The time course of this increase in muscle n-3 PUFA composition and anabolic protein expression is currently unknown. In Chapter 2 of this thesis ten healthy male participants consumed 5g.d-1 of n-3 PUFA-enriched fish oil for 4 weeks. Muscle biopsies samples were collected in the fasted, rested state 2 weeks prior, immediately before (Week 0), at Week 1, Week 2 and Week 4 after initiation of fish oil supplementation for assessment of changes in lipid composition and expression of anabolic signalling proteins over time. Muscle lipid profile, (% total n-3 PUFA/total fatty acids) increased from W0 to W2 (3.8 ± 0.2 to 5.1 ± 0.3 %) and continued to rise at W4 (6.7 ± 0.4 %). Total protein content of FAK increased from W0 to W4 (3.9 ± 1.5 fold) whereas total mTOR was increased from W0 at W1 (2.4 ± 0.6 fold) with no further significant increases at W2 and W4. For the first time this study demonstrates that oral fish oil consumption results in an increase of n-3 PUFA levels in human skeletal muscle that is associated with increases in the expression of anabolic signalling proteins.
Our understanding of the anabolic signalling process that underpins muscle protein synthesis has been advanced by the application of the WB technique. However, the semi-quantitative nature and poor dynamic range associated with the WB technique may lead to incongruence regarding the molecular response of skeletal muscle to anabolic stimulation. Chapter 3 of this thesis developed and applied a quantitative in vitro [γ-32P] ATP kinase assay (KA) alongside a traditional WB methodology to assess p70S6K1 signalling responses in human skeletal muscle to RE and protein feeding. Following validation in tissue culture with rapamycin and optimization of the assay in human skeletal muscle, this methodology was tested in a physiologically relevant context. In this regard, six males performed unilateral resistance exercise (RE) followed by the consumption of 20 g of protein. Skeletal muscle biopsies were obtained at pre-RE, at 1 h and 3 h post-RE. In response to RE and protein consumption, p70S6K1 activity was significantly increased from pre-RE at 1 h and 3 h post-RE (8.84 ± 0.78 to 17.18 ± 2.62 and 15.62 ± 3.12 µU/mg). However, phosphorylated p70S6K1thr389 was not significantly elevated. To assess if a combined stimulus of RE and feeding can influence AMPK activity we directly measured AMPK activity. AMPK activity was suppressed from pre-RE at 3 h post-RE (24.15 ± 1.6 to 15.64 ± 1.07 mU/mg), whereas phosphorylated ACCser79 was unchanged. These data therefore highlight the utility of the KA to study skeletal muscle plasticity.
Previous studies have shown that ingestion of n-3 PUFA potentiates the phosphorylation of mTORC1 and associated kinases in response to nutrition. However, no study has identified whether n-3 PUFA supplementation potentiates anabolic kinase activity when RE is performed prior to nutrient provision. In Chapter 4 of this thesis, twenty healthy males consumed 5g.d-1 of either fish oil (FO) or coconut oil (CO) capsules for 8 weeks. Muscle biopsy samples were collected in the fasted, rested state before and after 8 weeks of supplementation for assessment of changes in lipid composition. Following 8 weeks of supplementation muscle samples also were obtained at rest (Rest), post RE in both the exercise leg (Post-RE) and the rested leg (Pre-FED) and also at 3 h post RE and protein feeding from both the exercise leg (3 h post-REF) and rested leg (3 h post-FED). There was a 2-fold increase in muscle (5.53 ± 0.3 to 11.16 ± 0.45 % of total fatty acids) n-3 PUFA composition after supplementation in the FO group but no change in the CO group. Following supplementation there was an increase in p70S6K1 activity at 3 h post-REF from Rest in the CO group (5.6 ± 1.4 to 12.2 ± 2.1 µU/mg) but no change in the FO group. In the CO group, AMPKα2 was significantly increased at Post-RE from Rest (3.7 ± 0.7 to 9.9 ± 2.0 mU/mg). These data show that 8 weeks of n-3 PUFA enriched fish oil supplementation suppresses the activity of p70S6K1 in response to RE and protein feeding
