Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have compared the effect of phorbol 12-myristate 13-acetate (PMA) with that of insulin on three targets of insulin action in H4IIEC3 (H4) rat hepatoma cells. These parameters are the phosphorylation state and tyrosine kinase activity of the insulin receptor, the activation state of glycogen synthase, and the accumulation of p33 mRNA. Under conditions where insulin treatment of H4 cells clearly activated receptor serine and tyrosine phosphorylation on the insulin receptor beta-subunit in situ, activated receptor tyrosine kinase activity in vitro, and activated glycogen synthase and p33 mRNA accumulation in situ, PMA alone did not influence the insulin receptor phosphorylation state or tyrosine kinase activity and did not affect glycogen synthase activity, but markedly increased p33 mRNA accumulation. When PMA was added in the presence of insulin, particularly if PMA was preincubated, the receptor phosphorylation state and the tyrosine kinase activity again were not affected, but insulin-activated glycogen synthase was significantly diminished or abolished. In contrast, increased p33 mRNA accumulation by PMA was additive with that of insulin. Thus, under conditions where no effect was observed on the insulin receptor phosphorylation state or the tyrosine kinase activity, PMA acted in an insulin-antagonistic manner on glycogen synthase and in an insulin-like manner on p33 mRNA accumulation, indicating that these actions of PMA are unrelated to early events in the pathway of the insulin action. Effects on glycogen synthase are most readily explained by an effect of protein kinase C-activated phosphorylation of glycogen synthase.
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PMID:Contrasting interactions between phorbol ester and insulin on the regulation of glycogen synthase activity and p33 mRNA accumulation in rat hepatoma cells. 312 51

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function of the insulin receptor.
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PMID:Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity. 312 81

Tyr(P)-containing proteins were purified from extracts of insulin-treated rat hepatoma cells (H4-II-E-C3) by antiphosphotyrosine immunoaffinity chromatography. Two major insulin-stimulated, Tyr(P) proteins were recovered: an Mr 95,000 protein (identified as the insulin receptor beta subunit by its immunoprecipitation by a patient-derived anti-insulin receptor serum and several anti-insulin receptor (peptide) antisera) and an Mr 180,000 protein (which was unreactive with all anti-insulin receptor antibodies). After purification and tryptic digestion of the Mr 95,000 protein, tryptic peptides containing Tyr(P) were purified by sequential antiphosphotyrosine immunoaffinity, reversed-phase, anion-exchange chromatography. The partial amino acid sequence obtained by gas- and solid-phase Edman degradation was compared to the amino acid sequence of the intracellular extension of the rat insulin receptor deduced from the genomic sequence. Approximately 80% of all beta subunit [32P]Tyr(P) resides on two tryptic peptides: 50-60% of [32P]Tyr(P) is found on the tryptic peptide Asp-Ile-Tyr-Glu-Thr-Asp-Tyr-Tyr-Arg from the tyrosine kinase domain, which is recovered mainly as the double phosphorylated species (predominantly in the form with Tyr(P) at residues 3 and 7 from the amino terminus; the remainder with Tyr(P) at residues 3 and 8), with 10-15% as the triple phosphorylated species. A second tryptic peptide is located near the carboxyl terminus, contains 2 tyrosines, and has the sequence, Thr-Tyr-Asp-Glu-His-Ile-Pro-Tyr-Thr-; this contains 20-30% of beta subunit [32P]Tyr(P) and is identified primarily in a double phosphorylated form. Approximately 10% of beta subunit [32P]Tyr(P) resides on an unidentified tryptic peptide of Mr 4,000-5,000. The insulin-stimulated tyrosine phosphorylation of the insulin receptor in intact rat hepatoma cells thus involves at least 6 of the 13 tyrosine residues located on the beta subunit intracellular extension. These tyrosines are clustered in several domains in a distribution virtually identical to that previously found for partially purified human insulin receptor autophosphorylated in vitro in the presence of insulin. This multisite regulatory tyrosine phosphorylation is the initial intracellular event in insulin action.
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PMID:Identification of the insulin receptor tyrosine residues undergoing insulin-stimulated phosphorylation in intact rat hepatoma cells. 327 43

Previous studies have demonstrated differences in the size of insulin receptor subunits in brain and adipocytes that appear to involve variations in glycosylation of the proteins. In this report, we examined the degree of homology in the protein backbones of insulin receptors in both tissues by peptide mapping and compared the mRNAs encoding the receptors by Northern blot analysis. Photoaffinity-labeled insulin receptors from rat brain and adipocytes were deglycosylated and then subjected to partial proteolysis by five different enzymes with differing substrate specificities. The intact receptors and their proteolytic fragments were analyzed by electrophoresis and autoradiography. Each enzyme yielded a unique pattern of fragments ranging from 70 to 11 kDa. In all cases, there was a striking similarity in the peptide maps generated from insulin receptors in brain and adipocytes. Northern hybridization experiments were carried out using poly(A)+ RNA from rat brain, rat adipocytes, and human hepatocarcinoma (HEP G2) cells. In rat brain, two bands of 9.5 and 7.4 kb were detected and, in rat adipocytes, the same two bands were observed. The two mRNA bands observed in rat tissues represented only two of the five mRNA species seen in human HEP G2 cells. The results indicate that the protein domains and the mRNAs encoding of insulin receptors in brain and adipocytes are very similar, if not identical.
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PMID:Peptide mapping on Northern blot analyses of insulin receptors in brain and adipocytes. 328 25

In a subline of Reuber H35 rat hepatoma cells that becomes quiescent under serum-deprived conditions, insulin acts as a growth factor. When added to serum-deprived H35 cells, physiologic concentrations of insulin stimulate DNA synthesis, demonstrating that insulin alone is capable of inducing a transition from G0/G1 into S phase. This response, which is induced by nanomolar concentrations of insulin, is mediated directly through the insulin receptor. Here we show that coincident with this growth response, insulin or serum induces dramatic increases in the steady-state levels of c-fos and c-myc mRNAs in serum-deprived H35 cells in a time course similar to that observed in the regenerating liver. Other growth factors, including epidermal growth factor, appear not to affect these cells either in terms of DNA synthesis or c-myc mRNA induction. The phorbol ester phorbol 12-myristate 13-acetate (PMA) also induces c-myc and c-fos mRNAs without inducing DNA synthesis. However, the mechanism of this induction appears to be different from the insulin-induced induction since pretreatment of cells with PMA blocks only the PMA-mediated, not the insulin-mediated, induction of c-myc and c-fos.
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PMID:Insulin as a growth factor in rat hepatoma cells. Stimulation of proto-oncogene expression. 330 43

To evaluate the synthesis and initial processing of the insulin receptor precursor, we compared cell-free translation of rat liver poly(A)+ RNA in a reticulocyte lysate system with metabolically labeled rat hepatoma (Fao) cells. In in vitro translation assays, the primary L-[35S]cysteine-labeled products of rat liver mRNA specifically immunoprecipitable with insulin receptor antiserum were two closely migrating polypeptides with a Mr range of 160,000-164,000 (n = 7). This is similar to the size predicted by the insulin receptor cDNA sequence. When heterologous microsomal membranes were included in the cell-free system to process newly synthesized proteins co-translationally, the receptor precursors migrated as larger species of 180 +/- 2 kDa (n = 3). For comparison, when Fao cells were treated with tunicamycin to block core N-glycosylation and pulse-labeled with L-[35S]methionine, two closely migrating precursors were labeled that co-migrated with the unprocessed in vitro translation products (approximately 160 kDa). Pulse labeling of Fao cells in the absence of tunicamycin revealed receptor precursor species of 188 and 198 kDa that rapidly disappeared (t1/2 = 54 min) as the receptor subunits were observed. Thus, the initial products of insulin receptor mRNA translation are two approximately 162-kDa polypeptides that are rapidly processed in intact cells and can only be observed by in vitro studies or by using inhibitors of core glycosylation. Insulin proreceptor species can also be partially glycosylated during cell-free translation by added microsomal membranes. This is the first description of cell-free translation of the insulin proreceptor in a system that will allow detailed characterization of the earliest steps in insulin receptor biogenesis.
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PMID:Initial processing of the insulin receptor precursor in vivo and in vitro. 341 32

A lipoprotein-induced resistance to the action of insulin has been postulated. To test this hypothesis, cultured rat-derived hepatoma cells, designated FAO, and human-derived hepatoma cells, designated HEP-G2, were incubated for 20 h in the presence or absence of lipoprotein; specific 125I-insulin receptor binding and labeled glucose incorporation into glycogen were then measured. Very low density lipoproteins (d less than 1.006 g/ml) in physiologic (0.5 mg/ml) or pathophysiologic (5 mg/ml) concentrations did not modify insulin receptor binding of FAO or HEP-G2 cells. This was true for very low density lipoproteins derived from normal human, diabetic human, and streptozotocin-diabetic rat plasma. Low density lipoproteins (d = 1.019 - 1.063 g/ml) isolated from normal human plasma similarly failed to modify insulin receptor binding. Concerning insulin action, the different very low density lipoprotein preparations did not modulate either basal or insulin-stimulated glucose incorporation into glycogen of the cells. Thus, very low density lipoproteins and low density lipoproteins did not induce insulin resistance in cultured hepatoma cells either at the insulin receptor level or at the post-receptor level.
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PMID:Lack of a lipoprotein-induced insulin resistance in hepatoma cells in culture. 352 46

To elucidate the mechanism of glucagon-insulin (G-I) therapy, the effect of insulin and/or glucagon on the insulin receptor was studied in an experiment utilizing cultured cells (JTC-16) of rat ascites hepatoma. Insulin specific receptors were present on JTC-16 cells and were similar in nature to the receptors of primary culture rat hepatocytes. There were two kinds of insulin receptors. One had a high insulin affinity and the other had low insulin affinity. In the experiment involving addition of insulin the number of insulin receptors decreased after 24 hrs incubation in proportion to the increase in added insulin concentration. On the other hand, the number of insulin receptors increased with glucagon addition and the increase in proportion to the concentration of glucagon added. In the experiment involving simultaneous addition of insulin and glucagon, a 20% decrease in the number of receptors induced with 10(-9) M insulin was restored to the control level with simultaneous glucagon addition of the same concentration. The number of insulin receptors increased as the concentration of additive glucagon increased. These results show that simultaneous addition of insulin and glucagon inhibits the decrease in number of insulin receptors with insulin alone. These facts may obtain more potent action of insulin in G-I therapy via insulin receptor.
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PMID:The effect of insulin and glucagon on the insulin receptor of cultured hepatoma cells. 353 98

This study utilized uniform incubation conditions and demonstrated heterogeneity in the effects of various agents on 125-insulin binding and intracellular accumulation in five cell types. The cells used in this study included rat adipocytes, H4IIEC3 cultured hepatoma cells, normal human fibroblasts, and 3T3-L1 preadipocytes and adipocytes. Bacitracin increased insulin binding to rat adipocytes but inhibited binding to all other cells. Chloroquine increased total cell-associated insulin in all cells except H4IIEC3 hepatoma cells. Methylamine and dansylcadaverine increased or decreased binding depending on cell type. Similar heterogeneity was found in the intracellular accumulation of insulin. Under control conditions, intracellular insulin at steady state varied from 16 to 52% of the total cell-associated insulin. Bacitracin decreased intracellular accumulation of insulin in 3T3-L1 adipocytes, preadipocytes, and human fibroblasts but increased accumulation in rat adipocytes. Chloroquine increased insulin accumulation in all cell types except H411EC3 hepatoma cells. Both methylamine and dansylcadaverine increased intracellular insulin in rat adipocytes and decreased accumulation in human fibroblasts. These results provide additional evidence of heterogeneity in insulin and/or insulin receptor processing among different cell types.
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PMID:Heterogeneous effects of inhibitors of receptor processing on insulin binding and intracellular accumulation in various cell types. 353 51

The effects of tumour-promoting phorbol esters on the receptor-mediated endocytosis of insulin were investigated in the human hepatoma cell line HepG2. Treatment of these cells with the biologically active phorbol 12-O-tetradecanoylphorbol 13-acetate (TPA), but not with the non-tumour-promoting analogue 4 alpha-phorbol 12,13-didecanoate, resulted in dramatic morphological changes, which were accompanied by a 1.5-2.5-fold increase in specific 125I-insulin association with the cells at 37 degrees C. This increase in insulin binding was not observed when the binding reaction was performed at 4 degrees C. The potentiation of 125I-insulin association with TPA-treated cells at 37 degrees C could be completely accounted for by an increase in the intracellular pool of internalized insulin; there was no concomitant increase in cell-surface insulin binding. Dissociation studies showed that the enhanced internalization of insulin by cells after treatment with TPA resulted from a decrease in the rate of intracellular processing of the insulin after receptor-mediated endocytosis. The phorbol-ester-induced enhancement of internalized insulin in HepG2 cells was additive with the potentiation of endocytosed insulin induced by both the lysosomotropic reagent chloroquine and the ionophore monensin; this indicates that TPA affects the intracellular processing of the insulin receptor at a point other than those disrupted by either of these two reagents. The potentiation of insulin receptor internalization by tumour-promoting phorbol esters could be completely mimicked by treatment with phospholipase C, but not with phospholipase A, and partially mimicked by treatment with the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol. By these criteria, the effects of phorbol esters on the insulin receptor in HepG2 cells appear to be mediated through protein kinase C. These results support the concept that the activation of protein kinase C by treatment with phorbol esters causes a perturbation of the insulin-receptor-mediated endocytotic pathway in HepG2 cells, reflected in a long-term decreased rate of dissociation of internalized insulin by the phorbol-ester-treated cells.
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PMID:Potentiation of specific association of insulin with HepG2 cells by phorbol esters. 353 1


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