Based on the results aforementioned, I went on in the second part of the study to examine the impact of aging on EDCF-mediated contractions - the alterations of COX-2-mediated endothelium-dependent contractions and the associated release of prostaglandin(s) in the aortae from aged (>18 month-old) hamsters. Endothelium-dependent contractions in the presence of NG-nitro-L-arginine methyl ester (L-NAME) were significantly greater in the aortae from aged hamsters and contractions could also be observed without L-NAME, which were sensitive to COX-2 inhibitors and TP receptor antagonists. The levels of COX-2 expression, the release of PGF2alpha and vascular sensitivity to PGF 2alpha were augmented in aortae of aged hamsters. The present results indicate a positive impact of aging on COX-2-derived PGF2alpha-mediated endothelium-dependent contractions. / In the first part of the study, I investigated whether COX-2 participated in the occurrence of endothelium-dependent contractions in the aortae from young (-3 month-old) hamsters and identified the most possible EDCF. Endothelium-dependent contractions were elicited by acetylcholine and abolished by COX-2 inhibitors (NS-398, DuP-697 and celecoxib) and thromboxane-prostanoid (TP) receptor antagonists (S 18886, L-655,240 and GR 32191), but not by COX-1 inhibitors (valeryl salicylate and sc 560). RT-PCR and Western blot analysis using aortae with and without endothelium revealed that the COX-2 expression was localized mainly in the endothelium. Levels of prostangladin F2alpha (PGF2alpha ) and prostacyclin (PGI2) increased in response to acetylcholine and the release of both prostaglandins was inhibited by COX-2 but not COX-1 inhibitors. Exogenous PGF2alpha but not PGI2 caused contractions at a concentration that corresponded to the amount released endogenously. The release of PGF2alpha was not affected by the presence of nitric oxide (NO). The results of the present study suggest that a novel constitutive role of COX-2 in endothelium-dependent contractions, with its metabolites PGF2alpha acting as a physiological EDCF in the young hamster aortae. / In the third part of the study, I investigated the relationship and the intracellular signaling cascades linking two pro-inflammatory factors Ang II and COX-2, and tested whether COX-2 mediated the Ang II-induced vascular pathogenesis. Eight hour-incubation with 100 nmol/L Ang II resulted in maximal COX-2 expression in primary rat endothelial cells and it was inhibited by losartan and RNA synthesis inhibitor (actinomycin-D). Inhibitors of either p38 MAPK or ERK1/2 (respectively SB 202190 and PD 98059) decreased the COX-2 expression, and co-treatment with both inhibitors caused an additive effect, suggesting a joint mediation through both kinases. Protein kinase C (PKC) inhibitor (GF109203X), and particularly, the specific PKCdelta inhibitor (rottlerin), prevented Ang II-induced phosphorylation of ERK1/2 and COX-2 expression, indicating an upstream regulation of ERK1/2 by PKC delta. A pivotal role of PKCdelta in Ang II-induced COX-2 expression was further supported by a similar stimulatory effect of PKC activator, signified by the Ang II-stimulated translocation of PKCdelta to the membrane and confirmed by its phosphorylation (Tyr311). Small interfering RNA targeting PKCdelta (siPKCdelta) diminished COX-2 expression, which was abrogated in siPKCdelta-treated cells treated with SB 202190, confirming the parallel pathways of PKC delta-ERK1/2 and p38 MAPK. Aortae and renal arteries from Ang II-infused rats exhibited an increased endothelial COX-2 expression and impaired acetylcholine-induced relaxation that was normalized by celecoxib. Human mesenteric arteries incubated with Ang II demonstrated elevated endothelial COX-2 and MCP-1 expressions, of which the former was inhibited by SB 202190 plus rottlerin and the latter prevented by COX-2 inhibitor celecoxib. Renal arteries from hypertensive or diabetic patients revealed an exaggerated expression of COX-2 and MCP-1 in the endothelium. The present novel findings indicate that the activation of PKCdelta-ERK1/2 and p38 MAPK is critical in Ang II-induced COX-2 up-regulation in endothelial cells, and identify a COX-2-dependent pro-atherosclerotic cytokine MCP-1. (Abstract shortened by UMI.) / Wong, Siu Ling. / Adviser: Huang Yu. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 192-228). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

胰腺的胰島具有重要的生理功能，表現在系列的荷爾蒙，特別是能夠控制血糖穩態的胰島素的合成和分泌。胰島素的功能受到各種分子信號及環境的調節。在過去的十年裡，腎素血管緊張素系統(RAS)被發現除了調節血壓和體液穩態之外還具有局部性的生理功能。根據我們最近的發現，胰島存在自有的腎素血管緊張素系統並且可能在胰島生理作用和糖尿病方面發揮新穎的作用。同時，越來越多的研究發現一些與臨床相闊的調節因子在胰島的功能和糖尿病中起著關鍵的作用。這些調節因子促進胰島素分泌並且可以調節胰島細胞的生長和凋亡。其中一些調節因子顯示出極大的研究價值。胰高血糖素樣肽-1(GLP-1)能通過它在胰島上的受體改善胰島的功能和血糖的控制;另一方面， 維生素D 也可以通過它在胰島B細胞上的受體來起到調節胰島素分泌及控制糖尿病的作用。像胰島局部RAS一樣， GLP-1 和維生素D 都可以通過它們在同一個靶器官--胰島細胞上的受體來發揮它們的功能。因此，不難想象這三種調節因子之前具有潛在的聯系並且直接或間接地影響胰島功能。此研究可以分為三部分以闡述這三種調節因子在胰島上的新穎作用(1) GLP-l 和RAS 在胰島功能上的潛在協同作用; (2)維生素D 對於胰島RAS 表達的調節作用及對膜島功能的影響；(3) 維生素D 缺乏下的胰島RAS 表達以及胰島功能的改變。 / 在第一部分的研究裡，我們檢測了阻斷血管緊張素一型受體(纈沙坦)和增強GLP-l 作用(DPPIV 抑制劑LAF237) 的復合效應對二型糖尿病小鼠(db/db) 血糖控制和胰島功能方面的影響。我們比較了接受單一給藥和聯合給藥的db/db 小鼠的胰島功能。所有的藥物處理都改善了db/db 小鼠的血糖穩態，而聯合給藥組在增加胰島B細胞面積，減少細胞凋亡，促進增殖以及降低膜島氧化應激和膜島纖維化方面體現出復合效應。另外，短期的聯合給藥顯著促進分離出來的胰島細胞的胰島素分泌。這些結果顯示了血管緊張素型受體阻斷劑和DPPIV 抑制劑在改善胰島的結構與功能以及治療二型糖尿病方面具有復合效應。 / 據研究，維生素D 是種具有抗糖尿病和高血壓作用的荷爾蒙，而不適合的RAS活性能夠減少胰島功能和糖耐量。維生素D 對腎臟腎素的直接抑制作用表明維生素D 可能可以調節胰島得局部RAS 活性進而調節胰島的生理作用。因此第二部分的實驗旨在研究維生素D 是否能夠抑制分離培養的胰島中非正常表達的胰島局部RAS組分並且改善胰島且細胞功能。維生素D 受體存在於胰島且細胞的核與質中，計量依賴性地調節受體對活性維他命D-骨化三醇的反應。骨化三醇的刺激可以通過增加維生素D24羥化黣激發胰島局部維他命D 系統的反饋機制。在分離的胰島中，長期處於高糖的環境，胰島局部RAS 的異常表達可以一定濃度的骨化三醇治療和預防。然而，骨化三醇的送科治療效果，並沒有在生理正常糖濃度的情況下被發現。另外，在高糖環境下，骨化三醇增加胰島素前體合成以及葡萄糖刺激的服島素分泌。這些結果顯示骨化三醇能夠調節以及保護高糖環境引起的異常胰島RAS 組分表達並通過增加胰島素的合成與分泌來改善胰島的功能，為在高血糖和糖尿病情況下的維生素D 與胰島功能關系提供了新的機制。 / 循環中的維生素D 水平與血糖濃度以及糖尿病的患病風險成反比。第二部分的實驗結果現實了維生素D 具有潛在的調節胰島RAS 進而調節胰島功能的作用。因此，在第三部分的實驗裡，我們假設不充足的維生素D 水平可能引起異常的胰島RAS 表達進而引起胰島功能障薇。為了這個目的，我們使用了維生素D 受體缺失的基因敲除小鼠和維生素D 缺乏小鼠來檢測糖代謝，膜島形態以及局部RAS 組分的表達。結果顯示，在缺乏維生素D 以及正常的維生素D 作用的情況下，胰島局部RAS 組分異常表達。而這個維生素D 導致的RAS 異常表達的作用可能發生在高血糖現象之前，從而導致了胰島功能障礙，異常的糖代謝以及減弱的胰島且細胞本身的胰島素作用。這些結果為在生理情況下，維生素D 可以通過調節胰島局部RAS 的表達進而調節胰島功能提供了有力的支持。 / 總括來說，胰島局部RAS 在持續高糖環境下的胰島功能中有著關鍵的作用。GLP-l 和維生素D 都與胰島RAS 具有潛在的生物相關性並可以影響RAS 的表達，進而調節胰島功能和自細胞體積。我們的實驗數據顯示了這三種調節系統共同作用並調節目突島細胞功能以及血糖穩態，進一步提議了它們在二型糖尿病治療中的價值。 / Pancreatic islets perfonn critical biological activities by means of synthesizing and releasing islet peptide honnones, notably insulin that controls our glucose homeostasis. The insulin secretory function is, in turn, governed by various conditions and signaling molecules. In the past decade, it is recognized that the renin-angiotensin system (RAS) has local function rather than the maintenance of blood pressure and fluid homeostasis. With our recent recognition of an islet RAS, it is believed that it has novel roles in islet physiology and diabetes. Meanwhile, more and more clinically relevant regulators that have pivotal roles in islet function and diabetes have been well investigated; such regulators have positive action on insulin secretion, B-cell replication and cell apoptosis/proliferation balance. Of great interest in this context is the glucagon-like peptide I (GLP-I) that improves islet function and glycemic control via its islet specific receptors located on the islets. On the other hand,vitamin D also regulates islet insulin secretion and diabetes via its mediation of receptors on islet B-cells. Like islet RAS, GLPI and vitamin D exert their biological effects via mediation of respective receptors located on the common target, i.e. the islet beta-cells. As such, it is plausible to propose that all these three regulators have potential interactions so as to affect islet functions in a direct or an indirect manner. Accordingly, the primary objective of this study is to examine the potential roles oflocal RAS, GLP-I and vitamin D system in pancreatic islet function. The present study is thus divided into three main parts addressing the issues of these three novel regulators in islet function: (1) the potential synergism of GLP-I and RAS in islet function; (2) the modulatory effects of vitamin D on islet RAS expression and function; (3) The altered islet RAS and islet function under a hypovitaminosis D condition. / In the first part of our study, we examined the combined effect of blocking islet A Tl receptor (ATl receptor blocker: valsartan) and enhancing GLP-l actions (DPP IV inhibitor: LAF237) on islet function and glycemic control in a mouse model with type 2 diabetes, db/db mice. We compared the islet function in db/db mice with either valsartan or LAF237 mono treatment or combined treatment. Consistently, all these treatments improved glucose homeostasis in db/db mice while combined treatment resulted in a significant increase in islet B-cell area by decreasing cell apoptosis and increasing proliferation, together with marked decreases of islet oxidative stress and fibrosis. In addition, a short-term effect on stimulating insulin secretion was also observed in isolated islets with combined treatment. These results indicate that the combination treatments with ATl receptor blocker and DPP IV inhibitor has beneficial additive effects on islet structure and function in type 2 diabetes, compared with their monotherapeutic treatments. / It is reported that vitamin D is a hormone with anti-diabetic and anti-hypertension effects in human while inappropriate RAS activity has been known to reduce islet function and glucose tolerance. The direct suppressive effect of vitamin D on renal renin activity indicates vitamin D may acts as a regulator in RAS activity thus modulate islet physiology. In the second part of our study, it was aimed to study whether vitamin D vitamin D downregulation of abnormal islet RAS activity improves B-cell function using an isolated pancreatic islet model. VDR was localized in islet B-cell nuclei and cytoplasm, mediated responses to active form of vitamin D calcitriol in a dose-dependent manner. This islet local vitamin D system may have its own feedback system as a marked increase ofCYP24 transcription was triggered by calcitriol stimulation. In isolated islets exposed to prolonged high glucose environment, abnormal expressed islet RAS components could be reversed or protected by calcitriol at a specific concentration. However, the inhibition effect of calcitriol on islet RAS were not observed at physiological glucose concentrations. In additon, calcitriol increased islet proinsulin synthesis and insulin secretion with hyperglycemia. These results indicated that calcitriol modulate or protect the abnormal isolated islet RAS component expression against hyperglycemia and improve islet function via increasing insulin synthesis and secretion, which might provide an alternative mechanism by which vitamin D availability enhances islet function in hyperglycemia or diabetes. / The circulating vitamin D level is inversely related to blood glucose level and risks of diabetes. Results in the second part of experiments suggested the potential RAS modulatory effect of vitamin D in isolated islets Therefore, in the third part of our study, we hypothesize that the insufficient vitamin D levels may lead to the inappropriate regulation of islet RAS expression and thus result in islet dysfunction. To achieve this, we examined the potential islet RAS-mediated effect of vitamin D on islet function by accessmg glucose homeostasis, islet histomorphology, and local RAS expression and function by means of using a vitamin D receptor knockout and diet-induced vitamin D deficiency mouse models. Results showed that the islet RAS components were abnormally expressed when lacking a sufficient vitamin D level and normal vitamin D action. These observed effects of insufficient vitamin D might occur prior to onset of hyperglycemia thus modulating islet RAS expression, which in turn lead to islet failure and dysfunctional glucose homeostasis, together with decreased insulin actions in islet B-cells. These results provide supports for the view that vitamin D physiologically exerts modulatory effects on islet function by downregulating islet RAS expression and function. / In summary, islet local RAS may have a central role in islet function under prolonged hyperglycemic stress. GLP-l and vitamin D have biological interactions with the islet RAS by downregulation of its expression and function, thereby affecting islet cell function and cell mass. Our data indicate that all three regulators work together in the regulation of pancreatic islet B-cell functions and glucose homeostasis, further suggestive of their potential values in the treatment of type 2 diabetes. / 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. / Cheng, Qianni. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves [205]-243). / 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.v / Acknowledgements --- p.viii / List of Publications --- p.x / Table of Contents --- p.xii / List of Abbreviations --- p.xvi / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Endocrine Pancreas --- p.2 / Chapter 1.1.1 --- The structure and composition of endocrine pancreas --- p.3 / Chapter 1.1.2 --- Functions of endocrine pancreas --- p.4 / Chapter 1.1.3 --- Insulin structure and insulin receptors --- p.8 / Chapter 1.1.4 --- Mechanisms of insulin secretion --- p.11 / Chapter 1.1.5 --- Mechanisms of insulin actions --- p.18 / Chapter 1.1.6 --- Disorders of the endocrine pancreas --- p.22 / Chapter 1.2 --- Diabetes mellitus --- p.23 / Chapter 1.2.1 --- Type 1 diabetes mellitus (TlDM) --- p.24 / Chapter 1.2.2 --- Type 2 diabetes mellitus (T2DM) --- p.26 / Chapter 1.2.3 --- Other types of diabetes mellitus --- p.29 / Chapter 1.2.4 --- Islet dysfunction and T2DM --- p.30 / Chapter 1.3 --- Renin-angiotensin system (RAS) --- p.33 / Chapter 1.3.1 --- Components ofRAS --- p.33 / Chapter 1.3.2 --- Tissue local RAS --- p.42 / Chapter 1.3.3 --- Pancreatic local RAS --- p.43 / Chapter 1.4 --- Glucagon like peptide-l (GLP-l) and pancreatic islet function --- p.54 / Chapter 1.4.1 --- Gastrointestinal incretin honnones --- p.54 / Chapter 1.4.2 --- GLP-l and pancreatic islet function --- p.56 / Chapter 1.4.3 --- Incretin based therapies for T2DM --- p.59 / Chapter 1.4.4 --- GLP-lIRAS axis and pancreatic islet function --- p.62 / Chapter 1.5 --- Vitamin D and pancreatic islet function --- p.64 / Chapter 1.5.1 --- Vitamin D synthesis and metabolism --- p.65 / Chapter 1.5.2 --- Vitamin D physiological functions and pancreatic islets --- p.67 / Chapter 1.5.3 --- Vitamin D and diabetes mellitus --- p.68 / Chapter 1.5.4 --- Vitamin D and RAS --- p.70 / Chapter 1.6 --- Objectives --- p.71 / Chapter Chapter 2 --- Materials and Methods --- p.73 / Chapter 2.1 --- Experimental animal models --- p.74 / Chapter 2.1.1 --- Animal model ofT2DM --- p.74 / Chapter 2.1.2 --- Animal model for pancreatic islet isolation --- p.75 / Chapter 2.1.3 --- Vitamin D receptor knockout mice (VDRKO mice) --- p.75 / Chapter 2.1.4 --- Animal model for vitamin D deficiency --- p.76 / Chapter 2.2 --- Pancreatic islet isolation and culture --- p.76 / Chapter 2.2.1 --- Mice pancreatic islet and single B-cell isolation --- p.77 / Chapter 2.2.2 --- Primary culture of isolated pancreatic islets: --- p.78 / Chapter 2.3 --- Physiological assay for pancreatic islet function --- p.78 / Chapter 2.3.1 --- Measurement of blood glucose and glucose tolerance test --- p.78 / Chapter 2.3.2 --- Measurement of glucose-induced insulin secretion --- p.79 / Chapter 2.3.3 --- Measurement of (pro )insulin biosynthesis --- p.80 / Chapter 2.4 --- Detection ofmRNA expression --- p.80 / Chapter 2.4.1 --- Design of primers --- p.81 / Chapter 2.4.2 --- mRNA extraction and cDNA synthesis --- p.82 / Chapter 2.4.3 --- Detection of mRN A expression by conventional peR --- p.83 / Chapter 2.4.4 --- SYBR Green real-time peR --- p.83 / Chapter 2.4.5 --- Real-time peR analysis using the comparative eT method --- p.84 / Chapter 2.5 --- Detection of protein expression --- p.84 / Chapter 2.5.1 --- Western blot analysis --- p.84 / Chapter 2.5.2 --- Immunostaining assessment --- p.85 / Chapter 2.6 --- In situ detection of oxidative stress, proliferation and apoptosis --- p.88 / Chapter 2.6.1 --- Detection of islet reactive oxygen species --- p.88 / Chapter 2.6.2 --- Detection of cell proliferation --- p.89 / Chapter 2.6.3 --- Measurement of cell apoptosis --- p.90 / Chapter 2.7 --- Statistical data analysis --- p.90 / Chapter Chapter 3 --- Combination of DPP-IV Inhibitor LAF237 with ATl Receptor Antagonist Valsartan Enhances Pancreatic Islet Morphology and Function in a Mouse Model of Type 2 Diabetes (This work has been published in J Pharmacal Exp Ther, 327: PI-9) --- p.91 / Chapter 3.1 --- Abstract --- p.92 / Chapter 3.2 --- Introduction --- p.94 / Chapter 3.3 --- Materials and Methods --- p.96 / Chapter 3.4 --- Results --- p.103 / Chapter 3.4.1 --- Effects of acute treatment with GLP-I and valsartan on insulin secretion in isolated islets --- p.103 / Chapter 3.4.2 --- Effects of LAF237 and valsartan on pancreatic --- p.105 / Chapter 3.4.3 --- Effects of LAF237 and valsartan on --- p.107 / Chapter 3.4.4 --- Effects ofLAF237 and valsartan on islet apoptosis --- p.109 / Chapter 3.4.5 --- Effects of LAF237 and valsartan on islet fibrosis --- p.110 / Chapter 3.4.6 --- Effects of LAF237 and valsartan on pancreatic islet superoxide and nitrotyrosine expression --- p.113 / Chapter 3.4.7 --- Effects of LAF237 and valsartan on bood glucose concentration and glucose tolerance in db/db diabetic mice --- p.116 / Chapter 3.5 --- Discussion --- p.119 / Chapter Chapter 4 --- The Role of Calcitriol in Modulating the Expression and Function of Islet Renin-Angiotensin System in Isolated Mouse Pancreatic Islets --- p.124 / Chapter 4.1 --- Abstract --- p.125 / Chapter 4.2 --- Introduction --- p.127 / Chapter 4.3 --- Materials and Methods --- p.130 / Chapter 4.4 --- Results --- p.135 / Chapter 4.4.1 --- The expression of islet VDR under different glucose conditions and the effects of calcitriol --- p.135 / Chapter 4.4.2 --- The effect of calcitriol on high glucose-modulated islet RAS component expression --- p.140 / Chapter 4.4.3 --- The protective effect of calcitriol against high glucose on islet RAS component expression --- p.144 / Chapter 4.4.4 --- The effect of calcitriol on (pro )insulin biosynthesis and insulin release in isolated islets --- p.148 / Chapter 4.5 --- Discussion --- p.151 / Chapter Chapter 5 --- Altered Islet Local Renin-Angiotensin System and Islet Function in Mice with Hypovitaminosis D --- p.158 / Chapter 5.1 --- Abstract --- p.159 / Chapter 5.2 --- Introduction --- p.160 / Chapter 5.3 --- Materials and methods --- p.163 / Chapter 5.4 --- Results --- p.168 / Chapter 5.4.1 --- Glucose homeostasis and islet morphology in VDR KO mice --- p.168 / Chapter 5.4.2 --- Expression of vitamin D receptor and major RAS components in the pancreatic islets of WT and VDR KO mice --- p.170 / Chapter 5.4.3 --- Vitamin D deficiency in mice on a vitamin D deficient diet --- p.172 / Chapter 5.4.4 --- Altered glucose homeostasis in vitamin D deficient mice --- p.174 / Chapter 5.4.5 --- Islet histomorphology in vitamin D deficient mice --- p.176 / Chapter 5.4.6 --- Regulation of islet RAS components expression in vitamin D deficient mice --- p.179 / Chapter 5.4.7 --- Transcriptional regulation of islet insulin receptor and its substrates in vitamin D deficient mice --- p.181 / Chapter 5.4.8 --- Effect of calcitriol treatment on glucose tolerance in vitamin D deficient mice --- p.183 / Chapter 5.5 --- Discussion --- p.185 / Chapter Chapter 6 --- General Discussion --- p.191 / Chapter 6.1 --- Combination effects of blocking islet RAS components and enhancing incretin activity on improving islet function in type 2 diabetes --- p.193 / Chapter 6.2 --- Potential modulatory effect of vitamin D on islet RAS expression and action --- p.196 / Chapter 6.3 --- The role of vitamin D in modulating islet RAS in glucose homeostasis and islet function --- p.199 / Chapter 6.4 --- The significance ofRAS, GLP-l and vitamin D in the management of T2DM --- p.201 / Chapter 6.5 --- Conclusion --- p.202 / Chapter 6.6 --- Future studies --- p.202 / Chapter Chapter 7 --- Bibliography --- p.205

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