This increase in hepatic copper in six weekold Commd1Dhep mice probably results from residual copper pools accumulated in the preweaning period. Dietary studies have not been reported in Bedlington terriers with the homozygous COMMD1 deletion, but since most commercial dog food contains copper levels that exceed the minimum recommended daily intake, together with the presented data, suggest that reducing the gastrointestinal copper uptake by decreasing the dietary copper content would be beneficial to the liver pathology of affected dogs. Although our mouse model partially recapitulates the copper CHIR-99021 abmole accumulation phenotype of Bedlington terriers affected with CT, the exact mode of COMMD1 action in regulating hepatic copper metabolism remains elusive. However, several assumptions can be drawn from our data. Similar to Bedlington terriers, hepatic Commd1 deficiency in mice does not affect the incorporation of copper into Cp by Atp7b. Importantly, probably due to the increased bioavailable hepatic copper, the biosynthesis of holoceruloplasmin was even enhanced in middle-aged Commd1Dhep mice fed a copper-enriched diet compared to controls. Together with the observation that the copper-induced trafficking of ATP7B to the cell periphery is unaffected in COMMD1-deficient cells, it is tempting to speculate that, in excess copper, COMMD1 acts downstream of ATP7B and might be involved in the final step of the secretory pathway to efficiently release copper into the bile. This idea is further supported by the fact that COMMD1 partly localizes to vesicles of the endocytic pathway and cellular membranes, and shows only limited co-localization with ATP7B in HepG2 cells. However, COMMD1 is also implicated in regulating the protein levels of ATP7B. Whereas we previously demonstrated that COMMD1 expression augments the protein degradation of ATP7B in vitro, others have shown a decline in Atp7b expression after depletion of Commd1 in the mouse hepatoma Hepa1-6 cells. In line with this latter observation, a marked decrease in hepatic Atp7b in six week-old Commd1Dhep mice was observed, and may account for the increased hepatic copper levels observed in these animals. However, no correlation was seen between the degree of copper accumulation and Atp7b levels in Commd1Dhep mice fed a copperenriched diet, which argues against the role of impaired Atp7b protein stability in progressive copper accumulation in Commd1- deficient hepatocytes. Additionally, no discrepancies in Atp7b stability in primary Commd1-deficient hepatocytes compared to WT control cells were seen. Altogether, our data indicate that COMMD1 controls hepatic copper homeostasis downstream of ATP7B and may participate in the release of copper into the bile. Further studies are however needed to complete our understanding on the molecular function of COMMD1 in hepatic copper homeostasis. Interestingly, although Commd1Dhep mice fed a copper-enriched diet displayed a progressive increase in hepatic copper, no obvious liver pathology using histological analysis were seen, even after chronic exposure to high dietary copper. These data, supported by biochemical parameters and together with the observation that the mRNA expression of the copper-responsive genes Mt-I and Mt-II was only increased.