Doctoral Degrees (Medical Physiology)

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Browsing Doctoral Degrees (Medical Physiology) by Author "Abrahams, Yoonus"

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    Effect of an obesogenic diet on the epigenetic profile of livers from Wistar rats and treatment with an aspalathin-rich rooibos extract
    (Stellenbosch : Stellenbosch University, 2021-12) Abrahams, Yoonus; Pheiffer, Carmen; Johnson, Rabia; Samodien, Mugammad Ebrahim; Windvogel, Shantal; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.
    Background There is a scarcity of knowledge about obesity and metabolic syndrome development and progression in females, who are often under-represented in preclinical research. The aims of this study were thus to investigate the effects of high fat, high sugar (HFHS) feeding on gene expression and DNA methylation patterns in the livers of female and male Wistar rats and to determine whether an Aspalathin-rich rooibos extract, Afriplex® GRT, could prevent HFHS diet-induced metabolic dysregulation. Methods This study employed different animal models. Female and/or male Wistar rats were fed a HFHS diet for 12, 24 or 36 weeks. The effectiveness of Afriplex® GRT was investigated by daily supplementation with 60 mg/kg of bodyweight co-administered with diets for 36 weeks. Food and water intake, bodyweight, fasting blood glucose concentrations and glucose tolerance were measured regularly. After termination, organ weights, liver histology, fasting serum insulin, high- and low-density lipoprotein and cholesterol concentrations were measured. Gene expression was assessed using RT2 Profiler Rat Fatty Liver PCR arrays and TaqMan® gene expression assays. DNA methylation was quantified by pyrosequencing. The effects of DNA methylation were further investigated in the HepG2/C3A hepatocarcinoma cell line. Cells were treated with 2.5 μM of the DNA methyl transferase inhibitor, 5-aza-2’-deoxycytidine for 24 hours to induce DNA hypomethylation. Thereafter, steatosis and insulin resistance were induced by exposing cells to a 1:1 mixture of 0.5 mM palmitic and oleic acids for 24 hours. Cell viability, mitochondrial function, mitochondrial membrane potential, lipid accumulation, reactive oxygen species accumulation and gene expression were assessed. Results The HFHS diet increased visceral adiposity and hypertriglyceridaemia in both female and male rats, while hyperinsulinaemia and sustained obesity were observed in males only. Males fed the HFHS diet for 12 weeks showed reduced peroxisome proliferator-activated receptor gamma coactivator-1 alpha (Pgc1a) expression and hypermethylation of the Pgc1a promoter, but this was not observed in males fed the same diet for 36 weeks. Females fed HFHS showed a sex-specific reduction in peroxisome proliferator-activated receptor gamma (Pparg) expression. Afriplex® GRT did not prevent obesity or metabolic dysregulation. In vitro analysis demonstrated that DNA hypomethylation reduced lipid accumulation but did not affect insulin sensitivity. Steatosis and insulin resistance in the presence of DNA hypomethylation was associated with increased expression of uncoupling protein 2 (UCP2) and CCAAT/enhancer-binding protein beta (C/EBPß). Conclusion The HFHS diet elicited different responses in female and male Wistar rats. Male rats exhibited obesity, hyperinsulinaemia, glucose intolerance and altered methylation of Pgc1a. However, females exhibited a dampened metabolic response, potentially mediated through sex-specific downregulation of hepatic Pparg. Prophylactic daily supplementation with 60 mg/kg Afriplex® GRT did not prevent diet-induced obesity or metabolic dysregulation. For the first time, we show that DNA hypomethylation in HepG2 hepatocarcinoma cells decreases lipid accumulation in vitro and show that hypermethylation of a conserved CpG site in the promoter of Pgc1a is associated with decreased expression in the livers of male Wistar rats. We observed sex-specific hepatic differences between females and males which may have important implications for the development of therapeutic targets. In future, this may allow for the development of effective treatments free of sex-biased adverse effects and which confer protection against metabolic disease, reduce mortality and improve the quality of life of affected persons globally.