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Hypoglycaemia is a very common side-effect of insulin therapy for diabetes and directly affects cognitive function. It can be identified by the onset of symptoms and by blood glucose monitoring. Impaired awareness of hypoglycaemia is an acquired syndrome in people with insulin-treated diabetes. The definitions, frequency, causes, treatment and prevention of clinical hypoglycaemia and the effects on, and moderators of, cognitive function will be discussed. Two studies have examined the effects of hypoglycaemia on tests of particular cognitive domains in subjects with and without type 1 diabetes. Three further studies have examined the frequency of hypoglycaemia in people with and without impaired awareness, the prevalence of impaired awareness of hypoglycaemia (IAH) and have compared methods of assessing awareness of hypoglycaemia. In study 1 the effect of acute hypoglycaemia on psychomotor function was examined in healthy volunteers (n =20) and adults with type 1 diabetes (n=16). Although acute hypoglycaemia caused significant impairment of several psychomotor functions in nondiabetic adults, a lower magnitude of impairment was observed in those with type 1 diabetes. The potential mechanisms behind this are discussed. In study 2 the effect of acute hypoglycaemia on a simple two-choice reaction time test, which has a model with validated performance parameters, was examined in 14 nondiabetic volunteers. Application of the validated model to the results of this task revealed that hypoglycaemia affected central processing and was not related to the amount of evidence required to make a decision or to peripheral and motor processes. This study is the first to use this method to dissect the effects of hypoglycaemia on cognition and enhances understanding of the mechanism underlying neuroglycopenia in adults. In Study 3 the methods of evaluating awareness of hypoglycaemia were compared in people with type 1 diabetes. Good concordance in clinical characteristics and frequency of biochemical hypoglycaemia was observed between the methods described by Gold et al and Clarke et al but not with a Danish method. In study 4 continuous glucose monitoring (CGM) and home blood glucose monitoring were performed prospectively for 12 months in people with and without IAH. Those with IAH had a 1.6-fold higher incidence of biochemical hypoglycaemia as demonstrated by blood glucose monitoring, but CGM did not identify patients with IAH. In study 5 the prevalence of IAH in a large clinic population with type 1 diabetes was estimated and compared with earlier assessments. The overall prevalence was 20%.

The basis of this thesis is intended to inform the reader about the general complications of acute diabetes mellitus and its consequences. It focuses on the general definitions of the diseases, etiology, morbidity, mortality, pathogenesis of the disease, clinical presentation, treatment, and future developments in hopes of treating the disease. The major focus highlights the differences between diabetic ketoacidosis and hyperglycemic hyperosmolar state, as well as understanding the complications of diabetic hypoglycemia. The most critical effects of these disorders are also emphasized; cerebral edema, vascular thrombosis, and hyperchloremic metabolic acidosis.

Thesis (M. Sc. (Biochemistry)) -- University of Limpopo, 2015 / Diabetes mellitus is one of the major diseases worldwide that is life threatening and is reaching an epidemic proportion. The most important approach in reducing the burden of the disease worldwide is to search for effective, low cost hypoglycaemic drugs with fewer side effects. Past experimental evidence confirmed the hypoglycemic activity of many indigenous African medicinal plants. S. italica (Fabaceae family) is widely used by traditional healers to treat a number of diseases such as sexually transmitted diseases and other forms of intestinal complications traditionally. The current study was aimed at evaluating the in vitro effects of root and leaf extracts of S. italica on GLUT4 translocation in NIH-3T3-L1 preadipocytes and C2C12 muscle cells. In order to address the aim of the study various methods were undertaken. The roots and leaves of S. italica collected from Zebediela sub-region of the Limpopo province, South Africa, were ground to fine powder and extracted using acetone, methanol, ethyl acetate and n-hexane. The various extracts of the root and leaf material were subjected to fingerprint profiling using TLC plates and different mobile phases (BEA, CEF, EMW and BAW). The chromatograms were visualized with vanillin-H2SO4 reagent, p-anisaldehyde and iodine vapour. The extracts were assayed for the type of secondary metabolites contained in the studied plant parts using chemical text and by TLC analysis. The total phenolic content of the root and leaf material were also evaluated. Evaluation for antioxidant activity was performed using 0.2% DPPH qualitatively and quantitatively with vitamin C as a positive control. Toxicity study was performed on C2C12 muscle cells using the MTT assay, with Curcumin as a positive control and untreated cells as a negative control. The CC50 values of the acetone root and leaf extracts were determined by linear regression. The effect of acetone root and leaf extracts on glucose uptake by C2C12 muscle cells was evaluated, also on western blot and immunofluorescence for NIH-3T3-L1 preadipocytes. The solvents employed for extraction in this study are commonly used to extract various biological active compounds from plants in research settings. Methanol extracted more compounds followed by acetone, then ethyl acetate and n-hexane the least. The constituents extracted by methanol may be mostly sugars, amino acids and glycosides due to the polarity of this solvent. Hydro-alcoholic solvents extract a variety of compounds that are mostly polar. Acetone extracts xxii mostly alkaloids, aglycones and glycosides while n-hexane in general extracts mostly waxes, fats and fixed oils. High yield was obtained with leaf extracts with all the solvent used for extraction as compared to the root. The TLC finger-print showed that good separation was achieved with the methanol and acetone extracts in CEF mobile phase, ethyl acetate extracts in CEF and EMW and n-hexane extracts in BEA respectively, especially with the leaf extract. Most compounds present in S. italica extracts were UV active. Some compounds that were not reactive with vanillin-H2SO4 reagent were shown to be reactive with p-anisaldehyde reagent and iodine vapour which revealed the presence of sugars or aromatic compounds. Chemical analysis for secondary metabolites of the acetone root and leaf extracts revealed the presence of flavonoids, terpenes, tannins, steroids, reducing sugars and alkaloids while glycosides were detected only in the leaf extract. The results obtained using TLC analyses were consistent with the results obtained in the chemical analysis. Thin layer chromatography revealed the presence of glycoflavones in the acetone root extract, alkaloids in the root and leaf extracts; and phytosterols and flavonoid aglycones in root and leaf extracts. The acetone root and leaf extracts revealed the presence of phenols. The leaf extract was shown to contain high total phenolic content as compared to the root. The methanol and acetone root and leaf extracts were shown to possess antioxidant activity. However, the concentration of the activity was higher in the acetone root than in the leaf extract. The least activity was observed with the ethyl acetate root and leaf extracts as compared to other extracts. The n-hexane extracts however, was not shown to contain any antioxidant compounds. Although activity observed with the methanol extracts was comparable to that of the acetone extracts in the quantitative assay, the acetone extracts were shown to possess more antioxidant activity in the qualitative assay. The concentration of extracts increased with increase in scavenging activity. The root extract exhibited a more potent antioxidant activity compared to leaf extract. These extracts were evaluated for their cytotoxicity on normal cells. The highest cytotoxic concentration (CC50) was obtained with the root extract with a CC50 value of 297 635 μg/ml at 48 hrs, followed by CC50 value of 21 544 μg/ml at 24 hrs. The CC50 value of the leaf extract at 24 hrs was 2 904 μg/ml with the least value at 48 hrs. The root extract at 24 and 48 hrs together with the leaf extract at 24 hrs were not toxic to C2C12 muscle cells at the concentration tested in this study. The acetone extracts were shown to possibly enhance proliferation of C2C12 muscle cells at a concentration of 0.001–1000 μg/ml. The non-cytotoxic concentration of 25 μg/ml of the leaf extract in combination with insulin showed more glucose uptake as compared to other extracts as well as the control. Prolonged incubation time was shown to increase glucose uptake with leaf extract while increase in concentration of root extract decreased glucose uptake at 24 hrs. At incubation time of 3 and 24 hrs, glucose uptake results at concentration of 2.5 μg/ml were comparable with that of the root extract, with a similar trend observed at 25 μg/ml, although with decrease in uptake. The qualitative and quantitative fluorescence results showed GLUT4 to be translocated to the cell membrane. The leaf extract at a concentration of 25 μg/ml had more fold as compared to other extracts, indicative that more GLUT4 was translocated at this concentration of the leaf extract. The acetone root and leaf extracts were shown to increase protein expression of GLUT4 at 3 hrs incubation time as compared to other incubation times in insulin-stimulated C2C12 muscle cells. The plant constituents of S. italica was shown to contain a variety of secondary metabolites that maybe be acting alone or in concert with each other to exert the various activities observed in this study. Different solvents used for extraction may be responsible for the extraction of different constituents with antioxidant activity observed in the study. The acetone extracts enhanced proliferation of C2C12 muscle cells at concentrations used in the study. However, there was no significant reduction on viability of normal cells. In addition, the extracts were shown to enhance the differentiation of NIH-3T3-L1 preadipocytes into adipocytes and C2C12 muscle cells into myocytes. These in turn induced the translocation of GLUT4 to the cell membrane and as a consequence facilitate glucose transport. Hence, the differentiation of adipose cells as well as glucose uptake of muscle cells and GLUT4 expression might have been enhanced by constituents contained in the acetone extracts. In conclusion, the acetone leaf extract may have a beneficial role in glucose metabolism of differentiated C2C12 muscle cells. Therefore, further studies are however required to elucidate the molecular mechanism by which the acetone leaf extract of S. italica influences the translocation of GLUT4.

No description available.

The proposed thesis will examine three areas of research: (1) the effects of hypoglycaemia on cognitive function in adults with and without T1DM, (2) the symptoms and awareness of hypoglycaemia in children and adolescents with T1DM and (3) hypoglycaemia and driving in people with insulin-treated diabetes: self-treatment and adherence to recommendations for avoidance. (1) Executive cognitive function governs organisation of thoughts, prioritisation of tasks, and time management. This study examined the effect of acute hypoglycaemia on executive function in adults with and without diabetes. Thirty-two adults with and without type 1 diabetes were studied. Two hyperinsulinaemic glucose clamps were performed at least 2 weeks apart in a single-blind, counterbalanced order. Executive functions were assessed with a validated test suite (Delis-Kaplan Executive Function). A general linear model (repeated-measures ANOVA) was used. Compared with euglycaemia, executive functions (with one exception) were significantly impaired during hypoglycaemia; lower test scores were recorded with more time required for completion. Large Cohen d values (>0.8) suggest that hypoglycaemia induces decrements in aspects of executive function with large effect sizes. In some tests, the performance of participants with diabetes was more impaired than those without diabetes. Executive cognitive function, which is necessary to carry out many everyday activities, is impaired during hypoglycaemia in adults with and without type 1 diabetes. (2) In children with type 1 diabetes mellitus (T1DM) the prevalence of impaired awareness of hypoglycaemia (IAH) is uncertain. Questionnaires were completed by 98 children with T1DM (mean age 10.6 years) and their parent(s); hospital admission data for the previous year were collected. Awareness of hypoglycaemia was assessed using two questionnaire-based methods that have been validated in adults. For 4 weeks, participants performed routine blood glucose measurements and completed questionnaires after each episode of hypoglycaemia. The ‘Gold’ questionnaire classified a greater proportion of the participants as having IAH than the ‘Clarke’ questionnaire (68.4 vs. 22.4%). Using the ‘Clarke’ method, but not the ‘Gold’ method, children with IAH were younger and more likely to require external assistance or hospital admission. In contrast to adults, behavioural symptoms were the best predictors of awareness status. IAH affects a substantial minority of children and impending hypoglycaemia may be heralded by behavioural symptoms. The ‘Clarke’ method was more effective at identifying those at increased risk. (3) A clinical survey of an outpatient clinic population to ascertain current knowledge and practice among drivers with insulin-treated diabetes. A representative sample of 202 current drivers with insulin-treated diabetes completed a structured questionnaire. A minimum blood glucose level of 4.0 mmol/L or higher was considered necessary for driving by 74.8%, and 87.1% reported always keeping carbohydrate in their vehicle. However, 38.1% reported never carrying a glucose meter when driving, and 59.9% that they never test blood glucose before driving, or test only if symptomatic of hypoglycaemia. Most participants 89% would stop driving to treat hypoglycaemia although only 13.9% would wait longer than 30 min. Compliance with statutory requirements to inform the licensing authority and motor insurer is good.

Hyperglycaemia caused by insulin deficiency is believed to play a major role in the de-velopment of neuropathy in diabetic patients. The clinical and pathological features of diabetic neuropathy vary considerably, although sensory and autonomic dysfunctions are the most common characteristics. Normalisation of the blood glucose level by ef-fective insulin treatment decreases the incidence of diabetic neuropathy in patients. However, intensive insulin therapy may result in more frequent hypoglycaemic epi-sodes than are provoked by less ambitious diabetes control. Neuropathy might also be induced by severe hypoglycaemia in diabetes or insulinoma. Accordingly, it seems that the diversity in clinical symptoms of diabetic neuropathy may be due to the combined effects of hyperglycaemia and hypoglycaemia. Based on that assumption, the general aim of this project was to study the relationship between severe hypoglycaemia and pe-ripheral neuropathy in diabetic rats. To understand how the development of neuropathy is related to glycaemic control, we needed to be aware of the glucose dynamics in the animal model that we used. The aim was to ascertain whether the diabetic rats were similar to type 1 diabetic patients with regard to such dynamics. To achieve that goal, we used a MiniMed continuous glucose monitoring system (CGMS®) to measure sub-cutaneous glucose in freely moving rats over a period of 72 hours. The glucose monitor worked well, and it showed that the insulin-treated diabetic BB/Wor rats with a hyper-glycaemic insulin regimen have a glycaemic status similar to that of type 1 diabetic patients with poor glycaemic control. The diabetic rats with a hypoglycaemic regimen generally had low blood glucose levels. Prolonged hypoglycaemia led to axonal de- and regeneration of large myelinated fibres in vagus nerve destined to the laryngeal muscle. Axonal de- and regeneration was also observed in the gastrocnemius and sural nerves, although the frequency of degeneration was much lower in the sural nerve. Small myelinated and unmyelinated nerve fibres were normal in these nerves. These results suggest that hypoglycaemia preferentially damages muscle-related nerve fibres. In contrast, in the diabetic rats exposed to pro-longed hyperglycaemia, only the sural nerve exhibited decreased myelinated fibre diameter in the absence of obvious axonal degeneration. In situ glucose measurements by microdialysis showed that the glucose concentrations in blood and subcutaneous tissue were similar in healthy, diabetic hyperglycaemic, and diabetic hypoglycaemic rats. In the healthy and hyperglycaemic animals, the lowest glucose level was found in the peripheral nerve. Moreover, in controls, the glucose level was lower in muscle than in blood. In hypoglycaemic rats, there were no signifi-cant differences in glucose concentrations between different tissues.

People with type 2 diabetes are at increased risk of age-related cognitive impairment. Previous literature has focused on case-control studies comparing rates of cognitive impairment in patients with and without diabetes. Investigations of potential risk factors for cognitive impairment (including those with increased prevalence in diabetes, such as macrovascular disease, and diabetes-specific factors such as hypoglycaemia) in study populations consisting exclusively of patients with type 2 diabetes have been largely neglected. Moreover, previous studies have failed to take advantage of the extensive characterisation and prospective nature of longitudinal cohort studies to investigate the relative predictive ability of a wider range of potential risk factors for cognitive decline. Using data from the prospective Edinburgh Type 2 Diabetes Study (ET2DS) the present thesis aimed (i) to determine associations of cognitive decline with macrovascular disease and with severe hypoglycaemia, and (ii) to compare a wider range of potential risk factors in their ability to predict cognitive decline. In 2006/2007, 1066 patients with type 2 diabetes (aged 60 to 75 years) attended the baseline ET2DS clinic and 831 returned for the follow-up at year 4. Subjects were extensively characterised for risk factor profiles at baseline, and at year 4 for incidence of severe hypoglycaemia. Socioeconomic status was estimated using postcode data. Scores on seven tests of age-sensitive ‘fluid’ cognitive function, which were administered at baseline and at year 4, were used to derive a general cognitive component (‘g’). A vocabulary-based test, administered at baseline, estimated pre-morbid ability. Findings are reported in three parts. 1.) Macrovascular disease and cognition: Subjects with higher levels of biomarkers indicative of subclinical macrovascular disease, including plasma N-terminal pro-brain natriuretic peptide and carotid intima-media thickness, had significantly steeper four-year cognitive decline, independent of traditional cardiovascular risk factors, stroke, socioeconomic status and estimated pre-morbid cognitive ability. For ankle-brachial pressure index, the association fell just short of statistical significance. Effect sizes were overall modest, with fully adjusted standardised beta coefficients ranging from 0.06 to -0.12. Little evidence was found for associations of the symptomatic markers of macrovascular disease with four-year change in cognitive function that was independent of participants’ pre-morbid ability and socioeconomic status. 2.) Severe hypoglycaemia and cognition: Subjects with lower cognitive ability at baseline were at two-fold increased risk of experiencing their first-ever incident severe hypoglycaemia during follow-up. The rate of four-year cognitive decline was significantly steeper in those exposed to hypoglycaemia compared with hypoglycaemia-free participants, independently of cardiovascular risk factors, microand macrovascular disease and of estimated pre-morbid cognitive ability. Effect sizes again were overall modest (Cohen’s d = 0.2 to 0.3 for statistically significant differences in four-year cognitive decline between subjects with and those without hypoglycaemia, following multivariable adjustment) 3.) Consideration of a wider range of risk factors and cognition: A stepwise linear regression model including a total of 15 metabolic and vascular risk factors identified inflammation, smoking and poorer glycaemic control (in addition to some of the subclinical markers of macrovascular disease) as predictive of a steeper four-year cognitive decline. Other traditional cardiovascular risk factors, diabetic retinopathy, clinical macrovascular disease and a baseline history of severe hypoglycaemia were not included in this model. The interpretation of the latter finding is limited, however, by the fact that the stepwise regression procedure may exclude true predictors from a model when they correlate with already included risk factors. This thesis has demonstrated associations of later-life cognitive decline in people with type 2 diabetes with markers of subclinical macrovascular disease and poor glycaemic control (including hypoglycaemia) as well as other cardiometabolic risk factors (inflammation, smoking). Findings suggest that associations are relatively weak and complex due to inter-relationships amongst risk factors, and indicate a role of pre-morbid ability and socioeconomic status (which as risk factors are difficult to modify) in the relationships of risk factors with cognitive decline. Future research including case-control studies to compare risk factor associations between people with type 2 diabetes and non-diabetic older adults and randomised controlled trials to evaluate potential causal effects of individual modifiable risk factors on cognitive decline, will help to evaluate the mechanisms underlying the observation that people with type 2 diabetes are at risk of cognitive impairment in later life.