• Nutrition Research and Practice
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• v.3 no.3
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• pp.192-199
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• 2009

Cadmium intoxication has been associated with the dysregulation of iron homeostasis. In the present study, we investigated the effect of cadmium on the expression of ferroportin 1 (FPN1), an important iron transporter protein that is involved in iron release from macrophages. When we incubated cadmium with J774 mouse macrophage cells, FPN1 mRNA levels were significantly increased in a dose- and time-dependent manner. Furthermore, the cadmium-induced FPN1 mRNA expression was associated with increased levels of FPN1 protein. On the other hand, cadmium-mediated FPN1 mRNA induction in J774 cells was completely blocked when cells were co-treated with a transcription inhibitor, acitomycin D. Also, cadmium directly stimulated the activity of the FPN1-promoter driven luciferase reporter, suggesting that the cadmium up-regulates FPN1 gene expression in a transcription-dependent manner. Finally, cadmium exposure to J774 macrophages increased intracellular reactive oxygen species (ROS) levels by ${\sim}2$-fold, compared to untreated controls. When J774 cells were co-treated with antioxidant N-acetylcystein, the cadmium-induced FPN1 mRNA induction was significantly attenuated. In summary, the results of this study clearly demonstrated that cadmium increased FPN1 expression in macrophages through a mechanism that involves ROS production, and suggests another important interaction between iron and cadmium metabolism.

• Nutrition Research and Practice
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• v.2 no.4
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• pp.317-321
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• 2008

Macrophages play a key role in iron metabolism by recycling iron through erythrophagocytosis. Ferroportin-l (FPN1) is a transporter protein that is known to mediate iron export from macrophages. Since divalent metals often interact with iron metabolism, we examined if divalent metals could regulate the expression of FPN1 in macrophages. J774 macrophage cells were treated with copper, manganese, zinc, or cobalt at 10, 50, or $100\;{\mu}M$ for 16 to 24 h. Then, FPN1 mRNA and protein levels were determined by quantitative real-time PCR and Western blot analyses, respectively. In addition, effects of divalent metals on FPN1 promoter activity were examined by luciferase reporter assays. Results showed that copper significantly increased FPN1 mRNA levels in a dose-dependent manner. The copper-induced expression of FPN1 mRNA was associated with a corresponding increase in FPN1 protein levels. Also, copper directly stimulated the activity of FPN1 promoter-driven reporter construct. In contrast, manganese and zinc had no effect on the FPN1 gene expression in J774 cells. Interestingly, cobalt treatment in J774 cells decreased FPN1 protein levels without affecting FPN1 mRNA levels. In conclusion, our study results demonstrate that divalent metals differentially regulate FPN1 expression in macrophages and indicate a potential interaction of divalent metals with the FPN1-mediated iron export in macrophages.

• Journal of Nutrition and Health
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• v.42 no.5
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• pp.434-441
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• 2009

Ferroportin-1 (FPN) is a transporter protein that is known to mediate iron export from macrophages. The purpose of this study was to investigate the effect of copper on the regulation of FPN gene expression in J774 mouse macrophage cells. J774 cells were treated with various concentrations of $CuSO_4$ and RT-PCR analyses were performed to measure the steady-state levels of mRNAs for FPN and divalent metal transporter 1 (DMT1, an iron importer). Copper treatment significantly increased FPN mRNAs in a dose-dependent manner, but didn't change the levels of DMT1 mRNA. Experiments with transcriptional inhibitor actinomycin D (0.5 ${\mu}g$/mL) revealed that copper treatment did not affect the half-life of FPN mRNAs in J774 cells. On the other hand, results from luciferase reporter assays showed that copper directly stimulated the promoter activity of FPN. In summary, our data showed copper induced FPN mRNA of macrophages via a transcriptional rather than post-transcriptional mechanisms.

• Nutrition Research and Practice
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• v.9 no.6
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• pp.613-618
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• 2015

BACKGROUND/OBJECTIVES: Iron deficiency in early life is associated with developmental problems, which may persist until later in life. The question of whether iron repletion after developmental iron deficiency could restore iron homeostasis is not well characterized. In the present study, we investigated the changes of iron transporters after iron depletion during the gestational-neonatal period and iron repletion during the post-weaning period. MATERIALS/METHODS: Pregnant rats were provided iron-deficient (< 6 ppm Fe) or control (36 ppm Fe) diets from gestational day 2. At weaning, pups from iron-deficient dams were fed either iron-deficient (ID group) or control (IDR group) diets for 4 week. Pups from control dams were continued to be fed with the control diet throughout the study period (CON). RESULTS: Compared to the CON, ID rats had significantly lower hemoglobin and hematocrits in the blood and significantly lower tissue iron in the liver and spleen. Hepatic hepcidin and BMP6 mRNA levels were also strongly down-regulated in the ID group. Developmental iron deficiency significantly increased iron transporters divalent metal transporter 1 (DMT1) and ferroportin (FPN) in the duodenum, but decreased DMT1 in the liver. Dietary iron repletion restored the levels of hemoglobin and hematocrit to a normal range, but the tissue iron levels and hepatic hepcidin mRNA levels were significantly lower than those in the CON group. Both FPN and DMT1 protein levels in the liver and in the duodenum were not different between the IDR and the CON. By contrast, DMT1 in the spleen was significantly lower in the IDR, compared to the CON. The splenic FPN was also decreased in the IDR more than in the CON, although the difference did not reach statistical significance. CONCLUSIONS: Our findings demonstrate that iron transporter proteins in the duodenum, liver and spleen are differentially regulated during developmental iron deficiency. Also, post-weaning iron repletion efficiently restores iron transporters in the duodenum and the liver but not in the spleen, which suggests that early-life iron deficiency may cause long term abnormalities in iron recycling from the spleen.

• The Korean Journal of Physiology and Pharmacology
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• v.28 no.2
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• pp.113-120
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• 2024

Solute carrier 40A1 (SLC40A1) encodes ferroportin, which is the only known transmembrane protein that exports elemental iron from mammalian cells and is essential for iron homeostasis. Mutations in SLC40A1 are associated with iron-overload disorders. In addition to ferroportin diseases, SLC40A1 expression is downregulated in various cancer types. Despite the clinical significance of the SLC40A1 transporter, only a few studies have investigated genetic variants in SLC40A1. The present study was performed to identify genetic variations in the SLC40A1 promoter and functionally characterize each variant using in vitro assays. We investigated four haplotypes and five variants in the SLC40A1 promoter. We observed that haplotype 3 (H3) had significantly lower promoter activity than H1, whereas the activity of H4 was significantly higher than that of H1. Luciferase activity of H2 was comparable to that of H1. In addition, four variants of SLC40A1, c.-1355G>C, c.-662C>T, c.-98G>C, and c.-8C>G, showed significantly increased luciferase activity compared to the wild type (WT), whereas c.-750G>A showed significantly decreased luciferase activity compared to the WT. Three transcription factors, cAMP response element-binding protein-1 (CREB-1), chicken ovalbumin upstream promoter transcription factor 1, and hepatic leukemia factor (HLF), were predicted to bind to the promoter regions of SLC40A1 near c.-662C>T, c.-98G>C, and c.-8C>G, respectively. Among these, CREB1 and HLF bound more strongly to the variant sequences than to the WT and functioned as activators of SLC40A1 transcription. Collectively, our findings indicate that the two SLC40A1 promoter haplotypes affect SLC40A1 transcription, which is regulated by CREB-1 and HLF.

• Journal of the Korean Society of Food Science and Nutrition
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• v.37 no.6
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• pp.721-728
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• 2008

Hepcidin is a peptide hormone produced by the liver, of which secretion is closely related to iron status in the body. However, little is known about the molecular mechanism(s) by which this peptide regulates body iron homeostasis. The purpose of this study was to determine the effects of hepcidin treatment within the physiological concentration range on the expressions of two different iron transporter proteins-ferroportin (FPN) and divalent metal transporter 1 (DMT1). Differentiated Caco-2 intestinal cells and macrophage J774 cells were treated with either synthetic hepcidin or hepcidin-rich fraction separated from human urine at the concentration of 10 nM and 100 nM for 24 hours. Results show that hepcidin treatment in differentiated Caco-2 cells or in J774 cells did not change the level of either FPN mRNA or DMT1 mRNA. On the other hand, hepcidin treatment at the dose of 100 nM significantly decreased the FPN protein levels and DMT1 protein levels in differentiated Caco-2 cells. Similarly, urinary hepcidin treatment (10 nM & 100 nM) also significantly decreased the levels of FPN and DMT1 proteins in J774 macrophage cells. These results showed that hepcidin might play an important role in the regulation of iron homeostasis by lowering the protein levels of iron transporter FPN and DMT1 both in enterocytes and in macrophage cells.

• Journal of Nutrition and Health
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• v.54 no.5
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• pp.435-447
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• 2021

Purpose: Body adiposity is negatively correlated with hepatic iron status, and Korean pine nut oil (PNO) has been reported to reduce adiposity. Therefore, we aimed to study the effects of PNO on adiposity, hepatic mineral status, and the expression of genes and proteins involved in iron absorption. Methods: Five-week-old male C57BL/6 mice were fed a control diet containing 10% kcal from PNO (PC) or soybean oil (SBO; SC), or a high-fat diet (HFD) containing 35% kcal from lard and 10% kcal from PNO (PHFD) or SBO (SHFD). Hepatic iron, copper, and zinc content; and expression of genes and proteins related to iron absorption were measured. Results: HFD-fed mice had a higher white fat mass (2-fold; p < 0.001), lower hepatic iron content (25% lower; p < 0.001), and lower hepatic Hamp (p = 0.028) and duodenal Dcytb mRNA levels (p = 0.037) compared to the control diet-fed mice. Hepatic iron status was negatively correlated with body weight (r = -0.607, p < 0.001) and white fat mass (r = -0.745, p < 0.001). Although the PHFD group gained less body weight (18% less; p < 0.05) and white fat mass (18% less; p < 0.05) than the SHFD group, the hepatic iron status impaired by the HFD feeding did not improve. The expression of hepatic and duodenal ferroportin protein was not affected by the fat amount or the oil type. PNO-fed mice had significantly lower Slc11a2 (p = 0.022) and Slc40a1 expression (p = 0.027) compared to SBO-fed mice. However, the PC group had a higher Heph expression than the SC group (p < 0.05). The hepatic copper and zinc content did not differ between the four diet groups, but hepatic copper content adjusted by body weight was significantly lower in the HFD-fed mice compared to the control diet-fed mice. Conclusion: HFD-induced obesity decreased hepatic iron storage by affecting the regulation of genes related to iron absorption; however, the 18% less white fat mass in the PHFD group was not enough to improve the iron status compared to the SHFD group. The hepatic copper and zinc status was not altered by the fat amount or the oil type.