Iron overload is frequently observed in type 2 diabetes mellitus (DM2), but the underlying mechanisms remain unclear. We hypothesize that hepcidin may be directly regulated by insulin and play an important role in iron overload in DM2. We therefore examined the hepatic iron content, serum iron parameters, intestinal iron absorption, and liver hepcidin expression in rats treated with streptozotocin (STZ), which was given alone or after insulin resistance induced by a high-fat diet. The direct effect of insulin on hepcidin and its molecular mechanisms were furthermore determined in vitro in HepG2 cells. STZ administration caused a significant reduction in liver hepcidin level and a marked increase in intestinal iron absorption and serum and hepatic iron content. Insulin obviously upregulated hepcidin expression in HepG2 cells and enhanced signal transducer and activator of transcription 3 protein synthesis and DNA binding activity. The effect of insulin on hepcidin disappeared when the signal transducer and activator of transcription 3 pathway was blocked and could be partially inhibited by U0126. In conclusion, the current study suggests that hepcidin can be directly regulated by insulin, and the suppressed liver hepcidin synthesis may be an important reason for the iron overload in DM2.
Body iron overload is frequently observed in patients with type 2 diabetes mellitus (DM2) (1,2) or impaired glucose tolerance (IGT) (3,4). Iron overload has been confirmed as an independent factor contributing to the development of DM2 by causing oxidative stress injury in hepatocytes and pancreatic β-cells (5), which may finally lead to insulin resistance (IR) and reduction in insulin extraction and secretion (6). Prospective clinical studies (7,8) have demonstrated that body iron storage is positively correlated with the prevalence of DM2. Bloodletting (9) and iron restriction diet (10) could obviously help control blood glucose, improve insulin sensitivity, and protect against DM2. Nevertheless, the reason for iron accumulation in DM2 patients remains unclear.
Emerging evidence suggests that iron overload in DM2 patients may be related to the loss of insulin signal. Drugs that improve insulin sensitivity can also reduce body iron level (11). In the case of the same iron intake, iron overload deteriorates following the development of DM (12). Besides, free Fe3+ and/or serum ferritin is significantly increased in DM2 patients with poor glycemic control compared with those with good glycemic control (13,14). However, the effect of insulin on body iron metabolism, especially hepatic iron storage, and the underlying mechanisms remain elusive. Hepcidin, a circulatory antimicrobial peptide mainly expressed in the liver, plays a critical role in the regulation of iron metabolism by negatively regulating intestinal iron absorption and macrophage iron release and lowering the level of circulating iron (15). Decreased serum prohepcidin was recently reported in DM2 patients with hyperferritinemia (16,17), suggesting that hepcidin plays a potential role in iron overload in DM2.
In the current study, we examined the hepatic iron content, blood iron parameters, intestinal iron absorption, and liver hepcidin expression in streptozotocin (STZ)-induced diabetic rats and a rat model of IR induced by high-fat diet (HFD) with or without low-dose STZ induction. In addition, we also determined, for the first time, the direct effect of insulin on hepcidin expression and its molecular mechanism in the HepG2 cell line. Our study may help in understanding the effect of insulin on body iron homeostasis and confirm whether and how hepcidin is regulated by insulin, which would help gain new insights into the etiology of iron overload in DM2 and other diseases frequently accompanied with IR, thus helping improve the protection and treatment of iron overload in those diseases.
Research Design and Methods
STZ-Induced Type 1 Diabetic Rats With or Without Insulin Therapy
The animal experiments were approved by the Animal Ethics Committee of the Second Military Medical University in Shanghai, China. Eighteen male Sprague-Dawley rats (160–180 g) were equally divided into three groups: normal control (control), diabetic (STZ), and insulin-treated diabetic group (STZ+Ins). Diabetes was induced by intraperitoneal injection of STZ (75 mg/kg; Sigma-Aldrich, St. Louis, MO). Rats in the control group were injected with the buffer alone. Diabetes was identified in 72 h by fasting blood glucose level >16.7 mmol/L (∼300 mg/dL). After the model was confirmed, rats were fed for another 4 weeks. Neutral insulin (16 units/kg; Novo Nordisk, Copenhagen, Denmark) was administered via subcutaneous injection twice a day at 8:00–10:00 a.m. and p.m. in the last 2 weeks, nonfasting glucose was monitored at the same time, right before the insulin injection, and the average level was determined for further statistical analysis. During the last week of the experiment, four rats in each group were transferred to metabolic cages (Comprehensive Lab Animal Monitoring System; Columbus Instruments, Columbus, OH) in which the amount of food intake and excrement was precisely recorded and/or collected between 9:00 and 10:00 a.m.
STZ-Induced Type 2 Diabetic Rats With or Without Insulin Therapy
The rats in this model were divided into four groups: control group, in which rats were fed a standard diet; the HFD group, in which rats were fed an HFD; the HFD+STZ group, in which rats were fed an HFD for 4 weeks and then injected intraperitoneally with 25 mg/kg STZ; and the HFD+STZ+Ins group, in which rats were first treated similarly as those in the HFD+STZ group and then given a subcutaneous insulin injection (12 units/kg; Novo Nordisk, Copenhagen, Denmark) twice a day at 8:00–10:00 a.m. and p.m. in the last 2 weeks, with nonfasting glucose monitored at the same time, right before the insulin injection, and the average level was determined for further statistical analysis. The HFD (Shanghai Laboratory Animal Center, Chinese Academy of Sciences, National Laboratory Animal Center, Shanghai, China) contained crude protein 22.3/100 g (20% kcal), fat 19.8/100 g (45% kcal), and carbohydrate 44.6/100 g (15% kcal). An intraperitoneal glucose tolerance test (IPGTT) was performed at end of the 4th week before STZ injection. After a 12-h starvation, rats in each group received a glucose solution (50%; 1 g/kg body weight) via intraperitoneal injection. Blood samples were obtained by retro-orbital puncture at 0, 30, 60, 90, and 120 min. Homeostasis model assessment of IR (fasting glycemia [mmol/L] × fasting insulinemia [μUI/mL]/22.5) was calculated, and blood iron parameters were also measured at 0 min. HFD+STZ and HFD+STZ+Ins rats exhibiting fasting glucose levels >7.8 mmol/L (∼140 mg/dL) were considered diabetic, resembling human DM2 (18). The experiment in metabolic cages was performed as described above.
Insulin, Glucose, Interleukin-6, and Interleukin-1β Analysis
Glucose was measured using fresh blood by cutting and pricking the tail (Glucometer Gluco Touch; Roche, Munich, Germany). Serum levels of insulin, interleukin (IL)-6, and IL-1β were measured using radioimmunoassay kits (North Biotechnology, Beijing, China).
Iron Status Parameters
Iron level in the liver, diet, and excrement was quantitated using an atomic absorption spectrophotometer (Z-8100; Hitachi, Tokyo, Japan). The measured diet and excrement iron was used to calculate the apparent iron absorption rate, and the liver iron content was normalized to the wet tissue weight for each sample, respectively. Serum iron concentrations and total iron-binding capacity (TIBC) were determined in nonhemolyzed serum samples using colorimetric analysis kits (Nanjing Jian Cheng Biotechnology Institute, Nanjing, China). The transferrin (Tf) saturation was calculated as plasmatic iron/TIBC and Tf as TIBC/25. Serum ferritin and soluble Tf receptor (sTfR) were determined in nonhemolyzed serum samples using ELISA kits (sTfR, MBS268897, MyBioSource, San Diego, CA; ferritin, SEA518Ra, Life Science Inc., Houston, TX). The apparent iron absorption rate was calculated as ([diet iron intake − iron output in excrement]/diet iron intake) × 100%.
Real-Time Quantitative PCR Analysis
Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA) and reversely transcribed to cDNA by RT Reagent Kit (Primerscript; Takara Bio Inc., Shiga, Japan). Quantitative PCR amplification was performed with the SYBR Green Kit (Takara Bio Inc.) using the Roto-gene RG3000 (Corbett Research, Sydney, Australia), and mRNA levels of specific genes were normalized to the β-actin levels of the same sample.
Homogenates of rat liver and intestine or HepG2 cell lysates 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 Signaling Technology, Danvers, MA); and anti–β-actin (Santa Cruz Biotechnology, Santa Cruz, CA). Signals quantified by densitometry were normalized to β-actin levels or, in the case of phosphoproteins, to the total levels of the same protein.
HepG2 Culture, Treatment, and Small Interfering RNA Transfection
HepG2 (Chinese Academy of Sciences, Shanghai, China) cells were grown in high-glucose DMEM supplemented with 10% FBS and 1% antibiotic solution (Gibco, Grand Island, NY) and incubated in a humidified 5% CO2 atmosphere at 37°C. Cells were transferred to six-well plates and treated with 0.1, 1, 10, and 100 nmol/L insulin for 24 h or with 100 nmol/L insulin for 4, 8, 12, and 24 h. For SMAD4 and STAT3 silence, cells were transfected with SMAD4 and STAT3 small interfering RNA (siRNA) products (sc-29484 and sc-29493; Santa Cruz Biotechnology) that generally consist of pools of three to five target-specific 19–25 nucleotide siRNAs designed to knock down gene expression or negative control oligonucleotides (Santa Cruz Biotechnology) in the presence of Lipofectamine RNAiMAX, according to the manufacturer’s instructions (Invitrogen). Cells were maintained for 24 h after transfection and given 100 nmol/L insulin for another 24 h. For insulin pathway inhibitors, HepG2 was pretreated with LY294002 (10 μmol/L) or U0126 (1 μmol/L) for 30 min and then treated with 100 nmol/L insulin for 24 h.
Chromatin immunoprecipitation (ChIP) assay was performed as described (19). The antibodies against SMAD4 and STAT3 were purchased from Cell Signaling Technology. The primers used for PCR were Hepc forward, 5′-CGCT CTGT TCCC GCTT AT-3′, and Hepc reverse, 5′-CGAG TGAC AGTC GCTT TTAT G-3′, that amplified 129 bp 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 hepcidin promoter was performed by obtaining genomic DNA from HepG2 cells and cloning the proximal 942 bp of a human HAMP promoter into the pGL3-basic luciferase reporter vector, as described by Courselaud et al. (20). Site-directed mutagenesis (Quickchange II; Stratagene, Stockport, U.K.) on signal transducer and activator of transcription (STAT3) response element and putative bone morphogenetic protein responsive element were as described by Matak et al. (21). HepG2 cells were transfected with certain HAMP reporter constructs or the empty pGL3-basic vector using Superfectin II (Invitrogen), according to the manufacturer’s instructions. To normalize for the transfection efficiency, an internal control–pRL-SV40 Renilla luciferase plasmid (Promega) was cotransfected alongside the HAMP constructs in a 1:200 ratio in serum-free medium. After 24-h equilibration, cells were treated with insulin for an additional 24 h, and luciferase activity was determined in triplicate in at least two independent experiments using the Dual Luciferase Reporter Assay, according to the manufacturer’s instructions (Promega).
Data are represented as mean ± SEM. Statistical analysis was performed using the Statview software (SAS Institute Inc., Cary, NC). Statistical difference between two groups was assessed by the independent t test. One-way ANOVA, followed by least significant difference (LSD) t and Student-Newman-Keuls (SNK) post hoc test, was performed to analyze the difference between the three or more groups. Repeated-measures ANOVA was performed to estimate the effect of group and time (date) on values obtained during the experiments of IPGTT or metabolic cages. Differences were considered as significant at P < 0.05.
Effect of STZ Administration and HFD on Body Weight, Fasting Glucose, and/or Glucose Tolerance
Rats exhibited diabetic profiles with decreased body weights, hyperglycemia (Fig. 1A and B), and hypoinsulinemia (Table 1) 72 h after simple STZ injection. Supplementation of insulin restored the fasting blood glucose and body weight to the normal levels (Fig. 1A). All rats in the model of STZ-induced DM2 received an IPGTT at the end of the 4th week, right before the additional STZ injection, and serum glucose and insulin were measured before and 30, 60, 90, and 120 min after injection of 50% glucose solution (1 g/kg body weight). Except for the normal fasting blood glucose, a significant increase in glycemia and insulinemia was observed at different IPGTT times (Fig. 2C and D), and the homeostasis model assessment of IR index increased by about twofold in the rats of the HFD, HFD+STZ, and HFD+STZ+Ins groups (Supplementary Table 1). After establishment of the IR model, rats in the HFD+STZ and HFD+STZ+Ins groups developed hyperglycemia and continuous weight reduction 72 h after intraperitoneal injection of 25 mg/kg body weight STZ (Fig. 2A and B). Insulin therapy obviously increased the body weight and controlled the blood glucose at a normal level (Fig. 2A and B). Serum ketone body was also measured in the current study (Supplementary Fig. 1). STZ rats exhibited a significantly higher serum level of ketone body than the control and STZ+Ins rats (P < 0.001), and the latter two groups showed no difference. In the animal model of DM2, we found no significant change among the four groups (Supplementary Fig. 1).
Effect of STZ Administration and HFD on Iron Parameters, Serum IL-6, IL-1β, or Hemoglobin
Both liver iron content and serum iron were increased in STZ-induced diabetic rats (148.44 ± 30.28 μg/g and 97.43 ± 4.59 μmol/L vs. 68.98 ± 18.96 μg/g and 69.45 ± 11.32 μmol/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 similar change was also found in serum ferritin, which was obviously 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 serum sTfR 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 range without lowering the high level of Tf in STZ rats, making Tf saturation (serum iron/TIBC ratio) in insulin-treated rats even lower than that in normal control rats (P < 0.01; Table 1). Although a correlation was suggested between IL-6 and DM2 (22), we failed to observe a significant difference in serum IL-6 level or IL-1β, a more sensitive indicator of inflammation, among the three groups (Table 1), and we only found a twofold increase in liver mRNA expression of both genes (Supplementary Fig. 2).
Serum samples of control, HFD, HFD+STZ, and HFD+STZ+Ins rats before STZ injection were gained in the IPGTT at the point of 0 min, and the iron status was analyzed. HFD+STZ and HFD+STZ+Ins rats showed no difference in blood iron parameters before receiving an STZ injection (Supplementary Table 2). However, there was a 96% increase in hepatic iron content and a 65% increase in serum iron in HFD+STZ rats as compared with the control group (147.98 ± 25.98 vs. 75.33 ± 14.68 μg/g, P < 0.001; 67.40 ± 4.52 vs. 40.78 ± 2.16 μmol/L, P < 0.001; Table 2) when the whole experiment was finished, and insulin therapy could significantly lower the level of liver iron (P < 0.05) and serum iron (P < 0.001; Table 2). Serum ferritin and sTfR were significantly 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 significantly reduced by insulin supplement (HFD+STZ+Ins vs. HFD+STZ, P < 0.001; Table 2), and sTfR also began to recover, although with no statistical significance by the end of the experiment. HFD+STZ exhibited higher Tf saturation (vs. control, P = 0.001; Table 2), but had no change on TIBC and serum Tf. In addition, although enhanced erythropoiesis was once observed in HFD-fed rats (23), we found no change in hemoglobin (Hb) in either the HFD or HFD+STZ group (Table 2).
Alteration of Hepatic Hepcidin, STAT3, Intestinal Fpn, and Apparent Intestinal Iron Absorption Rate in Different Animal Models
According to the WB results, we found that STZ rats had a twofold increase in intestinal Fpn expression as compared with the control group (P < 0.01; Fig. 1C), which was accompanied by a 40% decrease in both hepcidin and STAT3 content in liver (P < 0.01 and P < 0.05, respectively; Fig. 1C), and no alteration in these three proteins was found in the insulin treatment group (Fig. 1C). Intestinal Fpn expression of HFD+STZ rats increased by 70% (P < 0.01; Fig. 2E), followed by a 30 and 50% reduction in liver STAT3 and hepcidin content (P < 0.05 and P < 0.001, respectively; Fig. 2E). There was no significant difference in the three proteins among the other three groups (Fig. 2E).
Apparent iron absorption rate was calculated by the amount of food intake and excrement, iron content in diet (110 and 130 mg/kg in the control natural diet and the HFD), and iron content in the excrement. There was a mean of 50% increase (range 65–80%) in intestinal iron absorption in STZ rats as compared with the normal and insulin-treated rats, and the difference was not significant between the normal and insulin-treated rats (P > 0.05; Fig. 1D). Intestinal iron apparent absorption in HFD rats was similar to that in control rats, at a level between 40 and 60%, significantly lower than the mean value of 75% in HFD+STZ rats (P < 0.05; Fig. 2F). Insulin treatment markedly controlled the high rate of intestinal iron absorption and kept it at a normal range from 35 to 55%, with no statistical difference from the control and HFD rats.
Insulin Upregulates Hepcidin and Total STAT3 Expression but Has No Effect on SMAD4 in HepG2 Cells
HepG2 cells were treated with insulin in both time and concentration gradient. Compared with the blank control group, hepcidin protein and mRNA expression was markedly increased by insulin treatment (P < 0.001, Fig. 3A; P < 0.001, Fig. 3B). SMAD4 and STAT3 are classic positive transcription regulators of hepcidin that are activated by binding to p-SMAD1/5/8 and phosphorylation (24,25), respectively. Insulin markedly increased the level of both phosphorylated and total STAT3 (P < 0.001; Fig. 4A and B), while it did not influence the phosphorylation ratio of STAT3 and had no effect on protein expression of SMAD4, p-SMAD1/5/8, and SMAD1 (Fig. 4A and B).
Insulin Stimulates Hepcidin Synthesis Through Activation of STAT3 but Not SMAD4, Partially Mediated by the Extracellular Signal–Related Kinase Pathway
The result of ChIP showed that the amount of STAT3 binding to hepcidin promoter increased by twofold as compared with the control group (P < 0.05; Fig. 5A) when HepG2 cells were treated with 100 nmol/L insulin for 24 h, but the amount of SMAD4 remained unchanged (Fig. 5A).
Basal fluorescence activity was reduced substantially because of the mutant STAT3 or SMAD4 binding site (P < 0.001; Fig. 5B). After treatment with 100 nmol/L insulin for 24 h, the fluorescence activity of the reporter gene of full-length hepcidin promoter and that with mutant SMAD4 binding sites was significantly stimulated (P < 0.05, Fig. 5B), but there was no increase in that with mutation of STAT3 response elements (Fig. 5B).
We interfered SMAD4 and STAT3 protein synthesis by transfecting specific siRNA into HepG2 cells (ΔD, for SMAD4 siRNA, compared with control, P < 0.001; ΔT, for STAT3 siRNA, compared with control, P < 0.001; Fig. 5C) and then treated the cells with 100 nmol/L insulin for 24 h. Hepcidin continued to elevate when SMAD4 was silenced, but remained unchanged after knockdown of STAT3 (Fig. 5C).
To observe the mediating pathway of insulin regulation on total STAT3 and hepcidin, HepG2 cells were pretreated with LY294002 (10 μmol/L) or U0126 (1 μmol/L) for 30 min and then exposed to 100 nmol/L insulin for 24 h (Fig. 6). Upregulation of hepcidin, phosphorylated, and total STAT3 by insulin was partially lowered by U0126 (U0126+Ins vs. Ins, P < 0.001; U0126+Ins vs. U0126, P < 0.01 or P < 0.05), but not suppressed in the presence of LY294002 (LY294002+Ins vs. Ins, P > 0.05).
Iron overload was frequently observed in patients with DM2 (1,2) and even those with IGT (3,4), in close association with the development of DM2 and its complications (26). However, pathways underlying iron accumulation in DM2 are still poorly understood. In the current study, we demonstrated that STZ-induced hypoinsulinemia significantly reduced hepcidin expression in the liver, leading to an abnormally high content of Fpn in the intestine and elevation of serum iron level and hepatic iron content, which could be conversed by recovering the liver hepcidin level through insulin therapy, suggesting that hepcidin is decreased by loss of insulin signal and plays an important role in iron overload in DM2. We also found that insulin could directly stimulate hepcidin expression in HepG2 cells through activation of STAT3 but not SMAD4, which was partially mediated by the extracellular signal–related kinase (ERK) pathway and independent of the phosphatidylinositol 3-kinase (PI-3K) pathway.
Rats with STZ-induced diabetes (STZ or HFD+STZ group) exhibited mild to moderate hepatic iron overload (HIO), accompanied with significantly elevated serum iron and ferritin and markedly decreased serum sTfR. STZ-induced elevation of iron storage was also observed in the proximal tubular lysosomes (27), myocardium (28), and artery (29), while studies of Silva et al. (30) and Saravanan et al. (31) showed that serum iron was increased in STZ rats. Additionally, Dogukan et al. (32) reported that STZ injection following HFD induction markedly increased the hepatic and serum iron levels. Our results demonstrated that insulin therapy effectively released HIO, manifested by the recovery of liver iron content and serum ferritin and the improvement tendency of serum sTfR, and lowered serum iron to normal level in STZ-induced both type 1 and type 2 diabetic rats. In addition, simple HFD induction that led to IR but did not elevate fasting blood glucose had no influence on liver or serum iron, which was not reported in other studies (23,33). These findings suggest that body iron homeostasis may be closely related to insulin signaling. Deficiency of insulin signaling could lead to HIO, probably through loss of restricted control of serum iron level, and an HFD seemed to have no impact on iron metabolism if it did not cause insulin decompensation, as reflected by increased fasting blood glucose. Different from 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 also reported not changed (34). Autoimmune gastritis in type 1 diabetes mellitus, caused by its specific autoimmune disorder (36), takes the most responsibility for the iron deficiency (37), and the inflammation may contribute to maintain hepcidin synthesis (24). Time of HFD induction was once considered a factor that may restrict its effect on liver iron content (23). However, hepatic iron level remained unchanged when the induction period was prolonged to 8 weeks in our experiment or 12 weeks in other studies (38). Other factors may include the difference in the species of rats or mice used in research or methods used to assay liver iron content, which may to some extent explain the finding of increased liver iron content induced by HFD in a report by Tsuchiya et al. (39).
Hepatic hepcidin was reduced significantly, while intestinal Fpn expression and apparent intestinal iron absorption were elevated significantly in STZ and HFD+STZ rats, but HFD rats showed no alteration on the above parameters. Insulin supplementation eliminated these abnormal changes in STZ and HFD+STZ rats, indicating that hepcidin, which negatively regulates Fpn and iron absorption and restricts the level of serum iron, may be positively regulated by insulin. It was also found in other HFD rat models that hepcidin expression remained unchanged (33,38) or began decreasing only when fasting blood glucose was increased (39). Clinical studies (16,17,34) showed that the prohepcidin or hepcidin level in blood was significantly decreased in DM2 patients with hyperferritinemia. All of these findings support the results of the current study. The failure to maintain or increase hepcidin synthesis, probably caused by loss of insulin signal, may be of critical importance in iron accumulation in DM2 patients. Sam et al. (34) demonstrated that IR, not insufficient insulin, caused the decrease of hepcidin and HIO in patients with DM2, in a recent clinical study including patients with polycystic ovary syndrome, type 1 diabetes mellitus, and DM2. Insulin treatment may allow us to gain a deeper insight into the hepcidin regulator in DM2, because it only replenishes insulin level but cannot radically improve IR. Insulin therapy may recover the decreased hepcidin level and release iron overload if it is caused by insufficient insulin signals, which has been already proved in our experiment. Effect of insulin therapy on iron overload and hepcidin reduction in patients with DM2 should be further determined in the next step. Hepcidin was also reported to be increased (40) or not changed (2) in DM2 patients. A bigger sample size and calculation of the hepcidin/ferritin ratio (41) may help us better understand the status of hepcidin synthesis. Erythropoiesis was once hypothesized to mediate insulin regulation on iron metabolism (23). However, no significant change in Hb was observed in the current study. Clinical studies (2,17) also reported that Hb remained unchanged in DM2 or IGT patients. In addition, hyperglycemia may be also involved in hepcidin regulation. It was recently reported that an oral glucose tolerance test could induce the elevation of serum hepcidin in healthy volunteers (42). This effect of glucose on hepcidin may partially be due to the boost of insulin activity. However, confounding factors such as hyperglycemia need to be further excluded by in vitro experiments so as to better understand the effect of insulin on hepcidin.
To further confirm the effect of insulin on hepcidin expression, we observed the regulatory effect of insulin on hepcidin in the HepG2 cell line in vitro and found that this effect was in a time- and dose-dependent manner. Hepcidin mRNA and protein were markedly upregulated by insulin treatment, indicating a direct and significant stimulatory effect of insulin on hepcidin expression. These results are consistent with the data that we previously found in animal experiments, which demonstrated that the reduced liver hepcidin content was correlated with STZ-induced hypoinsulinemia. To better understand how hepcidin was modulated by insulin, we also detected the levels of phosphorylated and total proteins of STAT3 in HepG2 cells treated with insulin. STAT3 is a novel transcription regulator of hepcidin and activated through phosphorylation by IL-6 (24). Our results showed that insulin could markedly enhance the protein expression of phosphorylated and total STAT3 and increase the DNA binding activity of STAT3, but it had no effect on the p-STAT3/STAT3 ratio. In addition, interruption of STAT3 expression by silencing RNA completely repressed the upregulatory effect of insulin on hepcidin, and the activity of the reporter gene of the hepcidin promoter with mutant STAT3 binding sites could no longer be stimulated by insulin, suggesting that insulin may regulate hepcidin specifically through activation of STAT3.
It was reported that insulin induced STAT3 phosphorylation (43) and enhanced its DNA binding activity (44). However, total STAT3 was not detectable (44) or showed no change (43). Some studies even argued that insulin could inhibit STAT3 protein expression as well as the activation of STAT3 via IL-6–induced phosphorylation (45). To further confirm how STAT3 was changed in hypoinsulinemia-induced iron overload, we supplemented WB analysis of total STAT3 in samples of STZ-induced diabetic rats. Liver total STAT3 expression was significantly decreased in STZ rats and entirely reversed by insulin therapy, which appeared thoroughly consistent with the alteration of hepcidin, suggesting that total STAT3 may be regulated by insulin in vivo and involved in its regulation on hepcidin. In addition, it is also worth mentioning that although some studies suggested a correlation between inflammation and DM (22), we found neither serum IL-6 nor IL-1β produced a significant change; only their mRNA expression levels had a twofold increase in the liver. These results indicate that STZ treatment may lead to inflammatory reaction in diabetic rats but, at least in the current study, only to a relatively mild extent.
ERK and PI-3K are believed to be involved in the activation of insulin on STAT3 (46). To clarify the pathway that mediates the effect of insulin on hepcidin and confirm the role of STAT3, we used inhibitors of the above two classical pathways and found that ERK inhibitor U0126 could significantly but partially suppress hepcidin response to insulin, while PI-3K inhibitor LY294002 had no effect on it. In addition, the increased phosphorylated and total STAT3 expression induced by insulin could be partially and significantly inhibited by U0126, but it was not affected by LY294002. This presentation was fully consistent with hepcidin, indicating that the regulatory effect of insulin on hepcidin may be mediated specifically by STAT3. It was found that Janus kinase 2 also participated in mediating insulin activation on STAT3 (47), which may explain the partial but not absolute inhibitory effect of U0126 on insulin-stimulated upregulation of total STAT3 and hepcidin.
In addition to STAT3, C/EBPα and SMADs are also classic hepcidin transcription regulators. It was found (48) that the expression and phosphorylation of C/EBPα was inhibited by insulin, suggesting that C/EBPα is not involved in the regulatory effect of insulin on hepcidin. In the current study, we found that insulin had no effect on the expression of total and/or p-SMAD1 and SMAD4 or on the DNA-binding activity of SMAD4. In addition, insulin kept increasing the hepcidin expression when SMAD4 protein synthesis was inhibited by siRNA, and point mutation of SMAD4 binding elements also showed no inhibitory effect on insulin enhancing the hepcidin promoter activity. Therefore, we conclude that STAT3, rather than the SMAD family, specifically and directly mediated the regulatory effect of insulin on hepcidin expression.
In conclusion, our study has demonstrated that hepcidin can be directly regulated by insulin selectively through STAT3 and partially through mediation of the ERK pathway, thus playing an important role in iron overload in DM2. Iron restriction should be considered once IGT or diabetes is confirmed. The results of the current study also suggest that, besides effective insulin treatment, hepcidin and/or a STAT3 stimulator may prove to be potential drug targets for the therapy of iron overload in DM2.
Acknowledgments. The authors thank Danghui Yu, the Editor-in-Chief of the Academic Journal of Second Military Medical University, for work on the modifications of the revised manuscript.
Funding. These studies were supported by funds from the National Natural Science Foundation of China (81273053).
Duality of Interest. No potential conflicts of interest relevant to this article were reported.
Author Contributions. H.W. was responsible for study concept and design, technical or material support, acquisition of data, analysis and interpretation 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 the manuscript for important intellectual content, and obtaining funding. M.L. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.