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Iron in fatty liver and in the metabolic syndrome: A promising therapeutic target

  • Paola Dongiovanni
    Affiliations
    Department of Internal Medicine, Centro Malattie Metaboliche del Fegato, Università degli Studi di Milano, and Fondazione IRCCS “Ca’ Granda” Ospedale Maggiore Policlinico, Milan, Italy
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  • Anna Ludovica Fracanzani
    Affiliations
    Department of Internal Medicine, Centro Malattie Metaboliche del Fegato, Università degli Studi di Milano, and Fondazione IRCCS “Ca’ Granda” Ospedale Maggiore Policlinico, Milan, Italy
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  • Silvia Fargion
    Affiliations
    Department of Internal Medicine, Centro Malattie Metaboliche del Fegato, Università degli Studi di Milano, and Fondazione IRCCS “Ca’ Granda” Ospedale Maggiore Policlinico, Milan, Italy
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  • Luca Valenti
    Correspondence
    Corresponding author. Address: Department of Internal Medicine, Università degli Studi di Milano, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Padiglione Granelli, via F Sforza 35, 20122 Milan, Italy. Tel.: +39 02 503 20278; fax: +39 02 503 2096.
    Affiliations
    Department of Internal Medicine, Centro Malattie Metaboliche del Fegato, Università degli Studi di Milano, and Fondazione IRCCS “Ca’ Granda” Ospedale Maggiore Policlinico, Milan, Italy
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Open AccessPublished:June 20, 2011DOI:https://doi.org/10.1016/j.jhep.2011.05.008

      Summary

      The dysmetabolic iron overload syndrome (DIOS) is now a frequent finding in the general population, as is detected in about one third of patients with nonalcoholic fatty liver disease (NAFLD) and the metabolic syndrome. The pathogenesis is related to altered regulation of iron transport associated with steatosis, insulin resistance, and subclinical inflammation, often in the presence of predisposing genetic factors. Evidence is accumulating that excessive body iron plays a causal role in insulin resistance through still undefined mechanisms that probably involve a reduced ability to burn carbohydrates and altered function of adipose tissue. Furthermore, DIOS may facilitate the evolution to type 2 diabetes by altering beta-cell function, the progression of cardiovascular disease by contributing to the recruitment and activation of macrophages within arterial lesions, and the natural history of liver disease by inducing oxidative stress in hepatocytes, activation of hepatic stellate cells, and malignant transformation by promotion of cell growth and DNA damage.
      Based on these premises, the association among DIOS, metabolic syndrome, and NAFLD is being investigated as a new risk factor to predict the development of overt cardiovascular and hepatic diseases, and possibly hepatocellular carcinoma, but most importantly, represents also a treatable condition. Indeed, iron depletion, most frequently achieved by phlebotomy, has been shown to decrease metabolic alterations and liver enzymes in controlled studies in NAFLD. Additional studies are warranted to evaluate the potential of iron reductive therapy on hard clinical outcomes in patients with DIOS.

      Abbreviations:

      DIOS (dysmetabolic iron overload syndrome), NAFLD (nonalcoholic fatty liver disease), HHC (hereditary hemochromatosis), MetS (metabolic syndrome), ID (iron depletion), IR (insulin resistance), NASH (nonalcoholic steatohepatitis), HFD (high-fat diet), FFAs (free fatty acids), ER (endoplasmic reticulum), T2D (type 2 diabetes), PCOS (polycystic ovary syndrome), Cp (ceruloplasmin), Fp-1 (ferroportin-1), HFE (hemochromatosis gene), AAT (alpha1-antitrypsin), ROS (reactive oxygen species), HO-1 (heme oxygenase-1), RBP4 (retinol binding protein-4), HSCs (hepatic stellate cells), 8-oxodG (7,8-dihydro-8-oxo-2′ deoxyguanosine), MCD (methionine choline deficient diet), InsR (insulin receptor)

      Keywords

      A strong association between iron overload unrelated to hereditary hemochromatosis (HHC) and several manifestations of the metabolic syndrome (MetS), including nonalcoholic fatty liver disease (NAFLD), has been demonstrated during the last years. Furthermore, iron stores have been linked to a heightened risk of metabolic complications, such as diabetes, and faster progression of organ damage, including hepatic and cardiovascular diseases. Although emerging evidence suggests that the association between iron, NAFLD, and MetS represents a clinically ominous condition, the mechanisms underpinning the dysmetabolic iron overload syndrome (DIOS) and the pathogenesis of organ damage are still debated, whereas the potential therapeutic role of iron depletion therapy (ID) for the prevention of clinical complications is just beginning to be evaluated in controlled trials. Here, we review the recent overall evidence on epidemiology, pathogenesis, genetics, natural history, and treatment of DIOS, and provide a hypothetical interpretation of contrasting findings, with possible lines of future research.

      Association between hyperferritinemia, MetS alterations, and NAFLD: the dysmetabolic iron overload syndrome

      Ferritin and increased body iron stores have been associated with insulin resistance (IR) and metabolic abnormalities defining MetS in population studies conducted both in Western and Eastern countries [
      • Jehn M.
      • Clark J.M.
      • Guallar E.
      Serum ferritin and risk of the metabolic syndrome in US adults.
      ,
      • Bozzini C.
      • Girelli D.
      • Olivieri O.
      • Martinelli N.
      • Bassi A.
      • De Matteis G.
      • et al.
      Prevalence of body iron excess in the metabolic syndrome.
      ,
      • Wrede C.E.
      • Buettner R.
      • Bollheimer L.C.
      • Scholmerich J.
      • Palitzsch K.D.
      • Hellerbrand C.
      Association between serum ferritin and the insulin resistance syndrome in a representative population.
      ,
      • Kim C.H.
      • Kim H.K.
      • Bae S.J.
      • Park J.Y.
      • Lee K.U.
      Association of elevated serum ferritin concentration with insulin resistance and impaired glucose metabolism in Korean men and women.
      ].
      Several studies confirmed the association between hyperferritinemia and type 2 diabetes (T2D). In a case–control study in Europe, subjects with hyperferritinemia had a 2.4-fold higher risk to develop T2D [
      • Salonen J.T.
      • Tuomainen T.P.
      • Salonen R.
      • Lakka T.A.
      • Nyyssonen K.
      Donation of blood is associated with reduced risk of myocardial infarction. The Kuopio Ischaemic Heart Disease Risk Factor Study.
      ], whereas in a cross-sectional study in 9486 US subjects, elevated ferritin was associated with T2D [
      • Ford E.S.
      • Cogswell M.E.
      Diabetes and serum ferritin concentration among US adults.
      ]. More recently, in a prospective nested case–control study in 32,826 healthy Chinese women, higher iron stores were associated with T2D independently of known risk factors [
      • Jiang R.
      • Manson J.E.
      • Meigs J.B.
      • Ma J.
      • Rifai N.
      • Hu F.B.
      Body iron stores in relation to risk of type 2 diabetes in apparently healthy women.
      ], and in a case–control study nested in the EPIC-Norfolk cohort, ferritin levels were again an independent predictor of incident T2D [
      • Forouhi N.G.
      • Harding A.H.
      • Allison M.
      • Sandhu M.S.
      • Welch A.
      • Luben R.
      • et al.
      Elevated serum ferritin levels predict new-onset type 2 diabetes: results from the EPIC-Norfolk prospective study.
      ]. In the HEIRS study considering 97,470 subjects belonging to six racial/ethnic groups, ferritin was independently associated with T2D [
      • Acton R.T.
      • Barton J.C.
      • Passmore L.V.
      • Adams P.C.
      • Speechley M.R.
      • Dawkins F.W.
      • et al.
      Relationships of serum ferritin, transferrin saturation, and HFE mutations and self-reported diabetes in the Hemochromatosis and Iron Overload Screening (HEIRS) study.
      ]. As concerning the relationship among iron stores, MetS and IR, in a cross-sectional study in 6044 US adults, ferritin was associated with MetS and IR [
      • Jehn M.
      • Clark J.M.
      • Guallar E.
      Serum ferritin and risk of the metabolic syndrome in US adults.
      ]. Other epidemiological studies showed a correlation between ferritin, MetS, and IR severity, which was independent of inflammation [
      • Bozzini C.
      • Girelli D.
      • Olivieri O.
      • Martinelli N.
      • Bassi A.
      • De Matteis G.
      • et al.
      Prevalence of body iron excess in the metabolic syndrome.
      ,
      • Wrede C.E.
      • Buettner R.
      • Bollheimer L.C.
      • Scholmerich J.
      • Palitzsch K.D.
      • Hellerbrand C.
      Association between serum ferritin and the insulin resistance syndrome in a representative population.
      ].
      An interesting disease model to dissect the relationship between iron and MetS is also provided by the polycystic ovary syndrome (PCOS). PCOS is characterized by overweight, IR, and increased ferritin levels, which are not related to inflammation, but likely to oligomenorrhea [
      • Escobar-Morreale H.F.
      • Luque-Ramirez M.
      • Alvarez-Blasco F.
      • Botella-Carretero J.I.
      • Sancho J.
      • San Millan J.L.
      Body iron stores are increased in overweight and obese women with polycystic ovary syndrome.
      ]. Increased iron stores have been suggested to contribute to IR frequently found in PCOS patients [
      • Ehrmann D.A.
      Polycystic ovary syndrome.
      ]. In PCOS, metformin reduced ferritin in parallel with an increase in insulin sensitivity, thus suggesting that hyperinsulinemia and IR play a role on the increased iron stores in these patients [
      • Luque-Ramirez M.
      • Alvarez-Blasco F.
      • Botella-Carretero J.I.
      • Sanchon R.
      • San Millan J.L.
      • Escobar-Morreale H.F.
      Increased body iron stores of obese women with polycystic ovary syndrome are a consequence of insulin resistance and hyperinsulinism and are not a result of reduced menstrual losses.
      ].
      Figure thumbnail fx2
      A different perspective focused on the liver: Mendler et al. first described a cohort of patients with unexplained hepatic iron overload characterized by the association with IR, and coined the term “insulin resistance-associated hepatic iron-overload syndrome” [
      • Mendler M.H.
      • Turlin B.
      • Moirand R.
      • Jouanolle A.M.
      • Sapey T.
      • Guyader D.
      • et al.
      Insulin resistance-associated hepatic iron overload.
      ]. On the other hand, increased ferritin is detected in about 30% of unselected patients with NAFLD [
      • Valenti L.
      • Dongiovanni P.
      • Fracanzani A.L.
      • Santorelli G.
      • Fatta E.
      • Bertelli C.
      • et al.
      Increased susceptibility to nonalcoholic fatty liver disease in heterozygotes for the mutation responsible for hereditary hemochromatosis.
      ], and in these subjects it has been associated with increased hepatic iron, as determined by histological and radiological assessment, and by quantitative phlebotomy [
      • Valenti L.
      • Dongiovanni P.
      • Fracanzani A.L.
      • Santorelli G.
      • Fatta E.
      • Bertelli C.
      • et al.
      Increased susceptibility to nonalcoholic fatty liver disease in heterozygotes for the mutation responsible for hereditary hemochromatosis.
      ,
      • Valenti L.
      • Fracanzani A.L.
      • Dongiovanni P.
      • Bugianesi E.
      • Marchesini G.
      • Manzini P.
      • et al.
      Iron depletion by phlebotomy improves insulin resistance in patients with nonalcoholic fatty liver disease and hyperferritinemia: evidence from a case–control study.
      ,
      • Valenti L.
      • Dongiovanni P.
      • Piperno A.
      • Fracanzani A.L.
      • Maggioni M.
      • Rametta R.
      • et al.
      Alpha1-antitrypsin mutations in NAFLD: high prevalence and association with altered iron metabolism but not with liver damage.
      ,
      • Valenti L.
      • Fracanzani A.L.
      • Bugianesi E.
      • Dongiovanni P.
      • Galmozzi E.
      • Vanni E.
      • et al.
      HFE genotype, parenchymal iron accumulation, and liver fibrosis in patients with nonalcoholic fatty liver disease.
      ]. The acronym NAFLD refers to a broad spectrum of liver diseases ranging from uncomplicated steatosis to nonalcoholic steatohepatitis (NASH), which may progress to cirrhosis and hepatocellular carcinoma [
      • Bugianesi E.
      • Leone N.
      • Vanni E.
      • Marchesini G.
      • Brunello F.
      • Carucci P.
      • et al.
      Expanding the natural history of nonalcoholic steatohepatitis: from cryptogenic cirrhosis to hepatocellular carcinoma.
      ], and poses a high risk of cardiovascular disease [
      • Fracanzani A.L.
      • Burdick L.
      • Raselli S.
      • Pedotti P.
      • Grigore L.
      • Santorelli G.
      • et al.
      Carotid artery intima-media thickness in nonalcoholic fatty liver disease.
      ]. Now the leading cause of liver disease in Western countries [
      • Browning J.D.
      • Szczepaniak L.S.
      • Dobbins R.
      • Nuremberg P.
      • Horton J.D.
      • Cohen J.C.
      • et al.
      Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity.
      ], NAFLD is characterized by hepatic insulin resistance (IR) and is considered a manifestation of the MetS [
      • Marchesini G.
      • Brizi M.
      • Bianchi G.
      • Tomassetti S.
      • Bugianesi E.
      • Lenzi M.
      • et al.
      Nonalcoholic fatty liver disease: a feature of the metabolic syndrome.
      ,
      • Angulo P.
      Nonalcoholic fatty liver disease.
      ,
      • Marchesini G.
      • Bugianesi E.
      • Forlani G.
      • Cerrelli F.
      • Lenzi M.
      • Manini R.
      • et al.
      Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome.
      ]. Progression of liver damage is more severe when fatty liver is complicated by NASH [
      • Marchesini G.
      • Bugianesi E.
      • Forlani G.
      • Cerrelli F.
      • Lenzi M.
      • Manini R.
      • et al.
      Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome.
      ,
      • Villanova N.
      • Moscatiello S.
      • Ramilli S.
      • Bugianesi E.
      • Magalotti D.
      • Vanni E.
      • et al.
      Endothelial dysfunction and cardiovascular risk profile in nonalcoholic fatty liver disease.
      ,
      • Ekstedt M.
      • Franzen L.E.
      • Mathiesen U.L.
      • Thorelius L.
      • Holmqvist M.
      • Bodemar G.
      • et al.
      Long-term follow-up of patients with NAFLD and elevated liver enzymes.
      ], which is thought to be provoked by lipid peroxidation and mitochondrial dysfunction determining oxidative stress and cytokine release [
      • Day C.P.
      From fat to inflammation.
      ].
      The condition characterized by hepatic iron overload involving both hepatocytes and macrophages [
      • Turlin B.
      • Mendler M.H.
      • Moirand R.
      • Guyader D.
      • Guillygomarc’h A.
      • Deugnier Y.
      Histologic features of the liver in insulin resistance-associated iron overload. A study of 139 patients.
      ], absence of inflammation, normal transferrin saturation, and associated with features of MetS is now more commonly referred as DIOS [
      • Barisani D.
      • Pelucchi S.
      • Mariani R.
      • Galimberti S.
      • Trombini P.
      • Fumagalli D.
      • et al.
      Hepcidin and iron-related gene expression in subjects with dysmetabolic hepatic iron overload.
      ,
      • Riva A.
      • Trombini P.
      • Mariani R.
      • Salvioni A.
      • Coletti S.
      • Bonfadini S.
      • et al.
      Revaluation of clinical and histological criteria for diagnosis of dysmetabolic iron overload syndrome.
      ]. Although the diagnostic criteria are not clearly defined, DIOS represents the most frequent iron overload condition, since, as the clinical presentation overlaps almost completely with that of hyperferritinemia associated with metabolic abnormalities, it is observed in 15% of patients with MetS [
      • Bozzini C.
      • Girelli D.
      • Olivieri O.
      • Martinelli N.
      • Bassi A.
      • De Matteis G.
      • et al.
      Prevalence of body iron excess in the metabolic syndrome.
      ], and it is associated in at least half of the cases with NAFLD [
      • Moirand R.
      • Mendler M.H.
      • Guillygomarc’h A.
      • Brissot P.
      • Deugnier Y.
      Non-alcoholic steatohepatitis with iron: part of insulin resistance-associated hepatic iron overload?.
      ]. DIOS patients have mild hepatic iron excess with a predominantly mixed sinusoidal/hepatocellular pattern [
      • Pietrangelo A.
      Hereditary hemochromatosis – a new look at an old disease.
      ], which presupposes macrophage iron retention and an iron recycling defect that is associated with the severity of inflammation and IR [
      • Nelson J.E.
      • Wilson L.
      • Brunt E.M.
      • Yeh M.M.
      • Kleiner D.E.
      • Unalp-Arida A.
      • et al.
      Relationship between the pattern of hepatic iron deposition and histological severity in nonalcoholic fatty liver disease.
      ].
      Recently, a more strict definition of DIOS has been proposed, based on the presence of two or more MetS components, steatosis, normal transferrin saturation, and mild hepatic iron overload, with typical involvement of the sinusoidal compartment [
      • Riva A.
      • Trombini P.
      • Mariani R.
      • Salvioni A.
      • Coletti S.
      • Bonfadini S.
      • et al.
      Revaluation of clinical and histological criteria for diagnosis of dysmetabolic iron overload syndrome.
      ]. However, this definition is not applicable to subjects who do not have an indication for liver biopsy.
      It has been reported that in DIOS iron absorption is decreased and hepcidin, the hormone that acts by decreasing intestinal iron absorption and recycling from macrophages [
      • Nemeth E.
      • Ganz T.
      Regulation of iron metabolism by hepcidin.
      ], is increased compared to healthy controls, indicating that iron compartmentalization in monocytes is likely related to a relatively preserved upregulation of hepcidin as an attempt to counteract iron excess [
      • Barisani D.
      • Pelucchi S.
      • Mariani R.
      • Galimberti S.
      • Trombini P.
      • Fumagalli D.
      • et al.
      Hepcidin and iron-related gene expression in subjects with dysmetabolic hepatic iron overload.
      ,
      • Ruivard M.
      • Laine F.
      • Ganz T.
      • Olbina G.
      • Westerman M.
      • Nemeth E.
      • et al.
      Iron absorption in dysmetabolic iron overload syndrome is decreased and correlates with increased plasma hepcidin.
      ].

      Metabolic hyperferritinemia and DIOS: different faces of the same problem?

      As “metabolic” hyperferritinemia associated with NAFLD and MetS and DIOS share the majority of clinical features (Table 1), we propose that they might be considered as two faces of the same health problem. In particular, both are characterized by (1) the presence of metabolic alterations typical of MetS [
      • Marchesini G.
      • Brizi M.
      • Bianchi G.
      • Tomassetti S.
      • Bugianesi E.
      • Lenzi M.
      • et al.
      Nonalcoholic fatty liver disease: a feature of the metabolic syndrome.
      ,
      • Riva A.
      • Trombini P.
      • Mariani R.
      • Salvioni A.
      • Coletti S.
      • Bonfadini S.
      • et al.
      Revaluation of clinical and histological criteria for diagnosis of dysmetabolic iron overload syndrome.
      ,
      • Fernandez-Real J.M.
      • Ricart-Engel W.
      • Arroyo E.
      • Balanca R.
      • Casamitjana-Abella R.
      • Cabrero D.
      • et al.
      Serum ferritin as a component of the insulin resistance syndrome.
      ]; (2) the presence of fatty liver [
      • Riva A.
      • Trombini P.
      • Mariani R.
      • Salvioni A.
      • Coletti S.
      • Bonfadini S.
      • et al.
      Revaluation of clinical and histological criteria for diagnosis of dysmetabolic iron overload syndrome.
      ]; (3) hyperferritinemia with normal or only slightly elevated transferrin saturation, reflecting physiological upregulation of hepcidin in response to increased iron stores [
      • Valenti L.
      • Dongiovanni P.
      • Fracanzani A.L.
      • Santorelli G.
      • Fatta E.
      • Bertelli C.
      • et al.
      Increased susceptibility to nonalcoholic fatty liver disease in heterozygotes for the mutation responsible for hereditary hemochromatosis.
      ,
      • Fernandez-Real J.M.
      • Ricart-Engel W.
      • Arroyo E.
      • Balanca R.
      • Casamitjana-Abella R.
      • Cabrero D.
      • et al.
      Serum ferritin as a component of the insulin resistance syndrome.
      ,
      • Adams P.C.
      • Reboussin D.M.
      • Barton J.C.
      • McLaren C.E.
      • Eckfeldt J.H.
      • McLaren G.D.
      • et al.
      Hemochromatosis and iron-overload screening in a racially diverse population.
      ]; (4) besides that in DIOS, mildly increased hepatic (as detected by histological scores after Perls’ stain for iron, and determination of liver iron concentration by atomic absorption spectrometry, or superconducting quantum interference device – SQUID) and body iron stores (indirectly estimated by the association with risk factors such as transferrin saturation, age, male gender, increased alcohol intake within normal limits, and HFE mutations, and quantitatively assessed by quantitative phlebotomy) have been demonstrated in patients with NAFLD associated with hyperferritinemia compared to those without hyperferritinemia, and the amount of body iron has been associated with serum ferritin [
      • Valenti L.
      • Dongiovanni P.
      • Fracanzani A.L.
      • Santorelli G.
      • Fatta E.
      • Bertelli C.
      • et al.
      Increased susceptibility to nonalcoholic fatty liver disease in heterozygotes for the mutation responsible for hereditary hemochromatosis.
      ,
      • Valenti L.
      • Fracanzani A.L.
      • Dongiovanni P.
      • Bugianesi E.
      • Marchesini G.
      • Manzini P.
      • et al.
      Iron depletion by phlebotomy improves insulin resistance in patients with nonalcoholic fatty liver disease and hyperferritinemia: evidence from a case–control study.
      ,
      • Haap M.
      • Machann J.
      • von Friedeburg C.
      • Schick F.
      • Stefan N.
      • Schwenzer N.F.
      • et al.
      Insulin sensitivity and liver fat: role of iron load.
      ,
      • Valenti L.
      • Swinkels D.W.
      • Burdick L.
      • Dongiovanni P.
      • Tjalsma H.
      • Motta B.M.
      • et al.
      Serum ferritin levels are associated with vascular damage in patients with nonalcoholic fatty liver disease.
      ]. Data obtained in recent studies by our group have been summarized in Supplementary Table 1. Furthermore, serum ferritin has been associated with the severity of insulin resistance [
      • Valenti L.
      • Swinkels D.W.
      • Burdick L.
      • Dongiovanni P.
      • Tjalsma H.
      • Motta B.M.
      • et al.
      Serum ferritin levels are associated with vascular damage in patients with nonalcoholic fatty liver disease.
      ,

      Manco M, Alisi A, Real JM, Equitani F, Devito R, Valenti L, et al. Early interplay of intra-hepatic iron and insulin resistance in children with non-alcoholic fatty liver disease. J Hepatol 2011. in press [PMID: 21168460].

      ,
      • Bugianesi E.
      • Manzini P.
      • D’Antico S.
      • Vanni E.
      • Longo F.
      • Leone N.
      • et al.
      Relative contribution of iron burden, HFE mutations, and insulin resistance to fibrosis in nonalcoholic fatty liver.
      ]. (5) Ferroportin-1 (Fp-1) mutations and polymorphisms have been excluded as a common cause of iron overload in both DIOS and NAFLD/MetS [
      • Valenti L.
      • Canavesi E.
      • Galmozzi E.
      • Dongiovanni P.
      • Rametta R.
      • Maggioni P.
      • et al.
      Beta-globin mutations are associated with parenchymal siderosis and fibrosis in patients with non-alcoholic fatty liver disease.
      ,
      • Pelucchi S.
      • Mariani R.
      • Salvioni A.
      • Bonfadini S.
      • Riva A.
      • Bertola F.
      • et al.
      Novel mutations of the ferroportin gene (SLC40A1): analysis of 56 consecutive patients with unexplained iron overload.
      ]. Unfortunately, the role of acquired factors, cytokines, and other genetic factors have been reported in details only for patients with a diagnosis of NAFLD and not in DIOS. Thus, comparative studies are clearly required to verify the aforementioned hypothesis.
      Table 1Comparison of clinical features of hyperferritinemia associated with NAFLD and MetS and of DIOS. Clinical features not referenced are present by definition
      • Riva A.
      • Trombini P.
      • Mariani R.
      • Salvioni A.
      • Coletti S.
      • Bonfadini S.
      • et al.
      Revaluation of clinical and histological criteria for diagnosis of dysmetabolic iron overload syndrome.
      .
      NAFLD, nonalcoholic fatty liver disease; MetS, metabolic syndrome; DIOS, dysmetabolic iron overload syndrome; muts, mutations; SNPs, single nucleotide polymorphisms; CRP, C reactive protein; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; MCP-1, macrophage chemoattractant protein-1 (CCL-2).

      Molecular mechanism underlying body iron accumulation in DIOS

      Iron accumulation in DIOS likely involves altered regulation of molecules involved in cellular iron export, such as ceruloplasmin (Cp) and Fp-1 induced by inflammation and micronutrients imbalance. A striking down-regulation of the cellular iron exporter Fp-1 has been observed in NASH, whereas hepcidin was physiologically increased in DIOS [
      • Aigner E.
      • Theurl I.
      • Theurl M.
      • Lederer D.
      • Haufe H.
      • Dietze O.
      • et al.
      Pathways underlying iron accumulation in human nonalcoholic fatty liver disease.
      ,
      • Pigeon C.
      • Ilyin G.
      • Courselaud B.
      • Leroyer P.
      • Turlin B.
      • Brissot P.
      • et al.
      A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload.
      ], confirming that altered iron trafficking underlies iron accumulation in NAFLD, whereas preserved hepcidin regulation inhibits Fp-1 protein activity, thus limiting further iron absorption and transferrin saturation. Since the Cu2+-dependent ferroxidase Cp, the physiological plasma Cu2+ transporter, is required for the mobilization of iron by Fp-1, it was hypothesized that low Cp may be a cause of iron accumulation in DIOS. Indeed, NAFLD patients with the lowest liver and serum Cu2+ and Cp levels were more likely to have iron overload [
      • Aigner E.
      • Theurl I.
      • Haufe H.
      • Seifert M.
      • Hohla F.
      • Scharinger L.
      • et al.
      Copper availability contributes to iron perturbations in human nonalcoholic fatty liver disease.
      ]. Moreover, Fp-1 mRNA was lower in patients with low hepatic Cu2+, and associated with steatosis and IR. As mentioned above, unbalanced oxidative stress is considered a trigger of NAFLD [
      • Sanyal A.J.
      Mechanisms of disease: pathogenesis of nonalcoholic fatty liver disease.
      ], and SOD1, one of the enzymes counteracting oxidative stress, depends on adequate Cu2+ availability [
      • Prohaska J.R.
      • Geissler J.
      • Brokate B.
      • Broderius M.
      Copper, zinc-superoxide dismutase protein but not mRNA is lower in copper-deficient mice and mice lacking the copper chaperone for superoxide dismutase.
      ]. Systemic Cu2+ deficiency causes mitochondrial dysfunction in mice [
      • Nose Y.
      • Kim B.E.
      • Thiele D.J.
      Ctr1 drives intestinal copper absorption and is essential for growth, iron metabolism, and neonatal cardiac function.
      ], with similar morphological and functional alterations to that described in NAFLD [
      • Wei Y.
      • Rector R.S.
      • Thyfault J.P.
      • Ibdah J.A.
      Nonalcoholic fatty liver disease and mitochondrial dysfunction.
      ], and patients with NASH had lower Cu2+ than those with simple steatosis [
      • Aigner E.
      • Strasser M.
      • Haufe H.
      • Sonnweber T.
      • Hohla F.
      • Stadlmayr A.
      • et al.
      A role for low hepatic copper concentrations in nonalcoholic fatty liver disease.
      ], suggesting a possible involvement of altered Cu2+metabolism in the pathophysiology of NASH by favoring both iron accumulation and reduced antioxidant activity.
      Other sources of increased serum ferritin in NAFLD are represented by subclinical inflammation and ferritin release by activated leukocytes, and hepatocellular necrosis. Hepatic iron accumulation is an early event in the natural history of NAFLD, and increased ferritin levels in pediatric patients correlate with IR and increased levels of cytokines [

      Manco M, Alisi A, Real JM, Equitani F, Devito R, Valenti L, et al. Early interplay of intra-hepatic iron and insulin resistance in children with non-alcoholic fatty liver disease. J Hepatol 2011. in press [PMID: 21168460].

      ]. An interesting hypothesis based on data obtained in the HFD model is that activated Kupffer cells may accumulate iron and release ferritin because of increased erythrophagocytosis, which would cause cytokines release and fibrogenesis [
      • Otogawa K.
      • Kinoshita K.
      • Fujii H.
      • Sakabe M.
      • Shiga R.
      • Nakatani K.
      • et al.
      Erythrophagocytosis by liver macrophages (Kupffer cells) promotes oxidative stress, inflammation, and fibrosis in a rabbit model of steatohepatitis: implications for the pathogenesis of human nonalcoholic steatohepatitis.
      ].

      Genetics of iron overload in NAFLD and the MetS

      In the attempt to explain the reason behind DIOS development in only a proportion of patients with NAFLD, several studies analyzed whether mutations in the HHC gene (HFE) may be involved, with conflicting results [
      • Bugianesi E.
      • Manzini P.
      • D’Antico S.
      • Vanni E.
      • Longo F.
      • Leone N.
      • et al.
      Relative contribution of iron burden, HFE mutations, and insulin resistance to fibrosis in nonalcoholic fatty liver.
      ,
      • Valenti L.
      • Dongiovanni P.
      • Fracanzani A.L.
      • Fargion S.
      HFE mutations in nonalcoholic fatty liver disease.
      ,
      • Chitturi S.
      • Weltman M.
      • Farrell G.C.
      • McDonald D.
      • Kench J.
      • Liddle C.
      • et al.
      HFE mutations, hepatic iron, and fibrosis: ethnic-specific association of NASH with C282Y but not with fibrotic severity.
      ,
      • Neri S.
      • Pulvirenti D.
      • Signorelli S.
      • Ignaccolo L.
      • Tsami A.
      • Mauceri B.
      • et al.
      The HFE gene heterozygosis H63D: a cofactor for liver damage in patients with steatohepatitis? Epidemiological and clinical considerations.
      ]. In a multicenter study in 587 Italian patients, we recently investigated whether the C282Y and H63D mutations predispose to iron overload in NAFLD [
      • Valenti L.
      • Fracanzani A.L.
      • Bugianesi E.
      • Dongiovanni P.
      • Galmozzi E.
      • Vanni E.
      • et al.
      HFE genotype, parenchymal iron accumulation, and liver fibrosis in patients with nonalcoholic fatty liver disease.
      ]. Both hepatocellular and non-parenchymal siderosis were associated with HFE mutations, but the penetrance of HFE mutations was relatively low, so that only one third of carriers had the hepatocellular iron accumulation typical of HHC, explaining less than half of the variability of this phenotype. Interestingly, we had previously shown that carriers of the C282Y mutation have lower insulin release, and develop NAFLD in the presence of less severe metabolic abnormalities (in particular the degree of adiposity), suggesting that heterozygosity for C282Y HFE mutation, responsible for mild iron overload, may increase the susceptibility to clinically overt NAFLD [
      • Valenti L.
      • Dongiovanni P.
      • Fracanzani A.L.
      • Santorelli G.
      • Fatta E.
      • Bertelli C.
      • et al.
      Increased susceptibility to nonalcoholic fatty liver disease in heterozygotes for the mutation responsible for hereditary hemochromatosis.
      ]. It could be hypothesized that the mechanism linking HFE mutations to iron accumulation in non-parenchymal cells may involve a relative hampering in hepcidin upregulation and facilitation of intestinal iron absorption, thus allowing the increase of body iron stores that will possibly localize in non-parenchymal cells because of defective iron export.
      Other genetic factors influencing hepatocellular damage, inflammation and iron handling might be involved. Alpha1-antitrypsin (AAT), the principal serum protease inhibitor synthesized by the liver, potentially represents one of such factors. The most common variants are the PiZ and PiS alleles, where amino acid substitutions lead to abnormal folding and spontaneous protein polymerization, determining endoplasmic reticulum (ER) stress and hepatocellular damage. Heterozygosity for the PiZ, and to a lesser extent for the PiS allele has been associated with cirrhosis and hepatocarcinoma [
      • Eigenbrodt M.L.
      • McCashland T.M.
      • Dy R.M.
      • Clark J.
      • Galati J.
      Heterozygous alpha 1-antitrypsin phenotypes in patients with end stage liver disease.
      ,
      • Serfaty L.
      • Chazouilleres O.
      • Poujol-Robert A.
      • Morand-Joubert L.
      • Dubois C.
      • Chretien Y.
      • et al.
      Risk factors for cirrhosis in patients with chronic hepatitis C virus infection: results of a case–control study.
      ,
      • Graziadei I.W.
      • Joseph J.J.
      • Wiesner R.H.
      • Therneau T.M.
      • Batts K.P.
      • Porayko M.K.
      Increased risk of chronic liver failure in adults with heterozygous alpha1-antitrypsin deficiency.
      ]. We found that the AAT mutations were highly prevalent in NAFLD, and associated with hyperferritinemia in the presence of normal transferrin saturation, and sinusoidal hepatic siderosis [
      • Valenti L.
      • Dongiovanni P.
      • Piperno A.
      • Fracanzani A.L.
      • Maggioni M.
      • Rametta R.
      • et al.
      Alpha1-antitrypsin mutations in NAFLD: high prevalence and association with altered iron metabolism but not with liver damage.
      ], the typical abnormalities of DIOS. It is thus possible that the coexistence of multiple genetic variants contributes to DIOS [
      • Mendler M.H.
      • Turlin B.
      • Moirand R.
      • Jouanolle A.M.
      • Sapey T.
      • Guyader D.
      • et al.
      Insulin resistance-associated hepatic iron overload.
      ,
      • Valenti L.
      • Dongiovanni P.
      • Fracanzani A.L.
      • Santorelli G.
      • Fatta E.
      • Bertelli C.
      • et al.
      Increased susceptibility to nonalcoholic fatty liver disease in heterozygotes for the mutation responsible for hereditary hemochromatosis.
      ,
      • Turlin B.
      • Mendler M.H.
      • Moirand R.
      • Guyader D.
      • Guillygomarc’h A.
      • Deugnier Y.
      Histologic features of the liver in insulin resistance-associated iron overload. A study of 139 patients.
      ,
      • Moirand R.
      • Mendler M.H.
      • Guillygomarc’h A.
      • Brissot P.
      • Deugnier Y.
      Non-alcoholic steatohepatitis with iron: part of insulin resistance-associated hepatic iron overload?.
      ], and it could be speculated that AAT modulate iron metabolism by inducing ER stress [
      • Vecchi C.
      • Montosi G.
      • Zhang K.
      • Lamberti I.
      • Duncan S.A.
      • Kaufman R.J.
      • et al.
      ER stress controls iron metabolism through induction of hepcidin.
      ].
      Since HFE and AAT mutations did not fully explain the variability of the phenotype, we next evaluated whether a wider panel of genetic variants reported to influence hepatic iron, including Fp-1 and beta-globin, might better predict DIOS and fibrosis progression [
      • Valenti L.
      • Canavesi E.
      • Galmozzi E.
      • Dongiovanni P.
      • Rametta R.
      • Maggioni P.
      • et al.
      Beta-globin mutations are associated with parenchymal siderosis and fibrosis in patients with non-alcoholic fatty liver disease.
      ]. The beta-thalassemia trait, commonly observed in the Mediterranean area, was more frequent in subjects with hyperferritinemia, and specifically associated with low hepcidin and parenchymal siderosis, leading to increased fibrosis. In Italian patients with NAFLD with predominantly parenchymal, non-parenchymal/mixed, or no hepatic siderosis, we observed a prevalence of H63D+/+ HFE genotype of 15%, 6%, and 3%; of the C282Y+/− HFE genotype of 25%, 12%, and 3%; of the PiS and PiZ AAT mutations of 22%, 15%, and 6%; and of beta-thalassemia trait of 32%, 8%, and 5%, respectively [
      • Valenti L.
      • Canavesi E.
      • Galmozzi E.
      • Dongiovanni P.
      • Rametta R.
      • Maggioni P.
      • et al.
      Beta-globin mutations are associated with parenchymal siderosis and fibrosis in patients with non-alcoholic fatty liver disease.
      ]. The prevalence of these genetic factors in patients without hepatic iron staining was superimposable to that of the general population. These differences were statistically significant, and 65% of patients with parenchymal iron accumulation carried at least one of these genetic factors vs. 9% of the control population [
      • Valenti L.
      • Canavesi E.
      • Galmozzi E.
      • Dongiovanni P.
      • Rametta R.
      • Maggioni P.
      • et al.
      Beta-globin mutations are associated with parenchymal siderosis and fibrosis in patients with non-alcoholic fatty liver disease.
      ]. Thus, these results support a preponderant effect of genetic factors, such as HFE and beta-globin mutations in the development of hepatocellular iron overload in NAFLD, suggesting it could represent a distinct and genetically determined sub-phenotype of DIOS at high risk of liver damage.

      Possible mechanisms of IR associated with metabolic hyperferritinemia/DIOS

      Clinical evidence suggests that iron might play a role in pathogenesis of IR [
      • Fargion S.
      • Dongiovanni P.
      • Guzzo A.
      • Colombo S.
      • Valenti L.
      • Fracanzani A.L.
      Iron and insulin resistance.
      ]. This hypothesis has been addressed in experimental studies including those determining an association of increased ferritin with iron stores [
      • Haap M.
      • Machann J.
      • von Friedeburg C.
      • Schick F.
      • Stefan N.
      • Schwenzer N.F.
      • et al.
      Insulin sensitivity and liver fat: role of iron load.
      ] and IR and amelioration of IR after ID [
      • Valenti L.
      • Fracanzani A.L.
      • Dongiovanni P.
      • Bugianesi E.
      • Marchesini G.
      • Manzini P.
      • et al.
      Iron depletion by phlebotomy improves insulin resistance in patients with nonalcoholic fatty liver disease and hyperferritinemia: evidence from a case–control study.
      ].
      As observed in HHC, in experimental models of obesity, iron accumulation within beta-cells alters insulin secretion, [
      • Cooksey R.C.
      • Jouihan H.A.
      • Ajioka R.S.
      • Hazel M.W.
      • Jones D.L.
      • Kushner J.P.
      • et al.
      Oxidative stress, beta-cell apoptosis, and decreased insulin secretory capacity in mouse models of hemochromatosis.
      ], whereas dietary iron restriction or chelation protects from diabetes [
      • Cooksey R.C.
      • Jones D.
      • Gabrielsen S.
      • Huang J.
      • Simcox J.A.
      • Luo B.
      • et al.
      Dietary iron restriction or iron chelation protects from diabetes and loss of beta-cell function in the obese (ob/ob lep−/−) mouse.
      ]. However, the relative deficit in insulin secretion is not sufficient to explain the metabolic alterations observed in DIOS.
      Supporting a causal role of iron overload in inducing IR, recent data indicate that manipulation of body iron stores by means of diet, genetic manipulation, or iron chelators is able to influence IR in different models of metabolic disease [
      • Cooksey R.C.
      • Jones D.
      • Gabrielsen S.
      • Huang J.
      • Simcox J.A.
      • Luo B.
      • et al.
      Dietary iron restriction or iron chelation protects from diabetes and loss of beta-cell function in the obese (ob/ob lep−/−) mouse.
      ,
      • Huang J.
      • Jones D.
      • Luo B.
      • Sanderson M.
      • Soto J.
      • Abel E.D.
      • et al.
      Iron overload and diabetes risk: a shift from glucose to fatty acid oxidation and increased hepatic glucose production in a mouse model of hereditary hemochromatosis.
      ,
      • Minamiyama Y.
      • Takemura S.
      • Kodai S.
      • Shinkawa H.
      • Tsukioka T.
      • Ichikawa H.
      • et al.
      Iron restriction improves type 2 diabetes mellitus in Otsuka Long-Evans Tokushima fatty rats.
      ], but the molecular mechanisms underlying the association between iron accumulation and IR and the tissues primarily involved are far from being clear.
      Iron overload has been hypothesized to induce IR by catalyzing oxidative stress [
      • Fernandez-Real J.M.
      • Lopez-Bermejo A.
      • Ricart W.
      Cross-talk between iron metabolism and diabetes.
      ,
      • Houstis N.
      • Rosen E.D.
      • Lander E.S.
      Reactive oxygen species have a causal role in multiple forms of insulin resistance.
      ]. Reactive oxygen species (ROS) have been implicated in IR pathogenesis on the basis of two types of indirect evidence: (1) an association of oxidative stress markers with obesity and T2D [
      • Furukawa S.
      • Fujita T.
      • Shimabukuro M.
      • Iwaki M.
      • Yamada Y.
      • Nakajima Y.
      • et al.
      Increased oxidative stress in obesity and its impact on metabolic syndrome.
      ,
      • Urakawa H.
      • Katsuki A.
      • Sumida Y.
      • Gabazza E.C.
      • Murashima S.
      • Morioka K.
      • et al.
      Oxidative stress is associated with adiposity and insulin resistance in men.
      ] and (2) evidence that factors that increase ROS in adipocytes induce IR [
      • Houstis N.
      • Rosen E.D.
      • Lander E.S.
      Reactive oxygen species have a causal role in multiple forms of insulin resistance.
      ,
      • Lin J.
      • Yang R.
      • Tarr P.T.
      • Wu P.H.
      • Handschin C.
      • Li S.
      • et al.
      Hyperlipidemic effects of dietary saturated fats mediated through PGC-1beta coactivation of SREBP.
      ]. Inhibition of mitochondrial superoxide dismutase (SOD2) has been hypothesized to mediate iron dependent oxidative damage and metabolic dysfunction [
      • Valenti L.
      • Conte D.
      • Piperno A.
      • Dongiovanni P.
      • Fracanzani A.L.
      • Fraquelli M.
      • et al.
      The mitochondrial superoxide dismutase A16V polymorphism in the cardiomyopathy associated with hereditary haemochromatosis.
      ,
      • Jouihan H.A.
      • Cobine P.A.
      • Cooksey R.C.
      • Hoagland E.A.
      • Boudina S.
      • Abel E.D.
      • et al.
      Iron-mediated inhibition of mitochondrial manganese uptake mediates mitochondrial dysfunction in a mouse model of hemochromatosis.
      ].
      Activation of NFκB in macrophages and Kupffer cells, and consequent release of TNFα [
      • Scaccabarozzi A.
      • Arosio P.
      • Weiss G.
      • Valenti L.
      • Dongiovanni P.
      • Fracanzani A.L.
      • et al.
      Relationship between TNF-alpha and iron metabolism in differentiating human monocytic THP-1 cells.
      ,
      • Fargion S.
      • Valenti L.
      • Dongiovanni P.
      • Scaccabarozzi A.
      • Fracanzani A.L.
      • Taioli E.
      • et al.
      Tumor necrosis factor alpha promoter polymorphisms influence the phenotypic expression of hereditary hemochromatosis.
      ,
      • She H.
      • Xiong S.
      • Lin M.
      • Zandi E.
      • Giulivi C.
      • Tsukamoto H.
      Iron activates NF-κB in Kupffer cells.
      ], a major player in IR in MetS and NAFLD by means of its ability to downregulate insulin signaling and decrease adiponectin levels [

      Manco M, Alisi A, Real JM, Equitani F, Devito R, Valenti L, et al. Early interplay of intra-hepatic iron and insulin resistance in children with non-alcoholic fatty liver disease. J Hepatol 2011. in press [PMID: 21168460].

      ,
      • Valenti L.
      • Fracanzani A.L.
      • Dongiovanni P.
      • Santorelli G.
      • Branchi A.
      • Taioli E.
      • et al.
      Tumor necrosis factor alpha promoter polymorphisms and insulin resistance in nonalcoholic fatty liver disease.
      ,
      • Kern P.A.
      • Di Gregorio G.B.
      • Lu T.
      • Rassouli N.
      • Ranganathan G.
      Adiponectin expression from human adipose tissue: relation to obesity, insulin resistance, and tumor necrosis factor-alpha expression.
      ,
      • Hotamisligil G.S.
      • Peraldi P.
      • Budavari A.
      • Ellis R.
      • White M.F.
      • Spiegelman B.M.
      IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-alpha- and obesity-induced insulin resistance.
      ], may also be implicated in the pathogenesis of IR associated with DIOS, which is typically characterized by iron accumulation in this cellular compartment.
      Supporting a role for iron in the induction of IR and a possible involvement of adipose tissue, Green et al. [
      • Green A.
      • Basile R.
      • Rumberger J.M.
      Transferrin and iron induce insulin resistance of glucose transport in adipocytes.
      ] demonstrated that isolated adipocytes treated with iron become insulin resistant, as detected by decreased insulin-stimulated glucose transport and increased lipolysis. If confirmed in vivo, these metabolic alterations would promote IR, and raise the risk of T2D and steatosis [
      • Green A.
      • Basile R.
      • Rumberger J.M.
      Transferrin and iron induce insulin resistance of glucose transport in adipocytes.
      ], but the effect of body iron overload on adipose tissue in vivo, except for a few data on adipokines (see below), is still under definition. Further work is required to determine whether iron may directly accumulate in adipose tissue and alter its function.
      Despite the model does not reflect the typical pattern of iron overload of DIOS, novel insights into the pathogenesis of iron induced IR have been provided in vivo by the detailed metabolic characterization of iron overloaded mice due to the deletion of the Hfe gene of HHC. Despite higher glucose uptake, these mice had lower glucose oxidation in skeletal muscle, which was linked to Ampk- and Pdk4-mediated [
      • Huang J.
      • Gabrielsen J.S.
      • Cooksey R.C.
      • Luo B.
      • Boros L.G.
      • Jones D.L.
      • et al.
      Increased glucose disposal and AMP-dependent kinase signaling in a mouse model of hemochromatosis.
      ] decrease in pyruvate dehydrogenase activity, and higher hepatic glucose output and metabolic inflexibility (i.e. a decreased ability to transition between utilization of carbohydrate and lipid fuel sources), both of which are characteristics of T2D [
      • Huang J.
      • Jones D.
      • Luo B.
      • Sanderson M.
      • Soto J.
      • Abel E.D.
      • et al.
      Iron overload and diabetes risk: a shift from glucose to fatty acid oxidation and increased hepatic glucose production in a mouse model of hereditary hemochromatosis.
      ]. Contrary to what expected, the metabolic alterations described in this model did not depend on mitochondrial oxidative damage. As iron sufficiency and deletion of Hfe facilitate erythropoiesis [
      • Ramos P.
      • Guy E.
      • Chen N.
      • Proenca C.C.
      • Gardenghi S.
      • Casu C.
      • et al.
      Enhanced erythropoiesis in Hfe-KO mice indicates a role for Hfe in the modulation of erythroid iron homeostasis.
      ], it would seem advantageous for an iron-loaded mouse to shift to the more energy-efficient but oxygen-inefficient fuel source of fatty acids to make use of that full capacity for oxygen transport [
      • Huang J.
      • Jones D.
      • Luo B.
      • Sanderson M.
      • Soto J.
      • Abel E.D.
      • et al.
      Iron overload and diabetes risk: a shift from glucose to fatty acid oxidation and increased hepatic glucose production in a mouse model of hereditary hemochromatosis.
      ]. Preliminary data from our group also confirm that dietary iron overload induces IR in mice, and the mechanism might be related to iron accumulation within visceral adipose tissue resulting in altered release of adipocytokines [
      • Dongiovanni P.
      • Ruscica M.
      • Benedan L.
      • Borroni V.
      • Recalcati S.
      • Steffani L.
      • et al.
      Dietary iron overload induces visceral adipose tissue insulin resistance associated with hyper-resistinemia, and synergizes with obesity and fatty liver in inducing systemic insulin resistance.
      ]. A working model of the mechanisms underlying iron associated IR is proposed in Fig. 1.
      Figure thumbnail gr1
      Fig. 1Proposed mechanisms explaining iron induced insulin resistance and metabolic alterations. FFAs, free fatty acids; ER, endoplasmic reticulum.

      Iron, adipose tissue, and adipokines

      NAFLD is highly prevalent in obesity, associated with chronic inflammation in adipose tissue, and with abnormal release of adipocytokines that play an endocrine role in the progression to NASH, T2D, and cardiovascular disease. Thus, it is likely that the effect of iron on IR involves altered regulation of adipose tissue and of adipokines.
      Serum levels of adiponectin, the major anti-steatotic and anti-inflammatory adipocyte-derived mediator, are reduced in obesity, T2D, and IR, whereas weight loss and PPARγ activation by glitazones induce adiponectin [
      • Lutchman G.
      • Modi A.
      • Kleiner D.E.
      • Promrat K.
      • Heller T.
      • Ghany M.
      • et al.
      The effects of discontinuing pioglitazone in patients with nonalcoholic steatohepatitis.
      ]. In obese mice, deletion of adiponectin receptors induces inflammation, oxidative stress, and IR, whereas adiponectin overexpression improves IR and reverts the diabetic phenotype [
      • Kim J.Y.
      • van de Wall E.
      • Laplante M.
      • Azzara A.
      • Trujillo M.E.
      • Hofmann S.M.
      • et al.
      Obesity-associated improvements in metabolic profile through expansion of adipose tissue.
      ]. Patients with NAFLD have decreased adiponectin [
      • Musso G.
      • Gambino R.
      • Biroli G.
      • Carello M.
      • Faga E.
      • Pacini G.
      • et al.
      Hypoadiponectinemia predicts the severity of hepatic fibrosis and pancreatic beta-cell dysfunction in nondiabetic nonobese patients with nonalcoholic steatohepatitis.
      ], and hypo-adiponectinemia predicts the severity of inflammation and fibrosis in NASH [
      • Polyzos S.A.
      • Toulis K.A.
      • Goulis D.G.
      • Zavos C.
      • Kountouras J.
      Serum total adiponectin in nonalcoholic fatty liver disease: a systematic review and meta-analysis.
      ]. Interestingly, a negative correlation between adiponectin and ferritin levels has been reported in patients with type 2 diabetes and in the general population [
      • Aso Y.
      • Takebayashi K.
      • Wakabayashi S.
      • Momobayashi A.
      • Sugawara N.
      • Terasawa T.
      • et al.
      Relation between serum high molecular weight adiponectin and serum ferritin or prohepcidin in patients with type 2 diabetes.
      ,
      • Ku B.J.
      • Kim S.Y.
      • Lee T.Y.
      • Park K.S.
      Serum ferritin is inversely correlated with serum adiponectin level: population-based cross-sectional study.
      ], although no data are available in NAFLD. Induction of heme oxygenase-1 (HO-1) by adiponectin, mediated by AMPK-mediated PPARα activation, elicited an antiapoptotic effect by decreasing iron in hepatocytes [
      • Lin H.
      • Yu C.H.
      • Jen C.Y.
      • Cheng C.F.
      • Chou Y.
      • Chang C.C.
      • et al.
      Adiponectin-mediated heme oxygenase-1 induction protects against iron-induced liver injury via a PPAR{alpha}-dependent mechanism.
      ], thus linking adiponectin to iron related liver damage. Adiponectin also induced cyclooxygenase-2 expression in mouse hepatocytes, conferring further protection against iron injury [
      • Lee F.P.
      • Jen C.Y.
      • Chang C.C.
      • Chou Y.
      • Lin H.
      • Chou C.M.
      • et al.
      Mechanisms of adiponectin-mediated COX-2 induction and protection against iron injury in mouse hepatocytes.
      ].
      Leptin is another well-studied adipokine, which plays an important role in the regulation of body weight through inhibition of food intake and stimulation of energy expenditure [
      • Halaas J.L.
      • Boozer C.
      • Blair-West J.
      • Fidahusein N.
      • Denton D.A.
      • Friedman J.M.
      Physiological response to long-term peripheral and central leptin infusion in lean and obese mice.
      ]. High levels of leptin have been observed in obesity, indicating the development of leptin resistance [
      • Maffei M.
      • Halaas J.
      • Ravussin E.
      • Pratley R.E.
      • Lee G.H.
      • Zhang Y.
      • et al.
      Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects.
      ]. Indeed, both leptin-mutant (ob/ob) and leptin receptor-deficient (db/db) mice are severely obese and insulin resistant, due to increased food intake and decreased energy expenditure [
      • Friedman J.M.
      • Halaas J.L.
      Leptin and the regulation of body weight in mammals.
      ]. Leptin rapidly reverses steatosis induced by high sucrose diet in rats [
      • Huang W.
      • Dedousis N.
      • O’Doherty R.M.
      Hepatic steatosis and plasma dyslipidemia induced by a high-sucrose diet are corrected by an acute leptin infusion.
      ], promotes the proliferation and migration of hepatoma cells in vitro [
      • Saxena N.K.
      • Sharma D.
      • Ding X.
      • Lin S.
      • Marra F.
      • Merlin D.
      • et al.
      Concomitant activation of the JAK/STAT, PI3K/AKT, and ERK signaling is involved in leptin-mediated promotion of invasion and migration of hepatocellular carcinoma cells.
      ], and is thought to be involved to the progression from NASH to fibrosis and hepatocellular carcinoma [
      • Kitade M.
      • Yoshiji H.
      • Kojima H.
      • Ikenaka Y.
      • Noguchi R.
      • Kaji K.
      • et al.
      Leptin-mediated neovascularization is a prerequisite for progression of nonalcoholic steatohepatitis in rats.
      ]. Hepatoma cells exposure to leptin directly up-regulates hepcidin, resulting in decreased iron absorption and impaired iron recycling, possibly contributing to DIOS pathogenesis [
      • Chung B.
      • Matak P.
      • McKie A.T.
      • Sharp P.
      Leptin increases the expression of the iron regulatory hormone hepcidin in HuH7 human hepatoma cells.
      ]. Thus, increased hepcidin, partially related to hyper-leptinemia, may represent the missing link between obesity and DIOS [
      • del Giudice E.M.
      • Santoro N.
      • Amato A.
      • Brienza C.
      • Calabro P.
      • Wiegerinck E.T.
      • et al.
      Hepcidin in obese children as a potential mediator of the association between obesity and iron deficiency.
      ]. However, there are no data on the correlation between leptin and iron stores in patients with NAFLD and MetS.
      Resistin is a recently discovered adipokine secreted by adipose tissue and macrophages that circulates at increased levels in obesity [
      • Steppan C.M.
      • Bailey S.T.
      • Bhat S.
      • Brown E.J.
      • Banerjee R.R.
      • Wright C.M.
      • et al.
      The hormone resistin links obesity to diabetes.
      ]. Treatment of mice with recombinant resistin impairs glucose tolerance, and anti-resistin antibodies improve IR in obese mice. Incubation of 3T3-L1 adipocytes with resistin inhibits insulin-stimulated glucose uptake [
      • Steppan C.M.
      • Bailey S.T.
      • Bhat S.
      • Brown E.J.
      • Banerjee R.R.
      • Wright C.M.
      • et al.
      The hormone resistin links obesity to diabetes.
      ], whereas in skeletal muscle resistin reduces the uptake and metabolism of FFAs [
      • Palanivel R.
      • Sweeney G.
      Regulation of fatty acid uptake and metabolism in L6 skeletal muscle cells by resistin.
      ]. Moreover, it seems that resistin significantly induces the gene expression of suppressor of cytokine signaling 3 (SOCS3), a known inhibitor of insulin signaling [
      • Steppan C.M.
      • Wang J.
      • Whiteman E.L.
      • Birnbaum M.J.
      • Lazar M.A.
      Activation of SOCS-3 by resistin.
      ]. So far there are no data supporting a relationship between resistin and iron overload, but an interaction between a polymorphism in the promoter of human resistin and oxidative stress has been reported [
      • Smith S.R.
      • Bai F.
      • Charbonneau C.
      • Janderova L.
      • Argyropoulos G.
      A promoter genotype and oxidative stress potentially link resistin to human insulin resistance.
      ], and antioxidants inhibited the expression of resistin in mice [
      • Felipe F.
      • Bonet M.L.
      • Ribot J.
      • Palou A.
      Modulation of resistin expression by retinoic acid and vitamin A status.
      ]. In a randomized trial, short-term vitamin C supplementation reduced resistin levels independently of inflammation [
      • Bo S.
      • Ciccone G.
      • Durazzo M.
      • Gambino R.
      • Massarenti P.
      • Baldi I.
      • et al.
      Efficacy of antioxidant treatment in reducing resistin serum levels: a randomized study.
      ].
      Visfatin is another novel adipokine predominantly secreted by visceral adipose tissue and increased in T2D [
      • Fukuhara A.
      • Matsuda M.
      • Nishizawa M.
      • Segawa K.
      • Tanaka M.
      • Kishimoto K.
      • et al.
      Visfatin: a protein secreted by visceral fat that mimics the effects of insulin.
      ] that exerts adipogenic effects in vitro and is, therefore, a good candidate to explain the accumulation of visceral adipose tissue that is associated with IR. In men with hyperglycemia, serum prohepcidin was strongly associated with visfatin, suggesting that circulating visfatin is perhaps upregulated by increasing iron stores, but no data are available specifically in patients with NAFLD, although most of the subjects with impaired fasting glucose or diabetes have increased liver fat. Moreover, visfatin correlated negatively with serum transferrin receptor, a marker of iron deficient erythropoiesis [
      • Fernandez-Real J.M.
      • Moreno J.M.
      • Chico B.
      • Lopez-Bermejo A.
      • Ricart W.
      Circulating visfatin is associated with parameters of iron metabolism in subjects with altered glucose tolerance.
      ]. Finally, retinol binding protein-4 (RBP4), an adipokine associated with IR, was correlated with ferritin levels in middle aged men and in subjects with type 2 diabetes, and iron increased RBP4 release by adipocytes in vitro [
      • Fernandez-Real J.M.
      • Moreno J.M.
      • Ricart W.
      Circulating RBP4 concentration might reflect insulin resistance-associated iron overload.
      ].
      These data suggest that DIOS is associated with abnormal endocrine function of adipose tissue and adipokines signaling, potentially contributing to metabolic abnormalities, liver damage, and cardiovascular disease.

      Association between iron overload and vascular damage in MetS and NAFLD

      NAFLD has been associated both with increased susceptibility to develop increased iron stores (DIOS), and with heightened risk of vascular damage, independently of classic risk factors [
      • Fracanzani A.L.
      • Burdick L.
      • Raselli S.
      • Pedotti P.
      • Grigore L.
      • Santorelli G.
      • et al.
      Carotid artery intima-media thickness in nonalcoholic fatty liver disease.
      ,
      • Targher G.
      • Bertolini L.
      • Padovani R.
      • Rodella S.
      • Zoppini G.
      • Zenari L.
      • et al.
      Relations between carotid artery wall thickness and liver histology in subjects with nonalcoholic fatty liver disease.
      ], and cardiovascular disease represents the first cause of death in patients with NAFLD [
      • Jepsen P.
      • Vilstrup H.
      • Mellemkjaer L.
      • Thulstrup A.M.
      • Olsen J.H.
      • Baron J.A.
      • et al.
      Prognosis of patients with a diagnosis of fatty liver – a registry-based cohort study.
      ]. Iron deposition in arterial wall macrophages is increased in atherosclerotic lesions [
      • Lapenna D.
      • Pierdomenico S.D.
      • Ciofani G.
      • Ucchino S.
      • Neri M.
      • Giamberardino M.A.
      • et al.
      Association of body iron stores with low molecular weight iron and oxidant damage of human atherosclerotic plaques.
      ] and, although evidence is controversial [
      • van der A.D.
      • Peeters P.H.
      • Grobbee D.E.
      • Roest M.
      • Marx J.J.
      • Voorbij H.M.
      • et al.
      HFE mutations and risk of coronary heart disease in middle-aged women.
      ,
      • Pardo Silva M.C.
      • Njajou O.T.
      • Alizadeh B.Z.
      • Hofman A.
      • Witteman J.C.
      • van Duijn C.M.
      • et al.
      HFE gene mutations increase the risk of coronary heart disease in women.
      ,
      • Danesh J.
      • Appleby P.
      Coronary heart disease and iron status: meta-analyses of prospective studies.
      ], increased iron stores have been suggested as a marker of cardiovascular risk [
      • Sullivan J.L.
      Macrophage iron, hepcidin, and atherosclerotic plaque stability.
      ]. Indirect confirmation of the “iron hypothesis” comes from studies of atherosclerosis treatment. Indeed, ID decreased atherogenesis in experimental models [
      • Lapenna D.
      • Pierdomenico S.D.
      • Ciofani G.
      • Ucchino S.
      • Neri M.
      • Giamberardino M.A.
      • et al.
      Association of body iron stores with low molecular weight iron and oxidant damage of human atherosclerotic plaques.
      ,
      • Ganz T.
      • Nemeth E.
      Iron imports. IV. Hepcidin and regulation of body iron metabolism.
      ], blood donation was associated with lower risk of myocardial infarction [
      • Salonen J.T.
      • Tuomainen T.P.
      • Salonen R.
      • Lakka T.A.
      • Nyyssonen K.
      Donation of blood is associated with reduced risk of myocardial infarction. The Kuopio Ischaemic Heart Disease Risk Factor Study.
      ] and phlebotomy slowed the progression of vascular disease [
      • Lee T.S.
      • Shiao M.S.
      • Pan C.C.
      • Chau L.Y.
      Iron-deficient diet reduces atherosclerotic lesions in apoE-deficient mice.
      ,
      • Zacharski L.R.
      • Chow B.K.
      • Howes P.S.
      • Shamayeva G.
      • Baron J.A.
      • Dalman R.L.
      • et al.
      Reduction of iron stores and cardiovascular outcomes in patients with peripheral arterial disease: a randomized controlled trial.
      ], whereas the lack of association between HFE mutations with vascular damage might be explained by the decrease in hepcidin levels, which would paradoxically facilitate iron export from macrophages [
      • Nemeth E.
      • Ganz T.
      Regulation of iron metabolism by hepcidin.
      ,
      • Chen Y.H.
      • Chau L.Y.
      • Chen J.W.
      • Lin S.J.
      Serum bilirubin and ferritin levels link heme oxygenase-1 gene promoter polymorphism and susceptibility to coronary artery disease in diabetic patients.
      ], determining more rapid clearance of iron from arterial lesions. Indeed, besides iron overload, hepcidin is also induced by inflammation and obesity, and local production determines iron trapping into macrophages [
      • Zacharski L.R.
      • Chow B.K.
      • Howes P.S.
      • Shamayeva G.
      • Baron J.A.
      • Dalman R.L.
      • et al.
      Reduction of iron stores and cardiovascular outcomes in patients with peripheral arterial disease: a randomized controlled trial.
      ,
      • Theurl I.
      • Theurl M.
      • Seifert M.
      • Mair S.
      • Nairz M.
      • Rumpold H.
      • et al.
      Autocrine formation of hepcidin induces iron retention in human monocytes.
      ]. Thus, excessive iron in macrophages would increase oxidative stress and transformation into foam cells and hepcidin may be responsible for iron induced atherogenesis [
      • Lapenna D.
      • Pierdomenico S.D.
      • Ciofani G.
      • Ucchino S.
      • Neri M.
      • Giamberardino M.A.
      • et al.
      Association of body iron stores with low molecular weight iron and oxidant damage of human atherosclerotic plaques.
      ].
      The still unexplained association between the C282Y hemochromatosis mutation and low LDL cholesterol [
      • Adams P.C.
      • Pankow J.S.
      • Barton J.C.
      • Acton R.T.
      • Leiendecker-Foster C.
      • McLaren G.D.
      • et al.
      HFE C282Y homozygosity is associated with lower total and low-density lipoprotein cholesterol: the hemochromatosis and iron overload screening study.
      ,
      • Pankow J.S.
      • Boerwinkle E.
      • Adams P.C.
      • Guallar E.
      • Leiendecker-Foster C.
      • Rogowski J.
      • et al.
      HFE C282Y homozygotes have reduced low-density lipoprotein cholesterol: the Atherosclerosis Risk in Communities (ARIC) Study.
      ], which was confirmed in a meta-analysis of genome-wide association studies [
      • Teslovich T.M.
      • Musunuru K.
      • Smith A.V.
      • Edmondson A.C.
      • Stylianou I.M.
      • Koseki M.
      • et al.
      Biological, clinical and population relevance of 95 loci for blood lipids.
      ], may also contribute to explain atherosclerosis protection in individuals carrying HFE mutations. Interestingly, also the beta-thalassemia trait is strongly associated with reduced cholesterol levels and lower cardiovascular risk [
      • Deiana L.
      • Garuti R.
      • Pes G.M.
      • Carru C.
      • Errigo A.
      • Rolleri M.
      • et al.
      Influence of beta(0)-thalassemia on the phenotypic expression of heterozygous familial hypercholesterolemia: a study of patients with familial hypercholesterolemia from Sardinia.
      ,
      • Maioli M.
      • Vigna G.B.
      • Tonolo G.
      • Brizzi P.
      • Ciccarese M.
      • Donega P.
      • et al.
      Plasma lipoprotein composition, apolipoprotein(a) concentration and isoforms in beta-thalassemia.
      ], but the elucidation of the relationship between iron and cholesterol metabolism requires, therefore, further studies.
      Recently, our group has shown that serum ferritin and hepcidin levels predicted vascular damage in NAFLD, but only in patients negative for HFE genotypes or beta-globin mutations associated with low hepcidin [
      • Valenti L.
      • Swinkels D.W.
      • Burdick L.
      • Dongiovanni P.
      • Tjalsma H.
      • Motta B.M.
      • et al.
      Serum ferritin levels are associated with vascular damage in patients with nonalcoholic fatty liver disease.
      ]. The mechanism seems to involve upregulation of macrophage chemoattractant protein-1 (MCP-1/CCL2), a chemokine involved in the recruitment of leukocytes to plaques and correlated with the atherosclerotic burden, by intracellular iron in monocytes [
      • Valenti L.
      • Dongiovanni P.
      • Motta B.M.
      • Swinkels D.W.
      • Bonara P.
      • Rametta R.
      • et al.
      Serum hepcidin and macrophage iron correlate with MCP-1 release and vascular damage in patients with metabolic syndrome alterations.
      ]. However, whether the presence of iron overload is associated with an increased rate of cardiovascular events in NAFLD and the MetS is presently unproven.

      Association between iron overload and liver damage

      We have shown [
      • Valenti L.
      • Fracanzani A.L.
      • Bugianesi E.
      • Dongiovanni P.
      • Galmozzi E.
      • Vanni E.
      • et al.
      HFE genotype, parenchymal iron accumulation, and liver fibrosis in patients with nonalcoholic fatty liver disease.
      ] that, in NAFLD, hepatocellular iron accumulation was associated with a higher risk of fibrosis compared to the absence of siderosis or the non-parenchymal iron accumulation, which is more commonly observed and typical of DIOS. However, evidence that only parenchymal iron carries a higher risk of progressive liver disease is still conflicting, since in a large US cohort, non-parenchymal iron, related to more severe metabolic alterations, was associated with histological inflammation and more advanced fibrosis [
      • Nelson J.E.
      • Wilson L.
      • Brunt E.M.
      • Yeh M.M.
      • Kleiner D.E.
      • Unalp-Arida A.
      • et al.
      Relationship between the pattern of hepatic iron deposition and histological severity in nonalcoholic fatty liver disease.
      ]. Furthermore, non-parenchymal iron overload has been associated with hepatocellular carcinoma in Italian patients with NASH-related cirrhosis [
      • Sorrentino P.
      • D’Angelo S.
      • Ferbo U.
      • Micheli P.
      • Bracigliano A.
      • Vecchione R.
      Liver iron excess in patients with hepatocellular carcinoma developed on non-alcoholic steato-hepatitis.
      ]. It is, therefore, likely that the genetic background underlying iron accumulation influences the outcome in ethnically different populations. Due to incomplete association with iron overload, HFE mutations were not associated with liver fibrosis in Italian patients with NAFLD [
      • Valenti L.
      • Fracanzani A.L.
      • Bugianesi E.
      • Dongiovanni P.
      • Galmozzi E.
      • Vanni E.
      • et al.
      HFE genotype, parenchymal iron accumulation, and liver fibrosis in patients with nonalcoholic fatty liver disease.
      ], although they predicted liver damage in US Caucasian patients with NASH [
      • Nelson J.E.
      • Bhattacharya R.
      • Lindor K.D.
      • Chalasani N.
      • Raaka S.
      • Heathcote E.J.
      • et al.
      HFE C282Y mutations are associated with advanced hepatic fibrosis in Caucasians with nonalcoholic steatohepatitis.
      ], whereas beta-globin mutations, the best predictor of parenchymal iron overload in the Mediterranean area, were associated with an almost double risk of severe fibrosis [
      • Valenti L.
      • Canavesi E.
      • Galmozzi E.
      • Dongiovanni P.
      • Rametta R.
      • Maggioni P.
      • et al.
      Beta-globin mutations are associated with parenchymal siderosis and fibrosis in patients with non-alcoholic fatty liver disease.
      ].
      Nevertheless, longitudinal studies with follow-up liver biopsies are needed to investigate the relationship between iron overload, ID, and the progression of hepatic disease.

      Mechanism of iron induced liver damage

      Evidence is accumulating that mild hepatic iron overload promotes the progression of liver damage associated with fatty liver also independently of IR, and once again the mechanism involves increased oxidative stress. Iron is a potent catalyst of oxidative stress via the Fenton reaction and can directly cause lipid peroxidation generating malonyldialdehyde, which is capable to activate hepatic stellate cells (HSCs), a major player of fibrogenesis in NAFLD [
      • Lee K.S.
      • Buck M.
      • Houglum K.
      • Chojkier M.
      Activation of hepatic stellate cells by TGF alpha and collagen type I is mediated by oxidative stress through c-myb expression.
      ,
      • Marra F.
      • Bertolani C.
      Adipokines in liver diseases.
      ]. ROS cause peroxidation of polyunsaturated fatty acids and nucleic acids [
      • Sumida Y.
      • Yoshikawa T.
      • Okanoue T.
      Role of hepatic iron in non-alcoholic steatohepatitis.
      ,
      • Day C.P.
      Steatohepatitis: a tale of two “hits”?.
      ], and a lipophilic antioxidant, such as vitamin E reduced liver enzymes and hepatocellular damage in a large randomized controlled trial in NASH [
      • Sanyal A.J.
      • Chalasani N.
      • Kowdley K.V.
      • McCullough A.
      • Diehl A.M.
      • Bass N.M.
      • et al.
      Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis.
      ]. Iron overload may thus play a role in NASH by generating oxidative DNA damage; supporting this hypothesis, hepatic 7,8-dihydro-8-oxo-2′ deoxyguanosine (8-oxodG), a DNA base-modified product generated by hydroxyl radicals, was increased in NASH, and correlated with iron overload, IR, and severity of hepatic steatosis. Moreover, ID decreased oxidative stress and HSCs activation in experimental models of liver injury [
      • Otogawa K.
      • Ogawa T.
      • Shiga R.
      • Nakatani K.
      • Ikeda K.
      • Nakajima Y.
      • et al.
      Attenuation of acute and chronic liver injury in rats by iron-deficient diet.
      ], and after phlebotomy hepatic 8-oxodG levels decreased with concomitant reduction of serum transaminases in NASH patients [
      • Fujita N.
      • Miyachi H.
      • Tanaka H.
      • Takeo M.
      • Nakagawa N.
      • Kobayashi Y.
      • et al.
      Iron overload is associated with hepatic oxidative damage to DNA in nonalcoholic steatohepatitis.
      ].
      Iron can also directly induce fibrogenesis, as HSCs can be activated by the generation of ROS with ascorbate/FeSO4 and by malonyldialdehyde. In addition, HSCs activation by collagen type I and TGFα was blocked by antioxidants [
      • Lee K.S.
      • Buck M.
      • Houglum K.
      • Chojkier M.
      Activation of hepatic stellate cells by TGF alpha and collagen type I is mediated by oxidative stress through c-myb expression.
      ].
      A specific receptor for ferritin has been demonstrated on activated HSCs, and it has been proposed that ferritin acts as a cytokine with pro-inflammatory activity regulating fibrogenesis via NFκB-regulated signaling in HSCs [
      • Ruddell R.G.
      • Hoang-Le D.
      • Barwood J.M.
      • Rutherford P.S.
      • Piva T.J.
      • Watters D.J.
      • et al.
      Ferritin functions as a proinflammatory cytokine via iron-independent protein kinase C zeta/nuclear factor kappaB-regulated signaling in rat hepatic stellate cells.
      ].
      Is iron overload sufficient to trigger oxidative damage? In rats, iron accumulation is associated with induction of HO-1, a sensitive indicator of oxidative stress, but not with fibrosis [
      • Brown K.E.
      • Dennery P.A.
      • Ridnour L.A.
      • Fimmel C.J.
      • Kladney R.D.
      • Brunt E.M.
      • et al.
      Effect of iron overload and dietary fat on indices of oxidative stress and hepatic fibrogenesis in rats.
      ], highlighting the difference between oxidative stress and damage, and suggesting that the former is not sufficient to elicit overt fibrosis, at least in rodents. While hepatic iron overload leads to oxidative stress, there is an associated up-regulation of antioxidant defenses that may be a critical factor limiting the accumulation of oxidative damage. Probably, co-existing liver injury or nutritional/genetic factors, and in particular the coexistence of steatosis [
      • Powell E.E.
      • Ali A.
      • Clouston A.D.
      • Dixon J.L.
      • Lincoln D.J.
      • Purdie D.M.
      • et al.
      Steatosis is a cofactor in liver injury in hemochromatosis.
      ], may compromise the ability to mount an effective antioxidant defense, and thus predispose to fibrogenesis. In a rat model of T2D, lipid peroxidation and hepatic superoxide production decreased in rats fed an iron-deficient diet or treated with phlebotomy [
      • Minamiyama Y.
      • Takemura S.
      • Kodai S.
      • Shinkawa H.
      • Tsukioka T.
      • Ichikawa H.
      • et al.
      Iron restriction improves type 2 diabetes mellitus in Otsuka Long-Evans Tokushima fatty rats.
      ]. In the methionine choline-deficient (MCD) model of NASH, hepatic iron overload was associated with necroinflammation and a trend toward increased perivenular fibrosis [
      • Kirsch R.
      • Sijtsema H.P.
      • Tlali M.
      • Marais A.D.
      • Hall Pde L.
      Effects of iron overload in a rat nutritional model of non-alcoholic fatty liver disease.
      ], whereas in the same model, a single injection of iron induced fibrosis development and worsening of steatosis, thereby emphasizing the role of iron in the progression of nutritional NASH to the fibrotic stage [
      • Imeryuz N.
      • Tahan V.
      • Sonsuz A.
      • Eren F.
      • Uraz S.
      • Yuksel M.
      • et al.
      Iron preloading aggravates nutritional steatohepatitis in rats by increasing apoptotic cell death.
      ], which is possibly mediated by the facilitation of apoptosis [
      • Takehara T.
      • Tatsumi T.
      • Suzuki T.
      • Rucker 3rd, E.B.
      • Hennighausen L.
      • Jinushi M.
      • et al.
      Hepatocyte-specific disruption of Bcl-xL leads to continuous hepatocyte apoptosis and liver fibrotic responses.
      ]. Apoptotic hepatocytes may indeed stimulate HSCs either directly or indirectly via TGF-β production [
      • Feldstein A.E.
      • Canbay A.
      • Angulo P.
      • Taniai M.
      • Burgart L.J.
      • Lindor K.D.
      • et al.
      Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis.
      ].
      Thus, iron could represent a second hit in the progression of liver damage from simple uncomplicated steatosis to fibrotic NASH, but since fibrosis is not a constant feature of DIOS, other genetic or acquired conditions are necessary to trigger this process. In addition, iron may favor malignant transformation and hepatocellular carcinoma by promotion of cell growth and oxidative dependent DNA damage [
      • Fargion S.
      • Valenti L.
      • Fracanzani A.L.
      Hemochromatosis gene (HFE) mutations and cancer risk: expanding the clinical manifestations of hereditary iron overload.
      ,
      • Dongiovanni P.
      • Fracanzani A.
      • Cairo G.
      • Megazzini C.P.
      • Gatti S.
      • Rametta R.
      • et al.
      Iron dependent regulation of MDM2 influences p53 activity and hepatic carcinogenesis.
      ]. A model depicting the proposed mechanisms underlying liver damage associated with iron overload in steatosis and DIOS is shown in Fig. 2.
      Figure thumbnail gr2
      Fig. 2Proposed mechanisms explaining iron induced liver damage associated with steatosis and DIOS in hepatocytes (brown), macrophages (gray), and hepatic stellate cells (yellow). Cp, ceruloplasmin; Cu, copper; Fe-Tf, ferric-transferrin; Fp-1, ferroportin-1; HCC, hepatocellular carcinoma; HFE, hemochromatosis gene; HSCs, hepatic stellate cells; MDA, malonyl-dialdehyde; ROS, reactive oxygen species; SOD2, Mn superoxide dismutase; Tf-R, transferrin receptor.

      Iron depletion therapy in DIOS: experimental studies

      Experimental evidence suggests that ID not only is able to counteract the negative effect of iron overload, but that mild iron deficiency itself may further positively impact on IR. Recently, our group has investigated the effect of iron depletion on glucose metabolism in hepatocytes in vitro and in an in vivo model. The data obtained indicate that cellular ID induced by chelators induces glucose uptake and utilization, increasing insulin receptor (InsR) binding activity and signaling, and that the mechanism is probably associated with the hypoxia inducible factor-1α HIF-1α stabilization by reduced iron availability [
      • Dongiovanni P.
      • Valenti L.
      • Ludovica Fracanzani A.
      • Gatti S.
      • Cairo G.
      • Fargion S.
      Iron depletion by deferoxamine up-regulates glucose uptake and insulin signaling in hepatoma cells and in rat liver.
      ]. In line with these findings, as skeletal muscles play a major part in glucose utilization, it has been shown that L6 myocytes adapt to ID by increasing glucose utilization through enhanced expression of the main basal glucose transporter Glut-1 [
      • Potashnik R.
      • Kozlovsky N.
      • Ben-Ezra S.
      • Rudich A.
      • Bashan N.
      Regulation of glucose transport and GLUT-1 expression by iron chelators in muscle cells in culture.
      ]. Furthermore, increased insulin sensitivity in peripheral tissues has been shown in a rat model of iron deficiency anemia [
      • Borel M.J.
      • Beard J.L.
      • Farrell P.A.
      Hepatic glucose production and insulin sensitivity and responsiveness in iron-deficient anemic rats.
      ]. This response may represent a metabolic adaptation (which is specular to that observed during iron overload) to decreased oxygen availability secondary to a deficiency in hemoglobin, myoglobin, and cytochromes due to the scarcity of iron [
      • Huang J.
      • Jones D.
      • Luo B.
      • Sanderson M.
      • Soto J.
      • Abel E.D.
      • et al.
      Iron overload and diabetes risk: a shift from glucose to fatty acid oxidation and increased hepatic glucose production in a mouse model of hereditary hemochromatosis.
      ], which forces tissues to depend more heavily on the anaerobic catabolism of glucose for their energy supply. A model of the proposed mechanisms underlying improved glucose clearance and insulin sensitivity under ID is shown in Fig. 3.
      Figure thumbnail gr3
      Fig. 3Proposed additional mechanisms by which iron depletion improves glucose disposal and insulin sensitivity. InsR, insulin receptor; HIF-1α, hypoxia inducible factor-1α; Glut1, glucose transporter 1.

      Iron depletion therapy in patients with NAFLD, MetS, and DIOS

      Several reports indicate that ID may be beneficial in patients with DIOS. ID has been first reported to be well tolerated in patients with DIOS [
      • Guillygomarc’h A.
      • Mendler M.H.
      • Moirand R.
      • Laine F.
      • Quentin V.
      • David V.
      • et al.
      Venesection therapy of insulin resistance-associated hepatic iron overload.
      ], and to improve insulin sensitivity in the short term (without changes in body weight) in patients with NAFLD with and without increased ferritin levels, in two uncontrolled studies conducted in 17 patients with impaired glucose tolerance [
      • Facchini F.S.
      • Hua N.W.
      • Stoohs R.A.
      Effect of iron depletion in carbohydrate-intolerant patients with clinical evidence of nonalcoholic fatty liver disease.
      ] and in 12 patients with normal glucose tolerance [
      • Valenti L.
      • Fracanzani A.L.
      • Fargion S.
      Effect of iron depletion in patients with nonalcoholic fatty liver disease without carbohydrate intolerance.
      ]. Phlebotomy led to decreased HbA1c levels, heightened insulin secretion and insulin sensitivity in a randomized controlled study in 28 patients with T2D and increased ferritin levels and stable body weight [
      • Fernandez-Real J.M.
      • Penarroja G.
      • Castro A.
      • Garcia-Bragado F.
      • Hernandez-Aguado I.
      • Ricart W.
      Blood letting in high-ferritin type 2 diabetes: effects on insulin sensitivity and beta-cell function.
      ]. In addition, ID improved insulin release in an uncontrolled study in 17 carriers of HFE mutations with steatosis [
      • Equitani F.
      • Fernandez-Real J.M.
      • Menichella G.
      • Koch M.
      • Calvani Menotti
      • Nobili V.
      • et al.
      Blood letting ameliorates insulin sensitivity and secretion in parallel to reduce iron overload in carriers of HFE mutations.
      ]. Regular blood donation was also associated with increased insulin sensitivity in 21 frequent donors compared to 66 healthy subjects, suggesting that stored iron impacts negatively on insulin action even in healthy people [
      • Fernandez-Real J.M.
      • Lopez-Bermejo A.
      • Ricart W.
      Iron stores, blood donation, and insulin sensitivity and secretion.
      ]. Both venesection therapy (in the absence of weight loss) and dietary treatment have been reported to improve serum ferritin, metabolic parameters, and liver function tests in 59 patients with DIOS in a controlled unmatched study [
      • Piperno A.
      • Vergani A.
      • Salvioni A.
      • Trombini P.
      • Vigano M.
      • Riva A.
      • et al.
      Effects of venesections and restricted diet in patients with the insulin-resistance hepatic iron overload syndrome.
      ]. However, in a case–control study in 128 patients (matched for age, sex, ferritin, and ALT levels) with diet-resistant NAFLD followed for 12 months, which took into account changes in body weight during the study, it was shown that ID reduced IR more than lifestyle modifications alone, independently of confounding factors [
      • Valenti L.
      • Fracanzani A.L.
      • Dongiovanni P.
      • Bugianesi E.
      • Marchesini G.
      • Manzini P.
      • et al.
      Iron depletion by phlebotomy improves insulin resistance in patients with nonalcoholic fatty liver disease and hyperferritinemia: evidence from a case–control study.
      ]. Lifestyle modifications were modestly effective on ferritin and liver enzymes, but did not improve IR, and the effect of ID was independent of changes in body weight and metabolic parameters [
      • Valenti L.
      • Fracanzani A.L.
      • Dongiovanni P.
      • Bugianesi E.
      • Marchesini G.
      • Manzini P.
      • et al.
      Iron depletion by phlebotomy improves insulin resistance in patients with nonalcoholic fatty liver disease and hyperferritinemia: evidence from a case–control study.
      ]. Of note, the advantage of ID by phlebotomy was more marked in patients with higher baseline iron stores (ferritin >320 ng/ml) [
      • Valenti L.
      • Fracanzani A.L.
      • Dongiovanni P.
      • Bugianesi E.
      • Marchesini G.
      • Manzini P.
      • et al.
      Iron depletion by phlebotomy improves insulin resistance in patients with nonalcoholic fatty liver disease and hyperferritinemia: evidence from a case–control study.
      ].
      Concerning the direct effect of ID on liver damage, in a relatively large multi-center prospectively enrolled observational study in 198 NAFLD patients without diabetes, after adjustment for propensity score (which is used to simulate the effect of randomization on treatment choice in observational studies), ID was associated with a higher probability of normalization not only of insulin resistance, but also of liver enzymes compared to lifestyle modifications alone during follow-up [
      • Valenti L.
      • Moscatiello S.
      • Vanni E.
      • Fracanzani A.L.
      • Bugianesi E.
      • Fargion S.
      • et al.
      Venesection for non-alcoholic fatty liver disease unresponsive to lifestyle counselling – a propensity score-adjusted observational study.
      ]. Furthermore, the analysis of a cardiovascular trial suggests that ID may also prevent cancer development and progression [
      • Zacharski L.R.
      • Chow B.K.
      • Howes P.S.
      • Shamayeva G.
      • Baron J.A.
      • Dalman R.L.
      • et al.
      Decreased cancer risk after iron reduction in patients with peripheral arterial disease: results from a randomized trial.
      ], indicating that in patients with liver disease, it might protect from hepatocellular carcinoma independently of fibrosis progression [
      • Sorrentino P.
      • D’Angelo S.
      • Ferbo U.
      • Micheli P.
      • Bracigliano A.
      • Vecchione R.
      Liver iron excess in patients with hepatocellular carcinoma developed on non-alcoholic steato-hepatitis.
      ,
      • Fargion S.
      • Valenti L.
      • Fracanzani A.L.
      Hemochromatosis gene (HFE) mutations and cancer risk: expanding the clinical manifestations of hereditary iron overload.
      ].
      Thus, ID associated with lifestyle modifications may represent an eligible therapy for patients with NAFLD in the presence of iron overload. However, while it is almost well established that ID may improve metabolic and biochemical parameters in patients with NAFLD, whether it also prevents progression to cirrhosis and hepatocellular carcinoma is not demonstrated. A randomized controlled trial is ongoing to evaluate the effect of ID on the progression of histologically evaluated liver damage in patients with NAFLD and increased iron stores (NCT00658164).

      Conclusions

      DIOS is now a frequent finding in the general population, and hyperferritinemia, which reflects fatty liver and hyperinsulinemia, but also mildly increased body iron stores, is also detected in about 20–30% of patients with NAFLD and the MetS. Excessive body iron may play a causal role in IR through mechanisms that involve a reduced ability to burn carbohydrates and altered function of adipose tissue and release of adipokines. Furthermore, DIOS may facilitate the evolution to T2D by altering beta-cell function, the progression of cardiovascular disease by contributing to the recruitment and activation of macrophages within arterial lesions, and the natural history of liver disease by inducing oxidative stress in hepatocytes, activation of HSCs, and malignant transformation by promotion of cell growth and DNA damage.
      Based on these premises, the association among DIOS, MetS, and NAFLD is being investigated as a new risk factor to predict the development of overt cardiovascular, and hepatic diseases, but most importantly represents also a treatable condition. Indeed, ID, most frequently achieved by phlebotomy, has already been reported to decrease IR, metabolic alterations, and liver enzymes in controlled studies in NAFLD. Additional, randomized controlled studies are warranted to evaluate the potential of ID on hard clinical outcomes in patients with hyperferritinemia, and results are awaited before iron depletion therapy can be recommended for the treatment of hyperferritinemia associated with NAFLD, MetS, and DIOS.

      Conflict of interest

      The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

      Financial support

      Bando Giovani Ricercatori 2007, Ministero della Salute e delle Politiche Sociali (GR-2007-683265), PUR 10% 2009 (2009-ATE0087) Università degli Studi di Milano to L.V.

      Acknowledgments

      We thank all the clinical and laboratory members of the Metabolic Liver Diseases Research Center, and our collaborators.

      Supplementary data

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