Deletion of hepatic Ctr1 reveals its function in copper acquisition and compensatory mechanisms for copper homeostasis

Abstract

Copper is a vital trace element required for normal growth and development of many organisms. To determine the roles for copper transporter 1 (Ctr1) in hepatic copper metabolism and the contribution of the liver to systemic copper homeostasis, we have generated and characterized mice in which Ctr1 is deleted specifically in the liver. These mice express less than 10% residual Ctr1 protein in the liver and exhibit a small but significant growth retardation, which disappears with age. Hepatic copper concentrations and the activities of copper-requiring enzymes are reduced; however, mild copper deficiency relative to Ctr1 protein deficit indicates compensatory mechanisms for copper metabolism. Copper concentrations of other organs did not alter despite the defect in hepatic copper uptake. Whereas biliary copper excretion is reduced, urinary copper concentration in these mice is higher than that of control mice. Our data indicate that Ctr1 plays a critical role in copper acquisition in the liver, and, when Ctr1 expression is compromised, compensatory mechanisms facilitate copper uptake and/or retention in the liver and excretion of copper via urine.

Fig. 1.

Deletion of Ctr1 in the liver. A: schematic depiction of chromosomal deletion of Ctr1 by recombination. Black triangles indicate loxp recombination sites that flank the four coding exons of Ctr1 (gray rectangles). A recombination event catalyzed by a Cre recombinase is indicated as dashed lines. P1, P2, and P3 indicate primers used for PCR genotyping of the loci. “X” indicates the intercross of mice possessing Ctr1^F/F or a Cre recombinase gene fused with the albumin gene promoter. B: PCR analysis of recombination. Genomic DNA samples extracted from the organs of mice at 2 mo of age were subjected to PCR using P1, P2, and P3 primers. The fragments of 241 bp (PCR product of P1/P2 primer) and 281 bp (PCR product of P1/P3 primer) reflect Ctr1^F and Ctr1 deletion (Δctr1), respectively. C: Northern blot analysis of Ctr1 transcripts. Total hepatic RNA isolated from 2 independent mice (1 male and 1 female) of each genotype was subjected to Northern blotting. A ³²P-labeled DNA probe specific to mouse Ctr1 was used to detect mouse Ctr1 mRNA. A GAPDH-specific probe was used as a loading control as shown in the bottom panel. D: determination of Ctr1 protein expression by immunoblotting. Membrane proteins extracted from the liver of 2-mo-old mice were subjected to Western blot analysis of Ctr1 using anti-Ctr1 antibodies. Transferrin receptor (TFR) was probed as a loading control.

Fig. 2.

Growth retardation of mice deleted for Ctr1 in the liver. A: body weights of Ctr1^F/F and Ctr1^F/FAlb-Cre mice were measured at 1, 2, 3, and 10 mo of age. B: relative wet weights of organs compared with total body weight were determined in mice at the age of 2 mo. Each bar represents the mean ± SD of the data of at least 10 male (M) or female (F) mice. *P < 0.05; **P < 0.005, Student's t-test.

Fig. 3.

Metal concentrations in the liver, blood, and serum. A portion of the large lob of the liver, whole blood, and serum were collected from 2-mo (female) and 10-mo-old (male) Ctr1^F/F and Ctr1^F/FAlb-Cre mice. Samples were digested in nitric acid, and then copper (Cu) (A), iron (Fe) (B), and zinc (Zn) (C) concentrations were measured by inductively coupled plasma mass spectrometry (ICP-MS). Each bar represents the mean ± SD of data obtained from at least 6 mice. *P < 0.005; **P < 0.001, Student's t-test.

Fig. 4.

Activities, copper incorporation, and expression levels of copper-dependent enzymes synthesized in the liver. Cu,Zn-superoxide dismutase (SOD1) (A) and cytochrome c oxidase (CCO) (B) activities were measured in cytosolic fractions and total protein extracts of the liver, respectively. Each bar represents the mean ± SD of 4 female mice of indicated genotypes at 2 mo of age. Immunoblots are shown below enzyme assay data. Tubulin was probed as a loading control. Data were compared with those of control mice (Ctr1^F/F) by Student's t-test. *P < 0.0001. C: determination of copper insertion into ceruloplasmin (Cp). Serum samples (2 μl) were subjected to Western blotting using anticeruloplasmin antibodies. To differentiate apo- and holoceruloplasmin, serum samples were diluted in a nonreducing SDS sample buffer at room temperature and then resolved by SDS-PAGE at 4°C (top). To probe total ceruloplasmin, serum samples were denatured in a SDS sample buffer containing DTT (100 mM) at 95°C for 5 min and then resolved by SDS-PAGE at room temperature (bottom).

Fig. 5.

Excretion of copper, iron, and zinc into bile and urine. Bile (A) and urine (B) samples of 2-mo and 10-mo-old mice were collected into acid-washed tubes, weighed, mixed with nitric acid, and then copper (Cu), iron (Fe), and zinc (Zn) levels were measured by ICP-MS. Each bar represents the mean ± SD of the data obtained from at least 6 mice. *P < 0.05; **P < 0.0001, Student's t-test.