The high affinity sodium/glucose cotransporter (SGLT1) couples transport of Na+ and glucose. Investigation of the structure/function relationships of the sodium/glucose transporter (SGLT1) is crucial to understanding co-transporter mechanism. In the first project, we used cysteine-scanning mutagenesis and chemical modification by methanethiosulphonate (MTS) derivatives to test whether predicted TM IV participates in sugar binding. Charged and polar residues and glucose/galactose malabsorption (GGM) missense mutations in TM IV were replaced with cysteine. Mutants exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes and COS-7 cells. The results from mutants T156C and K157C suggest that TM IV participates in sugar interaction with SGLT1. This work has been published in Am J Physiol Cell Physiol 295 (1), C64-72, 2008. The crystal structure of Vibrio parahaemolyticus SGLT (vSGLT) was recently published (1) and showed discrepancy with the predicted topology of mammalian SGLT1 in the region surrounding transmembrane segments IV-V. Therefore, in the second project, we investigated the topology in this region, thirty-eight residues from I143 to A180 in the N-terminal half of rabbit SGLT1 were individually replaced with cysteine and then expressed in COS-7 cells or Xenopus laevis oocytes. Based on the results from biotinylation of mutants in intact COS-7 cells, MTSES accessibility of cysteine mutants expressed in COS-7 cells, effect of substrate on the accessibility of mutant T156C in TM IV expressed in COS-7 cells, and characterization of cysteine mutants in TM V expressed in Xenopus laevis oocytes, we suggest that the region including residues 143-180 forms part of the Na+- and sugar substrate-binding cavity. Our results also suggest that TM IV of mammalian SGLT1 extends from residue 143-171 and support the crystal structure of vSGLT. This work has been published in Biochem Biophys Res Commun 378 (1), 133-138, 2009 Previous studies established that mutant Q457C human SGLT1 retains full activity, and sugar translocation is abolished in mutant Q457R or in mutant Q457C following reaction with methanethiosulfonate derivatives, but Na+ and sugar binding remain intact. Therefore, in the third project, we explored the mechanism by which modulation of Q457 abolishes transport, Q457C and Q457R of rabbit SGLT1 expressed in Xenopus laevis oocytes were studied using chemical modification, the two-electrode voltage-clamp technique and computer model simulations. Our results suggest that glutamine 457, in addition to being involved in sugar binding, is a residue that is sensitive to conformational changes of the carrier. This work has been published in Biophysical Journal 96 (2), 748-760, 2009. Taken together our study along with previous biochemical characterization of SGLT1 and crystal structure of vSGLT, we propose a limited structural model that attempts to bring together the functions of substrate binding (Na+ and sugar), coupling, and translocation. We propose that both Na+ and sugar enter a hydrophilic cavity formed by multiple transmembrane helices from both N-terminal half of SGLT1 and C-terminal half of SGLT1, analogous to all of the known crystal structures of ion-coupled transporters (the Na+/leucine transporter, Na+/aspartate transporter and lactose permease). The functionally important residues in SGLT1 (T156 and K157 in TM 4, D454 and Q457 in TM 11) are close to sugar binding sites.

Glukosetransporter spielen eine wichtige Rolle in der Versorgung des Gehirns mit Nährstoffen und somit für den Erhalt der physiologischen Zellintegrität. Glukose wird über die Blut-Hirn-Schranke (BHS) mittels spezifischen transmembranen Transportproteinen der SLC-Genfamilie (GLUT, SGLT) befördert. Dabei scheint während physiologischen Bedingungen hauptsächlich der Glukosetransporter GLUT1 (SLC2A1) für die Energieversorgung des Gehirns zuständig zu sein. Die Erforschung der SGLT-Expression ist in den letzten Jahren ein wichtiger Ansatzpunkt für neue Behandlungsstrategien vieler Erkrankungen, wie Diabetes Mellitus, maligne Neoplasien oder eines Herzinfarkts, geworden. Jedoch ist über deren Expression und Funktion im menschlichen Gehirn nur wenig bekannt. Besonders die Lokalisation entlang der BHS bleibt fraglich. Ein Großteil bisheriger Forschungsarbeiten beschäftigt sich hauptsächlich mit der Expressionsanalyse des Transporters SGLT1 im tierischen Gehirn in vivo (Poppe et al. 1997; Balen et al. 2008; Yu et al. 2013). Es konnte aufgezeigt werden, dass SGLT1 mRNA exklusiv in Neuronen und nicht an der BHS exprimiert wird. Dies wird durch in vitro Analysen einer humanen Hirnendothelzelllinie bestätigt. Demnach kann kein SGLT1 unter physiologischen Bedingungen nachgewiesen werden (Sajja et al. 2014). Im menschlichen Hirngewebe besitzen SGLTs somit keine zentrale Funktion für den Glukosetransport an der BHS. Im Gegensatz dazu konnte eine Expression von SGLT sowohl in vivo als auch in vitro während hypoglykämischen Bedingungen belegt werden (Vemula et al. 2009; Sajja et al. 2014). Die Expression der SGLT-Transporter während einer ischämischen Hypoglykämie führt zu der Annahme, dass diese Transporter für die Aufrechterhaltung der Energieversorgung des geschädigten Hirngewebes notwendig sind. Um die physiologischen Mechanismen nach einem Glukosemangel zu untersuchen, wurden SHT-Modelle etabliert (Salvador et al. 2013). In einem experimentellen Modell des Schädel-Hirn-Traumas im Rahmen eines DFG-gefördertes Projekts ist ein Expressionsverlauf von Glukosetransportern im Maushirn und in Hirnendothelzellen erarbeitet worden (Wais 2012; Salvador et al. 2015). Somit könnten SGLTs als Ansatzpunkt für den Nachweis der Überlebenszeit nach einem SHT fungieren. Die vorliegende Arbeit fokussiert sich auf die Expression der Natrium-abhängigen Glukosetransporter SGLT1 und SGLT2 im menschlichen Gehirn. Hierbei liegt das Hauptaugenmerk auf der Lokalisation dieser Transporter an der menschlichen BHS von post mortalem Hirngewebe. Weiterhin wird untersucht ob die Expressionsstärke von SGLT1 und SGLT2 eine Aussage über die Überlebenszeit von Verstorbenen nach einer traumatisch bedingten Hirnveränderung zulässt. Die Lokalisation von SGLT1 und SGLT2 an der menschlichen BHS konnte durch die Etablierung eines Protokolls zur Isolation von Hirnkapillaren erfolgen. Vorab wurden alle verwendeten Antikörper auf ihre Spezifität mittels siRNA Transfektion und Blockierung der Immunfluoreszenzsignale mittels immunisierten Peptids getestet. Somit ist die Spezifität der detektierten SGLT1- und SGLT2-Expression in menschlichen Hirnkapillaren gewährleistet. Anschließend wird untersucht, in welchen zeitlichem Verlauf nach einer traumatisch bedingten Hirnveränderung die verschiedenen Formen der Glukosetransporter exprimiert werden und ob ggf. der Umfang und die Verteilung von SGLT1, SGLT2 und GLUT1 sowie das Verhältnis zueinander Auskünfte über eine vitale bzw. postmortale Entstehung eines Traumas bzw. dessen Überlebenszeit zulässt. Hierfür wird ein Expressionsschema der Glukosetransporter generiert, abhängig von Todeszeitpunkt und Todesursache. Es konnte festgestellt werden, dass GLUT1 nicht als Target für die Ermittlung der Überlebenszeit nach einem Trauma geeignet ist. Dahingegen zeigen SGLT1 und SGLT2 eine signifikante Änderung der Expressionsstärke im contusionalen Gewebe in Abhängigkeit von der Überlebenszeit. Obwohl diese vorläufigen Daten einen neuen Ansatzpunkt für die forensische Fragestellung aufzeigen, müssen weitere Experimente mit einem erhöhten Umfang der Probenanzahl und kürzere Zeitspannen der Überlebenszeiträume durchgeführt werden. / The transport of glucose across the endothelial cells of the human blood-brain barrier (hBBB) plays a major role for energy supply of the brain and therefor for neuronal integrity. Glucose enters the brain cells through specific transmembrane transporter proteins of the SLC-gene family (GLUT, SGLT). Under physiological conditions glucose uptake across the BBB seems to be mediated primarily by facilitated diffusion through glucose transporter 1 (GLUT1). Although SGLTs are a known drug target for diabetes and furthermore play a role in other disease like cancer and cardiac ischemia, active glucose transport by SGLTs is hardly observed and very little is known about their expression or activity in human brain. Especially the function along the BBB remains uncertain. Up to now, expression analysis focused on SGLT1 and has been confirmed in vivo by analyzing brain tissue of animals (Poppe et al. 1997; Balen et al. 2008; Yu et al. 2013). Here detection mainly occurs in neurons, no SGLT1 mRNA in capillaries of the BBB could be found. Similarly in vitro experiments with a human brain microvascular endothelial cell line reveals no expression of SGLT1 under physiological conditions (Sajja et al. 2014). In human brain, SGLT1 is hardly expressed and so far could not be found along the BBB. In contrast to these findings, expression of SGLT1 could be detected in vivo as well as in vitro under hypoglycemic conditions (Vemula et al. 2009; Sajja et al. 2014). The occurrence of these transporters during ischemic hypoglycemia could lead to the conclusion that the secondary active glucose transport by SGLTs is necessary for additional glucose supply in injured brain. To investigate if SGLTs are required for the reconstruction of energy supply after glucose deficiency, traumatic brain injury (TBI) models were established to study secondary physiological mechanisms along the BBB (Salvador et al. 2013). In an experimental CCI (controlled cortical impact) mouse model within a DFG-funded project, an expression pattern of glucose transporters in the mouse brain and in brain endothelial cells has been developed (Wais 2012; Salvador et al. 2015). Thus it could lead as a Target for evidence of the time of survival after TBI. This study focuses on the sodium-dependent glucose transporters SGLT1 and SGLT2 expression in human brain. The main topic is to localize the sodium-dependent glucose transporters along the human BBB of post mortem brain tissue and to examine whether SGLT expression allow a conclusion to be drawn about the survival time of a patient after TBI. First of all the localization of SGLT1 and SGLT2 at the human BBB could be shown by establishment a capillary isolation protocol of human post mortem brain tissue. Therefore the antibody specificity was tested by a siRNA transfection protocol and blocking the immunofluorescence signal with an immunized peptide. Thus, specific SGLT1 and SGLT2 expression at the endothelial lining of the capillary lumen could be demonstrated. After attaching the value of SGLTs at the human BBB, the relationship of the glucose transporter expression in TBI tissue according to the survival time of the patient is presented. Hereby it should be clarified whether the expression and distribution of the transporters GLUT1, SGLT1 and SGLT2 as well as the relation to each other provide information on a vital or post mortal development of a trauma or its survival time. It could determine that GLUT1 is not suitable as a target for the representation of survival time after TBI. However, SGLT1 and SGLT2 show a significant change in the expression profile of traumatic brain regions. Here an increase according to the survival time after trauma can be shown. Although these preliminary data suggest a novel target for forensic questions, more experiments with an increased scope of survival time frames should be carried out.

The high affinity sodium/glucose cotransporter (SGLT1) couples transport of Na+ and glucose. Investigation of the structure/function relationships of the sodium/glucose transporter (SGLT1) is crucial to understanding co-transporter mechanism. In the first project, we used cysteine-scanning mutagenesis and chemical modification by methanethiosulphonate (MTS) derivatives to test whether predicted TM IV participates in sugar binding. Charged and polar residues and glucose/galactose malabsorption (GGM) missense mutations in TM IV were replaced with cysteine. Mutants exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes and COS-7 cells. The results from mutants T156C and K157C suggest that TM IV participates in sugar interaction with SGLT1. This work has been published in Am J Physiol Cell Physiol 295 (1), C64-72, 2008. The crystal structure of Vibrio parahaemolyticus SGLT (vSGLT) was recently published (1) and showed discrepancy with the predicted topology of mammalian SGLT1 in the region surrounding transmembrane segments IV-V. Therefore, in the second project, we investigated the topology in this region, thirty-eight residues from I143 to A180 in the N-terminal half of rabbit SGLT1 were individually replaced with cysteine and then expressed in COS-7 cells or Xenopus laevis oocytes. Based on the results from biotinylation of mutants in intact COS-7 cells, MTSES accessibility of cysteine mutants expressed in COS-7 cells, effect of substrate on the accessibility of mutant T156C in TM IV expressed in COS-7 cells, and characterization of cysteine mutants in TM V expressed in Xenopus laevis oocytes, we suggest that the region including residues 143-180 forms part of the Na+- and sugar substrate-binding cavity. Our results also suggest that TM IV of mammalian SGLT1 extends from residue 143-171 and support the crystal structure of vSGLT. This work has been published in Biochem Biophys Res Commun 378 (1), 133-138, 2009 Previous studies established that mutant Q457C human SGLT1 retains full activity, and sugar translocation is abolished in mutant Q457R or in mutant Q457C following reaction with methanethiosulfonate derivatives, but Na+ and sugar binding remain intact. Therefore, in the third project, we explored the mechanism by which modulation of Q457 abolishes transport, Q457C and Q457R of rabbit SGLT1 expressed in Xenopus laevis oocytes were studied using chemical modification, the two-electrode voltage-clamp technique and computer model simulations. Our results suggest that glutamine 457, in addition to being involved in sugar binding, is a residue that is sensitive to conformational changes of the carrier. This work has been published in Biophysical Journal 96 (2), 748-760, 2009. Taken together our study along with previous biochemical characterization of SGLT1 and crystal structure of vSGLT, we propose a limited structural model that attempts to bring together the functions of substrate binding (Na+ and sugar), coupling, and translocation. We propose that both Na+ and sugar enter a hydrophilic cavity formed by multiple transmembrane helices from both N-terminal half of SGLT1 and C-terminal half of SGLT1, analogous to all of the known crystal structures of ion-coupled transporters (the Na+/leucine transporter, Na+/aspartate transporter and lactose permease). The functionally important residues in SGLT1 (T156 and K157 in TM 4, D454 and Q457 in TM 11) are close to sugar binding sites.

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

A metformina é a droga de escolha no tratamento inicial do diabetes mellitus tipo 2 (DM2). Não existe consenso na literatura sobre qual seria a segunda melhor opção terapêutica após a falência desta em longo prazo. Objetivo: avaliar a eficácia em longo prazo de gliflozinas e gliptinas após a falência do tratamento primário com metformina no DM2. Material e métodos: foi realizada uma revisão sistemática para o maior tempo de tratamento nas bases de dados bases Embase, Pubmed (via Medline), Lilacs e Cochrane Library e metanálise em rede com as sulfoniluréias (glimepirida e glipizida) como meta comparador. Desfechos: eficácia da medicação (valor final da HbA1c e porcentagem de pacientes com HbA1c < 7%), variação de peso e frequência de pacientes com hipoglicemia. Resultados: O maior tempo de segmento foi de quatro anos. Foram selecionados um artigo com empagliflozina, um artigo com dapagliflozina e um artigo com saxagliptina com dados faltantes. Após um ano de tratamento, mais de 50% dos pacientes estavam com HbA1c > 7%. O perfil de eficácia em quatro anos da empagliflozina (23%) foi melhor que da dapagliflozina (5%) e saxagliptina (7%), porém com valores de HbA1c não estatisticamente significantes (7,4 e 7,3% entre as gliflozinas), sem dados para a saxagliptina. Entretanto, a empagliflozina foi superior à glimepirida no período de quatro anos (diferença média padronizada/DMP: 0,40, intervalo de confiança/IC95%: 0,23- 0,56). A variação de peso permaneceu estável após um ano de tratamento, com vantagem em quatro anos para a empa (DMP: 1,56, IC95%: 1,23- 1,88). A frequência de pacientes com hipoglicemia não diferiu entre empagliflozina e dapagliflozina (razão de chances: 1,53, IC95%: 0,80- 2,91) e foi significativamente menor do que em relação às sulfoniluréias. Conclusões: a falência da segunda terapia com gliflozinas ocorre em menos de um ano de tratamento ( > 50% dos pacientes com HbA1c > 7%). A empagliflozina obteve um controle glicêmico melhor em relação às sulfoniluréias, porém semelhante à dapagliflozina. A perda de peso foi mantida por quatro anos, com superioridade para empagliflozina. Houve uma baixa frequência de hipoglicemia nas gliflozinas em comparação com as sulfoniluréias. Mais estudos são necessários para avaliar a eficácia de gliptinas e gliflozinas em longo prazo, após a falência terapêutica com metformina / Metformin is the first-choice treatment in people with type 2 diabetes (TD2). There is no consensus in the medical literature about which drug would be a second-best option of treatment in the case of metformin failure in long-term. Objective: to assess the long-term efficacy of gliflozins and gliptins once metformin has failed as the primary treatment for TD2. Materials and methods: a systematic review was performed considering the longest period found in Embase, Pubmed (via Medline), Lilacs and Cochrane Library databases and also network meta-analyses using sulfonylureas (glimepiride and glipizide) as a meta comparator. Clinical outcomes where efficacy of medical treatment (final value of HbA1c and percentage of patients with HbA1c < 7%), weight variation and frequency of patients with hypoglycemia. Results: the longest period of the segment was 4 years. It was selected 1 article on empagliflozin, 1 article on dapagliflozin, and 1 article on saxagliptin with missing data. After one year of treatment, over 50% of the patients presented HbA1c > 7%. Efficacy rate in 4 years of empagliflozin (23%) was better than dapagliflozin (5%) and saxagliptin (7%), however presenting statistically non-significant values for HbA1c (7.4 and 7.3% between gliflozins), and missing data for the saxaglifozin. Nonetheless, empagliflozin performed better than glimepiride in the 4-year period (standardized mean difference SMD 0.4, confidence interval CI 95% 0.23 to 0.56). Weight variation remained stable after one year of treatment, presenting empagliflozin better results in the 4-year period (SMD 1.56, CI 95% 1.23 to 1.88). The frequency of patients with hyperglycemia did not vary for empagliflozin and dapagliflozin (odds ratio 1.53, CI 95% 0.8 to 2.91), and it was significantly lower when compared to the sulfonylureas. Conclusions: the failure of the secondary treatment using gliflozins occurs in less than one year of treatment (less than 50% of the patients presenting HbA1c > 7 %). Empagliflozin offered a better glycemic control compared to the sulfonylureas, but similar to dapagliflozin. The weight loss was maintained for 4 years, being empagliflozin the one with better results. There was a low frequency of hypoglycemia for the gliflozins when compared to the sulfonylureas. Further studies are required to evaluate the efficacy of gliptins and gliflozins in the long-term after metformin failure

A metformina é a droga de escolha no tratamento inicial do diabetes mellitus tipo 2 (DM2). Não existe consenso na literatura sobre qual seria a segunda melhor opção terapêutica após a falência desta em longo prazo. Objetivo: avaliar a eficácia em longo prazo de gliflozinas e gliptinas após a falência do tratamento primário com metformina no DM2. Material e métodos: foi realizada uma revisão sistemática para o maior tempo de tratamento nas bases de dados bases Embase, Pubmed (via Medline), Lilacs e Cochrane Library e metanálise em rede com as sulfoniluréias (glimepirida e glipizida) como meta comparador. Desfechos: eficácia da medicação (valor final da HbA1c e porcentagem de pacientes com HbA1c < 7%), variação de peso e frequência de pacientes com hipoglicemia. Resultados: O maior tempo de segmento foi de quatro anos. Foram selecionados um artigo com empagliflozina, um artigo com dapagliflozina e um artigo com saxagliptina com dados faltantes. Após um ano de tratamento, mais de 50% dos pacientes estavam com HbA1c > 7%. O perfil de eficácia em quatro anos da empagliflozina (23%) foi melhor que da dapagliflozina (5%) e saxagliptina (7%), porém com valores de HbA1c não estatisticamente significantes (7,4 e 7,3% entre as gliflozinas), sem dados para a saxagliptina. Entretanto, a empagliflozina foi superior à glimepirida no período de quatro anos (diferença média padronizada/DMP: 0,40, intervalo de confiança/IC95%: 0,23- 0,56). A variação de peso permaneceu estável após um ano de tratamento, com vantagem em quatro anos para a empa (DMP: 1,56, IC95%: 1,23- 1,88). A frequência de pacientes com hipoglicemia não diferiu entre empagliflozina e dapagliflozina (razão de chances: 1,53, IC95%: 0,80- 2,91) e foi significativamente menor do que em relação às sulfoniluréias. Conclusões: a falência da segunda terapia com gliflozinas ocorre em menos de um ano de tratamento ( > 50% dos pacientes com HbA1c > 7%). A empagliflozina obteve um controle glicêmico melhor em relação às sulfoniluréias, porém semelhante à dapagliflozina. A perda de peso foi mantida por quatro anos, com superioridade para empagliflozina. Houve uma baixa frequência de hipoglicemia nas gliflozinas em comparação com as sulfoniluréias. Mais estudos são necessários para avaliar a eficácia de gliptinas e gliflozinas em longo prazo, após a falência terapêutica com metformina / Metformin is the first-choice treatment in people with type 2 diabetes (TD2). There is no consensus in the medical literature about which drug would be a second-best option of treatment in the case of metformin failure in long-term. Objective: to assess the long-term efficacy of gliflozins and gliptins once metformin has failed as the primary treatment for TD2. Materials and methods: a systematic review was performed considering the longest period found in Embase, Pubmed (via Medline), Lilacs and Cochrane Library databases and also network meta-analyses using sulfonylureas (glimepiride and glipizide) as a meta comparator. Clinical outcomes where efficacy of medical treatment (final value of HbA1c and percentage of patients with HbA1c < 7%), weight variation and frequency of patients with hypoglycemia. Results: the longest period of the segment was 4 years. It was selected 1 article on empagliflozin, 1 article on dapagliflozin, and 1 article on saxagliptin with missing data. After one year of treatment, over 50% of the patients presented HbA1c > 7%. Efficacy rate in 4 years of empagliflozin (23%) was better than dapagliflozin (5%) and saxagliptin (7%), however presenting statistically non-significant values for HbA1c (7.4 and 7.3% between gliflozins), and missing data for the saxaglifozin. Nonetheless, empagliflozin performed better than glimepiride in the 4-year period (standardized mean difference SMD 0.4, confidence interval CI 95% 0.23 to 0.56). Weight variation remained stable after one year of treatment, presenting empagliflozin better results in the 4-year period (SMD 1.56, CI 95% 1.23 to 1.88). The frequency of patients with hyperglycemia did not vary for empagliflozin and dapagliflozin (odds ratio 1.53, CI 95% 0.8 to 2.91), and it was significantly lower when compared to the sulfonylureas. Conclusions: the failure of the secondary treatment using gliflozins occurs in less than one year of treatment (less than 50% of the patients presenting HbA1c > 7 %). Empagliflozin offered a better glycemic control compared to the sulfonylureas, but similar to dapagliflozin. The weight loss was maintained for 4 years, being empagliflozin the one with better results. There was a low frequency of hypoglycemia for the gliflozins when compared to the sulfonylureas. Further studies are required to evaluate the efficacy of gliptins and gliflozins in the long-term after metformin failure