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. Stewart Jeromson: PhD thesis II 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 Stewart Jeromson: PhD thesis III 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 Stewart Jeromson: PhD thesis IV 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 metabolism. Stewart Jeromson: PhD thesis V