This thesis has focussed on the expression of the different insulin like growth factor-I (IGF-I) isoforms (IGF-IEa, IGF-IEb and MGF) in human skeletal muscle, with specific reference to exercise and the ageing process. To enable this, a method of real time quantitative PCR was developed for the accurate detection and quantification of these splice variants. Studies were performed both in our laboratory and at the Copenhagen Muscle Research Centre, Denmark. The acute response of IGF-IEa and MGF to a single bout of high resistance exercise was studied in young (n 8, age 29.5 1.5 years) and old (n 7, age 74.4 1.8 years) subjects. At rest there were no significant differences between resting levels of either isoform between the young and old subjects. The study showed that when measured 2.5 hours after the end of the exercise bout, the mechano-sensitive isoform, MGF, was significantly upregulated in the young but not the old subjects (P 0.05). No change was observed after exercise in the IGF-IEa iso form in either age group suggesting that the isoforms were differentially regulated. Furthermore, in situ hybridisation using oligo probes specific for MGF mRNA confirmed its localisation within the muscle fibres. No significant correlation was observed between the change in MGF expression and the muscle myosin heavy chain isoform composition was observed. A second study in young (n 10, age 20-27 years) and old subjects (n 10, age 67-75 years) evaluated the effects of a single bout of prolonged (1 hour) eccentric muscle damaging cycling exercise. In this study, and in contrary to the weightlifting study, a significant increase was observed in MGF mRNA levels in both the young and old subjects after the end of the exercise challenge (P 0.05), whereas levels of IGF-IEa again showed no significant increase. Thus suggesting that older muscle might require some myofibrillar disruption and/or sarcolemmal damage caused by eccentric exercise as a prerequisite for stimulating MGF expression. The effects of a period of resistance training in older people (n 19, age 70-82 years) performed over 12 weeks revealed a significant increase in the mRNA of both MGF and IGF-IEa and also the third isoform detected, IGF-IEb (P 0.05). In addition, the role that growth hormone (GH) might play in regulating the IGF-I spice variants in muscle with training was also investigated in this study. Recombinant GH administered to subjects without exercise, favoured expression of the IGF-IEa isoform. When resistance training was combined with GH administration, enhanced levels of MGF were observed but not IGF-IEa. This suggests that GH administration may lead to an overall upregulation of the primary IGF-I transcript, which is spliced towards MGF when mechanical activity in the form of resistance training is imposed. The results of the work in this thesis have shown that it possible to detect and quantify IGF-I mRNA transcripts in human skeletal muscle, even those expressed at low levels. It has also shown that there may be differential regulation of the different isoforms in young and old muscle in response to exercise, which would support the idea that these isoforms of IGF-I have different physiological actions.

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Skeletal muscle phenotype, size and function respond to exercise, disease and ageing. The aim of this thesis was to investigate the signal transduction pathways responsible for selected skeletal muscle phenotype and size changes. Myostatin, recently identified as a negative regulator of muscle mass was exposed at 10 ng ml 4 to C2C12 cells, and using cDNA genome-wide profiling, was shown to act as a transcriptional suppressor. Furthermore, in these cells myostatin significantly (n8, p<0.05) reduced phosphorylation of components in the P1-3K pathway: PKB Ser473 -30 %, mTOR Ser2448 -50 %, p70 S6K Thr389 -60 %, whereas 4E-BP1 Thr37/46 remained unaffected. These data provide insights in to the mechanisms by which myostatin controls muscle mass, through negatively affecting transcription and translation. Differences in the concentrations of signalling proteins often alter cellular function and phenotype, as is evident from numerous heterozygous knock-out models. Whilst the levels of metabolic enzymes differ between fibre types, and are regulable by exercise, it is not known if this is also true of signal transduction proteins. Therefore, it was hypothesised that the relative levels of signalling proteins implicated in the adaptation to exercise in both fast rat extensor digitorum longus (EDL; 3% type I fibres) and slow Soleus (84% type I fibres) would be systematically different. Secondly, it was hypothesised that following 6 weeks of chronic electrical stimulation (CMNS) where the EDL undergoes a fast-to-slow transformation, the relative signalling protein concentrations between control EDL/stimulated EDL would mirror the differences shown in control EDL/Soleus. Finally, that CMNS would induce chronic signalling to produce, and maintain a slower phenotype. Western blots revealed that the concentrations of some proteins such as Calcineurin (2.6-fold) and p38 MAPK (1.36-fold) were higher in EDL, whilst others such as PGC-la (1.4-fold); and NFkB (3-fold) (all n=4, pc0.05) were higher in Soleus. CMNS of EDL also led to changes in protein levels between control EDL/stimulated EDL: AMPK which is higher in Soleus was actually 1.4-fold lower following stimulation of EDL, whereas other proteins such as PGC-la moved in the direction of that of Soleus. CMNS was also able to induce chronic phosphorylation of proteins involved in fibre type and mitochondrial biogenesis, such as AMPK 4 fold, and p38 -4.5-fold. These data show that signal transduction protein concentrations vary between fast and slow muscles, presumably reflecting differences at a fibre level. Furthermore, signalling proteins are regulated by CMNS of EDL, but do not always change in the direction of slow Soleus. Chronic phosphorylation of many signalling proteins can explain the characteristic phenotypic change in response to CMNS. Resistance training stimulates adaptive protein synthesis and hypertrophy whereas endurance training induces a partial fast-to-slow fibre phenotype transformation. To simulate these conditions, isolated rat muscles were stimulated at 25 °C with either high frequency (HFS; 6 x 10 repetitions, 3 s-bursts at 100 Hz to mimic resistance training) or low frequency (LFS; 3 h at 10 Hz to mimic endurance training). HFS significantly increased myofibrillar and sarcoplasmic protein synthesis 3 h after stimulation 5.3 and 2.7-fold, respectively (n=6, p<0.05). LFS had no significant effect on protein synthesis 3 h after stimulation, but increased UCP3 mRNA 11.7-fold, whereas HFS had no significant effect on UCP3 mRNA (n6, p<0.05). Only LFS increased AMPK phosphorylation significantly at Thr172 by 2-fold and increased POC- 1 a protein to 1.3-fold of control. LFS had no effect on PICB phosphorylation but reduced TSC2 phosphorylation at Thr1462 and deactivated translational regulators. In contrast, HFS acutely increased phosphorylation of PKB at Ser473 5.3-fold and the phosphorylation of TSC2, mTOR, GSK-3j3 at PKB-sensitive sites. HFS also caused a prolonged activation of the translational regulators p70 56k, 4E-BPI, eIF2B, and eEF2 (all n=8, p<0.05). This behaviour has been termed the AMPK-PICB switch, and is hypothesised to mediate specific adaptations to endurance and resistance training, respectively. Ageing is associated with a loss of muscle mass tenned sarcopenia. Essential amino acids (EAA) are potent stimulators of muscle protein synthesis (MPS), and therefore defects in EAA-induced anabolism might affect ability to maintain muscle mass in ageing and disease. MPS and signalling responses to EAA-stimulation of 20 fasted young versus 24 elderly subjects (age 28 ± 6 and 70 ± 6; BMI 24 ± 3, 25 ± 4 kg.m 2 respectively; means ± SD) and 8 fasted elderly versus 8 elderly with type II DM (age 66 ± 3 and 70 ± 6; BMI: 25 ± 4 vs. 32 ± 2 kg.m 2, respectively means ± SD) were measured using gas combustion mass spectrometry and Western blotting methods. Basal MPS rates were indistinguishable, but the elderly displayed a reduced anabolic responsiveness of MPS to EAA, possibly due to decreased intramuscular phosphorylation after EAA, of amino acid sensing/signalling proteins mTOR, p70 S6 kinase, 4E-BPI and eIF2Bs by —50 %. This was further exacerbated in elderly with type II DM whom exhibited reduced Ser2448 phosphorylation of mTOR by —50 %, reflecting decreased downstream signalling. Associated with the anabolic deficits were — 4-fold increases in NFiB protein, the inflammation-associated transcription factor, as well as —50 % and —20 % decreases in protein expression of p70 S6K of healthy elderly and elderly with type II DM, respectively. These results suggest that the elderly are unable to mount a full anabolic response to EAA and that this blunting is further pronounced in type II DM.