Ultrastructural evidence for the accumulation of insulin in nuclei of intact 3T3-L1 adipocytes by an insulin-receptor mediated process.

Abstract

Monomeric ferritin-labeled insulin (Fm-Ins), a biologically active, electron-dense marker of occupied insulin receptors, was used to characterize the internalization of insulin in 3T3-L1 adipocytes. Fm-Ins bound specifically to insulin receptors and was internalized in a time-and temperature-dependent manner. Fm-Ins was found in cytoplasmic vesicles within 5-10 min at 3rC and subsequently was observed in multivesicular bodies and Iysosomes. In addition, small amounts of Fm-Ins were associated with nuclei after 30 min. The number of Fm-Ins particles observed in nuclei continued to increase in a time-dependent manner until at least 90 min. In the nucleus, several Fm-Ins particles usually were found in the same general location-near nuclear pores, associated with the periphery of the condensed chromatin. Addition of a 250-fold excess of unlabeled insulin or incubation at 15°C reduced the number of Fm-Ins particles found in nuclei after 90 min by 99% or 92%, respectively. Nuclear accumulation of unlabeled ferritin was only 2% of that found with Fm-Ins after 90 min at 37°C. Biochemical experiments utilizing 12'I-labeled insulin and subceliular fractionation indicated that intact 3T3-L1 adipocytes internalized insulin rapidly and that =3% of the internalized ligand accumulated in nuclei after 1 hr. These data provide biochemical and high-resolution ultrastructural evidence that 3T3-L1 adipocytes accumulate potentially significant amounts of insulin in nuclei by an insulin receptor-mediated process. The transport of insulin or the insulin-receptor complex to nuclei in this cell or in others may be directly involved in the long-term biological effects of insulin-in particular, in the control of DNA and RNA synthesis.Most of the effects of insulin are observed within a few seconds after the addition of the hormone to cells. As a result, it is accepted that most of insulin's biological effects are generated as a direct result of binding to specific plasma membrane receptors. However, some insulin-responsive processes are not affected for several minutes or hours (for a review, see ref. 1). The mechanisms by which insulin effects these processes are unknown, although several have been postulated (for a review, see ref.2), including intracellular insulin processing (3)(4)(5). Insulin has been shown by both biochemical and ultrastructural techniques to be internalized by numerous cell types (6-18) and to have effects on isolated nuclei (19,20), leading to the speculation that internalization, intracellular translocation, and/or processing may be physiologically significant, especially for the long-term effects of insulin.This laboratory has used high-resolution electron microscopic and biochemical analyses to investigate the binding and subsequent processing ofthe insulin-receptor complex in various cells (11,(21)(22)(23)(24)(25) and has observed cell-specific heterogeneity in receptor organization, distribution, mechanism of internalization, and routes of intracellular processing (for a review, see ref. 26). As part...

Mechanism of autocrine stimulation in hematopoietic cells producing interleukin-3 after retrovirus-mediated gene transfer.

“…Indeed, our 32D cells are hyperdiploid and our FDC-P1 cells are hypodiploid, and each cell line has several metacentric marker chromo- We also tested the possibility that internalized IL3 might be biologically active after release from the receptor. Several reports have appeared of nuclear localization after the internalization of polypeptide growth factors, including epidermal and nerve growth factors (34), basic fibroblast growth factor (4), platelet-derived growth factor (34,48), and insulin (33,43). IL3 need not be glycosylated to be biologically active.…”

“…Such systems are compatible with a mechanism whereby receptor association and even signal transduction occur internally. More detailed analysis is available for platelet-derived growth factor/v-sis (19,24) (4,33,34,43,48). Internal oncogenelike effects have been described for acidic and basic fibroblast growth factor (4, 23); however, these repair-mediating growth factors do not possess signal sequences and stimulate proliferation in neighboring cells only after lysis of the damaged producer cells (13).…”

“…transduction intracellularly, leading to sustained as well as potentially anomalous mitogenic signals (26). Several reports suggest a postinternalization role for certain glycoprotein growth factors, as demonstrated by immunological detection of factors within the nuclei of cells expressing the appropriate receptor (4,33,34,43,48). Internal oncogenelike effects have been described for acidic and basic fibroblast growth factor (4,23); however, these repair-mediating growth factors do not possess signal sequences and stimulate proliferation in neighboring cells only after lysis of the damaged producer cells (13).…”

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

“…However, other reports suggest that the internalization of zymatic activities (Purrello et al, 1982), and mRNA release from nuclei (Schumm and Webb, 1983). We have demosntrated that insulin, but not its plasma membrane receptor, was translocated to and accumulated in the cell nucleus in several different cell types (Heyner et al, 1989;Smith and Jarett, 1987;Soler et al, 1989). Insulin in the nucleus was intact and tightly attached to the nuclear matrix .…”

“…We have demonstrated that internalized insulin was translocated to and accumulated in the nucleus of several different types of cells (Smith and Jarett, 1987;Heyner et al, 1989;Soler et al, 1989). It has been reported that other hormones or growth factors including epidermal growth factor (EGF), nerve growth factor (NGF), plateletderived growth factor (PDGF), fibroblast growth factor (aFGF) (reviewed by Burwen and Jones, 1987), insulin-like growth factor type I (IGF-I (Soler et al, 1990), and prolactin (Clevenger et al, 1991) are also translocated to and accumulated in the nucleus.…”