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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52577 |
| Date | 12 1900 |
| Creators | Louw, Rehette |
| Contributors | Huisamen, B., Van Rooyen, J., Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 118 pages : illustrations |
| Rights | Stellenbosch University |
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