Development of analytical tools to study the signaling mechanisms of glucose homeostasis regulation in islets of langerhans.

摘要

The work in this dissertation presents methods for sensitive and selective assays for hormones and proteins involved in the regulation of glucose homeostasis. The capillary electrophoresis (CE) conditions for a competitive immunoassay of glucagon were optimized for highest sensitivity of the immunoassay and resolution of the electrophoretic peaks using a Box-Behnken design. Injection time, voltage ramp time, and separation voltage were varied between three levels and two responses, bound-to-free (B/F) ratio of the immunoassay peaks and resolution between the peaks, were measured. Analysis of variance was applied to fit a predictive model, and a desirability function was used to simultaneously optimize both responses. A 10-s injection, 1.6-min ramp time, and a 22-kV separation voltage were the conditions found when high B/F was given more emphasis than high resolution. To test the model, calibration curves of a glucagon immunoassay were measured at the optimum and least optimum CE conditions. Optimal conditions increased the sensitivity of the immunoassay by 388% compared to the least optimum conditions while maintaining adequate resolution.;A microfluidic device was developed for electrophoresis immunoassays to monitor the acute secretory dynamics of insulin and IAPP from multiple islets of Langerhans. A simple flow-switching feature on the microfluidic chip allowed rapid step changes in gravity-driven perfusion of islets with cell culture media at varying concentrations of glucose. The islet perfusate was continuously sampled by electroosmosis and mixed online with immunoassay reagents in an 8 cm reaction channel at 37 °C. The immunoassay mixture was injected for 0.3 sec onto a 1.5 cm separation channel at 12 sec intervals. A two-color detection strategy provided spectral resolution and independent gain control of the immunoassay product fluorescent signals, for simultaneous quantitation of insulin and IAPP secreted from mouse islets of Langerhans in response to dynamic glucose stimulation. The response times of the two immunoassays were corrected for electrophoretic bias and secretion profiles of the two peptides were monitored during multiple step changes in glucose stimulation. Insulin and IAPP from groups of 4 or 9 islets were secreted in an approximately 10:1 ratio and displayed similar responses to step changes from 3 mM to 11 mM or 20 mM glucose. The ability to monitor the secretory dynamics of multiple peptides from islets of Langerhans in a highly automated fashion should be useful for investigating the hormonal regulation of glucose homeostasis.;The endocrine hormone secreting cells in pancreatic islets of Langerhans are subject to glucose responsive regulation by the post translational protein modification O-linked N-acetylglucosamine (O-GlcNAc). Dysregulation of this signaling pathway during hyperglycemia is linked to type II diabetes, although changes in the protein targets and levels of O-GlcNAc modification are not well understood. A click chemistry reaction was used to label O-GlcNAc modified proteins with a chemoselective probe. Subsequent one- or two-dimensional gel electrophoresis resolution of labeled proteins in cell lysate showed qualitative differences in O-GlcNAc levels for a clonal cell line cultured in low and high glucose media. Proteins of interest from the clonal cell line or from islets of Langerhans were identified by peptide mass fingerprinting with matrix-assisted laser desorption ionization time-of-flight mass spectrometry or by accurate mass with Fourier transform ion cyclotron resonance mass spectrometry.