Search

In feedlot steers, estradiol-17β (E2) and combined E2 and trenbolone acetate (a testosterone analog) implants enhance rate and efficiency of muscle growth; and, consequently, these compounds are widely used as growth promoters in several countries. Treatment with E2 stimulates protein synthesis rate and suppresses protein degradation rate in fused bovine satellite cell (BSC) cultures; however, the mechanisms involved in these effects are not known with certainty. Although the genomic effects of E2 mediated through the classical estrogen receptors have been characterized, recent studies indicate that binding of E2 to the G protein-coupled estrogen receptor (GPER)-1 mediates nongenomic effects of E2 on cellular function. Our current data show that inhibition of GPER-1, matrix metalloproteinases 2 and 9 (MMP2/9), or heparin binding epidermal growth factor-like growth factor (hbEGF) suppresses E2 stimulate protein synthesis rate in cultured BSCs (P < 0.001) suggesting that all of these are required in order for E2 to stimulate protein synthesis in these cultures. In contrast, inhibition of GPER-1, MMP2/9, or hbEGF has no effect on the ability of E2 to suppress protein degradation rates in fused BSC cultures indicating that these factors are not required in order for E2 to suppress protein degradation rate in these cells. Furthermore, treatment of fused BSC cultures with E2 increased (P < 0.05) pAKT levels indicating that the pAKT pathway may play a role in E2-stimulated effects on cultured BSC. In summary, our current data show that active GPER-1, MMP2/9, and hbEGF are necessary for E2-stimulated protein synthesis but not for E2-simulated suppression of protein degradation in cultured BSC. In addition, E2 treatment increases pAKT levels in cultured BSC., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Trenbolone acetate (TBA), a testosterone analog, increases protein synthesis and decreases protein degradation in fused bovine satellite cell (BSC) cultures. However, the mechanism through which TBA alters these processes remains unknown. Recent studies indicate that androgens improve rate and extent of muscle growth through a nongenomic mechanism involving G protein-coupled receptors (GPCR), matrix metalloproteinases (MMP), heparin-binding epidermal growth factor (hbEGF), the epidermal growth factor receptor (EGFR), erbB2, and the insulin-like growth factor-1 receptor (IGF-1R). We hypothesized that TBA activates GPCR, resulting in activation of MMP2/9 that releases hbEGF, which activates the EGFR and/or erbB2. To determine whether the proposed nongenomic pathway is involved in TBA-mediated alterations in protein turnover, fused BSC cultures were treated with TBA in the presence or absence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R, and resultant protein synthesis and degradation rates were analyzed. Assays were replicated at least 9 times for each inhibitor experiment utilizing BSC cultures obtained from at least 3 different steers that had no previous exposure to steroid compounds. As expected, fused BSC cultures treated with 10 n TBA exhibited increased ( < 0.05) protein synthesis rates and decreased ( < 0.05) protein degradation rates when compared to control cultures. Treatment of fused BSC cultures with 10 n TBA in the presence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R suppressed ( < 0.05) TBA-mediated increases in protein synthesis rate. Alternatively, inhibition of GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R in the presence of 10 n TBA each had no ( > 0.05) effect on TBA-mediated decreases in protein degradation. However, inhibition of both EGFR and erbB2 in the presence of 10 n TBA resulted in decreased ( < 0.05) ability of TBA to decrease protein degradation rate. Additionally, fused BSC cultures treated with 10 n TBA exhibit increased ( < 0.05) pAKT protein levels. These data indicate the TBA-mediated increases in protein synthesis likely involve GPCR, MMP2/9, hbEGF, EGFR, erbB2, and IGF-1R. However, the mechanism through which TBA mediates changes in protein degradation is different and appears to involve only the EGFR and erbB2. Furthermore, it appears the protein kinase B pathway is involved in TBA's effects on fused BSC cultures.

Implanting cattle with steroids significantly enhances feed efficiency, rate of gain, and muscle growth. However, the mechanisms responsible for these improvements in muscle growth have not been fully elucidated. Trenbolone acetate (TBA), a testosterone analog, has been shown to increase proliferation rate in bovine satellite cell (BSC) cultures. The classical genomic actions of testosterone have been well characterized; however, our results indicate that TBA may also initiate a quicker, nongenomic response that involves activation of G protein-coupled receptors (GPCR) resulting in activation of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) that release membrane-bound heparin-binding epidermal growth factor-like growth factor (hbEGF), which then binds to and activates the epidermal growth factor receptor (EGFR) and/or erbB2. Furthermore, the EGFR has been shown to regulate expression of the IGF-1 receptor (IGF-1R), which is well known for its role in modulating muscle growth. To determine whether this nongenomic pathway is potentially involved in TBA-stimulated BSC proliferation, we analyzed the effects of treating BSC with guanosine 5'-O-2-thiodiphosphate (GDPβS), an inhibitor of all GPCR; a MMP2 and MMP9 inhibitor (MMPI); CRM19, a specific inhibitor of hbEGF; AG1478, a specific EGFR tyrosine kinase inhibitor; AG879, a specific erbB2 kinase inhibitor; and AG1024, an IGF-1R tyrosine kinase inhibitor on TBA-stimulated proliferation rate (H-thymidine incorporation). Assays were replicated at least 9 times for each inhibitor experiment using BSC cultures obtained from at least 3 different animals. Bovine satellite cell cultures were obtained from yearling steers that had no previous exposure to androgenic or estrogenic compounds. As expected, BSC cultures treated with 10 n TBA showed ( < 0.05) increased proliferation rate when compared with control cultures. Additionally, treatment with 5 ng hbEGF/mL stimulated proliferation in BSC cultures ( < 0.05). Treatment with GDPβS, MMPI, CRM197, AG1024, AG1478, and/or AG879 all suppressed ( < 0.05) TBA-induced increases in proliferation. These data indicate that TBA likely initiates a nongenomic response involving GPCR, MMP2 and MMP9, hbEGF, EGFR, erbB2, and IGF-1R, which may play a role in TBA-mediated increases in BSC proliferation.

In feedlot steers, estradiol-17β (E2) and combined E2 and trenbolone acetate (a testosterone analog) implants enhance rate and efficiency of muscle growth; and, consequently, these compounds are widely used as growth promoters. Although the positive effects of E2 on rate and efficiency of bovine muscle growth are well established, the mechanisms involved in these effects are not well understood. Combined E2 and trenbolone acetate implants result in significantly increased muscle satellite cell number in feedlot steers. Additionally, E2 treatment stimulates proliferation of cultured bovine satellite cells (BSC). Studies in nonmuscle cells have shown that binding of E2 to G protein-coupled estrogen receptor (GPER)-1 results in activation of matrix metalloproteinases 2 and 9 (MMP2/9) resulting in proteolytic release of heparin binding epidermal growth factor-like growth factor (hbEGF) from the cell surface. Released hbEGF binds to and activates the epidermal growth factor receptor resulting in increased proliferation. To assess if GPER-1, MMP2/9, and/or hbEGF are involved in the mechanism of E2-stimulated BSC proliferation, we have examined the effects of G36 (a specific inhibitor of GPER-1), CRM197 (a specific inhibitor of hbEGF), and MMP-2/MMP-9 Inhibitor II (an inhibitor of MMP2/9 activity) on E2-stimulated BSC proliferation. Inhibition of GPER-1, MMP2/9, or hbEGF suppresses E2-stimulated BSC proliferation (P < 0.001) suggesting that all these are required in order for E2 to stimulate BSC proliferation. These results strongly suggest that E2 may stimulate BSC proliferation by binding to GPER-1 resulting in MMP2/9-catalyzed release of cell membrane-bound hbEGF and subsequent activation of epidermal growth factor receptor by binding of released hbEGF., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The objective of this study was to assess the role of the epidermal growth factor receptor (EGFR) in estradiol-17β (E2)-stimulated proliferation of cultured bovine satellite cells (BSCs). Treatment of BSC cultures with AG1478 (a specific inhibitor of EGFR tyrosine kinase activity) suppresses E2-stimulated BSC proliferation (P < 0.05). In addition, E2-stimulated proliferation is completely suppressed (P < 0.05) in BSCs in which EGFR expression is silenced by treatment with EGFR small interfering RNA (siRNA). These results indicate that EGFR is required for E2 to stimulate proliferation in BSC cultures. Both AG1478 treatment and EGFR silencing also suppress proliferation stimulated by LR3-IGF-1 (an IGF1 analogue that binds normally to the insulin-like growth factor receptor (IGFR)-1 but has little or no affinity for IGF binding proteins) in cultured BSCs (P < 0.05). Even though EGFR siRNA treatment has no effect on IGFR-1β mRNA expression in cultured BSCs, IGFR-1β protein level is substantially reduced in BSCs treated with EGFR siRNA. These data suggest that EGFR silencing results in post-transcriptional modifications that result in decreased IGFR-1β protein levels. Although it is clear that functional EGFR is necessary for E2-stimulated proliferation of BSCs, the role of EGFR is not clear. Transactivation of EGFR may directly stimulate proliferation, or EGFR may function to maintain the level of IGFR-1β which is necessary for E2-stimulated proliferation. It also is possible that the role of EGFR in E2-stimulated BSC proliferation may involve both of these mechanisms., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Both androgenic and estrogenic steroids are widely used as growth promoters in feedlot steers because they significantly enhance feed efficiency, rate of gain, and muscle growth. However, despite their widespread use relatively little is known about the biological mechanism by which androgenic and estrogenic steroids enhance rate and efficiency of muscle growth in cattle. Treatment of feedlot steers with a combined estradiol (E2) and trenbolone acetate (TBA) implant results in an increased number of muscle satellite cells, increased expression of IGF-1 mRNA in muscle tissue, and increased levels of circulating IGF-1. Similarly, treatment of bovine satellite cell (BSC) cultures with either TBA or E2 results in increased expression of IGF-1 mRNA, increased rates of proliferation and protein synthesis, and decreased rates of protein degradation. Effects of E2 on BSC are mediated at least in part through the classical E2 receptor, estrogen receptor-α (ESR1), the IGF-1 receptor (IGFR1), and the G protein-coupled estrogen receptor-1 (GPER-1), formerly known as G protein-coupled receptor-30 (GPR30). The effects of TBA appear to be primarily mediated through the androgen receptor. Based on current research results, it is becoming clear that anabolic steroid-enhanced bovine muscle growth involves a complex interaction of numerous pathways and receptors. Consequently, additional in vivo and in vitro studies are necessary to understand the mechanisms involved in this complex process. The fundamental information generated by this research will help in developing future, safe, and effective strategies to increase rate and efficiency of muscle growth in beef cattle.

Although the exact mechanism(s) by which estradiol (E(2)) enhances muscle growth in a number of species, including humans and cattle, is not known, E(2) treatment has been shown to stimulate proliferation of cultured bovine satellite cells (BSCs). This is particularly significant because satellite cells are the source of nuclei needed to support postnatal muscle fiber hypertrophy and are thus crucial in determining the rate and extent of muscle growth. The objective of this study was to assess the role of estrogen receptor-α (ESR1) and the type 1 insulin-like growth factor receptor (IGFR1) in E(2)-stimulated proliferation of cultured BSCs. To accomplish this, we have used small interfering RNA (siRNA) to silence expression of ESR1 or IGFR1 and assessed the effects on E(2)-stimulated proliferation in BSC cultures. In BSCs treated with nonspecific siRNA, E(2) significantly (P < 0.05) stimulates proliferation under conditions in which neither IGF-1 nor IGF-2 expression is increased; however, treatment of ESR1- or IGFR1-silenced cells with E(2) does not significantly stimulate proliferation. These results indicate that both ESR1 and IGFR1 are required for E(2) to stimulate proliferation in BSC cultures. The fact that this occurs under culture conditions in which neither IGF-1 nor IGF-2 mRNA expression is increased strongly suggests that E(2) activates IGFR1 via a mechanism that does not involve increased IGF-1 or IGF-2 binding to the receptor., (Copyright © 2013 Elsevier Inc. All rights reserved.)

It is well established that heat stress (HS) negatively affects growth rate in swine. Although reduced feed intake undoubtedly plays a significant role in this reduction, studies in laboratory animals and other nonswine species indicate muscle growth also is affected by HS-related alterations in muscle physiology. Evidence is now emerging that heat shock proteins (Hsp), produced in response to HS and other types of cellular stress, may play an important role in regulating the rate and efficiency of muscle growth. Because muscle satellite cells play a crucial role in postnatal muscle growth, the effects of HS on rates of satellite cell proliferation, protein synthesis, and protein degradation play an important role in determining the rate and extent of muscle growth. Consequently, in the current study we have examined the effects of mild HS (40.5°C for 48 h) on the rates of proliferation, protein synthesis, and protein degradation and on quantities of Hsp90, Hsp70, and Hsp25/27 mRNA and protein in cultured porcine muscle satellite cells (PSC). Mild HS of PSC cultures resulted in 2.5-, 1.4-, and 6.5-fold increases (P < 0.05) in the abundance of Hsp90, Hsp70, and Hsp25/27 mRNA, respectively, relative to control cultures. Abundance of Hsp 90, 70, and 25/27 proteins was also increased in HS PSC cultures compared with those in control cultures. Proliferation rates in HS PSC cultures were 35% less (P < 0.05) than those in control cultures. Protein synthesis rates in HS-fused PSC cultures were 85% greater (P < 0.05) than those in control cultures, and protein degradation rates in HS-fused PSC were 23% less (P < 0.05) than those in control cultures. In light of the crucial role satellite cells play in postnatal muscle growth, the HS-induced changes we have observed in rates of proliferation, protein turnover, and abundance of Hsp mRNA and Hsp protein in PSC cultures indicate that mild HS affects the physiology of PSC in ways that could affect muscle growth in swine.

Insulin-like growth factor binding protein-3 (IGFBP-3) suppresses proliferation of numerous cell types, including myogenic cells, via both insulin-like growth factor (IGF)-dependent and IGF-independent mechanisms; however, the mechanism of IGF-independent suppression of proliferation is not clearly defined. In nonmuscle cells, binding of IGFBP-3 to the low-density lipoprotein receptor-related protein-1 (LRP-1)/activated α(2)M receptor is reportedly required for IGFBP-3 to inhibit proliferation. These findings suggest that binding to this receptor also may be required for IGFBP-3 to suppress proliferation of cultured myogenic cells. To investigate the role of the LRP-1 receptor in suppression of myogenic cell proliferation by IGFBP-3, we have examined the effect of receptor-associated protein, an LRP-1 receptor antagonist, on recombinant porcine (rp)IGFBP-3 inhibition of L6 myogenic cell proliferation. Treatment with receptor-associated protein results in a 37% decrease (P < 0.05) in the ability of rpIGFBP-3 to inhibit L6-cell proliferation. In L6 cells subjected to LRP-1 small interfering RNA treatment for 48 h (LRP-1 silenced), LRP-1 mRNA levels were reduced by greater than 80% compared with control cultures treated with nonsense small interfering RNA (mock silenced). In addition, the 85-kDa transmembrane subunit of LRP-1 was undetectable in Western immunoblots of total protein lysates from LRP-1-silenced cells. Even though LRP-1 mRNA and protein levels were dramatically reduced in LRP-1-silenced L6 cells compared with mock-silenced controls, rpIGFPB-3 suppressed proliferation rate to the same extent in both LRP-1-silenced and mock-silenced cultures. Our results strongly suggest that, in contrast to data obtained for nonmuscle cell lines, the LRP-1 receptor is not required for IGFBP-3 to suppress proliferation of L6 myogenic cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Although androgenic and estrogenic steroids are widely used to enhance muscle growth and increase feed efficiency in feedlot cattle, their mechanism of action is not well understood. Although in vivo studies have indicated that androgens affect protein synthesis and protein degradation rate in muscle, results from in vitro studies have been inconsistent. We have examined the effects of trenbolone acetate (TBA), a synthetic androgen, on protein synthesis and degradation rates in fused bovine satellite cell (BSC) cultures. Additionally, we have examined the effects of compounds that interfere with binding of TBA or insulin-like growth factor-1 (IGF-1) to their respective receptors on TBA-induced alterations in protein synthesis and degradation rates in BSC cultures. Treatment of fused BSC cultures with TBA results in a concentration-dependent increase (P < 0.05) in protein synthesis rate and a decrease (P < 0.05) in degradation rate, establishing that TBA directly affects these parameters. Flutamide, a compound that prevents androgen binding to the androgen receptor, suppresses (P < 0.05) TBA-induced alterations in protein synthesis and degradation in fused BSC cultures, indicating the androgen receptor is involved. JB1, a competitive inhibitor of IGF-1 binding to the type 1 IGF receptor (IGF1R), suppresses (P < 0.05) TBA-induced alterations in protein synthesis and degradation, indicating that this receptor also is involved in the actions of TBA on both synthesis and degradation. In summary, our data show that TBA acts directly to alter both protein synthesis and degradation rates in fused BSC cultures via mechanisms involving both the androgen receptor and IGF1R., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Although androgenic and estrogenic steroids are widely used to enhance muscle growth and increase feed efficiency in feedlot cattle, their mechanism of action is not well understood. Further, in vivo studies indicate that estradiol (E2) affects muscle protein synthesis and/or degradation, but in vitro results are inconsistent. We have examined the effects of E2 treatment on protein synthesis and degradation rates in fused bovine satellite cell (BSC) cultures. Additionally, to learn more about the mechanisms involved in E2-enhanced muscle growth, we have examined the effects of compounds that interfere with binding of E2 or insulin-like growth factor (IGF)-1 to their respective receptors on E2-induced alterations in protein synthesis and degradation rates in BSC cultures. Treatment of fused BSC cultures with E2 results in a concentration-dependent increase (P < 0.05) in protein synthesis rate and a decrease (P < 0.05) in protein degradation rate. The pure estrogen antagonist ICI 182 780 suppresses (P < 0.05) E2-induced alterations in protein synthesis and degradation in fused BSC cultures. The G-protein coupled receptor (GPR)-30 agonist G1 does not affect either synthesis or degradation rate, which establishes that GPR30 does not play a role in E2-induced alterations in protein synthesis or degradation. JB1, a competitive inhibitor of IGF-1 binding to the Type 1 insulin-like growth factor receptor (IGFR-1), suppresses (P < 0.05) E2-induced alterations in protein synthesis and degradation. In summary, our data show that E2 treatment directly alters both protein synthesis and degradation rates in fused BSC cultures via mechanisms involving both the classical estrogen receptor (ER) and IGFR-1.

We previously showed that a combined trenbolone acetate (TBA)/estradiol-17beta (E2) implant significantly increases IGF-I mRNA levels in the LM of feedlot steers by 28 d after implantation. Here we compare the effects of E2 (25.7 mg), TBA (120 mg), and combined TBA (120 mg)/E2 (24 mg) implants on IGF-I, IGF-I receptor (IGFR-1), estrogen receptor (ER)-alpha and androgen receptor (AR) mRNA levels in the LM of steers. Twenty yearling crossbred steers with an average initial BW of 421.1 +/- 3.6 kg were stratified by BW and randomly assigned to 1 of 4 treatments: 1) nonimplanted, control; 2) implanted with TBA and E2; 3) implanted with E2; or 4) implanted with TBA. Steers were weighed weekly starting on d 0, and muscle biopsy samples were taken from each steer on d 0 (before implantation), 7, 14, and 28. Ribonucleic acid was prepared from each sample and real-time reverse transcription-PCR was used to determine the levels of IGF-I, IGFR-1, ER-alpha, and AR mRNA. Body weight of implanted steers, adjusted by using d-0 BW as a covariant, tended (P = 0.09) to be greater than that of control steers. On d 7 and 28, IGF-I mRNA levels were greater (58 and 78%, respectively; P < 0.009) in E2-implanted animals than in control steers. Similarly, on d 28 the LM IGF-I mRNA level was 65% greater (P = 0.017) in TBA/E2-implanted steers than in control animals. In contrast, the TBA implant did not increase (P = 0.99) LM IGF-I mRNA levels after 28 d of implantation. Muscle IGFR-1, AR, and ER-alpha mRNA levels were not different (P > 0.47) in any of the treated groups compared with the control group. These data suggest that E2 is responsible for the increased muscle IGF-I mRNA level observed in steers implanted with a combined TBA/E2 implant.

Androgenic and estrogenic steroids enhance muscle growth in animals and humans. Estradiol-17beta (E2) and trenbolone acetate (TBA) (a synthetic testosterone analog) increased IGF-I mRNA expression in bovine muscle satellite cell (BSC) cultures. The goal of this study was to evaluate the mechanisms responsible for this increase by evaluating the effects of ICI 182 780 (an E2 receptor antagonist), flutamide (an androgen receptor inhibitor), G1 (a GPR30 agonist), and BSA-conjugated E2 on E2 and/or TBA-stimulated IGF-I mRNA expression in BSC cultures. Flutamide completely suppressed TBA-stimulated IGF-I mRNA expression in BSC cultures. ICI 182 780 did not suppress E2-stimulated IGF-I mRNA expression and 100 nM ICI 182 780 enhanced (93%, p<0.05) IGF-I mRNA levels in BSC cultures. G1 (100 nM) stimulated IGF-I mRNA expression (100%, p<0.05) but had no effect on proliferation in BSC cultures. E2-BSA, which cannot cross the cell membrane, stimulated IGF-I mRNA expression (approximately 100%, p<0.05) in BSC but even at extremely high concentrations had no effect on proliferation. In summary, our data indicate the E2-stimulation of proliferation and E2-stimulation of IGF-I mRNA expression in BSC cultures occur via different mechanisms. Our previous results showing that ICI 182 780 inhibited BSC proliferation and results of the current study showing lack of response to E2-BSA or G1 suggest that E2-stimulated proliferation in BSC cultures is mediated through classical estrogen receptors. Stimulation by ICI 182 780, G1 and E2-BSA suggests the E2-stimulated IGF-I mRNA expression in BSC cultures is mediated through the GPR30 receptor.

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.

Although in vivo and in vitro studies have established that anabolic steroids, transforming growth factor-beta (TGF-beta), and myostatin affect muscle growth in meat-producing animals, their mechanisms of action are not completely understood. Anabolic steroids have been widely used as growth promoters in feedlot cattle for over 50 yr. A growing body of evidence suggests that increased muscle levels of IGF-I and increased muscle satellite cell numbers play a role in anabolic steroid enhanced muscle growth. In contrast to anabolic steroids, the members of the TGF-beta-myostatin family suppress muscle growth in vivo and suppress both proliferation and differentiation of cultured myogenic cells. Recent evidence suggests that IGFBP-3 and IGFBP-5 play a role in mediating the proliferation-suppressing actions of both TGF-beta and myostatin on cultured myogenic cells. Consequently, this review will focus on the roles of IGF-I and IGFBP in the cellular and molecular mechanisms of action of anabolic steroids and TGF-beta and myostatin, respectively.

Insulin-like growth factor binding protein (IGFBP)-3 binds IGFs with high affinity and affects their biological activity. IGFBP-3 that is not bound to IGF also affects cells via mechanisms involving binding to specific cell surface receptors and/or transport into the cell. IGFBP-3 is produced by porcine embryonic myogenic cell (PEMC) cultures. Additionally, IGFBP-3 facilitates the proliferation-suppressing actions of TGF-beta(1) and myostatin in PEMC cultures via mechanisms that do not involve IGF binding. Moreover, these mechanisms do not involve preventing myostatin or TGF-beta(1)-induced increases in phosphosmad2 or phosphosmad3 level. Consequently, the mechanism(s) by which IGFBP-3 facilitates the proliferation-suppressing actions of TGF-beta(1) and myostatin in PEMC is unclear. Since IGFBP-3 reportedly interacts with nuclear proteins that regulate transcription, TGF-beta(1) or myostatin-induced translocation of IGFBP-3 into the nucleus may facilitate the proliferation-suppressing actions of these cytokines. Here, we show that IGFBP-3 is localized in cells containing the muscle specific protein desmin, thus establishing the presence of this IGFBP in myogenic cells. IGFBP-3 is present in the cytoplasm of all myogenic cells and approximately 50% of the nuclei of proliferating PEMC. IGFBP-3 is also detectable in fused myotubes. IGFBP-3 suppresses IGF-I-stimulated differentiation of PEMC but has no affect on Long-R3-IGF-I-stimulated differentiation of PEMC. Treatment of PEMC for 24h with TGF-beta(1) (20 ng/ml) results in a 78% (p<0.01) increase in the number of nuclei that contain detectable IGFBP-3. These results suggest that translocation of IGFBP-3 into the nucleus of PEMC could play a role in mediating the proliferation-suppressing action of TGF-beta(1).

The growth factors, IGF-I and II, their binding proteins, IGFBP, and members of the transforming growth factor (TGF) superfamily (myostatin and TGFbeta1) are known to regulate proliferation and differentiation of myogenic cells. We hypothesized that changes in the relative expression of members of the IGF and TGFbeta systems play a significant role in regulating myogenesis in porcine embryonic myogenic cell (PEMC) cultures. Therefore, determining the expression patterns of these factors during PEMC myogenesis is important. Consequently, we used real-time PCR to explore the pattern of IGF-I; IGF-II; IGFBP-2, -3, and -5; IGF-type-I receptor; myogenin; myostatin; and TGFbeta1 mRNA expression during PEMC myogenesis. The progression of differentiation was assessed using creatine kinase activity and myogenin mRNA expression. As anticipated, creatine kinase activity was low in PEMC cultures at 48 h and increased 20-fold (P < 0.0001) between 48 h and its peak at 144 h. Similarly, myogenin mRNA was low at 48 h and increased approximately 5-fold (P < 0.0001) as differentiation progressed, peaking at 120 h and decreasing at 144 h. The patterns of IGF-I and IGFBP-2 mRNA expression were similar and were relatively lower in 48-h PEMC cultures, increasing approximately 5-fold (P < 0.0001) to their greatest levels at 120 h. In contrast, IGF-II and IGFBP-5 mRNA levels were relatively high at 48 h, peaking at 72 h, and steadily decreasing by 60 and 80%, respectively (P < 0.001), at 144 h. The level of IGF-type-I receptor mRNA was relatively high until 96 h of culture, after which it decreased 40% (P < 0.01), reaching a low at 144 h. Levels of IGFBP-3 mRNA were relatively high at 48 h, dropped approximately 40% to their lowest level at 72 h (P < 0.001), and then increased approximately 60% (P < 0.001) to their greatest levels at 144 h. Levels of TGFbeta1 mRNA decreased approximately 30% (P < 0.0001) between 48 and 96 h, then quickly rebounded to a peak at 120 h, and by 144 h had dropped to the levels seen at 72 h. Myostatin mRNA was at its greatest level at 48 h and declined rapidly between 72 and 96 h, finally decreasing by approximately 80% at 144 h (P < 0.0001). Our data demonstrate that these factors are differentially regulated during PEMC myogenesis and provide new information about their pattern of mRNA expression in cultured porcine muscle cells.

We have previously shown that exogenous recombinant porcine IGFBP-3 (rpIGFBP-3) suppresses proliferation and differentiation of L6 myogenic cells in an IGF-I-dependent manner and suppresses proliferation of L6 myogenic cells via an IGF-I-independent mechanism. In order to assess the effects of endogenously produced IGFBP-3, we have transfected L6 myogenic cells with a pEF6/V5 vector containing pIGFBP-3 cDNA under the control of the human elongation factor 1alpha (hEF-1alpha) promoter and with the empty vector. We have isolated a cell population that constitutively produces porcine IGFBP-3 (tL6 cells) and a stable mock transfected cell population containing the empty vector (mtL6 cells). Constitutive expression of IGFBP-3 slightly reduced the expression of IGFBP-5 but had no effect on IGFBP-4 production by L6 myogenic cells. Immunoneutralization of IGFBP-3 increased both IGF-I- and Long-R3-IGF-I-stimulated proliferation of tL6 cells (58 and 33%, respectively) (P<0.01). These data indicate endogenous pIGFBP-3, like exogenous rpIGFBP-3, suppresses the proliferation of L6 myogenic cells via both IGF-I-dependent and -independent pathways. Immunoneutralization of IGFBP-3 also increased IGF-I-stimulated differentiation (21%, P<0.05) but had no effect on Long-R3-IGF-I stimulated differentiation of tL6 myogenic cells. Results indicate that exogenous and endogenous IGFBP-3 affect proliferation and differentiation of L6 myogenic cells in a similar way. Immunohistochemical localization data reveal that pre-incubation with anti-pIGFBP-3 dramatically reduces the level of intracellular IGFBP-3 in tL6 myogenic cells indicating that endogenously produced IGFBP-3 must first be secreted before it is internalized and that anti-pIGFBP-3 prevents internalization of IGFBP-3. TL6 and mtL6 cells provide a good system to further investigate the mechanisms by which IGFBP-3 affects proliferation and differentiation of myogenic cells.

We have previously shown that cultured porcine embryonic myogenic cells (PEMC) produce both insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 and secrete these proteins into their media. Exogenously added recombinant porcine (rp) IGFBP-3 and rpIGFBP-5 act via IGF-dependent and IGF-independent mechanisms to suppress proliferation of PEMC cultures. Furthermore, immunoneutralization of endogenous IGFBP-3 and IGFBP-5 in the PEMC culture medium results in increased DNA synthesis rate suggesting that endogenous IGFBP-3 and IGFBP-5 suppress PEMC proliferation. TGF-beta superfamily members myostatin and TGF-beta1 have also been shown to suppress proliferation of myogenic cells, and treatment of cultured PEMC with either TGF-beta1 or myostatin significantly (P < 0.01) increases levels of IGFBP-3 and -5 mRNA. We have previously shown that immunoneutralization of IGFBP-3 decreases the proliferation-suppressing activity of TGF-beta1 and myostatin. Here, we show that immunoneutralization of IGFBP-5 also significantly (P < 0.05) decreases the DNA synthesis-suppressing activity of these molecules. Simultaneous immunoneutralization of both IGFBP-3 and IGFBP-5 in TGF-beta1 or myostatin-treated PEMC cultures restores Long-R3-IGF-I-stimulated DNA synthesis rates to 90% of the levels observed in control cultures receiving no TGF-beta1 or myostatin treatment (P < 0.05). Even though immunoneutralization of IGFBP-3 and -5 increased DNA synthesis rates in TGF-beta1 or myostatin-treated PEMC cultures, phosphosmad2 levels in these cultures were not affected. These findings strongly suggest that IGFBP-3 and IGFBP-5 affect processes downstream from receptor-mediated Smad phosphorylation that facilitate the ability of TGF-beta and myostatin to suppress proliferation of PEMC.

IGF-binding protein-5 (IGFBP-5) is produced by porcine embryonic myogenic cell (PEMC) cultures and is secreted into the medium. IGFBP-5 may play some role in myogenesis and/or in changes in myogenic cell proliferation that accompany differentiation. IGFBP-5 reportedly may either suppress or stimulate proliferation or differentiation of cultured cells depending on cell type and culture conditions. Additionally, IGFBP-5 has been shown to possess both IGF-dependent and IGF-independent actions in some cell types. The goal of this study was to produce recombinant porcine IGFBP-5 (rpIGFBP-5) and assess its IGF-I-dependent and IGF-I-independent actions on the proliferation of PEMCs. To accomplish this, we have expressed porcine IGFBP-5 in the baculovirus system, purified and characterized the expressed rpIGFBP-5 and produced an anti-porcine IGFBP-5 antibody that neutralizes the biological activity of porcine IGFBP-5. rpIGFBP-5, purified to 98% homogeneity using nickel affinity chromatography and IGF-I affinity chromatography, suppressed IGF-I-stimulated proliferation of PEMCs in a concentration-dependent manner (P>0.05). rpIGFBP-5 also suppressed Long-R3-IGF-I-stimulated proliferation of PEMCs (P>0.05), even in the presence of significant molar excess of Long-R3-IGF-I compared with rpIGFBP-5, demonstrating the IGF-independent activity that rpIGFBP-5 possesses in PEMCs, since Long-R3-IGF-I is an IGF analog that has very low affinity for the IGFBPs but retains its ability to bind to the type I IGF receptor and thereby can stimulate proliferation. The anti-rpIGFBP-5 IgY produced against rpIGFBP-5 specifically recognized native porcine IGFBP-5 in PEMC media that also contained porcine IGFBP-2, -3, and -4. This antibody is capable of neutralizing the effects of both rpIGFBP-5 and endogenously produced porcine IGFBP-5 on PEMCs as well as detecting IGFBP-5 in Western blots. The production of rpIGFBP-5 and a neutralizing antibody to porcine IGFBP-5 provides a powerful tool to investigate the role of IGFBP-5 in porcine myogenic cell proliferation and differentiation. The data provided here demonstrated that IGFBP-5 has the potential to affect proliferation of PEMCs during critical periods of in vitro muscle cell development and therefore may impact the capacity for ultimate postnatal muscle mass development in vivo.

Androgenic and estrogenic steroids enhance muscle growth in a number of species; however, the mechanism by which anabolic steroids enhance muscle growth is not known. Castrated male cattle (steers) provide a particularly good model system in which to study the effects of anabolic steroids on muscle growth because they respond dramatically to treatment with both estrogens and androgens. The goal of this study was to determine if treatment of bovine satellite cell (BSC) cultures with 17beta-estradiol (E(2)) or trenbolone (a synthetic androgen) directly affects proliferation rate or level of mRNA for estrogen receptor (ER)-alpha, androgen receptor, and growth factors that have been shown to affect muscle growth (insulin-like growth factor (IGF)-I, IGF binding protein (IGFBP)-3, and myostatin). BSC cultures were established from the semimembranosus muscles of steers and then treated for 48 h with various concentrations of E(2) or trenbolone ranging from 0.001 to 10 nM. IGF-I mRNA levels in proliferating BSC cultures were significantly increased at 0.01 (1.9-times control values, P < 0.02) and at 0.1, 1, and 10 nM E(2) (2.9-, 3.5-, and 3.5-times control values, respectively, P < 0.0001). Additionally both 1 and 10 nM trenbolone increased IGF-I mRNA levels to 1.7-times control values (P < 0.02). ER-alpha mRNA was detectable in BSC cultures, and levels were increased (2.3-times control levels, P < 0.001) in cultures treated with 0.001 nM E(2) but not in cultures treated with higher concentrations of E(2). Androgen receptor mRNA levels also were increased (1.5-times control levels, P < 0.02) in cultures treated with 0.001 nM trenbolone but not by treatment with higher concentrations of trenbolone. Levels of IGFBP-3 were increased (1.4-times control values, P < 0.02) by treatment with 0.001 nM E(2) but not by treatment with high concentrations of E(2). Myostatin mRNA levels were not affected by any concentration of either of the steroids. Although, levels of IGF-I mRNA were 10-times greater (P < 0.02) in fused BSC cultures than in proliferating cultures, treatment of fused cultures for 48 h with 10 nM E(2) increased IGF-I mRNA levels (2.5-times control levels, P < 0.02). Both E(2) and trenbolone increased (3)H-thymidine incorporation rate (1.5-times control levels, P < 0.001) in BSC cultures in media containing serum from which IGFBP-3 had been removed by anti-IGFBP-3 affinity chromatography. In summary, treatment of BSC cultures with either E(2) or trenbolone increased IGF-I mRNA level and proliferation rate, thus, establishing that these steroids have direct anabolic effects on cells present in the BSC culture., (Copyright 2004 Wiley-Liss, Inc.)

Insulin-like growth factor (IGF)-I stimulates both proliferation and differentiation of myogenic precursor cells. In vivo, IGFs are bound to one of the members of a family of six high-affinity IGF binding proteins (IGFBP 1-6) that regulate their biological activity. One of these binding proteins, IGFBP-3, affects cell proliferation via both IGF-dependent and IGF-independent mechanisms and it has generally been shown to suppress proliferation of cultured cells; however, it also may stimulate proliferation depending upon the cell type and the assay conditions. Cultured porcine embryonic myogenic cells (PEMCs) produce IGFBP-3 and its level drops significantly immediately prior to differentiation. Additionally, IGFBP-3 suppresses both IGF-I and Long-R3-IGF-I-stimulated proliferation of embryonic porcine myogenic cells. In this study, we have examined the effects of recombinant porcine IGFBP-3 (rpIGFBP-3) on IGF-I- and Long-R3-IGF-I-stimulated proliferation and differentiation of the L6 myogenic cell line. L6 cells potentially provide a good model for studying the actions of IGFBP-3 on muscle because they contain no non-muscle cells and they do not produce detectable levels of IGFBP-3. RpIGFBP-3 suppresses both IGF-I and Long-R3-IGF-I-stimulated proliferation of L6 cells, indicating that it suppresses proliferation via both IGF-dependent and IGF-independent mechanisms. Our data also show that rpIGFBP-3 causes IGF-independent suppression of proliferation without increasing the level of phosphosmad-2 in L6 cultures. Additionally, rpIGFBP-3 suppresses IGF-I-stimulated differentiation of L6 cells. In contrast, however, rpIGFBP-3 does not suppress Long-R3-IGF-I-stimulated differentiation. This suggests that rpIGFBP-3 does not have IGF-independent effects on L6 cell differentiation., (Copyright 2004 Wiley-Liss, Inc.)

Both transforming growth factor (TGF-beta) and growth and development factor (GDF)-8 (myostatin) affect muscle differentiation by suppressing proliferation and differentiation of myogenic cells. In contrast, insulin-like growth factors (IGFs) stimulate both proliferation and differentiation of myogenic cells. In vivo, IGFs are found in association with a family of high-affinity insulin-like growth factor binding proteins (IGFBP 1-6) that affect their biological activity. Treatment of porcine embryonic myogenic cell (PEMC) cultures with either TGF-beta(1) or GDF-8 suppressed proliferation and increased production of IGFBP-3 protein and mRNA (P < 0.005). An anti-IGFBP-3 antibody that neutralizes the biological activity of IGFBP-3 reduced the ability of either TGF-beta(1) or GDF-8 to suppress PEMC proliferation (P < 0.005). However, this antibody did not affect proliferation rate in the presence of both TGF-beta(1) and GDF-8. These data show that IGFBP-3 plays a role in mediating the activity of either TGF-beta(1) or GDF-8 alone but not when both TGF-beta(1) and GDF-8 are present. In contrast to findings in T47D breast cancer cells, treatment of PEMC cultures with IGFBP-3 did not result in increased levels of phosphosmad-2. Since TGF-beta and GDF-8 are believed to play a significant role in regulating proliferation and differentiation of myogenic cells, our current data showing that IGFBP-3 plays a role in mediating the activity of these growth factors in muscle cell cultures strongly suggest that IGFBP-3 also may be involved in regulating these processes in myogenic cells.

We used a muscle biopsy technique in conjunction with real-time PCR analysis to examine the time course of changes in muscle IGF-I, IGFBP-3, myostatin, and hepatocyte growth factor (HGF) mRNA in the longissimus muscles of Revalor-S-implanted and nonimplanted steers on d 0, 7, 12, and 26 after implantation (nine steers/treatment group). Administration of a Revalor-S implant increased (P < 0.01) ADG and improved (P < 0.05) feed efficiency, 36 and 34%, respectively, compared with steers that received no implant during the 26-d trial. Daily dry matter intake did not differ (P > 0.15) between nonimplanted and implanted steers. Steers receiving the Revalor-S implant had increased (P < 0.001) circulating IGF-I concentrations compared with nonimplanted steers. The longissimus muscles of steers receiving the Revalor-S implant contained increased (P < 0.001) IGF-I mRNA levels compared with longissimus muscles of nonimplanted steers over the 26-d duration of the study. Longissimus muscle IGF-I mRNA levels in implanted steers were increased (P < 0.003) relative to d-0 concentrations on d 7 and 12 (101% and 128%, respectively), and byd 26, longissimus muscle mRNA levels were more than three times (P < 0.0001) those in the longissimus muscles of the same steers on d 0. There was no treatment effect on the level of IGFBP-3, myostatin, or HGF mRNA in the longissimus muscle at any time point; however, levels of IGFBP-3, myostatin, and HGF mRNA increased with time on feed. Based on current and previous studies, we hypothesize that the increased IGF-I level in muscle of implanted steers by d 7 of implantation stimulates satellite cell proliferation and maintains a high number of proliferating satellite cells at a point in the growth curve where satellite cell numbers and activity are normally dropping off. This would prolong the period of rapid muscle growth, resulting in the observed increased rate and efficiency of muscle deposition in implanted steers.

The effects of subtherapeutic antimicrobial supplementation and weaning on serum levels of IGF-I and insulin-like growth factor binding proteins (IGFBP)-2, -3 and -4 were determined in crossbred weanling pigs. At weaning, pigs were allotted to a diet containing 21.8% crude protein and 1.15% lysine with or without Aureozol (110 mg/kg of Aureomycin chlortetracycline, 110 mg/kg of sulfathiazole, and 55 mg/kg of penicillin) for 4 wk. Insulin-like growth factor-binding proteins and IGF-I analyses were performed on blood samples that were drawn weekly. Weaning decreased serum IGFBP-3 levels in both control and Aureozol-treated groups on d 6 and d 14 (P < 0.05) relative to preweaning levels. The IGFBP-3 values returned to preweaning levels by d 21. Although the circulating levels of both the 43-kDa and the 39-kDa glycosylation variants of IGFBP-3 were affected by weaning, the level of the 39-kDa IGFBP-3 was affected relatively more than that of the 43-kDa IGFBP-3 (P < 0.05). Compared with circulating IGFBP-3 levels in control pigs, Aureozol-treated pigs had higher circulating IGFBP-3 levels on d 21 (43%, P < 0.05) and d 27 (46%, P < 0.05). In direct contrast to the effect of weaning on serum IGFBP-3 level, serum IGFBP-2 levels increased on d 6 and d 14 after weaning (P < 0.05) and decreased to preweaning levels by d 21. The IGFBP-2 levels continued to decline and were less than preweaning levels by d 27 (P < 0.05). Aureozol treatment had no effect on serum IGFBP-2 levels at any time. Serum levels of nonglycosylated IGFBP-4 were not affected by either weaning or Aureozol supplementation. Weaning decreased circulating IGF-I concentration on d 6 in both control and Aureozol-treated pigs (76 and 73%, respectively, P < 0.05) and on d 14 (62%, P < 0.05) and d 21 (32%, P < 0.05) in control pigs. Aureozol-supplemented pigs had higher serum IGF-I concentrations than control pigs on d 14 (82%, P < 0.05), d 21 (55%, P < 0.05), and d 27 (36%, P < 0.05). The Aureozol-fed pigs had a 14.2% increase in BW gain (P < 0.05) and a 59.6% increase in ADG (P < 0.05) compared with pigs fed the control diet. Both Aureozol-supplementation and weaning cause changes in serum IGFBP levels and IGF-I concentrations that might be involved in regulating rate and efficiency of growth.

Ribonuclease protection assays were used to measure steady-state semimembranosus muscle and/or hepatic levels of IGF-I, IGFBP-3, IGFBP-5, hepatocyte growth factor (HGF), and myostatin messenger RNA (mRNA) in steers implanted from 32 to 38 d with Revalor-S, a combined trenbolone acetate and estradiol implant. Insulin-like growth factor-ImRNA levels were 69% higher (P < 0.01, n = 7) in the livers of implanted steers than in the livers of nonimplanted steers. Similarly, IGF-I mRNA levels were 50% higher (P < 0.05, n = 7) in the semimembranosus muscles of implanted steers than in the same muscles from nonimplanted steers. Hepatic IGFBP-3 mRNA levels were 24% higher (P < 0.07, n = 7) in implanted steers than in nonimplanted steers. Hepatic HGF and IGFBP-5 mRNA levels did not differ between implanted and nonimplanted steers. Similarly, muscle IGFBP-3, IGFBP-5, HGF, and myostatin mRNA levels were not affected by treatment. Previous data from these same steers have shown that circulating IGF-I and IGFBP-3 concentrations were 30 to 40% higher (P < 0.01, n = 7) in implanted steers than in nonimplanted, control steers. Additionally, the number of actively proliferating satellite cells that could be isolated from the semimembranosus muscle was 45% higher (P < 0.01, n = 7) for implanted steers than for nonimplanted steers. Viewed together, these data suggest that increased muscle IGF-I levels stimulate increased satellite cell proliferation, resulting in the increased muscle growth observed in Revalor-S implanted steers.

Insulin-like growth factor binding proteins (IGFBPs) have been shown to affect proliferation of several cell types via insulin-like growth factor (IGF)-dependent and IGF-independent mechanisms. The goal of this study was to determine if levels of IGFBP-2, -3, -4 and -5 mRNA changed during differentiation of cultured porcine embryonic myogenic cells. Total RNA was isolated from muscle cultures at various stages of differentiation and Northern blots of this RNA were probed with 32P-labeled cDNA probes specific for individual IGFBPs. Fusion, myogenin mRNA, and creatine phosphokinase activity were used as markers of differentiation. The level of IGFBP-3 mRNA in differentiating cultures (120 h in culture) was only one-third of the level in myogenin negative, nonfused cultures (72 h in culture) (P < 0.05, n = 4). In contrast, the level of IGFBP-3 mRNA in extensively fused cultures (144 h in culture) was increased by three-fold as compared to the level in myogenin negative, nonfused cultures (P < 0.05, n = 4) and approximately seven-fold as compared to the 120-h cultures (P < 0.05, n = 4). No significant change in the level of IGFBP-5 mRNA was observed during differentiation of myogenic cultures. IGFBP-2 mRNA levels were not significantly different at 72, 96 and 120 h, but at 144 h IGFBP-2 mRNA level was increased three-fold as compared to nonfused cultures (72 h) (P < 0.05, n = 4). IGFBP-4 mRNA was not detectable on Northern blots of total RNA from porcine myogenic cultures at any stage of differentiation. Changes in IGFBP-3 and IGFBP-2 mRNA levels are associated with differentiation of embryonic porcine myogenic cells in culture and this may indicate that these IGFBPs play a role in differentiation of these cells., (Copyright 2002 Elsevier Science Inc.)

Sixty-three Angus x Simmental calves were allotted to a bull or a steer group based on sire, birth date, and birth weight to determine effects of castration status on performance, carcass characteristics, and circulating insulin-like growth factor I (IGF-I) concentrations in early-weaned cattle. At 75 d of age, calves in the steer group were castrated. Calves were not creep-fed prior to weaning. All calves were weaned and weighed at an average age of 115 d and transported by truck to the OARDC feedlot in Wooster, OH. Performance and carcass characteristics were measured in three phases. Phase 1 was from 115 to 200 d of age, phase 2 was from 201 to 277 d of age, and phase 3 was from 278 d of age to slaughter. Before implantation, four bulls and four steers were selected for serial slaughter and carcass evaluation. Steers were implanted with Synovex-C at 130 d of age and with Revalor-S at 200 and 277 d of age. Serum samples were collected from all calves on the day of implantation, 28 and 42 d after implantation, and at slaughter and analyzed for circulating IGF-I concentration. Bulls gained 9.7% faster (1.75 vs 1.60 kg/d; P < 0.01), consumed 25 kg more DM (521 vs 496 kg; P = 0.11), and were 3.3% more efficient (282 vs 273 g/kg, P < 0.10) than steers in phase 1. However, steers gained 10.5% faster (1.62 vs 1.46 kg/d; P < 0.02), consumed similar amounts of DM, and were 6.5% more efficient than bulls (214 vs 201 g/kg; P < 0.06) in phase 2. Overall gains and efficiency were similar between bulls and steers; however, bulls consumed 140 kg more DM (P < 0.05), were 27 kg heavier (P < 0.05), and had to stay in the feedlot 18 more days (P < 0.05) than steers to achieve a similar amount of fat thickness. Implanted steers had greater concentrations of circulating IGF-I than bulls (P < 0.01), and the pattern of IGF-I concentration over time was affected by castration status (castration status x time interaction; P < 0.01). Synovex-C had a lower impact on circulating IGF-I concentration (implant effect, P < 0.01) than either Revalor-S implant. Eighty-five percent of both bulls and steers had marbling scores sufficient to grade low Choice or better. Bulls achieved their target fat thickness later, increased muscle growth, and deposited fat more favorably than steers, possibly due to a gradual increase in IGF-I concentration as the testicles grew rather than the large fluctuations in IGF-I concentration observed in steers following implantation.

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.

This study was conducted to determine whether an antimicrobially induced (ASP-250) increase in serum IGF-I was the result of differences in feed intake. Serum IGF-I concentrations were measured in crossbred pigs that were fed a control diet or a diet supplemented with ASP-250 either for ad libitum consumption or limited to 85% of the control pigs' consumption. The pigs that consumed either diet ad libitum, control or ASP-250, consumed similar quantities of feed. The ASP-250 ad libitum-intake pigs had serum IGF-I concentrations that were greater (P<.01) than those of their ad libitum-intake control littermates. Similarly, the ASP-250 limit-fed pigs had serum IGF-I concentrations that were greater (P<.01) than those of the controls. Although the serum IGF-I concentrations of pigs fed the ASP-250-supplemented diet for ad libitum intake were greater than the serum IGF-I concentrations of the pigs limit-fed the ASP-250-supplemented diet, the differences were not significant (P<.08). The ASP-250-fed pigs had higher serum IGF binding protein (BP)-3 concentrations than did their control littermates (P<.003). A time course of antimicrobially induced alterations in serum IGF-I concentrations revealed that the effect of increased serum IGF-I levels in ASP-250-supplemented pigs (P<.02) was observed within 4 d and was maintained throughout the 4-wk study. These findings show that feed intake is not responsible for the increase in serum IGF-I observed with ASP-250 supplementation. Additionally, the antimicrobially induced increase in serum IGF-I concentrations occurs within a few days after initiation of the treatment.

Objective: To determine insulin-like growth factor-I (IGF-I) concentrations in canine mammary secretions and serum during lactation and to compare them between small and giant breeds of dogs., Animals: 7 gestating Beagles and 4 gestating Great Danes., Procedure: Dogs were fed a common nutritionally complete and adequate gestation and lactation diet. Milk samples were collected at postpartum hour 12 and postpartum days 3, 7, 14, 21, and 28 after IV oxytocin administration. Two puppies/litter were identified at whelping for collection of blood samples corresponding to the days of milk sample collection plus days 35 and 42. Maternal blood samples were obtained on days 1, 7, and 42 from Beagles and days 1, 7, and 28 from Great Danes and were acid/ethanol extracted and analyzed by use of a radioimmunoassay., Results: Maternal serum IGF-I concentration was greater in Great Danes at all sample collection times. Similarly, colostrum from Great Danes contained more IGF-I, compared with that of Beagles (70 ng/ml vs 40 ng/ml, respectively). These values decreased to approximately 10 ng/ml by day 3 in both breeds and remained between 10 and 20 ng/ml for the duration of lactation. Growth rate and serum IGF-I concentration were greater in Great Dane puppies at birth to day 42., Conclusions and Clinical Relevance: High IGF-I concentration in colostrum may be biologically important for newborn puppies. Body mass and serum IGF-I concentration are directly correlated in growing Beagle and Great Dane puppies. Serum IGF-I concentration may be an indicator of growth potential in dogs.

Insulin-like growth factor binding proteins (IGFBPs) affect the biological activity of IGF-I in several cell types, including cultured muscle cells. Additionally, at least one of the IGFBPs, IGFBP-3, has been shown to have IGF-independent effects on cell proliferation. Numerous studies have shown that immortalized muscle cell lines produce various IGFBPs, but to date no muscle cell line has been reported to produce IGFBP-3 protein or mRNA. Unlike muscle cell lines, primary cultures of porcine embryonic myogenic cells express IGFBP-3 mRNA and secrete a protein that is immunologically identifiable as IGFBP-3. Additionally, steady-state IGFBP-3 levels change significantly during differentiation. Here we report that differentiation of porcine myogenic cells in an IGFBP-3-free medium is accompanied by reduced steady-state IGFBP-3 mRNA levels. Steady-state levels of IGFBP-3 mRNA decreased approximately sevenfold (P < .05) during differentiation and then increased to predifferentiation levels once differentiation was complete. Addition of TGF-beta1 (0.5 ng/ml) to porcine myogenic cultures suppressed fusion and resulted in a sevenfold increase in steady-state IGFBP-3 mRNA and a 1.8-fold increase in IGFBP-3 protein levels as compared to untreated control cultures (P < .05). Results suggest that alterations in IGFBP-3 mRNA and protein may play a role in differentiation of porcine embryonic muscle cells.

Insulin-like growth factor binding protein (IGFBP)-3 effects proliferation and differentiation of numerous cell types by binding to insulin-like growth factors (IGF) and attenuating their activity or by directly affecting cells in an IGF-independent manner. Consequently, IGFBPs produced by specific cells may affect their differentiation and proliferation. In this study we show that embryonic porcine myogenic cells, unlike murine muscle cell lines, produce significant quantities of a binding protein immunologically identified as IGFBP-3. Nonfusing cells subcultured from highly fused porcine myogenic cell cultures do not produce detectable IGFBP-3 protein or mRNA, thus suggesting the IGFBP-3 is produced by muscle cells in the porcine myogenic cell cultures. Treatment of porcine myogenic cultures with 20 ng of IGF-I or 20 ng of Des (1-3) IGF-I/ml serum-free media for 24 h results in a threefold reduction in the level of IGFBP-3 in conditioned media. This reduction is not affected by cell density over a sixfold range. Additionally, treatment for 24 h with 20 ng of IGF-I/ml media results in a sevenfold decrease in the steady-state level of IGFBP-3 mRNA. This IGF-I-induced decrease in IGFBP-3 mRNA level appears to be relatively unique to myogenic cells. IGF-I treatment also causes a fourfold increase in the steady-state level of myogenin mRNA. This increase in myogenin mRNA suggests that, as expected, IGF-I treatment accelerates differentiation of myogenic cells. The simultaneous decrease in IGFBP-3 mRNA and protein that accompanies IGF-I-induced myogenin expression suggests that differentiation of myogenic cells may be preceded or accompanied by decreased production of IGFBP-3.

Muscle satellite cells were isolated from seven yearling steers implanted for 31 d with a combined implant that contained 120 mg of trenbolone acetate (TBA) and 24 mg of estradiol (E2) and from seven nonimplanted, control steers. Implanted steers had a 28% greater ADG and a 23% greater feed efficiency than did nonimplanted steers. Implanted steers had increased (P<.001) circulating IGF-I concentrations on d 6, 14, and 31 after implantation, and circulating IGF-I concentrations in control steers remained constant or decreased (P<.05) at these times. Maximum fusion percentage was greater (P< .005) in satellite cell cultures isolated from implanted steers (ISC cultures) than in satellite cell cultures isolated from control steers (NSC cultures) (72.8% vs 54.8%, respectively). Satellite cell cultures isolated from implanted steers (ISC cultures) also contained a greater (P<.001) number of myotube nuclei than did NSC cultures (7,998 nuclei/cm2 vs 5,150 nuclei/cm2, respectively). After 72 h in culture, the number of cells (corrected for plating density) was 43% greater (P<.05) in ISC cultures than in NSC cultures. [3H]Thymidine incorporation rates per 10(5) cells at 24 and 34 h after plating were greater (P<.05) in ISC cultures than in NSC cultures; however, incorporation rates did not differ at 72 h. These data indicate that TBA + E2 implantation may result in an in vivo activation of muscle satellite cell proliferation that can be detected in cell culture. This activation may play an important role in TBA + E2-enhanced muscle growth.

Insulin-like growth factors (IGFs) are potent stimulators of cellular growth and their half-life and biological activity are regulated by specific IGF binding proteins (IGFBPs). Western ligand blots of non-reduced human, bovine, ovine and porcine sera reveal an IGFBP-2 band at approximately 34,000 M(r). However, canine sera appear to contain a unique 37,000 M(r) IGFBP and lack the 34,000 M(r) IGFBP-2 band. In order to identify and characterize the 37,000 M(r) IGFBP, adult canine serum was subjected to non-reducing SDS polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose paper, followed by [125I]-IGF-1 ligand blotting or immunoblotting with commercially available IGFBP antibodies. The 37,000 M(r) canine IGFBP reacted with an anti-IGFBP-2 antibody indicating that it is a canine analogue of IGFBP-2. However, the large difference in apparent molecular size indicates that this is a unique molecular form of IGFBP-2. N- or O-glycanase treatment of canine sera did not alter the molecular size of canine IGFBP-2 indicating that it is not a glycosylated variant of the IGFBP. Subjecting canine sera to reducing SDS-PAGE followed by anti-IGFBP-2 western immunoblotting revealed that the actual molecular weight of the canine IGFBP-2 is similar to that of reduced IGFBP-2 from other species indicating similar peptide lengths. Thus, the increased non-reduced size of the canine 37,000 M(r) IGFBP-2 is possibly due to a unique secondary structure.

Treatment of lambs (initial BW 28 kg) for 24 d with a combined implant containing 40 mg of trenbolone acetate (TBA) and 8 mg of estradiol (E2) increased ADG 25% (P < .05, n = 8) and feed efficiency 23% (P < .05, n = 2) compared with unimplanted lambs. By d 3 following implantation, sera from wethers implanted with TBA + E2 showed 32% (307 vs 233 ng/mL) increases (P < .001, n = 8) in IGF-I concentration compared with sera from unimplanted wethers. This increase was maintained throughout the entire 24-d study. Steady-state hepatic IGF-I mRNA levels were increased approximately 150% in implanted lambs compared with unimplanted lambs (P < .05, n = 4). These data suggest that liver may be the source of at least part of the increased circulating IGF-I in TBA + E2-implanted sheep. In steers implanted with Revalor-S (120 mg of TBA and 24 mg of E2) for 40 d, the steady-state concentration of IGF-I mRNA in the longissimus muscle was 68% greater than in the longissimus muscle of unimplanted steers (P = .013, n = 4). Consequently, increased local production of IGF-I by muscle tissue may play a role in increasing circulating IGF-I concentrations as well as an autocrine or paracrine role in stimulating muscle growth in steers implanted with Revalor-S.

Insulin-like growth factor binding proteins (IGFBP) may act locally as autocrine or paracrine regulators of insulin-like growth factor activity in specific tissues such as muscle. Although secretion of IGFBP by cultured myogenic cell lines has been examined, little is known about secretion of IGFBP by primary myogenic cell cultures. This may be because primary myogenic cultures contain non-muscle cells (fibroblasts) that complicate interpretation of IGFBP determinations. We have circumvented this problem by subculturing nonfusing cells from extensively fused porcine myogenic cultures and comparing the IGFBP production of these nonfusing, porcine muscle-derived cells with that of primary porcine myogenic cell cultures. Immunoprecipitation with specific antibodies and 125I-IGF-I ligand blot analysis showed that myogenic cultures secreted IGFBP-3 (doublet band, 43 kDa and 39 kDa), IGFBP-2 (34 kDa), IGFBP-4 (30 and 24 kDa), and IGFBP-5 (30 and 28 kDa). Muscle-derived fibroblasts secreted no detectable IGFBP-3 but approximately 10 times more IGFBP-2 than did myogenic cell cultures. Treatment of myogenic cultures for 24 h with transforming growth factor (TGF) beta-1 caused a concentration-dependent increase in IGFBP-3 secretion with a maximum 1.5-fold increase occurring at .5 ng of TGF beta-1/mL. In contrast, TGF beta-1 treatment did not stimulate detectable IGFBP-3 secretion by muscle-derived fibroblast cultures. Northern analysis of total RNA using a porcine IGFBP-3 probe revealed that TGF beta-1 treatment resulted in a fourfold increase in the steady-state level of IGFBP-3 mRNA in myogenic cultures. Insulin-like growth factor binding protein-3 mRNA was not detectable in fibroblast cultures either before or after TGF beta-1 treatment. This is the first report of IGFBP-3 secretion by cultured myogenic cells.

The effect of antimicrobial supplementation on the sera concentrations of IGF-I was determined in crossbred weanling pigs. Pigs were allotted by weight, litter, and sex to either a control diet or a diet supplemented with ASP-250 (22.7 ppm of chlortetracycline, 22.7 ppm of sulfamethazine, and 11.4 ppm of penicillin) for 5 wk. The diets contained 21.8% crude protein and 1.15% lysine. Growth performance data were collected weekly. Insulin-like growth factor I and insulin-like growth factor binding protein (IGFBP) analyses were performed on blood samples that were drawn during the final week of the trial. Feeding ASP-250 to young pigs increased their sera IGF-I concentrations by 24.8% (P < .001). A 59% increase in sera IGFBP-3 levels also was observed. The pigs fed ASP-250 had a 26% increase in average daily gain (P < .01), a 6.7% improvement in gain:feed ratio (P < .05), and a 18.5% increase in feed consumption (P < .01) compared with pigs fed the control diet. Increased serum IGF-I concentrations with antimicrobial feeding may be involved in the enhanced growth performance observed.

Objectives of this study were to determine the influence of trenbolone acetate (TBA) and estradiol (E2) in a combined implant on feedlot performance, carcass characteristics, and carcass composition in finishing steers. Sixty-four large-framed (394.1 kg) crossbred steers were randomly assigned to one of four pens. Subsequently, pens were randomly assigned to one of two treatments, implanted (120 mg of TBA and 24 mg of E2) and nonimplanted. Eight steers/treatment were slaughtered for initial carcass composition. Remaining steers were assigned to one of three serial slaughter dates (d 40, 115, or 143). Implantation increased circulating trenbolone (TBOH) and E2 concentrations throughout the trial. Implantation increased ADG 18% (P < .001) during d 0 to 40, 21% (P < .001) from d 0 to 115, and 16% for the entire 143 d. Implant status had no effect (P > .05) on dry matter intake. Feed efficiency was improved 13% during d 0 to 40 (P < .01) and from d 41 to 115 (P = .07). Longissimus muscle area was larger (P < .05) in implanted steers than in nonimplanted steers on d 115. Carcasses from implanted steers had a smaller (P < .05) percentage of kidney, pelvic, and heart (KPH) fat on d 143 than those from nonimplanted steers. Carcasses from implanted steers possessed more carcass protein (P < .05) on d 40. Implanted steers had an 82% increase (P < .05) in daily carcass protein accretion during the first 40 d. Implantation increased (P < .01) carcass water but did not affect carcass fat accumulation throughout the feeding period. The combined TBA+E2 implant improved feedlot performance and stimulated carcass protein accretion in feedlot steers.

Objectives of this study were to analyze alterations in circulating IGF-I and insulin-like growth factor binding protein (IGFBP) concentrations due to administration of a combined trenbolone acetate (TBA) and estradiol (E2) implant. This study was part of a larger serial slaughter study in which 64 large-framed (394.1 kg) crossbred steers were randomly assigned to one of four pens. Pens were assigned to one of two treatments: implanted (120 mg of TBA and 24 mg of E2) and nonimplanted. After d 2, 24 steers/treatment remained on the study. These steers were assigned to one of three serial slaughter dates (d 40, 115, and 143). Blood samples were obtained on d 0, 2, 21, 40, 115, and 143 from remaining steers. Serum was harvested and analyzed for IGF-I, IGFBP, and mitogenic activity. Glycyl-glycine (GG) extraction of serum was performed to reduce IGFBP interference in the IGF-I RIA. Implantation with TBA+E2 interference in the IGF-I RIA. Implantation with TBA+E2 increased (P < .001) circulating IGF-I concentrations during the period from d 0 to d 40. On d 21 and 40, steers implanted with TBA+E2 had 16 and 22%, respectively, greater (P < .001) circulating IGF-I concentrations than nonimplanted steers. For steers in the study for at least 115 d, TBA+E2 increased (P < .05) IGF-I concentrations 9, 13, and 19% on d 21, 40, and 115, respectively, compared with nonimplanted steers. Implantation with TBA+E2 resulted in greater (P < .05) serum concentration of a 49/39-kDa IGFBP (IGFBP-3) on d 21 and 40 after implantation. Sera from steers implanted with TBA+E2 stimulated proliferation of cultured muscle satellite cells to a greater extent (P < .05) than did sera from nonimplanted steers on d 21, 40, 115, and 143 after implantation. In summary, TBA+E2 increased serum concentrations of both IGF-I and IGFBP-3. Additionally, implantation increased mitogenic activity of sera from implanted as compared to nonimplanted steers. These alterations may be partially responsible for the positive effects of TBA+E2 implants on feedlot performance and rate of protein accretion in steers.

We report here for the first time the isolation of a cDNA clone containing the open reading frame sequence for porcine insulin-like growth factor binding protein-5 (pIGFBP-5) and the complete deduced amino acid sequence for this porcine IGFBP. The cDNA sequence shares 94%, 90% and 91% identity to its human, mouse and rat counterparts, respectively. The deduced amino acid sequence consists of 252 amino acids and a putative 19 amino acid signal and shares 97%, 96%, and 96% identity to the human, mouse and rat peptides, respectively. The mature peptide contains the 18 conserved cysteines found in all of the IGFBPs. Northern blot analysis of total RNA isolated from porcine heart, muscle and spleen using a 315 base pair cDNA insert derived from the pIGFBP-5 open reading frame sequence revealed a single mRNA transcript of 6.0 kilobases.

Myogenic cells isolated from lamb fetuses (approximately mid-gestation) exhibited a concentration-dependent decrease in myogenic cell proliferation in response to transforming growth factor (TGF) beta-1 (P < .001). Half-maximal inhibition of proliferation occurred at approximately .05 ng of TGF beta-1/mL and maximal inhibition of proliferation occurred at approximately .1 ng of TGF beta-1/mL. The specificity of this inhibition was confirmed by neutralization of the activity following exposure to a TGF beta antibody. The TGF beta-1 also suppressed proliferation of ovine satellite cells isolated from 5-d-old lambs (P < .0035), but to a lesser extent than observed for embryonic cells. In contrast, TGF beta-1 did not significantly suppress serum-stimulated proliferation of ovine satellite cells isolated from 30- or 150-d-old lambs. Similarly, TGF beta-1 did not suppress proliferation of skeletal muscle fibroblast-like cells isolated from either fetal lambs or 150-d-old lambs. In fact, proliferation of fibroblast-like cells derived from embryonic ovine muscle was enhanced by exposure to TGF beta-1 at all levels tested; however, a concentration-dependent response was not observed. Media transfer experiments showed that conditioning of culture media by postnatally derived cells did not render TGF beta-1 inactive. The studies described in this manuscript suggest that sensitivity of ovine myogenic cells to the antiproliferative effect of TGF-beta may vary with the stage of development.(ABSTRACT TRUNCATED AT 250 WORDS)

We have examined the efficacy of various methods for reducing the interference of insulin-like growth factor-binding proteins (IGFBPs) with insulin-like growth factor-I (IGF-I) radioimmunoassays (RIAs) run on porcine sera. Acid-ethanol (AE) extraction, AE extraction followed by cryoprecipitation, glycyl-glycine (GG) extraction, GG extraction followed by Sephadex G-50 chromatography in 1 mol acetic acid/l (GG/G-50), and Sep-Pak chromatography were analysed. To provide a range of IGF-I and IGFBP levels, sera obtained from control, hypophysectomized, diabetic and somatotrophin-treated pigs were used. Recoveries of IGF-I added to sera prior to treatments other than Sep-Pak chromatography ranged from 85 to 105% and were not significantly different. In contrast, Sep-Pak chromatography gave extremely variable recoveries. 125I-Labelled IGF-I ligand blotting showed that GG extraction followed by acid G-50 chromatography was by far the most effective method of removing or inactivating IGFBPs in porcine sera. Consequently, this procedure was used as a standard against which to compare other extraction procedures. GG extraction alone removed or inactivated low molecular weight binding proteins but appeared to have little effect on IGFBP-3. AE extraction reduced the level of IGFBP-3 but had little effect on lower molecular weight binding proteins. Even though none of the tested procedures completely removed or inactivated the binding proteins, all samples yielded IGF-I displacement curves that were parallel to that obtained for IGF-I standard. Despite yielding parallel displacement curves, sera extracted by various methods gave dramatically different apparent IGF-I levels when subjected to IGF-I RIA.(ABSTRACT TRUNCATED AT 250 WORDS)

It is generally thought that growth factors play a major role in regulating proliferation and differentiation of myogenic cells. Cell culture studies indicate that of the known growth factors, the fibroblast growth factors, the transforming growth factor beta, and the insulin-like growth factor families play the most significant roles in this process. The fibroblast growth factors stimulate proliferation and inhibit differentiation of most cultured myogenic cells. Insulin-like growth factors also stimulate proliferation of myogenic cells, but, in contrast to the fibroblast growth factors, the insulin-like growth factors also stimulate differentiation. Transforming growth factor beta inhibits differentiation of cultured myogenic cells. There are conflicting reports as to its effect on proliferation. The combined effects of these growth factors in vivo may play a major role in determining the rate of proliferation and differentiation of muscle tissue.

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.

Both mu- and m-calpain (the micro- and millimolar Ca(2+)-requiring Ca(2+)-dependent proteinases) can completely remove Z-disks from skeletal muscle myofibrils and leave a space devoid of filaments in the Z-disk area. alpha-Actinin, a principal protein component of Z-disks, is removed from myofibrils by the calpains, and a 100-kDa polypeptide that comigrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the alpha-actinin subunit is released into the supernatant. Purified calpain does not degrade purified actin or purified alpha-actinin as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by N- and C-terminal amino acid analysis of calpain-treated and untreated alpha-actinin and actin. The 100-kDa polypeptide released from myofibrils by calpain elutes identically with native alpha-actinin off DEAE-cellulose and hydroxyapatite columns and, after purification, binds to pure F-actin in the same manner that untreated, native alpha-actinin binds. Calpain-released alpha-actinin also accelerates the rate of superprecipitation of reconstituted actomyosin, a sensitive property characteristic of native alpha-actinin. Consequently, the calpains release alpha-actinin from the Z-disk of myofibrils without degrading it or without altering its ability to bind to actin. These results indicate that alpha-actinin does not simply cross-link thin filaments across the Z-disk but that at least one additional protein (or perhaps an altered actin or alpha-actinin) is involved in the alpha-actinin/actin interaction in Z-disks.

We have evaluated the effect of transforming growth factor beta-1 (TGF beta-1) on proliferation and fusion of cultured ovine satellite cells isolated from 5-month-old wether lambs. The isolation and culture protocols were validated by clonal analysis of the original cell preparation and assessment of proliferation and fusion of control cultures. Approximately 85% of the original cells isolated were myogenic as assessed by clonal analysis. The ovine cells doubled approximately every 18 hours during their exponential growth period and achieved a maximum percent fusion of 39.5% after 144 hours in culture. TGF beta-1 inhibited fusion of these cells in a dose-dependent manner with half-maximal inhibition occurring at .08 ng/ml. Maximal inhibition (95% suppression) occurred between .1 and .5 ng/ml. TGF Beta-1 (.05-3.0 ng/ml) did not inhibit proliferation of cultured ovine satellite cells in serum-containing medium or in serum-free defined medium. In contrast, TGF beta-1 did significantly suppress serum-stimulated proliferation of either porcine or bovine satellite cells that were isolated by using a procedure identical to that used to isolate the ovine satellite cells. Thus, proliferation of ovine satellite cells appears to respond differently to TGF beta-1 than does proliferation of either porcine or bovine satellite cells.

Sera obtained from pigs before and after subtherapeutic levels of ASP250 supplementation (pre and post serum pools) have been subjected to comparative fractionation by using gel filtration and affinity chromatography on immobilized Cibacron Blue F3G-A. Comparable serum fractions obtained from pre- and post-ASP250 blood sera were assayed in muscle cell culture bioassays designed to measure their effect on proliferation. Pre- and post-ASP250 sera were subjected to gel filtration and divided into the following fractions: fraction 1, Kav less than .17; fraction 2, Kav = .17 to .41; fraction 3, Kav = .41 to .59. Post-ASP250 fractions 2 and 3 increased proliferation rate in cultured muscle cells to a greater extent than comparable pre-ASP250 fractions (P less than .001). Chromatography of fraction 3 on immobilized Cibacron Blue F3G-A showed that both pre- and post-ASP250 fraction 3 contained a putative inhibitor of myogenic cell proliferation as well as mitogenic factors. However, negative growth factor activity was greater in pre-ASP250 fraction 3 than in post-ASP250 fraction 3 (P less than .05). Additionally, positive growth factor activity was lower in pre-ASP250 fraction 3 than in post-ASP250 fraction 3 (P less than .05). These data suggest that levels and(or) activities of both positive and negative muscle growth factors in serum may be altered by the addition of antimicrobials to the diets of growing pigs.

The effect of antimicrobial supplementation of pigs on the capacity of their sera to influence proliferation and protein turnover in cultured muscle cells was evaluated. Mitogenic activity of sera increased when pigs were fed ASP250 (P less than .005) or carbadox (P less than .001), whereas the mitogenic activity of serum from pigs receiving the basal diet remained unchanged (P = .5). Additionally, sera from ASP250-fed pigs significantly decreased (P less than .001) total cellular protein degradation compared with sera obtained from the same pigs prior to supplementation. Neither ASP250 nor carbadox stimulated proliferation of myogenic cells when added to the culture media. Inclusion of ASP250 in swine diets altered the composition of their sera in a way that stimulated muscle cell proliferation and reduced the rate of protein degradation in cultured myogenic cells. Likewise, the inclusion of carbadox in swine diets increased the ability of their sera to stimulate cultured muscle cell proliferation.