The effect of dietary Red Palm Oil on the functional recovery and the PKB/Akt pathway in the ischaemic/reperfused isolated rat heart

Odendaal, Louise (Stellenbosch : Stellenbosch University, 2007-12)

Thesis

ENGLISH ABSTRACT: Introduction Cardiovascular disease is one of the leading causes of death in the world. Formation of harmful reactive oxygen species (ROS) is associated with several pathological conditions, and contributes to ischaemia/reperfusion injury. Antioxidants can be added to the diet in an attempt to decrease the prevalence of cardiovascular disease by decreasing the harmful effects of ischaemia/reperfusion injury. Red Palm Oil (RPO) consists of saturated, monounsaturated and polyunsaturated fatty acids and is rich in antioxidants such as -carotene, tocopherols and tocotrienols. It has previously been shown that RPO-supplementation improved reperfusion mechanical function. In these studies it was found that RPO might exert its beneficial effects during reperfusion through increased PKB/Akt pathway activity, which may lead to inhibition of apoptosis and improved mechanical function. Aims The aims of this study were: 1) to determine whether RPO-supplementation protected against ischaemia/reperfusion injury in the isolated perfused rat heart, 2) to confirm RPO-supplementation’s effect on the PKB/Akt pathway activity and, 3) to elucidate the regulators in the PKB/Akt pathway that RPOsupplementation influenced. Methods Male Wistar rats were divided into 4 groups, 2 control groups and 2 experimental groups. The 2 control groups were fed a standard rat chow (SRC) for 4 weeks. The two experimental groups received SRC and RPOsupplementation for 4 weeks. Hearts were excised and transferred to a Langendorff perfusion apparatus and perfused with Krebs-Henseleit buffer. Mechanical functional recovery was measured after 25 min of total global noflow ischaemia. The following parameters were also measured during various time points in the protocol: left ventricular develop pressure, heart rate, coronary flow, rate pressure product. Hearts were also freeze-clamped for biochemical analysis at 10 min during reperfusion. The biochemical analysis was aimed at determining PKB/Akt involvement. In a second protocol, hearts were subjected to the same perfusion protocol, but wortmannin was also added to the perfusion fluid, in order to inhibit PI3- kinase. Results Hearts from the RPO-supplemented rats showed an improved RPP recovery (92.26 ± 5.89 % vs 63.86 ± 7.74 %) after 10 min of reperfusion. This finding corroborated the findings of previous studies. Hearts of the RPOsupplemented rats perfused with wortmannin, showed increased RPP recoveries at several time points. Biochemical results showed that wortmannin did indeed inhibit PI3-K phosphorylation in the RPO-supplemented group, as was expected. The RPO-supplemented group that was perfused with wortmannin had an increased PKB/Akt (Ser473) phosphoyrylation, when compared to the wortmannin control group. It was also found that the combination of RPO and wortmannin had prosurvival effects. Discussion This study showed that RPO-supplementation offered protection against ischaemia/reperfusion injury in the Langendorff-perfusion apparatus at 10 min into reperfusion. Thereafter the significance of the protection was lost. This protection has been confirmed in several previous studies and several mechanisms have been proposed for this protection. Since no conclusive evidence exists on the precise mechanism of protection, our investigation focused on the regulators of the pro-survival PKB/Akt pathway. An improved functional recovery was also seen in the RPO-supplemented group that was perfused with wortmannin. This was an unexpected finding, because Wortmannin is a known PI3-kinase inhibitor (as was confirmed by our biochemical data). PI3-kinase phosphorylation leads to PKB/Akt phosphorylation and therefore, activation of a pro-survival pathway. It would be expected that wortmannin would inhibit PKB/Akt and thus decrease the survival of the cells. The RPO-supplementation thus reversed wortmannin’s detrimental effect to such an extent that the functional recovery was far better than RPO-supplementation alone. In the RPO + wortmannin group, PKB/Akt (Ser473) phosphorylation was increased, contrary to previous findings. This is an indication that RPO may have the ability to override wortmannin’s inhibitory effect on PI3-kinase, or that PKB/Akt (Ser473) may be phosphorylated independently of PI3-kinase.

AFRIKAANSE OPSOMMING: Inleiding Kardiovaskulêre siektes is een van die hoof oorsake van sterftes in die wêreld. Die vorming van skadelike reaktiewe suurstof spesies word geassosieer met verskeie patologiese kondisies en dra ook by tot isgemie/reperfusie skade. ‘n Moontlike manier om die voorkoms van isgemie/herperfusie skade asook kardiovaskulêre siektes te voorkom, is om antioksidante by die dieet te voeg. Rooi Palm Olie (RPO) bevat versadigde, mono-onversadigde en polionversadigde vetsure. RPO bevat ook ‘n oorvloed van antioksidante soos β- karoteen en tokoferole en tokotriënole. Dit is bewys in vorige studies dat RPO-aanvulling verbeter funksionele herstel. Hierdie voordelige effekte mag dalk wees agv verhoogde PKB/Akt pad aktiwiteit. Die PKB/Akt pad word geassosieer met die inhibisie van apoptose en verhoogde meganiese funksie. Doelwitte Die doelwitte van hierdie studie was om te bepaal of 1) RPO-aanvulling beskermende effekte teen isgemie/herperfusie skade in die geisoleerde rotharte het, 2) Bevestig of RPO-aanvulling wel die PKB/Akt pad beïnvloed 3). om die effekte wat RPO-aanvulling het op die reguleerders van die PKB/Akt pad te onthul. Metodes Manlike Wistar rotte is in 4 groepe verdeel. 2 Groepe kontrole rotte is ‘n standaard rotkosmengsel gevoer vir 4 weke. Die 2 eksperimentele groepe het ook ‘n standaard rotkosmengsel gekry plus ‘n RPO-aanvulling vir 4 weke. Harte is uitgesny en op ‘n Langendorff perfusie sisteem gemonteer en met Krebs-Henseleit buffer geperfuseer. Meganiese funksie herstel is gemeet na 25 min totale globale geen-vloei isgemie. Linker ventrikulêre ontwikkelde druk, harttempo, koronêre vloei en tempo druk produk is gemeet by verskillende tydpunte. Sommige harte is na 10 min herperfusie vir biochemiese analiese gevriesklamp. Die biochemiese analisiese was beoog om die PKB/Akt pad betrokkenheid te bepaal. ‘n Tweede stel harte is aan dieselfde perfusie protokol blootgestel, maar wortmannin (PI3-kinase inhibitor) is ook bygevoeg by die perfusie vloeistof. Resultate Die groep wat met RPO aangevul is, het na 10 min herperfusie, ‘n verbeterde tempo druk produk herstel getoon (92.26 ± 5.89 % vs 63.86 ± 7.74. Hierdie bevinding is ook met ander studies bevestig. ‘n Interessante bevinding was dat die groep wat met RPO aangevul is en met wortmannin geperfuseer is, ‘n verbeterde meganiese funksionele herstel getoon het. Biochemiese resultate het getoon dat wortmannin wel PI3-K fosforilering geinhibeer het. Die harte van die rotte in die groep wat aangevul is met RPO en daarna met wortmannin geperfuseer is, het ‘n toename in PKB/Akt (Ser473) fosforilering getoon, relatief tot die wortmannin geperfuseerde harte van die rotte in die kontrole groep. Hierdie groep (RPO-aanvulling en wortmannin perfusie) het beskermende effekte getoon. Bespreking Hierdie studie het getoon dat RPO-aanvulling beskerming gebied het teen isgemie/herperfusie skade in die Langendorff geperfuseerde rothart na 10 min herperfusie. Daarna is die beduidenheid van die beskerming verloor. Hierdie bevindings ondersteun die resultate van vorige studies. Verskeie moontlike meganismes is voorgestel vir die beskerming, maar die presiese meganisme is nog nie duidelik nie. In hierdie studie is daar gekyk na die reguleerders van die PKB/Akt pad. Geen vorige studies het al gefokus op RPO-aanvulling en sy effek op die reguleerders van die PKB/Akt pad nie. ‘n Onverwagte bevinding is dat harte van die rotte in die RPO + wortmannin groep ‘n verbeterde funksionele herstel getoon het. Wortmannin is ‘n PI3- kinase inhibitor. PI3-K fosforilering lei tot PKB/Akt fosforilering, wat tot sel beskerming lei. Dus, aangesien wortmannin PI3-K inhibeer, sou dit verwag word dat wortmannin sel beskerming sal verminder. Die RPO het egter die wortmannin se nadelige effekte tot so ‘n mate oorskrei dat die funksionele herstel baie beter was as die RPO-aanvulling alleen. Die verhoogde PKB/Akt (Ser473) fosforilering, wat gesien is in die RPO + wortmannin groep kan toegeskryf word aan RPO se vermoë om wortmannin se nadelige effekte te oorskrei. ‘n Moontlike verduideliking vir hierdie bevinding mag wees dat rooi palm olie PKB/Akt (Ser473) op ‘n PI3-K onafhanklike manier fosforileer.

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