Doctoral Degrees (Medical Physiology)
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- ItemEvaluation of mitochondrial and molecular derangements in cardiac adipose tissue during type 2 diabetes and relationship with cardiovascular risk(Stellenbosch : Stellenbosch University, 2023-06) Nyawo, Thembeka Amanda; Pheiffer, Carmen; Mazibuko-Mbeje, Sithandiwe; Phiwayinkosi, Dludla; Hanel, Sadie Van Gijsen; Hans, Strijdom; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.ENGLISH ABSTRACT: Cardiovascular disease (CVD) affects hundreds of millions of people globally, and 18.6 million deaths were attributed to CVD during 2019 alone. Type 2 diabetes (T2D) and obesity contribute significantly towards the increasing prevalence of CVD. Ageing and adipose tissue dysfunction are important mechanisms in the pathology of these metabolic diseases and their downstream cardiovascular complications. The roles of visceral (VAT) and subcutaneous (SAT) adipose depots in T2D and CVD development have been well documented; however, there is limited evidence on the pathological contribution of cardiac fat (CF) to CVD. The aim of this study was to elucidate the role of CF, in comparison to retroperitoneal (RF) and inguinal (IF) fat depots, representatives of VAT and SAT, respectively, in the development and progression of CVD in an experimental mouse model of obesity and diabetes. The study used male obese, diabetic db/db mice and their lean db/+ counterparts to explore morphological features of CF, gene expression signatures, mitochondrial bioenergetics, and associations with CVD risk factors. Briefly, mice were monitored for 8, 12 and 18 weeks, during which body weight and fasting blood glucose concentrations were measured weekly. Glucose tolerance was assessed using the oral glucose tolerance test one week prior to euthanasia. Blood and tissue samples of the heart, CF, RF, and IF were collected for assessment of biochemical markers, histological examination using Haematoxylin and Eosin staining, and gene expression analysis using quantitative real time PCR. In addition, CF, RF and IF were harvested for the assessment of mitochondrial function in adipose-derived stromal cells (ADSCs). Phenotypic and metabolic parameters deteriorated with disease and age, where db/db mice displayed conditions of hyperglycaemia, hyperinsulinaemia, hyperlipidaemia and glucose intolerance with ageing. In addition, an age-related increase in adiponectin serum levels were observed in db/+ mice, while in db/db mice, adiponectin levels decreased with age. Furthermore, histological analysis showed that adipocyte size in all depots increased over time in both the non-diabetic and diabetic state. Adipose depot-, disease- and age-related changes in gene expression signatures in CF, RF and IF were observed, with RF and IF in db/db mice exhibiting upregulation of genes involved in inflammation and oxidative stress, while CF appears to possess increased expression of genes representing thermogenic capacity. Disease- and age-related differential regulation of circulating CVD risk markers were observed. Dysregulation of markers such as metalloproteinase 9 (MMP9), intercellular adhesion molecule 1 (ICAM1), platelet endothelial cell adhesion molecule 1 (Pecam1) and Thrombomodulin (THBD) suggested vascular remodelling and dysfunction during CVD progression. Moreover, circulating levels of MMP9, ICAM and P-Selectin positively correlated with CF adipocyte size. In heart tissue, signs of histological myocardial changes with microarchitecture disruption and the presence of intramyocardial lipid droplets in db/db mice were observed in with ageing. Moreover, gene expression analysis in heart tissue revealed a disease- and age- related downregulation of brain natriuretic peptide (BNP) levels in db/db mice. Furthermore, ADSCs from CF had higher mitochondrial bioenergetics parameters compared to ADSCs from RF. This may be attributed to the higher uncoupling protein 1 (UCP1) expression in CF which reportedly decreases oxidative phosphorylation through thermogenesis. In conclusion, the positive correlation between CVD risk markers with CF adipocyte size in the diabetic state indicates a relationship with CF, supporting the notion that increased CF adiposity is associated with increased CVD risk. Importantly, CF, unlike RF and IF, displays beige-like adipocytes and thermogenic capacity, which may help mitigate the harmful effects of diabetes and its cardiovascular complications.
- ItemEffect 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.
- ItemIdentification of novel DNA methylation signatures in the development of cardiovascular disease(2021) Jooste, Tracey; Johnson, Rabia; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.ENGLISH ABSTRACT: Cardiovascular diseases (CVDs) remain the leading cause of death worldwide, claiming approximately 17.9 million lives annually. The last few decades have seen an exponential increase in the prevalence of major CVD risk factors, such as obesity, insulin resistance (IR), and type 2 diabetes mellitus (T2DM) in underdeveloped countries, including South Africa. This increase is concomitant to escalating CVD incidence and in part due to increased consumption of diets high in fat and sugar, and sedentary lifestyles. Several mechanisms have been implicated in the pathogenesis of diabetic-induced CVD and recently, increasing evidence suggests that dysregulation of the epigenome may play an important role in the development of T2DM and related cardiac complications. More specifically, aberrant DNA methylation has been extensively investigated and implicated in the development of chronic diseases such as obesity, T2DM and CVD. Nonetheless, it has been suggested that some active dietary compounds may reverse this epigenetic phenomenon which allows for the modification of the transcription of critical genes associated with the progression of CVD. Several plant-polyphenols have been reported to influence gene transcription by altering DNA methylation status. Thus, targeting DNA methylation could provide a promising approach for alternative prevention strategies. This study explored the gene expression networks activated during diet-induced CVD and the ability of a green rooibos extract, Afriplex GRT, to alter this consequence. In addition, the study aimed to evaluate aberrant DNA methylation associated with diet-induced CVD to further elucidate pathophysiology. To this end, cardiomyocytes exposed to high glucose and palmitate (HG + Pal) displayed a diminished antioxidant defence system, mitochondrial dysfunction and increased apoptosis, indicative of cardiac stress. Additionally, the combinatory treatment with HG + Pal induced transcriptional changes associated with inflammation, oxidative stress, altered lipid metabolism and increased contractile dysfunction, ultimately promoting the development of atherosclerosis and hypertrophic cardiomyopathy. Interestingly, post treatment with Afriplex GRT or Aspalathin had no significant effect on the metabolic and molecular derangements induced under HG + Pal stress. Similarly, RNA sequencing conducted on cardiac tissue of Wistar rats that received a high fat, high sugar (HFHS) diet, revealed the downregulation of differentially expressed genes (DEGs) involved in host antioxidant activity and inflammatory response, accompanied with an increase in hypertrophic gene expression possibly affecting cardiac muscle functionality. Supplementation with Afriplex GRT™ yielded no high confidence results for the amelioration of the transcriptomic signatures resulting from HFHS diet feeding. To profile DNA methylation throughout disease progression, cardiac tissue of male Wistar rats maintained on a HFHS diet were subjected to whole genome bisulfite sequencing (WGBS). The latter revealed aberrant DNA methylation of genes linked to the phagosome, platelet activation, toll-like receptor signalling and diabetic cardiomyopathy. Furthermore, hypomethylation within the intergenic and gene body regions of several differentially methylated genes (DMGs) overlapped with DEGs identified in the RNA sequencing analysis. Collectively these results demonstrate the ability of the HFHS diet to act as a pathological stimulus capable of inducing altered gene expression and DNA methylation associated with a heightened proinflammatory and lipid metabolism response that increased the risk of CVD development.
- ItemCharacterisation of high fat, high sugar diet-induced epigenetic changes in skeletal muscle of wistar rats and metabolic effects of an aspalathin-rich rooibos extract(Stellenbosch : Stellenbosch University, 2021-12) Myataza, Asive; Carmen, Pheiffer; Tarryn, Willmer; Shantal, Windvogel; Rabia, Johnson; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.Background Animal models are widely used to elucidate the pathophysiological mechanisms that underlie obesity and to test the efficacy of anti-obesity therapeutics. However, these models have been largely biased towards males, partly due to the complexity of hormonal fluctuations in females. The primary aim of this study was to elucidate DNA methylation profiles and gene regulatory networks that are altered in the skeletal muscle during the development of obesity in female and male Wistar rats, and to explore whether Afriplex-GRT™ could prevent aberrant DNA methylation patterns and the progression of metabolic disease. Methods Different animal models were employed where female and/or male Wistar rats were fed a standard or a high fat, high sugar (HFHS) diet for 12, 3 or 9 months. The effect of rooibos was investigated in the latter model, where 60 mg/kg of bodyweight Afriplex-GRTTM was co-administered with the diets. Parameters measured included food and water intake, bodyweight, and glucose, insulin, lipid and cytokine concentrations. Skeletal muscle was harvested for histology, gene expression measured using RT2 Profiler™ PCR arrays and Taqman® assays and global and gene-specific DNA methylation were quantified using pyrosequencing. To further explore the effects of DNA methylation, high glucose and fatty acids on skeletal muscle, C2C12 myocytes were differentiated with 7 μM 5-azacytidine, a global DNA methylation inhibitor, 33 mM glucose and 0.5 mM palmitate. Mitochondrial activity and oxidative stress were measured using appropriate assays. Myoblast differentiation and the expression of myoblast determination protein 1 (MyoD), myosin heavy chain 1 (Myh1), insulin growth factor 2 (Igf2), sterol regulatory element binding transcription factor 1 (Srebf1) and DNA methyltransferase 1 (Dnmt1) were assessed. Results The HFHS diet induced visceral adiposity and hypertriglyceridaemia in both male and female rats, while hyperinsulinaemia and significant bodyweight gain was observed in male rats, and systemic inflammation in females only. These changes were accompanied by increased expression of Igf2 and decreased expression of Srebfb1 and Dnmt1 in skeletal muscle of male, but not female rats. No differences in DNA methylation patterns were observed. Treatment with Afriplex-GRT™ did not ameliorate HFHS diet-induced metabolic dysregulation. In C2C12 cells, treatment with 5-azacytidine induced myoblast differentiation and MyoD and Myh1 expression, while palmitate inhibited differentiation and decreased the expression of MyoD, Myh1, Igf2 and Srebf1, which was restored by 5-azacytidine. High glucose increased Igf2 expression but did not affect Srebf1 expression, while a combination of high glucose and 5- azacytidine decreased Srebf1 and Dnmt1 expression. Conclusion The HFHS diet induced different metabolic responses and gene expression patterns in females and males. In general, females exhibited a dampened metabolic response, which we presume may be due to the intrinsic protective effects of female reproductive hormones. Afriplex-GRTTM did not prevent HFHS diet-induced weight gain, which contrasts previous findings where treatment with 60 mg/kg Afriplex-GRTTM decreased bodyweight in obese male rats, suggesting that Afriplex-GRTTM may be better targeted as a therapeutic than a preventative nutraceutical. This study provides novel information on how molecular differences in skeletal muscle may contribute to sex differences in response to HFHS feeding and may have important implications for the identification of therapeutic targets.
- ItemAn investigation of obesity as an etiology of male infertility in a rat model(Stellenbosch : Stellenbosch University, 2021-03) Skosana, Bongekile Trisha; Du Plessis, Stefan S.; Aboua, Yapo Guillaume; Van der Horst, Gerhard; Marais, Erna; Stellenbosch University. Faculty of Medicine and Health Science. Dept. of Biomedical Sciences: Medical Physiology.ENGLISH ABSTRACT: Until recent decades, the inability to produce offspring has been seen as a female-related issue, but better understanding has made it evident that male fertility is l ikewise an essential determinant of couple fertility. Infertility in males has numerous causative factors, including lifestyle factors and obesity. Obesity has been shown to compromise fertility through changes in several aspects of reproductive function, including dysregulation ofhormones (the HPG axis) and changes in the structure of the reproductive organs. There is, however, a great deal that is still unknown about how obesity and infertility interchangeably affect each other. New molecular techniques such as proteomics have been shown to provide insights into disease-causing mechanisms. These techniques may provide an avenue to discover more intrinsic changes that obesity may give rise to; it can also aid in the discovery of mechanisms through which obesity can act to bring about changes in male fertility. The overall aims of this study were: (1) To examine the effects of obesity on male fertility by observing specific macroscopic (anthropometric), microscopic (sperm parameters, histology of the testis and epididymis) and molecular (antioxidant enzyme, reproductive hormones) changes in a diet-induced obesity animal model; (2) To examine changes in protein expression within the reproductive tissues of obese animals, quantify these changes, and identify the affected molecular pathways. This study made use of an animal model of diet-induced obesity (DIO) to assess the effects of obesity on male reproductive organs and sperm parameters. Male Wistar rats (n=40) were randomly and equally divided into control (age-matched) and DIO groups and received standard rat chow or a high caloric diet ad libitum for 54 weeks, respectively. A long-term diet was chosen to mimic the gradual and chronic onset of obesity. At 60 weeks of age, rats were sacrificed by euthanasia. Each animal had their body weight measured and immediately examined post-mortem to determine visceral fat weight, testis weight and non-fasting blood glucose. Blood was collected from the thoracic cavity and used for plasma extraction and haematocrit analysis. Testis and epididymides were excised, weighed and preserved appropriately for subsequent sperm parameter evaluations (morphology and viability), histological analysis (H & E staining), protein determination, antioxidant evaluation (catalase, superoxide dismutase, glutathione, lipid peroxidation), and proteomics analysis (Liquid Chromatography Mass Spectrometry (LC-MS/MS)). The chronic diet elevated body and visceral fat weights significantly in the DIO group compared to controls. Sperm morphology and viability, as well as estradiol production were negatively altered in the DIO group. These changes were associated with alterations in several macroscopic, microscopic and molecular changes including changes in relative testicular weight, histological aberrations, and a reduction in antioxidant enzymes within the testis and epididymis respectively. Interestingly, testosterone was not significantly reduced, as seen in experiments with a shorter DIO feeding duration. This points towards a compensatory mechanism to counteract chronically increased testosterone concentrations. Protein expression profiles of the DIO and control groups suggest that the predominant molecular pathways affected by th treatment were related to metabolism. These seem to be the possible driver of changes in other proteins including those involved in the production of reactive oxygen species (ROS). Some lesser researched antioxidant proteins were increased in expression to counteract ROS. The negative histological changes observed in the DIO group were linked to the underexpression of structural proteins involved in cell-to-cell adhesion. Reproductive proteins including those involved in sperm production, fertilization and the early stages of embryonic development were reduced in expression in the DIO group. These negative changes were possibly instigated by the observed increases in stress proteins, redox and inflammatory proteins. It is therefore evident that long-term obesity can impair male reproductive parameters and could be a contributing factor to the decline in male fertility by affecting sperm and reproductive parameters. Proteomic analysis of the epididymis and sperm showed that proteins essential in metabolism, ROS production, stress, inflammation and in the regulation of reproductive function as well as sperm and epididymis structure were negatively affected. In addition, long-term obesity can mask detrimental changes in physiology due to compensatory mechanisms, making changes in reproductive parameters difficult to explain.