Investigating the divergent regulation of skeletal muscle metabolism by different acyl chain structures

In recent decades, the prevalence of obesity and type 2 diabetes has risen dramatically. Strategies to reduce the incidence of these diseases are of great clinical relevance. The contribution of dietary fat has been central to that debate. In particular, the composition of dietary fat can influence skeletal muscle metabolism and the sensitivity to feeding and exercise adaptations. Polyunsaturated omega-3 fatty acids are linked with beneficial effects on skeletal muscle metabolism and function while saturated fatty acids have been linked with metabolic dysfunction. It is still poorly understood how differences in fatty acid structure can have contrasting effects in skeletal muscle. It is known that omega-3 fatty acids are incorporated into skeletal muscle lipid pools, however, it is unknown what specific lipid species omega-3 fatty acids are incorporated into. In Chapter 2 of this thesis, the effects of EPA and DHA on the lipidomic profile of skeletal muscle myotubes were explored. Although similar in structure, EPA and DHA treatment resulted in divergent lipid profiles. EPA increased the content of DPA, while DHA reduced arachidonic acid. Both omega-3 fatty acids significantly increased the saturated fatty acid content. EPA and DHA incorporated into myotubes were largely directed towards phospholipid species. The changes in lipid profiles following EPA treatment were associated with increased basal and insulin dependent glucose uptake. This increase in glucose uptake was not driven by changes in the protein abundance of glucose transporters or mitochondrial respiration. DHA did not have any impact on the metabolic measures made. These data show for the first time that EPA and DHA differentially affect glucose uptake in skeletal muscle and this effect may be associated with the differential changes in lipid profiles. Previous studies have shown that omega-3 supplementation increase mTORC1 signalling and protein anabolism following feeding. There is evidence to suggest that EPA is the dominant n-3 fatty acid that drives muscle anabolism. These anabolic effects of omega-3 fatty acids may be driven by changes in the proteomic profile which increases sensitivity to extracellular stimuli. In chapter 3, the effects of EPA and DHA on protein turnover and protein expression are explored. Neither, EPA or DHA did not altered basal protein synthesis. The activation of the mTORC1 pathway in response to a combined stimulus of amino acids + serum was not altered by either EPA or DHA. EPA reduced protein breakdown and this was not related to a reduction in ubiquitinated proteins. Proteomic analysis showed that EPA and DHA differentially altered the abundance of a number of proteins. Given the significant incorporation into phospholipids, we explored how changes in membrane lipid content altered the proteins associated with membrane compartments. DHA treatment resulted in the decreased association of ribosomal proteins with the membrane while EPA induced a small increase in ribosomal proteins associated with the membrane. Gene ontology analysis showed that proteins involved in protein folding associated with cell membranes were enhanced following EPA treatment of myotubes. These results led us to hypothesis that EPA may enhance myotube protein content by altering protein fidelity In contrast to omega-3 fatty acids, saturated fatty acids such as palmitate are linked with the dysfunction of a number of metabolic systems. A number of studies have demonstrated that palmitate causes skeletal muscle insulin resistance through the generation of lipid intermediates, such as ceramides and diacylglycerols, which inhibit insulin action. Palmitoleate, a fatty acid analogous to palimate with the addition of a single double bond, can protect against the deleterious effect of palmitate and intrinsically improve glucose uptake. To date, no study has assessed the impact of palmitate and palmitoleate on lipid profiles. Treatment of myotubes with palmitate and palmitoleate respectively significantly increased the content of each fatty acid with myotubes, while, only modestly altering the abundance of other fatty acid species. Palmitate reduced insulin dependent glucose uptake and palmitoleate did not have any effect. PKB activation in response to insulin was unaltered by either fatty acid. Both palmitate and palmitate increased maximal mitochondrial respiration when used at a dose of 250µM but increasing concentration substantially reduced coupled respiration and increased proton leak. These data show that accumulation of palmitate specifically and not general lipid accumulation attenuates normal insulin action. The data also suggests that reduction in PKB activation may not be the critical mechanism for the loss of insulin stimulated glucose uptake following palmitate incorporation. Obesity and acute increases in circulating fatty acids are linked with a reduction in the muscle protein synthetic response to insulin and amino acids. It was hypothesised that PA and PAO would have different effects on protein turnover through changes in protein synthesis and breakdown. Incorporation of palmitate into myotubes resulted in the significant decrease in the basal protein synthesis while protein breakdown was unchanged. PAO did not alter protein synthesis or breakdown. Palmitate increased phosphorylation of ribosomal protein S6, a readout of P70S6K1 activity despite reduced protein synthesis. Eif2α phosphorylation was not altered by either fatty acid, indication no changes in endoplasmic reticulum stress. Proteomic analysis revealed that both fatty acids altered the protein abundance of a number of different proteins but no changes in proteins associated with muscle anabolism were detected. Despite increasing anabolic signalling, increasing palmitate accumulation resulted in depressed protein synthesis independent of changes in ER stress. Collectively, these data show that minor differences in fatty acid structure can elicit divergent metabolic activities in skeletal muscle. This may occur by altering the cell microenvironment through changes in lipid profiles, protein abundance and associated with cell membranes. The addition of a just a single double bond in the fatty acyl chain can prevent deleterious effects on glucose and protein metabolis

Differential localization and anabolic responsiveness of mTOR complexes in human skeletal muscle in response to feeding and exercise

Mechanistic target of rapamycin (mTOR) resides as two complexes within skeletal muscle. mTOR complex 1 (mTORC1-Raptor positive) regulates skeletal muscle growth, whereas mTORC2 (Rictor positive) regulates insulin sensitivity. To examine the regulation of these complexes in human skeletal muscle, we utilised immunohistochemical analysis to study the localisation of mTOR complexes prior to and following protein-carbohydrate feeding (FED) and resistance exercise plus protein-carbohydrate feeding (EXFED) in unilateral exercise model. In basal samples, mTOR and the lysosomal marker LAMP2 were highly co-localized and remained so throughout. In the FED and EXFED states, mTOR/LAMP2 complexes were redistributed to the cell periphery (WGA positive staining) (time effect; p=.025), with 39\% (FED) and 26\% (EXFED) increases in mTOR/WGA association observed 1h post-feeding/exercise. mTOR/WGA colocalisation continued to increase in EXFED at 3h (48\% above baseline) whereas colocalisation decreased in FED (21\% above baseline). A significant effect of condition (p=.05) was noted suggesting mTOR/WGA co-localization was greater during EXFED. This pattern was replicated in Raptor/WGA association, where a significant difference between EXFED and FED was noted at 3h post-exercise/feeding (p=.014). Rictor/WGA colocalization remained unaltered throughout the trial. Alterations in mTORC1 cellular location coincided with elevated S6K1 kinase activity, which rose to a greater extent in EXFED compared to FED at 1h post-exercise/feeding (p<.001), and only remained elevated in EXFED at the 3h time point (p=.037). Collectively these data suggest that mTORC1 redistribution within the cell is a fundamental response to resistance exercise and feeding, whereas mTORC2 is predominantly situated at the sarcolemma and does not alter localisation

Multiple AMPK activators inhibit L-Carnitine uptake in C2C12 skeletal muscle myotubes

Mutations in the gene that encodes the principal L-Carnitine transporter, OCTN2, can lead to a reduced intracellular L-Carnitine pool and the disease Primary Carnitine Deficiency. L-Carnitine supplementation is used therapeutically to increase intracellular L-Carnitine. As AMPK and insulin regulate fat metabolism and substrate uptake we hypothesised that AMPK activating compounds and insulin would increase L-Carnitine uptake in C2C12myotubes. The cells express all three OCTN transporters at the mRNA level and immunohistochemistry confirmed expression at the protein level. Contrary to our hypothesis, despite significant activation of PKB and 2DG uptake, insulin did not increase L-Carnitine uptake at 100nM. However, L-Carnitine uptake was modestly increased at a dose of 150nM insulin. A range of AMPK activators that increase intracellular calcium content [caffeine (10mM, 5mM, 1mM, 0.5mM), A23187 (10μM)], inhibit mitochondrial function [Sodium Azide (75μM), Rotenone (1μM), Berberine (100μM), DNP (500μM)] or directly activate AMPK [AICAR (250μM)] were assessed for their ability to regulate L-Carnitine uptake. All compounds tested significantly inhibited L-Carnitine uptake. Inhibition by caffeine was not dantrolene (10μM) sensitive. Saturation curve analysis suggested that caffeine did not competitively inhibit L-Carnitine transport. However, the AMPK inhibitor Compound C (10μM) partially rescued the effect of caffeine suggesting that AMPK may play a role in the inhibitory effects of caffeine. However, caffeine likely inhibits L-Carnitine uptake by alternative mechanisms independently of calcium release. PKA activation or direct interference with transporter function may play a role

Omega-3 Fatty Acids and Skeletal Muscle Health

Skeletal muscle is a plastic tissue capable of adapting and mal-adapting to physical activity and diet. The response of skeletal muscle to adaptive stimuli, such as exercise, can be modified by the prior nutritional status of the muscle. The influence of nutrition on skeletal muscle has the potential to substantially impact physical function and whole body metabolism. Animal and cell based models show that omega-3 fatty acids, in particular those of marine origin, can influence skeletal muscle metabolism. Furthermore, recent human studies demonstrate that omega-3 fatty acids of marine origin can influence the exercise and nutritional response of skeletal muscle. These studies show that the prior omega-3 status influences not only the metabolic response of muscle to nutrition, but also the functional response to a period of exercise training. Omega-3 fatty acids of marine origin therefore have the potential to alter the trajectory of a number of human diseases including the physical decline associated with aging. We explore the potential molecular mechanisms by which omega-3 fatty acids may act in skeletal muscle, considering then-3/n-6 ratio, inflammation and lipidomic remodelling as possible mechanisms of action. Finally, we suggest some avenues for further research to clarify how omega-3 fatty acids may be exerting their biological action in skeletal muscle

Lipid remodelling and an altered membrane proteome may drive the effects of EPA and DHA treatment on skeletal muscle glucose uptake and protein accretion

In striated muscle, EPA and DHA have differential effects on the metabolism of glucose and differential effects on the metabolism of protein. We have shown that, despite similar incorporation, treatment of C2C12 myotubes (CM) with EPA but not DHA improves glucose uptake and protein accretion. We hypothesized that these differential effects of EPA and DHA may be due to divergent shifts in lipidomic profiles leading to altered proteomic profiles. We therefore carried out an assessment on the impact of treating CM with EPA and DHA on lipidomic and proteomic profiles. FAME analysis revealed that both EPA and DHA led to similar but substantial changes in fatty acid profiles. Global lipidomic analysis showed that EPA and DHA induced large alterations in the cellular lipid profiles and in particular, the phospholipid classes. Subsequent targeted analysis confirmed that the most differentially regulated species were phosphatidylcholines and phosphatidylethanolamines containing long chain fatty acids with 5 (EPA treatment) or 6 (DHA treatment) double bonds. As these are typically membrane associated lipid species we hypothesized that these treatments differentially altered the membrane-associated proteome. SILAC based proteomics of the membrane fraction revealed significant divergence in the effects of EPA and DHA on the membrane associated proteome. We conclude that the EPA specific increase in polyunsaturated long chain fatty acids in the phospholipid fraction is associated with an altered membrane associated proteome and these may be critical events in the metabolic remodelling induced by EPA treatment

Postexercise High-Fat Feeding Supresses p70S6K1 Activity in Human Skeletal Muscle.

PURPOSE: To examine the effects of reduced CHO but high post-exercise fat availability on cell signalling and expression of genes with putative roles in regulation of mitochondrial biogenesis, lipid metabolism and muscle protein synthesis (MPS). METHODS: Ten males completed a twice per day exercise model (3.5 h between sessions) comprising morning high-intensity interval (HIT) (8 x 5-min at 85% VO2peak) and afternoon steady-state (SS) running (60 min at 70% VO2peak). In a repeated measures design, runners exercised under different isoenergetic dietary conditions consisting of high CHO (HCHO: 10 CHO, 2.5 Protein and 0.8 Fat g.kg per whole trial period) or reduced CHO but high fat availability in the post-exercise recovery periods (HFAT: 2.5 CHO, 2.5 Protein and 3.5 Fat g.kg per whole trial period). RESULTS: Muscle glycogen was lower (P<0.05) at 3 (251 vs 301 mmol.kgdw) and 15 h (182 vs 312 mmol.kgdw) post-SS exercise in HFAT compared to HCHO. AMPK-α2 activity was not increased post-SS in either condition (P=0.41) though comparable increases (all P<0.05) in PGC-1α, p53, CS, Tfam, PPAR and ERRα mRNA were observed in HCHO and HFAT. In contrast, PDK4 (P=0.003), CD36 (P=0.05) and CPT1 (P=0.03) mRNA were greater in HFAT in the recovery period from SS exercise compared with HCHO. p70S6K activity was higher (P=0.08) at 3 h post-SS exercise in HCHO versus HFAT (72.7 ± 51.9 vs 44.7 ± 27 fmol.min mg). CONCLUSION: Post-exercise high fat feeding does not augment mRNA expression of genes associated with regulatory roles in mitochondrial biogenesis though it does increase lipid gene expression. However, post-exercise p70S6K1 activity is reduced under conditions of high fat feeding thus potentially impairing skeletal muscle remodelling processes

Kv1.3 inhibitors have differential effects on glucose uptake and AMPK activity in skeletal muscle cell lines and mouse ex vivo skeletal muscle

Knockout of Kv1.3 improves glucose homeostasis and confers resistance to obesity. Additionally, Kv1.3 inhibition enhances glucose uptake. This is thought to occur through calcium release. Kv1.3 inhibition in T-lymphocytes alters mitochondrial membrane potential, and, as many agents that induce Ca2+release or inhibit mitochondrial function activate AMPK, we hypothesised that Kv1.3 inhibition would activate AMPK and increase glucose uptake. We screened cultured muscle with a range of Kv1.3 inhibitors for their ability to alter glucose uptake. Only Psora4 increased glucose uptake in C2C12myotubes. None of the inhibitors had any impact on L6 myotubes. Magratoxin activated AMPK in C2C12myotubes and only Pap1 activated AMPK in the SOL. Kv1.3 inhibitors did not alter cellular respiration, indicating a lack of effect on mitochondrial function. In conclusion, AMPK does not mediate the effects of Kv1.3 inhibitors and they display differential effects in different skeletal muscle cell lines without impairing mitochondrial function

Rapamycin does not prevent increases in myofibrillar or mitochondrial protein synthesis following endurance exercise

The present study aimed to investigate the role of the mechanistic target of rapamycin complex 1 (mTORC1) in the regulation of myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis following endurance exercise. Forty-two female C57BL/6 mice performed 1h of treadmill running (18mmin&minus;1; 5&deg; grade), 1h afteri.p.administration of rapamycin (1.5mg &middot; kg&minus;1) or vehicle. To quantify skeletal muscle protein fractional synthesis rates, a flooding dose (50mg &middot; kg&minus;1) ofl-[ring-13C6]phenylalanine was administered viai.p.injection. Blood and gastrocnemius muscle were collected in non-exercised control mice, as well as at 0.5, 3 and 6h after completing exercise (n=4 per time point). Skeletal muscle MyoPS and MitoPS were determined by measuring isotope incorporation in their respective protein pools. Activation of the mTORC1-signalling cascade was measured via direct kinase activity assay and immunoblotting, whereas genes related to mitochondrial biogenesis were measured via a quantitative RT-PCR. MyoPS increased rapidly in the vehicle group post-exercise and remained elevated for 6h, whereas this response was transiently blunted (30min post-exercise) by rapamycin. By contrast, MitoPS was unaffected by rapamycin, and was increased over the entire post-exercise recovery period in both groups (P&lt;0.05). Despite rapid increases in both MyoPS and MitoPS, mTORC1 activation was suppressed in both groups post-exercise for the entire 6h recovery period. Peroxisome proliferator activated receptor-&gamma; coactivator-1&alpha;, pyruvate dehydrogenase kinase 4 and mitochondrial transcription factor A mRNA increased post-exercise (P&lt;0.05) and this response was augmented by rapamycin (P&lt;0.05). Collectively, these data suggest that endurance exercise stimulates MyoPS and MitoPS in skeletal muscle independently of mTORC1 activation