Title page, summary and table of contents only. The complete thesis in print form is available from the University of Adelaide Library. / The insulin and insulin-like growth factor receptor (IR and IGF-lR respectively) networks are ancient and fundamental systems that control growth and metabolism in multicellular organisms. This thesis has examined several aspects of this field focusing on mammalian receptor biology and a comparison of the similarities and differences between the insulin and IGF receptor signalling systems. The insulin receptor family of proteins consist of eleven structural domains, of which the extracellular domains contain all the ligand binding and specificity determinants. The insert domain, within the extracellular region is the least understood of all the domains, and it has no similarity to any other protein sequence. It does however contain the cleavage site which separates the receptor into two subunits and also a small stretch of residues shown to directly contact bound ligand and which is absolutely required for ligand binding in short recombinant forms of the receptor. In addition, the human insulin receptor, expressed as one of two isoforms, A and B, results in the exclusion or inclusion of 12 amino acids directly adjacent to the ligand contacting amino acids in the insert domain. The A isoform lacking exon11 is expressed ubiquitously and the B isoform containing exon11 is co-expressed mainly in the traditional insulin responsive tissues of liver, muscle, adipocytes and kidney, where it is the dominant isoform. In this thesis recombinant insert domain was expressed in a bacterial system in an attempt to purify folded protein suitable for NMR structural analysis. The results of the expression studies indicated that the insert domain was unstructured in isolation and was unable to be adequately refolded by all conditions tried, although hydrophobic conditions appeared to partially stabilize the structure. The overall conclusions of this project were that the Insert domain is likely to have limited structure, and probably buried within the receptor, and therefore requires the presence of the rest of the extracellular domains to adopt its correct structure. A comparison of the ligand binding and phsophorylation potential between the two human isoforms of the insulin receptor was made. A competition binding assay using europium labelled insulin was developed, that found that both IGF-l and IGF-2 had an increased affinity for the hIR-A, but insulin had a slightly reduced affinity. These results differ from the established literature in the raw values, however the relative ratios of binding strength are consistent. The most likely reason for this is that the europium labelled insulin has a different mode of binding the receptors due to the location of the europium chelate. Interestingly, using europium labelled IGF-l produced results nearly identical to those of conventional competition assays. Phosphorylation assays indicated that the hIR-B isoform was more responsive than hIR-A. Even though IGF-2 and IGF-l had improved affinity for hIR-A, the level of phosphorylation was not as high. The ability of each growth factor to promote cellular proliferation correlated well with the relative strength of binding and activation of the receptor. The regions of the IR and IGF-1R involved in binding substrates and regulators are predominantly found in the juxtamembrane domain and the C-terminal domain, which contain several potential tyrosine and serine phosphorylation target sequences. In this study the effect of mutations in unique tyrosine residues and other residues in the C-terminal domain of the hIGF-lR was investigated. Results of time-course phosphorylation assays showed that mutation of Tyrosine¹²⁵¹ to phenylalanine caused hyperphosphorylation of the receptor and increased proliferation, which was caused by deregulation of a tyrosine phosphatase. A Tyrosine¹²⁵⁰ to phenylalanine mutation had altered kinetics of phosphorylation, displaying an unchanging rate of phosphorylation over time after ligand stimulation. However, proliferation was unaltered, indicating that even under extended exposure to ligand, the initial strength of receptor activation is more critical to affecting the biological response. The Caenorhabditis elegans insulin-like peptide family is a very large family consisting of possibly 38 peptides likely to be both agonists and antagonists of Daf-2 Receptor (IR homologue) signalling. Comparative modelling of all 38 peptides was performed based on the known structures of mammalian peptides. The overall results indicated that good quality models of ins peptides could be made despite the low sequence similarity with the templates. This suggested that it is the conformational shape of the molecule allowable by the individual residues that is most important when modelling and not having a perfect sequence match. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1200443 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2005

Title page, summary and table of contents only. The complete thesis in print form is available from the University of Adelaide Library. / The insulin and insulin-like growth factor receptor (IR and IGF-lR respectively) networks are ancient and fundamental systems that control growth and metabolism in multicellular organisms. This thesis has examined several aspects of this field focusing on mammalian receptor biology and a comparison of the similarities and differences between the insulin and IGF receptor signalling systems. The insulin receptor family of proteins consist of eleven structural domains, of which the extracellular domains contain all the ligand binding and specificity determinants. The insert domain, within the extracellular region is the least understood of all the domains, and it has no similarity to any other protein sequence. It does however contain the cleavage site which separates the receptor into two subunits and also a small stretch of residues shown to directly contact bound ligand and which is absolutely required for ligand binding in short recombinant forms of the receptor. In addition, the human insulin receptor, expressed as one of two isoforms, A and B, results in the exclusion or inclusion of 12 amino acids directly adjacent to the ligand contacting amino acids in the insert domain. The A isoform lacking exon11 is expressed ubiquitously and the B isoform containing exon11 is co-expressed mainly in the traditional insulin responsive tissues of liver, muscle, adipocytes and kidney, where it is the dominant isoform. In this thesis recombinant insert domain was expressed in a bacterial system in an attempt to purify folded protein suitable for NMR structural analysis. The results of the expression studies indicated that the insert domain was unstructured in isolation and was unable to be adequately refolded by all conditions tried, although hydrophobic conditions appeared to partially stabilize the structure. The overall conclusions of this project were that the Insert domain is likely to have limited structure, and probably buried within the receptor, and therefore requires the presence of the rest of the extracellular domains to adopt its correct structure. A comparison of the ligand binding and phsophorylation potential between the two human isoforms of the insulin receptor was made. A competition binding assay using europium labelled insulin was developed, that found that both IGF-l and IGF-2 had an increased affinity for the hIR-A, but insulin had a slightly reduced affinity. These results differ from the established literature in the raw values, however the relative ratios of binding strength are consistent. The most likely reason for this is that the europium labelled insulin has a different mode of binding the receptors due to the location of the europium chelate. Interestingly, using europium labelled IGF-l produced results nearly identical to those of conventional competition assays. Phosphorylation assays indicated that the hIR-B isoform was more responsive than hIR-A. Even though IGF-2 and IGF-l had improved affinity for hIR-A, the level of phosphorylation was not as high. The ability of each growth factor to promote cellular proliferation correlated well with the relative strength of binding and activation of the receptor. The regions of the IR and IGF-1R involved in binding substrates and regulators are predominantly found in the juxtamembrane domain and the C-terminal domain, which contain several potential tyrosine and serine phosphorylation target sequences. In this study the effect of mutations in unique tyrosine residues and other residues in the C-terminal domain of the hIGF-lR was investigated. Results of time-course phosphorylation assays showed that mutation of Tyrosine¹²⁵¹ to phenylalanine caused hyperphosphorylation of the receptor and increased proliferation, which was caused by deregulation of a tyrosine phosphatase. A Tyrosine¹²⁵⁰ to phenylalanine mutation had altered kinetics of phosphorylation, displaying an unchanging rate of phosphorylation over time after ligand stimulation. However, proliferation was unaltered, indicating that even under extended exposure to ligand, the initial strength of receptor activation is more critical to affecting the biological response. The Caenorhabditis elegans insulin-like peptide family is a very large family consisting of possibly 38 peptides likely to be both agonists and antagonists of Daf-2 Receptor (IR homologue) signalling. Comparative modelling of all 38 peptides was performed based on the known structures of mammalian peptides. The overall results indicated that good quality models of ins peptides could be made despite the low sequence similarity with the templates. This suggested that it is the conformational shape of the molecule allowable by the individual residues that is most important when modelling and not having a perfect sequence match. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1200443 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2005

No description available.

No description available.

Type 2 diabetes or adult onset diabetes, has been a global epidemic for the past two decades, and the number of new cases accelerates every year. Insulin resistance is one of the major factors behind this, wherein the insulin receptor, which signals to regulate glucose levels, based on the hormone insulin, loses its sensitivity. Obesity is one other major concern which is caused due to the improper balance between the caloric intake and the energy utilized. Gastric bypass surgeries (GBP) are performed to avert obesity. However, a beneficial side-effect is that the state of insulin resistance is reset to near baseline levels within a few days after the procedure. The reason behind this remains unexplained, with possible humoral effects, hypothesized to occur after the bariatric procedure. In this work, high-five insect cell line was utilized to recombinantly produce full length insulin receptors (IR). However commercially sourced IR ectodomains (eIR – soluble version of the full length IR with the completely extracellular α subunits along with extracellular and transmembrane regions of the β subunit), were used to study the interaction. Measuring the kinetics of IR-insulin interactions is critical to improving our understanding of this disease. In this study, a multiplex surface plasmon resonance (SPR) assay was developed for studying the interaction between insulin and the eIR. A scaffold approach was used in which anti-insulin receptor monoclonal antibody 83–7 (Abcam, Cambridge, UK) was first immobilized on the SPR sensorchip by amine coupling, followed by eIR capture. The multiplex SPR system (ProteOn XPR36TM, Bio-Rad Laboratories, Hercules, CA) enabled measurement of replicate interactions with a single, parallel set of analyte injections, whereas repeated regeneration of the scaffold between measurements caused variable loss of antibody activity. The main approach was to replicate the physiological IR-insulin interaction using this assay. It was also observed that insulin at higher concentrations tend to form dimers and hexamers in solution. This was tested using size exclusion chromatography analysis and proved to be true. Therefore an alternative analyte with the similar binding properties and affinity of insulin and at the same time with reduced self- association characteristics was explored. Lispro, the analogue of insulin with reduced self-association properties (generated by swapping of residue 28 and 29 with Lys and Pro respectively) was finally used to study the interaction with eIR. Interactions between recombinant human insulin with eIR-A (A isoform of the insulin receptor ectodomain) followed a two-site binding pattern (consistent with the literature), with a high-affinity site (dissociation constant KD1 = 38.1 ± 0.9 nM) and a low-affinity site (KD2 = 166.3 ± 7.3 nM). The predominantly monomeric insulin analogue Lispro had corresponding dissociation constants KD1 =73.2 ± 1.8 nM and KD2 =148.9 ± 6.1 nM, but the fit to kinetic data was improved when conformational change factor was included in which the high-affinity site was converted to the low-affinity site. Kinetics of interaction of insulin with eIR-A and eIR-B isoforms were then compared. eIR-A bound insulin with apparently higher affinity (with both the binding sites) when compared with eIR-B. This was again consistent with literature that IR-A had two-fold higher affinity for binding insulin than IR-B. The assay was further extended to study the effect of external factors such as glucose, visfatin on this interaction. Glucose (the main biomolecule which is regulated by the IR-insulin interaction) was tested, if it had any direct effect on the interaction. It was observed that glucose did not have any effect on eIR-insulin interactions. Visfatin, an adipocytokine which has been highly debated in literature for its insulin mimetic effects and IR binding properties, was then tested. The standard assay did not provide much insights as the reference channel immobilized with 83-7 monoclonal antibody to the receptor had much affinity for visfatin, leading to non-specific binding and negative responses. Therefore, in an alternative methodology was used - visfatin, Lispro and insulin were immobilized on separate channels along with bovine serum albumin immobilized on reference channel and eIR isoforms used as analyte to study the effect of visfatin on IR. This study showed that visfatin, a higher molecular weight protein compared to insulin, bound both the eIR isoforms. This is consistent with literature that visfatin binds IR at a site distinct from insulin, but the assay described here could not confirm the fact that it mimicked the signalling carried out by IR-insulin binding. Further studies are required to interpret the kinetics of visfatin-eIR interaction. To my knowledge, this is the first SPR assay developed to study eIR-insulin interactions in real-time. This could potentially be extended to study the interaction of insulin with full length insulin receptors and the effect of humoral and other external factors on the interaction, without the need for insulin labelling.

No description available.

In Drosophila, eight insulin-like peptides (DILP1-8) are produced and secreted in different locations. They regulate many aspects of development and physiology, such as organism growth, metabolic homeostasis, reproduction, stress resistance and life span. DILP2, 3 and 5 are mainly produced by a cluster of median neurosecretory cells in the brain known as insulin producing cells (IPCs). Here we showed that IPCs are under tight regulation of two G-protein coupled receptors (GPCRs), serotonin receptor 5-HT1A and octopamine receptor OAMB. Genetic manipulations of these two receptors in IPCs affected transcription levels of DILPs, hence altered feeding, carbohydrate levels, and resistance to stress (Paper I and II). Moreover, we showed that the insulin receptor (dInR) is strongly expressed in leucokininergic neurons (LK neurons), and selectively regulates growth of around 300 neuropeptidergic neurons expressing the bHLH transcription factor DIMMED. Overexpression of dInR in DIMM-positive neurons led to substantial neuronal growth, including cell body size, golgi apparatus and nuclear size, while knockdown of dInR had the opposite effect (Paper III). Manipulations of components in the insulin signaling pathway in LK neurons resulted in the similar cell size phenotypes. Furthermore, dInR regulated size scaling of DIMM-postive neurons is nutrient-dependent and partially requires the presence of DIMM (Paper III). Finally, we investigated the roles of DILPs (2, 3, 5 and 7) and LK neurons in regulation of feeding and diuresis at the adult stage (Paper IV).  In summary, we have identified two more regulators for IPC activity and demonstrated developmental roles of  DILPs and dInR in regulating neuronal size. Moreover, DILPs regulate water homeostasis together with a diuretic hormone leucokinin and as a consequence affects feeding behavior. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: In press. Paper 4: Manuscript.</p>

In Drosophila, the insulin signaling pathway is at the interface between dietary conditions and control of growth and development, reproduction, stress responses and life span. Eight insulin like peptides (Dilp1-8), an insulin tyrosine kinase receptor (dInR) and its downstream components, as well as a relaxin-like receptor type (Lgr3) form the core of this signaling. Here we showed that the dInR mediates post-mitotic cell growth specifically in about 300 peptidergic neurons expressing the basic helix loop helix (bHLH) transcription factor Dimmed (Paper I).  Overexpression of dInR in Dimm positive neurons leads to increased size of cell body, Golgi apparatus and nucleus, whereas dInR knockdown causes an opposite effect. Manipulation of downstream components of insulin signaling induces similar changes in Dimm positive neurons. This mechanism is nutrient dependent. In Paper II, we further investigate the relation between Dimmed and dInR for regulation of cell growth. Coexpressing Dimm and dInR in a range of Dimm negative neurons results in increased cell size in both larval and adult stages. We provide further evidence that dInR regulates cell growth in a Dimm dependent manner and that DILP6 from glia cells is involved in this regulation. In addition, we find that Dimm alone is capable of triggering cell growth in certain neuron types at different developmental stages. Furthermore, ectopic Dimm alone can block apoptosis.  Dimm is a known master regulator of peptidergic cell fate. In paper III we find that ectopic expression of Dimm in Dimm negative motor neurons results in transformation the neurons towards a neuroendocrine phenotype. They acquire enlarged axon terminations and boutons, lose both pre- and postsynaptic markers, and display diminished levels of wingless and its receptor dFrizzled. Furthermore they show increased expression of several Dimm targets. Finally, combined ectopic Dimm and dInR expression gives rise to stronger phenotypes. In paper IV we studied another DILP possibly involved in growth regulation, the under-investigated DILP1. We generated Dilp1-Gal4 lines and anti DILP1 antibodies and found that DILP1 is transiently expressed in brain insulin producing cells (IPCs) from pupal stages to newly hatched adult flies. Diapausing virgin female flies display a high expression level of dilp1/DILP1 over at least 9 weeks of adult life. DILP1 expression is also correlated with the persistence of larval/pupal fat body and its expression is regulated by other DILPs and short neuropeptide F (sNPF). Flies mutant in dilp1 display increased food intake, but decreased stress resistance and life span. We found no obvious role of DILP1 in growth regulation. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript. Paper 4: Manuscript.</p>

The goal of the "Quantitative Trait Gene" (QTG) program is to identify genes and mutations that underlie natural phenotypic variation. My goal with this work was to contribute an additional model to the program: ovariole number evolution in Drosophila. In this thesis I describe the progress I have made towards identifying a specific genetic change that contributed to the divergence of ovariole number between two Drosophila lineages. I identify specific developmental mechanisms relevant to establishing ovariole number in different Drosophila lineages by detailing ovarian cell-type specific specification, proliferation, and differentiation. I test specific candidates of genetic regulators of these developmental mechanisms with mutational analysis in D. melanogaster. I show that independent evolution of ovariole number has resulted from changes in distinct developmental mechanisms, each of which may have a different underlying genetic basis in Drosophila. I use the interspecies comparison of D. melanogaster versus D. sechellia to test for functional differences in insulin/insulin-like growth factor (IIS) signaling between the two species. I show that IIS activity levels and sensitivity have diverged between species, leading to both species-specific ovariole number and species-specific nutritional plasticity in ovariole number. Moreover, plastic range of ovariole number correlates with ecological niche, suggesting that the degree of nutritional plasticity may be an adaptive trait. My work and quantitative genetic analyses strongly support the hypothesis that evolution of the Drosophila insulin-like receptor (InR) gene, specifically, is at least partially responsible for the divergence in ovariole number and nutritional plasticity of ovariole number between D. melanogaster and D. sechellia. I detail ongoing experiments to test this hypothesis explicitly via cross-species transgenesis.

Adult schistosomes depend for growth and development on hormonal signals from the mammalian host, which may include the insulin signalling pathway. In this project, I firstly used microarray analysis to demonstrate that human insulin can be utilised by adult S. japonicum in culture, resulting in the modulation of distinct metabolic effects as reflected in transcriptional levels of parasite genes. The addition of insulin resulted in the differential expression of 1,101 genes with many related to functions corresponding to the biological and metabolic effects of insulin reported for mammalian cells. Those identified genes in male or female S. japonicum worms that were up or down regulated after exposure to insulin were predominantly involved in growth and development, with significant sex-specific responses evident. Insulin appeared to play a similar role in male parasites as those seen in classical mammalian systems including an increase in protein synthesis though gene transcription and the stimulation of mRNA translation and control protein degradation via the ubiquitin proteasome pathway. Microarray analysis indicated that insulin not only leads to increased gene expression of the PI3-K pathway, which enhances parasite growth, but may also play a role in the sexual differentiation and fecundity of female worms by activating the MAPK pathway. As the insulin target proteins, two types of insulin receptors from Schistosoma japonicum were isolated, S. japonicum insulin receptors 1 (SjIR-1) and 2 (SjIR-2), with features similar to insulin receptors from other taxa. The sequences share 70% and 74% identity to S. mansoni insulin receptor 1 and 2 (SmIR-1 and SmIR-2), respectively. SjIR-1 and SjIR-2 are highly conserved in their tyrosine kinase domain to other IRs from Homo, Mus musculus and Drosophila melanogaster. SjIR-2 is located in the parenchyma in males and in the vitelline glands of female worms, which occupy most of male or female tissue and play an important role in growth or fecundity. In contrast, SjIR-1 was located in the tegument and intestinal epithelium of adult worms, representing much smaller cellular regions compared with the voluminous vitelline tissue or parenchyma. This observation was further confirmed by real time PCR showing that SjIR-2 was more abundantly expressed in S. japonicum adult worm than SjIR-1. Phylogenetic analysis showed that SjIR-2 and SmIR-2 are closer to EmIR than to SjIR-1 and SmIR-1, indicating that SjIR-1 and SmIR-1 might perform specific functions in schistosomes, while SjIR-2, SmIR-2 and EmIR might share similar roles in parasite growth and development in the three parasitic flatworms. Structure modelling recovered the conserved structure between the SjIRs and Homo sapiens IR (HIR) implying a common predicted binding mechanism in the ligand domain and the same downstream signal transduction processing in the tyrosine kinase domain as in HIR. Two-hybrid analysis was used to confirm that the ligand domains of SjIR-1 and SjIR-2 contain the insulin binding site. Incubation of adult worms in vitro, both with a specific insulin receptor inhibitor and anti-SjIRs antibodies, resulted in a significant decrease in worm glucose levels, suggesting again the same function for SjIRs in regulating glucose uptake as described for mammalian cells. Adult worms of S. japonicum possess insulin receptors that can specifically bind to insulin, indicating that the parasite can utilize host insulin for development and growth by sharing the same pathway as mammalian cells in regulating glucose uptake. In vaccination/challenge trials, there was no significant reduction in adult worm burdens with either of the SjLD vaccines. However, there were significant reductions in mean lengths of adult worms ranging from 22-25% in the SjLD1 vaccinated group to 37-42% in the SjLD2 vaccinated groups, significant reductions in faecal eggs in both the SjLD1 (66%) and SjLD2 (68%) vaccinated groups, and a reduction in liver egg numbers in the SjLD1(33%) vaccinated group. These results show that although the SjLDs vaccines were unable to reduce adult worm numbers by clearing them from the vaccinated mice, nevertheless, they significantly depressed the growth of male and female adult worms and affected female egg production. The protective efficacy obtained in terms of the substantial decrease in faecal eggs exceeded that of many of the recently available schistosome antigens and prototype vaccine formulations, which, at best, elicit 40–50% protection in animals using the standard readouts of reduced worm burden or egg production and viability. Overall, disruption of this insulin pathway leading to parasite starvation through the prevention of glucose uptake thereby affecting parasite growth, development and female fecundity, provides a new intervention target and transmission blocking approach to combat schistosomiasis and may be applicable for the control of other debilitating parasitic infections as well.