Anabolic Response Research Articles

Pea and soy fortified with leucine stimulates muscle protein synthesis comparable to whey in a murine ageing model

PurposeTo meet the global dietary protein demands, a trend towards plant-based protein (PBP) sources to replace animal-derived protein is currently ongoing. However, PBPs may not have the same anabolic capacity to stimulate muscle protein synthesis (MPS) as dairy proteins. For vulnerable populations with specific medical needs, it is especially important to validate the anabolic properties of PBPs. In this study, a blend of pea and soy protein isolate, with or without additional leucine, was compared to whey protein isolate on MPS in aged mice.Methods25-Months aged C57BL/6J-mice received an oral gavage with 70 mg of whey protein isolate (W), PS protein isolate (PS; ratio 51:49), PS fortified with 19% leucine (PS + L), or 0.5mL water (F). Mice were subcutaneously injected with puromycin (0.04 µmol/g body weight, t = 30 min) and sacrificed 60 min thereafter. Left m. tibialis anterior (TA) was used to analyse MPS by the SUnSET method and mTOR signal transduction proteins. Amino acid concentrations were determined in plasma and right TA. Dried blood spots (DBS) were analysed for postprandial dynamics of amino acids at 10-20-45-60-min.ResultsMPS was significantly increased by W and PS + L (p < 0.003), however not by PS. Pathway protein 4EBP1 showed significant increases with W, PS and PS + L to F (p < 0.0002). W and PS + L increased plasma and muscle free leucine equally, which was confirmed by DBS.ConclusionA PS blend fortified with leucine stimulates MPS comparable to whey protein in this acute murine ageing model. Leucine appears to be the main driver for the anabolic responses observed.

Timing of cartilage articulation following impact injury affects the response of surface zone chondrocytes.

Post-traumatic osteoarthritis develops following an inciting injury to a joint and results in cartilage degeneration. Mechanical loading, including articulation, drives anabolic responses in cartilage clinically, in vivo, and in vitro. Tribological articulation, or sliding of cartilage on a glass counterface, has long been used as an in vitro tool to study cartilage tissue behavior. However, it is unclear if tribological articulation affects chondrocyte fate following injury, and if the timing of articulation impacts the resultant effect. The goal of this study was to investigate the effect of tribological articulation on injured cartilage tissue at two time points: (i) performed immediately after injury and (ii) 24 h after injury. Neonatal bovine femoral cartilage explants were injured using a rapid spring-loaded impactor and subsequently subjected to tribological articulation. Cell death due to impact injury was highest near the articular surface, suggesting a strain-dependent mechanism. Immediate articulation following injury mitigated cell death compared to injury alone or delayed articulation; markers for both general cell death and early-stage apoptosis were markedly decreased in the explants that were immediately slid. Interestingly, mitigation of cell death due to sliding was most predominant at the cartilage surface. Tribological articulation is known to create fluid flow within the tissue, predominantly at the articular surface, which could drive the protective response seen here. Altogether, this work shows that perturbations to the cellular environment immediately following cartilage injury significantly impact chondrocyte fate.

Underpowered studies in muscle metabolism research: Determinants and considerations

Biomedical research frequently employs null hypothesis testing to determine whether an observed difference in a sample is likely to exist in the broader population. Null hypothesis testing generally assumes that differences between groups or interventions are non-existent, unless proven otherwise. Because biomedical studies with human subjects are often limited by financial and logistical resources, they tend to have low statistical power, i.e. a low probability of statistically confirming a true difference. As a result, small but potentially clinically important differences may be overseen or ignored simply due to the absence of a statistically significant difference. This absence is often misinterpreted as ‘equivalence’ of treatments. In this educational paper, we will use practical examples related to the effects of exercise and nutrition on muscle protein metabolism to illustrate the most important determinants of statistical power, as well as their implications for both investigators and readers of scientific articles.Changes in muscle mass occur at a relatively slow rate, making it practically challenging to detect differences between treatment groups in a long-term setting. One way to make it ‘easier’ to differentiate between groups and hence increase statistical power is to have a sufficiently long study duration to allow treatment effects to become apparent. This is especially relevant when comparing treatments with relatively small expected differences such as the effect of modest changes in daily protein intake. Secondly, one could try to minimize the variance and response heterogeneity within groups, for example by using strict inclusion criteria and standardization protocols (e.g., meal provision), by using cross-over designs, or even within-subject designs where two interventions are compared simultaneously (e.g., studying an exercised limb vs a contralateral control limb) although this might limit the generalizability of the findings (e.g. such single-limb exercise training is not common in practice). In terms of data interpretation, investigators should obviously refrain from drawing strong conclusions from underpowered studies. Yet, such studies still provide valuable data for meta-analyses. Finally, because muscle protein synthesis rates are highly responsive to anabolic stimuli, acute metabolic studies are more sensitive to detect potentially clinically relevant differences in the anabolic response between treatments. Apart from further elaborating on these topics, this educational article encourages readers to more critically question null findings and scientists to more clearly discuss limitations that may have compromised statistical power.

Profiling growth performance, insulin-like growth factors (IGFs), and IGF-binding proteins (IGFBPs) in rainbow trout lacking IGFBP-2b.

Insulin-like growth factor binding proteins (IGFBP) regulate insulin-like growth factor (IGF) signaling, but IGFBP-specific functions are not well characterized in fishes. A line of rainbow trout (Oncorhynchus mykiss) lacking a functional IGFBP-2b was produced using gene editing and subsequent breeding to an F2 generation. This loss-of-function model (2bKO) was subjected to either continuous feeding or feed deprivation (3 wk) followed by refeeding (1 wk). During continuous feeding, the 2bKO line displayed faster specific growth rate for both body weight and fork length, higher feed intake, and reduced feed conversion ratio compared to a wild type (WT) line. However, loss of IGFBP-2b did not affect the feed deprivation or refeeding response in terms of weight loss or weight gain, respectively. Several components of the IGF/IGFBP system were affected by loss of IGFBP2b. Total serum IGF-1 in the 2bKO line was reduced to 0.5 - 0.8-fold of the WT line although the concentration of free serum IGF-1 was not affected. Gene expression differences include reduced abundance of igfbp1a1, igfbp1b2, igfbp5b2, and igfbp6b1 transcripts, and elevated igf2 and igfbp6b2 transcripts in liver of the 2bKO line. Collectively, these findings suggest that although IGFBP-2b is a carrier of circulating IGF-1 in salmonids, the presence of IGFBP-2a and compensatory responses of other IGF/IGFBP system components support an anabolic response that improved growth performance in the loss-of-function model.

Bioavailable testosterone and androgen receptor activation, but not total testosterone, are associated with muscle mass and strength in females.

Testosterone, the major androgen, influences the reproductive and non-reproductive systems in males and females via binding to the androgen receptor (AR). Both circulating endogenous testosterone and muscle AR protein content are positively associated with muscle mass and strength in males, but there is no such evidence in females. Here, we tested whether circulating testosterone levels were associated with muscle mass, function, or the muscle anabolic response to resistance training in pre-menopausal females. Twenty-seven pre-menopausal, untrained females (aged 23.5 ± 4.8 years) underwent a 12-week resistance training programme. Muscle strength, size, power, and plasma and urine androgen hormone levels were measured. Skeletal muscle biopsies were collected before and after the training programme to quantify the effect of resistance training on AR content and nuclear localisation. Primary muscle cell lines were cultured from a subset (n=6) of the participants' biopsies and treated with testosterone to investigate its effect on myotube diameter, markers of muscle protein synthesis and AR cellular localisation. Physiological levels of total testosterone were not associated with muscle mass or strength at baseline or with the changes in muscle mass and strength that occurred in response to resistance training in our cohort of pre-menopausal females. In contrast, bioavailable testosterone and the proportion of nuclear-localised AR were positively associated with skeletal muscle mass and strength in pre-menopausal females. In vitro, supra-physiological doses of testosterone increased myocyte diameter, but this did not occur via the Akt/mTOR pathway as previously suggested. Instead, we show a marked increase in AR nuclear localisation with testosterone administration in vitro. KEY POINTS: Total circulating testosterone was not related to muscle mass or strength before or after resistance training in pre-menopausal females. Bioavailable testosterone was positively related to exercise-induced muscle hypertrophy in pre-menopausal females. In vivo nuclear localisation of the androgen receptor was positively related to muscle mass in pre-menopausal females at baseline, but not to resistance training-induced hypertrophy. Testosterone treatment induced androgen receptor nuclear translocation but did not induce mTOR signalling in primary skeletal myocytes cultured from pre-menopausal female muscle.

Beta2-Adrenergic Stimulation Induces Resistance Training-Like Adaptations in Human Skeletal Muscle: Potential Role of KLHL41.

Skeletal muscle mass plays a pivotal role in metabolic function, but conditions such as bed rest or injury often render resistance training impractical. The beta2-adrenergic receptor has been highlighted as a potential target to promote muscle hypertrophy and treat atrophic conditions. Here, we investigate the proteomic changes associated with beta2-adrenergic-mediated muscle hypertrophy, using resistance training as a hypertrophic comparator. We utilize MS-based proteomics to map skeletal muscle proteome remodeling in response to beta2-adrenergic stimulation or resistance training as well as cell model validation. We report that beta2-adrenergic stimulation mimics multiple features of resistance training in proteome-wide remodeling, comprising systematic upregulation of ribosomal subunits and concomitant downregulation of mitochondrial proteins. Approximately 20% of proteins were regulated in both conditions, comprising proteins involved in steroid metabolism (AKR1C1, AKR1C2, AKRC1C3), protein-folding (SERPINB1), and extracellular matrix organization (COL1A1, COL1A2). Among overall most significantly upregulated proteins were kelch-like family members (KLHL) 40 and 41. In follow-up experiments, we identify KLHL41 as having novel implications for beta2-adrenergic-mediated muscle hypertrophy. Treating C2C12 cells with beta2-agonist for 96 h increased myotube diameter by 48% (p < 0.001). This anabolic effect was abolished by prior knockdown of KLHL41. Using siRNA, KLHL41 abundance was decreased by 60%, and the anabolic response to beta2-agonist was diminished (+ 15%, i.e., greater in the presence of KLHL41, knock-down × treatment: p = 0.004). In conclusion, protein-wide remodeling induced by beta2-adrenergic stimulation mimics multiple features of resistance training, and thus the beta2-adrenergic receptor may be a target with therapeutic potential in the treatment of muscle wasting conditions without imposing mechanical load.

PSII-18 Prematurity alters nutrient signaling and protein synthesis in skeletal muscle of neonatal piglets

Abstract Extrauterine growth restriction is common in infants born preterm, and negatively affects lean mass accretion and health. Prematurity has been shown to inhibit the feeding-induced stimulation of protein synthesis in skeletal muscle. We hypothesized that nutrient signaling, activation of the mechanistic target of rapamycin complex 1 (mTORC1), and protein synthesis in the muscle are limited in preterm infants which negatively impacts muscle growth. The objective of this study was to examine how prematurity influences the mechanisms involved in insulin- and amino acid-induced signaling and protein synthesis in skeletal muscle of piglets delivered either 9 d preterm (104 d, n = 25) or at term (112 d, n = 26) by cesarean section. After resuscitation, they were surgically implanted with jugular vein and carotid artery catheters and placed in individual incubators. They were randomly allotted to one of three treatments within each preterm and term birth groups: euaminoacidemic-euglycemic (FAST), hyperinsulinemic-euaminoacidemic-euglycemic (INS), or euinsulinemic-hyperaminoacidemic-euglycemic (AA) clamps on 4 d after birth. After the clamp procedure, the piglets were euthanized to collect longissimus dorsi muscle for estimating in vivo fractional protein synthesis rates and the abundance and activation of components related to insulin and amino acid signaling. Data were analyzed using the MIXED procedure of SAS. The leucine sensor, Sestrin1 bound to GATOR2 (P &amp;lt; 0.05) was reduced in response to AA whereas the abundance of the mTOR-RagA and mTOR-RagC complexes increased (P &amp;lt; 0.05). The phosphorylation of Akt was increased (P &amp;lt; 0.05) in response to INS and the phosphorylation of mTORC1 and protein synthesis increased (P &amp;lt; 0.05) in response to both AA and INS. Prematurity did not affect the protein abundances of the AA transporters for glutamine (SLC38A2), leucine (SLC7A5), and arginine (SLC38A9). However, prematurity reduced (P &amp;lt; 0.05) the abundances of the leucine sensors SAR1B, the Sestrin1-GATOR complex, the AA sensor, RAB1A, and the threonine sensor, TARS2 (P &amp;lt; 0.05). The abundances of the glutamine sensor, ARF1, the arginine sensor, CASTOR, and the methionine-SAMTOR GATOR complex were similar. Prematurity reduced (P &amp;lt; 0.05) the abundance of the mTOR-RagA and mTOR-RagC complexes, the phosphorylation of mTORC1 and muscle protein synthesis (P &amp;lt; 0.05). In conclusion, prematurity negatively affects protein synthesis in skeletal muscle of neonatal preterm piglets by blunting anabolic pathways responsible for nutrient-sensing and mTORC1 activation. The reduced anabolic response likely contributes to reduced lean growth and extrauterine growth restriction following preterm birth.

Focal adhesion kinase regulates tendon cell mechanoresponse and physiological tendon development.

Tendons enable locomotion by transmitting high tensile mechanical forces between muscle and bone via their dense extracellular matrix (ECM). The application of extrinsic mechanical stimuli via muscle contraction is necessary to regulate healthy tendon function. Specifically, applied physiological levels of mechanical loading elicit an anabolic tendon cell response, while decreased mechanical loading evokes a degradative tendon state. Although the tendon response to mechanical stimuli has implications in disease pathogenesis and clinical treatment strategies, the cell signaling mechanisms by which tendon cells sense and respond to mechanical stimuli within the native tendon ECM remain largely unknown. Therefore, we explored the role of cell-ECM adhesions in regulating tendon cell mechanotransduction by perturbing the genetic expression and signaling activity of focal adhesion kinase (FAK) through both invitro and invivo approaches. We determined that FAK regulates tendon cell spreading behavior and focal adhesion morphology, nuclear deformation in response to applied mechanical strain, and mechanosensitive gene expression. In addition, our data reveal that FAK signaling plays an essential role in invivo tendon development and postnatal growth, as FAK-knockout mouse tendons demonstrated reduced tendon size, altered mechanical properties, differences in cellular composition, and reduced maturity of the deposited ECM. These data provide a foundational understanding of the role of FAK signaling as a critical regulator of insitu tendon cell mechanotransduction. Importantly, an increased understanding of tendon cell mechanotransductive mechanisms may inform clinical practice as well as lead to the discovery of diagnostic and/or therapeutic molecular targets.

The Anabolic Response to Protein Ingestion During Recovery From Exercise Has No Upper Limit in Magnitude and Duration In Vivo in Humans: A Commentary.

A comprehensive recent study by Trommelen et al. demonstrated that muscle tissue exhibits a greater capacity to incorporate exogenous exogenous protein-derived amino acids into bound muscle protein than was previously appreciated, at least when measured in "anabolically sensitive," recreationally active (but not resistance-trained), young men following resistance exercise. Moreover, this study demonstrated that the duration of the postprandial period is modulated by the dose of ingested protein contained within a meal, that is, the postexercise muscle protein synthesis response to protein ingestion was more prolonged in 100PRO than 25PRO. Both observations represent important scientific advances in the field of protein metabolism. However, we respectfully caution that the practical implications of these findings may have been misinterpreted, at least in terms of dismissing the concept of protein meal distribution as an important factor in optimizing muscle tissue anabolism and/or metabolic health. Moreover, based on emerging evidence, this idea that the anabolic response to protein ingestion has no upper limit does not appear to translate to resistance-trained young women.

The anabolic response to a ground beef patty and soy-based meat alternative: a randomized controlled trial

BackgroundSoy-based meat alternatives (SBMA) are becoming increasingly popular, but it is unclear if they have the same anabolic effect on skeletal muscle as animal meat. ObjectivesWe aimed to compare the stimulation of skeletal muscle protein synthesis by consumption of 1 or two 4 oz patties of SBMA with 4 oz (80% protein/20% fat) beef. MethodsThe study design was a randomized controlled trial. Participants were aged 18–40 y of age and in good general health with a body mass index (kg/m2) between 20 and 32. Stable isotope tracer methods were used (L-[ring-2H5] phenylalanine, [U-13C9-15N]- tyrosine, and L-[ring-2H4] tyrosine) to quantify the response of muscle protein fractional synthetic rate (FSR) to consumption of a single beef (4 oz), single SBMA (4 oz), or two 4 oz SBMA patties (8 oz). Whole-body rates of protein synthesis, breakdown, and net balance, as well as plasma essential amino acid concentrations, were also measured. ResultsThe increase above basal in muscle protein FSR following consumption of the 4 oz beef patty (0.020 ± 0.016%/h) was significantly greater than the increase following consumption of 4 oz SBMA (P = 0.021; 0.003 ± 0.010%/h) but not 8 oz SBMA (P = 0.454; 0.013 ± 0.016%/h). The maximal essential amino acid concentration was significantly correlated (P = 0.046; r = 0.411) with the change in muscle FSR from the basal to the postprandial period. In addition, the change in muscle FSR from the basal to postprandial period was significantly correlated (P = 0.046; r = 0.412) with the corresponding change in whole-body protein synthesis. ConclusionsConsumption of a 4 oz beef patty stimulates muscle and whole-body protein synthesis >4 oz SBMA patty and similarly to 8 oz of SBMA.This trial was registered at clinicaltrials.gov as NCT05197140.

Mitigating aging and doxorubicin induced bone loss in mature mice via mechanobiology based treatments

Aging leads to a reduced anabolic response to mechanical stimuli and a loss of bone mass and structural integrity. Chemotherapy agents such as doxorubicin exacerbate the degeneration of aging skeleton and further subject older cancer patients to a higher fracture risk. To alleviate this clinical problem, we proposed and tested a novel mechanobiology-based therapy. Building upon prior findings that i) Yoda1, the Piezo1 agonist, promoted bone growth in young adult mice and suppressed bone resorption markers in aged mice, and ii) moderate tibial loading protected bone from breast cancer-induced osteolysis, we hypothesized that combined Yoda1 and moderate loading would improve the structural integrity of adult and aged skeletons in vivo and protect bones from deterioration after chemotherapy. We first examined the effects of 4-week Yoda1 (dose 5 mg/kg, 5 times/week) and moderate tibial loading (4.5 N peak load, 4 Hz, 300 cycles for 5 days/week), individually and combined, on mature mice (∼50 weeks of age). Combined Yoda1 and loading was found to mitigate age-associated cortical and trabecular bone loss better than individual interventions. As expected, the non-treated controls experienced an average drop of cortical polar moment of inertia (Ct.pMOI) by −4.3 % over four weeks and the bone deterioration occurred in the majority (64 %) of the samples. Relative to no treatment, loading alone, Yoda1 alone, and combined Yoda1 and loading increased Ct.pMOI by +7.3 %, +9.5 %, +12.0 % and increased the % of samples with positive Ct.pMOI changes by +32 %, +26 %, and +43 %, respectively, suggesting an additive protection of aging-related bone loss for the combined therapy. We further tested if the treatment efficacy was preserved in mature mice following two weeks (six injections) of doxorubicin at the dose of 2.5 or 5 mg/kg. As expected, doxorubicin increased osteocyte apoptosis, altered bone remodeling, and impaired bone structure. However, the effects induced by DOX were too severe to be rescued by Yoda1 and loading, alone or combined, although loading and Yoda1 individually, or combined, increased the number of mice showing positive responsiveness by 0 %, +15 %, and +29 % relative to no intervention after doxorubicin exposure. Overall, this study supported the potentials and challenges of the Yoda1-based strategy in mitigating the detrimental skeletal effects caused by aging and doxorubicin.

Critical variables regulating age-related anabolic responses to protein nutrition in skeletal muscle.

Protein nutrition is critical for the maintenance of skeletal muscle mass across the lifecourse and for the growth of muscle in response to resistance exercise - both acting via the stimulation of protein synthesis. The transient anabolic response to protein feeding may vary in magnitude and duration, depending on, e.g., timing, dose, amino acid composition and delivery mode, which are in turn influenced by physical activity and age. This review aims to: (i) summarise the fundamental metabolic responses of muscle to protein feeding, (ii) discuss key variables regulating muscle anabolic responses to protein feeding, and (iii) explore how these variables can be optimised for muscle anabolism in response to physical activity and ageing.

Effects of different combinations of mechanical loading intensity, duration, and frequency on the articular cartilage in mice

BackgroundUnderstanding how healthy articular cartilage responds to mechanical loading is critical. Moderate mechanical loading has positive effects on the cartilage, such as maintaining cartilage homeostasis. The degree of mechanical loading is determined by a combination of intensity, frequency, and duration; however, the best combination of these parameters for knee cartilage remains unclear. This study aimed to determine which combination of intensity, frequency, and duration provides the best mechanical loading on healthy knee articular cartilage in vitro and in vivo.Methods and resultsIn this study, 33 male mice were used. Chondrocytes isolated from mouse knee joints were subjected to different cyclic tensile strains (CTSs) and assessed by measuring the expression of cartilage matrix-related genes. Furthermore, the histological characteristics of mouse tibial cartilages were quantified using different treadmill exercises. Chondrocytes and mice were divided into the control group and eight intervention groups: high-intensity, high-frequency, and long-duration; high-intensity, high-frequency, and short-duration; high-intensity, low-frequency, and long-duration; high-intensity, low-frequency, and short-duration; low-intensity, high-frequency, and long-duration; low-intensity, high-frequency, and short-duration; low-intensity, low-frequency, and long-duration; low-intensity, low-frequency, and short-duration. In low-intensity CTSs, chondrocytes showed anabolic responses by altering the mRNA expression of COL2A1 in short durations and SOX9 in long durations. Furthermore, low-intensity, low-frequency, and long-duration treadmill exercises minimized chondrocyte hypertrophy and enhanced aggrecan synthesis in tibial cartilages.ConclusionLow-intensity, low-frequency, and long-duration mechanical loading is the best combination for healthy knee cartilage to maintain homeostasis and activate anabolic responses. Our findings provide a significant scientific basis for exercise and lifestyle instructions.

Dietary protein splanchnic uptake and digestibility via stable isotope tracers.

Dietary proteins are broken down into peptides across the gastrointestinal tract, with skeletal muscle being a primary deposition site for amino acids in the form of incorporation into, for example, metabolic and structural proteins. It follows that key research questions remain as to the role of amino acid bioavailability, of which protein digestibility and splanchnic sequestration (absorption and utilization) of amino acids are determining factors, impact upon muscle protein synthesis (MPS) in clinical states. Elevated splanchnic amino acid uptake has been implicated in anabolic resistance (i.e. attenuated anabolic responses to protein intake) observed in ageing, though it is unclear whether this limits MPS. The novel 'dual stable isotope tracer technique' offers a promising, minimally invasive approach to quantify the digestion of any protein source(s). Current work is focused on the validation of this technique against established methods, with scope to apply this to clinical and elderly populations to help inform mechanistic and interventional insights. Considerations should be made for all facets of protein quality; digestibility of the protein, absorption/utilization and subsequent peripheral bioavailability of amino acids, and resultant stimulation of MPS. Stable isotope tracer techniques offer a minimally invasive approach to achieve this, with wide-ranging clinical application.

Fava Bean Hydrolysate Augments Anabolic Response to Leucine in a Model of Aged Muscle

Fava Bean Hydrolysate Augments Anabolic Response to Leucine in a Model of Aged Muscle

The Influence of Aging on the Unfolded Protein Response in Human Skeletal Muscle at Rest and after Acute Exercise.

The unfolded protein response (UPR) is a proteostatic process that is activated in response to endoplasmic reticulum stress. It is currently unclear how aging influences the chronic and adaptive UPR in human skeletal muscle. Here we determined the effect of aging on UPR activation at rest, in response to exercise, and the associations with muscle function. Thirty young (20-35 yr) and 50 older (65-85 yr) individuals were enrolled. Vastus lateralis biopsies were performed at rest and 3 and 48 h after a single bout of resistance exercise. The abundance of UPR-related transcripts and proteins was measured by RNA sequencing and Western blotting, respectively. Fractional synthetic rates of muscle protein were determined by mass spectrometry after intravenous infusion of 13 C 6 phenylalanine. Older adults demonstrated elevated transcriptional and proteomic markers of UPR activation in resting muscle. Resting UPR gene expression was negatively associated with muscle strength and power in older adults. The UPR is similarly activated by acute resistance exercise in young and older adults and positively associated with muscle function but not the anabolic response to exercise. Skeletal muscle from older adults exhibits chronically activated UPR, which accompanies functional decline. The adaptive UPR is a proteostatic mechanism that is upregulated in response to exercise in young and older adults and positively associated with muscle function.

Nitric oxide contributes to rapid sclerostin protein loss following mechanical load

In response to mechanical loading of bone, osteocytes produce nitric oxide (NO•) and decrease sclerostin protein expression, leading to an increase in bone mass. However, it is unclear whether NO• production and sclerostin protein loss are mechanistically linked, and, if so, the nature of their hierarchical relationship within an established mechano-transduction pathway. Prior work showed that following fluid-shear stress (FSS), osteocytes produce NOX2-derived reactive oxygen species, inducing calcium (Ca2+) influx. Increased intracellular Ca2+ results in calcium-calmodulin dependent protein kinase II (CaMKII) activation, which regulates the lysosomal degradation of sclerostin protein. Here, we extend our discoveries, identifying NO• as a regulator of sclerostin degradation downstream of mechano-activated CaMKII.Pharmacological inhibition of nitric oxide synthase (NOS) activity in Ocy454 osteocyte-like cells prevented FSS-induced sclerostin protein loss. Conversely, short-term treatment with a NO• donor in Ocy454 cells or isolated murine long bones was sufficient to induce the rapid decrease in sclerostin protein abundance, independent of changes in Sost gene expression. Ocy454 cells express all three NOS genes, and transfection with siRNAs targeting eNOS/Nos3 was sufficient to prevent FSS-induced loss of sclerostin protein, while siRNAs targeting iNOS/Nos2 mildly blunted the loss of sclerostin but did not reach statistical significance. Similarly, siRNAs targeting both eNOS/Nos3 and iNOS/Nos2 prevented FSS-induced NO• production. Together, these data show iNOS/Nos2 and eNOS/Nos3 are the primary producers of FSS-dependent NO•, and that NO• is necessary and sufficient for sclerostin protein control.Further, selective inhibition of elements within this sclerostin-controlling mechano-transduction pathway indicated that NO• production occurs downstream of CaMKII activation. Targeting Camk2d and Camk2g with siRNA in Ocy454 cells prevented NO• production following FSS, indicating that CaMKII is needed for NO• production. However, NO• donation (1min) resulted in a significant increase in CaMKII activation, suggesting that NO• may have the ability to tune CaMKII response. Together, these data support that CaMKII is necessary for, and may be modulated by NO•, and that the interaction of these two signals is involved in the control of sclerostin protein abundance, consistent with a role in bone anabolic responses.

Continued use of alcohol accelerates cancer cachexia and impairs ability to induce 4E-BP1 but not p70S6K phosphorylation following muscle contraction

A primary co-morbidity of cancer is cancer cachexia characterized by the wasting of adipose tissue and skeletal muscle. Up to 80% of cancer patients may suffer from this wasting condition leading to reduced quality of life, poor treatment effectiveness and increased morbidity and mortality. The risk of cancer cachexia is enhanced in those with poor lifestyle habits including the habitual consumption of alcohol both prior to a cancer diagnosis and when consumption is continued during disease progression. The use of muscle contraction to improve outcomes in cachexia has been explored in alcohol naïve models with some success, however alcohol at high doses induces anabolic resistance preventing protein synthesis in response to an anabolic stimuli like muscle contraction. Therefore, muscle contraction may not be a suffcient stimuli to overcome cachexia induced catabolism when alcohol has been or is currently being consumed. The purpose of this work was to determine if alcohol induces anabolic resistance in tumor bearing mice or whether it can be used as a therapeutic intervention. It was hypothesized that alcohol would inhibit the stimulation of markers of the anabolic mTORC1 pathway in tumor bearing mice. METHODS: Female BALB/c mice, 12 weeks of age, were randomized into 4 groups (n = 12-14/group): control diet (CON), control diet-cancer (Cancer), Prior EtOH diet-cancer (PE-Cancer), and EtOH diet-cancer (E-cancer). All mice received the isocaloric Lieber DeCarli liquid diet with EtOH mice consuming 20% kcal/daily EtOH for 6 weeks. After 6 weeks, PE-cancer switched to the non-alcohol diet and all cancer groups had C26 carcinoma cells implanted. A single bout of maximal stimulated muscle contraction (10sets of 6 repetitions at 100Hz) was performed ~2 weeks later and muscles were collected 4 hours post contraction. RESULTS: E-cancer lost ~10% body mass, while no other cancer groups exhibited a significant loss in body mass indicating early-stage cachexia. Spleen mass was elevated in all cancer groups relative to control, while quadricep and gastrocnemius muscle weights were reduced in PE-cancer and E-cancer compared to CON. Muscle contraction significantly increased the phosphorylation of S6K1 Thr389 in all of groups except for PE-cancer where a strong trend was still detected (p=0.06). In contrast, phosphorylation of 4EBP1 Ser65 was only increased by muscle contraction in CON, as cancer prevented its stimulation. Therefore, despite accelerating body weight loss, continued alcohol consumption or prior alcohol consumption did not completely impair the anabolic response to maximally stimulated muscle contraction during the early stages of cancer cachexia. However, measurement of rates of degradation and synthesis over time are required to make further conclusions as to its therapeutic potential. Florida Department of Health Grant # 9BC03. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Endocrine Responses to Heated Resistance Exercise in Men and Women.

Pryor, JL, Sweet, DK, Rosbrook, P, Qiao, J, Looney, DP, Mahmood, S, and Rideout, T. Endocrine responses to heated resistance exercise in men and women. J Strength Cond Res 38(7): 1248-1255, 2024-We examined the endocrine responses of 16 (female = 8) resistance trained volunteers to a single bout of whole-body high-volume load resistance exercise in hot (HOT; 40° C) and temperate (TEMP; 20° C) environmental conditions. Thermoregulatory and heart rate (HR) data were recorded, and venous blood was acquired before and after resistance exercise to assess serum anabolic and catabolic hormones. In men, testosterone increased after resistance exercise in HOT and TEMP ( p < 0.01), but postexercise testosterone was not different between condition ( p = 0.51). In women, human growth hormone was different between condition at pre-exercise ( p = 0.02) and postexercise ( p = 0.03). After controlling for pre-exercise values, the between-condition postexercise difference was abolished ( p = 0.16). There were no differences in insulin-like growth factor-1 for either sex ( p ≥ 0.06). In women, cortisol increased from pre-exercise to postexercise in HOT ( p = 0.04) but not TEMP ( p = 0.19), generating a between-condition difference at postexercise ( p < 0.01). In men, cortisol increased from pre-exercise to postexercise in HOT only ( p < 0.01). Rectal temperature increased to a greater extent in HOT compared with TEMP in both men ( p = 0.01) and women ( p = 0.02). Heart rate increased after exercise under both conditions in men and women ( p = 0.01), but only women experience greater postexercise HR in HOT vs. TEMP ( p = 0.04). The addition of heat stress to resistance exercise session did not overtly shift the endocrine response toward an anabolic or catabolic response. When acute program variables are prescribed to increase postresistance exercise anabolic hormones, adding heat stress is not synergistic but does increase physiologic strain (i.e., elevated HR and rectal temperature).

Interleukin-15 Contributes to the Benefits of Βeta-Alanine Supplementation on the Performance of Elite Volleyball Players

Background: Volleyball involves intermittent activities closely linked to explosive power and muscular strength. Utilizing the ergogenic supplement beta-alanine can positively influence the balance between anabolic and catabolic factors, potentially enhancing the benefits of sports activities. Objectives: This study aimed to examine the effect of 4 weeks of beta-alanine supplementation on serum myokine levels, as well as aerobic and anaerobic power in elite volleyball players aged 22 - 28 years. Methods: In this quasi-experimental study, 22 male volleyball players from the Division 1 Volleyball League, aged between 22 and 28, were selected and randomly divided into two groups of 11: A supplement group and a control group. The supplement group received 6.4 g/day of beta-alanine (in 8 doses of 800 mg) for 4 weeks. The ANCOVA test was used for data analysis, with a significance level set at P ≤ 0.05. Results: Beta-alanine supplementation significantly increased serum myokine levels (interleukin-15, myonectin, and irisin) compared to the control group (P &lt; 0.05). Additionally, 4 weeks of beta-alanine supplementation was associated with a significant increase in measures of aerobic and anaerobic power (Bruce and Wingate tests) compared to the control group (P &lt; 0.05). Conclusions: Supplementation with Beta-Alanine led to improved performance (aerobic and anaerobic power) by increasing anabolic responses (irisin) and indicators of muscle hypertrophy (IL-15 and myonectin) in elite volleyball players.