Institut für Pharmakologie, Toxikologie und Klinische Pharmazie
der TU Braunschweig, AG Rustenbeck
The scientific interest of our group is directed towards the mechanisms which shape the biphasic kinetic of insulin secretion. The biphasic kinetic is regarded as indispensable for the maintenance of the glucose homeostasis of the organism. The diminution or even loss of the first phase secretion in response to a glucose challenge is the hallmark of the transition from impaired glucose tolerance to overt type 2 diabetes. There are two theoretical models to explain the biphasic nature. One postulates a sequence of different compartments which shape secretion kinetics, whereas the second one postulates the interaction of stimulatory and inhibitory signals. Currently, a derivative of the compartmental model is predominant, in which the role of the compartments is played by different pools of secretory granules which become differentially activated by a set of plasma membrane ion channels. New live cell imaging techniques for the visualisation of secretory granules and the growing perception that signals circumventing plasma membrane depolarisation are involved in early secretion events have put this "pool size hypothesis" into question. The pathophysiological perspective of our work is to clarify which events lead to the loss of the first phase and the pharmacological perspective is the reconstruction of the first phase by a beta cell-directed therapy.
Our experimental approach is based on the perifusion of isolated pancreatic islets and primary beta cells to register the consequences of experimental manipulations on insulin secretion and putative signalling compounds. By stimulating the islets or beta cells in a closely comparable manner in a variety of measuring stands we intend to create an encompassing view of the intracellular events underlying the kinetics of insulin secretion. Thus the batch perifusion of pancreatic islets (freshly isolated or cultured) to determine the insulin content in the fractionated effluate (by ELISA) is the starting point. The plasma membrane potential, whole cell currents and KATP channel currents can be registered by two electrophysiological stands. One is for work with single cells, the other one with an upright microscope for work on islets within vital pancreatic slices. Two epifluorescence microscopes can be used for conventional live cell measurements. One is equipped with a cooled CCD camera and coupled to a fraction collector to enable simultaneous determination of cytosolic calcium and insulin secretion. The other one is used for multiparameter measurements of mitochondrial function (NAD(P)H- and FAD autofluorescence, TMRE fluorescence for the mitochondrial membrane potential). Endpoint measurement of ATP, ADP and AMP complete the assessment of beta cell energy metabolism. Finally, an objective-based TIRF-microscope with a thermostated hood and an integrated perifusion system permits the visualization of submembrane secretory granule mobility and fusion. An observer-independent quantification is achieved by an in-house developed program. Standard molecular biology techniques are employed to generate fluorescent fusion proteins as granule labels.