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.

Low fat loss response after medium-term supervised exercise in obese is associated with exercise-induced increase in food reward

Objective: To examine exercise-induced changes in the reward value of food during medium-term supervised exercise in obese individuals. ---------- Subjects/Methods: The study was a 12-week supervised exercise intervention prescribed to expend 500 kcal/day, 5 d/week. 34 sedentary obese males and females were identified as responders (R) or non-responders (NR) to the intervention according to changes in body composition relative to measured energy expended during exercise. Food reward (ratings of liking and wanting, and relative preference by forced choice pairs) for an array of food images was assessed before and after an acute exercise bout. ---------- Results. 20 responders and 14 non-responders were identified. R lost 5.2 kg±2.4 of total fat mass and NR lost 1.7 kg±1.4. After acute exercise, liking for all foods increased in NR compared to no change in R. Furthermore, NR showed an increase in wanting and relative preference for high-fat sweet foods. These differences were independent of 12-weeks regular exercise and weight loss. ---------- Conclusion. Individuals who showed an immediate post-exercise increase in liking and increased wanting and preference for high-fat sweet foods displayed a smaller reduction in fat mass with exercise. For some individuals, exercise increases the reward value of food and diminishes the impact of exercise on fat loss.

Flexural behaviour and design of the new built-up LiteSteel beams

LiteSteel Beam (LSB) is a new cold-formed steel beam produced by OneSteel Australian Tube Mills. The new beam is effectively a channel section with two rectangular hollow flanges and a slender web, and is manufactured using a combined cold-forming and electric resistance welding process. OneSteel Australian Tube Mills is promoting the use of LSBs as flexural members in a range of applications, such as floor bearers. When LSBs are used as back to back built-up sections, they are likely to improve their moment capacity and thus extend their applications further. However, the structural behaviour of built-up beams is not well understood. Many steel design codes include guidelines for connecting two channels to form a built-up I-section including the required longitudinal spacing of connections. But these rules were found to be inadequate in some applications. Currently the safe spans of builtup beams are determined based on twice the moment capacity of a single section. Research has shown that these guidelines are conservative. Therefore large scale lateral buckling tests and advanced numerical analyses were undertaken to investigate the flexural behaviour of back to back LSBs connected by fasteners (bolts) at various longitudinal spacings under uniform moment conditions. In this research an experimental investigation was first undertaken to study the flexural behaviour of back to back LSBs including its buckling characteristics. This experimental study included tensile coupon tests, initial geometric imperfection measurements and lateral buckling tests. The initial geometric imperfection measurements taken on several back to back LSB specimens showed that the back to back bolting process is not likely to alter the imperfections, and the measured imperfections are well below the fabrication tolerance limits. Twelve large scale lateral buckling tests were conducted to investigate the behaviour of back to back built-up LSBs with various longitudinal fastener spacings under uniform moment conditions. Tests also included two single LSB specimens. Test results showed that the back to back LSBs gave higher moment capacities in comparison with single LSBs, and the fastener spacing influenced the ultimate moment capacities. As the fastener spacing was reduced the ultimate moment capacities of back to back LSBs increased. Finite element models of back to back LSBs with varying fastener spacings were then developed to conduct a detailed parametric study on the flexural behaviour of back to back built-up LSBs. Two finite element models were developed, namely experimental and ideal finite element models. The models included the complex contact behaviour between LSB web elements and intermittently fastened bolted connections along the web elements. They were validated by comparing their results with experimental results and numerical results obtained from an established buckling analysis program called THIN-WALL. These comparisons showed that the developed models could accurately predict both the elastic lateral distortional buckling moments and the non-linear ultimate moment capacities of back to back LSBs. Therefore the ideal finite element models incorporating ideal simply supported boundary conditions and uniform moment conditions were used in a detailed parametric study on the flexural behaviour of back to back LSB members. In the detailed parametric study, both elastic buckling and nonlinear analyses of back to back LSBs were conducted for 13 LSB sections with varying spans and fastener spacings. Finite element analysis results confirmed that the current design rules in AS/NZS 4600 (SA, 2005) are very conservative while the new design rules developed by Anapayan and Mahendran (2009a) for single LSB members were also found to be conservative. Thus new member capacity design rules were developed for back to back LSB members as a function of non-dimensional member slenderness. New empirical equations were also developed to aid in the calculation of elastic lateral distortional buckling moments of intermittently fastened back to back LSBs. Design guidelines were developed for the maximum fastener spacing of back to back LSBs in order to optimise the use of fasteners. A closer fastener spacing of span/6 was recommended for intermediate spans and some long spans where the influence of fastener spacing was found to be high. In the last phase of this research, a detailed investigation was conducted to investigate the potential use of different types of connections and stiffeners in improving the flexural strength of back to back LSB members. It was found that using transverse web stiffeners was the most cost-effective and simple strengthening method. It is recommended that web stiffeners are used at the supports and every third points within the span, and their thickness is in the range of 3 to 5 mm depending on the size of LSB section. The use of web stiffeners eliminated most of the lateral distortional buckling effects and hence improved the ultimate moment capacities. A suitable design equation was developed to calculate the elastic lateral buckling moments of back to back LSBs with the above recommended web stiffener configuration while the same design rules developed for unstiffened back to back LSBs were recommended to calculate the ultimate moment capacities.

The development of a serological-based diagnostic test for Dasheen mosaic potyvirus (DsMV)

Dasheen mosaic potyvirus (DsMV) is an important virus affecting taro. The virus has been found wherever taro is grown and infects both the edible and ornamental aroids, causing yield losses of up to 60%. The presence of DsMV, and other viruses,prevents the international movement of taro germplasm between countries. This has a significant negative impact on taro production in many countries due to the inability to access improved taro lines produced in breeding programs. To overcome this problem, sensitive and reliable virus diagnostic tests need to be developed to enable the indexing of taro germplasm. The aim of this study was to generate an antiserum against a recombinant DsMV coat protein (CP) and to develop a serological-based diagnostic test that would detect Pacific Island isolates of the virus. The CP-coding region of 16 DsMV isolates from Papua New Guinea, Samoa, Solomon Islands, French Polynesia, New Caledonia and Vietnam were amplified,cloned and sequenced. The size of the CP-coding region ranged from 939 to 1038 nucleotides and encoded putative proteins ranged from 313 to 346 amino acids, with the molecular mass ranging from 34 to 38 kDa. Analysis ofthe amino acid sequences revealed the presence of several amino acid motifs typically found in potyviruses,including DAG, WCIE/DN, RQ and AFDF. When the amino acid sequences were compared with each other and the DsMV sequences on the database, the maximum variability was21.9%. When the core region ofthe CP was analysed, the maximum variability dropped to 6% indicating most variability was present in the N terminus. Within seven PNG isolates ofDsMV, the maximum variability was 16.9% and 3.9% over the entire CP-coding region and core region, respectively. The sequence ofPNG isolate P1 was most similar to all other sequences. Phylogenetic analysis indicated that almost all isolates grouped according to their provenance. Further, the seven PNG isolates were grouped according to the region within PNG from which they were obtained. Due to the extensive variability over the entire CP-coding region, the core region ofthe CP ofPNG isolate Pl was cloned into a protein expression vector and expressed as a recombinant protein. The protein was purified by chromatography and SDS-PAGE and used as an antigen to generate antiserum in a rabbit. In western blots, the antiserum reacted with bands of approximately 45-47 kDa in extracts from purified DsMV and from known DsMV -infected plants from PNG; no bands were observed using healthy plant extracts. The antiserum was subsequently incorporated into an indirect ELISA. This procedure was found to be very sensitive and detected DsMV in sap diluted at least 1:1,000. Using both western blot and ELISA formats,the antiserum was able to detect a wide range ofDsMV isolates including those from Australia, New Zealand, Fiji, French Polynesia, New Caledonia, Papua New Guinea, Samoa, Solomon Islands and Vanuatu. These plants were verified to be infected with DsMV by RT-PCR. In specificity tests, the antiserum was also found to react with sap from plants infected with SCMV, PRSV-P, PRSV-W, but not with PVY or CMV -infected plants.