Effects of phenethanolamines and propranolol on the proliferation of cultured chick breast muscle satellite cells

Satellite cells were isolated from 20-d embryonic chick breast muscle via a Percoll density gradient fractionation technique. Culturing of these cells gave rise to at least 89% fusion (myotube nuclei number/total nuclei number). Proliferation of cultured satellite cells (indicated by myotube nuclei number) was increased in a dose-dependent manner when fibroblast growth factor (FGF) was included in the medium (25 to 200 ng/ml). Similar cultures were used to examine the effects of ractopamine and isoproterenol on satellite cell proliferation. Ractopamine and isoproterenol were added to culture medium (10(-11) to 10(-4) M) 24 h after initial plating. After a 72-h treatment period, the treatments were removed and replaced with a medium to promote fusion for 48 h. Cells then were fixed and stained, and myotube and total nuclei were counted. In later experiments, ractopamine and isoproterenol each increased (P less than .01) myotube nuclei number vs that observed in control cultures by 2.3 and 2.1 times, respectively. Similar differences were observed with total nuclei number. The number of myotube nuclei observed in cultures treated with 10(-6) M ractopamine or isoproterenol was reduced (P less than .01) by 25.4 and 23.6%, respectively, when propranolol, a beta adrenergic antagonist, was included at 10(-5) M with the respective agonist. These results indicate that ractopamine and isoproterenol each enhance the proliferative activity of chick satellite cells in culture and that the beta adrenergic receptor mediates this proliferative effect.     

Insulin-like growth factor I receptor analysis of satellite cell-derived myotube membranes established from two lines of Targhee rams selected for growth rate

Ovine-derived fibroblasts were used to validate an insulin-like growth factor I (IGF-I) membrane-receptor binding assay system. Competitive binding using fibroblasts revealed that half-maximal inhibition of 125I-IGF-I binding by IGF-I was 2.3 nM. SDS-polyacrylamide gel electrophoresis analysis of specific protein-associated 125I-IGF-I was consistent with the migration of 125I-IGF-I-labeled Type I IGF receptor alpha-subunits at Mr 133,000 daltons. Further, the efficiency of two cell solubilization methods was examined and time-dependent binding equilibrium was determined for the membrane assay system. Satellite cell-derived myotubes were subsequently isolated from primary satellite cell cultures established from the semimembranosus muscles of high and low efficiency-of-gain (EOG) Targhee rams, and IGF-I receptor dynamics were measured. A membrane competitive binding study revealed that half-maximal inhibition of 125I-IGF-I binding was achieved by 1-ng IGF-I for low, and 10-ng IGF-I for high, EOG myotube membrane preparations. Kd values were similar between the high EOG (4.78 nM) and low EOG (2.95 nM) groups; however, receptor concentrations (Bmax) appeared to differ between groups. High EOG membrane receptor Bmax was 3.88 pmole/micrograms protein (19.87 pmole/micrograms DNA), whereas low EOG membrane receptor Bmax was 1.22 pmole/micrograms protein (9.28 pmole/micrograms DNA). These preliminary findings support the hypothesis that genetic selection for EOG results in altered satellite cell responsiveness to IGF-I.     

Zilpaterol hydrochloride alters abundance of β-adrenergic receptors in bovine muscle cells but has little effect on de novo fatty acid biosynthesis in bovine subcutaneous adipose tissue explants

We predicted that zilpaterol hydrochloride (ZH), a β-adrenergic receptor (AR) agonist, would depress mRNA and protein abundance of β-AR in bovine satellite cells. We also predicted that ZH would decrease total lipid synthesis in bovine adipose tissue. Bovine satellite cells isolated from the semimembranosus muscle were plated on tissue culture plates coated with reduced growth factor matrigel or collagen. Real-time quantitative PCR was used to measure specific gene expression after 48 h of ZH exposure in proliferating satellite cells and fused myoblasts. There was no effect of ZH dose on [(3)H]thymidine incorporation into DNA in proliferating myoblasts. Zilpaterol hydrochloride at 1 μM decreased (P < 0.05) β1-AR mRNA, and 0.01 and 1 μM ZH decreased (P < 0.05) β2-AR and β3-AR mRNA in myoblasts. The expression of IGF-I mRNA tended to increase (P = 0.07) with 1 μM ZH. There was no effect (P > 0.10) of ZH on the β-AR or IGF-I gene expression in fused myotube cultures at 192 h or on fusion percentage. The β2-AR antagonist ICI-118, 551 at 0.1 μM attenuated (P < 0.05) the effect of 0.1 μM ZH to reduce expression of β1- and β2-AR mRNA. The combination of 0.01 μM ZH and 0.1 μM ICI-118, 551 caused an increase (P < 0.05) in β1-AR gene expression. There was no effect (P > 0.10) of ICI-118, 551 or ZH on β3-AR or IGF-I. Western blot analysis revealed that the protein content of β2-AR in ZH-treated myotube cultures decreased (P < 0.05) relative to control. Total lipid synthesis from acetate was increased by ZH in bovine subcutaneous adipose tissue explants in the absence of theophylline but was decreased by ZH when theophylline was included in the incubation medium. These data indicate that ZH alters mRNA and protein concentrations of β-AR in satellite cell cultures, which in turn could affect responsiveness of cells to prolonged ZH exposure in vivo. Similar to other β-adrenergic agonists, ZH had only modest effects on lipid metabolism in adipose tissue explants.     

Isolation and culture of fetal porcine myogenic cells and the effect of insulin, IGF-I, and sera on protein turnover in porcine myotube cultures

Porcine myogenic cells isolated from 50 to 55-d porcine fetuses were frozen and stored in liquid nitrogen until they were needed to establish cultures. Approximately 75.8 +/- .59% of the clonal cultures established from these frozen stocks produced myotubes and 60.8 +/- 2.3% of the nuclei in differentiated mass cultures were in myotubes. Differentiated cultures contained higher levels of creatine phosphokinase activity than undifferentiated cultures. Additionally, differentiated cultures incorporated [35S]methionine into putative myosin heavy chain, alpha-actinin, and actin more rapidly than did undifferentiated cultures. Insulin, insulin-like growth factor I, and sera stimulated total protein synthesis rate and decreased total protein degradation rate in myotube cultures. Based on our initial characterization, we believe that we have developed an effective and practical procedure for isolating and culturing fetal porcine myogenic cells.     

Regulation of bovine satellite cell proliferation and differentiation by insulin and triiodothyronine

Satellite cells activity contributes to postnatal muscle growth. Herein, we have studied the respective influence of insulin and triiodothyronine (T3) on the proliferation and differentiation of primary bovine satellite cells isolated from Semitendinosus muscle of Montbéliard steers. Under basal conditions, satellite cells proliferated until the fifth day of culture, began to fuse into myotubes and expressed differentiation markers such as connectin, myogenin, and myosin heavy chain (MHC) isoforms. Insulin behaved as an effective mitogen. Moreover, it promoted extensive myotube formation and enhanced differentiation as shown by an increase in the accumulation of differentiation markers. Maximal differentiation occurred with insulin physiological range concentrations. A delay in the stimulation of differentiation was registered with a high dose that promoted maximal proliferation. Conversely, T3 decreased cell proliferation in a dose-dependent manner. In addition, fusion and biochemical differentiation (accumulation of connectin, MyoD1, myogenin, and myosin heavy chain isoforms) were also enhanced. Bovine satellite cells seemed to respond differentially to insulin and T3 for proliferation. Interestingly, both hormones displayed a myogenic influence. Our observations suggest that both hormones could influence bovine satellite cells in vivo and contribute to the regulation of postnatal muscle growth.     

Abundance of message for insulin-like growth factors-I and -II and for receptors for growth hormone,insulin-like growth factors-I and -II,and insulin in the intestine and liver of pre- and full-term calves

The somatotropic axis and insulin are involved in pre- and postnatal development. In pre- and full-term calves (GrP0 and GrN0; born after 277 and 290 d of pregnancy, respectively) and in preterm calves on d 8 of life after being fed for 7 d (GrP8), we studied whether there are differences in the abundance of messenger RNA (mRNA) of IGF-I and IGF-II and of receptors for GH, IGF-I, IGF-II, and insulin among different intestinal sites (duodenum, jejunum, ileum, and colon) and whether there are ontogenetic differences during the perinatal period in intestine and liver. Intestinal site differences (P < 0.05) existed in mRNA levels of IGF-I and IGF-II and receptors for GH, IGF-I, IGF-II, and insulin. Abundance of mRNA of IGF-I and -II and of receptors for IGF-I and GH was highest (P < 0.05) in the colon, abundance of the receptor for IGF-II was comparably high in the colon and ileum, and that of the receptor for insulin was similarly high in colon, ileum, and jejunum. Among GrP0, GrN0, and GrP8 groups, there were differences (P < 0.05) in mRNA levels of IGF-I and IGF-II and of receptors for GH, IGF-I, IGF-II and insulin. Abundance of mRNA of IGF-I and IGF-II and of receptors for GH, IGF-I, IGF-II and insulin was highest (P < 0.05) in GrP0 calves immediately after birth and was primarily seen in the ileum. In liver, the mRNA levels differed (P < 0.05) among groups for IGF-II and receptors for IGF-I, IGF-II, and insulin, and were highest (P < 0.05) for IGF-II in GrP0, for receptors of IGF-I in GrN0, and were higher (P < 0.05) in GrP0 than GrP8 for receptors of IGF-II. In conclusion, mRNA levels of IGF-I and IGF-II and of receptors for GH, IGF-I, IGF-II, and insulin were different at different intestinal sites and in intestine and liver and changed during the perinatal period.     

Interaction of insulin-like growth factors (IGF) with isolated sheep hepatocytes. II. Binding, internalization and degradation of IGF-II

This study examines the binding and degradation of IGF-II by the ovine liver. Binding and degradation of 125I-IGF-II to isolated hepatocytes was time, temperature and cell number dependent. Ovine and human IGF-II were 2-5 times more effective in inhibiting 125I-hIGF-II binding than were the IGF-I preparations. Insulin did not affect binding. Autoradiographs of 125I-hIGF-II affinity cross-linked to hepatocytes showed a major band of molecular weight 271,000 under reduced conditions. This band was eliminated by 100 nM hIGF-II or oIGF-II but not by excess hIGF-I, oIGF-I or insulin. The internalization of IGF-II was examined by treating the cells with trypsin or sodium acetate to remove surface-bound IGF-II. Both treatments showed that 20-25% of 125I-hIGF-II was internalized. Mannose-6-phosphate at 1, 2 and 4 mM enhanced the binding of 125I-hIGF-II to hepatocytes 3.5, 12.8 and 16.4%, respectively. The lysosomal inhibitors ammonium chloride, chloroquine and leupeptin had no effect on 125I-hIGF-II degradation or cell-associated radioactivity indicating a nonlysosomal pathway of degradation for 125I-hIGF-II in the ovine hepatocyte. The low molecular weight sheep serum IGF binding protein inhibited binding of 125I-hIGF-II in a dose-dependent manner but had no effect on degradation, which suggests that degradation of 125I-hIGF-II is independent of receptor interaction. These studies demonstrate that IGF-II binds to specific high affinity sites in sheep hepatocytes which display the characteristics of type II IGF receptors. A significant fraction of the receptor bound IGF-II is internalized but not degraded by these cells, which suggests that the biological actions of IGF-II may be exerted by an intracellular pathway in sheep hepatocytes.     

Characterization of type II insulin-like growth factor (IGF) receptors in sheep liver plasma membranes

Interactions of insulin-like growth factors (IGFs) from recombinant human and natural ovine sources with sheep liver plasma membranes have been studied. Total specific binding of 125I-hIGF-II (40%) to liver plasma membranes greatly exceeded that of 125I-hIGF-I (1.5%) after incubation at 20 C for 90 min. Binding of 125I-hIGF-II to the plasma membranes was dependent upon time, temperature and membrane concentration of the incubation. Binding of 125I-hIGF-II was only partially reversed by addition of 100 nM IGF-II (18%) or by dilution with excess buffer (36%). Competitive inhibition studies of 125I-hIGF-II binding demonstrated that IGF-II from ovine or recombinant human sources was more effective at inhibiting binding than ovine or human IGF-I. Insulin did not affect binding of 125I-hIGF-II. Plasma membranes were affinity cross-linked to 125I-IGF-II followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence and absence of the reducing agent dithiothreitol. Following autoradiography, radioactive bands were localized at 274,000 Mr and 210,000-215,000 Mr in the presence and absence of reducing agent, respectively. This pattern was unaffected by 100 nM human or ovine IGF-I or 1,000 nM insulin, but coincubation with 100 nM human or ovine IGF-II eliminated the radioactive band. These data indicate that an IGF-II specific receptor is present in sheep liver plasma membranes which has characteristics similar to those of nonruminant Type II receptors.     

Effects of growth factors on insulin-like growth factor binding protein (IGFBP) secretion by primary porcine satellite cell cultures.

Insulin-like growth factor-binding proteins (IGFBP) regulate the biological functions of insulin-like growth factors (IGF) and may affect cell growth through IGF-independent actions. Growth factors and hormones have been shown to alter IGFBP production by target cells suggesting that the effects of these factors may be partially mediated by the local production of IGFBP. Growth factors, including IGF-I, transforming growth factor-beta1 (TGF-beta1), and basic fibroblast growth factor (bFGF) have potent effects on satellite cell proliferation and differentiation, and some of these factors have been shown to alter IGFBP production in various cell types. Consequently, some of their actions on muscle satellite cells may be mediated by the local production of IGFBP. In this study, we measured the effects of IGF-I, bFGF, and TGF-beta1 on IGFBP production by primary porcine satellite cell (PSC) cultures after first determining physiologically active concentrations of these growth factors to use according to [3H]thymidine incorporation dose responses. There is little information on the effects of these growth factors on IGFBP production in primary porcine myogenic cells due to the confounding affects of contaminating nonmuscle fibroblasts. Comparative studies show that primary porcine satellite cells produce IGFBP-3 and -5 whereas porcine muscle-derived nonfusing cells (FIB) produce IGFBP-2 and -4 but not IGFBP-3 or -5. Because of this, our investigations have focused on growth factor-induced production of IGFBP-3 and -5 in primary porcine satellite cells cultures. Both IGF-I and bFGF exhibited dose-dependent increases in [3H]thymidine incorporation with increasing concentration from 1 to 50 ng/mL (P < 0.05), whereas TGF-beta1 caused a dose-dependent decrease from 0.01 to 0.5 ng/mL (P < 0.05). When 20 ng/ mL of IGF-I was added to the media, IGFBP-3 was increased approximately 65% (P < 0.05) and IGFBP-5 was increased approximately twofold (P < 0.05). The addition of 0.5 ng/mL TGF-beta1 caused more than a two-fold increase in IGFBP-3 (P < 0.05) and approximately an 80% increase in IGFBP-5 (P < 0.05), whereas 50 ng/ mL of bFGF caused approximately 40% (P < 0.05) and 70% (P < 0.05) increases in IGFBP-3 and -5, respectively. Neither IGFBP-3 nor -5 was detectable in the conditioned media from fibroblasts whether or not IGF-I, TGF- beta1 or bFGF were present. These data suggest that the effects of IGF-I, TGF- beta1 and bFGF on porcine satellite cells may in part be through the autocrine/ paracrine production of IGFBP-3 and -5 by porcine satellite cells.     

The effect of the beta-adrenergic agonist clenbuterol on growth and protein metabolism in rat muscle cell cultures.

Cultures were established from neonatal rat muscle cells, satellite cells, and L6 myoblasts and changes in protein metabolism were determined as development proceeded. For all three cell types, culture protein content increased with increasing myotube content. The beta-adrenergic agonist clenbuterol (added to a final concentration of 10(-7) M) significantly stimulated fusion (as indicated by creatine kinase activity) in neonatal muscle cultures and also increased culture protein content. This was associated with a stimulation in both the fractional (ks, percentage/day, +13%, P less than .05) and absolute (As, micrograms/day, +19%, P less than .05) rates of protein synthesis within 24 h after drug administration. At 48 h, As was increased by 42% above that of controls (P less than .01). In contrast, in satellite cell cultures, clenbuterol had no consistent effects on either protein accretion, creatine kinase activity, or protein synthesis (ks and As). Similarly, the drug had no stimulatory effect on protein synthesis and protein accretion in L6 myoblast or L6 myotube cultures (and no effect in neonatally derived fibroblast cultures). It is concluded that the fusion response to clenbuterol and, therefore, changes in protein metabolism and protein accretion are greatly dependent on the origin and genetic integrity of muscle cells.     

A comparison of thiazolidinedione-induced adipogenesis and myogenesis in stromal-vascular cells from subcutaneous adipose tissue or semitendinosus muscle of postnatal pigs

This study compared the adipogenic potential of porcine stromal-vascular (S-V) cells from semitendinosus muscles and s.c. adipose tissue using thiazolidinediones. Stromal-vascular cells were obtained from s.c. adipose tissue and both semitendinosus muscles from 5- to 7-d-old pigs after collagenase digestion. Preadipocyte recruitment was measured using immunohistological evaluation for AD-3, a preadipocyte antibody. Ciglitazone increased the number of preadipocytes in adipose tissue but not semitendinosus muscle S-V cell cultures, whereas 10 microM troglitazone increased preadipocyte abundance in both adipose and muscle S-V cultures by approximately 3-fold (P < 0.05). Increasing troglitazone doses did not further increase preadipocyte number. Increases in preadipocytes were paralleled by increases in CCAAT/enhancer-binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma) positive cells in adipose tissue S-V cultures, whereas PPARgamma-reactive but not C/EBPalpha-reactive cells were increased in muscle S-V cultures treated with 10 microM troglitazone. Additionally, troglitazone treatment did not increase lipid content in s.c. adipose tissue or muscle S-V cell cultures. Cells plated on laminin-precoated culture dishes were used to determine whether troglitazone influenced adipogenesis or myogenesis in cocultures from muscle S-V cells. There was no effect on the number of myotubes or the average number of nuclei per myotube, suggesting myogenesis was not impaired by troglitazone treatment. These results suggest that regulation of intramuscular adipogenesis differs from that of subcutaneous adipogenesis.     

牛骨骼肌卫星细胞的分离培养及诱导分化方法的建立

为了给牛骨骼肌卫星细胞的分离培养及诱导分化方法及进一步揭示肌肉分化的机理及转基因肉牛的研究提供重要帮助,试验以新生胎牛的骨骼肌为试验材料,分别采用胶原酶Ⅰ和胶原酶Ⅺ与胰蛋白酶结合对其进行消化,并通过差速贴壁法对骨骼肌卫星细胞进行分离纯化,同时采用免疫荧光染色、RT-PCR、Western-blot法对骨骼肌卫星细胞进行鉴定。结果表明:研究成功获得了大量的牛骨骼肌卫星细胞;该细胞的标志性分子的mRNA及其蛋白表达的纯度在98%以上;细胞生长状态良好,可稳定传至90代并保持旺盛的增殖活力;细胞分化效率高,2%的马血清能够诱导细胞分化使其融合形成多核肌管,数量众多的肌管可自发融合为更粗的肌管,并可观察到其具有收缩现象。     

Protein metabolism in chicken muscle cell cultures treated with cimaterol

Primary muscle cell cultures were prepared from the leg muscle of 12-d broiler chicken embryos. The partitioning agent cimaterol (10(-6) to 10(-10) M) was added on d 1 and each day thereafter, and cells were studied after 7 d in culture. Cimaterol had no effect at any level either on the percentage of nuclei within multinucleated myotubes or on the total number of nuclei within myotubes. At 10(-7) M cimaterol, the quantity of the myofibrillar protein fraction was increased by 25.1 +/- 8.0% (P less than .05) and the quantity of myosin heavy chain was increased by 30.9 +/- 4.5% (P less than .05). To understand the basis for the increase in myofibrillar protein, the incorporation rate of [3H]Leu was measured in pulse labeling experiments. The apparent synthesis rate of the soluble protein fraction and the crude myofibrillar fraction was not significantly increased by cimaterol; however, cimaterol levels greater than 10(-8) M caused a 10 to 12% increase (P less than .05) in the incorporation rate of [3H]Leu into myosin heavy chain. The effect of cimaterol on release of [3H]Leu from prelabeled protein also was assessed in pulse-chase experiments; the apparent rate of protein degradation was inhibited by 10 to 15% (P less than .05) at the higher levels of cimaterol. Dot blot analysis indicated that the quantity of myosin heavy chain mRNA was elevated in cimaterol-treated cultures. Thus, the increased quantity of myofibrillar proteins in embryonic broiler muscle cell cultures is the combined result of a stimulation in the rate of protein synthesis and an inhibition in the rate of protein degradation.     

Growth factor regulation of bovine satellite cell growth in vitro

This study examined the effects of basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF-I) and transforming growth factor beta (TGF-beta) on the proliferation and differentiation of primary bovine satellite cells (BSC) in vitro. Individually, these three factors had the following effects on satellite cells: bFGF stimulated proliferation (P less than .01) but inhibited differentiation (P less than .05); IGF-I had no effect on proliferation but stimulated differentiation (P less than .01); and TGF-beta inhibited both proliferation and differentiation (P less than .01). When combined, the following effects were observed: maximum stimulation of proliferation (P less than .01) occurred in the presence of bFGF and IGF-I and differentiation was not stimulated; TGF-beta and bFGF continued to inhibit differentiation (P less than .01), but in the presence of bFGF, TGF-beta stimulated proliferation (P less than .01). No stimulation was observed in the presence of TGF-beta and IGF-I. Bovine satellite cells respond to these three growth factors that have been shown to regulate the activity of other myogenic cells, and in most instances, the responses among cells from various species are similar. These experiments indicate that the interactions of growth factors may be critical in regulating bovine satellite cell activity.     

Roles of IGF-I and the estrogen, androgen and IGF-I receptors in estradiol-17beta- and trenbolone acetate-stimulated proliferation of cultured bovine satellite cells

Although numerous studies have shown that both androgenic and estrogenic steroids increase rate and efficiency of muscle growth in steers, there is little consensus as to their mechanism of action. A combined estradiol 17beta (E2)/trenbolone acetate (TBA) implant causes a significant increase in muscle IGF-I mRNA and both E2 and TBA stimulate a significant increase in IGF-I mRNA level in bovine satellite cell (BSC) cultures in media containing 10% fetal bovine serum (FBS). Consequently, increased IGF-I expression may play a role in anabolic-steroid-enhanced muscle growth. However, even though treatment of cultured BSC with E2 or TBA in media containing 1% IGFBP-3-free swine serum (SS) results in increased proliferation there is no effect on IGF-I mRNA expression, suggesting that increased IGF-I expression may not be responsible for anabolic-steroid-enhanced BSC proliferation. To further examine the role of estrogen, androgen and IGF-I receptors and their respective ligands in E2- and TBA-stimulated BSC proliferation, we assessed the effects of specific inhibitors on E2- or TBA-stimulated proliferation of BSC. Both ICI 182 780 (an estrogen receptor blocker) and flutamide (an inhibitor of androgen receptor) suppressed (p<0.05) E2- and TBA-stimulated BSC proliferation, respectively. JB1 (a competitive inhibitor of IGF-I binding to type I IGF receptor) reduced (p<0.05) both E2- and TBA-stimulated proliferation in BSC cultures. Both the Raf-1/MAPK kinase (MEK)1/2/ERK1/2, and the phosphatidylinositol 3-kinase (PI3K)/Akt pathways play significant roles in the actions of IGF-I on proliferation and differentiation of myogenic cells. PD98059, an inhibitor of the MAPK pathway, and wortmannin, an inhibitor of the PI3K pathway, both suppressed (p<0.05) E2- and TBA-stimulated proliferation of cultured BSC. Our data suggest that IGF-I plays a role in E2- and TBA-stimulated proliferation of cultured BSC even in the absence of increased IGF-I expression.     

Proliferation of myogenic cells in the diaphragm of the fetal rabbit.

Diaphragms obtained from 40 fetal rabbits at gestational ages of 20, 22, 25 and 30 days were examined by light and transmission electron microscopes. The percentage of myogenic cells undergoing mitosis was calculated by counting mitotic nuclei in 1 micron sections. At 20 and 22 days gestation, myogenic cells (myoblasts and satellite cells) were actively proliferating with 2.66 +/- 0.41% (n = 15) and 2.18 +/- 0.20% (n = 23), of the cells in division, respectively. The myogenic cells undergoing mitosis appeared to be of both myoblasts and satellite cells at these stages of the development. The myotubes on day 20 of gestation contained a large number of lipid droplets and an abundance of glycogen particles which were reduced by day 22 of gestation. The mitotic rate on day 25 of gestation was reduced to 1.16 +/- 0.11% (n = 28). The muscle fibers were well differentiated at this stage and the majority of the dividing cells were considered to be satellite cells. In diaphragms from the full term fetuses (day 30), the mitotic rate was reduced to 0.31 +/- 0.05% (n = 24). It was suggested that myoblasts of the fetal rabbit diaphragm proliferated for the myotube formation during the earliest stage of the development (day 20 of gestation) and then the number of satellite cells increased after day 22 of gestation. Growth of the organ after day 25 of gestation appeared to be mostly due primarily to the hypertrophy and differentiation of the muscle fibers rather than proliferation of myoblasts.     

Effects of the beta-adrenergic agonist isoproterenol on protein accretion, synthesis, and degradation in primary chicken muscle cell cultures

Seven-day-old primary myotube cultures derived from embryonic chicken limb muscles were used to determine the effects of the beta-adrenergic agonist isoproterenol (ISO) on muscle protein metabolism in vitro. Isoproterenol increased (P less than .05) total protein accumulation after 2 h of acute exposure and after chronic exposure for 24 and 48 h. Isoproterenol did not consistently retard rate of protein degradation of the total protein (TP), myofibrillar protein (MFP) pools, and myosin heavy-chain subunit (MHC); degradation of these protein pools tended to be slowed by inclusion of ISO in the culture medium. After acute treatment of 1 X 10(-4) M ISO for 2 h, TP, but not MFP and MHC, synthesis rate was increased, and after chronic exposure to 1 X 10(-4), 1 X 10(-5), and 1 X 10(-6) M ISO, TP, MFP, and MHC synthesis rates and net accumulation of TP, cytoplasmic protein, and MHC fractions were enhanced (P less than .05). The beta-adrenergic antagonist propranolol (1 X 10(-5) M) blocked chronic stimulatory effects of ISO. Furthermore, after 48 h of exposure to ISO, effects on protein synthesis were less pronounced than those observed after 24 h of exposure. Isoproterenol imparted a more pronounced effect on protein synthesis than on protein degradation, indicating that increased muscle protein accretion observed in animals after ISO treatment is likely a function of enhanced protein synthesis.     

Basic principles of muscle development and growth in meat-producing mammals as affected by the insulin-like growth factor (IGF) system

This presentation aims to describe how the basic events in prenatal muscle development and postnatal muscle growth are controlled by the insulin-like growth factor system (IGF). The prenatal events (myogenesis) cover the rate of proliferation, the rate and extent of fusion, and the differentiation of three myoblast populations, giving rise to primary fibers, secondary fibers, and a satellite cell population, respectively. The number of muscle fibers, a key determinant of the postnatal growth rate, is fixed late in gestation. The postnatal events contributing to myofiber hypertrophy comprise satellite cell proliferation and differentiation, and protein turnover. Muscle cell cultures produce IGFs and IGF binding proteins (IGFBPs) in various degrees depending on the origin (species, muscle type) and state of development of these cells, suggesting an autocrine/paracrine mode of action of IGF-related factors. In vivo studies and results based on cell lines or primary cell cultures show that IGF-I and IGF-II stimulate both proliferation and differentiation of myoblasts and satellite cells in a time and concentration-dependent way, via interaction with type I IGF receptors. However, IGF binding proteins (IGFBP) may either inhibit or potentiate the stimulating effects of IGFs on proliferation or differentiation. During postnatal growth in vivo or in fully differentiated muscle cells in culture, IGF-I stimulates the rate of protein synthesis and inhibits the rate of protein degradation, thereby enhancing myofiber hypertrophy. The possible roles and actions of the IGF system in regulating and determining muscle growth as affected by developmental stage and age, muscle type, feeding levels, treatment with growth hormone and selection for growth performance are discussed.     

The effect of insulin on primary cultures of rat preadipocytes grown in fetal or postnatal pig serum

Stromal vascular cell cultures, prepared from the inguinal pads of 50-g Sprague Dawley rats, were exposed to media with 10% fetal pig serum which is inherently low in insulin, for the first 3 to 5 d of culture. Insulin was supplemented to media for periods of 2 to 6 d. In cultures treated (2 to 4 d) with 10(-9), to 10(-10) or 10(-11) M insulin, differentiated cells (lipid and esterase staining) appeared 1.5 to 2 times wider than differentiated cells in control cultures. At 10(-9) M insulin (4 to 5 d), in cultures grown in the presence of fetal pig serum the number of esterase reactive cells was increased twofold to threefold. The percentage of total cells that were esterase reactive was elevated 50 to 300% relative to control cultures. Insulin-treated preadipocytes were more reactive for lipoprotein lipase activity (histochemical assay) compared with reactivity of control cells. Quantitative analysis of percentage of light transmittance (Zeiss photometer) through stained cells indicated an increase (P less than .001) in lipoprotein lipase staining at 10(-9), 10(-11) and 10(-13) M insulin (2 d). The specific activity of glycerol phosphate dehydrogenase was elevated twofold to threefold (P less than .05) and soluble protein elevated 50 to 100% (P less than .05) in cultures treated (3 to 6 d) with 10(-9) M insulin. Decreasing the cell plating density (50%) in cultures grown in the presence of pig serum reduced the elevation in enzyme activity induced by insulin in preadipocyte cultures. Physiological levels of insulin enhanced lipogenic enzyme activity in preadipocytes and may enhance the conversion of stromal cells to preadipocytes.