類胰島素生長因子1(IGF1)是一種負責代謝、細胞生長、身體發展的多肽激素。微衛星和單核苷酸多態性(SNP), 與循環IGF1水平顯著相關。然而，目前沒有研究指出微衛星和SNPs的綜合影響，且這些遺傳變異對IGF1的調控機制仍是未知。本研究的假設是，微衛星和SNPs在啟動子區域可能有相互作用從而調控IGF1水平。因此，本研究的目的是找出影響IGF1表達的主要元素，並研究每個基因變異的作用。 / 在這項研究中，整個IGF1的基因，包括上游和下游的5萬鹼基對(bp)，可分為4個單體型區段，而IGF1的調控區在第3和第4個區段。與其它重復序列的微衛星相比，有21個重復序列的微衛星(IGF1上游969bp)與一套獨特的SNPs有關連。此外，有19個重復序列的微衛星有較低的循環IGF1。 / 功能性細胞分析進一步分析在生長激素(GH)依賴模型和GH獨立模型中，每個基因變異的角色。在GH獨立模型中，常見的單體型之間有不同的轉錄活性。與以前的研究結果相一致的是，有19個重複序列的單倍型轉錄活性最低。當單倍型為C-T-T，啓動子的轉錄活性受微衛星長度影響，較長的單體型有較低的轉錄活性。微衛星的長度效應或倚賴功能性SNP 1411C> T（rs35767）和叉頭蛋白A3（FOXA3）。以前研究發現在不同基因調控中，一個只結合C等位點並含有CCAAT /增強子結合蛋白delta（CEBPD）的轉錄激活複合物與FOXA3並存。因此，CEBPD可能與FOXA3相互作用從而調控IGF1的表達。而微衛星長度可能通過調節上游CEBPD轉錄複雜和下游FOXA3的相互作用從而影響IGF1的表達。單倍型T-C-A可能採取另一種調控機制，該機制或許被長約178鹼基對，含有“CA“部分的片段調控。GH依賴模型是模擬幼年時期IGF1的表達。在這個階段中，常見的單體型之間有不同的轉錄活性，但每個基因變異的調節作用均不強。 / 總括而言，IGF1的表達主要是由微衛星和SNPs組成的單體型調控。在幼年和成年，常見的單體型之間有差別顯著的轉錄活動。然而，GH獨立模型和GH依賴模型的調控機制是不同的。 / Insulin-like growth factor 1 (IGF1) is a polypeptide hormone responsible for metabolism, cell growth, and somatic development. Microsatellite and SNPs have been demonstrated to be significantly associated with circulating IGF1 level. However, no studies have ever investigated the combined effects of microsatellite and SNPs, and regulatory mechanisms of IGF1 expression by these genetic variants are yet unknown. The hypotheses of this study were that the microsatellite and SNPs may have certain regulatory functions in the promoter region, and interact with each other in the regulation. Therefore, the objectives were to identify the primary regulatory element in the regulation of IGF1 expression and to investigate the role of each genetic variant. / In this study, the whole IGF1 gene, including 50kb upstream and downstream, was divided into four haplotype blocks, in which the regulatory region of IGF1 lied in haploblock 3 and 4. Results of high-resolution melting analysis showed that a microsatellite (969bp upstream) with 21 repeats was associated with a different set of SNPs, compared to microsatellite with other repeat numbers. Also, haplotype with 19 CA repeats was significantly associated with a lower level of circulating IGF1. / Functional cellular assays were performed to further analyze the roles of each genetic variant in growth hormone (GH)-independent and GH-dependent models. In GH-independent model, it was found that common haplotypes showed differential transcriptional activities, and, consistent with previous findings, haplotype with 19 repeats was the least activated. On the background of haplotype C-T-T, transcriptional activity was regulated by microsatellite length, in which the haplotype with a longer microsatellite length tended to have a lower transcriptional activity. Further analysis showed that the microsatellite length effect depended on a functional polymorphism -1411C>T (rs35767) and forkhead box A3 (FOXA3), whose binding sites were several base pairs upstream of IGF1 transcription start site. Telgmann et al found a transcription activator complex containing CCAAT/enhancer binding protein delta (CEBPD) bound exclusively to the C allele and CEBPD often coexisted with FOXA3 in the regulation of various genes. Therefore, in the activation of IGF1, microsatellite length might regulate the interaction between the upstream CEBPD transcription complex and the downstream FOXA3. Haplotype T-C-A showed a yet unknown regulatory mechanism of IGF1 expression, which might be accounted for by the “C-A“ portion. In GH-dependent model, common haplotypes also showed differential transcriptional activities. However, further analysis revealed that the regulatory effects of each genetic variant alone (microsatellite or SNPs) were not strong. / To conclude, haplotype effect, which was contributed by both microsatellite and SNPs, played an important role in the regulation of IGF1 expression. Common haplotypes showed significantly differential transcriptional activities. However, the regulatory mechanisms were different in GH-independent model and GH-dependent model. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Yu Holly. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 127-140). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgement --- p.v / LIST OF ABBREVIATIONS --- p.vi / LIST OF FIGURES --- p.viii / LIST OF TABLES --- p.x / PUBLICATIONS --- p.xi / Chapter CHAPTER 1 --- INTRODUCTION TO INSULIN-LIKE GROWTH FACTOR 1 (IGF1) --- p.1 / Chapter 1.1 --- Production of IGF1 --- p.1 / Chapter 1.2 --- Other factors affecting IGF1 level --- p.5 / Chapter 1.2.1 --- Nutritional status --- p.5 / Chapter 1.2.2 --- Ethnicity --- p.5 / Chapter 1.2.3 --- Age --- p.8 / Chapter 1.2.4 --- Gender --- p.8 / Chapter 1.2.5 --- IGFBP --- p.9 / Chapter 1.2.6 --- Other growth factors --- p.10 / Chapter 1.3 --- Cellular functions of IGF1 --- p.10 / Chapter 1.4 --- Physiological functions of IGF1 --- p.13 / Chapter 1.4.1 --- Metabolism --- p.14 / Chapter 1.4.2 --- Somatic growth --- p.17 / Chapter 1.4.3 --- Longevity --- p.18 / Chapter CHAPTER 2 --- PATHOLOGY OF IGF1 --- p.20 / Chapter 2.1 --- IGF1 and cancer predisposition --- p.20 / Chapter 2.1.1 --- Evidences in genetic studies --- p.21 / Chapter 2.1.2 --- Evidences in lifestyle factors --- p.21 / Chapter 2.1.3 --- Evidences from population studies --- p.22 / Chapter 2.1.4 --- Miscellaneous evidence --- p.22 / Chapter 2.2 --- IGF1 and diabetes mellitus (DM) --- p.23 / Chapter 2.3 --- IGF1 and other diseases --- p.24 / Chapter CHAPTER 3 --- HYPOTHESES AND AIMS OF THE STUDY --- p.26 / Chapter 3.1 --- Hypotheses of the study --- p.26 / Chapter 3.2 --- Aims of the study --- p.26 / Chapter CHAPTER 4 --- RELATIONSHIP BETWEEN GENETIC VARIANTS AND IGF1 EXPRESSION --- p.28 / Chapter 4.1 --- Introduction --- p.28 / Chapter 4.2 --- Materials and methods --- p.31 / Chapter 4.2.1 --- Study subjects --- p.31 / Chapter 4.2.2 --- tagSNP selection and haplotype block construction --- p.32 / Chapter 4.2.3 --- Genescan analysis of the CA repeat microsatellite --- p.32 / Chapter 4.2.4 --- Genotyping assay of tagSNPs --- p.34 / Chapter 4.2.5 --- Statistical analysis --- p.36 / Chapter 4.3 --- Results --- p.37 / Chapter 4.3.1 --- Characteristics of the subjects --- p.37 / Chapter 4.3.2 --- Determination of haplotype blocks --- p.38 / Chapter 4.3.3 --- Selection of tagSNPs --- p.41 / Chapter 4.3.4 --- Genotyping analysis of tagSNPs --- p.43 / Chapter 4.3.5 --- Genescan analysis of -969bp CA repeat microsatellite --- p.46 / Chapter 4.3.6 --- Phased haplotype consisting of SNP / SNP and microsatellite --- p.48 / Chapter 4.3.7 --- Correlation between haplotypes in IGF1 promoter and circulating IGF1 level --- p.50 / Chapter 4.4 --- Discussion --- p.53 / Chapter CHAPTER 5 --- Transcriptional regulation of GENETIC VARIANTS IN different haplotypeS --- p.57 / Chapter 5.1 --- Introduction --- p.57 / Chapter 5.1.1 --- IGF1 gene structure --- p.57 / Chapter 5.1.2 --- Regulatory elements in IGF1 promoter --- p.58 / Chapter 5.1.3 --- Functional variant -1411C>T (rs35767) in IGF1 promoter --- p.60 / Chapter 5.1.5 --- Objectives of the study --- p.62 / Chapter 5.2 --- Materials and methods --- p.64 / Chapter 5.2.1 --- Comparative genomics --- p.64 / Chapter 5.2.2 --- Study subjects --- p.64 / Chapter 5.2.3 --- tagSNP selection and genotyping assay --- p.64 / Chapter 5.2.4 --- Primers and standard polymerase chain reaction (PCR) --- p.65 / Chapter 5.2.5 --- Enzyme digestion --- p.68 / Chapter 5.2.6 --- Ligation --- p.68 / Chapter 5.2.7 --- Transformation of DNA ligation products --- p.68 / Chapter 5.2.8 --- Preparation of E.coli supercompetent cells --- p.69 / Chapter 5.2.9 --- Construction of plasmids --- p.70 / Chapter 5.2.10 --- Cell lines --- p.71 / Chapter 5.2.11 --- Nucleic acid extraction --- p.72 / Chapter 5.2.12 --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.73 / Chapter 5.2.13 --- Transient transfection --- p.73 / Chapter 5.2.14 --- Luciferase reporter assay --- p.73 / Chapter 5.2.15 --- Optimization of a saturated luciferase reporter system --- p.74 / Chapter 5.2.16 --- Electrophoretic mobility shift assay (EMSA) --- p.74 / Chapter 5.2.17 --- Western blot analysis --- p.74 / Chapter 5.2.18 --- Prediction of putative functional SNPs --- p.76 / Chapter 5.2.19 --- Statistical analysis --- p.77 / Chapter 5.3 --- Results --- p.77 / Chapter 5.3.1 --- Evolutionarily conserved region (ECR) --- p.77 / Chapter 5.3.2 --- Frequency distribution of haplotypes of IGF1 promoter in the Chinese population --- p.79 / Chapter 5.3.3 --- Optimization of luciferase reporter system --- p.81 / Chapter 5.3.3.1 --- Gene expression level of different cell lines --- p.81 / Chapter 5.3.3.2 --- Cell line selection --- p.81 / Chapter 5.3.3.3 --- Saturation of expression plasmids in the luciferase reporter system --- p.83 / Chapter 5.3.3.4 --- Western blot analysis of gene expression level after transfection --- p.86 / Chapter 5.3.4 --- Possible functional SNPs in IGF1 regulatory region beyond ECR --- p.89 / Chapter 5.3.4.1 --- In silico analysis of putative functional SNPs --- p.89 / Chapter 5.3.4.2 --- Binding capacity of possible functional SNPs --- p.92 / Chapter 5.3.5 --- Transcriptional activities of common haplotypes and their derivatives --- p.95 / Chapter 5.3.5.1 --- GH-independent (GH-) model --- p.95 / Chapter 5.3.5.1.1 --- Common haplotypes --- p.95 / Chapter 5.3.5.1.2 --- Effect of microsatellite length on transcriptional activity of IGF1 promoter --- p.97 / Chapter 5.3.5.1.3 --- Effect of SNP on transcriptional activity of IGF1 promoter --- p.99 / Chapter 5.3.5.1.4 --- Summary --- p.101 / Chapter 5.3.5.2 --- GH-dependent (GH+) model --- p.101 / Chapter 5.3.5.2.1 --- Common haplotypes --- p.101 / Chapter 5.3.5.2.2 --- Effect of microsatellite length on transcriptional activity of IGF1 promoter --- p.103 / Chapter 5.3.5.2.3 --- Effect of SNP on transcriptional activity of IGF1 promoter --- p.105 / Chapter 5.3.5.2.4 --- Summary --- p.106 / Chapter 5.3.6 --- Putative mechanism of the interaction between microsatellite and SNPs --- p.106 / Chapter 5.3.6.1 --- Microsatellite length effect in C-T-T haplotype relied on rs35767 (-1411C>T) --- p.107 / Chapter 5.3.6.2 --- The interaction of SNP and microsatellite was dependent on FOXA3 --- p.110 / Chapter 5.3.6.3 --- Summary --- p.112 / Chapter 5.3.7 --- Serial deletion of IGF1 promoter fragment --- p.112 / Chapter 5.4 --- Discussion --- p.116 / Chapter 5.4.1 --- Distal regulatory mechanism of IGF1 expression --- p.116 / Chapter 5.4.2 --- Localized regulatory mechanism of IGF1 expression --- p.117 / Chapter CHAPTER 6 --- CONCLUSIONS AND FUTURE STUDIES --- p.125 / Chapter 6.1. --- Conclusions --- p.125 / Chapter 6.2. --- Future studies --- p.126 / Reference --- p.127

Ho Sin Ka Janice. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 105-119). / Abstracts in English and Chinese. / Chapter CHAPTER 1. --- Introduction / Chapter 1.1. --- Epidemiology of Type 2 Diabetes --- p.1 / Chapter 1.2. --- Risk factors contributing to Type 2 Diabetes --- p.3 / Chapter 1.2.1. --- Environmental and physiological factors --- p.3 / Chapter 1.2.2. --- Genetic factors --- p.3 / Chapter 1.3. --- Disruption of energy homeostasis in the pathogenesis of type 2 diabetes --- p.6 / Chapter 1.3.1. --- Clinical spectrum of diabetes --- p.6 / Chapter 1.3.2. --- Insulin as a key regulator of energy homeostasis --- p.7 / Chapter 1.3.3. --- Insulin secretion and glucose metabolism --- p.8 / Chapter 1.3.4. --- Insulin action and lipid metabolism --- p.9 / Chapter 1.3.5. --- Lipotoxicity and glucotoxicity --- p.12 / Chapter 1.3.6. --- Role of transcription factors as metabolic switch --- p.13 / Chapter 1.4. --- Candidate genes implicated in type 2 diabetes susceptibility --- p.15 / Chapter 1.4.1. --- Candidate genes involved in insulin secretion pathway --- p.15 / Chapter 1.4.1.1. --- HNF4A --- p.15 / Chapter 1.4.1.2. --- HNF1A --- p.16 / Chapter 1.4.1.3. --- PDX1/PBX1 --- p.17 / Chapter 1.4.1.4. --- NEUROD1 --- p.17 / Chapter 1.4.1.5. --- GCK --- p.17 / Chapter 1.4.1.6. --- KCNJ11/ABCC8 --- p.18 / Chapter 1.4.2 --- Candidate genes involved in insulin action pathway --- p.19 / Chapter 1.4.2.1. --- PPARG --- p.19 / Chapter 1.4.2.2. --- PPARA --- p.20 / Chapter 1.4.2.3. --- PPARGC1A --- p.20 / Chapter 1.4.2.4. --- ADIP0Q --- p.21 / Chapter 1.4.2.5. --- LPL --- p.21 / Chapter 1.4.2.6. --- UPC --- p.22 / Chapter 1.5. --- Hypothesis and objectives of the study --- p.23 / Chapter CHAPTER 2. --- Materials and methods / Chapter 2.1. --- Study design --- p.25 / Chapter 2.1.1. --- Two-stage candidate gene association design --- p.25 / Chapter 2.1.2. --- Power calculation --- p.27 / Chapter 2.2. --- Study cohort --- p.29 / Chapter 2.2.1. --- Subject recruitment --- p.29 / Chapter 2.2.2. --- Clinical and biochemical measurements --- p.30 / Chapter 2.2.3. --- Clinical definitions --- p.31 / Chapter 2.3. --- Genetic study --- p.32 / Chapter 2.3.1. --- Candidate gene selection --- p.32 / Chapter 2.3.2. --- SNP selection --- p.32 / Chapter 2.3.3. --- DNA sample preparation --- p.35 / Chapter 2.3.4. --- Genotyping methods --- p.36 / Chapter 2.3.4.1. --- Allele specific Tm shift assay --- p.36 / Chapter 2.3.4.2. --- Mass spectrometry assay --- p.40 / Chapter 2.4. --- Data quality control --- p.42 / Chapter 2.4.1. --- Stage 1 --- p.42 / Chapter 2.4.2. --- Stage 2 --- p.42 / Chapter 2.5. --- Statistical analysis --- p.45 / Chapter 2.5.1. --- Stage 1 analysis --- p.45 / Chapter 2.5.2. --- Stage 2 analysis --- p.45 / Chapter 2.5.3. --- Stage 1 and 2 combined analysis --- p.46 / Chapter CHAPTER 3. --- Results / Chapter 3.1. --- Clinical characteristics of subjects in stages 1 and 2 studies --- p.48 / Chapter 3.2. --- Case-control associations in stage 1 --- p.51 / Chapter 3.2.1. --- Association with T2D --- p.51 / Chapter 3.2.2. --- Association with T2D subset by metabolic syndrome --- p.54 / Chapter 3.3. --- Case-control associations in stage 2 --- p.60 / Chapter 3.3.1. --- SNP selection for genotyping --- p.60 / Chapter 3.3.2. --- Association with T2D --- p.63 / Chapter 3.3.3. --- Association with T2D subset by metabolic syndrome --- p.64 / Chapter 3.4. --- Case-control associations in combined stages 1 and 2 --- p.66 / Chapter 3.4.1. --- Association with T2D --- p.66 / Chapter 3.4.2. --- Association with T2D subset by metabolic syndrome --- p.70 / Chapter 3.4.3. --- Association with T2D subset by age at diagnosis --- p.74 / Chapter 3.4.4. --- Association with T2D subset by gender --- p.76 / Chapter 3.4.5. --- Genetic epistasis for T2D association --- p.79 / Chapter 3.5. --- Metabolic traits associations in control subjects in combined stages 1 and 2 studies --- p.83 / Chapter CHAPTER 4. --- Discussion --- p.86 / Chapter 4.1. --- Role of insulin secretion genes in type 2 diabetes --- p.87 / Chapter 4.2. --- Role of insulin action genes in type 2 diabetes --- p.92 / Chapter 4.3. --- Combined genetic effects on risk for type 2 diabetes --- p.97 / Chapter 4.4. --- Summary --- p.98 / Chapter 4.5. --- Limitation of this study and future direction --- p.101 / REFERENCES --- p.104 / APPENDICES --- p.119 / Chapter Appendix 1: --- Gene structure and linkage disequilibrium of genotyped SNPs of candidate genes --- p.119 / Chapter Appendix 2: --- Information of SNPs genotyped in stage 1 --- p.130 / Chapter Appendix 3: --- T2D association results (additive model) of 152 SNPs for stage 1 case- control samples --- p.137 / Chapter Appendix 4: --- T2D association results (additive model) of 152 SNPs for stage 1 case- control samples subset by metabolic syndrome status in cases --- p.144 / Chapter Appendix 5: --- T2D association results (additive model) of 22 SNPs for stage 2 case- control samples --- p.151 / Chapter Appendix 6: --- T2D association results (additive model) of 22 SNPs for stage 2 case- control samples subset by metabolic syndrome status in cases --- p.153

The development of type 2 diabetes over time involves defects in insulin action and insulin secretion. Defects in insulin action alone can be compensated with appropriate hyperinsulinemia. However, the progressive loss of pancreatic beta-cell function leads eventually to the development of persistent hyperglycemia that characterizes type 2 diabetes. Insulin secretion patterns reflect two phases when beta-cells are exposed to acute and sustained glucose stimulation. Through the study and understanding of the roles of these two phases in the regulation of glucose homeostasis, it is clear that insulin must not only be secreted in sufficient amounts, but also at the right time. In type 2 diabetes, the timing and magnitude of insulin secretion are altered, and an abnormal first-phase release initiates before the onset of the disease. Only a few pharmacokinetic/pharmacodynamic (PK/PD) models have considered the biphasic nature of insulin secretion. This study is aimed at describing the biphasic dynamics of insulin secretion through developing a PK/PD model based on current knowledge of the cellular mechanism of biphasic insulin secretion. The objectives of this work are to 1) evaluate the insulin-glucose kinetics using nonparametric analysis, 2) develop a physiologically based mechanistic PK/PD model to dynamically describe the biphasic insulin secretion, 3) evaluate the impact of ethnicity on insulin secretion kinetics following an intravenous glucose administration using population analysis and 4) extend the proposed model to oral glucose administration and utilize the co-secretion kinetics of insulin and C-peptide in a population PK/PD analysis of the prehepatic insulin secretion. Population analysis was done using a nonlinear mixed-effects model combined with the proposed PK/PD model to estimate population parameters and their variations between- and within-subjects and the covariates' effects on model parameters. The proposed model describes biphasic insulin behavior, accounts for first-phase insulin secretion, and also applies to oral glucose administration for estimating prehepatic insulin secretion in vivo and in liver extraction. This is done by an analysis that simultaneously uses plasma insulin and C-peptide concentrations. A significant higher first-phase insulin secretion was identified in healthy youths of African-American compared to Caucasians. The analysis showed no significant differences in the clearance of insulin from the plasma and the liver extraction of insulin between subjects with various levels of glucose tolerance. Obesity leads to a higher insulin production rate and lower elimination rate from the plasma than normal weight subjects. Also, type 2 diabetes and impaired glucose tolerance were found to reduce insulin production rate and resulted in a delayed insulin secretion from the beta-cells.

The aim of the present thesis was to further increase our understanding of mechanisms contributing to and maintaining cellular insulin resistance in type 2 diabetes (T2D). For this reason, the effects of high glucose and insulin levels on glucose transport capacity and insulin signaling, with emphasis on insulin receptor substrate 1 (IRS-1) were assessed in fat cells. Altered levels of IRS-1 have previously been observed in adipose tissue from insulin-resistant and T2D subjects. A high glucose level (≥15 mM) for 24 h exerted only a minor impairment on glucose transport capacity in human adipocytes, as opposed to rat adipocytes. However, when combined with a high insulin level (104 µU/ml), basal and insulin-stimulated glucose transport was significantly impaired in both human and rat adipocytes. This was associated with a depletion of IRS-1 and IRS-2 protein levels in rat adipocytes, as a result of post-translational changes and altered gene transcription, respectively. In human adipocytes was only IRS-1 protein levels reduced. The high glucose/high insulin setting achieved maximal impairment of glucose transport within 6 h. Subsequent incubations of rat adipocytes under physiological conditions could partially restore insulin sensitivity. Interestingly, in both human and rat fat cells, decreased levels of IRSs occurred after the establishment of impaired glucose transport, suggesting that the observed depletion of IRSs is a consequence rather than a cause of insulin resistance. Nonetheless, IRS depletion is likely to further aggravate insulin resistance. Tyrosine phosphorylation of IRS-1 upon insulin stimulation activates the signaling pathway that mediates glucose transport. Pre-treatment of human adipocytes with high glucose and insulin levels was not associated with any alterations in the total IRS-1 Tyr612 phosphorylation following 10 min insulin stimulation. However, a significant increase in basal Tyr612 phosphorylation was observed. Furthermore, a rise in basal IRS-1 Ser312 phosphorylation was found. This is associated with reduced IRS-1 function and is considered to target IRS-1 to degradation pathways, and thus could potentially explain the observed decrease in IRS-1 protein levels. Our results imply an enhanced activation of insulin’s negative-feedback control mechanism that inhibit IRS-1 function. This could potentially have contributed to the observed impairment of insulin action on glucose transport in these cells. Accordingly, we have also shown that the downstream activation of protein kinase B upon insulin-stimulation is significantly impaired in human adipocytes exposed to the high glucose/high insulin setting, indicating a defect in the signaling pathway mediating glucose transport. We also investigated whether there are humoral factors in the circulation of T2D patients that contribute to peripheral insulin resistance. Human adipocytes cultured for 24 h in medium supplemented with 25% serum from T2D subjects, as compared to serum from non-diabetic subjects, displayed significantly reduced insulin-stimulated glucose uptake capacity. The effect could neither be attributed to glucose, insulin, FFA, TNF-α or IL-6 levels in the serum, but other circulating factor(s) seem to be of importance. In conclusion, chronic conditions of elevated glucose and/or insulin levels all impair insulin action on glucose turnover, but to different extents. A clear distinction between rat and human fat cells in the response to these different milieus was also observed. Alterations in the function of the key insulin signaling protein IRS-1 might be involved in the mechanisms underlying the impaired glucose uptake capacity. IRS-1 reduction however, occurs after but probably aggravates the existing insulin resistance. The effects of high glucose and/or insulin levels may be of importance in T2D, but additional novel factors present in the circulation of T2D patients seem to contribute to cellular insulin resistance.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 79-87).

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.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Oct. 13, 2008). Includes bibliographical references (p. 56-64).

A angiotensina II (AngII) pode ativar as vias de sinalização das proteínas quinases ativadas por mitógenos (MAPKs). Investigamos o papel da obesidade e das MAPKs na resposta à AngII em ratos obesos por injeção de glutamato monossódico (Ob). Artérias mesentéricas de resistência com endotélio intacto e não as artérias sem endotélio, isoladas de Ob, respondem menos à AngII. As respostas à noradrenalina e ao cloreto de potássio estavam inalteradas. Aumento da expressão do receptor AT2 (AT2R), da óxido nítrico sintase (eNOS) e da ERK1/2 podem estar envolvidos na menor resposta pois a inibição do AT2R, da eNOS e da ERK1/2 corrigiram-na. A maior ativação da ERK1/2 nos Ob levou à maior ativação da eNOS e maior geração de NO, diminuindo a resposta à Ang II. Concluímos que na obesidade, a resposta contrátil à Ang II é menor, como possível mecanismo adaptativo frente ao aumento da ativação do sistema renina-angiotensina. Esse mecanismo envolve a participação do endotélio com maior liberação de NO, aumento do número de AT2R, e da fosforilação da eNOS e da ERK1/2. / Angiotensin II (AngII) can activate mitogen-activated protein kinases (MAPKs) pathways. We investigated the role of obesity and MAPKs in AngII response in monosodium glutamate-induced obese rats (Ob). Endothelium-intact but not endothelium-denuded mesenteric resistance arteries isolated from Ob exhibited a lower response to AngII. The response to nordrenaline and potassium chloride were unaltered. Increased expression of AT2 receptor, nitric oxide synthase (eNOS) and ERK1/2 might be involved in the reduced response since inhibition of AT2R, eNOS and ERK1/2 corrected it. Increased activation of ERK 1/2 in Ob might activate eNOS, generating more NO and vasodilation that contributed to reduce the contraction to AngII. We concluded that, in obesity, the lower response to AngII might be an adaptive mechanism against the increased activation of the renin-angiotensin system. This mechanism involves the participation of the endothelium through a greater release of NO, increased AT2R, eNOS and ERK1/2 expressions.

The ability of insulin to stimulate glucose uptake into muscle and adipose tissue is central to the maintenance of whole-body glucose homeostasis. Deregulation of insulin action manifests itself as insulin resistance, a key component of type 2 diabetes. Insulin resistance is also observed in HIV patients receiving protease inhibitors. An agent that can reversibly induce an insulin-resistant state would be a very useful tool in developing model systems that mimic the pathogenesis of type 2 diabetes. Insulin resistance can arise from defects in insulin signal transduction, changes in the expression of proteins or genes that are targets of insulin action, cross talk from other hormonal systems or metabolic abnormalities. Deterioration of the insulin-receptor-signalling pathway at different levels leading to decreased levels of signalling pathway intermediates and/or decreased activation through phosphorylation accounts for the evolution from an insulin-resistant state to type 2 diabetes. In addition, defects in GLUT4 glucose transporter translocation are observed, further fuelling impairments in skeletal muscle glucose uptake. Levels of insulin-induced GLUT4 translocation in the skeletal muscle of type 2 diabetic patients are typically reduced by 90%. Many cellular pathways & their intermediates are in some way or another linked to insulin signalling. This study focused on three of these namely the PI3-kinase/Akt pathway, the Mitogen Activated Protein Kinase (MAPK) cascade and the AMP Kinase pathway, with successful monitoring of the PI3-K pathway. Investigations involved observing and evaluating the effects of various compounds as well as an indigenous medicinal plant, Sutherlandia frutescens on the activities of key insulin signalling pathway intermediates within the three fore mentioned pathways including Akt, AMPK and MEK1/2 as well as membrane surface GLUT4 levels. Scientific research has in the past leant heavily on Western blotting as the method of choice for gaining vital information relating to signal transduction pathways, however for research into cellular mechanisms the negatives of this method outweigh the positives. The drawbacks include a need for large amount of cells, multiple washing steps which may be disadvantageous to any weak and transient interactions as well as lysing of cells which may interfere with the maintenance of the subcellular localisation of a specific signalling event. Based on these, the need for a better method in terms of speed & reliability to monitor phosphorylation states of signal transduction pathway intermediates & GLUT4 translocation was evident and was one VII of the main aims & successes of this study. The method created used the mouse muscle cell line C2C12 in conjunction with the quick, sensitive method of flow cytometry which allowed us to monitor these processes in these cells through immune-labelling. Adherent cell cultures such as the C2C12 cell line pose the problem of possible damage to plasma membrane receptors (including insulin receptors) during harvesting to obtain a cell suspension for flow cytometry. We however used C2C12 mouse myocytes to optimize a method yielding insulin responsive cells in suspension that were successfully used for flow cytometry after immunelabelling of insulin signalling intermediates. Insulin (0.1μM) significantly raised the levels of both P-Akt and GLUT4 above basal levels. This effect was shown to be dose dependent. At a concentration of 50μg/ml, Sutherlandia frutescens was able to act as an insulin-mimetic in terms of its ability to increase P-Akt levels, GLUT4 translocation and glucose utilisation in an acute manner. These increases could be reduced with the addition of wortmannin, a PI3-K inhibitor. Therefore, these results suggest the mechanism of the plant extract’s insulin-like activity may be in part due to the activation of the insulin signalling pathway leading to GLUT4 translocation, which involves the phosphorylation of insulin receptor- and subsequent PI3-K activity, leading to P-Akt activity. These results provide further evidence of this plant extract’s anti-diabetic potential. The effect of Sutherlandia frutescens on insulin secretion, calcium signalling and proliferation in INS-1 rat pancreatic cells was also investigated and it was found to increase the activities of all of these processes. However no change in the levels of GLUT2 glucose transporter was seen. Ritonavir is prescribed by the South African Department of Health in co-formulation with other protease inhibitors within its second regime in the treatment of HIV and AIDS. Using C2C12 cells, ritonavir decreased glucose uptake acutely and had no effect on GLUT4 translocation however surprisingly increased P-Akt levels. In conclusion, it was found that Sutherlandia frutescens has antidiabetic benefits, diverse in nature depending on tissue type as well as length of time administered. The establishment of novel flow cytometry techniques to assess antidiabetic properties using in vitro cell culture was achieved. These methods will be useful in the future for the assessment of insulin sensitivity and in the identification of novel compounds that stimulate the insulin signalling pathways.

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