Maintenance of Pancreatic Endocrine Cells of Neonatal Rats: Effect of 2-Deoxyglucose on Insulin Biosynthesis

Summary

The relationship between insulin biosynthesis and proinsulin mRNA activity was examined in pancreatic monolayer cultures of the neonatal rat. Monolayer cultures were maintained in TCM 199 medium containing 16.7 mM of either glucose or 2-deoxyglucose or in a basal medium not containing glucose for 24 hr. The fractions containing mRNA extracted from these cultures were translated in a cell-free protein synthesizing system of rabbit reticulocyte lysate. The proinsulin mRNA activity was determined by the radioactivity of the translation product immunoprecipitated using anti-insulin serum, and identified as preproinsulin with a molecular weight of 11,500 on gel electrophoresis. The amount of insulin biosynthesis was determined by the incorporation of [³H]-leucine into (pro)insulin. In islet B cells cultured in a medium with 16.7 mM glucose, insulin biosynthesis induced by 16.7 mM glucose was enhanced by 158% when cultured in basal medium without glucose. This increment corresponded nicely with a 185% increase in the proinsulin mRNA activity. However, the addition of 16.7 mM 2-deoxyglucose to the basal medium resulted in a 293% increase in glucose-induced insulin biosynthesis despite a 16% drop in the proinsulin mRNA activity, and primed a dose-dependent increase of insulin biosynthesis over the concentration range of 0 to 16.7 mM glucose. These results suggest first that in neonatal B cells insulin biosynthesis may be Tegulated at the transcriptional level, and second, that 2-deoxyglucose may cause the transition of the neonatal B cell to an adult-type response.

Key words

Monolayer culture - 2-Deoxyglucose - Insulin biosynthesis - Proinsulin mRNA activity