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.
Adipose tissue as a possible therapeutic target for polyphenols : a case for Cyclopia extracts as anti-obesity nutraceuticals
(Elsevier, 2019) Jack, Babalwa U.; Malherbe, Christiaan J.; Mamushi, Mokadi Peggy; Muller, Christo J. F.; Joubert, Elizabeth; Louwa, Johan; Pheiffer, Carmen
ENGLISH ABSTRACT: Obesity is a significant contributor to increased morbidity and premature mortality due to increasing the risk of many chronic metabolic diseases such as type 2 diabetes, cardiovascular disease and certain types of cancer. Lifestyle modifications such as energy restriction and increased physical activity are highly effective first-line treatment strategies used in the management of obesity. However, adherence to these behavioral changes is poor, with an increased reliance on synthetic drugs, which unfortunately are plagued by adverse effects. The identification of new and safer anti-obesity agents is thus of significant interest. In recent years, plants and their phenolic constituents have attracted increased attention due to their health-promoting properties. Amongst these, Cyclopia, an endemic South African plant commonly consumed as a herbal tea (honeybush), has been shown to possess modulating properties against oxidative stress, hyperglycemia, and obesity. Likewise, several studies have reported that some of the major phenolic compounds present in Cyclopia spp. exhibit anti-obesity effects, particularly by targeting adipose tissue. These phenolic compounds belong to the xanthone, flavonoid and benzophenone classes. The aim of this review is to assess the potential of Cyclopia extracts as an anti-obesity nutraceutical as underpinned by in vitro and in vivo studies and the underlying cellular mechanisms and biological pathways regulated by their phenolic compounds.
Ameliorative potentials of quercetin against cotinine-induced toxic effects on human spermatozoa
(Hainan Medical University Journal Publisher, 2016) Goss, Dale; Oyeyipo, Ibukun P.; Skosana, Bongekile T.; Ayad, Bashir M.; Du Plessis, Stefan S.
Objectives: Cotinine, the principal metabolite of nicotine found in smokers' seminal plasma, has been shown to adversely affect sperm functionality while quercetin, a flavonoid with diverse properties is associated with several in vivo and in vitro health benefits. The aim of this study was to investigate the potential benefits of quercetin supplementation against damage caused by the by-products of tobacco smoke in human sperm cells. Methods: Washed human spermatozoa from 10 normozoospermic donors were treated with nutrient medium (control), quercetin (30 mmol/L) and cotinine (190 mg/mL, 300 ng/mL) with or without quercetin for 60 and 180 min incubation periods. Computer-aided sperm analysis was used to assess sperm motility while acrosomereacted cells were identified under a fluorescent microscope using fluorescein isothiocyanate-labelled Pisum Sativum Agglutinin as a probe, viability was assessed by means of a dye exclusion staining technique (eosin/nigrosin) and oxidative stress by flow cytometry using dihydroethidium as a probe. Values were expressed as mean ± S.E.M. as compared by ANOVA. Results: Higher cotinine concentrations reduced the number of viable cells after 60 and 180 min of exposure while viability of cells was increased in the cotinine aliquots supplemented with quercetin after 180 min of exposure when compared with cotinine only treated group. Conclusion: This study indicates that the ameliorating ability of quercetin on cotinineinduced decline in sperm function is associated with increased number of viable cells.
AMP kinase activation and glut4 translocation in isolated cardiomyocytes
(Clinics Cardive Publishing, 2010-04) Webste, Ingrid; Friedrich, Sven O.; Lochner, Amanda; Huisamen, Barbara
Activation of AMP-activated protein kinase (AMPK) results in glucose transporter 4 (GLUT4) translocation from the cytosol to the cell membrane, and glucose uptake in the skeletal muscles. This increased activation of AMPK can be stimulated by a pharmacological agent, AICAR (5’-aminoimidazole-4-carboxamide ribonucleoside), which is converted intracellularly into ZMP (5’-aminoimidazole-4-carboxamideribonucleosidephosphate), an AMP analogue. We utilised AICAR and ZMP to study GLUT4 translocation and glucose uptake in isolated cardiomyocytes. Adult ventricular cardiomyocytes were treated with AICAR or ZMP, and glucose uptake was measured via [3H]-2-deoxyglucose accumulation. PKB/Akt, AMPK and acetyl-CoA-carboxylase phosphorylation and GLUT4 translocation were detected by Western blotting or flow cytometry. AICAR and ZMP promoted AMPK phosphorylation. Neither drug increased glucose uptake but on the contrary, inhibited basal glucose uptake, although GLUT4 translocation from the cytosol to the membrane occurred. Using flow cytometry to detect the exofacial loop of the GLUT4 protein, we showed ineffective insertion in the membrane under these conditions. Supplementing with nitric oxide improved insertion in the membrane but not glucose uptake. We concluded that activation of AMPK via AICAR or ZMP was not sufficient to induce GLUT4-mediated glucose uptake in isolated cardiomyocytes. Nitric oxide plays a role in proper insertion of the protein in the membrane but not in glucose uptake.
AMP kinase activation and glut4 translocation in isolated cardiomyocytes
(http://www.cvja.co.za/index.php, 2010-03) Webster, Ingrid; Huisamen, Barbara; Lochner, Amanda; Friedrich, Sven O; Biomedical Sciences: Medical Physiology
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 activities of Basella alba aqueous leave extract in blood, pancreas, and gonadal tissues of diabetic male Wistar rats
(Wolters Kluwer, 2018) Arokoyo, Dennis Seyi; Oyeyipo, Ibukun Peter; Du Plessis, Stefan Simon; Aboua, Yapo Guillaume
Background: Oxidative stress is frequently identified as a key element in the pathophysiology of many complications of diabetes mellitus, including reproductive complications. The antioxidant potential of medicinal plants have been suggested for therapeutic focus of diseases in recent reports. Objective: To investigate the effect of Basella alba (Ba) aqueous leave extract on diabetes‑induced oxidative stress. Materials and Methods: Forty male Wistar rats (8–10 weeks) were randomly divided into four groups (n = 10) and treated as follows; Control (C + Ns) and Diabetic (D + Ns) animals received oral normal saline 0.5 ml/100 g body weight daily, while Healthy Treatment (H + Ba) and Diabetic Treatment (D + Ba) rats were given Ba extract at an oral dose of 200 mg/kg body weight daily. Treatment was by gavage and lasted 4 weeks in all groups. Diabetes was induced in D + Ns and D + Ba rats by single intraperitoneal injection of streptozotocin (55 mg/kg) and fasting blood sugar (FBS) recorded weekly in all rats afterwards. Animals were euthanized at the end of the experiment and blood samples, pancreas, testes, and epididymis were preserved for analysis of oxidative stress biomarkers. Results: Oral administration of aqueous leave extract of Ba significantly (P < 0.0001) lowered FBS in D + Ba rats. There was significantly higher blood superoxide dismutase activity and serum ferric reducing antioxidant power, but lower serum concentration of conjugated dienes and thiobarbituric acid reactive substances in D + Ba compared to D + Ns rats (P < 0.05). Conclusion: Ba exerts antioxidant effects in the gonads by enhancing antioxidant parameters in circulating blood, but not necessarily in the gonadal tissues.
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.
Ascorbic acid causes spuriously low blood glucose measurements
(Health & Medical Publishing Group, 1993-01) Strijdom J. G.; Marais B. J.; Koeslag J. H.
[No abstract available]
Aspalathin reverts doxorubicin-induced cardiotoxicity through increased autophagy and decreased expression of p53/mTOR/p62 signaling
(MDPI, 2017-11-01) Johnson, Rabia; Shabalala, Samukelisiwe; Louw, Johan; Kappo, Abidemi Paul; Muller, Christo John Frederick
ENGLISH ABSTRACT: Doxorubicin (Dox) is an effective chemotherapeutic agent used in the treatment of various cancers. Its clinical use is often limited due to its potentially fatal cardiotoxic side effect. Increasing evidence indicates that tumour protein p53 (p53), adenosine monophosphate-activated protein kinase (AMPK), nucleoporin p62 (p62), and the mammalian target of rapamycin (mTOR) are critical mediators of Dox-induced apoptosis, and subsequent dysregulation of autophagy. Aspalathin, a polyphenolic dihydrochalcone C-glucoside has been shown to activate AMPK while decreasing the expression of p53. However, the role that aspalathin could play in the inhibition of Dox-induced cardiotoxicity through increased autophagy flux remained unexplored. H9c2 cardiomyocytes and Caov-3 ovarian cancer cells were cultured in Dulbecco’s Modified Eagle’s medium and treated with or without Dox for five days. Thereafter, cells exposed to 0.2 µM Dox were co-treated with either 20 µM Dexrazozane (Dexra) or 0.2 µM aspalathin (ASP) daily for 5 days. Results obtained showed that ASP mediates its cytoprotective effect in a p53-dependent manner, by increasing the Bcl-2/Bax ratio and decreasing apoptosis. The latter effect was diminished through ASP-induced activation of autophagy-related genes (Atgs) with an associated decrease in p62 through induction of AMPK and Fox01. Furthermore, we showed that ASP was able to potentiate this effect without decreasing the anti-cancer efficacy of Dox, as could be observed in Caov-3 ovarian cancer cells. Taken together, the data presented in this study provides a credible mechanism by which ASP co-treatment could protect the myocardium from Dox-induced cardiotoxicity.
Aspalathin, a C-glucosyldihydrochalcone from rooibos improves the hypoglycemic potential of metformin in type 2 diabetic (db/db) mice
(Institute of Physiology, The Czech Academy of Sciences, 2018-07-25) Dludla, P. V.; Gabuza, K. B.; Muller, C. J. F.; Joubert, E.; Louw, J.; Johnson, R.
ENGLISH ABSTRACT: Metformin is the first line therapy of type 2 diabetics, but continued reduction of their life expectancy warrants further investigation into alternative treatment strategies. This study reports on the combinational use of metformin with aspalathin, a C-glucosyl dihydrochalcone with known glucose lowering and antioxidant properties, as an effective hypoglycemic therapy in a type 2 diabetic (db/db) mouse model. When tested as a monotherapy, a low dose of aspalathin (13 mg/kg) showed no effect, while a high dose (130 mg/kg) has already displayed a better potential than metformin in protecting against diabetes associated symptoms in db/db mice. Thus, it remains of interest to determine whether this dihydrochalcone can improve the efficacy of metformin. The results showed that this combination therapy was more effective than the use of metformin as a monotherapy in ameliorating diabetes associated symptoms, including abnormal raised fasting plasma glucose levels, impaired glucose tolerance, as well as excessively increased body weights and fat content. The treated mice also had reduced food and water consumption when compared to untreated controls, with a pronounced effect evident in the last week of treatment. Therefore, this study supports further investigations into the ameliorative effect of combination therapy of metformin and aspalathin against diabetes associated symptoms.
Aspalathin, a natural product with the potential to reverse hepatic insulin resistance by improving energy metabolism and mitochondrial respiration
(Public Library of Science, 2019) Mazibuko-Mbeje, Sithandiwe E.; Dludla, Phiwayinkosi V.; Johnson, Rabia; Joubert, Elizabeth; Louw, Johan; Ziqubu, Khanyisani; Tiano, Luca; Silvestri, Sonia; Orlando, Patrick; Opoku, Andy R.; Muller, Christo J. F.
ENGLISH ABSTRACT: Aspalathin is a rooibos flavonoid with established blood glucose lowering properties, however, its efficacy to moderate complications associated with hepatic insulin resistance is unknown. To study such effects, C3A liver cells exposed to palmitate were used as a model of hepatic insulin resistance. These hepatocytes displayed impaired substrate metabolism, including reduced glucose transport and free fatty acid uptake. These defects included impaired insulin signaling, evident through reduced phosphatidylinositol-4,5-bisphosphate 3-kinase/ protein kinase B (PI3K/AKT) protein expression, and mitochondrial dysfunction, depicted by a lower mitochondrial respiration rate. Aspalathin was able to ameliorate these defects by correcting altered substrate metabolism, improving insulin signaling and mitochondrial bioenergetics. Activation of 5´-adenosine monophosphate-activated protein kinase (AMPK) may be a plausible mechanism by which aspalathin increases hepatic energy expenditure. Overall, these results encourage further studies assessing the potential use of aspalathin as a nutraceutical to improve hepatocellular energy expenditure, and reverse metabolic disease-associated complications.
Ataxia telangiectasia mutated protein kinase : a potential master puppeteer of oxidative stress-induced metabolic recycling
(Hindawi, 2021) Blignaut, Marguerite; Harries, Sarah; Lochner, Amanda; Huisamen, Barbara
ENGLISH ABSTRACT: Ataxia Telangiectasia Mutated protein kinase (ATM) has recently come to the fore as a regulatory protein fulfilling many roles in the fine balancing act of metabolic homeostasis. Best known for its role as a transducer of DNA damage repair, the activity of ATM in the cytosol is enjoying increasing attention, where it plays a central role in general cellular recycling (macroautophagy) as well as the targeted clearance (selective autophagy) of damaged mitochondria and peroxisomes in response to oxidative stress, independently of the DNA damage response. The importance of ATM activation by oxidative stress has also recently been highlighted in the clearance of protein aggregates, where the expression of a functional ATM construct that cannot be activated by oxidative stress resulted in widespread accumulation of protein aggregates. This review will discuss the role of ATM in general autophagy, mitophagy, and pexophagy as well as aggrephagy and crosstalk between oxidative stress as an activator of ATM and its potential role as a master regulator of these processes.
Ataxia-Telangiectasia Mutated is located in cardiac mitochondria and impacts oxidative phosphorylation
(Springer Nature, 2019) Blignaut, Marguerite; Loos, Ben; Botchway, Stanley W.; Parker, Anthony W.; Huisamen, Barbara
The absence of Ataxia-Telangiectasia mutated protein kinase (ATM) is associated with neurological, metabolic and cardiovascular defects. The protein has been associated with mitochondria and its absence results in mitochondrial dysfunction. Furthermore, it can be activated in the cytosol by mitochondrial oxidative stress and mediates a cellular anti-oxidant response through the pentose phosphate pathway (PPP). However, the precise location and function of ATM within mitochondria and its role in oxidative phosphorylation is still unknown. We show that ATM is found endogenously within cardiac myocyte mitochondria under normoxic conditions and is consistently associated with the inner mitochondrial membrane. Acute ex vivo inhibition of ATM protein kinase significantly decreased mitochondrial electron transfer chain complex I-mediated oxidative phosphorylation rate but did not decrease coupling efficiency or oxygen consumption rate during β-oxidation. Chemical inhibition of ATM in rat cardiomyoblast cells (H9c2) significantly decreased the excited-state autofluorescence lifetime of enzyme-bound reduced NADH and its phosphorylated form, NADPH (NAD(P)H; 2.77 ± 0.26 ns compared to 2.57 ± 0.14 ns in KU60019-treated cells). This suggests an interaction between ATM and the electron transfer chain in the mitochondria, and hence may have an important role in oxidative phosphorylation in terminally differentiated cells such as cardiomyocytes.
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.
Basic semen parameters assisted by Computer-Aided sperm analysis (CASA) and their correlations with advanced semen parameters in normozoospermic men with different abstinence periods
(Stellenbosch : Stellenbosch University, 2018-03) Ayad, Bashir Mohamed; Du Plessis, Stefan S.; Van der Horst, Gerhard; Stellenbonsch university. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences : Medical Physiology
Introduction: Affordable basic semen analysis remains a fundamental procedure to be performed routinely during the diagnosis of male infertility. Advanced semen analyses, provide valuable clinical insights in treatment related decision-making, but these are highly expensive and lack universal standardization. The World Health Organization (WHO) guidelines for semen analysis have been adopted by most human andrology and fertility laboratories around the world for more than thirty years. According to the most recent prescribed guidelines of the WHO, subjects must remain abstinent for a minimum period of two days, but not longer than seven days before collecting a sample for a standard semen analysis. Several studies have sought to determine the optimal period for ejaculatory abstinence. However, the results are often found to be contradictory. The aims of this study are two-fold: Aim I: To investigate the effect of short (4 hours) and long (4 days) abstinence periods on sperm quality based on functional and biochemical parameters in a population of normozoospermic men, in addition to the prediction of various basic and advanced semen parameters of the second (4 hours) ejaculate from a set of basic parameters obtained from the first (4 days) ejaculate. Aim II: Establishing a correlation between basic semen parameters assisted by Computer-aided sperm analysis (CASA) and a set of advanced semen analysis tests. To determine cut-off values for advanced semen parameters from various basic parameters based on WHO defined reference values. Methods: Semen samples were collected from one hundred potentially fertile, normozoospermic men (20 to 30 years) who abstained for a period of exactly 4 days and 4 hours prior to collection of the first and second ejaculates respectively. Semen samples were analysed according to the WHO guidelines. Sperm concentration, total sperm count (T.S.C.), total and progressive motility and kinematic/velocity parameters were analysed by CASA. Sperm viability was performed by dye exclusion and morphology via SpermBlueTM staining techniques using Computer Aided Sperm Morphology Analysis (CASMA). Sperm acrosome status was evealuated by fluorescence microscopy. Sperm DNA fragmentation and intracellular superoxide (O2−•) levels were assessed by flow cytometry. Seminal antioxidant status [superoxide dismutase (SOD), catalase (CAT), thiobarbituric acid reactive substances (TBARS)] were measured by means of spectrophotometry. Statistical comparisons between short and long abstinence periods were performed using paired Student’s t-tests on GraphPad Prism™ software, while the prediction of various basic and advanced semen parameters of the second ejaculate from a set of basic semen parameters of the first ejaculate was performed using linear regression models. Correlations were performed using Spearman rank correlation coefficients, while receiver operating characteristic (ROC) curves were used to determine cut-off values. Statistical significance was set at p<0.05. Results I: A significant increase in total and progressive motility as well as in the velocity parameters were observed after short (4 hours) abstinence compared to long (4 days) abstinence periods. DNA fragmentation and intracellular O2−• levels were not significantly different between short and long abstinence periods. Despite the observed decrease in semen volume, sperm concentration and T.S.C. after the short abstinence period, all mean values of the conventional semen parameters still remained above the lower reference limits as recommended by the WHO 5th edition. We were also able to make predictions of various basic (semen volume, sperm concentration, total motility, progressive motility, viability and normal morphology) and advanced (DNA fragmentation, seminal plasma CAT activity and TBARS) parameters of the second ejaculate from a set of basic semen parameters obtained from the first ejaculate with relative certainty. Results II: The proportions of total and progressively motile as well as rapid spermatozoa were positively correlated with CAT activity (p<0.05). A significant negative correlation was observed between VCL, VSL, VAP and both intracellular O2−• and TBARS levels. ALH was significantly and negatively correlated with intracellular O2−• levels and DNA fragmentation, while its correlation with SOD activity was positive (p < 0.05). A negative correlation was also found between the percentage of viable spermatozoa and both O2−• levels and DNA fragmentation, whereas the percentage of normal morphology was negatively correlated with O2−• levels and positively with CAT activity (p < 0.05). The optimal intracellular O2−• cut-off value to differentiate between asthenozoospermic and normozoospermic men was calculated to be 227 median DHE fluorescence intensity [MFI] (p < 0.01). At this cut-off value, the test was 80% sensitive and 86% specific. Sperm viability was associated with a seminal plasma TBARS cut-off value of 9.86 Umol/L (p = 0.02) with sensitivity and specificity of 81% and 80% respectively. Conclusion: Our data challenges the generally accepted guidelines regarding the prescribed prolonged abstinence periods since the results show that 4 hours of sexual abstinence yielded significantly better samples from a sperm functional point of view. The results obtained from this study further support the validity of some CASA parameters as sensitive indicators of changes in sperm oxidative status and DNA integrity. This study also enabled defining the cut-off values and prediction of certain advanced variables from the basic semen analysis.
Beneficial effects of adenosine triphosphate-MgC12 administered intravenously to rabbits subjected to haemorrhagic shock
(Health & Medical Publishing Group, 1986-10) Engelbrecht, F. M.; Mattheyse, F. J.
ENGLISH ABSTRACT: The beneficial effects of adenosine triphosphate (ATP)-MgCl2 administered as a bolus following fluid infusion or in combination with the infusion fluid were investigated in rabbits subjected to severe but reversible haemorrhagic shock. ATP-MgCl2 treatment led to a significant improvement of the metabolic functions of lung and liver tissue. Kidney tissue showed the same tendency, but the improvement did not reach significant levels. The release of lysosomal enzymes in vivo was retarded after treatment but not stopped. The mean arterial pressure was kept at a relatively constant level when ATP-MgCl2 was infused slowly. Administration as a bolus resulted in an immediate dramatic drop in pressure, followed by recovery and then a gradual decrease to levels which appeared to be incompatible with survival.
Blood-based DNA methylation biomarkers for type 2 diabetes : potential for clinical applications
(Frontiers Media, 2018) Willmer, Tarryn; Johnson, Rabia; Louw, Johan; Pheiffer, Carmen
ENGLISH ABSTRACT: Type 2 diabetes (T2D) is a leading cause of death and disability worldwide. It is a chronic metabolic disorder that develops due to an interplay of genetic, lifestyle, and environmental factors. The biological onset of the disease occurs long before clinical symptoms develop, thus the search for early diagnostic and prognostic biomarkers, which could facilitate intervention strategies to prevent or delay disease progression, has increased considerably in recent years. Epigenetic modifications represent important links between genetic, environmental and lifestyle cues and increasing evidence implicate altered epigenetic marks such as DNA methylation, the most characterized and widely studied epigenetic mechanism, in the pathogenesis of T2D. This review provides an update of the current status of DNA methylation as a biomarker for T2D. Four databases, Scopus, Pubmed, Cochrane Central, and Google Scholar were searched for studies investigating DNA methylation in blood. Thirty-seven studies were identified, and are summarized with respect to population characteristics, biological source, and method of DNA methylation quantification (global, candidate gene or genome-wide). We highlight that differential methylation of the TCF7L2, KCNQ1, ABCG1, TXNIP, PHOSPHO1, SREBF1, SLC30A8, and FTO genes in blood are reproducibly associated with T2D in different population groups. These genes should be prioritized and replicated in longitudinal studies across more populations in future studies. Finally, we discuss the limitations faced by DNA methylation studies, which include including interpatient variability, cellular heterogeneity, and lack of accounting for study confounders. These limitations and challenges must be overcome before the implementation of blood-based DNA methylation biomarkers into a clinical setting. We emphasize the need for longitudinal prospective studies to support the robustness of the current findings of this review.
Body composition in women with HIV/AIDS : the relevance of exercise
(Health and Medical Publishing Group (HMPG), 2008-07) Myburgh, Kathryn H.; De Bruto, Petro C.
Untreated infection with the human immunodeficiency virus (HIV) leads to severe physical debilitation, culminating in the acquired immune deficiency syndrome (AIDS). Multiple infections, body mass loss, physical weakness and wasting are characteristic manifestations of each of the four stages of HIV/AIDS, respectively (Table I). The latter two impact especially on the affected person’s ability to function, as well as on social and economic levels. However, even though antiretroviral treatment (ART) is now available at many selected public clinics in South Africa, it only complicates the issues surrounding body composition and physical function.

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