Development and Application of Analytical Instrumentation for Monitoring Function of Islets of Langerhans.

Abstract

Impaired insulin secretion is a hallmark of type 2 diabetes. Numerous reports of long-term kinetics of insulin secretion from islets have been reported, but potentially underlying fast kinetics of the phenomena are difficult to study using conventional insulin measurement techniques. To monitor long-term insulin secretion from islets of Langerhans, a microfluidic chip capable of long-term operation was designed. The device performed a capillary electrophoresis-based competitive immunoassay every 6 s and operated for 24 h, resulting in the completion of 14,400 assays in 24 h. A microfluidic chip was developed to perform intracellular calcium measurements on islets. With the chip, rapid changes were made to the islet environment by perfusing glucose, peptides or other stimulants over the islets. The chip was used in a collaborative effort to investigate the membrane-disrupting effects of islet amyloid polypeptide. Application of the peptide to islets resulted in the flux of calcium into islets, presumably due to the membrane-disrupting effects of the peptide. The results, corroborated by results from experiments performed on model membranes, identify a role of the His18 residue of the peptide for the demonstrated membrane-disrupting effects. The intracellular calcium chip was also used in a collaborative effort to investigate leptin signaling in islets. By monitoring a number of parameters, including islet cell mass, insulin secretion, and intracellular calcium concentration, islets from pancreas-specific leptin receptor knock-out mice were compared with control islets. The effects of leptin, palmitic acid, and glibenclamide on intracellular calcium concentration were explored. The results from the investigation demonstrate the inhibitory effect of leptin on calcium flux into islets and on glucose-stimulated insulin secretion and suggest a role of leptin-signaling on islet behavior. Oscillations in oxygen consumption by islets have been observed to correlate with insulin release levels. To investigate the link between metabolism and glucose-stimulated insulin secretion, a fiber optic nanosensor was developed to monitor oxygen consumption by islets. The submicron dimensions of the sensor enabled high spatial resolution for oxygen measurements, and the sensor demonstrated good reversibility, good stability, and high sensitivity compared with previously-reported optical oxygen sensors, making the sensor potentially suitable for oxygen measurements in islets.