Copper Transport and Disease: What Can We Learn from Organoids?

Abstract

Many metals have biological functions and play important roles in human health. Copper (Cu) is an essential metal that supports normal cellular physiology. Significant research efforts have focused on identifying the molecules and pathways involved in dietary Cu uptake in the digestive tract. The lack of an adequate in vitro model for assessing Cu transport processes in the gut has led to contradictory data and gaps in our understanding of the mechanisms involved in dietary Cu acquisition. The recent development of organoid technology has provided a tractable model system for assessing the detailed mechanistic processes involved in Cu utilization and transport in the context of nutrition. Enteroid (intestinal epithelial organoid)-based studies have identified new links between intestinal Cu metabolism and dietary fat processing. Evidence for a metabolic coupling between the dietary uptake of Cu and uptake of fat (which were previously thought to be independent) is a new and exciting finding that highlights the utility of these three-dimensional primary culture systems. This review has three goals: (a) to critically discuss the roles of key Cu transport enzymes in dietary Cu uptake; (b) to assess the use, utility, and limitations of organoid technology in research into nutritional Cu transport and Cu-based diseases; and (c) to highlight emerging connections between nutritional Cu homeostasis and fat metabolism.

Keywords: ATP7B; Wilson disease; copper; enteroid; fat; intestine; nutrition; organoid.

Figure 1

Model of dietary copper (Cu) acquisition and storage in human absorptive epithelium. Dietary Cu is in the Cu²⁺ oxidation state. Enzymes at the brush border reduce Cu²⁺ to Cu⁺ prior to importation into the cell (80). Reduced Cu is bound by Cu chaperones. Atox1 is a Cu chaperone that delivers Cu to ATP7A and ATP7B (30). Under low-Cu conditions, ATP7A resides in the trans-Golgi network (78). When intracellular Cu loads are high, ATP7A traffics to the basolateral membrane and transports Cu⁺ into portal circulation. ATP7B sequesters Cu in vesicles, presumably to buffer cytosolic Cu levels or deliver Cu to yet-to-be-identified Cu-dependent enzymes located in the vesicles.

Figure 2

Expression and distribution of ATP7A, ATP7B, and CTR1 in murine small intestine. Duodenal intestinal tissues from 2-week-old sex-matched mice were used to assess the expression and intracellular distribution of the key players in intestinal copper (Cu) homeostasis. (a) In wild-type tissues, ATP7A (probed with anti-ATP7A sc376467, Santa Cruz Biotechnology) showed strong basolateral staining consistent with previous reports. ATP7B (probed with anti-ATP7b ab124973, Abcam) had a strong vesicular pattern, and CTR1 (probed with 8G11-F10, hybridoma monoclonal antibody; H. Pierson, H. Yang & S. Lutsenko, unpublished data) was detected specifically at the basolateral membrane. F-actin (blue) marks the apical cell border. (b) Tissues with intestine-specific deletion of CTR1 in enterocytes (Int-CTR1^−/−) specifically lack CTR1 expression in the intestinal epithelium and show no basolateral staining of CTR1 in the epithelial layer. However, sparse epithelial cells did show CTR1 expression in the apical membrane in the context of the Int-CTR1^−/− tissues (yellow arrows). All scale bars: 25 μm.

Figure 3

Three-dimensional (3D) enteroids: sophisticated model systems for investigating intestinal epithelium. (a) Schematic of an enteroid. Small crypt-like projections decorate the surface of the enteroid. The apical cell surface faces the lumen, analogous to the morphology of the digestive tract. Panel a adapted with permission from Reference . (b) Bright-field image of a single, live differentiated mouse enteroid in culture. (c) 3D projection of a single differentiated mouse enteroid with the apical membranes stained for F-actin demonstrates apical–inward polarity.