consequently, UPEC causes cystitis and more severe form of pyelonephritis. To further understand the virulence characteristics of UPEC, we investigated the roles of BarA-UvrY two-component system (TCS) in regulating UPEC virulence. Our results showed that mutation of BarA-UvrY TCS significantly decreased the virulence of UPEC CFT073, as assessed by mouse urinary tract infection, chicken embryo killing assay, and cytotoxicity assay on human kidney and uroepithelial cell lines. Furthermore, mutation of either barA or uvrY gene reduced the production of hemolysin, lipopolysaccharide (LPS), proinflammatory cytokines (TNF-α and IL-6) and chemokine (IL-8). The virulence phenotype was restored similar to that of wild-type by complementation of either barA or uvrY gene in trans. In addition, we discussed a possible link between the BarA-UvrY TCS and CsrA in positively and negatively controlling virulence in UPEC. Overall, this study provides the evidences for BarA-UvrY TCS regulates the virulence of UPEC CFT073 and may point to mechanisms by which virulence regulations are observed in different ways may control the long-term survival of UPEC in the urinary tract

Optimized Hydrophobic Interactions and Hydrogen Bonding at the Target-Ligand Interface Leads the Pathways of Drug-Designing

Weak intermolecular interactions such as hydrogen bonding and hydrophobic interactions are key players in stabilizing energetically-favored ligands, in an open conformational environment of protein structures. However, it is still poorly understood how the binding parameters associated with these interactions facilitate a drug-lead to recognize a specific target and improve drugs efficacy. To understand this, comprehensive analysis of hydrophobic interactions, hydrogen bonding and binding affinity have been analyzed at the interface of c-Src and c-Abl kinases and 4-amino substituted 1H-pyrazolo [3, 4-d] pyrimidine compounds.In-silico docking studies were performed, using Discovery Studio software modules LigandFit, CDOCKER and ZDOCK, to investigate the role of ligand binding affinity at the hydrophobic pocket of c-Src and c-Abl kinase. Hydrophobic and hydrogen bonding interactions of docked molecules were compared using LigPlot program. Furthermore, 3D-QSAR and MFA calculations were scrutinized to quantify the role of weak interactions in binding affinity and drug efficacy.The in-silico method has enabled us to reveal that a multi-targeted small molecule binds with low affinity to its respective targets. But its binding affinity can be altered by integrating the conformationally favored functional groups at the active site of the ligand-target interface. Docking studies of 4-amino-substituted molecules at the bioactive cascade of the c-Src and c-Abl have concluded that 3D structural folding at the protein-ligand groove is also a hallmark for molecular recognition of multi-targeted compounds and for predicting their biological activity. The results presented here demonstrate that hydrogen bonding and optimized hydrophobic interactions both stabilize the ligands at the target site, and help alter binding affinity and drug efficacy