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The Xenopus laevis oocyte was examined as a model system for investigating insulin receptor function. The role of extracellular calcium on insulin-stimulated deoxyglucose uptake (ISDU) in the 𝘟𝘦𝘯𝘰𝘱𝘶𝘴 𝘭𝘢𝘦𝘷𝘪𝘴 oocyte was investigated. It was determined that removal of calcium from the medium did not alter the rate of ⁴⁵Ca²⁺ release from oocytes preloaded with ⁴⁵CaCl₂ In contrast to earlier reports using tissue explants and cultured cells, the insulin response in oocytes is not sensitive to a range of extracellular calcium concentrations from 1 μM to 10 mM. However, treatment of oocytes with 1 mM EGTA, in the absence of Ca²⁺, prior to, during or within 5 minutes of insulin addition resulted in a 2-4 fold inhibition of ISDU. To further investigate the event(s) in insulin signalling inhibited by EGTA, the number of receptors for insulin on the oocyte must be increased. To this end we have investigated the effects of the 5' and 3' untranslated regions as well as the coding region of mRNA on translational efficiency in reticulocyte lysate and oocytes. The results obtained in Xenopus oocytes are consistent with earlier cell-free data (Falcone and Andrews, 1991). We have demonstrated that replacing the cognate 5' UTR with the Xenopus beta globin 5' UTR appropriately linked to a consensus sequence for efficient translation initiation (ACCATGG) results in increased in translation in Xenopus oocytes. In vitro synthesized preprolactin transcript injected into oocytes was found to be functionally stable for several days (D. Andrews unpublished data). Stabilization of the preprolactin 3' transcript was localized to the UTR. Furthermore, inserting the preprolactin 3' UTR downstream of another coding region resulted in stabilization of the modified transcript. These results provided a basis for improving expression of cloned human insulin receptor in Xenopus oocytes. By optimizing the 5' and 3' UTR's of the insulin receptor clone we were successful in expressing high levels of insulin receptors in Xenopus oocytes. Effects of the coding region on translation were also investigated and we provide evidence that sequences in the coding region modulate translational efficiency. / Thesis / Master of Science (MSc)

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Blood glucose management in Type 1 diabetes is crucial in preventing several diabetic complications. Blood glucose management is a complex task requiring diabetics too carefully administer the correct dosages of insulin by taking their blood glucose levels, food consumption, exercise, stress, illnesses and several other factors into account. Improved bolus calculation greatly aids in controlling blood glucose levels within a tight range. This study investigates how the ets-concept (Equivalent Teaspoons Sugar-concept) can be used to develop products to calculate insulin boluses. A cellular phone based software application was developed to calculate insulin boluses using the ets-concept. This product was tested in a clinical trial. A blood glucose characterization procedure was also developed to characterize the blood glucose response of a Type 1 diabetic to carbohydrate ingestion and insulin administration. The characterization procedure was used during the clinical trial to characterize patients in order to customize the bolus calculation products for the specific diabetic user. / Thesis (Ph.D. (Mechanical Engineering)--North-West University, Potchefstroom Campus, 2006

Blood glucose management in Type 1 diabetes is crucial in preventing several diabetic complications. Blood glucose management is a complex task requiring diabetics too carefully administer the correct dosages of insulin by taking their blood glucose levels, food consumption, exercise, stress, illnesses and several other factors into account. Improved bolus calculation greatly aids in controlling blood glucose levels within a tight range. This study investigates how the ets-concept (Equivalent Teaspoons Sugar-concept) can be used to develop products to calculate insulin boluses. A cellular phone based software application was developed to calculate insulin boluses using the ets-concept. This product was tested in a clinical trial. A blood glucose characterization procedure was also developed to characterize the blood glucose response of a Type 1 diabetic to carbohydrate ingestion and insulin administration. The characterization procedure was used during the clinical trial to characterize patients in order to customize the bolus calculation products for the specific diabetic user. / Thesis (Ph.D. (Mechanical Engineering)--North-West University, Potchefstroom Campus, 2006

In der vorliegenden Arbeit wurde der Einfluss von Insulin und IGF-1 auf den transepithelialen Natriumtransport über die Zellmembran von fetalen distalen Lungenepithelzellen der Ratte (fetal distal lung epithelia, FDLE) als Modell der Pneumozyten vom Typ II des späten Frühgeborenen untersucht. In Ussing-Kammer Messungen konnte eine insulinabhängige schnelle Steigerung des transepithelialen Natriumstroms gezeigt werden. Durch Western Blot-Untersuchungen sowie Inhibition spezifischer Kinasen wurden die intrazellulären Signaltransduktionsmechanismen der Insulin-induzierten Stimulation des Natriumtransports weiter charakterisiert. Es konnte eine Beteiligung der Phosphatidylinositol 3-Kinase, der Proteinkinase B, sowie von mTORC2 an den Signalwegen in den untersuchten Zellen nachgewiesen werden. Ähnliche Wirkungen auf den Natriumtransport wie Insulin hatte der Wachstumsfaktor IGF-1. Somit wurde der akute Einfluss des Insulin/IGF-1 Systems auf den epithelialen Natriumtransport in fetalen Alveolarzellen charakterisiert.

Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: Introduction: It is well documented that insulin offers cardioprotection under ischaemic stress. In the past it was believed that the protective effects of insulin, such as the (a) recruitment of glucose transporters to enhance glucose entry into the cell, (b) stimulation of glycolysis, (c) enhancement of glycogen synthesis, (d) improved protein synthesis, and (e) positive inotropic and chronotropic properties, were metabolic of origin, but lately the emphasis has shifted towards the diverse signal transduction pathways elicited by insulin. Although these beneficial effects of insulin on ischaemia/reperfusion induced injury have been studied for many years, the exact protective mechanism is still not resolved. Aim: To investigate the influence of insulin on the signaling pathways as a possible protective mechanism against ischaemia/reperfusion and therefore to investigate the possible roles and cross signaling of cyclic adenosine monophosphate (cAMP), protein kinase B (PKB) and p38 mitogen activated protein kinase (p38 MAPK) in the cardioprotection offered by insulin to the reperfused, ischaemic myocardium. Materials and methods: Isolated rat hearts were perfused retrogradely in accordance with the Langendorff technique (95%02, 5% C02). After 30 min of stabilization, hearts were subjected to 30 min global low flow ischaemia (0,2 ml/min), followed by 30 min of reperfusion. Hearts perfused with standard Krebs Henseleit solution containing 5 mM glucose were compared to hearts perfused with a perfusion solution containing 5 mM glucose and 0,3 IlIU/ml insulin. Wortmannin was added during either ischaemia or reperfusion. Left ventricular developed pressure (LVDP), rate pressure product (RPP), tissue cAMP and PKB and p38 MAPK activation were measured. Results: Insulin treated hearts showed improved functional recovery (P<0.05) during reperfusion after ischaemia vs. non-insulin treated hearts (85.5±4.6% vs. 44.8±4.9%). However, the addition of wortmannin (a Pl3-kinase inhibitor) to the perfusion solution during either ischaemia or reperfusion abolished the improved recovery. At the end of ischaemia, cAMP levels of the insulin treated hearts were elevated significantly, while the cAMP content in the non-insulin treated hearts returned to control levels. Addition of wortmannin during ischaemia abolished this rise in cAMP. Wortmannin added during reperfusion only did not alter the levels of cAMP at the end of reperfusion. Activation of p38 MAPK was transient during ischaemia for both insulin and non-insulin treated hearts. Addition of wortmannin during ischaemia did not alter p38 MAPK levels at the end of ischaemia. P38 MAPK was activated significantly (P<0.001) in the non-insulin treated hearts vs. insulin treated hearts during reperfusion. Wortmannin, added at the onset of reperfusion, could partially abolish the effects of insulin to suppress p38 MAPK activation after 30 min of reperfusion. Activation of PKB in insulin treated hearts was significantly higher than non-insulin treated hearts during stabilization and early ischaemia. This activity was depressed by 30 min of ischaemia in both presence and absence of insulin. Wortmannin, when added before induction of ischaemia did not further lower this. The presence of insulin resulted in occurrence of strong PKB activation during reperfusion, peaking at 15 minutes and diminishing at 30 minutes. Wortmannin, added at the onset of reperfusion, abolished PKB activity measured at the end of reperfusion. Conclusion: Insulin exerted a positive inotropic effect and delayed the onset to ischaemic contracture. Inhibition of Pl3-kinase by wortmannin abolished the protective effects of insulin, arguing for an insulin stimulated PKB involvement in cardiac protection. Insulin also increased cAMP production and attenuated activation of p38 MAPK, both associated with improved recovery. This evidence suggested possible cross signaling between different signaling pathways. / AFRIKAANSE OPSOMMING: Agtergrond: Insulin beskerm harte wat aan isgemiese stres blootgestel word. Alhoewel hierdie voordelige effekte van insulien reeds vir verskeie jare bestudeer is, is die presiese meganisme waarmee insulien die hart beskerm steeds nie duidelik nie. Navorsers het die beskermende effekte van insulien aan metaboliese gevolge soos: (a) verhoogde glukose transport d.m.v. inspanning van meer glukose transporters (b), stimulering van glikolise, (c) vebeterde glikogeensintese, (d) verhoogde proteiensintese, en (e) die positiewe inotropiese en chronotropiese eienskappe van insulien toegeskryf. Onlangs het die fokus verskuif na ander diverse seintransduksiepaaie. Doel: Die doel van hierdie studie was dus om die moontlike betrokkenheid van hierdie sientransduksiepaaie asook die interaksie tussen sikliese adenomonofosfaat (cAMP), proteïn kinase B (PKB) en p38 MAPK in die beskerming wat insulien aan die isgemiese, gereperfuseerde miokardium bied, te bestudeer. Materiale en Metodes: Geïsoleerde rotharte is geperfuseer in ooreenstemming met die Langendorff metode. Na 30 min van stabilisasie is harte blootgestel aan 30 min. globale lae vloei isgemie (0,2 ml/min), en daarna is harte vir 30 min. geherperfuseer. Harte wat geperfuseer is met 'n perfusaat wat 5mM glukose bevat is vergelyk met harte wat geperfuseer is met 'n perfusaat wat 5mM glukose en 0,3 ~IU/ml insulien bevat. Sommige harte is geperfuseer met 'n perfusie oplossing waar wortmannin bygevoeg is tydens óf isgemie óf tydens herperfusie. Linker ventrikulêre ontwikkelde druk (LVDP), tempo-druk produk (RPP), weefsel cAMP-vlakke asook PKB en p38 MAPK aktiwiteit is gemeet. Resultate: Insulien-behandelde harte het funksioneel beduidend beter herstel tydens herperfusie na isgemie as harte wat nie met insulien behandel is nie (85.5±4.6% vs. 44.8±4.9%). Byvoeging van wortmannin by die perfusie oplossing tydens óf isgemie óf reperfusie, het die toename in herstel wat gesien is in die insulien-behandelde harte, opgehef. Die cAMP vlakke in die insulienbehandelde harte het aan die einde van isgemie beduidend gestyg (P<0.001), terwyl vlakke in harte wat nie met insulien behandel is nie, na kontrole vlakke teruggekeer het. Die teenwoordigheid van wortmannin in die perfusie oplossing tydens isgemie, het die styging in cAMP voorkom , terwyl die byvoeging van wortmannin tydens herperfusie. nie die cAMP vlakke beïnvloed het nie. Die aktivering van p38 MAPK tydens isgemie was van verbygaande aard in beide die insulien-behandelde harte en harte wat nie met insulien behandel is nie. Die byvoeging van wortmannin tydens isgemie het nie die p38 MAPK aktivering beïnvloed nie. P38 MAPK is beduidend geaktiveer tydens herperfusie in harte wat nie met insulien behandel is nie vergeleke met die insulien-behandelde harte. Die byvoeging van wortmannin tydens reperfusie kon die effek van insulien om p38 MAPK aktivering te onderdruk, gedeeltelik ophef. PKB aktivering tydens die stabilisasie fase en vroeë isgemie was beduidend hoër in die insulien-behandelde harte vs. die harte wat nie met isulien behandel is nie. Die aktiwiteit is onderdruk deur 30 min isgemie ongeag die teenwoordigheid van insulien. Die byvoeging van wortmannin tydens isgemie het PKB aktivering nie verder verlaag nie. Die teenwoordigheid van insulien het 'n sterk aktivering van PKB tydens herperfusie veroorsaak met 'n piek na 15 min en 'n verlaging na 30 min. Wortmannin bygevoeg aan die begin van herperfusie, het PKB aktiwiteit opgehef aan die einde van reperfusie. Opsomming: Insulien het 'n positiewe inotropiese invloed gehad, en het die begin van isgemiese kontraksie vertraag. Die inhibisie van Pl3-kinase deur wortmannin het die beskermende effekte van insulin opgehef, wat 'n insulin gestimuleerde PKB betrokkenheid aandui. Insulien het ook verhoogte cAMP produksie en verlaagde p38 MAPK aktivering tot gevolg gehad, en beide is geassosieer met verbeterde herstel. Hierdie resultate dui dus op moontlike interaksie tussen die verskillende seintransduksiepaaie.

Wong, Mei Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 207-251). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / PUBLICATIONS BASED ON WORK IN THIS THESIS --- p.vii / ABBREVIATIONS --- p.viii / TABLE OF CONTENTS --- p.x / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Diabetes Mellitus --- p.1 / Chapter 1.2 --- Structure and Functions of the Pancreas --- p.2 / Chapter 1.2.1 --- Size of Pancreatic β-Cells --- p.4 / Chapter 1.2.2 --- Signaling Pathways of Insulin Secretion from Pancreatic β-Cells --- p.4 / Chapter 1.3 --- Classification of Diabetes --- p.6 / Chapter 1.3.1 --- Type 1 Diabetes --- p.6 / Chapter 1.3.2 --- Type 2 Diabetes --- p.8 / Chapter 1.4 --- Pathologies of Type 2 Diabetes --- p.9 / Chapter 1.4.1 --- Hyperglycemia --- p.9 / Chapter 1.4.1.1 --- A dvanced Glycosylation End Products --- p.11 / Chapter 1.4.1.2 --- Protein Kinase C Activation --- p.13 / Chapter 1.4.1.3 --- The Glucosamine Pathway --- p.14 / Chapter 1.4.1.4 --- Oxidative Stress --- p.15 / Chapter 1.4.2 --- Insulin Resistance --- p.15 / Chapter 1.4.3 --- Loss of β-Cell Mass and β-Cell Dysfunction --- p.18 / Chapter 1.5 --- Complications of Diabetes Mellitus --- p.21 / Chapter 1.5.1 --- Cardiovascular Diseases --- p.21 / Chapter 1.5.2 --- Diabetic Retinopathy --- p.22 / Chapter 1.5.3 --- Diabetic Nephropathy --- p.23 / Chapter 1.5.4 --- Neuropathy --- p.24 / Chapter 1.6 --- Anti-Diabetic Drugs for Type 2 Diabetes Mellitus --- p.25 / Chapter 1.6.1 --- Secretagogues --- p.25 / Chapter 1.6.2 --- Sensitizers --- p.26 / Chapter 1.6.3 --- Alpha-Glucosidase Inhibitors --- p.27 / Chapter 1.6.4 --- Peptide Analogs --- p.27 / Chapter 1.6.4.1 --- Incretin Mimetics --- p.27 / Chapter 1.6.4.2 --- Dipeptidyl Peptidase-4 Inhibitors --- p.28 / Chapter 1.7 --- Insights of Porcine Islets in Treatment of Diabetics --- p.28 / Chapter 1.8 3 --- -Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMG CoA Reductase) --- p.31 / Chapter 1.8.1 --- Statins --- p.32 / Chapter 1.8.2 --- Pleiotropic Effects of Statins --- p.36 / Chapter 1.8.2.1 --- Statins and Isoprenylated Proteins --- p.36 / Chapter 1.8.2.2 --- Statins and Endothelial Functions --- p.38 / Chapter 1.8.2.3 --- Statins and Platelet Functions --- p.39 / Chapter 1.8.2.4 --- Statins and Plaque Stability --- p.39 / Chapter 1.8.2.5 --- Statins and Vascular Inflammation --- p.40 / Chapter 1.9 --- Clinical Studies of Statins on Diabetics --- p.41 / Chapter 1.10 --- Possible Factors Involved in Simvastatin-Regulated Insulin Secretion --- p.44 / Chapter 1.10.1 --- AMP-Activated Protein Kinase --- p.44 / Chapter 1.10.2 --- Caveolin-1 --- p.46 / Chapter 1.10.3 --- Sterol-Regulatory Elementary Binding Protein --- p.50 / Chapter 1.10.4 --- Protein Phosphatase 2A --- p.52 / Chapter 1.10.5 --- Calcium Sensing Receptor --- p.55 / Chapter 1.11 --- Objectives of Study --- p.59 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.60 / Chapter 2.1 --- Materials --- p.60 / Chapter 2.1.1 --- Solutions --- p.60 / Chapter 2.1.2 --- Antibodies --- p.63 / Chapter 2.2 --- Methods --- p.64 / Chapter 2.2.1 --- Maintenance of Pancreas Function --- p.64 / Chapter 2.2.2 --- Islet Isolation --- p.65 / Chapter 2.2.3 --- Hematoxylin and Eosin (H&E) Staining --- p.65 / Chapter 2.2.4 --- Simvastatin and Simvastatin-Na+ --- p.66 / Chapter 2.2.5 --- AICAR --- p.67 / Chapter 2.2.6 --- Compound C --- p.67 / Chapter 2.2.7 --- Incubation of Islets --- p.67 / Chapter 2.2.8 --- Western Blot --- p.68 / Chapter 2.2.9 --- Enzyme-Linked Immunosorbent Assay (ELISA) --- p.69 / Chapter 2.2.10 --- Statistical Analysis --- p.71 / Chapter CHAPTER 3 --- HISTOLOGY OF PORCINE PANCREATIC ISLETS OF LANGERHANS --- p.72 / Chapter 3.1 --- Comparison of Sizes of Porcine Pancreatic Islets in Histological Sections of Pancreas --- p.72 / Chapter CHAPTER 4 --- PROTEIN EXPRESSION OF HMG COA REDUCTASE --- p.75 / Chapter 4.1 --- Effect of Incubation Time on HMG CoA Reductase Expression --- p.75 / Chapter 4.2 --- Short-Term Effect of Simvastatin on HMG CoA Reductase Expression --- p.78 / Chapter 4.3 --- Long-Term Effect of Simvastatin on HMG CoA Reductase Expression --- p.81 / Chapter 4.4 --- Effect of Osmolality on HMG CoA Reductase Expression --- p.83 / Chapter 4.5 --- Effect of Simvastatin on Ser871 p-HMG CoA Reductase Expression --- p.87 / Chapter CHAPTER 5 --- EVALUATION OF THE ROLE OF SIMVASTATIN IN INSULIN SECRETION VIA HMG CO A REDUCTASE REGULATION --- p.90 / Chapter 5.1 --- Effect of Simvastatin on Insulin Secretion --- p.90 / Chapter 5.2 --- Effect of Different Concentrations of Simvastatin on Insulin Secretion --- p.94 / Chapter 5.3 --- Effect of Simvastatin on Insulin Content --- p.96 / Chapter CHAPTER 6 --- ROLE OF AMPK EXPRESSION IN INSULIN SECRETION PATHWAY --- p.100 / Chapter 6.1 --- Effect of Simvastatin on Thr172 p-AMPK α and AMPK α1 Expressions --- p.100 / Chapter 6.2 --- Evaluation of the Role of Simvastatin in AMPK Regulation --- p.104 / Chapter 6.3 --- Evaluation of the Role of PP2A in AMPK Regulation --- p.108 / Chapter 6.4 --- Evaluation of the Role of Simvastatin on Insulin Secretion via AMPK Regulation --- p.111 / Chapter 6.4.1 --- AMPK Regulation on Releasable Insulin Secretion --- p.111 / Chapter 6.4.2 --- AMPK Regulation on Non-Releasable Insulin Content and Total Insulin Content --- p.112 / Chapter CHAPTER 7 --- EFFECT OF SIMVASTATIN ON THE EXPRESSION OF REGULATORY PROTEINS INVOLVED IN INSULIN SECRETION --- p.119 / Chapter 7.1 --- Effect of Simvastatin on SREBP-2 Expression --- p.119 / Chapter 7.2 --- Effect of Simvastatin on Caveolin-1 Expression --- p.121 / Chapter 7.3 --- Effect of Simvastatin on Calcium Sensing Receptor Expression --- p.123 / Chapter CHAPTER 8 --- EFFECT OF SIMVASTATIN-NA+ ON INSULIN SECRETION --- p.126 / Chapter 8.1 --- Effect of Simvastatin-Na+ on HMG CoA Reductase Expression --- p.126 / Chapter 8.2 --- Effect of Simvastatin-Na+ on Insulin Secretion --- p.128 / Chapter 8.3 --- Effect of Different Concentrations of Simvastatin-Na+ on Insulin Secretion --- p.130 / Chapter 8.4 --- Effect of Simvastatin-Na+ on Insulin Content --- p.132 / Chapter CHAPTER 9 --- EFFECT OF PRAVASTATIN ON INSULIN SECRETION --- p.136 / Chapter 9.1 --- Effect of Pravastatin on Insulin Secretion --- p.136 / Chapter 9.2 --- Effect of Pravastatin on Insulin Content --- p.138 / Chapter CHAPTER 10 --- EFFECT OF METHYL-B-CYCLODEXTRIN ON INSULIN SECRETION --- p.142 / Chapter 10.1 --- Effect of Methyl-β-cyclodextrin on Insulin Secretion --- p.142 / Chapter 10.2 --- Effect of Methyl-β-cyclodextrin on Insulin Content --- p.144 / Chapter CHAPTER 11 --- DISCUSSION --- p.149 / Chapter 11.1 --- Importance of Studying Porcine Pancreatic Islets and Islet Distribution --- p.150 / Chapter 11.2 --- Screening of Concentration and Incubation Time of Simvastatin on Porcine Pancreatic Islets --- p.152 / Chapter 11.3 --- Glucose-Independent Effect of Simvastatin on Protein Expression of HMG CoA Reductase --- p.154 / Chapter 11.4 --- Role of AMPK in HMG CoA Reductase-Modulated Insulin Secretion --- p.159 / Chapter 11.5 --- Role of SREBP-2 in Simvastatin-Modulated Regulation --- p.174 / Chapter 11.6 --- Role of Calcium Sensing Receptor in Simvastatin-Modulated Regulation --- p.175 / Chapter 11.7 --- Role of Caveolin-1 in Simvastatin-Modulated Regulation --- p.179 / Chapter 11.8 --- "Effects of Simvastatin-Na+， Pravastatin and Methyl-β-cyclodextrin, and Importance of Endoplasmic Reticulum in Insulin Secretion" --- p.183 / Chapter CHAPTER 12 --- CONCLUSIONS AND FURTHER STUDIES --- p.197 / Chapter 12.1 --- Conclusions --- p.197 / Chapter 12.2 --- Further Studies --- p.203 / REFERENCES --- p.207

糖尿病是一种呈现流行趋势的代谢紊乱综合症，现如今，全球大约有3.46亿糖尿病患者， 这庞大的数字给各国的公共健康安全支出带来了严重的财政负担。 其中，二型糖尿病（T2DM）占90%。其特点是周围组织的胰岛素抵抗以及后期损伤的胰岛β细胞的功能。在饮食后，小肠会分泌两种肠促胰岛素，葡萄糖依赖性促胰岛素多肽（GIP）和胰高血糖素样肽-1（GLP-1）。两种多肽的主要功能是促进餐后胰岛细胞中胰岛素的分泌，另外他们还可以通过其自身的G蛋白偶联受体，GIPR和GLP-1R发挥其他作用，如葡萄糖依赖性的刺激胰岛素的生成，刺激胰岛β细胞的增殖，抑制细胞的凋亡等。这些功能也使肠促胰岛素成为糖尿病治疗的一种手段，比如Exendin-4和DPP4抑制剂。 然而，除了在胰岛中的作用，肠促胰岛还可能和脂质代谢相关，其中GIP和脂质代谢的报导研究的更加深入。在肥胖的状态下，血液中GIP含量高于正常水平；GIPR基因敲除老鼠和GIPR的抑制剂喂养的小鼠可以抵抗高脂饮食诱导的肥胖和2型糖尿病；GIP还可以直接调节脂肪细胞的脂肪生成和脂解。这些数据表明GIP在肥胖和糖尿病的发生过程中可能存在促进作用,这使得GIP治疗药物的开发需要谨慎的对待。 / 为了进一步研究GIP在脂肪细胞中发挥的生物学效应，在本研究中，我们利用腺病毒介导技术通过在脂肪细胞中过表达GIPR来增加GIP的活性，然后检查GIP在脂肪细胞中所起的作用。实验结果表明,GIP可以通过cAMP-PKA信号通路迅速并且长期的刺激脂肪细胞的炎症反应，增强IKKβ-NFκB信号通路和增加炎症基因的表达。更深入的机制研究表明，JNK 信号通路也参与GIP诱导的炎症反应，抑制JNK通路可以大部分恢复GIP增加的炎症因子的表达和IKKβ的磷酸化水平。由于长期的炎症反应，脂肪细胞的胰岛素信号通路受到GIP的损伤，在GIPR过表达的脂肪细胞中，胰岛素刺激的AKT磷酸化水平和葡萄糖吸收能力都被GIP降低，葡萄糖转运蛋白4（Glut-4）的表达水平也同时减少。因此，本研究结果表明GIP可能在肥胖的发展过程中，通过诱导脂肪细胞的炎症反应来损伤胰岛素敏感性而最终导致2型糖尿病的发生。 / Diabetes mellitus is a type of metabolic syndrome that has prevailed all over the world with the development of economic and over-nutrient lifestyle. It is estimated to 346 million diabetes patients in the worldwide most recently. The huge population put a major burden on the cost of public health care to all the countries. Among the types of diabetes, type 2 diabetes (T2DM) makes up 90% of recorded cases. The characteristics of T2DM are insulin resistance of peripheral tissues and impaired pancreatic cell function and mass. Two major incretins GIP (glucose-dependent insulinotropic peptide) and GLP-1 (glucagon-like peptide 1) are secreted from gut in response to food ingestion. The prominent role of GIP and GLP-1 is to stimulate glucose-dependent insulin release in pancreatic β cell. In addition, they both exert multiple biological effects via their relative G-protein coupled receptors, GIPR and GLP-1R, including glucose-stimulated insulin production, cell proliferation and anti-apoptosis in pancreatic β cells. The beneficent effects of incretins potentiate them as targets for the treatment of diabetes. GLP-1 analog, exendin-4 and DDP4 (dipeptidyl peptidase-4) inhibitors (to prevent GIP and GLP-1 from degradation) have been already used in clinical research. However, in addition to their effects on pancreatic β cell, both peptides are also related to lipid metabolism. The role of GIP has been studied more extensively. In obese state, the circulating level of GIP is elevated. GIPR knockout (KO) mice are resistant to high fat diet (HFD) induced obesity, a similar phenotype is found in GIPR antagonist administrated HFD-mice. Moreover, GIP also directly promotes lipogenesis and lipolysis in adipocytes. The rising evidence suggests a potential role of GIP in adipocyte biology and lipid metabolism, which diminishes the enthusiasm of GIP as a candidate therapeutic reagent for T2DM. / In order to further understand the biological effects of GIP in adipocytes, here, we over-expressed GIPR in 3T3-L1 CAR adipocytes via adenovirus-mediated gene transfer technology to enhance the activity of GIP. The results demonstrate that GIP impairs the physiological functions of adipocytes as a consequence of increasing the production of inflammatory cytokines, chemokines, and phosphorylation of IkB kinase (IKK) β through activation of the cyclic AMP-protein kinase A (cAMP-PKA) pathway. Activation of Jun N-terminal Kinase (JNK) pathway is also observed in GIP-induced inflammatory responses in adipocytes. An inhibitor of JNK blocks GIP-stimulated secretion of inflammatory cytokines and chemokines, as well as phosphorylation of IKKβ. The chronic inflammatory response eventually impairs insulin signaling in adipocytes, as demonstrated by reduction of protein kinase B (PKB/AKT) phosphorylation. The subsequently physiological analysis also indicates that GIP inhibits insulin-stimulated glucose uptake, and gene expression analysis reveals a decrease of glucose transporter 4 (Glut-4) in the meanwhile. The results suggest that GIP may be one of stimuli attributable to obesity induced insulin resistance via induction of adipocyte inflammation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Nie, Yaohui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 95-111). / 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 / Acknowledgements --- p.v / INTRODUCTION --- p.1 / Chapter Part 1 --- Obesity and Type 2 diabetes --- p.1 / Chapter 1.1 --- Introduction to diabetes --- p.1 / Chapter 1.1.2 --- Physiology of adipocyte --- p.4 / Chapter 1.1.3 --- Mechanism of obesity induced diabetes --- p.10 / Chapter Part 2 --- Incretins and T2DM --- p.12 / Chapter 2.1 --- History of incretins --- p.12 / Chapter 2.2 --- Physiological actions of incretins --- p.14 / Chapter 2.3 --- Molecular mechanism of incretin actions in pancreas --- p.16 / Chapter 2.4 --- Incretins and T2DM --- p.19 / Chapter Part 3 --- Incretins and lipid metabolism --- p.23 / Objective --- p.26 / Methods and materials --- p.28 / Chapter 1 --- Cell culture --- p.28 / Chapter 1.1 --- 3T3-L1 culture and differentiation --- p.28 / Chapter 1.2 --- 3T3-L1 CAR culture and differentiation --- p.29 / Chapter 2 --- Cloning and recombinant adenovirus construction --- p.30 / Chapter 2.1 --- Plasmid construct --- p.30 / Chapter 2.2 --- Construct of recombinant adenoviruses --- p.30 / Chapter 2.3 --- Generation and infection of the adenoviruses --- p.31 / Chapter 3 --- Physiological and morphological assays --- p.32 / Chapter 3.1 --- Lipolysis assay --- p.32 / Chapter 3.2 --- TUNEL assay --- p.32 / Chapter 3.3 --- Glucose uptake --- p.33 / Chapter 3.4 --- Glut-4 localization --- p.33 / Chapter 4 --- Gene expression analysis --- p.35 / Chapter 4.1 --- Quantitative real-time PCR --- p.35 / Chapter 4.2 --- Immunoblot analysis --- p.35 / Chapter 4.3 --- ELISA assay --- p.36 / Chapter 5 --- Isolation of primary adipocytes --- p.37 / Results --- p.38 / Chapter Part 1 --- Role of GIP in 3T3-L1 cells --- p.38 / Chapter 1.1 --- Differentiation of 3T3-L1 adipocytes --- p.38 / Chapter 1.2 --- GIP slightly stimulates phosphorylation of p-CREB and lipolysis in 3T3-L1 cells. --- p.40 / Chapter 1.3 --- Analysis of gene expression in GIP-treated adipocytes --- p.42 / Chapter 1.4 --- Discussion --- p.44 / Chapter Part 2 --- Role of GIP in GIPR over-expressing 3T3-L1 CAR adipocytes --- p.46 / Chapter 2.1 --- Differentiation of 3T3-L1 CAR adipocytes --- p.46 / Chapter 2.2 --- Functional tests in GIPR over-expressing 3T3-L1 CAR adipocytes. --- p.48 / Chapter 2.3 --- Effect of GIP on cell viability --- p.50 / Chapter 2.4 --- Analysis of gene expression in GIP-treated adipocytes --- p.52 / Chapter 2.5 --- GIP activates inflammatory responses in GIPR over-expressing adipocytes --- p.54 / Chapter 2.6 --- Inhibition of IKKb pathway restores GIP-induced inflammatory responses --- p.56 / Chapter 2.7 --- Effects of GIP on adipocytes are partially dependent on the cAMP-PKA pathway --- p.58 / Chapter 2.8 --- Activation of cAMP-PKA pathway induces adipocyte inflammation. --- p.60 / Chapter 2.9 --- cAMP-Epac pathway is not involved in GIP-induced inflammation --- p.62 / Chapter 2.10 --- GIP stimulates cell stress activated kinases --- p.64 / Chapter 2.11 --- JNK partially mediates GIP-induced adipocyte inflammation --- p.65 / Chapter 2.12 --- Inhibition of JNK pathway partially restores GIP-induced inflammatory responses --- p.67 / Chapter 2.13 --- GIP impairs insulin signaling in GIPR over-expressing 3T3-L1 CAR adipocytes via inducing inflammatory response --- p.69 / Chapter 2.14 --- GIP enhances basal glucose uptake but impairs insulin stimulated glucose uptake in 3T3-L1 CAR GIPR over-expressing adipocytes --- p.71 / Chapter 2.15 --- Discussion --- p.73 / Chapter Part 3 --- Role of GIP in primary adipocytes --- p.78 / Chapter 3.1 --- GIPR expression level in primary adipocytes --- p.78 / Chapter 3.2 --- Analysis of gene expression in primary adipocytes after GIP treatment --- p.80 / Chapter 3.3 --- Discussion --- p.81 / SUMMARY --- p.82 / Chapter Future investigation --- p.83 / Chapter Appendix 1: --- Abbreviations --- p.86 / Chapter Appendix 2: --- Protocols --- p.90 / Preparation of competent cells --- p.90 / Outlines of recombinant adenovirus preparation --- p.91 / Virus titering (TCID50) --- p.92 / Primers for real-time PCR --- p.93 / Chapter Publications and Scientfic activities --- p.94 / Thesis related publication: --- p.94 / Other pubiliations: --- p.94 / Scientific activities: --- p.94 / References --- p.95

Methylglyoxal (MG) is a reactive metabolite presents in all biological systems. The accumulation of MG in diabetic patients and animals has been long recognized. Recently, studies have shown that MG levels are elevated in hypertensive rats. However, the pathological effects of MG in diabetes and related insulin resistance syndrome such as obesity are currently unknown. In the present study, the role of MG in the pathogenesis of insulin resistance was investigated.<p> First, it was observed that MG induced structural and functional changes of insulin. Incubation of human insulin with MG in vitro yielded MG-insulin adducts, as evidenced by additional peaks observed upon mass spectrometric (MS) analysis. Tandem MS analysis of insulin B-chain adducts confirmed attachment of MG at an arginine residue. [3H]-2-deoxyglucose uptake ([3H]-2-DOG) by 3T3-L1 adipocytes was significantly and concentration-dependently decreased after treatment with MG-insulin adducts, in comparison with the effect of native insulin at the same concentration. A significant decrease of glucose uptake induced by MG-insulin adducts was also observed in L8 skeletal muscle cells. Unlike native insulin, MG-insulin adducts did not inhibit insulin release from pancreatic â-cells. The degradation of MG-insulin by cultured liver cells was also decreased. In conclusion, MG modifies insulin by attaching to internal arginine residue in the â-chain of insulin. The formation of this MG-insulin adduct decreases insulin-mediated glucose uptake, impairs autocrine control of insulin secretion, and decreases insulin clearance. These structural and functional abnormalities of the insulin molecule may contribute to the pathogenesis of insulin resistance.<p> Second, the effects of MG on the insulin signaling pathway were investigated. After 9 weeks of fructose treatment, an insulin resistant state was developed in Sprague-Dawley (SD) rats, demonstrated as increased triglyceride and insulin levels, elevated blood pressure, and decreased insulin-stimulated glucose uptake by adipose tissue. A close correlation between insulin resistance and the elevated MG accumulation in adipose and skeletal muscle tissues was observed. The insulin resistant state and the elevated MG level were reversed by the MG scavenger, N-acetyl cysteine (NAC) and metformin. In cultured adipose cells, MG treatment impaired insulin signaling as measured by decreased tyrosine phosphorylation of insulin-receptor substrate-1 (IRS-1) and the decreased kinase activity of phosphatidylinositol 3-kinase (PI3K). The ability of NAC to block MG-impairment of PI3K activity and IRS-1 phosphorylation further confirmed the role of MG in the development of insulin resistance. In cultured skeletal muscle cells, MG treatment significantly reduced the expression of IRS-1 and PI3K at the mRNA level. Similar to adipose cells, MG also decreased tyrosine phosphorylation of IRS-1 and PI3K activity. We also examined the mechanism of metformin to inhibit AGEs. Using mass spectrometry, stable metformin-MG adducts were identified.<p> In addition, we investigated the causative effect of MG in the pathogenesis of obesity, another form of insulin resistance. This study revealed a previously unrecognized effect of MG in stimulating adipogenesis by up-regulating Akt signaling. In Zucker fatty rats, dramatically increased MG accumulations in serum and different tissues were identified. The serum MG level increased age. In 10 and 12 week-old obese rats, MG was 144±50% and 171±15% of the age-matched control Zucker rats; this value increased to 241±7 % and 329±10% by 14 and 16 weeks (P<0.05, n=4). Further study suggested that MG accumulation stimulates the phosphorylation of Akt and its effectors p21 and p27. The activated Akt pathway then increased the activity of Cdk2 and accelerates the cell cycle progression and proliferation of pre-adipocytes. The effects of MG were efficiently reversed by both alagebrium, and Akt inhibitor SH-6.<p> Overall, the current study investigated the effect of MG during the pathogenesis of insulin resistance syndrome. MG, as the most potent precursor of AGEs, impairs the activity of insulin signaling pathway by glycating the insulin molecule and other insulin signaling proteins. Moreover, this study observed a previously unrecognized causative effect of MG in the proliferation of adipocytes by up-regulating the Akt signaling pathway. The results from this study offer new mechanisms to explain the development of insulin resistance and to prevent the related diseases.