Non‐CTR1 mechanisms of intestinal copper absorption determined with the Caco2 cell culture model
Authors: Linder Maria Christine , Ricarte Adrian , Kidane Theodros Z. , Corona Luis , Azenon Jonathan , Jaime Alexis 
Published: April 2020

Much is still unknown about how dietary copper is absorbed by the small intestine. The current view is that Cu(II) is reduced to Cu(I) by reductases in the brush border of enterocytes, and then taken up by copper transporter 1 (CTR1), the only as yet identified copper uptake transporter in mammals. Uptake of Cu(I) by CTR1 is inhibited by Ag(I) and can thus be used to assess the extent to which CTR1 contributes to copper uptake. Using Caco2 cell monolayers with tight junctions grown in bicameral chambers, we determined that, whether given as Cu(I) or Cu(II), only about one third of copper uptake was inhibited by an excess of Ag(I). So CTR1 is not the only copper uptake system in enterocytes. Our goals were thus to document the contributions of CTR1 and a potential chloride‐dependent process (reported by Zimnicka et al.
[1]) to copper absorption, and to explore whether cellular iron status or the ability to reduce Cu(II) impacts these processes. Caco2 cell monolayers were grown on collagen‐coated filters in Transwell plates until trans‐epithelial electrical resistance reached or exceeded 250 Ohms. Rates of Cu uptake were measured by following accumulation of radioactive Cu over 60 min in cells and the basal medium (equivalent to blood fluid) after application of 67Cu‐labeled Cu(II)‐nitrilotriacetate (5 uM) to the apical surface, and were calculated in terms of % dose/h/mg cell protein. Ascorbate (1 mM) was added to produce Cu(I). Control uptake medium contained all chloride salts. The contribution of CTR1 to uptake was measured by the fall in uptake rate due to 50–100 uM Ag(I). Cu(II) reduction was inhibited by an excess of Fe(III). Chloride dependent uptake was determined by the fall in uptake rate when sulfate was substituted for chloride. Effects of substituting other halide ions for chloride were also recorded. Cellular Fe status was varied by preincubating monolayers for 3 days with 5 uM ferric ammonium citrate, or depleting them with desferrioxamine. Levels of mRNA for CTR1, DMT1, and reductases (STEAPs 2 and 3) were determined by qPCR, relative to 18S rRNA. We found that excess Ag(I) decreased rates of Cu(I) and Cu(II) uptake about 30%. The same was true when sulfate was substituted for chloride. Silver and sulfate effects were additive. Substituting bromide for chloride made no difference, while iodide substitution doubled uptake rates. Fe pretreatment reduced expression of CTR1 and DMT1 mRNAs but had little effect on rates of copper uptake. Fe deficiency greatly enhanced expression not only of DMT1 but also of CTR1 mRNA, and increased rates of copper uptake as well. Inhibition of Cu/Fe reductases with excess Fe(III) did not reduce rates of Cu(II) uptake. These results indicate that at least in this intestinal model, CTR1 and a halide‐dependent uptake process each account for about one third of Cu uptake; that CTR1 expression is enhanced by iron deficiency – which would explain increased uptake of copper in that condition; and that copper can be absorbed not only in the Cu(I) state but also as Cu(II).