In this thesis it is demonstrated that leptin and insulin hyperpolarise hypothalamic glucose responsive neurones via the activation of the large conductance ATP-sensitive (K_APT) channel. This channel was potassium selective, had a single channel conductance of 150 pS and channel activity was inhibited by micromolar tolbutamide and millimolar internal ATP. Brain cortical cell bodies and nerve terminals possess a large-conductance calcium-activated potassium (BK) channel. The nerve terminal BK channel switched from high to low activity modes, whereas cell body BK channel activity inactivates during depolarisation. Furthermore, BK channel inactivation was abolished by internal trypsin treatment, suggesting an inactivating particle was associated with the channel. Internal application of alkaline phosphatase irreversibly removed mode switching and inactivation of cortical BK channels. Blocking the cell body BK channel pore with 100 mM intracellular tetraethylammonium (TEA) prevented alkaline phosphatase removal of inactivation, indicating that the phosphatase site of action was located close to the pore. Finally, protein kinase A (PKA) increased the occurrence of the high BK channel activity mode whereas PKA retarded the full recovery of BK inactivation induced by hyperpolarisation. In a separate study it was demonstrated that stably expressed human brain BK (<I>hSlo</I>) channels inactivate in a trypsin-insensitive manner. This inactivation was not due to barium contamination, since 5 μM internal barium blocked <I>hSlo</I> channels only during strong depolarisations, yet inactivation was observed at less positive potentials. Furthermore co-expression of either <I>hSlo</I>β-1, or voltage-gated potassium (Kv) β1.1 or β2.1 subunits with the <I>hSlo</I> α-subunit did not affect the extent or rate of channel inactivation. Finally, the Kvβ-subunits moved the calcium and voltage curves of <I>hSlo</I> to more negative voltages and altered the activation and deactivation kinetics in a manner almost identical to that observed on co-expression of <I>hSlo</I>β-1 subunit with <I>hSlo</I> or by increasing the internal calcium concentration.

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This thesis gives insight into the way that transcription of the insulin gene is regulated by nutrients. This is achieved primarily by characterising a MAP kinase pathway which links glucose metabolism to the activation of a beta cell transcription factor IUF1. An understanding of the precise mechanisms by which nutrients control beta cell function may be invaluable for the development of artificial cell lines that can be used for gene replacement therapy. A study of the E2 element of the rat II promoter illustrated that at least three factors bound to the region. These were identified as IUF1 (complex D5), USF (complex D4) and an uncharacterised factor D3. IUF1 is a beta cell specific transcription factor that has been implicated previously in glucose responsive insulin gene transcription. IUF1 binds to the insulin promoter in response to high levels of extracellular glucose. USF has been shown to be involved in the carbohydrate responsive transcription of various hepatic genes. The recently characterised stress activated (Reactivating Kinase) MAP kinase pathway was clearly shown to be involved in mediating the link between glucose metabolism within the beta cell and the binding activity of IUF1. Phosphorylation of the factor serves to induce an alteration in protein structure, which converts the factor to an active form that shows a high affinity for its DNA binding site, thus activating transcription. The RK pathway may prove to be a crucial link between nutrient metabolism and the activity of other physiological processes.

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This study examined the effects of exercise on glucose metabolism in adipocytes from female rats. Female rats were exercised by swimming six hours per day, five days per week for eight weeks. There was no variation in body weight gain (P > 0.05) between the exercise and control animals through the experimental period. The swimmers' fat cells were smaller (P < 0.05) than those of the sedentary controls of the same age. The rates of glucose oxidation of both C-1 and C-6 glucose were significantly higher (P < 0.05) in the exercise rats' adipocytes at all insulin concentrations. The sedentary control rats' adipocytes showed no significant response at any insulin concentration. Thus, exercise is a significant stimulus to cause increased oxidation rates in the adipocytes from exercising rats. These data also indicate that glucose transport, not defective glucose oxidation, is the limiting mechanism that accounts for the decreased responsiveness of adipocytes from sedentary control animals.

The purpose of this investigation was to determine the relationship between calcium and glucose uptake following muscle contraction with the use of the calcium channel blocker dantrolene. In previous studies an exercise model has been used to investigate the role of calcium during post-exercise glucose uptake. This study utilized electrical stimulation. It has been shown that exercise-induced glucose uptake is calciummediated, but to date no one has shown that glucose transport induced by electrical stimulation is calcium-mediated. Twenty four male Sprague Dawley rats weighing 140 g were sacrificed and their epitrochlearis muscles were removed. Four treatment groups were established: control, muscle incubated in glucose (4mM); insulin, muscles incubated in glucose (4mM) and insulin (1000uU/ml); electrical stimulation, at 50 Hz for two five minute intervals separated by one minute rest periods; insulin (1000uU/ml) and electrical stimulation at 50 Hz for two five minute intervals separated by one minute intervals. Each group consisted of contain 8-10 muscle preparations. Glucose uptake was measured through the use of a double label of radioactive mannitol and 3-O-methylglucose and analyzed using liquid scintillation. This project followed a randomized group design. Treatments were measured with a one way ANOVA. / School of Physical Education

One of the hallmarks of postprandial glucose homeostasis is the ability of insulin to promote glucose uptake into skeletal muscles. Insulin achieves this feat by enhancing the recruitment of glucose transporter 4 (GLUT4) from an intracellular compartment to the plasma membrane of muscles in order to create a net increase in surface GLUT4, which results in elevated glucose uptake. From a molecular perspective, this insulin-regulated GLUT4 traffic action requires the independent activation of Akt and Rac-1 in muscle cells because perturbation of either molecule results in an impaired response. Although Rac-1 has been validated as key component of insulin response, its downstream signalling capacity contributing to GLUT4 translocation remains unexplored. Studies on Rac-1 have shown that it is responsible for the formation of cortical remodelled actin that facilitates GLUT4 translocation following insulin stimulation. However, the downstream Rac-dependent molecules governing this actin remodelling are undetermined. Here we identified Arp2/3 and cofilin as the Rac-dependent regulators of insulin-induced actin remodelling in muscle cells. While Arp2/3 acts to initiate a burst of actin polymerization, cofilin balances out the actin dynamics through its severing/depolymerizing activity. Inhibition of either molecule’s function leads to defective GLUT4 translocation mediated by insulin in muscle cells, suggesting the requirement of actin dynamics to facilitate GLUT4 traffic to the plasma membrane. Furthermore, given the importance of Rac-1 in insulin-mediate GLUT4 traffic, its application potential to reverse insulin resistance has never been explored. We discovered that providing muscle cells with additional Rac-1 activity produces an insulin-independent gain in surface GLUT4 with magnitude comparable to that normally elicited by insulin. This phenotype is accomplished because of the concomitant cross-activation of Akt pathway when supplying the cells with active Rac-1. Interestingly, this response can bypass signalling defects imposed by cellular insulin resistance conditions, leading to restoration of GLUT4 translocation in muscle cells. Overall, these results not only reinforce the functional impact of Rac-1 on GLUT4 traffic but also identify additional molecules governed by Rac-1 contributing to the integrity of this insulin-mediated response in muscle cells.

This purpose of this study was to determine the effects of vitamin E and/or resistance exercise on insulin resistance and glucose uptake. Nine subjects, who were currently active but not strength training, were assigned to either the vitamin E or placebo group based on their prescreening measurements. Subjects underwent a 3-week vitamin E washout period before testing. At baseline testing subjects were given a 75-gram glucose load and blood was drawn every 15-minutes for 2-hours to analyze insulin and glucose response. Another oral glucose tolerance test (OGTT) was performed 45minutes after a 50-minute full body progressive resistance training session to determine insulin and glucose response to exercise. Subjects ingested either the placebo (3 capsules of olive oil) or 1200 IU vitamin E (3 capsules) for 9-weeks. After 3-weeks of supplementation the subjects returned for another exercising OGTT. After this session the subjects began a 6-week progressive resistance exercise program, in which they performed another OGTT after the last session. Both groups showed a significant increase in strength gains pre and post resistance training. The statistical analysis failed to demonstrate any differences between groups in insulin or glucose response in any of the four OGTT trials, but there were multiple trends present. Combining vitamin E supplementation with resistance training increased insulin sensitivity and the disposal of glucose. Both groups also had significant strength gains from pre to post study. Future research is needed for verification of these trends. / School of Physical Education

Motivation: The detrimental effects of obesity and insulin resistance in Caucasians and African-Americans have been the focus of many recent publications, and the association between PAI-1act and markers of the metabolic syndrome is well established but data on African subjects are still lacking. Objectives: To investigate possible differences between the association of PAI-1act with markers of the metabolic syndrome in Caucasian and African women. Methods We used cross-sectional data from the POWIRS I and II studies, involving 95 African and 114 Caucasian women respectively in the Potchefstroom district of the North West Province, South Africa. Results: Mean plasma PAI-1act was significantly higher in the Caucasian than in the African subjects (p < 0.001). Markers for the metabolic syndrome explained 60% of the variance of PAI-1act in the Caucasian group, but only 2.8% of the variance of PAI-1act in the African group. Waist circumference emerged as the strongest independent predictor of PAI-1act in the Caucasian (34%) as well as the African subjects (11%). Conclusion: This study showed clear differences in PAI-1act between African and Caucasian subjects, along with differences in the association of PAI-1act with markers of the metabolic syndrome. Apparent genetic differences between the two groups (especially the role of the 4G/5G genotype) may have an important influence on PAI-1act The role of PAI-1act in the metabolic syndrome may differ between Caucasians and Africans. / Thesis (M.Sc. (Nutrition))--North-West University, Potchefstroom Campus, 2005.

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