Concomitant Enzyme-Linked Immunosorbent Assay Measurements of Rat Insulin, Rat C-Peptide, and Rat Proinsulin from Rat Pancreatic Islets: Effects of Prolonged Exposure to Different Glucose Concentrations

Abstract

Until now, there have been few assays to measure C-peptide and proinsulin in the rat. We used a well-established rat insulin ELISA and validated two novel ELISAs for rat C-peptide and rat/mouse proinsulin to examine secretion and content of insulin, proinsulin, and C-peptide from rat islets cultured for 72 h at different glucose concentrations in culture medium. To examine long-term effects in vitro rather than short-term effects of exposure to low, normal, and high glucose, the exposure time to the different glucose concentrations was set to 72 h. The measurement uncertainty of the values obtainable from the ELISAs was determined by calculation of the variation pattern from the intraassay variation generated by unknown samples, and repeatability was determined by analysis of controls. The precision study and the analysis of controls confirm that the validated ELISAs for rat C-peptide and proinsulin would be useful for further studies on the effects of preculture in different glucose concentrations. The higher the glucose concentration used during the 72-h culture period of rat islets, the higher insulin, C-peptide and proinsulin values were obtained in a subsequent short-term glucose-challenge experiment. The proportion of proinsulin to insulin secreted increased, as did islet content, with increasing glucose concentration during preculture. We also observed a nonequimolar, glucose-dependent secretion and content of rat insulin over rat C-peptide after culture at 11.1 and 28 mM glucose.