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ist of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Blixt, M., Niklasson, B., Sandler, S. (2007) Characterization of -cell function of pancreatic islets isolated from bank voles de- veloping glucose intolerance/diabetes: an animal model show- ing features of both type 1 and type 2 diabetes mellitus, and a possible role of the Ljungan virus. General and Comparative Endocrinology, 154(1-3):41–47 II Blixt, M., Niklasson, B., Sandler, S. (2009) Suppression of bank vole pancreatic islet function by proinflammatory cytokines. Molecular and Cellular Endocrinology, 305(1-2):1–5 III Blixt, M., Niklasson, B., Sandler, S. (2010) Pancreatic islets of bank vole show signs of dysfunction after prolonged exposure to high glucose in vitro. Submitted IV Blixt, M., Niklasson, B., Sandler, S. (2010) Morphologic inves- tigation of the endocrine pancreas in diabetic bank voles indi- cates a type 2 diabetes profile. Manuscript Reprints were made with permission from the respective publishers. Contents Introduction ..................................................................................................... 9 Historical aspects of diabetes mellitus ................................................... 9 Background ................................................................................................... 11 Animal models used in diabetes research ................................................. 11 The bank vole ........................................................................................... 12 The Ljungan virus ................................................................................ 13 Proinflammatory cytokines ...................................................................... 14 Effects of high glucose ............................................................................. 15 Morphological changes of pancreatic islets in diabetes ........................... 16 Aim ............................................................................................................... 18 Materials and methods .................................................................................. 19 Animal and sample preparations (I – IV) ............................................ 19 Islet isolation and culture condition (I – III) ........................................ 20 Islet (pro)insulin biosynthesis rate (I – III) .......................................... 20 Islet glucose oxidation rate (I – III) ..................................................... 21 Medium insulin and proinsulin accumulation, islet insulin release and insulin content (I– III) .......................................................................... 21 Medium nitrite accumulation (II) ........................................................ 22 RNA isolation, cDNA synthesis and Real Time PCR (II, III) ............. 22 DNA quantification (III) ...................................................................... 23 Islet cell viability (II) ........................................................................... 23 Paraffin removal and antigen retrieval (IV) ......................................... 23 Haematoxylin staining (I, IV) .............................................................. 24 Fluorescence staining (IV) ................................................................... 24 Morphological evaluation (IV) ............................................................ 24 Statistical analysis (I –IV) ................................................................... 25 Results and discussion .................................................................................. 26 Glucose intolerance in bank voles (I) ....................................................... 26 NO independent reduction in insulin biosynthesis (II) ............................. 27 Prolonged exposure to high glucose (III) ................................................. 28 Islet morphology and function (IV) .......................................................... 30 Ljungan virus (I–IV) ................................................................................ 31 Conclusions ................................................................................................... 32 Acknowledgements ....................................................................................... 33 References ..................................................................................................... 35 Abbreviations AMV Avian myelobalstosis virus ANOVA Analysis of variance BB Bio-breeding CD-1 Inbred mouse strain derived from ICR mouse cDNA Complementary deoxyribonucleic acid Ct Cycle threshold CY2 Fluorescent dye fluorescein isothiocyanate Db/db–model Monogenic mouse model of obesity –leptin resistant DNAse Enzyme degrading deoxyribonucleic acid dNTP Deoxyribonucleotide triphosphate ELISA Enzyme–linked immunosorbent assay Fa/fa–model Monogenic rat model of obesity –leptin resistant GAD65 Glutamic acid carboxylase isoform 65 GK Goto Kakizaki inbred rat strain HBSS Hanks’ balanced salt solution IA–2 Islet antigen–2 IFN– Interferon gamma IL–1 Interleukin one beta iNOS Inducible nitric oxide synthase IPGGT Intraperitoneal glucose tolerance test Jc1–ICR Inbred mouse strain from Jackson laboratories KK Inbred mouse stain from Kasukabe in Saintama, Japan KRBH Krebs’ Ringer bicarbonate HEPES buffer LADA Latent autoimmune diabetes in adults LV Ljungan virus NOD Non obese diabetic inbred mouse strain NYS Nagoya–Shibata–Yasuda inbred mouse strain Ob/ob–model Monogenic mouse model of obesity –leptin deficient PDX–1 Pancreatic–duodenal homeobox – 1 RNAse Enzyme degrading ribonucleic acid RPMI Roswell Park Memorial Institute RT–PCR Reverse transcriptase polymerase chain reaction S.E.M. Standard error of the mean TBS Tris buffered saline TCA Trichloroacetic acid TNF– Tumor necrosis factor alfa UV Ultraviolet Introduction Diabetes mellitus is a group of metabolic disorders where the control of blood glucose homeostasis has been lost due to reduced ability to use carbo- hydrates as energy source. This is a consequence of a failing insulin hor- monal signaling system. The exact etiology of diabetes mellitus is still un- clear however, several theories have been presented arguing that genetic predisposition and environmental factors e.g. viral infections, dietary pro- teins, toxins as well as lifestyle may influence disease development [1-7]. The prevalence of diabetes mellitus is increasing rapidly. At present approx- imately 280 million adult individuals suffer from the disease. It is estimated that within 20 years this number will rise to over 400 million [8]. Diabetes mellitus can be divided into several variants of which type 1 and type 2 are the major diagnostic groups [9]. Type 1 diabetes mellitus is a chronic au- toimmune disease that affects the –cells in the pancreatic islet of Langer- hans, which results in an insufficient endogenous insulin production [10]. Type 2 diabetes mellitus evolves from relative insulin deficiency that is a result of an increasing desensitization of insulin sensitivity in the peripheral tissue and an impaired –cell function [11]. Ultimately both these conditions leads to increased blood glucose concentration and a failing –cell mass. The diabetic patients’ incapacity to metabolize sufficient carbohydrates leads to increasing use of other fuels, preferably fat and protein. This shift in meta- bolism will lead to loss of normal physiological conditions and eventually result in presentation of the classical symptoms of diabetes; fatigue, polydip- sia and polyuria depending on the graveness of the disease. Historical aspects of diabetes mellitus The first documented observation of diabetes is from the ancient Egypt pre- served in the Ebers’ papyrus dated to 1552 BC [12]. In this papyrus there are descriptions of many diseases, symptoms and treatments including a phrase regarding a remedy that has been translated in two ways “"A medicine to drive away the passing of too much urine," and "To eliminate urine which is too plentiful.". These sentences indicate that diabetes may have been present at this point of time in history. Ebers was not the author but the man who became the owner of the papyrus in 1862 AD. The first known user of the word diabetes, which is Greek, “to pass through”, was Demetrios that lived in the late 2nd century BC in Apameia, today a part of Turkey [13]. It was 9 not until the late 18th century that William Cullen used the word mellitus that is “honey” in Latin [14]. One century later the German histologist Paul Langerhans published a description of distinct