Dynamic measurements of metabolism and secretion in islets of Langerhans by amperometry and fluorescence microscopy.

Abstract

Insulin is secreted from microorgans in the pancreas known as islets of Langerhans in response to stimuli. The dynamics of secretion and metabolism in islets are interesting since defects in insulin secretion and glucose responsiveness are prevalent in type 2 diabetics. An early sign of type 2 diabetes is loss of oscillatory secretion. The small size of the islets and the period of the oscillations require microanalytical techniques with high sensitivity and high temporal resolution. Microelectrodes were used to monitor metabolic oscillations. These sensors are 3--8 mum in diameter, allowing for implantation into islets to monitor changes in oxygen and glucose consumption. In pancreatic beta-cells, Zn<super>2+</super> is co-released from secretory vesicles with insulin. Using confocal fluorescence microscopy and a Zn<super>2+</super> specific dye, Fluozin-3, secretion from islets was monitored. Advantages of this technique over conventional methods for monitoring secretion are decreased labor, and improved temporal and spatial resolution. The combination of these techniques and intracellular calcium concentration ([Ca<super>2+</super>]_i) measurements allowed for study of the nature and source of metabolic and secretory oscillations. Glucose gave rise to three oscillatory patterns in all parameters; slow (3--4 min.), fast (&sim;20 s), and slow oscillations with fast superimposed. Other fuels were unable to cause slow oscillations. Glyceraldehyde gave rise to fast oscillations, and in combination with sub-stimulator glucose slow oscillations were observed, showing that glucokinase feedback was required for slow oscillation. However, oscillatory [Ca<super>2+</super>]_i is also required for metabolic oscillations. Single cell electroporation with an electrolyte filled capillary allowed for changes in concentration of cellular compounds independently of each other and glucose. These measurements showed that increase in ATP caused changes in [Ca<super>2+</super>]_i, while changing ADP had no effects. We were also able to show that increased glucose-6-phosphate concentration halted slow oscillations, further supporting the importance of glucokinase. Knockout models of insulin and IGF-1 receptors show that these receptors are required for oscillations and glucose homeostasis. Imaging showed secretion to be heterogeneous surrounding an islet, which decreased with increasing glucose concentration. Our observations have expanded the knowledge of the dynamics of oscillation in islets by the development and applications of novel analytical techniques.