Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: INTRODUCTION: Obesity and its associated complications such as diabetes and cardiovascular disease are escalating worldwide. Cardiovascular mortality and the occurrence of heart failure following a myocardial infarction are increased among diabetics. A high caloric diet has been associated with specific metabolic alterations such as increased FA utilization and decreased glucose utilization. We therefore hypothesized that, in a rat model of diet-induced obesity, pathways involved in myocardial glucose utilization would be down regulated with simultaneous up regulation of FA utilization pathways and that this will lead to certain metabolic adaptations which will eventually become maladaptive. We aimed to elucidate mitochondrial oxidative capacity, biogenesis, and signaling pathways involved in substrate utilization and energy production in rats on the obesity inducing diet for a period of 8 or 16 weeks. METHODS: The diet of male Wistar rats (180-200 g) was supplemented with sucrose and condensed milk for 8 or 16 weeks (DIO) and compared to age-matched controls. We determined the fasting blood glucose and serum insulin levels, which was used to calculate the HOMA index. Furthermore, (i) A set of hearts were removed and freeze-clamped immediately. (ii) Isolated hearts were perfused with Krebs-Henseleit buffer (10 mM glucose), subjected to regional ischaemia by ligating the left anterior descending artery (35 min). After 10 min reperfusion, the infarcted and non-infarcted zones were freeze-clamped separately. Isolated mitochondria prepared from fresh tissue were used to measure oxidative capacity using either glutamate or palmitoyl-L-carnitine as substrates and exposed to anoxia (20 min) /reperfusion and used in e- transport chain complex analysis. Additionally we determined (i) ATP production (HPLC), (ii) citrate synthase activity and quantity as measure of active mitochondria per mg of protein and (iii) PDH complex expression and activity (ELISA). Levels of GLUT1, GLUT 4, PGC-1α, PPARα, PKB/Akt, GSK-3, PTEN, AMPK and PI3K activity were measured via Western blotting and Real-time PCR was used to measure the expression of PDK 4 and FAT/CD36. RESULTS: The blood glucose and serum insulin levels were significantly elevated in the diet group after 8 weeks of DIO. The PPARα, PGC-1α and PDK 4 levels were also significantly elevated in the diet group with no significant difference in the levels of any of the other proteins measured or the level of citrate synthase activity. After 16 weeks of DIO the citrate synthase, PTEN and p-PI3K activity was significantly reduced and the %recovery after anoxia/reperfusion when using palmitoyl was significantly increased in the diet group. There was no change in mitochondrial oxidative function in both groups after 8 and 16 weeks, as well as no difference in ATP production during state 3 respiration. CONCLUSION: The increased blood glucose and serum insulin levels as well as the increase in the HOMA index in the diet group after 8 weeks of DIO indicates that these obese animals were insulin resistant. An increase in the level of PPARα and PGC-1α expression indicates an early compensatory effect which facilitates enhanced fatty acid utilization. This is underscored by elevated levels of PDK 4. We could find no significant difference in the quantity of the PDH enzyme but there was a significant increase in the level of PDH activity in the diet group after 16 weeks of DIO. Mitochondria from the 16 weeks DIO animals were able to withstand anoxia/reperfusion and showed no malfunctioning of the electron transport chain, despite a reduction in PI3K & PTEN activity and the presence of insulin resistance. Mitochondrial biogenesis does not seem to play a significant role in the heart‟s adaptive response as there was no increase in the citrate synthase activity in both groups. We conclude that the hearts from these obese and insulin resistant rats are coping well and have adapted metabolically to compensate for any reduction in glucose oxidation. It is plausible that this initial metabolic adaptation may become maladaptive as obesity progresses. / AFRIKAANSE OPSOMMING: INLEIDING: Vetsug en die geassosieerde komplikasies, naamlik diabetes en kardiovaskulêre siektes is besig om wêreldwyd toe te neem. Kardiovaskulêre sterftes en die voorkoms van hartversaking ná 'n miokardiale infarksie is verhoog onder diabete. 'n Dieet met 'n hoë vetinname word ook geassosieer met spesifieke metabolise veranderings, soos verhoogde vetsuur gebruik en 'n afname in glukoseverbruik. Ons het dus vermoed dat seintransduksie paaie betrokke by miokardiale glukose verbruik afgereguleer sal wees met tesame opreguleering van seintransduksie paaie betrokke by vetsuur verbruik, en dat dit sal lei tot sekere metaboliese aanpassings wat uiteindelik wanaanpaslik sal word. Ons DOESTELLING in hierdie studie was dus om meer lig te werp op mitokondriale oksidatiewe kapasiteit, biogenese, en seintransduksie betrokke by substraatverbruik en energieproduksie in rotte op die vetsugveroorsakende-dieet (vir 'n tydperk van 8 tot 16 weke). METODE: Die dieet van die manlike Wistar rotte (180-200 g) is aangevul met sucrose en gekondenseerde melk vir 8 of 16 weke (DIO) en is dan vergelyk met kontrole rotte van dieselfde ouderdom. Die vastende bloedglukose-en insulienvlakke is bepaal en hierdie waardes is gebruik om die HOMA indeks te bereken. Verder is, (i) 'n stel harte verwyder en onmiddelik gevriesklamp (ii) geïsoleerde harte met Krebs-Henseleit buffer (10 mM glukose) geperfuseer en dan aan 35 min streeksiskemie en 10 min herperfusie blottgestel. Na 10 min herperfusie, is die infarct en nie-infarktsones apart gevriesklamp. Geïsoleerde mitokonderieë is voorberei van vars weefsel en is gebruik a) om oksidatiewe kapasiteit te meet met behulp van glutamaat of palmitoiel-L-karnitien as substrate of b) blootgestel aan 20 min anoksie gevolg deur heroksigenasie of c) is gebruik in elektronvervoerkettingkompleks analise. Verder is die volgende ook bepaal: (i) ATP-produksie (HPLC), (ii) sitraat sintase aktiwiteit en hoeveelheid, gemeet as maatstaf van die aktiewe mitkondrieë per mg van proteïen en (iii) PDH-komplekuitdrukking en aktiwiteit (ELISA). Vlakke van GLUT 1, GLUT 4, PGC-1 alpha, PPAR alpha, PKB/Akt, GSK-3, PTEN, AMPK, en PI 3 kinase is bepaal deur Western klad analise en “Real –time PCR” is gebruik om die uitdrukking van PDK 4 en FAT/CD 36 te bepaal. RESULTATE: Die bloedglukose- en serum insulinvlakke was beduidend verhoog in die dieetgroep na 8 weke van DIO. Die PPAR alpha, PGC-1 alpha en PDK 4 vlakke was ook beduidend verhoog in die dieetgroep met geen beduidende verskil in die vlakke van enige van die ander proteïene gemeet of die vlakke van sitraat-sintase aktiwiteit nie. Na 16 weke van DIO het die vlakke van sitraat-sintase, PTEN en p-PI 3 kinase beduidend verlaag en die % herstel na anoksie/heroksigenering (met die gebruik van palmitoiel-L-karnitien) het beduidend toegeneem in die dieetgroep. Geen ander mitokondriale veranderinge kon waargeneem word nie. Ons kon geen verandering vind in die mitokondriale oksidatiewe funksie tussen die twee groepe na 8 en 16 weke van DIO. Daar was ook geen verandering in die hoeveelheid ATP geproduseer gedurende staat 3 respirasie. GEVOLGTREKKING: Die verhoogde bloedglukose en serum insulienvlakke, sowel as die toename in die HOMA indeks in die dieetgroep na 8 weke van DIO, dui daarop dat hierdie oorgewig diere insulienweerstandig is. 'n Toename in die vlak van PPAR alpha en PGC-1 alpha uitdrukking dui op 'n vroeë kompenserende effek, wat die verhoogde verbruik van vetsure fasiliteer. Dit is beklemtoon deur verhoogde vlakke van PDK 4. Ons kon geen beduidende verskil in die hoeveelheid PDH-ensiem vind nie, maar daar was wel 'n beduidende toename in die vlak van PDH aktiwiteit in die dieetgroep na 16 weke van DIO. Mitokondrieë van die 16 weke DIO diere kon anoksie/heroksigenering weestaan en het geen wanfunksionering van die elektronvervoerketting getoon nie, ten spyte van 'n vermindering in PI 3 kinase en PTEN aktiwiteit en die teenwoordigheid van insulienweerstandigheid. Mitokondriale biogenese blyk nie 'n beduidende rol te speel in die hart se aanpassingreaksie nie, want daar was geen verhoging in die sitraat-sintase aktiwiteit in beide groepe nie. Na aanluiding van die resultate verkry met hierdie studie, maak ons ook die gevolgtrekking dat die harte van rotte na 16 weke se blootstelling aan 'n hoë vet omgewing, metabolies kon aanpas en goed funksioneer. Ons gevolgtrekking is dus dat die harte van diere wat lei aan vetsug en wat insulien weerstandig is die verlaaging in glukose metabolisme goed hanteer en metabolies aangepas het hierby. Dit is moontlik dat hierdie aanvanklike aanpasing wanaanpaslik kan word soos vetsug vorder.