In the studies about ovarian cancer cells, basal iNOS expression in the chemosensitive OV2008 cells was significantly higher than in the chemoresistant C13* cells. Cisplatin further increased iNOS expression in OV2008 cells, but had no effect in C13* cells. Furthermore, cisplatin dramatically reduced the expression levels of eNOS and nNOS, but again only in OV2008 cells. The data suggest that failure of cisplatin to upregulate iNOS and downregulate eNOS and nNOS in C13* cells could be an etiological factor in chemoresistance. Addition of exogenous NO at high levels, using SNAP, significantly increased p53 protein levels and caused apoptosis in both cell types. Specific iNOS inhibitor (1400W) partially blocked the pro-apoptotic effects of cisplatin in OV2008 cells, suggesting involvement of iNOS in cisplatin-induced apoptosis. However, blocking of all three isoforms of NOS with NG-amino-L-arginine in C13* cells dramatically changed these cells from chemoresistant to chemosensitive, greatly potentiating the pro-apoptotic effects of cisplatin. / Inhibition of Src-kinase activity reduces DNA synthesis in ovarian cancer cells. In an in vitro experiment, Src phosphorylated PKG on a tyrosine residue and PKG, presumable via serine-phosphorylation of Src, enhanced Src auto(tyrosine)phosphorylation. In ovarian cancer cells, inhibition of basal PKG activity with DT-2 decreased both basal and EGF-stimulated Src kinase activation and DNA synthesis. The data suggest that PKG at basal activity, is necessary for both basal and growth factor-stimulated Src kinase activation and enhanced DNA synthesis in human ovarian cancer cells. / The novel role of sGC/cGMP/PKG pathway on stimulating cell proliferation, potentially via interaction with the Src kinase pathway in human ovarian cancer cells, was demonstrated. ODQ dramatically reduced DNA synthesis rates, suggesting that basal sGC activity and basal cGMP levels are needed for ovarian cancer cell proliferation. DT-2 also reduced cell proliferation, suggesting the direct involvement of PKG. ANP and BNP had no effect on cell proliferation, suggesting that further activation of cGMP/PKG pathway above basal levels does not further enhance cell proliferation. / The present study also demonstrated that elevating cGMP slightly above the basal levels further protects pancreatic islet cells against spontaneous onset of apoptosis. The results showed that natriuretic peptides (both ANP and BNP) and low-level NO (i.e. physiological levels) as supply by NO donor, S-nitroso-N-acetylpenicilamine (SNAP) further prevented spontaneous apoptosis in pancreatic islets after isolation, whereas NO at high concentrations (i.e. pathological levels) promoted apoptosis in pancreatic islet cells. The commonly-used PKG inhibitor KT5823 and the newly-developed specific PKG inhibitor DT-2 completely prevented anti-apoptosic effect of ANP, suggesting the direct involvement of PKG in protection against spontaneous apoptosis. / The present study demonstrated that basal activity of sGC/cGMP/PKG signaling pathway is essential for partially limiting spontaneous apoptosis in pancreatic islet cells. The sGC inhibitor ODQ caused induction of apoptosis, which was completely blocked by co-treatment with ANP or BNP, agents that elevate cGMP via pGC, bypassing the ODQ block. Co-treatment with 8-Br-cGMP, a direct activator of PKG also completely prevented ODQ-induced apoptosis in islets. / Leung Lai-han. / "July 2006." / Adviser: Ronald Ray Fiscus. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1483. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 175-191). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

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

As espécies reativas de oxigênio são capazes de influenciar a secreção de insulina, porém ainda não está clara a influência da glicose, principal secretagogo deste hormônio, sobre a homeostase redox das células beta pancreáticas. Incubações por 1 e 48 horas com diferentes concentrações de glicose (2,8; 5,6; 8,3; 11,1; 16,7 e 20 mM) demonstraram que esta é capaz de alterar não só o conteúdo de superóxido, produzido pela mitocôndria e NADPH oxidase, mas também o sistema antioxidante, alterando a concentração de GSH e a expressão das enzimas antioxidantes. Além disso, aumenta a interação Rac1/Sod1, que mantém a NADPH oxidase ativa. Porém, não apresenta endossomas de sinalização redox, os redoxossomas, em resposta a glicose. Estas alterações podem afetar eventos chave para este tecido endócrino, como a secreção de insulina e a morte celular. / ROS production in pancreatic beta cells has been associated with the insulin secretion process but the mechanism by which glucose affects the redox state in these cells remains unknown. In order to address this issue, we evaluated the effect of 1 or 48 hours incubation of pancreatic beta cells with various glucose concentrations (2.8, 5.6, 8.3, 11.1, 16.7 and 20 mM). Glucose loading induced superoxide production by mitochondria and NADPH oxidase complex, and enhanced the antioxidant capacity by increasing GSH content and modulate expression of antioxidant enzymes. Glucose also promoted Rac1/Sod1 interaction that maintains NADPH oxidase activated. These cells however did not present redox endosomes, the redoxosomes, in response to glucose loading. These effects might be associated with the process of insulin secretion and pancreatic beta cell death.

O Diabetes Mellitus tipo 2 (DM 2) é uma doença crônica na qual os pacientes apresentam capacidade secretória de insulina inadequada para suplantar a resistência insulínica concomitante e, como resultado, advém a hiperglicemia. Os mecanismos que explicam a diminuição da secreção insulínica não são completamente conhecidos e acredita-se que o depósito de amilina, um achado histopatológico freqüente nesses pacientes, esteja envolvido. A amilina humana é uma proteína co-secretada com a insulina capaz de se agregar e se depositar nas ilhotas pancreáticas. Ainda não está totalmente estabelecido se a toxicidade da amilina humana é mediada pelas fibrilas maduras, conforme demonstrado em trabalhos mais antigos, ou por oligômeros de tamanho intermediário, como tem sido aventado nos trabalhos mais recentes. O objetivo deste estudo foi avaliar o perfil de genes modulados por oligômeros, bem como por fibrilas maduras de amilina, em ilhotas pancreáticas de rato. As ilhotas foram isoladas a partir de ratos Wistar, mantidas em cultura por 24 horas e a seguir tratadas com 10 M de oligômeros ou de fibrilas maduras de amilina por 24 horas adicionais em concentração fisiológica ou suprafisiológica de glicose. O RNA total foi extraído e utilizado para análise da expressão gênica por microarranjos de DNA. O conteúdo de RNA de alguns genes modulados nas condições experimentais estudadas também foi avaliado por RT-qPCR, a fim de validar os resultados obtidos pela análise de microarranjos. A análise das vias significativamente afetadas pelas preparações de amilina demonstrou que, em ilhotas mantidas em concentração fisiológica de glicose, os oligômeros de amilina modularam, entre outros, processos relacionados à Resposta ao Estresse e à Apoptose, processos não modulados pelas fibrilas maduras de amilina. Em concentração suprafisiológica de glicose, o tratamento com oligômeros de amilina deixou de modular as vias relacionadas a Estresse e Apoptose, surgindo como moduladas vias relacionadas aos processos de Regulação da endocitose e Biossíntese de óxido nítrico. Os resultados do RT-qPCR sugeriram que somente os oligômeros (e não as fibrilas maduras) de amilina modulam genes relacionados a apoptose (Anxa1, Rab5a) e ao estresse oxidativo (Nos2 e Xdh), o que vai ao encontro dos estudos mais recentes que atribuem às fibrilas intermediárias um papel na citotoxicidade das células . Um achado novo do presente estudo foi a identificação do mRNA do Gipr (receptor de polipeptídeo inibitório gástrico) como alvo de regulação negativa pelos oligômeros de amilina, o que sugere que esse possa ser um mecanismo adicional pelo qual essas fibrilas intermediárias de amilina sejam deletérias para a célula pancreática. / Type 2 diabetes mellitus is a chronic disease in which there is inability of pancreatic cells to secrete sufficient insulin to overcome the insulin resistance in the peripheral tissues with resultant hyperglycemia. Mechanisms leading to diminished insulin secretion are not completely known and the amyloid deposit, a frequent histopathological finding in patients with type 2 diabetes, is believed to be involved. Human amylin, a protein co-secreted with insulin, is capable of aggregating and forming deposits in the pancreatic islets. It is not fully established whether amylin cytotoxicity is mediated by mature amylin fibrils or by soluble oligomers. The objective of this study was to evaluate the gene profiling modulated by oligomers as well as by mature amylin fibrils in rat pancreatic islets. The islets were isolated from Wistar rats, maintained in culture for 24 hours and then treated with 10 M of oligomers or mature amylin fibrils for additional 24 hour in physiologic and supraphysiologic glucose concentrations. Total RNA was extracted and used for gene expression analysis by microarray. RNA content of some modulated genes was evaluated by RT-qPCR in order to validate the results obtained from the microarray analysis. The analysis of the pathways significantly affected by the two amylin preparations demonstrated that, in islets maintained in physiological glucose concentration, amylin oligomers modulated, among others, processes related to Response to stress and to Apoptosis, which were not modulated by mature amylin fibrils. In supraphysiological glucose concentration, treatment with oligomers did not modulate the pathways related to Stress and Apoptosis, which were replaced by processes related to Endocytosis regulation and Nitric oxide biosynthesis. RT-qPCR results suggested that only amylin oligomers modulate genes related to apoptosis (Anxa1, Rab5a) and oxidative stress (Nos2 e Xdh), which is in agreement with studies indicating a role for oligomers in the cytotoxicity of cells. A new finding of the present study was the identification of the Gipr (gastric inhibitory polypeptide receptor) mRNA as a target for downregulation by amylin oligomers, which suggests that this might be an additional mechanism by which these oligomers are deleterious to the pancreatic cells.

<p>The process of β-cell destruction that leads to type 1 diabetes (T1D) is incompletely understood and it is believed to be a result of both genetic and environmental factors. Enterovirus (EV) infections of the β-cells have been proposed to be involved, however, the effects of EV infections on human β-cells have been little investigated. This thesis summarises studies of three different Coxsackie B4 virus strains that have previously been shown to infect human islets. The effects of infections with these EV were studied <i>in vitro</i> in human islets and in a rat insulin-producing cell line. In addition, a pilot study was performed on isolated human islets to investigate the ability to treat such infections with an antiviral compound.</p><p>It was found that one of the virus strains replicated in human β-cells without affecting their main function for at least seven days, which <i>in vivo</i> may increase a virus’s ability to persist in islets.</p><p>Nitric oxide was induced by synthetic dsRNA, poly(IC), but not by viral dsRNA in rat insulinoma cells in the presence of IFN-γ, suggesting that this mediator is not induced by EV infection in β-cells and that poly(IC) does not mimic an EV infection in this respect.</p><p>All three virus strains were able to induce production of the T-cell chemoattractant interferon-γ-inducible protein 10 (IP-10) during infection of human islets, suggesting that an EV infection of the islets might trigger insulitis <i>in vivo</i>.</p><p>Antiviral treatment was feasible in human islets, but one strain was resistant to the antiviral compound used in this study.</p><p>To conclude, a potential mechanism is suggested for the involvement of EV infections in T1D. If EV infections induce IP-10 production in human islet cells <i>in vivo</i>, they might recruit immune cells to the islets. Together with viral persistence and/or virus-induced β-cell damage, this might trigger further immune-mediated β-cell destruction <i>in vivo</i>.</p>

<p>Type 1 diabetes mellitus affects millions of people worldwide. Islet transplantation is a minimal invasive surgical procedure that restores euglycemia and halts the progression of diabetic complications. However, despite transplantation of islets from multiple donors most patients reverse to hyperglycemia within five years. New strategies to improve long-term outcome of islet transplantation are indispensable. This thesis studied differences in the microvasculature between endogenous and transplanted pancreatic islets, and investigated means to improve islet graft revascularization and function. Islet graft microvessels were similar to endogenous islets responsive to adenosine, angiotensin II and nitric oxide (NO). Recipient hyperglycemia induced a higher basal islet graft blood flow, which also was less dependent on NO than in normoglycemic recipients. Transplantation of freshly isolated instead of cultured islets improved graft revascularization, oxygenation and function. Pretreatment of islets with vascular endothelial growth factor decreased their expression of matrix metalloproteinase-9 (MMP-9) and impaired graft revascularization. Moreover, MMP-9 pretreatment <i>per se</i> improved graft revascularization. <i>In vivo</i>, 20-25% of all endogenous rat islets was low oxygenated (pO₂ <10 mmHg). Changes in the islet mass, by means of whole-pancreas transplantation, doubled the fraction of low oxygenated islets in the endogenous pancreas of transplanted animals, whereas this fraction almost completely disappeared after a 60% partial pancreatectomy. Interestingly, oxygenation was related to metabolism, since well oxygenated islets <i>in vivo</i> had 50% higher leucine-dependent protein biosynthesis, which includes (pro)insulin biosynthesis. In intraportally transplanted islets, the low oxygenated fraction of islets was markedly increased one day post-transplantation, and the oxygenation remained low following revascularization. In summary, these data suggest that a better revascularization of transplanted islets can improve graft function. Furthermore, the oxygenation and metabolism of endogenous islets is tightly regulated. This regulation seems to be disturbed following transplantation, which may contribute to long-term islet graft failure. </p>

<p>Intraportal transplantation of isolated islets of Langerhans is a procedure approaching clinical acceptance as a treatment for patients with type I diabetes mellitus. One major problem with this treatment is that large amounts of cells are lost at the time of infusion into the portal vein, resulting in a low level of engraftment of the islets. One likely explanation for this loss is the instant blood-mediated inflammatory reaction (IBMIR), a thrombotic/inflammatory reaction occurring when islets come in contact with blood. The IBMIR is characterized by coagulation and complement activation, leading to platelet consumption, leukocyte infiltration of the islets, and disruption of islet integrity.</p><p>In this thesis, the IBMIR is shown to be triggered by tissue factor (TF), the main initiator of blood coagulation<i> in vivo</i>. TF is expressed in two forms by the endocrine cells of the pancreas, a full-length membrane-bound and an alternatively spliced soluble form. Blocking TF <i>in vitro</i> efficiently reduces the macroscopic clotting, expression of coagulation activation markers, and leukocyte infiltration. This blockade can be achieved by adding either an active site-specific anti-TF antibody or site-inactivated FVIIa that competes with active FVIIa in the blood. TF may be secreted from the islets, since it is colocalized with insulin and glucagon in their granules. The IBMIR has also been demonstrated <i>in vivo</i> in patients transplanted with isolated islets.</p><p>There are two ways to block the IBMIR in transplantation: systemic treatment of the patients, or islet pretreatment before transplantation to reduce their thrombogenicity. In this thesis, low molecular weight dextran sulfate (LMW-DS) is shown to reduce activation of the complement and coagulation systems and decrease the cell infiltration into the islets <i>in vitro</i> and<i> in vivo</i>, in both a xenogenic and an allogenic setting. Based on these results, LMW-DS is now in clinical trials. </p>

The process of β-cell destruction that leads to type 1 diabetes (T1D) is incompletely understood and it is believed to be a result of both genetic and environmental factors. Enterovirus (EV) infections of the β-cells have been proposed to be involved, however, the effects of EV infections on human β-cells have been little investigated. This thesis summarises studies of three different Coxsackie B4 virus strains that have previously been shown to infect human islets. The effects of infections with these EV were studied in vitro in human islets and in a rat insulin-producing cell line. In addition, a pilot study was performed on isolated human islets to investigate the ability to treat such infections with an antiviral compound. It was found that one of the virus strains replicated in human β-cells without affecting their main function for at least seven days, which in vivo may increase a virus’s ability to persist in islets. Nitric oxide was induced by synthetic dsRNA, poly(IC), but not by viral dsRNA in rat insulinoma cells in the presence of IFN-γ, suggesting that this mediator is not induced by EV infection in β-cells and that poly(IC) does not mimic an EV infection in this respect. All three virus strains were able to induce production of the T-cell chemoattractant interferon-γ-inducible protein 10 (IP-10) during infection of human islets, suggesting that an EV infection of the islets might trigger insulitis in vivo. Antiviral treatment was feasible in human islets, but one strain was resistant to the antiviral compound used in this study. To conclude, a potential mechanism is suggested for the involvement of EV infections in T1D. If EV infections induce IP-10 production in human islet cells in vivo, they might recruit immune cells to the islets. Together with viral persistence and/or virus-induced β-cell damage, this might trigger further immune-mediated β-cell destruction in vivo.

Type 1 diabetes mellitus affects millions of people worldwide. Islet transplantation is a minimal invasive surgical procedure that restores euglycemia and halts the progression of diabetic complications. However, despite transplantation of islets from multiple donors most patients reverse to hyperglycemia within five years. New strategies to improve long-term outcome of islet transplantation are indispensable. This thesis studied differences in the microvasculature between endogenous and transplanted pancreatic islets, and investigated means to improve islet graft revascularization and function. Islet graft microvessels were similar to endogenous islets responsive to adenosine, angiotensin II and nitric oxide (NO). Recipient hyperglycemia induced a higher basal islet graft blood flow, which also was less dependent on NO than in normoglycemic recipients. Transplantation of freshly isolated instead of cultured islets improved graft revascularization, oxygenation and function. Pretreatment of islets with vascular endothelial growth factor decreased their expression of matrix metalloproteinase-9 (MMP-9) and impaired graft revascularization. Moreover, MMP-9 pretreatment per se improved graft revascularization. In vivo, 20-25% of all endogenous rat islets was low oxygenated (pO2 &lt;10 mmHg). Changes in the islet mass, by means of whole-pancreas transplantation, doubled the fraction of low oxygenated islets in the endogenous pancreas of transplanted animals, whereas this fraction almost completely disappeared after a 60% partial pancreatectomy. Interestingly, oxygenation was related to metabolism, since well oxygenated islets in vivo had 50% higher leucine-dependent protein biosynthesis, which includes (pro)insulin biosynthesis. In intraportally transplanted islets, the low oxygenated fraction of islets was markedly increased one day post-transplantation, and the oxygenation remained low following revascularization. In summary, these data suggest that a better revascularization of transplanted islets can improve graft function. Furthermore, the oxygenation and metabolism of endogenous islets is tightly regulated. This regulation seems to be disturbed following transplantation, which may contribute to long-term islet graft failure.