New role of copper transporter ATP7B in lipid metabolism of enterocytes

Mutation of the copper exporter ATP7B is the cause of Wilson's disease and is associated with toxic accumulation of copper (Cu) in different tissues. The role of ATP7B in the intestine is far from being understood.

Our aim is the analysis of an intestinal ATP7B KO cell line and the characterization of Cu/iron (Fe) and lipid metabolism.

The CRISPR/Cas9 system was applied to human intestinal CaCo-2 cells. Single cell cloning as well as bacterial cloning were performed for sequencing analysis. Toxicity was determined by MTT assay and determination of Cu by AAS analysis. Gene expression analysis (RT-qPCR) was performed. Lipid metabolism was assessed by EM, RT-qPCR, ELISA, triglyceride quantification and staining.

About 50% of the CaCo-2 cell clones subjected to CRISPR/Cas9 mediated ATP7B KO showed increased sensitivity to Cu. At 0.15 – 0.75 mM Cu, the sensitivity in the KO cells was significantly elevated. Knockin confirmed the role of ATP7B, whereas Fe showed no impairment of cell survival. DCYTB metal reductase (-4.9 ± 1), oxidative stress regulating HMOX1 (+14.8 ± 5) and metallothionein 1 (+56.2 ± 16) were significantly affected. A significant downregulation of the chylomicron structural protein ApoB48 (-3.5 ± 0.4) and APOE were observed in KO cells. Intracellular triglyceride levels decreased after Cu treatment.

The characterization of a novel intestinal ATP7B knockout cell line showed impairment of Cu homeostasis and lipid metabolism. The results extend our understanding of the lipid and Cu transport in enterocytes.