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J. Anim. Sci. 2003. 81:965-972
© 2003 American Society of Animal Science

Growth factor messenger RNA levels in muscle and liver of steroid-implanted and nonimplanted steers1,2

M. E. White, B. J. Johnson{dagger}, M. R. Hathaway* and W. R. Dayton*,3

* Animal Growth and Development Laboratory, Department of Animal Science, University of Minnesota, St. Paul 55108 and and {dagger} Department of Animal Sciences and Industry, Kansas State University, Manhattan 66506

3 Correspondence:
348 ABLMS, 1334 Eckles Ave. (phone: 612-624-2234; fax: 612-624-3677; E-mail:
wdayton{at}umn.edu).


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
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-I mRNA 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.

Key Words: Estradiol • Growth Promoters • Insulin-Like Growth Factor • Muscles • Steers • Trenbolone


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
Feedlot steers implanted with Revalor-S, a combined implant containing 120 mg of trenbolone acetate (TBA) and 24 mg of estradiol (E2), exhibited a 20 to 25% increase in rate of gain, a 15 to 20% increase in feed efficiency, increased carcass protein, and increased longissimus muscle area compared with nonimplanted steers (Johnson et al., 1996a). Implantation of feedlot steers with Revalor-S also resulted in an increase in the number of actively proliferating satellite cells that could be isolated from the semimembranosus muscle (SM) (Johnson et al., 1998a). Steady-state IGF-I messenger RNA (mRNA) levels were increased in the longissimus muscle of implanted steers (Johnson et al., 1998b) and IGF-I levels were increased in the diaphragm muscle of nandrolone-treated rats (Lewis et al., 2002). Additionally, virally induced overexpression of IGF-I in muscle tissue resulted in a 15% increase in muscle mass in young adult mice (Barton-Davis et al., 1998), and overexpression of IGF-I extends the replicative lifespan of satellite cells (Barton-Davis et al., 1999; Chakravarthy et al., 2000). Viewed together, these data suggest that steroid-induced muscle growth may result from increased muscle IGF-I levels that maintain satellite cells in a proliferative state as steers approach maturity. Additionally, IGFBP (James et al., 1993; Damon et al., 1998; Yang et al., 1999), hepatocyte growth factor (HGF) (Allen et al., 1995; Tatsumi et al., 1998; Sheehan and Allen, 1999), or myostatin could play a role in anabolic steroid-induced muscle growth (McPherron et al., 1997; Carlson et al., 1999; Sakuma et al., 2000). Consequently, we have measured steady-state HGF, myostatin, IGF-I, IGFBP-3, and IGFBP-5 mRNA levels in SM and IGF-I, IGFBP-3, IGFBP-5, and HGF mRNA levels in livers from Revalor-S-treated steers and nonimplanted steers.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Implications
 Literature Cited
 
Animals
The University of Minnesota Animal Care Committee approved all experimental procedures. Fourteen crossbred yearling steers, matched for age and frame size, were used in this study. Average weight of the steers at the beginning of the experiment was 382 kg. Prior to implantation, steers were maintained on a high-roughage backgrounding diet (80% corn silage, 15% cracked corn, and 5% supplement). Beginning 14 d before implantation, steers were fed a step-up diet, so that by the day of implantation, they were consuming a high-concentrate (85% cracked corn, 10% corn silage, and 5% supplement) diet. This diet (77% DM; 13.2% CP) was fed ad libitum for the duration of the study. Steers were allocated into four pens (two pens of four steers/pen and two pens of three steers/pen), and steers in two of the pens (one pen containing four steers and one pen containing three steers) were implanted with Revalor-S on d 0 of the study. Steers were weighed weekly and feed intake of each pen was determined daily. Blood samples for IGF-I radioimmunoassays were taken from the left jugular vein on d 0, 6, 14, 20, 31, and on the day of slaughter. Insulin-like growth factor-I RIA data obtained from this study have been reported previously (Johnson et al., 1998a). Because facilities allowed preparation of satellite cells from two steers per day, one randomly selected implanted steer and one randomly selected nonimplanted steer were sacrificed on d 32 through 38 and satellite cells and RNA were isolated from the SM of each animal.

Ribonuclease Protection Assays (RPA) for IGF-I, IGFBP-3, IGFBP-5 Myostatin, and HGF.
Semimembranosus muscle and liver tissues were obtained immediately after death from steers used for satellite cell isolation and flash-frozen in liquid nitrogen. Total RNA was isolated using the single-step guanidinium thiocyanate procedure (Chomczynski and Sacchi, 1987). Briefly, total muscle tissue RNA (30 µg) or total liver RNA (10 µg) was hybridized with 5 x 105 counts/min of a bovine IGF-I, IGFBP-3, IGFBP-5 myostatin, or HGF antisense complementary RNA (cRNA) probe at 45°C overnight in hybridization solution (80% deionized formamide, 100 mM sodium citrate, 300 mM sodium acetate, pH 6.4, 1 mM EDTA). Following hybridization, samples were treated with a mixture of RNase A and T1 (Ambion, Inc., Austin, TX) for 30 min at 37°C. Protected RNA fragments were precipitated, dissolved in loading buffer, denatured at 90°C for 5 min, and resolved by electrophoresis on a 5% acrylamide, 8 M urea denaturing gel. The gel was then dried and subjected to autoradiography. In order to control for variation in RNA loading in the RPA, a labeled cRNA fragment of bovine cyclophilin was included in each hybridization reaction and the cyclophilin signal intensity was used to normalize the IGF-I, IGFBP-3, IGFBP-5, HGF, myostatin, and HGF signal intensities. The relative signal intensities of the protected fragment bands were measured using a Biorad GS-670 imaging densitometer in conjunction with Biorad Molecular Analyst software (Biorad Laboratories, Hercules, CA).

Bovine IGF-I, IGFBP-3, IGFBP-5, and Myostatin cDNA.
Bovine IGF-I, IGFBP-3, and IGFBP-5 cDNA were prepared in our laboratory by reverse transcription-PCR (RT-PCR) from bovine liver total RNA, and bovine myostatin cDNA was prepared by RT-PCR from bovine semimembranosus muscle total RNA.

Insulin-like growth factor-I primers were designed to amplify a 348-nucleotide (nt) sequence (209 to 556, accession number X15726) of the coding region of IGF-I and to add the T3 (5' end) and T7 (3' end) RNA polymerase promoter sequences to the amplified sequence. The forward primer was 5'-TTATTAACCCTCACTAAAGGGAAGGCTTTTATTTCAACAAGCCC-3' and the reverse primer was 5'-TAATACGACTCACTATAGGGCGATTTTGGTA-GGTCTTCTGGTG-3'. The sequence of the IGF-I cDNA amplicon was verified to be a 100% match with the appropriate section of the published bovine IGF-I cDNA (accession number X15726). The amplicon was then inserted into a TA cloning vector (pT-Adv, Clontech, Palo Alto, CA), and a clone with inverted insertion was isolated and cut with StyI (Promega Corp., Madison, WI) to remove an unwanted portion of the IGF-I sequence. The resulting plasmid, containing the T7 polymerase site, nucleotides 389 to 556 from the IGF-I sequence, and a portion of the vector sequence, was allowed to self-ligate. To generate an antisense IGF-I cRNA probe, this construct was cut with AvaII and transcribed with T7 polymerase. This yielded a 257-nt antisense probe that protected a 168-nt portion of IGF-I mRNA.

Bovine IGFBP-3 cDNA (nt 321 to 841, accession number M76478) was produced by RT-PCR of bovine liver total RNA using the forward primer 5'-GGCTGCGGTTGCTGTCTCA-3' and the reverse primer 5'-TCGCAGTTGGGAATGTGGATG-3'. The amplicon was inserted into a PCR cloning vector PT-Adv (Clontech) in the antisense orientation. When cut with StyI and transcribed with T7 polymerase, this construct yielded a 248-nt cRNA probe that protects a 180-nt (662 to 841) fragment of the bovine IGFBP-3 mRNA.

Bovine IGFBP-5 cDNA (corresponding closely to nt 351 to 680 of human IGFBP-5 cDNA) was produced by RT-PCR of bovine liver total RNA using the forward primer 5'-GGTTTGCCTCAACGAAAAGAG-3' and reverse primer 5'-GCGTGGGCTGGCTTTG -3' designed from the human cDNA sequence (accession number M62782). The amplicon was cut with BglII (Promega Corp.) and PstI (Promega Corp.) to yield a 199-nt fragment (nt 402 to 600) that was inserted into pBluescript SK+. When cut with SpeI (Promega Corp.) and transcribed with T7 polymerase, this construct yielded a 264-nt cRNA probe that protected a 199-nt fragment of the bovine IGFBP-5 mRNA.

Bovine myostatin cDNA was produced using RT-PCR of bovine SM total RNA. Primers were chosen from the published bovine myostatin cDNA sequence (accession number AF019620) to yield a 945-nt amplicon (nt 56 to 1,000). The forward primer was 5'-GCCCAGTGGATCTGA-ATGAGA-3' and the reverse primer was 5'-CTCTGGGGTTTGCTTGGTG-3'. The amplicon, whose nucleotide sequence was identical to the published sequence of bovine myostatin cDNA, was inserted into a TA cloning vector (pT-Adv, Clontech.). This construct was then cut with SpeI and the resulting 581-nt fragment was ligated into the SpeI site of pBluescript. When cut with Bgl4 (Promega Corp.) and transcribed with T7 polymerase, this construct yielded a 293-nt antisense cRNA probe that protected a 208-nt fragment of the myostatin mRNA (nt 793 to 1,000).

Bovine Hepatocyte Growth Factor cDNA.
Bovine HGF cDNA was produced using RT-PCR of total RNA isolated from bovine liver. Primers were chosen from the published genomic sequence of bovine ovarian thecal cell HGF (accession number S72476). Comparison of the published bovine genomic HGF sequence with the human HGF cDNA sequence (M60718) revealed that the human sequence contained 40 nt (656 to 695) that were not present in the bovine sequence. Consequently, we chose forward and reverse primers from regions of the bovine sequence that were present in the human cDNA sequence. The forward primer was 5'-TGCTATCGGGGTAAAGACCTAC-3' and the reverse primer was 5'-CCCATCAAAGCCCTTGT-3'. Based on the published bovine sequence and the location of the primers, we anticipated an amplicon of 235 nt. Instead, the amplicon we obtained contained 275 nt. The additional nucleotides in the amplicon corresponded to the 40 nt present in the human HGF cDNA sequence, but missing from the published bovine genomic sequence; in addition, nt 3 to 275 in the amplicon were identical to nucleotides 569 to 841 in the human HGF cDNA. Whereas it is unclear why there is a discrepancy between our sequence and the published bovine genomic sequence, our sequence agrees closely with the human HGF cDNA sequence. The amplicon was inserted into a TA cloning vector (pT-Adv, Clontech) and this construct was then cut with XhoI (Promega Corp.) and Kpn1 (Promega Corp.) to yield a restriction fragment that was ligated into the multiple cloning site of pBluescript. When cut with XhoI (Promega Corp.) and transcribed with T7 polymerase, this construct yielded a 296-nt antisense cRNA probe that protected a 233-nt fragment of the HGF mRNA.

Statistical Analysis.
Data were analyzed by ANOVA with the GLM procedure of SAS (SAS Inst., Inc., Cary, NC). Total RNA isolated from each steer was analyzed in three separate ribonuclease protection assays (RPA) for each growth factor. Data shown for each growth factor are from one of these assays that was representative of the results obtained in each individual RPA. In cases where main effects were significant, differences between means for preplanned comparisons were tested using Fisher’s LSD test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Implications
 Literature Cited
 
Hepatic IGF-I mRNA Levels in Control and Implanted Steers.
Since serum IGF-I levels were elevated in implanted steers, and the liver was thought to be the source of much of the circulating IGF-I, we were interested in determining if hepatic steady-state IGF-I mRNA levels were also elevated by implantation. Ribonuclease protection assays were used to measure the steady-state IGF-I mRNA concentrations in total RNA isolated from liver samples collected from control and implanted steers on d 32 to38 postimplantation. Figure 1Go shows the full length and protected fragments of the probes used in the RPA. Figure 2AGo shows the results of an IGF-I ribonuclease protection assay of total RNA isolated from the liver of representative implanted and nonimplanted steers. Densitometric analysis of RPA data (Figure 2BGo) from seven control and seven implanted steers showed that steady-state IGF-I mRNA levels were 69% higher (P < 0.01, n = 7) in the livers of implanted steers than in the livers of nonimplanted animals. This suggests that the liver may be a source of at least part of the increased circulating IGF-I in steroid-implanted animals.



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  		Figure 1. Autoradiogram showing the complementary RNA (cRNA) probes and protected fragments: A) cRNA probe and protected fragment for IGF-I: IGF-I (IGF), cyclophilin (Cyclo); B) cRNA probe and protected fragment for IGFBP-3: IGFBP-3 (BP-3), cyclophilin (Cyclo); C) cRNA probe and protected fragment for IGFBP-5: IGFBP-5 (BP-5), cyclophilin (Cyclo); D) cRNA probe and protected fragment for myostatin: myostatin (Myostatin), cyclophilin (Cyclo); E) cRNA probe and protected fragment for HGF: HGF (HGF), cyclophilin (Cyclo). For each growth factor, the full-length probes for the specific growth factor and cyclophilin are shown on the left, and the protected probe fragments are shown on the right.

 


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  		Figure 2. A) Autoradiogram of an IGF-I ribonuclease protection assay (RPA) showing the levels of IGF-I messenger RNA (mRNA) in 10 µg of total RNA isolated from livers of representative nonimplanted (C) and implanted (I) steers. Cyclophilin mRNA was measured in all samples to standardize for differences in recovery and loading. A lane (far left) containing labeled cRNA probes is also shown. The IGF-I protected fragment and the cyclophilin protected fragment are labeled in the C and I lanes. B) Average steady-state IGF-I mRNA levels in livers from nonimplanted steers (Control) and from steers implanted for 32 to 38 d with trenbolone acetate/estradiol2 (Implant). Levels were corrected for load and recovery by using bovine cyclophilin mRNA signal density. Average steady-state IGF-I mRNA levels in the livers of implanted steers were 69% higher (P < 0.01, n = 7) than those in non-implanted control steers. Data shown are from a single RPA done on all steers and are representative of results obtained from three separate RPA, each of which included all steers.

 
Insulin-Like Growth Factor-I mRNA Levels in Semimembranosus Muscles of Implanted and Nonimplanted Steers.
Several studies have established that IGF-I is produced locally in muscle tissue and that it may function in an autocrine or paracrine manner to stimulate muscle growth. Additionally, our previous studies with the same steers used in the current study have shown that more satellite cells can be isolated from the SM of implanted steers than from the SM of nonimplanted steers. Consequently, it was of interest to determine if TBA/E2 implantation increased steady-state concentration of IGF-I mRNA in muscle tissue. Figure 3AGo shows the results of an IGF-I ribonuclease protection assay of total RNA isolated from the semimembranosus muscles of a representative implanted and nonimplanted steers. Densitometric analysis of RPA data from seven control and seven implanted steers showed that IGF-I mRNA concentrations were higher in semimembranosus (50%, P < 0.05, n = 7) muscles of implanted steers than in the corresponding muscles of nonimplanted steers (Figure 3BGo). Viewed in light of reports that increased local IGF-I levels increase muscle mass, and in light of results from the same steers used in the current study showing that the SM from implanted steers contained more satellite cells than the SM from nonimplanted steers, these data suggest that TBA/E2-induced increases in muscle IGF-I may be at least partially responsible for the increased cell number and muscle growth observed in implanted steers.



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  		Figure 3. Autoradiogram of an IGF-I ribonuclease protection assay (RPA) showing the levels of IGF-I messenger RNA (mRNA) in 30 µg of total RNA isolated from the semimembranosus muscles of representative nonimplanted (C) and implanted (I) steers. Cyclophilin mRNA was measured in all samples to standardize for differences in recovery and loading. A lane (far left) containing labeled cRNA probes is also shown. The IGF-I protected fragment and the cyclophilin protected fragment are labeled in the C and I lanes. b) Average steady-state IGF-I mRNA levels in semimembranosus muscles from nonimplanted steers (Control) and from steers implanted for 32 to 38 d with trenbolone acetate (TBA)/estradiol (Implant). Levels were corrected for load and recovery by using bovine cyclophilin mRNA signal density. Average steady-state IGF-I mRNA levels in the semimembranosus muscles of implanted steers were 50% higher (P < 0.05, n = 7) than those in nonimplanted control steers. Data shown are from a single RPA done on all steers and are representative of results obtained from three separate RPA each of which included all steers.

 
Insulin-Like Growth Factor Binding Protein-5, HGF, and Myostatin mRNA Levels in the SM and/or Livers of Implanted and Nonimplanted Steers.
Steady-state IGFBP-5, HGF and myostatin mRNA levels in the SM of implanted and nonimplanted steers were compared. There were no significant differences in the levels of these mRNA in the muscle of implanted vs. nonimplanted steers; however, muscle HGF mRNA tended to be lower (P < 0.1, n = 7) in implanted steers (Figure 4Go). Additionally, the levels of hepatic IGFBP-5 and HGF mRNA were not different in implanted vs. nonimplanted steers (Figure 5Go).



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  		Figure 4. Average steady-state IGFBP-3, IGFBP-5, myostatin, and hepatocyte growth factor (HGF) messenger RNA (mRNA) levels in semimembranosus muscles from nonimplanted steers (Nonimplanted) and from steers implanted for 32 to 38 d with trenbolone acetate/estradiol (Implanted). Levels were corrected for load and recovery by using bovine cyclophilin mRNA signal density. Average steady-state HGF mRNA levels in the semimembranosus muscles of implanted steers were 27% lower (P < 0.1, n = 7) than those in nonimplanted control steers. Data shown are from a single ribonuclease protection assay (RPA) done on all steers and are representative of results obtained from three separate RPA, each of which included all steers.

 


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  		Figure 5. Average steady-state IGFBP-3, IGFBP-5, and hepatocyte growth factor (HGF) messenger RNA (mRNA) levels in livers from nonimplanted steers (Nonimplanted) and from steers implanted for 32 to 38 d with trenbolone acetate/estradiol (Implanted). Levels were corrected for load and recovery by using bovine cyclophilin mRNA signal density. Average steady-state IGFBP-3 mRNA levels in the livers of implanted steers were 24% higher (P < 0.07, n = 7) than those in nonimplanted control steers. Data shown are from a single ribonuclease protection assay (RPA) done on all steers and are representative of results obtained from three separate RPA, each of which included all steers.

 
Insulin-Like Growth Factor Binding Protein-3 mRNA levels in SM and Livers of Implanted and Nonimplanted Steers.
Since we have previously shown that circulating IGFBP-3 levels are increased in implanted vs. nonimplanted steers, we measured steady-state IGFBP-3 mRNA levels in SM and livers of implanted and nonimplanted steers in order to determine the source of this increased circulating IGFBP-3. Steady-state hepatic IGFBP-3 mRNA levels are 24% higher (P < 0.07, n = 7) in implanted steers than in nonimplanted steers (Figure 5Go). Steady-state IGFBP-3 mRNA levels in the SM of implanted and nonimplanted steers are not different (Figure 4Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Implications
 Literature Cited
 
We have previously shown that a greater number of actively proliferating satellite cells can be isolated from the SM of implanted steers than from the corresponding muscles of nonimplanted steers (Johnson et al., 1998a). Here we examine the steady-state levels of IGFBP-3, IGFBP-5, IGF-I, HGF, and myostatin in the SM of those same animals to determine if changes in the levels of any of these growth factors or binding proteins could be responsible for the increased number of proliferating satellite cells in the SM of implanted steers. Additionally, we have measured steady-state hepatic IGFBP-3, IGFBP-5, IGF-I, and HGF levels in these same steers.

Myostatin is a member of the transforming growth factor (TGF)-ß super family and has been shown to suppress muscle growth in mice (McPherron et al., 1997). Additionally, mutations in the myostatin gene are responsible for the double muscling phenomenon observed in beef cattle (Grobet et al., 1997). Recent reports indicate that the level of myostatin protein and/or mRNA is altered in muscle tissue undergoing hypertrophy, repair, or atrophy (Carlson et al., 1999; Kirk et al., 2000; Sakuma et al., 2000). These results led us to speculate that myostatin mRNA levels might be altered (decreased) by Revalor-S implantation, thus allowing increased proliferation of muscle satellite cells. However, our results show that after 32 to 38 d of implantation, the myostatin mRNA levels in the SM of implanted steers are not significantly different than those in the SM of nonimplanted steers.

We have shown previously that a greater number of actively proliferating satellite cells can be isolated from the SM of implanted steers than from the corresponding muscles of nonimplanted steers. The greater yield of proliferating satellite cells could result from increased activation of quiescent satellite cells or from maintaining satellite cells in an actively proliferating state. Because HGF has been reported to be a crucial element in the activation of quiescent satellite cells (Allen et al., 1995; Tatsumi et al., 1998; Sheehan et al., 2000), our finding that HGF mRNA levels are not increased and may even be lower in the SM of Revalor-S-implanted steers than in the corresponding muscles of nonimplanted steers could be interpreted to mean that activation of quiescent cells does not play a significant role in the Revalor-S-induced increase in proliferating satellite cells. However, several factors need to be considered before drawing this conclusion. Our study measured HGF mRNA after 32 to 38 d of implantation. It is possible that transient elevations in HGF mRNA and protein levels that are sufficient to activate quiescent satellite cells have already occurred by this time. Alternatively, it is possible that HGF levels are increased by releasing HGF that is stored in muscle tissue or by increasing the translation rate of HGF mRNA. Neither of these alternatives would require increases in HGF mRNA levels. Consequently, although our current results raise questions as to the role of increased HGF and satellite cell activation in the Revalor-S-induced increase in actively proliferating satellite cells in the SM, these results do not unequivocally eliminate activation of quiescent satellite cells as a mechanism. Further studies evaluating the time course of changes in muscle HGF and myostatin mRNA levels and satellite cell response to Revalor-S implantation will be required to ascertain the role of these growth factors and satellite cell activation in the Revalor-S-induced increase in actively proliferating satellite cells.

Hepatic IGFBP-3 mRNA levels are slightly elevated in implanted steers vs. nonimplanted controls. Although this elevation is slight, it is consistent with our previous observation that circulating IGFBP-3 levels are increased by Revalor-S implantation (Johnson et al., 1996b). The biological significance of this increase in circulating IGFBP-3 is not clear.

Steady-state IGF-I mRNA levels are significantly increased in the livers of steers implanted for 32 to 38 d with Revalor-S. Previous studies with these same steers have shown that circulating IGF-I concentrations are 33% higher (P < 0.01, n = 7) in steers implanted for 30 d with Revalor-S than in nonimplanted steers (Johnson et al., 1998a). These data are consistent with previous studies showing that hepatic IGF-I mRNA levels and circulating IGF-I concentrations are elevated (40% and 68%, respectively) in Revalor-S-implanted lambs (Johnson et al., 1998b). Thus, in both lambs and steers, Revalor-S implantation increases both hepatic IGF-I mRNA levels and circulating IGF-I levels. Since liver is thought to be a major source of circulating IGF-I, it is likely that increased production of IGF-I by the liver is at least partially responsible for increased circulating IGF-I concentrations in Revalor-S-implanted animals. However, recent reports of normal growth in mice in which the hepatic IGF-I gene has been specifically knocked out, put in question the role of circulating IGF-I in growth (Sjogren et al., 1999).

In addition to a Revalor-S-induced increase in steady-state hepatic IGF-I mRNA, our current study has shown a Revalor-S-induced increase in IGF-I mRNA in the SM of implanted steers. These data are consistent with our previous results demonstrating increased levels of IGF mRNA in the longissimus muscles of Revalor-S-implanted steers (Johnson et al., 1998b). The mechanism by which Revalor-S elevates muscle and liver IGF-I mRNA is not clear at present. Based on our current knowledge of how IGF-I levels are regulated, it is reasonable to speculate that Revalor-S-induced changes in GH levels or GH receptor levels/affinities may be responsible for the increased muscle and liver IGF-I mRNA (Breier et al., 1988). Growth hormone has been shown to play a major role in regulating serum IGF-I levels. Additionally, GH treatment of pigs raises the level of IGF-I mRNA in the liver and SM, but not in the longissimus muscle (Grant et al., 1991; Coleman et al., 1994; Brameld et al., 1996). However, since GH levels are reportedly not increased by Revalor-S treatment of steers (Hunt et al., 1991; Hayden et al., 1992), it appears unlikely that alterations in GH levels are responsible for Revalor-S-induced increases in muscle IGF-I mRNA level. This does not preclude the possibility that Revalor-S alters the number and/or affinity of GH receptors in liver and muscle tissues. These alterations could enhance the responsiveness of liver and muscle to GH. However, the fact that GH levels are not increased by Revalor-S implantation also raises the possibility that Revalor-S may increase liver and muscle IGF-I mRNA level via a mechanism that does not involve GH.

Since we have observed Revalor-S-induced increases in IGF-I mRNA in both the SM and longissimus muscles, we believe that increased muscle production of IGF-I may be a general response to Revalor-S implantation of steers. Additionally, we have previously shown that Revalor-S implantation increased the number of actively proliferating satellite cells in the SM of these same steers (Johnson et al., 1998a). Both the IGF-I and satellite cell results are very significant in view of a recent report that virally induced overexpression of IGF-I in the muscle tissue of mice resulted in a 15% increase in muscle mass in young adult mice and maintenance of muscle mass in old mice that would otherwise be losing muscle mass (Barton-Davis et al., 1998). These workers hypothesized that increased muscle growth resulted from increased activation and proliferation of satellite cells in muscles that are producing higher levels of IGF-I. This hypothesis is supported by a recent report that overexpression of IGF-I in muscle tissue increases the proliferative capacity of satellite cells (Chakravarthy et al., 2000). Other researchers also have suggested that IGF-I acts in an autocrine and/or paracrine manner to enhance muscle growth (Grant et al., 1991; Czerwinski et al., 1994; Brameld et al., 1996). It is possible that the increased IGF-I level in muscle of implanted steers 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. Whereas there is evidence that elevated IGF-I levels may increase the proliferative life of satellite cells, several studies have shown that IGF-I alone is not able to activate quiescent satellite cells (Allen et al., 1995). Thus, it is unlikely that the increased number of actively proliferating satellite cells in the semimembranosus muscle of implanted steers results from IGF-I stimulated activation of quiescent cells. Time course studies are needed to determine whether muscle levels of other growth factors such as HGF, myostatin, or fibroblast growth factor are elevated immediately after implantation.


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
Literature Cited
 
In feedlot steers, steady-state insulin-like growth factor-I messenger ribonucleic acid level in the semimembranosus muscle is increased after 30 d of implantation with a combined estradiol and trenbolone acetate implant. Because overexpression of insulin-like growth factor-I in muscle has been shown to increase muscle growth in young adult mice, it seems likely that increased production of this growth factor in anabolic steroid-implanted steers may play a role in the enhanced muscle growth observed in these animals. Although myostatin, hepatocyte growth factor, and insulin-like growth factor binding proteins-3 and -5 may also affect muscle growth and differentiation, their steady-state messenger ribonucleic acid levels were not altered by implantation, suggesting they may not be involved in the enhanced muscle growth observed in steers implanted with a combined estradiol and trenbolone acetate implant.


   Footnotes
 
1 This research has been funded in part by the Univ. of Minnesota Agric. Exp. Stn. Back

2 This research was funded in part by NRI Competitive Grants Program/USDA Grant No. 99-03256 and by a gift from Intervet, Inc. Back

Received for publication June 17, 2002. Accepted for publication December 3, 2002.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 


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