The mechanism controlling insulin accumulation in nuclei of H35 hepatoma cells was investigated by incubating intact cells with 125|-labeled insulin in the presence or absence of agents that perturb different intracellular sites involved in the processing of ligand-receptor complexes. Purified nuclei were isolated, and nuclear-associated 125|-insulin was determined. Insulin accumulation in the nuclei was time and temperature dependent. Nuclear accumulation was linear and insulin-concentration dependent between 5 and 50 ng insulin/ml. However, pharmacological concentrations of insulin increased the amount of insulin translocated to the nucleus to a far greater extent than it increased total cell-associated insulin. Chloroquine, an acidotrophic agent, increased total cell-associated and intracellular insulin but had no effect on nuclear accumulation. The monovalent ionophores monensin and nigericin inhibited nuclear accumulation of insulin at low concentrations (0.5–5.0 μM) without affecting total insulin binding or intracellular accumulation. At 10 or 25 μM, monensin and nigericin also acted as acidotrophic agents and increased total insulin binding and intracellular accumulation but inhibited nuclear accumulation by a maximum of 50%. Low concentrations of monensin and nigericin were additive; maximal concentrations were not. A 23187 and valinomycin did not affect insulin binding or intracellular and nuclear accumulation of insulin. Neither depletion of ATP by sodium azide, 2, 4-dinitrophenol, sodium cyanide, or oligomycin nor disruption of cytoskeletal elements by cytochalasin D or colchicine had any effect on nuclear accumulation of insulin. The results of this study suggest that 7) lysosomal processing of insulin does not precede translocation of the hormone to nuclei, 2) some of the insulin translocated to the nucleus may be routed through the Golgi, 3) there may be more than one intracellular pathway resulting in the accumulation of insulin in nuclei (but none are energy dependent or require cytoskeletal elements), and 4)pharmacological concentrations of insulin may affect the intracellular processing of the hormone-receptor complex. Nuclear accumulation of insulin may allow insulin to serve as its own second messenger in nuclear events involved in regulating cell growth and gene transcription.

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