| Summary: | 博士 === 國立臺灣大學 === 農業化學研究所 === 89 === Intracellular iron homeostasis maintained by iron uptake, functional uses, and storage is performed by transferrin receptor (TfR), iron-containing proteins, and ferritin. Iron regulatory proteins (IRPs: IRP1 and IRP2) are cytoplasmic RNA-binding proteins that are central components of a sensory and regulatory network of iron homeostasis by binding to iron responsive element(s) (IREs) in the 5’ or 3’ untranslated region of ferritin, mitochondrial aconitase (m-ACO) or TfR mRNAs. Although structurally and functionally similar, the two IRPs are different in their model of regulation by numerous factors such as iron, hydrogen peroxide and nitric oxide. Differential modulation of this two IRPs has been proposed in cellular models. We tested this hypothesis in a rat model using dietary treatment of non-iron components: oxidized frying soybean oil, fish oil and hypolipidemic drug clofibrate. Diets were formulated according to AIN-76 but oil content was increased to 15% (w/w). In all the experiments, Wistar rats were used unless specified otherwise. Blood parameters measured included: hemoglobin, serum iron, total iron binding capacity, serum copper and ceruloplasmin (Cp). RNA binding activities of IRP1 and IRP2 were measured using electrophoretic mobility-shift assays. Ferritin and m-ACO proteins were measured using Western blot analysis. Message RNA levels of transferrin, TfR, Cp and Wilson''s disease gene (ATP7B) were measured using Northern blot analysis.
First, in a time-course study of the effect of oxidized frying oil on IRPs, liver IRP1 but not IRP2 activity increased rapidly from day 2 of the treatment. The interaction of oxidized frying oil and dietary iron levels on IRPs were investigated in a 2×2 design, using two iron levels (control at 35 ppm Fe and low iron at 15 ppm Fe) and fresh soybean oil as reference oil for 6-wks feeding. Rats fed the diet of low iron in combination with oxidized oil had the lowest levels of hemoglobin, serum iron and transferrin saturation among the four groups. Based on results of two-way ANOVA, IRP1 activity was significantly increased by oxidized frying oil treatment, IRP2 activity was increased at low dietary Fe level. In response to IRP2, TfR mRNA increased significantly. Hepatic Tf and HNF-4mRNA levels also decreased by oxidized oil. It is concluded that consumption of high level of oxidized frying oil will aggravate iron deficiency in rats fed low iron diet.
Second, the effect of fish oil and coconut oil on liver iron was compared in weanling male Sprague-Dawley rats, and it was observed that liver iron and ferritin concentration decreased significantly in rats fed fish oil. The interaction of fish oil and dietary iron levels on IRPs were investigated in a 2×2 design, using two iron levels (35 ppm and 15 ppm Fe) and fresh soybean oil as reference oil for 6-wks feeding. In rats fed fish oil, serum iron, hepatic iron and ferritin concentration were significantly lower; hepatic IRP1 but not IRP2 activity increased significantly; serum copper content and Cp activity were significanly elevated in consistent with the increase mRNA level of heaptic Cp regardless of iron status.
Finally, the effects and interaction of clofibrate (0.5%) and dietary iron levels (35 ppm and 15 ppm) on proteins related to iron and copper metabolism were studied in a rat model using 2 x 2 experimental design. Wistar rats (6 each group) were assigned to the four diets and fed for 6 wks. In clofibrate-treated rats, serum iron and total iron binding capacity, and serum copper and Cp were significantly reduced by 50% and 20%, respectively, which were consistant with the reduced mRNA levels of transferrin and Cp in the liver. Reduced Tf and Cp may be resposible for impaired hepatic iron release. Hepatic iron and DFO-chelatable iron concentrations were significantly elevated by clofibrate treatment. In clofibrate-treated rats, the RNA-binding activity of IRP1 but not IRP2 were significantly elevated even in the presence of increased iron concentration, indicating differential modulation of IRP1 and IRP2 activities. Hepatic TfR mRNA was increased by low dietary iron but inhibited by clofibrate. The activity and protein of m-ACO were elevated by clofibrate treatment regardless of dietary iron levels. Copper accumulated in the liver of clofibrate-treated rats, which may result from impaired coppper efflux due to reduction of Wilson’s disease gene (ATP7B) mRNA. We conclude that clofibrate treatment altered molecular regulation of iron and copper metabolism in the liver and resulted in impaired iron and copper transport. These observations may have clinical implications.
In summary, dietary factors other than iron affect RNA binding activities of hepatic IRP1 and IRP2. IRP2 but not IRP1 activity increased in response to moderately decreased dietary iron and plays an essential role in the regulation of iron homeostatsis. IRP1 activity was significantly elevated in the liver of rats fed oxidized frying oil or clofibate even though their hepatic iron concentrations were not depleted or higher than that of the normal rats. The physiological implication of this phenomenon is not understood. These results support that differential modulation of hepatic IRP1 and IRP2 activities occurs in vivo.
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