The signaling pathways involved in the cardioprotection offered by insulin to the global low flow ischaemic/reperfused myocardium

Louw, Rehette (2001-12)

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.

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