The pathophysiology of cytokines released by infiltrating white blood cells upon pancreatic beta cells is not fully understood. Early growth response gene-1 (Egr-1) expression is specifically and transiently up regulated in pancreatic beta cells in response to glucose. We hypothesized that interleukin-1 beta (IL-1▀) induction of nitric oxide alters glucose induced Egr-1 transcription levels. Egr-1 levels were assessed via western blot, nitric oxide was measured with a Griess Reagent kit and insulin levels via ELISA. Glucose induced both insulin and Egr-1 production in INS-1 cells. IL-1▀ dose dependently increased nitric oxide production over time and significantly attenuated glucose induced Egr-1 expression. Sodium nitroprusside dose dependently reduced glucose induced Egr-1 production. The data suggest a strong relationship between IL-1▀ induced nitric oxide production and the reduction of glucose stimulated Egr-1 production. The pathways altered by this cytokine could provide a better understanding of the pathophysiology leading to pancreatic beta cell death.

PrediXcan, an imputed gene expression-trait association method, was compared to multiple linear regressions (MLR) of single nucleotide polymorphisms (SNPs) using the quantitative phenotypes serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL) and triglycerides (TG). The gene expression prediction models were trained using transcriptome- and genome-wide data from Depression Genes and Networks (DGN whole blood) and Genotype-Tissue Expression (GTEx) Project (GTEx whole blood, GTEx pancreas and GTEx liver). Linear combinations of the effect sizes derived using elastic net or least absolute shrinkage and selection operator (LASSO) with genotypes from 1304 European patients from the Diabetes Control and Complications Trial (DCCT) were used to estimate the genetically regulated expression (GReX) for genes. Different gene expression predictors were present in each training set. The 10-fold cross-validated predictive performance, estimated GReX, and p values from associations for matched genes were weakly correlated across training sets and strongly correlated for models derived using elastic net and LASSO. MLR models had more significant associations than PrediXcan models and larger inflation factors for p values. A comparison of p values for matched genes between PrediXcan and MLR models showed weak correlations but strong evidence for LDL and HDL associations with genes at locus 1p13.3 and 16q13, respectively. / Thesis / Master of Science (MSc)

Objective: To identify whether perceived stress is a risk-factor for higher cortisol levels and greater insulin resistance in Type 2 diabetic patients, using data from participants with and without diabetes in the National Survey of Midlife Development in the United States (MIDUS), specifically MIDUS II, Project 4. The following hypotheses were tested: (H1a) greater perceived stress would be associated with higher cortisol for Type 2 diabetic participants, (H1b) the perceived stress/cortisol relationship would be stronger for people with Type 2 diabetes than for those without it, (H2) greater perceived stress would be associated with higher Homeostatic Model Assessment-Insulin Resistance (HOMA-IR, insulin-resistance) for Type 2 diabetic participants, (H3a) subjective well-being would moderate the perceived stress/insulin resistance relationship for Type 2 diabetic participants, and (H3b) depression would moderate the perceived stress/insulin resistance relationship for Type 2 diabetic participants. Method: MIDUS, a longitudinal study of over 7,000 American adults, explores biopsychosocial factors that could contribute to variance in mental/physical health. Only complete data were utilized. Type 2 participants (n=115) consisted of 54 males and 62 females ranging in age from 36 to 81 years. Non-diabetic participants (n=1097) consisted of 470 males and 627 females ranging in age from 34 to 84 years. Results: None of the predicted relationships were statistically significant. Waist to hip ratio was significantly related to insulin resistance (r = .31, p = .001). Conclusions: Future studies should collect information about the type and duration of stressors in addition to perceptions about stress for those with Type 2 diabetes.

Dados prévios ja demostram que o controle intensivo da glicemia diminui o risco de eventos microvasculares em pacientes com diabetes mellitus. No entanto, seu efeito cardiovascular é incerto. Nós sumarizamos os dados de estudos das principais bases de dados. 2 revisores extraíram dados de estudos randomizados de pacientes com diabetes tipo 2, que visavam 2 níveis de intensidade da glicemia. Investigou-se as retinopatia, neuropatias, nefropatias, mortalidade cardiocascular e total, infarto do miocárdio (IAM), acidente vascular cerebral, amputação de membros e episódios de hipoglicemia. Realizamos a meta-análise para obter o risco relativo (RR). Foram incluídos 7 estudos com 27.814 pacientes. O controle intensivo reduziu o RR de IAM e amputação, além progressão da retinopatia, incidência de neuropatia periférica, incidência e progressão de nefropatia e microalbuminúria. Entretanto, dobrou o risco de episódios de hipoglicemia. Não houve diferenças quanto à mortalidade e outros resultados. Conclui-se que controle intensivo reduziu o risco de alguns eventos, sem reduzir a mortalidade, porém as custas do dobro da incidência de de hipoglicemia. / Previous data already show that intensive glucose control reduces the risk of microvascular events in patients with diabetes mellitus. However, its cardiovascular effect is uncertain. We summarize data from studies of the major databases. 2 reviewers extracted data from randomized studies of patients with type 2 diabetes, aimed at two intensity levels of blood glucose. We investigated the retinopathy, neuropathy, nephropathy, and total mortality cardiocascular, myocardial infarction (IAM), stroke, limb amputation and episodes of hypoglycemia. We conducted a meta-analysis to obtain the relative risk (RR). We included seven studies with 27.814 patients. The intensive control reduced the RR of IAM, and amputation, and progression of retinopathy, incidence of peripheral neuropathy, incidence and progression of nephropathy and microalbuminuria. However, it doubled the risk of hypoglycemia. There were no differences in mortality and other outcomes. We conclude that intensive control reduced the risk of some events without reducing mortality, but the expense of twice the incidence of hypoglycemia.

Fan, Rongrong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 130-150). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Kwan, Yee Mei. / Thesis (D.Nurs.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 152-171). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese; appendixes includes Chinese.

Cheng, Suosuo. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 159-173). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts aslo in English.

Poon, Chui Wa Christina. / "August 2011." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 123-131). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 論文摘要 --- p.vi / ACKNOWLEDGEMENTS --- p.ix / PUBLICATIONS --- p.x / Abstracts --- p.x / ABBREVIATIONS --- p.xii / Chapter 1. --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1. --- Diabetes --- p.1 / Chapter 1.1.1. --- Overview --- p.1 / Chapter 1.1.2. --- Diagnostic Criteria of Type-2 Diabetes --- p.2 / Chapter 1.1.3. --- Type-2 Diabetes (T2DM) --- p.3 / Chapter 1.1.3.1. --- Impaired Insulin Synthesis and Insulin Secretory Defects in Type-2 Diabetes --- p.3 / Chapter 1.1.3.2. --- β-Cell Dysfunction --- p.5 / Chapter 1.1.3.3. --- Insulin Resistance --- p.5 / Chapter 1.1.4. --- Glucose Toxicity --- p.6 / Chapter 1.1.4.1. --- Fasting Hyperglycemia --- p.8 / Chapter 1.1.4.2. --- Postprandial Hyperglycemia --- p.8 / Chapter 1.2. --- Oxidative Stress --- p.8 / Chapter 1.2.1. --- ROS and Mitochondria --- p.8 / Chapter 1.2.2. --- ROS Production by Mitochondria --- p.9 / Chapter 1.2.3. --- The Relationship of Glucose Recognition by β-cells and Oxidative Stress --- p.11 / Chapter 1.2.4. --- Important Roles of Glutathione in Pancreatic β-cells and Glutathione Synthesis --- p.14 / Chapter 1.2.5. --- N-acetyl-L-cysteine - A Potential Drug Treatment for Type-2 Diabetes? --- p.17 / Chapter 1.3. --- Role of F-actin Cytoskeleton on Glucose-induced Insulin Secretion --- p.18 / Chapter 1.4. --- Current Clinical Treatments for Type-2 Diabetes Mellitus --- p.21 / Chapter 1.4.1. --- Metformin --- p.22 / Chapter 1.4.2. --- Sulfonylureas --- p.22 / Chapter 1.4.3. --- Thiazolidinediones --- p.23 / Chapter 1.4.4. --- Glinides (Meglitinide Analogues) --- p.23 / Chapter 1.4.5. --- α-Glucosidase (AG) Inhibitors --- p.24 / Chapter 1.4.6. --- Dipeptidyl Peptidase-4 (DPP-4) Inhibitors --- p.24 / Chapter 1.4.7. --- (Clinical) Antioxidant Treatment --- p.24 / Chapter 1.5. --- Animal Models Used in Type-2 Diabetes Research --- p.25 / Chapter 1.6. --- Aims of Study --- p.27 / Chapter 2. --- RESEARCH DESIGN & METHODS --- p.28 / Chapter 2.1. --- Materials --- p.28 / Table 1. Sources and concentrations of drugs tested in this study: --- p.28 / Culture Medium - --- p.29 / General Reagents --- p.29 / Chapter 2.2. --- Isolation of Islets of Langerhans and Single Pancreatic β-Cells --- p.31 / Chapter 2.3. --- Measurement of Mitochondrial ROS Levels --- p.32 / Chapter 2.4. --- Measurement of Islets Insulin Release and Insulin Content --- p.34 / Chapter 2.4.1. --- Preparation of Samples --- p.34 / Chapter 2.4.2. --- Enzyme-Link Immunosorbent Assay (ELISA) --- p.35 / Chapter 2.5. --- Immunocytochemistry --- p.35 / Chapter 2.6. --- Data and Statistical Analysis --- p.37 / Chapter 3. --- RESULTS --- p.38 / Chapter 3.1. --- "Effects of L-NAC, Various Oxidative Stress Inducers/Reducers and Actin Polymerisation/Depolymerisation Inducers on Releasable Insulin Levels and Insulin Contents in Response to Low Glucose (5 mM) and High Glucose (15 mM) of Isolated Pancreatic Islets of (db+/m+) and (db+/db+) Mice" --- p.38 / Chapter 3.1.1. --- Effect of L-NAC on Insulin Secretion and Insulin Contents --- p.38 / Chapter 3.1.2. --- Effect of Cytochalasin B on Insulin Secretion and Insulin Contents --- p.39 / Chapter 3.1.3. --- Effect of 4-Phenyl Butyric Acid on Insulin Secretion and Insulin Contents --- p.43 / Chapter 3.1.4. --- Effect of Ursodeoxycholic Acid on Insulin Secretion and Insulin Contents --- p.46 / Chapter 3.1.5. --- Effect of Hydrogen Peroxide on Insulin Secretion and Insulin Contents --- p.49 / Chapter 3.1.6. --- Effect of Jasplakinolide on Insulin Secretion and Insulin Contents --- p.53 / Chapter 3.1.7. --- Effect of Thapsigargin on Insulin Secretion and Insulin Contents --- p.57 / Chapter 3.1.8. --- Effect of BSO on Insulin Secretion and Insulin Contents --- p.61 / Chapter 3.2. --- "Effects of L-NAC, Various Oxidative Stress Inducers/Reducers and Actin Polymerisation/Depolymerisation Inducers on Mitochondrial ROS Levels in Response to High Glucose (15 mM) Challenge in Isolated Single Pancreatic β-Cells of (db +/m+) and (db +/db +) Mice" --- p.65 / Chapter 3.2.1. --- "Effects of L-NAC (20 mM), 4-Phenyl Butyric Acid (4-PBA) (1 mM), Ursodeoxycholic Acid (UA) (500 μg/ml), H202 (200 μM), Thapsigargin (0.5 μM) and DL-Buthionine-[S,R]-Sulfoximine (BSO) (0.1 μM) Pre-treatments on Mitochondrial ROS Level in Response to High Glucose (15 mM) Challenge" --- p.65 / Chapter 3.2.2. --- "Effects of L-NAC (20 mM), Cytochalasin B (10 μM) and Jasplakinolide (5 μM) Pre-treatments on Mitochondrial ROS Level in Response to High Glucose (15 mM) Challenge_" --- p.76 / Chapter 3.3. --- "Effects of L-NAC, Various Oxidative Stress Inducers/Reducers and Actin Polymerisation/Depolymerisation Inducers on F-actin Cytoskeleton Levels Incubated in Low Glucose (5 mM) and High Glucose (15 mM) Medium in Single Pancreatic β-Cells of Non-Diabetic (db +/m+) and Diabetic (db +/db +) Mice" --- p.81 / Chapter 4. --- DISCUSSION --- p.100 / Chapter 4.1. --- General Discussion --- p.100 / Chapter 5. --- SUMMARY --- p.120 / Chapter 6. --- FUTURE PERSPECTIVES --- p.121 / Chapter 7. --- REFERENCES --- p.123

囊性纖維化（CF）是由囊性纖維化跨膜電導調節器（CFTR）的突變引起的一種隱性遺傳病。CF病人的肺、肝、胰腺、腸道與生殖道受到嚴重影響，其中有50%的成年病人患有糖尿病。由CF引起的糖尿病被稱為CF相關糖尿病（CFRD）, 关于它的病因至今仍然存有爭議。2007年，人們發現CFTR在分泌胰島素的胰島β細胞上有表達。儘管如此，β細胞上的CFTR与糖尿病发病的关系却一直被忽略。我們的研究目標是闡述β細胞上的CFTR在胰島素分泌中的作用。 / 在β細胞上，葡萄糖刺激的胰島素分泌伴隨著複雜的電活動，這種電活動被描述為細胞膜電位去极化疊加的動作電位的爆發。葡萄糖引起的ATP敏感的鉀離子通道（K[subscript Asubscript Tsubscript P]）的關閉被普遍認為是β細胞去極化的初始事件，初始的去極化啟動了電壓依賴的鈣離子通道，由此產生的鈣離子內流成為構成動作電位的去極化電流，引起了細胞內鈣離子的震盪，從而引起胰島素的釋放。雖然氯離子電流被認為參與了β細胞去極化電流，但是，人們仍然不能確定是哪一種氯離子通道介導了這個去極化電流。在我們研究的第一部分，CFTR被證明功能性的表達在β細胞上，並且可以被葡萄糖激活。CFTR可以被葡萄糖激活这一性质，在CFTR超表達的CHO 细胞上被進一步驗證。在原代培養的β細胞與β細胞株RIN-5F细胞中的葡萄糖引起的全細胞電流、膜電位的去極化、動作電位的幅度與頻率、鈣震盪和胰島素的分泌可以被CFTR的抑制劑或缺陷所降低。與野生型小鼠相比，CFTR基因敲除的小鼠，禁食之後，具有更高的血糖濃度，然而其胰島素的濃度低。 / 我們研究中的第二部分，利用了數學模型去闡明CFTR 在胰島素分泌的電活動中的角色。結果顯示， CFTR電導的減低可以使細胞的細胞膜去極化，從而導致需要更高的電刺激去引發動作電位，这些結果證明了CFTR對於维持細胞膜電位的貢獻。同時增加細胞內氯離子濃度和CFTR的電導可以引起更大頻率的膜電位的震盪，這一點證明了氯離子對於細胞膜電位震盪有著重要的作用。在数学模型中，CFTR電導的降低可以消除通過改變ATP/ADP值所引起的電火花， 這與我們在試驗中發現的CFTR參與了葡萄糖引起的動作電位是一致的。總而言之，我們的数学模型證明了CFTR對於胰島素的分泌是非常重要的，它通過介導氯離子外流對細胞膜電位的產生貢獻並且參與了電火花的產生，所有這些都進一步驗證了我們在實驗部分的發現。 / 综上所述，現有的研究揭示了CFTR，通過對β細胞膜電位作用與参与了動作電位的產生，在葡萄糖刺激胰島素分泌过程中的鮮為人知的重要角色。這個發現為揭示CFRD的病理機制提供了全新的視角，並且可能為開發治療CFRD的方法帶来了曙光。 / Cystic fibrosis (CF) is a recessive autosomal genetic disease resulted from mutations of cystic fibrosis transmembrane conductance regulator (CFTR). CF affects critically the lung, liver, pancreas, intestine and reproductive tract. CF patients also exhibit a high percentage of diabetes, which almost reach 50% in adult. The pathological cause of diabetes in CF patients, also called CF related diabetes (CFRD), is still controversial. It has been reported that CFTR expressed in the islet β cells, which is responsible for insulin secretion. However, the exact role of CFTR in islet β-cell and its relation to diabetes have been ignored. The present study aims to elucidate the role of CFTR in the process of insulin secretion by pancreatic islet β cells. / Glucose-stimulated insulin secretion is associated with a complex electrical activity in the pancreatic islet β-cell, which is characterized by a slow membrane depolarization superimposed with bursts of action potentials. Closing ATP-sensitive K⁺ channels (K[subscript Asubscript Tsubscript P]) in response to glucose increase is generally considered the initial event that depolarizes the β-cell membrane and activates the voltage-dependent Ca²⁺ channels, which constitutes the major depolarizing component of the bursting action potentials giving rise to the cytosolic calcium oscillations that trigger insulin release. While Cl⁻ has been implicated in an unknown depolarization current of the β-cell, the responsible Cl⁻ channel remains unidentified. In the first part of our study, we show functional expression of CFTR and its activation by glucose in the β-cell. Activation of CFTR by glucose was also demonstrated in CHO cell over-expression system. The glucose-elicited whole-cell currents, membrane depolarization, electrical bursts (both magnitude and frequency), Ca²⁺ oscillations and insulin secretion could be abolished or reduced by inhibitors/knockdown of CFTR in primary mouse β-cells or RIN-5F β-cell line, or significantly attenuated in isolated mouse islet β-cells from CFTR mutant mice compared to that of wildtype. Significantly increased blood glucose level accompanied with reduced level of insulin is found in CFTR mutant mice compared to the wildtype. The results strongly indicate a role of CFTR in the process of insulin secretion. / In the second part of our study, mathematical model is built up to clarify the role of CFTR in the electrical activity during insulin secretion. It is shown that reduction of CFTR conductance hyperpolarizes the membrane of the β-cell, for which it requires a larger electrical stimulus to evoke an action potential, indicating the contribution of CFTR to the membrane potential as demonstrated by our experimental results. Increase in intracellular Cl⁻ concentration and the conductance of CFTR result in higher frequency of membrane potential oscillations, demonstrating that Cl⁻ is crucial for the membrane potential oscillations. The electrical spikes induced by increase of ATP/ADP in the model are abolished by decreasing CFTR conductance, which is consistent with our findings that CFTR is involved in the generation of action potentials induced by glucose. In other word, our model demonstrates that CFTR is crucial for insulin secretion by its contribution to membrane potential and participating in the generation of electrical spikes via conducting Cl⁻ efflux, which confirms our findings in the experimental study. / Taken together, the present study reveals a previously unrecognized important role of CFTR in glucose-stimulated insulin secretion via contributing to the membrane potential and the participating in the generation of action potential in islet β cells. This finding sheds new light into the understanding of the pathogenesis of CFRD and may provide grounds for the development of new therapeutic approaches for CFRD. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Guo, Jinghui. / "December 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 156-164). / 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 PUBLICATIONS --- p.vi / Declaration --- p.viii / ABBREVIATIONS --- p.xi / LIST OF FIGURES --- p.xiii / Chapter Chapter 1: --- General introduction --- p.1 / Chapter 1.1 --- The function of islet β cells and diabetes --- p.1 / Chapter 1.1.1 --- The introduction of the pancreas --- p.1 / Chapter 1.1.2. --- Glucose metabolism and blood glucose regulation --- p.6 / Chapter 1.1.2.2 --- Blood glucose regulation --- p.7 / Chapter 1.1.3 --- Insulin secretion by the islet β cell --- p.10 / Chapter 1.1.4 --- Diabetes --- p.14 / Chapter 1.2 --- Cystic fibrosis-related diabetes --- p.17 / Chapter 1.2.1 --- Cystic fibrosis --- p.17 / Chapter 1.2.2 --- CFTR --- p.19 / Chapter 1.3 --- Mathematical model for insulin secretion --- p.25 / Chapter 1.4 --- Aim and hypothesis --- p.27 / Chapter 1.4.1 --- CFTR may be activated by glucose --- p.27 / Chapter 1.4.2 --- Activation of CFTR may depolarize the membrane potential --- p.28 / Chapter 1.4.3 --- CFTR-mediating Cl-efflux may be involved in the generation of electrical spikes --- p.28 / Chapter 1.4.4 --- Calcium oscillation depends on CFTR --- p.28 / Chapter 1.4.5 --- Insulin secretion --- p.29 / Chapter 1.5 --- Approaches to test the hypothesis --- p.29 / Chapter Chapter 2: --- Materials and Methods --- p.31 / Chapter 2.1 --- Cell culture --- p.31 / Chapter 2.1.1 --- RIN-5F cell --- p.31 / Chapter 2.1.2 --- CHO cell --- p.31 / Chapter 2.2 --- Islet isolation and culture --- p.32 / Chapter 2.3 --- CFTR knockdown --- p.33 / Chapter 2.4 --- Western blot --- p.35 / Chapter 2.5 --- Immunofluorescence --- p.37 / Chapter 2.6 --- Membrane potential (Vm) measurement --- p.38 / Chapter 2.7 --- Intracellular chloride imaging --- p.39 / Chapter 2.8 --- Intracellular calcium imaging --- p.40 / Chapter 2.9 --- Patch-clamp --- p.40 / Chapter 2.10 --- Blood glucose measurement --- p.42 / Chapter 2.11 --- Insulin ELISA --- p.42 / Chapter 2.12 --- Statistics --- p.42 / Chapter Chapter 3: --- Contribution of CFTR on the eletrophysiological properties in insulin secretion --- p.43 / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.2 --- Results --- p.45 / Chapter 3.2.1 --- Functional expression of CFTR in mouse islet β cells --- p.45 / Chapter 3.2.2 --- CFTR activation by glucose --- p.46 / Chapter 3.2.3 --- Involvement of CFTR in the maintenance of membrane potential of islet β cells --- p.47 / Chapter 3.2.4 --- CFTR is involved in the generation of spikes induced by glucose --- p.50 / Chapter 3.2.5 --- Generation of spikes burst in the β cell depends on intracellular chloride. --- p.52 / Chapter 3.2.6 --- Inhibition/mutation of CFTR attenuates calcium oscillation induced by glucose --- p.53 / Chapter 3.2.7 --- Inhibition/mutation of CFTR impairs insulin secretion --- p.53 / Chapter 3.3 --- Discussion --- p.71 / Chapter Chapter 4: --- Mathematical model for the role of CFTR in the process of insulin secretion in islet β cell --- p.74 / Chapter 4.1 --- Introduction to the mathematical modeling in the process of insulin secretion --- p.74 / Chapter 4.2 --- Methods --- p.77 / Chapter 4.2.1 --- Components in the model --- p.77 / Chapter 4.2.2 --- Assumptions and approaches in modeling --- p.78 / Chapter 4.2.3 --- Modeling ion channels and transporters --- p.79 / Chapter 4.2.3.1 --- KATP channel --- p.79 / Chapter 4.2.3.2 --- Sodium channel --- p.82 / Chapter 4.2.3.3 --- Voltage Dependent calcium channel --- p.83 / Chapter 4.2.3.4 --- NCX --- p.84 / Chapter 4.2.3.5 --- Na-K pump --- p.85 / Chapter 4.2.3.6 --- Kv channel --- p.87 / Chapter 4.2.3.7 --- Ca pump --- p.88 / Chapter 4.2.3.9 --- CFTR --- p.90 / Chapter 4.2.3.10 --- NKCC --- p.91 / Chapter 4.3 --- Results --- p.93 / Chapter 4.3.1 --- Role CFTR in regulation of the basal membrane potential in β cells --- p.93 / Chapter 4.3.2 --- Role of intracellular chloride concentration in the burst spikes induced by glucose --- p.95 / Chapter 4.3.3 --- Role of CFTR in the burst spikes induced by glucose --- p.96 / Chapter 4.4 --- Discussion --- p.105 / Chapter Chapter 5: --- General discussion and conclusion --- p.109 / Chapter 5.1 --- General discussion --- p.109 / Chapter 5.1.1 --- Role of CFTR in endocrine pancreas and diabetes --- p.109 / Chapter 5.1.2 --- Role of CFTR as a cell metabolic sensor --- p.111 / Chapter 5.1.3 --- Role of CFTR in excitable cells --- p.113 / Chapter 5.2 --- Conclusion --- p.114 / Appendix A --- p.115 / Appendix B --- p.118 / Reference: --- p.156

糖尿病是一種以不正常的高血糖為主要特徵的長期性的糖代謝紊亂疾病。二型糖尿病是常見的糖尿病類型，多於九成的糖尿病病人患有此種類型。各種引起糖尿病的病因最終都會導致血糖過高，並且最終會引起有關眼睛，腎臟，神經和血管系統的併發癥。迄今，糖尿病正影響著大約世界6%的人口，而現在患病率依然在逐年增加。在香港，由於高能量的食和缺乏運動，越來越多的老年人和青年人正在遭受著糖尿病的困擾。糖尿病不是一種致命性的疾病，但是如果沒有採取好的治療控制措施，糖尿病最終會引起一些併發癥，這些併發癥最終會使糖尿病患者走向死亡。高血糖癥不僅是糖尿病的主要特徵，而且也是引起各種糖尿病併發癥的重要因素，在二型糖尿病的治療當中，根據各種病理因素，市場上已經研製出了很多西藥來治療糖尿病。然而，它們都有一些副作用的限制。因此，我們需要通過綜合治療和通過新的途徑研製新的製劑來控制血糖水平，保護病人遠離長期併發癥的困擾。如今，腎臟在血糖平衡中的重要角色已經被很好的認知。 在過去的二十年裡， 通過減少血糖在腎臟的重吸收來增加尿液中血糖的排出，從而達到降低體內血糖水平的方法已經被提出并認為是治療糖尿病的一直新的途徑。 在腎臟中，鈉葡萄糖共轉運體2（SGLT 2）主要負責葡萄糖的重吸收，因此，鈉葡萄糖共轉運體2（SGLT 2）抑製劑被認為是一種有潛質的新型的治療糖尿病的製劑。然而，市場上至今沒有成功研製這種製劑。达格列嗪（dapagliflozin），作為一種最有潛質的鈉葡萄糖共轉運體2（SGLT 2）抑製劑，依然處於臨床三期實驗。至今，對具有鈉葡萄糖共轉運體2（SGLT 2）抑製作用的天然產物和傳統中醫藥的信息報導非常少。中醫中藥的治療理念強調整體治療，從此點看來，爲了使糖尿病患者遠離長期的糖尿病併發癥的困擾，中醫中藥可能比西藥更有優勢。 / 因此，本研究的目的是尋找那些具有體外能專門抑制鈉葡萄糖共轉運體2（SGLT 2）並且體內能通過增加尿糖排出來降低血糖水平的抗糖尿天然產物或傳統中藥。從文獻分析中找到了經常用於治療糖尿病的11種中藥和兩種天然產物。 / 試管實驗確立了五味子醇提物和丹皮酚對表達了人的鈉葡萄糖共轉運體2（SGLT 2）基因的COS 7細胞鏈中鈉葡萄糖共轉運體2對¹⁴C-α-甲基- D-葡萄糖苷的吸收作用具有很強的抑制作用。 / 生物活性引導的片段分析確立了五味子醇提物中的活性片段--乙酸乙酯：甲醇（4:6）（F8）片段具有明顯的專門抑制鈉葡萄糖共轉運體2的作用。本實驗也對F8進行了高效液相色譜和液質聯用色譜分析。五味子中三種常見的化合物：五味子甲素，五味子乙素和五味子醇甲存在于F8中，但濃度都很低。試管實驗顯示，這三種常見化合物均無抑制鈉葡萄糖共轉運體2的作用。因此得出結論，這三種常見的五味子化合物不是F8中有效的抑制鈉葡萄糖共轉運體2的活性成份。 / 本實驗也利用動物實驗調查了丹皮酚的抗糖尿作用。糖尿病大鼠被餵食了三個星期的丹皮酚，基礎血糖實驗和尿糖排出實驗均無陽性結果。 / Diabetes Mellitus (DM) is a chronic disorder of glucose metabolism characterized by abnormally high blood glucose level. Type 2 DM is the common form of diabetes which accounts for more than 90% of all DM cases. All causes of diabetes ultimately lead to hyperglycemia, and it can cause the late complications involving the eyes, kidneys, nerves and blood vessels, which are harmful to health. DM is now affecting about 6% population of the world, and the prevalence is still increasing quickly year by year. In Hong Kong, more and more elderly and youth are suffering from diabetes because of lacking of exercise and high energy diet. DM is not a fatal disease, but if no good action is taken, it can finally cause some kinds of complications, which can lead the patients to the end of their lives. Hyperglycemia is the major characteristics of diabetes, and it is also an important factor which induces all kinds of diabetic complications. In the therapy of type 2 diabetes, a lot of western medicine have been developed in the market according to various pathological causes. However, they have limitations such as existence of side effects. Therefore, combination therapy and development of new agents with novel mechanisms should be required to control the glycemic level and protect the patients from the long-term complications. Nowadays, the significance of the kidney's role in glucose homeostasis is well recognized. Glucose excretion with urine by reducing the renal glucose reabsorption to attenuate the glycemic level has been considered as a new mechanism to treat diabetes since the past two decades. Inhibitors on sodium glucose co-transporters 2 (SGLT 2) which are responsible for the glucose reabsorption in kidney are considered as a kind of new agents that have a potential on the treatment of diabetes. However, there is still no such kind of drug developed in the market, since the most potential one, dapagliflozin, is still on Phase III clinical trial. So far, only few information is found on natural products/traditional Chinese medicines (TCMs) that possess SGLT inhibitory action. Regarding the protection of patients from long-term complications, Chinese medicine which consider the body as a whole, may have advantages over western drugs. / Therefore, the aim of this study is to search for anti-diabetic TCM/natural products which specifically inhibit the activity of SGLT2 in vitro and attenuate plasma glucose level in vivo via increasing glucose excretion through urination. From literature review, 11 TCMs and 2 natural products frequently used in treating DM were selected for screening. / Using hSGLT 1 and hSGLT 2-expressed COS-7 cell lines as a model, in vitro study demonstrated that Fructus Schisandrae chinensis (ethanolic extract) and paeonol posses the most potent inhibitory effect on SGLT 2 in the in vitro ¹⁴C-α-methyl-D-glucopyranoside (¹⁴C-AMG) uptake assay. / The purification of active fraction(s) in ethanolic extract of Schisandrae chinensis fructus was carried out using the bioassay-guided fractionation assay. The ethyl acetate-methanol (4:6) fraction (F8) was selected with significant specific inhibitory effect on SGLT 2. UPLC and LC/MS-MS profiles of F8 were also given in this study. The concentrations of three common compounds of Fructus Shisansrae chinensis: deoxyschisandrin, schisandrin B (γ-schisandrin) and schisandrin were shown very low concentration in F8, the results of uptake assay showed none of these three compounds have inhibitory effects on SGLT 2. It is concluded that these three common compounds in Schisandrae chinensis fructus are not the effective ingredients in F8 which can specifically inhibit SGLT 2. / The anti-diabetic effects of paeonol in treating type 2 DM was investigated in animal study. Paeonol (200 and 300 mg/mL) was given to the type 2 diabetic rat model - Zucker Diabetic Fatty (ZDF) rats for three weeks, the results showed no positive effects on the basal glycaemia test and urinary glucose excretion test. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Qu, Yue. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 141-153). / Abstracts also in Chinese. / TABLE OF CONTENTS / ABSTRACT --- p.iv / 摘要 --- p.vii / ACKNOWLEDGEMENT --- p.ix / LIST OF ABBREVIATIONS --- p.x / LIST OF TABLES --- p.xiii / LIST OF FIGURES --- p.xiv / TABLE OF CONTENTS --- p.1 / Chapter CHAPTER 1 --- INTRODUCTION --- p.8 / Chapter 1.1 --- Definition, diagnosis, classification and epidemiology of Diabetes Mellitus --- p.8 / Chapter 1.1.1 --- Definition of Diabetes Mellitus --- p.8 / Chapter 1.1.2 --- Diagnosis of Diabetes Mellitus --- p.8 / Chapter 1.1.3 --- Classification of Diabetes Mellitus --- p.9 / Chapter 1.1.4 --- Prevalence of Diabetes Mellitus --- p.11 / Chapter 1.2 --- Glucose Homeostasis and Diabetes Mellitus --- p.12 / Chapter 1.2.1 --- General Description --- p.12 / Chapter 1.2.2 --- Kidney's role in Glucose Homeostasis --- p.14 / Chapter 1.2.2.1 --- Gluconeogenesis in the Kidney --- p.15 / Chapter 1.2.2.2 --- Glucose Reabsorption in the Kidney --- p.15 / Chapter 1.2.2.3 --- Renal glucose transporters --- p.17 / Chapter 1.2.2.4 --- Disorders with abnormal renal glucose transport --- p.19 / Chapter 1.3 --- Etiology of Diabetes Mellitus --- p.20 / Chapter 1.3.1 --- Pancreatic β cell dysfunction --- p.21 / Chapter 1.3.2 --- Insulin resistance --- p.21 / Chapter 1.4 --- Diabetic complications --- p.23 / Chapter 1.5 --- Treatment of type 2 Diabetes Mellitus --- p.25 / Chapter 1.5.1 --- Conventional therapy of type 2 Diabetes Mellitus --- p.25 / Chapter 1.5.2 --- New mechanism for the treatment of type 2 Diabetes Mellitus - Inhibition of glucose reabsorption by glucose transporters in Kidney --- p.29 / Chapter 1.6 --- Traditional Chinese Medicine for Diabetes Mellitus --- p.30 / Chapter 1.7 --- Project objective --- p.33 / Chapter CHAPTER 2 --- TRADITIONAL CHINESE HERBAL MATERIALS AND NATURAL PRODUCTS --- p.36 / Chapter 2.1 --- Materials --- p.36 / Chapter 2.2 --- General description and anti-diabetic effects of selected herbs/natural products --- p.38 / Chapter 2.3 --- Extraction Method --- p.45 / Chapter CHAPTER 3 --- IN VITRO STUDIES OF THE INHIBITORY EFFECT OF SELECTED TRADITIONAL CHINESE HERBS AND NATURAL PRODUCTS ON SODIUM GLUCOSE COTRANSPORTERS (SGLT) --- p.48 / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.2 --- Materials --- p.49 / Chapter 3.3 --- Methods and Methods --- p.52 / Chapter 3.3.1 --- In vitro model for screening of SGLT inhibitor --- p.52 / Chapter 3.3.1.1 --- Preparation of hSGLT1 and hSGLT2 Plasmid --- p.52 / Chapter 3.3.1.2 --- Transient Transfection of SGLT1 or SGLT2 clone --- p.53 / Chapter 3.3.1.3 --- Detection of mRNA expression level by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.54 / Chapter 3.3.1.4 --- Development of SGLT1 or SGLT2 stable cell lines --- p.56 / Chapter 3.3.1.5 --- Results --- p.56 / Chapter 3.3.2 --- Cell proliferation assay (MTT assay) --- p.57 / Chapter 3.3.2.1 --- Methods --- p.57 / Chapter 3.3.2.2 --- Results --- p.58 / Chapter 3.3.3 --- Uptake Assay of ¹⁴C-α-methyl-D-glucopyranoside (¹⁴C-AMG) in cultured COS-7 cells expressing SGLT1 or SGLT2 --- p.63 / Chapter 3.3.3.1 --- Methods --- p.63 / Chapter 3.3.3.2 --- Screening Results of Effective Chinese Herbs/Natural Products --- p.64 / Chapter 3.4 --- Discussion --- p.83 / Chapter CHAPTER 4 --- FRACTIONATION OF SCHISANDRAE CHINENSIS FRUCTUS --- p.86 / Chapter 4.1 --- Introduction --- p.86 / Chapter 4.2 --- Organic Extraction of Schisandrae Chinensis Fructus --- p.86 / Chapter 4.2.1 --- Material and Methods --- p.86 / Chapter 4.2.2 --- Result --- p.86 / Chapter 4.3 --- Bioassay-guided Fractionation of Ethanolic Extract of Schisandrae Chinensis Fructus --- p.87 / Chapter 4.3.1 --- Materials --- p.87 / Chapter 4.3.2 --- Methods --- p.87 / Chapter 4.3.2 --- Results --- p.89 / Chapter 4.4 --- ¹⁴C-α-methyl-D-glucopyranoside (¹⁴C-AMG) Uptake Assay of fractions in cultured COS-7 cells expressing SGLT1 or SGLT2 --- p.92 / Chapter 4.4.1 --- Methods --- p.92 / Chapter 4.4.2 --- Results --- p.93 / Chapter 4.5 --- Characterization of F8 of Schisandrae chinensis fructus using Ultra Performance Liquid Chromatography (UPLC) --- p.98 / Chapter 4.5.1 --- Introduction --- p.98 / Chapter 4.5.2 --- Materials and Methods --- p.98 / Chapter 4.5.3 --- UPLC chromatograms --- p.99 / Chapter 4.6 --- Characterization of F8 using Liquid Chromatography/Mass Spectrometry-Mass Spectrometry (LC/MS-MS) --- p.101 / Chapter 4.6.1. --- Materials --- p.101 / Chapter 4.6.2 --- Methods --- p.102 / Chapter 4.6.3 --- Results --- p.103 / Chapter 4.7 --- ¹⁴C-α-methyl-D-glucopyranoside (¹⁴C-AMG) Uptake Assay of three chemical standards in cultured COS-7 cells expressing SGLT1 or SLGT2 --- p.108 / Chapter 4.7.1 --- Methods --- p.108 / Chapter 4.7.2 --- Results --- p.108 / Chapter 4.8 --- Discussion --- p.111 / Chapter CHAPTER 5 --- IN VIVO STUDIES OF THE ANTI-DIABETIC EFFECT OF SELECTED TRADITIONAL CHINESE HERBS AND NATURAL PRODUCTS IN TYPE 2 DIABETIC RAT MODEL --- p.114 / Chapter 5.1 --- Introduction --- p.114 / Chapter 5.1.1 --- Diabetic Animal Models --- p.114 / Chapter 5.2 --- In vivo Study Tests --- p.117 / Chapter 5.2.1 --- Introduction --- p.117 / Chapter 5.2.2 --- Animals --- p.117 / Chapter 5.2.3 --- Methods --- p.118 / Chapter 5.2.4 --- Results --- p.120 / Chapter 5.3 --- Discussion --- p.125 / Chapter CHAPTER 6 --- GENERAL DISCUSSION --- p.128 / Chapter 6.1 --- Importance of SGLT --- p.128 / Chapter 6.2 --- Current developed SGLT 2 Inhibitors --- p.130 / Chapter 6.3 --- Importance and Treatment of DM by TCMs --- p.132 / Chapter 6.4 --- Screening and Developing drugs from Traditional Chinese medicinal plants --- p.134 / Chapter 6.5 --- Limitations and Improvements --- p.136 / Chapter 6.6 --- Future Works --- p.137 / Chapter 6.7 --- Conclusions --- p.139 / REFERENCES --- p.141