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1 Department of Surgery2 Department of Physiology, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK,3 Sports Medicine Research Unit, Bispebjerg Hospital, Copenhagen Muscle Research Centre, Copenhagen, Denmark4 Department of Molecular Muscle Biology, Copenhagen Muscle Research Centre, Copenhagen, Denmark
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Abstract |
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(Received 21 July 2003;
accepted after revision 14 October 2003;
first published online 18 October 2003)
Corresponding author M. Hameed: Department of Surgery, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK. Email: m.hameed{at}rfc.ucl.ac.uk
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Introduction |
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Alternative splicing of the IGF-I gene is known to generate three different isoforms (see Fig. 1) and at least two of these have recently been shown to be expressed in human skeletal muscle (Hameed et al. 2003). The first, IGF-IEa, is similar to the main isoform expressed by the liver and the second isoform, MGF (IGF-IEc), is expressed in a mechanosensitive manner (Yang et al. 1996; McKoy et al. 1999; Hameed et al. 2003). The third isoform, IGF-IEb, is thought to be predominately expressed in the liver and its role in muscle is still unknown (Rotwein, 1986). Local IGF-I synthesis is also thought to be partially growth hormone (GH)-dependent in some IGF-responsive tissues (Isaksson et al. 1982, 1987). Furthermore, in vivo footprinting studies have shown evidence of a GH-dependent change in chromatin structure of the rat IGF-I gene. The emergence of this GH-inducible deoxyribonuclease-I (DNase-I)-hypersensitivity site (HS), which is located within the second intron of the IGF-I gene, supports the idea of an enhanced IGF-I gene transcription by GH (Thomas et al. 1995). However, the extent to which these isoforms are regulated by GH in human skeletal muscle is not yet known.
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Later life is characterized by a loss of muscle strength and mass (sarcopenia), and also by a gradual decline in circulating GH levels (Rudman et al. 1981). It is possible that there is a relationship between these two phenomena, caused by an impairment to stimulate local IGF-I synthesis (Hameed et al. 2002). It is known that the muscles of even very elderly individuals can hypertrophy in response to resistance training (Fiatarone et al. 1990, 1994;Harridge et al. 1999; Singh et al. 1999), but the coupling to local growth factor expression still remains to be determined.
Sarcopenia may be linked to an impairment in satellite cell activation (Chakravarthy et al. 2000). Recent in vivo experiments that determined the timing of the mRNA expression of MGF, IGF-IEa and the marker of satellite cell activation, M-cadherin, following muscle damage, showed that the maximal peak in MGF mRNA expression occurred at an earlier time point and preceded the maximal peak in M-cadherin mRNA expression. There was an increase in the expression of IGF-IEa mRNA after this. These data are consistent with the idea that the MGF splice variant may play a role in the early stages of activation of these satellite cells (Hill & Goldspink, 2003). This is important, as it is clear that these cells are required to maintain the ongoing supply of muscle fibre nuclei in this postmitotic tissue in response to exercise and local tissue injury.
As the splice variants of the IGF-I gene differ in their biological action and in the way they are apparently induced, the present study was designed to determine the effects of GH and/or resistance training on local growth factor expression in the muscles of elderly subjects. The first aim of this study was to determine which isoforms of IGF-I in muscle are influenced by GH. The second was to determine how GH administration might interact with mechanical signals provided by high-resistance exercise. The final aim was to determine the extent to which the mRNA of the different isoforms of IGF-I could be up-regulated in elderly people in response to a period of strength training, with the addition of studying the effects of exercise training on an, as yet little studied, IGF-I isoform (IGF-IEb). To achieve these aims, muscle samples were studied which had been obtained from three groups of elderly men who had either (1) received daily injections of recombinant human GH (2) undertaken 12 weeks of resistance training in addition to receiving GH, or (3) undertaken 12 weeks of resistance training but received placebo instead of GH.
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Methods |
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Nineteen healthy, elderly male subjects participated in the study; age 74 ± 1 years (mean ±S.E.M.; range 70–82 years); height, 174 ± 1 cm (range 163–186 cm); body weight, 80.8 ± 1.8 kg (range 63.1–100.3 kg); body mass index, 26.7 ± 0.5 kg m-2 (range 20.9–32.8 kg m-2); body fat mass, 22.9 ± 1.2 kg (range 10.9–38.9 kg), body fat percentage 28.0 ± 1.0% (range 14.9–40.0%). Before inclusion, each subject underwent a medical evaluation, including medical history, physical examination, routine blood tests, and an exercise electrocardiogram. Exclusion criteria were metabolic, cardiac and malignant disease; anaemia; hormonal replacement therapy; and medication with 
or ß blockers. Further information regarding the characteristics of the subjects can be found in Lange et al. (2002). Informed consent was obtained from all subjects and procedures were performed according to the Declaration of Helsinki II. The study was approved by the Ethics Committee for Medical Research in Copenhagen and by the Danish National Board of Health.
Study design
Subjects were assigned to either resistance training (3 sessions per week, 3–5 sets of 8–12 repetition maximum per session) with placebo (RT group, n= 6), resistance training combined with rhGH administration (RT + GH group, n= 6) or rhGH alone (GH group, n= 7) in a randomised, placebo-controlled and double-blinded experimental design. No nutritional supplementation in addition to the subjects’ normal diet was provided for the duration of the study period.
Administration of recombinant human GH
Recombinant human GH or placebo (GH-Norditropin PenSet 24; placebo-Norditropin PenSet 24 Placebo; both from Novo Nordisk, Denmark) was administered daily through subcutaneous injection in the thigh. After careful instruction, subjects were able to self-administer either GH or placebo at home in the evening. The dose of GH administered was 0.5 IU m-2 rising to 1.5 IU m-2. Details of the administration procedures and any changes made to the dose as a consequence of side-effects caused by the GH are reported in a recently published paper (Lange et al. 2002).
IGF-I
Circulating IGF-I was measured at 0 and 12 weeks as previously described (Lange et al. 2002).
Magnetic resonance imaging (MRI)
MRI measurements were made at 0 and 12 weeks to determine quadriceps cross sectional area as previously described (Lange et al. 2002).
Muscle biopsies
Following local anaesthesia of the overlying skin (1% lidocaine), muscle biopsies were obtained from the right vastus lateralis muscle at baseline, 5 weeks and 12 weeks. In the two training groups biopsy samples were obtained 24 h after completion of the last training session. Samples were immediately frozen in liquid nitrogen and stored at -70°C.
First strand cDNA synthesis
Samples of 0.5 µg of total RNA were reverse transcribed into cDNA using Omniscript reverse transcriptase (Qiagen, Crawley, UK). Details of this procedure have been previously described (Hameed et al. 2003). Briefly, total RNA was mixed with diethyl-pyrocarbonate (DEPC) -treated water in a total volume of 10 µl and heated to 65°C for 5 min before transfer to ice. The samples were then mixed with 2 µl First Strand Buffer (10 x), 2 µl dNTPs (5 mM each), 15 pmol of sequence specific primer or 50 pmol random primers, 1 µl RNase inhibitor (10 units µl-1) and 1 µl Omniscript Reverse Transcriptase (4 units µl-1). The reaction volume was made up to 20 µl using DEPC-treated water. The samples were then incubated at 37°C for 1 h followed by 5 min at 93°C. To facilitate the efficiency of reverse transcription (RT) in transcripts expressed at low levels, such as MGF, short sequence specific dodecamers 50–100 base pairs downstream of the PCR reverse primers were used. The sequence for the specific RT primer used for MGF and IGF-IEa analysis was: 5'-GAAACGCCCATC-3'.
Real-time quantitative PCR
Quantification of the mRNA message encoding the three isoforms of IGF-I: IGF-IEa, IGF-IEb and IGF-IEc (MGF) was performed using LightCycler technology (Roche Diagnostics) with SYBR green I as the method of detection. Details of the method are as previously described (Hameed et al. 2003). Briefly, quantitative PCR was performed in a total reaction volume of 20 µl per capillary for the LightCycler format. This reaction mix contained 10 µl of a SYBR green mix (QuantiTect, Qiagen, UK), 0.5–10 pmol of each forward and reverse primer, 2 µl cDNA (made from 0.5 µg RNA) and nuclease-free water to make up the reaction volume. The primers used for real-time PCR were designed using Omiga version 2.0 software (Oxford Molecular, UK) and these were synthesized by Sigma Genosys (Cambridge, UK). Primer sequences for IGF-IEb are given in Table 1. Primer sequences for IGF-IEa and MGF are previously described in Hameed et al. (2003). Runs were performed in duplicate and mean values were subsequently used for analysis. The expression levels of the different targets are expressed as ng mRNA (1 µg total RNA)-1.
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Statistical analysis
Data are presented as means ±S.E.M ANOVA (Student-Newman-Keuls post hoc tests) were used to determine whether MGF and IGF-IEa expression were significantly different between the RT only, RT + GH and GH only groups and also within groups at each time point studied. Statistical significance was accepted at the P < 0.05 level. Linear regression studies were used to describe the relationship between levels of MGF mRNA and muscle cross sectional area (CSA) at baseline and between levels of serum IGF-I and IGF-IEa mRNA and MGF mRNA in muscle.
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Results |
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In contrast, after 5 weeks of resistance training only (RT group) MGF was increased by 163% from baseline, which was significantly greater (P < 0.05) than the increase observed in IGF-IEa (68% from baseline, P < 0.05). No further significant changes were observed at the 12 week time point. In the group where resistance training was combined with GH administration (RT + GH group) substantially greater increases in both IGF-IEa (167%) and particularly in MGF mRNA expression (456%) were observed at the 5 week time point. Again, there were no further significant changes seen after 12 weeks.
There were differences in the absolute levels of expression of the IGF-IEa and MGF transcripts. IGF-IEa was expressed at higher levels than MGF, by 2–3 orders of magnitude (10-5 ng versus 10-8 ng).
Due to a shortage of material, measurements of IGF-IEb mRNA were only made in the group that carried out RT (n= 5). Expression of this isoform is summarized in Fig. 3. Expression levels of this isoform were low and in the 10-8 ng range. Baseline levels of IGF-IEb mRNA (4.2 ± 0.6 x 10-8 ng) were significantly increased by approximately 75% (P < 0.01) after 5 weeks of training (7.1 ± 0.7 x 10-8 ng). By the 12 week time point they had declined to levels similar to those observed at baseline (4.7 ± 0.5 x 10-8 ng, P < 0.01).
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Discussion |
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GH stimulates postnatal growth of the body in general through induction of IGF-I gene expression (Sadowski et al. 2001). Although the liver is the major site of GH-regulated IGF-I production, there is evidence to suggest that GH is able to regulate IGF-I gene expression in other non-hepatic tissues including the kidney, bone, intestine and skeletal muscle in an autocrine/paracrine fashion (Isaksson et al. 1982, 1987). Increased IGF-I mRNA levels have been observed in the muscles of GH-treated hypophysectomised rats (Murphy et al. 1987; Isgaard et al. 1989; Gosteli-Peter et al. 1994) and the muscles of rats implanted with GH-secreting cells (Turner et al. 1988). In vitro studies have also shown that C2C12 skeletal muscle cells respond rapidly to GH administration with increased tyrosine phosphorylation of the GH receptor (GHR) and increases in IGF-I mRNA expression (Sadowski et al. 2001). However, whether GH directly stimulates IGF-I mRNA expression in human skeletal muscle and whether it differentially affects these isoforms of IGF-I remains unclear (Taaffe et al. 1996; Welle & Thornton, 1997).
Ageing is associated with reduced circulating levels of GH and IGF-I (Rudman et al. 1981; Corpas et al. 1993; Morley, 1995; Lamberts et al. 1997). At the age of 70 years, GH levels are approximately 20% of those seen at the age of 30 years. Furthermore, GH release in response to resistance exercise is markedly attenuated with increasing age both in the untrained and trained state (Pyka et al. 1994; Zaccaria et al. 1999). Several studies have successfully shown that GH administration in elderly people decreases fat mass and increases lean body mass (Rudman et al. 1990; Taaffe et al. 1994; Yarasheski et al. 1995; Papadakis et al. 1996; Lange et al. 2002). However, despite increasing lean body mass, GH administration has not been shown to increase muscle mass or strength in healthy elderly individuals (Papadakis et al. 1996; Lange et al. 2002). Furthermore, trials in elderly people where GH administration has been combined with resistance training, GH does not seem to augment the strength gains obtained from exercise training (Taaffe et al. 1994; Yarasheski et al. 1995; Lange et al. 2002). This is in contrast to studies in aged rats which have reported gains in muscle mass and tetanic tension when GH administration is combined with cycling exercise (Andersen et al. 2000).
The findings in the present study confirm the increase in IGF-I mRNA in the muscles of older people reported by Brill et al. (2002), who administered daily injections of GH over a one month period. However, the present study shows that there is an isoform-specific response, with IGF-IEa (the main liver isoform) being the most responsive to GH administration. Earlier studies investigating GH-regulated IGF-I mRNA expression in human skeletal muscle of elderly subjects had reported no consistent increases in IGF-I mRNA levels after 10 weeks of GH administration (Taaffe et al. 1996) or in muscle biopsy samples taken 10 h after a single subcutaneous injection of GH (Welle & Thornton, 1997). However, these previous studies did not distinguish between the different isoforms of IGF-I expressed in muscle. The IGF-I gene has two promoter regions, with transcripts initiating at promoter 2 believed to be GH sensitive (Lyall, 1996). Furthermore, the suggestion that the two isoforms are regulated differently, with IGF-IEa being GH responsive and MGF appearing relatively insensitive to GH would make sense as these isoforms have been reported to have different roles (Yang & Goldspink, 2002) and is also indicated by their different expression levels (Table 2). Levels of IGF-IEa mRNA were found to be 2–3 orders of magnitude higher than those of MGF mRNA. One possibility for this is that the IGF-IEa splice variant is required for the normal maintenance of muscle mass, but after injury or training, the MGF splice variant is required for satellite cell activation. It would therefore not make physiological sense to have continually high levels of MGF, as this would act to constantly activate the pool of satellite cells. Data obtained in this laboratory showed that the E peptide of MGF is biologically active and has a distinct activity compared with mature IGF-I. The E peptide of MGF was shown to increase myoblast proliferation and to maintain myoblasts in their mononucleated state. Furthermore, the use of an antibody to the IGF-I receptor did not inhibit the activities of this peptide. These data therefore suggests that the E domain of MGF exerts its biological action independently of the IGF-I receptor (Yang & Goldspink, 2002).
The evidence that both of these splice variants work independently of one another was further supported in a study in which it was shown that when rat muscles are exercised, MGF mRNA is expressed earlier than IGF-IEa (Haddad & Adams, 2002). Also, other recent work (Hill & Goldspink, 2003) showed that following local muscle injury the IGF-I gene is spliced to produce the MGF transcript, which precedes muscle satellite (stem) cell activation. After a few days, it appears that it is then spliced towards IGF-IEa. This indicates that these are different growth factors with different functions and expression kinetics.
There is now evidence from both animal and human studies to suggest that following mechanical loading, older muscle is less able to up-regulate MGF mRNA when compared to young muscle (Owino et al. 2001; Hameed et al. 2003). In the latter study, expression of this isoform in the young subjects was significantly increased 2.5 h after the end of a single bout of high-resistance exercise. Levels of IGF-IEa in both young and older subject groups in this study showed no significant change with exercise. However, it is clear from the present study that following a 12-week period of progressive resistance training, even muscles of elderly individuals are able to significantly up-regulate both IGF-IEa and MGF mRNAs. Levels of IGF-IEa mRNA were significantly increased after 5 weeks of resistance training and further increased at the 12 week time point when compared to baseline (68% and 103%). Indeed, MGF mRNA levels in the present study were more sensitive to the training regime than IGF-IEa.
Singh et al. (1999) investigated IGF-I peptide levels in human skeletal muscle following 10 weeks of strength training in old men and women (aged 72–98 years). This study reported a 
500% increase in levels of IGF-I within the muscle fibres of these subjects after the training period. However, the latter study did not differentiate between the different isoforms of IGF-I. Furthermore, as with the present study, young subjects were not evaluated to determine if there is an age-related impairment to the up-regulation of the different IGF-I isoforms in muscle with training.
The present study combined two stimuli (resistance training and GH supplementation). In this group, levels of IGF-IEa mRNA were no greater than those observed with either resistance training or GH administration. In contrast, MGF mRNA levels observed at 5 and 12 weeks were markedly higher when compared with both the RT only and GH only groups. This suggests that GH administration results in a greater up-regulation of the IGF-I gene in muscle which, when combined with high-resistance exercise causes splicing towards the MGF isoform. Indeed, even in the GH only group it is possible that the increased levels of MGF mRNA observed after 12 weeks may have resulted from more of the primary transcript being available for splicing as a result of the mechanical activity caused by habitual levels of physical activity. In the present study we observed significantly higher levels of MGF mRNA at rest (pretraining) in the group that received GH only. This finding is difficult to explain with certainty as subjects were randomly assigned to each group and muscle biopsies were obtained before subjects had received any GH or undertaken the resistance exercise programme. However, interestingly, a significant association was observed between basal levels of MGF mRNA and quadriceps CSA as determined by MRI (Fig. 5). This might provide some tentative evidence for the long-term regulation of muscle mass by the expression of local growth factors.
Circulating IGF-I is almost exclusively derived from the liver in response to GH secretion from the anterior pituitary. However, it has also been shown that the exercising muscle itself takes up GH during exercise and consequently releases IGF-I, thus providing a local contribution to circulating IGF-I (Brahm et al. 1997). In the present study, an increase in circulating levels of total IGF-I was seen from baseline to 12 weeks in subjects receiving exogenous GH (Lange et al. 2002), but no further increase in IGF-I levels were seen in the RT + GH groups. Interestingly, the change in circulating IGF-I levels in the GH only group correlated with the change in IGF-IEa mRNA (r = 0.81, P < 0.05), but not MGF mRNA (r = 0.43, ns), in muscle. There was no such relationship between the change in circulating IGF-I and the mRNA of IGF-IEa (r = 0.45, ns) or MGF (r =-0.53) in muscle in the RT only group. In contrast, in the RT + GH group the change in circulating IGF-I was related to the change in MGF (r = 0.83, P < 0.05), but not IGF-I Ea (r = 0.31). This suggests that GH administration may lead to an overall up-regulation of the IGF-I gene in muscle as well as in the circulation, and that isoform expression is regulated by physical activity, such that when combined with mechanical overload the IGF-I gene favours splicing towards the MGF isoform. The contribution of IGF-IEa and MGF to the circulating IGF-I levels may be a simple reflection of their increased expression in the different experimental groups, suggesting that under certain conditions MGF can contribute significantly to circulating IGF-I levels.
The fact that older muscle is adaptable to strength training regimens has been proven in studies which have examined the very oldest subjects (Fiatarone et al. 1990, 1994; Singh et al. 1999; Harridge et al. 1999) and this may be associated with up-regulation of local growth factors in muscle as a result of exercise. It is known that IGF-I has an anabolic action and increases rates of protein synthesis in muscle (Adams & McCue, 1998). However, the activation of satellite cells is also required for hypertrophied fibres to maintain their DNA to protein ratios (Kadi & Thornell, 2000) and it seems that MGF may play a role in the activation of satellite cells (Yang & Goldspink, 2002), thus the two isoforms act in tandem to promote muscle growth and repair.
After 12 weeks of training in the present study, the gain in muscle mass and strength was no different in the group that strength trained whilst receiving GH (RT + GH) compared with the RT only group. Muscle CSA increased significantly in both the RT group (6.3 ± 2.5%) and in the RT + GH group (10.4 ± 2.7%), whilst in terms of muscle function, subjects’ strength as determined by their 1 repetition maximum (1-RM) increased significantly and equally by approximately 65% in both the RT only and RT + GH groups (see Lange et al. 2002 for details). These findings are in agreement with previous studies in both old (Taaffe et al. 1994; Yarasheski et al. 1995) and young men (Yarasheski et al. 1992). Furthermore, both studies by Yarasheski et al. in young and old men reported that the increases in the rates of muscle protein synthesis were no greater when GH was combined with resistance exercise than when compared with resistance exercise alone. However, Welle et al. (1996) reported that GH treatment further improved training induced muscle strength in healthy elderly men. The possibility that a longer training period would have resulted in a greater increase in muscle mass and strength in RT + GH cannot be excluded, and might be suggested by the MGF mRNA data in the present study. Alternatively, it might be that the overload provided by training provides a sufficient stimulus for the up-regulation of MGF at levels which are optimal for muscle repair and adaptation, at least in healthy older people undergoing exercise training. In contrast to healthy older individuals, those frail elderly people who are unable to exercise and thus not increase expression of these local growth factors, may potentially benefit from therapeutic treatments that may increase their expression.
An additional finding in the present study is the change in the mRNA expression of a third isoform of the IGF-I gene, IGF-IEb. There are three alternative splicing sites at the 3, end of IGF-I transcripts, which generate different E peptides with a common 16 amino acid N-terminal sequence and alternative C-terminal sequences. Transcripts containing Eb are more abundant in the liver, whereas transcripts containing Ea are commonly expressed in extra-hepatic tissues (Adamo et al. 1993; Stewart & Rotwein, 1996). As with the mRNAs of MGF and IGF-IEa, IGF-IEb mRNA is up-regulated as a result of strength training exercise. The precise role of this isoform in muscle is unclear and further studies are required to elucidate its role.
In summary, this study demonstrates that elderly people can up-regulate the mRNAs of three isoforms of IGF-I in muscle as a result of high-resistance training. The administration of recombinant GH alone, and in combination with exercise, demonstrated that there is differential regulation of at least two mRNA isoforms of IGF-I, IGF-IEa and MGF. The data support the argument that the IGF-I gene is spliced towards the MGF isoform with increased mechanical activity provided by high-resistance exercise. Whether higher levels of the MGF splice variant, induced as a result of combining GH with exercise, induce further adaptations in muscle beyond 12 weeks remains to be determined.
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significant difference in percentage change between isoforms (P < 0.05).
