Comparison of the relative expression of caspase isoforms in different porcine skeletal muscles.

The present study investigated the relationship between muscle type and components of the caspase protease system in porcine trapezius (TZ), psoas (PS), longissimus dorsi (LD) and semitendinosus (ST) muscles. Muscles were classified according to slow and fast myosin heavy chain (MHC) content determined by western blotting. MHC slow, but not MHC fast protein expression was significantly different between muscles (p<0.001). Protein levels of caspases 3, 8 and 12 and the caspase inhibitor apoptosis repressor with caspase recruitment domain (ARC) were determined. In addition the level of caspase 3 mRNA and activity levels of caspase 3/7 were determined. There was a significant difference in protein levels and activity between muscles (p<0.01), although no difference was observed in mRNA abundance. The data show that multiple components of the caspase system are expressed in porcine skeletal muscle and that their levels are variable, but there is not a distinct association of expression with a particular muscle.

The kinetics of myofibrillar protein breakdown in perfused rat skeletal muscle.

Nt-Methylhistidine, a non-reutilised amino acid present in some myofibrillar proteins, was radioactively labelled in vivo with [Me-3H]methionine. The specific radioactivities of protein-bound methylhistidine and free methylhistidine in perfusate after perfusion of rat hind limbs taken from prelabelled rats was determined. The decrease in urinary methylhistidine activity with time was determined for rats similarly labelled. Comparison of the specific activities of free and bound methylhistidine and the non-linear semilogarithmic plot of urinary methylhistidine activity suggest that the myofibrillar protein catabolism, as indicated by methylhistidine release, may not be a simple exponential process. The possibility of non-random decay is discussed and an alternative model proposed.

Stearoyl-CoA desaturase mRNA is transcribed from a single gene in the ovine genome.

Clones corresponding to ovine stearoyl-CoA desaturase (SCD) cDNA were isolated from an adipose tissue cDNA library. All of these clones represented a single mRNA species as judged by restriction fragment and DNA sequence analysis. RNase protection analysis demonstrated that this SCD transcript is highly expressed in adipose tissue and liver, and in the mammary gland of lactating animals. A lower level of expression was detectable in a variety of other tissues including brain. Levels of the SCD transcript were decreased in adipose tissue during lactation, and this appears to be related to a marked decline in serum insulin and insulin-responsiveness of the tissue. Southern analysis of ovine and mouse genomic DNA demonstrated that the ovine SCD cDNA hybridised in a manner consistent with a single gene for SCD in ovine DNA; mouse genomic DNA produced a pattern of hybridisation consistent with the previously characterised mouse SCD-1 and SCD-2 genes. Three ovine cosmids were isolated that comprised the restriction fragments predicted by the genomic Southern analysis. The ovine SCD gene was predicted to be encompassed within a 23 kbp region that was present in all three cosmids. These results demonstrate that SCD is transcribed from a single gene in the ovine genome and this gene is insulin-responsive in ovine adipose tissue.

The relationship between slow and fast myosin heavy chain content, calpastatin and meat tenderness in different ovine skeletal muscles.

The present study investigated the relationship between fibre type distribution and slow (MHC-s) and fast (MHC-f) myosin heavy chain content on calpastatin and meat tenderness in longissimus dorsi (LD), tensor fasciae latae (TFL), semitendinosus (ST), trapezius (TZ) and supraspinatus (SS) muscles from six Mule×Charolais rams. Samples taken at slaughter were frozen either in liquid N(2) for analysis of MHC-s and MHC-f by immunoblotting, or in cooled isopentane for histochemical fibre typing. Calpastatin activity and an immunoreactive 135 kDa calpastatin band were analysed in samples taken 24 h postmortem. Shear force was determined on muscle chops taken at 24 h postmortem and conditioned until day 14. The intensity of MHC-s and MHC-f immunopositive bands correlated with %Type I and %Type II fibres identified histochemically (r(2)=0.612 and 0.366, respectively, p<0.001). Muscle specific differences were observed in MHC-s and MHC-f immunoreactivity, fibre type distribution, calpastatin activity, calpastatin 135 kDa immunoreactivity and shear force. MHC-s correlated positively with calpastatin activity (r(2)=0.725, p<0.001) and 135 kDa calpastatin (r(2)=0.228, p<0.01) across all muscle types. The data show that detection of MHC-s can be used to identify fibre type differences between ovine muscles and that this correlates with differences in calpastatin content and inhibitory activity, but not tenderness.

Differential effects of short-term ß agonist and growth hormone treatments on expression of myosin heavy chain IIB and associated metabolic genes in sheep muscle.

Growth hormone (GH) and ß agonists increase muscle mass, but the mechanisms for this response are unclear and the magnitude of response is thought to vary with age of animal. To investigate the mechanisms driving the muscle response to these agents, we examined the effects of short-term (6 day) administration of GH or cimaterol (a ß2-adrenergic agonist, BA) on skeletal muscle phenotype in both young (day 60) and mature (day 120) lambs. Expression of myosin heavy chain (MyHC) isoforms were measured in Longissimus dorsi (LD), Semitendinosus (ST) and Supraspinatus (SS) muscles as markers of fibre type and metabolic enzyme activities were measured in LD. To investigate potential mechanisms regulating the changes in fibre type/metabolism, expression or activity of a number of signalling molecules were examined in LD. There were no effects of GH administration on MyHC isoform expression at either the mRNA or protein level in any of the muscles. However, BA treatment induced a proportional change in MyHC mRNA expression at both ages, with the %MyHCI and/or IIA mRNA being significantly decreased in all three muscles and %MyHCIIX/IIB mRNA significantly increased in the LD and ST. BA treatment induced de novo expression of MyHCIIB mRNA in LD, the fastest isoform not normally expressed in sheep LD, as well as increasing expression in the other two muscles. In the LD, the increased expression of the fastest MyHC isoforms (IIX and IIB) was associated with a decrease in isocitrate dehydrogenase activity, but no change in lactate dehydrogenase activity, indicating a reduced capacity for oxidative metabolism. In both young and mature lambs, changes in expression of metabolic regulatory factors were observed that might induce these changes in muscle metabolism/fibre type. In particular, BA treatment decreased PPAR-γ coactivator-1ß mRNA and increased receptor-interacting protein 140 mRNA. The results suggest that the two agents work via different mechanisms or over different timescales, with only BA inducing changes in muscle mass and transitions to a faster, less oxidative fibre type after a 6-day treatment.

Effect of genotype and nutrition on calpastatin inhibitory activity and mRNA abundance in milk-fed lambs.

Muscle proteolysis is controlled by a wide range of enzyme systems. The reported effects of the calcium dependent proteinases (calpain I and II) and its specific inhibitor (calpastatin) on myofibrillar structure, has led to the speculation that this system may have a pivotal role in regulating protein turnover and muscle growth. The present study highlights the possibility of protein degradation being subject to genetic variation. The relationship between genotype, level of nutrition, muscle protein turnover and the calpain system in young milk-fed lambs was assessed. Male lambs which had been selected for 10 generations for high (W+) and low (W-) weight at weaning were used in the study. Lambs were removed from their mothers 4 days after birth and surgically fitted with abomasal catheters and infused with reconstituted milk replacer at a high or a low rate. At 8 weeks of age, measurements of muscle protein gain, synthesis and degradation were performed, the animals were slaughtered and samples rapidly removed for subsequent chemical analysis. The liveweight gain and weight of the m vastus lateralis was reflected (P < 0.001) in the designed differences in nutrient supply. The weight of the m vastus lateralis was greater (P < 0.01) in the W+ compared to the W- lambs. The rate of protein synthesis and calculated degradation were greater (P < 0.05) in W+ than W- lambs. Calpain I and II and calpastatin activity were not significantly altered by genotype or nutrition. Calpastatin mRNA abundance increased significantly (P < 0.05) between 1 and 8 weeks of age. Regression analysis revealed genotype-specific responses with respect to calpastatin activity and mRNA abundance.(ABSTRACT TRUNCATED AT 250 WORDS)

Transient changes in growth and in calpain and calpastatin expression in ovine skeletal muscle after short-term dietary inclusion of cimaterol.

Prolonged dietary inclusion of beta-adrenergic agonists can induce skeletal muscle hypertrophy in meat animals, by a mechanism probably related to the calcium-dependent proteolytic enzymes, or calpains, and in particular to their specific inhibitor calpastatin. Calpain and calpastatin activities are also believed to be important factors during post-mortem tenderisation of meat. beta-Agonist treatment is generally associated with increased calpastatin activity, which may lead to meat toughness. The aim of the present study was to examine the effect of a short period of cimaterol (feeding for 8 days, followed by reversion to a normal diet for a further 24 days) on muscle growth and on calpain isoform and calpastatin activities and specific mRNA abundance in the longissimus dorsi (LD) muscle. Significant changes were detected in LD wet weight and in calpastatin activity and mRNA after only 8 days treatment with cimaterol. After 24 further days on a control diet, both LD wet weight and calpastatin activity were not significantly different (P > 0.05) from untreated controls of the same age, although calpastatin mRNA stayed surprisingly high. In contrast to several earlier studies, changes in calpain I (or mu-calpain) and calpain II (or m-calpain) activity and calpain I mRNA were not significantly different (P > 0.05) from controls in any groups. These data suggest that calpastatin activity rather than the activity of either calpain isoform is closely linked to beta-agonist-induced muscle hypertrophy. Changes in calpastatin mRNA are not directly proportional to inhibitory activity, suggesting that variable mRNA species may be transcribed, spliced or stabilised, but not necessarily translated as part of the beta-agonist response.

Effect of beta-agonists on expression of calpain and calpastatin activity in skeletal muscle.

Administration of beta-adrenergic agonists to domestic species can lead to skeletal muscle hypertrophy, probably by reducing the rate of myofibrillar protein breakdown. Myofibrillar breakdown is associated with the calcium-dependent proteinase system (calpains I,II and calpastatin) whose activity also changes during beta-agonist treatment. A number of growth trials using the agonists cimaterol and clenbuterol with cattle, sheep, chicken and rat are reported which suggest a general mechanism whereby beta-agonists reduce calpain I activity, but increase calpain II and calpastatin activity in skeletal muscle. Parallel changes in specific mRNAs indicate that changes in gene expression or stabilisation of mRNA could in part explain the changes in activity.

The porcine insulin-like growth factor-I gene: characterization and expression of alternate transcription sites.

Genomic DNA encoding the 5' region of the porcine IGF-I gene was cloned and sequenced and shown to be highly homologous to that of man, rats and sheep. Two leader exons (exons 1 and 2), which are alternately spliced to exon 3 (encoding part of the mature IGF-I molecule), were identified by RNase protection analysis. In both cases, transcription initiates upstream from exons 1 and 2 at multiple dispersed start sites to yield two distinct IGF-I mRNA transcript classes (1 and 2) which differ in the precursor peptides predicted from their individual leader sequences. The expression of class 1 and 2 transcripts was measured in liver and muscle RNA from two groups of 2-month-old pigs whose energy status had been manipulated within physiological limits to produce marked differences in plasma IGF-I levels and growth rates. For this purpose, RNase protection probes were developed that contained the individual leader exons 1 and 2 linked separately to the common exon 3, so that class-specific and total IGF-I gene expression could be determined in a single assay. At normal plasma IGF-I concentrations (200 ng/ml), class 1 and 2 transcripts comprised 81 and 19% respectively of total liver IGF-I mRNA, while at a lower plasma concentration (90 ng/ml) the corresponding values were 95 and 5% respectively. Although both classes of transcript declined with the decrease in plasma IGF-I, the relative drop in levels of class 2 transcripts (84%) was substantially greater than that of class 1 (54%). In longissimus dorsi, cardiac and soleus muscles IGF-I mRNA was predominantly of class 1 and did not change in response to decreased plasma IGF-I. This suggests that liver-derived endocrine IGF-I has an important function in the regulation of muscle growth and that class 2 IGF-I transcripts are more sensitive to conditions that promote optimal growth.

Protein metabolism in ovine primary muscle cultures derived from satellite cells--effects of selected peptide hormones and growth factors.

Methods were developed for the isolation and culture of satellite cells from adult sheep muscle. Differentiated cultures of these cells were used to investigate the effects of four hormones and growth factors on protein synthesis and degradation. Insulin was found to have no effect except at supraphysiological concentrations (100 nmol/l and 1 mumol/l) where it is probably crossreacting with the insulin-like growth factor (IGF) type-I receptor. IGF-I was found to be anabolic at lower concentrations (1-3 nmol/l). Epidermal growth factor (EGF) had a smaller effect on protein synthesis and degradation than insulin or IGF-I. The specific activity of the muscle-specific enzyme creatine phosphokinase (CPK) was increased by treatment with EGF. When both IGF-I and EGF were present in the test media an additive effect on protein synthesis was observed. However, no additive effect of IGF-I and insulin was noted. No effects of bovine GH were seen.

Changes in protein metabolism of ovine primary muscle cultures on treatment with growth hormone, insulin, insulin-like growth factor I or epidermal growth factor.

Methods for the primary culture of muscle cells from fetal sheep were developed which gave high yields of cells. Myoblasts were grown in vitro, and allowed to fuse to form contractile multinucleate myotubes; these could be maintained in a good condition for at least 2 weeks. Protein turnover in these differentiated cultures was examined for sensitivity to each of four potentially anabolic peptide hormones and growth factors: insulin, insulin-like growth factor I (somatomedin C), epidermal growth factor and growth hormone. Insulin was found to have no effect except at high concentrations (1 mumol/l), compatible with its role as a somatomedin analogue. Insulin-like growth factor I was active at lower levels (1 nmol/l) but the cultures were not as responsive to it as were primary rat muscle cultures or differentiated L6 cells, which were tested in similar experiments. The maximum stimulation of protein synthesis observed with the ruminant system was only 16%. Epidermal growth factor was highly anabolic for primary cultures from sheep muscle, and the cells were very sensitive to it, half-maximal stimulation of protein synthesis being seen with concentrations as low as 20 pmol/l. No effects of bovine growth hormone were seen in the ovine system. However, an inhibition of protein breakdown was found with high concentrations (0.1 mumol/l) in the L6 rat myoblast cell line. It was found that the culture conditions used could affect the observed responses of protein synthesis and degradation, despite withdrawal of serum from the incubation media 22 h before testing.

Effect of lactation on insulin signal transduction in sheep adipose tissue and skeletal muscle.

The molecular basis of the insulin resistance of adipocytes and skeletal muscle during lactation has been investigated in sheep. The number of insulin receptors per adipocyte or per unit membrane protein for skeletal muscle is unchanged by lactation. The ability of insulin to stimulate autophosphorylation of its beta-subunit was enhanced in adipocytes but not in skeletal muscle during lactation. This increased autophosphorylation was due, at least in part, to enhanced tyrosine phosphorylation and was found when both solubilised, immunoprecipitated insulin receptors and intact adipocytes were incubated with insulin. The ability of the insulin receptor kinase to phosphorylate other proteins did not appear to be altered by lactation; this was shown with lectin-purified insulin receptors using the artificial substrate, polyglutamyl tyrosine, and in intact adipocytes. Lactation had no effect on the ability of insulin to activate two key downstream kinases, mitogen-activated protein kinase and phosphatidyl inositol-3-kinase in adipocytes. The study thus shows that the insulin resistance of lactation in sheep is due to changes downstream of the receptor in both adipocytes and skeletal muscle.

Hormonal control of insulin-like growth factor-I and growth hormone receptor mRNA expression by porcine hepatocytes in culture.

The effects of various hormones commonly added to hepatocyte culture media upon the expression of the GH receptor (GHR) and insulin-like growth factor-I (IGF-I) genes in cultured porcine hepatocytes were investigated. Preliminary investigations indicated that there was an absolute requirement only for insulin, with high losses of cell viability upon long term exclusion of insulin from the culture medium. The decline in GHR expression with time in culture was found to be less when high levels of glucose were included in the medium. Therefore the basal culture medium used in these studies was Williams' medium E supplemented with 0.2% (w/v) BSA, 5000 mg glucose/l and 100 nmol porcine insulin/l. The addition of dexamethasone (10 nmol/l) increased the expression of both GHR and IGF-I (class 1 transcripts only) mRNA (p < 0.001 and P < 0.05 respectively), and resulted in an increased responsiveness of IGF-I mRNA expression to GH (1 microgram/ml), when the two were added in combination (although only class 1 transcripts were shown to be statistically significant, P < 0.01). The addition of either thyroid hormone (1 nmol/l T3 or T4) alone also increased the expression of GHR mRNA (P < 0.01) in addition to the dexamethasone stimulated expression, with T4 appearing to decrease IGF-I expression slightly (P < 0.05) (either on its own or with T3). As with dexamethasone, the thyroid hormones increased the response of IGF-I mRNA expression to GH (1 microgram/ml) when added in combination with GH (P < 0.001). These observations demonstrate one possible mechanism for the interactions of glucocorticoids and thyroid hormones with the GH-IGF axis.

Maternal nutrition alters the expression of insulin-like growth factors in fetal sheep liver and skeletal muscle.

We investigated the influence of maternal dietary restriction between days 28 and 80 of gestation followed by re-feeding to the intake of well-fed ewes up to 140 days of gestation (term is 147 days) in sheep, on expression of mRNA for insulin-like growth factor (IGF)-I, IGF-II and growth hormone receptor (GHR) in fetal liver and skeletal muscle. Singleton bearing ewes either consumed 3.2-3.8 MJ/day of metabolisable energy (ME) (i.e. nutrient restricted - approximately 60% of ME requirements, taking into account requirements for both ewe maintenance and growth of the conceptus) or 8.7-9.9 MJ/day (i.e. well fed - approximately 150% of ME requirements) between days 28 and 80 of gestation. All ewes were then well fed (150% of ME requirements) up to day 140 of gestation and consumed 8-10.9 MJ/day. At days 80 and 140 of gestation, five ewes were sampled from each group and fetal tissues taken. There was no difference in fetal body weight or liver weights between groups at either sampling date, or skeletal muscle (quadriceps) weight at 140 days. IGF-I mRNA abundance was lower in livers of nutrient-restricted fetuses at day 80 of gestation (nutrient restricted 2.35; well fed 3.70 arbitrary units), but was higher than well-fed fetuses at day 140 of gestation, after 60 days of re-feeding (restricted/re-fed 4.27; well fed 2.83;s.e.d. 0.98 arbitrary units, P=0.061 for dietxage interaction). IGF-II mRNA abundance was consistently higher in livers of nutrient-restricted fetuses (80 days: nutrient restricted 7.78; well fed 5.91; 140 days: restricted/re-fed 7.23; well fed 6.01;s.e.d. 1.09 arbitrary units, P=0.061 for diet). Nutrient restriction had no effect on hepatic GHR mRNA abundance, but re-feeding of previously nutrient-restricted fetuses increased GHR mRNA compared with continuously well-fed fetuses (80 days: nutrient restricted 70.6; well fed 75.1; 140 days: restricted/re-fed 115.7; well fed 89.4;s.e.d. 10.13 arbitrary units, P=0.047 for dietxage interaction). In fetal skeletal muscle, IGF-I mRNA abundance was not influenced by maternal nutrition and decreased with gestation age (P<0.01). IGF-II mRNA abundance was higher in skeletal muscle of nutrient-restricted fetuses compared with well-fed fetuses at day 80 of gestation (nutrient restricted 16.72; well fed 10.53 arbitrary units), but was lower than well-fed fetuses after 60 days of re-feeding (restricted/re-fed 7.77; well fed 13.72;s.e.d. 1.98 arbitrary units, P<0.001 for dietxage interaction). There was no effect of maternal nutrition or gestation age on fetal skeletal muscle GHR expression. In conclusion, maternal nutrient restriction in early to mid gestation with re-feeding thereafter results in alterations in hepatic and skeletal muscle expression of IGF-I, IGF-II and/or GHR in the fetus which may subsequently relate to altered organ and tissue function.

Beta-adrenoceptors and the effect of beta-agonists on protein metabolism in ovine primary muscle cultures.

The beta-adrenergic receptors of differentiated ovine muscle cultures derived from either fetal or pre-pubertal lambs were characterized by binding of (+/-)-[3H]CGP-12177, directly to intact cells in monolayer. Fetal muscle cells contained a single class of specific and saturable binding sites which had a dissociation constant (Kd) of 0.38 x 10(-9) M and a binding capacity of 55.2 fmol/micrograms protein. beta-Adrenergic agonists competed for the specific binding sites with a typical beta 2-adrenergic specificity. Satellite muscle cells derived from pre-pubertal lambs contained two classes of binding site. The high affinity site had a Kd of 1.02 x 10(-9) M and a binding capacity of 28.4 fmol/micrograms protein and the low affinity site a Kd of 12.1 x 10(-9) M and a binding capacity of 389 fmol/micrograms protein. beta-Adrenergic agonists competed for the specific binding sites with a typical beta 1-adrenergic specificity. The beta-agonist cimaterol had no effect on either protein synthesis or degradation in fetal muscle cells. In cultures derived from satellite cells cimaterol significantly stimulated protein synthesis at concentrations of 10(-8) - 10(-7) M and at 10(-8) - 10(-6) M in the presence of serum. These effects were maintained if 10(-5) M propranolol was added to the incubation media, but were blocked by 10(-6) M isoproterenol. Propranolol and isoproterenol had no stimulatory effects on protein synthesis. Cimaterol also had no detectable effects on protein degradation or the transport of amino acids or glucose. It is concluded that although beta-adrenergic receptors are present in ovine muscle cultures they may not play a role in the anabolic effect of beta-agonists observed in cultured muscle cells.

Effects of gastric inhibitory polypeptide, somatostatin and epidermal growth factor on lipogenesis in ovine adipose explants.

Feeding raises the plasma concentrations of a number of gut-related hormones that may, in turn, influence the metabolism of peripheral tissues. This study investigated the effects of gut-related hormones on lipogenesis in explants from three differing adipose depots in lambs (aged 4-9 months). Incorporation of [14C]-acetate into lipid was measured over a 2-h period, following 24 h pre-incubation in the presence of hormone combinations. In perirenal fat explants, gastric inhibitory polypeptide (GIP) in the concentration range 0.01-10 nM stimulated lipogenesis. Maximal effects were seen at 1 nM (an average increase of 64% over basal values). In contrast, in the presence of insulin (0.1 nM), a dose-dependent decrease in lipogenesis was seen with increasing GIP concentration (P < 0.001 for the insulin x GIP interaction). Epidermal growth factor (EGF) and somatostatin in the same concentration range each inhibited lipogenesis. both in the presence and the absence of insulin (P < 0.001 in each case). Subcutaneous (back) fat and intermuscular (popliteal) fat responded similarly to each other, but significantly differently from the perirenal depot (P < 0.001). Here GIP, somatostatin or EGF (each at 1 nM) all separately stimulated lipogenesis.

Effect of gastric inhibitory polypeptide on bovine fat metabolism.

Gastric inhibitory polypeptide (GIP), a hormone secreted by the gastrointestinal tract in response to nutrient absorption, exerts anabolic effects on adipose tissue in some species. Cattle fed on grass silage diets tend to deposit more fat than animals fed on dried forages. We investigated the effect of diet on blood GIP concentrations in cattle. Plasma concentrations tended to be higher in cattle fed grass silage alone or supplemented with fishmeal (0.61 ng/ml) compared with animals fed dried grass/barley (0.43 ng/ml, P > 0.1) and were inversely correlated with plasma insulin concentrations (r = -0.727, P < 0.01). The effects of increasing concentrations (0, 0.1, 1.0, 10.0 nM) of GIP and glucagon-like peptide-1 (GLP-1) on basal and insulin-stimulated lipogenesis in bovine adipose tissue in vitro were investigated after 4 and 24 h of incubation. No conclusive effects were obtained with either peptide. Subsequently, the effect of exogenous administration of GIP (10 or 50 ng/kg liveweight per min) on whole-body fat metabolism was investigated in two steers in vivo. Plasma concentration and flux rate of palmitate was increased by GIP only at the higher infusion level suggesting lipolysis and possibly fatty acid re-esterification was stimulated at high concentration. We conclude that although gut peptides may regulate nutrient utilisation, it is unlikely that they play a major role in promoting fat accretion in cattle. However, the small number of animals used in these studies indicates the need for caution and further studies are warranted.

Glucocorticoids and insulin but not growth hormone modulate insulin binding to adipocyte membranes from sheep.

Mechanisms by which the glucocorticoid analogue dexamethasone and growth hormone modulate insulin action in sheep adipose tissue have been investigated. Maintenance of sheep adipose tissue in culture for 48 hr in the absence of exogenous hormones resulted in a decrease in insulin binding to adipocyte membranes; this was prevented by the inclusion of 10 nM dexamethasone during culture. Tissue culture for 48 hr with insulin itself decreased insulin binding to adipocyte membranes, whereas tissue culture with growth hormone had no effect on insulin binding. Neither dexamethasone nor growth hormone altered the ability of insulin to decrease insulin binding to its receptor. The study shows that the insulin-antagonistic effects of growth hormone on sheep adipose tissue metabolism are due to an effect subsequent to insulin binding to its receptor, whereas some of the effects of dexamethasone may be due to an increase in the insulin-binding capacity of membranes.

Effects of EGF receptor ligands on fetal ovine myoblasts.

Epidermal growth factor (EGF) receptors are widely distributed in mammalian tissues, including muscle. One ligand of these receptors, heparin-binding epidermal growth factor-like growth factor (HB-EGF) is also strongly expressed in adult muscle. However, in vitro studies of EGF action in cultured muscle cells of different species have yielded conflicting results. The purpose of this study was to investigate the potential role of EGF and related factors in the growth and development of fetal ovine muscle. High affinity EGF receptors were detected on clonally purified ovine fetal myoblasts, using [(125)I] human EGF as a ligand (K(d) values of 47 and 54 pM in separate experiments). Competitive binding studies in mixed secondary cultures showed that EGF had the highest affinity for the fetal ovine receptor, followed by HB-EGF and transforming growth factor alpha (TGF-alpha). These ligands all stimulated DNA synthesis in clonally purified ovine myoblasts, with their relative potencies at 0.1 nM reflecting their receptor binding affinities. Maximal effects were seen at 1-10 nM. EGF (10 nM) did not significantly inhibit the differentiation of clonally purified fetal ovine myoblasts, although there was increased proliferation of nondifferentiating cells. Hence a variety of EGF receptor ligands have the potential to influence the proliferation ovine muscle cell precursors in utero, but it is unlikely that they promote differentiation.

The effects of beta agonists on muscle cells in culture.

Increases in protein synthesis of 12% were found with two myogenic cell lines (L6 and G8-1) on treatment for 6 hr with the beta-adrenergic agonist cimaterol. In L6 cells, propranolol blocked the effect. Protein breakdown measured over 18-24 hr was unchanged. The Kd for cimaterol binding to the L6 beta-receptor was 26 nM which was compatible with its EC50 for the stimulation of protein synthesis (approx 5 nM). Evidence provided with muscle cell lines indicates a direct effect of cimaterol on protein synthesis, which may contribute to muscle accretion in cimaterol-fed animals.