Androgens and the molecular regulation of skeletal muscle mass

Muscle atrophy or the unintended loss of skeletal muscle mass can occur as a consequence of pathological disorders e.g. muscular dystrophies, chronic diseases (cachexia), malnutrition, immobilisation (disuse), as well as from normal ageing (sarcopenia). As skeletal muscles play vital roles in maintaining body posture, controlling movement and regulating whole body protein metabolism, muscle atrophy can have debilitating consequences for the patient, often leading to a reduced quality of life. The prevention or therapy of skeletal muscle atrophy has been a topic of interest for many years and its applications are spreading from sport sciences to ageing and clinical medicine. Physical exercise, whether or not combined with nutritional supplements, is an effective and safe countermeasure to attenuate or prevent muscle wasting. However, this approach is not always feasible in seriously ill patients, elderly people or those who have suffered severe injuries. Therefore, a thorough understanding of the etiology and underlying mechanisms of different muscle wasting conditions, together with the identification of factors determining muscle mass in healthy adults, is important to improve the clinical outcome for people suffering from skeletal muscle atrophy. Androgens are considered to be the main sex steroids regulating body composition, with decades of research highlighting their anabolic effect on muscle mass. However, their clinical application in the treatment of muscle wasting is limited because of severe side effects. Therapeutic agents that could achieve anabolic effects on skeletal muscle without androgenic activities on other pheripheral tissues are of great clinical interest. Enhancing our understanding of the androgenic regulation of skeletal muscle mass and of the molecular factors and signalling pathways modulated by androgens, may help in the identification of novel muscle-specific therapeutic targets to combat the devastating effects of muscle wasting. A first aim of this thesis was to gain more insight into the interindividual variation in skeletal muscle mass. In a population-based cross-sectional study (study 1), we extensively investigated the determinants of muscle mass and function in a cohort of 677 healthy young men (25-45 years). Moreover we tried to indentify genetic variations in the androgen receptor (AR) that are associated with serum testosterone (T) levels and muscle mass and function. Our results mainly confirmed previous findings that skeletal muscle mass and strength are highly heritable and are influenced by age, anthropometrics, body composition, physical activity and sex steroid levels. Next to the number of CAG repeats, we identified two single nucleotide polymorphisms (SNPs) (rs5965433 and rs5919392) in the AR gene that are associated with serum T levels. However, we could not provide evidence that these genetic variations in the AR gene also affect muscle mass or function. In a second part of this thesis, we investigated the effects of T and estradiol (E2) administration on the signalling pathway regulating muscle protein degradation in an “androgen deprivation-induced muscle atrophy mice model” (study 2). The gene and protein expression levels of muscle atrophy-inducing targets including Atrogin-1, MuRF1 and myostatin were measured at 3 different time-points (1, 7, and 30 days) and in 3 different muscle types [extensor digitorum longus (EDL), soleus (SOL) and the levator ani/bulcocavernosus muscles (LA/BC)] following orchidectomy of male mice. Our results showed important differences in atrophy signalling response between the LA/BC and the locomotor muscles. In the LA/BC, androgen deprivation resulted in a rapid and persistent upregulation of Atrogin-1 and MuRF1 mRNA and a downregulation of myostatin mRNA during the 30 day period, effects which were fully reversed by T. In the SOL and EDL muscle, a less pronounced upregulation of both atrogenes was only detectable early after orchidectomy (day 1), while myostatin mRNA levels were upregulated in the EDL only. No changes in the protein levels of Atrogin-1, MuRF1 and myostatin in EDL were found at any time point following orchidectomy, questioning their role in the androgenic regulation of the locomotor muscle mass. Furthermore, our results demonstrated that E2-treatment during androgen deprivation has anabolic effects on the LA/BC, which was associated with a partial suppression of Atrogin-1 and MuRF1 gene expression, indicating that further experiments examining the effects of E2 on skeletal muscle mass are of potential significance. In study 3, the regulation of atrophy and hypertrophy signalling molecules by T were examined in a “disuse atrophy mice model”. Following 1, 5 and 14 days of tail suspension, gene and protein expression levels of IGF1/Akt/p70S6K as well as myostatin, REDD1, Atrogin-1 and MuRF1 were examined in the SOL and EDL muscle. Because previous studies have shown that disuse atrophy is associated with reductions in serum T levels, we hypothesized that the therapeutic use of androgens in this setting would be beneficial. Tail suspension resulted in an increase, followed by a transient drop, in T levels and a decrease in muscle mass. IGF1 mRNA levels were downregulated during 1 and 5 days of tail suspension, and a subsequent reduction in the phosphorylated levels of Akt after 14 days of tail suspension was observed. Atrogin-1, MuRF1, myostatin and REDD1 gene expression levels were rapidly and transiently upregulated as early as 1 day following immobilization, even before muscle atrophy was observed. However, T treatment during tail suspension was not able to ameliorate muscle mass and did not restore the alterations in expression levels of catabolic and anabolic signaling molecules, indicating that the known anabolic effects of T are not sufficient to maintain muscle mass during muscle disuse. In conclusion, this thesis contributed to the better understanding of the molecular regulation of skeletal muscle mass by androgens and estrogens using 3 different models: a cohort of eugonadal men, an androgen deprivation-induced muscle atrophy mice model (orchidectomy), and a disuse atrophy mice model (tail suspension). Our results provided evidence for a complex mechanism by which T regulates muscle mass, with important time- and muscle type dependent differences

Genetic variations in the androgen receptor are associated with steroid concentrations and anthropometrics but not with muscle mass in healthy young men

OBJECTIVE: The relationship between serum testosterone (T) levels, muscle mass and muscle force in eugonadal men is incompletely understood. As polymorphisms in the androgen receptor (AR) gene cause differences in androgen sensitivity, no straightforward correlation can be observed between the interindividual variation in T levels and different phenotypes. Therefore, we aim to investigate the relationship between genetic variations in the AR, circulating androgens and muscle mass and function in young healthy male siblings. DESIGN: 677 men (25-45 years) were recruited in a cross-sectional, population-based sibling pair study. METHODS: Relations between genetic variation in the AR gene (CAGn, GGNn, SNPs), sex steroid levels (by LC-MS/MS), body composition (by DXA), muscle cross-sectional area (CSA) (by pQCT), muscle force (isokinetic peak torque, grip strength) and anthropometrics were studied using linear mixed-effect modelling. RESULTS: Muscle mass and force were highly heritable and related to age, physical activity, body composition and anthropometrics. Total T (TT) and free T (FT) levels were positively related to muscle CSA, whereas estradiol (E2) and free E2 (FE2) concentrations were negatively associated with muscle force. Subjects with longer CAG repeat length had higher circulating TT, FT, and higher E2 and FE2 concentrations. Weak associations with TT and FT were found for the rs5965433 and rs5919392 SNP in the AR, whereas no association between GGN repeat polymorphism and T concentrations were found. Arm span and 2D:4D finger length ratio were inversely associated, whereas muscle mass and force were not associated with the number of CAG repeats. CONCLUSIONS: Age, physical activity, body composition, sex steroid levels and anthropometrics are determinants of muscle mass and function in young men. Although the number of CAG repeats of the AR are related to sex steroid levels and anthropometrics, we have no evidence that these variations in the AR gene also affect muscle mass or function

Different approaches for bladder neck dissection during robot-assisted radical prostatectomy: the Aalst technique

ABSTRACT Introduction: Bladder neck dissection is one of the most delicate surgical steps of robotic-assisted radical prostatectomy (RARP) [1, 2], and it may affect surgical margins rate and functional outcomes [3, 4]. Given the relationship between outcomes and surgical experience [5–7], it is crucial to implement a step-by-step approach for each surgical step of the procedure, especially in the most challenging part of the intervention. In this video compilation, we described the techniques for bladder neck dissection utilized at OLV Hospital (Aalst, Belgium). Surgical Technique: We illustrated five different techniques for bladder neck dissection during RARP. The anterior technique tackles the bladder neck from above until the urethral catheter is visualized, and then the dissection is completed posteriorly. The lateral and postero-lateral approaches involve the identification of a weakness point at the prostate-vesical junction and aim to develop the posterior plane – virtually until the seminal vesicles – prior to the opening of the urethra anteriorly. Finally, we described our techniques for bladder neck dissection in more challenging cases such as in patients with bulky middle lobes and prior surgery for benign prostatic hyperplasia. All approaches follow anatomic landmarks to minimize positive surgical margins and aim to preserve the bladder neck in order to promote optimal functional recovery. All procedures were performed with DaVinci robotic platforms using a 3-instruments configuration (scissors, fenestrated bipolar, and needle driver). As standard protocol at our Institution, urinary catheter was removed on postoperative day two [8]. Conclusions: Five different approaches for bladder neck dissection during RARP were described in this video compilation. We believe that the technical details provided here might be of help for clinicians who are starting their practice with this surgical intervention

Gene expression of carnosine-related enzymes and transporters in skeletal muscle

Chronic oral beta-alanine supplementation can elevate muscle carnosine (beta-alanyl-L-histidine) content and improve high-intensity exercise performance. However, the regulation of muscle carnosine levels is poorly understood. The uptake of the rate-limiting precursor betaalanine and the enzyme catalyzing the dipeptide synthesis are thought to be key steps. The aims of this study were to investigate the expression of possible carnosine-related enzymes and transporters in both human and mouse skeletal muscle in response to carnosine-altering stimuli. Human gastrocnemius lateralis and mouse tibialis anterior muscle samples were subjected to HPLC and qPCR analysis. Mice were subjected to chronic oral supplementation of beta-alanine and carnosine or to orchidectomy (7 and 30 days, with or without testosterone replacement), stimuli known to, respectively, increase and decrease muscle carnosine and anserine. The following carnosine-related enzymes and transporters were expressed in human and/or mouse muscles: carnosine synthase (CARNS), carnosinase- 2 (CNDP2), the carnosine/histidine transporters PHT1 and PHT2, the beta-alanine transporters TauT and PAT1, betaalanine transaminase (ABAT) and histidine decarboxylase (HDC). Six of these genes showed altered expression in the investigated interventions. Orchidectomy led to decreased muscle carnosine content, which was paralleled with decreased TauT expression, whereas CARNS expression was surprisingly increased. Beta-alanine supplementation increased both muscle carnosine content and TauT, CARNS and ABAT expression, suggesting that muscles increase beta-alanine utilization through both dipeptide synthesis (CARNS) and deamination (ABAT) and further oxidation, in conditions of excess availability. Collectively, these data show that muscle carnosine homeostasis is regulated by nutritional and hormonal stimuli in a complex interplay between related transporters and enzymes

Birth weight in relation to sex steroid status and body composition in young healthy male siblings

Context: Sex steroid concentrations have a strong genetic determination, but environmental factors and body composition play an important role. From studies in children with intrauterine growth restriction, low birth weight has been associated with altered gonadotropin concentrations. Objective: We aim to investigate sex steroid concentrations in healthy young brothers in relation to birth weight (normal gestational age), body composition, and parental steroid concentrations. Design and Setting: We conducted a cross-sectional, population-based sibling pair study with inclusion of parental data. Participants: A total of 677 men (25-45 yr old) were included in this study, with 296 independent pairs of brothers and 122 fathers. Main Outcomes: We measured testosterone, estradiol, leptin, adiponectin, IGF-I (immunoassays), and free steroid hormones (calculated) in relation to birth weight and changes in body composition (dual-energy x-ray absorptiometry). Results: Birth weight was associated with serum testosterone (P = 0.0004)and SHBG (P = 0.0001), independent from weight, age, or fat mass, whereas no association with (free) estradiol, LH, or FSH was found. Paternal testosterone (P = 0.02), estradiol (P = 0.04), and SHBG (P = 0.0004) were associated with the respective sex steroid concentrations in the brothers. Weight increase (population rank) during life, was associated with lower testosterone (-15%; P < 0.001), independent from current weight and with higher free estradiol concentrations (+8%; P = 0.002), whereas weight decrease was associated with higher testosterone (+13%; P < 0.001). Conclusion: Birth weight and paternal steroid concentrations are associated with testosterone concentrations, independent from adult weight. These findings support the concept of in utero programming across the range of birth weight. (J Clin Endocrinol Metab 95: 1587-1594, 2010

Effects of sprint training combined with vegetarian or mixed diet on muscle carnosine content and buffering capacity

Carnosine is an abundant dipeptide in human skeletal muscle with proton buffering capacity. There is controversy as to whether training can increase muscle carnosine and thereby provide a mechanism for increased buffering capacity. This study investigated the effects of 5 weeks sprint training combined with a vegetarian or mixed diet on muscle carnosine, carnosine synthase mRNA expression and muscle buffering capacity. Twenty omnivorous subjects participated in a 5 week sprint training intervention (2-3 times per week). They were randomized into a vegetarian and mixed diet group. Measurements (before and after the intervention period) included carnosine content in soleus, gastrocnemius lateralis and tibialis anterior by proton magnetic resonance spectroscopy ((1)H-MRS), true-cut biopsy of the gastrocnemius lateralis to determine in vitro non-bicarbonate muscle buffering capacity, carnosine content (HPLC method) and carnosine synthase (CARNS) mRNA expression and 6 x 6 s repeated sprint ability (RSA) test. There was a significant diet x training interaction in soleus carnosine content, which was non-significantly increased (+11%) with mixed diet and non-significantly decreased (-9%) with vegetarian diet. Carnosine content in other muscles and gastrocnemius buffer capacity were not influenced by training. CARNS mRNA expression was independent of training, but decreased significantly in the vegetarian group. The performance during the RSA test improved by training, without difference between groups. We found a positive correlation (r = 0.517; p = 0.002) between an invasive and non-invasive method for muscle carnosine quantification. In conclusion, this study shows that 5 weeks sprint training has no effect on the muscle carnosine content and carnosine synthase mRNA

Androgen receptor polymorphisms in relation to circulating gonadal steroids and muscle parameters.

<p>Data are presented as standardized estimate ± SD (p-value). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086235#s3" target="_blank">Results</a> from mixed effects accounted for family structure and adjusted for age, height and weight.</p

Heritability estimates of selected muscle parameters.

<p>Heritability estimates of selected muscle parameters.</p

Anthropometrics according to quartiles of AR CAG repeat polymorphism.

<p>P-values result from ANOVA (overall difference between categories). Each bar represents the mean ± standard deviation (SD).</p

Combined gene and protein expression of hormone-sensitive lipase and adipose triglyceride lipase, mitochondrial content, and adipocyte size in subcutaneous and visceral adipose tissue of morbidly obese men

Aims: Lipotoxicity in obesity might be a failure of adipocytes to respond sufficiently adequate to persistent energy surplus. To evaluate the role of lipolytic enzymes or mitochondria in lipotoxicity, we studied expression levels of genes and proteins involved in lipolysis and mitochondrial DNA (mtDNA) content. Methods: As differences in lipid metabolism between men and women are extremely complex, we recruited only men (lean and morbidly obese) and collected subcutaneous and visceral adipose tissue during abdominal surgery for real-time PCR gene expression, protein expression, and microscopic study. Results: Although mRNA levels of hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) were increased in visceral adipose tissue of morbidly obese men, this was not paralleled by alterations in protein expression and phosphorylation of HSL and ATGL. mtDNA content of visceral adipose tissue was increased in morbidly obese men as compared to lean controls (p < 0.013). Positive correlations were observed between visceral adipocyte size and serum triacylglycerol (r = 0.6, p < 0.007) as well as between visceral adipocyte size and CRP (r = 0.6, p < 0.009) in analyses performed separately in obese men. Conclusion: Lipotoxicity of morbidly obese men might be related to the quantitative impact of the visceral fat depot rather than to important dysregulation of involved lipolytic enzymes or adipocyte mitochondria