Abstract
Different mutations in the copper transporter gene Atp7b are identified as the primary cause of Wilson’s disease. These changes result in high copper concentrations especially in the liver and brain, and consequently lead to a dysfunction of these organs. The Atp7(−/−) mouse is an established animal model for Wilson’s disease and characterized by an abnormal copper accumulation, a low serum oxidase activity and an increased copper excretion in urine. Metallothionein (MT) proteins are low molecular weight metal-binding proteins and essential for the zinc homeostasis but also play a role for the regulation of other metals, e.g. copper. However the molecular mechanisms of MT in regard to Atp7b remain still elusive. In this study we investigate the expression of MT in the liver and duodenum of Atp7b(−/−) mice and wildtype mice. Hepatic and duodenal expression of MT was measured by real-time reverse transcription-polymerase chain reaction and post-translational expression was analyzed by immunoblot and immunofluorescence. Expression of MT in liver und duodenum was significantly higher in Atp7b(−/−) mice than in controls. Hepatic and duodenal copper, iron and zinc content were also studied. Compared to control hepatic copper and iron content was significantly higher while hepatic zinc content was significantly lower in Atp7b(−/−) mice. In the duodenum copper and zinc content of Atp7b(−/−) mice was significantly lower than in controls. Duodenal iron content was also lower in Atp7b(−/−) mice, but did not reach statistical significance. The results of our study suggest that metallothionein is elevated in the liver and duodenum of Atp7b(−/−) mice.
Highlights
Wilson’s disease is an autosomal recessively inherited disorder of copper metabolism, caused by mutations in the Atp7b gene on chromosome 13 (Bull et al 1993; Tanzi et al 1993)
Metallothionein is elevated in the liver and duodenum of Atp7b(-/-) mice
To determine whether metallothionein is involved in our previous findings we measured the hepatic metallothionein expression on mRNA level by means of quantitative real-time RT-PCR in relation to the expression levels of the housekeeping gene actin and we could show that the hepatic MT-1 expression of Atp7b(-/-) mice was more than 400-fold higher than in wildtype mice (356.8 ± 134.37 MT-1/actin ratio (9 10-1 vs. 0.84 ± 0.31 MT-1/actin ratio (9 10-1); P \ 0.001; n = 5 per group) (Fig. 1a)
Summary
Wilson’s disease is an autosomal recessively inherited disorder of copper metabolism, caused by mutations in the Atp7b gene on chromosome 13 (Bull et al 1993; Tanzi et al 1993). Established animal models for Wilson’s disease are the Long Evans Cinnamon (LEC) rat (Wu et al 1994), the toxic milk mouse (Theophilos et al 1996) and the Atp7b(-/-) mouse (Buiakova et al 1999; Huster et al 2006). All these animal models are characterized by hepatic copper accumulation and liver damage (Buiakova et al 1999; Okayasu et al 1992).
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