Effect of dietary iron deficiency and overload on the expression of ZIP metal-ion transporters in rat liver

Abstract

The mammalian ZIP (Zrt-, Irt-like Protein) family of transmembrane transport proteins consists of 14 members that share considerable homology. ZIP proteins have been shown to mediate the cellular uptake of the essential trace elements zinc, iron, and manganese. The aim of the present study was to determine the effect of dietary iron deficiency and overload on the expression of all 14 ZIP transporters in the liver, the main site of iron storage. Weanling male rats (n = 6/group) were fed iron-deficient (FeD), iron-adequate (FeA), or iron-overloaded (FeO) diets in two independent feeding studies. In study 1, diets were based on the TestDiet 5755 formulation and contained iron at 9 ppm (FeD), 215 ppm (FeA), and 27,974 ppm (3% FeO). In study 2, diets were based on the AIN-93G formulation and contained iron at 9 ppm Fe (FeD), 50 ppm Fe (FeA), or 18916 ppm (2% FeO). After 3 weeks, the FeD diets depleted liver non-heme iron stores and induced anemia, whereas FeO diets resulted in hepatic iron overload. Quantitative RT-PCR revealed that ZIP5 mRNA levels were 3- and 8-fold higher in 2% FeO and 3% FeO livers, respectively, compared with FeA controls. In both studies, a consistent downregulation of ZIP6, ZIP7, and ZIP10 was also observed in FeO liver relative to FeA controls. Studies in H4IIE hepatoma cells further documented that iron loading affects the expression of these ZIP transporters. Overall, our data suggest that ZIP5, ZIP6, ZIP7, and ZIP10 are regulated by iron, indicating that they may play a role in hepatic iron/metal homeostasis during iron deficiency and overload.

Fig. 1

Body weights of rats fed iron-adequate (FeA), iron-deficient (FeD) and iron-overloaded (FeO) diets. (A) In study 1, weanling male Sprague-Dawley rats were fed modified TestDiet^® 5755 basal diets that contained 9 ppm Fe (FeD), 215 ppm Fe (FeA) or 27,974 ppm Fe (3% FeO) for 3 weeks. (B) In study 2, weanling male Sprague-Dawley rats were fed modified AIN-93G diets that contained 9 ppm Fe (FeD), 50 ppm Fe (FeA) or 18916 ppm Fe (2% FeO) for 3 weeks. Body weights were measured every third day. Final body weights were compared by one-way ANOVA followed by Tukey’s multiple comparison test. Values represent mean ± SE, n=6. Means without a common letter differ, P < 0.05.

Fig. 2

Effect of FeD and FeO on the expression of hepatic ZIP transporters in (A) study 1 and (B) study 2. Total RNA was isolated from liver and transcript abundances of all fourteen ZIP transporters were determined by using qRT-PCR. Shown above are the ZIP transporters whose expression differed compared to FeA controls. Relative transcript abundances of each gene were normalized to the average of two housekeeping genes (cyclophilin B and RPL13A). Transcript levels of BMP6, an iron-regulated gene, were measured to demonstrate iron-related changes in gene expression. Values are means ± SE, n=6. Asterisks indicate difference relative to FeA controls (*P < 0.05, **P < 0.01).

Fig. 3

Effect of iron loading on the expression of ZIP5, ZIP6, ZIP7, ZIP10, and ZIP3 in H4IIE cells. Cells were incubated in medium supplemented with or without 50 μM Fe-NTA for 8 hours. Total RNA was isolated and levels of ZIP5, ZIP6, ZIP7, ZIP10, and ZIP3 mRNA were determined by qRT-PCR and expressed as relative to control. Levels of TFR1 were measured to confirm iron loading of cells. Values are means ± SE of 3 independent experiments.