Browsing by Author "Odendaal, Louise"

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    The effect of dietary Red Palm Oil on the functional recovery and the PKB/Akt pathway in the ischaemic/reperfused isolated rat heart
    (Stellenbosch : Stellenbosch University, 2007-12) Odendaal, Louise; Engelbrecht, A. M.; Van Rooyen, J.; Du Toit, E. F.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    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.
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    The effect of dietary red palm oil on the functional recovery of the ischaemic/reperfused isolated rat heart : the involvement of the PI3-Kinase signaling pathway
    (BioMed Central, 2009-05) Engelbrecht, Anna-Mart; Odendaal, Louise; Du Toit, Eugene F.; Kupai, Krisztina; Czont, Tamas; Ferdinandy, Peter; Van Rooyen, Jacques
    We have previously shown that dietary red palm oil (RPO) supplementation improves functional recovery in hearts subjected to ischaemia/reperfusion-induced injury. Unfortunately, the cellular and molecular mechanisms responsible for this phenomenon are still poorly understood and no knowledge exists regarding the effects of RPO supplementation on the phosphoinositide 3-kinase (PI3-K) signaling pathway and apoptosis during ischaemia/reperfusion injury. Therefore, the aims of the present study were three fold: (i) to establish the effect of RPO on the functional recovery of the heart after ischaemia/reperfuion injury; (ii) to determine the effect of the PI3-K pathway in RPO-induced protection with the aid of an inhibitor (wortmannin); and (iii) to evaluate apoptosis in our model. Wistar rats were fed a standard rat chow control diet or a control diet plus 7 g RPO/kg for six weeks. Hearts were excised and mounted on a Langendorff perfusion apparatus. Mechanical function was measured after a 25 min period of total global ischaemia followed by 30 minutes of reperfusion. Hearts subjected to the same conditions were freeze-clamped for biochemical analysis at 10 min during reperfusion to determine the involvement of the PI3-Kinase signaling pathway and apoptosis in our model. Dietary RPO supplementation significantly increased % rate pressure product recovery during reperfusion (71.0 ± 6.3% in control vs 92.36 ± 4.489% in RPO; p < 0.05). The % rate pressure product recovery was significantly reduced when wortmannin was added during perfusion (92.36 ± 4.489% in the RPO group vs 75.21 ± 5.26% in RPO + Wm). RPO + Wm also significantly attenuated PI3-K induction compared with the RPO group (59.2 ± 2.8 pixels in RPO vs 37.9 ± 3.4 pixels in RPO + Wm). We have also demonstrated that PI3-K inhibition induced PARP cleavage (marker of apoptosis) in the hearts during ischaemia/reperfusion injury and that RPO supplementation counteracted this effect.