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Philippeos, Christina. "Insulin signalling in endothelial cells." Thesis, King's College London (University of London), 2014. http://kclpure.kcl.ac.uk/portal/en/theses/insulin-signalling-in-endothelial-cells(8e35db48-dc9c-41be-b1aa-1fbe241fc356).html.
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Insulin is known to act as an anti-inflammatory agent and protect vascular endothelial cells during ischaemic damage in vivo. Although it is known that insulin signals in part through phosphatidylinositol 3-kinases (PI3Ks) and Akt, its effects on endothelial junctions and actin cytoskeleton are unknown. This study aimed to characterise endothelial responses to insulin, identify endothelial insulin-‐induced changes in protein phosphorylation and determine the roles of these changes in regulating endothelial functions. Insulin stimulation induced dose-dependent Akt activation in both primary human umbilical vein endothelial cells (HUVECs) and an endothelial cell line, human bone-marrow endothelial cells (HBMECs). Insulin decreased basal HUVEC permeability, increased angiogenic loop formation in vitro and increased cell migration in a wound-healing model, compared to untreated cells. Insulin-stimulated changes in protein phosphorylation were identified using a 14-3-3 affinity purification proteomic screen, as 14-3-3 proteins interact specifically with phosphorylated Ser/Thr residues within 14-3-3-binding motifs. A total of 390 14-3-3-binding proteins were identified from insulin-stimulated HBMECs, from which 12 proteins were selected based on predicted roles in endothelial cytoskeleton regulation. Validation of these hits, performed by Far-Western overlay analysis, identified 4 IGF-I-regulated 14-3-3-binding proteins: Parg1 (ARHGAP29), RICH-1 (ARHGAP17), LMO7 and Epsin2. Parg1 depletion in HUVECs induced stress fibre formation, increased endothelial permeability, severely decreased angiogenic loop formation and decreased cell migration, compared to siRNA control-treated cells. This suggests that Parg1 regulates contractility and hence could affect endothelial cell-cell junctional stability. Depletion of RICH-1 and LMO7 in HUVECs resulted in mislocalisation of the tight junction protein ZO-1. However, this did not affect endothelial permeability, suggesting that these proteins are important for maintaining tight junction integrity. LMO7 and Epsin2 depletion each resulted in an increase in angiogenic loop formation, but did not detectably affect cell migration. Insulin stimulation of Epsin2 might increase lamellipodium formation, although further studies are required to establish the mechanisms involved. In conclusion, this thesis describes a 14-3-3-based proteomic screen that identified novel regulators of endothelial function. These proteins could contribute to the anti-inflammatory roles of insulin.
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Joharatnam, Jalini. "Insulin signalling in granulosa cells." Thesis, Imperial College London, 2012. http://hdl.handle.net/10044/1/9784.
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Polycystic ovary syndrome (PCOS) is characterised by hyperandrogenism and insulin resistance. Granulosa cells in PCOS demonstrate impaired insulin-induced glucose uptake and lactate accumulation, suggesting a post receptor, signalling pathway-specific impairment of insulin action. Gonadotrophins are also important in the regulation of glucose metabolism by of granulosa cells. The first objective of this project was to use KK1 cells an immortalised mouse granulosa cell line, to characterise insulin, androgen and FSH signalling as well as glucose metabolism. Cell lysates were subjected to western immunoblotting for key proteins in the insulin signalling pathways. Glucose metabolism of KK1 cells was also measured. Surprisingly, androgen alone stimulated glucose uptake and lactate production and augmented insulin-induced glucose metabolism. This suggests that the insulin resistance observed in granulosa cells from women with PCOS is not a direct effect of exposure to androgen. The main part of the thesis was examination of insulin action on human primary ovarian granulosa-lutein (GL) cells to investigate the mechanism of insulin resistance in PCOS. Insulin and FSH signalling in GL cells from women with anovulatory PCOS (anovPCO, n=11) was compared to that in GL cells from ovulatory women with (ovPCO, n=8) and without polycystic ovaries (controls n=12). Primary GL cells were incubated with insulin or FSH and analysed for glucose metabolism and progesterone production. Cell lysates were prepared for identification of signalling pathways, using western immunoblotting. The results confirmed selective impairment of glucose metabolism in cells from anovulatory PCOS. No significant impairment of insulin stimulated PI3K signalling was observed. However there was a reduction of p42/44ERK phosphorylation in the ovulatory PCOS group compared to controls. The significance of this finding with respect to impaired glucose metabolism in granulosa cells remains to be determined. We also showed FSH induced glucose metabolism, but without clear evidence of activation of the PI3-kinase pathway.
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Collison, Mary Williamson. "Insulin signalling in insulin resistance and cardiovascular disease syndromes." Thesis, University of Glasgow, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366184.
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Alves, Steven Ribeiro. "The relevence of insulin signalling in Alzheimer's disease." Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/22020.
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Mestrado em Biologia Molecular e CelularAlzheimer’s disease (AD) is the most common type of dementia worldwide. It is molecularly characterized by deposition of extracellular senile plaques (SPs) composed by aggregated amyloid beta (Aβ) peptide, the formation of neurofibrillary tangles (NFTs) derived from hyperphosphorylation of the microtubule-associated protein Tau, synaptic dysfunction due to the deposits of SPs and NFTs and oxidative stress induced by impaired metabolic pathways. The insulin signalling pathway can play a major role in diverse AD related pathways, such as APP cleavage, Tau hyperphosphorylation, Apolipoprotein E (ApoE) influence in insulin signalling efficiency and the insulin degrading enzyme, which is also the major Aβ degrading enzyme. Growing evidence links AD with type 2 diabetes (T2D) due to impaired insulin signalling (IS) and brain insulin resistance. In a cohort based study in the Aveiro region, a correlation between diabetes and poor cognitive scores in the Mini Mental State Examination (MMSE) test were observed, with a p-value of 0.072. Additionally, carriers of the allele ApoE-ɛ2 appeared to be protective against diabetes, in the literature the same allele appears to be protective for AD. Posteriorly, the analysis of protein interactions, via the development of interactome networks, identified several proteins involved in both AD and the IS pathways. Also, by correlating these pathways with the synapse proteome, a very high overlap was observed (88% for AD, 79% for IS and 96% for AD and IS coincident proteins), enforcing the importance of both pathways in synaptic signalling and plasticity. From gene ontology studies, it was possible to assess the principal biological processes and molecular functions of the dataset of proteins. For AD, response to stimulus, cellular component organization, cell communication, signalling, protein binding, receptor binding and kinase binding were categories with elevated representation. Regarding coincident proteins between AD and IS pathways, an increase in all categories was observed, meaning that insulin plays a pivotal role in many AD events. Finally, the analysis of SH-SY5Y differentiated cells treated with 0, 1, 10 and 100 nM of insulin for 0, 10 and 60 minutes, showed a decrease on the intracellular total levels of protein Tau and an increase in the phosphorylation at serine 396. Regarding the amyloid precursor protein (APP), increases in intracellular levels were observed, when treated with insulin for 10 minutes, followed by a decrease for 60 minutes exposure. The phosphorylation of APP at threonine 668, has previously been related to increased production of Aβ, by promoting APP cleavage via the amyloidogenic pathway. In cells treated with insulin, a clear increase was detected at the 10-minute time point. At 60 minutes, the levels of phosphorylation were low probably due to low total APP levels.
A doença de Alzheimer (DA) é o tipo mais comum de demência no mundo. É caracterizada molecularmente pela deposição extracelular de placas senis (PS) compostas por agregados do péptido amiloide beta (Aβ), pela formação de emaranhados neurofibrilares (EN) derivados da hiperfosforilação da proteína Tau, pela disfunção sináptica devido aos depósitos de PS e EN e também pelo stress oxidativo induzido pelo enfraquecimento das vias metabólicas. A via de sinalização da insulina desempenha um papel principal em diversas vias da DA, tal como na clivagem da APP, hiperfosforilação da proteína Tau, eficiência da sinalização da insulina influenciada pela Apolipoproteína E (ApoE) e pela enzima envolvida na degradação de insulina que também é a enzima principal na degradação de Aβ. Crescente evidência relaciona a DA com a diabetes de tipo 2 (T2D) devido ao mau funcionamento da sinalização pela insulina e da resistência cerebral à mesma. Num estudo baseado num cohort da região de Aveiro, foi observada uma correlação entre a diabetes e um mau resultado no teste do ‘Mini Mental State Examination’. Adicionalmente, também foi observada uma correlação entre os portadores do alelo ApoE-ɛ2 e um estado protetor contra a T2D. Este alelo também foi observado na literatura como sendo protetor contra a DA. Posteriormente, uma análise de interações entre proteínas, identificou várias proteínas envolvidas tanto na DA como na sinalização da insulina. Correlacionando estes dados com o proteoma da sinapse, foi possível observar que existe uma grande representação das duas condições e também das proteínas coincidentes às duas (88% para a DA, 79% para a sinalização da insulina e 96% para as proteínas relacionadas com ambas), reforçando o papel de ambas as vias na sinalização e plasticidade sináptica. Do estudo de ontologia genética para a DA, foi possível identificar diversas vias importantes, tais como, resposta a um estímulo, organização de componentes celulares, comunicação celular, ligação proteica e ligação a uma cinase. Em relação à sinalização da insulina, as mesmas categorias apareciam com maior representação, significando que a insulina tem um papel importante em muitos eventos da DA. Por fim, o tratamento de SH-SY5Y diferenciadas com 0, 1, 10 e 100 nM de insulina por 0, 10 e 60 minutos mostraram uma diminuição nos níveis intracelulares da proteína Tau e um aumento na sua fosforilação na serina 396. Em relação à proteína percursora amiloide (APP), o tratamento de insulina levou a um aumento nos níveis intracelulares, quando exposta por 10 minutos seguido por uma diminuição aos 60 minutos. Quanto à fosforilação da treonina 668 da APP, foi previamente demonstrado que um aumento na fosforilação desse resíduo, promove a clivagem pela via amiloidogénica, levando à produção de Aβ. Nas células tratadas com insulina, um aumento claro da fosforilação desse resíduo da APP foi observado aos 10 minutos. Aos 60 minutos, os níveis da fosforilação eram baixos provavelmente devido aos baixos níveis de APP total.
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Ng, Foong Loo Yvonne Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Insulin action: unravelling AKT signalling in Adipocytes." Awarded by:University of New South Wales. Biotechnology & Biomolecular Sciences, 2009. http://handle.unsw.edu.au/1959.4/44628.
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The Ser/Thr kinase Akt plays an important role in many of insulin's actions including GLUT4 translocation to the plasma membrane (PM). However, there are several features of Akt's regulation of GLUT4 translocation that remain unclear. The goal of my thesis was to resolve some of the following questions: Is activation of Akt sufficient to stimulate GLUT4 translocation? What is the quantitative relationship in signal transmission between individual components within the Akt cascade? What is the role of Akt in insulin resistance? To determine if activation of Akt is sufficient to mediate GLUT4 translocation, I developed a drug-inducible heterodimerisation strategy to activate Akt2 independently of other potential insulin signalling pathways. These studies revealed that activation of Akt2 resulted in rapid stimulation of GLUT4 translocation to a similar extent with maximum insulin, indicating that Akt2 is sufficient for this event. It was previously observed that maximum effect of insulin on GLUT4 translocation was obtained with minimum activation of Akt. To resolve this discrepancy, the relationship between Akt signalling components was examined using a quantitative kinetic and dose response approach combined with hierarchical cluster analysis. Most notably I observed a strong relationship between Akt at the PM, but not Akt in the whole cell lysate, with its substrate phosphorylation. Active pools of phospho-Akt and -AS160, a major substrate involved in GLUT4 translocation, were found in the lipid raft, highlighting the importance of subcellular partitioning of key signalling components for achieving biological specificity. The involvement of Akt in insulin resistance was investigated using the heterodimerisation strategy. These studies revealed that insulin itself initiates a pathway that causes insulin resistance by converging on target(s) downstream of Akt. This inhibitory pathway emanates from PI3-kinase and is likely induced by a range of insults including chronic insulin and dexamethasone. In conclusion, Akt is a crucial element in the insulin action pathway that exhibits precise spatial regulation. While the role of this nanoregulation of Akt in disease remains to be evaluated, my studies suggest that the major defect contributing to insulin resistance occurs downstream of Akt. The elucidation of this target will have major implications for metabolic diseases.
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Bray, Jonathan Alexander. "Comparing insulin and insulin-like growth factor-1 signalling in myoblasts." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596876.
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In this study a chimeric receptor system was employed in which the extracellular domain of the TrkC receptor was fused to the intracellular portion of either the insulin (TIR) or IGF-1 (TIGFR) receptor. These chimeric receptors were expressed in separate populations of the skeletal muscle cell line L6. Initial analysis of individual downstream signalling components and assessment of cell proliferation, induced by TIR or TIGFR stimulation revealed little difference between the two chimeras. To more comprehensively screen for potential differences, microarray analysis was used to compare regulation of gene expression by the two chimeric receptors. This led to the identification of several differentially regulated genes. Whilst it was initially hypothesised that skeletal muscle cells might yield several selectively insulin-sensitive genes, the majority of genes selectively regulated by one receptor were preferentially IGF-1 responsive, consistent with previous studies in other cell types. This perhaps reflects the more mitogenic effect of this ligand in vivo, manifest as an increased ability to regulate transcription per se. Of the differentially regulated genes, that encoding Fit-1m was found to be preferentially induced through activation of the TIGFR rather than the TIR. Further characterisation using real-time PCR established that induction of Fit-1 expression required an intact MAPK signalling pathway. Similar effects were observed when the regulation of Fit-1 expression by insulin and IGF-1 was examined. Subsequent work attempted to establish regions of promoter responsible for the preferential induction of Fit-1m expression by IGF-1. Despite defining promoter and putative enhancer regions which are important for Fit-1m transcription, no region was found which confers a response to stimulation with various ligands, including IGF-1. Rather, a high level of constitutive expression was driven by these DNA sequences, suggesting that an IGF-1 response inhibitory factor may control expression of this gene, binding outside the regions examined. Fit-1 joins an increasing list of genes preferentially regulated by the IGF-1R over the IR and provides and end point with which to analyse potential inherent differences in the signalling capacity of these two highly homologous receptors.
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Mercer, Ben N. "Does altered insulin signalling modulate vascular regeneration?" Thesis, University of Leeds, 2014. http://etheses.whiterose.ac.uk/7069/.
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The prevalence of insulin resistant syndromes is rising worldwide. Affected individuals are at increased risk of morbidity and premature mortality, much of which is driven by cardiovascular disease (CVD). Enhancement of vascular regeneration, using pharmacological or cell-based therapies, has been suggested as a strategy to help address these issues. Although many pathophysiological processes associated with insulin resistant syndromes are likely to impair vascular regeneration, the effect of insulin resistance per se is not established. South Asian (SA) ethnicity is associated with increased risk of CVD, and insulin resistance is thought to be a major contributor to this. We compared the angiogenic capacity of late outgrowth endothelial progenitor cells (LEPCs) from young SA men, with those from a matched group white European (WE) men. LEPC have previously been shown to offer potential as an autologous cell therapy in preclinical models of ischaemic CVD. Both groups were well matched, and free of classical cardiovascular risk factors, but the SA group were relatively insulin resistant. SA LEPCs did not augment vascular regeneration in a murine model of limb ischaemia, in contrast with WE LEPC. Akt activity, a critical modulator of angiogenesis, was reduced in SA LEPC, and we were able to rescue SA LEPC dysfunction by enhancing Akt activity. We then established the impact of insulin resistance per se on vascular regeneration, using insulin receptor haploinsufficient mice (IRKO). Indices of angiogenesis were reduced in isolated endothelial cells, aortic ring 5 segments, and ischaemic hind limb muscle. Moreover, this was associated with functional resistance to vascular endothelial growth factor (VEGF), which may have mechanistically contributed to our observations. Together, these data provide insight into how insulin resistance may promote the development of premature CVD, and show that by manipulating key growth factor signalling nodes, we can rescue impaired vascular regeneration. Furthermore, we have established that insulin resistance negatively impacts on the functional response to VEGF, and it will be important to explore the mechanisms underlying this phenomenon in future studies. It is hoped that these findings will help lead to the development of strategies to mitigate the effects of CVD in individuals with insulin resistance.
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Musial, Barbara. "Regulation of insulin signalling during mouse pregnancy." Thesis, University of Cambridge, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708844.
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Wuttke, Anne. "Lipid Signalling Dynamics in Insulin-secreting β-cells." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk cellbiologi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-198046.
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Certain membrane lipids are involved in intracellular signalling processes, among them phosphoinositides and diacylglycerol (DAG). They mediate a variety of functions, including the effects of nutrients and neurohormonal stimuli on insulin secretion from pancreatic β-cells. To ensure specificity of the signal, their concentrations are maintained under tight spatial and temporal control. Here, live-cell imaging techniques were employed to investigate spatio-temporal aspects of lipid signalling in the plasma membrane of insulin-secreting β-cells. The concentration of phosphatidylinositol 4-phosphate [PtdIns(4)P] increased after stimulation with glucose or Gq protein-coupled receptor agonists. The glucose effect was Ca2+-dependent, whereas the receptor response was mediated by isoforms of novel protein kinase C (PKC). The increases in PtdIns(4)P were paralleled by lowerings of the phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] concentration. This relationship was not caused by conversion of PtdIns(4,5)P2 to PtdIns(4)P but rather reflected independent regulation of the two lipids. Stimulation of β-cells with glucose or a high K+ concentration induced pronounced, repetitive increases in plasma-membrane DAG concentration, which were locally restricted and lasted only for a few seconds. This pattern was caused by exocytotic release of ATP, which feedback-activates purinergic P2Y1-receptors and stimulates local phospholipase C-mediated DAG generation. Despite their short durations the DAG spikes triggered local activation of PKC. Novel PKCs were recruited to the plasma membrane both after glucose and muscarinic receptor stimulation. While the glucose-induced translocation was synchronized with DAG spiking, muscarinic stimulation induced sustained elevation of the DAG concentration and stable membrane association of the kinase. Also conventional PKCs translocated to the membrane after glucose and receptor stimulation. The glucose-induced response was complex with sustained membrane association mirroring the cytoplasmic Ca2+ concentration, and superimposed brief recurring translocations caused by DAG. Interruption of the purinergic feedback loop underlying DAG spiking suppressed insulin secretion. Since the DAG spikes reflected exocytosis events, a single-cell secretion assay was established, which allowed continuous recording of secretion dynamics from many cells in parallel over extended periods of time. With this approach it was possible to demonstrate that insulin exerts negative feedback on its own release via a phosphatidylinositol 3,4,5-trisphosphate-dependent mechanism.
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Wiggins, Emma Louise. "Regulation of myogenesis by IGF- and insulin-signalling." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608747.
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Holt, L. J. "The role of Grb10 in insulin receptor signalling." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604199.
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To facilitate the study of Grb10 function, polyclonal antibodies were produced. They were directed against the SH2 domain of Grb10, which is common to all isoforms. The purified antibody was highly specific for Grb10 and able to detect both mouse and human forms of the protein. To examine the effects of Grb10 proteins on the insulin signalling pathway, they were expressed exogenously in CHO-T cells, in both a transient and stable manner. Comparison were made of the mouse and human isoforms of Grb10 at the genomic, RNA and protein levels. Differential expression of isoforms was observed in murine cells. Use of the information generated by the Human Genome Project allowed the genomic organisation of the human Grb10 isoforms to be determined. Bioinformatics tools were also employed to predict binding motifs and phosphorylation sites within Grb10. These elucidated similarities between the isoforms, and differences, in their potential regulation. In examining its effects on the insulin signalling system in CHO-T cells, hGrb10x was shown to associate with the activated insulin receptor in a rapid and sustained manner. Various isoforms of Grb10 bound equally well to the receptor. The global tyrosine phosphorylation state of the insulin receptor was not altered in the presence of hGrb10x. However, insulin-stimulated activation of IRS-1 and ERK were inhibited. The tyrosine phosphorylation state of a broad array of proteins was also affected by the expression of hGrb10x in CHO-T cells. hGrb10x induced an apparent increase in total IRS-1 protein level. The studies presented in this thesis are consistent with hGrb10x acting as a negative regulator of the insulin signalling pathway in CHO-T cells.
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McCulloch, Laura Jade. "The molecular genetics of insulin secretion and signalling." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:eb170e91-7b3c-453f-af58-7058909de435.
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Type 2 diabetes (T2D) and fasting plasma glucose (fpg) levels have distinct genetic components which are as yet only modestly understood. Understanding the genetics of this complex disorder and its related traits is likely to be of significant benefit to the field. Not only will it shed light on critical genes, pathways and mechanisms of regulation, but it may also contribute to the development of pharmaceutical anti-hyperglycaemic agents via the identification of key therapeutic targets. Therefore the aim of this thesis was to utilise a broad, multidisciplinary approach to study the genetics of insulin secretion and signalling. Traditionally genes which harbour rare variants causing monogenic beta-cell dysfunction have also been found to harbour common variants associated with T2D and fpg. As genome-wide association studies (GWAS) identify an increasing number of common variants and genes, they also increase the number of genes which act as monogenic candidates. I screened G6PC2, a novel fpg associated gene, in patients with monogenic forms of beta-cell dysfunction and demonstrated that rare variants in this gene are unlikely to be a common cause of monogenic beta-cell dysfunction. Although GWAS have been of considerable benefit to our understanding of complex disease genetics, they are not without their own limitations, primarily concerning signal refinement. To try to overcome this barrier for T2D and fpg signals I established a pipeline for fluorescence activated cell sorting of human islets to obtain pure beta-cells. In these cells, I performed transcript profiling of genes falling within T2D and fpg associated loci, demonstrating how this approach, alongside physiological analysis, can be of benefit for GWAS researchers and provide a starting point for fine mapping. Access to human beta-cells also enabled me to follow up one novel fpg association signal, SLC2A2. Through analysis within this metabolically relevant tissue I was able to establish that the mechanism for increased fpg levels is unlikely to be mediated via a beta-cell pathway. Although GWAS have highlighted a number of key genes associated with beta-cell dysfunction; they have been far less successful at identifying genes associated with insulin resistance, another key component of T2D pathogenesis. Additional approaches, including the study of rodent models, may be required to study this aspect of T2D. PTEN is known to negatively regulate the insulin signalling pathway and adipose tissue specific Pten-/- animals were shown to be markedly insulin sensitive. To assess the role of PTEN in human insulin sensitivity I performed mRNA expression profiling of PTEN in human adipose tissue biopsies from subjects with T2D and matched controls, demonstrating that PTEN is significantly reduced in the subcutaneous adipose tissue of the former. This response is likely to be a compensatory mechanism to counteract muscular insulin resistance although further investigation needs to be performed to determine the mechanism of compensatory downregulation. These data provide insights into a number of aspects of T2D genetics, and demonstrate how a multidisciplinary approach is of benefit to T2D genetic research.
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Wong, Martin Kin Lok. "Unravelling the insulin signalling pathway using mechanistic modelling." Thesis, The University of Sydney, 2016. http://hdl.handle.net/2123/16879.
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Type two diabetes affects 5% of the world's population and is increasing in prevalence. A key precursor to this disease is insulin resistance, which is characterised by a loss of responsiveness to insulin in liver, muscle and adipose tissue. This thesis focuses on understanding insulin signalling using the 3T3-L1 adipocyte cell model. Computational modelling was used to generate quantitative predictions in the signalling pathways of the adipocyte, many of which are mediated by enzymatic reactions. This study began by comparing existing enzyme kinetic models and evaluating their applicability to insulin signalling in particular. From this understanding, we developed an improved enzyme kinetic model, the differential quasi-steady state model (dQSSA), that avoids the reactant stationary assumption used in the Michaelis Menten model. The dQSSA was found to more accurately model the behaviours of enzymes in large in silico systems, and in various coenzyme inhibited and non-inhibited reactions in vitro. To apply the dQSSA, the SigMat software package was developed in the MATLAB environment to construct mathematical models from qualitative descriptions of networks. After the robustness of the package was verified, it was used to construct a basic model of the insulin signalling pathway. This model was trained against experimental temporal data at 1 nM and 100 nM doses of insulin. It revealed that the simple description of Akt activation, which displays an overshoot behaviour, was insufficient to describe the kinetics of substrate phosphorylation, which does not display the overshoot behaviour. The model was expanded to include Akt translocation and the individual phosphorylation at the 308 and 473 residues. This model resolved the discrepancy and predicts that Akt substrates are only accessible to Akt localised in the cytosol and that PIP3 sequestration of cytosolic Akt acts as a negative feedback.
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Pryor, Paul Robert. "Insulin-regulated signalling proteins involved in GLUT4 trafficking." Thesis, University of Bath, 1999. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.311250.
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Ribé, David Raymond. "Insulin signalling and membrane fusion in adipose cells." Thesis, University of Bath, 2005. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.415763.
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Insulin regulates glucose uptake in responsive cells by trafficking GLUcose Transporter 4 (GLUT4) molecules to the plasma membranes of these cells. This thesis focuses on events at the plasma membrane, including membrane fusion and activation of GLUT4. Wortmannin inhibition experiments in rat adipocytes did not show the trafficking-independent inhibition of insulin-stimulated glucose transport activity at low wortmannin concentrations seen in other cell types. The p38 MAP kinase inhibitor SB203580 inhibited labelling of cell surface GLUT4 as well as glucose transport activity. Purified recombinant protein standards were used to quantify SyNaptosomeAssociated Protein (SNAP) REceptor (SNARE) proteins in rat adipocytes. There were approximately 2,500,000 copies of syntaxin4, 8,900,000 copies of SNAP23, 1,900,000 copies of VAMP2 and 1,000,000 copies of Muncl8c in each adipose cell. Syntaxin4, SNAP23 and Muncl8 were predominantly localised to the plasma membrane. SNAP23 was in large excess compared to the other proteins in plasma membranes, suggesting that its availability was tightly regulated. VAMP2 was distributed between the plasma membrane and intracellular membranes. Insulin caused a two-fold increase in VAMP2 at the plasma membrane. The small amounts of syntaxin4 in intracellular membranes were in large excess over Muncl8c in the same membranes. These results suggest that Muncl8c is unlikely to play a major role in trafficking syntaxin4 to the plasma membrane as has been suggested. A number of approaches were applied to expressing and purifying recombinant SNAREs. The purified proteins were reconstituted into liposomes. This system is intended to be the future basis for experiments on SNARE function and their regulation by insulin.
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Danielsson, Anna. "Insulin signalling in human adipocytes : mechanisms of insulin resistance in type 2 diabetes." Doctoral thesis, Linköping : Univ, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-10327.
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Nield, Alex. "The role of Zinc Transporters in modulating Insulin signalling." Thesis, Federation University Australia, 2015. http://researchonline.federation.edu.au/vital/access/HandleResolver/1959.17/99999.
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Zinc is a cell impermeable transition metal with a large number of biological functions, and is an essential component of the insulin signalling pathway. Cellular free zinc increases insulin sensitivity though it is also toxic at high levels, making it essential for cells to tightly regulate bioavailable levels. This homeostasis is maintained by three groups of proteins known as Zips, ZnTs and metallothioneins (MTs). Zips and ZnTs are zinc transporters with Zips increasing cytosolic zinc by pumping it outside the cell or from organelle stores, while the ZnTs decrease cytosolic zinc. The MTs bind to free zinc in the cytosol, reducing its bioavailability. The rapid release of zinc mediated by these proteins has been implicated as a mechanism of signal pathway activation, through zinc activating and deactivating various signalling proteins. This thesis investigated one zinc transporter in particular, known as Zip7. Zip7 is a novel Zip transporter localised to the endoplasmic reticulum and has been implicated in cell signalling in breast cancer cells through release of zinc from cellular stores in response to extracellular stimuli. The aim of this thesis was to investigate the potential role of this zinc transporter in modulating the insulin signalling pathway. The human Zip7 protein sequence was analysed using various bioinformatics tools to identify regions that may contribute to the proposed novel function of this transporter. The loop regions of Zip7 were found to be poorly conserved between species with the exception of histidine rich regions, which showed a high level of conservation when compared to a diverse series of species and so are suspected to have an essential role in modulating the transport function of Zip7 by binding to zinc. These findings implicate histidine residues as an important functional component of Zip7. In order to identify whether Zip7 expression is essential for a normal insulin response, Zip7 mRNA was reduced via transfection of siRNA in mouse skeletal muscle cells and measurement of markers of insulin signalling. When Zip7 expression was reduced there was a subsequent decrease in the expression of several markers of insulin signalling including Glut4 protein levels, Akt phosphorylation and insulin-mediated glycogen synthesis, indicating that the cells were insulin resistant compared to the control. It was hypothesised that given the proposed role for Zip7 in mediating rapid zinc release and that Zip7 expression is important for normal insulin signalling, Zip7 activation is stimulated by insulin treatment to temporarily increase cytosolic zinc bioavailability as a positive feedback mechanism for prolonging pathway activation. To test this, live cell imaging of zinc flux in cells was performed in cells with reduced Zip7 expression compared to controls. Insulin was shown to cause an increase in cytosolic zinc in C2C12 cells. However when Zip7 expression was reduced, even though the cells showed signs of insulin resistance, there was still an increase in zinc levels mediated by insulin. Insulin treatment is known to induce cellular ROS production and hydrogen peroxide has been suggested to cause a release of zinc due to oxidation of MTs leading to a release of bound zinc. These findings indicate that insulin-stimulated zinc flux is the result of MT oxidation rather than Zip7 activation. Taken together, these results highlight an important role for Zip7 in the insulin signalling pathway and show a previously undescribed positive feedback loop whereby insulin mediates a release of zinc to potentially inhibit PTP1B and other phosphatases to prolong insulin signalling activation. Further work is needed to fully elucidate the role of Zip7 in this pathway.Doctor of Philosophy
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Wadley, Glenn, and mikewood@deakin edu au. "Regulation of insulin signalling by exercise in skeletal muscle." Deakin University. School of Health Sciences, 2003. http://tux.lib.deakin.edu.au./adt-VDU/public/adt-VDU20050826.111050.
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Regular physical activity improves insulin action and is an effective therapy for the treatment and prevention of type 2 diabetes. However, little is known of the mechanisms by which exercise improves insulin action in muscle. These studies investigate the actions of a single bout of exercise and short-term endurance training on insulin signalling. Twenty-four hours following the completion of a single bout of endurance exercise insulin action improved, although greater enhancement of insulin action was demonstrated following the completion of endurance training, implying that cumulative bouts of exercise substantially increase insulin action above that seen from the residual effects of an acute bout of prior exercise. No alteration in the abundance and phosphorylation of proximal members of the insulin-signalling cascade in skeletal muscle, including the insulin receptor and IRS-1 were found. A major finding however, was the significant increase in the serine phosphorylation of a known downstream signalling protein, Akt (1.5 fold, p ≤0.05) following an acute bout of exercise and exercise training. This was matched by the observed increase in protein abundance of SHPTP2 (1.6 fold, p ≤0.05) a protein tyrosine phosphatase, in the cytosolic fraction of skeletal muscle following endurance exercise. These data suggest a small positive role for SHPTP2 on insulin stimulated glucose transport consistent with transgenic mice models. Further studies were aimed at examining the gene expression following a single bout of either resistance or endurance exercise. There were significant transient increases in IRS-2 mRNA concentration in the few hours following a single bout of both endurance and resistance exercise. IRS-2 protein abundance was also observed to significantly increase 24-hours following a single bout of endurance exercise indicating transcriptional regulation of IRS-2 following muscular contraction. One final component of this PhD project was to examine a second novel insulin-signalling pathway via c-Cbl tyrosine phosphorylation that has recently been shown to be essential for insulin stimulated glucose uptake in adipocytes. No evidence was found for the tyrosine phosphorylation of c-Cbl in the skeletal muscle of Zucker rats despite demonstrating significant phosphorylation of the insulin receptor and Akt by insulin treatment and successfully immunoprecipitating c-Cbl protein. Surprisingly, there was a small but significant increase in c-Cbl protein expression following insulin-stimulation, however c-Cbl tyrosine phosphorylation does not appear to be associated with insulin or exercise-mediated glucose transport in skeletal muscle.
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Karlsson, Margareta. "Caveolae in insulin signalling in human and rat adipocytes /." Linköping : Univ, 2003. http://www.bibl.liu.se/liupubl/disp/disp2003/med782s.pdf.
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Hopkinson, Helen Elizabeth. "Betaâ†2-adrenoceptor signalling and the effect of insulin." Thesis, University of Nottingham, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301070.
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Hudson, Claire Anna. "Studying isoproterenol and insulin signalling in primary rat adipocytes." Thesis, University of Bristol, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.411025.
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Tyler-Rubinstein, Nadia. "The role of insulin receptor substrate signalling in metabolism." Thesis, Imperial College London, 2015. http://hdl.handle.net/10044/1/54894.
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Background/Aims: In order to mediate a diverse range of growth and metabolic functions, insulin receptor substrate (IRS) proteins recruit a complex network of intracellular signalling molecules including PI3-kinase and the Grb2 adaptor protein. These studies proposed to answer the question to what extent are the different phenotypes observed in Irs1 and Irs2 knockout mice due to the loss of the whole protein or due to the loss of specific signalling interactions between IRS proteins and either p85 (the regulatory subunit of PI3K) or Grb2? Determining which interactions mediate which biological responses is of interest to our understanding of insulin resistance and its associated pathologies. Methods: The strategy utilised for these studies was to mutagenise the IRS proteins in vivo with specific signalling defects with the aim of disrupting IRS1 or IRS2 signalling via two major insulin mediated pathways, the PI3K-Akt axis and the Grb2-Ras-Raf-MAPK cascade. The phenotypes of the mice were assessed in five main areas: growth, glucose homeostasis, energy homeostasis, cognitive behaviour and fertility. Results: The IRS1-PI3K mutants presented with a severe growth-defect and acute insulin resistance, though through compensatory β-cell expansion maintained normal glycaemia. They were also lean with low circulating leptin levels and displayed a severe defect in learning and memory. IRS2-PI3K mutants were glucose intolerant and insulin resistant with a reduction in β-cell area that resulted in hyperglycaemia and onset of diabetes around 3-months of age. Additionally, the mice had increased fat mass and high circulating leptin levels. In contrast, the Grb2 mutants displayed normal metabolic phenotypes. Conclusion: The data presented here revealed a primary role for IRS signalling via PI3K in regulating metabolic functions. Both the IRS-PI3K mutants largely phenocopy the corresponding Irs knockout mice. In contrast, the Grb2 mutants appeared metabolically normal, suggesting a relatively minor role for IRS-Grb2 interactions in metabolic and growth control.
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Turner, Mark C. "Cell culture models of insulin signalling and glucose uptake." Thesis, Loughborough University, 2015. https://dspace.lboro.ac.uk/2134/19582.
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Insulin maintains glucose homeostasis through its binding of the insulin receptor and activation of the insulin signalling cascade in insulin sensitive tissues. Skeletal muscle is a major endocrine organ, and is responsible for the majority of post-prandial glucose disposal. The maintenance of glucose homeostasis is a delicate balance and impairments in glucose disposal can have significant physiological effects, resulting in the onset of metabolic diseases such as diabetes mellitus. Insulin stimulated glucose uptake involves a number of signalling proteins to enable uptake to occur. In order to understand the complexities associated with the insulin signalling cascade, cell culture models have provided a controlled and easily manipulated environment in which to investigate insulin stimulated glucose uptake in skeletal muscle. While the majority of these experiments have been conducted in conventional monolayer cultures, the growing field of three-dimensional tissue engineering provides an alternative environment in which skeletal muscle cells can be grown to investigate their physiological function. The purpose of this thesis was to investigate the use of different cell culture models for investigating the effects of acute and chronic insulin exposure on skeletal muscle. Initial investigations aimed to establish glucose uptake in tissue engineering skeletal muscle constructs using tritium labelled (H3) 2-deoxy-d-glucose. Monolayer cultures were used to developed base line conditions. In these cultures, concentrations greater than 0.5 μCi for 15 minutes of insulin stimulation suggested an initial assay window for investigating insulin stimulated glucose uptake. However, the duration of insulin stimulation was not effective in measuring uptake in tissue engineered skeletal muscle constructs based upon western blot experiments of Akt phosphorylation, therefore insulin stimulation in skeletal muscle tissue engineered constructs was increased to 30 minutes. Glucose uptake is mediated via specific glucose transporter protein, GLUT1 and GLUT4. Therefore, the transcriptional profile of these transporters was elucidated in monolayer culture and tissue engineered skeletal muscle constructs. Time course experiments showed an increase in GLUT4 transcription in tissue engineered and monolayer culture systems which is associated with an increase in the transcription of skeletal muscle development and myogenic genes. In two dimensional culture, skeletal muscle cells were exposed to insulin during differentiation and in post-mitotic skeletal muscle myotubes to investigating the potential effects upon metabolic genes and proteins involved in insulin signalling. Chronic exposure to insulin during skeletal muscle differentiation reduced insulin signalling and resulted in an increase in basal glucose uptake and ablated insulin stimulated glucose uptake. In contrast, post-mitotic skeletal muscle myotubes did not shown similar changes and were not as responsive to acute insulin exposure. Therefore future experiments exposed skeletal muscle to insulin during differentiation. Using the previous findings as a basis for experimentation, the effects of chronic and acute insulin exposure upon three dimensional skeletal muscle constructs were investigated. Fibrin and collagen constructs were grown for a total period of 14 days. Constructs were exposed to insulin during differentiation and acutely stimulated for 30 minutes at day 14. Although there was a mean increase in Akt protein phosphorylation in both types of tissue-engineered constructs, these changes were not significant following acute insulin stimulation. In addition, glucose uptake in fibrin skeletal muscle constructs increased as a result of acute insulin stimulation however was not significantly difference to unstimulated constructs. The work presented in this thesis provides initial experimental data of the use of different skeletal muscle cell culture models for investigating insulin signalling and glucose uptake. Further research should further characterise these in vitro models for investigating skeletal muscle metabolism.
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Ahmed, Zamal. "The role of SH2-Bα and APS in insulin signalling." Thesis, University of Nottingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.269693.
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Goodfellow, Mark. "Type 1 insulin-like growth factor signalling in malignant melanoma." Thesis, University of Oxford, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.599998.
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Novel therapeutic strategies are sorely required for malignant melanoma, which has been shown to overexpress the type 1 insulin-like growth factor receptor (IGFIR) compared benign naevi. The 10Ft R mediates many features of the malignant phenotype and is an attractive anti-cancer therapeutic target. Inoculation with lGFlR depleted tumour cells has been shown to upregulate MHC Class 1 expression and confer protection from tumour rechallenge in vivo. The initial aim of this project was to investigate effects of IGFlR depletion on MHC Class I expression and its functional significance in melanoma cells IGF 1 R gene silencing, but not IGFIR inhibition, caused a significant increase in surface MHC Class 1 expression in one murine cell line, B16.FIO, but 0/8 human melanoma cell lines. In B16.FI0 cells, increased MHC Class 1 did not affect cell lysis or cytokine production by, or proliferation of, activated T lymphocytes in vitro. These results indicate that the increase in MHC Class I in melanoma cells is not of functional significance. The main focus of this project then addressed a key issue relevant to clinical trials of IGF I R inhibition. Identifying features which render melanoma cells sensitive to IGF1R inhibitors is important to select patients suitable to receive this therapy. The approach was to define changes in gene expression after IGFl or IGflR inhibitor treatment of melanoma cells. Initial experiments were performed to select concentrations of IGF1 and IGFlR inhibitor AZ1225380 I, the latter based on GI50 concentrations. An IGF gene signature was created which was significantly correlated to Ki67 expression, a proliferation marker, in clinical datasets. This suggests a rationale for IGFlR targeting in clinical melanoma. Gene expression profiling identified that IGF and IGflR inhibition had reciprocal effects on expression of HBP1, a transcriptional repressor of prognostic significance in breast cancer. HBPl was validated as an IGFI regulated gene, and experiments using signalling inhibitors indicated that IGFlR regulates HBP I via the P13K pathway. HBPI depletion did not influence effects of IGF IR inhibition on proliferation. However HBP1 induction appeared to mediate effects of AZ12253801 on inhibiting the WNT pathway, measure1 by TOPIFOP reporters, and HBP l depletion enhanced basal and WNT-induced NMYC expression. NMYC has not previously been reported to be a Wnt/β catenin target gene in neoplastic cells. These results encourage further investigation into the effects of IOF 1.K! In regulating HBPl, particularly with respect to dual lGFlR Wnt/β catenin targeting
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Armstrong, Jane Louise. "Insulin signalling to glycogen synthesis in cultured human muscle cells." Thesis, University of Newcastle Upon Tyne, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.340684.
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Scott, Mary Thomson. "Enhancement of insulin signalling in adipose tissue by malarial extracts." Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.286183.
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Maier, Michelle. "The role of Zn2+ in insulin signalling and muscle atrophy." Thesis, Federation University Australia, 2019. http://researchonline.federation.edu.au/vital/access/HandleResolver/1959.17/171022.
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Zn2+ is a broadly utilised ion in biology that has important catalytic, structural and regulatory roles within the cell. Zn2+ distribution in cells is maintained by zinc transporters, Zips and ZnTs, and disruptions in levels of Zn2+ have been associated with insulin resistance and muscle atrophy disorders. Zn2+ and reactive oxygen species (ROS) interact through inhibition of protein tyrosine phosphatases and ROS-mediated oxidation of the metal-binding metallothioneins (Mts) causing release of bound Zn2+, however the precise mechanisms are unclear. In the first study of this thesis addition of inhibitors of ROS-generating enzymes, superoxide dismutase 1 (SOD1) and NADPH oxidase 1 (NOX1) showed that only SOD1 inhibition increased short-term insulin-mediated Zn2+ release and increased the expression of Mt1 and 2. These results may suggest that ROS, in particular O2- accumulation through inhibition of SOD1, plays a role in insulin-mediated Zn2+ release. Inhibiting SOD1 prevents the conversion of O2- to H2O2 causing an accumulation of O2- in the cell which oxidises Mts to release Zn2+, thereby increasing Zn2+ levels within the cell. Manipulation of the expression of the zinc transporter Zip-7 has previously been shown to modulate cell signalling and glucose metabolism in C2C12 skeletal muscle cells, warranting further investigation into the role of Zn2+ within insulin signalling. Reducing Zip-7 expression when NOX1 was inhibited caused a decrease in Mt2 expression in response to insulin suggesting an interaction between insulin, Zip-7 and NOX1 activity but this requires further investigation. Skeletal muscle atrophy is a clinical symptom of insulin resistance and diabetes. Muscle atrophy is associated with increases in circulating glucocorticoid levels and accumulation of Zn2+ in muscle. This study investigates if Zn2+ homeostasis is disrupted in glucocorticoid-induced atrophy using C2C12 skeletal muscle cells treated with Dexamethasone (DEX) and iv insulin. Results demonstrate DEX-induced atrophy significantly increased the gene expression of the Mt1&2 and decreased glycogen accumulation when treated with insulin. Both confocal microscopy and flow cytometry showed significant increases in free cellular Zn2+ after DEX treatment. Notably, free Zn2+ levels observed with confocal microscopy increased after insulin treatment in control cells but decreased in DEX treated cells. Total cellular Zn2+ was increased by DEX treatment. This demonstrates that DEX causes Zn2+ accumulation in muscle cells and disrupts both Zn2+ homeostasis through blocking insulin-induced Zn2+ release, and insulin-induced glycogen synthesis. This raised the question of whether the same effects of atrophy on Zn2+ homeostasis apply to other cell systems. To investigate this, we examined adipose cells given that these too are involved in insulin resistance and muscle atrophy disorders. In this study we found similar increases in mRNA abundance of Mt1 & 2. Confocal microscopy revealed that DEX treatment caused changes in the distribution of free Zn2+ within peri-nuclear and cytosolic regions of the cell upon stimulation with insulin. Furthermore, investigation into morphometric changes using Oil Red O staining and particle analysis through Coherent Anti-Stokes Ramen Spectrophotometry (CARS) microscopy showed changes in cell and lipid droplet size consistent with reduced lipid turnover in DEX treated cells. These results highlight a potential mechanistic role for Zn2+ in the development of atrophy in 3T3-L1 adipocytes where increased free Zn2+ and its redistribution in cells may inhibit lipid metabolism downstream of insulin signalling. These findings show that insulin-induced Zn2+ release is disrupted by glucocorticoids and this is associated with insulin resistance. Restoring control of Zn2+ homeostasis, possibly through controlling oxidation or manipulating Zn2+ levels directly, may prove beneficial in metabolic disease states such as diabetes.Doctor of Philosophy
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Liu, Simon C. H. "Post-receptor signalling in the insulin regulation of glucose uptake." Thesis, University of Bath, 1997. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.245879.
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Sarkar, Phoebe Lorraine. "Characterizing the role of insulin signalling in advanced prostate cancer." Thesis, Queensland University of Technology, 2017. https://eprints.qut.edu.au/110524/2/Phoebe_Lorraine_Sarkar_Thesis.pdf.
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The majority of prostate cancer patients receiving hormone therapy become insulin resistant and studies show this metabolic dysfunction is associated with more rapid treatment failure, yet the effect of insulin on prostate cancer is not fully known. This thesis discovered the mechanisms by which insulin increases the propensity of prostate cancer cells to migrate which is required for cancer cells to disseminate and metastasise, giving a possible explanation for the more aggressive disease in prostate cancer patients with insulin resistance. The results provide a strong rationale for specifically monitoring and treating insulin resistance in prostate cancer patients, which is not current clinical practice. The results suggest that anti-diabetes drugs may be useful as adjuvant therapy in prostate cancer.
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Carter, Wayne Grant. "Site specificity and purification of an insulin receptor associated serine kinase from human placenta and rat liver." Thesis, University of Southampton, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.295913.
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Laberge, Marie-Kristine. "Nck1 is required for ER stress-induced insulin resistance and regulation of IRS1-dependent insulin signalling." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111950.
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Activation of the Unfolded Protein Response (UPR) following stress in the Endoplasmic Reticulum (ER) is an important mechanism by which obesity results in insulin resistance and type II diabetes. We uncovered a role for the adaptor protein Nck in modulating the UPR. In this study, we report that obese Nck1-/- mice, which show lower levels of UPR in liver and adipose tissue, present improved insulin signalling in these tissues. We established that the effect of Nck1 is cell autonomous by showing that HepG2 cells treated with Nck1 siRNA have reduced ER stress-induced UPR and Insulin Receptor Substrate-1 (IRS-1) serine phosphorylation. In these cells, we observed that the IRS-1 levels and activation of signalling components downstream of the insulin receptor were increased. This correlates with enhanced cell survival to stress and insulin stimulated glycogen synthesis. Overall, we demonstrated that Nck1 participates in ER-stress-induced insulin resistance and regulation of IRS-1-dependent signalling.
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Stenkula, Karin. "A molecular approach to insulin signalling and caveolae in primary adipocytes /." Linköping : Univ, 2006. http://www.bibl.liu.se/liupubl/disp/disp2007/med977s.pdf.
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Stenkula, Karin. "A molecular approach to insulin signalling and caveolae in primary adipocytes." Doctoral thesis, Linköpings universitet, Cellbiologi, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-8960.
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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.
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Sweeney, Gary. "Protein kinase C isoforms : insulin signalling, cyclic amp metabolism and diabetes." Thesis, University of Glasgow, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.306884.
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Parmar, Amanda. "Comparison of insulin and IGF-1 signalling : studies using chimeric receptors." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620683.
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Shaheen, Fozia. "Interactions between telomerase reverse transcriptase (TERT) and insulin signalling in muscle." Thesis, University of Warwick, 2010. http://wrap.warwick.ac.uk/49182/.
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Ageing is a well established risk factor for the development of the metabolic syndrome and type 2 diabetes (T2DM). It is well-recognised that there is an increase in insulin resistance with age, and disturbances in insulin signalling have been shown to affect both insulin sensitivity and lifespan. A decrease in mean telomere length provides a marker for biological age at the cellular level. Accelerated telomere shortening in both human and animal models has been documented in conditions associated with insulin resistance, including T2DM. Maintenance of telomere length is dependent on the activity of the enzyme telomerase (which is dependent on the expression of its catalytic protein TERT) which can help reverse this process and extend the lifespan of cells. When acting in this capacity, TERT is a nuclear protein, but recently it has been shown to possess the ability to shuttle between the nucleus and cytoplasm. The present study was performed with the aim of determining a possible role of TERT in insulin signalling in C2C12, primary mouse and human skeletal muscle cells. Confocal microscopy was used to study protein localisation; in parallel, changes in glucose uptake were determined by measuring 2-deoxyglucose. TERT protein in C2C12 cells was mostly non-nuclear (cytosolic) in localisation, and translocated to the plasma membrane in response to insulin. This translocation was inhibited by wortmannin and rapamycin indicating that this process was dependent on PI3K signalling (as is the insulin induced translocation of GLUT4) and also dependent on mTOR signalling. Altering TERT expression, through over-expression or RNAi-induced knock-down changed the intracellular distribution of GLUT1, GLUT4 and GLUT12, in a similar manner to the effect of insulin. GLUT1 and GLUT4 responded to insulin as expected in all the three cell systems, but there was a difference in the response of the recently identified GLUT12. GLUT12 in the C2C12 cells in the basal state was distributed throughout the cytoplasm and following insulin addition, became localised to peri-nuclear punctuate structures. By contrast, GLUT12 was localised to the plasma membrane (with some cytoplasmic presence) in primary mouse skeletal muscle tissue sections. Interestingly, under conditions of reduced plasma insulin levels in these mouse muscle sections GLUT12 remained abundantly present at the plasma membrane. Contrary to its actions in the C2C12 cells, insulin caused GLUT12 to mobilise from sub-plasma membrane pools and to translocate towards the plasma membrane in the human skeletal muscle cells. Furthermore, knocking-down GLUT12 expression in the C2C12 and primary mouse skeletal muscle cells decreased both basal and insulin dependent 2-deoxyglucose uptake. These observations suggest that this transporter may contribute towards overall glucose transport, but responds differently to an insulin stimulus in the C2C12 cells than in the primary mouse and human skeletal muscle cells. Importantly, GLUT12 distribution and expression were affected by a reduction in mTOR expression, but again there was a variation in the response between the C2C12 cells and both the other cell systems. However, over-expressing TERT increased 2-deoxyglucose uptake synergistically with the effect of insulin in all three cell systems. This increase in glucose transport was shown to be partly through the involvement of GLUT12, as knocking-down GLUT12 expression accounted for part of the increase in 2-deoxyglucose uptake seen in response to TERT over-expression. TERT protein was found to co-immunoprecipitate with all three glucose transporter proteins studied, suggesting that the effects of changes in TERT expression on GLUT intracellular localisation and glucose transport may be due to TERT-GLUT molecular interactions. Importantly, exposure to inhibitors of mTOR, PI3K and TERT did not diminish the ability of TERT to co-immunoprecipitate with GLUT1 and GLUT4 protein(s). However, geldanamycin, a Hsp90 inhibitor diminished the TERT-GLUT12 interaction, implying that the TERT-Hsp90-RAPTOR-mTOR complex formation may be important for this interaction. These findings suggest the existence of a novel extra-nuclear function of TERT and a possible mechanism that appears to regulate glucose transport, acting synergistically with insulin signalling. Although, TERT interacts with all the GLUT proteins studied, it appears to affect GLUT12 differently to GLUT1 and GLUT4 to regulate glucose transport and mTOR signalling appears to be important for GLUT12 function. Therefore, these results raise the prospect of the existence of a link between alterations in the extra-nuclear distribution of TERT and changes in insulin-dependent glucose transport.
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Norris, Dougall. "Mechanistic Insights into Insulin-Stimulated Akt Activation and GLUT4 Response Heterogeneity." Thesis, The University of Sydney, 2017. http://hdl.handle.net/2123/17943.
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Glucose Transporter 4 (GLUT4) undergoes membrane translocation in response to insulin, facilitating uptake of glucose from the blood into muscle and fat, a process that is central to blood-glucose homeostasis. This trafficking step is underpinned by extensive intercellular heterogeneity. Elucidating the upstream mechanisms, such as Akt activation, which give rise to this heterogeneity is likely to inform us about insulin signalling and its dysregulation in metabolic disease. In this thesis, the development of a novel fluorescent reporter of Akt recapitulated the behaviour of endogenous Akt and provided unique insight into Akt signalling dynamics including the identification of several previously undescribed behaviours. Concurrent assessment of signalling outcomes provided insight into the relationship between signalling nodes. It was evident that variability in GLUT4 responsiveness cannot be explained solely by activity at individual upstream signalling nodes. This is counter to the dogma that signalling pathways are linear processes. It would appear that the control of GLUT4 heterogeneity is dependent on multiple factors. The use of systems modelling approaches provided insight into the complex mechanisms contributing to Akt activation, which were experimentally validated highlighting the independence of T308 and S473 phosphorylation. In addition to this, iterative modelling was instrumental in the discovery of a novel Akt-dependent, mTORC1- independent negative feedback mechanism, which is crucial for the regulation of Akt recruitment. Akt phosphorylated at T308 alone proved indispensable for this novel feedback mechanism. Using a combination of complementary techniques, we have demonstrated the importance of fluorophore selection, the lack of linearity of signalling pathways and elucidated the complex mechanisms contributing to Akt activation. The findings that have arisen from this study will have a significant impact on the field of insulin signalling.
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Millar, Iona M. "Investigation of the functional roles of specific protein kinase C isoforms in 3T3-F442A adipocyte development and function." Thesis, University of Glasgow, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266679.
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Fatani, Sameer Hasan M. "The effects of diet-induced obesity on metabolic and vascular functions : role of insulin signalling and insulin resistance." Thesis, University of Liverpool, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.437519.
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Correia, Catarina Mendes. "Identification of transcriptional enhancers regulated by insulin signalling in mouse liver tissue." Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/19147.
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Mestrado em Bioquímica - Bioquímica ClínicaA diabetes mellitus de tipo 2 (DMT2) afeta cerca de 8% da população adulta mundial, representando 90% de todos os casos de diabetes. Esta doença é causada por uma resposta diminuída à ação da insulina, levando a hiperglicemia e hiperinsulinemia compensatória, que resultam numa diminuição severa da qualidade e esperança de vida dos pacientes. Apesar de ter sido associada à dieta e estilo de vida como fatores de risco principais, foram já propostos fatores genéticos da doença, com crescente apoio científico. Para estudar os mecanismos da transcrição envolvidos na resposta à insulina, analisei os fígados de ratinhos sujeitos a um programa de jejum/realimentação, após injeção do antagonista do recetor de insulina S961 (para simular resistência à insulina) ou PBS (usado como controlo). Para investigar e identificar enhancers regulados pela ingestão de alimentos e pela insulina, foi analisado o recrutamento do complexo Mediador (MED). Os dados obtidos sugerem que o complexo Mediador tem maior ocupação na proximidade de genes sobre-regulados pela ingestão de alimentos. No entanto, não foi possível identificar novos enhancers associados ao recrutamento do Mediador, e mais análises serão necessárias para melhorar a qualidade dos dados. Paralelamente, a conformidade do S961 enquanto antagonista do recetor de insulina foi avaliada. Apesar de os efeitos sistémicos e transcricionais da insulina serem bloqueados por este composto, os estados de fosforilação do Akt e da GSK, alvos da via de sinalização da insulina, são indicativos de um comportamento parcialmente agonista que deverá ser elucidado em estudos futuros.
Type 2 diabetes mellitus (T2DM) affects about 8% of the adult population worldwide, accounting for 90% of diabetes cases. This disease is caused by impairment of insulin signalling, leading to hyperglycaemia and compensatory hyperinsulinemia that result in a decrease in life quality and expectancy of patients. Even though it has mostly been associated with dietary and lifestyle risk factors, genetic factors of T2DM have been proposed, with increasing evidence. In order to study the transcriptional mechanisms involved in insulin signalling, I have analysed the livers of mice subjected to a fasting/refeeding program, after injection with insulin receptor antagonist S961 (to mimic insulin resistance) or PBS (control). To probe for feeding and insulin-regulated enhancers, Mediator (MED) complex recruitment was analysed. Data obtained suggest that Mediator complex occupancy is increased in the vicinity of feeding-upregulated genes. However, no novel enhancers were identified in association with recruited Mediator and further analyses are necessary to improve data quality. In parallel, S961 was assessed regarding its suitability as an insulin receptor antagonist. Although systemic and transcriptional effects of insulin signalling were blocked by S961, phosphorylation of Akt and GSK, downstream targets of the signalling pathway, suggested a partial agonist behaviour that should be clarified in future studies.
Type 2 diabetes mellitus (T2DM) rammer omkring 8% af alle voksne verden over og udgør endvidere 90% af alle diabetes tilfælde. Sygdommen er forårsaget af forringelse af insulin signaleringen, hvilket fører til hyperglycæmia og kompensatorisk hyperinsulinemia, resulterende i forværring af livskvaliteten samt formindskelse af den forventede levealder hos patienterne. Selvom T2DM mest er associeret med diæt og livstil, er der i stigende grad beviser for at genetiske faktorer også er involveret. For at undersøge de transkriptionelle mekanismer involveret i insulin signalering har jeg analyseret levere udtaget fra mus, der har været udsat for et faste/fodringsprogram og inden da er blevet injiceret med en insulin receptor antagonist S961 (for at simulere insulin resistens) eller PBS (kontrol). For desuden at identificere fodrings- og insulinregulerede enhancere blev rekruttering af mediator (MED) komplekset analyseret. Det indsamlede data antyder, at forekomsten af mediator komplekset er øget i nærheden af fodrings-opreguleret gener. Der er dog ikke identificeret nogle nye enhancere, der er associeret med rekrutteret mediator, og yderligere analyser vil være nødvendige for at forbedre kvaliteten af det indsamlede data. Desuden blev S961 undersøgt for sin evne som en insulin receptor antagonist. Selvom systematiske og transkriptionelle effekter af insulin signaleringen var blokeret af S961, så antyder fosforyleringen af Akt og GSK, der indgår i insulin signaleringskaskaden, at S961 opfører sig som en partiel agonist. Dette bør blive belyst yderligere i fremtidige studier.
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Rainey, Paul. "Studies into the antiapoptotic signalling of protein kinase B#gamma#." Thesis, University of Southampton, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342643.
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Sögård, Peter. "Mathematical modelling of insulin signalling : effects on glucose metabolism in skeletal muscle /." Stockholm, 2010. http://diss.kib.ki.se/2010/978-91-7409-834-1/.
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Lavin, Deborah Philomena. "Mechanisms and models of insulin receptor substrate-2 signalling in the kidney." Thesis, Queen's University Belfast, 2017. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.727417.
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Diabetic nephropathy (DN) is one of three interrelated microvascular complications of diabetes. DN is characterised by renal fibrosis and the progressive decline in renal function partly due to the incessant loss of operational nephrons. Current treatments for DN such as controlling blood pressure and blood glucose levels, delay, but do not prevent patient progression to end stage renal disease (ESRD). Diabetes is a major risk factor for the development of acute kidney injury (AKI) which can be pre-renal, intrinsic/ intra-renal, or post-renal, and is characterised by a swift reduction in renal function. The molecular mechanisms that underpin these renal diseases remain to be elucidated. Insulin receptor substrate-2 (IRS2) is a cellular scaffolding protein which mediates signalling from the activated insulin receptor to downstream signalling molecules such as those involved in the PI3K/MAPK pathway. IRS2 is pressed in the tubular epithelium and collecting ducts of the kidney, and upregulated in DN patients where it correlates with poor renal function. The overarching aim of this thesis was to elucidate mechanisms and models of IRS2 signalling in the kidney, its regulation by hypoxia and high glucose among other mediators, and the role of IRS2 in AKI. We demonstrate that chemical-induced HIF stabilisation via DMOG and subsequent IRS2 accumulation occurs in a number of cell types in the kidney, namely epithelial cells, and podocytes. However, we also showed that this was not a global effect in all kidney cells. We confirmed these data by exposing cells to 1 % 02 and we also detected differential patterns of HIF-1a, HIF-2a, and IRS2 protein expression in hypoxia. siRNA targeting HIF-1a or HIF-2a showed that both HIF-1a and HIF-2a influence IRS2 accumulation as a result of chemical-induced or physiological HIF stabilisation. In vivo, DMOG did not stabilise HIF or cause IRS2 accumulation. We observed that combination of DMOG or hypoxia with ITS stimulation induced an upwards shift in IRS proteins and increased downstream insulin signalling, evidenced by increased Akt phosphorylation. We determined that DMOG- or hypoxia-induced IRS2 accumulation was not dependent on the proteasome. We observed TGFpi signalling via IRS2, and found that high glucose prevented IRS2 accumulation in hypoxia. We found that the morphology of the Irs2-/- kidney was similar to the wild-type kidney, yet we observed elevated p- catenin staining and increased Akt signalling in the Irs2-/- kidney. Increases in fibrosis associated genes were also detected in Irs2-/- kidneys. Lastly, we determined that in a folic-acid induced model of AKI, Irs2-/- mice presented with somewhat exacerbated renal fibrosis as evidenced by higher inflammatory F4/80 staining. These data provide preliminary evidence for IRS2 as a potential therapeutic target in DN, through modulation of HIF stabilisation. However, further work is required to develop strategies to successfully increase renal IRS2 expression in vivo and examine what effect this will have on insulin signalling in the kidney. The long-term impact of these approaches would be improving or increasing insulin signalling in the diabetic kidney as a means of preventing DN development or progression.
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Bolukbasi, Ekin. "Characterization of Poly : a novel mediator of insulin receptor signalling in Drosophila." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5700.
Full textAbstract:
Poly is a novel, essential protein in Drosophila melanogaster, loss of function of which results in late larval lethality. Importantly, Poly is evolutionarily conserved with a human homologue. poly mutation was isolated in a P-element mutagenesis screen that aimed to generate a larger collection of single P-element induced mutants. Mutant poly larvae are characterized by extreme larval longevity without pupation, formation of melanotic masses, smaller imaginal discs and brains, and abnormal nuclear morphology in neuroblasts. During the course of my project, I attempted to identify cellular processes and pathways that Poly might be involved in. Interestingly, my data suggest that Poly is a novel interactor and regulator of Insulin receptor/target of rapamycin (InR/TOR) signalling in Drosophila. Linking environmental cues to cell growth and metabolism is an essential process that multicellular organisms need to accomplish successfully for normal development. InR/TOR signalling is a highly conserved pathway that mediates the link between the environment and cellular processes such as growth, metabolism and ageing. My analysis in Drosophila suggests that Poly interacts physically with the InR and mutation of Poly leads to an overall down-regulation of InR/TOR signalling in Drosophila as revealed by decreases in the phosphorylation levels of Akt, S6K and 4E-BP - all downstream effectors of this pathway. In addition, loss of poly results in constitutive activation of autophagy in Drosophila fat body and a decrease in stored triglyceride levels. Furthermore, I show that localisation and levels of Poly protein are dependent on insulin action in both Drosophila and human cells. Together, these data suggest that Poly is a novel mediator of InR signalling that promotes an increase in cell growth and metabolism. Taking into consideration the observed poly mutant phenotype, I also investigated the potential involvement of Poly during cell cycle progression and the Drosophila innate immune response. While my analysis suggests that poly loss of function does not have a direct effect on cell cycle progression, alteration of Poly has consequences on various aspects of the Drosophila innate immune response. Therefore, I conclude that the Drosophila innate immune response is a cellular process in which Poly plays a crucial role.
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McClean, Paula. "Inhibition of GIP signalling alleviates obesity, insulin resistance and type 2 diabetes." Thesis, University of Ulster, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.494369.
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Al-Abri, Abdulrahim. "Investigating the effect of PIP4K2a overexpression in insulin signalling in L6 myotubes." Thesis, University of Manchester, 2018. https://www.research.manchester.ac.uk/portal/en/theses/investigating-the-effect-of-pip4k2a-overexpression-in-insulin-signalling-in-l6-myotubes(1dd2d1dd-c765-4830-9b66-cf32a64d7de9).html.
Full textAbstract:
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.
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Roche, Lucy Mary. "Investigation of Rab-GAPs as links between insulin signalling and GLUT4 translocation." Thesis, University of Bath, 2013. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607627.
Full textAbstract:
TBC1D1 and TBC1D4 are Rab-GTPase Activating Proteins (Rab-GAPs) expressed in insulin-responsive tissues. Both proteins are involved in mechanisms which regulate basal levels of glucose transport and have been identified as targets of insulin and AMP-dependant kinase (AMPK) signalling pathways, which regulate GLUT4 translocation to the plasma membrane in muscle. We have characterised the C2C12 muscle cell model retrovirally expressing HA-epitope tagged GLUT4 in order to investigate how distinct signalling pathways regulate GLUT4 trafficking. Insulin-stimulation and treatment with the AMPK-activator (AICAR) increased the levels of GLUT4 at the plasma membrane by two-fold in C2C12 myotubes. Insulin-stimulation and activation of AMPK mobilised GLUT4 in to the actively cycling pool. However, our data revealed that insulin-stimulation or AMPK activation resulted in distinct effects on GLUT4 trafficking parameters at steady-state. Insulin increased GLUT4 exocytosis (kex) of this cycling pool. Activation of AMPK inhibited GLUT4 internalisation (ken). The combined effect of insulin-stimulation and AMPK-activation was synergistic and led to increased GLUT4 cell surface levels above those obtained with either treatment alone. Insulin-stimulation and AMPK activation in combination resulted in a partially additive effect on the size of the actively recycling GLUT4 pool and further enhanced kex of this cycling pool. Kinetic studies were performed to measure the effect of TBC1D1 and TBC1D4 knockdown on GLUT4 trafficking in C2C12 myotubes. siRNA-mediated knockdown of TBC1D4 did not affect the basal levels of cell surface GLUT4. Knockdown of TBC1D1 increased cell surface levels of GLUT4 in basal and in insulin-stimulated C2C12 myotubes. The knockdown increased the release of GLUT4 in to the actively recycling pool. By contrast TBC1D1 knockdown did not change the levels of GLUT4 at the plasma membrane that occur in the presence of the AMPK-activator (AICAR). Our results support a model whereby TBC1D1 inactivation by signalling-dependant phosphorylation is required for GLUT4 translocation, but with insulin and AICAR having separate and distinguishable effects on the released GLUT4.
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Kalloo-Hosein, Heidi Elisa. "Development of TrkC chimaerae to compare insulin and IGF-I receptor signalling." Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624695.
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Wilcox, Andrew. "Cloning of novel insulin-signalling proteins using the yeast two-hybrid system." Thesis, University of Nottingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.272849.
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