An investigation into the mechanisms whereby insulin protects the rat heart in a model of Low-Flow Ischemia
| dc.contributor.advisor | Huisamen, B. | en_ZA |
| dc.contributor.advisor | Van Rooyen, J. | en_ZA |
| dc.contributor.author | Lopes, John | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences : Medical Physiology. | en_ZA |
| dc.date.accessioned | 2008-12-01T16:15:08Z | en_ZA |
| dc.date.accessioned | 2010-10-28T18:51:02Z | |
| dc.date.available | 2012-12-31T22:10:04Z | |
| dc.date.issued | 2008-03 | |
| dc.description | Thesis (PhD)--Stellenbosch University, 2008. | en_ZA |
| dc.description.abstract | ENGLISH ABSTRACT: BACKGROUND: Insulin has been shown to protect the heart against ischemia/reperfusion (I/R) damage by activating the reperfusion injury salvage kinase (RISK) pathway during reperfusion (Jonassen et al 2001 and 2004, Hausenloy and Yellon 2004). The main focus of investigation has since been on insulin-treatment during reperfusion as therapy for acute myocardial infarction. This is based on the premise that reperfusion is the only opportunity for therapy, which negates the possible therapeutic value of insulin during ischemia. The objective of this study was therefore to investigate if insulin-mediated protection during ischemia contributes to protection during reperfusion, and if so, to identify the mechanisms involved. AIMS: - To set up a model of low-flow ischemia (LFI) that could be used to investigate the mechanisms whereby insulin protects the rat heart against I/R. - Determine if insulin-mediated cardioprotection during ischemia contributes to cardioprotection during reperfusion. - Establish the effect of insulin signaling on cyclic AMP (cAMP) accumulation during LFI due to its association with ß-adrenergic receptor (ß-AR) induced ischemic damage. - Identify the role of the ß-AR and its downstream targets, adenylyl cyclase (AC), cAMP activated protein kinase (PKA) and phospholamban (PLN) in protection by insulin during LFI. - Determine the role of phosphoinositide 3-kinase (PI3K) and external glucose availability in cardioprotection elicited during ischemia by insulin. METHODS: Male wistar rats were perfused in Langendorff mode in the absence and presence of insulin (0.3 mIU/ml), which was added to the Krebs-Henseleit perfusion buffer after 10 min stabilization. The perfusion protocol was as follows: 30 min stabilization, 30-45 min LFI (0.2 ml/min) and 30 min reperfusion. Inhibitors for ß-AR (propranolol), AC (MDL-12,330A), PKA (Rp-8-CPT-cAMPS) and PI3K (wortmannin) were respectively administered 5 min before LFI and were present until the end of ischemia. To determine the role of glucose in protection, glucose was omitted during LFI in the insulin and non-insulin groups. RESULTS: Insulin protected the heart during ischemia by delaying and alleviating contracture development that was followed by reduced hypercontracture and improved function during reperfusion without insulin. Thus protection during ischemia contributes to protection during reperfusion. cAMP was unexpectedly higher in the insulin-protected hearts during LFI. AC inhibition prevented cAMP accumulation and abolished cardioprotection. Notably, the rate of contracture progression in non-insulin treated hearts was enhanced by AC inhibition. cAMP production by AC might therefore be cardioprotective during ischemia and is enhanced by insulin. Respective inhibition of the ß-AR and PKA protected the non-insulin treated hearts, but could not enhance insulin-induced protection. PLN phosphorylation was equally reduced in PKA-inhibited hearts and insulin treated hearts during LFI. Interestingly, ß-blockade did not alter the levels of cAMP accumulation regardless of insulin, yet PKA activation is cAMP-dependent. Finally, external glucose was found to be a prerequisite for insulin-mediated protection, but this not dependent on PI3K activation. CONCLUSION: We conclude that insulin protects the rat heart during LFI, which contributes to protection during reperfusion. This protection is dependent on AC activation and cAMP accumulation during ischemia, which may be an innate mechanism of protection that is enhanced by insulin. An additional mechanism of protection by insulin involves the inhibition of theß-AR/PKA pathway, which is however mediated by cAMP. We therefore propose a dual role for cAMP in protection and pathology during ischemia, which is possibly determined by the subcellular localization of cAMP in the ischemic cardiomyocyte. This proposes a possible role for the phosphodiesterases, which regulate compartmentalization of cAMP and should therefore be investigated during LFI protection by insulin. Insulin-induced protection during ischemia is largely dependent on glucose availability and therefore also anaerobic metabolism, but does not require PI3K. | |
| dc.description.abstract | AFRIKAANSE OPSOMMING: AGTERGROND: Dit is onlangs bewys dat insulien die hart teen iskemie/herperfusie (I/R) beskerm deur aktivering van sekere kinases soos die eksterne gereguleerde kinase (ERK) en protein kinase B (PKB/Akt) tydens herperfusie (Jonassen et al 2001 and 2004, Hausenloy and Yellon 2004). As gevolg van hierdie waarneming en die feit dat herperfusie die enigste periode vir terapeutiese intervensie is, word daar tans klem gelê op die identifisering van die meganisme(s) van insulien beskerming. Daar word dus algemeen aangeneem dat daar geen terapeutiese moontlikhede vir insulien tydens iskemie bestaan nie, ten spyte van die feit dat insulien se beskerming tydens iskemie reeds voorheen aangetoon is (Van Rooyen et al 2002, Cave et al 2000). Die doel vir ons studie was dus om te bepaal of beskerming deur insulien tydens iskemie enige bydrae lewer tot beskerming tydens herperfusie, en indien wel, wat die mechanisms is. DOELWITTE: - Ontwikkel ‘n lae-vloei iskemie (LFI) model van die geperfiseerde rothart wat gebruik kan word om die meganismes te bepaal waarop insulien die hart teen I/R besering beskerm. - Bepaling van insulien gemedieerde beskerming tydens iskemie tot beskerming tydens herperfusie bydra. - Identifisering van die effek van insulien op die akkumulasie van die tweede boodskapper, sikliese AMP (cAMP) tydens LFI. - Bepaling van die rol van die beta-adrenerge reseptor (ß-AR) seintransduksie pad, met verwysing na die ß-AR, adenilaat siklase (AC), cAMP geaktiveerde protein kinase (PKA) en fosfolamban (PLN), in insulien gemedieerde beskerming tydens LFI. - Bepaling van die rol van fosfo-inositiel 3-kinase (PI3K) en eksterne glucose as substraat tydens LFI in insulin gemedieerde beskerming. METODES: Harte van manlike wistar rotte is retrograads (Langendorff metode) perfiseer sonder en met insulin (0.3 mIU/ml). Die perfuserings protokol was soos volg: 30 min stabilisasie, 30-45 min LFI (koronêre vloei 0.2 ml/min) en 30 min herperfusie. Inhibitore vir ß-AR (propranolol), AC (MDL-12,330A), PKA (Rp-8-CPT-cAMPS) en PI3K (wortmannin) is afsonderlik toegedien, vanaf 5 min voor LFI tot aan die einde daarvan. Die rol van glukose is ondersoek deur substraatvrye perfusie tydens LFI in harte met en sonder insulin behandeling. RESULTATE: Beskerming met insulin tydens LFI is aangetoon deur die vertraging in die ontwikkeling en afname in kontraktuur. Herperfusie sonder insulin van hierdie harte het gelei tot die afname in hiperkontraktuurontwikkeling en verbetering in funksionele herstel. Hierdie resultaat het getoon dat die beskermende effekte van insulin tydens LFI tot beskerming tydens reperfusie bydra. cAMP vlakke tydens iskemie was onverwags beduidend hoër in die insulin geperfuseerde harte. Inhibisie van AC het die akkumulasie van cAMP onderdruk, asook die beskerming deur insulin. Dis opmerlik dat die tempo waarteen kontraktuur ontwikkel het, ook vinniger was tydens AC inhibisie in insulin-vrye harte. Ons lei dus af dat die produksie van cAMP deur AC ‘n beskermingsmeganisme mag wees wat tydens iskemie aangeskakel word en deur insulin gestimuleer word. Inhibisie van die ß-AR en PKA onderskeidelik het gelei tot beskerming van insulin-vrye iskemiese harte, maar het geen effek op die beskerming van insulin gehad nie. Soortgelyke onderdrukking van PLN fosforilasie is in insulin beskermde harte gevind, sowel as in harte wat met die PKA inhibitor behandel was. Dis interessant dat ß-AR inhibisie geen effek op weefsel cAMP gehad het in enige van die groepe nie, terwyl PKA aktivering cAMP afhanklik is. Laastens, insulien beskerming is glucose-afhanklik, maar benodig nie PI3K aktivering tydens LFI nie. GEVOLGTREKKING: Beskerming deur insulien tydens LFI lei ook tot inhibisie van herperfusie-besering. Hierdie beskerming is van AC aktivering en die gepaardgaande cAMP toename afhanklik, en is dalk ‘n natuurlike meganisme van beskerming wat deur insulin bevorder word. Insulien beskerm ook deur inhibisie van die ß-AR/PKA seintransduksie pad, wat ook cAMP gemedieerd is. Die gevolgtrekking is dat cAMP ‘n dubbele rol tydens iskemie speel wat tot beskerming of beskadiging kan lei, wat op sy beurt deur kompartementalisering van cAMP beinvloed word. Die rol van fosfodiesterase (PDEs) in die distribusie en kompartementalisering van cAMP maak hierdie protein ‘n moontlike kandidaat in insulien beskerming. Insulien gemedieerde beskerming tydens iskemie is grootliks van glukose en dus anarobiese metabolisme afhanklik, maar nie PI3K aktivering nie. | |
| dc.description.version | Doctoral | en_ZA |
| dc.embargo.terms | 2012-12-31 | en_ZA |
| dc.format.extent | 207 pages | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/10019.1/4843 | |
| dc.language.iso | en_ZA | en_ZA |
| dc.publisher | Stellenbosch : University of tellenbsoch | en_ZA |
| dc.rights.holder | Stellenbosch University | en_ZA |
| dc.subject | Insulin | en_ZA |
| dc.subject | Cardioprotective agents | en_ZA |
| dc.subject | Ischemia | en_ZA |
| dc.subject | Receptors, Adrenergic | en_ZA |
| dc.subject.other | Biomedical Sciences | en_ZA |
| dc.title | An investigation into the mechanisms whereby insulin protects the rat heart in a model of Low-Flow Ischemia | en_ZA |
| dc.type | Thesis | en_ZA |