Translational control of insulin biosynthesis. Evidence for regulation of elongation, initiation and signal-recognition-particle-mediated translational arrest by glucose

Abstract

The biosynthesis of insulin in the islets of Langerhans is strongly controlled at the translational level by glucose. We have used a variety of experimental approaches in efforts to dissect the mechanisms underlying the stimulatory effect of glucose. To assess its effects on rates of peptide-chain elongation, isolated rat islets were labelled with [3H]leucine at different glucose concentrations in the presence or absence of low concentrations of cycloheximide. Under these conditions, at glucose concentrations up to 5.6 mM, endogenous insulin mRNA did not become rate-limiting for the synthesis of insulin, whereas stimulation of non-insulin protein synthesis was abolished by cycloheximide at all glucose concentrations, indicating either that insulin synthesis is selectively regulated at the level of elongation at glucose concentrations up to 5.6 mM, or that at these concentrations inactive insulin mRNA is transferred to an actively translating pool. Glucose-induced changes in the intracellular distribution of insulin mRNA in cultured islets were assessed by subcellular fractionation and blot-hybridization using insulin cDNA probes. At glucose concentrations above 3.3 mM, cytoplasmic insulin mRNA was increasingly transferred to fractions co-sedimenting with ribosomes, and relatively more of the ribosome-associated insulin mRNA became membrane-associated, consistent with effects of glucose above 3.3 mM on both the initiation of insulin mRNA and SRP (signal recognition particle)-mediated transfer of cytosolic nascent preproinsulin to the endoplasmic reticulum. When freshly isolated islets were homogenized and incubated with 125I-Tyr-tRNA, run-off incorporation of 125I into preproinsulin was increased by prior incubation of the islets of 16.7 mM-glucose. The addition of purified SRP receptor increased the run-off incorporation of [125I]iodotyrosine into preproinsulin, especially when the islets had been preincubated at 16.7 mM-glucose. These findings taken together suggest that glucose may stimulate elongation rates of nascent preproinsulin at concentrations up to 5.6 mM, stimulates initiation of protein synthesis involving both insulin and non-insulin mRNA at concentrations above 3.3 mM, and increases the transfer of initiated insulin mRNA molecules from the cytoplasm to microsomal membranes by an SRP-mediated mechanism that involves the modification of interactions between SRP and its receptor.