Dietary fat level affects tissue iron levels but not the iron regulatory gene HAMP in rats.

Because dietary fats affect the regulation and use of body iron, we hypothesized that iron regulatory and transport genes may be affected by dietary fat. A model of early-stage I to II, nonalcoholic fatty liver was used in which rats were fed standard (35% energy from fat) or high-fat (71% energy from fat) liquid diets with normal iron content (STD/HF groups). In addition, intraperitoneal injections of iron dextran were given to iron-loaded (STD+/HF+ groups) and iron-deficient diets to STD-/HF- groups. Plasma osmolality, hemoglobin level, and mean corpuscular hemoglobin concentration were increased in all STD diet groups compared with all HF diet groups. Plasma iron and transferrin saturation were affected by an interaction between dietary fat and iron. They were high in the STD group (normal iron) compared with their respective HF group. Similarly, this group also showed a 4-fold increase in the messenger RNA expression of the hepatic hemochromatosis gene. Spleen iron was high in the iron-loaded STD+ group compared with all other groups. Hepatic iron and messenger RNA expression of peroxisome proliferator-activated receptor-γ, CCAAT/enhancer binding protein α, interleukin-6, and iron transport genes (transferrin receptor 2, divalent metal transporter 1 iron-responsive element, and divalent metal transporter 1 non-iron-responsive element) were increased, whereas tumor necrosis factor α was decreased in the HF diet groups. The expression of iron regulatory gene HAMP was not increased in the HF diet groups. Iron regulatory and transport genes involved in cellular and systemic iron homeostasis may be affected by the macronutrient composition of the diet.

Interactions between hepatic iron and lipid metabolism with possible relevance to steatohepatitis.

The liver is an important site for iron and lipid metabolism and the main site for the interactions between these two metabolic pathways. Although conflicting results have been obtained, most studies support the hypothesis that iron plays a role in hepatic lipogenesis. Iron is an integral part of some enzymes and transporters involved in lipid metabolism and, as such, may exert a direct effect on hepatic lipid load, intrahepatic metabolic pathways and hepatic lipid secretion. On the other hand, iron in its ferrous form may indirectly affect lipid metabolism through its ability to induce oxidative stress and inflammation, a hypothesis which is currently the focus of much research in the field of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH). The present review will first discuss how iron might directly interact with the metabolism of hepatic lipids and then consider a new perspective on the way in which iron may have a role in the two hit hypothesis for the progression of NAFLD via ferroportin and the iron regulatory molecule hepcidin. The review concludes that iron has important interactions with lipid metabolism in the liver that can impact on the development of NAFLD/NASH. More defined studies are required to improve our understanding of these effects.

Effect of dietary fat to produce non-alcoholic fatty liver in the rat.

BACKGROUND AND AIM: Non-alcoholic steatohepatitis (NASH) belongs to a spectrum of non-alcoholic fatty liver disease (NAFLD). Oxidative stress is hypothesized to play an important role in the progression of the disease. We used the Lieber/DeCarli model for NASH to investigate the mechanisms involved in its progression. METHODS: Male Sprague-Dawley rats were fed standard (35% of energy from fat) or high fat (71% of energy from fat) liquid diets, ad libitum or two-thirds of the amount consumed ad libitum initially for 3 weeks and then extended to 5 weeks. RESULTS: Steatosis was absent in rats at 3 weeks feeding, but by 5 weeks, the high fat/ad lib group showed microvesicular steatosis and foci of macrovesicular steatosis without inflammation. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were not different. By 5 weeks feeding, hepatic triglycerides were highest in the high fat ad lib group and the ad lib groups were higher compared with their restricted groups. The oxidative stress marker, hydroxyalkenal (HAE) was decreased in the standard ad lib compared with the high fat ad lib group. Liver mRNA of interleukin-6, haem oxygenase-1, and markers of endoplasmic stress: C/EBP homologous protein (CHOP), glucose responsive protein-78 (GRP78) and spliced X-box DNA binding protein (spliced XBP1) were similar in the ad lib groups. CONCLUSIONS: Extending the feeding period of the high fat/ad lib diet for 5 weeks placed our rats with Type I to II NAFLD compared to the more progressed Type III state previously obtained after 3 weeks feeding. The milder condition obtained raised the prospect of genetic modifiers present in our rats that resist disease progression.

Mechanisms of heme iron absorption: current questions and controversies.

Iron is a critical micronutrient, and iron derived from heme contributes a large proportion of the total iron absorbed in a typical Western diet. Heme iron is absorbed by different mechanisms than non-heme iron, but despite considerable study over many years these mechanisms remain poorly understood. This review provides an overview of the importance of heme iron in the diet and discusses the two prevailing hypotheses of heme absorption; namely receptor mediated endocytosis of heme, and direct transport into the intestinal enterocyte by recently discovered heme transporters. A specific emphasis is placed on the questions surrounding the site of heme catabolism and the identity of the enzyme that performs this task. Additionally, we present the hypothesis that a non-heme iron transport protein may be required for heme iron absorption and discuss the experiences of our laboratory in examining this hypothesis.

Subcellular location of heme oxygenase 1 and 2 and divalent metal transporter 1 in relation to endocytotic markers during heme iron absorption.

BACKGROUND AND AIM: Heme is an important dietary micronutrient, although its absorptive mechanisms are poorly understood. One hypothesis suggests enterocytes take up heme by receptor-mediated endocytosis (RME) which then undergoes catabolism by heme oxygenase (HO) inside internalized vesicles. This would require the translocation of HO-1 or HO-2 to endosomes and/or lysosomes and the presence of a transporter, possibly divalent metal transporter 1 (DMT1), to transfer released iron to the cytoplasm. Currently, the location of HO-1 and HO-2 in enterocytes is unknown. METHODS: We studied the subcellular location of HO-1, HO-2, and DMT1 in the proximal small intestine of rats by confocal immunofluorescence microscopy up to 4 h after a dose of heme or ferrous iron. Double-labeling was performed with endocytotic (EEA1, Lamp1) and structural markers (F-actin). RESULTS: HO-1 was distributed evenly throughout the cytoplasm of enterocytes and did not colocalize with endocytotic markers in any condition. HO-2 staining remained constant with dosing, presenting as a dense band in the apical cytoplasm that colocalized extensively with endosomes. DMT1 staining was markedly reduced by ferrous iron, but not heme and did not exhibit colocalization with endocytotic markers. CONCLUSION: The subcellular location of HO-2 is consistent with the RME hypothesis for heme uptake and may suggest a possible role for this enzyme in heme degradation. The lack of translocation of DMT1 with heme dosing suggests another protein may be present to transport iron released from heme.

Molecular regulation of hepatic expression of iron regulatory hormone hepcidin.

Iron is a micronutrient that is an essential component that drives many metabolic reactions. Too little iron leads to anemia and too much iron increases the oxidative stress of body tissues leading to inflammation, cell death, and system organ dysfunction, including cancer. Maintaining normal iron balance is achieved by rigorous control of the amount absorbed by the intestine, that released from macrophages following erythrophagocytosis of effete red cells and by either release or uptake from hepatocytes. Hepcidin is a recently characterized molecule that appears to play a key role in the regulation of iron efflux from enterocytes, macrophages, and hepatocytes. It is produced by hepatocytes under basal conditions, in response to alterations in increased iron stores or reduced requirement for erythropoiesis and by inflammation. The proteins that regulate hepcidin expression are presently being defined, albeit that our present understanding is still far from complete. This review focuses on the molecules which regulate hepcidin expression. The subsequent characterization of these proteins using molecular, cellular, and physiological approaches also is discussed along with inflammatory signals and receptors involved in hepcidin expression.

The relevance of the intestinal crypt and enterocyte in regulating iron absorption.

Rigorous regulation of iron absorption is required to meet the requirements of the body and to limit excess iron accumulation that can produce oxidative stress. Regulation of iron absorption is controlled by hepcidin and probably by the crypt program. Hepcidin is a humoral mediator of iron absorption that interacts with the basolateral transporter, ferroportin. High levels of hepcidin reduce iron absorption by targeting ferroportin to lysosomes for destruction. It is also proposed that ferroportin is expressed on the apical membrane and coordinates with ferroportin-hepcidin derived from the basal surface to modulate the uptake phase of iron absorption. The crypt program suggests that as crypt cells differentiate and migrate into the absorptive zone they absorb iron from the diet at levels inverse to the amount of iron taken up from transferrin. Under most circumstances, intestinal iron absorption is controlled at multiple levels that lead to hepcidin/ferroportin modulation of the enterocyte labile iron pool (LIP). It is likely that transcription of iron transport proteins involved in the apical and basolateral transport of iron are differentially regulated by separate LIPs. Iron-responsive protein (IRP) 1 and IRP2 do not appear to play a significant role in the expression of iron transport proteins, although IRP2 regulates L- and H-ferritin expression. Despite the importance of hepcidin, there is evidence of hepcidin-independent regulation of iron absorption possibly involving haemojuvelin (HJV) and neogenin, which may be up-regulated during ineffective erythropoiesis.

Heme in intestinal epithelial cell turnover, differentiation, detoxification, inflammation, carcinogenesis, absorption and motility.

The gastrointestinal tract is lined by a simple epithelium that undergoes constant renewal involving cell division, differentiation and cell death. In addition, the epithelial lining separates the hostile processes of digestion and absorption that occur in the intestinal lumen from the aseptic environment of the internal milieu by defensive mechanisms that protect the epithelium from being breached. Central to these defensive processes is the synthesis of heme and its catabolism by heme oxygenase (HO). Dietary heme is also an important source of iron for the body which is taken up intact by the enterocyte. This review describes the recent literature on the diverse properties of heme/HO in the intestine tract. The roles of heme/HO in the regulation of the cell cycle/apoptosis, detoxification of xenobiotics, oxidative stress, inflammation, development of colon cancer, heme-iron absorption and intestinal motility are specifically examined.

Augmented internalisation of ferroportin to late endosomes impairs iron uptake by enterocyte-like IEC-6 cells.

Absorption of iron occurs by duodenal enterocytes, involving uptake by the divalent metal transporter-1 (DMT1) and release by ferroportin. Ferroportin responds to the hepatocyte-produced 25-amino-acid-peptide hepcidin-25 by undergoing internalisation to late endosomes that impair iron release. Ferroportin is also expressed on the apical membrane of polarised Caco-2 cells, rat intestinal cells and in IEC-6 cells (an intestinal epithelial cell line). A blocking antibody to ferroportin also impairs the uptake, but not the release, of iron. In this study IEC-6 cells were used to study the mechanism of impairment or recovery from impairment produced by the blocking antibody and the fate of DMT1 and ferroportin. Uptake of 1 muM Fe(II) was studied by adding the antibody from time 0 and after adding or removing the antibody once a steady state had been reached. Surface binding, maximum iron transport rate V(max) and transporter affinity (K(m)) were measured after impairment of iron uptake. Ferroportin and DMT1 distribution were assessed by immunofluorescence microscopy. Antibody-mediated impairment, or recovery from impairment, of Fe(II) uptake occurred within minutes. Impairment was lost when the antibody was combined with the immunizing peptide. DMT1 and ferroportin undergo internalisation to late endosomes and, in the presence of the antibody, augmented internalisation of DMT1 and ferroportin caused swelling of late endosomes. Surface binding of Fe(II) and iron transport V(max) were reduced by 50%, indicating that the antibody removed membrane-bound DMT1. The ferroportin antibody induced rapid turnover of membrane ferroportin and DMT1 and its internalisation to late endosomes, resulting in impaired Fe(II) uptake.

Decreased sucrase and lactase activity in iron deficiency is accompanied by reduced gene expression and upregulation of the transcriptional repressor PDX-1.

Disaccharidases are important digestive enzymes whose activities can be reduced by iron deficiency. We hypothesise that this is due to reduced gene expression, either by impairment to enterocyte differentiation or by iron-sensitive mechanisms that regulate mRNA levels in enterocytes. Iron-deficient Wistar rats were generated by dietary means. The enzyme activities and kinetics of sucrase and lactase were tested as well as the activity of intestinal alkaline phosphatase (IAP)-II because it is unrelated to carbohydrate digestion. mRNA levels of beta-actin, sucrase, lactase, and the associated transcription factors pancreatic duodenal homeobox (PDX)-1, caudal-related homeobox (CDX)-2, GATA-binding protein (GATA)-4, and hepatocyte nuclear factor (HNF)-1 were measured by real-time PCR. Spatial patterns of protein and gene expression were assessed by immunofluorescence and in situ hybridization, respectively. It was found that iron-deficient rats had significantly lower sucrase (19.5% lower) and lactase (56.8% lower) but not IAP-II activity than control rats. Kinetic properties of both enzymes remained unchanged from controls, suggesting a decrease in the quantity of enzyme present. Sucrase and lactase mRNA levels were reduced by 44.5% and 67.9%, respectively, by iron deficiency, suggesting that enzyme activity is controlled primarily by gene expression. Iron deficiency did not affect the pattern of protein and gene expression along the crypt to villus axis. Expression of PDX-1, a repressor of sucrase and lactase promoters, was 4.5-fold higher in iron deficiency, whereas CDX-2, GATA-4, and HNF-1 levels were not significantly different. These data suggest that decreases in sucrase and lactase activities result from a reduction in gene expression, following from increased levels of the transcriptional repressor PDX-1.

Differences in the uptake of iron from Fe(II) ascorbate and Fe(III) citrate by IEC-6 cells and the involvement of ferroportin/IREG-1/MTP-1/SLC40A1.

Dietary iron is present in the intestine as Fe(II) and Fe(III). Since enterocytes take up Fe(II) by the divalent metal transporter (DMT1), Fe(III) must be reduced. Whether other Fe(III) transport processes are present is unknown. Release of iron from the enterocyte into the plasma involves the iron-regulated transporter-1/metal transporter protein-1 (IREG-1/MTP-1, ferroportin) but ferroportin is also found on the apical membrane. We compared the uptake of iron from Fe(II):ascorbate and Fe(III):citrate using the rat intestinal enterocyte cell line-6 (IEC-6), in the presence of ferrous chelators, a blocking antibody to ferroportin, at different pH and during the over-expression of DMT1. Firstly, surface ferrireduction was absent. Secondly, blocking ferroportin partly and totally reduced Fe(II) and Fe(III) uptake, respectively. Thirdly, optimal Fe(II) uptake occurred at pH 5.5 but Fe(III) uptake was unaffected by pH and, fourthly, over-expression of DMT1 increased uptake of Fe(II) and Fe(III). This indicates that an increased extracellular H+ concentration facilitates DMT1-mediated Fe(II) uptake at the cell membrane. However, since Fe(III) uptake required DMT1, but not cell surface ferrireduction, and was independent of variations in extracellular pH, it appears that Fe(III) is internalised before ferrireduction and transport by DMT1. Ferroportin may function as a modulator of DMT1 activity and play a role in Fe(III) uptake, possibly by affecting the number or affinity of citrate binding sites.

Effects of cell proliferation on the uptake of transferrin-bound iron by human hepatoma cells.

The effects of cellular proliferation on the uptake of transferrin-bound iron (Tf-Fe) and expression of transferrin receptor-1 (TfR1) and transferrin receptor-2 (TfR2) were investigated using a human hepatoma (HuH7) cell line stably transfected with TfR1 antisense RNA expression vector to suppress TfR1 expression. At transferrin (Tf) concentrations of 50 nmol/L and 5 micromol/L, when Tf-Fe uptake occurs by the TfR1- and TfR1-independent (NTfR1)-mediated process, respectively, the rate of Fe uptake by proliferating cells was approximately 250% that of stationary cells. The maximum rate of Fe uptake by the TfR1- and NTfR1-mediated process by proliferating cells was increased to 200% and 300% that of stationary cells, respectively. The maximum binding of Tf by both TfR1- and NTfR1-mediated processes by proliferating cells was increased significantly to 160% that of stationary cells. TfR1 and TfR2-alpha protein levels expressed by proliferating cells was observed to be approximately 300% and 200% greater than the stationary cells, respectively. During the proliferating growth phase, expression of TfR1 messenger RNA (mRNA) increased to 300% whereas TfR2-alpha mRNA decreased to 50% that of stationary cells. In conclusion, an increase in Tf-Fe uptake by TfR1-mediated pathway by proliferating cells was associated with increased TfR1 mRNA and protein expression. An increase in Tf-Fe uptake by NTfR1-mediated pathway was correlated with an increase in TfR2-alpha protein expression but not TfR2-alpha mRNA. In conclusion, TfR2-alpha protein is likely to have a role in the mediation of Tf-Fe uptake by the NTfR1 process by HuH7 hepatoma cell in proliferating and stationary stages of growth.

Copper deficiency increases iron absorption in the rat.

Release of iron from enterocytes and hepatocytes is thought to require the copper-dependent ferroxidase activity of hephaestin (Hp) and ceruloplasmin (Cp), respectively. In swine, copper deficiency (CD) impairs iron absorption, but whether this occurs in rats is unclear. By feeding a diet deficient in copper, CD was produced, as evidenced by the loss of copper-dependent plasma ferroxidase I activity, and in enterocytes, CD reduced copper levels and copper-dependent oxidase activity. Hematocrit was reduced, and liver iron was doubled. CD reduced duodenal mucosal iron and ferritin, whereas CD increased iron absorption. Duodenal mucosal DMT1-IRE and ferroportin1 expression remained constant with CD. When absorption in CD rats was compared with that seen normally and in iron-deficient anemic animals, strong correlations were found among mucosal iron, ferritin, and iron absorption, suggesting that the level of iron absorption was appropriate given that the erythroid and stores stimulators of iron absorption are opposed in CD. Because CD reduced the activity of Cp, as evidenced by copper-dependent plasma ferroxidase I activity and hepatocyte iron accumulation, but iron absorption increased, it is unlikely that the ferroxidase activity of Hp is important and suggests another function for this protein in the export of iron from the enterocyte during iron absorption. Also, the copper-dependent ferroxidase activity of Cp does not appear important for iron efflux from macrophages, because Kupffer cells of the liver and nonheme iron levels of the spleen were normal during copper deficiency, suggesting another role for Cp in these cells.

IEC-6 cells are an appropriate model of intestinal iron absorption in rats.

Regulation of iron absorption, which is the primary mechanism for maintaining body iron stores, occurs primarily in the proximal small intestine. Recent identification of proteins that are involved in iron absorption such as the uptake transporter-divalent metal transporter (DMT1), the basolateral transporter, IREG1, and the ferroxidase-hephaestin provide new opportunities to study this process. We evaluated the rat intestinal cell line, IEC-6, as a model of intestinal iron transport. This involved measuring the expression of DMT1 and IREG1 by Western blot analysis and confocal microscopy, and hephaestin by protein-dependent copper oxidase activity. DMT1 and IREG1 were expressed in IEC-6 cells. The uptake of 1 micromol/L ferrous iron [Fe(II)]:ascorbate and its efflux also was associated with the expression of DMT1 under different levels of iron loading. The expression of DMT1 changed inversely with iron levels as did the uptake of Fe(II). However, with different levels of cellular iron, IREG1 expression remained constant, as did the release of iron from the cells, suggesting that they could be related. Ceruloplasmin and apotransferrin did not enhance the rate or extent of iron release. Copper oxidase activity, considered to indicate hephaestin activity, was detected only intracellularly. Confocal microscopy showed DMT1 and IREG1 on the cell membrane of IEC-6 cells at 4 degrees C but at intracellular locations at 37 degrees C, indicating that these proteins can function at the cell membrane and intracellularly. In terms of iron absorption, IEC-6 cells have a villous enterocyte phenotype and are regulated by iron stores as occurs in vivo; therefore, they represent an appropriate cell model with which to study this process.

Mechanisms and regulation of intestinal iron absorption.

Iron absorption from the small intestine is regulated according to the body's needs, increasing in iron deficiency and decreasing in iron overload. It has been proposed that the efficiency of absorption is determined by the amount of iron acquired by developing enterocytes when they are in the crypts of Lieberkuhn and that this regulates expression of iron transporters such as DMT1 in mature enterocytes of the intestinal villi. In the crypts the cells take up iron from plasma transferrin by receptor-mediated endocytosis, a process that is influenced by the hemochromatosis protein, HFE. Hence, the availability of plasma transferrin-bound iron and the expression and function of transferrin receptors (TfR1), HFE and DMT1 should all contribute to the absorptive capacity of villus enterocytes. These aspects of the regulation and mechanism of iron absorption were investigated in genetically normal rats and mice, and in Belgrade anemic (b/b) rats and HFE knockout mice. In most experiments the function of the TfR1 was assessed by the uptake of radiolabeled transferrin-bound iron given intravenously. Absorption of non-heme iron was measured using closed in situ duodenal loops. The expression and cellular distribution of DMT1 and TfR1 were determined by in situ hybridisation and immunohistochemistry. The uptake of transferrin-bound iron and expression of functional TfR1 was shown to occur mainly in crypt cells and to be proportional to the plasma concentration of iron. It was not impaired by the mutation of DMT1 that occurs in b/b rats but was impaired in HFE knockout mice. Iron absorption was increased in these mice but was still influenced by the level of iron stores, as in normal mice. These results are in accordance with the proposed regulation of iron absorption and suggest that DMT1 is not the only iron transporter operating within endosomes of crypt cells. This view was supported by the failure to detect DMT1 mRNA or protein in crypt cells. Expression of DMT1 mRNA and protein started at the crypt-villus junction and increased to reach highest levels in the mid-villus region. Greater expression was found in iron deficiency and less in iron loaded animals than in controls and in the iron deficient rats most of the protein was present on the brush border membrane. In normal rats the efficiency of iron absorption parallelled the level of DMT1 expression, but in b/b rats absorption was very low and independent of dietary iron content even though DMT1 was present in villus enterocytes. The results confirm the essential role of DMT1 in the uptake phase of non-heme iron absorption. When normal rats previously fed a low iron diet were given a bolus of iron by stomach tube, the subsequent absorption of iron from a test dose placed in the duodenum diminished in parallel with the expression of DMT1 mRNA and protein, commencing within 1hour and reaching low levels by 7 hours. The margination of DMT1 to the brush border membrane disappeared. These results show the level of expression and intracellular distribution and function of DMT1 respond very quickly to the iron content of the diet as well as being affected by storage iron levels.