Circulating Biomarkers of Testosterone's Anabolic Effects on Fat-Free Mass.

BACKGROUND: Biomarkers that predict response to anabolic therapies could expedite the development of function-promoting anabolic drugs. This study aimed to identify serum biomarkers that are responsive to testosterone administration and associated with increases in fat-free mass (FFM). METHODS: Serum samples were obtained from the 5α-Reductase Trial, a randomized trial that compared the effects of graded doses of testosterone enanthate for 20 weeks in healthy men randomized with placebo or dutasteride (dual SRD5A inhibitor). Testosterone's effects on FFM or strength measures did not differ between placebo vs dutasteride groups. Accordingly, 54 subjects treated with testosterone plus placebo were included in the discovery cohort, and 48 subjects randomized to dutasteride were included in the validation cohort. A total of 1162 biomarkers were evaluated using prespecified criteria. RESULTS: In the discovery cohort, testosterone administration increased propeptide of type III collagen (PRO-C3) and propeptide of type VI collagen (PRO-C6) levels in a dose- and concentration-dependent manner; increases in these biomarkers from baseline to week 12 were associated with changes in FFM from baseline to week 20 (PRO-C3: r2 = 0.437, P < 0.001; PRO-C6: r2 = 0.434, P < 0.001). Changes in PRO-C3 and PRO-C6 levels were significantly associated with changes in chest press strength (PRO-C3: r2 = 0.394, P < 0.001; PRO-C6: r2 = 0.530, P < 0.001). In the SOMAscan, changes in IGF binding protein-6 (IGFBP6) and glypican 3 (GPC3) were associated with changes in total and free testosterone levels and FFM. These findings were replicated in the Validation cohort. CONCLUSION: PRO-C3, PRO-C6, IGFBP6, and GPC3 fulfilled the prespecified criteria for biomarkers of testosterone-induced muscle anabolism. Changes in these biomarkers were associated with changes in total and free testosterone concentrations and with testosterone-induced gains in FFM.

Collagen Type III and VI Turnover in Response to Long-Term Immobilization.

BACKGROUND: Muscle mass and function are perturbed by immobilization and remobilization. When muscle mass changes, the quality and quantity of the extracellular matrix protein, particularly the collagens, change with it. In this study, we investigated the temporal profile of three peptide biomarkers derived from turnover of collagen type III and type VI in a long-term immobilization and remobilization study. We also compared individual biomarker levels with Lean body Mass (LBM) and changes therein, hypothesizing that these biomarkers would be biomarkers of the remodeling processes associated with immobilization and/or remobilization. METHODS: In the Berlin bed rest study, 20 young men were recruited and randomly assigned to 8-week's strict bed rest with or without resistive vibration exercise countermeasure. We measured three neo-epitope ELISA kits in the serum samples of this study: Pro-C3, measured the synthesis of collagen type III; Pro-C6, measured the synthesis of collagen type VI; and C6M measured the degradation of collagen type VI induced by MMP-2 and MMP-9 cleavage. RESULTS: Pro-C3 and Pro-C6 biomarkers are up-regulated with both immobilization and remobilization, whereas C6M is hardly affected at all. We found that Pro-C3 and C6M levels are related to LBM at baseline and that high levels of Pro-C6 are associated with smaller changes in muscle mass during both immobilization and remobilization. CONCLUSION: The Pro-C3 and-C6 biomarkers change likely reflect remodeling changes in response to unloading or reloading, whereas C6M does not appear to respond to unloading. Pro-C3 and C6M levels correlate with LBM at baseline, while Pro-C6 is related to the anabolic and catabolic responses to unloading and reloading.

Collagen fragment biomarkers as serological biomarkers of lean body mass - a biomarker pilot study from the DAHANCA25B cohort and matched controls.

BACKGROUND: Loss of muscle mass and function is an important complication to ageing and a range of pathologies, including, but not restricted to, cancer, organ failures, and sepsis. A number of interventions have been proposed ranging from exercise to anabolic pharmacological therapy, with varying success. Easily applicable serological biomarkers of lean and/or muscle mass and change therein would benefit monitoring of muscle mass during muscle atrophy as well as during recovery. We set out to validate if novel peptide biomarkers derived from Collagen III and VI were markers of lean body mass (LBM) or change therein in head and neck cancer patients in the Danish Head and Neck Cancer Group(DAHANCA) 25B cohort subjected to resistance training as well as in an age-matched and gender-matched control group. METHODS: Blood samples and dual X-ray absorptiometry data were measured at baseline, after 12 and 24 weeks in 41 HNSCC subjects of the DAHANCA 25B cohort of subjects recovering from neck and head cancer (stages provided in Table 1), and at baseline only in 21 healthy age-matched and gender-matched controls. Serum from blood was analyzed for the ProC3, IC6, and C6M peptide biomarkers and LBM were derived from the dual X-ray absorptiometry scans. RESULTS: We were not able to show any correlation between biomarkers and LBM or C6M and anabolic response to exercise in recovering head and neck cancer patients. However, we did find that the biomarkers IC6, IC6/C6M, and ProC3 are biomarkers of LBM in the control group subjects (R (2)/P of 0.249/0.035, 0.416/0.007 and 0.178 and P = 0.057, respectively). CONCLUSION: In conclusion, the IC6, ProC3, and IC6/C6M biomarkers are indeed biomarkers of LBM in healthy individuals of both genders, but not in HNSCC patients.

3-D ultrastructure and collagen composition of healthy and overloaded human tendon: evidence of tenocyte and matrix buckling.

Achilles tendinopathies display focal tissue thickening with pain and ultrasonography changes. Whilst complete rupture might be expected to induce changes in tissue organization and protein composition, little is known about the consequences of non-rupture-associated tendinopathies, especially with regards to changes in the content of collagen type I and III (the major collagens in tendon), and changes in tendon fibroblast (tenocyte) shape and organization of the extracellular matrix (ECM). To gain new insights, we took biopsies from the tendinopathic region and flanking healthy region of Achilles tendons of six individuals with clinically diagnosed tendinopathy who had no evidence of cholesterol, uric acid and amyloid accumulation. Biochemical analyses of collagen III/I ratio were performed on all six individuals, and electron microscope analysis using transmission electron microscopy and serial block face-scanning electron microscopy were made on two individuals. In the tendinopathic regions, compared with the flanking healthy tissue, we observed: (i) an increase in the ratio of collagen III : I proteins; (ii) buckling of the collagen fascicles in the ECM; (iii) buckling of tenocytes and their nuclei; and (iv) an increase in the ratio of small-diameter : large-diameter collagen fibrils. In summary, load-induced non-rupture tendinopathy in humans is associated with localized biochemical changes, a shift from large- to small-diameter fibrils, buckling of the tendon ECM, and buckling of the cells and their nuclei.

Type VI collagen turnover-related peptides-novel serological biomarkers of muscle mass and anabolic response to loading in young men.

BACKGROUND: Immobilization-induced loss of muscle mass is a complex phenomenon with several parallels to sarcopenic and cachectic muscle loss. Muscle is a large organ with a protein turnover that is orders of magnitude larger than most other tissues. Thus, we hypothesize that muscle loss and regain is reflected by peptide biomarkers derived from type VI collagen processing released in the circulation. METHODS: In order to test this hypothesis, we set out to develop an ELISA assay against an type VI collagen N-terminal globular domain epitope (IC6) and measured the levels of IC6 and an MMP-generated degradation fragment of collagen 6, (C6M) in a human immobilization-remobilization study setup with young (n = 11) and old (n = 9) men. They were subjected to 2 weeks of unilateral lower limb immobilization followed by 4 weeks of remobilization including thrice weekly resistance training, using the contralateral leg as internal controls. Subjects were sampled for strength, quadriceps muscle volume and blood at baseline (PRE), post-immobilization (2W), and post-remobilization (4W). Blood were subsequently analyzed for levels of the C6M and IC6 biomarkers. We subsequently tested if there was any correlation between C6M, IC6, or the C6M/IC6 ratio and muscle mass or strength at baseline. We also tested whether there was any relation between these biomarkers and changes in muscle mass or strength with immobilization or remobilization. RESULTS: The model produced significant loss of muscle mass and strength in the immobilized leg. This loss was bigger in young subjects than in elderly, but whereas the young recovered almost fully, the elderly had limited regrowth of muscle. We found a significant correlation between IC6 and muscle mass at baseline in young subjects (R (2) = 0.6563, p = 0.0045), but none in the elderly. We also found a significant correlation between C6M measured at the 4W time point and the change in muscle mass during remobilization, again only manifesting in the young men(R (2) = 0.6523, p = 0.0085). This discrepancy between the young and the elderly may be caused in part by much smaller changes in muscle mass in the elderly and partly by the relative small sample size. CONCLUSION: While we cannot rule out the possibility that these biomarkers in part stem from other tissues, our results strongly indicate that these markers represent novel biomarkers of muscle mass or change in muscle mass in young men.

Musculoskeletal ageing and primary prevention.

Loss of musculoskeletal mass and function is a natural ageing trait, reinforced by an unhealthy life style. Loss of bone (osteoporosis) and muscle (sarcopaenia) are conditions whose prevalence are increasing because of the change in population distribution in the western world towards an older mean age. Improvements in lifestyle factors, such as diet, smoking and exercise, are the most powerful tools to combat this decline efficiently; however, public health interventions aimed at tackling these problems have shown abysmal success at the population level, mostly due to failure in compliance. With these issues in mind, we believe that the primary prevention modality in coming decades will be pharmacological. We review the basic biology of musculoskeletal ageing and what measures can be taken to prevent ageing-associated loss of musculoskeletal mass and function, with particular emphasis on pharmacological means.

The neo-epitope specific PRO-C3 ELISA measures true formation of type III collagen associated with liver and muscle parameters.

AIM: The present study describes the assessment of true formation of type III collagen in different pathologies using a neo-epitope specific competitive Enzyme-linked immunosorbent assay (ELISA) towards the N-terminal propeptide of type III collagen (PRO-C3). METHODS: The monoclonal antibody was raised against the N-protease mediated cleavage site of the N-terminal propeptide of type III collagen and a competitive ELISA was developed using the selected antibody. The assay was evaluated in relation to neo-epitope specificity, technical performance, and as a marker for liver fibrosis and muscle mass using the rat carbon tetrachloride (CCl4) model and a study of immobilization induced muscle loss in humans, respectively. RESULTS: The ELISA was neo-epitope specific, technically stable and can be assessed in serum and plasma samples. In the CCl4 liver fibrosis model it was observed that serum PRO-C3 were significantly elevated in rats with liver fibrosis as seen by histology (56% elevated in the highest quartile of total hepatic collagen compared to control rats, p<0.001) and correlated significantly to total hepatic collagen in the diseased rats (r=0.46, p<0.01) and not in control rats, suggesting the pathological origin of the epitope. Human plasma PRO-C3 correlated significantly to muscle mass at baseline (R(2)=0.44, p=0.036). CONCLUSION: The developed neo-epitope specific serum ELISA for type III procollagen (PRO-C3) reflects true formation as it is specific for the propeptide cleaved off the intact collagen molecule. In a clinical and in a rodent study we showed that this marker was highly related to liver fibrosis and muscle mass.

Menopause, estrogens and frailty.

The controversy surrounding the results from the Women's Health Initiative (WHI) trials published a decade ago caused a significant decline in the use of menopausal hormone replacement therapy. However, these results have been vehemently contested and several lines of evidence suggest that in perimenopausal and non-obese women, estrogen therapy may indeed be of benefit. There is ample proof that menopause causes a loss of musculoskeletal tissue mass and quality, thereby causing a loss of health and quality of life. There is also solid evidence that hormone replacement therapy in itself prevents most of these effects in connective tissue in itself. Besides the independent, direct effects on the musculoskeletal tissues, estrogen deficiency also reduces the ability to adequately respond and adapt to external mechanical and metabolic stressors, e.g. exercise, which are otherwise the main stimuli that should maintain musculoskeletal integrity and metabolic function. Thus, normophysiological estrogen levels appear to exert a permissive effect on musculoskeletal adaptations to loading, thereby likely improving the outcome of rehabilitation following critical illness, musculoskeletal trauma or orthopedic surgical therapy. These effects add to the evidence supporting the use of estrogen therapy, particularly accelerated gain of functional capacity and independence following musculoskeletal disuse.

Serological muscle loss biomarkers: an overview of current concepts and future possibilities.

BACKGROUND: The skeletal muscle mass is the largest organ in the healthy body, comprising 30-40 % of the body weight of an adult man. It confers protection from trauma, locomotion, ventilation, and it represents a "sink" in glucose metabolism and a reservoir of amino acids to other tissues such as the brain and blood cells. Naturally, loss of muscle has dire consequences for health as well as functionality. Muscle loss is a natural consequence of especially aging, inactivity, and their associated metabolic dysfunction, but it is strongly accelerated in critical illness such as organ failure, sepsis, or cancer. Whether this muscle loss is considered a primary or secondary condition, it is known that muscle loss is a symptom that predicts morbidity and mortality and one that is known to impact quality of life and independence. Therefore, monitoring of muscle mass is relevant in a number of pathologies as well as in clinical trials as measures of efficacy as well as safety. METHODS AND RESULTS: Existing biomarkers of muscle mass or muscle loss have shown to be either too unreliable or too impractical in relation to the perceived clinical benefit to reach regular clinical research or use. We suggest serological neoepitope biomarkers as a possible technology to address some of these problems. Blood biomarkers of this kind have previously been shown to respond with high sensitivity and shorter time to minimum significant change than available biomarkers of muscle mass. We provide brief reviews of existing muscle mass or function biomarker technologies, muscle protein biology, and existing neoepitope biomarkers and proceed to present tentative recommendations on how to select and detect neoepitope biomarkers. CONCLUSION: We suggest that serological peptide biomarkers whose tissue and pathology specificity are derived from post-translational modification of proteins in tissues of interest, presenting so-called neoepitopes, represents an exciting candidate technology to fill out an empty niche in biomarker technology.

Differences in transcriptional patterns of extracellular matrix, inflammatory, and myogenic regulatory genes in myofibroblasts, fibroblasts, and muscle precursor cells isolated from old male rat skeletal muscle using a novel cell isolation procedure.

Aged skeletal muscle displays increased fibrosis and impaired regeneration. While it is not well characterized how skeletal muscle fibroblasts contribute to these phenomena, transforming growth factor-ß1 (TGF-ß1) and Delta/Notch signaling have been implicated to influence muscle regeneration. In this study, a unique combination of aging phenotypes is identified in differentiating fibroblasts (myofibroblasts), proliferating fibroblasts, and muscle precursor cells (MPCs) that characterize an impaired regenerative potential observed in aged skeletal muscle. Using a novel dual-isolation technique, that isolates fibroblasts and MPCs from the same rat skeletal muscle sample, and cell culture conditions of 5 % O(2) and 5 % CO(2), we report for the first time that myofibroblasts from 32-mo-old skeletal muscle, compared to 3-mo-old, display increased levels of mRNA for the essential extracellular matrix (ECM) genes, collagen 4α1 (83 % increase), collagen 4α2 (98 % increase), and laminin 2 (113 % increase), as well as increased levels of mRNA for the inflammatory markers, interleukin-6 (4.3-fold increase) and tumor necrosis factor α (3.2-fold increase), and TGF-ß1 (84 % increase), whose protein controls proliferation and differentiation. Additionally, we demonstrate that proliferating fibroblasts from 32-mo-old skeletal muscle display increased levels of mRNA for the Notch ligand, Delta 1 (≥2.0-fold increase). Together, these findings suggest that increased expression of ECM and inflammatory genes in myofibroblasts from 32-mo-old skeletal muscle may contribute to the fibrogenic phenotype that impairs regeneration in aged skeletal muscle. Furthermore, we believe the novel dual-isolation technique developed here may be useful in studies that investigate communications among MPCs, fibroblasts, and myofibroblasts in skeletal muscle.

Effects of 2 weeks lower limb immobilization and two separate rehabilitation regimens on gastrocnemius muscle protein turnover signaling and normalization genes.

BACKGROUND: Limb immobilization causes a rapid loss of muscle mass and strength that requires appropriate rehabilitation to ensure restoration of normal function. Whereas the knowledge of muscle mass signaling with immobilization has increased in recent years, the molecular regulation in the rehabilitation of immobilization-induced muscle atrophy is only sparsely studied. To investigate the phosphorylation and expression of candidate key molecular muscle mass regulators after immobilization and subsequent rehabilitation we performed two separate studies. METHODS: We immobilized the lower limb for 2 weeks followed by the in-house hospital standard physiotherapy rehabilitation (Study 1). Secondly, we conducted an intervention study using the same 2 weeks immobilization protocol during which protein/carbohydrate supplementation was given. This was followed by 6 weeks of rehabilitation in the form of resistance training and continued protein/carbohydrate supplementation (Study 2). We obtained muscle biopsies from the medial gastrocnemius prior to immobilization (PRE), post-immobilization (IMMO) and post-rehabilitation (REHAB) and measured protein expression and phosphorylation of Akt, mTOR, S6k, 4E-BP1, GSK3ß, ubiquitin and MURF1 and mRNA expression of Atrogin-1, MURF1, FOXO1, 3 and 4 as well as appropriate housekeeping genes. RESULTS: In both studies, no changes in protein expression or phosphorylation for any measured protein were observed. In Study 1, FOXO3 and FOXO4 mRNA expression decreased after IMMO and REHAB compared to PRE, whereas other mRNAs remained unchanged. Interestingly, we found significant changes in expression of the putative housekeeping genes GAPDH, HADHA and S26 with immobilization in both studies. CONCLUSIONS: In neither study, the changes in muscle mass associated with immobilization and rehabilitation were accompanied by expected changes in expression of atrophy-related genes or phosphorylation along the Akt axis. Unexpectedly, we observed significant changes in several of the so-called housekeeping genes GAPDH, HADHA and S26 with immobilization in both studies, thereby questioning the usefulness of these genes for normalization of RNA data purposes in muscle immobilization studies.

Activated protein synthesis and suppressed protein breakdown signaling in skeletal muscle of critically ill patients.

BACKGROUND: Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR), glycogen synthase kinase 3ß (GSK3ß) and forkhead box O (FoxO) pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors involved in muscle mass regulation, we investigated the phosphorylation and expression of key factors in these protein synthesis and breakdown signaling pathways in thigh skeletal muscle of critically ill intensive care unit (ICU) patients compared with healthy controls. METHODOLOGY/PRINCIPAL FINDINGS: ICU patients were systemically inflamed, moderately hyperglycemic, received insulin therapy, and showed a tendency to lower plasma branched chain amino acids compared with controls. Using Western blotting we measured Akt, GSK3ß, mTOR, ribosomal protein S6 kinase (S6k), eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), and muscle ring finger protein 1 (MuRF1); and by RT-PCR we determined mRNA expression of, among others, insulin-like growth factor 1 (IGF-1), FoxO 1, 3 and 4, atrogin1, MuRF1, interleukin-6 (IL-6), tumor necrosis factor α (TNF-α) and myostatin. Unexpectedly, in critically ill ICU patients Akt-mTOR-S6k signaling was substantially higher compared with controls. FoxO1 mRNA was higher in patients, whereas FoxO3, atrogin1 and myostatin mRNAs and MuRF1 protein were lower compared with controls. A moderate correlation (r2=0.36, p<0.05) between insulin infusion dose and phosphorylated Akt was demonstrated. CONCLUSIONS/SIGNIFICANCE: We present for the first time muscle protein turnover signaling in critically ill ICU patients, and we show signaling pathway activity towards a stimulation of muscle protein synthesis and a somewhat inhibited proteolysis.

Effects of concentric and repeated eccentric exercise on muscle damage and calpain-calpastatin gene expression in human skeletal muscle.

The purpose of this study was to compare the responsiveness of changes in Ca(2+)-content and calpain-calpastatin gene expression to concentric and eccentric single-bout and repeated exercise. An exercise group (n = 14) performed two bouts of bench-stepping exercise with 8 weeks between exercise bouts, and was compared to a control-group (n = 6). Muscle strength and soreness and plasma creatine kinase and myoglobin were measured before and during 7 days following exercise bouts. Muscle biopsies were collected from m. vastus lateralis of both legs prior to and at 3, 24 h and 7 days after exercise and quantified for muscle Ca(2+)-content and mRNA levels for calpain isoforms and calpastatin. Exercise reduced muscle strength and increased muscle soreness predominantly in the eccentric leg (P < 0.05). These responses as well as plasma levels of creatine kinase and myoglobin were all attenuated after the repeated eccentric exercise bout (P < 0.05). Total muscle Ca(2+)-content did not differ between interventions. mRNA levels for calpain 2 and calpastatin were upregulated exclusively by eccentric exercise 24 h post-exercise (P < 0.05), with no alteration in expression between bouts. Calpain 1 and calpain 3 mRNA did not change at any specific time point post-exercise for either intervention. Our mRNA results suggest a regulation on the calpain-calpastatin expression response to muscle damaging eccentric exercise, but not concentric exercise. Although a repeated bout effect was demonstrated in terms of muscle function, no immediate support was provided to suggest that regulation of expression of specific system components is involved in the repeated bout adaptation.