Medicine (St Vincent's) - Theses
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ItemMetabolic consequences of lipid-oversupply in key glucoregulatory tissues.( 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.
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ItemOver-expression of human CD39 in mouse liver protects against ischemia reperfusion injury in a model of liver transplantation( 2009)Primary graft non-function is one of the major limitations of organ transplantation increasing the risk of rejection and early graft failure. A major cause of primary non-function is ischemia reperfusion injury (IRI), an obligatory insult in transplantation. During procurement, the donor is subjected to a period of ischemia inducing the release of tissue-damaging factors such as nitric oxide and reactive oxygen species. Upon engraftment and reperfusion with the recipient blood, these ischemia-induced factors cause rapid cell death and amplification of the inflammatory response leading to further tissue damage. CD39 is an integral vascular and immune ectonucleotidase. CD39 hydrolyses extracellular nucleotides ATP and ADP into AMP, which is then hydrolysed into adenosine by CD73. Extracellular adenosine produced by the concerted action of CD39 and CD73 has potent anti-inflammatory and anti-coagulation effects acting principally via the purinergic adenosine receptor A2a. NKT cells have only recently been recognised and constitute an important subset of T lymphocytes that display both effector and suppressive functions. NKT cells are found in high proportion in the liver of mice and are implicated by depletion studies in protection against hepatic IRI. We have generated mice transgenic for human CD39 (hCD39) and have shown they have an anti-coagulant phenotype. As CD39 is also critical to immune regulation we hypothesised that transgenic expression of hCD39 would modify lymphocyte development and/or function and consequently impact on ischemia reperfusion injury. Flow cytometric analysis was used to assess the number and phenotype of lymphocytes within the thymus and in the periphery of hCD39 transgenic mice. In vitro and in vivo assays were used to test the function of CD4+ T cells and invariant NKT cells from hCD39 transgenic mice. Bone marrow adoptive transfers experiments defined the role of hCD39 expression on bone marrow progenitor cells in comparison to tissue expression. The importance of adenosine signalling through the A2a receptor was studied by crossing hCD39 transgenic mice with A2a receptor knock-out (KO) mice. The effect of hCD39 expression on ischemia reperfusion injury was evaluated in a model of murine liver transplantation A high level of hCD39 expression in the transgenic thymus resulted in lymphocyte maturation blockade and peripheral lymphopenia of CD4+ T cells and invariant NKT cells. Both lymphocyte populations were functionally deficient. The observed phenotype resulted from the expression of hCD39 on bone marrow progenitor cells but was independent of A2a receptor signalling. Over-expression of hCD39 in transgenic livers was protective against ischemia reperfusion injury induced by cold storage and liver transplantation.