RESUMEN
Spinal cord injury (SCI) causes severe and resistant sublesional disuse bone loss. Abaloparatide, a modified parathyroid hormone related peptide, is an FDA approved drug for treatment of severe osteoporosis with potent anabolic activity. The effects of abaloparatide on SCI-induced bone loss remain undefined. Thus, female mice underwent sham or severe contusion thoracic SCI causing hindlimb paralysis. Mice then received subcutaneous injection of vehicle or 20 µg/kg/day abaloparatide for 35 days. Micro-computed tomography (micro-CT) analysis of the distal and midshaft femoral regions of the SCI-vehicle mice revealed reduced trabecular fractional bone volume (56%), thickness (75%), and cortical thickness (80%) compared to sham-vehicle controls. Treatment with abaloparatide did not prevent SCI-induced changes in trabecular or cortical bone. However, histomorphometry evaluation of the SCI-abaloparatide mice demonstrated that abaloparatide treatment increased osteoblast (241%) and osteoclast (247%) numbers and the mineral apposition rate (131%) compared to SCI-vehicle animals. In another independent experiment, treatment with 80 µg/kg/day abaloparatide significantly attenuated SCI-induced loss in cortical bone thickness (93%) when compared to SCI-vehicle mice (79%) but did not prevent SCI-induced trabecular bone loss or elevation in cortical porosity. Biochemical analysis of the bone marrow supernatants of the femurs showed that SCI-abaloparatide animals had 2.3-fold increase in procollagen type I N-terminal propeptide, a bone formation marker than SCI-vehicle animals. SCI groups had 70% higher levels of cross-linked C-telopeptide of type I collagen, a bone resorption marker, than sham-vehicle mice. These findings suggest that abaloparatide protects the cortical bone against the deleterious effects of SCI by promoting bone formation.
Asunto(s)
Enfermedades Óseas Metabólicas , Traumatismos de la Médula Espinal , Femenino , Animales , Ratones , Proteína Relacionada con la Hormona Paratiroidea , Microtomografía por Rayos XRESUMEN
The purpose of the study was to identify potential predictors of muscle hypertrophy responsiveness following neuromuscular electrical stimulation resistance training (NMES-RT) in persons with chronic spinal cord injury (SCI). Data for twenty individuals with motor complete SCI who completed twice weekly NMES-RT lasting 12-16 weeks as part of their participation in one of two separate clinical trials were pooled and retrospectively analyzed. Magnetic resonance imaging (MRI) was used to measure muscle cross-sectional area (CSA) of the whole thigh and knee extensor muscle before and after NMES-RT. Muscle biopsies and fasting biomarkers were also measured. Following the completion of the respective NMES-RT trials, participants were classified into either high-responders (n = 8; muscle CSA > 20%) or low-responders (n = 12; muscle CSA < 20%) based on whole thigh muscle CSA hypertrophy. Whole thigh muscle and knee extensors CSAs were significantly greater (P < 0.0001) in high-responders (29 ± 7% and 47 ± 15%, respectively) compared to low-responders (12 ± 3% and 19 ± 6%, respectively). There were no differences in total caloric intake or macronutrient intake between groups. Extensor spasticity was lower in the high-responders compared to the low-responders as was the dosage of baclofen. Prior to the intervention, the high-responders had greater body mass compared to the low-responders with SCI (87.8 ± 13.7 vs. 70.4 ± 15.8 kg; P = 0.012), body mass index (BMI: 27.6 ± 2.7 vs. 22.9 ± 6.0 kg/m2; P = 0.04), as well as greater percentage in whole body and regional fat mass (P < 0.05). Furthermore, high-responders had a 69% greater increase (P = 0.086) in total Akt protein expression than low-responders. High-responders also exhibited reduced circulating IGF-1 with a concomitant increase in IGFBP-3. Exploratory analyses revealed upregulation of mRNAs for muscle hypertrophy markers [IRS-1, Akt, mTOR] and downregulation of protein degradation markers [myostatin, MurF-1, and PDK4] in the high-responders compared to low-responders. The findings indicate that body composition, spasticity, baclofen usage, and multiple signaling pathways (anabolic and catabolic) are involved in the differential muscle hypertrophy response to NMES-RT in persons with chronic SCI.
Asunto(s)
Terapia por Estimulación Eléctrica , Entrenamiento de Fuerza , Traumatismos de la Médula Espinal , Humanos , Baclofeno/metabolismo , Entrenamiento de Fuerza/métodos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Estudios Retrospectivos , Músculo Esquelético/fisiología , Espasticidad Muscular , Traumatismos de la Médula Espinal/metabolismo , Hipertrofia/patología , Terapia por Estimulación Eléctrica/métodosRESUMEN
Despite national and international efforts for the prevention of metabolic syndrome and its underlying diseases/disorders, its prevalence is still rising, especially in the middle-aged population. In this study, we explore the effect of high fat diet on the development of metabolic syndrome in middle-aged mice and to evaluate the potential benefits of voluntary physical exercise on the periphery as well as brain cognitive function, and to explore the potential mechanisms. We found that metabolic syndrome developed at middle age significantly impairs cognitive function and the impairment is associated with gene dysregulation in metabolic pathways that are largely affecting astrocytes in the brain. Eight-week voluntary wheel running at a frequency of three times a week, not only improves peripheral glucose control but also significantly improves learning and memory. The improvement of cognitive function is associated with restoration of gene expression involved in energy metabolism in the brain. Our study suggests that voluntary physical exercise is beneficial for metabolic syndrome-induced peripheral as well as cognitive dysfunction and can be recommended as therapeutic intervention for metabolic syndrome and associated diseases.
Asunto(s)
Síndrome Metabólico , Condicionamiento Físico Animal , Animales , Cognición , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Síndrome Metabólico/terapia , Ratones , Actividad Motora , Condicionamiento Físico Animal/fisiologíaRESUMEN
BACKGROUND: The role of Numb, a protein that is important for cell fate and development and that, in human muscle, is expressed at reduced levels with advanced age, was investigated; adult mice skeletal muscle and its localization and function within myofibres were determined. METHODS: Numb expression was evaluated by western blot. Numb localization was determined by confocal microscopy. The effects of conditional knock out (cKO) of Numb and the closely related gene Numb-like in skeletal muscle fibres were evaluated by in situ physiology, transmission and focused ion beam scanning electron microscopy, three-dimensional reconstruction of mitochondria, lipidomics, and bulk RNA sequencing. Additional studies using primary mouse myotubes investigated the effects of Numb knockdown on cell fusion, mitochondrial function, and calcium transients. RESULTS: Numb protein expression was reduced by ~70% (P < 0.01) at 24 as compared with 3 months of age in gastrocnemius and tibialis anterior muscle. Numb was localized within muscle fibres as bands traversing fibres at regularly spaced intervals in close proximity to dihydropyridine receptors. The cKO of Numb and Numb-like reduced specific tetanic force by 36% (P < 0.01), altered mitochondrial spatial relationships to sarcomeric structures, increased Z-line spacing by 30% (P < 0.0001), perturbed sarcoplasmic reticulum organization and reduced mitochondrial volume by over 80% (P < 0.01). Only six genes were differentially expressed in cKO mice: Itga4, Sema7a, Irgm2, Vezf1, Mib1, and Tmem132a. Several lipid mediators derived from polyunsaturated fatty acids through lipoxygenases were up-regulated in Numb cKO skeletal muscle: 12-HEPE was increased by ~250% (P < 0.05) and 17,18-EpETE by ~240% (P < 0.05). In mouse primary myotubes, Numb knockdown reduced cell fusion (~20%, P < 0.01) and delayed the caffeine-induced rise in cytosolic calcium concentrations by more than 100% (P < 0.01). CONCLUSIONS: These findings implicate Numb as a critical factor in skeletal muscle structure and function and suggest that Numb is critical for calcium release. We therefore speculate that Numb plays critical roles in excitation-contraction coupling, one of the putative targets of aged skeletal muscles. These findings provide new insights into the molecular underpinnings of the loss of muscle function observed with sarcopenia.
Asunto(s)
Proteínas de la Membrana , Músculo Esquelético , Proteínas del Tejido Nervioso , Retículo Sarcoplasmático , Animales , Calcio/metabolismo , Acoplamiento Excitación-Contracción , Técnicas de Inactivación de Genes , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
Spinal cord injury (SCI) results in dysregulation of carbohydrate and lipid metabolism; the underlying cellular and physiological mechanisms remain unclear. Fibroblast growth factor 21 (FGF21) is a circulating protein primarily secreted by the liver that lowers blood glucose levels, corrects abnormal lipid profiles, and mitigates non-alcoholic fatty liver disease. FGF21 acts via activating FGF receptor 1 and ß-klotho in adipose tissue and stimulating release of adiponectin from adipose tissue which in turn signals in the liver and skeletal muscle. We examined FGF21/adiponectin signaling after spinal cord transection in mice fed a high fat diet (HFD) or a standard mouse chow. Tissues were collected at 84 days after spinal cord transection or a sham SCI surgery. SCI reduced serum FGF21 levels and hepatic FGF21 expression, as well as ß-klotho and FGF receptor-1 (FGFR1) mRNA expression in adipose tissue. SCI also reduced serum levels and adipose tissue mRNA expression of adiponectin and leptin, two major adipokines. In addition, SCI suppressed hepatic type 2 adiponectin receptor (AdipoR2) mRNA expression and PPARα activation in the liver. Post-SCI mice fed a HFD had further suppression of serum FGF21 levels and hepatic FGF21 expression. Elevated serum free fatty acid (FFA) levels after HFD feeding were observed in post-SCI mice but not in sham-mice, suggesting defective FFA uptake after SCI. Moreover, after SCI several genes that are implicated in insulin's action had reduced expression in tissues of interest. These findings suggest that downregulated FGF21/adiponectin signaling and impaired responsiveness of adipose tissues to FGF21 may, at least in part, contribute to the overall picture of metabolic dysfunction after SCI.
Asunto(s)
Tejido Adiposo/patología , Factores de Crecimiento de Fibroblastos/sangre , Inflamación/patología , Hígado/patología , Enfermedad del Hígado Graso no Alcohólico/patología , Traumatismos de la Médula Espinal/complicaciones , Tejido Adiposo/metabolismo , Animales , Dieta Alta en Grasa , Inflamación/sangre , Inflamación/etiología , Resistencia a la Insulina , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/sangre , Enfermedad del Hígado Graso no Alcohólico/etiología , Transducción de SeñalRESUMEN
Spinal cord injury (SCI) results in marked atrophy of sublesional skeletal muscle and substantial loss of bone. In this study, the effects of prolonged electrical stimulation (ES) and/or testosterone enanthate (TE) on muscle mass and bone formation in a rat model of SCI were tested. Compared to sham-transected animals, a significant reduction of the mass of soleus, plantaris and extensor digitorum longus (EDL) muscles was observed in animals 6 weeks post-SCI. Notably, ES or ES + TE resulted in the increased mass of the EDL muscles. ES or ES + TE significantly decreased mRNA levels of muscle atrophy markers (e.g., MAFbx and MurF1) in the EDL. Significant decreases in bone mineral density (BMD) (-27%) and trabecular bone volume (-49.3%) at the distal femur were observed in animals 6 weeks post injury. TE, ES and ES + TE treatment significantly increased BMD by +6.4%, +5.4%, +8.5% and bone volume by +22.2%, and +56.2% and+ 60.2%, respectively. Notably, ES alone or ES + TE resulted in almost complete restoration of cortical stiffness estimated by finite element analysis in SCI animals. Osteoblastogenesis was evaluated by colony-forming unit-fibroblastic (CFU-F) staining using bone marrow mesenchymal stem cells obtained from the femur. SCI decreased the CFU-F+ cells by -56.8% compared to sham animals. TE or ES + TE treatment after SCI increased osteoblastogenesis by +74.6% and +67.2%, respectively. An osteoclastogenesis assay revealed significantly increased TRAP+ multinucleated cells (+34.8%) in SCI animals compared to sham animals. TE, ES and TE + ES treatment following SCI markedly decreased TRAP+ cells by -51.3%, -40.3% and -46.9%, respectively. Each intervention greatly reduced the ratio of RANKL to OPG mRNA of sublesional long bone. Collectively, our findings demonstrate that after neurologically complete paralysis, dynamic muscle resistance exercise by ES reduced muscle atrophy, downregulated genes involved in muscle wasting, and restored mechanical loading to sublesional bone to a degree that allowed for the preservation of bone by inhibition of bone resorption and/or by facilitating bone formation.
Asunto(s)
Traumatismos de la Médula Espinal , Animales , Densidad Ósea , Huesos , Estimulación Eléctrica , Miembro Posterior , Músculo Esquelético , Ratas , Traumatismos de la Médula Espinal/terapiaRESUMEN
Spinal cord injury (SCI) is associated with obesity and is a risk factor for type 2 diabetes mellitus (T2DM). Immobilization, muscle atrophy, obesity, and loss of sympathetic innervation to the liver are believed to contribute to risks of these abnormalities. Systematic study of the mechanisms underlying SCI-induced metabolic disorders has been limited by a lack of animal models of insulin resistance following SCI. Therefore, the effects of a high-fat diet (HFD), which causes weight gain and glucose intolerance in neurologically intact mice, was tested in mice that had undergone a spinal cord transection at thoracic vertebra 10 (T10) or a sham-transection. At 84 days after surgery, Sham-HFD and SCI-HFD mice showed impaired intraperitoneal glucose tolerance when compared with Sham control (Sham-Con) or SCI control (SCI-Con) mice fed a standard control chow. Glucose tolerance in SCI-Con mice was comparable to that of Sham-Con mice. The mass of paralyzed skeletal muscle, liver, and epididymal, inguinal, and omental fat deposits were lower in SCI versus Sham groups, with lower liver mass present in SCI-HFD versus SCI-Con animals. SCI also produced sublesional bone loss, with no differences between SCI-Con and SCI-HFD groups. The results suggest that administration of a HFD to mice after SCI may provide a model to better understand mechanisms leading to insulin resistance post-SCI, as well as an approach to study pathogenesis of glucose intolerance that is independent of obesity.
RESUMEN
To date, no efficacious therapy exists that will prevent or treat the severe osteoporosis in individuals with neurologically motor-complete spinal cord injury (SCI). Recent preclinical studies have demonstrated that sclerostin antibody (Scl-Ab) can prevent sublesional bone loss after acute SCI in rats. However, it remains unknown whether sclerostin inhibition reverses substantial bone loss in the vast majority of the SCI population who have been injured for several years. This preclinical study tested the efficacy of Scl-Ab to reverse the bone loss that has occurred in a rodent model after chronic motor-complete SCI. Male Wistar rats underwent either complete spinal cord transection or only laminectomy. Twelve weeks after SCI, the rats were treated with Scl-Ab at 25 mg/kg/week or vehicle for 8 weeks. In the SCI group that did not receive Scl-Ab, 20 weeks of SCI resulted in a significant reduction of bone mineral density (BMD) and estimated bone strength, and deterioration of bone structure at the distal femoral metaphysis. Treatment with Scl-Ab largely restored BMD, bone structure, and bone mechanical strength. Histomorphometric analysis showed that Scl-Ab increased bone formation in animals with chronic SCI. In ex vivo cultures of bone marrow cells, Scl-Ab inhibited osteoclastogenesis, and promoted osteoblastogenesis accompanied by increased Tcf7, ENC1, and the OPG/RANKL ratio expression, and decreased SOST expression. Our findings demonstrate for the first time that Scl-Ab reverses the sublesional bone loss when therapy is begun after relatively prolonged spinal cord transection. The study suggests that, in addition to being a treatment option to prevent bone loss after acute SCI, sclerostin antagonism may be a valid clinical approach to reverse the severe bone loss that invariably occurs in patients with chronic SCI.
Asunto(s)
Densidad Ósea/efectos de los fármacos , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Resorción Ósea/etiología , Traumatismos de la Médula Espinal/complicaciones , Animales , Anticuerpos/farmacología , Enfermedad Crónica , Marcadores Genéticos , Masculino , Osteogénesis/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Anabolic androgens have been shown to reduce muscle loss due to immobilization, paralysis and many other medical conditions, but the molecular basis for these actions is poorly understood. We have recently demonstrated that nandrolone, a synthetic androgen, slows muscle atrophy after nerve transection associated with down-regulation of regulator of calcineurin 2 (RCAN2), a calcineurin inhibitor, suggesting a possible role of calcineurin-NFAT signaling. To test this possibility, rat gastrocnemius muscle was analyzed at 56 days after denervation. In denervated muscle, calcineurin activity declined and NFATc4 was excluded from the nucleus and these effects were reversed by nandrolone. Similarly, nandrolone increased calcineurin activity and nuclear NFATc4 levels in cultured L6 myotubes. Nandrolone also induced cell hypertrophy that was blocked by cyclosporin A or overexpression of RCAN2. Finally protection against denervation atrophy by nandrolone in rats was blocked by cyclosporin A. These results demonstrate for the first time that nandrolone activates calcineurin-NFAT signaling, and that such signaling is important in nandrolone-induced cell hypertrophy and protection against paralysis-induced muscle atrophy.
Asunto(s)
Anabolizantes/farmacología , Calcineurina/metabolismo , Desnervación Muscular , Fibras Musculares Esqueléticas/metabolismo , Atrofia Muscular/metabolismo , Factores de Transcripción NFATC/metabolismo , Nandrolona/farmacología , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Núcleo Celular/metabolismo , Núcleo Celular/patología , Ciclosporina/farmacología , Inmunosupresores/farmacología , Masculino , Fibras Musculares Esqueléticas/patología , Atrofia Muscular/patología , Ratas , Ratas WistarRESUMEN
Glucocorticoids stimulate muscle atrophy through a cascade of signals that includes activation of FoxO transcription factors which then upregulate multiple genes to promote degradation of myofibrillar and other muscle proteins and inhibit protein synthesis. Our previous finding that glucocorticoids upregulate mRNA levels for FoxO1 in skeletal muscle led us to hypothesize that the FoxO1 gene contains one or more glucocorticoid response elements (GREs). Here we show that upregulation of FoxO1 expression by glucocorticoids requires the glucocorticoid receptor (GR) and binding of hormones to it. In cultured C2C12 myoblasts dexamethasone did not alter FoxO1 mRNA stability. Computational analysis predicted that the proximal promoter of the FoxO1 gene contained a cluster of eight GRE half sites and one highly conserved near-consensus SRE; the cluster is found between -800 and -2000bp upstream of the first codon of the FoxO1 gene. A reporter gene constructed using the first 2kb of the FoxO1 promoter was stimulated by dexamethasone. Removal of a 5' domain containing half of the GREs reduced reporter gene activity and removal of all GREs in this region ablated activation by dexamethasone. Restriction fragments of the cluster of 8 upstream GREs bound recombinant GR in gel shift assays. Collectively, the data demonstrate that the proximal promoter of the FoxO1 gene contains multiple functional GREs, indicating that upregulation of FoxO1 expression by glucocorticoids through GREs represents an additional mechanism by which the GR drives glucocorticoid-mediated muscle atrophy. These findings are also relevant to other physiological roles of FoxO1 such as regulation of hepatic metabolism.
Asunto(s)
Factores de Transcripción Forkhead/genética , Glucocorticoides/metabolismo , Regiones Promotoras Genéticas , Elementos de Respuesta , Animales , Células Cultivadas , Dexametasona/metabolismo , Dexametasona/farmacología , Ensayo de Cambio de Movilidad Electroforética , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Glucocorticoides/farmacología , Humanos , Ratones , Mioblastos Esqueléticos/efectos de los fármacos , Mioblastos Esqueléticos/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Transcripción GenéticaRESUMEN
BACKGROUND: Spinal cord injury (SCI) causes severe bone loss. At present, there is no practical treatment to delay or prevent bone loss in individuals with motor-complete SCI. Hypogonadism is common in men after SCI and may exacerbate bone loss. The anabolic steroid nandrolone reduces bone loss due to microgravity or nerve transection. OBJECTIVE: To determine whether nandrolone reduced bone loss after SCI and, if so, to explore the mechanisms of nandrolone action. METHODS: Male rats with complete transection of the spinal cord were administered nandrolone combined with a physiological replacement dose of testosterone, or vehicle, beginning on day 29 after SCI and continued for 28 days. RESULTS: SCI reduced distal femoral and proximal tibial bone mineral density (BMD) by 25 and 16%, respectively, at 56 days. This bone loss was attenuated by nandrolone. In ex vivo osteoclasts cultures, SCI increased mRNA levels for tartrate-resistant acid phosphatase (TRAP) and calcitonin receptor; nandrolone-normalized expression levels of these transcripts. In ex vivo osteoblast cultures, SCI increased receptor activator of NF-kB ligand (RANKL) mRNA levels but did not alter osteoprotegerin (OPG) mRNA expression; nandrolone-increased expression of OPG and OPG/RANKL ratio. SCI reduced mRNA levels of Wnt signaling-related genes Wnt3a, low-density lipoprotein receptor-related protein 5 (LRP5), Fzd5, Tcf7, and ectodermal-neural cortex 1 (ENC1) in osteoblasts, whereas nandrolone increased expression of each of these genes. CONCLUSIONS: The results demonstrate that nandrolone reduces bone loss after SCI. A potential mechanism is suggested by our findings wherein nandrolone modulates genes for differentiation and activity of osteoclasts and osteoblasts, at least in part, through the activation of Wnt signaling.
Asunto(s)
Anabolizantes/farmacología , Resorción Ósea/prevención & control , Nandrolona/farmacología , Traumatismos de la Médula Espinal/complicaciones , Vía de Señalización Wnt/fisiología , Animales , Resorción Ósea/etiología , Resorción Ósea/metabolismo , Diferenciación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Masculino , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Traumatismos de la Médula Espinal/metabolismoRESUMEN
Nandrolone, an anabolic steroid, slows denervation atrophy of rat muscle, prevents denervation-induced nuclear accumulation of intracellular domain of the Notch receptor, and elevates expression of Numb. Numb acts as an inhibitor of Notch signaling and promotes myogenic differentiation of satellite cells. Turnover of Numb is regulated by mdm2, an E3 ubiquitin ligase. With these considerations in mind, we investigated the effects of nandrolone on the expression of Numb and mdm2 proteins and determined the effect of mdm2 on nandrolone-induced alterations in Numb protein in C2C12 myoblasts. When C2C12 cells were cultured in a medium favoring differentiation (Dulbecco modified Eagle medium containing 2% horse serum), nandrolone up-regulated Numb protein levels in a time-dependent manner and prolonged Numb protein half-life from 10 to 18 hours. In contrast, nandrolone reduced the expression of mdm2 protein. To determine whether the decreased mdm2 expression induced by nandrolone was responsible for the increased levels and prolonged half-life of Numb protein in this cell line, mdm2-small interfering RNA (siRNA) was employed to inhibit mdm2 expression. Compared to cells transfected with scrambled siRNA (negative control), transfection with mdm2-siRNA increased basal Numb protein expression but abolished the further increase in Numb protein levels by nandrolone. In addition, transfection of mdm2-siRNA mimicked the effect of nandrolone to prolong the half-life of Numb protein. Moreover, when C2C12 cells were forced to overexpress mdm2, there was a significant decline in the expression of both basal and inducible Numb protein. Our data suggest that nandrolone, by a novel mechanism for this agent in a muscle cell type, increases Numb protein levels in C2C12 myoblasts by stabilizing Numb protein against degradation, at least in part, via suppression of mdm2 expression.
Asunto(s)
Proteínas de la Membrana/metabolismo , Nandrolona/farmacología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Animales , Línea Celular , Ratones , Mioblastos/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/biosíntesis , Regulación hacia ArribaRESUMEN
Spinal cord injury (SCI) results in atrophy of skeletal muscle and changes from slow oxidative to fast glycolytic fibers, which may reflect reduced levels of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), increased myostatin signaling, or both. In animals, testosterone reduces loss of muscle fiber cross-sectional area and activity of enzymes of energy metabolism. To identify the molecular mechanisms behind the benefits of androgens on paralyzed muscle, male rats were spinal cord transected and treated for 8 weeks with vehicle, testosterone at a physiological replacement dose, or testosterone plus nandrolone, an anabolic steroid. Treatments were initiated immediately after SCI and continued until the day animals were euthanized. In the SCI animals, gastrocnemius muscle mass was significantly increased by testosterone plus nandrolone, but not by testosterone alone. Both treatments significantly reduced nuclear content of Smad2/3 and mRNA levels of activin receptor IIB and follistatin-like 3. Testosterone alone or with nandrolone reversed SCI-induced declines in cellular and nuclear levels of PGC-1α protein and PGC-1α mRNA levels. For PGC-1α target genes, testosterone plus nandrolone partially reversed SCI-induced decreases in levels of proteins without corresponding increases in their mRNA levels. Thus, the findings demonstrate that following SCI, signaling through activin receptors and Smad2/3 is increased, and that androgens suppress activation of this signaling pathway. The findings also indicate that androgens upregulate PGC-1α in paralyzed muscle and promote its nuclear localization, but that these effects are insufficient to fully activate transcription of PGC-1α target genes. Furthermore, the transcription of these genes is not tightly coupled with their translation.
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Andrógenos/farmacología , Músculo Esquelético/efectos de los fármacos , Nandrolona/farmacología , Traumatismos de la Médula Espinal/patología , Animales , Atrofia/metabolismo , Atrofia/patología , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Miostatina/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Proteínas de Unión al ARN/metabolismo , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Testosterona/farmacología , Factores de Transcripción/metabolismoRESUMEN
Nandrolone, an anabolic steroid, slows denervation-atrophy in rat muscle. The molecular mechanisms responsible for this effect are not well understood. Androgens and anabolic steroids activate Notch signaling in animal models of aging and thereby mitigate sarcopenia. To explore the molecular mechanisms by which nandrolone prevents denervation-atrophy, we investigated the effects of nandrolone on Notch signaling in denervated rat gastrocnemius muscle. Denervation significantly increased Notch activity reflected by elevated levels of nuclear Notch intracellular domain (NICD) and expression of Hey1 (a Notch target gene). Activation was greatest at 7 and 35 days after denervation but remained present at 56 days after denervation. Activation of Notch in denervated muscle was prevented by nandrolone associated with upregulated expression of Numb mRNA and protein. These data demonstrate that denervation activates Notch signaling, and that nandrolone abrogates this response associated with increased expression of Numb, suggesting a potential mechanism by which nandrolone reduces denervation-atrophy.
Asunto(s)
Anabolizantes/administración & dosificación , Andrógenos/administración & dosificación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Desnervación Muscular , Músculo Esquelético/inervación , Atrofia Muscular/prevención & control , Nandrolona/administración & dosificación , Receptores Notch/metabolismo , Animales , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
Glucocorticoid-induced muscle atrophy results from muscle protein catabolism and reduced protein synthesis, associated with increased expression of two muscle-specific ubiquitin ligases (MAFbx and MuRF1), and of two inhibitors of protein synthesis, REDD1 and 4EBP1. MAFbx, MuRF1, REDD1 and 4EBP1 are up-regulated by the transcription factors FOXO1 and FOXO3A. The transcriptional co-activator PGC-1α has been shown to attenuate many forms of muscle atrophy and to repress FOXO3A-mediated transcription of atrophy-specific genes. Dexamethasone-induced muscle atrophy can be prevented by testosterone, which blocks up-regulation by dexamethasone of FOXO1. Here, an animal model of dexamethasone-induced muscle atrophy was used to further characterize effects of testosterone to abrogate adverse actions of dexamethasone on FOXO1 levels and nuclear localization, and to determine how these agents affect PGC-1α, and its upstream activators, p38 MAPK and AMPK. In rat gastrocnemius muscle, testosterone blunted the dexamethasone-mediated increase in levels of FOXO1 mRNA, and FOXO1 total and nuclear protein. Dexamethasone reduced total and nuclear PGC-1α protein levels in the gastrocnemius; co-administration of testosterone with dexamethasone increased total and nuclear PGC-1α levels above those present in untreated controls. Testosterone blocked dexamethasone-induced decreases in activity of p38 MAPK in the gastrocnemius muscle. Regulation of FOXO1, PGC-1α and p38 MAPK by testosterone may represent a novel mechanism by which this agent protects against dexamethasone-induced muscle atrophy.
Asunto(s)
Dexametasona/efectos adversos , Factores de Transcripción Forkhead/metabolismo , Glucocorticoides/administración & dosificación , Glucocorticoides/efectos adversos , Atrofia Muscular/prevención & control , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN/metabolismo , Testosterona/administración & dosificación , Factores de Transcripción/metabolismo , Animales , Dexametasona/administración & dosificación , Atrofia Muscular/inducido químicamente , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , RatasRESUMEN
BACKGROUND: Anabolic steroids, such as nandrolone, slow muscle atrophy, but the mechanisms responsible for this effect are largely unknown. Their effects on muscle size and gene expression depend upon time, and the cause of muscle atrophy. Administration of nandrolone for 7 days beginning either concomitantly with sciatic nerve transection (7 days) or 29 days later (35 days) attenuated denervation atrophy at 35 but not 7 days. We reasoned that this model could be used to identify genes that are regulated by nandrolone and slow denervation atrophy, as well as genes that might explain the time-dependence of nandrolone effects on such atrophy. Affymetrix microarrays were used to profile gene expression changes due to nandrolone at 7 and 35 days and to identify major gene expression changes in denervated muscle between 7 and 35 days. RESULTS: Nandrolone selectively altered expression of 124 genes at 7 days and 122 genes at 35 days, with only 20 genes being regulated at both time points. Marked differences in biological function of genes regulated by nandrolone at 7 and 35 days were observed. At 35, but not 7 days, nandrolone reduced mRNA and protein levels for FOXO1, the mTOR inhibitor REDD2, and the calcineurin inhibitor RCAN2 and increased those for ApoD. At 35 days, correlations between mRNA levels and the size of denervated muscle were negative for RCAN2, and positive for ApoD. Nandrolone also regulated genes for Wnt signaling molecules. Comparison of gene expression at 7 and 35 days after denervation revealed marked alterations in the expression of 9 transcriptional coregulators, including Ankrd1 and 2, and many transcription factors and kinases. CONCLUSIONS: Genes regulated in denervated muscle after 7 days administration of nandrolone are almost entirely different at 7 versus 35 days. Alterations in levels of FOXO1, and of genes involved in signaling through calcineurin, mTOR and Wnt may be linked to the favorable action of nandrolone on denervated muscle. Marked changes in the expression of genes regulating transcription and intracellular signaling may contribute to the time-dependent effects of nandrolone on gene expression.
Asunto(s)
Perfilación de la Expresión Génica , Desnervación Muscular , Músculo Esquelético/inervación , Músculo Esquelético/patología , Atrofia Muscular/genética , Atrofia Muscular/prevención & control , Nandrolona/farmacología , Animales , Western Blotting , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/cirugía , Análisis de Secuencia por Matrices de Oligonucleótidos , Tamaño de los Órganos/efectos de los fármacos , Tamaño de los Órganos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
Nandrolone is an anabolic steroid that has been demonstrated to reduce the loss of bone and muscle from hindlimb unweighting and to slow muscle atrophy after nerve transection. To determine whether nandrolone has the ability to protect bone against loss due to disuse after denervation, male rats underwent sciatic nerve transaction, followed 28 days later by treatment with nandrolone or vehicle for 28 days. Bone mineral density (BMD) was determined 28 days later or 56 days after nerve transection. Denervation led to reductions in BMD of 7% and 12% for femur and tibia, respectively. Nandrolone preserved 80% and 60% of BMD in femur and tibia, respectively, demonstrating that nandrolone administration significantly reduced loss of BMD from denervation. This study offers a potential novel pharmacological strategy for use of nandrolone to reduce bone loss in severe disuse- and denervation-related bone loss, such as that which occurs after spinal cord injury.
Asunto(s)
Resorción Ósea/prevención & control , Desnervación , Miembro Posterior , Nandrolona/uso terapéutico , Anabolizantes/farmacología , Anabolizantes/uso terapéutico , Animales , Densidad Ósea/efectos de los fármacos , Resorción Ósea/etiología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Fémur/inervación , Fémur/patología , Miembro Posterior/efectos de los fármacos , Miembro Posterior/inervación , Miembro Posterior/patología , Masculino , Nandrolona/farmacología , Ratas , Ratas Wistar , Nervio Ciático/cirugía , Tibia/inervación , Tibia/patologíaRESUMEN
Glucocorticoids are a well-recognized and common cause of muscle atrophy that can be prevented by testosterone. However, the molecular mechanisms underlying such protection have not been described. Thus, the global effects of testosterone on dexamethasone-induced changes in gene expression were evaluated in rat gastrocnemius muscle using DNA microarrays. Gene expression was analyzed after 7-d administration of dexamethasone, dexamethasone plus testosterone, or vehicle. Dexamethasone changed expression of 876 probe sets by at least 2-fold. Among these, 474 probe sets were changed by at least 2-fold in the opposite direction in the dexamethasone plus testosterone group (genes in opposition). Major biological themes represented by genes in opposition included IGF-I signaling, myogenesis and muscle development, and cell cycle progression. Testosterone completely prevented the 22-fold increase in expression of the mammalian target of rapamycin (mTOR) inhibitor regulated in development and DNA damage responses 1 (REDD1), and attenuated dexamethasone induced increased expression of eIF4E binding protein 1, Forkhead box O1, and the p85 regulatory subunit of the IGF-I receptor but prevented decreased expression of IRS-1. Testosterone attenuated increases in REDD1 protein in skeletal muscle and L6 myoblasts and prevented dephosphorylation of p70S6 kinase at the mTOR-dependent site Thr389 in L6 myoblast cells. Effects of testosterone on REDD1 mRNA levels occurred within 1 h, required the androgen receptor, were blocked by bicalutamide, and were due to inhibition of transcriptional activation of REDD1 by dexamethasone. These data suggest that testosterone blocks dexamethasone-induced changes in expression of REDD1 and other genes that collectively would otherwise down-regulate mTOR activity and hence also down-regulate protein synthesis.
Asunto(s)
Andrógenos/metabolismo , Atrofia Muscular/metabolismo , Proteínas Represoras/metabolismo , Testosterona/metabolismo , Andrógenos/uso terapéutico , Animales , Proteínas Portadoras/metabolismo , Dexametasona/efectos adversos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Glucocorticoides/efectos adversos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Masculino , Atrofia Muscular/inducido químicamente , Atrofia Muscular/prevención & control , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfoproteínas/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores Androgénicos/metabolismo , Receptores de Glucocorticoides/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal , Testosterona/uso terapéutico , Factores de TranscripciónRESUMEN
Causes of disuse atrophy include loss of upper motor neurons, which occurs in spinal cord injury (SCI) or lower motor neurons (denervation). Whereas denervation quickly results in muscle fibrillations, SCI causes delayed onset of muscle spasticity. To compare the influence of denervation or SCI on muscle atrophy and atrophy-related gene expression, male rats had transection of either the spinal cord or sciatic nerve and were sacrificed 3, 7, or 14 days later. Rates of atrophy increased gradually over the first week after denervation and then were constant. In contrast, atrophy after SCI peaked at 1 week, then declined sharply. The greater atrophy after SCI compared to denervation was preceded by high levels of ubiquitin ligase genes, MAFbx and MuRF1, which then also markedly declined. After denervation, however, expression of these genes remained elevated at lower levels throughout the 2-week time course. Interestingly, expression of the muscle growth factor, IGF-1 was increased at 3 days after denervation when fibrillation also peaks compared to SCI. Expression of IGF-1R, GADD45, myogenin, and Runx1 were also initially increased after denervation or SCI, with later declines in expression levels which correlated less well with rates of atrophy. Thus, there were significant time-dependent differences in muscle atrophy and MAFbx, MuRF1, and IGF-1 expression following SCI or denervation which may result from distinct temporal patterns of spontaneous muscle contractile activity due to injury to upper versus lower motor neurons.
Asunto(s)
Regulación de la Expresión Génica , Neuronas Motoras/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/inervación , Músculo Esquelético/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Animales , Masculino , Ratas , Ratas Wistar , Vértebras Torácicas/lesionesRESUMEN
The muscle ubiquitin ligases MAFbx and MuRF1 are upregulated in and promote muscle atrophy. Upregulation of MAFbx and MuRF1 by glucocorticoids has been linked to activation of FOXO1 and FOXO3A resulting from reduced Akt activity. We determined the requirements for the glucocorticoid receptor (GR) in these biological responses in C2C12 cells in which GR expression was knocked down by stable expression of an shRNA. Loss of GR prevented dexamethasone-induced increases in protein catabolism. Loss of GR, or inhibition of ligand binding to GR with RU486, prevented upregulation of MAFbx and MuRF1 by dexamethasone. Loss of GR also prevented dexamethasone-induced decreases in Akt phosphorylation, and increases in the fraction of FOXO1 that was unphosphorylated. The findings establish a requirement for the GR in activating molecular signals that promote muscle protein catabolism.