Background & Aims

Wilson disease is a disorder of copper (Cu) misbalance caused by mutations in ATP7B. ATP7B is highly expressed in the liver—the major site of Cu accumulation in patients with Wilson disease. The intestine also expresses ATP7B, but little is known about the contribution of intestinal ATP7B to normal intestinal copper homeostasis or to Wilson disease manifestations. We characterized the role of ATP7B in mouse intestinal organoids and tissues.

Methods

We collected intestinal tissues from ATP7B-knockout (Atp7b^−/−) and control mice, and established 3-dimensional enteroids. Immunohistochemistry and x-ray fluorescence were used to characterize the distribution of ATP7B and Cu in tissues. Electron microscopy, histologic analyses, and immunoblotting were used to determine the effects of ATP7B loss. Enteroids derived from control and ATP7B-knockout mice were incubated with excess Cu or with Cu-chelating reagents; effects on cell fat content and ATP7B levels and localization were determined by fluorescent confocal microscopy.

Results

ATP7B maintains a Cu gradient along the duodenal crypt−villus axis and buffers Cu levels in the cytosol of enterocytes. These functions are mediated by rapid Cu-dependent enlargement of ATP7B-containing vesicles and increased levels of ATP7B. Intestines of Atp7b^−/− mice had reduced Cu storage pools in intestine, Cu depletion, accumulation of triglyceride-filled vesicles in enterocytes, mislocalization of apolipoprotein B, and loss of chylomicrons. In primary 3-dimensional enteroids, administration of excess Cu or Cu chelators impaired assembly of chylomicrons.

Conclusions

ATP7B regulates vesicular storage of Cu in mouse intestine. ATP7B buffers Cu levels in enterocytes to maintain a range necessary for formation of chylomicrons. Misbalance of Cu and lipid in the intestine could account for gastrointestinal manifestations of Wilson disease.

中文翻译：

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ATPase铜转运蛋白ATP7B在肠中的功能

背景与目标

威尔逊病是由ATP7B突变引起的铜（Cu）失衡疾病。ATP7B在肝脏中高表达，而肝脏是Wilson病患者中Cu积累的主要部位。肠也表达ATP7B，但对于肠ATP7B对正常肠内铜稳态或对Wilson疾病表现的贡献知之甚少。我们表征了ATP7B在小鼠肠类器官和组织中的作用。

方法

我们从ATP7B敲除（Atp7b ^-/-）和对照小鼠收集了肠道组织，并建立了3维小肠。免疫组织化学和X射线荧光用于表征组织中ATP7B和Cu的分布。使用电子显微镜，组织学分析和免疫印迹来确定ATP7B丢失的影响。将来自对照组和ATP7B敲除小鼠的类固醇与过量的Cu或与Cu螯合剂一起孵育；通过荧光共聚焦显微镜确定对细胞脂肪含量，ATP7B水平和定位的影响。

结果

ATP7B沿十二指肠隐窝-绒毛轴维持Cu梯度，并缓冲肠上皮细胞胞质中的Cu含量。这些功能是由含铜的ATP7B囊泡的快速铜依赖性增大和ATP7B的水平升高介导的。的肠ATP7B ^{/ - -}小鼠中肠，铜耗尽，在肠上皮细胞中甘油三酯的填充的囊泡的累积，载脂蛋白B的错误定位，和乳糜微粒的损失减少了铜的存储池。在主要的3维小肠类固醇中，过量使用Cu或Cu螯合剂会损害乳糜微粒的组装。

结论

ATP7B调节小鼠小肠中Cu的囊泡贮藏。ATP7B缓冲肠细胞中的铜水平，以维持形成乳糜微粒所需的范围。肠道中铜和脂质的失衡可能导致了威尔逊病的胃肠道表现。