Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Introduction: It is well documented that insulin offers cardioprotection
against the consequences of ischaemia/reperfusion injury. Insulin-induced
improvements in cardiac functions are widely investigated in models of
ischaemia and reperfusion. It has been shown that many signalling pathways
may be involved in the cardioprotection properties of insulin under those
conditions. These pathways include PI3-K, PKB/Akt, p70S6k, ERK and many
others. However, little data exists on the effects of insulin on the heart under
normoxic condition. Some evidence has been presented that insulin has a
positive inotropic effect on the normoxic perfused rat heart, but no precise
cellular mechanism has been investigated or described in this regard. We
believe that an investigation into the effects of insulin on cardiac function and
pathways involved under normoxic conditions may help us to better
understand the mechanisms of insulin-induced cardioprotection. Aims: To
determine a suitable dose of insulin at which a positive inotropic response
could be detectable under normoxic conditions, to investigate the possible
mechanisms involved in insulin-induced increases in contractility with specific
reference to the vasculature and the coronary flow and to investigate a
possible involvement of PI3-K and its downstream effectors on the insulin
effects on cardiac functions under normoxic conditions. Materials and
methods: Isolated rat hearts were perfused retrogradely using the
Langendorff technique. After 10 minutes of stabilization hearts were perfused
for 30 minutes either with standard perfusion solution i.e. Krebs-Henseleit
buffer + glucose gassed with 95%O2, 5%CO2 (control hearts), or with
standard perfusion solution plus insulin alone or insulin together with the nitric oxide synthase inhibitor L-NAME or the PI3-K inhibitor wortmannin. Left
ventricular developed pressure (LVDevP), heart rate (HR) and coronary flow
(CF) as well as phosphorylated PI3-K and PKB/Akt in heart were measured.
Results: Administration of insulin alone at physiological concentrations
showed improved cardiac function compared to hearts in the control group.
Hearts that received insulin+L-NAME showed a significant decrease in
function compared to the control hearts and the hearts that received insulin
alone (p<0.05). Phosphorylated PKB/Akt (Thr308) was increased in hearts
that received insulin alone and insulin+L-NAME compared to the control
hearts. Phosphorylated PI3-K tended to be higher in hearts where insulin was
administered alone compared to the hearts that received insulin+L-NAME or
insulin+wortmannin. Conclusion: This study confirmed that physiological
concentrations of insulin exert positive inotropic effects on cardiac function in
normoxic perfused rat hearts as seen with the improved LVDevP. Inhibition of
PI3-K by wortmannin induced a decrease in phosphorylated PKB/Akt in
hearts that received insulin+wortmannin and administration of L-NAME
impaired the beneficial effects of insulin on cardiac functions. Therefore these
results may indicate that nitric oxide may have a role in the positive effect of
insulin on cardiac function in the healthy heart perfused under normoxic
conditions. L-NAME as well as wortmannin reversed the positive inotropic
effects of insulin. Both inhibitors also unmasked effects of insulin via nitric
oxide and PI3-K on heart rate and coronary flow. / AFRIKAANSE OPSOMMING: Inleiding: Dit is welbekend dat toediening van insulien die hart beskerm
teen ischemie/reperfusie-beserings, wat lei tot verbeterde hartfunksie.
Hierdie effek word wyd ondersoek in modelle van ischemie en reperfusie. Dit
is bewys dat ‘n verskeidenheid seintransduksie paaie, insluitend PI3-K,
PKB/Akt, p70S6k en ERK, betrokke is by hierdie beskermende effek van
insulien op die hart. Baie min data is egter beskikbaar rakende die effek van
insulien tydens normoksiese toestande. Alhoewel dit bekend is dat insulien ’n
inotropiese effek op die normale geperfuseerde hart het, is die presiese
sellulêre meganismes wat dit bewerkstellig nog nie nagevors nie. Om dus ‘n
beter begrip van hierdie meganismes te verkry is dit dus noodsaaklik om die
effekte van insulien onder normoksiese perfusie toestande na te vors.
Doelstellings: Om ‘n geskikte dosis, waarby insulien sy positiewe
inotropiese effek onder normale toestande het, vas te stel, om die moontlike
meganismes betrokke by insulien-geïnduseerde verbetering in
hartsametrekbaarheid te bestudeer, met spesifieke verwysing na die
bloedvoorsiening en koronêre vloei, en om die moontlike betrokkenheid van
die PI3-K pad en sy teiken effektore onder normale suurstof-toestande te
ondersoek. Materiaal en metodes: Geïsoleerde rotharte is geperfuseer
deur gebruik te maak van die Langendorff tegniek. Na ‘n stabilisasie periode
van 10 minute is rotharte blootgestel aan 30 minute perfusie met een van vier
oplossings: ‘n standaard perfusie oplossing (Krebs-Henseleit buffer met
glukose onder spesifieke gaskondisies van 95% O2, 5% CO2 – kontrole
harte); standaard perfusie oplossing en insulien; standaard perfusie oplossing met insulien en die stikstofoksied sintase inhibitor L-NAME, of
standaard perfusie oplossing, met insulien en die PI3-K inhibitor wortmannin.
Met verloop van die perfusie protokol, is ontwikkelde linker ventrikulêre druk
(LVDevP), harttempo (HR) en koronêre vloei (CF), sowel as PI3-K en
PKB/Akt fosforilasie, gemeet. Resultate: Toediening van insulien teen
fisiologiese konsentrasies het ‘n verbeterde hartfunksie tot gevolg, in
vergelyking met harte in die kontrole groep. In teenstelling hiermee het harte
wat insulien+L-NAME ontvang het ‘n betekenisvolle verlaagde funksie getoon
in vergelyking met die kontrole harte en harte wat slegs insulien ontvang het
(p<0.05). Harte wat slegs insulien, of insulien+L-NAME ontvang het, het ‘n
verhoging in gefosforileerde PKB/Akt (Thr308) getoon in vergelyking met
kontrole harte. Gefosforileerde PI3-K het ook geneig om hoër te wees in
harte wat insulien+L-NAME of insulien+wortmannin ontvang het, as in harte
wat slegs insulien ontvang het. Gevolgtrekking: Hierdie studie bewys dat
fisiologiese konsentrasies van insulien, onder normale suurstof-toestande, ‘n
positiewe inotropiese effek op hartfunksie uitoefen, soos gesien in die
verbeterde LVDevP. Wortmannin-geïnduseerde inhibering van die PI3-K pad
het ‘n verlaagde PKB/Akt fosforilasie tot gevolg gehad in harte wat
insulien+wortmannin ontvang het, terwyl die toediening van L-NAME die
voordelige effekte van insulien op hartfunksie onderdruk het. Hierdie
resultate dui dus aan dat stikstofoksied ‘n rolspeler is in die positiewe
inotropsiese effek van insulien op hartfunksie tydens normoksiese toestande,
aangesien beide inhibitore hierdie effek onderdruk het. Beide inhibitore het
ook die betrokkenheid van stikstofoksied en die PI3-K pad by die effek van
insulien op harttempo en koronêre vloei onthul.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/17380 |
| Date | 12 1900 |
| Creators | Manga-Manguiya, Edith Sylvie |
| Contributors | Van Rooyen, Jacques, Smith, Rob, University of Stellenbosch. Faculty of Science. Dept. of Physiological Sciences. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 164 leaves : ill. |
| Rights | University of Stellenbosch |
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