Browsing by Author "Smit, Sybrand Engelbrecht"
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- ItemThe effect of green rooibos extract on rat hearts in a pre-diabetic model : an evaluation of the function and mechanisms involved(Stellenbosch : Stellenbosch University, 2019-03) Smit, Sybrand Engelbrecht; Huisamen, Barbara; Marais, Erna; Johnson, Rabia; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.ENGLISH ABSTRACT: Background— Cardiovascular diseases (CVD) remains the leading cause of death globally, with a rising prevalence of individual risk factors such as obesity and insulin resistance. Furthermore, diabetic patients are particularly at risk for developing ischemic heart disease and strokes. In patient suffering an ischemic event, most of the myocardial damage incurred happens once blood flow is restored – a phenomena known as reperfusion injury. Mitochondrial ROS production is implicated as one of the major contributors to cell death in the reperfused heart and the importance of mitophagic processes (mitochondrial housekeeping) could be a potential therapeutic target to prevent ischemia/reperfusion injury (I/R-I). Aspalathus linearis (commonly known as rooibos) is an indigenous South African plant grown exclusively in the Western Cape fynbos region. Rooibos is rich in bioactive phenolic compounds, including aspalathin, a C-linked dihydrochalcone glucoside unique to rooibos, known for its hypoglycemic and strong antioxidant potential. Methods— The present study made use of 300 Wistar rats randomly allocated into controls and rats receiving a 16-week high-fat, high-caloric diet (HCD). After 10 weeks on the respective diets, half of each group received 60 mg/kg/day Afriplex green rooibos extract (GRT), containing 12% aspalathin, for 6 weeks. The primary aim was to investigate its therapeutic potential in treating 20 min global ischemia/reperfusion injury (I/R-I) in rats with increased susceptibility for CVD, as well as determine the mitochondrial oxidative phosphorylation (OxPhos) during four stages of I/R-I. The secondary aim was to elucidate GRT’s effect on cardiac signaling and mitophagy with regards to I/R-I. Results— HCD over 16 weeks resulted in daily increased food intake (22.30±1.27 g vs 18.00±0.54 g = ↑24%; p<0.001), decreased daily water intake (19.55±0.58 mL vs 29.74±0.74 mL = ↓34%; p<0.001), leading to increased body weight (403.9±5.1 vs373.6±4.8 g = ↑8%; p<0.001) and increased intraperitoneal fat (23.69±0.89 vs 13.98±0.43 g = ↑69%; p<0.001) compared to age-matched controls. Furthermore, HCD increased fasting blood glucose (5.75±0.16 vs 5.21±0.16 mM = ↑10%; p<0.05), fasting blood insulin (6.20±0.57 vs 4.31±0.54 ng/mL = ↑44%; p<0.05) and insulin resistance through raised HOMA-IR (3.52±0.35 vs 2.30±0.28 = ↑53%; p<0.05) compared to age-matched controls. GRT supplementation for 6 weeks had no significant effect on biometric parameters in either controls or HCD. Pre-ischemia, HCD rats presented with worse functional heart parameters, such as diastolic and systolic pressure, aortic output (AO), cardiac output (CO) and total work (WT) (10.69±0.14 vs 12.6±0.19 mW = ↓15%; p<0.001) compared to controls, while GRT supplementation was able to significantly improve these parameters in both controls (13.91±0.23 mW = ↑10%; p<0.001) and HCD (11.98±0.20 mW = ↑12%; p<0.001). GRT administration also lowered the heart rate during stabilization in both control and HCD by an average 10 bpms (p<0.05). Post-ischemia, HCD hearts were weaker than controls and had a lower WT recovery (55.51±2.28 vs 64.8±2.04% = ↓14%; p<0.01), while GRT restored WT recovery in HCD (63.35±2.49% = ↑14%; p<0.05). HCD rats also had a greater infarct size compared to controls (34.99±11.56 vs 16.42±8.71% = ↑113%; p<0.01) following a 35 min regional ischemia protocol. GRT supplementation led to a remarkable reduction in infarct size (13.22±6.66% = ↓62%; p<0.001), while conferring no added protection to controls (14.58±5.43%). Regarding cardiac and mitochondrial signaling, prior to ischemia HCD heart had a decreased dependence on insulin-dependent AMPK and increased inflammation via upregulated p38, whereas GRT treatment presented with decreased insulindependent PKB and AS160 signaling (together with increased FA OxPhos compared to carbohydrates, however showed more mitochondrial uncoupling, decreasing basal metabolic rate and thereby potentially less ROS production), inhibited GSK3β and conferred an anti-inflammatory effect by significantly reducing p38 activation. HCD also had higher mitophagy rates through Parkin and LC3 signaling. After 20 min ischemia, in HCD, ATM and AMPK were upregulated and insulin-dependent PKB downregulated with GSK3β. Mitophagy signals, such as PINK1 and p62 were elevated, but autophagic flux remained low during ischemia in all groups. GRT supplementation resulted in an opposite profile with AMPK downregulated and showing inhibition of ERK1/2. In early reperfusion, all protective signaling were downregulated in HCD including AMPK, PKB, AS160, GSK3b and ATM. Mitophagy was also activated through Parkin, p62 and LC3 while having increased OxPhos potential in FA compared to carbohydrates. GRT supplementation reduced oxidative stress and inflammation through downregulation of JNK1/2 and p38, and initiated mitophagy through an AMPKdependent mechanism and Parkin. GRT supplementation also inhibited mitochondrial OxPhos after reperfusion culminating in a potentially decreased ROS production. Conclusion— This study showed the cardioprotective effect of Afriplex GRT supplementation by improving functional heart recovery and reducing infarct size post-ischemia in rats with elevated risk for CVD. Another novel find is the reduction in heart rate induced by GRT treatment through inhibition of pacemaking cells. This could have potential therapeutic application in patients suffering from ischemic heart disease. GRT ilicits a cardiotonic effect in I/R-I through anti-inflammatory mechanisms pre- and post-ischemia. In early reperfusion, GRT treatment resulted in decreased oxidative stress, inhibition of mitochondrial OxPhos and enabled AMPKdependent mitophagy. GRT shows promise as a strong antioxidant and anti-inflammatory agent in managing adverse outcomes in patients at risk for CVD.
- ItemAn investigation into the effects of aspalathin on myocardial glucose transport using cardiomyocytes from control and obesity-induced insulin resistant rats, and terminally differentiated H9C2 cells(Stellenbosch : Stellenbosch University, 2016-03) Smit, Sybrand Engelbrecht; Huisamen, Barbara; Johnson, Rabia; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Medical PhysiologyENGLISH ABSTRACT: Introduction: Rooibos is an indigenous South African plant ingested as herbal tea and well-known for its strong anti-oxidant effects. Rooibos has shown to have cardioprotective properties in vitro and in vivo, but the role of individual Rooibos flavonoids in cardioprotection still remains unclear. This in vitro study investigated Aspalathin, a dihydrochalcone unique to rooibos, for (i) cardioprotective effects in the context of age- and obesity-induced insulin resistance, known to attenuate myocardial glucose uptake and utilization and (ii) the applicable signaling pathways involved. Methods: Male Wistar rats were allocated into three groups: 16-30 weeks feeding with either standard rat chow (C) or a high-fat, high-caloric diet (HFD), or 6-7 weeks feeding with C. Cardiomyocytes were isolated by collagenase perfusion digestion, using a Langendorff apparatus and glucose uptake determined by 2-[3H]-deoxyglucose (2DG) accumulation using liquid scinitillation analysis. In addition, H9C2 cells were differentiated into cardiomyocyte analogs and also used. Viability was tested by either Trypan-blue exclusion, JC-1-staining or PI-staining and FACS analysis, and metabolic activity determined with an ATP assay. Intracellular signaling was evaluated using Western blot analysis and commercially available antibodies to PKB and AMPK. Results: HFD caused significant increases in body weight gain, visceral adiposity, fasting and non-fasting blood glucose, serum insulin levels and an elevated HOMA-IR index. HFD cardiomyocytes were glucose uptake resistant to increasing concentration of insulin (1-100nM). Aspalathin (10uM) and insulin (10nM) co-incubation for 45mins induced 2DG uptake in younger control cardiomyocytes, while incubation for longer than 90 mins with aspalathin (10uM) induced 2DG uptake independent of insulin in younger control cardiomyocytes and differentiated H9C2 cells. Aspalathin improved metabolic activity and membrane integrity in cultured, differentiated H9C2 cells. Aspalathin also enhanced insulin-mediated 2DG uptake in older control cells, but failed to induce 2DG uptake in HFD cells. Acute treatment with aspalathin (15min) in conjunction with insulin in vitro significantly increased PKB activation and AMPK expression. Extended treatment with aspalathin (90mins) in young cells resulted in significantly increased AMPK activation/expression ratio, whereas aspalathin co-treatment with insulin resulted in increased PKB activation. Aged rats had significantly higher AMPK expression and activation compared to young rats. Conclusions: A high-fat, high-sucrose diet of at least 16 weeks is an effective model to induce insulin-resistant, obese rats. Aspalathin and insulin co-treatment for 45 mins in cardiomyocytes isolated from young rats, and co-treatment for 90 mins in aged, control rats, induced glucose uptake. Aspalathin of at least 90 mins induced glucose uptake in cardiomyocytes from young Wistar rats, and differentiated H9C2 cells. In addition, it resensitized the insulin-signaling pathway in cardiomyocytes, possibly through activation of PKB and AMPK, resulting in an additive response. These beneficial effects of aspalathin may ultimately be due to its antioxidant capacity, receptor-mediated actions or role in GLUT4 translocation, but this remains to be established.