M. E. White

Roles of IGF-I and the estrogen, androgen and IGF-I receptors in estradiol-17β- 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.

IGF-I mRNA levels in bovine satellite cell cultures: Effects of fusion and anabolic steroid treatment

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 17β‐estradiol (E2) or trenbolone (a synthetic androgen) directly affects proliferation rate or level of mRNA for estrogen receptor (ER)‐α, 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 E2 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 E2 (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‐α 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 E2 but not in cultures treated with higher concentrations of E2. 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 E2 but not by treatment with high concentrations of E2. 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 E2 increased IGF‐I mRNA levels (2.5‐times control levels, P < 0.02). Both E2 and trenbolone increased 3H‐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 E2 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.

Potential role of G-protein-coupled receptor 30 (GPR30) in estradiol-17β-stimulated IGF-I mRNA expression in bovine satellite cell cultures

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.

Role of insulin-like growth factor binding protein (IGFBP)-3 in TGF-β- and GDF-8 (myostatin)-induced suppression of proliferation in porcine embryonic myogenic cell cultures

Both transforming growth factor (TGF‐β) 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‐β1 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‐β1 or GDF‐8 to suppress PEMC proliferation (P < 0.005). However, this antibody did not affect proliferation rate in the presence of both TGF‐β1 and GDF‐8. These data show that IGFBP‐3 plays a role in mediating the activity of either TGF‐β1 or GDF‐8 alone but not when both TGF‐β1 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‐β 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. J. Cell. Physiol. 197: 225–231, 2003© 2003 Wiley‐Liss, Inc.

Effect of Estradiol-17β on protein synthesis and degradation rates in fused bovine satellite cell cultures

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.

Effects of implants of trenbolone acetate, estradiol, or both, on muscle insulin-like growth factor-I, insulin-like growth factor-I receptor, estrogen receptor-α, and androgen receptor messenger ribonucleic acid levels in feedlot steers

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.

Effect of insulin-like growth factor (IGF)-I and Des (1-3) IGF-I on the level of IGF binding protein-3 and IGF binding protein-3 mRNA in cultured 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. J Cell Physiol 178:227–234, 1999.

Decreased steady-state insulin-like growth factor binding protein-3 (IGFBP-3) mRNA level is associated with differentiation of cultured porcine myogenic cells

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‐β1 (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. J. Cell. Physiol. 179:237–243, 1999.

Effect of recombinant porcine IGFBP-3 on IGF-I and long-R3-IGF-I-stimulated proliferation and differentiation of L6 myogenic cells

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‐stimualted 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.

Effect of differentiation on levels of insulin-like growth factor binding protein mRNAs in cultured porcine embryonic myogenic cells.

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.