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To combat diabetes mellitus(DM), a chronicle metabolic disease, from which more than 400 million people suffered in the world, the patients must check the blood glucose level 4-5 times daily with an enzyme-based blood glucose meter and adjust blood glucose levels accordingly. The problem is that enzymatic glucose meters become unstable in the tropics. Therefore, the non-enzymatic method has been urged for blood glucose monitoring, among which the transition metal oxide electrode was found to be promising. However, despite the prolonged effort, its linear detection range is usually much smaller than the glucose level of diabetic patients, calling for an effective solution. Despite many previous attempts, none have solved the problem. Such a challenge has now been conquered by raising the NaOH concentration in the electrolyte, where amperometry, X-ray diffraction, Fourier-transform infrared spectroscopy, and Nuclear magnetic resonance measurements have been conducted. The linear range has been successfully enhanced to 40 mM in 1000 mM NaOH solution, and it was also found that NaOH affected the degree of glucose oxidation, which influenced the current response during sensing. It was expected that the alkaline concentration must be 25 times higher than the glucose concentration to enhance the linear range, much contrary to prior understanding. / Thesis / Candidate in Philosophy

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Two experiments were conducted to investigate the effects of added fat in late gestation cow and heifer diets on thermogenic and neonatal metabolic responses. In Experiment 1, the effects of source of fat in late gestation diets on serum glucose and thermogenic response during short-term cold stress were examined in fall-born neonatal beef calves. Pregnant fall-calving heifers (n = 15) were randomly assigned to three dietary treatments: Control (CON, n=5), Safflower seed (SAF, n=5), or Cottonseed (COT, n=5) supplement. Hay-based isonitrogenous and isocaloric diets met NRC requirements while containing 1.53%, 4.0% and 5.0% fat for CON, SAF and COT diets, respectively. Diets were fed for 47.5 ( 5.4 d before calving. Heifers were weighed weekly and at parturition. At parturition, colostrum samples were taken from the dam, calves weighed, and vigor scores recorded. Calves remained with their dams for 5 h to nurse. At 5.5 h of age, calves were fitted with an indwelling jugular catheter. At 6.5 h of age, calves were placed in a 5°C cold room for 90 min. Shivering scores (1= no shivering, 2 = slight shivering 3 = muscle shivering, 4 = severe muscle shivering), rectal temperatures and blood samples were taken every 15 min. Colostrum samples were analyzed for fat, solids, protein, lactose and IgG concentrations. BW and BCS of heifers at calving, and birth weights and vigor scores of calves were unaffected by diet (P > .5). Mean fat, lactose and IgG concentrations in colostrum were not different (P > .3) among treatments. SAF tended to increase colostral solids (P = .11) and protein (P = .13) compared to COT or CON. During cold stress, calf body temperature increased in a quadratic fashion (P < .03). Mean glucose levels tended (P = .12) to be greater and shivering scores were non-significantly increased in CON compared to SAF or COT calves. Glucose concentrations averaged 74.4, 51.9, and 60.0 ± 7.3 mg/dl, whereas shivering score averaged 2.14, 1.69, and 1.68 ± .24 in CON, SAF and COT calves, respectively. Shivering scores increased in all groups during cold exposure in a linear fashion (P < .001). Vigor scores increased in a linear fashion throughout cold exposure for all groups (P < .04). Cortisol concentrations decreased in a cubic fashion throughout cold exposure for all groups (P < .02). Cortisol concentrations averaged 28.62, 37.7, and 35.65 ± 3.58 ng/ml in CON, SAF and COT calves, respectively. We conclude that calves from dams fed high fat diets containing safflower seeds or cottonseed respond similarly to cold stress, but these responses are not necessarily consistent with greater cold resistance. In Experiment two, pregnant spring-calving cows (n = 75) were randomly assigned to two dietary treatments: Control (CON, n=35) and Cottonseed (COT, n=40). Hay-based isonitrogenous and isocaloric diets met NRC requirements while containing 2.0% and 5.0% fat for CON and COT diets, respectively. Diets were fed for 60 ± 5 d before calving. At parturition, calves were weighed, ambient temperature was recorded and dystocia score was recorded. At 30 min of age, rectal temperature one was recorded and shivering scores (1= no shivering, 2 = slight shivering 3 = muscle shivering, 4 = severe muscle shivering) were recorded. At 180 min postpartum, two blood samples were drawn from each calf to determine blood glucose and cortisol concentrations. At 36 ± 4 h postpartum, two blood samples were again drawn from each calf to determine blood glucose and IgG concentrations. Calf birth weight, calf sex, vigor score, shivering score, time to stand, dystocia score, and serum IgG concentrations were unaffected (P > .5) by diet. Shivering score was affected by ambient temperature (P < .003) and time of calving (P < .006). Calf birth weights were unaffected by diet, calf sex, and the diet x calf sex interaction (P > .2). Mean time to nurse was non-significantly longer (101.2 vs 70.1 min), respectively, for COT calves compared to CON calves. At 30 min (P < .05) rectal temperatures were higher in male than female calves from dams on the COT diet (39.3 vs 39.1°C). Whereas rectal temperatures were lower in male calves than female calves from dams on the CON diet (39.1 vs 39.3°C; diet x calf/sex, P < .05). The same relationship among rectal temperatures was observed at 180 min (diet x calf/sex, P < .05). Changes in body temperature between 30 and 180 min were affected by diet (P < .05) as body temperatures for COT calves increased more from 30 min to 180 min than CON calves. Body temperature at 30 min was affected by time of calving (P < .01). Body temperature at 180 min was affected by ambient temperature at calving (P < .03) and there was a tendency for body temperature at 180 min to be affected by time of calving (P < .09). Serum glucose concentrations at time 180 min were unaffected by diet (P > .3). Serum glucose concentrations at time 36 ± 4 h tended to be affected by sex (P < .07). With glucose levels higher in females (127 mg/dl) than in males (119 mg/dl). Differences in serum glucose at time 180 min and 36 + 4 h were not affected by diet, sex, or diet x sex interaction (P > .7). Serum glucose at 36 ± 4 h was affected by ambient temperature at calving (P < .04). Mean serum cortisol concentrations tended to be higher (47.4 ng/ml vs 36.5 ng/ml) for COT calves compared to CON calves (P < .09). Differences in serum cortisol levels were unaffected by sex or diet x sex interaction (P > .5). When ambient temperature or time of calving were included as covariates, calf weight, calf vigor and serum IgG were unaffected by ambient temperature or time of calving (P > .05). / Master of Science

Obese individuals present with an increased inflammatory tone as compared to healthy, normal-weight individuals, which is associated with insulin resistance. One factor hypothesized to contribute to increased inflammation in obese and diabetic states is elevated blood endotoxin levels, also known as metabolic endotoxemia. In healthy rodents (non-obese and insulin sensitive), there is evidence that blood endotoxin levels fluctuate over the course of the day with elevations in the post-prandial state that return to baseline levels in the post-absorptive state. High-fat feeding in these animals altered these fluctuations causing endotoxin levels to remain high throughout the day. The effects of alterations in endotoxin levels on glucose metabolism are not understood. The goal of this study was to determine the effects of short-term and long-term increases in endotoxin of a low magnitude on insulin signaling in a human primary cell line as well as the effects of short-term endotoxin treatments on glucose homeostasis in a C57/Bl6 mouse model. First, we tested the hypothesis in cell culture that short-term low-dose endotoxin treatments would enhance insulin-signaling and glycogen synthesis while long-term treatments would have inhibitory effects. Under our second hypothesis, we examined whether short-term low-dose treatments of endotoxin would contribute to improvements in glucose tolerance in a mouse model. In contrast to our first hypothesis, short-term endotoxin treatments did not improve insulin signaling or glycogen synthesis although long-term treatments did contribute to decreases in glycogen synthesis. Interestingly, short-term endotoxin treatments resulted in significant improvements in glucose clearance in the mouse model; this is believed to be partly attributed to LPS inhibiting gluconeogenesis. Future studies are necessary to understand the mechanisms responsible for altered glucose metabolism in response to low magnitude changes in LPS levels. / Ph. D.

Alzheimer’s Disease is a particularly vicious illness, with currently no preventative or curative treatment available. This paper focuses on how impairments in glucose metabolism and insulin signaling contribute to the disease process and further, on potential treatment options that target these specific dysfunctional processes in hopes of finding an effective cure or prevention therapy. Glucose hypometabolism presents years prior to the clinical symptoms of Alzheimer’s Disease and promotes the accumulation of Aβ, oxidative stress, and mitochondrial dysfunction. Additionally, a downregulation of GLUTs, particularly GLUT1 and GLUT3 also serves to decrease neural glucose uptake as well as to escalate glucose hypometabolism. Moreover, insulin resistance promotes tau hyperphosphorylation and extracellular aggregation of Aβ42, contributing to the Alzheimer’s Disease pathology. Due to the central role of insulin and glucose neural dysfunction to Alzheimer’s Disease, these processes pose as strong potential targets for much needed Alzheimer’s Disease curative and preventative therapy. Specifically, antioxidants and antidiabetics such as Metformin, thiazolidinediones, sulfonylureas, incretins, and intranasal insulin have shown some potential as future treatment options for Alzheimer’s Disease but require further investigation. Some non-pharmacological approaches, such as the ketogenic diet, have also been proposed as viable treatment options and work via their effects on glucose and insulin pathways. Dysfunctional glucose metabolism and insulin resistance are incredibly important in the progression of the Alzheimer’s Disease stages and as such, present as viable potential targets of future drug therapies.

Chez les levures, organismes eucaryotes unicellulaires, le glucose est la source d'énergie préférée. La levure modèle Kluyveromyces lactis possède deux perméases au glucose. L'expression d'une de ces deux perméases, codée par le gène RAG1, est induite par la présence de glucose extracellulaire et cette régulation transcriptionnelle dépend de la détection du glucose par un senseur membranaire spécifique, Rag4. Cependant, la régulation de l'expression de RAG1 dépend également de la capacité des cellules à métaboliser le glucose via la glycolyse. En effet, l'expression de RAG1 est fortement affectée dans des mutants glycolytiques malgré la présence de glucose extracellulaire. Au cours de cette thèse, nous nous sommes attachés à déterminer les mécanismes via lesquels la glycolyse contrôle l'expression de RAG1. Grâce à l'utilisation de mutants glycolytiques ou d'inhibiteurs chimiques de la glycolyse chez K. lactis, nous avons démontré que la glycolyse régule la stabilité du senseur Rag4 à la membrane plasmique et contrôle ainsi la voie de signalisation du glucose et l'expression de RAG1. De plus, ce mécanisme de contrôle est conservé chez la levure modèle Saccharomyces cerevisiae. L'étude plus approfondie de Rag4 nous a permis de déterminer que la transmission du signal glucose requiert la queue C-terminale cytoplasmique de Rag4, qui sert de plateforme d'interaction protéique. La caractérisation fonctionnelle de Rag4 nous a permis de mettre en évidence que la protéine contient plusieurs domaines impliqués dans le contrôle de sa stabilité en fonction du type de signal induisant la déstabilisation: signal glycolytique ou changement de source de carbone. Enfin, la nature du signal issu de la glycolyse qui cible le senseur membranaire Rag4 a été étudiée en testant deux hypothèses : le signal est protéique (enzyme de la glycolyse) ou métabolique (métabolite intermédiaire de la glycolyse). Ces travaux de thèse ont permis de mettre en évidence un rôle inédit de la glycolyse dans le contrôle de la stabilité des senseurs membranaires du glucose chez les levures K. lactis et S. cerevisiae / Yeasts are unicellular eukaryotic organisms which prefer glucose as energy source. The yeast model Kluyveromyces lactis has two glucose permeases. The expression of one of its permeases, RAG1, is induced by extracellular glucose. The glucose signaling pathway responsible for RAG1 expression regulation is dependent upon glucose sensing through a specific membrane glucose sensor, Rag4. However, RAG1 expression is also dependent upon glucose metabolism by glycolysis. Indeed, in glycolytic mutants RAG1 expression is strongly affected even when glucose is present. During these doctoral studies, we characterized mechanisms involved in glycolytic control on glucose signaling. Using glycolytic mutant or glycolysis chemical inhibitors, we have demonstrated that, in K. lactis, glycolysis targets the stability of the glucose sensor Rag4, controlling glucose signaling and RAG1 expression. This glycolytic control appears to be conserved in the yeast model Saccharomyces cerevisiae. We have shown that the C-terminal cytoplasmic tail of glucose sensor Rag4 is necessary for glucose signaling and forms a protein interaction platform. Rag4 protein contains several domains controlling Rag4 stability in response to different destabilization signals: glycolytic signal or carbon source signal. Finally, the nature of the glycolytic signal was studied considering two hypotheses: protein nature (e.g. glycolytic enzyme) or metabolic nature (e.g. glycolysis metabolic intermediate). This doctoral thesis underlines a new role of glycolysis in controlling membrane glucose sensor stability in K. lactis and S. cerevisiae

Thesis (MSc (Biomedical Sciences))--Cape Peninsula University of Technology, 2018. / Background: Early detection of individuals who are at risk of developing Glucose Intolerance would decrease the morbidity and mortality associated with this disease. MicroRNA is one of the most widely studied biomolecules involved in epigenetic mechanisms, hence it offers unique opportunities in this regard. Circulating microRNAs are associated with disease pathogenesis during the asymptomatic stage of disease. This has therefore attracted a lot of attention as a potential biomarker for identifying individuals who have an increased risk of developing Glucose Intolerance. The identification of high risk biomarkers for Glucose Intolerance will go a long way to eliminate the possible complications that arise due to late diagnosis and treatment of Glucose Intolerance. This could ultimately lead to better ways to prevent, manage and control the Glucose Intolerance epidemic that is rampant worldwide. The aim of the study is to investigate expression of circulating microRNA’s in blood of mixed ancestry subjects with glucose intolerance. Methods: A quantitative cross-sectional study design involving 36 individuals [who were age, gender and BMI (Body Mass Index) matched] from a total population of 1989 participants of mixed ancestry descent, residing in Bellville South, South Africa was used. Participants were classified as controls (normoglycemic), pre-diabetic (preDM) and diabetic (DM) (screen detected diabetic) according to WHO criteria of 1998. MicroRNAs were extracted from serum using the Qiagen miRNeasy Serum/Plasma Kit (ThermoFisher). The purified micro RNAs were reverse-transcribed to cDNA (complementary deoxyribonucleic acid) using the Qiagen RT2 First Strand Kit. Then, using Qiagen miScript SYBR Green PCR kit and miScript miRNA PCR arrays (ThermoFisher), the real time polymerase chain reaction was done to determine the expression profile the circulating micro RNAs present in the serum of the participants. Results: The 36 participants were evenly divided into 3 groups of 12 participants each as mentioned earlier. There were significant differences between groups in the waist (cm) (p=0.0415) and waist/hip ratio (p=0.0011) with highest values in the DM group and lowest in the normal group. Clinical parameters varied significantly according to glycemic status. As expected, the FBG (mmol/L) (p<0.0001), 2 HRs Post Glucose (mmol/L) (p<0.0001), HbA1c (%) (p=0.0009), Fasting Insulin (mIU/L) (p=0.0039), were all highest in the DM and lowest in the control group. In contrast, the 2 HRs Post Insulin (mIU/L) (p = 0.0027) was highest in the preDM group and lowest in the normal group, while the Glucose/Insulin ratio (p=0.0477) was highest in the normal group and lowest in the preDM group. Triglycerides (mmol/L) (p=0.0043) and Total Chol (mmol/L) (p=0.0429) were significantly increased through the three groups, with highest values in the DM group and lowest in the normal group. Furthermore, 12 of the 84 miRNAs studied were expressed through all the 3 groups and they exhibited both inverse and positive correlations between the clinical parameters, especially the glucose parameters (Fasting blood glucose, 2 hours post glucose, Fasting blood insulin, 2 hours post insulin and Glycated Hemoglobin).