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

Abstract

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 microM) or vanadate (500 microM) 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 microM 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.

Treatment of streptozotocin-induced diabetic rats with vanadate and phlorizin prevents the over-expression of the liver insulin receptor gene

“…4). In agreement with a recent report (28), treatment of these cells with 0.5 mM vanadate for 4 h led to a marked decrease in the relative abundance of the 9.5 and 7.5 kb insulin receptor mRNA species compared with 18S rRNA. In contrast, treatment of Fao cells with 5 mM phlorizin for 4 h did not affect the level of the 7.5 kb transcript and caused a weak, albeit significant, increase in the level of the 9.5 kb transcript.…”

“…This was achieved by reverse transcription of RNA followed by radioactive PCR amplification of insulin receptor cDNA as described previously (28). As primers, oligonucleotides (Genset, Paris, France) spanning sequences 2530-2550 (sense primer) and 2822-2843 (antisense primer) were used; these primers generate 313 and 277 bp products encoding the B and A isoforms of the insulin receptor respectively.…”

“…Fao rat hepatoma cells (30) were cultured in Coon's modified Ham's F12 medium supplemented with 5% calf serum as recently described (28). At subconfluence, the medium was changed to a serum-free Ham's medium and 24 h later cells were treated with vanadate or phlorizin as indicated in legend to Fig.…”

“…4. Insulin receptor mRNA was analyzed by Northern blotting of total cellular RNA as described above for rat liver except that a 32 P-labeled insulin receptor cDNA probe was used for hybridization (28) and that insulin receptor signals were normalized to 18S rRNA signals as visualized by UV light after ethidium bromide staining. A and B insulin receptor mRNA isotypes were analyzed as described above.…”

“…Although evidence has been presented that vanadate treatment of diabetic rats normalizes insulin receptor number (22,23), it is unknown whether such treatment also normalizes insulin receptor mRNA level. It is also unclear whether vanadate acts directly on insulin receptor gene expression, as is suggested by its ability to affect receptor number (24)(25)(26)(27)(28) and to decrease receptor mRNA level (28) in isolated cell systems. To address these questions, the in vivo effects of vanadate and phlorizin on liver insulin receptor number, insulin receptor mRNA level and insulin receptor mRNA isotype expression in streptozotocin-induced diabetic rats have been comparatively examined.…”