Acetaminophen influences musculoskeletal signaling but not adaptations to endurance exercise training.

Acetaminophen (ACE) is a widely used analgesic and antipyretic drug with various applications, from pain relief to fever reduction. Recent studies have reported equivocal effects of habitual ACE intake on exercise performance, muscle growth, and risks to bone health. Thus, this study aimed to assess the impact of a 6-week, low-dose ACE regimen on muscle and bone adaptations in exercising and non-exercising rats. Nine-week-old Wistar rats (n = 40) were randomized to an exercise or control (no exercise) condition with ACE or without (placebo). For the exercise condition, rats ran 5 days per week for 6 weeks at a 5% incline for 2 min at 15 cm/s, 2 min at 20 cm/s, and 26 min at 25 cm/s. A human equivalent dose of ACE was administered (379 mg/kg body weight) in drinking water and adjusted each week based on body weight. Food, water intake, and body weight were measured daily. At the beginning of week 6, animals in the exercise group completed a maximal treadmill test. At the end of week 6, rats were euthanized, and muscle cross-sectional area (CSA), fiber type, and signaling pathways were measured. Additionally, three-point bending and microcomputer tomography were measured in the femur. Follow-up experiments in human primary muscle cells were used to explore supra-physiological effects of ACE. Data were analyzed using a two-way ANOVA for treatment (ACE or placebo) and condition (exercise or non-exercise) for all animal outcomes. Data for cell culture experiments were analyzed via ANOVA. If omnibus significance was found in either ANOVA, a post hoc analysis was completed, and a Tukey's adjustment was used. ACE did not alter body weight, water intake, food intake, or treadmill performance (p > .05). There was a treatment-by-condition effect for Young's Modulus where placebo exercise was significantly lower than placebo control (p < .05). There was no treatment by condition effects for microCT measures, muscle CSA, fiber type, or mRNA expression. Phosphorylated-AMPK was significantly increased with exercise (p < .05) and this was attenuated with ACE treatment. Furthermore, phospho-4EBP1 was depressed in the exercise group compared to the control (p < .05) and increased in the ACE control and ACE exercise group compared to placebo exercise (p < .05). A low dose of ACE did not influence chronic musculoskeletal adaptations in exercising rodents but acutely attenuated AMPK phosphorylation and 4EBP1 dephosphorylation post-exercise.

Sex Does Not Affect Changes in Body Composition and Insulin-Like Growth Factor-I During US Army Basic Combat Training.

ABSTRACT: Roberts, BM, Staab, JS, Caldwell, AR, Sczuroski, CE, Staab, JE, Lutz, LJ, Reynoso, M, Geddis, AV, Taylor, KM, Guerriere, KI, Walker, LA, Hughes, JM, and Foulis, SA. Sex does not affect changes in body composition and insulin-like growth factor-I during US army basic combat training. J Strength Cond Res XX(X): 000-000, 2023-Insulin-like growth factor 1 (IGF-I) has been implicated as a biomarker of health and body composition. However, whether changes in body composition are associated with changes in IGF-I is unclear. Therefore, we examined the relationship between body composition changes (i.e., fat mass and lean mass) and total serum IGF-I levels in a large cohort of young men (n = 809) and women (n = 397) attending US Army basic combat training (BCT). We measured body composition using dual energy x-ray absorptiometry and total serum IGF-I levels during week 1 and week 9 of BCT. We found that pre-BCT lean mass (r = 0.0504, p = 0.082) and fat mass (r = 0.0458, p = 0.082) were not associated with pre-BCT IGF-I. Body mass, body mass index, body fat percentage, and fat mass decreased, and lean mass increased during BCT (all p < 0.001). Mean (±SD) IGF-I increased from pre-BCT (176 ± 50 ng·ml-1) to post-BCT (200 ± 50 ng·ml-1, p < 0.001). Inspection of the partial correlations indicated that even when considering the unique contributions of other variables, increases in IGF-I during BCT were associated with both increased lean mass (r = 0.0769, p = 0.023) and increased fat mass (r = 0.1055, p < 0.001) with no sex differences. Taken together, our data suggest that although changes in IGF-I weakly correlated with changes in body composition, IGF-I, in isolation, is not an adequate biomarker for predicting changes in body composition during BCT in US Army trainees.

The dose-response effects of flurbiprofen, indomethacin, ibuprofen, and naproxen on primary skeletal muscle cells.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) like ibuprofen, flurbiprofen, naproxen sodium, and indomethacin are commonly employed for their pain-relieving and inflammation-reducing qualities. NSAIDs work by blocking COX-1 and/or COX-2, enzymes which play roles in inflammation, fever, and pain. The main difference among NSAIDs lies in their affinity to these enzymes, which in turn, influences prostaglandin secretion, and skeletal muscle growth and regeneration. The current study investigated the effects of NSAIDs on human skeletal muscle cells, focusing on myoblast proliferation, differentiation, and muscle protein synthesis signaling. METHODS: Using human primary muscle cells, we examined the dose-response impact of flurbiprofen (25-200 µM), indomethacin (25-200 µM), ibuprofen (25-200 µM), and naproxen sodium (25-200 µM), on myoblast viability, myotube area, fusion, and prostaglandin production. RESULTS: We found that supraphysiological concentrations of indomethacin inhibited myoblast proliferation (-74 ± 2% with 200 µM; -53 ± 3% with 100 µM; both p < 0.05) compared to control cells and impaired protein synthesis signaling pathways in myotubes, but only attenuated myotube fusion at the highest concentrations (-18 ± 2% with 200 µM, p < 0.05) compared to control myotubes. On the other hand, ibuprofen had no such effects. Naproxen sodium only increased cell proliferation at low concentrations (+36 ± 2% with 25 µM, p < 0.05), and flurbiprofen exhibited divergent impacts depending on the concentration whereby low concentrations improved cell proliferation (+17 ± 1% with 25 µM, p < 0.05) but high concentrations inhibited cell proliferation (-32 ± 1% with 200 µM, p < 0.05). CONCLUSION: Our findings suggest that indomethacin, at high concentrations, may detrimentally affect myoblast proliferation and differentiation via an AKT-dependent mechanism, and thus provide new understanding of NSAIDs' effects on skeletal muscle cell development.

Differential activation of AKT isoforms by growth factors in human myotubes.

AKT signaling plays a crucial role in muscle physiology, and is activated by stimuli, including insulin, growth factors, and exercise. Three AKT isoforms have been identified in mammals, and they possess both distinct and redundant functions. However, it is currently unknown what the predominant AKT isoform is in primary human skeletal myotubes, and very little is known regarding the effects of insulin and insulin-like growth factor-I (IGF-I) on AKT isoforms activation in human myotubes. Thus, we sought to determine the abundances of each AKT isoform in primary human skeletal myotubes and their responses to insulin or IGF-I. Analysis of protein lysates by liquid chromatography-parallel reaction monitoring/mass spectrometry revealed that AKT1 was the most abundant AKT isoform and AKT3 was the least-abundant isoform. Next, myotubes were treated with either 100 nM insulin or 10 nM IGF-I for 5, 20, 45, or 60 min. In response to insulin, there was a significant treatment effect on phosphorylation of AKT1 and AKT2, but not AKT3 (p < 0.01). In response to IGF-I, there was a significant treatment effect on phosphorylation of pan-AKT at all timepoints compared to control (p < 0.01). Next, we determined how much of the total AKT isoform pool was phosphorylated. For insulin stimulation, AKT1 was significantly higher than AKT2 at 5 min and 60 min posttreatment (p < 0.05 both) and significantly different than AKT3 at all timepoints (p < 0.05). For IGF-I stimulation, AKT1 was significantly higher than AKT2 at 45 and 60 min posttreatment (p < 0.05 both) and significantly higher than AKT3 at all timepoints (p < 0.05). Our findings reveal the differential phosphorylation patterns among the AKT isoforms and suggest a potential explanation for the regulatory role of AKT1 in skeletal muscle.

The dose-response effects of arachidonic acid on primary human skeletal myoblasts and myotubes.

Background: Cellular inflammatory response, mediated by arachidonic acid (AA) and cyclooxygenase, is a highly regulated process that leads to the repair of damaged tissue. Recent studies on murine C2C12 cells have demonstrated that AA supplementation leads to myotube hypertrophy. However, AA has not been tested on primary human muscle cells. Therefore, the purpose of this study was to determine whether AA supplementation has similar effects on human muscle cells. Methods: Proliferating and differentiating human myoblasts were exposed to AA in a dose-dependent manner (50-0.80 µM) for 48 (myoblasts) or 72 (myotubes) hours. Cell viability was tested using a 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay and cell counting; myotube area was determined by immunocytochemistry and confocal microscopy; and anabolic signaling pathways were evaluated by western blot and RT-PCR. Results: Our data show that the treatment of primary human myoblasts treated with 50 µM and 25 µM of AA led to the release of PGE2 and PGF2α at levels higher than those of control-treated cells (p < 0.001 for all concentrations). Additionally, 50 µM and 25 µM of AA suppressed myoblast proliferation, myotube area, and myotube fusion. Anabolic signaling indicated reductions in total and phosphorylated TSC2, AKT, S6, and 4EBP1 in myoblasts at 50 µM of AA (p < 0.01 for all), but not in myotubes. These changes were not affected by COX-2 inhibition with celecoxib. Conclusion: Together, our data demonstrate that high concentrations of AA inhibit myoblast proliferation, myotube fusion, and myotube hypertrophy, thus revealing potential deleterious effects of AA on human skeletal muscle cell health and viability.

Celecoxib impairs primary human myoblast proliferation and differentiation independent of cyclooxygenase 2 inhibition.

The use of non-steroidal anti-inflammatory drugs (NSAIDs) for treatment of musculoskeletal injuries is commonplace in the general, athletic, and military populations. While NSAIDs have been studied in a variety of tissues, the effects of NSAIDs on skeletal muscle have not been fully defined. To address this, we investigated the degree to which the cyclooxygenase (COX)-2-selective NSAID celecoxib affects muscle cell proliferation, differentiation, anabolic signaling, and mitochondrial function in primary human skeletal myoblasts and myotubes. Primary muscle cells were treated with celecoxib or NS-398 (a pharmacological inhibitor of COX-2) as a control. Celecoxib administration significantly reduced myoblast proliferation, viability, fusion, and myotube area in a dose-dependent manner, whereas NS-398 had no effect on any of these outcomes. Celecoxib treatment was also associated with reduced phosphorylation of ribosomal protein S6 in myoblasts, and reduced phosphorylation of AKT, p70S6K, S6, and ERK in myotubes. In contrast, NS-398 did not alter phosphorylation of these molecules in myoblasts or myotubes. In myoblasts, celecoxib significantly reduced mitochondrial membrane potential and respiration, as evidenced by the decreased citric acid cycle (CAC) intermediates cis-aconitic acid, alpha-keto-glutarate acid, succinate acid, and malic acid. Similar results were observed in myotubes, although celecoxib also reduced pyruvic acid, citric acid, and fumaric acid. NS-398 did not affect CAC intermediates in myoblasts or myotubes. Together, these data reveal that celecoxib inhibits proliferation, differentiation, intracellular signaling, and mitochondrial function in primary human myoblasts and myotubes independent of its function as a COX-2 inhibitor.

LPS-stimulated NF-κB p65 dynamic response marks the initiation of TNF expression and transition to IL-10 expression in RAW 264.7 macrophages.

During injury and infection, inflammation is a response by macrophages to effect healing and repair. The kinetics of the responses of proinflammatory TNFα, anti-inflammatory IL-10, and inflammatory master regulator NF-κB elicited by lipopolysaccharide (LPS) may be critical determinants of the inflammatory response by macrophages; however, there is a lack of homogeneous kinetic data in this pathway. To address this gap, we used the RAW 264.7 macrophage cell line to define intracellular signaling kinetics and cytokine expression in cells treated with LPS for 15 min to 72 h. The abundance of IκBα was maximally reduced 45-min following LPS treatment, but expression increased at 10-h, reaching a maximum at 16 h. NF-κB phosphorylation was significantly increased 45-min following LPS treatment, maximal at 2-h, and decreased to basal levels by 6-h. Nuclear NF-κB expression was elevated 30-min following LPS treatment, maximal by 45-min, and returned to basal levels by 24-h. Binding of nuclear NF-κB to consensus oligonucleotide sequences followed a similar pattern to that observed for p-NF-κB, but lasted slightly longer. Following LPS treatment, TNFα mRNA expression began at 1-h, was maximal at 6-h, and decreased starting at 10-h. TNFα protein secretion in conditioned growth medium began at 4-h and was maximal by 16-h. IL-10 mRNA expression was induced by LPS at 10-h, and was maximal at 16-h. IL-10 protein secretion was induced at 16-h and was maximal at 24-h. Our data reveal the temporal kinetics of pro- and anti-inflammatory signaling events that may be important therapeutic targets for inflammatory diseases.

AKT2 is the predominant AKT isoform expressed in human skeletal muscle.

Skeletal muscle physiology and metabolism are regulated by complex networks of intracellular signaling pathways. Among many of these pathways, the protein kinase AKT plays a prominent role. While three AKT isoforms have been identified (AKT1, AKT2, and AKT3), surprisingly little is known regarding isoform-specific expression of AKT in human skeletal muscle. To address this, we examined the expressions of each AKT isoform in muscle biopsy samples collected from the vastus lateralis of healthy male adults at rest. In muscle, AKT2 was the most highly expressed AKT transcript, exhibiting a 15.4-fold increase over AKT1 and AKT3 transcripts. Next, the abundance of AKT protein isoforms was determined using antibody immunoprecipitation followed by Liquid Chromatography-Parallel Reaction Monitoring/Mass Spectrometry. Immunoprecipitation was performed using either mouse or rabbit pan AKT antibodies that were immunoreactive with all three AKT isoforms. We found that AKT2 was the most abundant AKT isoform in human skeletal muscle (4.2-fold greater than AKT1 using the rabbit antibody and 1.6-fold greater than AKT1 using the mouse antibody). AKT3 was virtually undetectable. Next, cultured primary human myoblasts were virally-transduced with cDNAs encoding either wild-type (WT) or kinase-inactive AKT1 (AKT1-K179M) or AKT2 (AKT2-K181M) and allowed to terminally differentiate. Myotubes expressing WT-AKT1 or WT-AKT2 showed enhanced fusion compared to control myotubes, while myotubes expressing AKT1-K179M showed a 14% reduction in fusion. Myotubes expressing AKT2-K181M displayed 63% decreased fusion compared to control. Together, these data identify AKT2 as the most highly-expressed AKT isoform in human skeletal muscle and as the principal AKT isoform regulating human myoblast differentiation.

Skeletal muscle PI3K p110ß regulates expression of AMP-activated protein kinase.

Skeletal muscle metabolic homeostasis is maintained through numerous biochemical and physiological processes. Two principal molecular regulators of skeletal muscle metabolism include AMP-activated protein kinase (AMPK) and phosphatidylinositol 3-kinase (PI3K); however, PI3K exists as multiple isoforms, and specific metabolic actions of each isoform have not yet been fully elucidated in skeletal muscle. Given this lack of knowledge, we performed a series of experiments to define the extent to which PI3K p110ß mediated expression and (or) activation of AMPK in skeletal muscle. To determine the effect of p110ß inhibition on AMPK expression and phosphorylation in cultured cells, C2C12 myoblasts were treated with a pharmacological inhibitor of p110ß (TGX-221), siRNA against p110ß, or overexpression of kinase-dead p110ß. Expression and phosphorylation of AMPK were unaffected in myoblasts treated with TGX-221 or expressing kinase-dead p110ß. However, expressions of total and phosphorylated AMPK at T172 were reduced in myoblasts treated with p110ß siRNA. When normalized to expression of total AMPK, phosphorylation of AMPK S485/491 was elevated in p110ß-deficient myoblasts. Similar results were observed in tibialis anterior muscle from mice with conditional deletion of p110ß (p110ß-mKO mice). Analysis of AMPK transcript expression revealed decreased expression of Prkaa2 in p110ß-deficient myoblasts and in p110ß-mKO muscle. Loss of p110ß had no effect on oligomycin-stimulated phosphorylation of AMPK or phosphorylated Acetyl-CoA carboxylase (ACC), although oligomycin-induced AMPK and ACC phosphorylation were increased in p110ß-deficient myoblasts compared to oligomycin-stimulated control myoblasts when normalized to levels of total AMPK or ACC. Overall, these results suggest that p110ß positively regulates expression of AMPK in cultured myoblasts and in skeletal muscle in vivo; moreover, despite the reduced abundance of AMPK in p110ß-deficient myoblasts, loss of p110ß does not appear to impair AMPK activation following stimulus. These findings thus reveal a novel role for p110ß in mediating skeletal muscle metabolic signaling.

RNA transcript expression of IGF-I/PI3K pathway components in regenerating skeletal muscle is sensitive to initial injury intensity.

OBJECTIVE: Skeletal muscle regeneration is a complex process involving the coordinated input from multiple stimuli. Of these processes, actions of the insulin-like growth factor-I (IGF-I) and phosphoinositide 3-kinase (PI3K) pathways are vital; however, whether IGF-I or PI3K expression is modified during regeneration relative to initial damage intensity is unknown. The objective of this study was to determine whether mRNA expression of IGF-I/PI3K pathway components was differentially regulated during muscle regeneration in mice in response to traumatic injury induced by freezing of two different durations. DESIGN: Traumatic injury was imposed by applying a 6-mm diameter cylindrical steel probe, cooled to the temperature of dry ice (-79°C), to the belly of the left tibialis anterior muscle of 12-week-old C57BL/6J mice for either 5s (5s) or 10s (10s). The right leg served as the uninjured control. RNA was obtained from injured and control muscles following 3, 7, and 21days recovery and examined by real-time PCR. Expression of transcripts within the IGF, PI3K, and Akt families, as well as for myogenic regulatory factors and micro-RNAs were studied. RESULTS: Three days following injury, there was significantly increased expression of Igf1, Igf2, Igf1r, Igf2r, Pik3cb, Pik3cd, Pik3cg, Pik3r1, Pik3r5, Akt1, and Akt3 in response to either 5s or 10s injury compared to uninjured control muscle. There was a significantly greater expression of Pik3cb, Pik3cd, Pik3cg, Pik3r5, Akt1, and Akt3 in 10s injured muscle compared to 5s injured muscle. Seven days following injury, we observed significantly increased expression of Igf1, Igf2, Pik3cd, and Pik3cg in injured muscle compared to control muscle in response to 10s freeze injury. We also observed significantly reduced expression of Igf1r and miR-133a in response to 5s freeze injury compared to control muscle, and significantly reduced expression of Ckm, miR-1 and miR-133a in response to 10s freeze injury as compared to control. Twenty-one days following injury, 5s freeze-injured muscle exhibited significantly increased expression of Igf2, Igf2r, Pik3cg, Akt3, Myod1, Myog, Myf5, and miR-206 compared to control muscle, while 10s freeze-injured muscles showed significantly increased expression of Igf2, Igf2r, Pik3cb, Pik3cd, Pik3r5, Akt1, Akt3, and Myog compared to control. Expression of miR-1 was significantly reduced in 10s freeze-injured muscle compared to control muscle at this time. There were no significant differences in RNA expression between 5s and 10s injury at either 7d or 21d recovery in any transcript examined. CONCLUSIONS: During early skeletal muscle regeneration in mice, transcript expressions for some components of the IGF-I/PI3K pathway are sensitive to initial injury intensity induced by freeze damage.

Phosphatidylinositol 3-kinase p110α mediates phosphorylation of AMP-activated protein kinase in myoblasts.

AMP-activated protein kinase (AMPK) is a serine/threonine kinase that functions as a sensor of intracellular energy. Activation of AMPK is associated with increased phosphorylation of the α-subunit at threonine 172 (T172) and decreased phosphorylation at serine 485 in AMPKα1 and serine 491 in AMPKα2 (S485/491). One potential mediator of AMPK phosphorylation is phosphatidylinositol 3-kinase (PI3K); however, the mechanism and the identities of the specific PI3K isoforms that regulate AMPK activation are not known. To determine whether PI3K p110α regulated AMPK activation in muscle cells, C2C12 myoblasts were subjected to pharmacological inhibition of p110α, siRNA directed against p110α, or overexpression of constitutively-active or dominant negative p110α. Chemical inhibition, siRNA, and expression of dominant-negative p110α were all associated with increased AMPK T172 phosphorylation, whereas expression of constitutively-active p110α reduced T172 phosphorylation. Conversely, pharmacological inhibition of p110α reduced AMPK S485/491 phosphorylation, while constitutively-active p110α increased AMPK S485/491 phosphorylation. This p110α-mediated increase in AMPK S485/491 phosphorylation was eliminated in the presence of the Akt inhibitor MK2206, suggesting that p110α-mediated phosphorylation of AMPKα at S485/491 is Akt-dependent. In response to oligomycin or serum-starvation, AMPK T172 phosphorylation was elevated in p110α-deficient myoblasts compared to control myoblasts. Overall, our findings identify PI3K p110α as a mediator of AMPK phosphorylation in myoblasts.

Role of phosphoinositide 3-OH kinase p110ß in skeletal myogenesis.

Phosphoinositide 3-OH kinase (PI3K) regulates a number of developmental and physiologic processes in skeletal muscle; however, the contributions of individual PI3K p110 catalytic subunits to these processes are not well-defined. To address this question, we investigated the role of the 110-kDa PI3K catalytic subunit ß (p110ß) in myogenesis and metabolism. In C2C12 cells, pharmacological inhibition of p110ß delayed differentiation. We next generated mice with conditional deletion of p110ß in skeletal muscle (p110ß muscle knockout [p110ß-mKO] mice). While young p110ß-mKO mice possessed a lower quadriceps mass and exhibited less strength than control littermates, no differences in muscle mass or strength were observed between genotypes in old mice. However, old p110ß-mKO mice were less glucose tolerant than old control mice. Overexpression of p110ß accelerated differentiation in C2C12 cells and primary human myoblasts through an Akt-dependent mechanism, while expression of kinase-inactive p110ß had the opposite effect. p110ß overexpression was unable to promote myoblast differentiation under conditions of p110α inhibition, but expression of p110α was able to promote differentiation under conditions of p110ß inhibition. These findings reveal a role for p110ß during myogenesis and demonstrate that long-term reduction of skeletal muscle p110ß impairs whole-body glucose tolerance without affecting skeletal muscle size or strength in old mice.