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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The polypeptide hormone insulin and the binding unit of cholera toxin (CTB) were coupled via a disulfide bond. This hybrid molecule had 1/30 the ability of native insulin to bind to the insulin receptor and 1/30 the biological activity of native insulin in H35 rat hepatoma cells and rat adipocytes. Thus, in these two cell types that are very sensitive to insulin, the biological activity of the hybrid molecule was as predicted on the basis of the ability of the molecule to interact with the insulin receptor. In contrast, in HTC rat hepatoma cells and rat thymocytes, two poorly responsive cell types, the insulin-CTB conjugate had 1/3 the biological activity of native insulin, a value 10 times greater than its insulin receptor binding potency. This increased activity of the conjugate did not appear to be due to cholera toxin in the preparation, since a control of uncoupled CTB had no biological activity. Furthermore, native cholera toxin increased intracellular levels of cAMP by 20-fold, whereas the conjugate had no effect on cAMP levels. The CTB moiety did, however, contribute to the biological activity of the conjugate, since the activity of the hybrid molecule, like cholera toxin, was inhibited by gangliosides, whereas the activity of native insulin was not. Finally, the binding to thymocytes of insulin-CTB conjugate, but not insulin, was inhibited by gangliosides. Thus, a hybrid hormone molecule has been constructed which has insulin-like biological activity with the receptor specificity of cholera toxin in poorly responsive cells.
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PMID:Insulin-cholera toxin binding unit conjugate: a hybrid molecule with insulin biological activity and cholera toxin binding specificity. 613 23

Hepatoma cells in culture exhibit a range of differentiated functions. Well differentiated hepatoma cells retain most of the functions of the adult liver, whereas dedifferentiated cells have lost most of them. In the present study, insulin binding and insulin effects were studied in four differentiated and two dedifferentiated cell lines derived from the Reuber hepatoma and in a partially differentiated cell line, HTC, derived from the Morris 7288C hepatoma. Specific insulin binding was lower in dedifferentiated cells than in partially differentiated and differentiated ones. In all cell lines, analysis of insulin binding yielded linear Scatchard plots. Although some variations in affinity of insulin for its receptor were observed, most of the differences in binding were accounted for by differences in insulin receptor number. In differentiated hepatoma cells, tyrosine aminotransferase (TAT)-specific activity was easily detectable, and insulin produced a 2- to 3-fold increase in enzyme activity within 4-6 h. By contrast, TAT activity in the dedifferentiated cells was low and did not respond to insulin. In the partially differentiated hepatoma HTC, insulin stimulated TAT only after basal TAT activity was induced by glucocorticoid treatment. Glycogen synthase (I and D forms) activities were detectable in all cell lines. In both differentiated and dedifferentiated Reuber hepatoma cells, insulin increased the I-form of glycogen synthase within 15 min. This effect of insulin was observed at lower insulin concentrations than stimulation of TAT activity. By contrast, insulin at any concentration was totally ineffective in stimulating glycogen synthase in glucocorticoid-treated and untreated HTC cells. These results indicate that in hepatoma cells, 1) insulin receptor number and insulin effect on TAT are modulated by the degree of cell differentiation; 2) the pathways of insulin action on TAT and glycogen synthase diverge in some postreceptor step(s) which is under independent control in differentiation; and 3) receptor and postreceptor defects exist in hepatoma cell lines which may be useful in dissecting the pathways of insulin action.
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PMID:Analysis of insulin action using differentiated and dedifferentiated hepatoma cells. 613 58

We have reported previously that insulin causes a complete but reversible desensitization to insulin action in rat hepatoma HTC cells in tissue culture, and that this insulin resistance is mediated by postbinding mechanisms rather than receptor down-regulation (Heaton, J. H., and Gelehrter, T. D. (1981) J. Biol. Chem. 256, 12257-12262). We report here that insulin causes a similar desensitization to the induction of tyrosine aminotransferase by the insulin-like growth factors IGF-I and IGF-II isolated from human plasma, and by multiplication-stimulating activity, the rat homologue of IGF-II. The results of both competition-binding studies and affinity cross-linking experiments indicate that insulin-like growth factors (IGFs) bind primarily to IGF receptors rather than to insulin receptors. The low concentrations at which these factors induce transaminase is consistent with their acting primarily via IGF receptors. This is confirmed by experiments utilizing anti-insulin receptor antibody which both inhibits 125I-insulin binding and shifts the concentration dependence of insulin induction of tyrosine aminotransferase to the right. This same immunoglobulin does not inhibit 125I-multiplication-stimulating activity binding and only minimally inhibits 125I-IGF-I binding. Anti-insulin receptor antibody also does not significantly shift the concentration dependence for the IGFs, suggesting that IGFs induce transaminase by acting via IGF receptors. Although insulin down regulates insulin receptors, it does not decrease IGF-I or IGF-II binding. We conclude that insulin causes desensitization of HTC cells to IGFs by affecting a postbinding step in IGF action, which may be common to the actions of both insulin and insulin-like growth factors.
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PMID:Insulin regulation of insulin-like growth factor action in rat hepatoma cells. 614 41

Insulin is able to stimulate a growth response in a variety of different cell types. However, the role of the insulin receptor in mediating this response is not clear. Indeed, it has been reported that the ability of insulin to stimulate a growth response is a result of its interaction with other growth factor receptors rather than the insulin receptor. We have previously reported that the H-35 hepatoma cell line responded to physiological concentrations of insulin as a growth factor and that the relative potency of proinsulin suggested that this response was mediated by the insulin receptor. In this report, two experimental approaches are used to demonstrate the involvement of the insulin receptor in mediating the growth response. Two different preparations of antibody to the insulin receptor are found to be capable of stimulating this response. In addition, the human insulin-like growth factors (IGF-I and II) show very low cross-reactivity with the insulin receptor and are significantly less potent than insulin in stimulating the growth response.
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PMID:The role of the insulin receptor in mediating the insulin-stimulated growth response in Reuber H-35 cells. 614 51

Rat hepatoma cells were labeled with [32P]orthophosphate and the insulin receptor subunits were identified by immunoprecipitation and sodium dodecyl sulfate-acrylamide gel electrophoresis. In the basal state, only the Mr = 95,000 (beta) subunit of the insulin receptor was phosphorylated. The covalent labeling with 32P of this subunit was stimulated about 3-fold by insulin (10(-6) M). This stimulation was due to an increase in the content of phosphoserine, the appearance of phosphotyrosine, and a possible increase in phosphothreonine as well. These results suggest phosphorylation of the insulin receptor at multiple sites is an early event in insulin action.
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PMID:Insulin stimulation of phosphorylation of the beta subunit of the insulin receptor. Formation of both phosphoserine and phosphotyrosine. 617 40

The development and application of affinity cross-linking methodology have allowed the identification of receptor subunits for at least seven different polypeptide growth factors. In the case of insulin, a complex disulfide-linked receptor structure has been deduced by this method in conjunction with results obtained from affinity purification of the receptor complex. The minimum subunit structure deduced for this receptor is (beta-S-S-alpha)-S-S-(alpha-S-S-beta), where alpha is a 125,000-dalton glycoprotein subunit and beta is a 90,000-dalton glycoprotein subunit. A receptor species with high affinity for insulinlike growth factor (IGF) I and low affinity for insulin exhibits striking homology to this insulin receptor structure. A third receptor structure has high affinity for IGF-II and lower affinity for IGF-I, with essentially no affinity for insulin. This IGF-II receptor structure has a molecular weight of about 250,000 and shows no evidence of disulfide linkage to other subunits. Receptor polypeptides with high affinity for epidermal growth factor, nerve growth factor, transformation growth factor, or platelet-derived growth factor have been linked to the respective 125I-labeled ligands and exhibit molecular weights of about 160,000, 140,000, 60,000, and 170,000, respectively. None of these receptors appears to be disulfide linked to other subunits. No apparent structural homology among these receptor types has been detected as yet. Recent evidence suggests that there may be important biochemical linkages between certain of the receptor systems. For example, an effect of insulin mediated through its own receptor in intact adipocytes or H-35 hepatoma cells rapidly results in a 5- to 10-fold increase in the affinity of the 250,000-dalton IGF-II receptor for 125I-labeled IGF-II. This may reflect an important mechanism by which insulin can simultaneously mediate rapid effects on cellular enzymes through its own receptor and indirectly promote cellular growth by potentiating growth factor action through this activation of the IGF-II receptor.
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PMID:Interrelationships among receptor structures for insulin and peptide growth factors. 630 63

An insulin-sensitive subcellular system was developed from rat adipocytes consisting of plasma membranes and mitochondria. Direct addition of insulin, concanavalin A or anti-insulin receptor antibody to this system resulted in the production of a mediator substance from the plasma membrane that caused dephosphorylation of the alpha subunit of pyruvate dehydrogenase in the mitochondria with concomitant activation of the enzyme. The mediator activated pyruvate dehydrogenase by activating the pyruvate dehydrogenase phosphatase and not by inhibiting the pyruvate dehydrogenase kinase. This was similar to the mechanism by which insulin causes activation of the enzyme in the intact cell. The insulin-sensitive mediator material from the adipocyte plasma membrane was acid-stable with a molecular weight of 1,000 to 1,500. Our laboratory has shown that the mediator that activates pyruvate dehydrogenase was present in intact adipocytes, hepatoma cells, and IM-9 lymphocytes. Insulin altered the amount or activity of the mediator consistent with the effect of the hormone on the cell. Other laboratories have shown similar effects on skeletal muscle and liver. We have shown the mediator to mimic insulin action on the low Km cyclic adenosine monophosphate (AMP) phosphodiesterase and the (calcium++-magnesium++)-adenosine triphosphatase (Ca++-Mg++)-ATPase of adipocyte plasma membranes in addition to pyruvate dehydrogenase. Other laboratories have shown the mediator to activate glycogen synthase. A body of direct and indirect evidence exists that demonstrates that more than one mediator exists. The chemical nature of the mediator is unknown but probably represents a new family of intracellular mediators of hormone action. These mediators may have clinical relevance in postreceptor defects of obesity and type II diabetes (noninsulin-dependent diabetes mellitus).
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PMID:The chemical mediators of insulin action: possible targets for postreceptor defects. 633 85

Insulin-ricin B chain conjugate, a hybrid molecule consisting of insulin covalently linked to the binding chain of ricin, was tested for insulin-like biological activity in HTC and H35 rat hepatoma cells, rat adipocytes, rat thymocytes, and human fibroblasts. In H35 cells and adipocytes, cells that have abundant insulin receptors and are very sensitive to insulin (ED50 of 30 pM and 50 pM, respectively), the conjugate had 5% the biological activity of native insulin (ED50 of 500 pM and 1000 pM, respectively). Since the insulin portion of the conjugate has 5% the potency of native insulin in binding to the insulin receptor, these observations suggested that (in these cells) the conjugate was acting via the insulin receptor. Moreover lactose and galactose, potent inhibitors of ricin binding to its receptor, had no effect on the action of the conjugate on H35 cells. In contrast, in thymocytes, HTC cells, and fibroblasts cells that have relatively few insulin receptors and require high concentrations of insulin to elicit biological actions (ED50 of 10 nM, 20 nM, and 1 nM, respectively), the conjugate had more biological activity than was predicted on the basis of its ability to bind to the insulin receptor. In addition, in these three insulin-insensitive cell types, the activity of the conjugate was inhibited by either lactose or galactose. Thus, these observations indicate that in cells which are relatively insensitive to insulin, the biological effects of the conjugate require the interaction of the ricin B chain moiety with the ricin receptor. In addition, in fibroblasts, the activity of the conjugate was inhibited by the addition of a monoclonal antibody to the insulin receptor which inhibits the response of fibroblasts to insulin. These data suggest, therefore, that in insulin-insensitive cells the binding of the ricin B chain moiety to its receptor enhances the interaction of the insulin portion of the conjugate with the insulin receptor.
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PMID:Insulin-ricin B chain conjugate has enhanced biological activity in insulin-insensitive cells. 634 61

Autophosphorylation of the insulin receptor was studied using a glycoprotein fraction solubilized and purified partially from the rat hepatoma cell line, Fao. Incubation of this receptor preparation with [gamma-32P] ATP, Mn2+, and insulin yielded a single insulin-stimulated phosphoprotein of Mr = 95,000 which corresponds to the beta-subunit of the insulin receptor. At 22 degrees C, incorporation of 32P was half-maximal at 30 s and about 90% complete after 2 min. At steady state, about 200 pmol of 32P were incorporated per mg of protein; this value corresponded to about 2 molecules of phosphate per insulin binding site estimated from Scatchard plots. Insulin increased the Vmax for autophosphorylation of the insulin receptor kinase nearly 20-fold with no effect on the Km for ATP. Mn2+ stimulated autophosphorylation by decreasing the Km of the kinase for ATP, whereas Mg2+ had no effect. Dilution of the insulin receptor over a 10-fold concentration range did not decrease the rate of autophosphorylation suggesting that it may occur by an intramolecular mechanism. When the phosphorylated beta-subunit of the insulin receptor was digested with trypsin, at least 5 phosphopeptides could be separated by high performance liquid chromatography on a mu Bondapak C18 reverse-phase column. Insulin stimulated the phosphorylation of all sites. These phosphate acceptor sites varied in their rate and degree of phosphorylation. Phosphopeptides pp4 and pp5 were phosphorylated very rapidly and reached steady state within 20 s, whereas phosphorylation of pp1 and pp2 required several minutes to reach steady state.
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PMID:Kinetic properties and sites of autophosphorylation of the partially purified insulin receptor from hepatoma cells. 636 36

We have previously reported that prolonged incubations of Fao cells, a cell line derived from the well-differentiated Reuber H35 rat hepatoma, with 10(-6) M insulin, induced a decrease in receptor number (down-regulation), an increase in receptor affinity for insulin, and a loss of insulin's biological effect (desensitization). In the present study, we have investigated the relationship between these changes in insulin binding and action and changes in the structure of the insulin receptor. Intact cells were surface labeled with Na125I and lactoperoxidase, and the 125I-labeled insulin receptor was immunoprecipitated using specific antibodies and analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of gels done under reducing conditions demonstrated the alpha (Mr = 135,000) and the beta (Mr = 95,000) subunits of the receptor. In nonreduced gels, free insulin receptor subunits were observed as well as four higher molecular weight bands with Mr = 210,000, 270,000, 350,000, and 520,000. Two-dimensional gel electrophoresis revealed that these bands correspond to alpha-beta heterodimer, alpha 2 homodimer, and two alpha-beta oligomers of high molecular weights, respectively. Cross-linking of 125I-insulin to intact cells with disuccinimidyl suberate revealed bands of Mr = 125,000, 210,000, 250,000 and 320,000, indicating that most of the forms of the receptor could bind insulin. After incubation with 10(-6) M insulin for 24 h, Fao cells revealed a marked decrease of the four oligomeric forms of the receptor, with little change in the level of the free alpha and beta subunits. A similar decrease of the oligomeric forms of the insulin receptor and an increase in the free subunits was observed when normal Fao cells are treated with 7 mM dithiothreitol. In dithiothreitol-treated cells, 125I-insulin binding was increased and this increase was accounted for by a change in affinity. In contrast to Fao cells, down-regulation of the insulin receptor in IM-9 lymphocytes occurs without a change in receptor affinity. In these cells, surface labeling revealed a decrease in total receptors after down-regulation, but not change in the proportion of the oligomeric forms to the free subunits of the receptor. These data suggest the following in Fao hepatoma cells. In the native state, the insulin receptor consists of free alpha and beta subunits and several kinds of disulfide-linked oligomers of these subunits.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Insulin receptor regulation and desensitization in rat hepatoma cells. The loss of the oligomeric forms of the receptor correlates with the change in receptor affinity. 638 1


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