Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Insulin binding and insulin stimulated amino acid and glucose uptake were determined in cultured HTC hepatoma cells in the presence of Ca2+ and ruthenium red (RR) in order to further characterise the putative calcium binding site on the receptor. These ions increased insulin receptor high affinity binding and the sensitivity of these responses to insulin. The insulin concentration required to half-maximally stimulate amino acid uptake decreased significantly from 26.9 +/- 5.8 ng/ml to 6.0 +/- 1.3 ng/ml in the presence of 10 mM Ca2+ and to 1.3 +/- 0.5 ng/ml in the presence of RR. The effect of Ca2+ and RR was more pronounced on insulin stimulated glucose uptake. These agents also increased receptor-effector coupling, reducing the percentage of occupied receptors required for maximal insulin stimulation of amino acid uptake from 10.8% in control cells to 3.4 and 1.4% in the presence of Ca2+ and RR respectively. The receptor occupancy required to produce maximal insulin responses on glucose uptake decreased from 20% (control) to 3.8% (Ca2+ and RR). We hypothesize that since Ca2+ and RR have similar effects, that occupation of Ca2+ binding sites on the receptor produces a conformational change in the insulin receptor which increases insulin receptor affinity, insulin sensitivity and acts on an early post-receptor event responsible for coupling binding to insulin action.
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PMID:High affinity insulin binding and insulin receptor-effector coupling: modulation by Ca2+. 170 65

1. Insulin receptors were partially purified from rat liver by chromatography on wheat-germ-lectin-Sepharose. Incubation with [gamma-32P]ATP in the presence of insulin resulted in increased phosphorylation of the beta-subunit on both tyrosine and serine residues. Two-dimensional mapping of tryptic peptides showed that, in agreement with previous studies using preparations of receptors from other sources, the tyrosine residues involved were the three tyrosines in the kinase domain (corresponding to tyrosines 1158, 1162 and 1163 of the human receptor) plus two tyrosines close to the C-terminus (corresponding to tyrosines 1328 and 1334). 2. The effects of insulin on the phosphorylation of receptors within intact rat liver cells were determined by incubating cells in the presence of [32P]Pi for 50 min and then with or without insulin for a further 10 min. The labelled receptors were then rapidly isolated by sequential use of wheat-germ-lectin-Sepharose chromatography and immuno-isolation using a monoclonal antibody to the C-terminal end of the beta-subunit. 3. Insulin was found to increase overall phosphorylation of the receptor nearly 3-fold. Two-dimensional mapping was then carried out in combination with phosphoamino acid analysis. This revealed that the pattern of phosphorylation of the receptors in cells incubated in the absence and presence of insulin exhibited a number of marked differences from that observed in previous studies on intact cells, which had been restricted to cells expressing very high levels of insulin receptors such as certain hepatoma-derived cells or cells transfected with insulin receptor cDNA. The differences in the effects of insulin included a larger increase in the proportion of receptors being phosphorylated on the three tyrosine residues of the kinase domain, no apparent phosphorylation of the two tyrosine residues close to the C-terminus and no increase in either threonine or overall serine phosphorylation. 4. The receptors appeared to be phosphorylated on a number of different serine residues in cells incubated in the absence of insulin. Evidence for both increases and decreases in the phosphorylation of specific serine residues on addition of insulin was obtained. 5. It is concluded that care should be taken when extrapolating findings on the phosphorylation of the insulin receptor within cultured cells to more physiological situations.
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PMID:Analysis of insulin receptor phosphorylation sites in intact rat liver cells by two-dimensional phosphopeptide mapping. Predominance of the tris-phosphorylated form of the kinase domain after stimulation by insulin. 170 33

The insulin-like growth factor-binding proteins (IGFBPs) are thought to determine the distribution of IGF-I and IGF-II between the blood and tissue compartments and to modulate their biological activities. A dynamic metabolic role for one of the IGFBPs, IGFBP-1, is suggested by the fact that plasma IGFBP-1 was increased after fasting and diabetes and rapidly decreased by refeeding or insulin treatment, respectively. IGFBP-1 mRNA also is increased in the livers of diabetic rats and decreased by insulin treatment. To understand the molecular basis for this regulation, we have examined the effects of insulin on IGFBP-1 and IGFBP-1 mRNA in the H4-II-E cell line derived from the well differentiated H35 rat hepatoma. IGFBP-1, identified by ligand blotting and immunoblotting, is the major IGFBP in H4-II-E cells. Incubation of H4-II-E cells with insulin for 24 h decreased IGFBP-1 in the culture medium by approximately 50%. Inhibition was observed at physiological concentrations of insulin (ED50, less than 0.5 nM), but not at higher concentrations of IGF-II. These results, together with the fact that H4-II-E cells do not possess IGF-I receptors with which insulin might cross-react, suggest that insulin acts via the insulin receptor. Insulin inhibited IGFBP-1 in the medium by 80% in the absence of glucose, suggesting that the inhibition is a direct effect of insulin; glucose exerted a smaller independent effect in the absence of insulin. Insulin decreased IGFBP-1 mRNA in H4-II-E cells by 50% within 1 h and by 90% after 2-12 h of incubation. Nuclear run-on transcription assays indicated a corresponding decrease in the rate of IGFBP-1 gene transcription. Pretreatment of H4-II-E cells with dexamethasone stimulated IGFBP-1 transcription and increased steady state IGFBP-1 mRNA; stimulation was abolished by insulin treatment, indicating that inhibition by insulin was dominant over induction by dexamethasone. Thus, insulin, acting through the insulin receptor, rapidly decreases the abundance of IGFBP-1 mRNA in H4-II-E cells. Regulation occurs at least in part at the level of gene transcription. We propose that regulation of IGFBP-1 synthesis is an important component of the regulation of IGFBP-1 by insulin in vivo.
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PMID:Insulin rapidly inhibits insulin-like growth factor-binding protein-1 gene expression in H4-II-E rat hepatoma cells. 171 86

We have shown previously that experimental modifications of the cellular lipid composition of an insulin-sensitive rat hepatoma cell line (Zajdela Hepatoma Culture, ZHC) affect both binding and biological actions of insulin. Discrepancies between insulin binding and actions implied a postbinding defect, responsible for the observed insulin resistance in lipid-treated cells. To elucidate the mechanism for this defect, we have studied insulin binding and insulin receptor kinase activity in partially purified receptor preparations from ZHC cells grown either in normal medium or in medium supplemented with linoleic acid or 25-hydroxycholesterol. Insulin binding to the lectin-purified insulin receptor showed only a small alteration in receptor affinity for the preparations from lipid-treated cells. Insulin-stimulated autophosphorylation of the beta-subunit of the insulin receptor, as well as insulin-induced phosphorylation of the artificial substrate poly(Glu,Tyr)4:1, was significantly decreased in the preparations from lipid-modified cells. Although differences in basal levels were observed, the magnitude of the insulin-stimulated kinase activity was significantly decreased in receptor preparations from lipid-treated cells. These findings indicate that experimental modification of the lipids of cultured hepatoma cells can produce in insulin receptor kinase activity changes that are proportional to the reduced insulin action observed in these cells.
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PMID:Lipid-induced insulin resistance in cultured hepatoma cells is associated with a decreased insulin receptor tyrosine kinase activity. 184 94

The gene (PP63) encoding the inhibitor (PP63) of the insulin receptor tyrosine kinase was isolated from a rat genomic library. The intron/exon organization was deduced from Southern-blot analysis and sequence data (i.e., the exons + the boundaries). The PP63 gene, which maps to chromosome 11, spans approx. 8 kb and contains seven exons separated by six introns of different sizes. All of the boundaries match the consensus GT/AG sequence for donor and acceptor splice sites. Primer extension and S1 mapping experiments were used to locate the transcription start point (tsp) 73 nt upstream from the translational initiator. Both in vitro transcription assays and transcription of a chimeric gene in intact hepatoma cells indicated that the sequence located immediately upstream from the tsp contained a promoter. Several putative cis-regulatory elements, including a TATA box and a C/EBP-binding site were found within the 250 bp preceding the tsp.
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PMID:Primary structure of the rat gene encoding an inhibitor of the insulin receptor tyrosine kinase. 184 62

In order to study the role of tyrosine autophosphorylation in insulin receptor signalling, we investigated a mutant human insulin receptor whereby the three major tyrosine autophosphorylation sites at positions 1158, 1162, and 1163 in the receptor beta-subunit were mutated to phenylalanines. When these mutant receptors were expressed in HTC rat hepatoma cells, there was no enhanced beta-subunit autophosphorylation and tyrosine kinase activity. In these cells there was enhanced insulin stimulation of [3H]AIB uptake and [3H]thymidine incorporation when compared to wild type HTC cells. The present study suggests therefore that the presence of the major insulin autophosphorylation sites is not a requirement for insulin stimulation of amino acid transport and mitogenesis.
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PMID:Transmembrane signalling by insulin via an insulin receptor mutated at tyrosines 1158, 1162, and 1163. 189 12

The mechanism of action of CS-045, a new orally active antidiabetic agent, was studied in vitro using cultured hepatoma cells (Hep G2) and muscle cells (BC3H-1). Treatment of both types of cultured cells with varying doses of CS-045 did not significantly alter insulin receptor binding. Basal and insulin-stimulated glucose transport in BC3H-1 cells was also unaltered by the drug. In contrast, CS-045 increased glycogen synthase I activity in both cell types. This effect was maximal after 24 hours and in Hep G2 cells was associated with a threefold increase in the apparent affinity of the enzyme for glucose-6-phosphate. Gluconeogenesis from lactate in Hep G2 cells was greatly reduced by CS-045 treatment. We conclude that CS-045 may act directly on muscle and liver cells to increase glucose utilization. It is also effective in reducing glucose production. These multiple effects may account in part for the ability of CS-045 to reduce blood sugar levels in vivo.
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PMID:In vitro studies on the action of CS-045, a new antidiabetic agent. 212 May 48

The delivery of insulin and the insulin receptor into an intracellular compartment may be important for eliciting some of the biologic responses of the cell to the hormone. Internalization of insulin-receptor complexes in cells from hyperinsulinemic type II diabetic patients is diminished, suggesting a possible role for this cellular process in insulin resistance. To examine whether hyperinsulinemia contributes to defective insulin-receptor processing in vitro, cultured hepatoma cells (HepG2) were incubated with high concentrations of (500 ng/ml) insulin from 1-3 days. Insulin induced a decrease in the number of total and surface insulin receptors within 24 hours; however, the hormone did not mediate a change in the number of intracellular receptors. The cellular itinerary of control and down-regulated receptors were then compared. Insulin mediated internalization of down-regulated receptors was impaired compared to control receptors; however, the down-regulated receptors that were internalized recycled back to the plasma membrane more efficiently. By covalently labeling the insulin receptor with the photoactive insulin derivative, 125I-NAPA-DP-insulin, it was demonstrated that the rates of receptor degradation of down-regulated and control receptors were similar. These results suggest that incubating HepG2 cells with high concentrations of insulin alters the cellular itinerary of the insulin receptor.
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PMID:Down-regulated insulin receptors in HepG2 cells have an altered intracellular itinerary. 215 9

Vanadate, at concentrations between 0.5 and 2 mM, rapidly decreased the basal level of P-enolpyruvate carboxykinase (GTP) (EC 4.1.1.32) mRNA and blocked the dibutyryl cyclic AMP (Bt2cAMP)-induced increase in enzyme mRNA in both FTO-2B and H4IIE rat hepatoma cells. The concentration of vanadate necessary to inhibit the expression of this gene was similar to that required for the vanadate-mediated activation of the insulin receptor tyrosine kinase. To determine whether vanadate could inhibit PEPCK gene transcription, a series of chimeric genes containing several deletions in the P-enolypyruvate carboxykinase promoter between -550 and -68 was linked to the structural genes for either amino-3-glycosyl phosphotransferase (neo) or chloramphenicol acetyltransferase and introduced into hepatoma cells using three methods: (a) infection with a Moloney murine leukemia virus-based retrovirus, (b) transfection and stable selection for neo expression, or (c) transient expression of chloroamphenicol acetyltransferase. In FTO-2B hepatoma cells infected with retrovirus, vanadate rapidly (within 1 h) inhibited transcription of the PEPCK-neo gene and blocked induction of gene expression caused by the addition of either Bt2cAMP or dexamethasone to the cells. Vanadate was not a general transcription inhibitor since, it like insulin, stimulated the expression of the c-fos gene. Also, the inhibitory effect of vanadate was rapidly reversible in FTO-2B cells since PEPCK gene expression could be stimulated by Bt2cAMP and dexamethasone after removal of vanadate. A series of 5' deletions in the P-enolpyruvate carboxykinase promoter (-550 to +73) was ligated to the structural gene for neo and stably transfected into hepatoma cells. Sequences responsive to vanadate were detected between -109 and -68. This result was confirmed using H4IIE hepatoma cells transiently expressing the PEPCK-CAT gene. The most likely target for vanadate in that region of the P-enolpyruvate carboxykinase promoter is cAMP regulatory element 1 which maps from -91 to -84. A comparison of the inhibitory effects of insulin and vanadate in this system indicated a major difference in the site of action of these two compounds on PEPCK gene transcription.
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PMID:Vanadate inhibits expression of the gene for phosphoenolpyruvate carboxykinase (GTP) in rat hepatoma cells. 216 40

To examine the cellular mechanism responsible for impaired insulin action in ageing, we determined various in-vitro parameters involved in the pathogenesis of insulin resistance, i.e. basal and insulin-stimulated [14C]3-O-methylglucose transport (3OMG), 125I-labelled insulin binding, activation of insulin receptor kinase (IRKA) in intact cells, and number and subcellular distribution of glucose transporters in subcellular membrane fractions of adipocytes from 6- (FR-6) and 24- (FR-24) month-old Fischer rats. Ageing had no effect on basal 3OMG (12 +/- 4 vs 13 +/- 3 fmol/5 x 10(4) cells, means +/- S.E.M.); in contrast, in FR-24 rats insulin-stimulated 3OMG was markedly decreased by 43% when compared with that in FR-6 rats (158 +/- 14 vs 90 +/- 8 fmol/5 x 10(4) cells; P less than 0.01). Insulin binding to adipocytes from FR-6 rats was 2.40 +/- 0.38% compared with 2.28 +/- 0.47% in FR-24 (P not significant). Moreover, ageing had no significant effect on IRKA, as determined by insulin-stimulated (0, 1, 4 and 500 ng insulin/ml) 32P-incorporation into histone 2B. In subcellular membrane fractions, low density microsomes and plasma membranes, glucose transporter numbers were determined using [3H]cytochalasin B binding and immunodetection using an antiserum against the C-terminal peptide of the hepatoma-G2-glucose transporter. Cytochalasin B binding revealed that in the basal state the intracellular pool of glucose transporters was depleted in FR-24 by about 39% compared with low density microsomes from FR-6: (48.6 +/- 7.2 vs 29.8 +/- 5.5 pmol/mg membrane protein; P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Potential mechanism of insulin resistance in ageing: impaired insulin-stimulated glucose transport due to a depletion of the intracellular pool of glucose transporters in Fischer rat adipocytes. 216 28


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