ATP7B knockout disturbs copper and lipid metabolism in Caco-2 cells

Intestinal cells control delivery of lipids to the body by adsorption, storage and secretion. Copper (Cu) is an important trace element and has been shown to modulate lipid metabolism. Mutation of the liver Cu exporter ATP7B is the cause of Wilson disease and is associated with Cu accumulation in different tissues. To determine the relationship of Cu and lipid homeostasis in intestinal cells, a CRISPR/Cas9 knockout of ATP7B (KO) was introduced in Caco-2 cells. KO cells showed increased sensitivity to Cu, elevated intracellular Cu storage, and induction of genes regulating oxidative stress. Chylomicron structural protein ApoB48 was significantly downregulated in KO cells by Cu. Apolipoproteins ApoA1, ApoC3 and ApoE were constitutively induced by loss of ATP7B. Formation of small sized lipid droplets (LDs) was enhanced by Cu, whereas large sized LDs were reduced. Cu reduced triglyceride (TG) storage and secretion. Exposure of KO cells to oleic acid (OA) resulted in enhanced TG storage. The findings suggest that Cu represses intestinal TG lipogenesis, while loss of ATP7B results in OA-induced TG storage.


Introduction
The absorption of lipids and essential trace elements, including copper (Cu), is predominantly mediated by specific cells of the small intestine. Dietary intake and processing of lipids has to be considered in metabolic diseases of Cu homeostasis, like Wilson disease (WD) and Menke disease (MD) [1,2]. Excess Cu is toxic and usually manifests with increased liver Cu load and Cu excretion. Low Cu is frequently associated with impairment of various biochemical processes and growth inhibition. The molecular mechanism that governs uptake and intracellular metabolism of Cu and lipids by intestinal cells is not fully understood. Infant rhesus monkeys revealed decreased Cu retention suggesting a reduced intestinal Cu absorption following Cu exposure [3]. MD patients suffer from Cu deficiency, caused by mutation of Cu transporter ATP7A, a ubiquitously expressed Cu exporter, resulting in deficiency of Cu in the body due to Cu accumulation in the enterocyte. In contrast, WD patients are characterized by Cu overload of various tissues, prominently liver and brain, due to mutation of Cu transporter ATP7B [4]. High accumulation of Cu in the liver is followed by increased oxidative stress (e.g. HMOX1, MT1, and SOD1), mitochondrial damage and reduced ceruloplasmin (CP) release [5,6]. In liver, Cu is mainly imported by Cu transporter 1 (CTR1) and divalent metal transporter 1 (DMT1), although a minor role of multidrug resistance protein (MDR1) was reported [7]. A CTR1-mediated uptake of intestinal Cu was shown in mice [8]. Cu inside the cell is distributed to other cell compartments, like mitochondria or via ATOX1 to the trans-Golgi-network (TGN). At the TGN, ATP7B provides Cu for incorporation into enzymes, e.g. CP and hephaestin (HEPH) in the intestine. Metallothionein (MT) is a low molecular metal-binding protein that is essential for the intracellular homeostasis of various metals resulting in cytoprotection and detoxification [9]. Excess Cu affects the intracellular localization of ATP7B and leads to vesicular storage or ultimately to excretion via bile [10]. ATP7A delivers Cu to lysyl oxidase, tyrosinase and peptidyl alpha-monooxygenase. In the enterocyte, ATP7A is commonly believed to represent the main Cu exporter, leading to the release of Cu into the blood for whole body distribution. Absence of ATP7A was shown to increase the intracellular accumulation of Cu in intestinal cells [11]. ATP7B is also expressed in enterocytes [12], however it´s functional role in human intestinal cells is largely unexplored and most evidence was previously derived from WD animal models. Lower Cu concentrations were observed in duodenal tissue of Atp7b -/mice as compared to wildtype suggesting that functional loss of ATP7B results in decreased uptake/storage [13,14]. Pierson et al. suggested that Atp7b mostly resides in the vesicular compartment suggesting a role in the cytosolic storage of Cu in intestinal cells rather than in the export of Cu as observed in the liver. Furthermore, a crosslink of iron and Cu homeostasis was previously suggested [15]. Cu loading of cells was implicated to affect lipid metabolism. Low hepatic Cu levels were correlated with liver steatosis in nonalcoholic fatty liver disease (NAFLD) [16]. In contrast, WD patients revealed normal serum triglyceride (TG) and cholesterol levels, but a reduction of cholesterol after hepatic manifestation [17]. Of note, studies of WD animal models revealed changes of TG and cholesterol levels in liver and intestine [14,[18][19][20]. For enterocytes of Atp7b -/mice, an impact of ATP7B on the chylomicron production was recently suggested [14]. High dietary fat increases the chylomicron production of enterocytes, which transport TGs into lymph and blood [21]. The synthesis of lipoproteins in the intestine, e.g. chylomicrons, VLDL, and HDL, depends on the availability of specific lipids, structural apolipoproteins (e.g. ApoB48 and ApoE), and export supporting proteins, like ABCA1. Cu was proposed to interfere with several processes of lipid metabolism; however the determination of the Cu impact needs further work.
The purpose of our study was the generation of a human intestinal ATP7B KO cell line to study the interrelation of Cu and lipid metabolism at the level of the enterocyte.

Cell culture
The human epithelial colorectal adenocarcinoma cell line Caco-2 was received from American Type Culture Collection (ATCC, Manassas, VA, USA). Caco-2 cell lines were grown in DMEM High Glucose (GE Healthcare, Chicago, IL, USA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and 100 U/mL penicillin/streptomycin (Hyclone, Logan, UT, USA). For differentiation, 10 5 cells were seeded on 24 mm diameter wells and grown to confluence for 14 days to allow cell differentiation [22]. Media change was performed 2-3 times a week. Cells were maintained in 5% CO 2 at 37˚C in a humidified chamber. 10 6 Caco-2 cells were seeded in standard cell culture medium. The next day, cells were transfected with 2 μg of plasmid pSpCas9(BB)−2A-Puro (PX459) V2.0 (Addgene plasmid no. 62988 was a gift from Feng Zhang [23]) containing Cas9 endonuclease and gRNA scaffold with an ATP7B-specific sgRNA sequence (5'-ATATCGGTGTCTTTGGCCGA-3'). After 24 h of transfection, a single cell dilution was performed. Standard medium containing 1.5 μg/ml puromycin was added for 72 h for selection.

Sequence analysis
DNA was purified using QIAamp DNA mini kit (Qiagen, Hilden, Germany). DNA sequencing was performed using Big Dye Version 3.1 (Life Technologies). For gross sequence analysis of the Caco-2 cell clones the primers 5'-AGAGGGCTATCGAGGCAC-3' / 5'-GGGCTCACC TATACCACCATC-3' were used. The respective PCR product derived from one clone (clone #1) was ligated into plasmid pCR2.1-TOPO (TOPO TA Cloning kit; Invitrogen). After transformation of the ligation mixture into E.coli, DNA from bacterial clones was isolated and forwarded to sequence analysis. The complete ATP7B coding region gene was also analyzed for Caco-2 cell clone #1 [24]. siRNA knockdown 50 nM small interfering RNA (siRNA) directed against ATP7A (AM16708; Ambion, Foster City, CA, USA) and 4 μl RNAiMax (Gibco) were incubated with cells for 24 h. A scrambled oligonucleotide with an unrelated sequence was used for control.

Retroviral transduction
Retroviral transduction was performed with vector pGCsamEN.ATP7B, expressing the coding region of human ATP7B using established protocols [24]. Selection of cells was performed with 1 μg/ml of blasticidin (Invitrogen) starting at day 1 after transduction.

Copper accumulation
0.1 mM CuCl 2 was added for 24 h in standard cell culture medium. Cells were washed five times with PBS and the cell pellet was collected. For measurement of subcellular Cu fractions differential centrifugation was used as described before [24]. Cu concentrations were determined by atomic absorption spectroscopy (Shimadzu AA-6300, Kyoto, Japan). Bradford protein assay (BioRad, Hercules, CA, USA) was used to normalize Cu measurements.

Triglyceride determination
0.1 mM CuCl 2 and oleic acid (125 μM; O3008, Sigma-Aldrich) were added in standard cell culture medium for 24 h. Cells were subjected to TG quantification (Triglyceride Quantification Assay, ab65336, Abcam) or AdipoRed Assay Reagent (PT-7009, Lonza) according to the protocol of the manufacturer. Total protein concentration was used for normalization. Cell culture supernatant was collected and triglycerides were determined by an automated cobas 8000 analyzer system (Roche Diagnostics GmbH, Mannheim, Germany).

Electron microscopy
Cells grown in standard cell culture medium were treated with 0.1 mM CuCl 2 for 24 h. Cell pellets were resuspended in 2.5% glutaraldehyd in Sorensen's Phosphate Buffer (0.133 M Na 2 HPO 4 , 0.133 M KH 2 PO 4 , pH 7.2) and fixed with 1% osmiumtetroxide. After dehydration, specimens were embedded and sixty nanometer ultrathin sections were prepared (Leica Ultra Cut E ultramicrotome, Vienna, Austria). Counterstaining was performed with uranyl acetate and lead. Samples were inspected on a transmission electron microscope EM 208S transmission electron microscope (Phillips, Hamburg, Germany). Measurement of lipid droplets sizes was performed using the software ImageJ according to the reference scale of each image. At least 20 cells were analyzed per condition from different experiments.

Real-time quantitative PCR
Total RNA was isolated by RNeasy kit (Qiagen, Hilden, Germany). Transcription was performed using 1 μg of RNA and SuperScript III (Invitrogen, Carlsbad, CA, USA). For quantitative real time PCR (qPCR) SYBR Green PCR Core Plus (Eurogentec, Liège, Belgium) and primers (S1 Table) were added. PCR was analyzed on the ABI Prism 7900 HT Sequence Detection System (Applied Biosystems, Carlsbad, CA, USA). Ct values were normalized to the expression of the house-keeping gene Actin (ΔΔCt method) and log 2 expression was calculated.

ApoE ELISA
ApoE protein was determined using Human ApoE ELISA Kit (EHAPOE, Thermo Scientific). Cell culture supernatants were used at 1:10 dilution and absorbance was assessed at 450 nm. Normalization was performed by total protein concentration.

Statistical analysis
Statistical analysis was performed by Kruskal-Wallis 1-way ANOVA and Wilcoxon Mann-Whitney-test using SPSS 22.0 software and GraphPad Prism 8. A p<0.05 value was used to indicate significance.

Generation of a human ATP7B CRISPR/Cas9 knockout intestinal cell line
An ATP7B knockout cell line was generated in human intestinal Caco-2 cells to study the impact of ATP7B on Cu and lipid metabolism. Caco-2 cells were transfected with a plasmid containing the endonuclease Cas9 and a guide RNA, targeting exon 2 of human ATP7B (Fig  1). Cell growth was observed in 13 clones after puromycin selection of CRISPR/Cas9 plasmid. Clones were characterized by gross sequence analysis of ATP7B exon 2 and subjected to MTT assay (S2 Table). Seven clones showed wildtype ATP7B and an almost identical viability as compared to parental cells when exposed to a toxic concentration of Cu (0.25 mM). Six clones showed significant reduced viability (<30%) and an ambiguous nucleotide sequence close to the PAM region after gross sequence analysis suggesting multiple compound deletions (S1A and S1B Fig, respectively). The cDNA of one Caco-2 cell clone (clone #1) was therefore further analyzed via bacterial cloning of the ATP7B exon 2 PCR product. Sequence analysis of the bacterial clones (n = 19) suggested a compound mutation consisting of altogether three deletions (p.L394F_A395del, p.L394FfsX9 and p.E396KfsX11) corroborating the reported polyploidy of the Caco-2 cell line (S3 Table) [25]. Importantly, a wildtype sequence could not be observed in the bacterial clones. The respective Caco-2 cell line was termed ATP7B KO (KO cells). The remaining coding sequence of ATP7B was unchanged suggesting that CRISPR/Cas9 mutagenesis induced a highly specific knockdown.

ATP7B knockout cells display disturbed copper homeostasis
The expression of ATP7B protein was determined in KO cells to evaluate the effect of deletions in exon 2. A knockin cell line, termed KI cells, expressing the wildtype ATP7B ORF was established from KO cells by retroviral vector. An ATP7B-specific protein band could not be detected by Western blot analysis of KO cells (S2 Fig). KI and parental cells showed similar high levels of ATP7B expression as determined by densitometric quantification of ATP7B-specific protein bands (Fig 2A). In order to characterize the sensitivity of KO cells to high Cu, different Cu concentrations were exposed to the cells. A significant drop of viable cells was observed at Cu concentrations ranging from 0.25 mM to 1.0 mM in KO cells as compared to parental and KI cells ( Fig 2B). Exposure to Cu concentrations above 0.5 mM resulted in an almost complete functional loss to evade Cu toxicity. In contrast, ATP7B knockin could fully restore survival suggesting that the CRISPR/Cas9 induced deletions are causative of the functional loss observed in KO cells. Since Caco-2 cells also express ATP7A, which was proposed to be a Cu exporter [26,27], we also assessed whether knockdown of ATP7A in KO cells further impairs the function to ATP7A knockdown did not modulate viability of KO cells (Fig 2C). However, the viability of parental cells was significantly affected by ATP7A knockdown to the same level of KO cells (48%±3 and 48%±2, respectively). The intracellular Cu storage of KO cells was determined (Fig 2D). When cells were propagated in standard cell culture medium (basal medium contained <2.5 μM Cu), an almost identical level of intracellular Cu was observed in KO and parental cells. However, KO cells revealed a significant increased level of intracellular Cu when incubated with 0.1 mM Cu for 24 h. Note, that cells cultivated at this Cu concentration showed no signs of apoptosis and proliferation arrest. After ultracentrifugation, most Cu (~80%) was observed in the supernatant of 15,000 xg fraction of the cell lysates suggesting low molecular weight, cytosolic storage (S4 Fig).
As Cu and iron homeostasis seems to be related [15], the sensitivity to toxic iron was determined in KO cells. In contrast to the reduced Cu sensitivity of KO cells, exposure to iron (10 mM to 30 mM) did not alter the cellular viability as compared to parental cells (S5 Fig).

Modulation of gene expression after intestinal ATP7B knockout
The question was addressed whether expression of genes related to Cu and lipid homeostasis was affected in the KO cell line. A set of 27 genes were selected from PubMed. Gene expression was analyzed by RT-qPCR analysis. Cells were grown in standard cell culture medium (basal medium) or incubated with 0.1 mM Cu. Expression was compared to parental Caco-2 cells grown in basal medium (control). 19/27 genes did not show significant changes of gene expression (� 1-log 2 expression relative to control) regardless whether treated with Cu or not (S4 Table). Genes not modulated were related to Cu metabolism (ATP7A, ATOX1, CTR1, MTF1 and SOD1), iron metabolism (DMT1, EPAS1, FPN1, HEPH, MRP1 and STEAP3), lipid metabolism (HMG-CoA, LDLR, PLN2, PPARα, PPARγ, and VLDLR), and apolipoprotein synthesis (ApoA4 and ApoB100). In contrast, expression of three genes involved in protection from oxidative stress, metallothionein 1 (MT1), duodenal cytochrome B metal reductase (DCYTB), and hemeoxygenase 1 (HMOX1), were affected in KO cells (Fig 3). After addition of Cu, MT1 was significantly induced, however to similar high levels as compared to parental cells. DCYTB was significantly downregulated in KO cells regardless of Cu addition. HMOX1 expression was induced in KO cells with highest levels after addition of Cu, suggesting that a high anti-oxidative response is induced after knockdown of ATP7B in the intestinal cell line. Of note, five genes related to lipid and apolipoprotein synthesis were modulated in KO cells (Fig 3). ABCA1, a cholesterol efflux pump belonging to the ATP-binding cassette (ABC) transporters [28], was significantly downregulated in KO and parental cells after Cu treatment. APOA1, APOC3 and APOE were upregulated in KO cells regardless whether or not Cu was exposed to the cells. APOB48 was downregulated in KO cells after Cu treatment, whereas parental cells receiving the same treatment did not show modulation of mRNA level as compared to control.

ATP7B knockout affects lipid metabolism in intestinal cells
Cu transport was proposed to affect lipid metabolism of intestinal cells. The number of intracellular lipid droplets (LDs) was investigated in KO cells by electron microscopy (Fig 4). According to their size, two classes of LDs were defined, having large (>600 nm) and small diameters (<600 nm). The numbers of LDs were similar in KO and parental cells when cultivated in standard cell culture media (basal media contains~10 mg TG/dl) (Table 1). However, addition of Cu (0.1 mM) increased (~3-fold) the number of smaller sized LDs in KO cells and to a somewhat lesser extent (~2-fold) in parental cells. In parallel, the number of larger sized LDs decreased (~3-fold) in both cell lines after addition of Cu suggesting that maturation of large sized LDs is impaired by Cu.
In order to assess the storage and secretion of lipids in the cells, the concentrations of intracellular and secreted TGs were determined (Fig 5). Levels of intracellular and secreted TGs were similar in KO cells and parental cells when grown in basal medium (Fig 5A and 5B). Addition of Cu repressed intracellular TG storage and secretion. Secretion of ApoE, a

PLOS ONE
determinant of various lipoproteins, was secreted at significant higher levels by KO cells as compared to parental cells (Fig 5C). Oleic acid (OA), a common long chain unsaturated fatty acid, was used to further investigate lipid metabolism of KO cells. After OA exposure, KO cells significantly accumulated higher amounts (~2-fold) of intracellular TGs as compared to parental cells (Fig 5D). However, total secretion of TGs was similar in KO cells and parental cells after OA addition, suggesting that high intracellular lipid storage does not implement an TG export of similar levels when ATP7B is lacking (Fig 5E). Addition of Cu did not affect lipid storage and secretion of parental cells following OA treatment.

Discussion
Cu is an important molecule of many basic metabolic processes, including inflammatory response, antioxidant defense, and lipid peroxidation. The availability of Cu was recently linked to lipid metabolism and implicated in the pathogenesis of epidemic nutritional disorders, e.g. nonalcoholic fatty liver disease (NAFLD) and obesity [16,29,30]. In this scenario, the enterocyte of the proximal intestine has a central role to accommodate the body's specific physiologic needs, including the uptake and processing of Cu and lipids. We explored the impact of copper transporter ATP7B to mediate Cu and lipid metabolism in the enterocyte, a role that was previously characterized for the hepatocyte [18,19,31]. Using CRISPR/Cas9 technology, a novel KO cell line was established from Caco-2 cells, one of the most studied human model with many biochemical and morphological characteristics of enterocytes [22,32].
The CRISPR/Cas9 mutational approach targeting exon 2 of ATP7B resulted in the generation of several cell clones carrying compound heterozygous mutations around amino acid position 395, the targeted putative Cas9 cleavage site. In contrast to a previously reported mutational approach of ATP7B, a homozygous mutation was not observed here [33]. However, only a small number of cell clones was analyzed following an undirected mutational approach. While the deleterious variants observed in the KO cell line were not previously reported from patients, exon 2 is known to harbor numerous disease-causing mutations. Nevertheless, all variants of the KO cell line caused deletion/frame-shift mutations and resulted in absence of ATP7B protein expression and impaired ATP7B function.
Functional loss was indicated by Cu sensitivity of the KO cells, which could be specifically restored after re-expression of wildtype sequence. A significant elevation of intracellular Cu was observed in KO cells after exposition to 0.1 mM Cu suggesting that intracellular homeostasis is impaired in the situation of elevated Cu. The daily Cu intake by food can significantly vary and is estimated to be around 2 mg for a typical adult, suggesting that the Cu exposure of cells used in this study is within physiological ranges. Most Cu was found in the lower molecular weight fraction of the enterocyte cell lysates indicating that intracellular Cu storage is likely associated with the cytosol. Of note, at basal Cu concentrations we did not observe a higher Cu accumulation in KO cells as compared to parental cells. This finding parallels previous observations of high Cu accumulation in cell models and WD liver suggesting that the export of Cu is impaired after loss of ATP7B [34][35][36]. For hepatocytes, it is commonly viewed that the main function of ATP7B is the transport of excess Cu into the bile [1]. The role of ATP7B for Cu homeostasis in the enterocyte is however far less understood as compared to the liver. Our determination of Cu resistance in the KO cell line are suggestive of an ATP7B exporter function in intestinal cells, however the Cu efflux was not directly assessed. In addition, while reversal of high Cu sensitivity was observed after ATP7B knockin, other mechanisms, e.g. increased vesicular Cu sequestration, could be envisaged to cause evasion from toxicity. In LEC rats, where ATP7B is nonfunctional due to a deletion, high Cu levels were found in the intestine following a Cu diet corroborating our findings [37]. In contrast, AAS analysis and Xray fluorescence microscopy of intestinal cells suggested reduced Cu levels in ATP7B [13,14,38]. Differences of the Cu content in diets and cell culture media, methodology of Cu determination, and differentiation status of the intestinal cells may account for some of the controversial findings. Further work is however required to address the exact role of ATP7B in the intestine, e.g. during polarized Cu trafficking. MT1 belongs to the first line defense antioxidant and HMOX1 is an important defender of oxidative stress [39][40][41]. Downregulation of DCYTB, a metal reductase, is considered as prevention of oxidative stress [42]. Expression values of these genes indicate that KO cells do not show elevated oxidative stress at low (basal) Cu concentrations, but respond to Cu treatment by induction of an oxidative stress defense mechanism that is not mounted in parental cells. Various other genes, implicated to maintain intracellular Cu homeostasis, were not modulated by the low/high Cu exposure of KO cells suggesting that the regulation of basic Cu transporters, importantly ATP7A, CTR1, DMT1 and SOD1, is preserved under the experimental conditions used in the study. In duodenal samples of WD patients ATP7A was increased and CTR1 was reduced, whereas expression of DMT1 and ATP7B was unaffected suggesting that some genes may be regulated by additional factors in patients [43]. ATP7A was suggested as a major Cu exporter of enterocytes [2]. High intestinal Cu concentrations were observed in patients having Menkes disease and in IEC-6 intestinal cells [11,44]. In this regard it is of interest that our simultaneously impairment of ATP7A and ATP7B did not synergistically increase the Cu sensitivity of Caco-2 cells suggesting that both transporters are involved in the escape from toxic Cu. This result is surprising, since the double knockout cells, which have no option to export Cu either by ATP7A or ATP7B, show the same resistance as cells with only one downregulated transporter. We did not assess gene expression and Cu accumulation in double knockout cells and it could be argued that other genes might be modulated, e.g. CTR1 is downregulated/endocytosed resulting in an overall reduced Cu loading. However, CTR1 mRNA was not downregulated in the KO cell line. Further experiments, also addressing expression levels of proteins are needed. Taken together, our data suggest that similar to liver, where ATP7B presents exporting functions, this is also operative in the enterocyte, extending previous findings in knockout mice [14].
Cu metabolism was suggested to affect iron homeostasis [11,42,45]. A modulation of genes related to iron homeostasis was observed for intestinal cells [11,42]. Our results on gene expression related to iron homeostasis as well as toxicity of KO cells after iron exposure did not support a role of ATP7B in iron homeostasis. However additional studies, including intestinal double knockouts, are needed to determine the role of the two Cu transporters for regulation of iron homeostasis in the enterocyte.
In a portion of WD patients, alterations in serum TG/cholesterol and appearance of liver steatosis suggested interrelation of Cu homeostasis and lipid metabolism [1,17,18,31]. To gain more insights in the molecular mechanism, different aspects of lipid metabolism were investigated in KO cells. One important finding of our study was the increased level of intracellular TGs in KO cells after exposition to OA, one of the most commonly found long-chain monounsaturated omega-9 fatty acids in nature. TG storage is proposed as a protective mechanism against fatty acid-induced lipotoxicity [46]. OA was previously shown to be readily takenup by Caco-2 cells which resulted in high-efficient secretion of TG-rich particles, prominently chylomicrons and VLDLs [47][48][49][50]. In hepatic cells, OA induces synthesis of hepassocin (HPS), a cytokine implicated to be reduced in NAFDL [51]. It is therefore tempting to speculate that such pathways are involved in the establishment of NAFDL in WD patients. The overall level of TG secretion by KO cells was unchanged as compared to parental cells suggesting that the release of excess intracellular TGs is hampered in the absence of ATP7B. In skeletal muscle cells, OA was found to accelerate lipid oxidation in a SIRT1-PGC1alpha-dependent mechanism [52]. Although we did not address lipid oxidation in our study, our finding of increased TG accumulation in KO cells suggests that ATP7B is involved in OA-induced lipid metabolism, at least in the enterocyte.
Our findings of increased levels of lipoprotein gene expression, namely ApoA1, ApoC3, and ApoE, are suggestive of an increased lipoprotein secretion in the absence of ATP7B. The level of ApoB100 expression, one prominent protein of various lipoprotein classes [53], was however not affected in KO cells. Of note, ApoE, found in highest quantities in VLDL, was induced in KO cells. Several studies suggested a direct link between Cu deficiency and dyslipidemia with increased concentrations of ApoE [54,55]. Similarly, overexpression of ApoE resulted in a combined hyperlipidemia phenotype [56]. However, ApoE expression of Caco-2 cells might be higher as compared to intestine and the relative role of intestine-secreted ApoE is not known [57,58]. It is however conceivable that secretion of lipoproteins, which prominently carry TGs rather than cholesterol, like VLDL, is increased in enterocytes in the absence of ATP7B. This is in line with findings of reduced cholesterol levels in animal models of WD [18,20]; however contradicting results were reported by others [19].
Cu displayed significant effects on the processing of lipids in KO and parental Caco-2 cells. Whereas the number of large size LDs was reduced, numbers of smaller sized LDs were increased suggesting that maturation of small sized LDs to large size LD, including chylomicrons [59], is hampered by Cu regardless whether or not ATP7B was expressed. The Cu-induced effect on LD generation may not be directly related to ApoB48 mRNA expression, since parental Caco-2 cells expressed normal levels. Lipid transporter ABCA1, reported to contribute to the production of HDL particles via release of phospholipid and free, unesterified cholesterol from the plasma membrane [28], was repressed by Cu in intestinal cells regardless of ATP7B expression. Downregulation of ABCA1 might therefore represent an intestinal response to Cu that may manifest in WD patients resulting in low cholesterol [17,20,60]. We also found a reduced intracellular TG storage and secretion that was independent of ATP7B expression when Cu treatment of the cells was performed in basal cell culture medium. Exposition of intestinal cells with Cu may thus prevent overall cellular lipid uptake resulting in decreased TG storage and secretion. Interference of Cu absorption and long-chain free fatty acid has been observed in rats. It was speculated that Cu may directly bind to free fatty acids and/or interact with the glycocalyx of the intestine [61,62]. In a situation of Cu deficiency, rats showed increased intestinal levels of TGs suggesting that low intestinal Cu can stimulate lipogenesis [63,64]. In contrast, Cu treatment resulted in decreased intestinal TG storage of enterocytes corroborating the findings of this study [51]. Conversely, when a high fat diet was used for animals lacking ATP7B, increased hepatic enzymes for cholesterol synthesis but normal values of serum TGs and cholesterol were found [60]. Taken together, our findings reveal a crucial role of Cu and ATP7B in the storage, processing, and secretion of lipids in a human enterocyte model. Supporting information S1 Fig. ATP7B CRISPR/Cas9 treatment of Caco-2 cells. (A) Cu resistance of cell clones (green) was examined by MTT assay. Clone #1 revealed compound deletion and clone #2 harboured wildtype ATP7B. WT cells (black) were used as control. Viability of cells was determined relative to untreated cells (100%). Mean ± SD are given (n = 3). � P < 0.05. ns, not significant. (B) Gross sequence analysis of clone #1 before bacterial cloning showed ambiguous nucleotide sequences between position 1181 and 1185. Note, that nucleotide sequence could be analyzed up to a certain position, whereas thereafter the sequence was unreadable (N) due to deletions. The PAM motif is marked in yellow. Forward (top) and reverse (bottom) sequence analysis is depicted.  Table. Gene expression analysis of KO cells before and after copper load. Genes related to the Cu, iron (Fe) or lipid metabolism were examined. Cells were analyzed before and after Cu exposure. Log 2 gene expression is given relative to parental (WT) cells prior Cu treatment. Mean ± SE is given (n = 3). (DOCX)