Roheeth D. Delima

Hepatic Iron Loading in Mice Increases Cholesterol Biosynthesis

Iron and cholesterol are both essential metabolites in mammalian systems, and too much or too little of either can have serious clinical consequences. In addition, both have been associated with steatosis and its progression, contributing, inter alia, to an increase in hepatic oxidative stress. The interaction between iron and cholesterol is unclear, with no consistent evidence emerging with respect to changes in plasma cholesterol on the basis of iron status. We sought to clarify the role of iron in lipid metabolism by studying the effects of iron status on hepatic cholesterol synthesis in mice with differing iron status. Transcripts of seven enzymes in the cholesterol biosynthesis pathway were significantly up‐regulated with increasing hepatic iron (R2 between 0.602 and 0.164), including those of the rate‐limiting enzyme, 3‐hydroxy‐3‐methylglutarate‐coenzyme A reductase (Hmgcr; R2 = 0.362, P < 0.002). Hepatic cholesterol content correlated positively with hepatic iron (R2 = 0.255, P < 0.007). There was no significant relationship between plasma cholesterol and either hepatic cholesterol or iron (R2 = 0.101 and 0.014, respectively). Hepatic iron did not correlate with a number of known regulators of cholesterol synthesis, including sterol‐regulatory element binding factor 2 (Srebf2; R2 = 0.015), suggesting that the increases seen in the cholesterol biosynthesis pathway are independent of Srebf2. Transcripts of genes involved in bile acid synthesis, transport, or regulation did not increase with increasing hepatic iron. Conclusion: This study suggests that hepatic iron loading increases liver cholesterol synthesis and provides a new and potentially important additional mechanism by which iron could contribute to the development of fatty liver disease or lipotoxicity. (HEPATOLOGY 2010;)

Disruption of hemochromatosis protein and transferrin receptor 2 causes iron-induced liver injury in mice†

Mutations in hemochromatosis protein (HFE) or transferrin receptor 2 (TFR2) cause hereditary hemochromatosis (HH) by impeding production of the liver iron‐regulatory hormone, hepcidin (HAMP). This study examined the effects of disruption of Hfe or Tfr2, either alone or together, on liver iron loading and injury in mouse models of HH. Iron status was determined in Hfe knockout (Hfe−/−), Tfr2 Y245X mutant (Tfr2mut), and double‐mutant (Hfe−/−×Tfr2mut) mice by measuring plasma and liver iron levels. Plasma alanine transaminase (ALT) activity, liver histology, and collagen deposition were evaluated to assess liver injury. Hepatic oxidative stress was assessed by measuring superoxide dismutase (SOD) activity and F2‐isoprostane levels. Gene expression was measured by real‐time polymerase chain reaction. Hfe−/−×Tfr2mut mice had elevated hepatic iron with a periportal distribution and increased plasma iron, transferrin saturation, and non‐transferrin‐bound iron, compared with Hfe−/−, Tfr2mut, and wild‐type (WT) mice. Hamp1 expression was reduced to 40% (Hfe−/− and Tfr2mut) and 1% (Hfe−/−×Tfr2mut) of WT values. Hfe−/− ×Tfr2mut mice had elevated plasma ALT activity and mild hepatic inflammation with scattered aggregates of infiltrating inflammatory cluster of differentiation 45 (CD45)–positive cells. Increased hepatic hydoxyproline levels as well as Sirius red and Massons Trichrome staining demonstrated advanced portal collagen deposition. Hfe−/− and Tfr2mut mice had less hepatic inflammation and collagen deposition. Liver F2‐isoprostane levels were elevated, and copper/zinc and manganese SOD activities decreased in Hfe−/−×Tfr2mut, Tfr2mut, and Hfe−/− mice, compared with WT mice. Conclusion: Disruption of both Hfe and Tfr2 caused more severe hepatic iron overload with more advanced lipid peroxidation, inflammation, and portal fibrosis than was observed with the disruption of either gene alone. The Hfe−/−×Tfr2mut mouse model of iron‐induced liver injury reflects the liver injury phenotype observed in human HH. (HEPATOLOGY 2012)

Iron uptake from plasma transferrin by a transferrin receptor 2 mutant mouse model of haemochromatosis

BACKGROUND & AIMS Hereditary haemochromatosis type 3 is caused by mutations in transferrin receptor (TFR) 2. TFR2 has been shown to mediate iron transport in vitro and regulate iron homeostasis. The aim of this study was to determine the role of Tfr2 in iron transport in vivo using a Tfr2 mutant mouse. METHODS Tfr2 mutant and wild-type mice were injected intravenously with (59)Fe-transferrin and tissue (59)Fe uptake was measured. Tfr1, Tfr2 and ferroportin expression was measured by real-time PCR and Western blot. Cellular localisation of ferroportin was determined by immunohistochemistry. RESULTS Transferrin-bound iron uptake by the liver and spleen in Tfr2 mutant mice was reduced by 20% and 65%, respectively, whilst duodenal and renal uptake was unchanged compared with iron-loaded wild-type mice. In Tfr2 mutant mice, liver Tfr2 protein was absent, whilst ferroportin protein was increased in non-parenchymal cells and there was a low level of expression in hepatocytes. Tfr1 expression was unchanged compared with iron-loaded wild-type mice. Splenic Tfr2 protein expression was absent whilst Tfr1 and ferroportin protein expression was increased in Tfr2 mutant mice compared with iron-loaded wild-type mice. CONCLUSIONS A small reduction in hepatic transferrin-bound iron uptake in Tfr2 mutant mice suggests that Tfr2 plays a minor role in liver iron transport and its primary role is to regulate iron metabolism. Increased ferroportin expression due to decreased hepcidin mRNA levels is likely to be responsible for impaired splenic iron uptake in Tfr2 mutant mice.

Brain transcriptome perturbations in the Hfe−/− mouse model of genetic iron loading

Severe disruption of brain iron homeostasis can cause fatal neurodegenerative disease, however debate surrounds the neurologic effects of milder, more common iron loading disorders such as hereditary hemochromatosis, which is usually caused by loss-of-function polymorphisms in the HFE gene. There is evidence from both human and animal studies that HFE gene variants may affect brain function and modify risks of brain disease. To investigate how disruption of HFE influences brain transcript levels, we used microarray and real-time reverse transcription polymerase chain reaction to assess the brain transcriptome in Hfe(-/-) mice relative to wildtype AKR controls (age 10 weeks, n≥4/group). The Hfe(-/-) mouse brain showed numerous significant changes in transcript levels (p<0.05) although few of these related to proteins directly involved in iron homeostasis. There were robust changes of at least 2-fold in levels of transcripts for prominent genes relating to transcriptional regulation (FBJ osteosarcoma oncogene Fos, early growth response genes), neurotransmission (glutamate NMDA receptor Grin1, GABA receptor Gabbr1) and synaptic plasticity and memory (calcium/calmodulin-dependent protein kinase IIα Camk2a). As previously reported for dietary iron-supplemented mice, there were altered levels of transcripts for genes linked to neuronal ceroid lipofuscinosis, a disease characterized by excessive lipofuscin deposition. Labile iron is known to enhance lipofuscin generation which may accelerate brain aging. The findings provide evidence that iron loading disorders can considerably perturb levels of transcripts for genes essential for normal brain function and may help explain some of the neurologic signs and symptoms reported in hemochromatosis patients.

Brain changes in iron loading disorders

Abnormal iron accumulation within the brain is associated with various neurodegenerative diseases; however, there is debate about whether milder disorders of systemic iron loading, such as haemochromatosis, affect the brain. Arguments on both sides of the debate are often based on some common assumptions that have not been rigorously tested by appropriate experimentation. Recent research from our lab has applied high-throughput molecular techniques such as microarray to models of dietary and genetic iron loading to identify subtle but important effects on molecular systems in the brain that may go undetected by other methods commonly used in the field. In this chapter, we review the existing research in animal models and human patients and discuss the strengths and limitations of the different approaches commonly used. Using our findings as an example, we argue that transcriptomic methods can provide unique insights into how systemic iron loading can affect the brain and suggest some basic guidelines for extracting the most robust and reliable information from microarray studies.

Potential protective effects of zinc in iron overload

Zinc is considered an anti-oxidant owing to its ability to protect the cell from the effects of oxidative damage through its interaction with cellular thiols, preventing their oxidative inactivation, and by competing with metal ions that produce reactive oxygen species. There are chronic and acute mechanisms by which zinc acts as an anti-oxidant. The chronic anti-oxidant effects of zinc involve long-term exposure to zinc, resulting in the induction of the anti-oxidant protein metallothionein (MT). The acute effects of zinc operate via two mechanisms; firstly, zinc can provide protection of protein sulfhydryls and secondly, zinc can prevent the formation of hydroxide ions from hydrogen peroxide through the antagonism of redoxactive metals such as iron and copper (1). Although there is compelling evidence for the anti-oxidant properties of zinc (1, 2), the mechanisms by which it works are yet to be fully elucidated. Metal storage diseases of the liver such as Wilson’s disease and hereditary hemochromatosis are well known. Wilson’s disease is an autosomal recessive genetic disorder of copper metabolism, which results in the aberrant accumulation of copper in the tissues of the human body. The major symptoms of Wilson’s disease include liver damage, neurological symptoms and Kayser–Fleischer corneal rings. In liver disease experienced by patients suffering from Wilson’s disease, copper accumulation in hepatocytes initially causes mitochondrial damage through lipid oxidation, leading to chronic liver damage and even cirrhosis. It is suggested that mitochondrial DNA undergoes premature oxidative ageing, most likely mediated through mitochondrial copper accumulation. Current treatments for Wilson’s disease involve chelation therapy or zinc supplementation. Zinc is thought to interfere with the pathogenesis of Wilson’s disease in two ways; firstly, zinc may inhibit duodenal absorption of copper through the induction of MT; and secondly, zinc may act as an anti-oxidant which minimizes tissue injury. A study in this issue of Liver International has explored the possible anti-oxidant potential of zinc in ironinduced toxicity (3). To determine the anti-oxidant properties of zinc, MT, glutathione, zinc and iron accumulation were studied following administration of iron either with or without zinc to a rat hepatoma cell line; the distribution of MTand cell death index were also measured. It was found that the iron content was approximately 50% lower in cells treated with zinc–iron than compared with cells treated with iron alone, indicating a possible interaction with a shared zinc–iron transporter, and Formigari et al. suggest that divalent metal transporter 1 (DMT1) is a possible candidate zinc–iron transporter. Though studies have shown that zinc treatment increases expression of DMT1 (4), it is unlikely to account for the decreased iron content in the cells treated with zinc–iron observed by Formigari et al. as any regulation of DMT1 would effect both metals. It would be more likely that there may have been competition between zinc and iron for access to a transporter. There is also mounting evidence that zinc may not be a major substrate for DMT1 (5, 6), and thus full understanding of the transport system for zinc remains unclear. Zip proteins have been shown to transport zinc across cell and intracellular organelle membranes (7, 8). Since the discovery of Zip transporters, members of the Zip family have been shown to transport iron as well as other cations. Upregulation of Zip14 by Il-6 appears to contribute to the hepatic zinc accumulation and hypozincemia of inflammation and was subsequently analyzed for its capability to mediate nontransferrin bound iron uptake. It was found that zinc competed for iron uptake in cells overexpressing Zip14, suggesting they share a common pathway (7). This was supported by the fact that in hepatocytes endogenous Zip14 when suppressed by targeted siRNA resulted in decreased uptake of both zinc and iron. It is for these reasons that Zip14 is also a possible candidate for the transport of both zinc and iron in the liver, and may also explain the inhibition of uptake of iron by zinc observed by Formigari et al. Formigari et al. (3) also found that MT content was higher in both the zinc and zinc–iron-treated cells than in cells treated with iron alone. They suggest that this maybe owing to zinc-induced metal-response-element transcription factor 1 (MTF-1) binding to a metal response element (MRE) in MT and upregulating MT mRNA expression. This is supported by other studies that have observed zinc-induced activation of MTF-1 resulting in increased MTexpression (9). Although MT is predominantly a cytoplasmic protein it can also be found

Changes in gene expression of cholesterol metabolism pathways in mouse models of haemochromatosis

Introduction: The liver is central to the metabolism of both iron and cholesterol. Cholesterol is synthesised and further metabolised to bile acids in the liver and the liver plays an important role in regulation of iron metabolism. It is also the organ in which excess iron is stored. Clinically, links have been noted between lipid and iron metabolism, with approximately one - third of patients with non - alcoholic fatty liver disease exhibiting altered iron parameters. On a molecular level, we have previously reported that wild - type mice fed iron - deficient, normal or iron - loaded diets exhibited increased hepatic cholesterol and increased hepatic gene expression of enzymes in the cholesterol biosynthesis pathway with increasing hepatic iron burden. In the genetic disorder, haemochromatosis, the liver can become overloaded with iron; however, clinical studies have suggested that lipid metabolism may not be perturbed in haemochromatosis. Methods and Materials: We investigated hepatic cholesterol metabolis m in three mouse models of hereditary haemochromatosis: Hfe - / - , Tfr2 Y245X single mutant and Hfe - / - x Tfr2 Y245X double mutant animals as well as wild - type controls. Mice were fed normal mouse chow and sacrificed at 10 weeks of age. Hepatic gene expression, total cholesterol and non – haem iron were measured. Liver non - haem iron was similar in Hfe - / - and Tfr2 Y245X mice (16.6±0.8 and 17±1 μmol Fe /g liver, respectively) and significantly higher in the double mutant animals (22.4±0.7 μmol Fe /g liver ; P<0.004) than either of the single mutant mice. Results: Only one group of genes increased significantly with increasing hepatic iron: those involved in cholesterol transport. Gene expression of apolipoproteins A4, C1, C2, C3 and E increased significantly with increasing hepatic iron as did expression of VLDL receptor. In contrast to our findings in wild - type mice, gene expression of cholesterol biosynthetic enzymes did not increase significantly with liver iron burden and there were no differences in hepatic cholesterol between the groups of mutant mice. We also measured expression of genes involved in cholesterol regulation, which similarly, did not increase with increasing hepatic iron. Approximately 50% of cholesterol synthesised in the liver is directed to bile acid synthesis; however, gene expression of bile acid pathway enzymes did not change with respect to hepatic iron burden. Conclusion: These results suggest that iron - associated cholesterol regulation may be ameliorated by the genetic changes which occur in haemochromatosis.Poster presented at Fifth Congress of the International BioIron Society that took place in University College London (London, United Kingdom) during 14-18th April 2013.