在2型糖尿病病人身上，常常併發高膽固醇血症，HMG CoA 還原酶的抑制劑常常用作治療這類病症。由於高膽固醇血症與胰島素抵抗和2型糖尿病有著密切關係，我們推測辛伐他汀對於2型糖尿病的發展有著保護和有利的作用。在這項研究中，我們主要測試了辛伐他汀 （10 nM； 24 hr）對於胰島β細胞主要功能的影響，包括其對於葡萄糖的胰島素分泌功能影響。我們假設，在肥胖/糖尿病（db⁺/ db⁺）小鼠分離的胰島，辛伐他汀可以恢復葡萄糖 （5 mM和15 mM）引起的胰島素分泌（加上降低的胰島素含量）。 / 在這個項目中，我們運用24周大的基因糖尿糖C57BL/KSJ +db/+db (db⁺/db⁺)肥鼠和相同年齡的無糖尿病C57BL/KSJ +m/+m (db⁺/m⁺)小鼠作為動物模型。通過應用obese/diabetic (db+/db+)和lean/non-diabetic (db+/m+)中分離的胰腺胰島和胰島β細胞，我們研究了胰腺胰島功能性障礙的潛在機理以及辛伐他汀對於恢復葡萄糖 （5 mM和15 mM）引起的胰島素分泌（加上降低的胰島素含量）的有利作用。資料清晰的顯示，葡萄糖引起的胰島素分泌和胰島素含量在obese/diabetic (db+/db+)的胰腺胰島中明顯低於在lean/non-diabetic (db⁺/m⁺)的胰腺胰島中。在24hr的辛伐他汀處理後，辛伐他汀恢復了葡萄糖 （5 mM和15 mM）引起的胰島素分泌（加上降低的胰島素含量）及葡萄糖 （15 mM）引起的胞內鈣離子變化。 / 在這個項目中，我們證明鈣敏感受體 （CaSR）在obese/diabetic (db⁺/db⁺)中的表達量明顯較低，而辛伐他汀的處理可以顯著性增加鈣敏感受體在obese/diabetic (db⁺/db⁺)胰島中的表達。有人建議說，obese/diabetic (db⁺/db⁺)的胰島中被抑制的鈣敏感受體表達與胰島β細胞的胰島分泌功能障礙有關。這暗示了辛伐他汀可能通過變構啟動鈣敏感受體來恢復obese/diabetic (db⁺/db⁺)胰島中葡萄糖引起的胰島素分泌和胰島含量。實驗也同樣証明辛伐他汀調節的PLA₂信號通路對於辛伐他汀改善obese/diabetic (db⁺/db⁺)胰島β細胞的胰島素分泌功能起著至關重要的作用。除此之外，我們的實驗結果證明高濃度的葡萄糖處理顯著的增加了obese/diabetic (db⁺/db⁺)細胞膜肌動蛋白骨架的密度，而辛伐他汀顯著的減少了這一變化。因此，obese/diabetic (db⁺/db⁺)胰島β細胞的胰島素分泌障礙是由肌動蛋白細胞骨架聚集阻礙胰島素顆粒胞吐引起的。而辛伐他汀通過解聚和重組肌動蛋白細胞骨架來改善obese/diabetic (db⁺/db⁺)胰島β細胞的胰島素分泌功能。 / 在這項研究中，我們的實驗結果證明葡萄糖可以顯著提高obese/diabetic (db⁺/db⁺)胰島β細胞內ROS的含量。而辛伐他汀處理部分降低了胰島β細胞內ROS的含量。除此之外，我們還研究了5 mM和15 mM葡萄糖對於內質網應力（ER-stress）相關的蛋白比如PERK, eIF2α 和IRE1表達的影響。這些內質網跨膜蛋白可以感應ER-stress從而啟動應力感測器來開啟複雜的信號通路。與lean/non-diabetic (db⁺/m⁺)相比，PERK and eIF2α在obese/diabetic (db⁺/db⁺)的胰島中表達量更低，這表明obese/diabetic (db⁺/db⁺)胰島β細胞的功能性障礙可能與ER-stress有關。而辛伐他汀的處理明顯的增加了這些蛋白的表達量，由此證明辛伐他汀還通過對抗ER-stress來保護obese/diabetic (db⁺/db⁺)胰島β細胞。 / 總而言之，我們的資料第一次證明了辛伐他汀通過PLA₂信號通路變構啟動鈣敏感受體來保護obese/diabetic (db⁺/db⁺)胰島β細胞（比如：恢復葡萄糖引發的胰島素分泌和提高減少的胰島素含量），還通過提高obese/diabetic (db⁺/db⁺)胰島β細胞中被抑制的ER-stress相關蛋白的表達量來抵抗ER-stress帶來的損傷。 / Diabetics often have hyperlipidemia as a co-morbidity. Despite the well-documented cholesterol-lowering properties of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG CoA) reductase inhibitors (statins) in treating hypercholesterolemia, the beneficial effects of statins consumption in T2DM treatment are confusing. In the current study, we examined the effects of the simvastatin (10 nM; 24 hr) on β-cell function leading to insulin secretory response to glucose. We hypothesized that statins restore the blunted glucose (5 mM and 15 mM)-induced insulin secretion (plus the reduced insulin content) of isolated pancreatic islets of obese/diabetic (db⁺/db⁺) mice. / In the present study, genetically diabetic C57BL/KSJ +db/+db (db⁺/db⁺) mice at 24 week of age and their age-matched non-diabetic littermates C57BL/KSJ +m/+m (db⁺/m⁺) were used. Our results clearly showed that the suppressed glucose (5 mM and 15 mM)-induced insulin release (plus insulin content) and glucose (15 mM)-induced [Ca²⁺]i changes of isolated pancreatic islets of obese/diabetic (db⁺/db⁺) was restored after simvastatin (10 nM; 24 hr) treatment. / The biochemical existence of CaR in pancreatic islets of lean/non-diabetic (db⁺/m⁺) and obese/diabetic (db⁺/db⁺) mice was confirmed. The suppressed/down-regulated expression of CaR was associated to the blunted insulin secretion in pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice, and it was markedly up-regulated by simvastatin (10 nM; 24 hr). The involvement of CaR-mediated PLA₂ signaling in simvastatin (10 nM; 24 hr)-induced restoration of glucose (15 mM)-induced insulin secretion in pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice was investigated. Our results also showed that the increased density of plasma membrane actin cytoskeleton of obese/diabetic (db⁺/db⁺) mice was significantly decreased by simvastatin (10 nM; 24 hr) treatment. The simvastatin-induced depolymerization and remodeling of actin cytoskeleton may improve insulin secretion capability in pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice. / The glucose (15 mM)-induced intracellular ROS level was significantly higher in pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice. The elevated ROS level was partially diminished by simvastatin (10 nM; 24 hr) treatment. The protein expressions of PERK and eIF2α (ER stress proteins) were lower in pancreatic islet cells isolated from obese/diabetic (db⁺/db⁺) mice, suggesting that abnormal expresstion/activity of PERK and eIF2α would be coupled to the ER-stress mediated failure of pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice. As simvastatin (10 nM; 24 hr) up-regulated the protein expression of these proteins, this drug exerted protective effect on pancreatic β-cells against ER stress and restored the blunted glucose (15 mM)-induced insulin secretion (plus the reduced insulin content) in obese/diabetic (db⁺/db⁺) mice. / In conclusion, our results demonstrate, for the first time, that simvasatatin (a HMG-CoA reductase inhibitor) (10 nM; 24 hr) provides beneficial effects (i.e. restoration of the blunted glucose-induced insulin release plus the reduced insulin content) in pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice via the allosteric modification/up-regulation of extracellular calcium-sensing receptor through the PLA₂ signaling pathway, and provides protective/antioxidant effects against oxidative stress caused by chronic hyperglycemia in pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice by up-regulating protein expression of the suppressed ER stress sensors and antioxidant enzyme. / 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. / Au, Lai Shan. / Thesis (Ph.D.) Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 458-532). / Abstracts also in Chinese.

胰腺內的胰島具有極其重要的功能，通過產生并分泌一系列的胰島荷爾蒙，特別是能控制機體葡萄糖利用的胰島素，來調節體內血糖穩態。胰島素的分泌受到多種因素或信號通路的調節。据信，在臨床上表現出來明顯的高血糖症的時候，胰島細胞的分泌功能已經出現典型性的缺陷。由此，大量的研究證據指出，2 型糖尿病表現出來的代謝型缺陷主要為胰島功能紊亂，而并不是周圍組織胰島素抵抗。這表明，胰島素功能缺陷是早於高血糖症的發生的。另一方面，大量證據表明長期性的高血糖會導致胰島　細胞功能紊亂。鑒於此，揭示胰島功能調節的潛在機理并闡明胰島功能与高血糖症之間的關係變得尤為重要。 / 在臨床上表現出能調節胰島功能和血糖控制的相關因子正與日俱增。其中極具研究價值的是一種多肽，稱作胰高血糖素様肽（GLP-1），其作用表現在通過增強胰島素分泌和胰島素敏感性來增強胰島　細胞的功能和增值。GLP-1 在體內的降解能被DPP-4 的抑製劑所延阻。同時，通過對一種名為SGLT2 的葡萄糖轉運蛋白的抑制，機體內的血糖水平能被顯著降低。這一作用是通過阻止腎臟對葡萄糖的重吸收來實現的，並且是不依賴于胰島素的。由於DPP-4 抑制所表現的最終生理作用需要通過胰島素的信號通路來實現，但SGLT2 的抑制卻不依賴於胰島素，由此不難想象，對SGLT2 和DPP-4 的聯合抑制在2 型糖尿病的血糖控制方面具有潛在的協同效應。即通過對SGLT2 的抑制來顯著降低血糖水平，從而促進GLP-1 在體內的作用效應。因此，本研究的第一部分研究SGLT2 和DPP-4 的單一或聯合抑制（利用SGLT2 抑製劑BI-38335 和DPP-4 抑製劑linagliptin）在二型糖尿病動物模型db/db老鼠種對胰島功能和體內葡萄糖穩態的作用。在此研究中，我們比較了SGLT2 和DPP-4 單一抑制或聯合抑制對db/db 老鼠胰島功能的影嚮。研究發現，所有的實驗組都能顯著降低血糖以及糖化血紅蛋白（HbA1c）的水平，而且聯合抑制組表現出更叫顯著的效應。聯合抑制組增強了胰島細胞的胰島素分泌功能，改善葡萄糖耐受并增加胰島素的敏感性。於此一致的是，聯合抑制組降低了β細胞凋亡和胰島免疫細胞標記物，並且抑制了与TLR2 信號通路相關的一系列炎症分子，通過則一系列作用實現對胰島的保護。上述研究表明，對SGLT2 和DPP-4 的聯合抑制在對胰島功能和胰島形態學上的保護至少能夠表現出加性效應，從而更好實現對血糖的調控。 / 在第一部分的工作中，我們利用的動物模型db/db 老鼠是一類較嚴重的糖尿病動物模型，它表現出及其嚴重的高血糖症，糖耐受失調同β細胞缺陷。我們集中于研究SGLT2 和DPP-4 的抑制對這類嚴重糖尿病的胰島功能的調節，具體表現在對胰島β細胞功能的正向調節，包括胰島素分泌功能的增強和β細胞質量的增加。廣為接受的一點是，胰島素抵抗和胰島素分泌功能的缺失最能表徵從正常葡萄糖耐受發展到2 型糖尿病的這一進程。這一進程的早期主要表現為由肥胖或衰老而引起的代償性的胰島素抵抗，此時伴有正常或受損的葡萄糖耐受以及正常的胰島素分泌功能。此時，任何能影響胰島功能的因素都會減緩或加速2 型糖尿病的發生。鑒於此，研究此类因素從而到达阻止2 型糖尿病的发生就显得尤为重要。因此，在本研究的第二部分，我们研究利用高脂飼料诱导的肥胖老鼠模型和老化的老鼠模型来分别研究煙酸（niacin 或 nicotinic acid）对胰岛功能的影響。煙酸是一種臨床上廣汎使用的降血脂藥物，但近年來的研究發現長期或高劑量的使用會導致高血糖症和血糖控制失調的出現，然而這一現象產生的具體機製並不清楚。因此，我們第二部分的研究集中於揭示煙酸引起的高血糖症是否歸因於其對胰島功能的破壞，以及潛在的分子機制。我們的研究發現，在肥胖老鼠和老齡鼠中，煙酸能夠引起高血糖症，破壞葡萄糖體內穩態並且降低胰島素分泌能力；另一方面，煙酸增加饑餓血清胰島素水平並且引起葡萄糖耐受實驗中第一期胰島素分泌缺陷。體內和體外實驗還發現煙酸誘導煙酸受體GPR109a，UCP2 和PPARγ的表達增加以及SIRT1 的表達和NAD,NAD/NADH 降低。通過基因沉默技術降低GPR109a 在β細胞中的表達，我們發現煙酸的上述作用都被極大的減弱，從而揭示了煙酸引起的胰島功能降低是由其受體GPR109a 介導的。 / 總闊來說，我們的研究揭示了DPP-4 同SGLT2 的聯合抑制在增強胰島功能和胰島形態學上的保護以及改善胰島素抵抗等方面能夠表現出加性效應，從而更好實現對血糖的調控。另一方面，我們的研究闡述了煙酸通過它的受體GPR109a 以及其下游信號通路如PPARγ和SIRT1 來損害胰島細胞功能。綜上所述，我們當前的研究證實了一系列因素對胰島功能的調控，從而充實并擴展了我們對胰島功能和血糖控制以及2 型糖尿病之間關係的認識。 / Pancreatic islets are of great importance to govern glucose homeostasis through production and secretion of islet peptide hormones, notably insulin, which functions as a master regulator to control glucose disposal in the body. Insulin secretion is regulated by various factors and signaling pathways. It is well known that islet insulin secretory function is typically lost by the time when signs of hyperglycemia that becomes clinically apparent. Thus, it has been pointed out that islet dysfunction, rather than peripheral insulin resistance, is the primary defect of type 2 diabetes mellitus (T2DM), indicating that deficiencies in islet function are prior to the onset of hyperglycemia. On the other hand, it is also widely accepted that chronic hyperglycemia results in islet β cells dysfunction. In this regard, it is of great importance to unravel the underlying mechanisms by which islet function is regulated, thus elucidating the relationship between hyperglycemia and islet function. / There are ever increasing candidates of clinically relevant factors identified as criticalregulators for islet function and glycemic control. Of great interest is the glucagon-like peptide 1 (GLP-1) that improves β cell function and proliferation and its degradation can be delayed by dipeptidyl peptidase-4 (DPP-4) inhibition. Meanwhile, plasma glucose levels can be remarkably lowered by inhibition of sodium-glucose co-transporter 2 (SGLT2), through blockade of renal glucose reabsorption. In this regard, since the mode of action of SGLT2 inhibition is independent of insulin but the efficacy of DPP-4 inhibition relies on the insulin signalling, it is plausible to hypothesize that sustained lowering of plasma glucose by SGLT2 inhibition can facilitate the actions of GLP-1 from DPP-4 inhibition, thus leading to a potential synergistic effect on islet function and glycemic control. Accordingly, the first part of this study was to investigate the combination effects of SGLT2 and DPP-4 blockade on islet function and glucose homeostasis using an animal model of T2DM, the db/db mice. We compared the effects of either DPP-4 inhibition (by a DPP-4 inhibitor, linagliptin) or SGLT2 inhibition (by an SGLT2 inhibitor, BI-38335) individually and in combination on islet function and glycemic control in db/db mice. Active treatments markedly enhanced islet function, improved glycemic control and reduced islet and peripheral tissue inflammation, with the combined treatment showing the greater effects. These data indicate that combined SGLT2 inhibition with DPP-4 inhibition work additively to exhibit benefits to islet function, inflammation and insulin resistance, thus improving glycemic control. / In the first part, we investigated a positive regulation of islet function in overt diabetic mice, in which there are severe hyperglycemia and β cell failure. It is widely accepted that the progression from normal glucose tolerance to T2DM is characterized by dual defects that include insulin resistance and an insulin secretory defect caused by β cell dysfunction. In the early stage, there is compensated insulin resistance resulting from obesity or aging with normal or even impaired glucose tolerance as well as nearly normal insulin secretory capacity. As such, any factors that affect islet function in this stage may delay or accelerate the onset of diabetes. In this regard, it is noteworthy to study the regulation of such factors in islet function in order to prevent the development of T2DM. Thus, in the second part, we investigated how islet function was regulated by a widely used lipid-lowering drug, niacin (nicotinic acid), in obese mice and aged mice. Niacin has been known to impair euglycemic control during prolonged and high dose treatments but the underlying mechanism(s) whereby the islets are involved remains unclear. As such, we aimed at elucidating whether this hyperglycemic effect is due to the dysfunction of pancreatic islet and, if so, the underlying mechanism(s) involved. We investigated the direct effects of niacin on islet function and insulin resistance in HFD-induced obese (DIO) mice and aged mice. Our results showed that eight-week treatments with niacin impaired glycemic control and islet function in DIO and aged mice. Moreover, niacin treatments significantly induced PPARγ and GPR109a expression but decreased SIRT1 expression in pancreatic islets, while islet morphology remained unchanged. In vitro studies showed that niacin decreased glucose-stimulated insulin secretion (GSIS), cAMP, NAD/NADH ratio, and mitochondrial membrane potential (ΔΨm) but increased reactive oxygen species (ROS) transiently, while upregulated expression levels of UCP2, PPARγ and GPR109a in INS-1E cells. In corroboration, the decrease in GSIS and cAMP levels were abolished by the knockdown of GPR109a. These data indicate that chronic treatment of niacin induces hyperglycemia, which is due, partly, to impaired pancreatic islet function, probably via the mediation of islet niacin receptor GPR109a. / Collectively, our study has revealed that inhibition of DPP-4 or SGLT2 alone can improve islet function, and combined inhibition of DPP-4 and SGLT2 works additively to exhibit benefits to islet cell function/morphology, inflammation and insulin resistance, thus improving glycemic control. On the other hand, we have also elucidated that niacin impairs islet β cell function through GPR109a and downstream signaling pathways such as PPARγ and SIRT1. Taken together, the present study has shown the regulation of is let β cell function by different factors, which has an added advance to our knowledge about the intricate relationship between islet function and hyperglycemia and T2DM. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Lihua. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 168-195). / Abstracts also in Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgement --- p.vii / List of Publications --- p.viii / List of Abbreviations / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Endocrine pancreas --- p.2 / Chapter 1.1.1 --- Structure and composition of endocrine pancreas --- p.3 / Chapter 1.1.2 --- Architecture and composition of the islet --- p.3 / Chapter 1.1.3 --- Endocrine cells and their function --- p.5 / Chapter 1.2 --- Disorders of the endocrine pancreas --- p.9 / Chapter 1.3 --- Insulin --- p.10 / Chapter 1.3.1 --- Insulin Structure --- p.10 / Chapter 1.3.2 --- Insulin actions and insulin receptor --- p.11 / Chapter 1.3.3 --- Insulin secretion --- p.12 / Chapter 1.3.3.1 --- Glucose-induced insulin secretion --- p.13 / Chapter 1.3.3.2 --- Phasic insulin secretion --- p.14 / Chapter 1.3.4 --- The regulation of insulin secretion --- p.16 / Chapter 1.3.5 --- Autocrine insulin feedback --- p.20 / Chapter 1.4 --- Diabetes mellitus --- p.21 / Chapter 1.4.1 --- Type 1 diabetes mellitus (T1DM) --- p.22 / Chapter 1.4.2 --- Type 2 diabetes mellitus (T2DM) --- p.23 / Chapter 1.4.3 --- Obesity and T2DM --- p.23 / Chapter 1.4.4 --- Islet dysfunction and T2DM --- p.25 / Chapter 1.5 --- Incretin hormones and DPP-4 inhibition --- p.27 / Chapter 1.5.1 --- Incretin hormones --- p.27 / Chapter 1.5.2 --- Functions of incretin hormones --- p.30 / Chapter 1.5.3 --- Regulation of GLP-1 --- p.34 / Chapter 1.5.4 --- Incretin-based therapy for T2DM --- p.35 / Chapter 1.6 --- Sodium-dependent glucose cotransporter 2 (SGLT2) and its inhibitors --- p.38 / Chapter 1.6.1 --- Sodium-dependent glucose cotransporter 2 (SGLT2) --- p.38 / Chapter 1.6.2 --- Rationale for SGLT2 inhibition --- p.40 / Chapter 1.6.3 --- Consequences of SGLT2 inhibition --- p.41 / Chapter 1.6.4 --- Strategies of SGLT2 inhibition --- p.43 / Chapter 1.6.4.1 --- SGLT2 inhibitors --- p.44 / Chapter 1.6.4.1 --- SGLT2 inhibitors --- p.47 / Chapter 1.7 --- Niacin (nicotinic acid) and its clinical usage --- p.49 / Chapter 1.7.1 --- Niacin general introduction --- p.49 / Chapter 1.7.2 --- General roles of niacin --- p.49 / Chapter 1.7.3 --- Anti-lipolytic effect --- p.50 / Chapter 1.7.4 --- Niacin receptor --- p.51 / Chapter 1.7.5 --- Hyperglycemic effect of niacin --- p.52 / Chapter 1.8 --- General hypothesis --- p.54 / Chapter Chapter 2 --- General Materials and Methods --- p.56 / Chapter 2.1 --- Experimental animal models --- p.57 / Chapter 2.1.1 --- Animal model of type 2 diabetes --- p.57 / Chapter 2.1.2 --- High-fat diet-induced obese mice --- p.58 / Chapter 2.1.3 --- Aged mice --- p.59 / Chapter 2.2 --- INS-1E cell culture and treatment --- p.59 / Chapter 2.2.1 --- Mouse pancreatic islet isolation --- p.59 / Chapter 2.2.2 --- Primary culture of isolated pancreatic islets --- p.60 / Chapter 2.3 --- Pancreatic islet isolation and culture --- p.60 / Chapter 2.4 --- Glucose-stimulated insulin secretion (GSIS) assay --- p.61 / Chapter 2.5 --- Assessment of glucose homeostasis --- p.61 / Chapter 2.6 --- Determination of mRNA expression --- p.62 / Chapter 2.6.1 --- Design of specific primers --- p.63 / Chapter 2.6.2 --- Total RNA extraction and cDNA synthesis --- p.63 / Chapter 2.6.3 --- Real-time PCR analysis --- p.64 / Chapter 2.7 --- Detection of protein expression --- p.64 / Chapter 2.7.1 --- Western blotting analysis --- p.64 / Chapter 2.7.2 --- Immunofluorescent staining --- p.65 / Chapter 2.8 --- Biochemical analyses --- p.65 / Chapter 2.8.1 --- Plasma insulin and blood HbA1c levels --- p.65 / Chapter 2.8.2 --- Detection of cAMP --- p.66 / Chapter 2.8.3 --- NAD and NADH determination --- p.66 / Chapter 2.9 --- Detection of intracellular ROS --- p.67 / Chapter 2.10 --- Detection of mitochondrial membrane potential --- p.67 / Chapter 2.11 --- Statistical analysis --- p.67 / Chapter Chapter 3 --- Effects of Combining Linagliptin Treatment with BI-38335, A Novel SGLT2 Inhibitor, on Pancreatic Islet Function and Inflammation in db/db Mice --- p.70 / Chapter 3.1 --- Abstract --- p.71 / Chapter 3.2 --- Introduction --- p.72 / Chapter 3.3 --- Materials and Methods --- p.74 / Chapter 3.3.1 --- Animal model and experimental design --- p.74 / Chapter 3.3.2 --- In vivo glucose homeostasis --- p.75 / Chapter 3.3.3 --- Pancreas and islet studies --- p.76 / Chapter 3.3.4 --- Biochemical analyses --- p.77 / Chapter 3.3.5 --- Real-time PCR analyses --- p.77 / Chapter 3.3.6 --- Statistical analysis. --- p.78 / Chapter 3.4 --- Results --- p.78 / Chapter 3.4.1 --- Treatments with DPP-4 and SGLT2 inhibitors lower plasma glucose --- p.78 / Chapter 3.4.2 --- Treatments with DPP-4 and SGLT2 inhibitors improve glycemic --- p.80 / Chapter 3.4.3 --- Pancreatic islet function in db/db mice --- p.83 / Chapter 3.4.4 --- Pancreatic islet and peripheral tissue inflammation --- p.86 / Chapter 3.4.5 --- Islet morphology and preserved beta cells --- p.89 / Chapter 3.5 --- Discussion --- p.93 / Chapter Chapter 4 --- Niacin-Induced Hyperglycemia Is Mediated via Niacin Receptor GPR109a in Pancreatic Islets --- p.98 / Chapter 4.1 --- Abstract --- p.99 / Chapter 4.2 --- Introduction --- p.100 / Chapter 4.3 --- Research design and methods --- p.102 / Chapter 4.3.1 --- Animal model and experimental design --- p.102 / Chapter 4.3.2 --- In vivo glucose homeostasis --- p.102 / Chapter 4.3.3 --- Pancreas and islet studies --- p.103 / Chapter 4.3.4 --- INS-1E cell culture and treatment --- p.103 / Chapter 4.3.5 --- Construction of small interfering RNA for GPR109a --- p.103 / Chapter 4.3.6 --- Real-time PCR analyses --- p.104 / Chapter 4.3.7 --- Western blotting assay --- p.104 / Chapter 4.3.8 --- Detection of intracellular and mitochondrial ROS --- p.105 / Chapter 4.3.9 --- Detection of mitochondrial membrane potential (ΔΨm) --- p.105 / Chapter 4.3.10 --- Measurement of cAMP levels --- p.105 / Chapter 4.3.11 --- Determination of NAD and NADH levels --- p.106 / Chapter 4.3.12 --- Measurement of cell viability --- p.106 / Chapter 4.3.13 --- Statistical analysis --- p.106 / Chapter 4.4 --- Results --- p.106 / Chapter 4.4.1 --- Glycemic control in HFD-induced obese mice --- p.106 / Chapter 4.4.2 --- Pancreatic islet function in HFD-induced obese mice --- p.110 / Chapter 4.4.3 --- Pancreatic islet morphology and gene expression --- p.112 / Chapter 4.4.4 --- INS-1E function and intracellular levels of cAMP, NAD, and NADH --- p.114 / Chapter 4.4.5 --- Gene expression in INS-1E cells --- p.117 / Chapter 4.4.6 --- Status of ROS and ΔΨm in INS-1E cells --- p.119 / Chapter 4.4.7 --- GPR109a knockdown in INS-1E cells --- p.122 / Chapter 4.5 --- Discussion --- p.129 / Chapter Chapter 5 --- Niacin Impairs Pancreatic Islet Glucose-Stimulated Insulin Secretion in Aged Mice through The Suppression of SIRT1 Signaling --- p.134 / Chapter 5.1 --- Abstract --- p.135 / Chapter 5.2 --- Introduction --- p.136 / Chapter 5.3 --- Research design and methods --- p.139 / Chapter 5.3.1 --- Animal model and experimental design --- p.139 / Chapter 5.3.2 --- In vivo glucose homeostasis --- p.139 / Chapter 5.3.3 --- Pancreas and islet studies --- p.140 / Chapter 5.3.4 --- Real-time PCR analyses --- p.140 / Chapter 5.3.5 --- Western blotting assay --- p.140 / Chapter 5.3.6 --- NAD and NADH determination --- p.141 / Chapter 5.3.7 --- NEFA determination --- p.141 / Chapter 5.3.8 --- Statistical analysis --- p.141 / Chapter 5.4 --- Results --- p.142 / Chapter 5.4.1 --- Glycemic control in middle aged mice --- p.142 / Chapter 5.4.2 --- Pancreatic islet function in HFD-induced obese mice --- p.147 / Chapter 5.4.3 --- NAD, NADH levels in pancreatic islet --- p.149 / Chapter 5.4.4 --- Genes expression in pancreatic islet --- p.151 / Chapter 5.5 --- Discussion --- p.150 / Chapter Chapter 6 --- General discussion --- p.156 / Chapter 6.1 --- Combined inhibition of DPP-4 with SGLT2 on islet function, inflammation and insulin resistance in T2DM --- p.158 / Chapter 6.2 --- Niacin impairs islet function in high-fat diet-induced obese mice and aged mice --- p.161 / Chapter 6.3 --- General conclusion --- p.164 / Chapter 6.4 --- Future directions --- p.166 / Chapter Chapter 7 --- Bibliography --- p.167

A radiação ultravioleta (UV) do sol é a principal responsável pelo desenvolvimento do câncer de pele não-melanoma. A radiação UV induz efeitos biológicos, que promovem a fotocarcinogênese, agindo diretamente sobre o DNA, provocando mutações. Porém, tudo indica que há um efeito indireto, que induz imunossupressão. O fenômeno fisiológico da imunotolerância, que é responsável pela prevenção de doenças autoimunes e pela modulação da resposta inflamatória, pode ser aproveitado por determinadas neoplasias para escaparem do controle imunológico e reforçado pela radiação UV. Os personagens principais da imunotolerância no ambiente tumoral são células dendríticas imaturas e linfócitos T reguladores (Treg), além de inúmeras citocinas imunossupressoras. Com o objetivo de avaliar a imunotolerância na fotocarcinogênese em lábio, foram analisadas, por meio da técnica de imuno-histoquímica, 75 amostras de material fixado e emblocado em parafina, sendo 44 casos de queilite actínica (QA) nos três graus de displasia epitelial (discreta, moderada e intensa), 18 casos de carcinoma epidermoide de lábio (CEL) e 12 espécimes representativos de lábio normal (LN). Tais amostras foram submetidas aos anticorpos anti-CD83, anti-DEC-205, anti-FOXP3, anti-CD1a e anti-CD207. Foi efetuada a contagem de células em 3 campos significativos, escolhidos aleatoriamente, e obtida a média de células contadas/campo. Assim, os resultados mostraram uma grande quantia de linfócitos Treg FOXP3+ tanto no CEL como na QA. Também houve uma expressão acentuada de DEC-205 nos casos estudados. Quanto à presença de células de Langerhans CD207+ e CD1a+, notou-se um acúmulo no epitélio das lesões de QA e nas ilhotas do CEL, além de algumas células estarem presentes no tecido conjuntivo. Já para o marcador CD83, a contagem de células positivas foi baixa em relação aos demais marcadores tanto no CEL como na QA. Portanto, sugere-se um microambiente imunotolerante tanto no início, quanto no estabelecimento do processo da fotocarcinogênese em lábio. / Ultraviolet radiation (UV) is the main cause of non-melanoma skin cancer and induces biological effects that promote photocarcinogenesis by causing mutations directly on DNA. However, there seems to be an indirect effect, which induces immunosuppession. The physiological phenomenon of immune tolerance, which is responsible for the prevention of autoimmune diseases and the modulation of inflammatory response, may be used by certain tumors to escape immune control. This phenomenon is enhanced by UV radiation. Immature dendritic cells, regulatory T cells (Treg), and also several immunosuppressive cytokines play a central role in immune tolerance associated to the tumor environment. In order to assess the immune tolerance in lip photocarcinogenesis, we analyzed 75 samples represented by 44 cases of actinic cheilitis (AC), 18 cases of lip squamous cell carcinoma (LSCC) and 12 specimens of normal lip. Sections were submitted by means of immunohistochemistry, to the antibodies against CD83, DEC-205, FOXP3, CD1a and CD207. Positive cells for each marker were counted in three high-power fields in each section and the results expressed by the mean number of cells per field. The results showed a large amount of Treg FOXP3+ in both AC and LSCC. There was also a strong staining for DEC-205 in the cases studied. There were an increased number of CD1a+/CD207+ Langerhans cells in the AC epithelium and LSCC islets, as well as a few cells in the connective tissue. For the marker CD83, the count of positive cells was low compared to other markers for both lesions. Therefore, we suggest an immune tolerant microenvironment both at the beginning, and in the establishment of the lip photocarcinogenesis process.

A doença enxerto contra hospedeiro (GVHD) é uma complicação comum nos pacientes submetidos ao transplante de células-tronco hematopoiéticas (TCTH), sendo considerada a maior causa de morbidade e mortalidade nesses pacientes. O principal objetivo do presente estudo foi relacionar a concentração de células de Langerhans em mucosa bucal de pacientes com GVHDc bucal com a expressão da quimiocina CCL20 e de seu receptor CCR6 no epitélio bucal, a fim de elucidar os mecanismos biológicos envolvidos no recrutamento das células de Langerhans na GVHDc. Foram selecionados fragmentos obtidos por biópsia de mucosa bucal de 60 pacientes onco-hematológicos e hematológicos submetidos previamente ao transplante de células tronco hematopoiéticas no Hospital Amaral Carvalho, Jaú SP, onde 30 pacientes desenvolveram GVHDc em mucosa bucal (Grupo 1) e 30 não desenvolveram GVHDc (Grupo 2). Amostras obtidas a partir de 30 biópsias de lesões não inflamatórias em mucosa bucal constituíram o Grupo Controle (Grupo 3). Cortes microscópicos foram avaliados em coloração de rotina Hematoxilina e Eosina, e submetidos à técnica imuno-histoquímica, utilizando-se anticorpos monoclonais anti-CD1a e anti-CCR6, e anticorpos policlonais anti-CCL20. As células de Langerhans CD1a+ foram quantificadas no epitélio da mucosa bucal, e os resultados demonstraram um maior número destas células nos pacientes com GVHDc quando comparados àqueles sem GVHDc e ao Grupo Controle (p<0,001). A análise da imunomarcação das moléculas CCR6 e CCL20 foi subjetiva com aplicação de escores. Quanto à molécula CCR6, houve maior expressão no Grupo 1 (p<0,001) em comparação aos outros Grupos; porém, quanto à expressão de CCL20, não houve diferença estatística entre os três Grupos (p=0,108). Estes resultados sugerem que o aumento das células de Langerhans, na doença enxerto contra hospedeiro crônica, em mucosa bucal, pode estar associado a maior expressão do receptor CCR6. Possivelmente, o maior recrutamento de células de Langerhans até a mucosa bucal, em pacientes transplantados de medula óssea, colabora para o desenvolvimento da GVHDc bucal. / The graft versus host disease (GVHD) is a common complication in patients undergoing hematopoietic stem cell transplantation (HSCT), and considered a major cause of morbidity and mortality in these patients. The main objective of this study was to compare the concentration of Langerhans cells in oral mucosa of patients with oral chronic GVHD (GVHDc) with the expression of the chemokine CCL20 and its receptor CCR6 in oral epithelium, in order to clarify the biological mechanisms involved in the recruitment of Langerhans cells in GVHDc. We selected 60 biopsies of oral mucosa from onco-hematological and hematological patients submitted to prior hematopoietic stem cell transplantation at Hospital Amaral Carvalho, Jaú - SP from which 30 patients developed GVHDc in the oral mucosa (Group 1) and 30 did not develop GVHDc (Group 2). The Control Group (Group 3) was obtained from 30 biopsies of non-inflammatory lesions of oral mucosa. Microscopic sections were evaluated in routine Hematoxylin and Eosin staining, and submitted to immunohistochemistry using anti-CD1a and anti-CCR6 monoclonal antibodies, and anti-CCL20 polyclonal antibody. The Langerhans cells (CD1a+) were quantified in the epithelium of the oral mucosa, and the results showed a greater number of these cells in patients with GVHDc compared to those without GVHDc and the Control Group (p<0.001). Analysis of immunostaining of molecules CCL20 and CCR6 were subjective with application of scores. The expression of CCR6 molecule was more significant in Group 1 (p<0.001) compared to other groups, but in relation to CCL20 expression, there was no statistical difference between the three groups (p=0.108). These results suggest that the increase of Langerhans cells in GVHDc affecting oral mucosa may be associated with increased expression of the receptor CCR6. We suggest that the increased recruitment of Langerhans cells to the oral mucosa in patients with transplanted bone marrow contributes to the development of oral GVHDc.

La métaplasie épithéliale est un phénomène d'adaptation tissulaire apparaissant à la suite d'une agression chronique. Caractérisées par le remplacement d'un épithélium par un autre, ces transformations métaplasiques peuvent apparaître dans différentes régions anatomiques (bronches, sophage, col de l'utérus, estomac et vessie). Bien que mieux adapté à résister à un agent irritatif, l'épithélium métaplasique présente cependant une susceptibilité accrue au développement cancéreux. Ainsi, la grande majorité (87%) des lésions (pré)néoplasiques cervicales se développe au sein du microenvironnement métaplasique de la zone de transformation du col utérin. Les travaux réalisés dans le cadre de cette thèse ont contribué à une meilleure compréhension des mécanismes responsables de la métaplasie épidermoïde cervicale et ont démontré, pour la première fois, une implication du TGF-β1 et de la PGE2 dans limmunodéficience locale observée dans les foyers de métaplasie mature et immature. Ainsi, en réduisant lexpression de E-cadhérine, le TGF-β1 altère indirectement la rétention épithéliale des cellules présentatrices dantigène en empêchant leurs interactions avec les cellules épithéliales. Quant à la PGE2, elle affecte la migration et le phénotype de ces cellules immunitaires, les rendant tolérogènes et, par conséquent, incapables dactiver les lymphocytes T naïfs. Par ailleurs, il est possible que la réduction de ΔNp63 par les facteurs de transcription Snail et Slug participe à la mise en place des épithélia métaplasiques ainsi quà leur transformation maligne.

La transplantation d’îlots de Langerhans microencapsulés est un traitement prometteur du diabète de type 1. La microcapsule protège l’îlot du système immunitaire, tout en permettant la diffusion de petites molécules. Comme la microcapsule empêche la revascularisation des îlots, leur oxygénation se fait par diffusion d’oxygène et ils sont exposés à l’hypoxie. Le manque d’oxygène est un facteur limitant dans la survie des îlots microencapsulés. Il est connu que les plus petits îlots sont plus résistant à l’hypoxie à cause d’une meilleure diffusion de l’oxygène. À cette fin, les agrégats de cellules dispersées d’îlots seront étudiés. Lorsque les cellules des îlots sont dispersées, elles ont la propriété de se ré-assembler dans une structure semblable à celle des îlots. La présente étude a permis de mettre au point une technique de formation des agrégats, de les caractériser et de comparer la résistance à l’hypoxie des îlots et des agrégats. Ceux-ci ont une structure semblable aux îlots et ils sont de plus petite taille. Pour cette raison, ils sont plus viables après un choc hypoxique tout en renversant efficacement l’hyperglycémie de souris diabétiques. Les agrégats sont une alternative intéressante pour la transplantation d’îlots microencapsulés puisque leur oxygénation est plus efficace. / Transplantation of microencapsulated islets of Langerhans is a promising treatment for type 1 diabetes mellitus. The microcapsule allows the diffusion of small molecules, while protecting the islet from the antibodies and immune cells. However, microcapsule prevents islet revascularization, thus oxygenation depends on diffusion and islets are exposed to hypoxia. Poor oxygenation is a major limitation in microencapsulated islet survival. It was shown that smaller islets are more resistant to hypoxia because of a better oxygen diffusion. In this study, dispersed islet cell aggregates will be used to improve the oxygenation. When islet cells are dispersed into single cells, they have the ability to re-associate into an islet-like structure. This study allowed to set up a technique to form aggregates, to characterized them and to compare the resistance to hypoxia of islets and aggregates. Aggregates have a similar structure than islets and they are smaller. For this reason, they survive better to a hypoxic treatment, while restoring efficiently normoglycemia in diabetic mices. Aggregates are an interesting solution for microencapsulated islet transplantation because they have a better oxygenation.

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 5 uppsatser.

Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 2 uppsatser.

Durante a gravidez, o pâncreas endócrino materno sofre alterações morfológicas e funcionais que resultam no aumento da massa de células beta e da secreção de insulina. Nos estágios finais da gestação ocorre aumento dos níveis plasmáticos de glicocorticóides que resulta na diminuição da secreção e da proliferação das células beta. Este fenômeno, que ocorre no período compreendido entre o final da gravidez e o inicio da lactação, promove a reversão fisiológica da adaptação funcional que se fez necessária durante a gravidez. Assim, estudamos mecanismos moleculares envolvidos na redução de proliferação destas células. As proteínas cinases reguladas por sinais extracelulares (ERK) estão envolvidas no crescimento e sobrevida celular. Os resultados mostram que o glicocorticóide sintético, dexametasona, diminui a proliferação de células beta e, para isto, induz diminuição da fosforilação das ERK-1/2 por meio do aumento da expressão de uma fosfatase de MAPK (MKP-1). Este mecanismo deve estar envolvido no remodelamento pancreático pós-natal induzido pelos glicocorticóides. / During pregnancy, maternal pancreatic islets undergo morphofunctional changes that increase beta cell mass and insulin secretion. At late stages of pregnancy there is an increase in plasma glucocorticoid levels that inhibit beta cell proliferation and beta cell function. This situation, which occurs in a period between late pregnancy and early stages of lactation, counteracts the functional gain established throughout pregnancy. In this work we studied the molecular mechanisms involved in the impaired beta cell proliferation. The extracellular regulated kinases (ERKs) are involved in cellular growth and survival. Our results show that dexametasone, a synthetic glucocorticoid, inhibits proliferation by a mechanism that includes up regulation of a dual specificity phosphatase (MKP1). This, by extension, impairs ERK1/2 activation. This mechanism could take part in the induced-glucocorticoid reestablishment of endocrine pancreatic mass after parturition.

O efeito de alterações no estado de óxido-redução (redox), tanto pelo aumento no estado oxidativo quanto pelo aumento no estado redutor, foi avaliado sobre a funcionalidade de ilhotas pancreáticas, através da análise da secreção de insulina estimulada pela glicose (GSIS), metabolismo da glicose e oscilações intracelulares de cálcio. O aumento no estado oxidativo inibiu a funcionalidade da célula pancreática. Entretanto, diminuição no estado oxidativo pela adição de antioxidantes exerceu efeito dual sobre a funcionalidade da célula <font face=\"Symbol\">&#946 pancreática, na qual pequenas alterações no estado redox estimularam a GSIS, enquanto alterações maiores suprimiram este efeito positivo. Adicionalmente, o conteúdo das espécies reativas de oxigênio (EROs) foi modulado por mudanças na concentração de glicose. Agudamente, o aumento na concentração de glicose suprimiu o conteúdo de EROs, que pôde ser correlacionada com o aumento na atividade da via de formação de NADPH, a via das pentoses-fosfato. Sob estes aspectos, alterações no estado redox podem ser parte do processo da GSIS. / The effect of changes in the oxidation/reduction (redox) state over pancreatic islet function was analyzed by shifts toward oxidative or reducing environments. Pancreatic cell function was analyzed by glucose-stimulated insulin secretion (GSIS), glucose metabolism and intracellular calcium oscillations. Redox modulation favoring the oxidative state inhibited pancreatic cell function. However, the suppression of the oxidative state by antioxidant treatment exerted a dual effect on pancreatic <font face=\"Symbol\">&#946 cell function, where small changes were positively correlated with an increase in insulin secretion, while higher changes suppressed GSIS. Additionally, the reactive oxygen species (ROS) content was modulated by changes in glucose concentration. Increasing concentrations of glucose acutely suppressed ROS content, what was correlated with the activation of the NADPH source, the pentose-phosphate pathway. Thus, the intracellular adjustment of ROS content may be part of the insulin secretion mechanism in response to glucose.