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

<p>This thesis examines the effect of surgical weight loss on gut and adipose tissue peptides involved in appetite regulation and energy homeostasis in morbidly obese humans. Roux-en-Y gastric bypass (RYGBP) is the gold standard operation used for effective long-term weight loss and improved health. The exact mechanisms for this outcome are under investigation.</p><p>We measured ghrelin, a recently discovered hunger hormone, insulin, adiponectin and leptin along with anthropometry measures in 66 morbidly obese patients prior to and 6 and 12 months after RYGBP. Impressive weight loss occurred postoperatively as did alterations in the peptides. Consistent correlations were found between weight, leptin, ghrelin and insulin. The main findings were low ghrelin concentrations in obesity and an increase after RYGBP.</p><p>We explored inflammatory proteins C-reactive protein (CRP), serum amyloid A and interleukin-6 before and during massive weight loss 6 and 12 months after RYGBP in morbidly obese subjects. The studied proteins declined after surgery and a correlation between CRP and homeostatic model of assessment for insulin resistance, independent of BMI, strongly linked insulin resistance and inflammation. CRP declined most in insulin-sensitive subjects.</p><p>We examined the excluded stomach mucosa and vagus nerve by measuring gastrin, pepsinogen I (PGI), pancreatic polypeptide (PP) and ghrelin levels during week 1 and year after RYGBP. Ghrelin levels rose with weight loss but declined 24-hours after surgery, like PP, indicating transient vagal nerve damage. Low levels of gastrin and PGI suggest a resting mucosa.</p><p>We evaluated gut peptides: peptide YY (PYY), glucaogon like peptide-1 (GLP-1), pro-neurotensin (pro-NT) and PP, in lean (young and middle-aged), obese and postoperative RYGBP subjects pre- and postprandially. RYGBP subjects had exaggerated levels of PYY and GLP-1 postprandially and higher basal proNT levels, implying a ‘satiety peptide tone’ that may contribute to the maintenance of weight loss.</p><p>In summary, RYGBP results in marked weight loss and alterations in gut and adipose tissue peptides involved in appetite regulation and energy homeostasis. These postoperative peptide changes may contribute to impressive weight loss observed after RYGBP.</p>

This thesis examines the effect of surgical weight loss on gut and adipose tissue peptides involved in appetite regulation and energy homeostasis in morbidly obese humans. Roux-en-Y gastric bypass (RYGBP) is the gold standard operation used for effective long-term weight loss and improved health. The exact mechanisms for this outcome are under investigation. We measured ghrelin, a recently discovered hunger hormone, insulin, adiponectin and leptin along with anthropometry measures in 66 morbidly obese patients prior to and 6 and 12 months after RYGBP. Impressive weight loss occurred postoperatively as did alterations in the peptides. Consistent correlations were found between weight, leptin, ghrelin and insulin. The main findings were low ghrelin concentrations in obesity and an increase after RYGBP. We explored inflammatory proteins C-reactive protein (CRP), serum amyloid A and interleukin-6 before and during massive weight loss 6 and 12 months after RYGBP in morbidly obese subjects. The studied proteins declined after surgery and a correlation between CRP and homeostatic model of assessment for insulin resistance, independent of BMI, strongly linked insulin resistance and inflammation. CRP declined most in insulin-sensitive subjects. We examined the excluded stomach mucosa and vagus nerve by measuring gastrin, pepsinogen I (PGI), pancreatic polypeptide (PP) and ghrelin levels during week 1 and year after RYGBP. Ghrelin levels rose with weight loss but declined 24-hours after surgery, like PP, indicating transient vagal nerve damage. Low levels of gastrin and PGI suggest a resting mucosa. We evaluated gut peptides: peptide YY (PYY), glucaogon like peptide-1 (GLP-1), pro-neurotensin (pro-NT) and PP, in lean (young and middle-aged), obese and postoperative RYGBP subjects pre- and postprandially. RYGBP subjects had exaggerated levels of PYY and GLP-1 postprandially and higher basal proNT levels, implying a ‘satiety peptide tone’ that may contribute to the maintenance of weight loss. In summary, RYGBP results in marked weight loss and alterations in gut and adipose tissue peptides involved in appetite regulation and energy homeostasis. These postoperative peptide changes may contribute to impressive weight loss observed after RYGBP.

The acute effects of exercise on appetite, ad libitum energy intake and gut hormone responses have received much attention over the past two decades. The experiments in this thesis have contributed to this research by examining appetite, acylated ghrelin, peptide-YY (PYY), leptin and ad libitum energy intake responses to two consecutive days of moderate-high intensity running. To achieve this 15 individuals aged 21 (2) y, with a BMI of 23.0 (1.9) kg·m-2 were recruited. Additionally, appetite, acylated ghrelin, PYY, glucagon-like peptide-1 (GLP-1), and ad libitum energy intake responses to an acute bout of moderate intensity treadmill exercise were compared in lean and overweight/obese (ow/ob) males and females. Two separate cohorts of individuals were recruited; 22 lean individuals and 25 ow/ob individuals (aged 38 (15) and 45 (12) y, with a BMI of 22 (2) and 29 (3) kg·m 2, for lean and ow/ob individuals, respectively). In Chapter 4, two consecutive days of 60 min treadmill running at 70% VO₂ peak did not produce compensatory changes in appetite or energy intake over two days. There were no main effects of trial for acylated ghrelin or leptin. However a main effect of trial for PYY indicated higher concentrations on the exercise than control trial. A meta-analysis was completed in Chapter 5, suggesting further research in the effects of acute exercise on appetite regulatory hormones in individuals who are ow/ob was necessary. In Chapter 6, 60 min of treadmill exercise at 60% VO₂ peak did not alter appetite sensations or energy intake in the 7 h after exercise in lean and ow/ob males and females. There were no main effects of sex, BMI or trial for acylated ghrelin; however, PYY and GLP-1 concentrations were higher in exercise than control trials. This thesis has demonstrated that over two days, high volume exercise does not stimulate compensatory appetite regulatory changes, in lean healthy males. In the short term, lean and ow/ob males and females respond similarly to acute exercise, showing no alterations in appetite or food intake responses, whilst PYY and GLP-1 concentrations are higher in exercise than control trials.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20983号 / 医博第4329号 / 新制||医||1027(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 杉田 昌彦, 教授 湊谷 謙司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Facing the growing prevalence of type 2 diabetes (T2D), the development of nutritionalinterventions allowing not only optimal glycemic control but also promoting postprandial satietyand overall satisfaction constitutes an interesting therapeutic avenue. This study was carried outin two parts, with the first part informing the second one.The first part was conducted in 61 middle-aged adults with or without prediabetes orT2D and aimed to assess the influences of gender/sex and health status on the relative rankingof the importance of eight common determinants of food choices as well as the sensoryevaluation and the frequency of consumption of almonds, pistachios, avocados, oatmeal, andeggs. Data analysis showed that 1) participants perceived “taste and own food preferences” ashaving the greatest influence on their food choices, 2) women attributed more importance to their“own food-related health beliefs” (p=0.040), while men reported a higher influence of the“recommendations of a health professional” (p=0.065), 3) almonds’ and pistachios’ taste wasrated the highest, and 4) taste ratings of pistachios (β=0.323, p=0.018) and avocados (β=0.604,p<0.001) were positively associated with their frequency of consumption by participants.Based on the sensory evaluation of the five foods, almonds were included in the testmeal of the second part of this study. The latter was conducted in 7 middle-aged men with T2Dand aimed to assess the effects of the types of macronutrient subtypes contained in isocaloricmacronutrient-matched meals on the postprandial glycemic, hormonal (insulin and glucagon-likepeptide-1 (GLP-1)) and appetite responses. The control meal contained white bread, butter andcheese, and the test meal contained white bread and almonds. Data analysis showed that thetest meal was associated with 1) lower postprandial glycemia (p=0.014), 2) higher postprandialGLP-1 serum concentrations (p=0.044) as well as 3) decreased hunger (p=0.032) and increasedfullness (p=0.014). There were no meal-associated differences in postprandial serum insulinconcentrations.Results highlight the importance of taste and food preferences and point out somegender/sex-related differences in the determinants of food choices. They also support thebeneficial effects of almonds, a food that seemed well appreciated by men and women, on keytherapeutic targets of T2D management.

No description available.

Diabetes Mellitus has continued to drastically affect the health of the world and many complications can prove fatal. As long as this metabolic disease persist, research discoveries will need to continue to be made so that patient outcomes and healthcare are dramatically enhanced. In recent years, GLP-1 has been the topic of conversation for diabetes research, due to its promising effects in promoting insulin sensitivity. Furthermore, bile acids and their receptors (TGR5 & FXR) have shown promise in their actions in the regulation of GLP-1, and thus glucose homeostasis. Here we have shown the detection and increased expression of TGR5 and GLP-1, and decreased expression of FXR in diabetic mouse intestinal mucosa tissues. We have also shown the detection and increased expression of these receptors in STC-1 cells. More importantly we have linked the connection of increased glucose concentration (hyperglycemia) to increased TGR5 activation to increased GLP-1 release, thus leading to increased insulin sensitivity and altered diabetic outcomes.

O diabetes mellitus do tipo 1 (DM1) é uma doença autoimune caracterizada pela destruição seletiva de células pancreáticas produtoras de insulina. Existem diversas formas de tratamento do DM1, tais como administração de insulina, imunossupressores, transplantes de pâncreas ou de ilhotas pancreáticas, porém todos se mostram ineficientes em algum aspecto. Recentemente, uma nova classe de medicamentos, os inibidores da enzima dipeptidil peptidase 4 (iDPP-4), demonstrou eficiência terapêutica e segurança no tratamento de pacientes com diabetes mellitus do tipo 2 devido ao aumento do hormônio peptídeo-1 semelhante ao glucagon (GLP-1, do inglês glucagon-like peptide-1). Além disso, o uso de inibidores de DPP-4 em modelos experimentais de DM1 demonstrou proteção das células pancreáticas contra apoptose, estimulação de neogênese de ilhotas pancreáticas e melhora do controle homeostático da glicose. Esse presente projeto teve como objetivo avaliar o perfil imunológico e endocrinológico da administração do inibidor de DPP-4 (MK0431) em DM1 experimental quimicamente induzido por estreptozotocina em camundongos C57Bl/6. Os animais diabéticos foram tratados com ração controle ou ração contendo inibidor de DPP-4 (4g MK0431/Kg de ração) ad libitum durante 30 e 90 dias. Durante o tratamento os animais tiveram glicemia, peso e teste de tolerância oral à glicose avaliados. Ao final do tratamento, os animais foram eutanasiados e o sangue, baço, timo, linfonodos pancreáticos e pâncreas foram coletados. Após 30 dias de tratamento com inibidor, foi observado um aumento do hormônio GLP-1 no soro, além de um padrão imunológico favorável. Dentre os mecanismos imunológicos, foi possível observar um aumento de células T reguladoras (CD4+CD25+Foxp3+) no baço e uma diminuição da citocina IFN- no homogenato pancreático. Após 90 dias de tratamento com inibidor, também foi detectado um aumento de insulina e GLP-1 séricos e uma diminuição nos níveis glicêmicos dos animais tratados. Observou-se uma redução no padrão inflamatório no microambiente pancreático, caracterizado pela diminuição das citocinas TNF- e IFN- no homogenato pancreático e por uma redução da freqüência de macrófagos CD11b+ nos linfonodos pancreáticos. Os resultados obtidos neste projeto contribuíram para validar a eficácia terapêutica da administração de inibidor de DPP-4 no tratamento do DM1 experimental, bem como os mecanismos imunológicos e endocrinológicos envolvidos. Sem a ocorrência de efeitos tóxicos relevantes, o uso de inibidores de DPP-4 pode se tornar uma alternativa terapêutica para o tratamento do DM1 em humanos, que constitui uma doença crônica associada à baixa qualidade de vida em longo prazo e necessidade de tratamento de alto custo. / Davanso, M.R. Immunoendocrinological analyses after administration of dipeptidyl-peptidase-4 inhibitor on experimental type 1 diabetes. 2012. 105p. Thesis (Masters Degree) School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, 2012. Type 1 Diabetes Mellitus (DM1) is an autoimmune disease characterized by the selective destruction of the insulin-producing pancreatic cells. Several forms of treatment for DM1 are current known such as insulin administration, immunosuppressors, pancreas or pancreatic islets transplantation, however, they all are inefficient in some aspect. Recently, a new class of drugs, the dipeptidyl-peptidase-4 inhibitors (iDPP-4) showed therapeutic efficacy and safety in the treatment with type 2 diabetes mellitus patients due to an increase in the glucagon-like peptide-1 (GLP-1). In addition, the use of DPP-4 inhibitors in experimental models of DM1 has demonstrated a protection of pancreatic cells against apoptosis, stimulation of pancreatic islets neogenesis and improvement in the glucose homeostatic control. This project evaluated the immunological and endocrinological profile of the DPP-4 (MK0431) inhibitor administration in experimental chemically induced DM1 by streptozotocin in C57BI/6 mice. The diabetic animals were treated with either a normal chow diet or diet containing DPP-4 inhibitor (4g MK0431/Kg of diet) ad libitum during 30 and 90 days. During the treatment the animals were evaluated regarding glycemia, weight, and oral glucose tolerance test. At the end of the treatment, the animals were killed and the blood, spleen, thymus, pancreatic lymph nodes and pancreas were collected. After 30 days of treatment with inhibitor, it was observed an increase in the hormone GLP-1 in the serum, besides a favorable immunological pattern. Among the immunologic mechanisms, it was possible to observe an increase in the regulator T cells (CD4+CD25+Foxp3+) of the spleen and a decrease in the cytokine IFN- in the pancreatic homogenate. After 90 days of treatment with inhibitor, it was also noticed an increase in the insulin and serum GLP-1 levels as well as a decrease in the glycemic levels in the treated animals. It was observed a reduction in the inflammatory pattern in the pancreatic microenvironment characterized by a decrease in the cytokines TNF- and IFN- in the pancreatic homogenate and by a reduction in the frequency of CD11b+ macrophages in the pancreatic lymph nodes. The results obtained in this project contributed to validate the therapeutic efficacy of the DPP-4 inhibitor administration in the treatment of experimental DM1, as well as the immunological and endocrinological mechanisms involved. Without the occurrence of relevant toxic effects, the use of DPP-4 inhibitors may become a therapeutic alternative for the treatment of DM1 in humans, which constitutes a chronic disease associated to low life quality and need for high cost treatment.

Introdução/Objetivo: Este estudo teve como objetivo testar a hipótese de que a atividade e/ou expressão da dipeptidil peptidase IV (DPPIV), uma enzima que inativa peptídeos com ações cardioreno protetoras, como o peptídeo-1 semelhante ao glucagon (GLP-1) e o peptídeo natriurético cerebral (BNP), estaria associada a um pior prognóstico na insuficiência cardíaca (HF). Métodos: Injúria do miocárdio foi realizada através da ablação do ventrículo esquerdo (VE) por radiofrequência em ratos Wistar machos (200-250 g). Os ratos foram divididos em três grupos: Sham, HF e HF + inibidor de DPPIV (sitagliptina 200mg/kg/b.i.d). Seis semanas após a cirurgia, os animais foram alojados individualmente em gaiolas metabólicas durante 3 dias para avaliação da função renal. Atividade e expressão da DPPIV no plasma e coração foram medidas por espectrofotometria e por immunoblotting, respectivamente. Para a avaliação da função cardíaca um cateter de pressão-volume foi posicionado dentro da cavidade do VE. A análise histológica foi realizada para os parâmetros morfométricos. A atividade da DPPIV no plasma também foi medida em pacientes com HF (n = 190). Resultados: A atividade DPPIV e sua abundância estavam aumentadas em animais com HF em comparação com Sham. Além disso, a atividade de DPPIV no plasma se correlacionou positivamente com o volume diastólico final (R = 0,517; p < 0,001) e o peso do pulmão/peso corporal (R = 0,492; p < 0,01). Uma correlação negativa entre a atividade DPPIV plasmática e a fração de ejeção também foi observada (R = 0,602; p < 0,001). Curiosamente, os animais HF também exibiram um aumento da expressão/atividade de DPPIV no tecido cardíaco, especialmente em células endoteliais. Seis semanas de tratamento com o inibidor de DPPIV sitagliptina atenuou a disfunção cardíaca, fibrose intersticial, congestão pulmonar e infiltração de macrófagos. O tratamento com sitagliptina também elevou os níveis plasmáticos de GLP-1 ativo, e aumentou a ativação de vias de sinalização cardioprotetoras como PKA e Akt; e reduziu os níveis de apoptose e marcadores pró-inflamatórios em comparação com ratos não tratados. Ratos com HF apresentaram maiores níveis circulantes de BNP, contudo a atividade da PKG renal foi mais baixa nesses animais em comparação com o grupo tratado com sitagliptina, sugerindo uma diminuição da razão BNP ativo/total. A função renal não diferiu entre os grupos, mas o ritmo de filtração glomerular estava ligeiramente aumentado no grupo tratado em comparação com os animais HF. Pacientes com HF apresentaram uma maior atividade plasmática da DPPIV e correlações foram encontradas com a com a fração de ejeção (R = -0,20; p = 0,009) e a quimiocina Ccl2 (R² =0,30; p < 0.01). Conclusões: Em conjunto, nossos resultados demonstram que a atividade plasmática da DPPIV se correlaciona com um pior prognóstico em pacientes e animais com HF e que a DPPIV possui um papel importante na fisiopatologia desta doença / Aim: The present study aimed to test the hypothesis that the activity and/or expression of dipeptidyl peptidase IV (DPPIV), an enzyme that inactivates cardiorenal protective peptides including glucagon-like peptide-1 (GLP-1) and brain natriuretic peptide (BNP), would be associated with poorer outcomes in heart failure (HF). Methods: Experimental HF was induced in male Wistar rats (200-250 g) by left ventricular (LV) myocardial injury after radiofrequency catheter ablation. Rats were divided in three groups: Sham, HF and HF+DPPIV inhibitor (sitagliptin 200mg/kg/b.i.d). Six weeks after surgery, animals were individually housed in metabolic cages during 3 days for assessment of renal function. Plasma and heart DPPIV activity/expression were measured spectrophotometrically and by immunoblotting respectively. For evaluation of cardiac function a pressure-volume catheter was positioned into the LV cavity. Histological analysis was performed for morphometric parameters. Plasma DPPIV activity was also measured in patients (n = 190) with heart failure. Results: Plasma DPPIV activity and abundance were increased in animals with HF compared to Sham. Additionally, plasma DPPIV activity positively correlated with ventricular end diastolic volume (R² =0.517; p < 0.001) and lung/body weight (R² =0.492; p < 0.01). A negative correlation between plasma DPPIV activity and ejection fraction was also observed (R² =0.602; p < 0.001). Interestingly, HF animals also exhibited an increase of expression and activity of DPPIV in heart tissue, especially in endothelial cells. Six-week treatment with the DPPIV inhibitor sitagliptin attenuated cardiac dysfunction, mitigated cardiac hypertrophy, interstitial fibrosis, lung congestion and macrophage infiltration. Sitagliptin also raised the plasma levels of active GLP-1, increased activation of cardioprotective signaling pathways including PKA, and Akt; and reduced the levels of apoptosis and pro-inflammatory biomarkers compared to non-treated HF rats. Despite the higher circulating total BNP, renal PKG activity was lower in HF rats compared with sham and sitagliptin-treated rats, suggesting a decrease in active/total BNP ratio. Renal function did not differ between groups, but glomerular filtration rate was modestly, but significantly increased by Sitagliptin compared to HF. Plasma DPPIV activity in patients was also increased compared to healthy subjects and correlations was found with ejection fraction (R² =-0.20; p=0.009) and the chemokine Ccl2 (R² =0.30; p < 0.01). Conclusions: Taken together, our results demonstrate that circulating DPPIV activity correlates with poorer cardiovascular outcomes in human and experimental HF and might play an important role in the pathophysiology of HF.