Transgenic overexpression of Niemann-Pick C2 protein promotes cholesterol gallstone formation in mice

Research Article

Transgenic overexpression of Niemann-Pick C2 protein promotes cholesterol gallstone formation in mice Mariana Acuña1,5,y, Lila González-Hódar1,y, Ludwig Amigo1, Juan Castro1, M. Gabriela Morales1, Gonzalo I. Cancino2, Albert K. Groen3, Juan Young4, Juan Francisco Miquel1,5, Silvana Zanlungo1,5,⇑ 1 Department of Gastroenterology, Faculty of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile; 2Neuroscience and Mental Health Program, The Hospital for Sick Children, Toronto, Canada; 3Departments of Pediatrics/Laboratory Medicine, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands; 4Centro de Estudios Científicos (CECs), Valdivia, Chile; 5FONDAP ‘‘Center for Genome Regulation” (CGR), Santiago, Chile

Background & Aims: Niemann-Pick C2 (NPC2) is a lysosomal protein involved in the egress of low-density lipoproteinderived cholesterol from lysosomes to other intracellular compartments. NPC2 has been detected in several tissues and is also secreted from the liver into bile. We have previously shown that NPC2-deficient mice fed a lithogenic diet showed reduced biliary cholesterol secretion as well as cholesterol crystal and gallstone formation. This study aimed to investigate the consequences of NPC2 hepatic overexpression on liver cholesterol metabolism, biliary lipid secretion, gallstone formation and the effect of NPC2 on cholesterol crystallization in model bile. Methods: We generated NPC2 transgenic mice (Npc2.Tg) and fed them either chow or lithogenic diets. We studied liver cholesterol metabolism, biliary lipid secretion, bile acid composition and gallstone formation. We performed cholesterol crystallization studies in model bile using a recombinant NPC2 protein. Results: No differences were observed in biliary cholesterol content or secretion between wild-type and Npc2.Tg mice fed the chow or lithogenic diets. Interestingly, Npc2.Tg mice showed an increased susceptibility to the lithogenic diet, developing more cholesterol gallstones at early times, but did not show differences in the bile acid hydrophobicity and gallbladder cholesterol saturation indices compared to wild-type mice. Finally, recombinant NPC2 decreased nucleation time in model bile.

Keywords: Lithiasis; NPC2; Lithogenic diet; Gallbladder. Received 8 January 2015; received in revised form 29 September 2015; accepted 1 October 2015 ⇑ Corresponding author. Address: Pontificia Universidad Católica de Chile, Departamento de Gastroenterología, Marcoleta 367, Casilla 114-D, Santiago, Chile. Tel.: +56 2 2354 3833; fax: +56 2 639 7780. E-mail address: [email protected] (S. Zanlungo). y Both authors equally contributed to this work. Abbreviations: NPC2, Niemann-Pick C2; NPC1, Niemann-Pick C1; NPC2(h/h), Niemann-Pick C2-deficient hypomorph mice; ABCG5/G8, adenosine triphosphate–binding cassettes G5 and G8; ConA, Concanavalin A; Npc2.Tg, Niemann-Pick C2 transgenic mice; PCR, polymerase chain reaction; qPCR, quantitative polymerase chain reaction; HEK, human embryonic kidney; BSA, bovine serum albumin; ALT, alanine transaminase; Abcb11, ATP-binding cassette, sub-family B member 11; Abcb4, ATP-binding cassette, sub-family B member 4; CSI, cholesterol saturation index; SDS, sodium dodecyl sulfate; PBS, phosphate buffered saline; DMEM, Dulbecco’s Modified Eagle’s medium; FBS, fetal bovine serum; IMAC, immobilized metal affinity chromatography; ELISA, enzyme-linked immunosorbent assay.

Conclusions: These results suggest that NPC2 promotes cholesterol gallstone formation by decreasing the cholesterol nucleation time, indicating a pro-nucleating function of NPC2 in bile. Ó 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Introduction Niemann-Pick C2 (NPC2) is a lysosomal soluble protein that binds cholesterol with nanomolar affinity and 1:1 stoichiometry through its aliphatic chain [1–3]. In vitro assays have shown that NPC2 mediates the transfer of cholesterol between liposomes and that this transfer is accelerated in the presence of anionic phospholipids, such as those present in lysosomes [4,5]. In cells, NPC2 participates with the transmembrane protein Niemann-Pick C1 (NPC1) in cholesterol efflux from low density lipoprotein receptor-mediated endocytosis [6]. In the currently accepted model NPC2 binds cholesterol obtained from the endocytic pathway during and/or after acid lipase hydrolysis and delivers the cholesterol to NPC1, which mediates transfer through the lysosomal membrane to other cell compartments [1,6]. In addition, NPC2 is found in several fluids, including epididymis fluid, milk, plasma and bile [7–9]. In mice epididymis, NPC2 is involved in adding cholesterol to spermatozoa during maturation and its deficiency in spermatozoa reduced their ability to fertilize cumulus-oocytes complexes [10]. In mammals, the liver is a key organ for cholesterol homeostasis. One of the major uptake mechanisms of lipoprotein cholesterol into the liver is the receptor-mediated endocytic pathway. Additionally, hepatocytes eliminate sterols through bile as unesterified cholesterol and bile acids [11]. Biliary cholesterol disposal is critical not only for normal body cholesterol homeostasis but also for the pathogenesis of cholesterol gallstones, a highly prevalent and costly disease condition in Western countries [12]. We have previously reported that NPC2 is expressed in the liver and is secreted into the bile in human and mice. In fact, biliary NPC2 levels were increased in gallstone-susceptible C57BL6/J mice compared to a gallstone-resistant BALB/c strain [9]. In addition, Yamanashi et al. found a positive association between NPC2 and cholesterol levels in human bile [13]. Recently, we described

Journal of Hepatology 2015 vol. xxx j xxx–xxx

Please cite this article in press as: Acuña M et al. Transgenic overexpression of Niemann-Pick C2 protein promotes cholesterol gallstone formation in mice. J Hepatol (2015), http://dx.doi.org/10.1016/j.jhep.2015.10.002

Research Article that NPC2-deficient hypomorph mice [NPC2(h/h)] fed a chow diet showed increased biliary cholesterol and phospholipid secretions. In contrast, NPC2(h/h) mice fed a lithogenic diet showed reduced biliary cholesterol secretion and gallbladder bile cholesterol saturation, leading to resistance to cholesterol gallstone formation [14]. This work indicates that NPC2 expression is an important factor in the regulation of diet-derived cholesterol metabolism and in diet-induced cholesterol gallstone formation in mice. Additionally, NPC2 was described as a positive regulator of biliary cholesterol secretion via stimulation of ABCG5/8mediated cholesterol transport using adenovirus infection in mice [13]. Liver cholesterol hypersecretion into bile is the primary pathogenic event for cholesterol crystallization, but other factors contribute to the development of gallstones, including gallbladder stasis [15,16], which refers to a diminution of gallbladder contraction, and the balance between anti-nucleating and pronucleating factors [17–19]. These pro-nucleating factors could explain why bile from patients with the same degree of cholesterol saturation has large differences in cholesterol crystallization speed and the number of gallstones [17,20,21]. Some proteins found in the bile, like IgG, haptoglobin, mucin and aminopeptidase N, promote cholesterol crystallization in vitro [22–24]. However, only mucin has been demonstrated to have pro-nucleating activity in vivo and patient correlations [25–28]. Interestingly, NPC2 is part of the group of bile glycoproteins that bind to Concanavalin A (ConA) [9], acting as a potent activator of cholesterol crystallization [20,29] and therefore, NPC2 could have a pronucleating function in bile. Of note, 59 different proteins were identified by ConA affinity purification, including haptoglobin and mucin-2, in a proteomic analysis of human bile [30]. However, NPC2 was not detected as a ConA binding protein in this study. The results obtained suggest that NPC2 has a function in the liver modulating lipids secretion, but its role in bile has not been determined. Here, we investigate whether overexpression of the Npc2 gene in the liver influences hepatic cholesterol metabolism, biliary lipid secretion and diet-induced gallstone formation. We developed a new Npc2 transgenic strain in a gallstonesusceptible C57BL6/J genetic background. In addition, to analyze the function of biliary NPC2, we studied the effect of NPC2 on cholesterol crystallization in model bile. No differences were observed in biliary cholesterol concentration or secretion between Npc2.Tg and wild-type mice fed chow or lithogenic diets. Interestingly, Npc2.Tg mice showed an increased susceptibility to the lithogenic diet, developing more cholesterol gallstones at early times, but showed no differences in the gallbladder cholesterol saturation index (CSI) compared to wild-type mice. In addition, recombinant NPC2 decreased the nucleation time in model bile.

All mice had free access to water and chow diet (0.02% cholesterol; Prolab RMH 3000, PMI Feeds, St. Louis, MO, USA) until they were used for the studies. For the experiments, 6 week-old Npc2.Tg and wild-type male mice were fed chow or lithogenic diets (TestDiet, St. Louis, MO, USA) (15% fat, 1.25% cholesterol and 0.5% cholic acid) for 14, 21 or 28 days. All animals were fasted for 2 h before bile, blood and liver sampling. All protocols were approved by our institution’s review board for animal studies and were in agreement with the US Public Health Service Policy on Humane Care and Use of Laboratory Animals recommended by the Institute for Laboratory Animal Research in its Guide for Care and Use of Laboratory Animals. Hepatic and gallbladder bile and liver sampling Mice were anesthetized by intraperitoneal injection of ketamine and xylazine at 80–100 and 5–10 mg/kg, respectively. Thereafter mice were euthanized and liver samples and bile were processed as described previously in detail [32]. Hepatic and biliary lipid analyzes Hepatic cholesterol content was analyzed after lipid extraction [32]. Hepatic and gallbladder bile cholesterol and bile acids were measured by enzymatic methods [33,34] and phospholipids were assessed by a colorimetric method [35]. Gallbladder bile CSI was calculated from Carey’s critical tables [36], using biliary lipid measurements performed after 14 days of feeding with a lithogenic diet and before mice developed gallstones. Bile acid composition was measured using liquid chromatography-mass spectrometry as described in [37] using hepatic bile obtained after 14 days of feeding with a chow or lithogenic diet. The bile acid hydrophobicity indices were calculated according to Heuman [38]. Gallstone Formation in vivo After 2 weeks of consuming a lithogenic diet, gallbladder bile was taken and evaluated for the presence of microscopic and macroscopic characteristics of gallstone formation. Genes and proteins expression analyzes Real-time polymerase chain reactions (qPCR), SDS-PAGE, immunoblotting immunofluorescence, quantification of total protein and NPC2 concentration in bile were performed as described in detail in the Supplementary materials and methods section. Cell transfection and purification of recombinant NPC2 protein HEK293 cells were transfected transiently with the plasmid containing Npc2 wild-type mouse cDNA donated by Dr. Matthew Scott (Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, USA) [3], or with the plasmid without insert. The conditioned medium was collected and used to purify the NPC2-myc-His protein, as detailed in Supplementary materials and methods. Preparation of model bile The model bile was prepared with sodium taurocholate, cholesterol and phosphatidylcholine, all obtained from Sigma-Aldrich (St. Louis, MO, USA). The details of the protocol are in Supplementary materials and methods. Nucleation assays

Materials and methods Animals and diet To generate NPC2 transgenic mice, mouse Npc2 cDNA was cloned into the pLIV7 plasmid, provided by Dr. John M. Taylor (Gladstone Institute Of Cardiovascular Disease, University of California, USA) [31]. The construct was used for zygote microinjection in Centro de Estudios Científicos, Valdivia, Chile. Transgenic mice were bred with C57BL6/J mice to produce wild-type and NPC2 transgenic mice (Npc2.Tg). The offspring were screened for the presence and orientation of the transgene via PCR of genomic DNA from mice tails and Southern blot, as detailed in Supplementary materials and methods.

2

Model bile was incubated with wild-type recombinant NPC2 protein, mucin, bovine serum albumin (BSA) and Ni-Affinity proteins obtained from cells transfected with an empty vector. The time of nucleation and the number of cholesterol crystals were evaluated, as detailed in Supplementary materials and methods. Immunoprecipitation experiments The purified recombinant NPC2 protein was immunoprecipitated with anti-NPC2 antibody (Sigma-Aldrich) and protein G PLUS-agarose sc-2002 (Santa Cruz Biotechnology, Dallas, TX, USA). The crystallization-promoting activity of the

Journal of Hepatology 2015 vol. xxx j xxx–xxx

Please cite this article in press as: Acuña M et al. Transgenic overexpression of Niemann-Pick C2 protein promotes cholesterol gallstone formation in mice. J Hepatol (2015), http://dx.doi.org/10.1016/j.jhep.2015.10.002

JOURNAL OF HEPATOLOGY

A

nI

Cloning site

Kp

supernatant was tested in the nucleation assays. For more details, see Supplementary materials and methods.

Intron1

Statistics

5` apoE

B

2 Npc2

WT

C

Npc2.Tg

Results

1 kb

Hepatic cholesterol content and biliary lipid secretion in wild-type and Npc2.Tg mice fed chow and lithogenic diets

300

Npc2 mRNA

200

100

0

D

Liver Lung Heart Brain Stomach Kidney C+ C- WT Tg WT Tg WT Tg WT Tg WT Tg WT Tg

NPC2

ε-cop

E kDa 130 100 70 55 40 35

Gallbladder bile WT Tg

Hepatic bile WT Tg

kDa 130 70 40 35 25 15

Liver C+ WT Tg

NPC2

25 10 NPC2 15

Cathepsin B

NPC2

Merge

ε-cop

Zoom

WT

F

35

NPC2.Tg

To analyze the role of NPC2, we generated Npc2 transgenic mice using a mouse Npc2 transgenic vector derived from the pLIV7 plasmid, which contains the apolipoprotein E genomic region and a hepatic control region, aiming for a high liver expression of the transgene [31] (Fig. 1A). The animals were viable and healthy with normal body weights and reproduction (data not shown). Correct insertion of the Npc2 transgene was corroborated by Southern blot analysis (Fig. 1B). With this method, we also determined that Npc2.Tg mice have 5 inserted copies of the transgene. Additionally, the transgenic mice have 200-fold more Npc2 mRNA compared to wild-type mice (Fig. 1C). Furthermore, immunoblot analysis showed high NPC2 expression in the liver and also in the kidneys and lungs (Fig. 1D). Increased levels of NPC2 in the liver were associated with high levels of NPC2 in bile (Fig. 1E). We also evaluated NPC2 intracellular localization in liver sections using Cathepsin B as a lysosomal marker. NPC2 and Cathepsin B co-localized suggesting that overexpressed NPC2 is destined for lysosomes and the intracellular pattern is consistent with an endosomal-lysosomal distribution, as described by Garver et al. [39] (Fig. 1F). Liver weight was increased in Npc2.Tg and wild-type mice fed a lithogenic diet, but no differences were found between the animals (Supplementary Table 2). In addition, transgenic and wildtype mice do not have significantly different serum alanine transaminase (ALT) levels, indicating that NPC2 overexpression does not produce hepatic damage (data not shown). Furthermore, hepatic histological analyzes do not show obvious differences between wild-type and Npc2.Tg mice fed chow or lithogenic diets (Supplementary Fig. 1). Only a small number of inflammatory foci were observed in the Npc2.Tg mice fed with the lithogenic diet and not in the wild-type mice. As expected, the lithogenic diet resulted in increased lipid accumulation, visualized by Oil Red O staining, in both wild-type and Npc2.Tg mice (Supplementary Fig. 1). No differences were observed in hepatic cholesterol content between Npc2.Tg and wild-type mice fed the chow diet (Supplementary Table 2). As expected, an increase in hepatic cholesterol content was observed when both animals were fed the lithogenic diet. However, this increase was smaller in Npc2.Tg mice (Supplementary Table 2). No differences were found between wild-type and Npc2.Tg mice fed the chow diet in biliary lipid secretions and composition of cholesterol, bile salts, phospholipids and total lipid concentration. As expected, the lithogenic diet induced an increase in biliary secretion of all lipids analyzed and also an increase in cholesterol composition in both types of animals (Table 1). A

HCR

3`apoE

Relative expression (Npc2.Tg/WT)

The statistical significance of differences between the means of experimental groups was evaluated using one-way analysis of variance with a post hoc Bonferroni multiple-comparison test using Prism 5.00 (GraphPad, La Jolla, CA, USA). A difference was considered to be statistically significant at p 60.05.

1

Fig. 1. NPC2 transgenic mice characterization. (A) A schematic illustration of the Npc2 transgene construct. HCR, hepatic control region. (B) Southern blot analysis of NheI-EcoRV-digested genomic DNA from Npc2 transgenic mice using a 155 bp-long [a-32P]dCTP-labeled cDNA probe that is part of exon 3 of the gene. (C) Real-time PCR analysis of Npc2 mRNA levels from the liver of mice. mRNA levels were normalized to 18S rRNA and were relative to wild-type (WT) mice. (D, E) NPC2 protein expression determined via immunoblotting analysis of different tissues (D); liver homogenates and hepatic and gallbladder bile (E) of Npc2.Tg and wild-type littermate adult males with anti-NPC2 and anti-e-cop antibodies. (F) Immunofluorescence analysis of liver sections using anti-NPC2 (red) and anticathepsin B (green) antibodies.

small difference in total lipids was found between the wildtype and Npc2.Tg mice fed the chow diet, but no differences were found between the animals fed the lithogenic diet. We also

Journal of Hepatology 2015 vol. xxx j xxx–xxx

3

Please cite this article in press as: Acuña M et al. Transgenic overexpression of Niemann-Pick C2 protein promotes cholesterol gallstone formation in mice. J Hepatol (2015), http://dx.doi.org/10.1016/j.jhep.2015.10.002

Research Article Table 1. Effect of lithogenic diet on hepatic cholesterol levels and biliary lipid secretion in wild-type and Npc2.Tg mice.

Biliary lipid secretion (nmol/min/g liver) Mice Wild-type Npc2.Tg

Diet

Biliary lipid composition

Ch

BS

PL

Mole% Ch

Mole% BS

Mole% PL

PL/ (BS + PL)

Total lipids (g/dl) 1.55 ± 0.34

Chow

0.99 ± 0.33

47.94 ± 19.55

8.39 ± 2.51

1.80 ± 0.30

83.00 ± 2.16

15.20 ± 2.03

0.155 ± 0.021

Lithogenic

5.10 ± 1.48a,b

194.23 ± 61.50a,b

31.57 ± 8.47a,b

2.16 ± 0.25a,b

84.07 ± 1.58

13.77 ± 1.56

0.141 ± 0.016

3.51 ± 0.25a,b

Chow

0.99 ± 0.25

63.27 ± 15.61

10.41 ± 2.25

1.35 ± 0.31

84.64 ± 1.81

14.00 ± 1.66

0.142 ± 0.017

2.36 ± 0.60b

Lithogenic

5.27 ± 1.48a,b

178.80 ± 61.51a,b

29.92 ± 7.52 a,b

2.48 ± 0.45a,b

82.99 ± 2.95

14.52 ± 2.61

0.149 ± 0.027

3.18 ± 0.41a,b

Effect of the lithogenic diet on gallstone formation in wild-type and Npc2.Tg mice C57BL6/J mice are gallstone-susceptible when they are fed the lithogenic diet containing high cholesterol, cholic acid and fats. Cholesterol, bile salts and phospholipid contents were measured in bile samples collected 5 and 14 days after lithogenic diet feeding was started and the CSI was calculated (Supplementary Table 3, Table 2 and Fig. 3A). After 5 days, no differences in the gallbladder lipids concentration and CSI between wild-type and Npc2.Tg mice were observed (Supplementary Table 3). Interestingly, a bile saturation index close to 100% was found in both groups after 14 days of lithogenic diet feeding, indicating that the gallbladder bile was very close to being saturated with cholesterol. However, no difference in the CSI between wild-type and Npc2.Tg mice was detected (Fig. 3A). Accordingly, the relative lipid compositions of gallbladder biles from wild-type and Npc2.Tg mice fed the lithogenic diet 4

a,b 10 5

di en og

th Li

et di ic

w

en

ho C

ic en og th Li

ic

di w ho 0

di

di

et

et

0

1

og

1

2

th

2

Li

3

a,b 3

et

a,b

Abcb4 4

di

4

C

og th Li

D

mRNA (relative expression)

en

WT Npc2.Tg

Abcb11 a,b

5

et

0

et

et

Abcg8 a,b

15

di ic

w ho C

C

mRNA (relative expression)

0

w

We also analyzed the hepatic expression of genes encoding canalicular lipid transporters in wild-type and Npc2.Tg mice fed chow and lithogenic diets. The expression of Abcg5/8, the cholesterol transporter and Abcb11, the bile salts transporter were significantly increased in both animals fed the lithogenic diet compared to those fed with the chow diet (Fig. 2A–C). The expression of the phospholipid transporter Abcb4 was significantly increased in wild-type mice fed the lithogenic diet compared to wild-type mice fed the chow diet, but this change was not significant in Npc2.Tg mice (Fig. 2D).

5

ho

Hepatic gene expression of canalicular transporters in wild-type and Npc2.Tg mice fed chow and lithogenic diets

a,b

10

C

Gallbladder bile concentrations of cholesterol, bile salts and phospholipids were measured in Npc2.Tg and wild-type mice fed chow and lithogenic diets (Table 2). No significant differences were observed in the lipid composition between both animals fed either diet, but an increase in phospholipid/(bile salt plus phospholipid) [PL/(BS+PL)] ratio is observed in Npc2.Tg mice fed the chow diet. As expected and in accordance with biliary lipid secretion measurements, a significant increase in gallbladder bile cholesterol composition and in the CSI was observed when both animals were fed the lithogenic diet.

B

a,b

15

di

Gallbladder bile lipid composition in wild-type and Npc2.Tg mice fed chow and lithogenic diets

Abcg5 20

et

A

mRNA (relative expression)

observed an increase in total lipids in animals fed on this diet, compared to the animals fed the chow diet.

mRNA (relative expression)

Values are expressed as means ± SD. Measurements were performed in 5 wild-type and 6 Npc2.Tg fed the chow diet and in 8 wild-type and 11 Npc2.Tg mice fed the lithogenic diet. aSignificantly different (p <0.05) from wild-type mice fed the chow diet; bSignificantly different from Npc2.Tg mice fed the chow diet (p <0.05). Ch, cholesterol; BS, bile salts; PL, phospholipids.

Fig. 2. Hepatic gene expression of canalicular lipid transporters in wild-type and Npc2.Tg mice fed with chow and lithogenic diets. (A–D) Real-time PCR analysis of mRNA levels from the livers of mice fed chow or lithogenic diets. mRNA levels were normalized to 18S rRNA and were relative to wild-type (WT) mice fed with a chow diet. Five animals were analyzed in each group. The following genes were analyzed: Abcg5 (A), Abcg8 (B), Abcb11 (C) and Abcb4 (D). a Significantly different (p <0.05) from wild-type mice fed the chow diet; b Significantly different (p <0.05) from Npc2.Tg mice fed the chow diet.

but without gallstones were both located in the one-phase area near the phase 2 area denoted region B (Fig. 3B). Despite the similar saturation indices, we found an increase in plate-like solid cholesterol monohydrate crystals in Npc2.Tg mice compared to wild-type mice littermates after 14 days of lithogenic diet feeding (Fig. 3C). Of note, we observed liquid crystals and solid tubule crystals more frequently in the bile of Npc2.Tg mice (Fig. 3C). Accordingly, we also found a significant increase of 73% in gallstone prevalence in Npc2. Tg mice compared to a 31% in wild-type mice (Fig. 3D). Therefore, Npc2.Tg mice had increased susceptibility to cholesterol gallstone formation compared with wild-type mice littermates. In addition, no significant differences were observed in gallbladder volume between wild-type and transgenic mice fed a lithogenic diet for 14 days (49.18 ± 24.27 ll and 38.00 ± 15.94 ll in 11 wild-type and 9 Npc2.Tg mice, respectively), suggesting that gallbladder contraction is similar in both sets of animals. We also analyzed

Journal of Hepatology 2015 vol. xxx j xxx–xxx

Please cite this article in press as: Acuña M et al. Transgenic overexpression of Niemann-Pick C2 protein promotes cholesterol gallstone formation in mice. J Hepatol (2015), http://dx.doi.org/10.1016/j.jhep.2015.10.002

JOURNAL OF HEPATOLOGY Table 2. Effect of lithogenic diet on gallbladder bile lipid composition in wild-type and Npc2.Tg mice.

Mice

Gallbladder bile lipid composition Mole% Ch Mole% BS Mole% PL

Diet

Wild-type Chow Lithogenic Npc2.Tg Chow Lithogenic

1.69 ± 0.51 3.80 ± 0.45a,b 1.47 ± 0.34 3.75 ± 1.06a,b

86.70 ± 4.12 85.38 ± 1.42 84.42 ± 7.49 85.93 ± 2.39

11.61 ± 3.83 10.81 ± 1.47 14.11 ± 7.18 10.32 ± 2.06

PL/ (BS + PL) 0.118 ± 0.039 0.112 ± 0.015b 0.143 ± 0.073a 0.107 ± 0.022b

Total lipids (g/dl) 7.27 ± 2.18 6.38 ± 0.98 8.17 ± 1.00 7.11 ± 1.98

Saturation index (%) 29.90 ± 19.88 95.52 ± 17.52a,b 30.55 ± 3.49 96.05 ± 34.48a,b

Values are expressed as means ± SD. Measurements were performed in 6 wild-type and 9 Npc2.Tg mice fed the chow diet and in 10 wild-type and 8 Npc2.Tg fed the lithogenic diet. aSignificantly different from wild-type mice fed the chow diet (p <0.05); bSignificantly different from Npc2.Tg mice fed the chow diet (p <0.05). Ch, cholesterol; BS, bile salts; PL, phospholipids.

A

B (% )

Cholesterol saturation index (%)

WT Npc2.Tg

ste

rol

150 Chow Lithogenic

2 PHASE

E

2 PHASE

Ch

50

1 PHASE

0 100

80 90 Bile salts (%)

0

70

Npc2.Tg

D

WT Npc2.Tg 100

Prevalence (%)

3 PHASE

10

*

75 50 25

ks

0.15

Chow Lithogenic

0 - 0.15 - 0.30

w

ee

ks

WT Npc2.Tg

4

ee w 3

2

w

ee

ks

0

E

Hydrophobicity index

C

ole

100

Phospholipids (%) 10 20 30 20 A B C D

Fig. 3. Cholesterol saturation index, prevalence of gallstone formation, and bile acid hydrophobicity index in wild-type and Npc2.Tg mice. (A) Cholesterol saturation index. (B) Relative lipid compositions of gallbladder biles of wild-type mice (n = 11) (r) and Npc2.Tg mice (n = 9) () fed with the lithogenic diet plotted on a condensed phase diagram according to Mole% of each (see Table 2). Onephase micellar zone is enclosed by a solid curved line. Above the micellar zone, two solid and two dashed lines divide the phase diagram into regions A to E with different crystallization sequences [44]. (C) Polarizing-light microscopy of gallbladder bile showing the crystallization sequences from lithogenic diet-fed Npc2.Tg mice. (D) Time-course cholesterol gallstone formation after feeding a lithogenic diet for 14 (n = 26 and 22 in wild-type (WT) and Npc2.Tg mice, respectively), 21 and 28 days (n = 9 mice in each experimental group). ⁄p <0.01 compared to wild-type mice fed for 14 days. (E) Bile salt hydrophobicity index in hepatic bile obtained from wild-type and Npc2.Tg mice fed with chow (n = 4 and 6, respectively for wild-type and NPC2.Tg mice) and lithogenic diets (n = 8 for wild-type and Npc2.Tg mice).

the formation of cholesterol gallstones after feeding the animals a lithogenic diet for 21 and 28 days, and we found that the differences between wild-type and Npc2.Tg mice decreased with increased feeding time. At 28 days, 100% of the animals of both groups had cholesterol gallstones (Fig. 3D). These results suggest that the differences observed in cholesterol gallstone formation between wild-type and Npc2.Tg are not

related to the cholesterol concentration in the bile and indicate that Npc2.Tg mice had increased susceptibility to cholesterol gallstone formation. Next, we analyzed biliary bile acid composition in the Npc2.Tg mice. No significant differences were found in the biliary bile salt hydrophobicity index between wild-type and Npc2.Tg mice fed the chow diet (Fig. 3E). As expected, after feeding the lithogenic diet there was an increase in the hydrophobicity of the biliary bile salts in both wild-type and Npc2.Tg mice (Fig. 3E), but no significant differences were detected between the two groups. In addition, no differences were observed in the ratio of muricholates to cholate in wild-type and Npc2.Tg mice fed the chow diet (Supplementary Fig. 2). These data show that the different crystallization behavior in wild-type and Npc2.Tg mice is not caused by the presence of more hydrophobic biliary bile salts in the Npc2.Tg mice on the lithogenic diet. We quantified the levels of NPC2 in bile using an ELISA assay. As expected, we found increased NPC2 levels in Npc2.Tg mice compared to wild-type mice fed the chow diet (105.18 ± 48.28 ng/ml and 561.88 ± 262.53a,b ng/ml in 6 wild-type and 6 Npc2.Tg mice, respectively) and the lithogenic diet (134.13 ± 53.92 ng/ml and 676.14 ± 282.35a,b ng/ml in 6 wild-type and 6 Npc2.Tg mice, respectively, ap <0.05 from wild-type mice fed the chow diet; b p <0.05 from wild-type mice fed the lithogenic diet). To analyze if the increased biliary NPC2 concentration in Npc2. Tg mice on the lithogenic diet is a specific phenomenon or if other proteins are also affected, we determined total protein and glycoprotein concentration in hepatic bile, and also levels of mucin-1 and albumin in gallbladder bile. No differences were observed in the biliary total protein concentration (2.35 ± 1.30 mg/ml and 2.50 ± 1.64 mg/ml in 8 wild-type and 9 Npc2.Tg mice, respectively) and the levels of mucin-1 and albumin analyzed by immunoblot were similar in both samples (Supplementary Fig. 3). In addition, no differences were observed in the concentration of the ConAbinding biliary glycoproteins (0.18 ± 0.07 mg/ml and 0.18 ± 0.06 mg/ml in 10 wild-type and 11 Npc2.Tg mice, respectively). In conclusion, our results strongly suggest that the higher concentration of NPC2 in bile is the main factor involved in the increased formation of biliary cholesterol gallstones observed in Npc2.Tg mice. NPC2 cholesterol crystal growth assays in model bile To corroborate the effect of NPC2 in gallstone formation, we performed cholesterol crystallization studies in model bile using a recombinant NPC2 protein. We purified a wild-type 6xHis tag recombinant NPC2, and filipin staining was used to evaluate if

Journal of Hepatology 2015 vol. xxx j xxx–xxx

5

Please cite this article in press as: Acuña M et al. Transgenic overexpression of Niemann-Pick C2 protein promotes cholesterol gallstone formation in mice. J Hepatol (2015), http://dx.doi.org/10.1016/j.jhep.2015.10.002

Research Article

1.5 1.0

a,b

0.5

+

N

y

N

N

Em

PC

2

pt

o N

an NP tib C o 2 N PC dy PC 2 2 wi an th tib ou od t y

2

in M

PC

ct ve

uc

A BS

ei pr

ot

or

0

3 2 1

an NP ti C N bod 2 N PC PC 2 y 2 wi an th tib ou od t y

+

N

PC

2

in M

N PC

2

y pt Em

o N

uc

r ve ct o

A BS

ot e

in

0

pr

Relative area under the curve

B

a,b a,b,c

n

A

Relative nucleation day

the recombinant protein was functional and capable of restoring normal cholesterol levels in NPC2-deficient fibroblasts (Supplementary Fig. 4). For nucleation assays, model bile was incubated with purified recombinant NPC2 at physiological concentrations. Nucleation assays were performed in supersaturated model bile in which the formation of crystals was reproducible in mode and time. The CSI of different model bile samples were near 100%. Different structures were observed as time progressed. First, we visualized liquid crystals, and then, we observed arcs, needles, spirals and tubular crystalline structures (data not shown). Later, the classic plate-like cholesterol monohydrate crystals and subsequently, the polymorphic and agglomerated cholesterol monohydrate crystals appeared (Supplementary Fig. 5). Wild-type NPC2 protein significantly accelerated cholesterol crystallization, similar to the mucin positive control (Fig. 4A). To confirm these results, we performed immunoprecipitation experiments incubating purified NPC2 protein with a NPC2 specific antibody, and the supernatant obtained from the immunoprecipitation was added to model bile. Bile without NPC2 was not able to accelerate the nucleation process, resulting in a nucleation time similar to that obtained with BSA, used as negative control (Fig. 4A). Among the

Fig. 4. Effect of wild-type recombinant NPC2 protein on nucleation time and crystal number in model bile. Model bile was incubated in different conditions: without protein, with BSA and with proteins obtained from cells transfected with an empty vector as negative controls; with mucin as a positive control, with recombinant NPC2, with NPC2 pre-incubated with a NPC2 antibody or elution buffer. (A) Relative day of nucleation (appearance of the first cholesterol monohydrate crystal in model bile with protein/appearance of the first cholesterol monohydrate crystal in model bile without protein). (B) Relative area under the crystal number curve (area under the curve of bile with protein/area under the curve of bile without protein). Each column in the graphs represent the average of five independent assays (each done in duplicate), with the exception of the immunoprecipitation experiments, which show the average of two independent assays (last two bars). aSignificantly different (p <0.02) from bile without protein; bSignificantly different (p <0.02) from bile with BSA and cSignificantly different (p <0.001) from bile with empty vector.

6

different samples of bile, there was no difference in size and shape of crystals. Fig. 4B shows the area under the curve of the crystal number, which represents the total number of cholesterol crystals obtained. Model bile without protein and with the empty vector had the lowest number of crystals, while the positive control mucin showed the highest number of crystals. Model bile with the recombinant NPC2 protein showed a tendency towards an increase number of crystals compared to the controls, although this change was not significant. There was no difference in CSI among model bile samples, so the difference in crystallization kinetics is most likely due to a pro-nucleating effect of the NPC2 protein and not to differences in the degree of bile saturation. These results indicate that recombinant NPC2 protein promotes cholesterol crystallization in model bile, suggesting that NPC2 acts as a pro-nucleating factor in bile.

Discussion The present study demonstrates that NPC2, a key protein involved in the hepatic lipoprotein endocytic pathway, is also relevant for controlling biliary cholesterol crystallization and gallstone formation induced by a lithogenic diet in mice. Using a well-established dietary model for lithogenesis, we showed that mice with hepatic NPC2 overexpression exhibited increased diet-induced gallstone formation without differences in bile acid hydrophobicity and gallbladder cholesterol saturation indices compared to wild-type mice. In addition, recombinant NPC2 decreased the nucleation time in model bile. To evaluate the role of NPC2 in hepatic cholesterol homeostasis and transport, we developed an Npc2 transgenic mouse with preferential expression in the liver. Npc2.Tg mice showed increased hepatic Npc2 mRNA and protein expression as well as increased NPC2 levels in bile, suggesting that the liver is the primary source of the biliary protein. This is consistent with earlier findings that suggest that some proteins found in bile are not found in homogenates derived from gallbladder and come mainly from the liver [18]. As expected, liver weight and hepatic cholesterol content were increased in wild-type and Npc2.Tg mice fed the lithogenic diet compared with animals fed the chow diet. Total cholesterol liver content was decreased in Npc2.Tg mice compared to wildtype mice when the animals were fed a lithogenic diet. One possible explanation is that increased NPC2 levels would produce a larger output of cholesterol from lysosomes, increasing the availability of cholesterol to be transported to other organs. It is noteworthy that this decrease in hepatic cholesterol was observed only in transgenic mice fed the lithogenic diet, suggesting that although NPC2 is necessary to mediate cholesterol efflux from lysosomes, it is not the limiting factor. In this sense, the NPC1 transmembrane protein that participates with NPC2 to mediate cholesterol efflux from lysosomes emerges as the limiting step in this pathway. Indeed, in vitro studies showed that NPC1 has a cholesterol transfer rate significantly lower than NPC2 [40]. Additionally, bidirectional transfer of cholesterol between NPC1 and model membranes is accelerated >100-fold by NPC2 [40]. NPC2 hepatic overexpression did not alter the secretion of bile lipids, but both animals responded to the lithogenic diet increasing their secretion of cholesterol, bile salts and phospholipids, as has been reported in several mouse models [14,32,41]. Accordingly, both animals fed a lithogenic diet have increased

Journal of Hepatology 2015 vol. xxx j xxx–xxx

Please cite this article in press as: Acuña M et al. Transgenic overexpression of Niemann-Pick C2 protein promotes cholesterol gallstone formation in mice. J Hepatol (2015), http://dx.doi.org/10.1016/j.jhep.2015.10.002

JOURNAL OF HEPATOLOGY expression of the cholesterol and bile salts canalicular transport proteins Abcg5/g8 and Abcb11. In addition, there was increased expression of the phospholipids transporter Abcb4 in both animals, but this increase was only significant for wild-type mice. These results differ from the work of Yamanashi et al. in which they overexpressed NPC2 using an adenovirus and found an increased cholesterol secretion into bile [13]. These differences may be due to the different mouse strains used and the transient expression with adenovirus compared to stable expression in the transgenic mice. On the other hand, we have previously shown a decrease in cholesterol secretion and crystallization in NPC2-deficient mice fed a lithogenic diet [14], whereas in this work we found that both cholesterol secretion and the CSI do not change in Npc2.Tg mice on the lithogenic diet. These results suggest, as previously discussed, that although NPC2 is necessary to mediate cholesterol efflux from lysosomes, it is not the limiting factor, being NPC1 the protein that has this property. Supporting this idea, hepatic NPC1 overexpression by adenovirus-mediated gene transfer increased biliary cholesterol secretion by 100–150% in both wild-type and cholesterol-fed Npc1-deficient mice [42]. Npc2.Tg animals exhibited a marked increase in cholesterol gallstone formation, indicating that they are clearly more susceptible to a lithogenic diet than wild-type mice. However, no differences in the concentration of biliary lipids, bile acid hydrophobicity and cholesterol saturation indices of gallbladder bile were detected between Npc2.Tg and wild-type mice after 14 days of feeding with the lithogenic diet. Remarkably, bile lipid composition of Npc2.Tg and wild-type mice on the lithogenic diet remains in the one phase zone of the ternary phase diagram, and not in the left two phase or central three phase regions in which bile lipid composition allows cholesterol crystallization [43,44]. These results appear to contrast with the cholesterol crystallization that we found. However, this could probably be explained because bile lipid composition measurements were performed on animals without cholesterol gallstones to avoid difficulties in obtaining reliable biliary lipid measurements due to the presence of excess precipitated cholesterol. Our results show that Npc2.Tg and wild-type mice have similar relative lipid compositions located in the one phase area near the phase 2 area denoted as region B. In addition, no differences were found in the total protein concentration in bile and the levels of specific proteins, such as albumin and the pro-nucleating mucin-1, suggesting that the increase in biliary NPC2 levels is a specific phenomenon in the Npc2.Tg mice. However, many other proteins, such as IgG, haptoglobin, aminopeptidase N and mucins (e.g. mucin-5ac), that have been claimed to promote biliary cholesterol crystallization, were not measured in this study [22–24,28]. In addition, proposed antinucleating proteins, such as apolipoproteins A-1 and A-2 could also be evaluated [45]. Other studies, including proteomic analysis of bile from Npc2.Tg mice, are required to further evaluate this matter. The increased susceptibility of the Npc2.Tg mice was observed at early times after feeding with a lithogenic diet; however after 28 days of feeding both the Npc2.Tg and wild-type mice had equal prevalence for gallstone formation (100% in both). The results obtained suggest that NPC2 in bile increases cholesterol precipitation and acts as a pro-nucleating protein. The relevance of cholesterol pro-nucleating proteins is evidenced in studies that analyze bile from patients who have the same

degree of cholesterol saturation but present large differences in cholesterol crystallization kinetics and in the number of gallstones [17,20,21]. It has been found that many non-lithiasic patients have cholesterol supersaturated gallbladder bile, suggesting that supersaturation of gallbladder bile is not the only determinant for cholesterol crystallization [17–19]. Other studies have shown that when ConA-binding biliary proteins isolated from patients with high lithiasis incidence were added to bile from patients with low lithiasis incidence, cholesterol crystallization was strongly induced [20]. Regarding this, we have previously shown that NPC2 is part of the ConA-binding fraction in human bile [9]. To better understand the function of NPC2 in bile and gallstone formation, we evaluated the effect of NPC2 in an in vitro cholesterol crystal formation assay [46,47]. Notably, recombinant NPC2 was able to accelerate nucleation in bile without affecting the total number of cholesterol crystals formed. There were no differences in CSI among the model bile samples, suggesting that the difference in crystallization kinetics was due to pronucleating effect of NPC2. Furthermore, immunoextraction of recombinant NPC2 protein increased the nucleation time, indicating that NPC2 was responsible for the acceleration in cholesterol crystallization. These results agree with those obtained with Npc2.Tg mice, suggesting that NPC2 has a pro-nucleating function in gallbladder bile. Additionally, our laboratory demonstrated that NPC2 content is significantly greater in the bile of gallstone-susceptible mice compared to gallstone-resistant mice, while hepatic NPC2 levels are similar [9]. Yamanashi et al. also described increased NPC2 levels in human bile that were positively correlated with biliary cholesterol concentration [13]. NPC2 has not been found in two proteomics analysis of biliary proteins related to cholesterol gallstones in patients [48,49]. It is possible that, in these patients, the pathogenesis of the disease is more related to hypersecretion/hypersaturation of gallbladder bile cholesterol rather than an increased amount of pro-nucleating factors. The crystallization sequences observed in this study in both native and model bile were in agreement with previous descriptions. The liquid crystals appear first, followed by arcs, needles, spirals and tubular crystalline structures. As time progressed, the classic plate-like cholesterol monohydrate crystals and subsequently, the polymorphic and agglomerated cholesterol monohydrate crystals appear [43]. It was postulated that phospholipid vesicles are the major vehicle for cholesterol precipitation in bile as well as an important determinant of the cholesterol nucleation time of bile [50]. In this model, cholesterol would precipitate from the vesicles and crystal formation would be a consequence of fusion and aggregation of these cholesterol rich vesicles [51]. These are two critical events in the complex pathophysiological cascade of cholesterol gallstone formation [52]. We postulate that NPC2 produces thermodynamic alterations in the model bile system, destabilizing the vesicles, promoting its fusion and aggregation, and therefore, accelerating the cholesterol crystallization process. Furthermore, there is a study that reported a fusogenic activity of NPC2, which means that NPC2 induces membrane fusion [53]. One possibility would be the direct interaction of NPC2 with the lipid bilayer of vesicles. Interestingly, Cheruku et al., using cholesterol transfer assays between purified NPC2 protein and phospholipids vesicles, demonstrated that NPC2 transports cholesterol rapidly to phospholipid vesicles via a collisional mechanism, which involves a direct interaction with the

Journal of Hepatology 2015 vol. xxx j xxx–xxx

7

Please cite this article in press as: Acuña M et al. Transgenic overexpression of Niemann-Pick C2 protein promotes cholesterol gallstone formation in mice. J Hepatol (2015), http://dx.doi.org/10.1016/j.jhep.2015.10.002

Research Article acceptor membrane. The effective interaction between the protein and the membrane is rate-limiting for ligand transfer [5,54]. The experimental findings of this work could be translated to human studies. We have previously reported that NPC2 is present in hepatic and gallbladder human bile [9]. In addition, Yamanashi et al. found a positive association between NPC2 and cholesterol levels in human bile [13]. Therefore, NPC2 could favor cholesterol crystallization in patients with increased cholesterol levels in bile and could also play an important role in patients who do not have or have little gallbladder bile cholesterol hypersaturation, where pro-nucleating factors are preponderant. Therefore, eventually, NPC2 levels in bile could be used as a marker for cholesterol pro-nucleating activity in patients. In conclusion, our in vivo and in vitro results strongly suggest that NPC2 plays an important role in cholesterol gallstone formation and acts as a pro-nucleating factor. This finding provides new knowledge regarding the pathogenesis of biliary lithiasis with potential human applications. Financial support This study was supported by grants from the Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) (grant numbers 1070622 and 1110310 to SZ, and 1130303 to JFM); Fondo Nacional de Desarrollo de Areas Prioritarias, FONDAP, Project no. 15090007, Center for Genome Regulation (CGR) to SZ and JFM; Comisión Nacional de ciencia y tecnología (CONICYT) PhD student grant # 21120490 to MA. Conflict of interest The authors declare that they have no conflict of interest in relation to this manuscript. Authors’ contributions Mariana Acuña, Lila González-Hódar, Juan Francisco Miquel and Silvana Zanlungo conceived, designed and coordinated the study. Mariana Acuña, Lila González-Hódar, M. Gabriela Morales, Juan Young, Ludwig Amigo and Juan Castro performed the experiments and were responsible for data acquisition and analysis. Mariana Acuña, Lila González-Hódar, Ludwig Amigo, Juan Castro, Gonzalo I. Cancino, Albert K. Groen, Juan Francisco Miquel and Silvana Zanlungo participated in the analysis and interpretation of data. Mariana Acuña, Lila González-Hódar, Gonzalo I. Cancino, Albert K. Groen, Juan Francisco Miquel and Silvana Zanlungo participated in the drafting of the article and/or in revising it critically.

Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2015.10.002. References [1] Naureckiene S, Sleat DE, Lackland H, Fensom A, Vanier MT, Wattiaux R, et al. Identification of HE1 as the second gene of Niemann-Pick C disease. Science 2000;290:2298–2301.

8

[2] Friedland N, Liou HL, Lobel P, Stock AM. Structure of a cholesterol-binding protein deficient in Niemann-Pick type C2 disease. Proc Natl Acad Sci U S A 2003;100:2512–2517. [3] Ko DC, Binkley J, Sidow A, Scott MP. The integrity of a cholesterol-binding pocket in Niemann-Pick C2 protein is necessary to control lysosome cholesterol levels. Proc Natl Acad Sci U S A 2003;100:2518–2525. [4] Babalola JO, Wendeler M, Breiden B, Arenz C, Schwarzmann G, LocatelliHoops S, et al. Development of an assay for the intermembrane transfer of cholesterol by Niemann-Pick C2 protein. Biol Chem 2007;388:617–626. [5] Cheruku SR, Xu Z, Dutia R, Lobel P, Storch J. Mechanism of cholesterol transfer from the Niemann-Pick type C2 protein to model membranes supports a role in lysosomal cholesterol transport. J Biol Chem 2006;281:31594–31604. [6] Kwon HJ, Abi-Mosleh L, Wang ML, Deisenhofer J, Goldstein JL, Brown MS, et al. Structure of N-terminal domain of NPC1 reveals distinct subdomains for binding and transfer of cholesterol. Cell 2009;137:1213–1224. [7] Kirchhoff C, Osterhoff C, Young L. Molecular cloning and characterization of HE1, a major secretory protein of the human epididymis. Biol Reprod 1996;54:847–856. [8] Larsen LB, Ravn P, Boisen A, Berglund L, Petersen TE. Primary structure of EPV20, a secretory glycoprotein containing a previously uncharacterized type of domain. Eur J Biochem 1997;243:437–441. [9] Klein A, Amigo L, Retamal MJ, Morales MG, Miquel JF, Rigotti A, et al. NPC2 is expressed in human and murine liver and secreted into bile: potential implications for body cholesterol homeostasis. Hepatology 2006;43: 126–133. [10] Busso D, Onate-Alvarado MJ, Balboa E, Castro J, Lizama C, Morales G, et al. Spermatozoa from mice deficient in Niemann-Pick disease type C2 (NPC2) protein have defective cholesterol content and reduced in vitro fertilising ability. Reprod Fertil Dev 2014;26:609–621. [11] Zanlungo S, Rigotti A. Determinants of transhepatic cholesterol flux and their relevance for gallstone formation. Liver Int 2009;29:323–330. [12] van der Velde AE, Brufau G, Groen AK. Transintestinal cholesterol efflux. Curr Opin Lipidol 2010;21:167–171. [13] Yamanashi Y, Takada T, Yoshikado T, Shoda J, Suzuki H. NPC2 regulates biliary cholesterol secretion via stimulation of ABCG5/G8-mediated cholesterol transport. Gastroenterology 2011;140:1664–1674. [14] Balboa E, Morales G, Aylwin P, Carrasco G, Amigo L, Castro J, et al. NiemannPick C2 protein expression regulates lithogenic diet-induced gallstone formation and dietary cholesterol metabolism in mice. Lipids 2012;47: 13–25. [15] Wang HH, Portincasa P, Liu M, Tso P, Samuelson LC, Wang DQ. Effect of gallbladder hypomotility on cholesterol crystallization and growth in CCKdeficient mice. Biochim Biophys Acta 2010;1801:138–146. [16] Portincasa P, Di Ciaula A, Vendemiale G, Palmieri V, Moschetta A, VanbergeHenegouwen GP, et al. Gallbladder motility and cholesterol crystallization in bile from patients with pigment and cholesterol gallstones. Eur J Clin Invest 2000;30:317–324. [17] Tudyka J, Kratzer W, Kuhn K, Mason R, Wechsler JG, Adler G. Solitary versus multiple gallstones: the importance of total biliary protein concentration and other factors. Hepatogastroenterology 1995;42:638–644. [18] van Erpecum KJ, Wang DQ, Lammert F, Paigen B, Groen AK, Carey MC. Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: soluble pronucleating proteins in gallbladder and hepatic biles. J Hepatol 2001;35:444–451. [19] Groen AK, Noordam C, Drapers JA, Egbers P, Jansen PL, Tytgat GN. Isolation of a potent cholesterol nucleation-promoting activity from human gallbladder bile: role in the pathogenesis of gallstone disease. Hepatology 1990;11: 525–533. [20] Miquel JF, Van Der Putten J, Pimentel F, Mok KS, Groen AK. Increased activity in the biliary Con A-binding fraction accounts for the difference in crystallization behavior in bile from Chilean gallstone patients compared with Dutch gallstone patients. Hepatology 2001;33:328–332. [21] Jirsa M, Groen AK. Role of biliary proteins and non-protein factors in kinetics of cholesterol crystallisation and gallstone growth. Front Biosci 2001;6: E154–E167. [22] Upadhya GA, Harvey PR, Strasberg SM. Effect of human biliary immunoglobulins on the nucleation of cholesterol. J Biol Chem 1993;268:5193–5200. [23] Yamashita G, Ginanni Corradini S, Secknus R, Takabayashi A, Williams C, Hays L, et al. Biliary haptoglobin, a potent promoter of cholesterol crystallization at physiological concentrations. J Lipid Res 1995;36: 1325–1333. [24] Nunez L, Amigo L, Rigotti A, Puglielli L, Mingrone G, Greco AV, et al. Cholesterol crystallization-promoting activity of aminopeptidase-N isolated from the vesicular carrier of biliary lipids. FEBS Lett 1993;329:84–88.

Journal of Hepatology 2015 vol. xxx j xxx–xxx

Please cite this article in press as: Acuña M et al. Transgenic overexpression of Niemann-Pick C2 protein promotes cholesterol gallstone formation in mice. J Hepatol (2015), http://dx.doi.org/10.1016/j.jhep.2015.10.002

JOURNAL OF HEPATOLOGY [25] van den Berg AA, van Buul JD, Tytgat GN, Groen AK, Ostrow JD. Mucins and calcium phosphate precipitates additively stimulate cholesterol crystallization. J Lipid Res 1998;39:1744–1751. [26] Lee TJ, Smith BF. Bovine gallbladder mucin promotes cholesterol crystal nucleation from cholesterol-transporting vesicles in supersaturated model bile. J Lipid Res 1989;30:491–498. [27] Levy PF, Smith BF, LaMont JT. Human gallbladder mucin accelerates nucleation of cholesterol in artificial bile. Gastroenterology 1984;87: 270–275. [28] Wang HH, Afdhal NH, Gendler SJ, Wang DQ. Targeted disruption of the murine mucin gene 1 decreases susceptibility to cholesterol gallstone formation. J Lipid Res 2004;45:438–447. [29] Keulemans YC, Mok KS, Gouma DJ, Groen AK. The role of the Concanavalin Abinding fraction in cholesterol crystallization in native human bile. J Hepatol 1997;27:1041–1050. [30] Kristiansen TZ, Bunkenborg J, Gronborg M, Molina H, Thuluvath PJ, Argani P, et al. A proteomic analysis of human bile. Mol Cell Proteomics 2004;3: 715–728. [31] Simonet WS, Bucay N, Lauer SJ, Taylor JM. A far-downstream hepatocytespecific control region directs expression of the linked human apolipoprotein E and C-I genes in transgenic mice. J Biol Chem 1993;268:8221–8229. [32] Morales MG, Amigo L, Balboa E, Acuna M, Castro J, Molina H, et al. Deficiency of Niemann-Pick C1 protein protects against diet-induced gallstone formation in mice. Liver Int 2010;30:887–897. [33] Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem 1974;20:470–475. [34] Talalay P. Enzymic analysis of steroid hormones. Methods Biochem Anal 1960;8:119–143. [35] Baginski ES, Foa PP, Zak B. Microdetermination of inorganic phosphate, phospholipids, and total phosphate in biologic materials. Clin Chem 1967;13:326–332. [36] Carey MC. Critical tables for calculating the cholesterol saturation of native bile. J Lipid Res 1978;19:945–955. [37] Vrieze A, Out C, Fuentes S, Jonker L, Reuling I, Kootte RS, et al. Impact of oral vancomycin on gut microbiota, bile acid metabolism, and insulin sensitivity. J Hepatol 2014;60:824–831. [38] Heuman DM. Quantitative estimation of the hydrophilic-hydrophobic balance of mixed bile salt solutions. J Lipid Res 1989;30:719–730. [39] Garver WS, Heidenreich RA, Erickson RP, Thomas MA, Wilson JM. Localization of the murine Niemann-Pick C1 protein to two distinct intracellular compartments. J Lipid Res 2000;41:673–687. [40] Infante RE, Wang ML, Radhakrishnan A, Kwon HJ, Brown MS, Goldstein JL. NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes. Proc Natl Acad Sci U S A 2008;105:15287–15292.

[41] Repa JJ, Berge KE, Pomajzl C, Richardson JA, Hobbs H, Mangelsdorf DJ. Regulation of ATP-binding cassette sterol transporters ABCG5 and ABCG8 by the liver X receptors alpha and beta. J Biol Chem 2002;277:18793–18800. [42] Amigo L, Mendoza H, Castro J, Quinones V, Miquel JF, Zanlungo S. Relevance of Niemann-Pick type C1 protein expression in controlling plasma cholesterol and biliary lipid secretion in mice. Hepatology 2002;36:819–828. [43] Wang DQ, Carey MC. Characterization of crystallization pathways during cholesterol precipitation from human gallbladder biles: identical pathways to corresponding model biles with three predominating sequences. J Lipid Res 1996;37:2539–2549. [44] Wang DQ, Carey MC. Complete mapping of crystallization pathways during cholesterol precipitation from model bile: influence of physical-chemical variables of pathophysiologic relevance and identification of a stable liquid crystalline state in cold, dilute and hydrophilic bile salt-containing systems. J Lipid Res 1996;37:606–630. [45] Kibe A, Holzbach RT, LaRusso NF, Mao SJ. Inhibition of cholesterol crystal formation by apolipoproteins in supersaturated model bile. Science 1984;225:514–516. [46] Wang DQ, Cohen DE, Carey MC. Biliary lipids and cholesterol gallstone disease. J Lipid Res 2009;50:S406–S411. [47] Portincasa P, Moschetta A, van Erpecum KJ, Calamita G, Margari A, vanBergeHenegouwen GP, et al. Pathways of cholesterol crystallization in model bile and native bile. Dig Liver Dis 2003;35:118–126. [48] Zhou H, Chen B, Li RX, Sheng QH, Li SJ, Zhang L, et al. Large-scale identification of human biliary proteins from a cholesterol stone patient using a proteomic approach. Rapid Commun Mass Spectrom 2005;19: 3569–3578. [49] Zhang D, Xiang J, Wang L, Xu Z, Sun L, Zhou F, et al. Comparative proteomic analysis of gallbladder bile proteins related to cholesterol gallstones. PLoS One 2013;8:e54489. [50] Peled Y, Halpern Z, Baruch R, Goldman G, Gilat T. Cholesterol nucleation from its carriers in human bile. Hepatology 1988;8:914–918. [51] Kibe A, Dudley MA, Halpern Z, Lynn MP, Breuer AC, Holzbach RT. Factors affecting cholesterol monohydrate crystal nucleation time in model systems of supersaturated bile. J Lipid Res 1985;26:1102–1111. [52] Miquel JF, Nunez L, Rigotti A, Amigo L, Brandan E, Nervi F. Isolation and partial characterization of cholesterol pronucleating hydrophobic glycoproteins associated to native biliary vesicles. FEBS Lett 1993;318:45–49. [53] Abdul-Hammed M, Breiden B, Adebayo MA, Babalola JO, Schwarzmann G, Sandhoff K. Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion. J Lipid Res 2010;51:1747–1760. [54] Xu Z, Farver W, Kodukula S, Storch J. Regulation of sterol transport between membranes and NPC2. Biochemistry 2008;47:11134–11143.

Journal of Hepatology 2015 vol. xxx j xxx–xxx

9

Please cite this article in press as: Acuña M et al. Transgenic overexpression of Niemann-Pick C2 protein promotes cholesterol gallstone formation in mice. J Hepatol (2015), http://dx.doi.org/10.1016/j.jhep.2015.10.002