MECHANISMS CONTROLLING IRON MOBILIZATION DURING IRON DEFICIENCY IN RAT BRAIN
Open Access
- Author:
- Han, Jian
- Graduate Program:
- Nutrition
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 16, 2001
- Committee Members:
- John Elliot Beard, Committee Chair/Co-Chair
Jonathan R Day, Committee Member
Byron C Jones, Committee Member
James Robert Connor, Committee Member
Michael Henry Green, Committee Member - Keywords:
- mRNA
ferritin
transferrin receptor
brain
iron-related proteins
iron deficiency
rat
transferrin - Abstract:
- The effect of iron deficiency on brain iron metabolism has not yet been thoroughly studied. A number of investigators have shown that iron and iron-related proteins (ferritin, transferrin, and transferrin receptor) are not uniformly distributed in the brain. As shown in various research models, iron deficiency changes the distribution of various iron-related proteins to different extents. While the iron-related protein responses to iron deficiency have been described, the mRNA expression of these proteins in the brain has not been determined. The present studies were conducted to investigate the mRNA expression of iron-related proteins in the rodent brain and to elucidate the possible mechanisms underlying iron mobilization by these proteins during iron deficiency. The overall hypothesis is that iron deficiency induces changes in brain iron, iron-related proteins and their mRNA and that these changes are heterogeneously distributed across brain regions. These responses describe the adaptive mechanisms in place in the brain when experiencing dietary iron deprivation. Twenty-one day old male and female Sprague-Dawley rats were randomly assigned to either an iron-deficient (3.5 mg Fe/kg diet) or a control diet (35 mg Fe/kg diet) for six weeks. At the time of killing, iron status was verified. Ferritin, transferrin, and transferrin receptor proteins were determined by ELISA, and the mRNA content and distribution were determined by Northern blot, RT-PCR and in situ hybridization. Regression analysis was used to study the relationships among iron, iron-related proteins and their mRNA content in the brain. ANOVA was used to determine significant differences in brain regional mRNA contents, iron protein contents, and iron contents between control and iron-deficient groups. The first objective was to test the hypothesis that dietary iron deficiency affects the mRNA content of H and L ferritin in rat brain regions (i.e., hippocampus, substantia nigra, striatum, pons, cerebellum, cortex, and thalamus) which subsequently affects the content of H and L ferritin subunit proteins. Results showed that the H to L ferritin mRNA ratio was 6:1. The ratio was not affected by dietary iron deficiency in contrast to a different effect of iron deficiency on liver ferritin mRNA. Iron deficiency changed neither the mRNA location nor the mRNA content of H and L ferritin in brain regions. Iron deficiency reduced brain H ferritin protein concentration significantly in all regions, whereas it only reduced L ferritin concentration in striatum, substantia nigra, and pons. The fact that iron deficiency had no effect on H and L ferritin mRNA content in contrast to the effect on the protein content indicates the predominance of post-transcriptional regulation of ferritin production in brain. Importantly, the regional variation in ferritin gene expression at the transcriptional level was not strongly dependent on local regional iron status, which suggests other roles for ferritin in brain biology. The second objective of this research was to test the hypothesis that iron deficiency affects transferrin, transferrin receptor protein, and their mRNA contents in rat brain regions. The iron-deficient diet significantly decreased brain iron concentration (22% - 63%) and increased transferrin concentration (22% to 130%) in a regional specific fashion. Iron-deficient rats exhibited significantly increased transferrin receptor levels in thalamus (74%) and cortex (40%). In situ hybridization studies showed that transferrin and transferrin receptor mRNAs had complementary distributions in the brain. Transferrin mRNA was most pronounced in corpus callosum, pons, gray matter of the cerebellum, lateral ventricles, caudate putamen, and thalamus. In contrast, transferrin receptor mRNA was lowest in these regions and highest in cortex, hippocampus, and the white matter of the cerebellum. Transferrin mRNA content decreased from 20% to 50% in all brain regions except cerebellum, while regional transferrin receptor mRNA content did not change in all regions except striatum in iron deficiency. Regression analysis showed a lack of correlation between transferrin protein content and its mRNA content (r= 0.6, p<0.05), indicating that transferrin protein in some brain regions must be derived from other sources. The opposite effect of iron deficiency on brain and liver transferrin mRNA expression suggests that liver could be the source of transferrin protein in the brain during iron deficiency. This study suggests that transferrin and transferrin receptor respond to iron deficiency in a coordinated fashion, and one could imply that transferrin and transferrin receptor-mediated movement of iron in the brain is regulated by dietary iron status and transferrin uptake from the plasma. The third objective was to test the hypothesis that iron-related proteins and their mRNA are expressed in the same type of cells in both control and iron-deficient rat brains. The cellular distribution of iron, H and L ferritin, transferring, and transferrin receptor protein was determined by peroxidase anti-peroxidase immunohistochemistry. The H ferritin protein was found in neurons and oligodendrocytes, while the mRNA was found in neurons, ependymal cells in the ventricle area, and epithelial cells in the choroid plexus. The H ferritin protein in the oligodendrocytes may be related to iron transport in this type of cells. The L ferritin protein and iron were found in the same types of cells, indicating an important role of L ferritin in long-term iron storage. Iron deficiency did not change the types of cells containing H and L ferritin. Transferrin protein was found in oligodendrocytes, epithelial cells in choroid plexus, and neurons, while its mRNA was found in oligodendrocytes, ependymal cells in ventricles, and epithelial cells in choroid plexus. The data indicate that transferrin protein found in neurons may be translocated from other cells such as epithelial cells or ependymal cells which contain transferrin mRNA. Iron deficiency did not change the cell types containing transferrin and transferrin receptor at either protein or mRNA level. In summary, the results of these studies contribute the following findings to current understanding of brain iron metabolism. 1) The dynamics of the regulation of L ferritin and transferrin message differs greatly between the brain and liver. 2) The brain adapts to iron deficiency by increasing transferrin protein uptake from the plasma in contrast to increased de novo synthesis. 3) Ependymal cells in the ventricles and epithelial cells in the choroid plexus are important for iron uptake and the synthesis of iron-related proteins. 4) H and L ferritin not only play important roles in iron storage but also likely contribute to iron transport within the brain. Overall, this study helps to clarify the adaptive strategy the brain utilizes when experiencing an imbalance in iron requirements and iron supply.