Cell-Specific Ca Response In Pancreatic ß-Cells

.............................................................................6 ORIGINAL PAPERS....................................................................7 ABBREVIATIONS.....................................................................8 INTRODUCTION.......................................................................9 The structure of a pancreatic islet........................................... ..........9 Stimulus-secretion coupling in ß-cells...............................................10 Ca response in ß-cells....................................................................11 Heterogeneity concept..................................................................12 Hyperglycemic ob/ob and db/db mice...............................................13 AIMS OF THE INVESTIGATION....................................................14 MATERIALS AND METHODS......................................................15 Animals.................................................................................15 Islet and ß-cell preparation.............................................................15 Measurement of cytoplasmic Ca with Fura-2......................................15 Measurement of cytoplasmic Ca with Fluo-3.....................................16 Measurement of NAD(P)H fluorescence............................................17 Experiment design and definition of response parameters........................17 Measurement of insulin release from intact islets.................................17 Measurement of insulin release from single ß-cells................................18 Measurement of MitoTracker intensity.............................................18 Chemicals................................................................................19 Statistical analysis......................................................................19 RESULTS................................................................................20 Ca response in single dispersed ß-cells (Paper I and III).......................20 Ca response in ß-cell clusters and islets (Paper II)...............................21 Ca response to nutrient and non-nutrient insulin secretagogues in lean, ob/ob and db/db mouse ß-cells (Paper III and IV)..................................23 NAD(P)H response to glucose and KIC in lean, ob/ob and db/db mouse ß-cells (Paper III).......................................................................26 Insulin secretion in isolated ß-cells (Paper IV).....................................27 Mitochondrial state and Ca response in ß-cells (Paper V).....................28 DISCUSSION..........................................................................29 SUMMARY............................................................................37 ACKNOWLEDGEMENTS............................................................38 REFERENCES.........................................................................40 PAPER I PAPER II PAPER III PAPER IV PAPER V