The Journal of Nutrition Vol. 128 No. 3 March 1998, pp. 525-535

Dietary Iron Intake Rapidly Influences Iron Regulatory Proteins, Ferritin Subunits and Mitochondrial Aconitase in Rat Liver^1,2,3

Opal S. Chen, Kenneth P. Blemings, Kevin L. Schalinske, and Richard S. Eisenstein⁴

Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, WI 53706

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Iron regulatory protein 1 (IRP1) and IRP2 are cytoplasmic RNA binding proteins that are central regulators of mammalian iron homeostasis. We investigated the time-dependent effect of dietary iron deficiency on liver IRP activity in relation to the abundance of ferritin and the iron-sulfur protein mitochondrial aconitase (m-acon), which are targets of IRP action. Rats were fed a diet containing 2 or 34 mg iron/kg diet for 1-28 d. Liver IRP activity increased rapidly in rats fed the iron-deficient diet with IRP1 stimulated by d 1 and IRP2 by d 2. The maximal activation of IRP2 was five-fold (d 7) and three-fold (d 4) for IRP1. By d 4, liver ferritin subunits were undetectable and m-acon abundance eventually fell by 50% (P < 0.05) in iron-deficient rats. m-Acon abundance declined most rapidly from d 1 to 11 and in a manner that was suggestive of a cause and effect type of relationship between IRP activity and m-acon abundance. In liver, iron deficiency did not decrease the activity of cytosolic aconitase, catalase or complex I of the electron transport chain nor was there an effect on the maximal rate of mitochondrial oxygen consumption with the use of malate and pyruvate as substrates. Thus, the decline in m-acon abundance in iron deficiency is not reflective of a global decrease in liver iron-sulfur proteins nor does it appear to limit ATP production. Our results suggest a novel role for m-acon in cellular iron metabolism. We conclude that, in liver, iron deficiency preferentially affects the activities of IRPs and the targets of IRP action.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Iron performs a central role in cellular and organismal physiology because it is a cofactor for a large number of proteins required for cell function (Beard et al. 1996, Eisenstein et al. 1997, Hentze and Kuhn 1996, Rouault and Klausner 1996, Theil 1994). Iron-containing proteins have essential roles in oxygen transport, ATP production, DNA synthesis and other physiologic processes. However, two problems associated with the use of iron in biological systems are its low solubility and its propensity to catalyze formation of toxic oxidants. To maintain iron levels within a range that meets the needs of the organism yet reduces the likelihood of iron-catalyzed cellular damage, mammals possess a number of proteins that promote the efficient and safe use of this essential nutrient.

The ability to modulate the abundance of proteins that function in the transport, uptake and metabolic utilization of iron is a central factor in the maintenance of iron homeostasis. In normal individuals, interorgan transport and uptake of non-heme iron is performed largely by the transferrin (Tf)⁵/transferrin receptor (TfR) system. Serum Tf, which binds two iron atoms, transports iron between tissues. Iron-loaded Tf (Fe₂Tf) binds to cell surface TfR, and receptor-mediated endocytosis carries the Fe₂Tf/TfR complex into the cell, ultimately resulting in iron delivery to the cytoplasm. The iron-free form of transferrin (apoTf)/TfR complex returns to the cell surface where apo-Tf is released to pick up additional iron elsewhere in the body.

View larger version (22K): [in this window] [in a new window]   Fig 2. Time-dependent effect of ingestion of an iron-deficient or iron-adequate diet on the RNA binding activity of iron regulatory protein (IRP)1 and IRP2 in rat liver. Panel A shows the effect of dietary iron intake on RNA binding activity of IRP1 in liver cytosol from rats fed the iron-deficient (group ID) or fed the control diet on a pair-fed (groups PF) or unrestricted (group C) basis. For panel A each point represents the mean ± SEM (n = 4) except on d 21 where n = 2 for group ID and d 28 where n = 3 for group PF. At time points at which n = 4, the honestly significant difference (HSD) value, represented by the bar, equals 0.024, whereas for d 21, HSD equals 0.035 and for d 28 HSD equals 0.028. Panel B shows IRP2 RNA binding activity measured in the same samples as those used for panel A. For panel B, each point represents the mean ± SEM (n = 4) except on d 21 where n = 2 for group ID and d 28 where n = 3 for group PF. At time points at which n = 4, the HSD value, represented by the bar, equals 0.042, whereas for d 21 HSD equals 0.059 and for d 28 HSD equals 0.049. Panel C represents the total (IRP1 + IRP2) iron responsive element (IRE) binding activity in rat liver cytosol as a function of time for the three different dietary treatments. For panel C, each point represents the mean ± SEM (n = 4) except on d 21 where n = 2 for group ID and d 28 where n = 3 for group PF. At time points at which n = 4, the HSD value equals 0.058, whereas for d 21, HSD equals 0.082 and for d 28 HSD equals 0.067. For all three panels, the RNA binding activity was determined by electrophoretic mobility shift assay as described in Materials and Methods.

View larger version (23K): [in this window] [in a new window]   Fig 1. Time-dependent effect of ingesting an iron-deficient or iron-adequate diet on growth rate, liver weight and blood hemoglobin level in rats. The weight of rats as a function of time is shown in panel A. The arrows indicate the day on which the weight of rats in the iron-deficient (group ID) or pair-fed control (group PF) became significantly different from rats fed the control diet (group C). Panel B shows the liver weight of rats from groups C, PF and ID for the days on which four rats from each group were killed. Liver weight is expressed as a percentage of body weight. The bars shown in panels B and C represent the minimal difference [5% honestly significant difference (HSD)] for statistical significance by Tukey's test at  = 5%. For panel B, each point represents the mean ± SEM (n = 4) except on d 28 where n = 3 for group PF. At time points at which n = 4, the HSD value, represented by the vertical bar, equals 0.52, whereas for d 28, the HSD value equals 0.60. Panel C shows the effect of diet on blood hemoglobin concentration (g/L) for the rats from groups ID, PF and C. For panel C each point represents the mean ± SEM (n = 4) except on d 28 where n = 3 for group PF. At time points at which n = 4 for each group, the HSD value, represented by the vertical bar, equals 20.5, whereas for d 28, the HSD value equals 23.7.

Cytoplasmic iron has a number of metabolic fates. First, it can be incorporated as a component of various iron-containing proteins. In this regard, formation of hemoglobin during red blood cell maturation represents the major daily use of iron within the body. Second, iron can be stored in a safe yet available form within the iron storage protein ferritin. Ferritin, a macromolecule composed of 24 subunits of two types termed H and L, stores up to 4500 iron atoms. Third, tissues such as intestine or liver export iron and thus facilitate its acquisition by other organs. A major mechanism by which iron homeostasis is maintained occurs through the action of iron regulatory proteins (IRPs).

IRPs are cytoplasmic iron-regulated RNA binding proteins that bind to and regulate the utilization of mRNAs encoding proteins that function in the uptake, metabolic utilization or storage of iron (Eisenstein et al. 1997, Hentze and Kuhn 1996, Rouault and Klausner 1996, Theil 1994). IRPs bind to specific stem-loop structures, iron responsive elements (IREs), in the 5 or 3 untranslated region (UTR) of certain mRNAs. A single IRE is present in the 5UTR of the mRNAs encoding the ferritin subunits and the erythroid isoform of 5-aminolevulinate synthase (eALAS), the rate-limiting enzyme in heme formation. Under low iron conditions, IRPs bind to ferritin or eALAS mRNAs and repress their translation. In contrast, TfR mRNA contains multiple IREs in its 3UTR where IRP binding protects the mRNA from degradation. Iron excess leads to inactivation of the RNA binding activity of IRPs, presumably causing their dissociation from the targeted mRNAs and ultimately enhancing iron storage capacity while reducing uptake of Tf-bound iron. Because they regulate the utilization of mRNAs encoding proteins that function in the uptake and metabolic fate of iron, IRPs are considered central regulators of iron metabolism.

Two IRPs, IRP1 and IRP2, have been identified and they have similar affinities for IREs in known IRE-containing mRNAs (referenced in Hentze and Kuhn 1996, Rouault and Klausner 1996). However, iron regulates the function of IRP1 and IRP2 through different mechanisms. IRP1 is a bifunctional protein. In the apo- or iron-free form, IRP1 binds IRE-containing RNA with high affinity. In contrast, when fully iron loaded, the holo form of IRP1 contains a [4Fe-4S] iron-sulfur cluster, has enzymatic activity as an aconitase but binds RNA with much lower affinity. In fact, IRP1 is the cytosolic isoform of the tricarboxylic acid (TCA) cycle enzyme aconitase (c-acon), which converts citrate to isocitrate. Thus, assembly or disassembly of the iron-sulfur cluster is believed to regulate the RNA binding activity of IRP1 in the absence of alterations in the total level of the protein. In contrast, IRP2 is not believed to be an iron-sulfur protein, and it contains a novel 73 amino acid region that confers iron-dependent degradation of the protein (Guo et al. 1995, Iwai et al. 1995). Although the role of IRP in modulating iron uptake and storage is reasonably well understood, the apparent identification of new mRNA targets for IRP action potentially expands the scope of cellular processes affected by these regulatory RNA binding proteins (Dandekar et al. 1991, Gunshin et al. 1997).

The TCA cycle enzyme mitochondrial aconitase (m-acon) contains a putative IRE in the 5UTR of its mRNA (Dandekar et al. 1991, Kim et al. 1996, Zheng et al. 1992). Several lines of evidence indicate that the IRE in m-acon mRNA confers iron-dependent regulation of its translation. Either IRP1 or IRP2 will repress translation of m-acon mRNA in a cell-free protein synthesis system (Gray et al. 1996, Kim et al. 1996). We showed that liver cytosolic IRE binding activity was inversely correlated with m-acon abundance (Chen et al. 1997). Furthermore, we have recently shown that iron regulates m-acon synthesis in cultured cells through a translational mechanism (Schalinske et al. 1998). Therefore the evidence strongly suggests that m-acon mRNA is a target of IRP action.

Several proposals have been advanced with the aim of explaining the physiologic purpose(s) of IRP-mediated modulation of m-acon abundance. These include the potential role of IRPs in regulating mitochondrial production of free radicals and other toxic oxidants by modulating TCA cycle activity and hence flux through the electron transport chain (Gray et al. 1996). Another possibility is that IRP-mediated changes in citrate metabolism reflect a use of this organic acid, or its metabolite(s), in cellular iron trafficking or release (Schalinske et al. 1998). It has also been suggested that IRP mediation of m-acon synthesis may indicate that iron-availability controls the synthesis of many iron-sulfur proteins through an IRP-dependent mechanism (Kim et al. 1996). To further address these questions, we have examined the kinetics with which IRP activity and m-acon abundance respond to iron deficiency in rat liver in comparison with another target of IRP action, ferritin protein abundance. We compare these changes in m-acon and ferritin with the effect of iron intake on other iron-containing proteins in liver as well as the capacity of mitochondria to consume oxygen. Our results indicate that dietary iron intake rapidly affects IRP activity as well as ferritin and m-acon abundance and that these changes represent selective effects of dietary iron on liver function.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals and diets.  Weanling male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) initially weighing 44 ± 0.4 g (n = 88) were placed in individual stainless steel wire-mesh cages in a room with a 12-h light:dark cycle and were given free access to distilled-deionized water. The dark cycle was from 1900 to 0700 h. Body weight was measured daily. Rats were randomly assigned to three diet groups. During the first 7 d, all rats were fed the control diet, which contained 34 mg Fe/kg diet, a level approximately equal to the NRC dietary iron requirement for rats (35 mg Fe/kg diet) (NRC 1995). On the eighth day, they were fed one of the three diets. One group (group ID) was given free access to an iron-deficient diet (2 mg Fe/kg diet), a second group (group PF) was pair-fed the control diet (34 mg Fe/kg diet) to the level of food consumed by the group ID and a third group (group C) was given free access to the control diet. Hereafter, the day on which the rats were divided into these three groups is referred to as d 0 of the 29-d experimental period. On d 0, four rats fed the control diet were killed. On d 1, 2, 4, 7, 11, 21 and 28, between 0900 and 1100 h, four rats from each group were anesthetized with ketamine and xylazine (80 and 10 mg/kg body weight, respectively). Blood was collected by heart puncture after which the liver was excised and weighed. All use of animals met the standards of the University of Wisconsin Research Animal Resource Center.

The composition of the basal iron-deficient diet, based on AIN-76 formulation (AIN 1977), is summarized in Table 1. Iron concentration of the basal iron-deficient diet was determined to be 2 mg iron/kg diet by atomic absorption (UW-Madison, Department of Soil Science Mineral Analysis Facility). A diet containing 34 mg iron/kg diet was produced by the addition of ferrous sulfate (FeSO₄·7H₂O; J. T. Baker, Phillipsburg, NJ) and the iron concentration was confirmed by atomic absorption.

Blood analyses.  Hemoglobin concentration was determined in heparinized blood as described (Chen et al. 1997).

Subcellular fraction of liver mitochondria and cytosol.  For analysis of IRP RNA binding activity as well as for measurement of ferritin subunit or c- and m-acon abundance and/or activity, the homogenates were prepared and fractionated as described previously (Chen et al. 1997). Fresh or previously frozen subcellular fractions were used as indicated below. Recovery and cross-contamination of cytosol and mitochondria was calculated on the basis of the amount of activity of the marker enzymes for cytosol (lactate dehydrogenase) (LDH, EC 1.1.1.27) and mitochondria (glutamate dehydrogenase (GDH, EC 1.1.4.3) recovered in each fraction compared with the amount of activity present in the homogenate (Chen et al. 1997). Determination of the extent to which these two fractions were cross-contaminated is an important issue with regard to accurate quantification of c- and m-acon activities. Mitochondrial contamination of cytosol was 3.2 ± 0.3% (mean ± SEM) and the recovery of cytosol was 74 ± 1%. Cytosolic contamination of mitochondria was 0.3 ± 0.02%. Recovery of mitochondria was 39 ± 1%. To determine the c- and m-acon activity per gram of liver, the aconitase activity measured in cytosol or mitochondria was normalized on the basis of the recovery of cytosol or mitochondria from the homogenate. The summation of the c- and m-acon activities (units/g liver) was equal to 108 ± 1.4% of the activity in detergent-treated homogenate (units/g liver).

For assay of mitochondrial oxygen consumption, a different isolation procedure had to be used because our standard method (Chen et al. 1997) gave poorly coupled mitochondria as a result of the use of glycerol in the buffer (Bleming, K. P., University of Wisconsin-Madison, unpublished observations). This alternative procedure was also used for determination of complex I (EC 1.6.5.3) and catalase (EC 1.11.1.6) activity. We used a HEPES/sucrose/mannitol/EGTA buffer in this procedure (Blemings et al. 1994). Recovery of mitochondria, with the use of GDH, ranged from 5 to 25%.

Enzyme assays.  Assays for GDH, LDH and aconitase were done with the use of previously frozen cytosol or mitochondria and have been described previously (Chen et al. 1997). Activity of the heme protein catalase was measured spectrophotometrically in previously frozen homogenates (Beers and Sizer 1952). Activity of complex I of the mitochondrial electron transport chain was measured spectrophotometrically in sonicated previously frozen mitochondria using ubiquinone-1 (generously supplied by Hoffman LaRoche, Basel, Switzerland) as the electron acceptor. The method of Finel et al. (1992) was used except that the NADH concentration was raised to 0.25 mmol/L and the ubiquinone-1 concentration was varied from 5 to 80 µmol/L to determine the apparent K_m (K_m) and V_max (V_max) for complex I activity from each rat. Ubiquinone-1 stock solutions were prepared in ethanol and stored at 20°C. The ubiquinone-1 concentration of the stock solution was determined spectrophotometrically after reduction with potassium borohydride (Crane et al. 1959). For d 0 through 11, insufficient liver tissue was available to determine complex I activity.

Gel electrophoresis and Western blot analysis.  Tissue ferritin and m-acon abundance were determined by SDS-PAGE followed by Western blot analysis using rabbit polyclonal antibodies raised against rat liver ferritin and bovine heart m-acon, respectively (Chen et al. 1997). A representative immunoblot appears in Chen et al. (1997).

Oxygen consumption by isolated mitochondria.  Oxygen consumption was measured in freshly isolated mitochondria using a Clark electrode (Gilson Medical Electronics, Middleton, WI) (Greenawalt 1974). Succinate (10 mmol/L) with rotenone (5 µmol/L) or pyruvate (10 mmol/L) with malate (1 mmol/L) were used as substrates. The use of pyruvate and malate as substrates permits electrons to flow into the electron transport chain through complex I and complex II. When succinate is used with rotenone, electrons are donated to complex II through the action of succinate dehydrogenase and activity of complex I is inhibited by rotenone. The maximal rate of oxygen consumption was measured with 0.1 mmol/L of the uncoupler 2,4-dinitrophenol (DNP). The oxygraph was calibrated by using the xanthine oxidase method (Billiar et al. 1970).

RNA binding activity assay.  IRE RNA binding activity was determined in previously frozen cytosol by electrophoretic mobility shift assay (EMSA) by using a [³²P]labeled RNA of the first 73 nucleotides of the rat L-ferritin 5UTR (Chen et al. 1997). Free and protein bound [³²P] RNA bands were cut from gels and quantified by scintillation counting (Chen et al. 1997).

Statistical analysis.  All values are reported as means ± SEM. ANOVA procedures appropriate for a two-factor design were used. The main effects of day and diet were analyzed by ANOVA. In the event of a significant F-test, Tukey's honestly significant difference test for multiple comparisons was used to determine significant differences across the three groups at each time point (Snedecor and Cochran 1980). When comparing the effect of diet over the 29-d experiment, the least significant difference test was used (Snedecor and Cochran 1980). Statistical analysis was performed using Minitab (release 10.51 Xtra, Minitab, State College, PA) and SAS (SAS version 6.11, SAS Institute, Cary, NC). Pearson correlation coefficients (r) were determined using Minitab. Differences were considered significant at P < 0.05.

	RESULTS

Abstract Introduction Methods Results Discussion References

Growth, liver weight and blood hemoglobin.  The time-dependent effect of diet on weight gain, blood hemoglobin and liver weight was examined (Fig. 1). No differences in weight gain were detected among the three groups for the first 7 d of the experimental period (Fig. 1A). Compared with group C, the body weight of rats from groups PF and ID was significantly lower by d 8 and 10, respectively. Over the 29-d experiment, rats in group ID showed no significant difference in weight gain compared with group PF.

Liver weight, expressed as a percentage of body weight, on d 0 and 1, was 5.4-5.6 g/100 g in all three groups of rats (Fig. 1B). However, by d 2 and thereafter, the relative liver size among rats from group ID was significantly lower than the size of the liver from rats in group C (Fig. 1B). A similar difference was noted between group PF and group C starting on d 4. By d 21 and 28, relative liver size among rats from group ID and PF varied between 3.8 and 4.2 g/100 g body weight, whereas rats from group C had a liver weight that was between 4.9 and 5.3 g/100 g body weight (Fig. 1B). Over the entire experimental period, a significant effect of diet on relative liver size was apparent between group C and group ID or PF but not between groups ID and PF (Table 2).

Blood hemoglobin (Hb) concentration ranged from 82 g/L on d 1 to 121 g/L on d 21 in rats from group C (Fig. 1C). Hb concentration in group PF was not different than that of group C on all days of the study (Fig. 1C; Table 2). In contrast, rats from group ID exhibited a time-dependent decline in blood Hb level, starting on d 2, that was significant compared with that of group PF (Fig. 1C). By d 7, group ID blood Hb was significantly lower than that of group C. Blood Hb concentration continued to decline in rats from group ID such that at d 28 it was 42 ± 3 g/L, which was 35% of the concentration in group C (121 ± 9 g/L) and 38% of the concentration in group PF (111 ± 11 g/L). For the 29-d experiment, there was a significant effect of diet on the Hb concentrations between groups PF and ID (Table 2).

Time-dependent effect of dietary iron intake on hepatic IRP1 and IRP2 RNA binding activity.  One of our aims was to determined how rapidly IRPs would be activated in liver when rats were fed an iron-deficient diet. For group C, hepatic IRP1 RNA binding activity was 0.061 ± 0.004 pmol/mg protein on d 0 (Fig. 2A). Hepatic IRP1 RNA binding activity declined by ~30% in group C up to d 11. Similar results were obtained for rats in group PF (Fig. 2A). In contrast, rats fed the iron-deficient diet exhibited a time-dependent increase in hepatic IRP1 RNA binding activity for the first 4 d of the experiment; at that point, the activity reached a plateau (Fig. 2A). After rats had received only one meal of the iron-deficient diet, the hepatic IRP1 activity was significantly greater in group ID compared with group C (Fig. 2A). Compared with rats from group PF, hepatic IRP1 RNA binding activity was significantly higher in group ID starting on d 2 (Fig. 2A). Iron deficiency ultimately caused a three-fold activation of hepatic IRP1 RNA binding activity compared with rats fed the control diet in an unrestricted (group C) or pair-fed (group PF) manner. When comparing the effect of diet over the 29-d experiment, the mean RNA binding activity of IRP1 was significantly higher in group ID compared with either group C or PF, whereas groups C and PF were not significantly different from each other (Table 2).

Hepatic IRP2 displayed a similar but not identical response to iron deficiency compared with IRP1 (Fig. 2B). Compared with the d 0 value, the RNA binding activity of IRP2 declined in the control (by 30%) and pair-fed (by 50%) groups during the 29-d experimental period. IRP2 RNA binding activity increased significantly and progressively starting on d 2 until d 7 in liver of rats from group ID as opposed to the IRP2 RNA binding activity in groups PF or C (Fig. 2B). For IRP2, iron deficiency resulted in a maximal activation of 3.2- and 5.2-fold compared with the amount of hepatic IRP2 RNA binding activity present in rats from groups C or PF, respectively (Fig. 2B). Over the entire experimental period, we found a significant effect of diet when comparing the means of the IRP2 RNA binding activity in group ID with those of groups PF and C (Table 2). There was a small difference in IRP2 RNA binding activity between groups PF and C (Table 2).

For rats fed the iron-deficient diet, total IRE binding activity (IRP1 + IRP2) in liver increased significantly starting at d 2 compared with group PF (Fig. 2C). Compared with the value on d 0, the mean total hepatic IRE binding activity in group ID increased progressively, reaching the maximal level at d 7, declining slightly on d 11 and remaining unchanged thereafter (Fig. 2C). For d 7 through 28, total IRE binding activity was about ~fourfold higher in rats fed the iron-deficient diet compared with group C or PF. When considered over the entire experimental period, there was a large effect of dietary iron content and a less extensive effect of food intake on total IRP activity (Table 2).

We also measured the amount of 2-mercaptoethanol (2-ME) inducible IRE binding activity. For IRP1, high levels of 2-ME convert the c-acon form of the protein, which binds RNA with low affinity, into a high affinity RNA binding form. The 2-ME induction assay provides a measure of the total amount of IRP1 protein present (Hentze et al. 1989). Starting at d 7 and proceeding through d 28, the hepatic 2-ME inducible IRE binding activity was significantly greater in group ID rats than in group PF (Fig. 3). Thus, even in severe iron deficiency, the amount of 2-ME inducible IRE binding activity remained relatively unchanged, whereas the level of activity observed in groups PF and C declined (Fig. 3). There was a small but significant effect of diet on the mean value of 2-ME inducible IRE binding activity over the 29-d experimental period (Table 2).

View larger version (23K): [in this window] [in a new window]   Fig 3. Time-dependent effect of dietary iron intake on 2-mercaptoethanol (2-ME) induced iron responsive element (IRE) binding activity in rat liver cytosol. The same samples of liver cytosol as used in Figure 2 were used for determination of 2-ME inducible IRE RNA binding activity. As noted in the text, the 2-ME induction assay provides a measure of the total amount of iron regulatory protein (IRP)1. Each point represents the mean ± SEM (n = 4) except on d 21 where n = 2 for group ID and d 28 where n = 3 for group PF. At time points at which n = 4, the honestly significant difference (HSD) value equals 1.20, whereas for d 21 HSD equals 1.70 and for d 28 HSD equals 1.39. The RNA binding activity was determined by electrophoretic mobility shift assay as described in Materials and Methods.

Time-dependent effect of iron deficiency on cytosolic and mitochondrial aconitase activity in liver.  Iron deficiency had a differential effect on the cytosolic and mitochondrial aconitases and appeared to alter their proportional contribution to total cellular aconitase activity. When expressed as units per gram of liver, c-acon comprised 61% and m-acon 39%, respectively, of total cellular aconitase activity in rats from group C on d 0 as measured under V_max conditions (compare Figs. 4A and 4C). Iron deficiency had no effect on liver c-acon activity when expressed as units per gram of liver (Fig. 4A; Table 2). On d 0, the activity of c-acon was 1.72 ± 0.04 unit/g liver, whereas by d 28 the activity of this enzyme was 1.94 ± 0.05 units/g liver in rats of group ID. When expressed as units per milligram protein, similar results were obtained for the effect of diet on c-acon activity with the exception that for d 11 and 21, the activity in group ID was greater than that in group PF (Fig. 4B). Because, as noted above, high levels of 2-ME convert the c-acon form of IRP1 into a high affinity RNA binding form that can be measured by EMSA, any changes in 2-ME inducible RNA binding activity should be reflected in the level of c-acon enzyme activity. The effect of iron intake on c-acon activity was similar to the effect of dietary iron on 2-ME inducible IRE binding activity with the exception of d 28. Overall, iron deficiency had little effect on c-acon activity or 2-ME inducible IRP activity (Table 2).

View larger version (28K): [in this window] [in a new window]   Fig 4. Time-dependent effect of dietary iron intake on the activity of cytosolic (c-acon) and mitochondrial aconitase (m-acon) in rat liver. Aconitase activity was determined in liver cytosol (panels A and B) or mitochondria (panels C and D) of rats fed the iron-deficient or control diets as described in Figure 1. Aconitase activity was determined by the coupled assay of aconitase and isocitrate dehydrogenase as described (Chen et al. 1997). One unit of enzyme activity is defined as 1 µmol of NADP+ reduced per minute. Quantitative data are the mean ± SEM (n = 4). Data are expressed as units/g liver to facilitate comparison of the contributions of the two aconitase isoforms to total cellular aconitase activity (panels A and C) or as units/mg protein (panels B and D) to facilitate comparison of the c-acon data with the 2-mercaptoethanol (2-ME) inducible iron regulatory protein (IRP) activity data. For all panels, each point represents the mean ± SEM (n = 4) except on d 28 where n = 3 for group PF. For panel A, there was no effect of treatment. For panel B, the honestly significant difference (HSD) value, represented by the vertical bar, equals 0.0041 for all time points except for d 28 where HSD equals 0.0047. For panel C, the HSD value was 0.26 for all time points except for d 28 where HSD equals 0.30. For panel D, the HSD value was 0.0028 for all time points except for d 28 where the HSD value was 0.0033.

Compared with c-acon, iron deficiency had a much larger effect on the activity of m-acon. Rats from group C displayed no significant change in m-acon activity (units/g liver) over the 29-d experiment (Fig. 4C). In contrast, rats from group ID displayed a progressive decline in m-acon activity (units/g liver) that was first apparent at d 4 (Fig. 4C), although on d 2, m-acon activity was significantly lower in group ID compared with group PF when expressed as units per milligram protein (Fig. 4D). Mitochondrial aconitase activity steadily declined up to d 11, after that, it reached a plateau (Fig. 4C). By d 28, m-acon accounted for 25% of the total cellular aconitase activity in group ID, which was lower than the 41% value for group C on d 28. In contrast to group ID or C, rats in group PF exhibited an increase in liver m-acon activity (Fig. 4C). By d 28, m-acon accounted for 47% of total cellular aconitase activity in group PF. A significant effect of diet on the mean m-acon activity was found for the entire experimental period (Table 2).

Steady-state level of m-acon and ferritin in rat liver.  Consumption of the iron-deficient diet resulted in a time-dependent decrease in the abundance of m-acon protein that reached a plateau at d 11 (Fig. 5A). The decline in m-acon abundance in iron-deficient rats (Fig. 5A) resembled the decline in m-acon activity (Fig. 4D) with the exception of d 7 when there appeared to be a transient increase in m-acon abundance. For group ID, the abundance of m-acon was significantly less than that in group C by d 4, whereas groups PF and ID became significantly different by d 7. Compared with group C, the abundance of m-acon in group ID declined by 30-40% and compared with group PF, the amount of m-acon protein differed by 50% by d 28. The average mean abundance of m-acon in group PF was higher than that in group C on d 7 through 28 and this difference was significant on d 21 and 28 (P < 0.05). There was an effect of diet on m-acon abundance over the entire 29-d experiment (Table 2). Overall, our results indicate that in iron-deficient rats, the reduction in m-acon activity was due largely to loss of m-acon protein as opposed to loss of the iron-sulfur cluster coupled with preservation of m-acon protein.

View larger version (25K): [in this window] [in a new window]   Fig 5. Time-dependent effect of dietary iron intake on the abundance of ferritin and mitochondrial aconitase (m-acon) in rat liver. The relative amount of ferritin or absolute amount of m-acon was determined by immunoblotting using antibodies against rat liver ferritin or bovine heart m-acon as described (Chen et al. 1997). Panel A shows the abundance of m-acon in liver mitochondria from rats in control (C), pair-fed (PF) and iron-deficient groups (ID). A standard curve of pure bovine heart m-acon was used on every blot to determine the absolute amount of m-acon protein present. For both panels, each point represents the mean ± SEM (n = 4) except on d 28 where n = 3 for group PF. For panel A, the honestly significant difference (HSD) value was 0.19 for all time points except for d 28 where the HSD value was 0.22. Panel B shows the relative abundance of ferritin in liver homogenate from rats in groups C, PF and ID. Fifty micrograms of homogenate protein was used to determine the relative abundance of ferritin protein. The HSD value was 0.46 for all time points except for d 28 where the HSD value was 0.53.

The abundance of both ferritin subunits was measured by immunoblotting and found to be strongly and rapidly affected by dietary iron intake (Fig. 5B). On d 2, the abundance of ferritin subunits in liver of rats fed the iron-deficient diet was significantly less than the ferritin subunit level in liver of rats in group PF (Fig. 5B). By d 4, ferritin subunits were no longer detectable in liver of the iron-deficient rats (Fig. 5B). In contrast, rats fed the control diet in an unrestricted or pair-fed manner exhibited a strong and significant rise in ferritin subunit abundance until about d 11, after which the level of ferritin subunits failed to increase further (Fig. 5B). The average mean level of ferritin subunits tended to be higher in the liver of rats pair-fed the control diet compared with the rats given free access to the control diet. Taken together, our results demonstrate a rapid effect of dietary iron intake on ferritin subunit abundance in rat liver. Over the 29-d experiment, there was a strong effect of iron intake and a less strong effect of food intake on ferritin subunit abundance (Table 2).

Indices of mitochondrial function.  We wished to determine whether certain other iron-containing proteins in liver were affected by iron deficiency, particularly those involved in mitochondrial respiration. Because ATP production by mitochondria requires the action of numerous iron-containing proteins, we assessed mitochondrial function by determining the maximal capacity of mitochondria to consume oxygen in the presence of different substrates. We used pyruvate and malate as substrates, which permits electrons to enter the electron transport chain through complex I and complex II. Succinate and rotenone were used to measure complex II activity. The maximal capacity for oxygen consumption was obtained by performing measurements in the presence of the uncoupler DNP.

Over the 29-d experimental period, the mean maximal rate of oxygen consumption in the presence of malate and pyruvate ranged from 1.5 to 1.7 µmol/(min·g liver) for the three treatment groups and was not affected by diet (Table 2). When oxygen consumption was measured in the presence of succinate with rotenone present, the rate varied between 4.5 and 5.8 µmol/(min·g liver) and there was a small effect of diet over the entire experimental period (Table 2). Although there was a significant effect of diet on the mean maximal rate oxygen consumption with succinate as a substrate, we feel it is of limited importance because it was variable on a day to day basis (results not shown).

We directly measured the activity of complex I in sonicated mitochondria with the use of ubiquinone-1 as the electron acceptor. Because complex I contains the NADH dehydrogenase component, which itself contains 11 different iron-sulfur centers, we felt that it was a good candidate for examining the effects of iron deficiency on the abundance of mitochondrial iron-sulfur proteins. Because of limitations in the amount of tissue available, we were able to measure complex I activity only on d 21 and 28. On these days, we found no effect of iron intake on the V_max or K_m values for complex I (Table 2).

Catalase activity.  We measured activity of the peroxisomal heme protein catalase as another indicator of the status of iron-containing enzymes. Liver catalase activity increased progressively in all three groups of rats during the course of the experiment (Fig. 6). Other investigators have observed a similar developmental change in liver catalase activity (Knox 1972, Mertens-Strijthagen et al. 1979). No significant effect of diet on catalase activity was observed at any of the time points examined (Fig. 6, Table 2). The lack of effect of dietary iron intake on catalase activity was similar to the failure of dietary iron to greatly affect the activity of c-acon and complex I or the rate of mitochondrial oxygen consumption in the presence of pyruvate and malate.

View larger version (20K): [in this window] [in a new window]   Fig 6. Time-dependent effect of dietary iron intake on the activity of catalase in rat liver. Catalase activity was determined in liver homogenates of rats fed the iron-deficient or control diets as described in Figure 1. The units used here are based on the change in µmol/min through a midpoint absorbance of 0.5. Quantitative data are the means ± SEM (n = 4). There are no significant differences among the three groups.

Time-dependent relationship between cytosolic IRE binding activity and m-acon, ferritin and blood hemoglobin level.  We examined the relationship between total liver cytosolic IRE RNA binding activity and liver m-acon abundance, ferritin abundance and blood hemoglobin level. The results for the iron-deficient group are shown because this more easily permits visualization of the relationship between the length of time the rats had been fed the iron-deficient diet and the individual measurement of interest (Fig. 7). There was a significant inverse linear relationship between total (IRP1 + IRP2) liver cytosolic RNA binding activity and liver m-acon abundance (Fig. 7, panel A, r = 0.653, P < 0.0001), liver ferritin subunit level (Fig. 7, panel B, r = 0.612, P < 0.001) and blood hemoglobin (Fig. 7, panel C, r = 0.614, P < 0.001). Similar results were obtained when data from all three groups were analyzed simultaneously (results not shown). When the relationship between liver cytosolic IRE binding activity and m-acon abundance was examined in groups C and PF, no significant relationship was found (results not shown). However, there were significant inverse relationships between IRP activity and ferritin abundance in group C (r = 0.567, P = 0.002) and group PF (r = 0.765, P < 0.0001) and between IRP activity and blood Hb in group PF (r = 0.666, P < 0.0001) (results not shown).

View larger version (19K): [in this window] [in a new window]   Fig 7. Relationships of iron regulatory protein (IRP) activities to liver mitochondrial-aconitase, blood hemoglobin and liver ferritin abundance obtained from rats fed the iron-deficient diet from d 0 through 28. The correlation coefficient (r) for panels A, B and C was 0.653, 0.612 and 0.614, respectively. All correlations were significant [P < 0.0001 (panel A) or 0.001 (panels B and C)].

	DISCUSSION

Abstract Introduction Methods Results Discussion References

This study was designed to further elucidate the extent to which dietary iron intake affects hepatic IRP function as well as the physiologic purposes of modulating m-acon abundance. First, we wanted to determine how rapidly liver IRPs responded to dietary iron deficiency. Second, we wanted to define the relationship between IRP activation in response to iron deficiency and alterations in the abundance of ferritin subunits and m-acon. Third, we compared m-acon abundance with the activity of other iron-containing mitochondrial or cytoplasmic proteins or protein complexes in response to dietary iron deficiency. This permitted us to determine whether the diet-dependent alteration of m-acon abundance was a selective or general effect of dietary iron intake on iron-containing proteins in liver. Our approach also allowed us to address whether the decrease in m-acon abundance was associated with changes in mitochondrial function and hence capacity to generate ATP.

We observed a rapid activation of the RNA binding activity of hepatic IRP1 and IRP2 in response to consumption of an iron-deficient diet. On d 1, IRP1 RNA binding activity was increased in rats from group ID and by d 2, the activities of both IRPs were stimulated. Our observations demonstrate that because IRPs respond rapidly to dietary iron they can serve as a key link between dietary iron intake and the storage of iron in liver.

The alterations we observed in IRP RNA binding activity were associated with changes in the abundance of ferritin and m-acon that are clearly consistent with these proteins being targets of IRP action (Chen et al. 1997). In rats fed the control diet, ferritin subunit level increased progressively throughout the study with the most rapid increase occurring in the first several days of the experiment. Others have noted similar findings with regard to the rise in liver ferritin iron in the first weeks after weaning (Linder et al. 1972). We found that the increase in ferritin subunit abundance in the liver of rats fed the control diet was associated with a small time-dependent decrease in IRP RNA binding activity, which was most apparent in group PF. We have previously noted that small changes in IRE binding activity were associated with relatively large changes in the steady-state level of ferritin, suggesting a threshold effect of IRPs on ferritin accumulation (Chen et al. 1997). In rats ingesting the iron-deficient diet, the significant increase in IRP RNA binding activity was associated with a complete loss of detectable ferritin subunits within 4 d. This repression of ferritin subunit expression in iron deficiency contrasted strongly with the time-dependent increase in the iron storage protein in rats from groups PF and C. Thus the iron content of the diet can have rapid and extensive effects on IRP activity as well as on the abundance of ferritin subunits in liver.

The abundance of m-acon also declined in iron deficiency, but the extent and kinetics of its regulation differed from that observed for ferritin subunits. We observed a clear time-dependent effect of iron intake on IRP RNA binding activity that had a strong inverse correlation with m-acon abundance. The alterations in IRP RNA binding and m-acon protein level induced by iron deficiency are consistent with a cause and effect type of relationship between these proteins. When comparing IRP RNA binding activity from rats in group ID with those from group PF, an increase in group ID was first apparent on d 2, whereas m-acon abundance was not significantly reduced until d 7, although on d 4 the value for group ID was less than group C. The continued increase in IRP RNA binding activity that occurred up to d 7 was associated with a further decline in m-acon abundance up to d 11. These results imply that activation of IRPs leads to a decline in m-acon abundance and also show that iron deficiency causes a differential degree of repression of ferritin and m-acon proteins.

Our results provide further support that the IRE in m-acon mRNA is functional in vivo and that IRPs act selectively in modulating the translation of IRE-containing mRNAs. Previous investigators have demonstrated that IRP modulate m-acon mRNA translation in a cell-free protein synthesis system (Gray et al. 1996, Kim et al. 1996). More recently, we have demonstrated that m-acon synthesis is iron-regulated in a number of mammalian cell lines and that iron acts at the translational level in HL60 cells to stimulate m-acon biosynthesis (Schalinske et al. 1998). Thus, there is strong evidence that m-acon mRNA is an in vivo target of IRP. However, our previous (Chen et al. 1997) and current study demonstrated a clear differential dose- and time-dependent effect of diet on m-acon and ferritin abundance. Our results, coupled with those of others, indicate that the differential effect of iron status on m-acon and ferritin abundance is likely to occur as a result of variations in the ability of IRPs to repress translation of these IRE-containing mRNAs (Kim et al. 1996, Schalinske et al. 1998) and possibly to enhanced degradation of ferritin protein in response to iron deficiency (Munro and Drysdale 1970).

Perhaps most importantly, our results demonstrate that dietary iron deficiency leads to relatively rapid and selective changes in liver IRP activity and in two targets of IRP action, ferritin and m-acon abundance. In contrast to m-acon and ferritin, the activity of other iron-containing proteins or protein complexes we examined such as catalase, c-acon and complex I of the electron transport chain was unaffected by iron intake. Numerous reports indicate that, in liver, there is little effect of iron deficiency on various iron-protein components of the mitochondrial pathways used for ATP production or on iron-containing proteins other than ferritin. In fact, liver proteins that require iron for their function (i.e., catalase, cytochrome P450 and SDH) are refractory to the effects of iron deficiency (Beutler and Blaisdell 1958, Cusack and Brown 1965, Dallman 1986, Dhur et al. 1989, Rao and Jagadeesan 1996). Iron deficiency does lead to altered morphology of liver mitochondria (Dallman and Goodman 1971) yet there appears to be no difference in mitochondrial function at least until the animal has been severely anemic for periods of time longer than that used in our study (Bailey-Wood et al. 1975, Massini et al. 1994). Subcellular fractions from liver of severely anemic rats displayed no change in succinate dehydrogenase activity or oxygen consumption in the presence of succinate (Bailey-Wood et al. 1975, Beutler and Blaisdell 1960, Evans and Mackler 1985). We observed a small reduction in oxygen consumption in the presence of succinate but no change when pyruvate and malate were substrates. Over our 29-d experiment, we observed clear variability among the three groups of rats with regard to the rate of oxygen consumption when succinate was the substrate (results not shown); others have noted similar variability (Beutler and Blaisdell 1960). Considered together, most reports find no significant perturbations in the in vitro energy-related functions of liver mitochondria from iron-deficient animals, and these observations are supported by direct measurements of the liver content of Fe-S proteins (Ohira et al. 1982). We conclude that in liver, the primary and initial effect of iron deficiency is to alter the activity of IRPs and the abundance of proteins encoded by IRE-containing mRNAs.

Several hypotheses have been advanced to explain why m-acon mRNA contains an IRE. These have included suggestions that IRPs may regulate flux through the TCA cycle to affect ATP production and/or mitochondrial generation of oxygen radicals or other toxic oxidants (Gray et al. 1996) and that the presence of an IRE in m-acon mRNA is an indicator of a general role for IRPs in regulating the synthesis of multiple Fe-S proteins (Kim et al. 1996). Our results indicate that, in liver, modulation of m-acon abundance by dietary iron is not related to the capacity of mitochondria to consume oxygen and by inference to generate ATP. Hence, it would appear that the alterations in m-acon activity we observed are not of a sufficient magnitude to alter TCA cycle function as it appears to do in other tissues (Costello et al. 1995). In this regard, using iron deficiency as a model, it also appears unlikely that m-acon is down-regulated, at least in liver, to reduce oxidative stress. A number of components of the cellular antioxidant defense system are not altered by iron deficiency, and indicators of radical-induced oxidative damage are reduced in iron deficiency (Rao and Jagadeesan 1996). However, alterations in cytoplasmic citrate level, perhaps induced by changes in m-acon abundance, may well allosterically influence the activity of enzymes involved in fatty acid formation (acetyl CoA carboxylase) and/or glucose oxidation (phosphofructokinase). Citrate may also contribute to the changes in interorgan interrelationships of liver and muscle with respect to carbohydrate metabolism that have been observed in chronic iron deficiency (Azevendo et al. 1989, Henderson et al. 1986, Klempa et al. 1989). Finally, it is apparent that there is no generalized decrease in Fe-S proteins in liver of iron-deficient animals despite the large increase in IRP activity. This finding argues against the suggestion that IRPs modulate the synthesis of Fe-S proteins in general and suggest that in mammalian liver, the Fe-S protein m-acon is uniquely regulated in this regard.

Our results suggest that IRPs modulate m-acon abundance and activity as a means to alter the use of citrate in cellular processes related to iron metabolism. This may include a role for this organic acid, or its metabolite(s), in iron metabolism such as the interaction of iron with ferritin or transferrin (Aisen and Leibman 1968). There is evidence supporting a role for citrate in iron metabolism, including the observations that citrate is effective at mobilizing iron from hepatocytes (Baker et al. 1981), ferric citrate accumulates in serum in iron overload (Grootveld et al. 1989), and ferric citrate transporters exist in procaryotic and possibly eucaryotic systems (deSilva et al. 1996). In this regard, it is relevant to note that little is known at the cellular or molecular level how ferritin iron is mobilized and released to extracellular apo-Tf in iron deficiency or other situations, and what compounds mediate this directed movement of iron (Young and Aisen 1981). To address the possibility that IRP-dependent changes in m-acon synthesis affect iron trafficking and/or release, or perhaps other cellular processes, our current efforts are aimed at defining how the changes in m-acon abundance influence cellular metabolism of citrate and related organic acids.

	ACKNOWLEDGMENTS

We thank Dan Steffen for excellent technical assistance and Melissa K. Schultz of the CALS Statistical Consulting service for excellent advice on statistical analyses.

	FOOTNOTES

Manuscript received 23 September 1997. Initial reviews completed 29 October 1997. Revision accepted 26 November 1997.

	LITERATURE CITED

Abstract Introduction Methods Results Discussion References

• Aisen P., Leibman A. Citrate-mediated exchange of Fe³⁺ among transferrin molecules. Biochem. Biophys. Res. Commun. 1968; 32:220-226 [Medline]
• American Institute of Nutrition Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J. Nutr. 1977; 107:1340-1348
• Azevedo J. L., Willis W. T., Turcotte I. P., Rovner A. S., Dallman P. R., Brooks G. A. Reciprocal changes of muscle oxidases and liver enzymes with recovery from iron deficiency. Am. J. Physiol. 1989; 256:E401-E405 [Abstract/Free Full Text]
• Bailey-Wood R., Blayney L. M., Muir J. R., Jacobs A. The effects of iron deficiency on rat liver enzymes. Br. J. Exp. Pathol 1975; 56:193-198 [Medline]
• Baker E., Vicary F. R., Huehns E. R. Iron release from isolated hepatocytes. Br. J. Haematol. 1981; 47:493-504 [Medline]
• Beard J. L., Dawson H., Pinero D. J. Iron metabolism: a comprehensive review. Nutr. Rev. 1996; 54:295-317 [Medline]
• Beers R. F., Sizer I. W. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem 1952; 195:133-140 ^{[Free Full Text]}
• Beutler E., Blaisdell R. K. Iron enzymes in iron deficiency. III. Catalase in rat red cells and liver with some further observations on cytochrome. C. J. Lab. Clin. Med. 1958; 52:694-699
• Beutler E., Blaisdell R. K. Iron enzymes in iron deficiency. V. Succinic dehydrogenase in rat liver, kidney and heart. Blood 1960; 15:30-35 ^{[Abstract/Free Full Text]}
• Billiar R. B., Knappenberger M., Little B. Xanthine oxidase for calibration of the oxygen electrode apparatus. Anal. Biochem. 1970; 36:101-104 [Medline]
• Blemings K. P., Crenshaw T. D., Swick R. W., Benevenga N. J. Lysine -ketoglutarate reductase and saccharopine dehydrogenase are located only in the mitochondrial matrix in rat liver. J. Nutr. 1994; 124:1215-1224
• Chen O. S., Schalinske K. L., Eisenstein R. S. Dietary iron intake modulates the activity of iron regulatory proteins and the abundance of ferritin and mitochondrial aconitase in rat liver. J. Nutr. 1997; 127:238-248 [Abstract/Free Full Text]
• Costello L. C., Liu Y., Franklin R. B. Testosterone stimulates the biosynthesis of m-aconitase and citrate oxidation in prostate epithelial cells. Mol. Cell. Endocrinol. 1995; 112:45-51 [Medline]
• Crane F. L., Lester R. L., Widmer C., Hatefi Y. Studies on the electron transport system. XVIII. Isolation of coenzyme Q (Q275) from beef heart and beef heart mitochondria. Biochim. Biophys. Acta 1959; 32:73-79
• Cusack R. P., Brown W. D. Iron deficiency in rats: changes in body and organ weights, plasma proteins, hemoglobins, myoglobins and catalase. J. Nutr. 1965; 86:383-393 ^[Medline]
• Dallman P. R. Biochemical basis for the manifestations of iron deficiency. Annu. Rev. Nutr. 1986; 6:13-40 [Medline]
• Dallman P. R., Goodman J. R. The effects of iron deficiency on the hepatocyte: a biochemical and ultrastructural study. J. Cell Biol. 1971; 48:79-90 [Abstract/Free Full Text]
• Dandekar T., Stripecke R., Gray N. K., Goosen B., Constable A., Hohansson H. E., Hentze M. W. Identification of a novel IRE in murine and human eALAS mRNA. EMBO J. 1991; 10:1903-1909 [Medline]
• deSilva D. M., Askwith C. C., Kaplan J. Molecular mechanisms of iron uptake in eukaryotes. Physiol. Rev. 1996; 76:31-47 ^{[Abstract/Free Full Text]}
• Dhur A., Galan P., Hercberg S. Effects of different degrees of iron deficiency on cytochrome P450 complex and pentose phosphate pathway dehydrogenases in the rat. J. Nutr. 1989; 119:40-47
• Eisenstein, R. S., Kennedy, M. C. & Beinert, H. (1997) The iron responsive element (IRE), the iron regulatory protein (IRP) and cytosolic aconitase: posttranscriptional regulation of mammalian iron metabolism. In: Metal Ions and Gene Regulation (Silver, S. & Walden, W., eds.), pp. 157-216. Chapman and Hall, New York, NY.
• Evans T. C., Mackler B. Effect of iron deficiency on energy conservation in rat liver and skeletal muscle submitochondrial particles. Biochem. Med. 1985; 34:93-99 [Medline]
• Finel M., Skehel J. M., Albracht S.P.J., Fearnley I. M., Walker J. E. Resolution of NADH:ubiquinone oxidoreductase from bovine heart mitochondria into two subcomplexes, one of which contains the redox center of the enzyme. Biochemistry 1992; 31:11425-11434 [Medline]
• Gray N. K., Pantopoulos K., Dandekar T., Ackrell B., Hentze M. W. Translational regulation of mammalian and drosophila citric acid cycle enzymes via iron-responsive elements. Proc. Natl. Acad. Sci. U.S.A. 1996; 93:4925-4930 [Abstract/Free Full Text]
• Greenawalt J. W. The isolation of outer and inner mitochondrial membranes. Methods Enzymol. 1974; 31:310-323 [Medline]
• Grootveld M., Bell J. D., Halliwell B., Aruoma O. I., Bomford A., Sadler P. J. Non-transferrin-bound iron in plasma or serum from patients with idiopathic hemochromatosis. J. Biol. Chem 1989; 264:4417-4422 [Abstract/Free Full Text]
• Gunshin H., Mackenzie B., Berger U. V., Gunshin Y., Romero M. F., Boron W. F., Nussberger S., Gollan J. L., Hediger M. A. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature (Lond.) 1997; 388:482-488 [Medline]
• Guo B., Phillips J. D., Yu Y., Leibold E. A. Iron regulates the intracellular degradation of iron regulatory protein 2 by the proteasome. J. Biol. Chem 1995; 270:21645-21651 [Abstract/Free Full Text]
• Henderson S. A., Dallman P. R., Brooks G. A. Glucose turnover and oxidation are increased in iron-deficient anemic rats. Am. J. Physiol. 1986; 250:E414-E421 [Abstract/Free Full Text]
• Hentze M. W., Kuhn L. C. Molecular control of vertebrate iron metabolism: mRNA-based circuits operated by iron, nitric oxide, and oxidative stress. Proc. Natl. Acad. Sci. U.S.A. 1996; 93:8175-8182 [Abstract/Free Full Text]
• Hentze, M. W., Rouault, T. A., Harford, J. B. & Klausner, R. D. (1989) Oxidation-reduction and the molecular mechanism of a regulatory RNA-protein interaction. Science (Washington, DC) 244: 357-359.
• Iwai K., Klausner R. D., Rouault T. A. Requirements for iron-regulated degradation of the RNA binding protein, iron regulatory protein 2. EMBO J. 1995; 21:5350-5357
• Kim H.-Y., LaVaute T., Iwai K., Klausner R. D., Rouault T. A. Identification of a conserved and functional iron-responsive element in the 5-untranslated region of mammalian mitochondrial aconitase. J. Biol. Chem 1996; 271:24226-24230 [Abstract/Free Full Text]
• Klempa K. L., Willis W. T., Chengson R., Dallman P. R., Brooks G. A. Iron deficiency decreases gluconeogenesis in isolated rat hepatocytes. J. Appl. Physiol. 1989; 67:1868-1872 ^{[Abstract/Free Full Text]}
• Knox, W. E. (1972) Enzyme Patterns in Fetal, Adult and Neoplastic Rat Tissues. S. Karger, Basel, Switzerland.
• Linder M. C., Moor J. R., Scott L. E., Munro H. N. Prenatal and postnatal changes in the content and species of ferritin in rat liver. Biochem. J. 1972; 129:455-462 [Medline]
• Masini A., Salvioli G., Cremonesi P., Botti B., Gallesi D., Ceccarelli D. Dietary iron deficiency in the rat. I. Abnormalities in energy metabolism of the hepatic tissue. Biochim. Biophys. Acta 1994; 1188:46-52 [Medline]
• Mertens-Strijthagen J., Schrijver C. D., Coninck S.W.-D., Wattiaux R. A centrifugation study of rat-liver mitochondria, lysosomes and peroxisomes during the perinatal period. Eur. J. Biochem. 1979; 98:339-352 [Medline]
• Munro H. N., Drysdale J. W. Role of iron in the regulation of ferritin metabolism. Fed. Proc. 1970; 29:1469-1473 [Medline]
• National Research Council (1995) Nutrient Requirements of Laboratory Animals, 4th rev. ed. National Academy Press, Washington, DC.
• Ohira Y., Hegenauer J., Strause L., Chen C.-S., Saltman P., Beinert H. Mitochondrial NADH dehydrogenase in iron-deficient and iron-repleted rat muscle: an EPR and work performance study. Br. J. Haematol. 1982; 52:623-630 [Medline]
• Rao J., Jagadeesan V. Lipid peroxidation and activities of antioxidant enzymes in iron deficiency and effect of carcinogen feeding. Free Radical Biol. Med. 1996; 21:103-108 [Medline]
• Rouault T. A., Klausner R. D. Post-transcriptional regulation of genes of iron metabolism in mammalian cells. J. Biol. Inorg. Chem. 1996; 1:494-499
• Schalinske, K. L., Chen O. S. & Eisenstein, R. S. (1998) Iron differentially stimulates translation of mitochondrial aconitase and ferritin mRNAs in mammalian cells. J. Biol. Chem. 273: (in press).
• Snedecor, G. W. & Cochran, W. G. (1980) Statistical Methods, 7th ed. The Iowa State University Press, Ames, IA.
• Theil E. C. Iron regulatory elements (IREs): a family of mRNA non-coding sequences. Biochem. J. 1994; 304:1-11
• Young S. P., Aisen P. Transferrin receptors and the uptake and release of iron by isolated hepatocytes. Hepatology 1981; 1:114-119 [Medline]
• Zheng L., Kennedy M. C., Blondin G. A., Beinert H., Zalkin H. Binding of cytosolic aconitase to the iron responsive element of porcine mitochondrial aconitase mRNA. Arch. Biochem. Biophys. 1992; 299:356-360 [Medline]

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