Correspondence to: Elmar Aigner, MD, First Department of Medicine, Paracelsus Medical University Salzburg, Müllner Hauptstrasse 48, Salzburg-5020, Austria. Tel.:(+43)-662-4482-58358; fax: +43-662-4482-881; e-mail: firstname.lastname@example.org
Christian Datz, MD, Department of Internal Medicine, General Hospital Oberndorf; Teaching Hospital of the Paracelsus Medical University Salzburg, Paracelsusstrasse 37, Oberndorf-5110, Austria. Tel.: (+43)-6272-4334-533; Fax: +43-6272-4334-200; e-mail: email@example.com
The metabolic syndrome (MetS) affects iron homeostasis in a many-faceted fashion. On the one side, hyperferritinaemia with normal or mildly elevated transferrin saturation is observed in approximately one-third of patients with non-alcoholic fatty liver disease (NAFLD) or the MetS. This constellation has been named the ‘dysmetabolic iron overload syndrome (DIOS)’. Current evidence suggests that elevated body iron stores exert a detrimental effect on the clinical course of obesity-related conditions and that iron removal improves insulin sensitivity and delays the onset of T2DM. On the other side, iron deficiency is a frequent finding in more progressed stages of obesity. The mechanisms underlying the DIOS and obesity-related iron deficiency appear strikingly similar as elevated hepcidin concentrations, low expression of duodenal ferroportin (FPN) and impaired iron absorption are found in both conditions. This review summarizes the current knowledge about the dysregulation of iron homeostasis in the MetS and particularly in its hepatic manifestation NAFLD.
Iron is essential to the life of all mammalian organisms. It has a key role in oxygen transport and in enzymes involved in mitochondrial respiration, DNA biosynthesis and the citric acid cycle via the capability to change its redox state. However, this characteristic also renders excess iron detrimental – mostly via the formation of reactive oxygen species (ROS), which may lead to severe organ damage. To both meet biological iron requirements and avoid systemic iron toxicity, iron stores are tightly equilibrated by a complex molecular network involving liver, gut, the erythropoietic bone marrow and reticuloendothelial system (RES). Iron perturbations are frequently observed in patients with obesity, insulin resistance or non-alcoholic fatty liver disease (NAFLD). Interestingly, both iron deficiency (particularly in morbidly obese patients) and iron excess the dysmetabolic iron overload syndrome, (DIOS) have been well documented in association with obesity-related conditions.
The physiology of iron metabolism
Many aspects of the physiological regulation of human iron homeostasis have been elucidated over the past decade. Iron is absorbed as Fe2+ in the proximal duodenum by the divalent metal transporter 1 (DMT1). Following its transfer through the duodenal basolateral membrane facilitated by the iron exporter ferroportin (FPN) , iron undergoes oxidation by the membrane-bound copper containing ferroxidase hephaestin before being incorporated into transferrin for further transport in the circulation. Most cells acquire iron via the uptake of transferrin-bound iron (Fe3+) by the transferrin receptor (TfR1). Iron is mainly required for haeme biosynthesis in the erythropoietic bone marrow and other haeme-containing enzymes (e.g. cytochromes), whereas excess iron is stored in the liver hepatocytes. Iron is exported from hepatocytes, macrophages and all other mammalian cells via FPN, which has so far been identified as the only iron exporter.
Systemic iron homeostasis is maintained in a hormone-like negative feedback mechanism by the 25-amino acid peptide hepcidin (hepatic bactericidal protein). Hepcidin is secreted from hepatocytes in response to iron overload, inflammation, hypoxia or anaemia. Hepcidin exerts its regulatory functions on iron homeostasis via binding to FPN, thereby leading to FPN phosphorylation, degradation and consecutively to blockage of cellular iron export which induces a decrease in serum iron. Although in quantitative terms the liver is the main source of circulating hepcidin, macrophages, pancreatic islet cells and adipose tissue can also excrete hepcidin.
Important upstream regulators of hepcidin expression include hemojuvelin (HJV), a bone-morphogenetic protein co-receptor; HFE, a non-classical MHC class –1 molecule; and TfR-2, a liver-specific iron uptake molecule. Mutations of these genes are associated with low hepcidin formation and iron overload. Matriptase-2 (TMPRSS6) is a serine protease which is required for adequate hepatic hepcidin down-regulation in response to iron loss or anaemia. Mutations of TMPRSS6 lead to inappropriately elevated hepcidin concentrations and cause rare iron-refractory iron-deficiency anaemia (IRIDA). In IRIDA, oral or parenteral iron cannot be adequately used for haemoglobin biosynthesis because persistently high levels of hepcidin result in defective duodenal iron uptake and macrophage iron recycling. As increased erythropoiesis leads to low hepcidin levels and increased intestinal iron absorption as found in thalassaemia or other haemolytic anaemias, erythropoiesis-derived factors are postulated to modulate hepatic hepcidin expression. Growth and differentiation factor 15 (GDF15) and twisted gastrulation (TWSG1), which are secreted during erythroid development, may represent links between erythropoiesis and systemic iron homeostasis. The systemic regulation of iron metabolism is illustrated in Figure 1.
Elevated ferritin and iron markers in patients with NAFLD
Metabolic syndrome alterations have been well established as a clinically important differential diagnosis underlying hyperferritinaemia once inflammatory conditions or true iron overload syndromes have been excluded. Elevated serum ferritin levels are the hallmark of dysregulated iron homeostasis in the metabolic syndrome, and transferrin saturation usually ranges in the upper range of normal or is slightly raised. The term ‘dysmetabolic iron overload syndrome’ (DIOS) is now most frequently used to describe the typical association of hepatic steatosis with mild to moderate iron deposition in liver biopsies (haemosiderosis) and increased serum ferritin in patients with IR[2, 16]. The cardinal characteristics of frequent iron overload and iron distribution disorders are summarized in Table 1. Taking liver and serum assessment of iron stores into consideration, body iron excess in obesity or IR appears relatively mild with a dysproportional elevation of ferritin. It seems reasonable that hyperferritinaemia in NAFLD patients is caused by true iron excess as well as obesity-associated low-grade inflammation; thus, elevated ferritin may be found in the metabolic syndrome without evidence of iron excess.
Table 1. Synopsis of the biochemical characteristics of true iron overload disease (hemochromatosis) compared to common iron distribution disorders
Iron distribution disorders are characterized by elevated serum ferritin, but total body iron stores are not relevantly elevated or may even be low in the case of long-standing ACD. Iron perturbations in NAFLD/IR resemble iron metabolism of ACD as in these conditions high hepcidin concentrations are found along with iron deposition also in macrophages which results in impaired iron mobilization from cells. Abbreviations: ACD – anaemia of chronic disease; NAFLD – non-alcoholic fatty liver disease; IR – insulin resistance; HCV – hepatitis C virus infection; Φ – macrophages (spleen, bone marrow and liver Kupffer cells); TfS – transferrin saturation; sFe – serum iron; ↑ – elevated; ↑↑ – markedly elevated; ↔ – unchanged; ↓ – decreased; ↓↓ – markedly decreased; + – present; - – absent.
The coincidence of elevated liver transaminases and hyperferritinaemia may be indicative of iron overload in hereditary haemochromatosis that represents the principal differential diagnosis. HFE genotyping, transferrin saturation, assessment of clinical manifestations of the metabolic syndrome, taking a detailed history on alcohol consumption and – rarely – liver biopsy are necessary components for the diagnostic work-up of the patient with hyperferritinaemia and will usually provide the diagnostic clues to the origin of elevated ferritin concentrations. The diagnostic approach to the patient with hype-rferritinaemia is summarized in Figure 4.
Evidence for a role of iron in IR
It was first raised by Sullivan in 1981 that the delayed occurrence of cardiovascular disease in women compared to men with a similar incidence after menopause might be due to reduced premenopausal iron stores. The association of higher iron stores with diabetes, IR and NAFLD has been repeatedly confirmed in many investigations. Ferritin levels were found to predict a higher rate of diabetes and gestational diabetes in prospective investigations and case–control cohorts[20-22]. Serum ferritin was positively associated with BMI, visceral fat mass, serum glucose levels and insulin sensitivity, blood pressure, the MetS and also related to cholesterol levels. Iron stores clustered with metabolic risk markers in a population of both obese  and apparently healthy, lean teenagers. These findings are clinically relevant due to the higher risk of developing cardiovascular or cerebrovascular disease in patients with IR. Similarly, iron stores are also increased in the polycystic ovary syndrome (PCOS). In these patients, increased iron stores were linked to IR and not explained by menstrual irregularities. In summary, these data reflect a relationship between elevated body iron stores and several clinical manifestations of IR or the MetS.
Evidence for a role of iron in NAFLD
The contribution of increased iron stores for NAFLD disease severity and progression has been intensely investigated. Several investigations found an association of iron with more progressed stages or higher incidence of NAFLD [32-35]; however, this association was not confirmed in subsequent studies[36, 37]. Data concerning the relevance of excess iron in NAFLD disease progression are mainly hampered by the lack of prospective investigations with serial liver biopsies in a cohort of patients large enough to correct for established co-factors for disease progression[36, 38]. Recently, in the so far largest investigation, iron deposition in NAFLD liver biopsies, especially in Kupffer cells, has been linked with more advanced stages of fibrosis and disease severity. Clinically important serum ferritin has been associated with increased mortality of patients on the waiting list as well as after transplantation[40, 41]. Excess liver iron may also be involved in the progression of NASH-associated fibrosis to hepatocellular carcinoma. Mutations in the most frequent iron overload–related HFE mutation are likely not related to iron excess in NAFLD. However, mutations in the βglobin- and α1-antitrypisin gene may be involved, at least in populations where the respective mutations occur frequently[44, 45].
Pathways linking iron to the aggravation of IR
It has long been known that in hemochromatosis, iron overload may cause diabetes. Hepatic and peripheral IR increase and pancreatic insulin secretion decreases as body iron rises. Liver and muscle insulin sensitivity as well as pancreatic insulin secretion are restored if iron is removed.
As iron is a potent catalyst for the formation of ROS, it is assumed that augmentation of oxidative stress is the key mechanism underlying iron-induced IR although direct evidence for this hypothesis is lacking. Oxidative stress is increased in T2DM and obesity and induces IR in fat cells. In vitro analyses demonstrated that intracellular iron chelation enhances insulin receptor signalling and that excess iron decreases insulin receptor activity. Intracellular iron depletion with desferoxamine leads to an increase in phosphorylation status of Akt/protein kinase B (Akt/PKB), forkhead transcription factor O1 (FoxO1) and glycogen synthase kinase 3β (GSK3β), which are mediators of the insulin effects on gluconeogenesis and glycogen synthesis. Likewise, genes involved in glucose utilization such as GLUT1 and hypoxia-inducible factor 1α (HIF1α) were up-regulated in hepatoma cells along with improved glucose clearance. Thus, iron overload may worsen IR by interfering with insulin receptor signalling and inhibiting the ability to burn carbohydrates in the liver and muscle.
In isolated adipocytes, iron treatment induced an insulin-resistant phenotype, characterized by increased lipolysis and impaired glucose uptake in response to insulin. Adipokines are a heterogenous group of peptide hormones mediating the metabolic effects of obese adipose tissue to other tissues, and associative links have been observed between adipokines and parameters of iron metabolism. Ferritin is inversely related to adiponectin serum concentrations in insulin-resistant and insulin-sensitive subjects. A positive association was found between iron stores and circulating retinol-binding protein 4 (RBP4), which both decreased in response to iron depletion. A similar relationship was observed between serum visfatin concentrations and parameters of iron metabolism. However, these observations may reflect the coincidence of increased iron stores with other surrogate markers of IR and did not provide evidence of a causative link between these adipokines and the modulation of iron homeostasis. However, the intriguing idea has been proposed that excess dietary iron may be re-routed to visceral adipose tissue and alter its secretion of adipokines, as has been reported for resistin. This may represent a mechanistic link between the observed associations of elevated ferritin concentrations with adipokines.
Among adipokines, leptin, which is increased in the insulin-resistant state, was found to up-regulate hepcidin transcription in hepatocyte cultures via JAK2/STAT3-dependent signalling pathways. Thus, leptin-induced hepcidin synthesis may contribute to iron perturbations in NAFLD.
Obesity-related iron deficiency
Although iron excess as indicated by hyperferritinaemia, high-normal transferrin saturation and liver iron deposition is a typical finding in NAFLD and the MetS, severe forms of obesity are frequently accompanied by iron deficiency and even anaemia. The association between adult obesity and low iron stores has been evaluated in a recent meta-analysis of all controlled studies. Although iron deficiency appears as a typical finding in severe obesity which has been documented in several investigations (citations), the review concluded that most studies demonstrated higher haemoglobin and ferritin concentrations, and transferrin saturation decreased as BMI increases. This has mainly been attributed to an increasing influence of obesity-related inflammation.
With regard to molecular changes of iron homeostasis, elevated serum hepcidin concentrations indicating the absence of true iron deficiency have been reported in severe obesity. Expression of hepcidin in the adipose tissue from anaemic, iron-poor, morbidly obese subjects has been demonstrated, which is absent in lean adipose tissue. Markedly decreased duodenal iron absorption has been observed in obese subjects compared to healthy lean controls. These are hallmarks of iron homeostasis also reported in patients with DIOS, suggesting that the DIOS and obesity-related iron deficiency may be the two sides of the same coin as will be discussed in more detail below.
Mechanisms of iron perturbation in obesity and NAFLD
The following section aims to delineate the current knowledge about changes of iron molecules linked to iron perturbations in obesity and IR-associated conditions. A summary thereof is given in Figure 2.
Several studies investigated the expression of the key iron regulator hepcidin in patients with the DIOS. Urinary, serum and liver hepcidin concentrations were increased in patients with DIOS compared to controls, hemochromatosis patients and insulin-resistant subjects without excess iron[56, 57]. Increased hepcidin production correlates directly with liver iron concentration, indicating an intact physiological response to full iron stores[56, 57]. Additionally, hepcidin was found to be synthesized in adipose tissue of morbidly obese patients, obesity is characterized by a low-grade inflammatory state, and highly significant association was observed in vivo in patients with DIOS between hepcidin concentrations and TNF-α, suggesting that besides the iron stimulus, the pro-inflammatory milieu also contributes to hepcidin production in NAFLD. Clinically, increased hepcidin and pro-inflammatory cytokines may be derived from both the expanding adipose tissue and the liver. We have recently identified glucose as a trigger for hepcidin secretion most likely from pancreatic β-cells. Our results demonstrated that in response to glucose ingestion, serum iron concentrations gradually declined along with an increase in serum hepcidin. In cell culture, β-cells exhibited hepcidin release in response to glucose, but HepG2 liver cells did not. Preliminary data show that the decline in serum iron is augmented in NAFLD patients, suggesting that hepcidin secreted in response to nutrient ingestion may contribute to iron perturbations observed in NAFLD.
Hepcidin exerts its influence on iron metabolism via down-regulation of FPN-mediated iron export. Expression of FPN is low compared to control subjects and hemochromatosis patients both in the duodenum and in liver tissue[56, 57, 60]. In duodenal biopsies from NAFLD patients without liver iron deposition, FPN expression was similar to healthy control subjects, whereas it was decreased in NAFLD patients with iron accumulation. In support of these molecular findings, dietary iron uptake was decreased in patients with the DIOS. Likewise, obesity has been identified as a risk factor for impaired response to dietary iron fortification, likely explained by low FPN expression in the duodenum.
Phagocytosis of fragile erythrocytes by liver Kupffer cells was observed in rabbits on a high-fat diet, and phagocytosis of these fragile erythrocytes was increased in cell culture. Aggregates of erythrocytes were documented in microscopic areas of increased inflammation in human NAFLD. Hence, haeme-iron uptake via hepatic erythrophagocytosis may also be involved in the deposition of iron in NAFLD, subsequently enhancing inflammation and oxidative stress.
Most cells acquire iron via transferrin receptor 1 (TfR1). However, TfR1 expression in liver biopsies from NAFLD patients with low iron was higher compared to patients with NAFLD and excess iron or patients with HFE-associated hemochromatosis, suggesting physiologically reduced TfR1 expression in response to iron accumulation rather than increased iron uptake via the TfR pathway.
Copper is an important modulator of iron homeostasis, and copper status is linked to iron perturbations in NAFLD. Copper is the key molecule for hephaestin ferroxidase activity in the duodenal enterocytes where it facilitates the loading of iron to apo-Tf. In a similar manner, copper is required for ceruloplasmin ferroxidase activity to mobilize iron from storage sites like the liver or the RES, and adequate copper supply induces FPN expression and membrane-bound ceruloplasmin expression is required for FPN expression and cell surface stability. Low liver and serum copper concentrations were found in NAFLD patients with iron accumulation. Low copper concentrations were linked to low serum activity of the ferroxidase ceruloplasmin. In addition, lower hepatic expression of FPN was detected in rats on a copper deficient diet. These observations demonstrate that besides low FPN expression associated with low-grade systemic inflammation, inadequate copper bioavailability further impairs iron export from liver cells in patients with NAFLD and likely also the MetS. The potential mechanisms reported so far are summarized in Figure 3.
The therapeutic potential of modulating iron stores in NAFLD
Removing excess iron may have a beneficial effect on the extent and course of IR-associated conditions. Iron depletion is generally well tolerated, with the exception that patients with DIOS generally respond with a quick decrease in transferrin saturation and are prone to develop anaemia during phlebotomy treatment. These findings are in accordance with the underlying molecular mechanisms summarized earlier. A lower incidence of diabetes, lower postprandial serum insulin concentrations and higher pancreatic insulin sensitivity as reflected by improved beta-cell function were found in subjects who underwent previous phlebotomy treatment. Iron depletion was also found to improve coronary vascular dysfunction in patients with T2DM and in patients with known coronary artery disease. Even in non-insulin-resistant healthy subjects, frequent blood donations were linked to improved insulin sensitivity. Investigations examining iron depletion via phlebotomy in human NAFLD have unequivocally shown benefits of iron removal with regard to systemic or hepatic insulin resistance and to pancreatic insulin sensitivity[65, 68]. In a randomized trial, improved HbA1c, insulin sensitivity and secretion were observed in those subjects who underwent phlebotomy treatment. The beneficial effects of iron depletion were even evident in addition to successful lifestyle modifications. Similarly, this association holds true for iron depletion and other cardiovascular risk factors, and iron depletion may also prevent cancer development and progression. Very recently, the first controlled clinical trial demonstrated the benefits of iron depletion of several components of the insulin resistance syndrome, further strengthening the evidence to offer iron reduction therapy to these patients. However, data are still lacking that iron reduction therapy improves clinical endpoints such as fibrosis in NAFLD or complications in T2DM. It is also currently not clear whether patients with NASH benefit from more aggressive treatment of iron excess with regard to the progression to cirrhosis or the development of hepatocellular carcinoma.
It is important to consider that hyperferritinaemia does not necessarily indicate the presence of true iron overload. Particularly in NAFLD and the MetS, moderately elevated serum ferritin may arise from concomitant subclinical inflammation without liver iron deposition. Hence, liver iron quantification/histological iron staining by either biopsy or MRI should be obtained prior to the initiation of phlebotomy treatment to avoid unnecessary or potentially even harmful therapeutic intervention treatment.
From a practical point of view, these studies suggest that iron depletion therapy via phlebotomy represents at least a safe add-on therapy for insulin-resistant patients with elevated ferritin. We have adopted the practice to perform biweekly phlebotomies in these subjects until serum ferritin concentrations are between 50 and 100 μg/L or patients become anaemic. In contrast to patients with hemochromatosis, biweekly phlebotomies are recommended in patients with NAFLD. Patients with NAFLD have impaired iron mobilization from storage sites and are likely to develop anaemia in response to phlebotomy treatment. This is in marked contrast to patients with hemochromatosis who tolerate phlebotomies up to twice weekly.
There are no data available which would currently allow recommendations on copper supplementation. As free copper may also function as a catalyst for ROS generation, detailed and cautious further animal and human investigations are required on this subject.
Hyperferritinaemia is a relevant finding in NAFLD and other insulin-resistant conditions, as excess iron appears to be associated with adverse outcomes, IR and thereby accelerated disease progression. Removal of excess iron via phlebotomy is a safe and beneficial add-on treatment, which can easily be offered to these patients and is linked to favourable effects on IR and inflammation. The mechanisms of liver iron deposition in NAFLD are related to elevated hepcidin concentrations and impaired iron export from liver cells in response to low expression of the iron export molecule FPN. Adipose tissue inflammation (TNF-α and IL-6) and adipokine secretion (leptin and resistin) or hepcidin may represent signals from obese adipose tissue which dysregulate iron homeostasis as well as glucose or lipid metabolism.
Key points for clinical practice
Iron status (ferritin, serum iron and transferrin saturation) should be determined in patients with NAFLD as iron overload may detrimentally affect the course of the disease.
If patients fulfil criteria for the DIOS and liver iron overload is confirmed, phlebotomy treatment should be considered after exclusion of underlying inflammation (infection, malignancy and autoimmune disease).
A beneficial effect on insulin resistance and transaminase levels can be expected from iron depletion.
Phlebotomy appears generally safe when performed biweekly until a ferritin concentration of 50–100 μg/l is reached. To control for therapeutic success and anaemia, serum ferritin and haemoglobin concentrations should be determined at every visit. A summary of the clinical approach to the patient with elevated ferritin concentration is given in Figure 4.
Conflict of interest
None of the authors has any potential financial conflicts of interest to disclose with regard to this manuscript.
Department of Internal Medicine, General Hospital Oberndorf, Paracelsusstrasse 37, 5110 Oberndorf, Austria (C. Datz, D. Niederseer); Department of Laboratory Medicine, Paracelsus Medical University, Müllner Hauptstrasse 48, 5020 Salzburg, Austria (T.K. Felder); First Department of Medicine, Paracelsus Medical University, Müllner Hauptstrasse 48, 5020 Salzburg, Austria (E. Aigner).