Heyang Wang, Hongxia Li, Xin Jiang, Wencai Shi, Zhilei Shen, and Min Li

Hepcidin Is Directly Regulatedby Insulin and Plays anImportant Role in Iron Overloadin Streptozotocin-InducedDiabetic Rats

Iron overload is frequently observed in type 2diabetes mellitus (DM2), but the underlyingmechanisms remain unclear. We hypothesize thathepcidin may be directly regulated by insulin andplay an important role in iron overload in DM2. Wetherefore examined the hepatic iron content, serumiron parameters, intestinal iron absorption, andliver hepcidin expression in rats treated withstreptozotocin (STZ), which was given alone or afterinsulin resistance induced by a high-fat diet. Thedirect effect of insulin on hepcidin and its molecularmechanisms were furthermore determined in vitro inHepG2 cells. STZ administration caused a significantreduction in liver hepcidin level and a markedincrease in intestinal iron absorption and serum andhepatic iron content. Insulin obviously upregulatedhepcidin expression in HepG2 cells and enhancedsignal transducer and activator of transcription 3protein synthesis and DNA binding activity. Theeffect of insulin on hepcidin disappeared when thesignal transducer and activator of transcription 3pathway was blocked and could be partially inhibitedby U0126. In conclusion, the current study suggeststhat hepcidin can be directly regulated by insulin,and the suppressed liver hepcidin synthesis may bean important reason for the iron overload in DM2.Diabetes 2014;63:1506–1518 | DOI: 10.2337/db13-1195

Body iron overload is frequently observed in patientswith type 2 diabetes mellitus (DM2) (1,2) or impairedglucose tolerance (IGT) (3,4). Iron overload has beenconfirmed as an independent factor contributing to thedevelopment of DM2 by causing oxidative stress injuryin hepatocytes and pancreatic b-cells (5), which mayfinally lead to insulin resistance (IR) and reduction ininsulin extraction and secretion (6). Prospective clinicalstudies (7,8) have demonstrated that body iron storageis positively correlated with the prevalence of DM2.Bloodletting (9) and iron restriction diet (10) couldobviously help control blood glucose, improve insulinsensitivity, and protect against DM2. Nevertheless, thereason for iron accumulation in DM2 patients remainsunclear.

Emerging evidence suggests that iron overload inDM2 patients may be related to the loss of insulin signal.Drugs that improve insulin sensitivity can also reducebody iron level (11). In the case of the same iron intake,iron overload deteriorates following the development ofDM (12). Besides, free Fe3+ and/or serum ferritin issignificantly increased in DM2 patients with poor glyce-mic control compared with those with good glycemiccontrol (13,14). However, the effect of insulin on bodyiron metabolism, especially hepatic iron storage, and theunderlying mechanisms remain elusive. Hepcidin, a cir-culatory antimicrobial peptide mainly expressed in the

Military Hygiene Department, Faculty of Naval Medicine, Second Military MedicalUniversity, Shanghai, China

Corresponding author: Min Li, linlimin115@126.com.

Received 11 August 2013 and accepted 23 December 2013.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1195/-/DC1.

© 2014 by the American Diabetes Association. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

1506 Diabetes Volume 63, May 2014

METABOLISM

liver, plays a critical role in the regulation of iron me-tabolism by negatively regulating intestinal iron absorp-tion and macrophage iron release and lowering the levelof circulating iron (15). Decreased serum prohepcidinwas recently reported in DM2 patients with hyper-ferritinemia (16,17), suggesting that hepcidin playsa potential role in iron overload in DM2.

In the current study, we examined the hepatic ironcontent, blood iron parameters, intestinal iron absorp-tion, and liver hepcidin expression in streptozotocin(STZ)-induced diabetic rats and a rat model of IR inducedby high-fat diet (HFD) with or without low-dose STZinduction. In addition, we also determined, for the firsttime, the direct effect of insulin on hepcidin expressionand its molecular mechanism in the HepG2 cell line. Ourstudy may help in understanding the effect of insulin onbody iron homeostasis and confirm whether and howhepcidin is regulated by insulin, which would help gainnew insights into the etiology of iron overload in DM2and other diseases frequently accompanied with IR, thushelping improve the protection and treatment of ironoverload in those diseases.

RESEARCH DESIGN AND METHODS

STZ-Induced Type 1 Diabetic Rats With or WithoutInsulin Therapy

The animal experiments were approved by the AnimalEthics Committee of the Second Military Medical Uni-versity in Shanghai, China. Eighteen male Sprague-Dawley rats (160–180 g) were equally divided into threegroups: normal control (control), diabetic (STZ), andinsulin-treated diabetic group (STZ+Ins). Diabetes wasinduced by intraperitoneal injection of STZ (75 mg/kg;Sigma-Aldrich, St. Louis, MO). Rats in the control groupwere injected with the buffer alone. Diabetes was iden-tified in 72 h by fasting blood glucose level.16.7 mmol/L(;300 mg/dL). After the model was confirmed, rats werefed for another 4 weeks. Neutral insulin (16 units/kg;Novo Nordisk, Copenhagen, Denmark) was administeredvia subcutaneous injection twice a day at 8:00–10:00 A.M.

and P.M. in the last 2 weeks, nonfasting glucose wasmonitored at the same time, right before the insulininjection, and the average level was determined for fur-ther statistical analysis. During the last week of the ex-periment, four rats in each group were transferred tometabolic cages (Comprehensive Lab Animal MonitoringSystem; Columbus Instruments, Columbus, OH) in whichthe amount of food intake and excrement was preciselyrecorded and/or collected between 9:00 and 10:00 A.M.

STZ-Induced Type 2 Diabetic Rats With or WithoutInsulin Therapy

The rats in this model were divided into four groups:control group, in which rats were fed a standard diet; theHFD group, in which rats were fed an HFD; the HFD+STZgroup, in which rats were fed an HFD for 4 weeksand then injected intraperitoneally with 25 mg/kg STZ;

and the HFD+STZ+Ins group, in which rats were firsttreated similarly as those in the HFD+STZ group and thengiven a subcutaneous insulin injection (12 units/kg; NovoNordisk, Copenhagen, Denmark) twice a day at 8:00–10:00 A.M. and P.M. in the last 2 weeks, with nonfastingglucose monitored at the same time, right before the in-sulin injection, and the average level was determined forfurther statistical analysis. The HFD (Shanghai LaboratoryAnimal Center, Chinese Academy of Sciences, NationalLaboratory Animal Center, Shanghai, China) containedcrude protein 22.3/100 g (20% kcal), fat 19.8/100 g (45%kcal), and carbohydrate 44.6/100 g (15% kcal). An in-traperitoneal glucose tolerance test (IPGTT) was per-formed at end of the 4th week before STZ injection. Aftera 12-h starvation, rats in each group received a glucosesolution (50%; 1 g/kg body weight) via intraperitonealinjection. Blood samples were obtained by retro-orbitalpuncture at 0, 30, 60, 90, and 120 min. Homeostasismodel assessment of IR (fasting glycemia [mmol/L] 3fasting insulinemia [mUI/mL]/22.5) was calculated, andblood iron parameters were also measured at 0 min.HFD+STZ and HFD+STZ+Ins rats exhibiting fasting glu-cose levels .7.8 mmol/L (;140 mg/dL) were considereddiabetic, resembling human DM2 (18). The experimentin metabolic cages was performed as described above.

Insulin, Glucose, Interleukin-6, and Interleukin-1bAnalysis

Glucose was measured using fresh blood by cutting andpricking the tail (Glucometer Gluco Touch; Roche,Munich, Germany). Serum levels of insulin, interleukin(IL)-6, and IL-1b were measured using radioimmunoas-say kits (North Biotechnology, Beijing, China).

Iron Status Parameters

Iron level in the liver, diet, and excrement was quanti-tated using an atomic absorption spectrophotometer(Z-8100; Hitachi, Tokyo, Japan). The measured dietand excrement iron was used to calculate the apparentiron absorption rate, and the liver iron content wasnormalized to the wet tissue weight for each sample,respectively. Serum iron concentrations and total iron-binding capacity (TIBC) were determined in non-hemolyzed serum samples using colorimetric analysis kits(Nanjing Jian Cheng Biotechnology Institute, Nanjing,China). The transferrin (Tf) saturation was calculated asplasmatic iron/TIBC and Tf as TIBC/25. Serum ferritinand soluble Tf receptor (sTfR) were determined in non-hemolyzed serum samples using ELISA kits (sTfR,MBS268897, MyBioSource, San Diego, CA; ferritin,SEA518Ra, Life Science Inc., Houston, TX). The apparentiron absorption rate was calculated as ([diet iron intake 2iron output in excrement]/diet iron intake) 3 100%.

Real-Time Quantitative PCR Analysis

Total RNA was extracted using TRIzol (Invitrogen,Carlsbad, CA) and reversely transcribed to cDNA by RTReagent Kit (Primerscript; Takara Bio Inc., Shiga, Japan).

diabetes.diabetesjournals.org Wang and Associates 1507

Quantitative PCR amplification was performed with theSYBR Green Kit (Takara Bio Inc.) using the Roto-geneRG3000 (Corbett Research, Sydney, Australia), andmRNA levels of specific genes were normalized to theb-actin levels of the same sample.

Western Blotting

Homogenates of rat liver and intestine or HepG2 celllysates were prepared for Western blot (WB) analysis.The following antibodies were used: antiferroportin(Fpn; Alpha Diagnostic International, San Antonio, TX);antihepcidin (ab30760; Abcam, Cambridge, MA); anti–phospho-signal transducer and activator of transcription 3(p-STAT3), anti-STAT3, anti–phospho-Smad1/5/8(p-Smad1/5/8), anti-Smad1, anti-Smad4 (Cell SignalingTechnology, Danvers, MA); and anti–b-actin (Santa CruzBiotechnology, Santa Cruz, CA). Signals quantified by den-sitometry were normalized to b-actin levels or, in the caseof phosphoproteins, to the total levels of the same protein.

HepG2 Culture, Treatment, and Small Interfering RNATransfection

HepG2 (Chinese Academy of Sciences, Shanghai, China)cells were grown in high-glucose DMEM supplementedwith 10% FBS and 1% antibiotic solution (Gibco, GrandIsland, NY) and incubated in a humidified 5% CO2 at-mosphere at 37°C. Cells were transferred to six-wellplates and treated with 0.1, 1, 10, and 100 nmol/L in-sulin for 24 h or with 100 nmol/L insulin for 4, 8, 12,and 24 h. For SMAD4 and STAT3 silence, cells weretransfected with SMAD4 and STAT3 small interferingRNA (siRNA) products (sc-29484 and sc-29493; SantaCruz Biotechnology) that generally consist of pools ofthree to five target-specific 19–25 nucleotide siRNAsdesigned to knock down gene expression or negativecontrol oligonucleotides (Santa Cruz Biotechnology) inthe presence of Lipofectamine RNAiMAX, according tothe manufacturer’s instructions (Invitrogen). Cells weremaintained for 24 h after transfection and given100 nmol/L insulin for another 24 h. For insulin pathwayinhibitors, HepG2 was pretreated with LY294002(10 mmol/L) or U0126 (1 mmol/L) for 30 min and thentreated with 100 nmol/L insulin for 24 h.

Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) assay was per-formed as described (19). The antibodies against SMAD4and STAT3 were purchased from Cell Signaling Tech-nology. The primers used for PCR were Hepc forward, 59-CGCT CTGT TCCC GCTT AT-39, and Hepc reverse, 59-CGAG TGAC AGTC GCTT TTAT G-39, that amplified 129bp of human hepcidin promoter.

Generation of Hepcidin Promoter Plasmid Constructs,Cell Transfection, and Luciferase Reporter Assays

Generation of pGL3-basic luciferase reporter (Promega,Southampton, U.K.) with full-length human hepcidinpromoter was performed by obtaining genomic DNAfrom HepG2 cells and cloning the proximal 942 bp of

a human HAMP promoter into the pGL3-basic luciferasereporter vector, as described by Courselaud et al. (20).Site-directed mutagenesis (Quickchange II; Stratagene,Stockport, U.K.) on signal transducer and activator oftranscription (STAT3) response element and putativebone morphogenetic protein responsive element were asdescribed by Matak et al. (21). HepG2 cells were trans-fected with certain HAMP reporter constructs or theempty pGL3-basic vector using Superfectin II (In-vitrogen), according to the manufacturer’s instructions.To normalize for the transfection efficiency, an internalcontrol–pRL-SV40 Renilla luciferase plasmid (Promega)was cotransfected alongside the HAMP constructs ina 1:200 ratio in serum-free medium. After 24-h equili-bration, cells were treated with insulin for an additional24 h, and luciferase activity was determined in triplicatein at least two independent experiments using the DualLuciferase Reporter Assay, according to the manu-facturer’s instructions (Promega).

Statistical Analysis

Data are represented as mean 6 SEM. Statistical analysiswas performed using the Statview software (SAS In-stitute Inc., Cary, NC). Statistical difference between twogroups was assessed by the independent t test. One-wayANOVA, followed by least significant difference (LSD) tand Student-Newman-Keuls (SNK) post hoc test, wasperformed to analyze the difference between the three ormore groups. Repeated-measures ANOVA was performedto estimate the effect of group and time (date) on valuesobtained during the experiments of IPGTT or meta-bolic cages. Differences were considered as significant atP , 0.05.

RESULTS

Effect of STZ Administration and HFD on Body Weight,Fasting Glucose, and/or Glucose Tolerance

Rats exhibited diabetic profiles with decreased bodyweights, hyperglycemia (Fig. 1A and B), and hypo-insulinemia (Table 1) 72 h after simple STZ injection.Supplementation of insulin restored the fasting bloodglucose and body weight to the normal levels (Fig. 1A).All rats in the model of STZ-induced DM2 receivedan IPGTT at the end of the 4th week, right before theadditional STZ injection, and serum glucose and insulinwere measured before and 30, 60, 90, and 120 min afterinjection of 50% glucose solution (1 g/kg body weight).Except for the normal fasting blood glucose, a significantincrease in glycemia and insulinemia was observed atdifferent IPGTT times (Fig. 2C and D), and the homeo-stasis model assessment of IR index increased by abouttwofold in the rats of the HFD, HFD+STZ, and HFD+STZ+Ins groups (Supplementary Table 1). After estab-lishment of the IR model, rats in the HFD+STZ andHFD+STZ+Ins groups developed hyperglycemia andcontinuous weight reduction 72 h after intraperitonealinjection of 25 mg/kg body weight STZ (Fig. 2A and B).

1508 Hepcidin Is Directly Regulated by Insulin Diabetes Volume 63, May 2014

Insulin therapy obviously increased the body weight andcontrolled the blood glucose at a normal level (Fig. 2Aand B). Serum ketone body was also measured in thecurrent study (Supplementary Fig. 1). STZ rats exhibi-ted a significantly higher serum level of ketone bodythan the control and STZ+Ins rats (P , 0.001), and thelatter two groups showed no difference. In the animalmodel of DM2, we found no significant change amongthe four groups (Supplementary Fig. 1).

Effect of STZ Administration and HFD on IronParameters, Serum IL-6, IL-1b, or Hemoglobin

Both liver iron content and serum iron were increased inSTZ-induced diabetic rats (148.44 6 30.28 mg/g and

97.43 6 4.59 mmol/L vs. 68.98 6 18.96 mg/g and69.45 6 11.32 mmol/L in control group, P , 0.001;Table 1) and significantly lowered by insulin treatment(P, 0.05 and P, 0.001, respectively; Table 1). A similarchange was also found in serum ferritin, which was ob-viously elevated in STZ rats (STZ vs. control, P , 0.001;Table 1) and markedly recovered by insulin supplement(STZ vs. STZ+Ins, P, 0.001; Table 1). The level of serumsTfR was lower in STZ rats (STZ vs. control, P , 0.05;Table 1) and upregulated by insulin therapy, however,with no statistical significance (Table 1). Interestingly,insulin therapy restored serum iron to the normal rangewithout lowering the high level of Tf in STZ rats, making

Figure 1—Body weight, blood glucose, and iron status in STZ-induced type 1 diabetic rats with or without insulin therapy. A and B:Sprague-Dawley rats (160–180 g) received intraperitoneal injection of STZ (75 mg/kg; Sigma-Aldrich) or citrate buffer alone. Body weightwas monitored during the whole experiment. Insulin therapy was performed in the last 2 weeks of the experiment. Subcutaneous insulininjection (16 units/kg body weight) was given two times every day at 8:00–10:00 A.M. and P.M., respectively. Blood glucose was measuredevery 2–3 days during the insulin therapy two times right before the insulin injection, and the average level was determined for furtherstatistical analysis. C: Protein levels of hepatic hepcidin, STAT3, and intestinal Fpn were analyzed by WB. n = 6/group. D: Rats weretransferred to metabolic cages in the last week of the experiment. The amounts of food intake and excrement were precisely recordedbetween 9:00 and 10:00 A.M., and the iron content was analyzed to calculate the apparent intestinal iron absorption. White circles, controlgroup; black circles, STZ group; black squares, STZ+Ins group. n = 6/group. Data are expressed as means 6 SEM for three groups of sixrats. One-way ANOVA, followed by LSD t and SNK post hoc tests, was performed to analyze differences among the three groups.Repeated-measures ANOVA was performed to estimate the effect of group and time (date) on values obtained during the experiments ofmetabolic cages. Differences were considered significant at P < 0.05. *Significantly different from controls, P < 0.05; **P < 0.01.

diabetes.diabetesjournals.org Wang and Associates 1509

Tf saturation (serum iron/TIBC ratio) in insulin-treatedrats even lower than that in normal control rats (P ,0.01; Table 1). Although a correlation was suggestedbetween IL-6 and DM2 (22), we failed to observe a sig-nificant difference in serum IL-6 level or IL-1b, a moresensitive indicator of inflammation, among the threegroups (Table 1), and we only found a twofold increase inliver mRNA expression of both genes (SupplementaryFig. 2).

Serum samples of control, HFD, HFD+STZ, andHFD+STZ+Ins rats before STZ injection were gained inthe IPGTT at the point of 0 min, and the iron status wasanalyzed. HFD+STZ and HFD+STZ+Ins rats showed nodifference in blood iron parameters before receiving anSTZ injection (Supplementary Table 2). However, therewas a 96% increase in hepatic iron content and a 65%increase in serum iron in HFD+STZ rats as comparedwith the control group (147.98 6 25.98 vs. 75.33 614.68 mg/g, P , 0.001; 67.40 6 4.52 vs. 40.78 6 2.16mmol/L, P , 0.001; Table 2) when the whole experimentwas finished, and insulin therapy could significantlylower the level of liver iron (P , 0.05) and serum iron(P , 0.001; Table 2). Serum ferritin and sTfR weresignificantly increased or decreased in HFD+STZ rats(vs. control, P, 0.001 and P, 0.01, respectively; Table 2)and remained normal in HFD rats. Serum ferritin was sig-nificantly reduced by insulin supplement (HFD+STZ+Insvs. HFD+STZ, P , 0.001; Table 2), and sTfR also beganto recover, although with no statistical significance bythe end of the experiment. HFD+STZ exhibited higherTf saturation (vs. control, P = 0.001; Table 2), but hadno change on TIBC and serum Tf. In addition, althoughenhanced erythropoiesis was once observed in HFD-fedrats (23), we found no change in hemoglobin (Hb) ineither the HFD or HFD+STZ group (Table 2).

Alteration of Hepatic Hepcidin, STAT3, Intestinal Fpn,and Apparent Intestinal Iron Absorption Rate inDifferent Animal Models

According to the WB results, we found that STZ rats hada twofold increase in intestinal Fpn expression as com-pared with the control group (P , 0.01; Fig. 1C), whichwas accompanied by a 40% decrease in both hepcidin andSTAT3 content in liver (P , 0.01 and P , 0.05, re-spectively; Fig. 1C), and no alteration in these threeproteins was found in the insulin treatment group (Fig.1C). Intestinal Fpn expression of HFD+STZ rats in-creased by 70% (P , 0.01; Fig. 2E), followed by a 30 and50% reduction in liver STAT3 and hepcidin content (P ,0.05 and P , 0.001, respectively; Fig. 2E). There was nosignificant difference in the three proteins among theother three groups (Fig. 2E).

Apparent iron absorption rate was calculated by theamount of food intake and excrement, iron content in diet(110 and 130 mg/kg in the control natural diet and theHFD), and iron content in the excrement. There wasa mean of 50% increase (range 65–80%) in intestinal ironabsorption in STZ rats as compared with the normal andinsulin-treated rats, and the difference was not significantbetween the normal and insulin-treated rats (P . 0.05;Fig. 1D). Intestinal iron apparent absorption in HFD ratswas similar to that in control rats, at a level between 40and 60%, significantly lower than the mean value of 75%in HFD+STZ rats (P , 0.05; Fig. 2F). Insulin treatmentmarkedly controlled the high rate of intestinal iron absorp-tion and kept it at a normal range from 35 to 55%, with nostatistical difference from the control and HFD rats.

Insulin Upregulates Hepcidin and Total STAT3Expression but Has No Effect on SMAD4 in HepG2 Cells

HepG2 cells were treated with insulin in both time andconcentration gradient. Compared with the blank control

Table 1—Fasting insulin, iron parameters, and serum IL-6 and IL-1b in STZ-induced type 1 diabetic rats with or without insulintherapy

Control (n = 6) STZ (n = 6) STZ+Ins (n = 6)

Insulin (mIU/mL) 24.87 6 6.22 10.50 6 2.16*** 23.60 6 7.44

Liver iron (mg/g) 68.98 6 18.96 148.44 6 30.28*** 105.78 6 12.76*,†

Serum iron (mmol/L) 69.45 6 11.32 97.43 6 4.59*** 69.84 6 7.75††

TIBC (mmol/L) 104.31 6 9.65 129.69 6 9.25*** 122.31 6 5.60***

Serum Tf (g/L) 4.17 6 0.39 5.19 6 0.37*** 4.89 6 0.22***

Tf saturation (%) 66.29 6 6.35 75.56 6 7.62** 56.95 6 4.05**,††

Serum ferritin (ng/mL) 69.30 6 5.39 163.95 6 8.69*** 100.27 6 12.90***,††

Serum sTfR (ng/mL) 31.69 6 7.79 17.73 6 2.37* 22.00 6 1.76*

IL-6 (pg/mL) 465.88 6 73.95 418.18 6 17.29 423.28 6 75.53

IL-1b (ng/mL) 0.22 6 0.05 0.24 6 0.04 0.23 6 0.04

Data are means6 SEM. One-way ANOVA, followed by LSD t and SNK post hoc test, was performed to analyze differences among thethree groups. Compared with controls: *P , 0.05; **P , 0.01; ***P , 0.001. Compared with STZ group: †P , 0.05; ††P , 0.001.

1510 Hepcidin Is Directly Regulated by Insulin Diabetes Volume 63, May 2014

group, hepcidin protein and mRNA expression wasmarkedly increased by insulin treatment (P , 0.001,Fig. 3A; P , 0.001, Fig. 3B). SMAD4 and STAT3 areclassic positive transcription regulators of hepcidin thatare activated by binding to p-SMAD1/5/8 and phos-phorylation (24,25), respectively. Insulin markedly in-creased the level of both phosphorylated and total STAT3(P , 0.001; Fig. 4A and B), while it did not influence thephosphorylation ratio of STAT3 and had no effect onprotein expression of SMAD4, p-SMAD1/5/8, andSMAD1 (Fig. 4A and B).

Insulin Stimulates Hepcidin Synthesis ThroughActivation of STAT3 but Not SMAD4, Partially Mediatedby the Extracellular Signal–Related Kinase Pathway

The result of ChIP showed that the amount of STAT3binding to hepcidin promoter increased by twofold ascompared with the control group (P , 0.05; Fig. 5A)when HepG2 cells were treated with 100 nmol/L insulinfor 24 h, but the amount of SMAD4 remained unchanged(Fig. 5A).

Basal fluorescence activity was reduced substantiallybecause of the mutant STAT3 or SMAD4 binding site(P , 0.001; Fig. 5B). After treatment with 100 nmol/L

Figure 2—Body weight, blood glucose, and iron status in STZ-induced type 2 diabetic rats with or without insulin therapy. A and B: Bodyweight was monitored throughout the experiment. Fasting glucose was monitored from 72 h after STZ injection to the start of insulintreatment, during which subcutaneous insulin injection (12 units/kg body weight) was given two times every day at 8:00–10:00 A.M. andP.M., respectively. Nonfasting glucose was measured every 2–3 days during the insulin therapy, two times right before the insulin injection,and the average level was determined for further statistical analysis. C and D: Blood glucose and serum insulin were measured in rats ofcontrol, HFD, HFD+STZ, and HFD+STZ+Ins groups (before STZ injection) at 0, 15, 30, 60, and 120 min after intraperitoneal injection of50% glucose (1 g/kg). E: Protein levels of hepatic hepcidin, STAT3, and intestinal Fpn were analyzed by WB. n = 6/group. F: Rats weretransferred to metabolic cages in the last week of the experiment. The amount of food intake and excrement was precisely recordedbetween 9:00 and 10:00 A.M., and the iron content was analyzed to calculate the apparent intestinal iron absorption. White circles, controlgroup; black circles, HFD group; white squares, HFD+STZ group; black squares, HFD+STZ+Ins group. n = 6/group. Data are expressed asmeans 6 SEM for four groups of six rats. One-way ANOVA, followed by LSD t and SNK post hoc test, was performed to analyze dif-ference between the four groups. Repeated-measures ANOVA was performed to estimate the effect of group and time (date) on valuesobtained during the experiments of IPGTT or metabolic cages. Differences were considered as significant at P < 0.05. *Significantlydifferent from controls, P < 0.05; **P < 0.01; ***P < 0.001. C, control; H, hepcidin; I, Insulin; S, STZ.

diabetes.diabetesjournals.org Wang and Associates 1511

insulin for 24 h, the fluorescence activity of the reportergene of full-length hepcidin promoter and that withmutant SMAD4 binding sites was significantly stimulated(P, 0.05, Fig. 5B), but there was no increase in that withmutation of STAT3 response elements (Fig. 5B).

We interfered SMAD4 and STAT3 protein synthesisby transfecting specific siRNA into HepG2 cells (DD, forSMAD4 siRNA, compared with control, P , 0.001; DT,for STAT3 siRNA, compared with control, P, 0.001; Fig.5C) and then treated the cells with 100 nmol/L insulinfor 24 h. Hepcidin continued to elevate when SMAD4was silenced, but remained unchanged after knockdownof STAT3 (Fig. 5C).

To observe the mediating pathway of insulin regula-tion on total STAT3 and hepcidin, HepG2 cells werepretreated with LY294002 (10 mmol/L) or U0126(1 mmol/L) for 30 min and then exposed to 100 nmol/Linsulin for 24 h (Fig. 6). Upregulation of hepcidin, phos-phorylated, and total STAT3 by insulin was partially loweredby U0126 (U0126+Ins vs. Ins, P , 0.001; U0126+Insvs. U0126, P, 0.01 or P, 0.05), but not suppressed in thepresence of LY294002 (LY294002+Ins vs. Ins, P . 0.05).

DISCUSSION

Iron overload was frequently observed in patients withDM2 (1,2) and even those with IGT (3,4), in close asso-ciation with the development of DM2 and its complica-tions (26). However, pathways underlying ironaccumulation in DM2 are still poorly understood. In thecurrent study, we demonstrated that STZ-inducedhypoinsulinemia significantly reduced hepcidin expres-sion in the liver, leading to an abnormally high content ofFpn in the intestine and elevation of serum iron level andhepatic iron content, which could be conversed by re-covering the liver hepcidin level through insulin therapy,suggesting that hepcidin is decreased by loss of insulinsignal and plays an important role in iron overload inDM2. We also found that insulin could directly stimulate

hepcidin expression in HepG2 cells through activation ofSTAT3 but not SMAD4, which was partially mediated bythe extracellular signal–related kinase (ERK) pathwayand independent of the phosphatidylinositol 3-kinase(PI-3K) pathway.

Rats with STZ-induced diabetes (STZ or HFD+STZgroup) exhibited mild to moderate hepatic iron overload(HIO), accompanied with significantly elevated serumiron and ferritin and markedly decreased serum sTfR.STZ-induced elevation of iron storage was also observedin the proximal tubular lysosomes (27), myocardium(28), and artery (29), while studies of Silva et al. (30) andSaravanan et al. (31) showed that serum iron was in-creased in STZ rats. Additionally, Dogukan et al. (32)reported that STZ injection following HFD inductionmarkedly increased the hepatic and serum iron levels.Our results demonstrated that insulin therapy effectivelyreleased HIO, manifested by the recovery of liver ironcontent and serum ferritin and the improvement ten-dency of serum sTfR, and lowered serum iron to normallevel in STZ-induced both type 1 and type 2 diabetic rats.In addition, simple HFD induction that led to IR but didnot elevate fasting blood glucose had no influence onliver or serum iron, which was not reported in otherstudies (23,33). These findings suggest that body ironhomeostasis may be closely related to insulin signaling.Deficiency of insulin signaling could lead to HIO, prob-ably through loss of restricted control of serum ironlevel, and an HFD seemed to have no impact on ironmetabolism if it did not cause insulin decompensation, asreflected by increased fasting blood glucose. Differentfrom DM2, in patients with type 1 diabetes mellitus,body iron store was not changed (34) or even deficient(35) as opposed to overload, and hepcidin was alsoreported not changed (34). Autoimmune gastritis in type 1diabetes mellitus, caused by its specific autoimmunedisorder (36), takes the most responsibility for the iron

Table 2—Serum insulin, iron parameters, and Hb in STZ-induced type 2 diabetic rats with or without insulin therapy

Control (n = 6) HFD (n = 6) HFD+STZ (n = 6) HFD+STZ+Ins (n = 6)

Insulin (mIU/mL) 34.35 6 4.94 59.36 6 8.62*** 24.33 6 1.90 53.70 6 6.07***

Liver iron (mg/g) 75.33 6 14.68 82.90 6 18.44 147.98 6 25.98*** 113.8 6 12.96*,‡

Serum iron (mmol/L) 40.78 6 2.16 37.58 6 4.23 67.40 6 4.52*** 39.04 6 16.49‡‡‡

TIBC (mmol/L) 88.83 6 2.05 83.47 6 4.74 90.01 6 14.93 90.68 6 3.03

Serum Tf (g/L) 3.55 6 0.08 3.34 6 0.19 3.60 6 0.60 3.63 6 0.12

Tf saturation (%) 45.96 6 3.41 45.25 6 7.15 76.25 6 10.15¶¶¶ 42.84 6 17.38

Serum ferritin (ng/mL) 70.58 6 6.82 74.21 6 13.98 178.50 6 9.26*** 107.91 6 14.91***,‡‡‡

Serum sTfR (ng/mL) 33.12 6 6.27 36.55 6 11.77 15.14 6 2.78** 22.27 6 1.34

Hb (g/L) 125.38 6 10.56 130.66 6 8.59 133.25 6 7.61 129.69 6 5.86

Data are mean 6 SEM. One-way ANOVA, followed by LSD t and SNK post hoc test, was performed to analyze differences among thefour groups. Compared with controls: *P , 0.05; **P , 0.01; ***P , 0.001. Compared with HFD+STZ: ‡P , 0.05; ‡‡‡P , 0.001.Compared with controls: ¶¶¶P = 0.001.

1512 Hepcidin Is Directly Regulated by Insulin Diabetes Volume 63, May 2014

deficiency (37), and the inflammation may contribute tomaintain hepcidin synthesis (24). Time of HFD inductionwas once considered a factor that may restrict its effecton liver iron content (23). However, hepatic iron levelremained unchanged when the induction period wasprolonged to 8 weeks in our experiment or 12 weeks inother studies (38). Other factors may include the dif-ference in the species of rats or mice used in research ormethods used to assay liver iron content, which mayto some extent explain the finding of increased liveriron content induced by HFD in a report by Tsuchiyaet al. (39).

Hepatic hepcidin was reduced significantly, while in-testinal Fpn expression and apparent intestinal iron ab-sorption were elevated significantly in STZ and HFD+STZrats, but HFD rats showed no alteration on the aboveparameters. Insulin supplementation eliminated theseabnormal changes in STZ and HFD+STZ rats, indicatingthat hepcidin, which negatively regulates Fpn and ironabsorption and restricts the level of serum iron, may bepositively regulated by insulin. It was also found in other

HFD rat models that hepcidin expression remained un-changed (33,38) or began decreasing only when fastingblood glucose was increased (39). Clinical studies(16,17,34) showed that the prohepcidin or hepcidin levelin blood was significantly decreased in DM2 patientswith hyperferritinemia. All of these findings support theresults of the current study. The failure to maintain orincrease hepcidin synthesis, probably caused by loss ofinsulin signal, may be of critical importance in ironaccumulation in DM2 patients. Sam et al. (34) dem-onstrated that IR, not insufficient insulin, caused thedecrease of hepcidin and HIO in patients with DM2, ina recent clinical study including patients with polycysticovary syndrome, type 1 diabetes mellitus, and DM2.Insulin treatment may allow us to gain a deeper insightinto the hepcidin regulator in DM2, because it onlyreplenishes insulin level but cannot radically improveIR. Insulin therapy may recover the decreased hepcidinlevel and release iron overload if it is caused by in-sufficient insulin signals, which has been alreadyproved in our experiment. Effect of insulin therapy on

Figure 3—Effect of insulin on hepcidin expression in HepG2 cells. A and B: HepG2 cells were treated with 100 nmol/L insulin for 4, 8, 12,and 24 h separately or insulin at the indicated concentrations of 0, 0.1, 1, 10, and 100 nmol/L for 24 h. Hepcidin protein (A) and mRNA (B)expression were determined by WB and real-time quantitative PCR, respectively. Data are expressed as means 6 SEM, determined inthree independent experiments. One-way ANOVA, followed by LSD t and SNK post hoc test, was performed to analyze difference be-tween the groups. *Significantly different from control group, P < 0.05; **P < 0.01; ***P < 0.001; §significantly different compared withcontrol group, P = 0.056.

diabetes.diabetesjournals.org Wang and Associates 1513

iron overload and hepcidin reduction in patients withDM2 should be further determined in the next step.Hepcidin was also reported to be increased (40) or notchanged (2) in DM2 patients. A bigger sample size andcalculation of the hepcidin/ferritin ratio (41) may helpus better understand the status of hepcidin synthesis.Erythropoiesis was once hypothesized to mediate in-sulin regulation on iron metabolism (23). However, nosignificant change in Hb was observed in the currentstudy. Clinical studies (2,17) also reported that Hbremained unchanged in DM2 or IGT patients. In addi-tion, hyperglycemia may be also involved in hepcidinregulation. It was recently reported that an oral glucosetolerance test could induce the elevation of serumhepcidin in healthy volunteers (42). This effect of glu-cose on hepcidin may partially be due to the boost ofinsulin activity. However, confounding factors such ashyperglycemia need to be further excluded by in vitroexperiments so as to better understand the effect ofinsulin on hepcidin.

To further confirm the effect of insulin on hepcidinexpression, we observed the regulatory effect of insulin

on hepcidin in the HepG2 cell line in vitro and found thatthis effect was in a time- and dose-dependent manner.Hepcidin mRNA and protein were markedly upregulatedby insulin treatment, indicating a direct and significantstimulatory effect of insulin on hepcidin expression.These results are consistent with the data that we pre-viously found in animal experiments, which demon-strated that the reduced liver hepcidin content wascorrelated with STZ-induced hypoinsulinemia. To betterunderstand how hepcidin was modulated by insulin, wealso detected the levels of phosphorylated and totalproteins of STAT3 in HepG2 cells treated with insulin.STAT3 is a novel transcription regulator of hepcidin andactivated through phosphorylation by IL-6 (24). Ourresults showed that insulin could markedly enhance theprotein expression of phosphorylated and total STAT3and increase the DNA binding activity of STAT3, but ithad no effect on the p-STAT3/STAT3 ratio. In addition,interruption of STAT3 expression by silencing RNAcompletely repressed the upregulatory effect of insulinon hepcidin, and the activity of the reporter gene of thehepcidin promoter with mutant STAT3 binding sites

Figure 4—Insulin upregulates STAT3 protein expression in HepG2 cells but has no effect on SMAD4. A: HepG2 cells were treated with100 nmol/L insulin for 4, 8, 12, and 24 h separately. B: HepG2 cells were incubated with the indicated concentrations of insulin for 24 h.Protein levels of p-STAT3, total STAT3, p-SMAD1, total SMAD1, and SMAD4 were detected by WB, and the phosphorylation ratio ofSTAT3 was calculated by p-STAT3/STAT3. Data are expressed as means 6 SEM, determined in three independent experiments. One-way ANOVA, followed by LSD t and SNK post hoc test, was performed to analyze difference between the groups. Significantly differentfrom control, ***P < 0.001.

1514 Hepcidin Is Directly Regulated by Insulin Diabetes Volume 63, May 2014

could no longer be stimulated by insulin, suggesting thatinsulin may regulate hepcidin specifically through acti-vation of STAT3.

It was reported that insulin induced STAT3 phos-phorylation (43) and enhanced its DNA binding activity(44). However, total STAT3 was not detectable (44) orshowed no change (43). Some studies even argued thatinsulin could inhibit STAT3 protein expression as well asthe activation of STAT3 via IL-6–induced phosphoryla-tion (45). To further confirm how STAT3 was changed inhypoinsulinemia-induced iron overload, we supple-mented WB analysis of total STAT3 in samples of STZ-induced diabetic rats. Liver total STAT3 expression wassignificantly decreased in STZ rats and entirely reversedby insulin therapy, which appeared thoroughly consistentwith the alteration of hepcidin, suggesting that total

STAT3 may be regulated by insulin in vivo and involvedin its regulation on hepcidin. In addition, it is also worthmentioning that although some studies suggested a cor-relation between inflammation and DM (22), we foundneither serum IL-6 nor IL-1b produced a significantchange; only their mRNA expression levels had a twofoldincrease in the liver. These results indicate that STZtreatment may lead to inflammatory reaction in diabeticrats but, at least in the current study, only to a relativelymild extent.

ERK and PI-3K are believed to be involved in the ac-tivation of insulin on STAT3 (46). To clarify the pathwaythat mediates the effect of insulin on hepcidin andconfirm the role of STAT3, we used inhibitors of theabove two classical pathways and found that ERK in-hibitor U0126 could significantly but partially suppress

Figure 5—The stimulatory effect of insulin on hepcidin is dependent on the activation of STAT3. A: DNA binding activity of SMAD4 andSTAT3 was determined by ChIP when HepG2 was treated with 100 nmol/L for 24 h. B: Mutant SMAD4- and STAT3-binding sites onhepcidin promoter refers to Matak et al. (21). HepG2 cells were transfected with luciferase reporter constructs of hepcidin promoter of full-length, SMAD4-, or STAT3-mutant, exposed 24 h later to 100 nmol/L insulin for 24 h, and luciferase activity was measured. C: HepG2 cellswere transfected with invalid, SMAD4, or STAT3 siRNA, were exposed 24 h later to 100 nmol/L insulin for 24 h, and hepcidin proteinexpression was analyzed by WB. Data are expressed as means 6 SEM, determined in three independent experiments. Statistical dif-ference between two or more groups was assessed by the independent t test or one-way ANOVA followed by LSD t and SNK post hoctest. *Significantly different from control, P< 0.05; **P< 0.01; ***P< 0.001; #compared with the full-length hepcidin promoter, P < 0.001.BMP, bone morphogenetic protein; C, control; I, Insulin; IgG, immunoglobulin G; TSS, transcription start site.

diabetes.diabetesjournals.org Wang and Associates 1515

hepcidin response to insulin, while PI-3K inhibitorLY294002 had no effect on it. In addition, the increasedphosphorylated and total STAT3 expression induced byinsulin could be partially and significantly inhibited byU0126, but it was not affected by LY294002. This pre-sentation was fully consistent with hepcidin, indicatingthat the regulatory effect of insulin on hepcidin may bemediated specifically by STAT3. It was found that Januskinase 2 also participated in mediating insulin activationon STAT3 (47), which may explain the partial but notabsolute inhibitory effect of U0126 on insulin-stimulatedupregulation of total STAT3 and hepcidin.

In addition to STAT3, C/EBPa and SMADs are alsoclassic hepcidin transcription regulators. It was found(48) that the expression and phosphorylation of C/EBPawas inhibited by insulin, suggesting that C/EBPa is notinvolved in the regulatory effect of insulin on hepcidin.In the current study, we found that insulin had no effecton the expression of total and/or p-SMAD1 and SMAD4or on the DNA-binding activity of SMAD4. In addition,insulin kept increasing the hepcidin expression when

SMAD4 protein synthesis was inhibited by siRNA, andpoint mutation of SMAD4 binding elements also showedno inhibitory effect on insulin enhancing the hepcidinpromoter activity. Therefore, we conclude that STAT3,rather than the SMAD family, specifically and directlymediated the regulatory effect of insulin on hepcidinexpression.

In conclusion, our study has demonstrated that hep-cidin can be directly regulated by insulin selectivelythrough STAT3 and partially through mediation of theERK pathway, thus playing an important role in ironoverload in DM2. Iron restriction should be consideredonce IGT or diabetes is confirmed. The results of thecurrent study also suggest that, besides effective insulintreatment, hepcidin and/or a STAT3 stimulator mayprove to be potential drug targets for the therapy of ironoverload in DM2.

Acknowledgments. The authors thank Danghui Yu, the Editor-in-Chiefof the Academic Journal of Second Military Medical University, for work on themodifications of the revised manuscript.

Figure 6—The effect of insulin on hepcidin and STAT3 expression is partially mediated by ERK pathway. HepG2 cells were pretreated withPI-3K inhibitor LY294002 (LY; 10 mmol/L) or ERK inhibitor U0126 (U; 1 mmol/L) for 30 min and exposed to 100 nmol/L insulin (I) for 24 h.Protein levels of p-STAT3, STAT3, and hepcidin were analyzed by WB. Data are expressed as means 6 SEM, determined in three in-dependent experiments. One-way ANOVA, followed by LSD t and SNK post hoc test, was performed to analyze difference between thegroups. Significantly different from control, ***P < 0.001; ###compared with the insulin group, P < 0.001; &compared with U group, P <0.05; &&P < 0.01. C, control.

1516 Hepcidin Is Directly Regulated by Insulin Diabetes Volume 63, May 2014

Funding. These studies were supported by funds from the National NaturalScience Foundation of China (81273053).

Duality of Interest. No potential conflicts of interest relevant to thisarticle were reported.

Author Contributions. H.W. was responsible for study concept anddesign, technical or material support, acquisition of data, analysis and inter-pretation of data, drafting of the manuscript, and statistical analysis. H.L., X.J.,W.S., and Z.S. were responsible for acquisition of data and technical support.M.L. was responsible for study concept and design, critical revision of themanuscript for important intellectual content, and obtaining funding. M.L. is theguarantor of this work and, as such, had full access to all the data in the studyand takes responsibility for the integrity of the data and the accuracy of thedata analysis.

References1. Rajpathak SN, Crandall JP, Wylie-Rosett J, Kabat GC, Rohan TE, Hu FB.

The role of iron in type 2 diabetes in humans. Biochim Biophys Acta 2009;1790:671–681

2. Zheng X, Jiang T, Wu H, et al. Hepatic iron stores are increased asassessed by magnetic resonance imaging in a Chinese population withaltered glucose homeostasis. Am J Clin Nutr 2011;94:1012–1019

3. Ren Y, Tian H, Li X, Liang J, Zhao G. Elevated serum ferritin concentrationsin a glucose-impaired population and in normal glucose tolerant first-degree relatives in familial type 2 diabetic pedigrees. Diabetes Care2004;27:622–623

4. Sharifi F, Nasab NM, Zadeh HJ. Elevated serum ferritin concentrations inprediabetic subjects. Diab Vasc Dis Res 2008;5:15–18

5. Silva M, Bonomo LdeF, Oliveira RdeP, Geraldo de Lima W, Silva ME,Pedrosa ML. Effects of the interaction of diabetes and iron supplementationon hepatic and pancreatic tissues, oxidative stress markers, and liverperoxisome proliferator-activated receptor-a expression. J Clin BiochemNutr 2011;49:102–108

6. Simcox JA, McClain DA. Iron and diabetes risk. Cell Metab 2013;17:329–341

7. Forouhi NG, Harding AH, Allison M, et al. Elevated serum ferritin levelspredict new-onset type 2 diabetes: results from the EPIC-Norfolk pro-spective study. Diabetologia 2007;50:949–956

8. Montonen J, Boeing H, Steffen A, et al. Body iron stores and risk of type 2diabetes: results from the European Prospective Investigation into Cancerand Nutrition (EPIC)-Potsdam study. Diabetologia 2012;55:2613–2621

9. Valenti L, Fracanzani AL, Dongiovanni P, et al. Iron depletion by phlebot-omy improves insulin resistance in patients with nonalcoholic fatty liverdisease and hyperferritinemia: evidence from a case-control study. Am JGastroenterol 2007;102:1251–1258

10. Cooksey RC, Jones D, Gabrielsen S, et al. Dietary iron restriction or ironchelation protects from diabetes and loss of b-cell function in the obese(ob/ob lep-/-) mouse. Am J Physiol Endocrinol Metab 2010;298:E1236–E1243

11. Luque-Ramírez M, Alvarez-Blasco F, Botella-Carretero JI, Sanchón R, SanMillán JL, Escobar-Morreale HF. Increased body iron stores of obesewomen with polycystic ovary syndrome are a consequence of insulin re-sistance and hyperinsulinism and are not a result of reduced menstruallosses. Diabetes Care 2007;30:2309–2313

12. Lee DH, Liu DY, Jacobs DR Jr, et al. Common presence of non-transferrin-bound iron among patients with type 2 diabetes. Diabetes Care 2006;29:1090–1095

13. Canturk Z, Cetinarslan B, Tarkun I, Canturk NZ. Serum ferritin levels inpoorly- and well-controlled diabetes mellitus. Endocr Res 2003;29:299–306

14. Shetty JK, Prakash M, Ibrahim MS. Relationship between free iron andglycated hemoglobin in uncontrolled type 2 diabetes patients associatedwith complications. Indian J Clin Biochem 2008;23:67–70

15. Ganz T. Hepcidin and iron regulation, 10 years later. Blood 2011;117:4425–4433

16. Aso Y, Takebayashi K, Wakabayashi S, et al. Relation between serum highmolecular weight adiponectin and serum ferritin or prohepcidin in patientswith type 2 diabetes. Diabetes Res Clin Pract 2010;90:250–255

17. Fernández-Real JM, Equitani F, Moreno JM, Manco M, Ortega F, Ricart W.Study of circulating prohepcidin in association with insulin sensitivity andchanging iron stores. J Clin Endocrinol Metab 2009;94:982–988

18. Reed MJ, Meszaros K, Entes LJ, et al. A new rat model of type 2 diabetes:the fat-fed, streptozotocin-treated rat. Metabolism 2000;49:1390–1394

19. Wang RH, Yu H, Deng CX. A requirement for breast-cancer-associatedgene 1 (BRCA1) in the spindle checkpoint. Proc Natl Acad Sci U S A 2004;101:17108–17113

20. Courselaud B, Pigeon C, Inoue Y, et al. C/EBPalpha regulates hepatictranscription of hepcidin, an antimicrobial peptide and regulator of ironmetabolism. Cross-talk between C/EBP pathway and iron metabolism.J Biol Chem 2002;277:41163–41170

21. Matak P, Chaston TB, Chung B, Srai SK, McKie AT, Sharp PA. Activatedmacrophages induce hepcidin expression in HuH7 hepatoma cells.Haematologica 2009;94:773–780

22. Mirza S, Hossain M, Mathews C, et al. Type 2-diabetes is associated withelevated levels of TNF-alpha, IL-6 and adiponectin and low levels of leptinin a population of Mexican Americans: a cross-sectional study. Cytokine2012;57:136–142

23. Le Guenno G, Chanséaume E, Ruivard M, Morio B, Mazur A. Study of ironmetabolism disturbances in an animal model of insulin resistance. Di-abetes Res Clin Pract 2007;77:363–370

24. Fleming MD. The regulation of hepcidin and its effects on systemic andcellular iron metabolism. Hematology Am Soc Hematol Educ Program2008;151–158

25. Wrighting DM, Andrews NC. Interleukin-6 induces hepcidin expressionthrough STAT3. Blood 2006;108:3204–3209

26. Swaminathan S, Fonseca VA, Alam MG, Shah SV. The role of iron indiabetes and its complications. Diabetes Care 2007;30:1926–1933

27. Nankivell BJ, Tay YC, Boadle RA, Harris DC. Lysosomal iron accumulationin diabetic nephropathy. Ren Fail 1994;16:367–381

28. Zhao N, Sun Z, Mao Y, et al. Myocardial iron metabolism in the regulationof cardiovascular diseases in rats. Cell Physiol Biochem 2010;25:587–594

29. Rowe PA, Kavanagh K, Zhang L, Harwood HJ Jr, Wagner JD. Short-termhyperglycemia increases arterial superoxide production and iron dysreg-ulation in atherosclerotic monkeys. Metabolism 2011;60:1070–1080

30. Silva M, de Brito Magalhães CL, de Paula Oliveira R, Silva ME, Pedrosa ML.Differential expression of iron metabolism proteins in diabetic and diabeticiron-supplemented rat liver. J Biochem Mol Toxicol 2012;26:123–129

31. Saravanan G, Ponmurugan P, Begum MS. Effect of S-allylcysteine, a sul-phur containing amino acid on iron metabolism in streptozotocin induceddiabetic rats. J Trace Elem Med Biol 2013;27:143–147

32. Dogukan A, Sahin N, Tuzcu M, et al. The effects of chromium histidinate onmineral status of serum and tissue in fat-fed and streptozotocin-treatedtype II diabetic rats. Biol Trace Elem Res 2009;131:124–132

33. Kishino Y, Tanaka Y, Ikeda T, et al. Ezetimibe increases hepatic iron levelsin mice fed a high-fat diet. J Pharmacol Exp Ther 2013;345:483–491

34. Sam AH, Busbridge M, Amin A, et al. Hepcidin levels in diabetes mellitusand polycystic ovary syndrome. Diabet Med 2013;30:1495–1499

diabetes.diabetesjournals.org Wang and Associates 1517

35. De Block CE, Van Campenhout CM, De Leeuw IH, et al. Soluble transferrinreceptor level: a new marker of iron deficiency anemia, a common man-ifestation of gastric autoimmunity in type 1 diabetes. Diabetes Care 2000;23:1384–1388

36. De Block CE, De Leeuw IH, Van Gaal LF. Autoimmune gastritis in type 1diabetes: a clinically oriented review. J Clin Endocrinol Metab 2008;93:363–371

37. Hermo JA, González L, Loureiro C. Autoimmune chronic gastritis and irondeficiency anemia. Am J Med 1999;107:401–403

38. Sonnweber T, Ress C, Nairz M, et al. High-fat diet causes iron deficiencyvia hepcidin-independent reduction of duodenal iron absorption. J NutrBiochem 2012;23:1600–1608

39. Tsuchiya H, Ebata Y, Sakabe T, Hama S, Kogure K, Shiota G. High-fat,high-fructose diet induces hepatic iron overload via a hepcidin-independentmechanism prior to the onset of liver steatosis and insulin resistance inmice. Metabolism 2013;62:62–69

40. Jiang F, Sun ZZ, Tang YT, Xu C, Jiao XY. Hepcidin expression and ironparameters change in Type 2 diabetic patients. Diabetes Res Clin Pract2011;93:43–48

41. Mitsuyoshi H, Yasui K, Harano Y, et al. Analysis of hepatic genes involvedin the metabolism of fatty acids and iron in nonalcoholic fatty liver disease.Hepatol Res 2009;39:366–373

42. Aigner E, Felder TK, Oberkofler H, et al. Glucose acts as a regulator ofserum iron by increasing serum hepcidin concentrations. J Nutr Biochem2013;24:112–117

43. Gartsbein M, Alt A, Hashimoto K, Nakajima K, Kuroki T, Tennenbaum T.The role of protein kinase C d activation and STAT3 Ser727 phosphory-lation in insulin-induced keratinocyte proliferation. J Cell Sci 2006;119:470–481

44. Coffer PJ, van Puijenbroek A, Burgering BM, et al. Insulin activates Stat3independently of p21ras-ERK and PI-3K signal transduction. Oncogene1997;15:2529–2539

45. Campos SP, Wang Y, Baumann H. Insulin modulates STAT3 protein acti-vation and gene transcription in hepatic cells. J Biol Chem 1996;271:24418–24424

46. Ihle JN. STATs and MAPKs: obligate or opportunistic partners in signaling.Bioessays 1996b;18:95–98

47. Carvalheira JB, Siloto RM, Ignacchitti I, et al. Insulin modulates leptin-inducedSTAT3 activation in rat hypothalamus. FEBS Lett 2001;500:119–124

48. Hemati N, Ross SE, Erickson RL, Groblewski GE, MacDougald OA. Sig-naling pathways through which insulin regulates CCAAT/enhancer bindingprotein alpha (C/EBPalpha) phosphorylation and gene expression in 3T3-L1adipocytes. Correlation with GLUT4 gene expression. J Biol Chem 1997;272:25913–25919

1518 Hepcidin Is Directly Regulated by Insulin Diabetes Volume 63, May 2014