Identification of a novel rat hepatic gene induced early by insulin, independently of glucose

Abstract

We used mRNA differential display to identify new genes induced early after exposure to insulin. Our screening strategy was based on the comparison of gene expression during the time course of insulin induction in the liver of 12-day-old suckling rats both in vivo and in vitro. A novel, early induced transcript, EIIH, was identified that encodes a 353-amino-acid protein with several features suggesting that it may be secreted or bound to membranes. EIIH is also distantly related to a variety of LRR (leucine-rich repeat) proteins. Insulin treatment increased EIIH mRNA levels in the hepatocytes of suckling, fasted adult and STZ (streptozotocin)-treated diabetic rats, where insulin was required to maintain the basal level of EIIH expression. EIIH expression was induced during the suckling/weaning transition, and remained detectable thereafter. Tissue distribution analysis in adult rats revealed a pattern of expression mainly in the liver, intestine and islets of Langerhans, closely following that of the Glut2 (glucose transporter 2), suggesting that it may play a role in carbohydrate metabolism. EIIH may be a primary target of the transcriptional regulation by insulin, and may therefore constitute a new model to study the mechanisms by which insulin acts on gene transcription.

Figure 1. Identification of EIIH by DD/RT-PCR and analysis of its induction pattern by insulin

(A) RNA blot analysis of GK mRNA level of 12-day-old rat hepatocytes in primary cultures stimulated by 100 nM insulin (left-hand panel) and in the liver of pups of the same age after glucose force-feeding (5 g/kg of body mass; right-hand panel) for the indicated times. Total RNA (10 μg) was hybridized with a 1.8 kb GK probe and autoradiographed for 20 h. Lower panels show ethidium-bromide-stained 18 S RNAs used as a control for RNA loading. (B) DD/RT-PCR cDNA pattern obtained with primers P6 and T11. The arrow points to EIIH cDNA. (C) RNA blot analysis of the induction pattern of EIIH mRNA by insulin. Hybridization of the same membrane as in (A) with the 600 bp EIIH probe. The duration of insulin treatment is indicated above each panel.

Figure 2. Time-course analysis of GK and EIIH mRNAs accumulation by RNA blot analysis

Cells were cultured in the absence of glucose and insulin for 16 h. Insulin (100 nM) was added, and total RNA was prepared at the times indicated above the panels. (A) 12-day-old suckling and (B) fasted adult rats after insulin treatment. The bottom panels of (A) and (B) show ethidium-bromide-stained 18 S RNA. It should be noted that the increase of EIIH mRNA at 24 h was not observed in other experiments. Blots are representative of three independent experiments.

Figure 3. EIIH mRNA expression in liver during development

Total RNA was extracted from the liver of rats at the days indicated above the blot, and was hybridized with EIIH and GK probes. Birth and weaning are indicated by vertical arrows. The rats were weaned at day 20 on to a high-carbohydrate, low-fat diet. Bottom panels, 18 S RNA stained with ethidium bromide. The blot is representative of two independent experiments.

Figure 4. Effect of insulin on EIIH mRNA levels in the liver of diabetic STZ-treated rats

Diabetes was induced by injection of STZ 5 days before the experiment. Intraperitoneal injection of insulin was performed 30 min before rats were killed. The bars represent the relative EIIH (A) and GK (B) mRNA levels as determined by real-time RT-PCR after normalization with cyclophillin expression levels. Results are means±S.E.M. of four (STZ rats treated by insulin) and six (controls and STZ rats) animals. Statistical significance was calculated using ANOVA between STZ-treated and control rats (*P<0.05), STZ-treated and STZ-treated insulin-injected rats (**P<0.05), and control and STZ-treated insulin-injected rats (#P<0.05) for EIIH mRNA. The real-time RT-PCR measurement was repeated twice with similar results.

Figure 5. Tissue distribution analysis of EIIH and Glut2 mRNAs by RT-PCR

RT-PCR products (10 μl) were analysed by electrophoresis on a 1.5% (w/v) agarose gel. (A) Whole-organ tissue distribution. Li, liver; Pa, pancreas; AT, adipose tissue; SM, skeletal muscle; K, kidney; S, spleen; D, duodenum; I, ileum; B, brain; Pl, placenta; Lu, lung; Te, testis; -, no cDNA. (B) Expression in whole pancreas (Pa) and isolated pancreatic islets of Langerhans (IsL). (C) Expression in whole brain (B), dissected hypothalamus (H) and pituitary gland (Pi). The sizes of the RT-PCR products were 2489 bp for EIIH, 717 bp for Glut2, 738 bp for GK and 568 bp for cyclophilin. Cyclophilin was used as an indication of cDNA input in the reactions.

Figure 6. Northern blot analysis of the oestradiol-treated adult rat hepatocytes in vitro

Adult rat hepatocytes were treated with 17β-oestradiol and insulin at the indicated concentrations, and RNAs were analysed by Northern blotting as described in the Experimental section. Blots are representative of three separate experiments. Dex, dexamethasone; E2, 17β-oestradiol.