Molecular markers of obesity and diabetes

Recently it has been shown that the consumption of a diet high in saturated fat is associated with impaired insulin sensitivity and increased incidence of type 2 diabetes. In contrast, diets that are high in monounsaturated fatty acids (MUFAs) or polyunsaturated fatty acids (PUFAs), especially very long chain n-3 fatty acids (FAs), are protective against disease. However, the molecular mechanisms by which saturated FAs induce the insulin resistance and hyperglycaemia associated with metabolic syndrome and type 2 diabetes are not clearly defined. It is possible that saturated FAs may act through alternative mechanisms compared to MUFA and PUFA to regulate of hepatic gene expression and metabolism. It is proposed that, like MUFA and PUFA, saturated FAs regulate the transcription of target genes. To test this hypothesis, hepatic gene expression analysis was undertaken in a human hepatoma cell line, Huh-7, after exposure to the saturated FA, palmitate. These experiments showed that palmitate is an effective regulator of gene expression for a wide variety of genes. A total of 162 genes were differentially expressed in response to palmitate. These changes not only affected the expression of genes related to nutrient transport and metabolism, they also extend to other cellular functions including, cytoskeletal architecture, cell growth, protein synthesis and oxidative stress response. In addition, this thesis has shown that palmitate exposure altered the expression patterns of several genes that have previously been identified in the literature as markers of risk of disease development, including CVD, hypertension, obesity and type 2 diabetes. The altered gene expression patterns associated with an increased risk of disease include apolipoprotein-B100 (apo-B100), apo-CIII, plasminogen activator inhibitor 1, insulin-like growth factor-I and insulin-like growth factor binding protein 3. This thesis reports the first observation that palmitate directly signals in cultured human hepatocytes to regulate expression of genes involved in energy metabolism as well as other important genes. Prolonged exposure to long-chain saturated FAs reduces glucose phosphorylation and glycogen synthesis in the liver. Decreased glucose metabolism leads to elevated rates of lipolysis, resulting in increased release of free FAs. Free FAs have a negative effect on insulin action on the liver, which in turn results in increased gluconeogenesis and systemic dyslipidaemia. It has been postulated that disruption of glucose transport and insulin secretion by prolonged excessive FA availability might be a non-genetic factor that has contributed to the staggering rise in prevalence of type 2 diabetes. As glucokinase (GK) is a key regulatory enzyme of hepatic glucose metabolism, changes in its activity may alter flux through the glycolytic and de novo lipogenic pathways and result in hyperglycaemia and ultimately insulin resistance. This thesis investigated the effects of saturated FA on the promoter activity of the glycolytic enzyme, GK, and various transcription factors that may influence the regulation of GK gene expression. These experiments have shown that the saturated FA, palmitate, is capable of decreasing GK promoter activity. In addition, quantitative real-time PCR has shown that palmitate incubation may also regulate GK gene expression through a known FA sensitive transcription factor, sterol regulatory element binding protein-1c (SREBP-1c), which upregulates GK transcription. To parallel the investigations into the mechanisms of FA molecular signalling, further studies of the effect of FAs on metabolic pathway flux were performed. Although certain FAs reduce SREBP-1c transcription in vitro, it is unclear whether this will result in decreased GK activity in vivo where positive effectors of SREBP-1c such as insulin are also present. Under these conditions, it is uncertain if the inhibitory effects of FAs would be overcome by insulin. The effects of a combination of FAs, insulin and glucose on glucose phosphorylation and metabolism in cultured primary rat hepatocytes at concentrations that mimic those in the portal circulation after a meal was examined. It was found that total GK activity was unaffected by an increased concentration of insulin, but palmitate and eicosapentaenoic acid significantly lowered total GK activity in the presence of insulin. Despite the fact that total GK enzyme activity was reduced in response to FA incubation, GK enzyme translocation from the inactive, nuclear bound, to active, cytoplasmic state was unaffected. Interestingly, none of the FAs tested inhibited glucose phosphorylation or the rate of glycolysis when insulin is present. These results suggest that in the presence of insulin the levels of the active, unbound cytoplasmic GK are sufficient to buffer a slight decrease in GK enzyme activity and decreased promoter activity caused by FA exposure. Although a high fat diet has been associated with impaired hepatic glucose metabolism, there is no evidence from this thesis that FAs themselves directly modulate flux through the glycolytic pathway in isolated primary hepatocytes when insulin is also present. Therefore, although FA affected expression of a wide range of genes, including GK, this did not affect glycolytic flux in the presence of insulin. However, it may be possible that a saturated FA-induced decrease in GK enzyme activity when combined with the onset of insulin resistance may promote the dys-regulation of glucose homeostasis and the subsequent development of hyperglycaemia, metabolic syndrome and type 2 diabetes.

The impact of outcomes-driven curriculum reform on teacher perspectives of professional practice

This thesis is the result of an investigation of a Queensland example of curriculum reform based on outcomes, a type of reform common to many parts of the world during the last decade. The purpose of the investigation was to determine the impact of outcomes on teacher perspectives of professional practice. The focus was chosen to permit investigation not only of changes in behaviour resulting from the reform but also of teachers' attitudes and beliefs developed during implementation. The study is based on qualitative methodology, chosen because of its suitability for the investigation of attitudes and perspectives. The study exploits the researcher's opportunities for prolonged, direct contact with groups of teachers through the selection of an over-arching ethnography approach, an approach designed to capture the holistic nature of the reform and to contextualise the data within a broad perspective. The selection of grounded theory as a basis for data analysis reflects the open nature of this inquiry and demonstrates the study's constructivist assumptions about the production of knowledge. The study also constitutes a multi-site case study by virtue of the choice of three individual school sites as objects to be studied and to form the basis of the report. Three primary school sites administered by Brisbane Catholic Education were chosen as the focus of data collection. Data were collected from three school sites as teachers engaged in the first year of implementation of Student Performance Standards, the Queensland version of English outcomes based on the current English syllabus. Teachers' experience of outcomes-driven curriculum reform was studied by means of group interviews conducted at individual school sites over a period of fourteen months, researcher observations and the collection of artefacts such as report cards. Analysis of data followed grounded theory guidelines based on a system of coding. Though classification systems were not generated prior to data analysis, the labelling of categories called on standard, non-idiosyncratic terminology and analytic frames and concepts from existing literature wherever practicable in order to permit possible comparisons with other related research. Data from school sites were examined individually and then combined to determine teacher understandings of the reform, changes that have been made to practice and teacher responses to these changes in terms of their perspectives of professionalism. Teachers in the study understood the reform as primarily an accountability mechanism. Though teachers demonstrated some acceptance of the intentions of the reform, their responses to its conceptualisation, supporting documentation and implications for changing work practices were generally characterised by reduced confidence, anger and frustration. Though the impact of outcomes-based curriculum reform must be interpreted through the inter-relationships of a broad range of elements which comprise teachers' work and their attitudes towards their work, it is proposed that the substantive findings of the study can be understood in terms of four broad themes. First, when the conceptual design of outcomes did not serve teachers' accountability requirements and outcomes were perceived to be expressed in unfamiliar technical language, most teachers in the study lost faith in the value of the reform and lost confidence in their own abilities to understand or implement it. Second, this reduction of confidence was intensified when the scope of outcomes was outside the scope of the teachers' existing curriculum and assessment planning and teachers were confronted with the necessity to include aspects of syllabuses or school programs which they had previously omitted because of a lack of understanding or appreciation. The corollary was that outcomes promoted greater syllabus fidelity when frameworks were closely aligned. Third, other benefits the teachers associated with outcomes included the development of whole school curriculum resources and greater opportunity for teacher collaboration, particularly among schools. The teachers, however, considered a wide range of factors when determining the overall impact of the reform, and perceived a number of them in terms of the costs of implementation. These included the emergence of ethical dilemmas concerning relationships with students, colleagues and parents, reduced individual autonomy, particularly with regard to the selection of valued curriculum content and intensification of workload with the capacity to erode the relationships with students which teachers strongly associated with the rewards of their profession. Finally, in banding together at the school level to resist aspects of implementation, some teachers showed growing awareness of a collective authority capable of being exercised in response to top-down reform. These findings imply that Student Performance Standards require review and, additional implementation resourcing to support teachers through times of reduced confidence in their own abilities. Outcomes prove an effective means of high-fidelity syllabus implementation, and, provided they are expressed in an accessible way and aligned with syllabus frameworks and terminology, should be considered for inclusion in future syllabuses across a range of learning areas. The study also identifies a range of unintended consequences of outcomes-based curriculum and acknowledges the complexity of relationships among all the aspects of teachers' work. It also notes that the impact of reform on teacher perspectives of professional practice may alter teacher-teacher and school-system relationships in ways that have the potential to influence the effectiveness of future curriculum reform.