Masters Degrees (Medical Physiology)

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Abnormalities of bone and mineral metabolism in patients with eating disorders
(Stellenbosch : Stellenbosch University, 2001) Conradie, Maria Martha; Hough, F. S.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division Medical Physiology.
ENGLISH ABSTRACT: Osteopenia is a well documented complication of anorexia nervosa (AN). The pathogenesis of this bone loss is presently poorly defined in the literature. Pathogenetic mechanisms that have been implicated include certain nutritional factors, exercise abuse, hypogonadism, hypercortisolism and/or vitamin 0 deficiency. We studied, 59 Caucasian eating disorder patients aged 15-45yr. The eating disorder was classified by a single, qualified psychiatrist according to OSM IV R criteria as either anorexia nervosa (AN: n =25), bulimia nervosa (BN: n = 17) or eating disorder not otherwise specified (EONOS: n = 17). All patients were subjected to a detailed dietary and general history. We assessed the prevalence and severity (OEXA), the nature (osteocalcin, deoxypyridinoline) and site (vertebral versus hip) of osteopenla in these patients. he role of nutritional factors (energy intake, weight, height, BMI, plasma albumin, lipids), physical activity, hypercortisolemia (plasma and urinary free cortisol), vitamin 0 deficiency (plasma 250HD) and hypogonadism (amenorrhoea, E2, LH, FSH) in the pathogenesis of bone loss were also evaluated. Mild osteopenia (BMO decreased by more than 1SO below age-matched controls) was documented in 46% of the total study population, with more marked osteopenia (Z-Score < -2 SO) present in 15%. Both vertebral and hip osteopenia were documented. In the study population those patients with AN (Lumbar BMO (q/cm") = 0.869 ± 0.121) were most likely to develop osteoporosis, although a significant percentage of patients with BN (Lumbar BMO (q/crn") = 0.975 ± 0.16) and EONOS (Lumbar BMO (g/cm2) = 0.936 ± 0.10) were also osteopenic (29% and 35% respectively). Twenty four percent (24%) of the total patient population had a history of fragility fractures. These fractures were reported more commonly amongst patients with AN and EONOS (28% and29.4%). Fracture prevalence was however similar in patients with normal and low bone mass. Conventional risk factors were similar in patients with normal and low bone mass, except for a significantly longer duration of amenorrhoea (p = 0.009), a lower BMI (p = 0.0001) and greater alcohol consumption (p = 0.05) in the osteopenic patients. Nutritional parameters (S-albumin, protein, Ca, and P04 intakes), physical activity, as well as 25(OH) vitamin D levels were similar in AN and BN subjects, as well as in patients with a low versus normal BMD. Plasma and urine cortisol levels were also similar in these subgroups. With the exception of two patients with borderline osteopenia, significant bone loss was only documented in those patients with a past or current history of amenorrhoea. In the total patient population the duration of amenorrhoea was significantly (p<0.009) longer in patients with osteopenia versus those with a normal bone mass. A significant negative correlation between BMD (Z-Score) and duration of amenorrhoea was also documented in the total patient population (r = -0.4, P = 0.001) as well as in all three eating disorder groups (AN r - -0.4, P = 0.03; BN r = - 0.6, P = 0.008; EDNOS r = -0.6, P = 0.005). In the total patient population, those patients with amenorrhoea, had lower BMD and BMI values and lower estrogen levels compared to those with a normal menstrual cycle. We conclude that osteopenia commonly attends AN, as well as BN and EDNOS. Nutritional (with the exception of alcohol consumption) and mechanical factors as well as hypercortisolemia did not appear to contribute significantly to bone loss in this study population. Hypogonadism appeared to be the main cause of the bone loss observed in these patients.
Acrosome size and kinematics of human spermatozoa
(Stellenbosch : University of Stellenbosch, 2007-03) Murray, George M.; Du Plessis, S. S.; Franken, D. R.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.
For spermatozoa to gain access to the oocyte for fertilization, lytic enzymes need to be released during the acrosome reaction. These enzymes, which are stored and transported within an organelle termed the acrosome, make it possible for spermatozoa to collectively penetrate the layers of cells and glycoproteins that surround and protect an oocyte. Acrosomes may thus be viewed as essential for fertilization and their shape, size and volume were examined morphometrically by utilizing automated morphometric analysis equipment. In addition to the acrosome being necessary for normal unassisted fertilization, spermatozoa also need the ability to migrate to the oocyte. Following zona pellucida binding, sperm tail thrust movement initiates zona penetration into the space created by the digestive action of the acrosomal enzymes. Therefore the motion characteristics of spermatozoa were also quantified in terms of kinematic properties. In the treatment of male sub fertility, assisted reproductive techniques are applied. In the application of such techniques, a motile sub-population of spermatozoa was obtained by employing a procedure (swim-up selection) that selects cells on the basis of their kinematic ability. This study presents an analysis of the morphometric and kinematic qualities of spermatozoa populations that are subjected to swim-up selection and investigates the relationship of these morphometrical and kinematic qualities. Computer-assisted semen analysis, swim-up selection and automated sperm morphology analysis tests were all used to evaluate spermatozoa populations. Results indicated that, irrespective of acrosome size, higher kinematic parameter measurements were observed post-swim-up. A significant inverse relationship between the population’s average acrosome size and a number of kinematic parameters was observed. Our results indicated that for a post-swim-up population of spermatozoa an increase in the average acrosome size was significantly related to a decrease in the kinematic parameters VAP, VCL and the VSL within the same population.
The anti-diabetic and insulin sensitizing potential of a watery extract of Agathosma tested in rat models of type 1 and type 2 diabetes
(Stellenbosch : Stellenbosch University, 2015-03) Jansen, Vereneque; Huisamen, Barbara; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.
ENGLISH SUMMARY: Introduction: Epidemiological data highlights that South Africa is currently facing a quadruple burden of disease of which non-communicable diseases (NCDs) are estimated to account for 29% of all deaths. These NCDs include, amongst others, cardiovascular disease, type 2 diabetes, hypertension, cancer, chronic lung disease and depression. Diabetes has become a major problem worldwide and in South Africa and is currently rated as the 5th largest cause of death by Statistics South Africa in 2011. This creates an enormous burden of disease on populations and the utilization of herbal remedies has escalated in popularity because of this. Buchu water is one of these herbal remedies advertised as having anti-diabetic properties. This water is a by-product of the extraction of the oil from the leaves of Agathosma and is already freely available to the public. The aim of this study was therefore to use animal models of type 1 diabetes, and obesity and insulin resistance to scientifically verify or refute these claims. Methods: We utilized male Wistar rats. Two different rat models were used: (i) a type 1 diabetic model; induced via a once-off intraperitoneal Streptozotocin (40mg/kg) injection ablating ~50% of pancreatic beta cells (T1D); (ii) A diet-induced obese model, rendering rats insulin resistant after receiving a high caloric diet for 16 weeks. Half of each experimental group was treated with diluted Buchu water for a period of 14 weeks and 16 weeks, respectively, while the rest consumed normal water. Water and food consumption were monitored, body weight and intraperitoneal fat measured, blood was collected to determine serum glucose and insulin levels, skeletal muscle was removed to test insulin sensitivity using radiolabelled deoxyglucose, pancreas and skeletal muscle harvested and stored in liquid nitrogen for further biochemical analysis. Results: One of the main findings of this study was that ingestion of Buchu water results in weight loss despite no decrease in food consumption. This occurred in both the pathological models and control animals. In the obese animals, this weight loss was due to a decrease in intra-peritoneal fat. A second important finding was that the ingestion of Buchu water in all instances, whether given as treatment (treated with Buchu water 3 weeks after the start of the experiment) or as prophylactic (treated with Buchu water from the start of the start of the experiment), resulted in normalization of glucose levels in a type-1 diabetic model with residual beta-cell mass. An insulin-sensitizing effect was not clearly established in skeletal muscle but this may be because of a large variation in values obtained, as well as the use of a slow-twitch muscle. A definite effect on pancreatic insulin secretion has been demonstrated by raised C-peptide levels in the diet-induced obese model. Conclusion: This study, with regard to the type 1 diabetic model, has confirmed the anti-diabetic effect of Buchu water by significantly lowering blood glucose levels of fasted and non-fasted blood and normalizing Intraperitoneal glucose tolerance test (IPGTT) curves. This was however not so evident in the obese model utilized. Despite this, animals lost weight which was mainly intra-peritoneal fat.
Antioxidant supplementation as protective strategy against stem cell impairment in Type 2 Diabetes
(Stellenbosch : Stellenbosch University, 2021-03) Maartens, Michelle; Van de Vyver, Mari; Marais, Erna; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences.
ENGLISH ABSTRACT: Type 2 Diabetes Mellitus (T2DM) is a global epidemic. It is a complex disorder that leads to cellular dysfunction and the development of co-morbidities. The underlying pathologic microenvironment in T2DM, associated with hyperglycaemia, include the accumulation of advanced glycation end products, excessive oxidative stress, and chronic inflammation. Bone marrow mesenchymal stem cells (MSC) are especially susceptible to this damaging microenvironment and as consequence the endogenous repair mechanisms within the body fail giving rise to secondary complications such as non-healing wounds, retinopathy, and neuropathy. There is thus a need for a holistic approach when it comes to disease management in which anti-diabetic drugs focussed on glucose control is complemented by supplementary treatment aimed at restoring homeostasis. Natural and synthetic antioxidants such as Ascorbic acid-2-phosphate (AAP) and N-acetyl-l-cysteine (NAC) are known to protect cells against oxidative stress-induced damage in vitro and have been shown to reduce inflammation in various models. The efficacy of these antioxidants to restore homeostasis and prevent cellular dysfunction in T2DM is however still unknown. The aim of this study was to investigate the protective effects of combined NAC and AAP supplementation against MSCs impairment using an animal model of obese diabetic (B6.C-Lepob/J) (ob/ob) (n=14) and wild type control (C57BL6/J) (n=20) mice. All mice received jelly cubes containing either antioxidants (NAC7.5mM+AAP0.6mM) or placebo (vehicle control) for a period of 6-weeks. Metabolic parameters (weight and blood glucose) were assessed on a weekly basis and the overall antioxidant status of animals assessed at the end of the 6-week period by analysing the total antioxidant capacity (TAC) and Malondialdehyde (MDA) levels in serum. Bone marrow MSCs were isolated from mice in each of the respective treatment groups and their ex vivo growth rate, viability, multi-lineage differentiation capacity and paracrine responsiveness upon stimulation with wound fluid assessed. Compared to the wild type (C) mice, excessive weight gain (weight: C 28.7±1.6 g; DM 44.3±3.5 g) (p<0.05) and hyperglycaemia (blood glucose: C 10.1±1.3 mmol/L; DM 23.6±5.7 mmol/L) (p<0.05) was evident in the DM animals validating the animal model as representative of T2DM. The antioxidant supplementation did not affect metabolic parameters indicating that differences observed between supplement and placebo treated mice were not due to weight loss or changes in glucose metabolism. NAC/AAP significantly (p<0.05) reduced lipid peroxidation in DM animals (DM:P 39.0±14.7 nmol/L; DM:S 21.5±11.6 nmol/L) to a level comparable to that of controls (C:P 22.9±11.4 nmol/L; C:S 30.5±3.3 nmol/L) and increased the overall TAC (C:P 3.7±1.3 U/mL; C:S 4.0±0.7 U/mL; DM:P 5.2±0.9 U/mL; DM:S 5.8±1.5 U/mL). The ex vivo growth rate of cells derived from DM animals were impaired (Time to confluence: C 8 days; DM 12 days). NAC/AAP supplementation did however improve the growth rate and viability of MSCs derived from both animal models. NAC/AAP furthermore reduced the adipogenic differentiation capacity of MSCs but could not restore osteogenesis in DM MSCs. Upon stimulation with wound fluid, slightly increased IL6 (DM:S+WF 1583.3±481.9 pg/mL) and IL10 (DM:S+WF 27.1±9.8 pg/mL) release was evident in MSCs derived from NAC/AAP supplemented animals. In conclusion, NAC/AAP supplementation was able to reduce oxidative stress in animals and improved MSCs viability.
Aspalathus linearis as a possible SGLT2 inhibitor : investigating the antihypertensive effects of green rooibos extracts
(Stellenbosch : Stellenbosch University, 2023-03) Willemse, Ilze Verona; Barbara, Huisamen; Erna, Marais; Shantal, Windvogel; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.
ENGLISH ABSTRACT: Introduction: Obesity-induced non-communicable disease development is a growing epidemic. SGLT2, located in kidneys, reabsorb glucose and sodium from urine. Studies suggest SGLT2 expression and activity are upregulated during chronic hyperglycaemia. SGLT2-inhibitors, a costly class of anti-diabetic treatment, are also cardio-protective and anti-hypertensive. An alternative treatment is Rooibos (Aspalathus linearis), a native South African plant. Afriplex GRT™ and E1CHA, are Rooibos extracts containing high levels of Aspalathin. It is hypothesized that Rooibos may reduce diet-induced hypertension and vascular dysfunction. Aim(s): To (i) verify whether different Rooibos extracts embody SGLT2- inhibitors as a key mechanism of action to counteract diet-induced metabolic changes, (ii) determine the possible expression levels of SGLT2 in aortic tissue and (iii) whether this is modulated by ingestion of Rooibos extracts. Methods: Adult male Wistar rats were randomly allocated into either Controls (rodent chow) or HFD (obesogenic diet)(n=96/group) and further subdivided into eight treatment groups (n=12-36/group). Animals were on diets for 16 weeks, with a 6-week treatment period (last 6 weeks) with 60mg/kg/day of either Afriplex GRT™ or E1CHA (SA Rooibos council) or Empagliflozin (EMPA) (10mg/kg/day) (positive control). Body weight, food and water consumption, blood glucose, blood pressure, IP fat weight, liver weight, and ex vivo vascular reactivity (on aorta’s without PVAT) was determined. Western blot analysis of aortic tissue was conducted to determine activation and expression levels of the proteins implicated in vascular function (AMPK, eNOS, PKB/Akt), SGLT2, ATM, mTOR,S6Kinase and markers of SGLT2 activation and expression (ERK and IĸB). Serum was used to determine aldosterone and endothelin 1 (ET-1) expression levels by means of ELISA kits. Results: HFD animals had amplified food consumption, body weight, glucose levels, IP fat, liver weight, and was hypertensive. Moreover, the aortas of HFD had decreased in the following: phosphorylated AMPK, total IĸB, total and phosphorylated ATM, total and phosphorylated mTOR, phosphorylated ERKp42. While the following were increased: total SGLT2, total ERK p42/p44. Treatment with GRT™ reduced the following: water intake, IP fat weight, fasting and non-fasting glucose levels, systolic and mean arterial blood pressure. Furthermore GRT™ increased total ERK p42/p44 expression and ERK p42 activation vs Untreated control. E1CHA treatment increased vasodilation, fasting blood glucose levels, and body weight. While it reduced vasoconstriction, water intake, IP fat weight, total ATM, phosphorylated ERK p42, total and phosphorylated mTOR and liver weight (compared to the Untreated HFD). Treatment with EMPA increased glucose tolerance, total ERKp42/p44 and total AMPK. The following were reduced: vasodilation, IP fat weight, fasting blood glucose and water consumption. Conclusion: The HFD model had an adverse influence on cardiovascular health. In HFD animals, treatment with E1CHA improved vascular function, while treatment with GRT™ improved glucose metabolism and blood pressure. Furthermore, EMPA treatment improved glucose tolerance in the HFD group. E1CHA and GRT™ also showed characteristics of EMPA. Therefore, E1CHA and GRT™ may be potential alternative therapeutic agents against obesity induced insulin resistance, hypertension and vascular dysfunction. We conclude that SGLT2 is present within the aortic tissue and that they are upregulated during insulin resistance. Furthermore, Rooibos extracts may mimic the mechanism of action of SGLT2-inhibitors.
ATM Expression in peripheral blood mononuclear cells as a biomarker of insulin resistance
(Stellenbosch : Stellenbosch University, 2019-04) Williams, Lois Esther; Huisamen, Barbara; Stellenbosch University. Faculty of Engineering. Dept. of Biomedical Sciences: Medical Physiology.
Introduction: The Ataxia Telangiectasia Mutated (ATM) gene codes for the 350 kDa ATM protein kinase. ATM gene mutations cause inactivity/deficiency of the ATM protein, resulting in the autosomal recessive disease Ataxia Telangiectasia (AT). AT patients are predisposed to developing insulin resistance or further progress to type 2 diabetes and are at high risk of developing ischaemic heart disease. Due to the prevalence of insulin resistance in AT patients, we investigated the relationship between ATM protein levels and the degree of insulin resistance and proposed it as a possible early diagnostic technique for insulin resistance. Aim: To determine whether peripheral blood mononuclear cells (PBMCs) can be used to determine ATM levels in insulin resistant subjects and subsequently used as a biomarker of insulin resistance. Objectives: (i) To standardise a protocol for the isolation of PBMCs from rat blood. (ii) To isolate rat PBMCs and determine the ATM levels using Western blotting. (iii) To determine differences between ATM levels in obese rats and compare them to controls. (iv) To analyse PBMCs from a female Black Xhosa population with different degrees of insulin resistance and to determine a relationship with ATM levels. Methods: Male Wistar rats were fed an obesogenic diet (od) for 16 weeks to induce obesity and insulin resistance and compared to age-matched and young controls fed standard rat chow. Body weight and intraperitoneal (IP) fat mass were determined and oral glucose tolerance test (OGTT) was performed. PBMCs were isolated according to the standardised protocol and Western blotted for ATM and P22phox. The Western blotting protocol was repeated with samples collected from patients. Results from the animal model: 1. Effects of obesity/insulin resistance vs. age-matched controls: (i) larger IP fat mass; (ii) increased area under the curve of OGTT’s; (iii) elevated basal glucose levels. (iv) The phospho-(P)/total-(T) ATM ratio was decreased. 2.Effects of age: (i) As expected, older animals weighed more while T-ATM was decreased, P-ATMincreased and the P-/T-ATM ratio increased with age. In PBMC’s from patients, the following were observed: (i) Body mass index (BMI) was significantly higher in obese and pre-diabetic vs. control patients. (ii) The waist-to-hip ratio (WHR) of obese and pre-diabetic women was higher vs. controls. (iii) Trunk-to-limb fat mass (TF/LF) was increased in obese and pre-diabetics vs. controls but (iv) no differences in the lipid profiles were observed except for increased triglyceride levels between young pre-diabetic patients vs. their controls. (v) Fasting blood glucose of obese and pre-diabetic patients was significantly increased vs. controls. (vi) Significantly higher P-ATM levels were seen for obese and pre-diabetic vs. control patients. T-ATM levels increased with the state of insulin resistance. Effects of age: (i) BMI was significantly higher between young (Y) and middle aged (MA) control, obese and pre-diabetic groups vs. their respective controls while (ii) WHR of Y obese and Y pre-diabetic vs. Y controls also increased significantly. (iii) The TF/LF ratio was increased between Y and MA control and Y obese, Y pre-diabetic, MA obese and MA pre-diabetic women vs. their respective controls. Furthermore (iv) the blood glucose levels of Y pre-diabetics were increased vs. Y control. (v) The P-ATM levels was increased in Y pre-diabetic vs. Y control and therefore did not increase with age but the T-ATM levels significantly increased with age. Conclusion: ATM levels can be measured in PBMCs and are affected by the insulin resistant state and age. Unfortunately, due to the variation in ATM levels under different degrees of insulin resistance, it would be difficult to use ATM as a biomarker of insulin resistance.
Cardio-metabolic risk profile of people living with HIV: Is retinal microvascular geometric morphology a marker of effect?
(Stellenbosch : Stellenbosch University, 2022-04) Kgokane, Boipelo Mirriam Lepetjia; Strijdom, Hans; Everson, Frans; Kamau, Festus; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.
Background and Aim Cardiovascular disease in people living with HIV has become of great concern. HI-viral factors, ART-toxicity and HIV/ART-associated cardiometabolic adverse effects have been implicated in the development of cardiovascular disease. Retinal microvascular geometric features may be potential useful markers of these effects. We aimed to investigate whether altered retinal microvascular geometric features are markers of HIV, ART and/or HIV/ART-associated cardiometabolic effects in a study population from the Western Cape Province. Methods The study followed a cross-sectional (HIV-free: n = 88 and HIV+ART: n = 122) and longitudinal (baseline vs. 18-month follow-up for HIV+ART only: n = 82) study design. Volunteering participants were recruited from health care clinics. Demographic, lifestyle, socioeconomic and anthropometric data were collected. Fasting blood and urine samples were collected and transported to the National Health Laboratory Services for biochemical analyses. Retinal images were obtained (Canon CR-2 camera) and vessel features quantified (MONA REVA 2.1.1 software). Linear stepwise regression (cross-sectional) and linear mixed model (longitudinal) analyses were applied to elucidate independent associations and statistical significance of p < 0.05. Results Population characteristics: The study population was relatively young (HIV-free:44.06±11.09 and HIV+ART:40.35±8.94years) and mostly women (HIV-free:80.7% and HIV+ART:63.1%). The baseline median/mean viral load (VL), CD4 cell count and ART-duration were 50 (10 to 675032) copies mRNA/mL, 539.92±237.16 cells/mm3 and 166 (1 to 707) weeks respectively. Cardiometabolic results: Body mass index (BMI) (24.50±6.65 vs. 28.25±7.68kg/m2, p < 0.001) was significantly lower in HIV+ART vs. HIV-free. ∆BMI in HIV+ART was significantly correlated with average arterial tree diameter (r = 0.323, p < 0.05), total length of skeletonised tree (r = 0.355, p < 0.01) and arteriolar branching angle (r = 0.234, p < 0.05). High density lipoprotein-cholesterol (1.59±0.74 vs. 1.39±0.45mmol/L, p = 0.019) and gamma-glutamyl transferase (GGT) (43.5 (14 to 494) vs. 27.0 (11 to 814)U/L, p < 0.001) were significantly higher in HIV+ART vs. HIV-free, but decreased in HIV+ART (Baseline vs. Follow-up HDL:1.62±0.77 vs. 1.44±0.64mmol/L, p = 0.017 and GGT:45 (14 to 494) vs. 41.50 (14 to 219)U/L, p = 0.004). HDL was significantly correlated with central retinal venular equivalent (CRVE) (r =-0.195, p < 0.01) and GGT with venular branching optimality (r = 0.180, p < 0.05). HIV and ART results: Cross-sectionally, HIV+ART status independently associated with CRVE (-0.146 (- 0.280 to -0.012), p = 0.033) and arteriolar and venular mother branch (D0), first daughter branch (D1) and second daughter branch (D2) (p < 0.05, respectively). VL (-0.198 (-0.025 to -0.001), p = 0.037) and ART- duration (0.188 (0.001 to 0.024), p = 0.047) were independently associated with arteriolar-venular ratio (AVR). Longitudinally, VL independently associated with CRVE (0.096 (0.017 to 0.175), p = 0.018) and AVR (-0.003 (-0.0006 to 0.000003), p = 0.046). CD4 cell count was independently associated with number of branchpoints 0.042 (-0.002 to 0.086), p=0.006) and endpoints (3.0 (0.750 to 5.250), p=0.010). HIV duration independently associated with lacunarity (-0.0080 (-0.0150 to -0.0010), p=0.036) and fractal analyses (0.011 (0.0001 to 0.021), p=0.045). 2nd-line ART was independently associated with CRVE (8.58 (0.35 to 16.81), p=0.041) and ART-duration with fractal analysis (-0.022 (-0.037 to -0.008), p=0.003). Discussion and conclusion HIV+ART appeared to have a more favourable cardiovascular risk profile vs. HIV-free. Various markers of HIV/ART and HIV-ART-associated cardiometabolic risk factors were associated with retinal vessel features and associations appeared mostly favourable/cardioprotective. These results indicate that retinal vessel geometric features may be potential markers of the effects of HIV/ART and/or associated cardiometabolic risk factors in the current study population.
Changes in hyo-laryngeal elevation post-pharyngeal electrical stimulation
(Stellenbosch : Stellenbosch University, 2015-04) Basson, Tobias Johannes; Pillay, Mershen; Du Plessis, Stefan S.; Stellenbosch University. Faculty of Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.
ENGLISH ABSTRACT: Swallowing disorders are prevalent in many elderly individuals and are common amongst individuals suffering from neurological diseases. These individuals are affected from slight swallowing difficulty to total swallowing inability. In severe cases this may cause aspiration pneumonia, dehydration, malnutrition and ultimately death. Swallowing disorders can be diagnosed and treated to increase quality of life. New treatment strategies to understand the pathophysiology and impaired swallowing response are needed. Neuromuscular electrical stimulation is used as rehabilitation method in various disciplines. This method of rehabilitation of physiological dysfunction is used in treating swallowing disorders and has become a focus for current research. To understand the effect of electrical stimulation to the swallowing centre it is proposed to study its mechanism on normal swallowing musculature. The outcome of the effect that electrical stimulation has on healthy individuals may possibly be used to extrapolate to clinical settings and its benefit for modern dysphagia rehabilitation. The purpose of this study was to report on the hyo-laryngeal movement pattern of young healthy, male and female, individuals and to measure the effect of a single neuromuscular electrical stimulation session on the hyo-laryngeal complex of 22 young healthy individuals. Lastly, the aim was to determine the detraining or lasting effect on the hyo-laryngeal swallowing complex of a single neuromuscular electrical stimulation session. The study reported on baseline hyo-laryngeal complex movement patterns by measuring the anterior movement and elevation of the hyo-laryngeal complex through the use of videofluoroscopy swallow study. Analysis of these measurements where done to report on the effect of electrical stimulation on the hyo-laryngeal complex movement pattern pre- and post- electrical stimulation. Significant changes were revealed with elevation of the hyo-laryngeal complex, however no significant effects could be found with anterior movement of the hyo-laryngeal complex pre- and post- electrical stimulation. It was found that elevation of the hyo-laryngeal complex lowered after a single electrical stimulation session. The hyo-laryngeal complex movement pattern remained similar between genders. Lastly it was found that a single electrical stimulation session showed significant reversibility towards baseline levels. This might be related to muscle fatigue and one would need to take into account muscle recovery for future research.
Characterising pathways that contribute to post-TB lung disease
(Stellenbosch : Stellenbosch University, 2024-02) Jacobs, Steve; Maarman, Gerald; Windvogel, Shantal; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.
ENGLISH ABSTRACT: Tuberculosis (TB) is a major global health challenge, especially in low- and middle-income countries (LMICs). Post-tuberculosis lung disease (PTLD) is a common and debilitating sequela of TB, which can lead to chronic respiratory symptoms and impaired lung function. The pathogenesis of PTLD is not well understood, and much remains to be discovered, although some other authors have hypothesized that inflammation may be a key contribution, despite successful completion of TB treatment. Moreover, accumulating data suggests that PTLD might also include pulmonary hypertension (PH). The latter is a multi-organ disease and highly morbid clinical condition, with a broad range of clinical presentations and is caused by a large spectrum of underlying conditions. The relationship between PTLD and PH is complex and multifactorial, involving host, environmental, and pathogenic factors. Given the inflammatory nature of TB, it is likely that pro-inflammation may contribute to the development of PH post-TB, however, there is currently no data on this topic. This is concerning, as millions of people live with TB, close to 60 000 people die of TB in South Africa per annum, while every year almost 600 new cases of TB are reported. Given this context, it is worrying that TB patients (whether previous or current, or treated) are at risk of developing debilitating PTLD and fatal PH. This project, aimed to delineate the involvement of pathwaysthat may contribute to the development of PH in a post-TB context, and to explore the pathways that specifically contribute to PH in the same context. In our pursuit, we managed to highlight the instrumental roles of inflammatory pathways as part of PTLD pathogenesis. Our novel findings suggest that there is a pro-inflammatory state in active TB patients on treatment that persists post-TB, regardless of being successfully treated for TB. The unusual circulation pattern of inflammatory cytokines could be ascribed to mitochondrial dysfunction in immune cells. Considering the destructive nature of these cytokines, there is a need for further research to explore the implications of a persistent pro-inflammatory state, as it may predispose patients to PTLD. In terms of PH, we explored myriad pathways that may cause PH, now a new key feature of PTLD. Our review of the literature demonstrated a link between melatonin and PH as part of PTLD. Our findings demonstrate that melatonin is an important link between the gut microbiota and the development of PH (where suppressed melatonin-crosstalk between the gut and lungs could promote the development of PH). More studies are needed to investigate the link between the gut microbiota, melatonin and PH. Studies could also investigate whether microbiota genes play a role in the epigenetic aspects of PH. This is relevant because, e.g., dysbiosis (caused by epigenetic factors) could reduce melatonin signalling between the gut and lungs, reduce subcellular melatonin concentrations in the gut/lungs, or reduce melatonin serum levels secondary to epigenetic factors. PH is a fatal disease, and this essentially means that despite successful TB treatment thousands of patients are at risk of developing PH. Yet, there is no cure for PH and most developing countries do not have specialised PH drugs. Therefore, there is a need for research on better treatments for PH, particularly, in the post-TB context. We explored the potential of the repurposing of drugs for the treatment of PH especially in countries with resource limitations. Studies have demonstrated the benefits of medicinal plants against PH, most of which are of Indian or Asian descent. Africa is a rich source of multiple medicinal plants scientifically proven to counteract myriad pathologies. When perusing these studies one can notice that African medicinal plants afford biological effects that counteract the same molecular pathways (e.g., proliferation, vasoconstriction, inflammation, oxidative stress, and mitochondrial dysfunction) also involved in the pathogenesis of PH. Viable options include Aspalathus linearis, Allium sativium, Trifolium pratense L, Mimosa pigra L, and Aloe ferox. However, most of these plants have never been tested in an experimental PH model, and https://scholar.sun.ac.za 4 therefore, our proposition is hypothetical at the most. Regardless, we believe that future studies should investigate these and other African medicinal plants in appropriate models of PH, to test their efficacy and effectiveness. The relationship between PTLD and PH is complex and still requires several puzzles to be placed to fully understand it. The pathophysiology of PTLD that leads to PH, epidemiological factors and potential treatment options remains largely unexplored and are key areas for future research. Research into alternative and novel therapies is especially crucial as this has the potential to improve patient’s quality of life and clinical outcomes. Ultimately, further research will hopefully pave the way for the development of a comprehensive approach to PH prevention, detection and diagnosis, and treatment strategies.
Characterization of a sonified peak flow monitor
(Stellenbosch : University of Stellenbosch, 2000-03) Vermeulen, M. O.; Wessels, J. A.; Von Backstrom, T. W.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences.
ENGLISH ABSTRACT: The Whistle Watch™, an innovative and commercialised peak flow monitor, inspired this study, with its abnormal and complex measuring behaviour. The Whistle Watch™ latter is an audible peak flow monitor with a threshold-activated whistle as the essential component. The whistle is calibrated for a certain flow, and then encased in a body with a variable exhaust valve to atmosphere. Using the Whistle Watch™, with the exhaust valve pre-set, executing a forced expiratory effort, the audible notification of the whistle would indicate a stable asthmatic condition at that setting. No audible notification would result in the use of medication as a preventative measure. Due to the absence of existing theories and literature on the mechanics of whistles, the Whistle Watch™ was empirically developed. This study therefore, focuses on the characterisation and consequent improved understanding ofthe mechanics ofa whistle, with the objective to monitor pulmonary function in a novel way. During this study, a novel technique was developed to determine the reed activation point, or onset of oscillation, in terms of pressure. This technique was then implemented throughout the study. The initial observation and experimentation underlined the whistle's activation sensitivity towards any irregularities of the reed surface. A statistical spread of reed activation pressures defined the reed's inherent non-linear properties. A high dependence of reed activation towards upstream geometry was noted, and a clarification hypothesis was formulated. The effect of reed dimensions on activation pressure was exposed as a complex unexplored field. Existing mathematical reed theories only accommodate steady state oscillations, whereas the completed study indicated a high sensitivity of the reed activation pressure towards different input envelopes. This sensitivity was encapsulated in a mathematical model, with initial support and proofprovided by a previous independent study. All the observed effects and phenomena had far reaching practical application towards the production and quality control ofthe Whistle Watch™.
Chronic stress and semen parameters
(Stellenbosch : Stellenbosch University, 2020-03) Van der Merwe, Esmari; Du Plessis, Stefan; Basson, E.; Stellenbosch University. Faculty of Health and Medical Sciences. Dept. of Biomedical Sciences: Medical Physiology.
ENGLISH ABSTRACT: It has been well documented that stress has adverse effects on the body and can lead to various health issues. Stress has been investigated as a cause for unexplained infertility in both men and women. Semen quality is a key indicator of male reproductive health. Numerous studies have been done on the effect of stress on semen parameters and an association between chronic psychological stress and poor semen parameters have been reported. Managing psychological stress can help to improve the health of an individual. In order to address the problem it is therefore important to determine if an individual experience high levels of stress. This can be established through psychological questionnaires and various biomarkers, such as the screening test for time urgency perfectionism (TUP) and alpha-amylase in saliva. In general, more or less 84% of couples are estimated to conceive naturally within a year. The remaining 16% of couples are affected by infertility. Within this group, it is estimated that male reproductive factors are the sole cause of one-third of cases and a contributing factor in another 20% of cases. Management of chronic stress in female patients has shown improved IVF rate of 67% or higher. However, as of yet no study has been performed on males to correlate the levels of TUP-stress, alpha-amylase to semen parameters as well as other seminal stress markers such as DNA fragmentation and oxidative stress (ROS). This study compared TUP-categories (Low, Moderate, High) with respect to semen parameters, alpha-amylase levels, age and BMI and investigated if increased alpha-amylase levels correlate with semen parameters, age and BMI. The experiments were performed at Medfem Fertility Clinic in Bryanston Johannesburg and the Division of Medical Physiology in the Department of Biomedical Sciences at Stellenbosch University. A total of 62 male patients of Medfem Fertility Clinic adhering to the basic requirements enrolled in the study. Results showed no significant difference between age, semen parameters and alpha-amylase between TUP categories. Men in the High TUP category had a significant higher BMI compared to those in the Low and Moderate categories. No significant correlation was found between alpha-amylase, age, BMI and semen parameters. This study was unsuccessful in proving a significant relationship between the TUP categories, age and semen parameters. The High TUP category did show a significantly higher BMI compared to the Low and Moderate TUP groups. This finding confirms that there is a link between psychological stress and elevated BMI. Although there was no significant difference between the TUP categories with regards to sORP values, the Moderate and High categories were both higher than the normal value for sORP in semen. This implies that chronic stress leads to elevated levels of oxidative stress in semen. No relationship was found between TUP categories and alpha-amylase levels. Although both are used to detect chronic stress, the TUP questionnaire is used to detect personality types who are prone to chronic stress, whilst salivary alpha-amylase is a biomarker for chronic stress and functions in a completely different way. It is possible that whilst both can be used to detect chronic stress it is not advised to attempt to establish a relationship between the two as the mechanisms of both are very different.
A comparison of motility and head morphology of sperm using different semen processing methods and three different staining techniques
(Stellenbosch : University of Stellenbosch, 2010-12) McAlister, Debra Ann; Du Plessis, Stefan; Van der Horst, Gerhard; Maree, Liana; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.
ENGLISH ABSTRACT: Sperm morphology remains an important parameter in the prediction of fertility, both in vivo and in vitro. However, there remains a considerable level of concern surrounding the true potential of this parameter due to the lack of standardization of differential staining techniques used for the evaluation of sperm morphology. This study aimed at investigating two commonly used staining techniques, Rapidiff® (RD) and Papanicolaou (PAP), along with a new commercially available stain, SpermBlue® (SB), in the evaluation of sperm morphometry and morphology. Results indicated that significant differences in sperm morphometry exist due to the use of the staining techniques. Findings further indicated that RD causes sperm head swelling while PAP causes sperm head shrinkage. Results obtained using the SB staining technique have indicated measurements closest to that which would be obtained through the evaluation of fresh, unstained sperm. The lack of standardization and the different effects various stains have on sperm structure and overall sperm morphology evaluation should raise a level of concern, particularly when evaluating patients with borderline morphology. Based on this, the use of the SB staining technique is recommended over RD and PAP for effective and accurate morphology evaluation. In further support of this technique, SB was shown to be quick and simple in method, and allowed for the easy detection of sperm by computer aided sperm analysis (CASA) systems such as the Sperm Class Analyzer (SCA®). The second aim of this study was to examine the concentration, morphology and motility of the resultant sperm populations following semen preparation using the PureSperm® density gradient and swim-up techniques. Semen preparation is an essential step in any fertility treatment protocol, and it is important that the sperm obtained following semen preparation has sperm morphology and motility characteristics capable of improving assisted fertility success rates. Currently, the PureSperm® density gradient and sperm swim-up are the most widely employed techniques in fertility clinics. Although there is sufficient evidence to suggest they are each effective at extracting sperm with improved quality from neat semen, there remains insufficient evidence to suggest which of these two techniques is superior. The present investigation revealed that both sperm preparation methods were effective at improving sperm morphology and motility, however to varying degrees. The swimup method yielded a population of sperm with superior motility and morphology when assessed according to World Health Organisation (WHO) criteria, while the PureSperm® density gradient technique isolated a higher percentage of normal sperm, according to both WHO and Tygerberg strict criteria, with motility better than that of neat semen. Although results obtained via the swim-up method suggest it would be best for use in in vitro fertilization (IVF), the very low concentration of sperm isolated via this method remains a significant draw-back. The PureSperm® density gradient separation technique on the other hand is capable of isolating larger quantities of sperm, which is likely to be of more benefit with fertility treatments requiring larger quantities of sperm. Based on these findings, the use of PureSperm® density gradient technique is recommended, due to its ability to isolate large quantities of good quality sperm. However, a swim-up may still be of use when performing fertility treatment using a sperm sample which possesses a high concentration and motility.
A critical analysis of mitochondrial functioning and associated proteins in obesity-related cardiomyopathy
(Stellenbosch : Stellenbosch University, 2013-03) George, Siddiqah; Huisamen, Barbara; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.
ENGLISH ABSTRACT: INTRODUCTION: The mechanism behind obesity-related cardiomyopathies is at present not completely known, however, cardiac insulin resistance has been implicated as one of the main arbitrators of obesity-related cardiovascular disease. A few studies have associated perturbations in the insulin-mediated PI3K/PKB/Akt pathway in mediating this insulin resistance. Moreover, this pathway has been shown to regulate myocardial apoptosis, which in turn has been implicated in a number of cardiovascular diseases. Currently, few studies have compared the early onset and advanced effects of obesity on the heart. AIMS: To compare the early and advanced stages of obesity in terms of myocardial (i) PI3K/PKB/Akt signalling, (ii) apoptotic signalling and (iii) mitochondrial integrity. Furthermore, we aim to assess the cardiac mitochondrial (i) PI3K/PKB/Akt signalling, (ii) apoptotic signalling and (iii) integrity during the advanced stages of obesity. METHODS: Male Wistar rats were randomly assigned to either a control or diet-induced obesity (DIO) group. Controls were fed a standard rat chow diet and the DIO group fed a high caloric diet (standard rat chow supplemented with sucrose and condensed milk). The diets were implemented for either 8 or 20 weeks and thereafter, the body weight, intra-peritoneal fat mass, and fasting blood glucose and insulin levels (including intra-peritoneal glucose tolerance tests (IPGTTs)) were determined. Freeze-clamped hearts from both groups were subjected to cytosolic western blot analysis for PI3K p85 subunit, PKB/Akt, GSK-3α/β, Bad, Bax and Bcl-2. A fraction of each heart was also subjected to WB analysis of the mitochondrial electron transport chain (ETC) complexes (I-V). Thereafter, the above mentioned proteins were also probed for in mitochondria isolated from the 20 weeks group after administering insulin and exposing the hearts to ischemia. Oxidative phosphorylation (OXPHOS) capacity analysis was then conducted on mitochondria isolated from 20 weeks DIO and control groups and thereafter a citrate synthase (CS) activity assay was performed on these mitochondria. RESULTS: After the 8 and 20 weeks diet, the DIOs had significantly increased intra-peritoneal fat mass, fasting plasma glucose and insulin levels, compared to their controls. Cytosolic WB analysis: The tp85, pp85 and pPKB/Akt levels were significantly higher in the DIOs in comparison to the controls after 8 weeks of diet. Furthermore, pBad and Bax expression were significantly elevated in these animals. After 20 weeks of diet, the DIOs had significantly decreased pp85, tPKB/Akt and pPKB/Akt levels. The tBad was significantly elevated, while the Bad phosphorylated over total expression (P/T) ratio was significantly decreased, in these animals. CS activity assay: CS activity was significantly decreased in the DIOs, versus the controls, at 20 weeks. Mitochondrial ETC WB analysis: The subunit expression in complexes I-III and V did not differ significantly after 8 weeks however, the expression was significantly lower in complexes I and II after 20 weeks. Interestingly, the complexes III and V expression was significantly elevated. Mitochondrial OXPHOS analysis: The ADP/O ratio with (1) glutamate or (2) palmitoyl-L- carnitine as substrate, showed a significant decrease in the DIOs at 20 weeks. Mitochondrial WB analysis: The pp85 subunit was significantly elevated in the control and DIO groups, exposed to insulin and ischemia, in comparison to the untreated controls. The Bcl-2 levels were significantly decreased in the insulin and ischemia DIOs, when matched against the untreated DIOs. The tBad expression did not differ significantly between the insulin and untreated controls, while the tBad was significantly augmented in the ischemia controls versus untreated controls. All significant differences were taken as p<0.05. CONCLUSION: The results indicate that the initial stage of diet-induced obesity is associated with cardioprotection as there is augmented PI3K/PKB/Akt pathway signalling and a decrease in apoptotic markers. In contrast, during the advanced stages of obesity a decreased activity in PI3K/PKB/Akt pathway is associated with myocardial apoptosis and decreased mitochondrial function and integrity.
A diffusion tensor imaging study in HIV patients with and without apathy
(Stellenbosch : University of Stellenbosch, 2010-12) Fouche, Jean-Paul; Strijdom, Hans; Carey, Paul Dermot; Spottiswoode, Bruce Shawn; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.
ENGLISH ABSTRACT: HIV/AIDS is a global epidemic that accounts for a large percentage of the mortality in South Africa every year. Since the implementation of anti-retroviral treatment, HIV positive individuals have been living longer, and the cognitive impairment associated with the disease is becoming increasingly apparent. During the initial systemic infection of HIV, the virus migrates through the blood-brain barrier and inflicts axonal injury by causing upregulation of cytokines and neurotoxic proteins. HIV-associated dementia is a neuropsychological classification of cognitive impairment in HIV and a variety of symptoms have been classified as a part of the dementia complex. One of these is apathy, which is thought to be a precursor for dementia in HIV patients. Three groups of individuals have been recruited and scanned using magnetic resonance imaging (MRI) to examine changes in the brain. These are an HIV non-apathetic cohort, an HIV apathetic cohort and a healthy control cohort. Diffusion tensor imaging (DTI) is an MRI technique used to quantitatively assess white matter (WM) integrity using metrics such as fractional anisotropy (FA). Voxel-based analysis, tract-based spatial statistics (TBSS) and tractography are three established DTI analysis methods that have been applied in numerous studies. However, there are certain methodological strengths and limitations associated with each technique and therefore all three of these techniques were used to compare WM differences across groups. The frontal-subcortical pathways are known to be abnormal in apathy, and this has been demonstrated in a number of imaging studies. Most of these studies have examined apathy in the context of neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s. However, to our knowledge this is the first DTI study in HIV apathetic patients. With the tractography method, the anterior thalamic radiation and the corpus callosum were reconstructed for each individual to determine whether there were any global changes in these tracts. No significant changes were found. However, a variety of regions in the WM were significantly abnormal in the HIV cohorts when comparing the data at a voxel-based level and using TBSS. This included areas such as the genu and splenium of the corpus callosum, the internal capsule and corona radiata. Changes in frontal WM for the HIV apathy group are an indication of dysfunction in the frontal-striatal circuits, and previous literature has implicated these circuits in the neuropathology of apathy in a variety of central nervous system (CNS) disorders.
Does sexual violence alter social behvaiour via a maladaptive HPA-axis?
(Stellenbosch : Stellenbosch University, 2023-03) Fortuin, Chelsi Shante; Qulu, Lihle; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.
ENGLISH ABSTRACT: Background and Aim: South Africa was identified as the rape capital of the world, recording one of the highest rates of sexual violence worldwide with 72.1 reported cases per 100 000 in the 2019/2020 year. The mental health and well-being of sexual assault survivors exhibit a dysregulated HPA-axis stress response, which may be a primary source of structural and functional alterations leading to PTSD symptoms, but they don’t always co-exist. Chronic stress and psychiatric diseases cause dysregulation of the HPA axis. A cortisol imbalance brought on by the dysregulation of the HPA-axis leads to a decrease in oxytocin secretion, which eliminates oxytocin's potential to attenuate HPA-axis activity. A lack of oxytocin has been linked to broken social and maternal bonds as well as losing relationship attachments as a result of trauma exposure such as sexual violence / rape. Oxytocin dysregulation has been associated in a large variety of dysregulated social behaviour and PTSD diagnosis. Additionally, when bound to cortisol, glucocorticoid receptor sensitivity modifies the stress response's equilibrium point, which in turn regulates the HPA axis's negative feedback loop. As a result, they have been linked to anticipating the effects of stress. Therefore, the aim of this study is to determine whether being subjected to sexual violence alters social behavior via a dysregulated HPA-axis. Methods: Data analysis was completed using PTSD, perceived stress and social support scores to investigate the effects of rape on mental health trajectory. Additionally, enzyme-linked immunosorbent assays were used, to explore the role of oxytocin and cortisol as indicators of PTSD in rape-exposed women. Both cortisol and oxytocin found within plasma samples were analysed over a one-year period at four time points (baseline [± twenty days], three months, six months and twelve months post rape) to assess whether these hormone levels, that are activated by sexual violence, were transient or long lasting, and whether these hormone levels may lead to the formation of PTSD. Mixed effect regression analysis were done to determine if cortisol and oxytocin concentrations predict PTSD outcomes. Results: PTSD symptoms significantly decreased overtime, p = 0.000. Similarly, perceived stress, p = 0.0023, and cortisol concentrations, p = 0.004, significantly decreased overtime. Whereas social support scores, p = 0.5851, and oxytocin concentrations, p = 0.995, had no significant changes. Additionally, cortisol concentrations, p = 0.1660, and social support, p = 0.827, were not able to predict PTSD outcome, however oxytocin concentrations, slope = 9.32, p = 0.002 and perceived stress scores, slope = -5.654, p = 0.033, could predict PTSD. Conclusion: These findings demonstrate a relationship between PTSD symptom severity and HPA-axis maladaptation, via augmented oxytocin responses in victims of rape. We conclude that these findings may have significant clinical ramifications for women who have been subjected to rape. Particularly, offering scientific based PTSD interventions to rape exposed victims may show promise in alleviating symptoms and "normalizing" HPA axis receptivity to stress related stimuli.
The drug interaction between N-acetylcysteine, ascorbic acid-2 phosphate and metformin
(Stellenbosch : Stellenbosch University, 2023-11) Gilbert, Keenen Gregory George; Van de Vyver, Mari; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Dept. of Medicine. Division of Clinical Pharmacology.
ENGLISH ABSTRACT: Background: Type 2 diabetes (T2DM) is a glucose metabolism disorder. Its prevalence is increasing rapidly in sub-Saharan Africa with it negatively impacting global health systems and the economy. The South African Department of Health management objectives for thetreatment of T2DM is to relieve symptoms, prevent acute metabolic and long-term complications, improve quality of life and productivity of patients and reduce the economic burden on individuals, family, and community. However, despite achieving glucose control, >80% of T2DM patients still develop co-morbidities because of persistent oxidative stress and inflammation. Adjuvant treatments using antioxidants are effective at counteracting oxidative stress, it is however unclear if these treatments will interfere with the insulin-sensitizing function of metformin, which is available as the first-line oral medication for the treatment of T2DM. This study therefore investigated if there is any drug interaction between metformin and the antioxidants, N-acetylcysteine (NAC) and ascorbic acid-2-phosphate (AAP). Methods: In vitro experiments were performed using C2C12 skeletal muscle myoblasts under different treatment periods and pretreatments to determine if the antioxidants NAC and AAP alters glucose uptake and thus the requirement for Metformin. Cellular growth and viability under low and high glucose culture conditions were assessed over a period of 48 hours. Additionally, a dose response experiment over a period of 6 days exposing cells to 5 different concentrations of NAC and AAP was performed to determine the optimal and non-toxic concentration of the specific antioxidants. Glucose uptake was assessed using fluorescent microscopy and the 2NBDG assay under various conditions (insulin, metformin) following pretreatment (24h) with either NAC and/or AAP. The beneficial effect(s) of combination therapy was also determined by assessing the Total Antioxidant Capacity (colorimetric assay) and reactive oxygen species (ROS) (fluorometric assay) within the culture supernatants with and without NAC and/or AAP pretreatment (24h). Results: Over the 48-hour period, cells cultured in high glucose conditions had a significantly (p<0,0001) higher cell number per 10mm2 plate surface area when compared to cells cultured in low glucose conditions. The optimal concentrations of NAC and AAP was determined to be 3.75mM and 0.6mM, respectively. The combination of AAP, NAC and metformin treatment significantly decreased ROS levels (2-fold, p<0.05) and increased the total antioxidant capacity (p<0.01) (11.57±5.66 U/mL) when compared to metformin treatment on its own (0.81±2.48 U/mL). The pretreatment (24h) of cells with a combination of AAP/NAC prior to glucose tarvation (2h) and exposure to either insulin (30min) and/or metformin (2h) significantly increased glucose uptake compared to cells without pre-treatment. Conclusion: There is a comparable effect between metformin, NAC and AAP when used in combination with each other, which reduces oxidative stress in vitro. Additionally, the combination of metformin, NAC and AAP improves glucose uptake in C2C12 mouse myoblasts in vitro that resulted in altered glucose profiles. Thus, patients taking adjuvant antioxidants may require glucose monitoring and changes in metformin requirements. This study warrants further investigation to determine the precise mechanism of action underlying the synergistic effect observed between NAC, AAP and metformin affecting glucose uptake. Screening the efficacy of other anti-diabetic agents and antioxidants that target both glucose homeostasis and oxidative stress within a diabetic microenvironment as well as its associated comorbidities is furthermore recommended.
The effect of 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and 5'-aminoimidazole-4-carboxamide-ribonucleoside-phosphate (ZMP) on myocardial glucose uptake
(Stellenbosch : Stellenbosch University, 2005-03) Webster, Ingrid; Huisamen, Barbara; Lochner, Amanda; Stellenbosch University. Faculty of Medicine & Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.
ENGLISH ABSTRACT: Introduction: Exercise increases skeletal muscle glucose uptake via AMP-activated protein kinase (AMPK) activation and GLUT4 translocation from cytosol to cell membrane. It also promotes glucose utilisation in type 2 diabetic patients via increased insulin sensitivity. Insulin stimulates GLUT4 translocation by activating P13- kinase and protein kinase B (PKB/Akt). We therefore postulated that a connection exists between these two pathways upstream of GLUT4 translocation. Understanding this connection is important in the development of treatment strategies for type 2 diabetes. This exercise-induced increase in AMP-activated protein kinase (AMPK) activation can be mimicked by a pharmacological agent, 5'-aminoimidazole-4- carboxamide ribonucleoside (AlGAR), which is converted intracellularly into 5'- aminoimidazole-4-carboxamide-ribonucleosidephosphate (ZMP), an AMP analogue. Aim: To investigate the effect of two pharmacological AMPK-activating compounds, ZMP and AlGAR, on the phosphorylation of AMPK, the phosphorylation of PKB/Akt as well as possible feedback on insulin-stimulated glucose uptake and GLUT4 translocation. Materials and Methods: Adult ventricular cardiomyocytes were isolated from male Wistar rats by collagenase perfusion and treated with 1 mM AlGAR or 1 mM ZMP in the presence or absence of 100 nM insulin or 100 nM wortmannin, an inhibitor of P13- kinase. Glucose uptake was measured via eH]-2-deoxyglucose (2DG) accumulation. PKB/Akt and AMPK phosphorylation and GLUT4 translocation was detected by Western blotting. Purinergic receptors were blocked with 8-cyclopentyl-1,3- dipropylxanthine (8CPT) and the effect on AMPK phosphorylation noted. Certain results were confinned or refuted by repeating experiments using the isolated rat heart model. Results: AICAR and ZMP promoted AMPK phosphorylation. Neither drug increased glucose uptake but in fact inhibited basal glucose uptake, although GLUT4 translocation from cytosol to membrane occurred. Both compounds also attenuated insulin stimulated glucose uptake. Wortmann in abolished glucose uptake and PKB/Akt phosphorylation elicited by insulin while, in the presence of wortmannin, AICAR and ZMP increased levels of PKB/Akt phosphorylation. Although AICAR and ZMP increased glucose uptake in skeletal muscle, this was not seen in cardiomyocytes. However both compounds increased GLUT4 translocation, clearly demonstrating that translocation and activation of GLUT4 are separate processes. 8CPT had no effect on the phosphorylation of AMPK by either AICAR or ZMP indicating that there was no involvement of the purinergic receptors. Conclusion: Although AICAR and ZMP increase glucose uptake in skeletal muscle, this was not seen in cardiomyocytes. Conversely, both compounds inhibited both basal and insulin stimulated glucose uptake despite increasing GLUT4 translocation. Inhibition of PI3-kinase in presence or absence of insulin unmasked hitherto unknown effects of AICAR and ZMP on PKB phosphorylation.
The effect of an aspalathin-rich rooibos extract on inflammatory crosstalk between adipocytes and muscle cells
(Stellenbosch : Stellenbosch University, 2021, 2021-12) Obonye, Nnini; Muller, Christo; Mazibuko-Mbeje, Sithandiwe; Strijdom, Hans; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.
ENGLISH ABSTRACT: The skeletal muscle not only plays a role in maintaining posture and generating force (contraction), but also is a dynamic metabolically active tissue that plays a central role in the regulation of glucose metabolism. Western diets and a sedentary lifestyle reduce the contribution of skeletal muscle to the regulation of blood glucose. In addition, inflammation resulting from chronic elevated levels of pro-inflammatory cytokines is implicated in the development of insulin resistance, metabolic disease and loss of skeletal muscle mass. The gut plays a central role in the maintenance of metabolic health and disease. Metabolic disease is associated with gut dysbiosis and “leaky” gut syndrome releasing lipopolysaccharide (LPS) into the circulation driving a chronic proinflammatory cytokine response. To date there have been numerous studies demonstrating that Aspalathus linearis (rooibos) plant polyphenols protect against the development of metabolic diseases. The aim of this study was three-fold. Firstly, study the effects of LPS on skeletal muscle growth and metabolism in vitro using murine C2C12 myoblasts. Secondly, to study the effect(s) of 3T3-L1 adipocyte derived adipokines on LPS-induced pro-inflammatory cytokine secretion by myoblasts, and thirdly, to investigate the effect of an aspalathin-rich green rooibos extract (Afriplex GRT™) on the LPSinduced immune responses. To establish the skeletal muscle model of inflammation, C2C12 myoblasts were exposed to LPS (0.1 μg/mL and 1 μg/mL) for 24 hours and treated with Afriplex GRTTM (GRT) (1 μg/mL and 10 μg/mL) for 24 hours. Cell viability, inflammation (IL-6 secretion), glucose uptake, and expression of relevant genes and proteins were assessed. Furthermore, C2C12 myoblasts were differentiated in the presence of LPS to assess the effects of inflammation on myogenesis. A coculture system using C2C12 myoblasts and 3T3-L1 pre-adipocytes and mature adipocytes were used to study the secretion of IL-6 and adiponectin. LPS was a potent inducer of pro-inflammatory IL-6 cytokine response, specifically in myoblasts as opposed to myotubules, suggesting that IL-6 is differentially regulated in myotubules. Myoblasts exposed to LPS during differentiation results in decreased myotube width and number suggesting that metabolic endotoxemia affects muscle mass and potentially affecting skeletal muscle energy metabolism. The myogenic regulatory factors, myogenin, MyoD and myostatin were downregulated by LPS during myoblast differentiation. In co-culture, LPS significantly increased IL-6 secretion in both myoblasts and 3T3-L1 pre-adipocytes, whereas IL-6 secretion was modulated by the differentiated 3T3-L1 adipocytes in co-culture. A dramatic increase in adiponectin levels secreted by the differentiated 3T3-L1 adipocytes compared to the preadipocytes could have accounted for the lower IL-6 secretion in the co-culture. GRT was unable to ameliorate the effects of LPS-induced inflammation.
Effect of Aspalathus linearis supplementation, during anti-retroviral treatment, on the heart and aortas of male Wistar rats and the effects of drinking rooibos on the cardiovascular profile of patients on ART.
(Stellenbosch : Stellenbosch University, 2017-03) Imperial, Emiliana Gomes; Webster, Ingrid; Westcott, Corli; Strijdom, Hans; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology
ENGLISH ABSTRACT : Introduction Improved survival rates in the human immunodeficiency virus (HIV) population, due to the use of antiretroviral therapy (ART), are associated with increased risk for cardiovascular disease (CVD). Although the cardiovascular effects of rooibos as an antioxidant have been well documented its nutraceutical properties have yet to be investigated as a possible supplement in ART. AIM To investigate the cardiovascular effects of Aspalathus linearis/ rooibos infusion supplementation during ART, on male Wistar rats and the effects of rooibos tea consumption on the cardiovascular profiles of patients on ART. Methods Male Wistar rats were randomly divided into groups and treated with a fixed dose combination (Efavirenz 600mg, emtricitabine 200mg, tenofovir 300mg) and a 2% (w/v) rooibos tea solution independently or in combination for 9 weeks. Fluid intake and weights were measured weekly. Hearts were perfused on the isolated working heart rig and subjected to 20min global or 35min regional ischemia. Functional parameters were recorded and percentage recovery and infarct size (IS) calculated. Using the aortic ring protocol for vascular reactivity, thoracic aortas (with and without perivascular adipose tissue (PVAT)) were subjected to phenylephrine-induced contraction as well as acetylcholine-induced relaxation. Rat serum was collected for biochemical analyses. Participants for the human study were recruited and blood samples collected by a phlebotomist. Blood pressure tests were conducted, anthropometric data recorded and samples prepared for biochemical analyses. Results ART increased IS compared to control [Control (% area at risk): 28.17±5.10 , ART: 50.56±4.08] however this was not seen in the Rooibos+ART group. In vascular reactivity experiments, without PVAT, ART significantly diminished relaxation compared to control (p=0.03) whilst this effect was not observed with Rooibos supplementation (p=0.003). Rooibos caused increased contraction compared to control (p=0.01), ART (p=0.0457) and the combination (p=0.0468). In the presence of PVAT all treatment groups relaxed significantly less than controls (p<0.01). Area under the curve (AUC): Control+PVAT, 0.00099; Rooibos+PVAT: 0.00079; ART+PVAT, 0.00091; ART+Rooibos+PVAT, 0.00088. Whilst Rooibos and ART showed significantly higher contraction than the control (p=0.006 and p=0.03 respectively). AUC: Control+PVAT, 31.45; Rooibos+PVAT: 43.42; ART+PVAT, 36.32; ART+Rooibos+PVAT, 33.34. Overall the presence of PVAT caused a significant decrease in contraction compared to the absence thereof; Control+PVAT versus Control -PVAT group (p<0.0001); rooibos +PVAT versus rooibos -PVAT (p<0.0001), ART+PVAT versus ART -PVAT (p<0.0001) and Rooibos+ART +PVAT versus Rooibos+ARTPVAT (p<0.0001). Rooibos caused a significant reduction in total cholesterol (TC) levels (p=0.048) compared to all other groups. Epidemiological data revealed that drinking rooibos had no significant effects on the cardiovascular parameters of patients irrespective of HIV status or ART adherence. Discussion and Conclusion In aortic rings anti-vasodilatory effects of ART were not observed with rooibos supplementation. ART induced significant increase in IS was not observed in the Rooibos + ART treated group. Rooibos exerted pro-contractile effects whilst the presence of PVAT reduced contraction of the aortic rings. Deleterious effects by the current ART regimen were not observed in the epidemiological study and rooibos consumption had no significant effects on the assessed cardiovascular parameters of all patient groups. Further scientific investigation is required.
The effect of chronic ingestion of afriplex grt™ on myocardial insulin resistance and mitochondrial function – a preclinical study.
(Stellenbosch : Stellenbosch University, 2018-03) Kroukamp, Marlouw; Huisamen, Barbara; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.
Introduction: Obesity is an excessive fat accumulation in the body, known to be a significant precursor to insulin resistance and type 2 diabetes. Complications associated with obesity can contribute to the development of cardiovascular disease which, in turn, is associated with abnormal mitochondrial energetics. Rooibos (Aspalathus linearis) has known antidiabetic, anti-obesity and cardiovascular benefits. We investigated these properties with a focus on mitochondrial function, using an aspalathin-rich green rooibos extract, Afriplex GRT Extract. Aims: (i) To determine whether Afriplex GRT Extract ingestion can improve the insulin resistance state elicited by a high fat diet using a rat model. (ii) To determine whether Afriplex GRT Extract ingestion has any effects on myocardial mitochondrial oxidative phosphorylation potential and the process of mitophagy Methods: Male Wistar rats were fed either a control or a high fat diet (HFD) for 16 weeks. Half of each group was treated with Afriplex GRT Extract (60mg/kg/day) from weeks 11 to 16. Body weight, food and water intake were monitored, and liver- and intraperitoneal fat (IP) weighed at sacrifice. After sacrifice, myocardial mitochondria were isolated. Half of these were used to measure respiration on an oxygraph and the other half for Western Blot analysis of proteins involved in mitophagy. In addition, hearts were freeze clamped immediately after sacrifice and used to determine levels of insulin signalling proteins. Results: The HFD caused increased body weight-, liver- and IP fat gain, and elevated leptin levels while it decreased insulin signalling protein expression (GLUT4, tAkt/PKB and tAMPK). Ingestion of Afriplex GRT Extract diminished the weight gain, liver weight gain, IP fat weight gain and leptin in HFD animals. Moreover, it improved oral glucose tolerance in control animals and, according to a borderline significance in a HOMA-IR calculation, insulin resistance in the control animals. However, an increased basal glucose level was seen in HFD animals treated with Afriplex GRT Extract. Afriplex GRT Extract improved mitochondrial coupling efficiency in HFD animals and oxidative phosphorylation in control animals. Afriplex GRT Extract decreased mitochondrial p62 and cytosolic Beclin-1 levels and BNIP3 dimerization in the mitochondria of the control animals, while increasing autophagic flux (LC3-II expression) in hearts from HFD animals. A decrease in Sesn2 levels indicated possible anti-oxidant effects. Conclusion: We conclude that Afriplex GRT Extract resulted in less weight gain in HFD animals, improved whole-body insulin sensitivity and enhanced mitochondrial function. At the dose used, no detrimental effects were observed.

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