In the human insulin gene, three regulatory sequences upstream of the transcription start site at -77 (the CT1 box), -210 (the CT2 box), and -315 (the CT3 box) bind a beta-cell-specific transcription factor, IUF1. Recent studies have mapped a glucose response element to a CT-like sequence in the rat insulin I gene. The present study was therefore undertaken to ascertain the role of IUF1 in glucose-stimulated insulin gene transcription. IUF1-binding activity was measured by electrophoretic mobility shift assay using the CT2 box as probe. When freshly isolated rat islets of Langerhans were incubated in medium containing low concentrations (3 mM) of glucose IUF1 activity fell to undetectable levels within 6 h. In high (20 mM) glucose IUF1 activity remained constant over a 24 h period. The loss of IUF1 activity was reversible. Thus when islets were incubated for 4 h in low glucose and transferred to high glucose, IUF1 levels recovered within 15 min. This effect was dependent on glucose metabolism as it was inhibited by mannoheptulose. Incubation of islets for 4 h in low concentrations of glucose supplemented with phosphatase inhibitors prevented the fall in IUF1 activity. No recovery in IUF1 activity was observed when islets were treated for 4 h with low glucose and then for a further 1 h with low glucose and dibutyryl cyclic AMP, or forskolin, or the phorbol ester phorbol 12-myristate 13-acetate. These results demonstrate that the IUF1-binding activity in islets of Langerhans is modulated by glucose in a phosphorylation-dependent manner, and that protein kinase A or protein kinase C are not involved. Finally, IUF1 was shown to be immunologically related to a recently cloned factor, IPF1, that binds to a CT-like sequence in the rat insulin I gene promoter.

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Over short time periods glucose controls insulin biosynthesis predominantly through effects on preexisting mRNA. However, the mechanisms underlying the translational control of insulin synthesis are unknown. The present study was carried out to determine the effect of glucose on the activity and/or phosphorylation status of eukaryotic initiation and elongation factors in islets. Glucose was found to increase the activity of the guanine nucleotide-exchange factor eIF-2B over a rapid time course (within 15 min) and over the same range of glucose concentrations as those that stimulate insulin synthesis (3-20 mM). A nonmetabolizable analogue of glucose (mannoheptulose), which does not stimulate insulin synthesis, failed to activate eIF-2B. The best characterized mechanism for modulating eIF-2B activity involves changes in the phosphorylation of the alpha-subunit of its substrate eIF-2. However, in islets, no change in eIF-2 alpha phosphorylation was seen under conditions where eIF-2B activity was increased, implying that glucose regulates eIF-2B via an alternative pathway. Glucose also did not affect the phosphorylation states of three other regulatory translation factors. These are the cap-binding factor eIF-4E, 4E-binding protein-1, and elongation factor eEF-2, which do not therefore seem likely to be involved modulating the translation of the preproinsulin mRNA under these conditions.