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.

Body composition changes during 8 weeks of military training are not accurately captured by circumference-based assessments.

In 1981, the US military adopted body fat standards to promote physical readiness and prevent obesity. Separate circumference-based equations were developed for women and men. Both predictive equations were known to underestimate %BF. However, it was not known how well these abdominal circumference-based methods tracked changes in %BF. This study examined the validity of the circumference-based %BF equations for assessing changes in %BF in young adult recruits during Army Basic Combat Training (BCT). Dual-energy X-ray absorptiometry (DXA) and circumference-based measures of %BF were obtained in women (n = 481) and men (n = 926) at the start (pre-BCT) and end (post-BCT) of 8 weeks of BCT. Repeated-measure ANOVAs were used to assess differences between DXA and circumference pre-BCT and for the change during BCT. Pre-BCT, circumferences underestimated %BF relative to DXA, with mean errors of -6.0% ± 4.4% for women and -6.0% ± 3.5% for men (both p < 0.01), and no difference between sexes was observed (p = 0.77). DXA detected a -4.0% ± 2.4% and -3.3% ± 2.8% change in %BF for women and men in response to BCT, respectively (both p < 0.01), whereas circumference estimates of %BF indicated a 0.0% ± 3.3% (p = 0.86) change in women and a -2.2% ± 3.3% (p < 0.01) change in men (sex difference by technique p < 0.01). In conclusion, circumference-based measures underestimated %BF at the start of BCT in both sexes as compared to DXA. Circumference measures underestimated changes in %BF during BCT in men and did not detect changes in women. These findings suggest that circumference-based %BF metrics may not be an appropriate tool to track changes in body composition during short duration training.

The Risk of Menstrual Dysfunction Increases for Women during U.S. Army Basic Combat Training.

PURPOSE: To determine whether changes in menstruation develop in female trainees during BCT and whether changes in body mass, body composition and/or physical activity are associated with menstrual interruption during BCT. METHODS: Female trainees grouped according to self-reported menstrual status in the 12 months before BCT as having regular cycles (RC; n = 352) or MD ( n = 97) completed height, body mass, and body composition assessments and questionnaires before and after BCT. Fisher's exact test and Mann-Whitney U test were used to compare between-group differences in categorical and continuous variables, respectively. Among RC trainees, odds ratios were calculated to examine the influence of changes in body mass, lean mass, and fat mass on a trainee's likelihood to miss a period during BCT. RESULTS: There were no differences in race, height, body mass, body mass index, or physical activity history at pre-BCT between RC and MD ( P > 0.05). Overall, 86% of trainees experienced changes to menstruation during BCT. RC were more likely than MD to have at least one period during BCT (81% vs 69%, respectively, P = 0.01). Among RC, gaining more body mass and lean mass and losing less fat mass were associated with increased odds of missing a period during BCT. CONCLUSIONS: These findings demonstrate that most female trainees experience menstrual changes during BCT. Menstrual cycle interruptions do not appear to align with loss of body or fat mass.

MyD88 and not TRIF knockout is sufficient to abolish LPS-induced inflammatory responses in bone-derived macrophages.

Macrophages play an important role in the response to infection and/or repair of injury in tissues. To examine the NF-κB pathway in response to an inflammatory stimulus, we used wild-type bone-marrow-derived macrophages (BMDMs) or BMDMs with knockout (KO) of myeloid differentiation primary response 88 (MyD88) and/or Toll/interleukin-1 receptor domain-containing adapter-inducing interferon-ß (TRIF) via CRISPR/Cas9. Following treatment of BMDMs with lipopolysaccharide (LPS) to induce an inflammatory response, translational signalling of NF-κB was quantified via immunoblot and cytokines were measured. Our findings reveal that MyD88 KO, but not TRIF KO, decreased LPS-induced NF-κB signalling, and 10% expression of basal MyD88 expression was sufficient to partially rescue the abolished inflammatory cytokine secretion observed upon MyD88 KO.

Comparison of CRISPR and adenovirus-mediated Myd88 knockdown in RAW 264.7 cells and responses to lipopolysaccharide stimulation.

Genomic manipulation offers the possibility for novel therapies in lieu of medical interventions in use today. The ability to genetically restore missing inflammatory genes will have a monumental impact on our current immunotherapy treatments. This study compared the efficacy of two different genetic manipulation techniques: clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) transfection to adenoviral transduction to determine which method would provide the most transient and stable knockdown of myeloid differentiation primary response 88 (MyD88). MyD88 is a major regulator of nuclear factor kappa light chain enhancer of activated B cells (NFκB) pathway in Raw 264.7 macrophages. Following genetic manipulation, cells were treated for 24 h with Lipopolysaccharide (LPS) to stimulate the inflammatory pathway. Confirmation of knockdown was determined by western immunoblotting and quantification of band density. Both CRISPR/Cas9 and adenoviral transduction produced similar knockdown efficiency (~64% and 60%, respectively) in MyD88 protein 48 h post adenoviral transduction. NFκB phosphorylation was increased in CRISPR/Cas9-mediated MyD88 knockdown and control cells, but not in adenovirus-mediated MyD88 knockdown cells, following LPS administration. CRISPR/Cas9-mediated MyD88 knockdown macrophages treated with LPS for 24 h showed a 65% reduction in tumor necrosis factor alpha (TNFα) secretion, and a 67% reduction in interleukin-10 (IL-10) secretion when compared to LPS-stimulated control cells (P ≤ 0.01 for both). LPS did not stimulate TNFα or IL-10 secretion in adenovirus-mediated control or MyD88 knockdown cells. These data demonstrate that Raw 264.7 macrophages maintain responsiveness to inflammatory stimuli following CRISPR/Cas9-mediated reductions in MyD88, but not following adenovirus-mediated MyD88 knockdown.

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.

Sea anemone model has a single Toll-like receptor that can function in pathogen detection, NF-κB signal transduction, and development.

In organisms from insects to vertebrates, Toll-like receptors (TLRs) are primary pathogen detectors that activate downstream pathways, specifically those that direct expression of innate immune effector genes. TLRs also have roles in development in many species. The sea anemone Nematostella vectensis is a useful cnidarian model to study the origins of TLR signaling because its genome encodes a single TLR and homologs of many downstream signaling components, including the NF-κB pathway. We have characterized the single N. vectensis TLR (Nv-TLR) and demonstrated that it can activate canonical NF-κB signaling in human cells. Furthermore, we show that the intracellular Toll/IL-1 receptor (TIR) domain of Nv-TLR can interact with the human TLR adapter proteins MAL and MYD88. We demonstrate that the coral pathogen Vibrio coralliilyticus causes a rapidly lethal disease in N. vectensis and that heat-inactivated V. coralliilyticus and bacterial flagellin can activate a reconstituted Nv-TLR-to-NF-κB pathway in human cells. By immunostaining of anemones, we show that Nv-TLR is expressed in a subset of cnidocytes and that many of these Nv-TLR-expressing cells also express Nv-NF-κB. Additionally, the nematosome, which is a Nematostella-specific multicellular structure, expresses Nv-TLR and many innate immune pathway homologs and can engulf V. coralliilyticus Morpholino knockdown indicates that Nv-TLR also has an essential role during early embryonic development. Our characterization of this primitive TLR and identification of a bacterial pathogen for N. vectensis reveal ancient TLR functions and provide a model for studying the molecular basis of cnidarian disease and immunity.