1,10-Phenanthroline Increases Nuclear Accumulation of Insulin in Response to Inhibiting Insulin Degradation but Has a Biphasic Effect on Insulin's Ability to Increase mRNA Levels

Previous reports demonstrated that insulin is translocated through the cytoplasm to the nucleus of H35 hepatoma cells and suggested that nuclear insulin may be involved in stimulating transcription of immediate-early genes. In a recent study, inhibition of insulin-degrading enzyme with 1,10-phenanthroline, a Zn²⁺ chelator, caused a significant increase in the nuclear accumulation of insulin. The present study characterized the effects of 1,10-phenanthroline and its nonchelating isomer, 1,7-phenanthroline, on insulin degradation, nuclear accumulation, and stimulation of immediate-early gene expression. 1,10- but not 1,7-phenanthroline inhibited insulin degradation and increased nuclear accumulation of insulin in a dose-dependent manner. 1,7-phenanthroline caused a dose-dependent decrease in the expression of insulin-stimulated immediate-early genes, but had no significant effect on α-tubulin mRNA levels. In the presence of insulin, Northern analysis revealed that 1,10-phenanthroline at all concentrations tested increased α-tubulin mRNA levels, but had a biphasic effect on insulin-stimulated immediate-early gene expression. At low concentrations (5–200 μM), 1,10-phenanthroline increased the expression of insulin-stimulated g33, c-fos, and Egr-1 mRNA. At concentrations greater than 1 mM, insulin-stimulated immediate-early gene expression was decreased similar to the effect seen with 1,7-phenanthroline. Nuclear run-on analysis demonstrated that high concentrations of 1,10-phenanthroline decreased insulin-stimulated immediate-early gene transcription but had no effect on transcription of α-tubulin. However, low concentrations of 1,10-phenanthroline did not increase transcription of any genes. Taken together, these results suggest that, in the presence of insulin, low concentrations of 1,10-phenanthroline inhibited degradation of mRNA through a zinc-dependent process. In contrast, at high concentrations both 1,10- and 1,7-phenanthroline block transcription stimulated by insulin by inhibiting binding between DNA and DNA-binding proteins or transcription factors. These actions of phenanthroline did not allow us to determine whether the increased amount of insulin in the nucleus proportionately affects immediate-early gene transcription.