Biochimica et Biophysica Acta 1832 (2013) 1482–1491

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Biochimica et Biophysica Acta

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Fasting reduces liver fibrosis in a mouse model forchronic cholangiopathies

Aleksandar Sokolović a, Cindy P.A.A. van Roomen b, Roelof Ottenhoff b, Saskia Scheij b, Johan K. Hiralall a,Nike Claessen c, Jan Aten c, Ronald P.J. Oude Elferink a, Albert K. Groen d, Milka Sokolović b,⁎a Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlandsb Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlandsc Pathology Department, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlandsd Department of Paediatrics, Centre for Liver, Digestive and Metabolic Diseases, University Medical Centre, Groningen, The Netherlands

Abbreviations: 36b4, ribosomal protein, large, P0; Apaffactor 1; Bax, BCL2-associated X protein; Bcl-xl, BCL2-lcontaining, mucin-like, hormone receptor-like sequence 1tein; Hprt, hypoxanthine guanine phosphoribosyl transfermonoclonal antibody Ki 67; Mac1, integrin alpha M;Mcp12; MFs, myofibroblasts; Mpo, myeloperoxidase; p21, cyc1A; Pcna, proliferating cell nuclear antigen; Plat, plasminominogen activator, urokinase; Tgfβ1, transforming growinhibitor of metalloproteinase; α-Sma, actin, alpha 2, smo⁎ Corresponding author at: Department of Medical Bi

Center, University of Amsterdam,Meibergdreef 15, 1105ATel.: +31 646412482.

E-mail addresses: asokolovic@gmail.com (A. Sokolov(C.P.A.A. van Roomen), r.ottenhoff@amc.uva.nl (R. Otten(S. Scheij), k.hiralall@amc.uva.nl (J.K. Hiralall), n.claessej.aten@amc.uva.nl (J. Aten), r.p.oude-elferink@amc.uva.na.k.groen@med.umcg.nl (A.K. Groen), milkasokolovic@g

0925-4439/$ – see front matter © 2013 Elsevier B.V. Alhttp://dx.doi.org/10.1016/j.bbadis.2013.05.012

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 November 2012Received in revised form 6 May 2013Accepted 14 May 2013Available online 22 May 2013

Keywords:Liver fibrosisFastingMatrix remodelingInflammation

Chronic cholangiopathies often lead to fibrosis, as a result of a perpetuated wound healing response, charac-terized by increased inflammation and excessive deposition of proteins of the extracellular matrix. Our pre-vious studies have shown that food deprivation suppresses the immune response, which led us to postulateits beneficial effects on pathology in liver fibrosis driven by portal inflammation. We investigated the conse-quences of fasting on liver fibrosis in Abcb4−/− mice that spontaneously develop it due to a lack of phospho-lipids in bile. The effect of up to 48 h of food deprivation was studied by gene expression profiling, (immuno)histochemistry, and biochemical assessments of biliary output, and hepatic and plasma lipid composition. Incontrast to increased biliary output in the wild type counterparts, bile composition in Abcb4−/− miceremained unchanged with fasting and did not influence the attenuation of fibrosis. Markers of inflammation,however, dramatically decreased in livers of Abcb4−/− mice already after 12 h of fasting. Reduced presenceof activated hepatic stellate cells and actively increased tissue remodeling further propelled a decrease inparenchymal fibrosis in fasting. This study is the first to show that food deprivation positively influencesliver pathology in a fibrotic mouse model for chronic cholangiopathies, opening a door for new strategiesto improve liver regeneration in chronic disease.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Chronic cholangiopathies, like primary sclerosing cholangitis (PSC),are caused by progressive inflammation and scarring of the bile ducts inthe liver [1]. The inflammation impedes the flow of bile to the gut,which can lead to liver fibrosis (and cirrhosis as its ultimate stage),

, apoptotic peptidase activatingike 1; F4/80, EGF-like module; Gfap, glial fibrillary acidic pro-ase; Ki67, antigen identified by, chemokine (C-Cmotif) ligandlin-dependent kinase inhibitorgen activator, tissue; Plau, plas-th factor, beta 1; Timp, tissueoth muscleochemistry, Academic MedicalZ Amsterdam, The Netherlands.

ić), c.vanroomen@amc.uva.nlhoff), s.scheij@amc.uva.nln@amc.uva.nl (N. Claessen),l (R.P.J. Oude Elferink),mail.com (M. Sokolović).

l rights reserved.

the final common phase of all chronic liver diseases. In general, liver in-jury is followed by a wound healing response, characterized by com-pensatory proliferation, inflammation and deposition of extracellularmatrix (ECM) [2]. Initially, fibrosis is reversible — normal architectureis restored by fibrolysis, and ECM producing cells are removed by apo-ptosis [3,4]. Recurrent chronic injury, however, results in an imbalancebetween fibrogenesis and fibrolysis, leading to scar formation, architec-tural distortion, cirrhosis and liver failure [5,6]. Liver transplantationis the only effective treatment, but surgical contraindications and thelack of donors urge for interventions that halt disease progression.

Starvation causes complex physiological reaction, involving bothcentral and peripheral responses orchestrated by the nervous, endo-crine and digestive systems [7,8]. The goal is to conserve energy, delaygrowth processes, preserve cellular ATP levels, and minimize oxidativedamage. The IGF-1/Akt/FOXO pathway plays a central role in the regu-lation of this conserved response [9,10], supported by nutrient-sensingmTOR signaling [11]. When the reserves are low, there is necessity fora trade-off between the risk of starvation and disease [12,13]. It is notsurprising therefore that the suppression of the immune response isone of the major adaptations to food deprivation [14–16]. Given thatinflammation is a driving force in the pathogenesis of biliary fibrosis

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[17], we postulated that starvation-induced immune suppression couldalleviate the pathology in sclerosing cholangitis.

Intermittent fasting and caloric restriction in general have benefi-cial effect on health status of laboratory animals [18,19]. In acutely in-duced liver fibrosis, however, dietary restriction causes exacerbationof the phenotype and increases mortality rate [20,21]. The boostedmetabolic efficiency caused by dietary restriction [22], which increasestoxicity of CCl4, is a possible cause. To our knowledge, the effect of com-plete foodwithdrawal on chronically induced liver fibrosis has not beenaddressed before. To assess it, we used the Abcb4−/−mice in which thelack of ABCB4 (ATP-binding cassette, subfamily b, member 4) leadsto an absence of phosphatidylcholine in the bile, making it toxic [23].Bile salts accumulate in the intrahepatic biliary system disruptingtight junctions and basementmembranes of bile ducts, and bile leakageto the portal tract. This triggers a cascade leading to non-suppurativeinflammatory cholangitis, periductal fibrosis shortly after birth, andductular proliferation [24]. These mice also spontaneously developcholesterol cholecysto- and hepatolithiasis as a result of impaired cho-lesterol solubility in PC-deficient bile, and, eventually, hepatocellularcarcinomas [23,25,26]. Macroscopic and microscopic features of scle-rosing cholangitis in Abcb4−/− mice closely resemble those of humanPSC [24,27–29]. Notable similarities with PSC were recently furtheraccentuated by gene expression profiling [30]. In general, Abcb4−/−

mice are considered a model for human MDR3 deficiency rangingfrom progressive familial intrahepatic cholestasis type 3 to adult livercirrhosis [31].

Results of our study of fibrotic Abcb4−/− mouse livers, indeed,confirmed the hypothesis that food deprivation influenced the pa-thology, and revealed an ameliorating effect of as short as 12 h offasting on chronically-induced fibrosis.

2. Materials and methods

2.1. Animals

We used 3 months old male Abcb4−/− (FVB background), andWTFVB mice (Charles River, Maastricht, The Netherlands). Four animalsper group were used. The animals were kept separately in regularcages, and in grid bottom cages 24 h prior to and during fasting, toprevent coprophagia and intake of bedding. The study was approvedby the AMC Animal Experiments Committee.

2.2. Plasma and tissue collection and analytical procedures

The gallbladders in anesthetized mice (Hypnorm and Diazepam,1 ml/kg and 10 mg/kg respectively) were cannulated, bile was col-lected for 15 min, and stored at−20 °C. Blood samples were collectedby cardiac puncture and plasmawas stored at−20 °C. The livers werequickly excised and parts were snap-frozen in liquid nitrogen andstored at −80 °C, or fixed in 4% buffered formalin and embeddedin paraffin. Total liver collagen was determined by measuring hy-droxyproline content [32]. Plasma concentrations of total cholesterol,triglycerides, FFA and choline-containing PL were determined usingthe equivalent colorimetric enzymatic kits (Wako Chemicals GmbH,Neuss, Germany and bioMérieux Benelux, Boxtel, The Netherlands).Biliary BS and PL contentswere analyzed as described [33]. Biliary cho-lesterol was measured fluorescently [34]. Tissue lipids were extractedusing a chloroform–methanol-based (2:1 by volume) method [35];cholesterol and triglycerides were measured using the CHOD-PAP(Ecoline 25 cholesterol, Merck, Darmstadt, Germany) and GPO-PAPmethod (Ecoline S+, DiaSys Diagnostic Systems, Holzheim, Germany),and PL was measured with an enzymatic colorimetric assay (Biolabo,Maizy, France). To correct the obtained lipid values for the amountof tissue, the protein content of the liver was measured using theBCA method (Pierce, Perbio Science Nederland BV, Etten-Leur, TheNetherlands).

2.3. Histology and immunohistochemistry

Four animals per time point and five sections per animal wereanalyzed. Paraffin sections were dewaxed and stained with hematox-ylin and eosin (HE) for general histology, or with 0.2% picro-sirius red(PSR) to detect fibrillar collagen. Immunohistology was performedas described [36]. Sections were incubated with rat IgG2b anti-mouseF4/80 monoclonal antibody (AbD Serotec, Oxford, UK) and Ki67 (Dako,Glostrup, Denmark). Primary antibodies were detected using appro-priate secondary horseradish peroxidase-conjugated polyclonal goatIgG antibodies, directed against mouse IgG2a, or rat IgG2a (SouthernBiotech, Birmingham, AL). Bound peroxidase activity was visualizedusing H2O2 and 3,3′-diaminobenzidine as chromogen. Sections werecounterstained with hematoxylin.

2.4. Reverse transcription, quantitative PCR and data analyses

Total RNA was extracted from frozen livers with TRIzol reagent(Invitrogen, Breda, The Netherlands), and its quality was assessedwith RNA 6000 Nano LabChip® Kit in an Agilent 2100 bioanalyzer(Agilent Technologies, Palo Alto, USA). qPCR was performed as de-scribed [37]. The average of three house-keeping genes (36b4, Hprtand cyclophilin b) was used to normalize the expression of the stud-ied genes. mRNA concentration was calculated using the LinRegPCRprogram [38,39].

2.5. Western blot analysis

Western blot analysis was performed as described [37], usingantibodies against MMP2 andMMP9 (goat anti-mouse; Bio-Rad Labo-ratories, Veenendaal, The Netherlands), with goat anti-rabbit IgGhorseradish peroxidase conjugated (Santa Cruz Biotechnology) as sec-ondary antibody. Chemiluminescence was quantified by Lumi-ImagerF1 using CDP-Star (Roche, Mannheim, Germany), using β-actin as areference.

2.6. Statistical analysis

ANOVA and two-tailed Student's t-test were used to determinestatistical significance. P b 0.05 was set as significance threshold. Inall figures asterisks denote significant differences induced by fastingwithin a strain, hashtags represent significant differences betweenAbcb4−/− and WT at baseline condition, while error bars show thestandard deviations.

3. Results

3.1. Fasting reduces ECM deposition in the livers of Abcb4−/− mice

Abcb4−/− mice lost 10, 15 and 20% of the initial body weight in re-sponse to 12, 24 and 48 h of fasting, respectively. The rate of bodyweight loss was the same as in the WT animals (SupplementaryFig. 1A). As expected in fed condition, fibrotic livers of Abcb4−/−

mice were macroscopically more robust and stiff than those of the WTmice (Supplementary Fig. 1B). As we reported previously [40] thesteatotic appearance characteristic for fasting occurred in Abcb4−/−

animals later than in the WT. The reason can lie in the fact that fibrotictissue, with closed fenestrae and ECM deposits, hampers the transportto hepatocytes. Upon 48 h of fasting, Abcb4−/− livers obtained a flat-tened shape similar to that of the WT animals, but remained palpablyharder. The characteristic lack of phospholipids and cholesterol in thebile was reflected in its lighter coloration, but no other differenceswere observed (Supplementary Fig. 1C).

To investigate the effects of fasting on the fibrotic livers in Abcb4−/−

mice, we first assessed the connective tissue by picro-Sirius red staining(Fig. 1A). Parenchymal fibrosis was clearly reduced already after 12 h of

Fig. 1. Food deprivation ameliorates parenchymal fibrosis in the livers of Abcb4−/− mice. A) Representative Picro-sirius red immunostaining of liver sections of wild-type (WT) andAbcb4−/− mice, fed or starved for 12, 24 and 48 h. Scale bar represents 100 μm. B) Hydroxyproline concentration (expressed in μg/mg wet tissue) in Abcb4−/− andWT livers, 0, 12,24 h and 48 h after starvation started. C–I) Relative mRNA expression level of collagens 1, 3, 4 and fibronectin 1 (components of ECM), α-Sma and vimentin (markers of activatedHSC), and Gfap (marker of quiescent HSC), in livers at 0, 12, 24 and 48 h of fasting. mRNa expression is normalized by 3 house-keeping genes and shown as fold change comparedwith WT control at 0 h. Asterisks denote significance compared to fed condition in the corresponding strain (P b 0.05), hashtags represent significance between the Abcb4−/− andWT mine at baseline, and error bars show SD.

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fasting, causing a decrease of inter-portal bridging. Total liver collagen,responsible for the structural integrity of the ECM and used as a markerfor liver fibrogenesis [41], was determined by measuring hydroxypro-line content. In Abcb4−/− mice it decreased 40, 70 and 80% at 12, 24and 48 h of fasting, respectively, and was not affected in WT animals(Fig. 1B). mRNA expression of collagens 1 and 3 was lowered by fastingin both Abcb4−/− and WT mice (Fig. 1C–D). Expression of Col4 and fi-bronectin decreased at 12 and 24 h (Fig. 1E–F). These data strongly sug-gest a decrease in matrix deposition in response to fasting in Abcb4−/−

mice.Hepatic stellate cells (HSC) play a central role in ECM remodeling

by production of MMPs and TIMPs [42,43], and by deposition of fibril-lar collagens type 1 and 3 [43]. The impact of fasting was analyzed bymeasuring α-Sma and vimentin, markers for activated HSC, and Gfap,a marker of early activation of HSCs [44]. At the baseline, Gfap was5-fold lower in Abcb4−/− livers than in the WT (Fig. 1I), implyingan ongoing chronic disease [45], with activated HSC dominating infibrotic Abcb4−/− livers (Fig. 1G–H).With fasting, a significant decreasein expression levels was observed for all three markers in Abcb4−/−

mice (α-Sma and vimentin; Fig. 1F–I). In WT mice, these markerswere also downregulated after 12 and 24 h of fasting. In support, theexpression of fibronectin, which decreases starvation-induced apopto-sis in rat HSC [46], was downregulated (Fig. 1F).

A clearly different response at 48 h compared to previous timepoints does not come as a surprise [47], since such a long stretch inmice (comparable to several weeks in humans) provokes a complexsurvival response. Although we mainly focus on the effect of shortand moderate fasting, we provide the later time point to completethe picture of the response in fibrotic livers.

3.2. Fibrotic Abcb4−/− livers respond to fasting by matrix remodeling

To assess the mechanism behind the reduced amount of ECM, westudied the components of the matrix remodeling machinery. Giventhe long mRNA half-life of MMP 2 and 9 (46 to 150 h, depending onpresence of TGFβ1 [48]), we determined the protein expression levelsof these two metalloproteinases. They both increased at 24 and 48 h(Fig. 2A), suggesting an actively ongoing tissue remodeling. Gene ex-pression level of the counteracting Mmp13, which degrades collagen1 [45] to accelerate fibrogenesis in cholestatic livers [49], was stronglydownregulated at all time points (Fig. 2B), pointing further towardspromotion of fibrolysis. The expression levels of Plau and Plat, whichactivate MMPs [50], increased in prolonged fasting in Abcb4−/−

mice (80% Plat and 30% Plau; Fig. 2C–D), after an initial decrease inshort fasting. Food deprivation did not affect their gene expressionin WT animals. Timp1, which inhibits most MMP activity [51] and

Fig. 2. Starvation causes an active matrix remodeling in Abcb4−/− livers. A) Protein expression of MMP2 and -9, as obtained by Western-blot analysis of hepatic tissue lysates after0, 24 and 48 h of fasting in Abcb4−/− mice. B–F) Relative gene expression of Mmp13, Plau, Plat, Timp1 and Tgfβ1 at 0, 12, 24 h and 48 h of fasting in Abcb4−/− and WT mice. mRNaexpression is normalized by 3 house-keeping genes and shown as fold change compared with WT control at 0 h. Asterisks denote significance compared to fed condition in thecorresponding strain (P b 0.05), hashtags represent significance between the Abcb4−/− and WT mine at baseline, and error bars show SD.

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promotes the survival of HSC [52], was significantly downregulatedby fasting in fibrotic livers at all time points (Fig. 2E). Tgfβ1, whichpromotes hepatic fibrosis by prompting HSC differentiation intomyofibroblasts, was 2-fold higher in fed fibrotic than in WT livers(Fig. 2F). Compared to fed Abcb4−/−, Tgfβ1 expression was 75, 70and 50% lower at three fasting time points, respectively. Matrixremodeling in fasting, therefore, seems to be regulated by reducedexpression of profibrotic cytokine Tgfβ1 and thereby reduced numberof activated HSC/MFs.

3.3. Hepatic cell turnover seems reduced by fasting

Abcb4−/− mice have an intrinsically high hepatocyte proliferation[53]. We therefore analyzed the effect of fasting on proliferationmarkers Pcna and Ki67. They decreased in both Abcb4−/− and WTmice (Fig. 3B–C) and Pcna returned to control values in prolongedfasting. Immunostaining (Fig. 3A) confirmed that the fibrotic liversof Abcb4−/− mice contained cycling hepatocytes and Ki67-positivecells in the portal tract. Ki67-positive cells were not found in fastedAbcb4−/− livers at any time point, or in any WT livers. Fasting, there-fore, seemed to have reduced proliferation in Abcb4−/− livers. Thiswas supported by the increased cell-cycle arrest, since the expression

of p21, involved in repair of DNA damage, was strongly upregulated inAbcb4−/− mice (even 700% at 48 h; Fig. 3D).

The frequency of apoptotic changes (Councilman bodies) did notchange dramatically in Abcb4−/− mice upon fasting, especially in aci-nar zones 2 (Fig. 4A). Similarly, mRNA concentrations of proapoptoticmarkers Bax and Apaf barely changed in Abcb4−/− mice (Fig. 4B–C),while in the WT they significantly decreased with fasting (40–60%).Expression of antiapoptotic Bcl-xlwas unaffected by fasting in fibroticlivers, though it has strongly increased in prolonged fasting in WTmice (Fig. 4D). This suggests a possible different mechanism behindapoptosis between the highly proliferative livers of fed Abcb4−/−

mice, and those affected by fasting.

3.4. Biliary output and hepatic and plasma lipid composition in fastedAbcb4−/− mice

In contrast to the earlier reported increased biliary output infasted WT mice [40], bile flow in Abcb4−/− mice tended to decreaseafter 12 h (40%, P b 0.06; Table 1, Supplementary Fig. 2). As expected,PL were barely measurable in Abcb4−/− animals. Cholesterol concen-tration was 2.7–6.5 fold lower during fasting compared to WT, whilenone of the measured biliary secretion rates – BS, cholesterol or PL –

Fig. 3. Fasting reduces originally high cell turnover in fibrotic livers of Abcb4−/− mice. A) Immunostaining for Ki67 in the liver sections of fed and 12 h fasted WT and Abcb4−/−

mice. Scale bar represents 100 μm. B–D) Gene expression of Ki67, Pcna and p21 upon 0, 12, 24 and 48 h of food deprivation. mRNa expression is normalized by 3 house-keepinggenes and shown as fold change compared with WT control at 0 h. Asterisks denote significance compared to fed condition in the corresponding strain (P b 0.05), hashtagsrepresent significance between the Abcb4−/− and WT mine at baseline, and error bars show SD.

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was affected by fasting, indicating that the Abcb4−/− mice had lostthe adaptive biliary response to fasting seen in FVB animals.

Cholesterol concentration in the liver of Abcb4−/− mice increasedduring fasting (Table 1, Supplementary Fig. 3), hepatic triglycerideconcentration was strongly increased, while that of PL did not change.Hepatic TG and cholesterol concentrations were somewhat lower infed Abcb4−/− mice than in their WT counterparts, but fasting alteredthem so that concentrations of both were higher in fibrotic mice. Thereason for this discrepancy could lay in the fact that lower amounts ofcholesterol are released into the bile of Abcb4−/− mice.

To assess the lipid status in the systemic circulation of fastedAbcb4−/− mice, we measured the plasma concentrations of choles-terol, TG, FFA and PL (Table 1, Supplementary Fig. 4). Similarly toWT, plasma cholesterol concentration increased after 12 and 24 h offasting, TG concentration strongly decreased in longer fasting, PLconcentrations remained stable, while FFA concentration increased(50 and 25% after 12 and 24 h). Initially, plasma cholesterol level infed Abcb4−/− mice was two-fold lower than in theWT, and remainedsuch in fasting.

3.5. Fasting reduces hepatic inflammation in Abcb4−/− mice

Bile duct proliferation and portal inflammation [23] are the mostprominent histopathological features of Abcb4−/− mice and arecaused by secretion of toxic bile [54,55]. To assess the effect of fastingon portal inflammation, we analyzed the presence of inflammatorycells in livers of Abcb4−/− andWT mice. Markers for activated mono-cytes andmacrophages (Cd11b; Cd11c), neutrophils (Mpo), circulating

monocytes (Mcp1), and liver-resident Kupffer cells (F4/80), as well asinflammatorymarker Tnfα, were clearly (and expectedly) higher in fi-brotic than in control livers (Fig. 5A–F;Mpo andMcp1 not shown). In-flammation seemed attenuated in fasted fibrotic livers by significantlylowered expression of general markers F4/80, Cd11b and Cd11c. In fi-brotic livers of fed Abcb4−/− mice, immunostaining for F4/80 showedareas with abundant periportal inflammation, with large numbers ofF4/80 positive macrophages and relatively few F4/80 positive Kupffercells in the adjacent lobules (Fig. 6, top right panels). More Kupffercells were present in the areas where periportal inflammation wasless abundant. A striking shift in localization of F4/80 macrophageswas observed at 12 and 24 h of fasting (Fig. 6, two mid-right panels).The periportal tracts contained far less F4/80 macrophages, whereastheir number was increased in acinar zone 2. Upon 24 and 48 h offasting the number of macrophages was further reduced, with adher-ent F4/80 macrophages now often present in central veins (Fig. 6, twobottom-right panels). In WT mice, the relative number of F4/80 posi-tive Kupffer cells decreased only after 48 h of starvation (Fig. 6, leftpanels). A decrease in expression of proinflammatory marker Irf5that promotes inflammatorymacrophage polarization [56], and an in-crease of Ym1, a marker for alternatively activated macrophages, indi-cate a shift towards alternatively activated macrophages in fastedfibrotic livers (Fig. 5E–F).

4. Discussion

This study reveals that fasting causes a reduction of liver fibrosis ina mouse model for biliary cholangiopathies. Rapid adaptive response

Fig. 4. Apoptosis in fasted fibrotic livers. A) Hematoxylin–eosin staining of liver sections of fed and fasted WT and Abcb4−/− mice. Scale bar represents 100 μm. B–D) Relative mRNAexpression level of Bax, Apaf and Bcl-xl, after 0, 12, 24 h and 48 h of fasting. mRNa expression is normalized by 3 house-keeping genes and shown as fold change compared with WTcontrol at 0 h Asterisks denote significance compared to fed condition in the corresponding strain (P b 0.05), hashtags represent significance between the Abcb4−/− and WT mineat baseline, and error bars show SD.

Table 1Biliary output and hepatic and plasma lipid composition in up to 48 h fasted Abcb4−/− and wild type mice. The values are given as mean ± S.D.

Abcb4−/− WT

0 h 12 h 24 h 48 h 0 h 12 h 24 h 48 h

Biliary lipidsBile flow (μl/min·100 g) 9.5 ± 1.64 5.6 ± 0.29⁎⁎ 6.6 ± 1.00 7.4 ± 1.14 3.7 ± 0.9 6.5 ± 0.4⁎ 6.2 ± 0.8⁎ 5.1 ± 0.6Bile salts (nmol/min·100 g) 272 ± 87 197 ± 30 260 ± 50 398 ± 85 168 ± 36 267 ± 27⁎ 228 ± 7 367 ± 107⁎

Cholesterol (nmol/min·100 g) 0.5 ± 0.16 0.3 ± 0.04 0.5 ± 0.15 0.8 ± 0.14 1.3 ± 0.2 2.2 ± 0.2⁎ 2.1 ± 0.1 3.0 ± 0.5⁎

Phospholipids (nmol/min·100 g) 0.16 ± 0.12 0.01 ± 0.01 0.08 ± 0.04 0.22 ± 0.04 3.0 ± 0.7 8.0 ± 1.3⁎ 6.5 ± 0.4⁎ 14.4 ± 2.6⁎

Hepatic lipidsTriglycerides (nmol/mg) 5.7 ± 0.8 15.2 ± 0.8⁎ 17.6 ± 1.2⁎ 12.8 ± 2.4⁎ 7.9 ± 1.1 11.7 ± 1.6⁎ 13.5 ± 2.3⁎ 12.2 ± 1.0⁎

Cholesterol (nmol/mg) 8.1 ± 0.2 11.9 ± 0.5⁎ 12.7 ± 1.0⁎ 13.2 ± 0.8⁎ 10.6 ± 0.3 10.5 ± 0.4 9.5 ± 0.4 17.4 ± 1.1⁎

Phospholipids (nmol/mg) 19.2 ± 1.2 21.2 ± 0.8 23.0 ± 1.2 21.8 ± 1.2 20.6 ± 0.6 19.2 ± 0.6 21.5 ± 0.6 21.0 ± 0.5

Plasma lipidsCholesterol (mmol/l) 2.2 ± 0.03 2.5 ± 0.1⁎ 2.5 ± 0.1⁎ 2.2 ± 0.2 3.8 ± 0.2 4.3 ± 0.1⁎ 4.5 ± 0.1⁎ 4.9 ± 0.2⁎

Triglycerides (mmol/l) 1.7 ± 0.1 1.8 ± 0.1 1.1 ± 0.07⁎ 0.55 ± 0.04⁎ 1.4 ± 0.2 0.9 ± 0.1⁎ 0.6 ± 0.1⁎ 0.8 ± 0.1⁎

Free fatty acids (mmol/l) 0.53 ± 0.02 0.77 ± 0.05⁎ 0.66 ± 0.04⁎ 0.64 ± 0.11 1.3 ± 0.3 1.0 ± 0.1 1.0 ± 0.1 1.4 ± 0.3Phospholipids (mmol/l) 2.0 ± 0.03 2.1 ± 0.1 2.0 ± 0.1 1.8 ± 0.1 3.6 ± 0.2 3.5 ± 0.1 3.6 ± 0.1 3.8 ± 0.2

⁎ Denotes significance (P b 0.05).⁎⁎ Denotes significance (P b 0.06).

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Fig. 5. Fasting affects inflammatory status in fibrotic livers. A–F) Relative mRNA level of F4/80, Tnfα, Cd11b, Cd11c, Irf5 and Ym1 upon 0, 12, 24 and 48 h of fasting. mRNa expression isnormalized by 3 house-keeping genes and shown as fold change compared with WT control at 0 h. Asterisks denote significance compared to fed condition in the correspondingstrain (P b 0.05), hashtags represent significance between the Abcb4−/− and WT mine at baseline, and error bars show SD.

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to food deprivation in Abcb4−/− mice (already after 12 h) loweredinflammation, decreased hepatocyte proliferation, lowered the num-ber of activated HSC/MFs, decreased production of ECM components,and increased expression of genes involved in tissue remodeling,leading ultimately to amelioration of liver fibrosis.

Collagen, responsible for the structural integrity of the ECM, wasstrongly affected by fasting. Within 48 h, total collagen synthesis inlivers of Abcb4−/− mice (measured by hydroxyproline) decreasedto 20%, similarly to the response found in the articular cartilage offasted guinea pigs [57]. This corresponds with a notion that collagenproduction is sensitive to changes in food intake, and that malnutri-tion may have profound effects on its production [58]. Amount andthe types of produced collagen are regulated by the nutritional stateof the animal or by the disease processes — either directly as inwound healing, inflammation, and fibrosis, or indirectly, as in starva-tion and diabetes. In advanced starvation, collagen is one of the twomajor sources of energy (proteins) in the body [59]. The regulationof collagen mass in fasted fibrotic livers occurred, however, alreadyin early stages of starvation, before the other energy sources weredepleted. This points to an active ECM remodeling, rather than to anad hoc degradation for mere provision of amino acids. The mostprominent drop was noticed in expression of fibril-forming collagens1 and 3 and vimentin, predominantly synthesized by activated HSC[60], suggesting that the process of liver remodeling was specificallyinduced by food deprivation.

Chronic liver diseases are characterized by aberrant matrix depo-sition, calling for our attention to the role of ECM in resolution of

liver fibrosis. Tissue remodeling is regulated by MMPs, involved inthe ECM degradation, and TIMPs, their endogenous inhibitors. Theirsubtle balance maintains liver fibrogenesis. Tissue homeostasis isfurther regulated by proteolytic activity of the PLAU/PLAT/plasmin,responsible for the maintenance of the physiologic levels of ECM[61]. PLAU promotes ECM degradation through activation of MMPs(MMP-2, -3 and -9; [62,63]), increases the differentiation of hepaticstem cells, and HGF-dependent regeneration of hepatocytes [64].PLAT protects ECM proteins from proteolytic degradation and helpsexpedite wound healing [65]. In fed Abcb4−/− mice, hepatic Plauand Plat expression levels were much higher than in their WT coun-terparts (3- and 8-fold, respectively). Short and moderate fastinginitially reduced the expression of both, but prolonged fasting poseda demand for higher mRNA concentrations, indicating an ongoingbalancing action between fibrolysis and fibrogenesis. Together withstrongly (6-fold) reduced Timp1 expression and an increase in MMP2and 9 protein levels, our data indicate that fibrotic livers responded tofood deprivation not only by decreased synthesis, but by an activeECM remodeling. Matrix remodeling in fasting was further supportedby reduced expression ofMmp13. Expression of this collagenasemainlyproduced by Kupffer cells is high in collagen 1a1 r/r mouse model thatfails to recover from fibrotic liver injury, due to resistance to MMPdegradation of ECM [66]. However, given the contradictory findingsby other groups [49], the role of Mmp13 in (resolution of) liver fibrosisremains controversial.

Apart from ECM synthesis and degradation, tissue remodeling com-prises cell proliferation, death, and migration. In general, hepatocyte

Fig. 6. Fasting affects macrophage presence and localization in fibrotic livers. A) Representative immunostainings for F4/80 in the livers of fed and fasted WT and Abcb4−/− mice.Bar represents 50 μm.

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proliferation is low under normal conditions, except to compensate fora loss of cells [67] and it gets further reducedwhen the energy supply islow [68]. Caloric restriction induces apoptosis and decreases hepatocyteproliferation in a murine strain with a high incidence of spontaneousliver tumors [69]. It seems that, as a part of tissue remodeling, fibroticlivers of Abcb4−/− mice respond to fasting by decreasing their high he-patocyte proliferation. In fed condition, gene expression levels of α-Smaand vimentin, markers for activated HSC [70], was considerably higherin fibrotic Abcb4−/− livers than in theWT. Consistently with previouslyshown increased apoptosis in HSCs in response to nutrient deprivation[46], this study shows a decrease in number of both activated stellatecells and those with early contractile phenotype [71], most likely in re-sponse to decreased production of fibronectin and TIMP1, which bothprevent HSC apoptosis [46,52].

The impressive decrease in inflammation in Abcb4−/− mice, themacrophage migration from the periportal tract towards the centralvein, and the shift from classically to alternatively activated macro-phages, seem a likely driving force for improved pathology. Overall,altered concentrations of cytokines, metabolic and hormonal trophicfactors induced by fasting could decrease inflammation in fibroticlivers. Notably, profibrotic cytokine leptin that increases TIMP1 ex-pression [72] is strongly reduced by fasting [73]. Fasting increasescirculating corticosterone [74,75], decreasing thereby the productionof cytokines and interleukins [76,77], with an immunosuppressiveeffects in humans [78,79] and mice [19,80]. In Abcb4−/− mice, persis-tent hepatic inflammation triggers profibrotic signaling via activationof cytokines, promoting the formation of MFs, which in turn synthetizeelevated amounts of ECM proteins [81,82]. TGF-β1, the most potentfibrogenic cytokine, prompts HSC differentiation into myofibroblasts,by enhancing expression of TIMPs (that block ECM degradation), andby directly stimulating synthesis of interstitial fibrillar collagens [17].Liver fibrosis in Abcb4−/− mice was attenuated in fasting, most likely

driven by downregulation of Tgfβ1, which reduced the number of acti-vated HSC. Downregulation of a number of proinflammatory markers,a.o. Tnfα and Irf5 (a transcriptional activator of pro-inflammatory cyto-kines and chemokines [56]), clearly points to fasting-induced suppres-sion of immune response. The change in number, but also localizationof resident and infiltrated macrophages, may be causal in resolutionof the parenchymal fibrosis (e.g. by altered production of MMPs[49,83]), especially given the pivotal but divergent roles of macro-phages in matrix remodeling — favoring ECM accumulation duringongoing injury, and enhancing matrix degradation during recovery[84]. In the starved animals, the high energy demand of the immuneresponse (e.g. for production of acute phase response proteins) leadsto a massive change in hepatic transcriptome directed towards its sup-pression [15,16]. The attenuated inflammation in Abcb4−/− mice iscompatible with our previous finding that the suppressed immune re-sponse (subserving energy preservation) is one of the highlights of thebody's adaptive response to fasting [15].

In contrast to this study, caloric restriction aggravates the pathol-ogy in rodent models of acutely induced liver fibrosis [20,21]. Thedifference in pathogenesis between the models may explain thediscrepancy in the effect of fasting. In a widely used BDLmodel, for in-stance, ligation of the bile ducts results in an acute interstitial (biliary)fibrosis, associated with massive proliferation of bile ducts that is onlyrarely observed in man. On the other hand, progression of fibrosis inAbcb4−/− mice is spontaneous due to cholangiocyte proliferationand massive upregulation of profibrogenic genes, and bears more re-semblance to human biliary fibrosis. We have recently demonstratedthat IGF1 overexpression induces liver fibrosis in Abcb4−/− mice(by increasing bile duct proliferation), but not in BDL-induced acutecholestasis (Sokolovic et al.; Insulin-like growth factor 1 enhancesbile-duct proliferation and fibrosis in abcb4−/− mice, BBADIS, 2013;In Press). Given the role of IGF1-signaling in starvation [10], this

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dissimilar effect of fasting in models of chronic and acute fibroticdisease may not come as a surprise.

Among chronic liver diseases, alcoholic liver disease and cirrhosisare linked to physiological starvation and malnutrition, but throughdifferent, multifactorial mechanisms. Though pathophysiology in-cludes reduced food intake, maldigestion and malabsorption, fastingis prescribed every so often, only to worsen the pathology. Nutritionalsupport seems to improve nutritional status and lean body mass inboth categories of patients [85,86]. On the other hand, food restrictionis beneficial in patients suffering from chronically non-alcohol-inducedliver injury. In NASH and NAFLD patients, histological improvements ofliver pathology positively correlate with the size of weight loss [87,88],though the mechanism is not yet fully understood. Composition oflipids that was recently critically associated with the reversal of the dis-ease phenotype in Abcb4−/−mice [30]may be a part of the explanation.Lipid storage has beneficial effects on the chronic wound healing re-sponse, while lipid compartmentalization or loss in TG storage capacityis linked to lipotoxicity and exacerbates liver injury [89,90]. In fibroticAbcb4−/− livers, the surge of TGs from adipose tissue provides thisprotective environment in fasting. Consistency with the effect of foodrestriction on liver pathology in NASH and NAFLD, suggests that the ef-fects observed in this study are specific for the chronically induced liverfibrosis.

In conclusion, this study demonstrates that fasting leads to allevi-ation of biliary fibrosis by decreased inflammation and by actively in-creased matrix remodeling. Fasting in the fibrotic Abcb4−/− modelmay help improve understanding of the mechanisms of resolution,and inform strategies to improve liver regeneration in chronic liverdisease.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbadis.2013.05.012.

Acknowledgements

The authors would like to thank Dr. D.R. de Waart for hydroxypro-line, and Ms. Anna Gruber for assistance in lipid measurements.

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