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>Magister Scientiae - MSc / The incidence of type 2 diabetes (T2D) is persistently increasing globally. T2D is associated with pancreatic β cell dysfunction and insulin resistance in peripheral tissues such as the liver and skeletal muscle. Skeletal muscle is the major site for insulin stimulated glucose uptake. Maintenance on a gestational high fat diet may programme insulin resistance. Programming is induced by the exposure of organisms to either a stimulus or insult during foetal and/or early neonatal life and alters offspring physiology and metabolism. The aim of the present study was therefore to investigate the effects of maternal diets, varying in fat content, on neonatal hepatic and skeletal muscle gene (mRNA) and protein (immunoreactivity) expression of proximal insulin signalling factors: insulin receptor alpha (IRα), insulin receptor substrate 2 (IRS2) and phosphoinositide 3-kinase-p110 alpha (PI3K-p110α), and to assess the therapeutic potential of Aspalathus linearis extract after high fat programming. Pregnant rats were randomised into groups maintained on diets with varying fat proportions: 10% (control), 20% (20F), 30% (30F) and 40% (40F) fat as energy throughout gestation. Neonatal liver and skeletal muscle were collected to determine the proximal insulin signalling expression profiles of the target factors: IRα, IRS2 and PI3K-p110α. Quantitative polymerase chain reaction (qPCR) was applied to determine mRNA expression of these target insulin signalling factors. Immunostaining of the target proteins in the liver and skeletal muscle was performed followed by relative quantification with image analysis software. Further, Aspalathus linearis (Al) extract was orally administered to mothers during gestation in the 10% (Control-Al) and 40% (HFD-Al) diets at a dose of 150 mg/kg. Body weight, food intake and blood glucose concentrations were monitored throughout gestation in mothers. Maternal diets, varying in the percentage of fat content, showed no significant effect on neonatal hepatic IR and IRS2 mRNA expression. However, hepatic PI3K mRNA expression was elevated in 30F neonates compared to 20F neonates. Skeletal muscle IR and PI3K mRNA expression were reduced in the 30F and 40F neonates compared to 20F neonates. There was reduced hepatic IRα immunoreactivity in 40F neonates compared to control and 20F neonates. Further, skeletal muscle IRα immunoreactivity was significantly reduced in 30F and 40F neonates compared to control neonates. Therefore foetal high fat programming reduced IRα in both the liver and skeletal muscle which may impair proximal insulin signalling in these glucose recipient organs. Aspalathus linearis had no effect on maternal serum insulin and glucagon concentrations. In addition, maternal caloric intake, body weight and organ weights (liver, brain and pancreas) were not altered amongst the groups. Further, HFD-Al neonates were heavier than control neonates. In conclusion, Aspalathus linearis, at a dose of 150 mg/kg, had neither harmful nor ameliorative effects in pregnant mothers fed high fat diet during gestation. In addition, Aspalathus linearis treatment had no ameliorative effects on neonates from mothers fed high fat diet throughout gestation.

The prevalence of type II diabetes is increasing at an alarming rate due to the western world lifestyle. Type II diabetes is characterized by an insulin resistance distinguished by impaired glucose uptake in adipose and muscle tissues. The molecular mechanisms behind the insulin recistance and also the knowledge considering normal insulin signalling in fat cells, especially in humans, are still unclear. Insulin receptor substrate (IRS) is known to be important for medating the insulin-induced signal from the insulin receptor into the cell. We developed and optimized a method for transfection of primary human adipocytes by electroporation. By recombinant expression of proteins, we found a proper IRS to be crucial for both mitogenic and metabolic signalling in human adipocytes. In human, but not rat, primary adipocytes we found IRS1 to be located at the plasma membrane in non-insulin stimulated cells. Insulin stimulation resulted in a two-fold increase of the amount of IRS1 at the plasma membrane in human cells, compared with a 12-fold increase in rat cells. By recombinant expression of IRS1 we found the species difference between human and rat IRS1 to depend on the IRS proteins and not on properties of the host cell. The adipocytes function as an energy store, critical for maintaining the energy balance, and obesity strongly correlates with insulin resistance. The insulin sensitivity of the adipocytes with regard to the size of the cells was examined by separating small and large cells from the same subject. We found no increase of the GLUT4 translocation to the plasma membrane following insulin stimulation in the large cells, whereas there was a two-fold increase in the small cells. This finding supports the idea of a causal relationship between the enlarged fat cells and reduced insulin sensitivity found in obese subjects. The insulin receptor is located and functional in a specific membrane structure, the caveola. The morphology of the caveola and the localization of the caveolar marker proteins caveolin-1 and -2 were examined. Caveolae were shown to be connected to the exterior by a narrow neck. Caveolin was found to be located at the neck region of caveolae, which imply importance of caveolin for maintaining and sequestering caveolae to the plasma membrane. In conclusion, the transfection technique proved to be highly useful for molecular biological studies of insulin signal transduction and morphology in primary adipocytes.

Emerging evidence suggests that an important mechanism for the negative feedback control of insulin signalling involves the inhibition of tyrosine phosphorylation of IRS-1 by its prior serine/threonine (ser/thr) phosphorylation. IRS-1 ser/thr phosphorylation has been linked to the dissociation of IRS-1 from the insulin receptor and PI3K, and its degradation via a proteasome-dependent pathway. Studies in animal models have shown that increases in plasma free fatty acids (FFAs) are associated with reduced IRS-1-signalling, and so it has been postulated that elevated FFA cause insulin resistance by activating pathways that negatively regulate insulin action, including hyper-phosphorylation of ser/thr residues in IRS-1. We have shown that in the case of linoleate-induced insulin resistance in L6 rat skeletal muscle cells, the inhibition of IRS-1-dependent signalling arises via effects on both the phosphorylation status and degradation of IRS-1, which are mediated, in part, by IKKb. In addition, the reduction of IRS-1 mRNA levels allude to transcriptional effects of linoleate treatment that also contribute to the observed reduction in the total levels of this protein. PtdOH, particularly dilinoleoyl PtdOH, was found to be significantly increased in linoleate treated L6 cells, and sufficient to induce at least some of the effects on insulin-signalling that are observed upon linoleate treatment. It is unlikely, however, that IKKb and PtdOH are components of the same inhibitory pathway, since inhibiting IKKb activity did not alleviate the effects of PtdOH on IRS-1 tyrosine (tyr) phosphorylation. Moreover, although an integral component of the mechanism by which linoleate induces insulin-resistance in L6 cells, it appears that restoring IRS-1 function in linoleate treated cells is not sufficient to reverse insulin resistance. Hence, we hypothesise that linoleate induces multiple inhibitory pathways in L6 cells, with at last two of these involving IKKb- and PtdOH-dependent inhibition of IRS-1 signalling, which act in parallel to reduce glucose disposal and cause insulin resistance in this model.

Insulin signalling is an essential process in humans by which the level of plasma glucose is maintained within the physiologically healthy range. Insulin activates the phosphoinositide 3 kinase (PI3K) signalling pathway that generates the phospholipid messenger PtdIns(3,4,5)P3, which in turn enhances the activity of two important proteins, AKT and Rac1. This then leads to increase the presence of the glucose transporter 4 (GLUT4) at the plasma membrane that enhances the intake of glucose, particularly in skeletal muscle cells and adipocytes. Insulin signalling also triggers interconversion of several other phosphoinositides (PIs) which play pivotal roles in different steps of glucose regulation. PtdIns5P is an important PI that is robustly increased after insulin treatment in the skeletal muscle cell line, L6 myotubes. Many of PtdIns5P`s functions are not fully understood. To gain more knowledge of the role of PtdIns5P in insulin signalling in muscle cells, the PtdIns5P kinase phosphatidylinositol-5-phosphate 4-kinase a (PIP4K2a) was over-expressed in L6 myotubes as a way of removing PtdIns5P, and the consequences in insulin signalling were studied. Although PtdIns5P is converted by PIP4K2a to PtdIns(4,5)P2 which is a precursor of the potent PI PtdIns(3,4,5)P3, previous studies revealed that the increase in PtdIns(3,4,5)P3 induced by insulin in control cells is diminished in cells overexpressing PIP4K2a, for unknown reasons. Additionally, although the phosphorylation of the serine/threonine protein kinase AKT was not affected in these L6 cells, glucose uptake was attenuated. The current study investigates the possible causes of attenuating glucose uptake in PIP4K overexpressing myotubes by examining the small GTPase Rac1 which plays an important role in the cytoskeleton re-arrangement that is necessary for GLUT4 translocation. Furthermore, the possible roles of PI phosphatases that may cause the disturbance on the levels of PIs in response to insulin were evaluated. Additionally, the potential role of PtdIns5P in Rac1 activation in L6 myotubes was further investigated by delivering synthetic PtdIns5P using a carrier-based delivery approach. The results showed that the attenuation of glucose uptake documented in previous studies occurred as a result of a defect in the process of translocating GLUT4 from intracellular storage to the plasma membrane. Rac1 activity was significantly reduced in cells expressing PIP4K2a. Quantifying the level of PIs suggested that PIP4K2a expression increases the removal of PtdIns(3,4,5)P3 by the PI 5-phosphatase, SKIP. Silencing the expression of SKIP by siRNA restored the level of PtdIns(3,4,5)P3 but Rac1 activity and the attenuation GLUT4 translocation were not rescued possibly as a result of removing PtdIns5P by PIP4K2a. On the other hand, exogenous delivery of PtdIns5P in L6 myotubes activates both Rac1 and GLUT4 translocation in the absence of insulin. However, activating GLUT4 translocation by the exogenous PtdIns5P requires PI3K activity since redistribution of GLUT4 to the plasma membrane is inhibited by the PI3K inhibitor, wortmannin. Removing PtdIns5P reduces Rac1 activity and stimulates SKIP that inhibits PtdIns(3,4,5)P3 increase which attenuates GLUT4 translocation and hence glucose uptake. These results emphasise the critical role played by PtdIns5P which seems to serve as a regulator of insulin signalling, both directly and/or by regulating other enzymes involved in the metabolism of PIs.

The primary function of adipose tissue is to store energy in the form of triacylglycerol, which is hydrolyzed to fatty acids to supply other tissues with energy. While insulin promotes the storage of triacylglycerol, catecholamines stimulate its hydrolysis. The development of type II diabetes is strongly associated with obesity, indicating a role of triacylglycerol metabolism in the pathogenesis of diabetes. Caveolae are plasma membrane invaginations found in most cells but are highly abundant in adipocytes. Insulin receptors are localized in caveolae and their function depends on intact caveolae structures. In the present thesis work, mass spectrometry-based methodology allowed identification of a number of new proteins and their posttranslational modifications in caveolae of human adipocytes. Variable N-terminal acetylation and phosphorylation of caveolin-1α and caveolin-1β were identified, which might regulate the function of caveolae. The transcription regulator protein PTRF was identified as the major caveolae associated protein. Specific proteolytic modifications of PTRF at the cytosolic surface of caveolae and phosphorylation on nine serine and one threonine residues were identified. Moreover, insulin induced translocation of PTRF from the plasma membrane to the nucleus. PTRF was previously shown to regulate the activity of both RNA polymerase I and polymerase II, thus a role of PTRF in mediating the anabolic action of insulin on protein synthesis and gene transcription is proposed. PTRF was also involved in an extranuclear function in the hormonal regulation of triacylglycerol metabolism in caveolae. PTRF was colocalized with the triacylglycerol regulator proteins perilipin and hormone-sensitive lipase (HSL) in the triacylglycerol-synthesizing caveolae subclass. We showed that, while perilipin was translocated to the plasma membrane, both PTRF and HSL were translocated from the plasma membrane to the cytosol as a complex in response to insulin. The perilipin recruited to the plasma membrane was highly threonine phosphorylated. By mass spectrometry, three phosphorylated threonine residues were identified and were located in an acidic domain in the lipid droplet targeting domain of perilipin. The insulin-induced recruitment of perilipin to the plasma membrane might, therefore be phosphorylation-dependent. Isoproterenol, which stimulates hydrolysis of triacylglycerol, induced a complete depletion of perilipin B from the plasma membrane, suggesting a function of perilipin B to protect newly synthesized triacylglycerol in caveolae from being hydrolyzed by HSL. The location of PTRF and HSL was not affected by isoproterenol, indicating that insulin is acting against a default presence of PTRF and HSL in caveolae. Taken together, this thesis expands our knowledge about caveolae and provided valuable information about their involvement in novel roles, particularly in the hormonal regulation of triacylglycerol metabolism.

Type II diabetes mellitus (T2DM) has been associated with the onset of diet-induced obesity, which is currently on the rise worldwide. T2DM is typically characterized by insulin resistance in peripheral tissues such as adipose tissue, liver, and skeletal muscle. In skeletal muscle it is widely accepted that the defective insulin action is due to the inability of the cell to sufficiently activate the insulin signalling pathway and promote systemic glucose uptake. The sarcolipin-null (KO) mouse is a potential novel model for diet-induced obesity and diabetes. KO mice become significantly more obese and display a greater glucose intolerance than wildtype (WT) mice following an 8-week high-fat diet (HFD; 42% calories from fat) but the underlying mechanisms are still unknown. In this study the role of defective skeletal muscle insulin signalling in the development of the impaired glucose tolerance in KO mice was investigated. It was hypothesized that the HFD fed KO mice would exhibit greater reductions in IRS1 tyr628 and Akt ser473 phosphorylation (i.e. decreased activation of the insulin signalling pathway) than controls. Furthermore, it was believed that KO mice would display increased phosphorylation of IRS1 ser307, which is commonly associated with insulin resistance. At 16-weeks of age KO mice and littermates were subdivided into two groups and placed on either a HFD (n=30) or chow diet (n=24) for an 8-week period. Changes in body weight, glucose tolerance, and insulin tolerance were assessed pre- and post-diet period. Following the completion of the diet intervention mice were treated with an intraperitoneal injection of insulin (0.75U/kg) or vehicle solution and sacrificed for tissue collection. Epididymal/inguinal and retroperitoneal fat pads were removed for assessment of whole body adiposity. Whole gastrocnemius muscle was excised and homogenized for Western blot analysis of several key proteins of the insulin signalling cascade. Following completion of the HFD KO mice (48.6 ± 1.6 g) weighed significantly more than HFD fed wildtype (WT) mice (41.5 ± 1.6 g), and all chow fed mice (KO: 36.8 ± 1.5 g; WT: 35.2 ± 1.2 g; p<0.001). Glucose tolerance testing showed that KO mice exhibited significantly greater glucose intolerance compared to control mice post-HFD (p<0.001). Insulin tolerance testing, however, revealed no change in insulin sensitivity in KO or WT mice post-HFD (p>0.05). The HFD fed KO mice (0.73 ± 0.06 g) had an elevated retroperitoneal fat pad weight than HFD fed WT (0.49 ± 0.05 g) and all chow fed mice (KO: 0.28 ± 0.04 g; WT: 0.24 ± 0.04 g; p<0.01). Western blot analysis revealed a similar reduction in insulin receptor substrate-1 (IRS1) tyr628 phosphorylation in both KO and WT mice following the HFD (Con WT: 2.82 ± 0.69; Con KO: 3.06 ± 0.73; HFD WT: 1.71 ± 0.28; HFD KO: 1.28 ± 0.11 fold increase over non-insulin stimulated mice; p<0.02). IRS1 ser307 phosphorylation was elevated in both genotypes post-HFD (HFD WT: 2.97 ± 1.19; HFD KO: 2.17 ± 0.59 fold increase over standard chow fed control mice; p<0.03). Insulin treatment did not stimulate phosphorylation of Akt ser473 in KO or WT mice regardless of diet (p>0.05). In summary there was no difference between KO and WT mice in skeletal muscle insulin sensitivity as assessed by the phosphorylation of insulin signalling intermediates. An increase in IRS1 ser307 phosphorylation appears to be the primary mechanism for the reduced activation of IRS1 following the HFD in both KO and WT mice. However, the results from the current investigation did not support the notion that impaired skeletal muscle insulin signalling is responsible for the more pronounced diet-induced glucose intolerance observed in KO mice. Future studies investigating the viability of skeletal muscle GLUT4 translocation and glucose uptake as well as the glucose-induced insulin secretion of pancreatic β-cells following consumption of a HFD would help elucidate the mechanism of glucose intolerance in KO mice.

Type II diabetes mellitus (T2DM) has been associated with the onset of diet-induced obesity, which is currently on the rise worldwide. T2DM is typically characterized by insulin resistance in peripheral tissues such as adipose tissue, liver, and skeletal muscle. In skeletal muscle it is widely accepted that the defective insulin action is due to the inability of the cell to sufficiently activate the insulin signalling pathway and promote systemic glucose uptake. The sarcolipin-null (KO) mouse is a potential novel model for diet-induced obesity and diabetes. KO mice become significantly more obese and display a greater glucose intolerance than wildtype (WT) mice following an 8-week high-fat diet (HFD; 42% calories from fat) but the underlying mechanisms are still unknown. In this study the role of defective skeletal muscle insulin signalling in the development of the impaired glucose tolerance in KO mice was investigated. It was hypothesized that the HFD fed KO mice would exhibit greater reductions in IRS1 tyr628 and Akt ser473 phosphorylation (i.e. decreased activation of the insulin signalling pathway) than controls. Furthermore, it was believed that KO mice would display increased phosphorylation of IRS1 ser307, which is commonly associated with insulin resistance. At 16-weeks of age KO mice and littermates were subdivided into two groups and placed on either a HFD (n=30) or chow diet (n=24) for an 8-week period. Changes in body weight, glucose tolerance, and insulin tolerance were assessed pre- and post-diet period. Following the completion of the diet intervention mice were treated with an intraperitoneal injection of insulin (0.75U/kg) or vehicle solution and sacrificed for tissue collection. Epididymal/inguinal and retroperitoneal fat pads were removed for assessment of whole body adiposity. Whole gastrocnemius muscle was excised and homogenized for Western blot analysis of several key proteins of the insulin signalling cascade. Following completion of the HFD KO mice (48.6 ± 1.6 g) weighed significantly more than HFD fed wildtype (WT) mice (41.5 ± 1.6 g), and all chow fed mice (KO: 36.8 ± 1.5 g; WT: 35.2 ± 1.2 g; p<0.001). Glucose tolerance testing showed that KO mice exhibited significantly greater glucose intolerance compared to control mice post-HFD (p<0.001). Insulin tolerance testing, however, revealed no change in insulin sensitivity in KO or WT mice post-HFD (p>0.05). The HFD fed KO mice (0.73 ± 0.06 g) had an elevated retroperitoneal fat pad weight than HFD fed WT (0.49 ± 0.05 g) and all chow fed mice (KO: 0.28 ± 0.04 g; WT: 0.24 ± 0.04 g; p<0.01). Western blot analysis revealed a similar reduction in insulin receptor substrate-1 (IRS1) tyr628 phosphorylation in both KO and WT mice following the HFD (Con WT: 2.82 ± 0.69; Con KO: 3.06 ± 0.73; HFD WT: 1.71 ± 0.28; HFD KO: 1.28 ± 0.11 fold increase over non-insulin stimulated mice; p<0.02). IRS1 ser307 phosphorylation was elevated in both genotypes post-HFD (HFD WT: 2.97 ± 1.19; HFD KO: 2.17 ± 0.59 fold increase over standard chow fed control mice; p<0.03). Insulin treatment did not stimulate phosphorylation of Akt ser473 in KO or WT mice regardless of diet (p>0.05). In summary there was no difference between KO and WT mice in skeletal muscle insulin sensitivity as assessed by the phosphorylation of insulin signalling intermediates. An increase in IRS1 ser307 phosphorylation appears to be the primary mechanism for the reduced activation of IRS1 following the HFD in both KO and WT mice. However, the results from the current investigation did not support the notion that impaired skeletal muscle insulin signalling is responsible for the more pronounced diet-induced glucose intolerance observed in KO mice. Future studies investigating the viability of skeletal muscle GLUT4 translocation and glucose uptake as well as the glucose-induced insulin secretion of pancreatic β-cells following consumption of a HFD would help elucidate the mechanism of glucose intolerance in KO mice.

<p>Menin is a tumour suppressor protein associated with the MEN1 hereditary cancer syndrome. Numerous protein interactions have been identified for menin but its specific function in tumour suppression remains enigmatic. Since the protein is well conserved, the <em>Drosophila</em> model system was used to study menin protein interactions in hopes of further elucidating menin function. In this study, two menin protein interactions were examined; the first was a novel interaction with <em>Drosophila</em> fas-associated death domain (dFADD), an important protein in innate immune signalling and the second was a conserved interaction with Trithorax (Trx), a histone methyltransferase important for activation of gene expression. Both these interactions were confirmed through co-immunoprecipitation in <em>Drosophila</em> S2 cells. Unlike <em>dFADD</em> mutants that are highly susceptible to bacterial infection, <em>Mnn1</em> mutants display normal resistance, suggesting that menin does not play an essential role in innate immune signalling. Moreover, <em>dFADD</em> mutants do not display a heat shock sensitivity phenotype, as previously reported for <em>Mnn1</em> mutants. The importance of the menin-Trx interaction in the regulation of heat shock gene expression was examined since both proteins were independently shown to be necessary for proper expression of <em>hsp70</em>. Chromatin immunoprecipitation analyses demonstrate that menin and Trx co-localize in the coding region of <em>hsp70</em>. Heat shock results in a loss of menin and an increase in Trx localization at <em>hsp70</em> and the proteins fail to co-immunoprecipitate with prolonged heat stress, suggesting a complex regulation of the interaction. Due to the recently identified interactions between mammalian menin and proteins of the insulin signalling pathway, a potential role for <em>Drosophila</em> menin in this pathway was examined. <em>Mnn1</em> mutants display increased desiccation and starvation resistance, similar to other positive regulators of insulin signalling. Overall, this thesis describes a novel interaction between menin and dFADD and a conserved interaction with Trx and also proposes a potential role for menin in the <em>Drosophila</em> insulin signalling pathway.</p> / Doctor of Philosophy (PhD)