Exploration of the copper-related compensatory response in the Belgrade rat model of genetic iron deficiency

Jiang, Lingli; Ranganathan, Perungavur N.; Lu, Yan; Kim, Changae; Collins, James F.

doi:10.1152/ajpgi.00261.2011

Cited by 16 publications

(21 citation statements)

References 54 publications

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“…At least one study has, however, shown that Dmt1 can transport cuprous copper (16). Moreover, rats expressing a mutant Dmt1 protein (Belgrade) (19), although iron deficient, were recently shown not to have alterations in hepatic or serum copper concentrations, whereas iron-deprived, phenotypically normal littermates did (consistent with previous studies in wild-type rats) (20). Given this background, our intent for this investigation was to test the hypothesis that Dmt1 can transport copper, particularly during iron deficiency.…”

Section: Introductionsupporting

confidence: 79%

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Divalent metal transporter 1 (Dmt1) Mediates Copper Transport in the Duodenum of Iron-Deficient Rats and When Overexpressed in Iron-Deprived HEK-293 Cells

Jiang

Garrick

et al. 2013

The Journal of Nutrition

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Intracellular copper-binding proteins (metallothionein I/II) and a copper exporter (Menkes copper-transporting ATPase) are upregulated in duodenal enterocytes from iron-deficient rats, consistent with copper accumulation in the intestinal mucosa. How copper enters enterocytes during iron deficiency is, however, not clear. Divalent metal transporter 1 (Dmt1), the predominant iron importer in the mammalian duodenum, also transports other metal ions, possibly including copper. Given this possibility and that Dmt1 expression is upregulated by iron deprivation, we sought to test the hypothesis that Dmt1 transports copper during iron deficiency. Two model systems were utilized: the Belgrade (b) rat, expressing mutant Dmt1, and an inducible Dmt1-overexpression cell culture system. Mutant rats (b/b) were fed a semipurified, AIN93G-based control diet and phenotypically normal littermates (+/b) were fed control or iron-deficient diets for ~14 wk. An everted gut sleeve technique and a colorimetric copper quantification assay were utilized to assess duodenal copper transport. The control diet-fed +/b rats had normal hematological parameters, whereas iron-deprived +/b and b/b rats were iron deficient and Dmt1 mRNA and protein levels increased. Importantly, duodenal copper transport was similar in the control +/b and b/b rats; however, it significantly increased (~4-fold) in the iron-deprived +/b rats. Additional experiments in Dmt1 overexpressing HEK-293 cells showed that copper ((64)Cu) uptake was stimulated (∼3-fold) in the presence of an iron chelator. Dmt1 transcript stabilization due to a 3' iron-responsive element was also documented, likely contributing to increased transport activity. In summary, these studies suggest that Dmt1 enhances copper uptake into duodenal enterocytes during iron deprivation.

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Section: Introductionsupporting

confidence: 79%

“…The upper small intestine and liver tissue were then collected for mRNA and protein analysis. In addition, hemoglobin and hematocrit were measured by standard methods (20) from blood obtained by cardiac puncture just before death. All animal studies were approved by the University of Florida Institutional Animal Care and Use Committee.…”

Section: Methodsmentioning

confidence: 99%

Divalent metal transporter 1 (Dmt1) Mediates Copper Transport in the Duodenum of Iron-Deficient Rats and When Overexpressed in Iron-Deprived HEK-293 Cells

Jiang

Garrick

et al. 2013

The Journal of Nutrition

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“…Enterocytes were isolated from duodenum and proximal jejunum of rats and mice as described (20). Approximately 15 cm of rat proximal intestine (∼12 cm for mice) was removed immediately after death and flushed with ice-cold PBS, cut into ∼5-cm lengths (∼4 cm for mice), and everted on individual wooden sticks.…”

Section: Methodsmentioning

confidence: 99%

“…Thirty micrograms of cytosolic or membrane proteins were resolved by SDS-7.5% PAGE, blotted, and immunoprobed as described earlier (21). The anti-Atp7a antibody (called "54-10") (20,22) and anti-ZnT1 (a kind gift from Robert Cousins, University of Florida, Gainesville, FL) (23) are well validated. Rabbit polyclonal anti-Heph Ab was from GeneTex (cat.…”

Section: Methodsmentioning

confidence: 99%

Discovery of a cytosolic/soluble ferroxidase in rodent enterocytes

Ranganathan

Fuqua

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Hephaestin (Heph), a membrane-bound multicopper ferroxidase (FOX) expressed in duodenal enterocytes, is required for optimal iron absorption. However, sex-linked anemia (sla) mice harboring a 194-amino acid deletion in the Heph protein are able to absorb dietary iron despite reduced expression and mislocalization of the mutant protein. Thus Heph may not be essential, and mice are able to compensate for the loss of its activity. The current studies were undertaken to search for undiscovered FOXs in rodent enterocytes. An experimental approach was developed to investigate intestinal FOXs in which separate membrane and cytosolic fractions were prepared and FOX activity was measured by a spectrophotometric transferrin-coupled assay. Unexpectedly, FOX activity was noted in membrane and cytosolic fractions of rat enterocytes. Different experimental approaches demonstrated that cytosolic FOX activity was not caused by contamination with membrane Heph or a method-induced artifact. Cytosolic FOX activity was abolished by SDS and heat (78°C), suggesting protein-mediated iron oxidation, and was also sensitive to Triton X-100. Furthermore, cytosolic FOX activity increased ∼30% in iron-deficient rats (compared with controls) but was unchanged in copper-deficient rats (in contrast to the reported dramatic reduction of Heph expression and activity during copper deficiency). Additional studies done in sla, Heph-knockout, and ceruloplasmin-knockout mice proved that cytosolic FOX activity could not be fully explained by Heph or ceruloplasmin. Therefore rodent enterocytes contain a previously undescribed soluble cytosolic FOX that may function in transepithelial iron transport and complement membranebound Heph.intestine | ferrozine | cyto-FOX

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“…Blood was collected by cardiac puncture, and hemoglobin and hematocrit were measured by routine methods (8). The duodenum was excised and inverted on a wooden stick after which enterocytes were isolated using a well established, previously published method (8,18). Duodenal enterocytes were used for mRNA isolation, Western blot analysis, and chromatin immunoprecipitation experiments.…”

Section: Methodsmentioning

confidence: 99%

Transcription Factors Sp1 and Hif2α Mediate Induction of the Copper-transporting ATPase (Atp7a) Gene in Intestinal Epithelial Cells during Hypoxia

Xie

Collins

2013

Journal of Biological Chemistry

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Exploration of the copper-related compensatory response in the Belgrade rat model of genetic iron deficiency

Abstract: Exploration of the copper-related compensatory response in the Belgrade rat model of genetic iron deficiency.

Cited by 16 publications

References 54 publications

Divalent metal transporter 1 (Dmt1) Mediates Copper Transport in the Duodenum of Iron-Deficient Rats and When Overexpressed in Iron-Deprived HEK-293 Cells

Divalent metal transporter 1 (Dmt1) Mediates Copper Transport in the Duodenum of Iron-Deficient Rats and When Overexpressed in Iron-Deprived HEK-293 Cells

Discovery of a cytosolic/soluble ferroxidase in rodent enterocytes

Transcription Factors Sp1 and Hif2α Mediate Induction of the Copper-transporting ATPase (Atp7a) Gene in Intestinal Epithelial Cells during Hypoxia

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