The global obesity epidemic is linked to an increased incidence of a number of metabolic disorders, including type 2 diabetes mellitus, the metabolic syndrome, and nonalcoholic fatty liver disease (NAFLD). The term NAFLD encompasses a number of pathological conditions ranging from hepatic steatosis (fatty liver), which is thought to be a largely benign condition, to more aggressive disease states, including nonalcoholic steatohepatitis (NASH) and cirrhosis; a number of patients may ultimately progress from cirrhosis to hepatic failure and hepatocellular carcinoma.1 Surveys suggest that the occurrence of NAFLD in the general population may be as high as 30%-35%,2 but this incidence may rise significantly in obese individuals.
The incidence of NAFLD is closely associated with insulin resistance (IR) and the metabolic syndrome.3 The development of NAFLD is often considered to be a two-stage process.4 Stage one arises from lipid accumulation in the liver; this could occur for a number of reasons, including increased uptake of fat (derived, for example, from either dietary sources or from the flow of free fatty acids [FFAs] released from the adipose tissue as a result of IR), increased lipid synthesis, or decreased hepatic lipid secretion. Stage two occurs as a result of inflammation, mitochondrial dysfunction, and oxidative stress, and leads to cellular injury, apoptosis, and fibrosis. There is good evidence that FFAs directly induce cellular damage via induction of oxidative stress and the production of proinflammatory cytokines.5 Therefore, the esterification of FFAs and their deposition in the liver as triglycerides may act as a protective mechanism to prevent further hepatocellular damage.6
Other factors that induce oxidative stress may also be involved in the development of NAFLD. In this context, there is some evidence that iron, a powerful pro-oxidant, may be an important factor in the progression of NAFLD; studies have found an increased frequency of hereditary hemochromatosis (HFE) gene mutations (which predispose to liver iron loading) in patients with NAFLD.7, 8 Given these potential links between iron, lipid metabolism, and the etiology of fatty liver disease, the study by Graham et al.9 in this issue of HEPATOLOGY is particularly timely. They studied mice fed diets containing different amounts of iron to explore further the role of iron in the development of NAFLD, focusing specifically on the effects of iron status on hepatic cholesterol synthesis. Cholesterol, like iron, is an essential factor for normal cellular physiology but is highly toxic in excess. A number of regulatory systems have therefore evolved to control cholesterol synthesis. The effects of iron loading and iron deficiency on the expression of enzymes coordinating the cholesterol biosynthetic pathway were studied through use of microarray technology. Using existing databases and other online resources, gene set enrichment analysis allowed Graham et al. to identify a number of differences between groups of genes with related biological functions.
The expression of 3-hydroxy-3-methylglutarate-CoA reductase (Hmgcr), the first and the rate-limiting enzyme in cholesterol synthesis, as well as the expression of a number of other genes encoding enzymes in the cholesterol biosynthetic pathway, were positively and significantly regulated by liver nonheme iron content. Liver cholesterol was also significantly correlated with liver nonheme iron levels, indicating that changes in biosynthetic enzyme expression were translated into functional increases in cholesterol production. Cholesterol metabolism is governed by a family of transcription factors termed sterol regulatory element binding proteins (SREBPs); SREBP-2 is particularly important in regulating many of the genes involved in the cholesterol biosynthetic pathway. However, in this study, the expression of SREBP-2 was not influenced by iron status. Taken together, these findings suggest a role for iron in cholesterol synthesis; however, the nature of the underlying molecular mechanisms remains elusive.
Excess cholesterol is cytotoxic and therefore it is essential that mechanisms are in place to either use or export cholesterol once it has been synthesized. Bile acid synthesis is the major metabolic route for hepatic cholesterol. However, the rate-limiting enzyme in the main bile acid synthetic pathway, cholesterol-7α-monooxygenase (Cyp7a1), as well as other key enzymes in this metabolic pathway, were not correlated with liver nonheme iron. This suggests that cholesterol synthesized in response to elevated liver iron is not diverted into the bile acid synthetic pathway. There was limited evidence that some cholesterol may be exported to other organs; however, the lack of correlation between plasma cholesterol and either liver iron or liver cholesterol levels suggests that much of the cholesterol synthesized in response to iron loading remains within the liver. These data contrast with previous findings by Brunet et al.,10 in which iron-loaded rats developed hypercholesterolemia but showed no significant change in hepatic cholesterol concentration. One explanation for these differences may be the feeding programs used in the respective studies. Graham et al. argue that the longer feeding regimen used by Brunet et al. (12 weeks on a high-iron diet) may have generated significant levels of oxidative stress resulting in inflammation. In contrast, in the study by Graham et al., in which mice were fed a high-iron diet for 3 weeks, there was no histological evidence of fatty deposits or inflammation in the livers of these animals.
The studies by Graham et al.9 provide important new insights into the relationship between iron, lipid metabolism, and the etiology of NAFLD/NASH. Recent work has revealed that the so-called unfolded protein response (UPR), which arises as a result of endoplasmic reticulum (ER) stress, may also be important in mediating aberrant changes in iron and lipid metabolism seen in a number of conditions. Hepatocytes are major storage and redistribution centers for a number of nutrients and have abundant networks of rough ER to facilitate the secretion or export of their cargo. Although the ER are highly adaptive, they come under enormous stress following overnutrition11 or inflammation.12 As a result, the secretory network can be compromised, leading to the accumulation of unfolded proteins within the lumen of the ER.13 The UPR results in a number of metabolic changes including increased production of cholesterol (and triglycerides) in hepatocytes.14 In addition, several recent pieces of evidence link the UPR to changes in iron metabolism. HFE mutations, which lead to hereditary hemochromatosis and iron overload, are associated with activation of the UPR.15 Furthermore, induction of the UPR stimulates the production of hepcidin,16, 17 the major regulator of iron homeostasis.18
Based on these recent advances, a hypothetical model for the development of NAFLD can be proposed, which encompasses the roles of both iron and cholesterol (Fig. 1). Overnutrition, a leading factor in the development of obesity, IR, and the metabolic syndrome, results in increased lipid deposition in the liver. IR is associated with an increased flux of FFAs from the adipose tissue to the liver, which further exacerbates steatosis. It is well documented that obesity is associated with chronic low-grade inflammation, impaired iron homeostasis,19 and elevated production of the adipokine leptin, which in turn increases hepatic hepcidin production.20 Both overnutrition and inflammation are associated with ER stress and the induction of the UPR.11, 12 Recent work has shown that this leads to enhanced production of hepcidin,16, 17 which, once released from hepatocytes into the circulation, interacts with the iron efflux protein ferroportin and blocks iron release from a number of cell types, including hepatocytes,18 resulting in elevated intracellular iron levels. The present study by Graham et al. shows that increased intracellular iron is significantly and positively associated with elevated hepatic cholesterol synthesis, further contributing to the liver lipid burden. The combination of steatosis and cellular iron loading (together with increased FFAs) could result in increased oxidative stress, which would exacerbate the progression from fatty liver to NASH, cirrhosis, and potentially hepatocellular carcinoma. Although many of these links and hypotheses remain to be proven, the study by Graham et al. opens up a number of new avenues for future investigation of the relation between iron and lipid metabolism.