Effect of physiological concentrations of insulin and antidiabetic drugs on RNA release from isolated liver nuclei

Schumm, Dorothy E.; Webb, Thomas E.

doi:10.1002/jcb.240230119

Cited by 13 publications

(10 citation statements)

References 26 publications

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“…Insulin failed to stimulate growth in the PG19 mouse melanoma cells, which had internalization-defective, but kinase-competent insulin receptors (51). When added to isolated nuclei, insulin affected various nuclear processes, such as nucleo-cytoplasmic transport of macromolecules (52), protein phosphorylation (53), enzymatic activities (54), and mRNA release from nuclei (55). Recently, we demonstrated that trypsin treatment, which resulted in undetectable insulin binding to the insulin receptor and phosphorylation of insulin receptor and IRS-1 (56), did not change insulin's ability to stimulate immediate early gene transcription in H35 rat hepatoma cells (57).…”

Section: Discussionmentioning

confidence: 99%

Insulin-induced egr-1 Expression in Chinese Hamster Ovary Cells Is Insulin Receptor and Insulin Receptor Substrate-1 Phosphorylation-independent

Harada

Smith

et al. 1995

Journal of Biological Chemistry

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Insulin's effects primarily are initiated by insulin binding to its plasma membrane receptor and the sequential tyrosine phosphorylation of the insulin receptor and intracellular substrates, such as insulin receptor substrate-1 (IRS-1). However, studies suggest some insulin effects, including those at the nucleus, may not be regulated by this pathway. The present study compared the levels of insulin binding, insulin receptor and IRS-1 tyrosine phosphorylation, and phosphatidylinosi- Insulin's effects primarily are initiated by insulin binding to its plasma membrane receptor and the sequential tyrosine phosphorylation of the insulin receptor and intracellular substrates, such as insulin receptor substrate-1 (IRS-1), 1 IRS-2, or Shc (reviewed in Ref. 1). These substrates bind to Src homology 2 domains of several cytoplasmic signal proteins through their tyrosine phosphorylation sites. These proteins include the 85-kDa subunit of phosphatidylinositol (PI) 3Ј-kinase, GRB-2, or Syp (tyrosine phosphatase) (1). Activation of these molecules and the following activation of other intracellular molecules, such as p21 ras , raf-1, mitogen-activated protein kinase, or S6 kinase is believed to be responsible for many of insulin's biological responses.It is well known that insulin affects nuclear events such as gene expression and cell growth (reviewed in Ref.2). One of insulin's effects on nuclear events is the stimulation or inhibition of a number of genes, specifically immediate early genes (3, 4). The immediate early genes are a large and diverse group, and the mechanisms involved in their regulation are complex. The induction of c-fos transcription, one of the well-characterized immediate early genes, by insulin or other growth factors is believed to require receptor phosphorylation and p21 ras activation. For instance, insulin induced c-fos mRNA accumulation in Chinese hamster ovary (CHO) cells overexpressing human insulin receptor but not in their parent cells (5). Inhibition of p21 ras activity by dominant inhibitory mutants suppressed insulin-induced activation of the c-fos promoter (6). However, Mundschau et al. (7) have shown that induction of expression of the immediate early gene egr-1, but not c-fos, c-myc, and JE, was independent of platelet-derived growth factor receptor autophosphorylation using three different conditions in which platelet-derived growth factor receptor autophosphorylation was blocked. In addition, Eldredge et al. (8) reported that epidermal growth factor (EGF) induced c-fos expression in the cells expressing kinase-deficient EGF receptors. These results indicate the existence of another signaling mechanism, which operates independently of growth factor receptor tyrosine kinase activity and affects some, but not all, nuclear responses to growth factor stimulation.In the present study, we tested the possibility of the existence of divergent pathways in insulin signal transduction mechanisms regulating immediate early gene expression. We utilized CHO cells stably transfected with only neomyci...

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Section: Discussionmentioning

confidence: 99%

Insulin-induced egr-1 Expression in Chinese Hamster Ovary Cells Is Insulin Receptor and Insulin Receptor Substrate-1 Phosphorylation-independent

Harada

Smith

et al. 1995

Journal of Biological Chemistry

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“…(12) and others (13,14) have used isolated nuclei to study some of insulin's responses. The present study utilized a simple system: an insulin-responsive cell in which the insulin receptors were proteolytically inactivated, which eliminated both insulin binding and receptor tyrosine kinase activity.…”

Section: Cellmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…Miller (11) demonstrated that insulin microinjected into the cytoplasm of frog oocytes stimulated RNA and protein synthesis. Others showed that insulin added to isolated nuclei effected enzymatic (12) and transport processes (13,14).In a series of biochemical and ultrastructural studies we have reported that intact insulin accumulated in nuclei of 3T3-L1 adipocytes (15) and H35 hepatoma cells (13, 16) and associated with the nuclear matrix, which is involved with gene expression (17). Nuclear accumulation of insulin was temperature-and time-dependent but energy-independent.…”

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confidence: 97%

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Direct stimulation of immediate-early genes by intranuclear insulin in trypsin-treated H35 hepatoma cells.

Lin

Harada

Loten

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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H35 hepatoma cells were treated with trypsin to abolish insulin binding and insulin-stimulated receptor kinase activity. Insulin was, however, internalized by fluidphase endocytosis in trypsin-treated cells. Furthermore, nuclear accumulation of insulin was similar in control and trypsin-treated hepatoma cells. Northern blot analysis revealed insulin increased g33 and c-fos mRNA concentrations identically in control and trypsin-treated cells but had no effect on P2-microglobulin mRNA. Actinomycin D treatment prior to or after insulin addition demonstrated that insulin increased gene transcription and had no effect on mRNA degradation. These studies suggest that the accumulation of intact insulin in cell nuclei may be directly involved in the increased transcription of immediate-early genes.The pleiotropic hormonal and growth-promoting effects of insulin are well known (1). These include insulin's stimulation of glucose, ion, and amino acid transport, effects on numerous enzymatic processes, and stimulation of protein synthesis and cellular proliferation. Also documented are insulin's positive or negative effects on the expression of a number of specific genes (2), several of which may be associated with cell growth and proliferation (3)(4)(5)(6)(7). The mechanisms by which insulin affects these cellular processes are relatively unknown. It is generally accepted that the first and obligatory step is insulin binding to its cell membrane receptor. The ensuing autophosphorylation of the receptor's ,B subunit and activation of the intrinsic tyrosine kinase activity (8) have been implicated, directly or indirectly, in some effects of insulin. However, recent evidence suggests that alternative second messenger-type mechanisms are involved in other insulin responses (9, 10). In addition, some insulin responses may be induced without insulin binding to the plasma membrane receptor. Miller (11) demonstrated that insulin microinjected into the cytoplasm of frog oocytes stimulated RNA and protein synthesis. Others showed that insulin added to isolated nuclei effected enzymatic (12) and transport processes (13,14).In a series of biochemical and ultrastructural studies we have reported that intact insulin accumulated in nuclei of 3T3-L1 adipocytes (15) and H35 hepatoma cells (13, 16) and associated with the nuclear matrix, which is involved with gene expression (17). Nuclear accumulation of insulin was temperature-and time-dependent but energy-independent. Although the nuclear accumulation of insulin resulted in part from receptor-mediated endocytosis, the plasma membrane receptor was not translocated to the nucleus (18). Interestingly, insulin accumulation in the nucleus continued to increase at media insulin concentrations in excess of those that saturated the plasma membrane receptors (16). This observation was, to our knowledge, the first indication that a non-receptor-mediated process also might be involved in insulin's nuclear translocation.In a recent study that investigated the nuclear translocation of insulin,...

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“…Although insulin has been shown to affect nuclear processes (20) and incubation of isolated nuclei with insulin affects biologic processes (38,39), previous studies have often failed to provide convincing evidence that a significant amount of internalized insulin can accumulate in the nuclei of intact cells. The current study provides that evidence.…”

Section: Methodsmentioning

confidence: 99%

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

Smith¹,

Jarett²

1987

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Effect of physiological concentrations of insulin and antidiabetic drugs on RNA release from isolated liver nuclei

Cited by 13 publications

References 26 publications

Insulin-induced egr-1 Expression in Chinese Hamster Ovary Cells Is Insulin Receptor and Insulin Receptor Substrate-1 Phosphorylation-independent

Insulin-induced egr-1 Expression in Chinese Hamster Ovary Cells Is Insulin Receptor and Insulin Receptor Substrate-1 Phosphorylation-independent

Direct stimulation of immediate-early genes by intranuclear insulin in trypsin-treated H35 hepatoma cells.

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

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