Glucose-regulated translational control of proinsulin biosynthesis with that of the proinsulin endopeptidases PC2 and PC3 in the insulin-producing MIN6 cell line

Abstract

In the short term (< 2 h), proinsulin biosynthesis is predominately glucose regulated at the translational level; however, the details at the molecular level behind this mechanism are not well defined. One of the major hindrances for gaining a better understanding of the proinsulin biosynthetic mechanism has been a lack of an abundant source of beta-cells that express a phenotype of regulated proinsulin biosynthesis in the appropriate 2.8-16.7 mmol/l glucose range as defined in normal pancreatic islets. In this study, we demonstrate that in the MIN6 cell line, specific glucose-regulated translational control of proinsulin biosynthesis is present in the appropriate glucose concentration range. In addition to that of proinsulin, the biosynthesis of the two proinsulin conversion endopeptidases, PC2 and PC3, was coordinately glucose regulated in MIN6 cells, whereas that of the exopeptidase, carboxypeptidase H, was unaffected by glucose. Proinsulin, PC2 and PC3 biosynthesis was specifically stimulated over that of total MIN6 cell protein synthesis above a threshold of 4 mmol/l glucose that reached a maximum rate between 8 and 10 mmol/l glucose. Glucose-induced proinsulin, PC2, and PC3 biosynthesis was rapid (occurring after a 20-min lag period but reaching a maximum by 60 min), unaffected by the presence of actinomycin D; and in parallel experiments, stimulatory glucose concentrations did not alter MIN6 cell total preproinsulin, PC2, or PC3 mRNA levels. Thus, short-term (< 2 h) glucose stimulation of proinsulin, PC2 and PC3 biosynthesis in MIN6 cells, like that in isolated islets, was mediated at the translational level. Intracellular signals for mediating glucose-stimulated proinsulin PC2 and PC3 biosynthesis translation in MIN6 cells also appeared to be similar to those in pancreatic islets, requiring glucose metabolism and a supporting role for protein kinase A. However, protein kinase C or a Ca(2+)-dependent protein kinase did not appear to be required for glucose-regulated proinsulin biosynthesis in MIN6 cells, as in islets. MIN6 cells are the first beta-cell line that indicate glucose-regulated proinsulin biosynthesis translation essentially identical to that in differentiated islet beta-cells and will be an important experimental model to better define the mechanism of proinsulin biosynthesis in detail.