• Nutrition Research and Practice
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• 제2권4호
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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.

• Nutrition Research and Practice
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• 제3권3호
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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.

• Journal of Nutrition and Health
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• 제42권5호
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• pp.434-441
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• 2009

본 연구는 J774 대식세포에서 FPN 유전자 발현 조절에 구리가 미치는 영향을 알아보기 위하여 수행되었으며 그 결과는 다음과 같다. J774 대식 세포에 구리를 처리하였을 때, iron exporter FPN의 mRNA 수준이 농도 의존적으로 증가하는 것으로 나타났다. 반면, iron importer DMT1의 mRNA 수준은 구리 처리에 의해 영향을 받지 않았다. Actinomycin D를 이용하여 mRNA 합성을 억제한 상태에서 FPN mRNA 분해 정도를 시간별로 추적한 결과, acitnomycin D 처리 후 9시간 경과시 FPN mRNA 수준이 처음 수준의 약 60% 정도로 감소하였다. 배양액에 구리를 첨가한 경우에도 FPN mRNA의 분해 정도는 아무것도 처리하지 않은 대조군과 유의적인 차이가 없었으며, 이로 볼 때 구리는 FPN mRNA의 안정성에 영향을 미치지 않는 것으로 생각된다. 한편, reporter assay 실험 결과 구리의 첨가는 FPN 프로모터 활성을 유의적으로 증가시키는 것으로 나타나, 구리가 FPN mRNA의 전사 과정을 직접적으로 촉진함을 알 수 있었다. 또한, FPN 5'-UTR에 위치하는 IRE (iron response element)의 존재 여부는 구리에 의한 FPN 전사 개시 활성에 영향을 주지 않는 것으로 나타났으며, 이로 볼 때 구리는 철분과는 독립적인 작용 기작에 의해 FPN 유전자 발현을 조절하는 것으로 사료된다. 이상의 결과를 종합해 볼 때, 구리는 대식 세포에서 전사개시 과정을 활성화함으로써 농도 의존적으로 FPN 유전자 발현을 촉진하는 것으로 생각되며, 이는 구리가 철분의 대사에 미치는 새로운 작용 기작을 제시한다. 앞으로, 구리와 철 분의 상호 작용이 FPN의 철분 및 다른 무기질 이온의 세포내 외 수송 (transport)에 어떤 영향을 미치는지에 대한 기능적 연구가 계속적으로 이루어져야 할 것으로 사료된다.

• The Korean Journal of Physiology and Pharmacology
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• 제28권2호
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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.

• 한국식품영양과학회지
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• 제37권6호
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• pp.721-728
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• 2008

본 연구에서는 소장세포(Caco-2)와 대식세포(J774)를 이용하여 FPN과 DMT1의 유전자 발현에 hepcidin 펩타이드 호르몬이 미치는 영향을 알아보기 위하여 수행되었으며 그 결과를 요약하면 다음과 같다. Caco-2 세포에서 FPN과 DMT1의 mRNA 및 단백질 수준은 분화 진행에 따라 비례하여 증가하였으며, 특히 DMT1 단백질은 분화 초기에는 거의 발현되지 않다가 분화 7일째에 비로소 발현되기 시작한 후 급격히 증가하여 분화 17일째에는 7일째에 비해 단백질 수준이 10배 이상 크게 증가되었다. 분화된 Caco-2 세포에서 소변 hepcidin과 합성 hepcidin을 100 nM 농도로 24시간 동안 처리하였을 때, FPN 단백질 수준이 대조군에 비해 각각 60%와 70% 수준으로 유의하게 감소하였다. DMT1 단백질의 경우, 소변 hepcidin 100 nM 농도에서만 대조군의 55% 수준으로 유의하게 감소되었다. J774 세포에 소변 hepcidin 혹은 합성 hepcidin을 24시간 처리한 결과, 10 nM과 100 nM 농도에서 모두 대조군에 비해 FPN 단백질 수준이 유의적으로 감소하는 것으로 나타났으며, DMT1 단백질 수준도 소변 hepcidin 10 nM과 100 nM 처리에 의해 각각 대조군의 40%와 37% 수준으로 유의하게 감소하였다. 분화된 Caco-2 세포와 J774 세포에서 10 nM 혹은 100 nM 농도의 hepcidin 처리 시 DMT1 mRNA와 FPN mRNA 수준에는 영향을 미치지 않는 것으로 나타났으며, 이로 볼 때 hepcidin은 전사과정의 조절보다는 DMT1과 FPN 단백질로의 번역과정을 억제하거나 분해 속도를 촉진함으로써 이들 단백질의 수준을 낮추는 것으로 보인다. 이상의 결과는, hepcidin 펩타이드 호르몬이 DMT1 단백질과 FPN 단백질의 수준을 억제함으로써 체내 철분 대사 조절에 중요하게 관여함을 나타낸다. 특히 소장세포와 대식세포에 동시에 작용함으로써, 소장에서의 철분 흡수와 대식세포에서의 철분 방출을 효율적으로 억제하는 조절 인자로 작용할 수 있음을 제시한다. 앞으로 hepcidin의 생성 및 분비를 조절하는 요인에 대한 연구와 hepcidin이 실제 세포 내외로의 철분의 수송이 미치는 영향에 대한 기능적 연구가 계속적으로 이루어져야 할 것으로 사료된다.

• Nutrition Research and Practice
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• 제9권6호
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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.

• Journal of Nutrition and Health
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• 제54권5호
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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.