Doctoral Degrees (Medical Physiology)

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Browsing Doctoral Degrees (Medical Physiology) by Subject "Ataxia telangiectasia"

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    An investigation into the importance of the ATM protein in the myocardial pathology associated with insulin resistance and type 2 diabetes
    (Stellenbosch : Stellenbosch University, 2017-03) Espach, Yolandi; Huisamen, Barbara; Strijdom, Hans; Engelbrecht, Anna-Mart; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology
    ENGLISH ABSTRACT : Introduction: Ataxia-telangiectasia (A-T) is an autosomal, recessive disorder that is caused by mutations in the ataxia-telangiectasia mutated (ATM) gene. The gene product, ATM, is a 350 kDa serine/threonine protein kinase with a large number of substrates in various pathways. Patients suffering from A-T display a high incidence of insulin resistance or type 2 diabetes mellitus (T2DM) and are more susceptible to ischaemic heart disease. Although it is known that the ATM protein is expressed in the heart and that structural and functional changes are observed in the hearts of ATM knock-out mice, very little research has been done on ATM and its role in insulin signalling in the cardiovascular context. This project aimed to identify and characterise an obese, insulin resistant animal model in which myocardial ATM expression is altered; to use this model to determine the effect of ATM manipulation on myocardial function and response to ischaemia/reperfusion injury (IRI); and to determine the effect of ATM manipulation on insulin signalling networks. Methodology: Male Wistar rats received a high caloric diet (HCD) or standard rat chow (control) for 16 weeks after which biometric data was collected. ATM was manipulated in perfusion studies prior to ischaemia using insulin (activator) and KU-60019 (inhibitor). Hearts perfused using the Langendorff balloon model were subjected to global ischaemia followed by reperfusion and Western blots were performed to evaluate insulin signalling intermediates. Hearts were also perfused using the working mode and subjected to regional ischaemia. Following reperfusion, infarct size (IFS) was determined. Aortic ring isometric tension studies were performed to determine the effect of KU-60019 on vasodilation. Results: The HCD resulted in significantly increased body mass, visceral fat mass, glucose levels, insulin levels and HOMA-IR index compared to the control diet and ATM expression was reduced in the HCD hearts. Cardiac function and IFS were comparable in the control and HCD hearts. In control hearts, insulin administration activated the insulin signalling network prior to ischaemia and cardiac function was improved during reperfusion. Insulin had no effect on the insulin signalling network or cardiac function in the insulin resistant HCD hearts. High concentrations of insulin increased IFS in both the control and HCD hearts. ATM inhibition improved cardiac function in control and HCD hearts during early reperfusion but had no effect on cardiac function during later reperfusion. ATM phosphorylation was increased by insulin and decreased by KU-60019 in control hearts, but could not be manipulated in HCD hearts. Insulin-stimulated PKB/Akt activation is not ATM-dependent in the heart. However, ATM inhibition appears to down-regulate insulin signalling via PI3K, PTEN and GSK-3β. ATM inhibition caused NO-dependent vasodilation in control hearts, suggesting a role for ATM in vasoconstriction. Conclusion: ATM is a complex signalling regulator with numerous substrates. In our study, we found that acute cardiac ATM inhibition did not result in significant cardiac dysfunction or complete abrogation of insulin signalling. However, we found that ATM possibly plays a significant role in vasoconstriction. More research needs to be done to fully understand the cardiac role of ATM in insulin signalling.
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    An investigation into the role of ATM protein in mitochondrial defects associated with cardiovascular pathology resulting from insulin resistance
    (Stellenbosch : Stellenbosch University, 2019-04) Blignaut, Marguerite; Huisamen, Barbara; Lochner, Amanda; Engelbrecht, Anna-Mart; Van Vuuren, Derick; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.
    Background: Ataxia-telangiectasia (A-T) is a rare, recessive disorder that develops in the absence of Ataxia-Telangiectasia Mutated protein kinase (ATM). This complex disease is characterised by neurodegeneration, increased risk of cancer, a high incidence of insulin resistance and Type 2 diabetes (T2D) as well as cardiovascular disease. Mitochondrial dysfunction is associated with the development of cardiomyopathy and T2D. Obesity and insulin resistance contribute towards the development of cardiac dysfunction, and have been linked with mitochondrial disturbances. ATM has previously been associated with mitochondria, and the absence thereof results in decreased mitochondrial respiration and ATP synthesis as well as structural abnormalities. This study aimed to investigate the role of ATM in mitochondrial oxidative phosphorylation and mitophagy as possible contributors towards cardiovascular dysfunction in obesity. Methodology: The effects of high palmitic and oleic acid, either alone or in combination with insulin or high glucose on ATM expression levels were investigated in an H9c2 cardiomyoblast model. Insulin signal transduction intermediates were determined by western blotting and glucose uptake by [3H]2-deoxyglucose accumulation. Mitoplasts were prepared from cardiac mitochondria of male Wistar rats with digitonin and the quality thereof confirmed with transmission electron microscopy (TEM). The location of ATM was determined with Super-resolution structured illumination microscopy (SR-SIM) and western blotting. Oxidative phosphorylation (oxphos) analysis was performed polarographically (Clark-type electrode) on mitochondria obtained from 1) young male Wistar rat hearts perfused with the ATM specific inhibitor, KU60019 or vehicle (DMSO), and 2) chow fed age-matched controls and diet induced obese (DIO) rat hearts perfused ex vivo with either DMSO or ATM-specific activators and its inhibitor (KU60019 ± insulin or KU60019 ± chloroquine). Oxphos was determined in carbohydrate (glutamate+malate) or fatty acid (palmitoyl-L-carnitine+malate) substrates while protein expression levels of ATM as well as markers of mitophagy and mitochondrial fission were measured (western blotting). The redox status of NAD(P)H was determined with 2-photon fluorescence lifetime imaging microscopy in H9c2 cells ± KU60019. Results: This study showed that: (i) high levels of fatty acids and insulin affects the expression levels of ATM; (ii) ATM is located on the inner mitochondrial membrane of cardiac mitochondria. iii) Inhibition of ATM decreased carbohydrate-stimulated oxidative phosphorylation in cardiac mitochondria (p=0.0024), potentially through Complex I. This was supported by the observation that ATM inhibition decreased NADPH (p=0.02) and increased NADH accumulation in H9c2 cells. iv) The effect of ATM inhibition on oxidative phosphorylation was not influenced by diet; v) ATP synthesis as well as respiratory control index improved with the addition of insulin (p<0.005). vi) The inhibition of ATM was associated with decreased fission (p=0.0038). vii) The decrease in the autophagosomal membrane marker, LC3-II (p<0.0001) seems to be associated with the cytosolic role of ATM. Conclusion: ATM is located on the inner mitochondrial membrane and inhibition thereof influences mitochondrial ATP synthesis, potentially through Complex I substrate oxidation. Inhibition of ATM did not affect oxidative phosphorylation in obesity, but resulted in mitochondrial autophagy disruption as well as decreased fission.