Insulin inhibits transcription of IRS-2 gene in rat liver through an insulin response element (IRE) that resembles IREs of other insulin-repressed genes

Abstract

Recent data indicate that sustained elevations in plasma insulin suppress the mRNA for IRS-2, a component of the insulin signaling pathway in liver, and that this deficiency contributes to hepatic insulin resistance and inappropriate gluconeogenesis. Here, we use nuclear run-on assays to show that insulin inhibits transcription of the IRS-2 gene in the livers of intact rats. Insulin also inhibited transcription of a reporter gene driven by the human IRS-2 promoter that was transfected into freshly isolated rat hepatocytes. The human promoter contains a heptanucleotide sequence, TGTTTTG, that is identical to the insulin response element (IRE) identified previously in the promoters of insulin-repressed genes. Single base pair substitutions in this IRE decreased transcription of the IRS-2-driven reporter in the absence of insulin and abolished insulin-mediated repression. We conclude that insulin represses transcription of the IRS-2 gene by blocking the action of a positive factor that binds to the IRE. Sustained repression of IRS-2, as occurs in chronic hyperinsulinemia, contributes to hepatic insulin resistance and accelerates the development of the diabetic state.

Figure 1

Insulin-mediated inhibition of IRS-2 gene transcription in rat liver. Nuclear run-on assays of nascent ³²P-labeled RNA transcripts were performed with nuclei isolated from the livers of streptozotocin-treated rats that were injected s.c. with or without insulin as indicated. Male Sprague–Dawley rats were treated with streptozotocin for 36 h and then injected with insulin or vehicle for 6 h, as described (19). For each experimental group, liver tissue from three rats was pooled, homogenized, and subjected to nuclear run-on assay, as described in Materials and Methods. After hybridization with the indicated cDNA probe, the filters for all probes were exposed to Kodak X-Omat film with Bio-Max (Kodak) intensifying screens at −80°C for 48 h, except for the PEPCK probe, which was exposed for 8 h. Relative levels of the transcripts for each gene were quantified with a Fuji Bio-Imaging analyzer. A blank value corresponding to the vector band was subtracted from all values.

Figure 2

Putative IRE sequence in promoter region of mouse and human IRS-2 genes as compared with rat PEPCK gene. Nucleotide numbering at the right refers to the translation start site, where +1 is the A of the ATG. Arrows below the human IRS-2 sequence denote the three base pair changes contained in the pIRS2-Luciferase(mut-3bp) reporter gene. Mouse IRS-2 sequence is from Sun et al. (23). Human IRS-2 sequence is from Vaβen et al. (17). Rat PEPCK sequence is from Beale et al. (32).

Figure 3

Insulin-mediated inhibition of IRS-2 promoter activity in transfected rat hepatocytes incubated in the absence (A) and presence (B) of dexamethasone. The wild-type construct, pIRS2-Luciferase(wt), contains a ≈1-kb fragment corresponding to base pairs −1051 to −116 of the human IRS-2 promoter linked to a luciferase reporter gene. The mutant pIRS2-Luciferase construct, pIRS2-Luciferase(mut-3bp), contains the same human IRS-2 promoter fragment except that the IRE sequence (base pairs −574 to −568) was mutated from 5′-TGTTTTG-3′ to 5′-AGATCTG-3′. Primary rat hepatocytes were isolated and plated as described in Materials and Methods. Hepatocytes were cotransfected with 0.1 μg per dish of the reference reporter plasmid pCMV-SEAP and 3 μg per dish of either pIRS2-Luciferase(wt) (▴) or pIRS2-Luciferase(mut-3bp) (●) as indicated. After transfection, the cells were switched from medium A to serum-free RPMI medium 1640 and incubated for 8 h at 37°C, after which the medium was supplemented with the indicated concentration of insulin in the absence (A) or presence (B) of 5 μM dexamethasone. After an additional incubation for 16 h, the cells and medium were harvested for measurement of luciferase and alkaline phosphatase activities, respectively. Alkaline phosphatase activity was used as the internal reference to normalize for transfection efficiency. The value for wild-type pIRS2-luciferase activity in the absence of insulin was arbitrarily set as 1. Each value represents the average of duplicate incubations. This experiment was repeated three times with similar results.

Figure 4

Time course of insulin-mediated inhibition of IRS-2 promoter activity in transfected rat hepatocytes. Primary rat hepatocytes were isolated and plated as described in Materials and Methods. Hepatocytes were cotransfected with pCMV-SEAP plasmids and either pIRS2-Luciferase(wt) (▴) or pIRS2-Luciferase(mut-3bp) (●) as described in the legend to Fig. 3. Six hours after transfection (zero time for the experiment), the serum-free RPMI medium 1640 in all dishes was supplemented with 5 μM dexamethasone, and the dishes were incubated an additional 24 h before harvest. Insulin (100 nM) was added to duplicate dishes at the indicated time before harvest. Luciferase and alkaline phosphatase activities were measured as described in the legend to Fig. 3. The value for wild-type pIRS2-Luciferase activity in the absence of insulin was arbitrarily set as 1. Each value represents the average of duplicate incubations. This experiment was repeated three times with similar results.

Figure 5

Single point mutation analysis of the IRE sequence in human IRS-2 promoter (A and B) and in rat PEPCK promoter (C). (A and B) Primary rat hepatocytes were isolated and plated as described in Materials and Methods. Cells were cotransfected with 0.1 μg per dish pCMV-SEAP and 3 μg per dish of either pIRS2-Luciferase(wt) or the indicated mutant version in which the indicated single base pair substitution was made in the IRE sequence. After incubation for 8 h at 37°C, the serum-free RPMI medium 1640 was supplemented with 5 μM dexamethasone in the absence (open bar) or presence (closed bar) of 100 nM insulin. After an additional 16-h incubation, the cells and medium were harvested for measurement of luciferase and alkaline phosphatase activities, respectively. (A) IRS-2-mediated luciferase activity in the absence and presence of insulin for each mutant construct is plotted above the corresponding mutated base pair. The bases in the heptanucleotide IRE are numbered 1–7. The value for the wild-type construct was arbitrarily set at 1. (B) The percent inhibition of IRS-2-mediated luciferase activity in mutant constructs by insulin is plotted below the corresponding mutated base pair. Results in A and B are plotted as the mean ± SEM of four independent experiments, each of which was done in duplicate. (C) The percent inhibition of PEPCK-mediated chloramphenicol acetyltransferase (CAT) activity in mutant constructs by insulin is plotted below the corresponding mutated base pair. These data are replotted from Hall et al. (9).