Expression of chemokine receptors in insulin-resistant human skeletal muscle cells.

Adipokines including chemokines are able to induce insulin resistance in human skeletal muscle cells, which may also be relevant for the observed link between obesity and diabetes. This study is aimed to analyze the expression of chemokine CC motif receptors (CCRs) in the insulin-resistant state in human skeletal muscle cells. Differentiated skeletal muscle cells were incubated for 24-72 hours with high concentrations of glucose and insulin (GI) or TNFalpha. In addition, myocytes were co-stimulated with monocyte chemotactic protein (MCP)-1 or adipocyte-conditioned medium (CM) and TNFalpha for 24 and 48 hours. Treatment with GI rapidly induced insulin resistance whereas TNFalpha impaired insulin signaling in a more chronic fashion (48-72 h). CM and MCP-1 also induced insulin resistance that was, however, not increased by co-stimulation with TNFalpha. Expression of CCR2 was decreased during differentiation but up-regulated in insulin-resistant myocytes after treatment with GI (24-72 h) and TNFalpha (72 h). Expression of CCR4 and CCR10 was down-regulated after treatment with TNFalpha, MCP-1, and CM. Our data show that the expression of CCR2, CCR4, and CCR10 is differentially regulated by different insulin resistance-inducing treatments in myotubes. However, we could not find a clear correlation between the level of insulin resistance and CCR expression in myotubes. In conclusion, we propose that upregulation of CCR2 in skeletal muscle does not represent a major step leading to muscle insulin resistance.     

Binding of glucocorticoid antagonists to androgen and glucocorticoid hormone receptors in rat skeletal muscle

The binding of ten steroids possessing antiglucocorticoid activity has been studied in rat skeletal muscle cytosol. The affinity of these steroids for both the androgen and the glucocorticoid receptors was determined by competition with radioactive R1881 (methyltrienolone, metribolone) and dexamethasone, respectively. The antiglucocorticoid activity of these compounds was assessed in rat hepatoma (HTC) cells by measuring their inhibitory effect on the glucocorticoid-induced tyrosine aminotransferase activity. This led to identification of five novel in vitro glucocorticoid antagonists. All the steroids tested bound to both the glucocorticoid and the androgen receptors in muscle. Four steroids had an affinity for the glucocorticoid receptor higher than for the androgen receptor. The assumption is made that the steroids tested also behave as antagonists when binding to the glucocorticoid receptor in muscle and behave as agonists when binding to the androgen receptor. On this basis, the data allow one to compute a potential anticatabolic (PAG) and a potential anabolic (PAA) index for each compound. These indices might be of predictive value to determine whether these steroids exert their anabolic action in muscle through the glucocorticoid receptor or through the androgen receptor. The data also make it unlikely that satellite cells are a preferential target for anabolic steroids in muscle.     

Leptin receptors in human skeletal muscle.

Human skeletal muscle expresses leptin receptor mRNA; however, it remains unknown whether leptin receptors (OB-R) are also expressed at the protein level. Fourteen healthy men (age = 33.1 +/- 2.0 yr, height = 175.9 +/- 1.7 cm, body mass = 81.2 +/- 3.8 kg, body fat = 22.5 +/- 1.9%; means +/- SE) participated in this investigation. The expression of OB-R protein was determined in skeletal muscle, subcutaneous adipose tissue, and hypothalamus using a polyclonal rabbit anti-human leptin receptor. Three bands with a molecular mass close to 170, 128, and 98 kDa were identified by Western blot with the anti-OB-R antibody. All three bands were identified in skeletal muscle: the 98-kDa and 170-kDa bands were detected in hypothalamus, and the 98-kDa and 128-kDa bands were detected in thigh subcutaneous adipose tissue. The 128-kDa isoform was not detected in four subjects, whereas in the rest its occurrence was fully explained by the presence of intermuscular adipose tissue, as demonstrated using an anti-perilipin A antibody. No relationship was observed between the basal concentration of leptin in serum and the 170-kDa band density. In conclusion, a long isoform of the leptin receptor with a molecular mass close to 170 kDa is expressed at the protein level in human skeletal muscle. The amount of 170-kDa protein appears to be independent of the basal concentration of leptin in serum.     

Em evidence of myoblast origin in regenerating human skeletal muscle explants

EM study of cultured human skeletal muscle explants on 10 consecutive days after incubation made possible a record for the first time, the early events occurring during regeneration. After incubation, normal myonuclei underwent activation and dense granulation. Some myonuclei showed early transformation to presumptive myoblasts. The conclusion was that myonuclei transformed into myoblasts which developed into satellite cells (SC). These SC of myonuclear origin, proliferated, and fused forming myotubes that matured into myofibres, replacing damaged muscle. The findings have new implications for the current myoblast / cell transplant and gene transfer therapy research which may provide possible answers for muscular dystrophy in the future.     

HIF-1alpha response to hypoxia is functionally separated from the glucocorticoid stress response in the in vitro regenerating human skeletal muscle

Injury of skeletal muscle is followed by muscle regeneration in which new muscle tissue is formed from the proliferating mononuclear myoblasts, and by systemic response to stress that exposes proliferating myoblasts to increased glucocorticoid (GC) concentration. Because of its various causes, hypoxia is a frequent condition affecting skeletal muscle, and therefore both processes, which importantly determine the outcome of the injury, often proceed under hypoxic conditions. It is therefore important to identify and characterize in proliferating human myoblasts: 1) response to hypoxia which is generally organized by hypoxia-inducible factor-1α (HIF-1α); 2) response to GCs which is mediated through the isoforms of glucocorticoid receptors (GRs) and 11β-hydroxysteroid dehydrogenases (11β-HSDs), and 3) the response to GCs under the hypoxic conditions and the influence of this combination on the factors controlling myoblast proliferation. Using real-time PCR, Western blotting, and HIF-1α small-interfering RNA silencing, we demonstrated that cultured human myoblasts possess both, the HIF-1α-based response to hypoxia, and the GC response system composed of GRα and types 1 and 2 11β-HSDs. However, using combined dexamethasone and hypoxia treatments, we demonstrated that these two systems operate practically without mutual interactions. A seemingly surprising separation of the two systems that both organize response to hypoxic stress can be explained on the evolutionary basis: the phylogenetically older HIF-1α response is a protection at the cellular level, whereas the GC stress response protects the organism as a whole. This necessitates actions, like downregulation of IL-6 secretion and vascular endothelial growth factor, that might not be of direct benefit for the affected myoblasts.     

Expression of myostatin gene in regenerating skeletal muscle of the rat and its localization

The expression of myostatin mRNA was examined in regenerating skeletal muscle of the rat. Skeletal muscle regeneration was induced by injecting bupivacaine or hypertonic saline solution into the femoral muscle, and the tissues were collected 48 h after the treatment. In situ hybridization analysis revealed that the cells positive for myostatin message were localized in the regenerating area of the bupivacaine-treated tissues, where a numerous number of mononucleated cells were present. The myostatin-positive mononucleated cells contained both myogenic and nonmyogenic cells, as revealed by immunohistochemical staining for desmin and vimentin. Bupivacaine treatment to the testes resulted in no myostatin message expression in the testicular vimentin-positive cells, suggesting that the expression of myostatin message in vimentin-positive cells is a skeletal muscle-specific phenomenon. Furthermore, crushed muscle extract prepared from regenerating skeletal muscle had induced myostatin mRNA expression in skeletal muscle-derived fibroblasts in a dose-dependent manner. These results indicated that myostatin is expressed during skeletal muscle regeneration both in myogenic and nonmyogenic cells, and suggested that some factor(s) capable of inducing myostatin expression in fibroblasts are present in regenerating skeletal muscle.     

Purinergic receptors expressed in human skeletal muscle fibres

Purinergic receptors are present in most tissues and thought to be involved in various signalling pathways, including neural signalling, cell metabolism and local regulation of the microcirculation in skeletal muscles. The present study aims to determine the distribution and intracellular content of purinergic receptors in skeletal muscle fibres in patients with type 2 diabetes and age-matched controls. Muscle biopsies from vastus lateralis were obtained from six type 2 diabetic patients and seven age-matched controls. Purinergic receptors were analysed using light and confocal microscopy in immunolabelled transverse sections of muscle biopsies. The receptors P2Y(4), P2Y(11) and likely P2X(1) were present intracellularly or in the plasma membrane of muscle fibres and were thus selected for further detailed morphological analysis. P2X(1) receptors were expressed in intracellular vesicles and sarcolemma. P2Y(4) receptors were present in sarcolemma. P2Y(11) receptors were abundantly and diffusely expressed intracellularly and were more explicitly expressed in type I than in type II fibres, whereas P2X(1) and P2Y(4) showed no fibre-type specificity. Both diabetic patients and healthy controls showed similar distribution of receptors. The current study demonstrates that purinergic receptors are located intracellularly in human skeletal muscle fibres. The similar cellular localization of receptors in healthy and diabetic subjects suggests that diabetes is not associated with an altered distribution of purinergic receptors in skeletal muscle fibres. We speculate that the intracellular localization of purinergic receptors may reflect a role in regulation of muscle metabolism; further studies are nevertheless needed to determine the function of the purinergic system in skeletal muscle cells.     

Characterization and quantification of the androgen and glucocorticoid receptors in cytosol from rat skeletal muscle

The binding of the radioactive synthetic hormonal steroids [3H]dexamethasone (9 alpha-fluoro-11 beta, 17 alpha, 21-trihydroxy-16 alpha-methyl-1,4-pregnadiene-3,20-dione) and [3H]methyltrienolone (17 beta-hydroxy-17 alpha-methyl-4,9,11-estratien-3-one) to cytosol from rat skeletal muscle was studied using dextran-coated charcoal to separate unbound and receptor-bound steroid. The rates of association, dissociation, and degradation of the complexes of dexamethasone and methyltrienolone with receptor were highly dependent on temperature. The temperature dependence of association was greater for dexamethasone, and that of degradation was greater for methyltrienolone. Dissociation rates were insignificant for both steroid-receptor complexes compared to association and degradation rates. The apparent equilibrium dissociation constants for the binding of dexamethasone and methyltrienolone to their receptor binding sites were about 7 and 0.3 nM, respectively, regardless of temperature (0. 15 or 23 degrees C). The lack of influence of temperature on the equilibrium constants indicate that the binding was of hydrophobic character, and the corresponding free energy changes upon binding of dexamethasone and methyltrienolone to their respective binding sites were -41 and -49 kJ mol-1 under equilibrium conditions at 0 degrees C. The apparent maximum number of binding sites determined from Scatchard plots under these conditions was about 1900 fmol/g of tissue, 3500 fmol/mg of DNA or 30 fmol/mg of protein in the case of the dexamethasone receptor, and the corresponding figures for the methyltrienolone were about 100 fmol/g of tissue, 200 fmol/mg of DNA or 2 fmol/mg of protein. The ligand specificities of the binding sites for dexamethasone and methyltrienolone were typical of a glucocorticoid and an androgen receptor, respectively. Both steroid-receptor complexes were retained on DNA-cellulose columns, and were eluted by NaCl at an ionic strength of 0.1. The DNA-cellulose step purified about 20 times, and was used to allow gel exclusion chromatography and electrofocusing. Both steroid-receptor complexes were excluded from a column of Sephadex G-150. Electrofocusing in preparative columns gave reproducible patterns consisting of three peaks for each receptor. The apparent isoelectric points were 5.4, 5.6 and 6.2 for the glucocorticoid receptor, and 5.9, 6.2 and 8.5 for the androgen receptor.     

Peroxisomes in regenerating human skeletal myofibers

Peroxisomes of the human regenerating skeletal myofibers were studied qualitatively and quantitatively by electron cytochemistry and were compared with those of the mature normal human skeletal muscle fibers. Peroxisomes visualized by electron cytochemistry with 3,3'-diaminobenzidine (DAB) were small round or oval bodies delimited by a single membrane and contained the electron-opaque, coarsely granular matrix. Muscle grafts of the regenerating normal human quadriceps obtained from 4 orthopedic patients were analyzed 2 weeks after transplantation into nude mice; they contained peroxisomes with a mean diameter of 0.25 microns, ranging from 0.12 to 0.67 microns. The group mean density of peroxisomes per 100 microns2 was 2.0 +/- 0.4 (SE), while that of histochemically normal mature human quadriceps femoris myofibers was 0. The cytochemical controls without DAB or with the presence of 3-amino 1,2,4-triazole in the solution containing DAB lacked the electron-opaque reaction, indicating that these reactions were on an enzymatic basis. The results of this study showed clearly that the regenerating normal human skeletal myofibers contained numerous peroxisomes differing from the mature normal human muscle fibers in which the peroxisomes were not observed.     

Increased phenylalanine incorporation in regenerating skeletal muscle grafts

Skeletal muscle regenerates following grafting, but little is known about protein synthesis and its regulation during regeneration. We determined the sequence of changes in protein synthesis in rat extensor digitorum longus (EDL) muscle by the measurement of phenylalanine (Phe) incorporation into muscle protein at various times after grafting. Compared with control EDL, Phe incorporation in grafts doubled in 1 day, was four- to eight-fold greater from days 2 to 10 after grafting, and then subsided. Tissue mass (wet weight) increased rapidly from days 7 to 20 in EDL grafts. The maximal increase in protein synthesis occurred 7-10 days after grafting, whether or not the nerve was left intact. Autoradiography indicated that incorporated radioactivity was associated with regenerating muscle fibers on day 10. Deficiencies of insulin, pituitary or testicular hormones, or chronic in vivo administration of insulin, growth hormone, testosterone, or tri-iodothyronine did not substantially alter the elevation in incorporation of the Phe into muscle protein 10 days after grafting. The breakdown of EDL protein, measured in vitro simultaneously with protein synthesis, was increased five-fold, and overall protein degradation was elevated six-fold 10 days after grafting. These findings indicate that Phe incorporation is rapidly elevated following grafting of the EDL, and that by days 7-10 reflects synthesis in regenerating muscle fibers. The increase in protein synthesis associated with muscle regeneration at this time appears to be independent of innervation and anabolic hormones.     

Expression of laminin subunits in human fetal skeletal muscle

Summary The laminin variant of adult skeletal muscle fibres and Schwann cells is known as merosin, and is composed of M-B1-B2 chains. Blood vessels and immature fibres express the A chain in association with B1 or S, and B2. The importance of merosin has recently been shown by its absence in one form of congenital muscular dystrophy and in the mutantdy/dy mouse, and by its partial deficiency in Fukuyama congenital muscular dystrophy. We have examined the immunocytochemical localization of the M, A, B1 and B2 laminin chains in human fetal muscle from 7 to 40 weeks' gestation to ascertain their developmental expression. The B1 and B2 chains were detected on muscle fibres at 7 weeks, but only traces of the A or M chain were seen. By 21 weeks maximal levels of all four subunits were observed on all fibres. This suggests that the basement membrane is still being assembled until this stage of development. Expression of the A chain on muscle fibres was not reduced until 34 weeks and low levels persisted at birth. The concomitant expression of the M and A chains at early stages may indicate a laminin variant, in addition to merosin, that is highly expressed in fetal muscle. Merosin was seen in intramuscular nerves at 11 weeks. B1 and B2 subunits were detected in blood vessels from 7 weeks' gestation and the A chain from 11 weeks. The capillary network, however, is not fully established in fetal muscle. Merosin is therefore detected early during human fetal muscle development, and this should be taken into account when assessing aborted fetuses at risk for congenital muscular dystrophy.     

Increased adipogenicity of cells from regenerating skeletal muscle

Adipose tissue development is observed in some muscle pathologies, however, mechanisms that induce accumulation of this tissue as well as its cellular origin are unknown. The adipogenicity of cells from bupivacaine hydrochloride (BPVC)-treated and untreated muscle was compared in vitro. Culturing cells from both BPVC-treated and untreated muscles in adipogenic differentiation medium (ADM) for 10 days resulted in the appearance of mature adipocytes, but their number was 3.5-fold higher in cells from BPVC-treated muscle. Temporal expressions of PPARgamma and the presence of lipid droplets during adipogenic differentiation were examined. On day 2 of culture in ADM, only cells from BPVC-treated muscle were positive both for PPARgamma and lipid droplets. Pref-1 was expressed in cells from untreated muscle, whereas its expression was absent in cells from BPVC-treated muscle. In ADM, the presence of insulin, which negates an inhibitory effect of Pref-1 on adipogenic differentiation, was required for PPARgamma2 expression in cells from untreated muscle, but not for cells from BPVC-treated muscle. These results indicate that BPVC-induced degenerative/regenerative changes in muscle lead to increased adipogenicity of cells, and suggest that this increased adipogenicity not only involves an increase in the number of cells having adipogenic potential, but also contributes to the progression of these cells toward adipogenic differentiation.     

beta-Adrenoceptor signaling in regenerating skeletal muscle after beta-agonist administration

Stimulating the beta-adrenoceptor (beta-AR) signaling pathway can enhance the functional repair of skeletal muscle after injury, but long-term use of beta-AR agonists causes beta-AR downregulation, which may limit their therapeutic effectiveness. The aim was to examine beta-AR signaling during early regeneration in rat fast-twitch [extensor digitorum longus (EDL)] and slow-twitch (soleus) muscles after bupivacaine injury and test the hypothesis that, during regeneration, beta-agonist administration does not cause beta-AR desensitization. Rats received either the beta-AR agonist fenoterol (1.4 mgxkg(-1)xday(-1) ip) or saline for 7 days postinjury. Fenoterol reduced beta-AR density in regenerating soleus muscles by 42%. Regenerating EDL muscles showed a threefold increase in beta-AR density, and, again, these values were 43% lower with fenoterol treatment. An amplified adenylate cyclase (AC) response to isoproterenol was observed in cell membrane fragments from EDL and soleus muscles 7 days postinjury. Fenoterol attenuated this increase in regenerating EDL muscles but not soleus muscles. beta-AR signaling mechanisms were assessed using AC stimulants (NaF, forskolin, and Mn(2+)). Although beta-agonist treatment reduces beta-AR density in regenerating muscles, these muscles can produce large cAMP responses relative to healthy (uninjured) muscles. Desensitization of beta-AR signaling in regenerating muscles is prevented by altered rates of beta-AR synthesis and/or degradation, changes in G protein populations and coupling efficiency, and altered AC activity. These mechanisms have important therapeutic implications for modulating beta-AR signaling to enhance muscle repair after injury.     

Reutilisation of tritiated thymidine in studies of regenerating skeletal muscle

Summary Two different aspects of tritiated thymidine (³H-Tdr) reutilisation in skeletal muscle were examined. Injection of a high dose (7 Ci/g) of ³H-Tdr into mice prior to crush injury of skeletal muscle resulted in heavy labelling (grain counts) of myotube nuclei 9 d later. In contrast, myotube nuclei were essentially unlabelled when a low dose (1 Ci/g) of ³H-Tdr was injected at similar times with respect to injury. It was concluded that labelling seen after the high dose was due to reutilisation of ³H-Tdr. (Such ³H-Tdr reutilisation can account for the results of Sloper et al. (1970) which previously supported the concept of a circulating muscle precursor cell.) When replicating muscle precursors were labelled directly with ³H-Tdr 48 h after injury, the percentages of labelled myotube nuclei and the distribution of nuclear grain counts were similar with either high or low dose.We also investigated whether the light labelling seen in regenerated myotube nuclei after 9 d, when ³H-Tdr had been injected before the onset of myogenesis (as found by McGeachie and Grounds 1987), was due to ³H-Tdr reutilisation or, alternatively, to proliferation of local cells in the wound which subsequently gave rise to muscle precursors. Labelling of myotube nuclei was compared in mice injected with ³H-Tdr either 2 h before, or 2 h after injury. In another experiment, mice were injected 12 h after injury and lesions sampled 1, 12 or 36 h later, to see whether local cells were replicating 12 h after injury, and what labelled cells subsequently entered to wound. No difference was found in myotube labelling between mice injected before or after injury, and no cells replicating locally in the wound at 12 h after injury were observed. The results clearly show that the light labelling was due to ³H-Tdr reutilisation.     

Quantitation of glucocorticoid receptors in bovine skeletal muscle: Topographical distribution, sex effect and breed comparisons

The concentration of the cytosolic glucocorticoid receptor (GR) was determined in skeletal muscles of calves in order to study possible differences in individual muscles from different parts of the body as well as the influence of sex and breed. In male and female Simmental calves the topographical distribution of GR was similar: the lowest concentrations were seen in abdominal muscle, whereas in neck, shoulder and hindleg the GR concentrations were higher; this difference was more pronounced in male than in female calves. In general, female calves had about 2-fold higher GR concentrations than males. The cytosolic cortisol concentrations were differing neither between individual muscles nor between sexes. The cortisol secretion during a 24-h sampling period 1 week prior to slaughter showed no sex difference. GR concentrations in neck muscle of female calves of four different German cattle breeds (Holstein Friesian, Brown Swiss, Simmental and German Gelbvieh) were rather similar; however, when Brown Swiss with the highest GR levels were compared to Holstein Friesian calves with the lowest concentrations, a significant difference was evident (P < 0.05).