Medicine (St Vincent's) - Theses
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ItemRole of AMP-activated protein kinase in regulating skeletal muscle metabolism during exerciseO'Neill, Hayley Maree ( 2013)AMP-activated protein kinase (AMPK) αβγ heterotrimer is an evolutionary conserved serine/ threonine stress sensing kinase that once activated, by low energy status (nutrient deprivation, exercise), restores energy balance by switching off ATP consuming pathways (fatty acid and cholesterol synthesis) and switching on ATP generating pathways (fatty acid oxidation, glucose uptake, mitochondrial biogenesis). Acetyl-CoA carboxylase (ACC; 1 and 2) catalyzes the carboxylation of acetyl-CoA to malonyl-CoA; a precursor for fatty acid synthesis, and an inhibitor of carnitine palmitoyltransferase-1, which controls transport of fatty acids into mitochondria for oxidation. ACC was one of the first identified substrates of AMPK, and phosphorylation of ACC1 at S79 (equivalent S212 site on ACC2) inhibits enzyme activity, reduces malonyl-CoA and suppresses de novo fatty acid synthesis in liver, where ACC1 is predominantly expressed. No studies have assessed the contribution of ACC2 S212 phosphorylation in regulating skeletal muscle fatty acid oxidation, where ACC2 is predominantly expressed. Skeletal muscle is a major contributor to whole-body energy expenditure and is responsible for ~80% of insulin-stimulated glucose disposal; therefore, metabolic alterations in this tissue could influence whole-body insulin sensitivity and substrate utilization. Pharmacological activation of AMPK with aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR) enhances fatty acid oxidation, glucose uptake, insulin sensitivity and mitochondrial biogenesis in skeletal muscle; however, AMPK deficient mouse models where a single subunit has been mutated or deleted show a relatively minor role for AMPK in regulating these processes at rest and during exercise. An important caveat of these studies is that AMPK activity is partially suppressed due to presence of the alternative subunit isoform. Interleukin-6 (IL-6) is produced and released from skeletal muscle during exercise, and like AICAR and contraction, increases AMPK activity and is Geffects of IL-6 on substrate utilization during exercise and insulin-sensitivity post-exercise. Therefore, to assess the contribution of AMPK, ACC2 and IL-6 in regulating substrate utilization at rest and during exercise we generated muscle-specific AMPK β1β2 null (M-KO) and whole-body ACC2 S212A knockin (KI) mice and utilized IL-6 KO mice. Under resting conditions, all mice had similar body and tissue weights, oxygen consumption, substrate utilization and activity levels compared to WT littermates. AMPK β1β2 M-KO mice had normal insulin sensitivity despite reduced mitochondria content (~30%) and impaired exercise tolerance (~95%) and glucose uptake during exercise/ muscle contraction (~55 to 70%). IL-KO mice had similar exercise tolerance and muscle glucose clearance during steady-state submaximal treadmill exercise. Muscles from ACC2 S212A KI mice tended to oxidize less fatty acids, which resulted in greater accumulation of muscle ceramides and whole-body and skeletal muscle insulin resistance. ACC2 KI mice were also resistant to AICAR effects on lowering ACC2 activity and enhancing fatty acid oxidation. When challenged with a high-fat diet (HFD) for 12 weeks or exercise, ACC2 KI responded similar to WT, suggesting that AMPK phosphorylation of ACC2 S212 is not essential for metabolic control in response exercise or a HFD. Collectively, these studies highlight that AMPK is important for mitochondrial content, exercise capacity and insulin-independent regulation of glucose uptake during exercise; however, IL-6 or AMPK/ACC2 signaling is not essential for regulating substrate utilization during exercise, and alternative pathways are involved.
ItemMetabolic consequences of lipid-oversupply in key glucoregulatory tissues.Turpin, Sarah Maggie ( 2009)Obesity and type 2 diabetes are the most prevalent metabolic diseases in the western world and affect over 50% of the world’s population. During obesity non-adipose tissues such as the liver and skeletal muscle take up and store excess fatty acids (FA) as lipids such as triacylglycerols (TAG) and diacylglycerols (DAG). Excessive lipid storage in non-adipose tissues can result in the dysfunction of cellular processes and lead to programmed cell death (apoptosis). Lipid-induced apoptosis was investigated in the key glucoregulatory tissues, the liver and skeletal muscle. Lipid-induced apoptosis was detected in vitro in both hepatocytes and myotubes but was not detected in the livers or skeletal muscles of genetically obese mice or high-fat fed mice. Further investigation discovered despite exacerbated TAG accumulation, endoplasmic reticulum stress (ER) was not activated in the liver and pathways of cellular remodelling (proteolysis and autophagy) were not initiated in skeletal muscle. These studies demonstrated that the liver and skeletal muscle are adaptable to increased lipid storage in physiological models but not isolated cell culture systems. In vitro experiments demonstrated unsaturated FAs could protect hepatocytes from lipoapoptosis and it has been suggested this is due to driving FA accumulation into TAG lipid droplets. Adipose triglyceride lipase (ATGL) is one of the primary TAG lipases. To explore TAG metabolism in the liver, primary hepatocytes were derived from ATGL null mice and ATGL was over-expressed in the livers of chronically obese mice. It was found that cellular FA uptake and TAG esterification was increased and TAG lipolysis and FA oxidation were decreased in the ATGL null hepatocytes. This resulted in exacerbated TAG and diacylglycerol (DAG) storage. The gene expression of metabolic regulators such as cytochrome c oxidase subunit 2 (COX2), medium chain acyl Co-A dehydrogenase (MCAD), peroxisome proliferators-activated receptor co-activator 1! (PGC1!), nuclear respiratory factor 1 (NRF1) and FA translocase/cluster of differentiation 36 (FAT/CD36) were increased in ATGL null hepatocytes compared with wild type hepatocytes, suggesting that the reduction in FA oxidation in the ATGL null hepatocytes was probably due to limited FA substrate availability. Interestingly, despite increased TAG and DAG, the hepatocytes remained insulin sensitive. To investigate hepatic ATGL over-expression an adenovirus containing an ATGL insert was injected into chronic high fat fed mice. Hepatic ATGL over-expression in the iii chronically obese mice reduced TAG, DAG and ceramide content in the liver. This resulted in improved hepatic insulin signalling and whole body insulin sensitivity. In summary, studies from this thesis suggested the use of in vitro systems are not a substitute for in vivo models when assessing the toxic effects of lipid oversupply, TAG accumulation may be a protective mechanism against cellular remodelling and programmed cell death, and increased ATGL expression in the liver can reduce hepatic steatosis and enhance whole body insulin sensitivity. Therefore, increasing hepatic ATGL expression could be a therapeutic approach to treat obesity and type 2 diabetes.