Assessment of pancreatic islet cell function and survival

Sammanfattning: Function and survival of pancreatic islet insulin-producing beta-cells (β-cells) and glucagonproducing alpha-cells (α-cells) were studied, and methods for this purpose were developed or refined. Dynamic control of glucose metabolism is essential for β-cell stimulus-secretion coupling. ATP is an important metabolic parameter and therefore we set up a technique to monitor dynamic changes of ATP in insulin-producing cells using luciferase bioluminescence at the level of single cells or groups of cells. We could detect a decrease in ATP in response to the mitochondrial uncoupler FCCP in HIT M2.2 cells and an increase in ATP in response to glucose in intact mouse islets. The glucose-induced stimulus-secretion coupling was also studied in a mutant mouse model with β-cell specific depletion of mtDNA and consequently disruption of the mitochondrial respiratory chain. In this model we could observe disrupted response in mitochondrial membrane potential (MMP), impaired response in the cytoplasmic free Ca2+ concentration ([Ca2+]i) as well as disrupted insulin release. The interrelation between [Ca2+]i and MMP was studied in response to glucose stimulation and to non-metabolic stimuli in mouse β-cells using a method for simultaneous detection of these two parameters. Our results indicate involvement of Ca2+-dependent activation of mitochondrial dehydrogenases, under low glucose conditions. MMP depolarization due to Ca2+-influx into mitochondria is consistent with effects under high glucose conditions. The latter phenomenon could also be observed subsequent to every peak of glucose-induced slow [Ca2+]i oscillations. A method for on-line detection of apoptosis at single-cell level was established with a FRETbased biosensor that was sensitive to caspase-3-like proteases, which act as executive caspases in several pathways of apoptotic cell-death. This was detected by two-photon laser scanning microscopy, a modality that is commonly used for in vivo microscopy. Therefore we conclude that the method has the potential to be used for in vivo detection of apoptosis. Methods for enrichment of human and rat pancreatic α-cells were established or modified using fluorescence-activated cell sorting (FACS) based on only intrinsic cellular properties such as light scatter and autofluorescence. The sorted cells showed high purity, high viability and also demonstrated functional [Ca2+]i responses. In conclusion, the methods that I have developed or refined are successfully used in studies of islet cell function and survival, and they will be useful also for future in vivo experiments. My results contribute to our understanding how islet cells function in physiology and how their function becomes compromised in diabetes.