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Both zinc and iron are essential minerals for vertebrates. Zinc could influence growth, reproduction and immune function by regulate enzyme activity and gene expression. Iron participates in oxygen transfer, electron transport and DNA synthesis. Cellular iron concentration was under tightly control because iron presents in excess will catalysis generation of free radicals to destroy biological macromolecules. Iron regulatory proteins can bind iron responsive elements in the 5''-untranslational region of ferritin, mitochondrial aconitase, d-ALA synthase and in the 3''-untranslational region of transferrin receptor mRNAs to regulate the expression of the upper proteins and coordinate the storage, utilization and uptake of iron. In cell culture, it has been shown that iron can convert IRP1 to cytosolic aconitase by Fe-S cluster insertion. Biological interactions between trace minerals have long been recognized by nutritionists. In previous studies, hepatic iron accumulation occurred repeatedly in zinc deficient rats liver. And the purpose of this study was to explore the factors that alter hepatic iron homeostasis in zinc deficient rats.
In the first experiment, we assessed the effect of dietary zinc deficiency on hepatic iron metabolism in rats. Ferritin, mitochondrial aconitase, and iron regulatory proteins activities were represented as cellular iron storage, utilization and regulating capacity respectively. Two groups of male wistar rats were given free access to either a control or a zinc deficient diet for 3 weeks. The final body weight, dietary intake and feed efficiency were significantly lower in zinc deficient group. Liver zinc concentration and serum alkaline phosphatase activity were significantly lower in zinc deficient group, indicating the zinc deficient status. The liver iron and ferritin concentration in the zinc deficient group were 1.9 and 1.96 fold higher than in the control group, but there was no significantly different in the ferritin mRNA content, demonstrating that the ferritin synthesis is regulated at the translational level by IRPs. Comparing with control group, the mitochondrial aconitase activity was significantly higher in the zinc deficient group. The protein content of mitochondrial aconitase detected by western blots was also higher in the deficient group and the magnitude was paralleled with the change in the activity, indicating the increase in enzyme activity comes from the increase of protein content. The specific activity of mitochondrial succinate dehydrogenase in the zinc deficient group was 1.4 fold higher than in the control group. These results showed that rats fed zinc deficient diet would enhance hepatocyte utilizing iron for synthesis of iron-related protein. Comparing with control group, the activity of cytosolic aconitase was significantly lower in the zinc deficient group imply that the total IRP1 content would be lower in zinc deficient group. The total but not the spontaneous IRPs activities were significantly lower in zinc deficient group than in the control group. So the active percentage ( spontaneous/total ) of IRPs was higher in zinc deficient group. There was positive correlation between the cytosolic aconitase and the total IRP1 activities ( r=0.70, p=0.001 ), supporting that the total IRP1 content was low in zinc deficient status. Take together, these results indicated that hepatic iron homeostasis was altered in rat fed zinc deficient diet.
Three factors that could contribute to the change in the amount of hepatic IRP1 in zinc deficient rats were 1) zinc deficiency, 2) iron overload and 3) food restriction. To investigate the effect of zinc deficiency on the hepatic iron homeostasis, zinc deficient rats were repleted with a single dose of zinc acetate ( 6.6 mg Zn ) by gavage for 24hr before killed. Ten Wistar male rats were fed zinc deficient diet ( ZnD ) and the other ten rats were given free access to a control diet ( C ) or limit its intake to 70% of control rats ( DR ). After 3 weeks, half of the zinc deficient rats were supplemented with zinc by gavage and designed as zinc supplement ( ZnS ) group. Liver zinc concentration and serum alkaline phosphatase activity were significantly lower in zinc deficient group comparing with C and DR groups, indicating the zinc deficient status. And the liver zinc concentration of ZnS group was not different with the C group, showing gavage a single dose of zinc can restore the zinc status of rats. The concentration of serum iron was high in ZnD group comparing with the C, DR and ZnS groups. The concentration of liver iron and ferritin were high in ZnS group and by the sequence of ZnD, DR and C groups to low. The results indicated that acute zinc repletion will enhance hepatocyte uptake more iron and store in ferritin in zinc deficient rats. The activities of mitochondrial aconitase and succinate dehydrogenase from high to low were ZnS, ZnD, DR and C groups and parallel change with liver iron concentration, implying that the change in iron homeostasis might be resulted from iron but not zinc status. The activity of cytosolic aconitase was lowest in the ZnS group and negative correlated ( r=0.50, p=0.02 ) with liver iron concentration. However, the spontaneous IRPs activities were not significantly different among groups. And like the cytosolic aconitase, the activity of total inducible IRP1 was lowest in the ZnS group and highest in the control group. These results demonstrated that iron but not zinc concentration would be the major factor contributing to the change in the iron homeostasis of zinc deficient rats.
To investigate the effect of iron accumulation on the IRP1 content, two groups of S. D. male rats were fed either a control ( C ) or an iron overloading diet ( IO, 2.5% carbonyl iron ) for 8 weeks. Serum iron concentration and transferrin saturation of IO group were significantly higher than the C group. The amount of iron accumulate in the liver was 19 times higher in the IO group comparing with that in the C group. The content of ferritin in the liver of IO groups was 10 times higher than the control group, indicating the dietary iron loading effect. Under this condition, the activity of cytosolic aconitase in the IO group was significantly lower than in the C group. The activities of spontaneous and total inducible IRP1 activities in the liver were also lower in the IO group comparing with that in the C group. The results indicated iron overloading would decrease the content of IRP1 in the liver.
To explore the effect of food restriction on the hepatic IRP1 content Seven Wistar male rats were fed zinc deficient diet ( ZnD ) and the other tweenty-one rats were given free access to a control diet ( C ) or limit its intake to 70% or 50% of control rats ( M, S ) for 3 weeks. Liver zinc concentration and serum alkaline phosphatase activity were significantly lower in ZnD group comparing with C groups, indicating the zinc deficient status. Serum and liver iron concentration of ZnD group were significantly higher than the C group. The growth indices of ZnD group was similar with the S group. So comparing with the S group, the ZnD group has higher liver iron, ferritin concentration, G6PDH and SDH activities.
In conclusion, this study demonstrated that the iron but not the zinc and energy was the major factor contributing to the change in hepatic IRP1 content in zinc deficient rats. In contrast to cell culture, iron would decrease IRP1 content in certain condition in animal model.
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