Effect of Glucose on Initiation and Elongation Rates in Isolated Rat Pancreatic Islets

Abstract

Abstract Isolated rat pancreatic islets incubated in media containing glucose at 16.6 mm incorporate [3H]leucine into protein at twice the rate of islets incubated in glucose at 2.8 mm. Increased incorporation was independent of new RNA synthesis, so the effects of glucose on initiation and elongation rates were determined. The relative rates of elongation at 2.8 and 16.6 mm glucose were determined by pulsing islets in [3H]leucine for 6 min and measuring the ratio of incorporation into soluble (S) and polysome associate (P) peptides. At 2.8 mm glucose, S/P = 3.41 ± 0.28, and at 16.6 mm glucose, S/P = 4.72 ± 0.27, an apparent 28% increase in elongation at the higher glucose. The ratio method assumes that the size of the proteins synthesized is the same. Islets proteins were labeled with [14C]leucine at 16.6 mm glucose and with [3H]leucine at 2.8 mm glucose and subjected to co-electrophoresis on sodium dodecyl sulfate polyacrylamide gels, and the average molecular weight of proteins synthesized at the higher glucose concentration was found to be about 26% smaller. After correction for differences in the size of proteins synthesized, there was no significant effect of glucose on elongation. Evidence that glucose regulates total islet protein synthesis at the level of initiation includes: (a) increased [3H]leucine incorporation into nascent peptide with the distribution on heavier polysome aggregates indicating an increased number of ribosomes per mRNA, even when new RNA synthesis is blocked; (b) a reduction in the number of ribosomes per mRNA by aurintricarboxylic acid (10-5 m) at 16.6 mm glucose with no significant change at 2.8 mm glucose; (c) when elongation is partially inhibited with low doses of cycloheximide (2 µg per ml), ribosomes accumulate on mRNA at 2.8 mm glucose but not at 16.6 mm glucose; under these conditions the amount of protein synthesized is proportional to the amount of mRNA available for translation. Total islet mRNA appeared to be the same at both glucose concentrations, and the doubling of protein synthesis in the absence of changes in elongation is most likely due to a doubling of the over-all initiation rate. Glucose regulation of proinsulin-insulin synthesis appears to be more complex. When elongation was partially blocked, it was determined that in 16.6 mm glucose about 2 to 3 times more proinsulin mRNA was available for translation. There was a preferential utilization of the available mRNA for proinsulin in addition, since there was about a 5 to 6-fold increase in over-all translation of proinsulin mRNA. Methods are being developed to determine whether the glucose effect on translation of proinsulin mRNA is at the level of initiation, elongation, or both.