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Endocrinology Vol. 138, No. 11 4821-4829
Copyright © 1997 by The Endocrine Society

ARTICLES

Vanadate, But Not Insulin, Inhibits Insulin Receptor Gene Expression in Rat Hepatoma Cells1

Sylvie Bortoli, Mohamed Amessou, Martine Collinet, Bernard Desbuquois and Soledad Lopez

Unité 30 INSERM, Hôpital Necker-Enfants Malades, 75015 Paris, France

Address all correspondence and requests for reprints to: Bernard Desbuquois, M.D., Unité 30 INSERM, Tour Lavoisier, Hôpital Necker-Enfants-Malades, 149 rue de Sèvres, 75015 Paris, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin and vanadate treatments have recently been shown to reverse the overexpression of the hepatic insulin receptor (IR) gene in streptozotocin-induced diabetic rats. To better understand the mechanisms underlying these effects, the abilities of insulin and vanadate to affect IR gene expression have been comparatively examined in Fao hepatoma cells, an insulin-responsive cell line. Exposure of Fao cells to insulin (1 µM) or vanadate (500 µM) for 24 h led to a 2-fold decrease in IR number in total cellular membranes. Insulin treatment did not affect IR messenger RNA (mRNA) level regardless of time of exposure and concentration. In contrast, vanadate treatment caused a time- and dose-dependent decrease in IR mRNA level, which was maximal (4-fold change) after a 24-h exposure to 500 µM vanadate and was fully reversible. Insulin treatment increased from 28 to 39% the relative expression of isotype A IR mRNA, but vanadate treatment did not significantly affect this parameter. Vanadate treatment did not modify mRNA half-life (3.5 h) in 5, 6 dichlorobenzimidazole riboside-treated cells but decreased by 4-fold the transcriptional activity of the IR gene. These data show for the first time that, although both insulin and vanadate decrease total cellular IR number in Fao cells, only vanadate decreases IR mRNA level. It does so by inhibiting transcription of the IR gene, suggesting an action on the gene promoter which could be mediated by changes in the level of expression and/or of phosphorylation of trans-acting factors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MULTIPLE pleiotropic effects of insulin on cellular growth and metabolism are mediated by a specific cell-surface receptor (1, 2). The insulin receptor (IR) is a transmembrane glycoprotein composed of two {alpha}-subunits and two ß-subunits linked by disulfide bonds. The {alpha}-subunit is located outside the cell membrane and contains the insulin-binding domain, whereas the ß-subunit spans the membrane and contains a cytoplasmic protein tyrosine kinase domain. Binding of insulin to the {alpha}-subunit activates the tyrosine kinase of the ß-subunit and catalyzes the autophosphorylation of specific tyrosine residues in this subunit, leading to the phosphorylation of downstream intracellular substrates involved in insulin signaling.

The {alpha}- and ß-subunits of the IR are encoded by a single gene composed of 22 exons (1, 2). The mature receptor occurs as two isoforms generated by alternative splicing of exon 11, which encodes a sequence of 12 amino acids at the carboxy terminus of the {alpha}-subunit. This sequence is present in the type B (exon 11+) isoform but is absent in the type A (exon 11-) isoform. Isoforms A and B exhibit slight differences with regard to insulin binding affinity and tyrosine kinase activity.

Although the IR is ubiquitously expressed, the level of receptors and the relative expression of receptor isotypes vary among cells and tissues (1, 2, 3). In addition, receptor expression is regulated by a variety of agents and environmental conditions including hormones, metabolic substrates, cell growth and differentiation, and development (3). Depending on the agent or condition, regulation occurs at transcriptional, posttranscriptional, and/or postranslational steps. For example, glucocorticoid hormones up-regulate the IR by stimulating transcription of the gene (4), whereas insulin down-regulates the receptor primarily by increasing the rate of degradation of the protein (5).

Previous studies have shown that the increased number of IRs in liver of streptozotocin-induced diabetic rats is reversed by treatments with insulin (6) and vanadate (7), an insulinomimetic agent. Insulin treatment has also been shown to reverse the increased level of IR messenger RNA (mRNA) level in liver of diabetic rats (8, 9). Although these effects may be mediated by the reversal of metabolic abnormalities potentially involved in IR overexpression, such as hyperglycemia, they are consistent with the ability of both insulin (3) and vanadate (10, 11) to decrease the number of cell surface IRs in isolated cells. However, studies on the effects of insulin on IR mRNA level in isolated cells have yielded conflicting results (12, 13, 14, 15, 16, 17) and, to the best of our knowledge, the effects of vanadate on this parameter have not been studied.

To better understand how insulin and vanadate regulate IR gene expression, we have analyzed the effects of these agents at different steps of expression in Fao hepatoma cells, a well differentiated insulin-responsive cell line. Our results show that, while both insulin and vanadate decrease IR number, only vanadate decreases IR mRNA level, and does so, at least in part, by reducing transcription of the IR gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and reagents
Coon’s modified Ham’s F-12 medium and FCS were purchased from Life Technologies Limited (Uxbridge, UK). Porcine insulin was from Novo-Nordisk (Bagsvaerd, Denmark). Sodium orthovanadate and 5,6-dichlorobenzimidazole riboside (DRB) were purchased from Sigma-Aldrich (St. Quentin-Fallavier, France). 125I, deoxycytidine 5'-[{alpha}-32P]-triphosphate ([{alpha}-32P]dCTP) (3000 Ci/mmol), uridine 5'-[{alpha}-32P]triphosphate ([{alpha}-32P]UTP) (3000 Ci/mmol) and the multiprime DNA labeling kit were from Amersham Life Science (Little Chalfont, UK). Human glyceraldehyde 3-phosphate dehydrogenase (G3PDH) complementary DNA (cDNA) probe was from Clontech (Palo Alto, CA). HI-Taq polymerase was from Bioprobe (Montreuil sous Bois, France). RNase inhibitor was from Boehringer/Mannheim (Meylan, France). Avian myeloblastosis virus reverse transcriptase was from Ozyme (Montigny-le-Bretonneux, France). Random hexamer primers were from Pharmacia (Upssala, Sweden). Cronex films were from Sterling Diagnostic Imaging Inc. (Newark, DE).

Cell culture
The Fao hepatoma cell line is a well differentiated subclone derived from the hepatoma H4IIEC3 line established in culture from the Reuber H35 rat hepatoma (18, 19). This cell line retains a number of characteristics of normal hepatocytes, including a high number of IRs and insulin-sensitive enzymes (20).

Fao cells were cultured attached to 10-cm Falcon plastic Petri dishes in Coon’s modified Ham’s F12 medium supplemented with 5% FCS. They were incubated at 37 C in a humidified atmosphere composed of 5% CO2 and 95% air. The culture medium was renewed three times a week. At each passage, cells were harvested with 0.05 mg/100 ml trypsin and 0.2 mg/ml EDTA. At subconfluence, the medium was changed to a serum-free Ham’s F12 medium and 24 h later cells were treated with insulin or vanadate as indicated.

Preparation of soluble cell extracts and assay of insulin binding activity
After rapid washing of the monolayers with 50 mM Tris-HCl buffer, pH 7.4, the cells were scraped off the plates in the same medium containing 10 µg/ml aprotinin and 0.2 mM phenylmethylsulfonyl fluoride, and homogenized in a 7 ml Dounce homogenizer (15 strokes of the B pestle). The homogenate was centrifuged at 150,000 x g for 60 min; the particulate material was resuspended into 5 mM Tris-HCl, pH 7.4, and solubilized by the addition of Triton X-100 (0.2%, wt/vol). Insoluble aggregates were removed by centrifugation at 15,000 x g for 10 min. The soluble extract was stored in aliquots at -80 C until used.

Insulin binding to solubilized receptors was measured using 125I-insulin (prepared using lactoperoxidase; 400 Ci/mmol) as ligand. Cell extracts (200 µg protein) were incubated with [125I]-insulin (0.05 pmol) in 0.3 ml of 50 mM Tris-HCl buffer, pH 7.4, containing 0.06% Triton X-100, 10 mg/ml BSA, 1 mg/ml bacitracin and, when indicated, different concentrations of unlabeled insulin. After 24–36 h at 4 C, free and receptor bound insulin were separated by polyethyleneglycol precipitation (21) using {gamma}-globulin as a carrier and collecting the precipitate by centrifugation (22). Results were corrected for nonspecific binding by performing parallel incubations in the presence of excess (0.5–1 nmol) unlabeled insulin.

RNA isolation and Northern blot analysis
Total cellular RNA was isolated by repeated low temperature extraction by guanidinium thiocyanate/phenol/chloroform followed by precipitation with cold isopropanol (23). After denaturation by heating at 65 C for 5 min, it was size-fractionated by electrophoresis on a 1% agarose/2.2 M formaldehyde gel. RNA was transferred by capillarity to Nylon membranes in 10 x SSC (1 x SSC = 150 mM NaCl, 15 mM sodium citrate) and fixed by baking at 80 C for 2 h. The integrity of the blotted RNA was assessed by UV visualization of the ethidium bromide-stained 28 S and 18 S ribosomal RNA bands.

cDNA probes and hybridization of RNA blots
The following cDNA probes were used for hybridization: a 2.3-kb EcoRI cDNA fragment coding for the rat IR {alpha}-subunit (24) (a kind gift from Dr. B. Goldstein, Joslin Diabetes Center, Boston, MA); a 2.6-kb EcoRI-BamHI cDNA fragment coding for rat phosphoenolpyruvate carboxykinase (PEPCK) (25); a 2.9-kb EcoRI cDNA fragment coding for the human poly A binding protein (pABP) (26); and a 1.1-kb EcoRI fragment encoding for the human glyceraldehyde 3-phosphate dehydrogenase (G3PDH). Probes were labeled (2 x 106 cpm/ng) with [{alpha}-32P] dCTP using a multiprime DNA labeling kit.

For studies on IR mRNA, blots were prehybridized for 4–5 h at 42 C in 20 ml of 42% formamide, 12.5% dextran sulfate, 3.5 mM sodium pyrophosphate, 0.8% SDS, 8 x Denhardt’s reagent, 0.35 M NaCl, 0.042 M Tris-HCl, pH 7.5, and 0.15 mg/ml of denatured salmon sperm DNA. Hybridization was performed overnight at 42 C by adding heat-denatured cDNA probes (106 cpm/ml final concentration) in the same medium, supplemented with 4 ml of 50% formamide, 1 mM sodium pyrophosphate, 1% SDS, 10 x Denhardt’s reagent, 0.05 M Tris-HCl, pH 7.5, and 0.8 mg/ml of denatured salmon sperm DNA. Blots were washed three times in 2 x SSC-0.1% SDS at room temperature and then once in 0.1 x SSC-0.1% SDS at 55 C. They were then subjected to autoradiography with intensifying screens for 18–72 h at -80 C. The intensities of the bands seen on autoradiograms were determined by scanning densitometry (Hewlett Packard, ScanJet II scanner connected to Macintosh computer). After hybridization with the IR probe, blots were rehybridized with the pABP probe and, for each treatment, values of IR signals were normalized to values of pABP signals. Hybridizations of the RNA blots to G3PDH and PEPCK probes were carried out basically as described for the IR.

RT and amplification of IR cDNA isoforms
RT was performed on 1 µg of total RNA with 1 U/µl of RNase inhibitor and 90 mU/ml of random hexamer primers in a volume of 10 µl. The annealing reaction was carried out for 5 min at 65 C, after which the incubation mixture was chilled on ice for 2 min. The first strand cDNA was synthesized by adding 1 U/µl of avian myeloblastosis virus reverse transcriptase in 1 x RT buffer(50 mM Tris-HCl, pH 8.3, 60 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol) supplemented with 0.5 mM deoxynucleotide triphosphates in a final volume of 20 µl. Incubations in the absence of reverse transcriptase were used as negative controls. After 2 h at 42 C, DNA/RNA hybrids were denatured at 95 C for 2 min.

Oligonucleotide primers spanning nucleotides 2530–2550 in exon 10 (sense primer, 5'-CATTCAGGAAGACCTTCGAGG-3') and nucleotides 2822–2843 in exon 12 (antisens primer, 5'-TGCAATCAGGACTCCCCAGA-3') were used to amplify a region of the rat IR cDNA. These primers generate a 313- or a 277-bp fragment after amplification of the cDNA encoding the B isoform or A isoform, respectively. Two microliters of the RT reaction were mixed with 1 x Taq buffer (10 mM Tris-HCl pH 8.8, 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100) supplemented with 1 µM sense primer, 1 µM antisense primer, 0.1 mM each 2'-deoxyadenosine 5'-triphosphate, 2'-deoxyguanosine 5'-triphosphate and 3'-deoxythymidine 5'-triphosphate and 0.06 µCi of [{alpha}-32P]-dCTP in a final volume of 50 µl. The reaction mixture were overlaid with mineral oil, heated for 5 min at 95 C in a thermal cycler (Perkin Elmer Instruments) and kept at 72 C. After addition of 0.2 U/µl HI-Taq DNA polymerase, twenty-five cycles of amplification were performed. Each cycle consisted of a 60-sec denaturation at 94 C, a 60-sec annealing at 57 C, and an 80-sec extension at 72 C. The two products of amplification were resolved by electrophoresis on a 8% polyacrylamide gel and visualized by autoradiography. They were quantitated by direct measurement of the radioactivity on the dried gel using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Measurement of IR mRNA half-life
The half-life of IR mRNA was estimated from sequential determinations of IR mRNA level after blocking gene transcription with 100 µM DRB (27). This agent has previously been shown not to affect Fao cell viability (28). RNA was quantitated as described above 0, 0.5, 1, 2, 4, and 8 h after DRB addition, and results were plotted semilogarithmically against time. First order decay rate constants were derived from these plots and used to calculate half-life values.

Isolation of nuclei and run-on transcription assay
Nuclei were prepared from 65 x 106 cells (five plates) as described previously (28). After scraping in phosphate-bufferred saline, cells were resuspended in 2 ml of a lysis solution consisting of 10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, and 0.5% Nonidet P40. Nuclei were sedimented by centrifugation at 450 x g for 5 min, and after resuspension in the lysis solution, sedimented again. The final sediment was suspended in 50 mM Tris-HCl, pH 8, 40% (vol/vol) glycerol, 5 mM MgCl2 and 0,1 mM EDTA, and stored at -80 C until used.

Transcription assays were performed by incubating 10–20 x 106 nuclei in 200 µl (final volume) of 10 mM Tris-HCl buffer, pH 7.9, 50 mM NaCl, 0.4 mM EDTA, 1.2 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 350 mM ammonium sulfate, 4 mM MnCl2, 10 mM phosphocreatine, 10 mg/ml creatine phosphokinase, 2 mM each CTP, ATP and GTP, 500 U/ml RNAsine, 1 mCi/ml [{alpha}-32P] UTP for 30 min at 30 C. Incubations were then continued at 37 C in the presence of first 10 µg/ml RNase-free DNase (15 min), then 40 µg/ml proteinase K (15 min), and finally 100 µg/ml proteinase K and 1% SDS (30 min). The radiolabeled RNA was isolated by sequential guanidinium chloride extraction and sodium acetate/ethanol precipitation. Labeled RNA (15 x 106 cpm/blot) was hybridized with 20 µg of various cDNA probes (IR, PEPCK, pUC) and genomic DNA that had been previously immobilized on a Nylon membrane using a slot blot apparatus. Hybridization was carried out in 50% formamide, 4.7% SDS, 0.2 mg/ml denatured salmon sperm DNA, 0.067 mg/ml tRNA carrier, 1 x Denhardt’s reagent, 3 x SSEP (1 x SSEP = 150 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA) for 65–70 h at 42 C. Blots were washed consecutively in: 1) 1 x SSC, 0.1% SDS, 3 x 20 min at 65 C; 2) 1 x SSC,2 x 15 min at 65 C; 3) 1 x SSC, 10 mg/ml RNase A, 30 min at 37 C; and 4) 1 x SSC, 0.1% SDS, 15 min at 65 C. Blots were then subjected to autoradiography and hybridization signals were quantitated by densitometric scanning. The relative transcription rates of the IR and PEPCK genes were referred to as the ratio between the intensity of the corresponding signals and the intensity of the genomic DNA signal after subtraction of the intensity of the pUC signal.

Statistical methods
Statistical analyses were carried out using the nonparametric Kruskal-Wallis test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of insulin and vanadate treatments on insulin binding activity and insulin receptor mRNA level in Fao hepatoma cells
Exposure of Fao cells to 1 µM insulin and 500 µM vanadate for 24 h led to a 40% decrease in the ability of insulin to bind, at tracer concentrations, to solubilized cell extracts (Fig. 1Go). Competition studies with unlabeled insulin showed that the decrease in insulin binding in treated cells was unaffected by insulin concentration, and that the concentrations required to inhibit binding of 125I-insulin by 50% were similar (0.8–0.9 nM) in insulin-treated, vanadate treated and control cells (results not shown). These results indicate that the main effects of insulin and vanadate were to decrease IR number. Scatchard analysis of the binding data using the two-site model showed that insulin and vanadate treatments decreased by at least 2-fold the number of both high affinity/low capacity and low affinity/high capacity sites (Table 1Go). In addition, vanadate and to a lesser extent insulin moderately increased the affinity of the two classes of binding sites.



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  		Figure 1. Effect of insulin and vanadate treatments on insulin binding activity in Fao cells. Cells were treated with 1 µM insulin (I) or 500 µM vanadate (V) or left untreated (C) for 24 h. A total particulate fraction was prepared, solubilized with Triton X-100, and examined for insulin binding activity, as described in Materials and Methods. Incubation mixtures contained, in a final volume of 0.3 ml, 200 µg protein and 0.15 nM [125I]-insulin. Results are expressed as mean ± SEM of four to five independent determinations; **, P < 0.05 vs. control cells.

 

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  		Table 1. Scatchard analysis of insulin binding to control, insulin-treated, and vanadate-treated cells

 
Total RNA prepared from control cells and from cells treated by insulin and vanadate as indicated above was analyzed by Northern blotting using a cDNA receptor probe. As previously observed in Fao hepatoma cells and normal rodent liver (29), two major mRNA species of 9.5 and 7.5 kb were found to hybridize to IR cDNA, the 7.5-kb species being the most abundant (Fig. 2Go). The abundance of these two species was decreased about 4-fold in vanadate-treated cells but was unchanged in insulin-treated cells. Vanadate treatment did not affect the abundance of the pABP and G3PDH transcripts, suggesting that it does not act as a general inhibitor of gene transcription.



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  		Figure 2. Northern blot analysis of IR mRNA in control, insulin-treated and vanadate-treated Fao cells. Cells were treated with 1 µM insulin (I) or 500 µM vanadate (V) or left untreated (C) for 24 h. Total RNA (20 µg per lane) was subjected to Northern blotting and hybridized with the indicated cDNA probes as described in Materials and Methods. Results are representative of several independent experiments.

 
Blots from untreated and treated cells were also probed with a cDNA for PEPCK, an insulin-sensitive enzyme that is highly expressed in hepatoma cells. In agreement with previous studies (30, 31), both insulin and vanadate led to a comparable decrease in the relative abundance of the PEPCK transcript. Thus, the lack of effect of insulin on the expression of the IR gene in Fao cells did not result from a general loss of insulin effects on gene expression.

Time and dose dependence of insulin and vanadate effects on IR mRNA level
Insulin failed to affect IR mRNA level regardless of dose and time of treatment (Fig. 3Go, A and B). Because glucose has been reported to increase IR mRNA level in several types of cells (32), a potential inhibitory effect of insulin could have been missed in cells cultured in the presence of glucose. However, when cells were preincubated in a glucose-deprived medium for 24 h and subsequently treated by insulin, no effects of insulin on receptor mRNA were observed whether glucose was present or not during insulin treatment (Fig. 3BGo). In addition, basal IR mRNA in cells incubated in a glucose-deprived medium were not affected by glucose at 5, 12.5, and 25 mM (Fig. 3CGo).



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  		Figure 3. Time course and dose dependence of insulin and glucose effects on IR mRNA level in Fao cells. A, Cells were treated with 1 µM insulin for the indicated times; B, cells were incubated in a glucose-free medium for 24 h then treated by insulin at the indicated concentrations in a medium containing (bottom) or not (top) 25 mM glucose for another 24 h; C, cells were incubated in a glucose-free medium for 24 h then in a medium containing glucose at the indicated concentration for another 24 h. In each of these conditions, IR mRNA was analyzed by Northern blot and quantitated by scanning densitometry of autoradiograms as described in Materials and Methods. Results, normalized for pABP mRNA content, are the mean ± SEM of three independent determinations, except in B, in which three separate RNA samples were pooled before analysis.

 
The ability of vanadate to decrease IR mRNA level in Fao hepatoma cells was time and dose dependent (Fig. 4Go). In time studies, there was a 2- to 4-h lag before any decrease was observed, and a significant effect was seen at 4 h on the 9.5-kb transcript and at 8 h on the 7.5-kb transcript. The lack of an apparent effect of vanadate on the 7.5-kb transcript at 4 h could be due to the higher expression of this transcript relative to the 9.5-kb transcript and to the nonlinearity of the autoradiographic signal at the exposition time used. The decrease in IR mRNA progressed gradually, with a lowest level (9.5 kb and 7.5 kb transcripts, 10% and 25% of control, respectively) at 24 h. Dose-response studies showed that only at concentrations above 100 µM did vanadate affect IR mRNA level, with a decrease to about 45–50% and 70–75% of control values at 250 µM and 500 µM, respectively. When cells treated by 500 µM vanadate for 24 h were washed free of vanadate, IR mRNA level was fully restored in 24 h (Fig. 5Go).



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  		Figure 4. Time course and dose dependence of vanadate effect on IR mRNA level in Fao cells. Cells were treated with 500 µM vanadate for the indicated times (A) or with vanadate at the indicated concentration for 24 h (B). IR mRNA was analyzed by Northern blot and quantitated by scanning densitometry of autoradiograms as described in Materials and Methods. Results, normalized for pABP mRNA content, are the mean ± SEM of three independent determinations. *, P < 0.05 vs. control cells; **, P < 0.01 vs. control cells; ***, P < 0.0001 vs. control cells.

 


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  		Figure 5. Reversibility of vanadate effect on IR mRNA. Fao cells were incubated with 500 µM vanadate or left untreated for 24 h. Vanadate-treated and control cells were then incubated in a vanadate-free medium for another 24 h (VR and CR, respectively). IR mRNA was analyzed by Northern blot and quantitated by scanning densitometry of autoradiograms as described in Materials and Methods. Results, normalized for pABP mRNA content, are the mean ± SEM of three to four independent determinations. **, P < 0.01 vs. control cells.

 
Changes in IR isotype expression in insulin- and vanadate-treated cells
The relative expression of the mRNAs encoding isotypes A (exon 11-) and B (exon 11+) of the IR has been shown to be tissue-specific and to be influenced by hormonal and environmental factors. To determine possible effects on insulin and vanadate on receptor isotype expression in Fao cells, total RNA from untreated and treated cells was extracted and subjected to RT and radioactive PCR amplification. The 317- and 277-bp radiolabeled products corresponding to the B and A isotype, respectively, were separated by electrophoresis, visualized by autoradiography, and quantified using a PhosphorImager (Fig. 6Go). Under basal conditions, the B mRNA isotype was predominant, with the A mRNA isotype accounting for only 28% of total products. Insulin treatment led to a moderate, albeit significant increase in the relative expression of the A mRNA isotype (39% of total products), but vanadate treatment did not elicit any significant effect.



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  		Figure 6. Effect of insulin and vanadate on the relative abundance of the isotype A and the isotype B IR mRNA. Total RNA was prepared from Fao cells that had been treated with 1 µM insulin (I) or 500 µM vanadate (V) for 24 h or left untreated (C). Partial sequences of IR cDNA including or not exon 11 were generated by RT-PCR amplification using specific oligonucleotide primers as described in Materials and Methods. The products of amplification corresponding to the A and B receptor isotypes were resolved by electrophoresis and quantified using a PhosphorImager. The results, expressed as the relative expression of the A isotype (ratio A/A + B x 100), are the mean ± SEM of three to five independent determinations; *, P < 0.05 vs. control cells.

 
Effect of vanadate on IR mRNA stability
To determine if vanadate decreased the stability of IR mRNA, the half-life of this mRNA was measured. To achieve this, cells were first preincubated in the presence or absence of 500 µM vanadate for 4 h. Ongoing transcription was then inhibited by the addition of 100 µM DRB, and at subsequent time points the 9.5 and 7.5 kb IR mRNA species were quantitated (Fig. 7Go). IR mRNA level decayed at similar rates whether cells had been treated or not with vanadate and regardless of the mRNA species considered (t1/2, 3.3–3.5 h). These results suggest that the decrease in steady-state IR mRNA level in vanadate-treated cells is not due to a decrease in mRNA stability.



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  		Figure 7. Effect of vanadate on the half-life of IR mRNA. Fao cells were treated with 500 µM vanadate or left untreated for 4 h. DRB (100 µM) was then added and at the indicated times after DRB addition total RNA was isolated. IR mRNA was analyzed by Northern blot and quantitated by scanning densitometry of autoradiograms as described in Materials and Methods. The first-order decay rate constants were derived and used to calculate half-life values. Results are representative of three independent determinations.

 
Effect of vanadate on IR gene transcriptional activity
The results described above suggest that the effect of vanadate on IR mRNA level occurred at the transcriptional level. Accordingly, run-on transcription experiments were performed with nuclei isolated from control and vanadate-treated cells. On autoradiography, a decrease in the intensity of the IR signal was clearly detectable with nuclei from vanadate-treated cells (Fig. 8Go). Densitometric analysis indicated a 4-fold decrease in relative transcriptional activity.



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  		Figure 8. Effect of vanadate on IR gene transcription in isolated nuclei. A, Nuclei were isolated from Fao cells left untreated (C) or treated with 500 µM vanadate (V) for 24 h and used for in vitro transcription as described in Materials and Methods. After incubation of nuclei with [{alpha}-32P] UTP, 32P-labeled RNA was isolated and hybridized (15 x106 cpm/blot) to filter-immobilized DNA complementary to IR, pUC, and PEPCK, and genomic DNA (20 µg each). Filters were washed and subjected to autoradiography. B, The signals seen in A were quantitated by scanning densitometry. The intensities of the IR and PEPCK signals were normalized to the genomic signal after subtraction of the intensity of the PUC signal. The results are expressed as the mean ± half the range of two independent determinations.

 
For comparison, the effect of vanadate on the transcriptional activity of the gene encoding PEPCK was also studied. Vanadate treatment also resulted in a marked decrease in the hybridization signal obtained with PEPCK cDNA (about 5-fold). A comparable degree of inhibition of PEPCK gene transcription in hepatoma cells by vanadate has been previously reported (31).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Vanadate, an inhibitor of phosphotyrosine phosphatases, has previously been shown to mimic a number of insulin’s biological effects, including down-regulation of the IR protein (33). However, the mechanisms underlying the effect of insulin and vanadate on the receptor protein appear to differ, at least in certain types of cells (11). In addition, despite the fact that insulin and vanadate regulate in a similar manner the expression of several genes, it is unknown whether, and how, vanadate affects the expression of the IR gene. In the present study, we show that insulin and vanadate cause a comparable decrease in the number of total cellular IRs in Fao hepatoma cells. However, only vanadate decreased the steady-state level of IR mRNA. It inhibited the transcriptional activity of the IR gene but did not affect the half-life of receptor mRNA.

Effects of insulin and vanadate on insulin binding activity
As with a number of cells (3), insulin treatment of Fao hepatoma cells caused a decrease in the number of IRs, which affected to the same extent high affinity/low capacity and low affinity/high capacity sites. A comparable decrease in IR number as judged on insulin binding activity (34), cell surface 125I-labeling of the {alpha}-subunit (35) and Western immunoblot of the ß-subunit (36) has previously been observed in insulin-treated Fao cells by Kahn and his colleagues. However, an additional effect of insulin described by these authors, attributed to a loss of the oligomeric forms of the receptor, was to increase receptor affinity (34, 35). The lesser effect of insulin treatment on receptor affinity in our study could be related to the fact that we measured insulin binding activity in Triton X-100 solubilized membranes and not in intact cells as Crettaz and Kahn (34) did. Conceivably, the relative proportions of the oligomeric forms of the receptor and their corresponding affinities for insulin may differ in solubilized extracts and in the intact membrane.

Exposure of Fao hepatoma cells to 0.5 mM vanadate for 24 h led to a decrease in total cellular receptor number comparable to that observed with insulin. In two previous studies, vanadate has been shown to decrease cell surface insulin binding activity in cultured rat adipocytes (10) and human IM9 lymphocytes (11). However, the effect of vanadate in Fao cells differed from that observed in IM9 lymphocytes, in which total cellular receptor number was increased as a result of an inhibition of receptor degradation. These findings suggest that the main effect of vanadate in Fao cells is probably to inhibit receptor synthesis, although a positive effect on receptor stability similar to that observed in IM9 lymphocytes cannot be excluded. The effect of vanadate on insulin binding activity described here also differed from that observed after a brief exposure of freshly isolated adipocytes to vanadate. Under these conditions, vanadate induced an increase in cell surface insulin binding by increasing receptor affinity (37) or number (38). The latter effect, also elicited by a brief exposure to insulin, has been attributed to a recruitment of receptors within the plasma membrane (38).

Effects of insulin and vanadate on IR mRNA level
Previous studies have given conflicting results with regard to the effect of insulin on IR mRNA level. In vivo, an increase in hepatic receptor mRNA level has been reported in hypoinsulinemias induced by streptozotocin treatment (8, 9) and fasting (8), as well as in hyperinsulinemias associated with genetic obesity (39) or induced by insulin infusion (40). In vitro, insulin treatment decreased IR mRNA level in HepG2 hepatoma (12, 13) and AR42J pancreatic acinar cells (14) but was without effect in IM9 lymphocytes (15, 16) and HepG2 cells at low concentration (17). In the present study, insulin did not affect IR mRNA level in Fao cells regardless of concentration and time of treatment. Insulin did also not affect receptor mRNA level in the mouse AT3F cell line and in primary cultures of rat hepatocytes (S. Bortoli, unpublished observations). These findings suggest that, in Fao cells, insulin decreases the expression of the receptor by acting postranslationally, presumably by increasing the rate of degradation of the receptor protein as it does in most cell types (3). They also suggest that other factors than insulin are implicated in the regulation of IR mRNA level in vivo.

Because glucose has been shown to increase IR mRNA level in 3T3 fibroblasts overexpressing the human IR (32), a potential inhibitory effect of insulin may have been masked by the presence of glucose in the medium. However, insulin failed to affect IR mRNA whether glucose was present or not, and addition of glucose to glucose-deprived cells was also ineffective. This could be related to the fact that Fao cells exhibit a high rate of neoglucogenesis and do not require glucose for growth.

Unlike insulin treatment, vanadate treatment of Fao hepatoma cells caused a marked decrease in IR mRNA. This effect coordinately affected the two receptor transcripts, occurred after a lag phase of 4 h, achieved a maximum by 24 h, and was fully reversible. The concentrations at which IR mRNA level was decreased (>200 µM) were closely similar to those required for previously described effects of vanadate in isolated cells (33). Vanadate treatment was found to also decrease IR mRNA level in AT3F cells and cultured hepatocytes (S. Bortoli, unpublished observations).

We have recently found that in vivo vanadate treatment normalizes hepatic IR mRNA level in streptozotocin-induced diabetic rats (Amessou et al., submitted for publication). The present results are consistent with these findings and suggest that, although vanadate may act in vivo in part by reversing metabolic abnormalities involved in receptor mRNA overexpression, such as hyperglycemia (40), it can also inhibit receptor gene expression via direct effects on insulin target cells. A number of other genes the expression of which is regulated by vanadate in isolated cells have been identified. Some of them, such as L-type pyruvate kinase (41), c-fos (31, 42), and gene 33 (43) are stimulated, whereas others, such as PEPCK and tyrosine aminotransferase (31), are inhibited. However, unlike the IR gene, most of these genes are similarly affected by insulin and vanadate.

Expression of IR mRNA isoforms
The IR occurs as two alternatively spliced isoforms, A (exon 11-) and B (exon 11+), which exhibit functional differences and are expressed in a tissue-specific manner (1, 2). In the present study, Fao hepatoma cells were found to express 70% isoform B, a level of expression close to that of normal liver cells (90%). At variance with these findings, the relative expression of isoform B in previous studies on Fao (44) and HepG2 (45, 46) hepatoma cells was only 30%. However, it has been shown that the phenotype of HepG2 hepatoma cells depends on cell density, and that glucocorticoid hormones, probably by eliciting a more adult phenotype, increase the expression of isoform B (45). In our study, cells were cultured in the absence of glucocorticoid hormones but used at subconfluency, a condition which may have favored the expression of isoform B.

Previous studies have shown that insulin treatment decreases the high level of receptor isotype A in Fao hepatoma cells (44) but does not affect isotype expression in HepG2 cells (46). In the present study, insulin treatment caused a moderate but significant increase in the relative expression of the A isoform. The latter result is consistent with the finding that the relative expression of isotype A is increased in skeletal muscle (47) and liver (48) of spontaneously obese diabetic rhesus monkeys, ant that, at least in muscle (47), the level of this isotype is significantly correlated with plasma insulin concentration. A positive correlation between the expression of the A isoform in skeletal muscle and plasma insulin has also been observed (48) in a cohort of obese and non insulin-dependent diabetic human subjects initially reported (49) as showing no change in isoform expression.

Site of action of vanadate on IR gene expression
In the present study, vanadate treatment of Fao hepatoma cells decreased the transcriptional activity of the IR gene but did not affect the half-life of IR mRNA, suggesting a transcriptional site of vanadate action. Previously, vanadate treatment has also been shown to inhibit the transcription of the PEPCK gene in FTO 2B hepatoma cells (31) and a vanadate-responsive sequence, distinct from the insulin-responsive sequence, has been identified in the PEPCK gene promoter (31). Because this sequence contains a cAMP-regulatory element (CRE), it has been proposed that vanadate may alter transcription of the PEPCK gene by modifying nuclear proteins such as CREB and C/EBP (30). Interestingly, C/EBP{alpha} and C/EBPß have been shown to bind and transactivate the human IR promoter (50), suggesting that these factors may also be involved in the transcriptional effect of vanadate on the IR. Owing to its ability to inhibit phosphotyrosine phosphatases, vanadate could act on the transcription factors by altering their level of tyrosine phosphorylation. In addition, the ability of orally administered vanadate to reverse the altered expression of C/EBPß (51) and hepatocyte nuclear factor 1 (52) in diabetic rats suggest that vanadate may also affect the cellular level of these trans-acting factors.

In summary, we have shown that, although both insulin and vanadate down-regulate the IR protein in Fao hepatoma cells, only vanadate decreases IR mRNA level, by inhibiting IR gene transcription. Further studies are required to identify the cis- and trans-acting factors involved in the transcriptional effect of vanadate, and to determine whether this agent affects the level of expression and/or phosphorylation of the trans-acting factors.


   Acknowledgments
 
We gratefully thank Dr. Robert Barouki and Dr. Claude Forest for helpful discussions and their critical review of this manuscript. We also thank Dr. Bénédicte Antoine (INSERM U129, Paris), Dr. Bernard Lardeux and Anne-Marie Durand-Schneider (INSERM U 327, Paris) who provided AT3F cells and hepatocytes in primary culture, respectively. We appreciate the excellent advice of Jean-François Pageaux (INSERM U352, Lyon) for Scatchard analyses.


   Footnotes
 
1 This study was supported in part by a grant of the Ministère de l’Enseignement Supérieur et de la Recherche No. 93162. Back

Received May 20, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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