Electron microscopic visualization of insulin translocation into the cytoplasm and nuclei of intact H35 hepatoma cells using covalently linked Nanogold-insulin.

Insulin affects numerous metabolic processes as well as nuclear events such as gene transcription. Our previous ultrastructural and biochemical studies demonstrated insulin accumulation in nuclei of cultured and rapidly proliferating cells, and biochemical evidence suggested that insulin entered the cell cytoplasm before accumulating in the nucleus. The present study was undertaken to develop a covalently linked electron-dense insulin complex that could be used to visualize the intracellular translocation of insulin and confirm that insulin enters the cytoplasm of cells. Insulin was cross-linked to 1.4-nm diameter Nanogold particles. The complex binds to the plasma membrane insulin receptor, is biologically active, and is degraded by cellular insulin-degradative enzymes. Ultrastructural analysis after silver intensification of the gold particles confirmed that insulin internalization culminates in the translocation of some internalized insulin to the cytoplasm and nuclei. When cytoplasmic insulin-degrading enzyme (IDE) activity was inhibited with 1,10-phenanthroline, an increase in the number of cytoplasmic and nuclear Nanogold-insulin particles was observed. The results of this and previous studies suggest that 1) the translocation of insulin to the cytoplasm, 2) the regulation of insulin degradation in the cytoplasm by IDE, 3) the possible interaction of insulin with cytoplasmic proteins other than IDE, and 4) the subsequent accumulation of intact insulin or insulin complexed with cytoplasmic proteins in nuclei may play a role in insulin's regulation of gene transcription and cell proliferation.