Anabolic Response Research Articles

Associations Of Skeletal Muscle Lipid Infiltration With Hypertrophy And Physical Performance Outcomes In Older Adults

Preserving muscle mass and quality is critical for long term health and longevity. Unfortunately, the hypertrophic potential of aged individuals is diminished, with some experiencing less than favorable outcomes from supervised resistance training programs. This has led investigators to explore the “poor” responder muscle phenotype. PURPOSE: The purpose of this study was to determine whether muscle lipid infiltration plays a role in anabolic adaptation responses, such as muscle growth and physical performance. METHODS: The effects of a 14-week progressive resistance training (PRT) program on muscle size and quality, strength, and physical function in 48 individuals aged 65 and older (mean age ± SD, 70.8 ± 4.5 yrs) was determined. Computed tomography (CT) imaging of cross-sectional mid-thigh regions was used to measure intermuscular adipose tissue (IMAT) and thigh muscle density (TMD) as measures of thigh muscle lipid content. Associations between these lipid depots and baseline function, as well as muscle adaptations to PRT, were made for muscle size (DXA muscle mass and CT muscle area) and physical function and performance (strength, power, SF-36, PROMIS) using multiple linear regression models adjusted for potential confounders such as sex, BMI, CT muscle area, and baseline muscle strength. The association of muscle lipid and physical activity were conducted as a secondary analysis. RESULTS: At baseline, TMD (mean Hounsfield unit ± SD, 42.1 ± 4.0 HU), but not IMAT (mean area ± SD, 12.5 ± 4.3 cm2), was significantly associated with all physical function and performance variables (R2 range 0.45-0.75, p < 0.05) except leg extension strength and power. Neither IMAT nor TMD was related to physical activity. Following PRT, IMAT was not associated with any exercise adaptation, whereas TMD was negatively associated with percent change in isometric strength (R2 0.17) and muscle power (R2 0.28, p < 0.05). CONCLUSIONS: Muscle fatty infiltration can impact strength and power gains following PRT in older persons. More work is needed to understand the dynamics of ectopic muscle fat accumulation and its influence on physical function and muscle metabolism/anabolism.

Membrane characteristics tune activities of endosomal and autophagic human VPS34 complexes.

The lipid kinase VPS34 orchestrates diverse processes, including autophagy, endocytic sorting, phagocytosis, anabolic responses and cell division. VPS34 forms various complexes that help adapt it to specific pathways, with complexes I and II being the most prominent ones. We found that physicochemical properties of membranes strongly modulate VPS34 activity. Greater unsaturation of both substrate and non-substrate lipids, negative charge and curvature activate VPS34 complexes, adapting them to their cellular compartments. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) of complexes I and II on membranes elucidated structural determinants that enable them to bind membranes. Among these are the Barkor/ATG14L autophagosome targeting sequence (BATS), which makes autophagy-specific complex I more active than the endocytic complex II, and the Beclin1 BARA domain. Interestingly, even though Beclin1 BARA is common to both complexes, its membrane-interacting loops are critical for complex II, but have only a minor role for complex I.

Hallmarks of frailty and osteosarcopenia in prematurely aged PolgA(D257A/D257A) mice.

BackgroundFrailty is a geriatric syndrome characterized by increased susceptibility to adverse health outcomes. One major determinant thereof is the gradual weakening of the musculoskeletal system and the associated osteosarcopenia. To improve our understanding of the underlying pathophysiology and, more importantly, to test potential interventions aimed at counteracting frailty, suitable animal models are needed.MethodsTo evaluate the relevance of prematurely aged PolgA(D257A/D257A) mice as a model for frailty and osteosarcopenia, we quantified the clinical mouse frailty index in PolgA(D257A/D257A) and wild‐type littermates (PolgA(+/+), WT) with age and concertedly assessed the quantity and quality of bone and muscle tissue. Lastly, the anabolic responsiveness of skeletal muscle, muscle progenitors, and bone was assessed.ResultsPolgA(D257A/D257A) accumulated health deficits at a higher rate compared with WT, resulting in a higher frailty index at 40 and 46 weeks of age (+166%, +278%, P < 0.0001), respectively, with no differences between genotypes at 34 weeks. Concomitantly, PolgA(D257A/D257A) displayed progressive musculoskeletal deterioration such as reduced bone and muscle mass as well as impaired functionality thereof. In addition to lower muscle weights (−14%, P < 0.05, −23%, P < 0.0001) and fibre area (−20%, P < 0.05, −22%, P < 0.0001) at 40 and 46 weeks, respectively, PolgA(D257A/D257A) showed impairments in grip strength and concentric muscle forces (P < 0.05). PolgA(D257A/D257A) mutation altered the acute response to various anabolic stimuli in skeletal muscle and muscle progenitors. While PolgA(D257A/D257A) muscles were hypersensitive to eccentric contractions as well as leucine administration, shown by larger downstream signalling response of the mechanistic target of rapamycin complex 1, myogenic progenitors cultured in vitro showed severe anabolic resistance to leucine and robust impairments in cell proliferation. Longitudinal micro‐computed tomography analysis of the sixth caudal vertebrae showed that PolgA(D257A/D257A) had lower bone morphometric parameters (e.g. bone volume fraction, trabecular, and cortical thickness, P < 0.05) as well as reduced remodelling activities (e.g. bone formation and resorption rate, P < 0.05) compared with WT. When subjected to 4 weeks of cyclic loading, young but not aged PolgA(D257A/D257A) caudal vertebrae showed load‐induced bone adaptation, suggesting reduced mechanosensitivity with age.ConclusionsPolgA(D257A/D257A) mutation leads to hallmarks of age‐related frailty and osteosarcopenia and provides a powerful model to better understand the relationship between frailty and the aging musculoskeletal system.

The mTORC2 Component Rictor Is Required for Load-Induced Bone Formation in Late-Stage Skeletal Cells.

ABSTRACTBone relies on mechanical cues to build and maintain tissue composition and architecture. Our understanding of bone cell mechanotransduction continues to evolve, with a few key signaling pathways emerging as vital. Wnt/β‐catenin, for example, is essential for proper anabolic response to mechanical stimulation. One key complex that regulates β‐catenin activity is the mammalian target of rapamycin complex 2 (mTORc2). mTORc2 is critical for actin cytoskeletal reorganization, an indispensable component in mechanotransduction in certain cell types. In this study, we probed the impact of the mTORc2 signaling pathway in osteocyte mechanotransduction by conditionally deleting the mTORc2 subunit Rictor in Dmp1‐expressing cells of C57BL/6 mice. Conditional deletion of the Rictor was achieved using the Dmp1–Cre driver to recombine Rictor floxed alleles. Rictor mutants exhibited a decrease in skeletal properties, as measured by DXA, μCT, and mechanical testing, compared with Cre‐negative floxed littermate controls. in vivo axial tibia loading conducted in adult mice revealed a deficiency in the osteogenic response to loading among Rictor mutants. Histological measurements of osteocyte morphology indicated fewer, shorter cell processes in Rictor mutants, which might explain the compromised response to mechanical stimulation. In summary, inhibition of the mTORc2 pathway in late osteoblasts/osteocytes leads to decreased bone mass and mechanically induced bone formation. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

P0871THE IMPACT OF ENDOGENOUS PTH/PTH1R/ATF4 AXIS ON TRABECULAR AND CORTICAL BONE REMODELING AND BONE GROWTH OF YOUNG RATS WITH EXPERIMENTAL CHRONIC KIDNEY DISEASES

Abstract Background and Aims Chronic kidney disease - mineral bone disorder (CKD–MBD) is one of the major clinical complications in patients with chronic kidney disease (CKD). Bone remodeling has been suggested to play a fundamental role in the maintenance of skeletal integrity via a balance between the bone formation/resorption process. The parathyroid hormone (PTH) is a key hormone controlling bone metabolism. PTH is known to affect bone with a net catabolic and anabolic effect but the mechanisms responsible for these differing effects are poorly understood. Previously, we demonstrated the distinct effect of mild degree of CKD on trabecular and cortical bone strength in rapidly growing rats (Pawlak et al. PlosOne, 2016). The aim of the present study was to evaluate the effect of endogenous PTH, its receptor PTH1R and activating transcription factor 4 (ATF4) – the major regulators of the anabolic PTH response in osteoblasts on bone remodeling and growth of young rats with experimental CKD. Method Four-week old Wistar male rats were divided into 2 groups: with CKD induced by surgical 5/6 subtotal nephrectomy, and sham-operated (CON). After one (CON-1; CKD-1) and three months (CON-3; CKD-3) of the surgery the femurs were collected and their lengths were measured. The activity of alkaline phosphatase (ALP), a bone formation marker, and tartrate-resistant acid phosphatase (TRACP5b) reflecting bone resorption were determined in homogenates from trabecular and cortical left femurs. The expression of PTH1R and ATF4 gene was determined by QRT-PCR in right femurs. Serum PTH was analyzed using ELISA kit. Computations were performed using Statistica ver.10 computer software. Results Serum PTH was increased in CKD-3 compared to CKD-1, moreover the slight increase in PTH levels was noted in CKD-3 compared to appropriate controls (both p&amp;lt;0.05). The activity of ALP and TRACP5b in trabecular bone tissue were significantly lower in CKD-3 compared to CKD-1 rats (p&amp;lt;0.01 and p&amp;lt;0.05; respectively). There was a strong positive correlation between ALP and TRACP5b in this bone region (R =0.624, p =0.002), whereas both ALP and TRACP5b were inversely related to serum PTH (R = -0.534, p =0.012 and R = -0.636, p =0.002; respectively). The activity of TRACP5b in cortical bone tissue was significantly higher in CKD-3 compared to CKD-1 group (p&amp;lt;0.05), and it was positively associated with PTH levels (R =0.597, p =0.004). There was no difference in ALP activity between the studied groups, and ALP was not associated with TRACP5b in this bone region. The expression of ATF4 and PTH1R genes was significantly increased in the CKD-3 group compared with the appropriate control (p&amp;lt;0.01 and p&amp;lt;0.05; respectively) and with the CKD-1 group (p&amp;lt;0.05 and p&amp;lt;0.01; respectively). The expression of ATF4 was inversely correlated with ALP and TRACP5b in trabecular bone (R = -0.534, and R = -0.528, both p&amp;lt;0.05), whereas it was positively related to TRACP5b in cortical bone region (R =0.418, p&amp;lt;0.05). The femoral length was significantly increased during 3-month of CKD development (p&amp;lt;0.000), and it was positively associated with PTH levels (R =0.571, p =0.007), cortical TRACP5b activity (R =0.609, p =0.002) and tended to be related with ATF4 gene expression (R =0.409, p =0.065). In contrast, the femoral length was inversely related to ALP activity in trabecular bone tissue (R= -0.656, p =0.0007). Conclusion The endogenous PTH, through PTH1R/ATF4 axis, inhibited trabecular bone remodeling. In contrary, PTH/PTH1R/ATF4 system intensified bone resorption in cortical bone region. This opposite effect of PTH on bone remodeling was associated with the intensification of growth process in the long bones of young rats with CKD.

PTH(1\u201334) treatment and/or mechanical loading have different osteogenic effects on the trabecular and cortical bone in the ovariectomized C57BL/6 mouse

In preclinical mouse models, a synergistic anabolic response to PTH(1–34) and tibia loading was shown. Whether combined treatment improves bone properties with oestrogen deficiency, a cardinal feature of osteoporosis, remains unknown. This study quantified the individual and combined longitudinal effects of PTH(1–34) and loading on the bone morphometric and densitometric properties in ovariectomised mice. C57BL/6 mice were ovariectomised at 14-weeks-old and treated either with injections of PTH(1–34); compressive loading of the right tibia; both interventions concurrently; or both interventions on alternating weeks. Right tibiae were microCT-scanned from 14 until 24-weeks-old. Trabecular metaphyseal and cortical midshaft morphometric properties, and bone mineral content (BMC) in 40 different regions of the tibia were measured. Mice treated only with loading showed the highest trabecular bone volume fraction at week 22. Cortical thickness was higher with co-treatment than in the mice treated with PTH alone. In the mid-diaphysis, increases in BMC were significantly higher with loading than PTH. In ovariectomised mice, the osteogenic benefits of co-treatment on the trabecular bone were lower than loading alone. However, combined interventions had increased, albeit regionally-dependent, benefits to cortical bone. Increased benefits were largest in the mid-diaphysis and postero-laterally, regions subjected to higher strains under compressive loads.

The Effects of a Protease Enzyme Blend on Post-Resistance Exercise Intramuscular Anabolic Signaling

Abstract Objectives Protease supplementation has been reported to decrease inflammation and indices of muscle damage while increasing functional recovery following strenuous resistance exercise compared to a placebo. While various mechanisms have been proposed, the effects of protease supplementation on the resistance exercise induced anabolic signaling response has not been reported in the literature. To examine the effects of a protease enzyme blend added to whey protein on post-resistance exercise intramuscular anabolic signaling. Methods Ten resistance-trained males (24.4 ± 4.1yrs, 1.79 ± 0.86 m, 92.6 ± 10.4 kg) were enrolled in this double-blind, cross-over, placebo controlled study and engaged in three separate bouts of resistance exercise. Each participant completed 4 sets of leg presses and leg extensions for 8–10 repetitions at 75% of their 1-repetition maximum with 90 seconds of rest between each set. Immediately following the resistance exercise protocol, participants consumed either 250 mg of a protease enzyme blend + 26 g of whey protein (PW), 26 g whey alone (W), or non-caloric control (CON) in a counterbalanced fashion. Skeletal muscle microbiopsies were obtained from the vastus lateralis pre-exercise (PRE), 1-hour (1H), and 3-hours (3H) post-exercise. Multiplex signaling assay kits were used to quantify the phosphorylation status of proteins specific to the mTOR (AKT, mTOR, p70S6K) and MAPK (ERK1/2, JNK, p38) signaling pathways using the MAGPIX® (Luminex, Austin, TX, USA). A 2-way repeated measures analysis of variance (ANOVA) was used to identify differences between treatments over time. Results A main effect for time (p &amp;lt; 0.05) revealed phosphorylation of AKT was decreased at 1H (p &amp;lt; 0.001), mTOR was increased at 1H (P = 0.025) and 3H (P = 0.009) post-exercise, while p70S6K remained unchanged (P &amp;gt; 0.05) from PRE. A main effect for time (p &amp;lt; 0.05) was found with increased phosphorylation at 1H for JNK (P = 0.001), and decreased phosphorylation at 3H for ERK 1/2 (P = 0.022) with respect to baseline. Additionally, there were no differences in any mTOR nor MAPK signaling proteins observed between treatments. Conclusions These data suggest that acute protease supplementation may not alter mTOR or MAPK signaling in skeletal muscle following acute resistance exercise. Funding Sources Deerland Enzymes, Kennesaw, GA.

SAT-392 The Role of β-arrestin2 in Bone Catabolic Response to Hyperparathyroidism In Vivo

Primary hyperparathyroidism (PHPT) is an endocrine disorder characterized by elevated parathyroid hormone (PTH) levels and hypercalcemia caused by the overactive parathyroid glands, resulting in negative impacts on the skeleton including bone loss and increased bone fragility1. PTH binds and activates parathyroid hormone type 1 receptor (PTH1R) which primary couples to Gαs, stimulating the downstream effectors that mediate bone remodeling processes2. PTH1R activity is regulated by arrestins, specially β-arrestin2 (β-arr2), through signal termination and receptor internalization2. Previously, we have seen anabolic effects of hyperparathyroidism (cPTH) on trabecular bone in mice overexpressing Gαs3. We hypothesized that increased Gαs protein levels in osteoblasts outcompete β-arr binding to PTH1R, leading to reduced signal termination and increased bone formation. To test this hypothesis, we are testing if the deletion of β-arr2 will also result in an anabolic response to cPTH in this study. The response of β-arr2 knockout (KO) mice to cPTH have yet to be documented. The hypothesis of this study is that β-arr2 KO mice treated with cPTH will exhibit anabolic effects on the trabecular bone. Nine-week-old wild-type (WT) C57BL/6 and β-arr2 KO mice were treated for 14 days with either rPTH1-34 (80ng/g/day) or saline (PBS) using micro-osmotic pumps to simulate hyperparathyroidism. There are 8 groups (n=10 per group) including both sexes, 2 genotypes (WT and KO), and 2 treatment groups (PTH and PBS). Two 30 mg/kg doses of 0.6% calcein green were administered subcutaneously to mice at 7 and 2 days prior to euthanasia to label bones. Decalcified tibiae were embedded in paraffin for histological analysis. Undecalcified tibiae were embedded in plastic for dynamic histomorphometry. Micro-computed tomography (μCT) was used to access bone microarchitecture of femurs and vertebrae followed by biomechanical testing of bone strength. The μCT data of distal femurs show that cPTH treatment increased bone volume in female KO mice (6.864 ± 2.318 vs 4.690 ± 1.555 %; P= 0.0328; n=9 per group) and maintained bone in male KO mice (13.37 ± 2.860 vs 13.38 ± 3.135; P= 0.9968, n= 10) compared to control. Histological analysis show higher osteoclastic activity in both sexes and genotypes when treated with cPTH, suggesting that the anabolic response may be at the level of osteoblasts and osteocytes. These promising results support our hypothesis that arrestin-mediated PTH receptor downregulation plays an importance role in bone weakness associated with hyperparathyroidism. These studies are important for understanding the clinical phenotype of PHPT patients and suggest that inhibition of β-arr2 in PHPT could be a path for drug therapy.

SAT-402 Investigating Analogues of Parathyroid Hormone (PTH) and PTH-Related Peptide (PTHrP) to Improve Anabolic: Catabolic Response Ratios Using UMR106-01 Osteocytic Cells

Osteoporosis, the most common bone disease in humans, is characterised by decreased bone mass and increased fracture risk1. Osteoporosis development is associated with an imbalance of bone resorption and deposition mediated by all three bone cell types; osteoblasts, osteocytes and osteoclasts2. Current treatments for osteoporosis either prevent further bone resorption (Bisphosphonates and anti-RANKL) or increase bone deposition through anabolism (Teriparatide and Abaloparatide)3. The two anabolic treatments are truncated analogues of endogenous peptides; parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP), respectively. Both are administered intermittently, act on PTH1R receptors on osteoblasts and osteocytes leading to an increase in bone mineral density and bone strength. However, both treatments also have side-effects of hypercalcemia and cortical bone porosity caused by osteoclast stimulation. We therefore compared an array of PTH and PTHrP analogues for potential stimulation of catabolic side effects and desired anabolic effects. Screening assays used are UMR106-01 osteocytic cells that have high levels of PTH1R to measure mRNAs involved in anabolic responses (suppression of WNT inhibitors SOST and Dkk1) and catabolic responses (stimulation of RANKL/OPG, IL6). Peptides were also analysed by real-time cellular impedance assays (xCELLigence) to measure unbiased receptor stimulation. xCELLigence assays showed PTH1-34, PTHrP1-34 and Abaloparatide had the highest potencies (3.7 nM,1.4 nM,1.7nM respectively) while Tyr1PTH1-34 and PTH2-34 had significantly decreased potencies (64nM and 130nM), the β-arrestin biased agonist, D-Trp12Tyr34PTH7–34, had no effect. PTH1-34 potently inhibited SOST (IC50 0.29nM), and catabolic genes (RANK/OPG EC50 0.5nM, IL6 EC50 2.5nM). PTHrP1-34 provided higher potencies for anabolic (inhibited SOST IC50 0.08nM) and catabolic (RANKL/OPG EC50 0.3nM, IL6 EC50 1nM) genes. Altering the first amino acid to Tyrosine; Tyr1PTH1-34 caused potent anabolic responses (SOST IC50 0.97nM) yet showed decreased potency for catabolic responses (RANKL/OPG EC50 20nM, IL6 EC50 > 100nM). Removing the first amino acid of PTH to PTH2-34 drastically decreases the effectiveness of the peptide (OPG, SOST and RANKL IC50 – no effect, IL6 EC50 > 100nM). These results indicate the importance of the N-terminal amino acid for PTH affinity and efficacy and suggest that Tyr1PTH1-34 may offer the best combination of bone stimulation without causing hypercalcemia.References 1 A.Gupta, L. March. (2016) Treating osteoporosis, Aust Prescr 2 Chen X, et al. (2018) Osteoblast–osteoclast interactions. Connect. Tissue Res 3 Tu KN, et al. (2018) Osteoporosis: A review of treatment options. P T

Acute Effect of the Timing of Resistance Exercise and Nutrient Intake on Muscle Protein Breakdown.

Background: Combining resistance exercise (RE) with nutrient intake stimulates muscle protein net balance. However, it is still unclear whether the optimal timing of nutrient intake is before or after RE, especially on muscle protein breakdown (MPB) for an augmented muscle anabolic response. The aim of this study was to investigate the effect of a substantial mixed meal (i.e., nutrient- and protein-dense whole foods) before or after RE, compared with RE without a meal on the acute response of MPB in a crossover-design study. Methods: Eight healthy young men performed three trials: (1) meal intake before RE (Pre), (2) meal intake after RE (Post), and (3) RE without meal intake (No). Plasma insulin and 3-methylhistidine (3-MH), an MPB marker, were measured. Results: Time course change in plasma insulin level after RE was significantly higher in the Post condition than in the Pre and No conditions. The area under the curve of 3-MH concentration was significantly lower in the Post condition than in the Pre and No conditions. Conclusions: These results suggest that a substantial mixed meal immediately after RE may effectively suppress MPB in the morning.

Disruption of the aldehyde dehydrogenase 2 gene increases the bone anabolic response to intermittent PTH treatment in an ovariectomized mouse model.

Aldehyde dehydrogenase 2 (ALDH2) is the enzyme that oxidizes the acetaldehyde produced by alcohol metabolism. This variant not only affects the response to alcohol but is also associated with several diseases, such as esophageal cancer, myocardial infarction, and particularly osteoporosis. In our previous study, we reported that compared to wild-type (WT) mice, Aldh2 knockout (KO) mice naturally have a strong bone formation ability, and high expression of parathyroid hormone receptor (PTHR1) in osteocytes. The effect of the Aldh2 gene on bone metabolism in response to intermittent PTH treatment is unknown. The purpose of this study was to clarify the effect of the Aldh2 gene on the bone anabolic response to intermittent PTH treatment in ovariectomized mice. Female KO and WT mice were ovariectomized at 8weeks of age. At 14weeks of age, the KO and WT mice were divided into vehicle-treated (Veh) and PTH-treated (PTH) groups (i.e., the WT-Veh, WT-PTH, KO-Veh and KO-PTH groups). PTH (1-34) and vehicle were subcutaneously administered to each group at a dose of 40μg/kg body weight (BW) five times per week for 4weeks. Micro-CT showed that the bone volume (BV), trabecular number (Tb.N), connectivity density (Conn.D), and cortical thickness (Ct.Th) values in the KO-PTH mice were significantly higher than those in the KO-Veh mice. Histomorphometric analysis showed that the BV, Tb.N, and mineral apposition rate (MAR) values in the KO-PTH group were significantly higher than those in the KO-Veh group. The mRNA expression level of PTHR1 in the KO-PTH group was significantly increased and that of p21 in the KO-PTH group was significantly decreased compared with the levels in the KO-Veh group. The expression of PTHR in osteocytes from the KO-PTH group was also significantly increased compared with that in osteocytes from the KO-Veh group. Furthermore, cell cultures revealed that the ALP+CFU-f/total CFU-f percentage was significantly higher in the KO-PTH group than in the KO-Veh group. We concluded that in ovariectomized Aldh2 KO mice, the bone anabolic response to intermittent PTH treatment was significantly enhanced compared to that in WT mice, which may be mediated by the high expression level of PTHR1.

Effect of Electrical Pulse Stimulation on Anabolic Signaling in L6 Myotubes

The mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase with a central role in controlling cellular anabolism. By phosphorylating two downstream targets, p70‐S6 kinase (p70‐S6K) and eIF4E binding protein 1 (4EBP1), mTOR induces an increase in mRNA translation, leading to an upregulation of protein synthesis. In muscle tissue, mTOR activation in response to an exercise stimulus is a key signaling event in the anabolic response to physical activity. As studying the signaling response to exercise at the cellular level is challenging in whole organisms due to difficulty in isolating muscle cell level effects, development of a cell culture model mimicking exercise would provide an avenue for understanding the intracellular cellular signaling response to exercise in skeletal muscle. The aim of this study was to determine whether 12 hours of electrical pulse stimulation (EPS) would alter the anabolic response in L6 myotubes.METHODSMurine L6 myoblasts were cultured on 10 cm plates in standard culture medium supplemented with 1% penicillin/streptomycin, and 10% fetal bovine serum until 60% confluence. At this point, differentiation was induced by switching to medium without sodium pyruvate supplemented with 1% penicillin/streptomycin, 2% horse serum, and 0.5% insulin‐transferrin‐selenium (ITS). After 7 days of differentiation, myotubes were stimulated, in culture, for 12 hours at a low frequency (1 Hz, 2ms, 60V). Differentiation media was changed in all plates immediately before stimulation and immediately after stimulation in half of the plates. Half of the cells were harvested immediately after stimulation to assess the acute effects and the remaining plates were harvested 24 hours post stimulation and re‐feeding. Protein expression of common anabolic markers was measured using western blot techniques.RESULTSOur results indicate an increase in phosphorylated 4EBP1 immediately after stimulation (EPS‐Ea) compared to control (CON‐Ea) (p = 0.05). Additionally, phosphorylation levels return to baseline 24 hours post stimulation. In contrast, phosphorylation levels of p70‐S6K immediately after stimulation (EPS‐Ea) were not different from control (CON‐Ea) but were elevated 24 hours post stimulation (EPS‐La) (p = 0.09). Finally, we determined the ratio of phosphorylated to total levels of 4EBP1 were two‐fold greater immediately after stimulation (EPS‐Ea) compared to 24 hours post stimulation (EPS‐La) (p = 0.09). Conversely, the ratio of phosphorylated to total levels of p70‐S6K were two times greater 24 hours post stimulation (EPS‐La) compared to immediately after stimulation (EPS‐Ea) (p = 0.09).CONCLUSIONWe were able to determine that electrical pulse stimulation of L6 myotubes could be used as a model to better understand anabolic signaling strategies following EPS in culture. This study represents a first step in using electrical pulse stimulation as an in culture exercise mimetic and elucidating the role of mTOR and downstream targets in cell growth.Support or Funding InformationThis work was funded in part by the College of Health and Education and the Huffines Institute for Sports Medicine at Texas A&amp;M University

Soluble markers of cartilage in the forward study - a prospective-retrospective hypothesis generating study for identification of a low cartilage repair endotype

Purpose: Data from UK biobank suggest that an endotype of low cartilage repair is associated with structural progression in osteoarthritis (OA) [1]. The anabolic or anti-catabolic response of different interventions could more pronounced in different endotypes of cartilage turnover, as observed with anti-resorptive treatments and bone turnover markers in the osteoporosis field. Sprifermin a truncated and recombinant FGF18 have been shown to induce chondrocyte proliferation and cartilage formation in in vitro settings [2-4].

Mechano‐therapy using high loads enhances regrowth of skeletal muscle in young, but induces muscle damage in old rats.

IntroductionOlder adults have an impaired ability to recover from skeletal muscle atrophy compared to young, predisposing them to loss of independence and poor quality of life. We have previously shown that mechano‐therapy in the form of cyclic, compressive loading (CCL) enhances regrowth of atrophied skeletal muscle in young adult rats, but not in old. Our preliminary data show that passive, transverse stiffness of muscles from old rats is elevated compared to young, potentially blunting the mechanical stimulus and subsequent growth response. In the current study, we hypothesized that an increased CCL load proportional to the higher tissue stiffness in old rats would enhance skeletal muscle regrowth when compared to atrophied muscles of old rats subjected to a lower load of CCL or ambulation alone.MethodsYoung (10 months; N = 6) and old (30 months; N = 6) adult male F344/Brown Norway rats underwent 14 days of hind limb suspension before being randomly assigned to one of the following: reloading for 7 days without CCL (RE); reloading with 4.5 N CCL (RE + CCL, 4.5 N); or reloading with 7.6 N CCL (RE + CCL, 7.6 N). Starting on day 0, four bouts of CCL were administered to the right gastrocnemius using a custom designed device capable of applying a precise load, with 48 hrs between bouts. Gastrocnemius muscle sections were analyzed for muscle fiber cross‐sectional area (CSA), satellite cell abundance (Pax7+/fiber), embryonic myosin heavy chain expression (eMyHC+/fiber) and centro‐nucleation using immunohistochemistry.ResultsMuscle fiber CSA was load dependent in young adult rats, but had no effect at either load in old rats. When compared to muscles from RE rats, 4.5 N CCL had no effect on satellite cell abundance regardless of age; however, satellite cell abundance was higher in both young and old adult muscle after 7.6 N CCL compared to RE with old rats having 2‐fold higher levels compared to RE. Regardless of load, CCL was not associated with differences in eMyHC expression or centrally located nuclei in young adult rat muscle, while muscles receiving 7.6 N CCL in old rats had a higher number of centrally located nuclei and were ~16‐fold higher in eMyHC+ fibers compared to RE.ConclusionsThese results show that CCL induces a load dependent anabolic response in young adult animals during muscle mass recovery following disuse, but has no regrowth effect on muscles from old animals at either load. Furthermore, using a higher CCL load, scaled for elevated transverse stiffness in aged muscle, induces damage in muscle of old but not young rats.Support or Funding InformationThis research was supported by NIH grant RO1AT009268 and R21AG042699.

YAP Involves in Reduced Anabolic Response to Functional Overload in Lewis Lung Carcinoma‐bearing Mice

Cancer‐associated weight loss, also known as cachexia, is connected to poor prognosis, reduced response to chemotherapy, and impaired patient quality of life. While resistance exercise/muscle contraction is a promising therapy, anabolic resistance, the suppressed ability to induce protein synthesis, is present and can’t be solely elucidated by the change of mTOR signaling. Yes‐associated protein (YAP) is a downstream effector of Hippo signaling and is known to control skeletal muscle size in response to mechanical stimulus. However, the role of YAP on anabolic resistance in cancer cachexia needs to be clarified. The purpose of this study was to determine whether the state of YAP activity after functional overload is altered in cachectic mice. Approximately three‐month‐old male C57BL6 mice were divided into two groups: Control group with PBS (100 μl) injection (Con, n=5) and tumor‐implantation group with Lewis lung Carcinoma (LLC) cells injection (2×105 cells in 100 μl PBS, LLC, n=5). Injection was made at the right flank under anesthesia. Four weeks after injection tumor growth was visible, synergist ablation surgery was performed on the left leg (Overload) and the right leg served as an internal control with sham surgery (Sham). Puromycin (0.04 μmol/g body weight, BW) was injected 30 minutes prior to sacrifice and plantaris muscles (PLAN) were removed and snap‐frozen at day 7 following the surgery. After total protein was extracted, routine Western blotting was performed using 60~120 μg of the total protein. Paired t‐test (Sham vs. Overload) and unpaired t‐test (Con vs. LLC) were used for statistical analysis and the levels of significance was set at 0.05. PLAN weights were comparable between Con and LLC with sham surgery (19.8±1.2 mg vs. 17.3±0.5 mg for Con and LLC, respectively, p=0.077). Seven‐day functional overload increased PLAN weights regardless of tumor inoculation, but the degree of hypertrophy was different (42.7±4.0% vs. 27.6±4.6% for Con and LLC, respectively, p&lt;0.05). LLC mice showed reduced levels of phosphorylated p70S6K (p‐p70) with sham surgery (1.0±0.3 vs. 0.4±0.1, for Con and LLC, respectively, p&lt;0.05) and those with SA surgery (4.4±0.7 vs. 1.6±0.7, for Con and LLC, p&lt;0.05). In harmony with these results, the basal levels of muscle protein synthesis in LLC mice were decreased by 51% compared to Con mice and the levels remained lower after functional overload (2.1±0.1 vs. 1.1±0.1 for Con and LLC, respectively, p&lt;0.05). Interestingly, the basal phosphorylated levels of YAP (p‐YAP) were elevated in LLC mice (1.0±0.1 vs. 2.2±0.1, for Con and LLC, respectively, p&lt;0.05). However, functional overload did not alter the levels of p‐YAP in LLC mice while Con mice significantly increased the levels by 55% (p&lt;0.05). In conclusion, the data suggest that YAP plays a role, at least in part, in decreased anabolic response to functional overload in LLC‐bearing mice.Support or Funding InformationSupported by Louisiana Board of Regents Support Fund (LEQSF(2017‐20)‐RD‐A‐22) to SS.

Design and in vitro evaluation of a novel synergic intra-articular treatment for post-traumatic osteoarthritis

Purpose: Post-traumatic (PT) OA, subsequent to joint injury, accounts for over 12% of the overall disease burden of OA. In the development of PTOA, the chondrocytes activity is progressively impaired with upregulation of proteolytic enzymes and downregulation of the most important growth factors involved in promoting chondrocyte anabolism. Based on the literature, we selected three promising target for PTOA treatment: (1) MMP3 is one of the metalloproteases mainly involved in collagen degradation, since several clinical studies reported an increase of synovial fluid MMP3 level in OA patients. In our previous work a Hyaluronic Acid (HA) partial hexadecylamide (HYADD®4) was selected as the strongest MMP inhibitor (MMPI) among a series of glycosaminoglycans. (2) TRPV4 channel is a Ca2+-permeable osmomechano-TRP channel, highly expressed in articular chondrocytes. It plays a pivotal role in chondrocyte mechanotransduction; if activated after a trauma, it is able to induce anabolic responses in vitro and chondroprotection in vivo. RN-1747, a small synthetic hydrophobic molecule was already reported as selective TRPV4 agonist. (3) mTOR (mammalian target of rapamycin) is a serine/threonine protein kinase that plays a vital role in cartilage development and homeostasis as well as contributing to the process of cartilage degeneration associated with OA. Rapamycin is an mTOR inhibitor already FDA-approved for the prevention of organ transplant rejection. The objective of these studies are: (1) to optimize the HYADD®4 chemical structures in order to increase the MMPI potency and selectivity; (2) to develop a stable formulation of the MMPI with an active molecule (RN-1747 or Rapamycin) in order to obtain a novel synergic intra-articular (i.a.) treatment for PTOA. Methods: A series of HA derivatives were synthesized, varying the polymer backbone and the hydrophobic side chain, chemically characterized by 1H-NMR and tested in vitro as MMP3 inhibitors. The inhibitor selected, FID-145 (a partial oleylamide of sulphated HA; patent pending), was screened in vitro against 10 different human MMPs and other targets. The efficacy of FID-145 was assessed in an in vitro model of inflammatory arthritis, where bovine cartilage biopsies were exposed to IL-1β/Oncostatin M (OSM) for 3 weeks; at the same time, cartilage explants were treated with FID-145. Collagen release in the surnatants was quantified and compared to controls. The formulation of FID-145 in PBS was characterized by TEM analysis and a Nile Red staining, where increasing concentrations of FID-145 were mixed with the same concentration of Nile Red and the fluorescence emission at 600 nm was measured. Micellar formulations of FID-145 with either RN-1747 or Rapamycin were obtained through mixing, sonication and sterilizing filtration. The loading degree in either RN-1747 or Rapamycin and the stability of both formulations were analyzed by HPLC-MS. The mTOR inhibition activity and the TRPV4 activation potency were assessed by two specific assays (K-Elisa mTOR Activity Kit, Merck, and Fluo 8 AM calcium assay, AAT Bioquest, in primary chondrocyte, respectively). Kinetics of RN-1747 release from FID-145 formulation was determined in vitro, in human synovial fluid, at 37°C. Results: Among the tested HA derivatives, FID-145 showed the highest inhibition potency against MMP3 and no activity against ADAMTS4 and elastase in vitro. In the cartilage explant model, a strong collagen release was detected in inflammatory conditions after 3 weeks; this tendency was reversed when FID-145 was added to the cartilage biopsies and collagen release decreased compared to control. FID-145 is an amphiphilic polymer and its ability to form micelles in water was confirmed by: 1) the TEM images showing the presence of spherical aggregates with diameter around 40 nm and 2) a Critical Micelle Concentration (CMC) of 0.2±0.1 mg/mL, measured with the Nile Red staining. RN-1747 and Rapamycin were successfully loaded at 0.15±0.05 mg/mL in 12 mg/mL FID-145 in PBS; both formulations proved to be stable, sterile and suitable for i.a. injection for OA treatment. The mTOR inhibition and the TRPV4 activation effect of the Rapamycin and TRPV4 formulations, respectively, was showed to be maintained in vitro. The controlled release of RN-1747 from the FID-145 micellar formulation showed that only about 50% of total loaded RN-1747 was released within 7 days. Conclusions: FID-145, a new amphiphilic HA derivative, was screened as a relatively potent and selective MMP3 inhibitor. When formulated in water, FID-145 yielded a stable micellar formulation easily loaded with hydrophobic, pharmacologically active molecules such as RN-1747 or Rapamycin, of promising relevance in the development of a novel synergic i.a. treatment for PT-OA.

Caloric restriction induces anabolic resistance to resistance exercise

PurposeWeight loss can result in the loss of muscle mass and bone mineral density. Resistance exercise is commonly prescribed to attenuate these effects. However, the anabolic endocrine response to resistance exercise during caloric restriction has not been characterized.MethodsParticipants underwent 3-day conditions of caloric restriction (15 kcal kg FFM−1) with post-exercise carbohydrate (CRC) and with post-exercise protein (CRP), and an energy balance control (40 kcal kg FFM−1) with post-exercise carbohydrate (CON). Serial blood draws were taken following five sets of five repetitions of the barbell back squat exercise on day 3 of each condition.ResultsIn CRC and CRP, respectively, growth hormone peaked at 2.6 ± 0.4 and 2.5 ± 0.9 times the peak concentrations observed during CON. Despite this, insulin-like growth factor-1 concentrations declined 18.3 ± 3.4% in CRC and 27.2 ± 3.8% in CRP, which was greater than the 7.6 ± 3.6% decline in CON, over the subsequent 24 h. Sclerostin increased over the first 2 days of each intervention by 19.2 ± 5.6% in CRC, 21.8 ± 6.2% in CRP and 13.4 ± 5.9% in CON, but following the resistance exercise bout, these increases were attenuated and no longer significant.ConclusionDuring caloric restriction, there is considerable endocrine anabolic resistance to a single bout of resistance exercise which persists in the presence of post-exercise whey protein supplementation. Alternative strategies to restore the sensitivity of insulin-like growth factor-1 to growth hormone need to be explored.

Connexin 43 Is Necessary for Murine Tendon Enthesis Formation and Response to Loading.

The enthesis is a mineralized fibrocartilage transition that attaches tendon to bone and is vital for musculoskeletal function. Despite recent studies demonstrating the necessity of muscle loading for enthesis formation, the mechanisms that regulate enthesis formation and mechanoresponsiveness remain unclear. Therefore, the current study investigated the role of the gap junction protein connexin 43 in these processes by deleting Gja1 (the Cx43 gene) in the tendon and enthesis. Compared with their wild-type (WT) counterparts, mice lacking Cx43 showed disrupted entheseal cell alignment, reduced mineralized fibrocartilage, and impaired biomechanical properties of the supraspinatus tendon entheses during postnatal development. Cx43-deficient mice also exhibited reduced ability to complete a treadmill running protocol but no apparent deficits in daily activity, metabolic indexes, shoulder muscle size, grip strength, and major trabecular bone properties of the adjacent humeral head. To examine enthesis mechanoresponsiveness, young adult mice were subjected to modest treadmill exercise. Gja1 deficiency in the tendon and enthesis reduced entheseal anabolic responses to treadmill exercise: WT mice had increased expression of Sox9, Ihh, and Gli1 and increased Brdu incorporation, whereas Cx43-deficient mice showed no changes or decreased levels with exercise. Collectively, the results demonstrated an essential role for Cx43 in postnatal tendon enthesis formation, function, and response to loading; results further provided evidence implicating a link between Cx43 function and the hedgehog signaling pathway. © 2020 American Society for Bone and Mineral Research.

Morphology and Anabolic Response of Skeletal Muscles Subjected to Eccentrically or Concentrically Biased Exercise.

Long-term eccentric exercise is known to promote muscle growth better than concentric exercise, but its acute effect on muscle is not well understood because of misinterpreted modeling and in situ and in vitro stretch protocols. Knowing if the initial bout of eccentric exercise promotes muscle growth and limits damage is critical to understanding the effect of this mode of exercise. To directly evaluate the immediate effects of eccentric and concentric exercises on untrained muscle when fiber strains were physiological and exercise doses were comparable. Controlled laboratory study. Laboratory. A total of 40 skeletally mature male Long-Evans rats (age = 16 weeks, mass = 452.1 ± 35.2 g) were randomly assigned to an eccentric exercise (downhill walking, n = 16), concentric exercise (uphill walking, n = 16), or control (no exercise, n = 8) group. Rats were exposed to a single 15-minute bout of eccentric or concentric exercise on a motorized treadmill and then were euthanized at 6 or 24 hours postexercise. We harvested the vastus lateralis muscle bilaterally. The percentage increase or decrease in protein abundance in exercised animals relative to that in unexercised control animals was evaluated as elevated phosphorylated p70S6k relative to total p70S6k. Fiber damage was quantified using immunoglobulin G permeability staining. One-way analysis of variance and post hoc Tukey tests were performed. Rats exposed to eccentric exercise and euthanized at 24 hours had higher percentage response protein synthesis rates than rats exposed to eccentric exercise and euthanized at 6 hours (P = .02) or to concentric exercise and euthanized at 6 (P = .03) or 24 (P = .03) hours. We assessed 9446 fibers for damage and found only 1 fiber was infiltrated (in the concentric exercise group euthanized at 6 hours). Furthermore, no between-groups differences in immunoglobulin G fluorescent intensity were detected (P = .94). Incorporating eccentric exercise is a simple, universally available therapeutic intervention for promoting muscle recovery. A single 15-minute dose of eccentric exercise to a novice muscle can better exert an anabolic effect than a comparable dose of concentric exercise, with very limited evidence of fiber damage.

Two dietary advice strategies to increase protein intake among community-dwelling older adults: A feasibility study

Expert groups propose that recommendations for protein intake for older adults should be increased to compensate for a blunted anabolic response to protein. As many older adults do not meet the (current and higher) recommendations, we examined the feasibility of two dietary advice strategies to increase protein intake following either an even distribution of protein over the day ('even' strategy) or a peak in protein during one meal moment ('peak' strategy). This randomized controlled trial examined the feasibility (effectiveness and appreciation) of an 'even' or 'peak' dietary advise strategy on increasing protein intake after four weeks among community-dwelling older adults aged≥65y. Participants (n=60) were randomized into one of the three groups; two intervention groups ('even' or 'peak' strategy) and one control group. Participants of both intervention groups with a baseline protein intake <0.9g/kg adjusted body weight (aBW)/day received personalized dietary advice and protein enriched food products to increase their protein intake to at least 1.2g/kg aBW/day, and those with an intake between 0.9 and 1.2g/kg aBW/day to increase their protein intake with 0.3g/kg aBW/day. When current intake was higher than 1.2g/kg aBW/day they received advice to follow the strategy without increasing protein intake. The 'even' group was advised to consume a maximum of 20g of protein during each meal or snack moment while the 'peak' group was advised to consume at least one daily meal with 35-45g of protein. Effectiveness to increase protein intake was assessed by three 24-hrecalls after four weeks and appreciation of the dietary strategies by a five point scale questionnaire. Baseline protein intake was 71.0±12.1g/day in participants with a protein intake <1.2g/kg aBW/day. Protein increased significantly more in both intervention groups (+28.9±22.9g/day for the 'even' group and+21.8±21.4g/day for the 'peak' group) versus control (+5.7±13.0g/day), and participants following the 'peak' strategy more often had at least one meal per day very high in protein (≥35g). Both intervention groups considered the advice to increase protein as (very) clear (100% and 93.6% for the 'even' and 'peak' strategy), and most reported they would (partly) continue following the advice (81% and 88% for the 'even' and 'peak' strategy). The 'even' and 'peak' dietary advice strategies were effective in increasing protein intake in four weeks and both well appreciated by community-dwelling older adults. The study is registered in The Netherlands National Trial Register (NTR6849).