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© 2007 American Society for Nutrition J. Nutr. 137:2366-2370, November 2007

Biochemical, Molecular, and Genetic Mechanisms

Leptin Increases the Expression of the Iron Regulatory Hormone Hepcidin in HuH7 Human Hepatoma Cells1,2

Bomee Chung, Pavle Matak, Andrew T. McKie and Paul Sharp*

Nutritional Sciences Division, King's College London, London SE1 9NH, UK

* To whom correspondence should be addressed. E-mail: paul.a.sharp{at}kcl.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Obesity is a major global health problem and is associated with low-grade inflammation and, in a number of cases, poor iron status. We speculated that the adipokine leptin might play a role in regulating iron metabolism in the overweight population because it shares a number of common biological features with IL-6, a major factor in the development of the anemia of chronic disease via its stimulatory actions on the production and release of the iron regulatory hormone hepcidin. To test this hypothesis, we exposed HuH7 human hepatoma cells to leptin and measured hepcidin mRNA expression by quantitative PCR. HuH7 cells were also transfected with a hepcidin promoter–luciferase reporter gene construct to investigate transcriptional regulation of hepcidin. In leptin-treated cells, hepcidin mRNA expression was enhanced significantly. Preincubation with a Janus kinase (JAK) 2 inhibitor significantly diminished this response. Hepcidin promoter activity was also increased in the presence of leptin. This effect was decreased either by mutation of the signal transducer and activator of transcription (STAT) 3 binding motif in the hepcidin promoter or by coexpressing a dominant-negative STAT3 mutant. These data suggest that leptin upregulates hepatic hepcidin expression through the JAK2/STAT3 signaling pathway. As a consequence, the increased production of leptin in overweight individuals might be a major contributor to the aberrant iron status observed in these population groups.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
The global incidence of obesity has increased dramatically over the past 50 y. Currently >1 billion people are thought to have a BMI >30 kg·m–2 (1), with the number of those affected projected to increase dramatically over the next 30 y. Obesity is characterized by the presence of chronic low-grade inflammation (2–4) and an increased risk of developing a number of chronic diseases, such as insulin resistance and type 2 diabetes (5).

The link between chronic diseases and anemia is well characterized (6). The expression of hepcidin, a 25 amino acid peptide hormone, in the liver is increased dramatically by inflammation and because of chronic disease (7–9). Proinflammatory cytokines, such as IL-6 and IL-1, (10–13) are thought to be central to this mechanism. IL-6 in particular has been shown to exert its effects on hepcidin gene transcription via Janus kinase (JAK)/signal transducer and activator of transcription (STAT)3 interactions (14–16), which are essential to cytokine receptor signaling (17). Once released, hepcidin is thought to bind to the iron efflux protein ferroportin (18) and thereby act as a negative regulator of body iron homeostasis, inhibiting the release of iron recycled from senescent red blood cells by reticuloendothelial macrophages (19) and the absorption of dietary iron by intestinal enterocytes (20,21).

A number of studies have noted an association between being overweight or obese and having poor iron status (22–28). Adipose tissue is an active endocrine organ and releases a number of cytokines and adipokines (29,30), which may in turn influence iron metabolism. Leptin, the first adipokine to be discovered (31), is intriguing in this regard for 3 reasons: 1) it belongs to the family of long-chain helical cytokines (32); 2) its circulating levels are proportional to fat mass (33); and 3) its membrane receptors exhibit structural similarity to class I cytokine receptors (34,35). Interestingly, this class of receptors also includes the gp130 subunit of the IL-6 receptor family, suggesting that IL-6 and leptin may operate via a similar mode of action (36). Therefore, leptin might be part of the axis that links obesity, inflammation, and hepcidin release with aberrant iron metabolism. As a first step in establishing a possible role for leptin in the regulation of iron metabolism, we have investigated its ability to influence hepcidin expression via JAK/STAT signaling in a well-characterized human hepatoma cell line.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Cell culture. HuH7 human hepatoma cells were grown in DMEM containing 10% fetal bovine serum and were used for experiments at 80% confluence. Cells were incubated in the absence or presence of leptin (7.5–125 nmol/L; R&D Systems) for up to 24 h. In some experiments, cells were preincubated overnight with the JAK2 inhibitor 1,2,3,4,5,6-hexabromocyclohexane (HBCH; 50 µmol/L; Merck Biosciences) prior to leptin treatment.

    Real-time quantitative PCR. Total RNA was isolated from HuH7 cells using Trizol reagent (Invitrogen). Following first strand synthesis, expression levels of hepcidin and 18S (used as a housekeeper gene) mRNA were analyzed by real-time PCR using an ABI Prism 7000HT PCR cycler with gene-specific primers (21,37) and a Quanti-Tect SYBR Green PCR kit (Qiagen), used according to the manufacturer's protocol. Quantitative measurements of each gene were derived from a standard curve constructed from known concentrations of PCR product. Because of the heterogeneity in group variances, data were log transformed prior to statistical analysis and expressed as the hepcidin:18S ratio.

Expression of leptin receptors (the long and short forms) in HuH7 cells was confirmed by RT-PCR amplification for 36 cycles, using an MJ Research PTC-200 DNA engine thermal cycler and the primer sequences published by Bennett et al. (38). Hypoxanthine phosphoribosyltransferase mRNA expression (21) was used as a housekeeper gene in these studies. PCR products were stained with ethidium bromide on 1% agarose gel and visualized under UV light.

    Western blotting. HuH7 cell monolayers were lysed with ice-cold lysis buffer (10 mmol/L Tris-HCl pH 7.5, 50 mmol/L NaCl, 1% Triton X-100, 20% glycerol, 1 mmol/L EDTA, 1 mmol/L Na3VO4, 10 mg/L protease inhibitor cocktail). Lysates were precleared of insoluble membrane material and nuclei by centrifugation (13,000 x g for 10 min) and subjected to Western blotting using an antiphospho-STAT3 antibody (1:1000 dilution; SC-8059, Santa Cruz Biotechnology). Following incubation with a horseradish peroxidase–conjugated secondary antibody (1:2000 dilution; Dako), cross-reactivity was detected using ECL Plus and Hyperfilm ECL (GE Healthcare), according to the manufacturer's instructions. At the end of the experiment, membranes were stripped (Western Stripping Buffer, Perbio Science) and reprobed with antibodies recognizing total cellular STAT3 (1:1000 dilution; SC-482, Santa Cruz Biotechnology), which acted as a control for protein loading.

Leptin receptor isoforms in HuH7 cells were also identified by Western blotting, using an antibody that recognized all receptor subtypes (1:1000 dilution; SC-1834, Santa Cruz Biotechnology).

Hepcidin peptide production by HuH7 cells in response to leptin treatment (75 nmol/L; 0–24 h) was assessed using an antihepcidin antibody (1:1000 dilution; a gift from Professor Kaila Srai, University College, London, UK) raised against a synthetic peptide corresponding to the 25 amino acid residues of the mature form of human hepcidin. This antibody was used previously to detect hepcidin protein in human tissue samples (26).

    Hepcidin promoter. A 624-base-pair (bp) nucleotide fragment of the 5' flanking region of the human hepcidin antimicrobial peptide gene was amplified from genomic DNA extracted from HepG2 cells using the following primers: forward 5'-GGCATACGCGTCTGTGCTGGGCCATATTACTGCTGTC-3' and reverse 5'-CATCGTAACGCGTGTACTCATCGGACTGTAGATGTTAGC-3'. The promoter fragment was subcloned into pGL3-basic vector (Promega) upstream of the firefly luciferase reporter gene between the MluI and XhoI restriction sites. A second hepcidin promoter construct was generated in which the STAT3 binding motif was mutated [TTC to GGA, (14)] using QuikChangeII Site-Directed Mutagenesis kit (Stratagene). Both the wild-type (wt) and STAT3 mutant promoters were sent for DNA sequencing (Cogenics) to ensure that they conformed to the published sequences (14,15).

    Cell transfection and reporter assays. HuH7 cells were transfected with either the wt or the STAT3 mutant hepcidin-luciferase construct or the empty pGL3-basic vector, using Fugene6 (Roche) according to the manufacturer's instructions. As a normalization control, the pRL-SV40 Renilla luciferase plasmid (Promega) was cotransfected alongside the hepcidin constructs in a 1:50 ratio. In some experiments, a dominant-negative STAT3 mutant (dnSTAT3) vector was cointroduced. After 24 h, cells were treated with leptin (for 4 h), and luciferase activity was determined in triplicate using the Promega Dual Luciferase Reporter Assay, according to the manufacturer's instructions.

    Statistical analysis. Statistical differences (P < 0.05) among groups were determined by either a 1-way or a 2-way ANOVA, followed by Tukey's post hoc test when F-test was significant at P < 0.05.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
HuH7 hepatoma cells incubated in the presence of leptin exhibited a rapid (evident within 1 h of leptin treatment) increase in hepcidin mRNA expression (Fig. 1A). However, hepcidin levels declined back toward baseline after 24 h of leptin treatment. All further measurements of hepcidin mRNA expression were made following leptin stimulation for 4 h. The leptin-induced increase in hepcidin mRNA level was dose dependent and was significantly increased at leptin concentrations above 50 nmol/L, compared with untreated control cells (Fig. 1B). In agreement with the effects of leptin on hepcidin mRNA expression, hepcidin protein levels in HuH7 cells were also increased in response to leptin treatment (Fig. 1C). The stimulatory action of leptin on hepcidin mRNA was still evident in the presence of the endotoxin inhibitor polymyxin B (1 µmol/L; data not shown), suggesting that potential contamination of the recombinant leptin with bacterial endotoxin was not responsible for these effects.


Figure 1
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  		FIGURE 1  Leptin increased hepcidin expression in HuH7 hepatoma cells. Leptin (75 nmol/L) induced a rapid and significant increase in hepcidin mRNA abundance. Hepcidin expression declined back to baseline levels after 24 h exposure to leptin (A). Hepcidin mRNA expression was significantly increased at leptin concentrations above 50 nmol/L compared with the untreated control cells (B). Hepcidin protein levels in HuH7 cells were increased in response to leptin (75 nmol/L) (C). Real-time PCR data are means ± SEM from 4 separate experiments. Means without a common letter differ, P < 0.05 (1-way ANOVA; Tukey's post hoc test).

 
Alternate splicing of the leptin receptor gene can give rise to a number of receptor transcripts (34). However, only the long receptor isoform, Ob-Rb, is capable of signaling via the classical cytokine-stimulated JAK/STAT pathway (34). To determine whether Ob-Rb was expressed in HuH7 cells, we used a combination of RT-PCR and Western blotting. Using receptor isoform-specific primers, we were able to detect the mRNA expression of both Ob-Rb and Ob-Ra (shorter receptor isoforms) in HuH7 cells (Fig. 2A). Similarly, Western blotting of HuH7 cell proteins with an antibody that recognized all leptin receptor isoforms produced 2 bands: a major band at 120 kDa that corresponded with the predicted molecular weight of Ob-Rb and a fainter band at ~83 kDa that was consistent with the molecular weight of the shorter Ob-Ra receptor (Fig. 2B).


Figure 2
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  		FIGURE 2  Leptin receptor isoforms in HuH7 cells. PCR analysis (36 cycles) of Ob receptor isoforms revealed that HuH7 cells express both the long form (Ob-Rb) and the short form (Ob-Ra) of the leptin receptor (A). Western blotting of membrane protein from 2 independent HuH7 cell cultures using an antileptin receptor antibody that cross reacts with all leptin receptor isoforms (B).

 
Because Ob-Rb is the predominant receptor protein isoform expressed in HuH7 cells, we next investigated whether leptin regulates hepcidin expression through Ob-Rb and JAK/STAT signaling. In support of the involvement of this pathway, incubation of HuH7 cells with leptin (75 nmol/L) for 20 min led to a rapid increase in the cytosolic levels of phosphorylated STAT3 (p-STAT3). This increase was diminished by preincubation with the JAK2 inhibitor HBCH (Fig. 3A). Similarly, the leptin-induced increase in hepcidin mRNA expression in HuH7 cells was significantly decreased by preincubation with HBCH (Fig. 3B). Taken together, these data suggest that leptin regulates hepcidin expression in HuH7 hepatoma cells by activating Ob-Rb and the JAK2/STAT3 pathway.


Figure 3
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  		FIGURE 3  Leptin activates the JAK2/STAT3 signaling pathway in HuH7 cells. Western blotting of HuH7 cell lysates revealed that incubation with leptin (75 nmol/L) for 20 min caused a dramatic rise in p-STAT3. Preincubation of HuH7 cells with the JAK2 inhibitor HBCH diminished this response. Blots were stripped and reprobed for total STAT3 protein, which acted as the protein loading control (A). Leptin (75 nmol/L for 4 h) significantly increased hepcidin mRNA expression. This effect was significantly diminished in the presence of HBCH. Incubation with HBCH alone had no affect on basal levels of hepcidin mRNA (B). Real-time PCR data are means ± SEM from 4 separate experiments. Means without a common letter differ, P < 0.05 (1-way ANOVA; Tukey's post hoc test).

 
In previous work, researchers have shown that the hepcidin promoter contains a consensus STAT3 binding site (14–16). To functionally test whether leptin promotes the binding of STAT3 to this known motif [present at position –72 to –64 bp relative to the start of gene transcription (15)], we cloned a 624-bp fragment of the hepcidin promoter upstream of a luciferase reporter gene. Two constructs were made by inserting either the wt promoter or a hepcidin promoter containing a mutation in the STAT3 binding motif. While leptin had no affect on cells transfected with the empty pGL3-basic vector (data not shown), it significantly activated luciferase transcription in HuH7 cells transfected with the 0.6 kb wt hepcidin promoter construct (Fig. 4B). In cells transfected with the STAT3 mutant hepcidin promoter construct, leptin (75 nmol/L) caused an increase in luciferase activity; however it was significantly diminished compared with the leptin-mediated increase in luciferase activity of the wt promoter (Fig. 4B). There was a significant affect of the promoter (P < 0.001), leptin treatment (P < 0.001), and promoter X leptin (P < 0.001) on luciferase activity.


Figure 4
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  		FIGURE 4  Leptin increased hepcidin promoter activity via a STAT3-dependent mechanism. Luciferase activity of the wt hepcidin promoter construct was significantly increased in the presence of leptin (75 nmol/L). When cells were transfected with the STAT3 mutant construct, luciferase activity was still significantly increased in the presence of leptin, but was significantly diminished compared with the activity of the wt promoter (A). Data were analyzed by 2-way ANOVA and Tukey's post hoc test, n = 4 per group. The response to leptin was inhibited when cells were cotransfected with increasing concentrations of a dnSTAT3 expressing vector (B). Data were analyzed using 1-way ANOVA and Tukey's post hoc test, n = 3 per group. Data are presented as means ± SEM. Means without a common letter differ, P < 0.05.

 
In addition, coexpression of a vector encoding a dnSTAT3 completely inhibited the response to leptin (Fig. 4C). Interestingly, with increasing concentrations of the dnSTAT3, luciferase activity was significantly lower than in untreated cells, supporting previous assertions that constitutive STAT3 activity is required for the basal expression of hepcidin (14–16).


   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
Sustained obesity is known to have serious pathogenic consequences in a number of body tissues and organs, including the liver, where it is associated with an increased incidence of nonalcoholic steatohepatitis and an elevated prevalence of hepatocellular carcinoma (39,40). These effects are thought to be mediated in part by elevated levels of leptin seen in obese subjects, which increase in proportion to fat mass (33). In vitro, leptin was shown to promote cell proliferation and migration in hepatoma cell lines (41) and is thought to be central to the progression from nonalcoholic steatohepatitis, through fibrosis, to hepatic cancer in obese subjects (42).

In addition to liver disease, there is an increasing body of evidence that suggests a direct link between being overweight and having poor iron status (22–28). The hypoferremia noted in obese subjects appeared to arise from a combination of 2 distinct mechanisms: 1) the development of iron deficiency (27,28) and 2) the presence of chronic low-grade inflammation that resulted from the enhanced production and release of a cocktail of proinflammatory cytokines and adipokines from the adipose tissue (29,30). These inflammatory stimuli in turn lead to an increase in the expression of hepcidin, which once released into the circulation, impaired the recycling of iron by reticuloendothelial macrophages (19) and the absorption of iron by duodenal enterocytes (20,21), resulting in hypoferremia (9,43).

In this study, we investigated whether leptin, an adipokine with cytokine-like actions, in addition to its proliferative role in the liver (41), might also regulate hepatic hepcidin expression. Cytokines exert their biological effect through the JAK/STAT signaling pathway (17). However, of the multiple leptin receptor subtypes, only the long form, Ob-Rb, is able to couple to the JAK/STAT pathway (34). Originally, it was believed that Ob-Rb was only present in the hypothalamus, where it mediates the effects of leptin on appetite suppression. It is now clear that Ob-Rb, as well as the shorter Ob-Ra receptor, are universally distributed in both neuronal and nonneuronal tissues, where they exert a wide array of biological functions (34). In keeping with this distribution pattern, we were able to detect abundant amounts of both the Ob-Rb and the Ob-Ra receptors in HuH7 cells.

Exposure of HuH7 cells to leptin produced a dramatic increase in p-STAT3 levels, suggesting that Ob-Rb was functional in this cell line. These findings are in agreement with previously published data showing that leptin can activate STAT3 in HuH7 cells overexpressing the Ob-Rb receptor isoform (44). Leptin treatment induced hepcidin expression in both a time- and a dose-dependent manner. Interestingly, the leptin-induced increases in both p-STAT3 and hepcidin mRNA were diminished by preincubation with the JAK2 inhibitor HBCH. In addition, in the hepcidin promoter–luciferase reporter-gene assays, either mutation of the STAT3 consensus binding motif or coexpression of a dnSTAT3 significantly reduced the leptin-induced increase in hepcidin promoter activity. Taken together, these data indicate that leptin is a powerful regulator of hepatic hepcidin expression and operates through the Ob-Rb receptor coupled to the JAK2/STAT3 signaling pathway.

In conclusion, we have shown that leptin can directly regulate hepatic hepcidin expression. Increased production of hepcidin in the presence of leptin was predicted to result in decreased duodenal iron absorption and impaired iron recycling from reticuloendothelial macrophages because of the inhibitory actions of hepcidin on ferroportin protein expression (18). Together with other stimuli, such as proinflammatory cytokines, leptin can now be added to the list of adipose-derived factors that may contribute to the hypoferremia observed in the overweight and obese population.


   FOOTNOTES
 
1 Supported by the Biotechnology and Biological Sciences Research Council. Bomee Chung is funded in part by the Overseas Research Student (ORS) Award Scheme. Pavle Matak is funded by a studentship from the Medical Research Council. Back

2 Author disclosures: B. Chung, P. Matak, A. T. McKie, and P. Sharp, no conflicts of interest. Back

3 Abbreviations used: bp, base-pairs; dnSTAT3, dominant-negative STAT3 mutant; HBCH, 1,2,3,4,5,6-hexabromocyclohexane; JAK, Janus kinase; p-STAT3, phosphorylated STAT3; STAT, signal transducer and activator of transcription; wt, wild type. Back

Manuscript received 1 August 2007. Initial review completed 29 August 2007. Revision accepted 10 September 2007.


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 Materials and Methods
 Results
 Discussion
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B. Chung, T. Chaston, J. Marks, S. K. Srai, and P. A. Sharp
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J. Nutr., August 1, 2009; 139(8): 1457 - 1462.
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P. Matak, T. B. Chaston, B. Chung, S. K. Srai, A. T. McKie, and P. A. Sharp
Activated macrophages induce hepcidin expression in HuH7 hepatoma cells
Haematologica, June 1, 2009; 94(6): 773 - 780.
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