M. R. Hathaway

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.

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.

Effect of dietary supplementation of oregano essential oils to sows on colostrum and milk composition, growth pattern and immune status of suckling pigs

This study evaluated the effects of supplementing sow diets with oregano essential oils (OEO) during gestation and lactation on sow colostrum and milk composition and on the growth pattern and immune status of suckling pigs. A total of 70 second-parity sows were randomly assigned to 1 of 2 gestation dietary treatments within 24 h after service: control (CON) or CON + 250 mg/kg of OEO (OREG). In lactation, sows were again assigned to either the CON or OREG dietary treatment. Thus, the lactation treatments were CON-CON, CON-OREG, OREG-CON, and OREG-OREG. Colostrum and blood samples were collected from 6 sows per lactation dietary treatment. Thymus lymphocyte (T lymphocyte) subpopulations (γδ, cluster of differentiation 8, and 32 cluster of differentiation 4) were enumerated in blood and mammary secretions along with IGF-1, IgG, and IgA concentrations. Piglet growth rate were determined from 18, 17, 17, and 18 litters from the CON-CON, CON-OREG, OREG-CON, and OREG-OREG lactation dietary treatments, respectively. Growth rates were determined in 630 piglets, and piglets were individually identified and weighed on 1, 5, 9, 12, 16, and 19 d of age. Oregano essential oil supplementation during gestation or lactation had no effect (P > 0.05) on GE, CP, GE:CP, GE:fat, and IGF-1 in sow milk. Reductions of the fat percentage in milk on d 7 (P < 0.05) and d 14 (P = 0.07) were found in sows supplemented with OEO during lactation compared with those in the CON treatment. Milk from sows supplemented with OEO during lactation had the greatest number of T lymphocytes compared with those in the lactation CON treatment on d 14 of lactation (P < 0.01). The number of T lymphocytes in milk was greater for sows in the CON-OREG treatment compared with those other treatments on d 14 of lactation (P < 0.05). Energy intake was greater on d 1 to 5 in piglets from sows fed OEO during gestation than those from sows in the CON treatment (P < 0.05). A trend (P = 0.10) for greater milk intake was observed in piglets from sows supplemented with OEO during gestation compared with those from sows in the CON treatment. Similarly, a tendency for an increase in ADG on d 1 to 5 was found in piglets from sows supplemented with OEO during gestation compared with those from sows in the CON treatment (P = 0.10). Insulin-like growth factor-1 at birth and on d 7 and 14 of lactation did not differ among piglets from sows assigned to the different dietary treatments. Oregano essential oil supplementation of sow diets did not affect (P > 0.05) immunoglobulin concentrations in piglets after suckling. Supplementing sow diets with OEO during gestation or lactation did not affect (P > 0.05) the T lymphocytes, percentage of T-lymphocyte subpopulations, and natural killer cell activity of piglets during lactation. Supplementing sow diets with 250 mg/kg of OEO during gestation and lactation did not affect the growth potential of and immune responses in suckling piglets.

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.

In vivo effect of a β-adrenergic agonist on activity of calcium-dependent proteinases, their specific inhibitor, and cathepsins B and H in skeletal muscle

DEAE-Sephacel and phenyl-Sepharose chromatography were compared as methods for separating and quantitatively isolating calpain I, calpain II, and calpastatin from lamb muscle extracts. DEAE-Sephacel chromatography gave greater than 90% recovery of all three proteins, while phenyl-Sepharose gave only 70, 66, and 48% of the DEAE recovery of calpain I, calpain II, and calpastatin, respectively. Additionally, DEAE-Sephacel chromatography was shown to effectively separate calpastatin and calpain I. Consequently DEAE-Sephacel appears to be superior to phenyl-Sepharose for quantitative isolation of the components of the calcium-dependent proteinase system from muscle extracts. Dietary administration of beta-agonist (L-644, 969; Merck Sharpe & Dohme Research Laboratories) decreases extractable calpain I activity in lamb longissimus dorsi (LD) muscle by 10-14% (P less than 0.05), increases calpain II activity by 34-42% (P less than 0.001), and increases calpastatin activity by 59-75% (P less than 0.001). Additionally, net cathepsin B activity is reduced by 30% (P less than 0.05) in the LD of beta-agonist-treated lambs. Reduced activity of the calcium-dependent or catheptic proteinase systems may contribute to the increased protein accretion in muscles of beta-agonist-treated lambs.

The effect of exogenous prolactin on lactation performance of first-litter sows given protein-deficient diets during the first pregnancy

Twenty-four sows were used to study the effects of dietary protein restriction during pregnancy and exogenous porcine prolactin (pPRL) during late pregnancy and throughout lactation on lactation performance. Eight sows were given a protein-adequate diet containing 179 g crude protein (CP)kg−1 during their first pregnancy while the remaining 16 sows received the same amount of a diet containing 80 g CP kg−1. Eight of the sows given 80 g CP kg−1 during pregnancy were injected with 15 mg pPRL i.m. twice daily at 08:00 and 20:00 between day (d) 102.1 (±0.3) of pregnancy and weaning after their first lactation. Pregnant sows offered the low protein diet gained significantly less body weight during gestation and tended to eat less in the subsequent lactation than sows given the protein-adequate diet. Dietary protein had no significant effect on birth weight, milk yield, milk composition or growth rate of the litter during lactation. Neither dietary protein intake during pregnancy nor exogenous prolactin affected the concentrations of plasma glucose, serum insulin, urea or non-esterified fatty acid (NEFA) during lactation. The concentration of lactose in plasma during lactation was unaffected by treatment, but at d 105 of pregnancy, plasma lactose levels were greater in sows which had received exogenous prolactin (32.4 vs. 6.2 mg l−1, P < 0.05). The concentrations of RNA and DNA in mammary tissue biopsies were unaffected by either dietary protein or pPRL. The concentration of RNA and DNA increased between d 70 and 90 from 0.66 to 2.77 mg g−1 and from 0.54 to 1.19 mg g−1, respectively. Thereafter, RNA increased to 4.40 mg g−1 at d 14 of lactation whilst DNA concentration remained at a similar level of 0.90 mg g−1. Milk yield of sows between d 5 and 8 and between d 19 and 22 of lactation was reduced from 8.36 to 7.00 kg day−1 and from 10.74 to 8.22 kg day−1, respectively, in sows given pPRL. The protein content of colostrum from sows treated with pPRL was reduced from 164 to 104 g kg−1 whereas the fat content increased from 47 to 127 g kg−1. These results indicate that the administration of exogenous pPRL during late pregnancy and throughout lactation initiated lactogenesis prematurely and reduced subsequent milk yield during established lactation.

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.