INTRODUCTION

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IRON IS THE TRACE MINERAL that has been studied most extensively with respect to exercise. It has been reported that strenuous exercise may adversely affect iron metabolism and contribute to low-iron status characterized by reduced blood hemoglobin, serum iron, and ferritin concentration in both animals and human athletes (5, 24, 33, 36, 40). However, it is still unknown whether the observed low-iron status indicates a true iron deficiency or a false impression of the iron-deficient status (24, 40). In addition, the mechanism by which strenuous exercise produces this change in iron status is not well determined. Several explanations for this low-iron status induced by exercise have been suggested, such as intravascular hemolysis due to the increased mechanical destruction of red blood cells; significant increase in plasma volume relative to red cell mass (blood dilution); negative iron balance between gain and loss (increased iron loss in urine, sweat, and feces and decreased intestinal iron absorption); iron redistribution from the reticuloendothelial system to the hepatocytes due to hemolysis; or suppression of erythropoiesis due to the decrease in erythropoietin synthesis (6, 24, 37, 40). However, these explanations do not appear to be valid, at least in some cases. For example, it was reported that mean serum ferritin decreased to 32 µg/l in elite male distance runners (10). However, median serum ferritin for adult men is 94 µg/l (7). If this low plasma ferritin is due to an expanded plasma volume, then this would require a threefold expansion of the plasma volume, which is highly unlikely (10). Magazanik et al. (22) also reported that the decrease in serum iron levels of subjects after the second week of training cannot be related to plasma volume expansion.

Until now, however, almost all research on the relationship between exercise and iron status has been focused on observations of the changes in the iron balance between gain and loss. The data generated were mainly obtained from the measurement of tissue iron and hematologic iron indexes (5, 8, 10, 22, 24, 27, 33, 34, 36, 39, 40), and little is known about the effect of strenuous exercise on the mechanism of iron uptake by some important cells in iron utilization and metabolism, such as bone marrow cells.

Therefore, it is necessary to investigate further the effect of strenuous exercise on iron metabolism, including studies at cellular and/or molecular levels to elucidate the precise mechanisms of strenuous-exercise-induced low-iron status. As in other iron-utilizing cells, the mechanism of iron uptake by the bone marrow cells also involves the processes of transferrin-bound iron and non-transferrin-bound iron uptake. It is generally believed that transferrin and transferrin-receptor-mediated iron delivery is the main route for cellular iron accumulation in vivo. This process is initiated with the binding of transferrin-iron to receptors on the cell membrane, and then transferrin-iron enters into the cell by endocytosis of the receptor-transferrin-iron complex (30, 31). The number of transferrin receptors on cell membrane is an important factor affecting the ability of cells to take up iron from transferrin (12). By controlling the level of transferrin-receptor expression, cells can determine the amount of iron acquired. Transferrin-receptor expression, in turn, is regulated by intracellular iron level. When the intracellular iron level falls, the transferrin-receptor level rises.

Bone marrow cells are the major iron-transporting and -utilizing cells in the body. Hence, this cell type was used in the present study to investigate the effect of strenuous exercise on the expression of transferrin receptor and the mechanism of transferrin-receptor-mediated iron uptake in rats.

	METHODS

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Materials. ⁵⁹Fe (FeCl₃, 5 µCi/µg) and [¹²⁵I]Na (carrier free) were purchased from Radiochemical Center (Amersham). Rat apotransferrin, bovine serum albumin, HEPES, Sephadex G50, hemoglobin assay kit, dibutyl phthalate, and Histopaque were obtained from Sigma Chemical (St. Louis, MO). Transferrin was labeled with ¹²⁵I and ⁵⁹Fe as previously described (29). Pronase was purchased from Boehringer-Mannheim (Mannheim, Germany).

Animals and exercise protocol. Female Sprague-Dawley rats (weighing 190 ± 20 g), supplied by the Animal House of The Hong Kong Polytechnic University, were housed in pairs in stainless steel rust-free cages at 21 ± 2°C, relative humidity of 60-65%, with 12:12-h dark-light periods. After being kept under the standard laboratory conditions for 1 wk, the animals were randomly assigned to either an exercise (n = 8) or sedentary control group (n = 6). Laboratory rodent diet for rats (PMI Nutrition, the Richmond Standard) and distilled water were provided ad libitum throughout the experimental period. Swimming was performed by a modification of the method of Ruckman and Sherman (34) and Prasad and Pratt (27).

Blood, tissue samples, and preparation of bone marrow mononuclear cells. At the end of the 3-mo experiment, animals were fasted for 24 h after the last exercise regimen and then heparinized and anesthetized with diethyl ether. Fasting blood samples were collected after death, and aliquots were taken immediately for hemoglobin, iron, and hematocrit determination. The liver, spleen, kidney, heart, and adrenal gland were removed, weighed, and stored in a freezing chamber at 70°C for subsequent determination of tissue nonheme iron concentrations. The bone marrow cells were isolated from both femurs and tibiae by rapidly splitting scraped bones and were suspended in ice-cold saline buffered with 20 mM HEPES (pH 7.4) at 4°C. The crude cell suspension was passed through two layers of nylon mesh (16). The mononuclear cells were obtained by Histopaque density gradient sedimentation. The cells were then washed three times with ice-cold Hanks' balanced salt solution (HBSS) buffered with 20 mM HEPES, pH 7.4, containing 2% BSA. Subsequently, the cells were suspended in the same buffer to a final concentration of 1 × 10⁸ cells/ml. The cell suspension was kept at 4°C for no longer than 3 h before use. It was found that ~1.8 × 10⁸ mononuclear cells were obtained in each rat. To determine the percentage of erythroblasts in cell suspensions, cell counts were carried out on dried cell smears stained with May-Grunwald-Giemsa stains.

Transferrin-binding assay. Binding assay was performed as described by Muta et al. (23). Briefly, 2 × 10⁵ mononuclear cells were incubated with labeled transferrin in various amounts, ranging from 0.12 to 70 nM in a total incubation volume of 0.1 ml HBSS buffered with HEPES (pH 7.4) containing 2 mg/ml of BSA at 4°C for 60 min. For nonspecific binding, 7 µM of unlabeled ferric transferrin were added. All assays were done in triplicate. At the end of the incubation period, the cell samples were placed on top of 120 µl of dibutyl phthalate-toluene, 4.5:1, in a 400-µl Eppendorf centrifuge tube and then centrifuged at 5,000 g for 1 min at 4°C. The bottom of the tube containing the cell pellet was cut off, and radioactivity was counted by a gamma counter. The transferrin-binding data were transformed to Scatchard plots. The number of transferrin receptors per cell and the apparent dissociation constants were calculated from Scatchard plot analysis.

Transferrin-mediated iron uptake. The cell suspensions were prewarmed in a shaking water bath for 10 min at 37°C. Labeled transferrin (⁵⁹Fe, ¹²⁵I) was then added at a final concentration of 0.5 µM in a total volume of 3.2 ml of HBSS containing 2 mg/ml BSA. After the desired incubation periods, 100 µl of incubation suspension containing 2 × 10⁶ mononuclear cells were removed. The cellular transferrin and iron uptakes were immediately stopped by diluting the cell suspension with 40 vol of ice-cold PBS (16). After being washed three times with 2 ml of cold PBS, the cells were incubated with 200 µl of Pronase (1 mg/ml) in ice-cold PBS for 30 min at 4°C. This led to the release of receptor-bound transferrin on the outer cell membrane and allowed separation of membrane-bound and intracellular radioactive transferrin and iron. For the measurement of transferrin and iron in the cytosolic and stromal fractions, the cell suspension was washed three times with ice-cold saline buffered to pH 7.4 with 20 mM HEPES, transferred to a new tube, and hemolyzed with buffer-detergent solution (Tris-buffered saline, pH 7.2, containing 0.1% Triton X-200). The cytosolic and stromal fractions were then separated by a centrifuge as described earlier (28). Each fraction was counted for radioactivity.

Analytic methods. Cell counts were made by using a hemocytometer. Hemoglobin concentration was determined by cyanmethemoglobin method (4), and hematocrit was measured by the microhematocrit centrifuge. Tissue nonheme iron concentrations were measured according to Kaldor (17). Plasma iron and total iron-binding capacity were determined by using commercial kits (Sigma Chemical).

	RESULTS

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Effect of strenuous exercise on body and organ weights, hematologic indexes, and tissue nonheme iron concentrations. The average body weights and most of the organ weights in the two groups did not differ significantly at the end of the 3-mo period (Table 1). Heart weight in the exercised rats was significantly higher (P < 0.01) than that in the control animals. This result confirmed the observations in several studies using swimming for exercising rats (8, 15). It indicated that exercised rats might have an adaptation resulting in an increase in the capacity of the heart to deliver more blood to the muscle and other tissues. Liver and spleen weights were not affected by exercise, and this is in keeping with the observations of Gagne et al. (8) and Ruckman and Sherman (34).

Binding of [¹²⁵I]transferrin to erythroblasts. The bone marrow mononuclear cells were incubated with radioactive iodinated transferrin at 4°C, a temperature at which only receptor binding takes place on the cell surface (18, 19). Our preliminary experiments showed that saturation of specific transferrin-binding sites was found to occur at a transferrin concentration of 35 nM. This concentration was similar to that obtained previously from human erythroblasts (23). Scatchard analysis of the transferrin-binding data was performed to determine the apparent dissociation constant and the number of specific transferrin receptors per cell (Fig. 1). The apparent dissociation constants were 12.7 ± 0.27 nM in the exercised rats and 11.7 ± 1.10 nM in the sedentary rats. Neither constant differed significantly from the other (P > 0.05), indicating no effects of exercise on the binding affinity of receptors for transferrin.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   A: specific [125I]transferrin (Tf) binding to rat erythroblasts of strenuously exercised rats () and control animals (). Saturation concentration of binding is ~35 nM of transferrin. For assay condition, see METHODS. Symbols represent means of assays in triplicate. B: Scatchard plots of saturation results shown in A.

Time course of [¹²⁵I]transferrin internalization and receptor-mediated iron uptake. The time courses of internalization of labeled transferrin and receptor-mediated iron uptake in erythroblasts were observed in the exercised and sedentary rats. The cells were incubated with 0.5 µM of labeled transferrin (⁵⁹Fe, ¹²⁵I) for the various periods at 37°C. The approaches employed by Karin and Mintz (18) were used here to release cell-surface-bound ligand with Pronase digestion and to determine the internalization of transferrin. Transferrin internalization increased with the incubation time in a linear manner during the initial incubation period and then reached a steady state (Fig. 2). After 30 min of incubation, intracellular transferrin approached a level of 8.40 ± 1.94 fmol/10⁶ cells in the erythroblasts of exercised rats. This was significantly different from the control level of 2.70 ± 0.38 fmol/10⁶ cells (P < 0.05). At other time points (i.e., 5, 10, 20 min) it was also observed that transferrin endocytosis in the erythroblasts of exercised rats was significantly higher than that in the sedentary animals (Fig. 2A). This indicated that the rate of transferrin internalization in the exercised rats increased in proportion to the increase in transferrin-receptor numbers on the membrane of the cells. By contrast, no significant difference was found between the mean transferrin endocytotic cycle time in the exercised rats (2.91 ± 1.16 min) and that in the control animals (2.60 ± 0.49 min). The mean cycle time of transferrin was calculated from the following relationship as described previously (29): mean cycle time (min) = transferrin uptake (fmol/10⁶ cells)/iron uptake rate (fmol · 10⁶ cells¹ · min¹). This calculation is based on the assumption that each molecule of diferric transferrin, which is endocytosed by a cell, donates both iron atoms to the cell (29).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Time course of transferrin internalization (A) and transferrin-bound 59Fe uptake (B) in strenuously exercised (, n = 8) and control (, n = 6) rat erythroblasts. Cells were incubated with 0.5 µM transferrin (59Fe, 125I) in Hanks' balanced salt solution at 37°C. At indicated time points, cells were washed and treated as described in text. Values are means ± SE. Equations of regression lines in B: y = 4.66x + 53.03, r2 = 0.993 (top line); and y = 1.62x + 24.70, r2 = 0.699 (bottom line). * P < 0.05 vs. control.

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Changes in radioactive iron in cytosolic and stromal fractions. After entering into the cell, iron is transported into the mitochondria for heme synthesis or stored in ferritin. Therefore, we further investigated the subcellular distribution of radioactive iron in two fractions, cytosolic and stromal. The latter consists of outer cell membrane plus intracellular organelles including mitochondria. The cells were preincubated with Pronase, and this treatment has been shown to give maximal release of surface-bound transferrin and iron. Thus it is reasonable to believe that the majority of radioactive iron in the stromal fraction reflects the radioactivity incorporated into heme (38). Figure 3 shows the time course of iron accumulation in both fractions [stromal (A), cytosolic (B)]. In the erythroblasts of exercised rats, the rate of stromal iron accumulation was 4.22 fmol · 10⁶ cells¹ · min¹ after the incubation period. The corresponding value in the sedentary rats was 1.15 fmol · 10⁶ cells¹ · min¹. Comparison of the slops of regression lines showed that there was significant difference in iron accumulation rates in the two groups (P < 0.05). However, in the cytosolic fraction, the iron accumulation rate in the erythroblasts of exercised rats was 1.28 fmol · 10⁶ cells¹ · min¹ and that in the control cells was 0.63 fmol · 10⁶ cells¹ · min¹. No significant difference was found. These results might reflect a mechanism by which the increased transferrin-bound iron uptake, because of the increased transferrin receptor, may provide more iron for heme synthesis in the immature erythroid cells in the exercised rats.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Time course of radioactive iron accumulation in stromal (A) and cytosolic (B) fractions of erythroblasts in strenuously exercised rats (, n = 8) and control animals (, n = 6). After incubation, cells were fractionated and radioactivity was counted. Equations of regression lines in A and B, respectively: y = 2.73x + 93.75, r2 = 0.675 (top line), and y = 1.11x + 23.31, r2 = 0.788 (bottom line); y = 1.63x + 31.33, r2 = 0.925 (top line), and y = 0.67x + 16.78, r2 = 0.864 (bottom line). * P < 0.05 vs. control.

	DISCUSSION

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In the present study, we compared the numbers of specific transferrin receptors on the membrane of bone marrow erythroblasts in strenuously exercised and control rats. It was found that the average number of surface transferrin receptors on erythroblasts was significantly increased in the strenuously exercised rats, reaching a level of about twofold more than that in the control animals.

The results also showed that the rate of radioactive transferrin internalization increased in the erythroblasts of the strenuously exercised rats proportional to the increase in the number of surface transferrin receptors. The parallel significant increase in the rate of radioactive iron uptake by the erythroblasts of exercised rats was also observed. Scatchard analysis demonstrated that the affinity of transferrin receptor to transferrin was not affected by exercise because the apparent dissociation constants of transferrin receptors were not found to have be significant different between the strenuously exercised and the control rats. The mean recycling time of transferrin was ~2.5-3 min in the erythroblasts of either strenuously exercised or control rats. This value was in good agreement with the previous results in several different types of immature erythroid cells (12, 13, 15, 29). All of these results demonstrated clearly that this increased iron uptake by the cells in exercised rats resulted directly from the increased expression of transferrin receptor on the cellular membrane rather than the changes in affinity of transferrin receptor and transferrin recycling time.

Bridges and Cudkowicz (2) demonstrated that the intracellular low iron availability might result in a parallel increase in surface and intracellular transferrin receptors. This increase is due to the increased biosynthesis of new receptors. The molecular mechanism of intracellular iron homeostasis has recently been well characterized (9). The amount of iron acquired by mammalian cells is determined by the level of transferrin-receptor expression on the cellular membrane, whereas the level of transferrin-receptor expression is mainly controlled by intracellular iron at the posttranscriptional level. Recent studies have established the existence and structures of iron response elements (IREs) and of iron regulatory proteins [(IRPs) or IRE-binding proteins], which specifically bind and modify expression of the mRNAs of which the IREs are a part (1, 3, 20, 21). The IREs are sequences of RNA found in the 5'-untranslated regions of the mRNAs for ferritin and in the 3'-untranslated regions of the mRNAs for transferrin receptor. They are bound by IRPs that lack one of four possible iron atoms in a cubic iron sulfur cluster. When IRPs bind IREs tightly, they inhibit translation of mRNAs of ferritin and also inhibit the degradation of the mRNAs of transferrin receptor. Thus, under the conditions of cellular iron deficiency, cells make less ferritin, whereas the number of transferrin receptor is increased (1). Hence, iron deficiency is compensated for by the increased transferrin-receptor levels, permitting a cell to absorb more iron by endocytosis of transferrin. Apparently, like iron storage, iron uptake is adjusted by a feedback-control loop, in which intracellular iron controls its own size (11). The increased transferrin-receptor expression, found in the erythroblasts of strenuously exercised rats in this study, implied that strenuous exercise could lead to a decrease in intracellular iron level as well as an increase in cellular iron demand. The low intracellular iron level stimulates the expression of transferrin receptor on the cellular membrane, which, in turn, increases cellular iron uptake. Probably, the increased iron acquired by the cells is mainly transported to the mitochondria for heme synthesis. This possibility was supported by the finding on the subcellular distribution of iron in cytosolic and stromal fractions in strenuously exercised rats. In addition, the increased cellular iron accumulation might be one of the causes of the low plasma iron concentration found in the strenuously exercised rats.

Iron deficiency progresses in three stages: 1) iron stores in the bone marrow, liver, and spleen are depleted; 2) erythropoiesis diminishes as the iron supply to the erythroid marrow is reduced; and 3) hemoglobin production falls, resulting in anemia (40). Our findings did not support the viewpoint that strenuous exercise could produce a "sport anemia" because no significant difference was found in hemoglobin concentrations in the strenuously exercised and the control rats. In the study on effects of exercise on iron metabolism in rats, Strause et al. (36) obtained similar results. They found that strenuous exercise (swimming) did not reduce hemoglobin concentration but did increase iron absorption. However, our results did show that strenuous exercise could induce a low-iron status, indicated by a low plasma iron concentration, transferrin saturation, and the marked decrease in nonheme iron concentrations of the liver and spleen in addition to the reduced intracellular iron level and enhanced transferrin-receptor expression. These results were in agreement with some previous results observed by others (8, 27, 36). Prasad and Pratt (27) and Strause et al. (36) observed lower iron stores in the liver and spleen of exercising compared with sedentary rats. Gagne et al. (8) reported that serum iron and transferrin saturation were significantly lower in exercised animals. Because all the measurements in the present study were made at the end of the 3-mo experimental period rather than at the earlier stage of the exercise, changes in the above indexes, including the increased transferrin-receptor expression and iron accumulation, might not be transitory physiological adaptations. They probably represented the early signs (the first stage) of iron deficiency. However, it should be pointed out that it is premature to conclude from these results that strenuous exercise can lead to iron deficiency in rats. More investigations are needed.

Our study demonstrated that 3 mo of strenuous exercise could not result in a true iron deficiency or sport anemia. However, it is unknown whether it could be developed after longer periods (6 mo or more) of strenuous exercise and/or of greater exercise intensity (3 h of swimming or more per day). It has been well documented that exercise increases erythropoiesis and erythrocyte turnover (35, 39), namely, iron turnover, which was supported by our results. It is reasonable to believe that, if iron turnover increased above the available iron provided from the body's iron stores or the diet, true iron deficiency might occur. Exercise intensity and length of exercise period should be considered as two important factors in determining the magnitude of the increase in iron turnover. On the basis of these considerations, therefore, it is worthy to investigate further the effect of longer periods and/or greater intensity of strenuous exercise on transferrin-receptor expression and cellular iron accumulation.