Doctoral Degrees (Medical Physiology)
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Browsing Doctoral Degrees (Medical Physiology) by Author "Fan, WenJun"
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- ItemEffect of different substrates on functional performance and kinase activation during reperfusion after ischaemia in hearts from obese insulin resistant rats(Stellenbosch : Stellenbosch University, 2017-03) Fan, WenJun; Huisamen, Barbara; Lochner, Amanda; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical PhysiologyENGLISH ABSTRACT : Obesity is an important risk factor for the development of insulin resistance, the metabolic syndrome and diabetes and has also been implicated as one of the major risk factors for coronary heart disease. Ischaemic heart disease impacts on both cardiac metabolism and function. During early reperfusion after ischaemia, several protein kinases are specifically activated, including PI3K/PKB, MAPKs (ERK, JNK and p38 MAPK), and the tyrosine kinases. Activation of PKB and ERK, the so-called reperfusion injury salvage kinase (RISK) pathway, is associated with a reduction in infarct size and/or improvement in functional recovery. PKB is an enzyme central also to insulin signalling and glucose uptake. Activation of the JNK signaling pathway has been suggested to be a prerequisite for PKB activation; however, its role in ischaemia/reperfusion (I/R) injury remains controversial. We hypothesize that obesity-induced insulin resistance will affect infarct size, functional recovery and interactions between PKB, JNK, ERK, p38MAPK and PTEN activation during reperfusion after exposure to ischaemia. The aim of the study was therefore to assess the effects of hyperphagia-induced obesity and insulin resistance in rats on the response of the heart to I/R injury, with particular attention to the intracellular signalling pathways during early reperfusion. To further elucidate the role of JNK, we used SP600125, a specific inhibitor of JNK. Methods: Insulin resistance was induced by feeding rats a high caloric diet for 16 weeks (DIO). Hearts from DIO and age-matched controls (C) were perfused in the working mode (preload 15cm H2O; afterload 100cm H2O) and subjected to (i) 15 min global ischaemia followed by different reperfusion times for evaluation of functional recovery and freeze-clamping of tissues for Western blot or (ii) 35 min regional ischaemia followed by 2 hours reperfusion for infarct size determination (IS), using tetrazolium staining. Substrates were glucose (G) (10mM), glucose (10mM) plus BSA (3%) (G+B), and glucose (10mM) plus fatty acid (1.2mM palmitate / 3% BSA) (G+FA). The JNK inhibitor, SP600125, was administered either before ischaemia or during reperfusion after ischaemia. Infarct size, functional recovery as well as expression and activation of PKB, ERK, JNK, p38MAPK and PTEN were used as endpoints. Results: (I) In the presence of glucose alone as substrate, the hearts from DIO rats exhibited an improved tolerance to ischaemia/reperfusion (I/R) injury as reflected by an increase in functional recovery (after exposure to 15 min global ischaemia) as well as a reduction in infarct size (after 35 min regional ischaemia) compared with the age-matched controls. This was associated with early activation of PKB and JNKp54/p46 at 10 min reperfusion, with down regulation of activation of these kinases after 30 min reperfusion. (II) Contrary to expectations, the combination of a high concentration of fatty acids and glucose as substrates (G+FA) afforded significantly more protection against I/R injury in hearts from both DIO and control rats, when compared with the respective groups perfused with glucose alone as substrate. This improved protection in both groups was associated with increased activation of the PKB pathway. Interestingly, perfusion with glucose and a high concentration of fatty acid maintained PKB activation throughout the reperfusion phase, in contrast to the transient activation seen with glucose alone as substrate. (III) SP600125 (10 uM), administrated either before ischaemia or during early reperfusion after ischaemia, almost completely inhibited the JNK pathway and exacerbated myocardial I/R injury, particularly in hearts from DIO rats. Conclusion: Our study demonstrates, in contrast to several other studies, that dietary-induced obesity and high perfusate fatty acid concentrations, increase the tolerance of the ex vivo myocardium to I/R injury. It was also found that, contrary to expectations, a high concentration of circulating fatty acid was not detrimental to hearts of normal rats during I/R, indicating the beneficial actions of fatty acids on the outcome of I/R injury. This protection was shown to be associated with activation of PKB and JNK during early reperfusion. Administration of the selective JNK inhibitor, SP600125, before or after myocardial ischaemia indicates that JNK and its downstream signalling pathways are critical in mediating protection against I/R in our study. SP-induced effects were also associated with lower activation of PKB. Our results suggest that the cross-talk between the JNK and PKB pathways in the post-ischaemic myocardium may be a major contributing factor to the outcome of I/R injury The data presented here, although seemingly dichotomous, actually solidify the hypothesis that JNK signalling specifically and simultaneously modulates pro- and antiapoptotic effector mechanisms within cardiomyocytes. They also reflect an extraordinary complexity of the heart‘s metabolic, functional, and structural changes in obesity. In addition, the results obtained showed that moderate hyperphagia-induced obesity does not have a harmful effect on the ischaemic-reperfused heart and in fact, reduced the sensitivity of the heart to I/R damage. This was further substantiated by the beneficial effects of fatty acids in the perfusate. Taken together, our results are potentially of clinical significance, and confirm the importance of events during early reperfusion as possible therapeutic targets.