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;)
Iron is an essential trace element and an important structural or functional component of many physiological systems. The liver is the major storage site for iron and is important in the regulation of iron metabolism, being the site of production of the hormone hepcidin, which regulates iron absorption.1 Iron metabolism is tightly regulated; nevertheless, iron deficiency and iron overload can occur and may have serious clinical consequences. The most common disorder associated with iron depletion is iron deficiency anemia, which affects more than 30% of the world's population.2 At the other end of the spectrum, iron overload can occur in subjects with hereditary hemochromatosis, which is caused by mutations in one of several genes, or secondary to iron administration.3 A range of biochemical disturbances may result from dysregulated iron metabolism; these include metabolic disorders affecting glucose and insulin, leading to diabetes,4 and to nonalcoholic fatty liver disease (NAFLD).5
Like iron, cholesterol is essential in normal physiological systems. It is required in cell membranes to maintain cellular integrity and for the formation of bile acids which aid in fat digestion. It is also a precursor of steroid hormones and vitamin D.6, 7 Also like iron, excesses and deficiencies of cholesterol can result in pathophysiological sequelae, including atherosclerosis and NAFLD, skeletal abnormalities, and mental health disorders.8-10
NAFLD is a collective term for chronic liver disorders which can range from fatty deposits in hepatocytes to nonalcoholic steatohepatitis (NASH) and which can progress to cirrhosis and hepatocellular carcinoma.11 It has been proposed that progression of NAFLD from steatosis to steatohepatitis occurs via a number of steps that result from the actions of additional factors upon the steatotic liver, a model known as the “two-hit hypothesis”.12 One of the factors identified as contributing the second hit is the presence of reactive oxygen species that cause oxidative stress.13 Iron is known to catalyze the production of reactive oxygen species which can then initiate cellular damage, including lipid peroxidation,14 and an increase in iron has been shown to increase the oxidation of cholesterol, particularly when the liver is already under conditions of oxidative stress.15 This is supported by a recent study which reported that hepatocyte iron loading was associated with liver fibrosis in patients with NAFLD.16 Thus, excess hepatic iron has been hypothesized to be a cofactor in the progression of steatosis to NASH and, indeed, several studies have reported an association between parameters of iron loading and NASH.17-19
Previous studies investigating the interaction between iron and cholesterol have focused on the plasma and present conflicting information. Administration of a high iron diet to animals has been found to result in an increase in plasma cholesterol in some studies but not in others,20, 21 and intraperitoneal administration of iron has been shown to lower plasma cholesterol.22 In humans homozygous for the Cys282Tyr (C282Y) mutation in HFE, which causes hemochromatosis, plasma low-density lipoprotein (LDL) cholesterol has been found to be reduced.23 In contrast, plasma total and LDL cholesterol were found to be reduced in anemic women.24
Further clarification of the potential role of iron in disordered lipid metabolism is required. To examine this, we studied the effects of iron status on hepatic cholesterol synthesis in mice with iron burdens ranging from deficient to overloaded. We show that increasing iron burden in mice results in an increase in the transcripts of approximately half of the enzymes of the cholesterol biosynthetic pathway, resulting in an increase in hepatic total cholesterol. These results provide a new and potentially important additional mechanism by which iron could contribute to the development of NAFLD or lipotoxicity.
Iron is an essential trace element which plays a role in many physiological systems. Perhaps not surprisingly, it has also been linked to changes in many metabolic processes, including disorders of lipid metabolism.5, 42 Here, we present data indicating a link between hepatic iron status and the production of cholesterol by the liver.
Hepatic iron stores were two-fold lower than normal in iron-deficient mice and eight-fold higher than normal in iron-loaded mice. This was reflected in the transcript levels of the iron hormone hepcidin-1, which were up-regulated in the presence of increasing iron. Conversely, transferrin receptor 1 transcript, which contains several iron-responsive elements in its 3′ untranslated region,43 was substantially up-regulated in iron deficiency. Hfe and transferrin receptor 2, neither of which are regulated by iron at the transcriptional level,44, 45 exhibited no regulation by hepatic iron levels.
Cholesterol is an important molecule in homeostasis. It is a component of lipid membranes and can be further metabolized either within the liver or extrahepatically. Like iron, excess cholesterol can be toxic, being deposited in arteries to form atherosclerotic plaques,8 or in the liver, where it may contribute to NALFD. The present study suggests a role for iron in cholesterol synthesis: increasing hepatic iron was positively associated with increasing hepatic cholesterol, and significant positive correlations of liver iron with transcript levels of enzymes involved in cholesterol biosynthesis were seen. Seven enzyme transcripts exhibited significant positive relationships with hepatic iron levels, including Hmgcr, which codes for the rate-limiting enzyme. Nine did not exhibit significant associations with hepatic iron and one, Hsd17b7, exhibited a significant negative correlation. It is unclear why transcript levels of Hsd17b7 decreased with increasing iron; however, the decrease did not appear to affect cholesterol production, because this increased with increasing hepatic iron.
Bile acid synthesis represents the major metabolic route for hepatic cholesterol.6, 7 The current results suggest that cholesterol produced in response to increased liver iron is not directed to bile acid synthesis. Cytochrome P450 7a1 (Cyp7a1) mRNA, which encodes the rate-limiting enzyme in bile acid synthesis,46 did not significantly correlate with liver iron and Hsd3b7 mRNA, which encodes another enzyme involved early in bile acid synthesis, declined in response to increasing iron. Additionally, transcript levels of the bile acid transporter Abcb11 and two regulators of bile acid synthesis, Hnf4a and Nr1h3, did not exhibit significant correlations with liver iron.
Cholesterol may also be exported to other organs for further processing, for example, for the manufacture of steroid hormones.7 Abca1 and Apoc3 mRNA exhibited significant positive correlations with liver iron. Abca1 is a transporter which exports cholesterol to ApoA147 and, recently, overexpression of Apoc3 has been shown to enhance VLDL secretion from a hepatoma cell line.48 Although these results may suggest that cholesterol produced in response to iron loading might be exported to other organs, the observation that plasma cholesterol levels showed no relationship with either liver iron or cholesterol raises the possibility that much of the cholesterol produced by the liver under these conditions remains there. This may also explain the lack of agreement in other studies which have examined iron status and plasma levels of cholesterol.
Cholesterol may also be exported directly into the canaliculus. Abcg5 is a half-transporter which dimerizes with Abcg8 to export cholesterol and plant sterols into the canaliculus,49 whereas Abcb4 is a transporter which exports cholesterol and phosphatidylcholine into the canaliculus.50 Investigation of these transporters revealed that Abcg5 mRNA correlated positively with liver iron, whereas Abcb4 mRNA correlated negatively. Superficially, this suggests up-regulation of cholesterol export into the bile, particularly given that the substrate preference for Abcb4 is phosphatidylcholine rather than cholesterol.51 However, Abcb4 knockout mice overexpressing Abcg5 and Abcg8 have only very low levels of cholesterol in the bile,52 and the presence of bile salt micelles is required to accept cholesterol.53 Thus, in the present study, despite the increase in Abcg5 transcript with increasing iron, the down-regulation of bile acid synthetic enzymes and Abcb4 mRNA under the same conditions suggests that transport of cholesterol to the bile does not increase to accommodate the increase in cholesterol production.
Both iron and cholesterol metabolism are under complex regulatory control. Hence, we investigated some of the potential regulators that may explain the observed up-regulation of cholesterol biosynthesis. Srebf2 preferentially activates many of the genes in the cholesterol biosynthesis pathway.35 In the present study, four of these genes—Hmgcr, Pmvk, Cyp51 and Sc5d—were significantly up-regulated in response to increasing liver iron levels; however, the mechanism leading to this up-regulation appears to be independent of Srebf2 expression, which did not change in response to iron status. Srebf2 is regulated both transcriptionally and posttranscriptionally35 and, although we cannot rule out a posttranscriptional response of Srebf2 to iron, we believe this to be unlikely given that the majority of known targets of Srebf2 measured in the present study were not up-regulated. Similarly, expression of several genes measured in the present study—Dhcr7, Fdps, Abcg5, and Apob—is known to be regulated by CCAAT/enhancer binding protein α (C/EBPα), which is induced by iron loading.54-56 However, of these genes, only Abcg5 increased with increasing hepatic iron concentration, suggesting that C/EBPα is also unlikely to be involved in the observed up-regulation of cholesterol synthesis. Finally, expression of Tmem97, a gene newly identified as being associated with cholesterol regulation and a target of Srebf2,36 was unchanged in the face of increasing iron burden. The same authors reported a number of other genes as potential regulators of cholesterol metabolism, including Bhmt2 and Vrk3, knockdown of which reduced intracellular cholesterol, and Psap, knockdown of which increased intracellular cholesterol.36 In the present study, neither Bhmt2, Vrk3, nor Psap correlated significantly with liver iron, suggesting that regulation of cholesterol metabolism by iron is independent of these three genes.
In a previous study, Brunet et al.57 observed a pronounced plasma hypercholesterolemia in iron-loaded rats compared with wild-type controls. Hepatic cholesterol concentration did not change and the activity of Hmgcr decreased. These parameters correlated significantly with hepatic malondialdehyde, a marker of oxidative stress, and led the authors to suggest that the changes were due to oxidative damage of the membranes in which the enzymes of cholesterol metabolism reside. It is interesting that in the present study, hepatic total cholesterol increased with increasing iron burden, suggesting that enzyme activity was not disrupted. The difference between the two studies is likely to be explained by the different feeding regimes employed: 12 weeks on a 3% carbonyl iron diet in the study by Brunet et al. compared with 3 weeks on a 2% carbonyl iron diet in the present study. The longer exposure to a higher iron diet is likely to have generated higher levels of oxidative stress than in the present study.
The current study may have important clinical implications for a role of iron in contributing to the pathogenesis of NAFLD. Alterations in cholesterol metabolism have been reported to be associated with iron parameters in many disease states, including iron overload,58 iron deficiency,24 peripheral artery disease,59 and NAFLD.60-62 In the present study, Apoc3 was seen to increase with increasing hepatic iron, and overexpression of Apoc3 has been reported to result in hepatic steatosis.63 Furthermore, a recent, large, multicenter study of patients with NAFLD reported that deposition of iron in hepatocytes was associated with increased risk of moderate to severe liver fibrosis16 and increasing hepatic iron has been shown to be associated with increased lipid peroxidation.14 It has been proposed that the development of NASH occurs in two stages: (1) the deposition of fat, resulting in steatosis and (2) the intervention of another factor which causes steatohepatitis.12 It has been hypothesized that the second stage involves oxidative stress, which can be caused by iron-generated reactive oxygen species. These reactive oxygen species can initiate lipid peroxidation which can lead to cellular damage.5 Increased production of cholesterol by the liver with increasing liver iron burden is, in itself, consistent with a contribution of iron loading to steatosis. The presence of iron may additionally contribute the reactive oxygen species, which can allow further progression of the disease.
In summary, the findings reported in this study suggest that hepatic iron loading increases the synthesis and deposition of cholesterol in the liver. The mechanism appears to be independent of Srebf2. The observations are consistent with a role for iron in the development of NAFLD, with iron contributing, first, to increased cholesterol production and, second, to increased oxidative stress leading to lipid peroxidation.