Testing anabolic activity, potency and mechanisms of action of the phyto-derived beta 2 agonist higenamine.

Higenamine (Hige), a plant derived alkaloid is classified as ß2 agonist by the World Anti-Doping Agency (WADA). However, pharmacologic mechanisms of its performance-enhancing activity have not been investigated so far. Therefore, we investigate the anabolic activity and associated molecular mechanisms of Hige in C2C12 myotubes. In differentiated C2C12 cells dose-dependent effects of Hige on myotube size were analyzed. The mRNA expression of genes involved in hypertrophy was measured. For mechanistic studies, ß2-adrenoceptor (ADRB2), androgen receptor (AR), and estrogen receptor (ER) inhibitors and dexamethasone (Dexa) were co-incubated and myotube diameter was evaluated. The interaction of Hige with the AR and ER was investigated. Hige treatment significantly increased myotube diameters and stimulated the mRNA expression of hypertrophy-involved genes. In contrast to the ADRB2 inhibitor (ICI 118551), the ER inhibitor ZK 191703, the AR inhibitor Flutamide (Flu), and treatment with Dexa were able to antagonize the Hige-induced increase of myotube diameter. Hige has antagonistic activity in the AR and ER yeast transactivation assay. Our results demonstrate that Hige induces anabolic effects in C2C12 cells but not via the ADRB2. There are indications for a cross talk between Hige and the AR and ER. Future studies are necessary to investigate the involved molecular mechanisms.

Combinatory in vitro effects of the ß2-agonists salbutamol and formoterol in skeletal muscle cells.

ß2-agonists are used for the treatment of bronchoconstriction, but also abused in doping. Beside an ergogenic activity ß2-agonists may have also anabolic activity. Therefore, we investigated the anabolic activity and associated molecular mechanisms of Salbutamol (SAL) and Formoterol (FOR) alone, as well as in combination in C2C12 myotubes. In differentiated C2C12 cells, dose-dependent effects of SAL and FOR (alone/in combination) on myotube diameter, myosin heavy chain (MHC) protein expression and the mRNA expression of genes involved in hypertrophy were analyzed. ß2-adrenoceptor 2 (ADRB2), androgen receptor (AR) and estrogen receptor (ER) inhibitors, as well as dexamethasone (Dexa) were co-incubated with the ß2-agonists and myotube diameter was determined. SAL and FOR treatment significantly induced hypertrophy and increased MHC expression and the mRNA expression of Igf1, mTOR, PIk3r1 and AMpKa2. In contrast to an ER inhibitor, the ADRB2 and AR inhibitors, as well as Dexa antagonized FOR and SAL induced hypertrophy. Combined treatment with SAL and FOR resulted in significant additive effects on myotube diameter and MHC expression. Future clinical studies are needed to prove this effect in humans and to evaluate this finding with respect to antidoping regulations.

Moderate intensity continuous training reverses the detrimental effects of ovariectomy on RyR1 phosphorylation in rat skeletal muscle.

High 17ß-Estradiol (E2) concentrations in isolated ventricular myocytes as well as a lack of ovarian hormones in cardiac muscle of ovariectomized (OVX) rodents has been shown to lead to arrhythmogenic effects by inducing post-translational modifications, including phosphorylation of the sarcoplasmic reticulum (SR) Ca2+ release channel ryanodine receptor-2 (RyR2). The effects of estrogens on the phosphorylation status of the RyR1 in skeletal muscle have not been investigated before. Furthermore, while high intensity exercise has been shown to increase RyR phosphorylation, there is no data on the effects of moderate intensity continuous training (MICT). The aims of the study were to investigate the effects of a 3-day treatment with low (1 nM, moderate (5 nM) and high (10 nM, 100 nM) E2 concentrations on RyR1 mRNA and protein expression and phosphorylation status (pRyRSer2844) in cultured C2C12 myotubes and to study the effects of OVX on RyR1 expression and phosphorylation in rat skeletal muscle in combination with 3 weeks of MICT. Treatment with low, physiological E2 concentrations reduced dihydropyridine receptor (DHPR) and RyR1 mRNA content in C2C12 myotubes compared to untreated control cells, whereas RyR1 protein phosphorylation (pRyRSer2844) was significantly increased after treatment with high, non-physiological E2 concentrations (p ≤ 0.05). RyR1 protein content (p ≤ 0.05) and pRyRSer2844 (p ≤ 0.05) were significantly elevated in skeletal muscle of OVX vs. sham-operated rats. Importantly, pRyRSer2844 levels were similar to sham-operated controls in OVX rats after MICT (OVX vs. OVX + MICT, p ≤ 0.05). Our results indicate, that one of the actions of estrogens is to alter skeletal muscle Ca2+ homeostasis by modulating the expression and phosphorylation of the RyR1 in skeletal muscle. Notably, regular MICT was able to counteract RyR1 phosphorylation in skeletal muscle of OVX rats.

Effects of isoflavones on breast tissue and the thyroid hormone system in humans: a comprehensive safety evaluation.

Isoflavones are secondary plant constituents of certain foods and feeds such as soy, linseeds, and red clover. Furthermore, isoflavone-containing preparations are marketed as food supplements and so-called dietary food for special medical purposes to alleviate health complaints of peri- and postmenopausal women. Based on the bioactivity of isoflavones, especially their hormonal properties, there is an ongoing discussion regarding their potential adverse effects on human health. This review evaluates and summarises the evidence from interventional and observational studies addressing potential unintended effects of isoflavones on the female breast in healthy women as well as in breast cancer patients and on the thyroid hormone system. In addition, evidence from animal and in vitro studies considered relevant in this context was taken into account along with their strengths and limitations. Key factors influencing the biological effects of isoflavones, e.g., bioavailability, plasma and tissue concentrations, metabolism, temporality (pre- vs. postmenopausal women), and duration of isoflavone exposure, were also addressed. Final conclusions on the safety of isoflavones are guided by the aim of precautionary consumer protection.

The role of sex, adiposity, and gonadectomy in the regulation of irisin secretion.

A sexual dimorphism has been reported for the adipo-myokine irisin at rest and in response to exercise. The effects of male and female sex, adiposity, and gonadectomy on irisin secretion have not been investigated before. The objective of this study was to elucidate the effects of sex, adiposity, and gonadectomy in the regulation of irisin secretion as well as PGC-1α/FNDC5 mRNA and protein expression. We hypothesized that a lack of female sex hormones by ovariectomy reduces irisin levels and inhibits skeletal muscle expression of PGC-1α and FNDC5. Circulating irisin was measured in vivo in serum samples of healthy and obese men and women at rest and in response to acute exercise. The effects of gonadectomy on serum irisin, PGC-1α and FNDC5 muscle mRNA, and protein expression were investigated in ovariectomized (OVX) and orchiectomized (ORX) Wistar rats. Serum irisin at rest was not significantly different between men and women (lean or obese). However, in response to acute aerobic exercise, irisin levels increased significantly more in lean women versus men (p ≤ 0.05). In obese individuals, resting irisin concentrations were significantly higher compared to lean subjects (p ≤ 0.001) and the irisin response to acute exercise was blunted. Only the lack of gonadal hormones in OVX but not ORX rats increased serum irisin levels (p ≤ 0.01) and resulted in significantly increased body weight (p ≤ 0.01), adipose tissue content (p ≤ 0.05), muscle FNDC5 mRNA (p ≤ 0.05), and protein (p ≤ 0.01) expression without altering PGC-1α expression. Testosterone treatment in ORX rats leads to increased PGC-1α mRNA content and reduced PGC-1α protein content without affecting FDNC5 expression or serum irisin levels. We show that a sexual dimorphism exists for the acute irisin response to exercise in normal-weight but not in obese subjects. OVX, which is associated with increased adiposity and insulin insensitivity, increases basal FNDC5 expression and serum irisin, without altering PGC-1α expression. This may be an early sign for metabolic disturbances associated with menopause, such as a developing irisin resistance or an attempt of the organism to improve glucose metabolism.

Ecdysteroids: A novel class of anabolic agents?

Increasing numbers of dietary supplements with ecdysteroids are marketed as "natural anabolic agents". Results of recent studies suggested that their anabolic effect is mediated by estrogen receptor (ER) binding. Within this study the anabolic potency of ecdysterone was compared to well characterized anabolic substances. Effects on the fiber sizes of the soleus muscle in rats as well the diameter of C2C12 derived myotubes were used as biological readouts. Ecdysterone exhibited a strong hypertrophic effect on the fiber size of rat soleus muscle that was found even stronger compared to the test compounds metandienone (dianabol), estradienedione (trenbolox), and SARM S 1, all administered in the same dose (5 mg/kg body weight, for 21 days). In C2C12 myotubes ecdysterone (1 µM) induced a significant increase of the diameter comparable to dihydrotestosterone (1 µM) and IGF 1 (1.3 nM). Molecular docking experiments supported the ERß mediated action of ecdysterone. To clarify its status in sports, ecdysterone should be considered to be included in the class "S1.2 Other Anabolic Agents" of the list of prohibited substances of the World Anti-Doping Agency.

[Dislocation of double-threaded screws after stabilization of the odontoid process with the Knöringer technique]. / Dislozierte Doppelgewindeschrauben nach Densosteosynthese in Knöringer-Technik.

A type III fracture of the odontoid process according to Anderson-D'Alonzo in a 92-year-old patient was stabilized anteriorly with two double-threaded screws using the technique of Knöringer. Postoperatively, cranial dislocation of the screws was evident and attempts to correct the screw position failed resulting in screw proximity to the brain stem. In a second revision the screws could be removed by an anterior approach and fusion was achieved by a posterior approach. Type III fractures of the odontoid process are usually stable and should be treated conservatively. In case of instability posterior stabilization techniques should be selected. Anterior osteosynthesis of the odontoid process with double-threaded screws in osteoporotic bone carries the risk of screw dislocation.

Modulation of follistatin and myostatin propeptide by anabolic steroids and gender.

The purpose of this pilot study was to investigate the impact of training, anabolic steroids and endogenous hormones on myostatin-interacting proteins in order to identify manipulations of myostatin signalling. To identify whether analysis of the myostatin interacting proteins follistatin and myostatin propeptide is suitable to detect the abuse of anabolic steroids, their serum concentrations were monitored in untrained males, bodybuilders using anabolic steroids and natural bodybuilders. In addition, we analysed follistatin and myostatin propeptide serum proteins in females during menstrual cycle. Our results showed increased follistatin concentrations in response to anabolic steroids. Furthermore, variations of sex steroid levels during the menstrual cycle had no impact on the expression of follistatin and myostatin propetide. In addition, we identified gender differences in the basal expression of the investigated proteins. In general, follistatin and myostatin propeptide concentrations were relatively stable within the same individual both in males and females. In conclusion, the current findings provide an insight into gender differences in myostatin-interacting proteins and their regulation in response to anabolic steroids and endogenous hormones. Therefore our data provide new aspects for the development of doping prevention strategies.

ERß-specific agonists and genistein inhibit proliferation and induce apoptosis in the large and small intestine.

Epidemiological data indicate that intake of estrogens and isoflavones may be beneficial for the prevention of colorectal cancer (CRC). Based on this data, the aim of the study was to investigate estrogen receptor (ER) subtype-specific effects on intestinal homeostasis. Ovariectomized (OVX) female Wistar rats were either treated with 17ß-estradiol (4 µg/kg body wt/day) (E2), an ERα-specific agonist (ALPHA) (10 µg/kg body wt/day), an ERß-specific agonist (BETA) (100 µg/kg body wt/day) or genistein (GEN) (10 mg/kg body wt/day) for three weeks. Vehicle-treated OVX and SHAM animals and those cotreated with BETA and the pure antiestrogen Fulvestrant (ICI 182780) (100 µg/kg body wt/day and 3 mg/kg body wt/day) served as controls. GEN and BETA treatment but not E2 and ALPHA administration reduced proliferation in ileal and colonic mucosa cells. The rate of apoptosis in the small intestine and colon was increased by treatment with BETA and GEN, but not by E2. BETA induced antiproliferative and proapoptotic activity also in SHAM animals. The effects were antagonized by the pure antiestrogen Fulvestrant. Polymerase chain reaction gene array analysis revealed that BETA resulted in the downregulation of the oncogene transformation-related protein 63 (p63). Our data indicate that activation of the ERß by specific ERß agonists and GEN induces antiproliferative and proapoptotic effects in the intestinal tract. This observation can be taken as an indication that intake of GEN and specific ERß agonists may protect the ileal and colonic epithelium from tumor development via modulation of tissue homeostasis.

Myosin heavy chain expression pattern as a marker for anabolic potency: desoxymethyltestosterone (madol), norandrostenedione and testosterone repress MHC-IIb expression and stimulate MHC-IId/x expression in orchiectomized rat gastrocnemius muscle.

Both 19-norandrostenedione (estr-4-ene-3,17-dione, NOR) and desoxymethyltestosterone (17alpha-methyl-5alpha-androst-2-en-17beta-ol, DMT or "madol") are 'designer steroids' misused for doping purposes in the bodybuilding scene. We have previously characterized the pharmacological profile of madol and identified potential adverse side effects. The aim of this study was to investigate the anabolic potency of NOR, madol and the reference substance testosterone propionate (TP). Besides wet weight of the M.levator ani (LA), we examined the effects on muscle fiber type composition and myosin heavy chain (MHC) expression in the M.gastrocnemius (Gas) muscle as additional markers for anabolic potency. A Hershberger assay was performed, where orchiectomized (orchi) male Wistar rats were treated subcutaneously with NOR, madol, TP or vehicle control (all 1 mg/kg BW/day) for 12 days. Wet weights of the Gas, LA, prostate and seminal vesicle were examined to determine anabolic and androgenic effects. Fiber type composition of the Gas muscle was analyzed using ATPase staining, and MHC protein profiles were determined by silver stain and Western blot analysis. NOR and madol exhibited strong anabolic and weak androgenic potency by stimulating growth of the LA but not the prostate and seminal vesicle. Skeletal muscle fiber type composition characterized by ATPase staining was not significantly altered between the treatment groups, although there was a tendency toward lower levels of type IIB and increased type IIA fibers in all treatment groups relative to orchi. MHC protein expression determined by Western blot and silver stain analysis revealed that MHC IId/x was significantly up-regulated, while MHC IIb was significantly down-regulated in NOR, madol and TP groups relative to orchi. There were no significant differences for MHC IIa and MHC I expression between groups. Results suggest that the observed MHC expression shift could serve as a molecular marker to determine anabolic activity of anabolic steroids at least in skeletal muscle of orchi rats. The molecular mechanisms as well as the androgen-dependent regulation of MHC expression in intact skeletal muscle remain to be further investigated.

Estradiol and genistein antagonize the ovariectomy effects on skeletal muscle myosin heavy chain expression via ER-beta mediated pathways.

The age-related decline in ovarian sex hormone production following the onset of menopause alters skeletal muscle metabolic, structural and functional characteristics. The myosin heavy chain (MHC) expression pattern defines skeletal muscle contraction velocity and is therefore an important factor in skeletal muscle function. The present study was designed to examine the effects of 17beta estradiol (E2), estrogen receptor (ER) subtype selective agonists (ERalpha, ERbeta) or genistein (Gen) following ovary removal (OVX) in female Wistar rats in combination with a high intensity treadmill-based exercise protocol (Ex) or normal cage-based activity (NoEx) on MHC protein expression patterns in the slow fiber type m.Soleus (Sol) and the fast fiber type m.Gastrocnemius (Gas). Gen and E2 in the Sol significantly stimulated MHC-I expression relative to OVX only in the absence of exercise (NoEx). MHC-IIb expression in the Gas was significantly increased relative to OVX in Gen Ex and E2 Ex and NoEx groups. The estrogenic effects in the Sol and Gas were both predominantly mediated via ERbeta pathways, since the ERbeta agonist induced greater MHC increases than OVX or ERalpha. We therefore propose that high intensity exercise in combination with exposure to E2, Gen, ERalpha or ERbeta agonists in OVX rats exerts differential effects on MHC expression in skeletal muscles composed of mainly slow type I MHC (Sol) or fast type II MHC (Gas). In summary, the data shows that MHC composition is affected by estrogens and exercise in a fiber type specific manner and that these effects are mainly mediated by ER-beta. This is of great importance with respect to skeletal muscle health and potential treatment with ER selective agonists.

Responses of estrogen sensitive tissues in female Wistar rats to pre- and postnatal isoflavone exposure.

Effects of isoflavones on estrogen sensitive tissues are discussed controversially. This study was designed to investigate tissue specific effects of an isoflavone exposure through different periods of life in female Wistar rats and to compare the effects of genistein (GEN) to those of mixed dietary isoflavones, GEN and daidzein (DAI). One group received an isoflavone-free diet (IDD), another was fed an isoflavone-rich diet (IRD) and the third group an IDD supplemented with GEN (GEN(d)) prior to mating, throughout pregnancy and up to weaning. The offspring were kept on the respective diets during growth, puberty and adulthood. The weight of the uterus, the height of the uterine and vaginal epithelium, the bone mineral density of the tibia, and the expression of the estrogen sensitive gene CaBP9K in the liver were determined. At d21, the uterine weight, the uterine epithelium and the expression of CaBP9K in the liver were significantly stimulated in GEN(d) animals compared to IDD and IRD. Interestingly, bone mineral density was increased in GEN(d) and in IRD animals. Around puberty (d50) neither uterine wet weights nor trabecular bone density differed significantly among the isoflavone groups and the IDD control. At d80 no significant differences in uterine weight were observed among IDD, GEN(d) and IRD animals. However, bone mineral density was increased in GEN(d) and IRD animals. In summary, our results demonstrate that lifelong dietary exposure to isoflavones can affect estrogen sensitive tissues, apparently in a tissue selective manner. With respect to health risk and benefit our data indicate that an increased bone mineral density can be achieved by lifelong exposure to an IRD, which, in contrast to GEN supplementation, does not seem to stimulate the proliferation of the uterine epithelium.

Anabolic and androgenic activity of 19-norandrostenedione after oral and subcutaneous administration--analysis of side effects and metabolism.

One of the most frequently misused steroid precursors (prohormones) is 19-norandrostenedione (estr-4-ene-3,17-dione, NOR). Recently we have show that NOR stimulates skeletal muscle growth after s.c. administration in a highly selective manner but exhibits only weak androgenic activity in rats. Because most abusers take NOR orally, the aim of this study was to compare the anabolic and androgenic potency of NOR between s.c. and oral application. Orchiectomised rats were treated with NOR either s.c. (1 mg/kg BW/day) or orally (0.1, 1 and 10 mg/kg BW/day). The tissue weights of the levator ani, the seminal vesicle and the prostate were analysed to determine the anabolic and androgenic activity. Heart and liver wet weights were examined to identify side effects. Serum concentrations of NOR and its metabolite nandrolone (NT) were determined. GCMC analysis revealed that free and glucuronidated NOR and NT were detectable in the serum after oral and s.c. administration and that NOR was converted to NT in comparable amounts independent of the route of administration. In agreement to our previous study s.c. application of NOR stimulates skeletal muscle growth but has only weak androgenic effects. In contrast, after oral administration of NOR neither stimulation of the prostate nor the levator ani could be observed in the doses administered in this study. Interestingly, and in contrast to s.c. treatment, oral administration of NOR resulted in a dose-dependent decrease of body weight. In summary, oral administration of NOR, at least in the rat, seems to be a very ineffective strategy for stimulating skeletal muscle mass increases but may be associated with side effects.

SWISSspine: a nationwide registry for health technology assessment of lumbar disc prostheses.

SWISSspine is a so-called pragmatic trial for assessment of safety and efficiency of total disc arthroplasty (TDA). It follows the new health technology assessment (HTA) principle of "coverage with evidence development". It is the first mandatory HTA registry of its kind in the history of Swiss orthopaedic surgery. Its goal is the generation of evidence for a decision by the Swiss federal office of health about reimbursement of the concerned technologies and treatments by the basic health insurance of Switzerland. During the time between March 2005 and 2008, 427 interventions with implantation of 497 lumbar total disc arthroplasties have been documented. Data was collected in a prospective, observational multicenter mode. The preliminary timeframe for the registry was 3 years and has already been extended. Data collection happens pre- and perioperatively, at the 3 months and 1-year follow-up and annually thereafter. Surgery, implant and follow-up case report forms are administered by spinal surgeons. Comorbidity questionnaires, NASS and EQ-5D forms are completed by the patients. Significant and clinically relevant reduction of low back pain VAS (70.3-29.4 points preop to 1-year postop, p < 0.0001) leg pain VAS (55.5-19.1 points preop to 1-year postop, p < 0.001), improvement of quality of life (EQ-5D, 0.32-0.73 points preop to 1-year postop, p < 0.001) and reduction of pain killer consumption was revealed at the 1-year follow-up. There were 14 (3.9%) complications and 7 (2.0%) revisions within the same hospitalization reported for monosegmental TDA; there were 6 (8.6%) complications and 8 (11.4%) revisions for bisegmental surgery. There were 35 patients (9.8%) with complications during followup in monosegmental and 9 (12.9%) in bisegmental surgery and 11 (3.1%) revisions with 1 [corrected] new hospitalization in monosegmental and 1 (1.4%) in bisegmental surgery. Regression analysis suggested a preoperative VAS "threshold value" of about 44 points for increased likelihood of a minimum clinically relevant back pain improvement. In a short-term perspective, lumbar TDA appears as a relatively safe and efficient procedure concerning pain reduction and improvement of quality of life. Nevertheless, no prediction about the long-term goals of TDA can be made yet. The SWISSspine registry proofs to be an excellent tool for collection of observational data in a nationwide framework whereby advantages and deficits of its design must be considered. It can act as a model for similar projects in other health-care domains.

Comparison of the bone protective effects of an isoflavone-rich diet with dietary and subcutaneous administrations of genistein in ovariectomized rats.

Administration of the isoflavone genistein (GEN) has been described to result in bone protection but also to induce uterotrophic responses. To compare bone protective effects of GEN with an isoflavone-rich diet (IRD) and to further elucidate molecular mechanisms involved in bone-protection, ovariectomized rats (OVX) received either a diet low in isoflavone content (IDD) enriched with GEN (42 mg kg(-1)b.wtd(-1)) (GEN(d)), an IRD (14 mg kg(-1)b.wtd(-1) GEN, 14 mg kg(-1)b.wtd(-1) daidzein) or were treated subcutaneously (s.c.) with GEN (10 mg kg(-1)b.wtd(-1)) (GEN(sc)) for 12 weeks. Intact (SHAM), vehicle treated OVX animals and those substituted with 17beta-estradiol (2microg kg(-1)b.wtd(-1)) (E(2)), served as controls. OVX-induced bone loss could be antagonized in E(2), GEN(sc), GEN(d) and IRD groups. Uterine wet weight (UWW) was only stimulated in E(2) and GEN(sc) animals. Serum biomarkers of bone-formation (osteocalcin, osteopontin) and bone-resorption (telopeptides of collagen type I, pyridinoline cross-links) were elevated in OVX compared to SHAM and E(2) animals. Feeding IRD stimulated bone-formation and inhibited bone-resorption, whereas s.c. or dietary administration of GEN only resulted in a stimulation of bone-formation. The results of the present study indicate that in contrast to s.c. administration, dietary intake of GEN resulted in bone protection without stimulation of UWW. Dietary intake of isoflavones by an IRD also did not result in a stimulation of UWW, yet IRD appeared to be more effective in bone protection than administration of pure GEN.

Estrogen receptor subtype-specific effects on markers of bone homeostasis.

To further elucidate the processes involved in the physiology of bone-protection by estrogens, ovariectomized (OVX) rats were treated subcutaneously with 17beta-estradiol (E(2)), the ERalpha-specific agonist (16alpha-LE2) and the ERbeta-specific agonist (8beta-VE2). OVX and intact animals served as controls. Biomarkers of bone-formation (osteocalcin (OC), osteopontin (OPN)) and bone-resorption (telopeptides of collagen type I (CTx), pyridinoline cross-links (Pyd)) were quantified. Bone mineral density was measured by computed tomography. OVX-induced bone loss could be antagonized by subcutaneous administration of 17beta-estradiol and 16alpha-LE2. Serum levels of CTx, OC and OPN were significantly elevated in OVX compared to intact animals and reduced by 17beta-estradiol and 16alpha-LE2. Treatment of OVX rats with 8beta-VE2 did not affect bone mineral density (BMD) or bone-marker serum levels. Taken together, the complex expression pattern of bone-markers in OVX rats following subcutaneous administration of ER subtype-specific agonists indicates that 17beta-estradiol exerts its bone-protective effects by modulating the activity of osteoclasts and osteoblasts via ERalpha.

C2C12 myoblastoma cell differentiation and proliferation is stimulated by androgens and associated with a modulation of myostatin and Pax7 expression.

Androgens are modulators of skeletal muscle adaptation and regeneration processes. The control of satellite cell activity is a key mechanism during this process. In this study, we analyzed the ability of dihydrotestosterone (DHT) and anabolic steroids to induce and modulate the differentiation of C2C12 myoblastoma cells toward myotubes. C2C12 cells were dose-dependently treated with DHT and anabolic steroids. The time-dependent effects on differentiation were measured and correlated with the expression of genes involved in the regulation of satellite cell activity. The distribution of C2C12 cells within the cell cycle was measured by flow cytometry and differentiation by creatine kinase (CK) activity. Gene expression was analyzed using quantitative real-time PCR and confocal microscopy. The treatment with DHT and anabolic steroids resulted in a stimulation of C2C12 cell proliferation and CK activity. The antiandrogen flutamide was able to antagonize this effect. The expression of the androgen receptor, SOX8, SOX9, Delta, Notch, myostatin, and paired box gene7 (Pax7) was modulated by androgens. The treatment with DHT and anabolic steroids resulted in a strong stimulation of myostatin expression not only in undifferentiated cells but also in myotubes. The stimulation could be antagonized by flutamide. The expression of Pax7 was detectable in C2C12 cells early after treatment with DHT. Our results demonstrate that the key mechanisms of satellite cell differentiation are modulated by androgens. Androgens stimulate the proliferation of C2C12 cells, accelerate the process of differentiation, and increase the expression of myostatin in undifferentiated and differentiated cells. Our findings may have implications not only for the treatment of muscular diseases but also for the improvement of doping analytical methods.

The prohormone 19-norandrostenedione displays selective androgen receptor modulator (SARM) like properties after subcutaneous administration.

One of the most frequently misused steroid precursors (prohormones) is 19-norandrostenedione (4-estrene-3,17-dione, NOR), which is, after oral administration, readily metabolised to nortestosterone, also known as nandrolone (durabolin). In this study we have characterised molecular mechanisms of its action determined its tissue specific androgenic and anabolic potency after subcutaneous (s.c.) administration and investigated potential adverse effects. Receptor binding tests demonstrate that NOR binds with high selectivity to the AR. The potency of NOR to transactivate androgen receptor (AR) dependent reporter gene expression was 10 times lower as compared to dihydrotestosterone (DHT). In vivo experiments in orchiectomised rats demonstrated that s.c. treatment with NOR resulted only in a stimulation of the weight of the levator ani muscle; the prostate and seminal vesicle weights remained completely unaffected. Like testosterone, administration of NOR resulted in a stimulation of AR and myostatin mRNA expression in the gastrocnemius muscle. NOR does not affect prostate proliferation, the liver weight and the expression of the tyrosine aminotransferase gene (TAT) in the liver. Summarizing these data it is obvious that NOR, if administrated s.c. and in contrast to its metabolite nandrolone, highly selectively stimulates the growth of the skeletal muscle but has only weak androgenic properties. This observation may have relevance with respect to therapeutic aspects but also doping prevention.

Analysis of the effects of oestrogen receptor alpha (ERalpha)- and ERbeta-selective ligands given in combination to ovariectomized rats.

BACKGROUND AND PURPOSE: Studies with oestrogen receptoralpha (ERalpha)- and ERbeta-selective compounds have already shown that the effects of 17beta-estradiol (E2) on body weight, movement drive and bone-protection are mediated via ERalpha. This study was based on the hypothesis that activation of ERbeta may antagonize ERalpha-mediated effects and designed to investigate potential effects of ERalpha/ERbeta heterodimers. EXPERIMENTAL APPROACH: Ovariectomized (OVX) female Wistar rats were treated with combinations of the ERalpha-specific agonist 16alpha-LE2 (ALPHA; 1 and 10 microg kg(-1) d(-1)), the ERbeta-specific agonist 8beta-VE2 (BETA; 100 microg kg(-1) d(-1)), the phytoestrogen, genistein (10 mg kg(-1) d(-1)) and with the anti-oestrogen compound, ICI 182,780 (3 mg kg(-1) d(-1)) for three weeks. The combined effects of the substances on body weight increase, tibial bone mineral density (BMD) and the influence on running wheel activity (RWA) were investigated. KEY RESULTS: OVX-induced body weight increase was reduced by co-administration of genistein and BETA. Co-application of BETA or genistein with ALPHA had no effect on ALPHA-mediated bone-protection. The RWA of OVX animals was significantly reduced by treatment with genistein but stimulated by application of ALPHA. The stimulatory effect of ALPHA on RWA could be antagonized by co-treatment with the pure antioestrogen ICI 182,780 but also by co-administration of genistein or BETA. CONCLUSIONS AND IMPLICATIONS: Our results indicate that activation of ERbeta may modulate ERalpha-mediated physiological effects in vivo. The observation that substances with selective affinity for ERbeta are able to antagonize distinct physiological functions, like RWA, may be of great relevance to the pharmaceutical use of such drugs.