Altered expression of iron regulatory proteins with aging is associated with transient hepatic iron accumulation after environmental heat stress.

An increasing body of evidence suggests that dysregulation of iron metabolism contributes to age-related pathologies. We have previously observed increased hepatic iron with aging, and that environmental heat stress stimulates a further increase in iron and oxidative liver injury in old rats. The purpose of this study was to determine a mechanism for the increase in hepatic iron in old rats after heat stress. Young (6 mo) and old (24 mo) Fischer 344 rats were exposed to two heating bouts separated by 24 h. Livers were harvested after the second heat stress, and protein levels of the iron import protein, transferrin receptor-1 (TFR1), and the iron export protein, ferroportin (Fpn) were determined by immunoblot. In the nonheated condition, old rats had lower TFR1 expression, and higher Fpn expression. After heat stress, TFR1 declined in the old rats, and iron chelation studies demonstrated that this decline was dependent on a hyperthermia-induced increase in iron. TFR1 did not change in the young rats after heat stress. Since TFR1 is inversely regulated by iron, our results suggest that the increase in intracellular iron with aging and heat stress lower TFR1 expression. Fpn expression increased in both age groups after heat stress, but this response was delayed in old rats. This delay in the induction of an iron exporter suggests a mechanism for the increase in hepatic iron and oxidative injury after heat stress in aged organisms.

Effect of bioactive dietary polyphenols on zinc transport across the intestinal Caco-2 cell monolayers.

Polyphenolic compounds are known to possess many beneficial health effects, including the antioxidative activities of scavenging reactive oxygen species and chelating metals, such as iron and zinc. Tea and red wine are thought to be important sources of these compounds. However, some polyphenolic compounds can also reduce the absorption of iron, and possibly other trace metals, when included in a diet. There is very little information on the effect of dietary polyphenolic compounds on the status of trace elements other than iron. The effects of epigallocatechin-3-gallate (EGCG), green tea extract (GT), and grape seed extract (GSE) on the absorption of (65)Zn were examined and compared with their effects on (55)Fe absorption in human intestinal Caco-2 cells grown on microporous membrane inserts. The levels of EGCG, GT, and GSE used in this study were within physiological ranges and did not affect the integrity of the Caco-2 cell monolayers. GSE significantly (P < 0.05) reduced zinc transport across the cell monolayer, and the decreased zinc transport was associated with a reduction in apical zinc uptake. However, EGCG and GT did not alter zinc absorption. In contrast, the polyphenolic compounds in EGCG, GT, and GSE almost completely blocked transepithelial iron transport across the cell monolayer. The effect of GSE on zinc absorption was very different from that on iron absorption. Whereas GSE decreased zinc absorption by reducing apical zinc uptake, the polyphenolic compounds inhibited iron absorption by enhancing apical iron uptake. GSE inhibited zinc absorption similarly to that observed for phytate. Phytate significantly (P < 0.05) decreased transepithelial zinc transport by reducing apical zinc uptake. The inhibition of zinc absorption may be due to the presence of procyanidins in GSE, which bind zinc with high affinity and block the transport of zinc across the apical membrane of enterocytes. Further research on the absorption of zinc as zinc-polyphenol complexes and free zinc should provide further insight into the process of dietary zinc absorption in the presence of GSE and other bioactive dietary polyphenols. The present study suggests that some individuals should consider their zinc status if they regularly consume procyanidin-containing foods in their diet. However, further studies, especially in vivo studies, are needed to confirm these results.

Ascorbic acid offsets the inhibitory effect of bioactive dietary polyphenolic compounds on transepithelial iron transport in Caco-2 intestinal cells.

We previously reported that (-)-epigallocatechin-3-gallate (EGCG) and grape seed extract (GSE) at high concentration nearly blocked intestinal iron transport across the enterocyte. In this study, we aimed to determine whether small amounts of EGCG, GSE, and green tea extract (GT) are capable of inhibiting iron absorption, to examine if ascorbic acid counteracts the inhibitory action of polyphenols on iron absorption, and to explore the mechanisms of polyphenol-mediated apical iron uptake and basolateral iron release. An(55)Fe absorption study was conducted by adding various concentrations of EGCG, GSE, and GT using Caco-2 intestinal cells. Polyphenols were found to inhibit the transepithelial (55)Fe transport in a dose-dependent manner. The addition of ascorbic acid offset the inhibitory effects of polyphenols on iron transport. Ascorbic acid modulated the transepithelial iron transport without changing the apical iron uptake and the expression of ferroportin-1 protein in the presence of EGCG. The polyphenol-mediated apical iron uptake was inhibited by membrane impermeable Fe(2+) chelators (P < 0.001), but at a low temperature (4°C), the apical iron uptake was still higher than the control values at 37°C (P < 0.001). These results suggest that polyphenols enhance the apical iron uptake partially by reducing the conversion of ferric to ferrous ions and possibly by increasing the uptake of polyphenol-iron complexes via the energy-independent pathway. The present results indicate that the inhibitory effects of dietary polyphenols on iron absorption can be offset by ascorbic acid. Further studies are needed to confirm the current findings in vivo.

Molecular mechanism of intestinal iron absorption.

Iron is an essential trace metal in the human diet because of its role in a number of metabolic processes including oxygen transport. In the diet, iron is present in two fundamental forms, heme and non-heme iron. This article presents a brief overview of the molecular mechanisms of intestinal iron absorption and its regulation. While many proteins that orchestrate iron transport pathway have been identified, a number of key factors that control the regulation of iron absorption still remain to be elucidated. This review also summarizes new and emerging information about iron metabolic regulators that coordinate regulation of intestinal iron absorption.

Bioactive dietary polyphenols inhibit heme iron absorption in a dose-dependent manner in human intestinal Caco-2 cells.

UNLABELLED: Although heme iron is an important form of dietary iron, its intestinal absorption mechanism remains elusive. Our previous study revealed that (-)-epigallocatechin-3-gallate (EGCG) and grape seed extract (GSE) markedly inhibited intestinal heme iron absorption by reducing the basolateral iron export in Caco-2 cells. The aim of this study was to examine whether small amounts of EGCG, GSE, and green tea extract (GT) could inhibit heme iron absorption, and to test whether the inhibitory action of polyphenols could be offset by ascorbic acid. A heme-55Fe absorption study was conducted by adding various concentrations of EGCG, GSE, and GT to Caco-2 cells in the absence and presence of ascorbic acid. Polyphenolic compounds significantly inhibited heme-55Fe absorption in a dose-dependent manner. The addition of ascorbic acid did not modulate the inhibitory effect of dietary polyphenols on heme iron absorption when the cells were treated with polyphenols at a concentration of 46 mg/L. However, ascorbic acid was able to offset or reverse the inhibitory effects of polyphenolic compounds when lower concentrations of polyphenols were added (≤ 4.6 mg/L). Ascorbic acid modulated the heme iron absorption without changing the apical heme uptake, the expression of the proteins involved in heme metabolism and basolateral iron transport, and heme oxygenase activity, indicating that ascorbic acid may enhance heme iron absorption by modulating the intracellular distribution of 55Fe. These results imply that the regular consumption of dietary ascorbic acid can easily counteract the inhibitory effects of low concentrations of dietary polyphenols on heme iron absorption but cannot counteract the inhibitory actions of high concentrations of polyphenols. PRACTICAL APPLICATION: Bioactive dietary polyphenols inhibit heme iron absorption in a dose-dependent manner. The small amounts of polyphenolic compounds present in foods are capable of reducing heme iron transport across the intestinal enterocyte. However, the inhibitory effects of dietary polyphenolic compounds on heme iron absorption can be offset by ascorbic acid and can possibly be avoided by decreasing the consumption of polyphenols while simultaneously taking ascorbic acid.

Bioactive dietary polyphenols decrease heme iron absorption by decreasing basolateral iron release in human intestinal Caco-2 cells.

Because dietary polyphenolic compounds have a wide range of effects in vivo and vitro, including chelation of metals such as iron, it is prudent to test whether the regular consumption of dietary bioactive polyphenols impair the utilization of dietary iron. Because our previous study showed the inhibitory effect of (-) -epigallocatechin-3-gallate (EGCG) and grape seed extract (GSE) on nonheme iron absorption, we investigated whether EGCG and GSE also affect iron absorption from heme. The fully differentiated intestinal Caco-2 cells grown on microporous membrane inserts were incubated with heme (55)Fe in uptake buffer containing EGCG or GSE in the apical compartment for 7 h. Both EGCG and GSE decreased (P < 0.05) transepithelial transport of heme-derived iron. However, apical heme iron uptake was increased (P < 0.05) by GSE. Despite the increased cellular levels of heme (55)Fe, the transfer of iron across the intestinal basolateral membrane was extremely low, indicating that basolateral export was impaired by GSE. In contrast, EGCG moderately decreased the cellular assimilation of heme (55)Fe, but the basolateral iron transfer was extremely low, suggesting that the basolateral efflux of heme iron was also inhibited by EGCG. Expression of heme oxygenase, ferroportin, and hephaestin protein was not changed by EGCG and GSE. The apical uptake of heme iron was temperature dependent and saturable in fully differentiated Caco-2 cells. Our data show that bioactive dietary polyphenols inhibit heme iron absorption mainly by reducing basolateral iron exit rather than decreasing apical heme iron uptake in intestinal cells.

Systemic iron status.

Iron is one of the essential micronutrients, and as such, is required for growth, development, and normal cellular functioning. In contrast to some other micronutrients such as water-soluble vitamins, there is a significant danger of toxicity if excessive amounts of iron accumulate in the body. A finely tuned feedback control system functions to limit this excessive accumulation by limiting absorption of iron. This chapter will discuss systemic and brain iron homeostasis.

Altered expression of iron regulatory genes in cirrhotic human livers: clues to the cause of hemosiderosis?

Hepatic iron deposition unrelated to hereditary hemochromatosis occurs commonly in cirrhosis but the pathogenesis of this condition is unknown. The aim of this study was to compare the expression of genes involved in the regulation of iron metabolism in cirrhotic (n=22) and control human livers (n=5). Transcripts were quantitated by real-time RT-PCR and protein levels were assessed by western blot. Hepatic iron concentrations (HICs) were measured by a spectrophotometric method. Levels of hepcidin mRNA did not differ between controls and cirrhotic livers; there was a highly significant correlation between hepcidin transcript levels and HIC in the latter group. Ferroportin, divalent metal transporter-1 (DMT1), and ferritin heavy chain mRNA levels were significantly higher in cirrhotic human livers than in controls (P=0.007, 0.039, and 0.025, respectively). By western blot, ferroportin and DMT1 levels were generally diminished in the cirrhotic livers compared to controls; neither correlated with HIC. In contrast, the abundance of ferritin increased with increasing HIC in the cirrhotic livers, whereas transferrin receptor decreased, indicating physiologically appropriate regulation. In conclusion, hepcidin expression appears to be appropriately responsive to iron status in cirrhosis. However, there are complex alterations in DMT1 and ferroportin expression in cirrhotic liver, including decreases in ferroportin and DMT1 at the protein level that may play a role in aberrant regulation of iron metabolism in cirrhosis.

Bioactive dietary polyphenolic compounds reduce nonheme iron transport across human intestinal cell monolayers.

There is persuasive epidemiological evidence that regular intake of dietary bioactive polyphenolic compounds promotes human health. Because dietary polyphenolic compounds have a wide range of effects in vivo and vitro, including chelation of metals such as iron, it is prudent to test whether the regular consumption of bioactive polyphenolic components impair the utilization of dietary iron. We examined the influence of the dietary polyphenols (-) -epigallocatechin-3-gallate (EGCG) and grape seed extract (GSE) on transepithelial iron transport in Caco-2 intestinal cells. The range of EGCG and GSE concentrations used in this study was within physiological levels and did not affect the integrity of differentiated Caco-2 cell monolayers. Both EGCG and GSE decreased (P < 0.001) transepithelial iron transport. However, apical iron uptake was increased (P < 0.001) by the addition of EGCG and GSE. The increased uptake of iron might be due in part to the reducing activity of EGCG and GSE. Both EGCG and GSE reduced approximately 15% of the applied Fe(3+) to Fe(2+) in the uptake buffer. Despite the increased cellular levels of (55)Fe, the transfer of iron across the basolateral membrane of the enterocyte was extremely low, indicating that basolateral exit via ferroportin-1 was impaired, possibly through formation of a nontransportable polyphenol-iron complex. Our data show that polyphenols inhibit nonheme iron absorption by reducing basolateral iron exit rather than by decreasing apical iron import in intestinal cells.

Colocalization of ferroportin-1 with hephaestin on the basolateral membrane of human intestinal absorptive cells.

An iron exporter ferroportin-1 (FPN-1) and a multi-copper oxidase hephaestin (Heph) are predicted to be expressed on the basolateral membrane of the enterocyte and involved in the processes of iron export across the basolateral membrane of the enterocyte. However, it is not clear where these proteins are exactly located in the intestinal absorptive cell. We examined cellular localization of FPN-1 and Heph in the intestinal absorptive cells using the fully differentiated Caco-2 cells. Confocal microscope study showed that FPN-1 and Heph are located on the basolateral membrane and they are associated with the transferrin receptor (TfR) in fully differentiated Caco-2 cells grown on microporous membrane inserts. However, Heph protein was not detected in the crypt cell-like proliferating Caco-2 cell. In stably transfected human intestinal absorptive cells expressing human FPN-1 modified by the addition of GFP at the C-terminus, we show that FPN-1-GFP is located on the basolateral membrane and it is associated with Heph suggesting the possibility that FPN-1 might associate and interact with Heph in the process of iron exit across the basolateral membrane of intestinal absorptive cell.

Copper repletion enhances apical iron uptake and transepithelial iron transport by Caco-2 cells.

The influence of copper status on Caco-2 cell apical iron uptake and transepithelial transport was examined. Cells grown for 7-8 days in media supplemented with 1 microM CuCl(2) had 10-fold higher cellular levels of copper compared with control. Copper supplementation did not affect the integrity of differentiated Caco-2 cell monolayers grown on microporous membranes. Copper-repleted cells displayed increased uptake of iron as well as increased transport of iron across the cell monolayer. Northern blot analysis revealed that expression of the apical iron transporter divalent metal transporter-1 (DMT1), the basolateral transporter ferroportin-1 (Fpn1), and the putative ferroxidase hephaestin (Heph) was upregulated by copper supplementation, whereas the recently identified ferrireductase duodenal cytochrome b (Dcytb) was not. These results suggest that DMT1, Fpn1, and Heph are involved in the iron uptake process modulated by copper status. Although a clear role for Dcytb was not identified, an apical surface ferrireductase was modulated by copper status, suggesting that its function also contributes to the enhanced iron uptake by copper-repleted cells. A model is proposed wherein copper promotes iron depletion of intestinal Caco-2 cells, creating a deficiency state that induces upregulation of iron transport factors.