Increases in serum and liver copper content are noted during iron deficiency in mammals, suggesting that copper-dependent processes participate during iron deprivation. One point of intersection between the 2 metals is the liver-derived, multicopper ferroxidase ceruloplasmin (Cp) that is important for iron release from certain tissues. The current study sought to explore Cp expression and activity during physiologic states in which hepatic copper loading occurs (eg, iron deficiency). Weanling rats were fed control or low iron diets containing low, normal, or high copper for ∼ 5 weeks, and parameters of iron homeostasis were measured. Liver copper increased in control and iron-deficient rats fed extra copper. Hepatic Cp mRNA levels did not change; however, serum Cp protein was higher during iron deprivation and with higher copper consumption. In-gel and spectrophotometric ferroxidase and amine oxidase assays demonstrated that Cp activity was enhanced when hepatic copper loading occurred. Interestingly, liver copper levels strongly correlated with Cp protein expression and activity. These observations support the possibility that liver copper loading increases metallation of the Cp protein, leading to increased production of the holo enzyme. Moreover, this phenomenon may play an important role in the compensatory response to maintain iron homeostasis during iron deficiency.

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

The Menkes copper ATPase (Atp7a) pumps copper into the trans-Golgi for cuproenzyme synthesis, and translocates to the basolateral membrane of enterocytes for copper export. Recent studies demonstrated that three 5’ end splice variants of the Atp7a transcript exist in rat duodenum, all of which are strongly induced during iron deprivation. To explore a possible role for Atp7a (and copper) in intestinal iron absorption, the current studies were undertaken to test the hypothesis that multiple Atp7a transcript and protein variants exist in intestinal epithelial cells. Northern blot analyses using probes generated from the full-length Atp7a cDNA revealed several specific hybridization bands, all of which were more intense in RNA samples extracted from duodenal enterocytes isolated from iron-deficient rats. A PCR-based approach, using forward primers specific for the alternative 5’ end splice variants and a reverse primer in exon 23, demonstrated that 3 full-length transcripts exist in rat IEC-6 cells. To identify possible Atp7a protein variants, three distinct polyclonal antisera were utilized. The specificity of the antisera was first established by western blotting and immunoprecipitation studies using samples derived from isolated rat enterocytes and Atp7a knockdown IEC-6 cells. Several specific immunoreactive bands were documented, and a unique Atp7a protein distribution in cytosolic vesicle-like structures was noted. In conclusion, multiple Atp7a transcript and protein variants exist in rodent intestinal epithelial cells and are induced by dietary iron deprivation. Further studies will be designed to determine the subcellular distribution of Atp7a protein variants and possible unique functions of each.

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