Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: Introduction: It is well documented that insulin offers cardioprotection under ischaemic stress. In the past it was believed that the protective effects of insulin, such as the (a) recruitment of glucose transporters to enhance glucose entry into the cell, (b) stimulation of glycolysis, (c) enhancement of glycogen synthesis, (d) improved protein synthesis, and (e) positive inotropic and chronotropic properties, were metabolic of origin, but lately the emphasis has shifted towards the diverse signal transduction pathways elicited by insulin. Although these beneficial effects of insulin on ischaemia/reperfusion induced injury have been studied for many years, the exact protective mechanism is still not resolved. Aim: To investigate the influence of insulin on the signaling pathways as a possible protective mechanism against ischaemia/reperfusion and therefore to investigate the possible roles and cross signaling of cyclic adenosine monophosphate (cAMP), protein kinase B (PKB) and p38 mitogen activated protein kinase (p38 MAPK) in the cardioprotection offered by insulin to the reperfused, ischaemic myocardium. Materials and methods: Isolated rat hearts were perfused retrogradely in accordance with the Langendorff technique (95%02, 5% C02). After 30 min of stabilization, hearts were subjected to 30 min global low flow ischaemia (0,2 ml/min), followed by 30 min of reperfusion. Hearts perfused with standard Krebs Henseleit solution containing 5 mM glucose were compared to hearts perfused with a perfusion solution containing 5 mM glucose and 0,3 IlIU/ml insulin. Wortmannin was added during either ischaemia or reperfusion. Left ventricular developed pressure (LVDP), rate pressure product (RPP), tissue cAMP and PKB and p38 MAPK activation were measured. Results: Insulin treated hearts showed improved functional recovery (P<0.05) during reperfusion after ischaemia vs. non-insulin treated hearts (85.5±4.6% vs. 44.8±4.9%). However, the addition of wortmannin (a Pl3-kinase inhibitor) to the perfusion solution during either ischaemia or reperfusion abolished the improved recovery. At the end of ischaemia, cAMP levels of the insulin treated hearts were elevated significantly, while the cAMP content in the non-insulin treated hearts returned to control levels. Addition of wortmannin during ischaemia abolished this rise in cAMP. Wortmannin added during reperfusion only did not alter the levels of cAMP at the end of reperfusion. Activation of p38 MAPK was transient during ischaemia for both insulin and non-insulin treated hearts. Addition of wortmannin during ischaemia did not alter p38 MAPK levels at the end of ischaemia. P38 MAPK was activated significantly (P<0.001) in the non-insulin treated hearts vs. insulin treated hearts during reperfusion. Wortmannin, added at the onset of reperfusion, could partially abolish the effects of insulin to suppress p38 MAPK activation after 30 min of reperfusion. Activation of PKB in insulin treated hearts was significantly higher than non-insulin treated hearts during stabilization and early ischaemia. This activity was depressed by 30 min of ischaemia in both presence and absence of insulin. Wortmannin, when added before induction of ischaemia did not further lower this. The presence of insulin resulted in occurrence of strong PKB activation during reperfusion, peaking at 15 minutes and diminishing at 30 minutes. Wortmannin, added at the onset of reperfusion, abolished PKB activity measured at the end of reperfusion. Conclusion: Insulin exerted a positive inotropic effect and delayed the onset to ischaemic contracture. Inhibition of Pl3-kinase by wortmannin abolished the protective effects of insulin, arguing for an insulin stimulated PKB involvement in cardiac protection. Insulin also increased cAMP production and attenuated activation of p38 MAPK, both associated with improved recovery. This evidence suggested possible cross signaling between different signaling pathways. / AFRIKAANSE OPSOMMING: Agtergrond: Insulin beskerm harte wat aan isgemiese stres blootgestel word. Alhoewel hierdie voordelige effekte van insulien reeds vir verskeie jare bestudeer is, is die presiese meganisme waarmee insulien die hart beskerm steeds nie duidelik nie. Navorsers het die beskermende effekte van insulien aan metaboliese gevolge soos: (a) verhoogde glukose transport d.m.v. inspanning van meer glukose transporters (b), stimulering van glikolise, (c) vebeterde glikogeensintese, (d) verhoogde proteiensintese, en (e) die positiewe inotropiese en chronotropiese eienskappe van insulien toegeskryf. Onlangs het die fokus verskuif na ander diverse seintransduksiepaaie. Doel: Die doel van hierdie studie was dus om die moontlike betrokkenheid van hierdie sientransduksiepaaie asook die interaksie tussen sikliese adenomonofosfaat (cAMP), proteïn kinase B (PKB) en p38 MAPK in die beskerming wat insulien aan die isgemiese, gereperfuseerde miokardium bied, te bestudeer. Materiale en Metodes: Geïsoleerde rotharte is geperfuseer in ooreenstemming met die Langendorff metode. Na 30 min van stabilisasie is harte blootgestel aan 30 min. globale lae vloei isgemie (0,2 ml/min), en daarna is harte vir 30 min. geherperfuseer. Harte wat geperfuseer is met 'n perfusaat wat 5mM glukose bevat is vergelyk met harte wat geperfuseer is met 'n perfusaat wat 5mM glukose en 0,3 ~IU/ml insulien bevat. Sommige harte is geperfuseer met 'n perfusie oplossing waar wortmannin bygevoeg is tydens óf isgemie óf tydens herperfusie. Linker ventrikulêre ontwikkelde druk (LVDP), tempo-druk produk (RPP), weefsel cAMP-vlakke asook PKB en p38 MAPK aktiwiteit is gemeet. Resultate: Insulien-behandelde harte het funksioneel beduidend beter herstel tydens herperfusie na isgemie as harte wat nie met insulien behandel is nie (85.5±4.6% vs. 44.8±4.9%). Byvoeging van wortmannin by die perfusie oplossing tydens óf isgemie óf reperfusie, het die toename in herstel wat gesien is in die insulien-behandelde harte, opgehef. Die cAMP vlakke in die insulienbehandelde harte het aan die einde van isgemie beduidend gestyg (P<0.001), terwyl vlakke in harte wat nie met insulien behandel is nie, na kontrole vlakke teruggekeer het. Die teenwoordigheid van wortmannin in die perfusie oplossing tydens isgemie, het die styging in cAMP voorkom , terwyl die byvoeging van wortmannin tydens herperfusie. nie die cAMP vlakke beïnvloed het nie. Die aktivering van p38 MAPK tydens isgemie was van verbygaande aard in beide die insulien-behandelde harte en harte wat nie met insulien behandel is nie. Die byvoeging van wortmannin tydens isgemie het nie die p38 MAPK aktivering beïnvloed nie. P38 MAPK is beduidend geaktiveer tydens herperfusie in harte wat nie met insulien behandel is nie vergeleke met die insulien-behandelde harte. Die byvoeging van wortmannin tydens reperfusie kon die effek van insulien om p38 MAPK aktivering te onderdruk, gedeeltelik ophef. PKB aktivering tydens die stabilisasie fase en vroeë isgemie was beduidend hoër in die insulien-behandelde harte vs. die harte wat nie met isulien behandel is nie. Die aktiwiteit is onderdruk deur 30 min isgemie ongeag die teenwoordigheid van insulien. Die byvoeging van wortmannin tydens isgemie het PKB aktivering nie verder verlaag nie. Die teenwoordigheid van insulien het 'n sterk aktivering van PKB tydens herperfusie veroorsaak met 'n piek na 15 min en 'n verlaging na 30 min. Wortmannin bygevoeg aan die begin van herperfusie, het PKB aktiwiteit opgehef aan die einde van reperfusie. Opsomming: Insulien het 'n positiewe inotropiese invloed gehad, en het die begin van isgemiese kontraksie vertraag. Die inhibisie van Pl3-kinase deur wortmannin het die beskermende effekte van insulin opgehef, wat 'n insulin gestimuleerde PKB betrokkenheid aandui. Insulien het ook verhoogte cAMP produksie en verlaagde p38 MAPK aktivering tot gevolg gehad, en beide is geassosieer met verbeterde herstel. Hierdie resultate dui dus op moontlike interaksie tussen die verskillende seintransduksiepaaie.

Thesis (MScMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: INTRODUCTION: The mechanism behind obesity-related cardiomyopathies is at present not completely known, however, cardiac insulin resistance has been implicated as one of the main arbitrators of obesity-related cardiovascular disease. A few studies have associated perturbations in the insulin-mediated PI3K/PKB/Akt pathway in mediating this insulin resistance. Moreover, this pathway has been shown to regulate myocardial apoptosis, which in turn has been implicated in a number of cardiovascular diseases. Currently, few studies have compared the early onset and advanced effects of obesity on the heart. AIMS: To compare the early and advanced stages of obesity in terms of myocardial (i) PI3K/PKB/Akt signalling, (ii) apoptotic signalling and (iii) mitochondrial integrity. Furthermore, we aim to assess the cardiac mitochondrial (i) PI3K/PKB/Akt signalling, (ii) apoptotic signalling and (iii) integrity during the advanced stages of obesity. METHODS: Male Wistar rats were randomly assigned to either a control or diet-induced obesity (DIO) group. Controls were fed a standard rat chow diet and the DIO group fed a high caloric diet (standard rat chow supplemented with sucrose and condensed milk). The diets were implemented for either 8 or 20 weeks and thereafter, the body weight, intra-peritoneal fat mass, and fasting blood glucose and insulin levels (including intra-peritoneal glucose tolerance tests (IPGTTs)) were determined. Freeze-clamped hearts from both groups were subjected to cytosolic western blot analysis for PI3K p85 subunit, PKB/Akt, GSK-3α/β, Bad, Bax and Bcl-2. A fraction of each heart was also subjected to WB analysis of the mitochondrial electron transport chain (ETC) complexes (I-V). Thereafter, the above mentioned proteins were also probed for in mitochondria isolated from the 20 weeks group after administering insulin and exposing the hearts to ischemia. Oxidative phosphorylation (OXPHOS) capacity analysis was then conducted on mitochondria isolated from 20 weeks DIO and control groups and thereafter a citrate synthase (CS) activity assay was performed on these mitochondria. RESULTS: After the 8 and 20 weeks diet, the DIOs had significantly increased intra-peritoneal fat mass, fasting plasma glucose and insulin levels, compared to their controls. Cytosolic WB analysis: The tp85, pp85 and pPKB/Akt levels were significantly higher in the DIOs in comparison to the controls after 8 weeks of diet. Furthermore, pBad and Bax expression were significantly elevated in these animals. After 20 weeks of diet, the DIOs had significantly decreased pp85, tPKB/Akt and pPKB/Akt levels. The tBad was significantly elevated, while the Bad phosphorylated over total expression (P/T) ratio was significantly decreased, in these animals. CS activity assay: CS activity was significantly decreased in the DIOs, versus the controls, at 20 weeks. Mitochondrial ETC WB analysis: The subunit expression in complexes I-III and V did not differ significantly after 8 weeks however, the expression was significantly lower in complexes I and II after 20 weeks. Interestingly, the complexes III and V expression was significantly elevated. Mitochondrial OXPHOS analysis: The ADP/O ratio with (1) glutamate or (2) palmitoyl-L- carnitine as substrate, showed a significant decrease in the DIOs at 20 weeks. Mitochondrial WB analysis: The pp85 subunit was significantly elevated in the control and DIO groups, exposed to insulin and ischemia, in comparison to the untreated controls. The Bcl-2 levels were significantly decreased in the insulin and ischemia DIOs, when matched against the untreated DIOs. The tBad expression did not differ significantly between the insulin and untreated controls, while the tBad was significantly augmented in the ischemia controls versus untreated controls. All significant differences were taken as p<0.05. CONCLUSION: The results indicate that the initial stage of diet-induced obesity is associated with cardioprotection as there is augmented PI3K/PKB/Akt pathway signalling and a decrease in apoptotic markers. In contrast, during the advanced stages of obesity a decreased activity in PI3K/PKB/Akt pathway is associated with myocardial apoptosis and decreased mitochondrial function and integrity. / AFRIKAANSE OPSOMMING: INLEIDING: Die meganisme verantwoordelik vir vetsug-verwante kardiomiopatieë is huidiglik nie bekend nie maar kardiale insulienweerstandigheid word geïmpliseer as een van die hoof bemiddelaars van vetsug-verwante hartsiektes. Verskeie studies het versteurings in die insulien-gemediëerde PI3K/PKB/Akt pad geassosieer met die bevordering van hierdie insulienweerstandigheid. Daarbenewens is dit getoon dat hierdie pad betrokke is in die regulering van miokardiale apoptose, wat op sy beurt geïmpliseer is in 'n aantal kardiovaskulêre siektes. Daar is tans min studies beskikbaar wat die vroeë en laat gevolge van obesiteit op die hart vergelyk. DOELWITTE: Om die vroeë en gevorderde stadiums van vetsug te vergelyk in terme van miokardiale (i) PI3K/PKB/Akt seintransduksie, (ii) apoptotiese seintransduksie en (iii) mitokondriale integriteit. Verder, het die studie ten doel om die kardiale mitokondriale (i) PI3K/PKB/Akt en (ii) apoptotiese seintransduksie en (iii) integriteit in die gevorderde stadiums van vetsug te bepaal. METODES: Manlike Wistar rotte is ewekansig toegewys aan óf 'n kontrole of dieet-geïnduseerde vetsug (DIO) groep. Kontroles is met 'n normale rotkos dieet en die DIO groep met 'n hoë kalorie dieet (normale rotkos aangevul met sukrose en kondensmelk) gevoed. Die dieet is vir 8 of 20 weke volgehou en daarna was die liggaamsgewig, intra-peritoneale vet massa, en vastende bloed glukose en insulien vlakke (insluitende intra-peritoneale glukose toleransie toets (IPGTT`s)) bepaal. Gevriesklampte harte van beide groepe is onderwerp aan sitosoliese WB-analise vir die PI3K p85 subeenheid, PKB / Akt, GSK-3α/β, Bad, Bax en Bcl-2. `n Fraksie van hierdie harte is ook onderwerp aan westerse klad analise (WK-analise) van die mitokondriale elektron vervoer ketting (EVK) komplekse (I-V). Daarna is bogenoemde proteïene ondersoek in mitokondrieë geïsoleer uit die 20 weke groep ná die toediening van insulien en die blootstelling van die harte aan iskemie. Die oksigraaf mitokondriale oksidatiewe fosforilering (OXPHOS) kapasiteit analise is dan op mitokondrieë van 20 weke DIO en kontrole groepe uitgevoer en daarna is 'n sitraatsintase (SS) aktiwiteitstoets gedoen. RESULTATE: Na die 8 en 20 weke dieet, het die intra-peritoneale vet massa, vastende plasma glukose en insulien vlakke in die DIOs aansienlik toegeneem, in vergelyking met hul kontroles. Sitosoliese WK-analise: Die tp85, pp85 en pPKB/Akt vlakke was beduidend hoër in die DIOs in vergelyking met die kontroles, na 8 weke van die dieet. Verder is die pBad en Bax vlakke beduidend verhoog in hierdie diere. Na 20 weke van die dieet, het die pp85, tPKB/Akt en pPKB/Akt vlakke beduidend afgeneem in die DIOs, in vergelyking met die kontroles. Die tBad was beduidend verhoog, terwyl die Bad verhouding van gefosforileerde oor die totale proteïen uitdrukking (P/T)-verhouding) beduidend verminder het in hierdie diere. SS aktiwiteitstoets: SS aktiwiteit is beduidend verminder in die DIOs, teenoor die kontroles, op 20 weke. Mitokondriale EVK WK-analise: Die subeenheid uitdrukking in komplekse I-III en V was nie beduidend verskillend na 8 weke nie. Na 20 weke egter, was die uitdrukking aansienlik laer in komplekse I en II. Interessant genoeg, is die uitdrukking aansienlik verhoog in komplekse III en V. Mitokondriale OXPHOS analise: Die ADP/O verhouding met (1) glutamaat of (2) palmitiel-L-karnitien as substraat, het beduidend afgeneem in die DIOs teen 20 weke. Mitokondriale WK-analise: Die pp85 subeenheid was beduidend verhoog in die kontrole en DIO groepe, blootgestel aan insulien en iskemie, in vergelyking met die onbehandelde kontroles. Die Bcl-2 vlakke was beduidend verminder in die insulien en isgemie DIOs, in vergelyking met onbehandelde DIOs. Die tBad uitdrukking het nie beduidend verskil tussen die insulien en onbehandelde kontroles nie, terwyl die tBad beduidend verhoog was in die isgemie kontroles versus onbehandelde kontroles. Alle beduidende verskille is geneem as p<0.05. GEVOLGTREKKING: Die resultate dui daarop dat die eerste fase van dieet-geïnduseerde obesiteit geassosieer is met kardiale beskerming want `n toename in PI3K/PKB/Akt seintransduksie en 'n afname in apoptotiese merkers is waargeneem. In teenstelling, in die gevorderde stadium van vetsug is daar 'n afname in aktiwiteit in die PI3K/PKB/Akt pad wat verband hou met verhoogde miokardiale apoptose en verminderde mitokondriale funksie en integriteit.