Urinary Proteomic Biomarkers of Trabecular Bone Volume Change during Army Basic Combat Training.

PURPOSE: Optimize a dMS-based urinary proteomic technique and evaluate the relationship between urinary proteome content and adaptive changes in bone microarchitecture during BCT. METHODS: Urinary proteomes were analyzed with an optimized dMS technique in two groups of 13 recruits (n = 26) at the beginning (Pre) and end (Post) of BCT. Matched by age (21 ± 4 yr), sex (16 W), and baseline tibial trabecular bone volume fractions (Tb.BV/TV), these groups were distinguished by the most substantial (High) and minimal (Low) improvements in Tb.BV/TV. Differential protein expression was analyzed with mixed permutation ANOVA and false discovery proportion-based adjustment for multiple comparisons. RESULTS: Tibial Tb.BV/TV increased from pre- to post-BCT in High (3.30 ± 1.64%, p < 0.0001) but not Low (-0.35 ± 1.25%, p = 0.4707). The optimized dMS technique identified 10,431 peptides from 1,368 protein groups that represented 165 integrative biological processes. 74 urinary proteins changed from pre- to post-BCT (p = 0.0019) and neutrophil mediated immunity was the most prominent ontology. Two proteins (Immunoglobulin heavy constant gamma 4 and C-type lectin domain family 4 member G) differed from pre- to post-BCT in High and Low (p = 0.0006). CONCLUSIONS: The dMS technique can identify more than 1000 urinary proteins. At least 74 proteins are responsive to BCT, and other principally immune system-related proteins show differential expression patterns that coincide with adaptive bone formation.

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.

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.

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.

Insulin-like growth factor-I biocompartmentalization across blood, interstitial fluid and muscle, before and after 3 months of chronic resistance exercise.

This investigation examined the influence of 12-week ballistic resistance training programs on the IGF-I system in circulation, interstitial fluid, and skeletal muscle, at rest and in response to acute exercise. Seventeen college-aged subjects (11 women/6 men; 21.7 ± 3.7 yr) completed an acute ballistic exercise bout before and after the training program. Blood samples were collected pre-, mid-, and postexercise and analyzed for serum total IGF-I, free IGF-I, and IGF binding proteins (IGFBPs) 1-4. Dialysate and interstitial free IGF-I were analyzed in vastus lateralis (VL) interstitial fluid collected pre- and postexercise via microdialysis. Pre- and postexercise VL muscle biopsies were analyzed for IGF-I protein expression, IGF-I receptor phosphorylation (p-IGF-IR), and AKT phosphorylation (p-AKT). Following training, basal serum IGF-I, free IGF-I, IGFBP-2, and IGFBP-3 decreased whereas IGFBP-1 and IGFBP-4 increased. Training reduced basal dialysate and interstitial free IGF-I but had no effect on basal skeletal muscle IGF-I, p-IGF-IR, or p-AKT. Acute exercise elicited transient changes in IGF-I system concentrations and downstream anabolic signaling both pre- and posttraining; training did not affect this acute exercise response. Posttraining, acute exercise-induced changes in dialysate/interstitial free IGF-I were strongly correlated with the changes in intramuscular IGF-I expression, p-IGF-IR, and p-AKT. The divergent influence of resistance training on circulating/interstitial and skeletal muscle IGF-I demonstrates the importance of concurrent, multiple biocompartment analysis when examining the IGF-I system. As training elicited muscle hypertrophy, these findings indicate that IGF-I's anabolic effects on skeletal muscle are mediated by local, rather than systemic mechanisms.NEW & NOTEWORTHY In the first investigation to assess resistance training's effects on the IGF-I system in serum, interstitial fluid, and skeletal muscle, training decreased basal circulating and interstitial IGF-I but did not alter basal intramuscular IGF-I protein activity. Posttraining, acute exercise-induced interstitial IGF-I increases were strongly correlated with intramuscular IGF-I expression and signaling. These findings highlight the importance of multibiocompartment measurement when analyzing IGF-I and suggest that IGF-I's role in hypertrophic adaptations is locally mediated.

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.

Emerging evidence that adaptive bone formation inhibition by non-steroidal anti-inflammatory drugs increases stress fracture risk.

There is mounting evidence suggesting that the commonly used analgesics, non-steroidal anti-inflammatory drugs (NSAIDs), may inhibit new bone formation with physical training and increase risk of stress fractures in physically active populations. Stress fractures are thought to occur when bones are subjected to repetitive mechanical loading, which can lead to a cycle of tissue microdamage, repair, and continued mechanical loading until fracture. Adaptive bone formation, particularly on the periosteal surface of long bones, is a concurrent adaptive response of bone to heightened mechanical loading that can improve the fatigue resistance of the skeletal structure, and therefore may play a critical role in offsetting the risk of stress fracture. Reports from animal studies suggest that NSAID administration may suppress this important adaptive response to mechanical loading. These observations have implications for populations such as endurance athletes and military recruits who are at risk of stress fracture and whose use of NSAIDs is widespread. However, results from human trials evaluating exercise and bone adaptation with NSAID consumption have been less conclusive. In this review, we identify knowledge gaps that must be addressed to further support NSAID-related guidelines intended for at-risk populations and individuals.

The Central Role of Osteocytes in the Four Adaptive Pathways of Bone's Mechanostat.

We review evidence supporting an updated mechanostat model in bone that highlights the central role of osteocytes within bone's four mechanoadaptive pathways: 1) formation modeling and 2) targeted remodeling, which occur with heightened mechanical loading, 3) resorption modeling, and 4) disuse-mediated remodeling, which occur with disuse. These four pathways regulate whole-bone stiffness in response to changing mechanical demands.

A prospective field study of U.S. Army trainees to identify the physiological bases and key factors influencing musculoskeletal injuries: a study protocol.

BACKGROUND: Musculoskeletal injuries (MSKIs) are common in military trainees and present a considerable threat to occupational fitness, deployability, and overall military readiness. Despite the negative effects of MSKIs on military readiness, comprehensive evaluations of the key known and possible risk factors for MSKIs are lacking. The U.S. Army Research Institute of Environmental Medicine (ARIEM) is initiating a large-scale research effort, the ARIEM Reduction in Musculoskeletal Injury (ARMI) Study, to better understand the interrelationships among a wide range of potential MSKI risk factors in U.S. Army trainees in order to identify those risk factors that most contribute to MSKI and may be best targeted for effective mitigation strategies. METHODS: This prospective study aims to enroll approximately 4000 (2000 male and 2000 female) U.S. Army trainees undergoing Basic Combat Training (BCT). Comprehensive in-person assessments will be completed at both the beginning and end of BCT. Participants will be asked to complete surveys of personal background information, medical history, physical activity, sleep behaviors, and personality traits. Physical measurements will be performed to assess anthropometrics, tibial microarchitecture and whole body bone mineral density, muscle cross-sectional area, body composition, and muscle function. Blood sampling will be also be conducted to assess musculoskeletal, genetic, and nutritional biomarkers of risk. In addition, participants will complete weekly surveys during BCT that examine MSKI events, lost training time, and discrete risk factors for injury. Participants' medical records will be tracked for the 2 years following graduation from training to identify MSKI events and related information. Research hypotheses focus on the development of a multivariate prediction model for MSKI. DISCUSSION: Results from this study are expected to inform current understanding of known and potential risk factors for MSKIs that can be incorporated into solutions that optimize Soldier health and enhance military readiness.

Nonsteroidal Anti-Inflammatory Drug Prescriptions Are Associated With Increased Stress Fracture Diagnosis in the US Army Population.

Stress fractures are common in military personnel and endurance athletes, and nonsteroidal anti-inflammatory drug (NSAID) use is widespread in these populations. NSAIDs inhibit prostaglandin synthesis, which blunts the anabolic response of bone to physical activity and could therefore increase risk of stress fracture. The objective of this study was to determine whether prescribed NSAIDs were associated with stress fracture diagnoses among US Army soldiers. We also aimed to establish whether acetaminophen, an analgesic alternative to NSAIDs, was associated with stress fracture risk. A nested case-control study was conducted using data from the Total Army Injury and Health Outcomes Database from 2002 to 2011 (n = 1,260,168). We identified soldiers with a diagnosis of stress fracture (n = 24,146) and selected 4 controls per case matched on length of military service (n = 96,584). We identified NSAID and acetaminophen prescriptions 180 to 30 days before injury (or match date). We also identified soldiers who participated in basic combat training (BCT), a 10-week period of heightened physical activity at the onset of Army service. Among these individuals, we identified 9088 cases and 36,878 matched controls. Conditional logistic regression was used to calculate incident rate ratios (RR) for stress fracture with adjustment for sex. NSAID prescription was associated with a 2.9-fold increase (RR = 2.9, 95% confidence interval [CI] 2.8-2.9) and acetaminophen prescription with a 2.1-fold increase (RR = 2.1, 95% CI 2.0-2.2) in stress fracture risk within the total Army population. The risk was more than 5-fold greater in soldiers prescribed NSAIDs (RR = 5.3, 95% CI 4.9-5.7) and more than 4-fold greater in soldiers prescribed acetaminophen (RR = 4.4, 95% CI 3.9-4.9) during BCT. Our results reveal an association between NSAID and acetaminophen prescriptions and stress fracture risk, particularly during periods of heightened physical activity. Prospective observational studies and randomized controlled trials are needed to support these findings before clinical recommendations can be made. © 2018 American Society for Bone and Mineral Research.

Characterization of growth hormone disulfide-linked molecular isoforms during post-exercise release vs nocturnal pulsatile release reveals similar milieu composition.

OBJECTIVE: To characterize the influence of mode (aerobic/resistance) and volume of exercise (moderate/high) on circulating GH immediately post-exercise as well as following the onset of sleep. DESIGN: This study used repeated measures in which subjects randomly completed 5 separate conditions: control (no exercise), moderate volume resistance exercise (MR), high-volume resistance exercise (HR), moderate volume aerobic exercise (MA), and high volume aerobic exercise (HA). METHODS: Subjects had two overnight stays on each of the 5 iterations. Serial blood draws began as soon as possible after the completion of the exercise session. Blood was obtained every 20 min for 24-h. GH was measured using a chemiluminescent immunoassay. Pooled samples representing post exercise (PE) and first nocturnal pulse (NP) were divided into two aliquots. One of these aliquots was chemically reduced by adding 10 mM glutathione (GSH) to break down disulfide-linked aggregates. RESULTS: No differences were observed when pooling GH response at post-exercise (2.02 ±â€¯0.21) and nocturnal pulse (2.63 ±â€¯0.51; p = .32). Pairwise comparisons revealed main effect differences between controls (1.19 ±â€¯0.29) and both MA (2.86 ±â€¯0.31; p = .009) and HA (3.73 ±â€¯0.71; p = .001). Both MA (p = .049) and HA (p = .035) responses were significantly larger than the MR stimulus (1.96 ±â€¯0.28). With GSH reduction, controls significantly differed from MA (p = .018) and HA (p = .003) during PE, but only differed from HA (p = .003) during NP. CONCLUSIONS: This study demonstrated similar GH responses to exercise and nocturnal pulse, indicating that mode and intensity of exercise does not proportionately affect GH dimeric isoform concentration.

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.

Paracellular calcium flux across Caco-2 cell monolayers: Effects of individual amino acids.

High-protein diets are generally considered beneficial for calcium (Ca) economy and bone health. Improved intestinal Ca absorption efficiency may be one mechanism by which higher-protein diets affect Ca homeostasis and bone health. The signaling pathways and individual amino acids (AA) responsible for this effect have not been fully elucidated and may involve the transcellular pathway, paracellular pathway or a combination. The primary aim of this study was to investigate whether a mixture of AA and/or functionally distinct individual AA directly affect paracellular Ca absorption across an intestinal epithelial cell model (Caco-2 Bbe). Using Ussing chambers, we examined the effect of six treatments - vehicle (Veh), 80 mM raffinose (Raf; positive control), 2× mixed AA(2×AA, twice the concentration in standard growth media), the branched-chain amino acid leucine (2-10 mM Leu), the aromatic amino acid phenylalanine (2-10 mM Phe) and the dibasic amino acid lysine (2-10 mM Lys) - on Ca flux. Leu (5 mM) increased Ca flux by 38% (+122 nmol Ca/cm2/h, P<.001) as compared to Veh, while 10 mM Phe reduced Ca flux. No other differences were observed. Leu increased Ca flux through cellular redistribution of the Ca permissive channel Cldn-2 to the tight junction membrane (P<.05). Inhibition of mTORC1 signaling did not abrogate the effect of Leu on Cldn-2 localization, indicating a non-mTORC1-dependent signaling pathway is involved. These data indicate that Leu may improve Ca absorption in a cell model, potentially contributing to the observed benefits of higher-protein diets on bone health in humans.

Changes in tibial bone microarchitecture in female recruits in response to 8 weeks of U.S. Army Basic Combat Training.

BACKGROUND: U.S. Army Basic Combat Training (BCT) is a physically-demanding program at the start of military service. Whereas animal studies have shown that increased mechanical loading rapidly alters bone structure, there is limited evidence of changes in bone density and structure in humans exposed to a brief period of unaccustomed physical activity. PURPOSE: We aimed to characterize changes in tibial bone density and microarchitecture and serum-based biochemical markers of bone metabolism in female recruits as a result of 8 weeks of BCT. METHODS: We collected high-resolution peripheral quantitative computed tomographic images of the distal tibial metaphysis and diaphysis (4% and 30% of tibia length from the distal growth plate, respectively) and serum markers of bone metabolism before and after BCT. Linear mixed models were used to estimate the mean difference for each outcome from pre- to post-BCT, while controlling for race/ethnicity, age, and body mass index. RESULTS: 91 female BCT recruits volunteered and completed this observational study (age = 21.5 ±â€¯3.3 yrs). At the distal tibial metaphysis, cortical thickness, trabecular thickness, trabecular number, bone volume/total volume, and total and trabecular volumetric bone density (vBMD) increased significantly by 1-2% (all p < 0.05) over the BCT period, whereas trabecular separation, cortical tissue mineral density (TMD), and cortical vBMD decreased significantly by 0.3-1.0% (all p < 0.05). At the tibial diaphysis, cortical vBMD and cortical TMD decreased significantly (both -0.7%, p < 0.001). Bone strength, estimated by micro finite element analysis, increased by 2.5% and 0.7% at the distal tibial metaphysis and diaphysis, respectively (both p < 0.05). Among the biochemical markers of bone metabolism, sclerostin decreased (-5.7%), whereas bone alkaline phosphatase, C-telopeptide cross-links of type 1 collagen, tartrate-resistance acid phosphatase, and 25(OH)D increased by 10-28% (all p < 0.05). CONCLUSION: BCT leads to improvements in trabecular bone microarchitecture and increases in serum bone formation markers indicative of new bone formation, as well as increases in serum bone resorption markers and decreases in cortical vBMD consistent with intracortical remodeling. Together, these results demonstrate specific changes in trabecular and cortical bone density and microarchitecture following 8 weeks of unaccustomed physical activity in women.

Circulating sclerostin is not suppressed following a single bout of exercise in young men.

The aim of this study was to determine whether an acute bout of exercise reduces serum sclerostin under diet-controlled conditions that stabilize the parathyroid hormone (PTH)-1,alpha-hydroxylase axis. Fourteen male volunteers (age, 22.1 years ± 4.05; BMI, 27.3 kg/m2  ± 3.8) completed a randomized crossover study in which they performed 10 sets of 10 repetitions of plyometric jumps at 40% of their estimated one-repetition maximum leg press or a nonexercise control period. A calcium-controlled diet (1000 mg/day) was implemented prior to, and throughout each study period. Blood was drawn for analysis of serum sclerostin, Dickkopf-1, markers of bone metabolism (PTH, calcium), markers of bone formation (bone alkaline phosphatase, BAP; osteocalcin, OCN), and markers of bone resorption (tartrate-resistant acid phosphatase 5b, TRAP5b; C-telopeptide cross-links of type I collagen, CTX) at baseline and 12, 24, 48, and 72 h following exercise or rest. Changes in serum concentrations were expressed as percentage change from individual baselines. Data were analyzed using a repeated measured linear mixed model to assess effects of time, physical activity status (rest or exercise condition), and the time by activity status interaction. There was a significant effect of exercise on OCN (P = 0.005) and a significant interaction effect for CTX (P = 0.001). There was no effect of exercise on any other biochemical marker of bone metabolism. A single bout of plyometric exercise did not induce demonstrable changes in biochemical markers of bone metabolism under conditions where dietary effects on PTH were controlled.

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.

Risk of Stress Fracture Varies by Race/Ethnic Origin in a Cohort Study of 1.3 Million US Army Soldiers.

Stress fractures (SF) are common and costly injuries in military personnel. Risk for SF has been shown to vary with race/ethnicity. Previous studies report increased SF risk in white and Hispanic Soldiers compared with black Soldiers. However, these studies did not account for the large ethnic diversity in the US military. We aimed to identify differences in SF risk among racial/ethnic groups within the US Army. A retrospective cohort study was conducted using data from the Total Army Injury and Health Outcomes Database from 2001 until 2011. SF diagnoses were identified from ICD-9 codes. We used Cox-proportional hazard models to calculate time to SF by racial/ethnic group after adjusting for age, education, and body mass index. We performed a sex-stratified analysis to determine whether the ethnic variation in SF risk depends on sex. We identified 21,549 SF cases in 1,299,332 Soldiers (more than 5,228,525 person-years of risk), revealing an overall incidence rate of 4.12 per 1000 person-years (7.47 and 2.05 per 1000 person-years in women and men, respectively). Using non-Hispanic blacks as the referent group, non-Hispanic white women had the highest risk of SF, with a 92% higher risk of SF than non-Hispanic black women (1.92 [1.81-2.03]), followed by American Indian/Native Alaskan women (1.72 [1.44-1.79]), Hispanic women (1.65 [1.53-1.79]), and Asian women (1.32 [1.16-1.49]). Similarly, non-Hispanic white men had the highest risk of SF, with a 59% higher risk of SF than non-Hispanic black men (1.59 [1.50-1.68]), followed by Hispanic men (1.19 [1.10-1.29]). When examining the total US Army population, we found substantial differences in the risk of stress fracture among racial/ethnic groups, with non-Hispanic white Soldiers at greatest risk and Hispanic, American Indian/Native Alaskan, and Asian Soldiers at an intermediate risk. Additional studies are needed to determine the factors underlying these race- and ethnic-related differences in stress fracture risk. © 2017 American Society for Bone and Mineral Research.

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.