Insulin Stimulates Accumulation and Efflux of Macromolecules in Isolated Nuclei From H35 Hepatoma Cells

This study used 10-nm gold particles with 5–7 insulin molecules attached (Au₁₀-Ins) to investigate the site of interaction of insulin with the nuclear envelope during insulin uptake into intact isolated nuclei. Despite its size, and in the absence of ATP, Au₁₀-Ins entered nuclei through the nuclear pore and associated with the heterochromatin. Because Au₁₀-Ins is essentially gold-bovine serum albumin (Au-BSA) with a few insulin molecules attached, the effect of insulin and other growth factors on the nuclear accumulation of BSA coupled to 10-, 15-, and 24-nm-diam colloidal gold particles (Au₁₀-BSA, Au₁₅-BSA, and Au₂₄-BSA) was determined. The Au-BSA complexes were excluded from nuclei in the absence of insulin. Insulin (0.5–100 ng/ml) caused a dose-dependent accumulation of Au₁₀-BSA in the nucleus. The nuclear membrane was shown to be intact by several criteria, therefore, accumulation of Au-BSA occurred via the nuclear pore and was not due to leakage across or through the membrane. Uptake of 15- and 24-nm Au-BSA molecules was not affected by insulin, suggesting the hormone had a limited effect in increasing the functional diameter of the nuclear pores. Glucagon, epidermal growth factor, platelet-derived growth factor, insulinlike growth factor I, and insulin A or B chains did not stimulate the accumulation of Au₁₀-BSA. The insulin-stimulated accumulation of Au₁₀-BSA was blocked by concanavalin A, mimicked by wheat-germ agglutinin, and did not require ATP. The Au₁₀-BSA in the nucleus was associated with heterochromatin, suggesting it bound to a nuclear element. Efflux of Au₁₀-BSA from the nucleus was studied in nuclei previously loaded with Au₁₀-BSA in the presence of insulin and reincubated with or without insulin in the absence or presence of 3 mM ATP or a nonhydrolyzable ATP analogue. Insulin stimulated the efflux of Au₁₀-BSA from the nucleus only in the presence of ATP, possibly indicating ATP freed bound Au₁₀-BSA, allowing the molecule to diffuse through the insulin-dilated nuclear pore. These data describe an insulin-sensitive route for macromolecular exchange through nuclear pores that could serve a role in insulin's control of cell growth and gene expression.