Effects Of Insulin On Hepatic Transferrin Receptors Expression And Its Mechanism

BackgroundDysmetabolic iron overload syndrome (DIOS) is now frequently found in patientswith metabolic disease[1]. DIOS has been confirmed to play an important role in the onsetand development of the chronic metabolic diseases by causing oxidative stress injury inliver, increasing cellular lipid burden through enhancing liver cholesterol synthesis[2-4].Hyperinsulinemia is a common character of DIOS[1]. Most recently, it was proposed thatenvironmentally induced hyperinsulinemia is the root cause rather than consequence ofinsulin resistance in chronic metabolic diseases[7]. There are interacting pathways linkingglucose and iron metabolism[6], and insulin plays an important role in glucose metabolism.Whether hyperinsulinemia may influence the hepatic iron metabolism has not beenreported. It has been found[9-10]for a long time that insulin could promote cellular ironuptake in adipocytes by stimulating the recycle of transferring receptor between cellmembrane and cytoplasm. Most recent studies demonstrated[11-12]that iron absorptionwas decreased in DIOS patients without altering the level of serum iron and TfR-1waselevated obviously[12-13]indicating that the hepatic iron overload may be associated withthe cellular iron uptake by the level of TfR-1. All the evidences imply thathyperinsulinemia might induce hepatic iron overload by up-regulating liver TfR-1.ObjectiveWe observed the effect of hyperinsulinemia on hepatic iron and hepatic TfRsexpression in rats, and further studied the molecular mechanisms of insulin regulation onTfRs in human liver cell line HL-7702. The result of our study helps understand the effectof insulin on hepatic iron homeostasis. Methods1. Animals study1.1Acute insulin administration and analysisTwelve female SD rats（weighing180-200g）were equally divided into two groups:normal control group (NC group) and acute insulin administration group (AI group). Therats in AI group received a single intraperitoneal (i.p.) injection of3units of insulin, andrats in NC group were injected with saline alone. All rats were killed4h after injection.Glucose was measured using fresh blood by cutting and pricking the tail(GlucometerGluco Touch).Serum levels of insulin and corticosterone were measured usingradioimmunoassay kits. Iron level in the liver was quantitated using an atomic absorptionspectrophotometer. Serum iron concentrations and TIBC were using colorimetric analysiskits. Protein levels of hepatic TfRs and IRPs were detected by Western blot.1.2Chronic insulin administration and analysisTwelve female SD rats （weighing180-200g）were equally divided into two groups:normal control group (NC group) and chronic insulin administration group (CI group).Alzet minipumps were implanted to deliver insulin at3U/day in CI group, and saline inNC group. All rats were killed5days after surgery. Glucose was measured using freshblood by cutting and pricking the tail(Glucometer Gluco Touch).Serum levels of insulinand corticosterone were measured using radioimmunoassay kits. Iron level in the liver wasquantitated using an atomic absorption spectrophotometer. Serum iron concentrations andTIBC were using colorimetric analysis kits. Protein levels of hepatic TfRs and IRPs weredetected by Western blot.2. Cell study2.1Cell cultureHL-7702(Chinese Academy of Sciences, Shanghai，China) were grown in1640medium supplemented with10%fetal bovine serum,100units/ml penicillin, and50ug/mlstreptomycin sulfatev at37℃in a humidified5%CO2atmosphere.2.2Effect of insulin time and concentration gradient on TfR-1mRNA and protein expressionHL-7702cells were transferred to12-well plates and then treated with0.1,1,10,100nM insulin for12h or with100nM insulin for4,8,12,24h mRNA levels of TfR-1and β-actin was detected by real-time quantitative PCR analysis.HL-7702cells were transferred to6-well plates and then treated with0.1,1,10,100nM insulin for12h or with100nM insulin for4,8,12,24h protein levels of TfR-1weredetected by Western blot.2.3Effect of insulin on TfR-1mRNA stabilityHL-7702cells were transferred to12-well plates and actinomycin D (5μg/ml) wasadded to the control and insulin treated cells, and total RNA was isolated at0,2and4h ofactinomycin D addition. RT-PCR was performed with specific primers of human TfR-1and β-actin.2.4Effect of insulin time and concentration gradient on TfR-2,IRP-1and IRP-2expressionHL-7702cells were transferred to6-well plates and then treated with0.1,1,10and100nM insulin for12h and then the total protein was extracted, protein levels ofTfR-2, IRP-1and IRP-2were detected by Western blot.HL-7702cells were transferred to6-well plates and then treated with100nM insulinfor4,8,12and24h. Protein levels of TfR-2, IRP-1and IRP-2were detected by Westernblot.2.5Effect of insulin on intracellular iron content in HL-7702cellsThe cells were transfected with specific siRNA products for TfR-1or negativecontrol siRNA for48h, and then incubated with30μM apo-transferrin or30μMholo-transferrin for6h with or without pre-treatment of100nM insulin for6h beforedetection of the fuorescence. After incubation, intracellular iron content was quantifiedusing PG-FLdiacetate (5μM).2.6The mechanism of insulin on TfR-1expression2.6.1The expression of TfR-1by using IRPs siRNAHL-7702cells were transfected with co siRNA (both IRP-1and IRP-2), IRP-1orIRP-2for48h,then given100nM insulin for another12h. Protein levels of TfR-1, IRP-1and IRP-2were detected by Western blot. 2.6.2The expression of TfR-1by using insulin pathway inhibitorHL-7702cells were pretreated with PI-3K inhibitor LY294002(10μM), MEKinhibitor U0126(1μM) or JNK inhibitor SP600125(10μM) for30min before exposure to100nM insulin for12h. protein levels of TfR-1and IRP-2were detected by Western blot.3. Statistical AnalysisValues are represented as Mean±SEM. Statistical analysis was performed using theStatview software. Statistical difference between two groups was assessed by theIndependent-t test. One way ANOVA, followed by LSD-t and SNK post-hoc test, wasperformed to analyze the difference between the three or more groups. Repeated measuresANOVA was performed to estimate the effect of group and time on values. Differenceswere considered significant at P<0.05.Results1. Animal study1.1Effect of acute insulin administration on iron parameters andhepatic TfRs and IRPs expression in ratsSD rats received acute insulin administration (AI) by a single injection. It was foundthat plasma glucose was decreased and insulin level was increased in AI rats (P<0.05)without significantly altering the serum corticosterone, iron concentrations, TIBC, ironlevel,TfRs and IRPs expression in the liver(P>0.05).1.2. Effect of chronic insulin administration on iron parameters andhepatic TfRs and IRPs expression in ratsSD rats received chronic insulin administration (CI) through subcutaneousimplantation of a mini-pump.The CI rats presented with significant hyperinsulinemia asrepresented by a4-fold increase in plasma insulin (P<0.01), and a mild to meditate hepaticiron overload as compared with the control rats (P<0.05), with no significant changedetected in serum corticosterone, iron concentrations and TIBC (P>0.05). The Western blot results showed that compared with the control group, TfR-1, but not TfR-2, wassignificantly elevated in CI rats (TfR-1,CI vs. NC, P<0.05; TfR-2, CI vs. NC, P>0.05),accompanied with an approximate1.5-fold increase in both IRP-1and IRP-2(IRP-1, CI vs.NC, P<0.05; IRP-2, CI vs. NC, P<0.05).2. Cell study2.1Effect of insulin time and concentration gradient on TfR-1mRNA and protein expressionHL-7702cells were treated with insulin in both time and concentration gradient. Inthe time gradient, TfR-1expression increased to the maximum of2-fold at12h (P<0.05),and in the concentration gradient, the maximal effect about2-fold elevation was detected at100nM (P<0.05).HL-7702cells were treated with insulin in both time and concentration gradient.Compared with the blank control group, TfR-1protein expressions were markedlyincreased after insulin treatment (P<0.05). In the time gradient, TfR-1expression increasedto the maximum of1.5-fold at12h (P<0.001), and in the concentration gradient, themaximal effect about1.5-fold elevation was detected at100nM (P<0.01).2.2Effect of insulin on TfR-1mRNA stabilityAnalysis of the half-life of TfR-1mRNA in HL-7702cells with or without insulintreatment showed that TfR-1mRNA stability was markedly increased in insulin-treatedcells than that in untreated control cells (P<0.05).2.3Effect of insulin time and concentration gradient on TfR-2,IRP-1and IRP-2expressionHL-7702cells were treated with insulin in both time and concentration gradient. Nosignificant alteration of TfR-2was observed in HL-7702cells treated with insulin of timeor concentration gradient (P>0.05).In the time gradient, insulin greatly stimulated IRP-1(P<0.01) and IRP-2(P<0.001) expression with the maximal effect present at8h. The mosteffective concentration of insulin on IRP-1and IRP-2expression were both found at100 nM (P<0.001).2.4Effect of insulin on intracellular iron content in HL-7702cellsThe fluorescence was significantly suppressed by holo-Tf (holo-Tf vs. control,p<0.01, holo-Tf+Ins vs control, P<0.05), and further decreased in HL-7702cells withinsulin pretreatment (holo-Tf+Ins vs. holo-Tf, P<0.05). We found that holo-Tf could nolonger repressed the fluorescence of HL-7702cells (TfR-1siRNA+holo-Tf vs. control,P>0.05), and the fluorescence was still not changed by additional pretreatment of insulin(TfR-1siRNA+holo-Tf+Insulin vs. TfR-1siRNA+holo-Tf, P>0.05).2.5The mechanism of insulin on TfR-1expression2.5.1The expression of TfR-1by using IRPs siRNAInsulin-induced expression of TfR-1was blocked in HL-7702cells lacked both IRPs(P>0.05). The expression of TfR-1was not influenced by IRP-1siRNA (P<0.05). Theinsulin-induced expression of TfR-1was blocked in HL-7702cells lacked only IRP-2(P>0.05).2.5.2The expression of TfR-1by using insulin pathway inhibitorHL-7702cells were pretreated with LY294002(10μM), U0126(1μM) or SP600125(10μM) and then exposed to insulin. Up-regulation of TfR-1and IRP-2by insulin wasentirely blocked by LY294002(LY+I vs. LY, P>0.05), but not suppressed in the presenceof U0126(U+I vs. U, P<0.01) or SP600125(SP+I vs. SP, P<0.01).ConclusionsIn our study we observed that acute and chronic insulin administration on hepaticiron homeostasis and TfRs, IRPs expression in rat. We also observed insulin changecellular iron homeostasis in HL-7702cells in vitro. IRPs siRNA and insulin inhibitor wereused to study the molecular mechanism of the change of iron homeostasis. Conclusionswere made as follows:1. Hyperinsulnemia could induce mild to moderate hepatic iron overload and increased TfR-1,IRP-1and IRP-2expression in the liver.2. Insulin increased cellular iron by up-regulating liver TfR-1in HL-7702cells.Insulin markedly up-regulated protein level of TfR-1by increasing its mRNA stability.Insulin-induced TfR-1expression was blocked by IRP-2siRNA, and disappeared when thePI-3K pathway was inhibited by LY294002.In conclusion, the findings of the present study suggest that hyperinsulnemia couldinduce hepatic iron overload by up-regulating liver TfR-1via the PI-3K/IRP-2pathway.