Dietary advanced glycation end-products aggravate non-alcoholic fatty liver disease

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1 Submit Mnusript: Help Desk: DOI: /wjg.v22.i World J Gstroenterol 2016 September 21; 22(35): ISSN (print) ISSN (online) 2016 Bishideng Publishing Group In. All rights reserved. Bsi Study Dietry dvned glytion end-produts ggrvte non-loholi ftty liver disese ORIGINAL ARTICLE Christopher Leung, Chndn B Herth, Zhiyun Ji, Sof Andrikopoulos, Bronwyn E Brown, Mihel J Dvies, Leni R River, John B Furness, Josephine M Forbes, Peter W Angus Christopher Leung, Chndn B Herth, Zhiyun Ji, Sof Andrikopoulos, Peter W Angus, Deprtment of Mediine, the University of Melbourne, Austin Helth, Heidelberg, Melbourne, Vitori 3084, Austrli Christopher Leung, Peter W Angus, Deprtment of Gstroenterology nd Heptology, Austin Helth, Austin Hospitl, Heidelberg, Melbourne, Vitori 3084, Austrli Bronwyn E Brown, Mihel J Dvies, Hert Reserh Institute, Newtown, New South Wles 2042, Austrli Leni R River, John B Furness, University of Melbourne, Vitori 3052, Austrli Leni R River, Metboli Reserh Unit, Shool of Mediine, Dekin University, Vitori 3216, Austrli Josephine M Forbes, Glytion nd Dibetes Complitions Group, Mter Medil Reserh Institute, South Brisbne, Queenslnd 4101, Austrli Author ontributions: Leung C, Herth CB, Forbes JM nd Angus PW substntilly ontributed to the oneption, design, nlysis, interprettion of dt; Leung C, Herth CB nd Ji Z performed the mjority of experiments; Andrikopoulos S, Brown BE, Dvies MJ nd River LR provided vitl nlytil tools nd experiments; ll uthors were involved in editing nd pproving the finl mnusript. Supported by Ntionl Helth nd Medil Reserh Counil of Austrli; NHMRC erly reer fellowship. Institutionl review bord sttement: The study ws reviewed nd pproved by the Austin Helth Animl Ethis Committee (Projet number A2011/04342). Institutionl niml re nd use ommittee sttement: All proedures involving nimls were reviewed nd pproved by the Institutionl Animl Cre nd Use Committee of the Austin Helth Animl Ethis Committee (Projet number A2011/04342). Conflit-of-interest sttement: To the best of our knowledge, no onflit of interest exists. Dt shring sttement: Dt set vilble from the orresponding uthor t hris.leung@y7mil.om. Open-Aess: This rtile is n open-ess rtile whih ws seleted by n in-house editor nd fully peer-reviewed by externl reviewers. It is distributed in ordne with the Cretive Commons Attribution Non Commeril (CC BY-NC 4.0) liense, whih permits others to distribute, remix, dpt, build upon this work non-ommerilly, nd liense their derivtive works on different terms, provided the originl work is properly ited nd the use is non-ommeril. See: lienses/by-n/4.0/ Mnusript soure: Invited mnusript Correspondene to: Christopher Leung, MBBS, FRACP, Deprtment of Gstroenterology nd Heptology, Austin Helth, Austin Hospitl, 145 Studley Rd, Heidelberg, Vitori 3084, Austrli. hris.leung@y7mil.om Telephone: Fx: Reeived: My 5, 2016 Peer-review strted: My 6, 2016 First deision: June 20, 2016 Revised: June 27, 2016 Aepted: August 10, 2016 Artile in press: August 10, 2016 Published online: September 21, 2016 Abstrt AIM To determine if mnipultion of dietry dvned glytion end produt (AGE), intke ffets nonloholi ftty liver disese (NAFLD) progression nd 8026 September 21, 2016 Volume 22 Issue 35

2 Leung C et l. Progression of NAFLD whether these effets re medited vi RAGE. METHODS Mle C57Bl6 mie were fed high ft, high frutose, high holesterol () diet for 33 wk nd ompred with nimls on norml how. A third group were given diet tht ws high in AGEs. Another group ws given diet tht ws mrinted in vinegr to prevent the formtion of AGEs. In seond experiment, RAGE KO nimls were fed diet or high AGE diet nd ompred with wildtype ontrols. Hepti biohemistry, histology, pirosirius red morphometry nd hepti mrna were determined. RESULTS Long-term onsumption of the diet generted signifint stetoheptitis nd fibrosis fter 33 wk. In this model, hepti 4-hydroxynonenl ontent ( mrker of hroni oxidtive stress), heptoyte bllooning, pirosirius red stining, α-smooth musle tin nd ollgen type 1A gene expression were ll signifintly inresed. Inresing the AGE ontent of the diet by bking further inresed these mrkers of liver dmge, but this ws brogted by pre-mrintion in eti id. In response to the diet, RAGE -/- nimls developed NASH of similr severity to RAGE +/+ nimls but were proteted from the dditionl hrmful effets of the high AGE ontining diet. Studies in isolted Kupffer ells showed tht AGEs inrese ell prolifertion nd oxidtive stress, providing likely mehnism through whih these ompounds ontribute to liver injury. CONCLUSION In the model of NAFLD, mnipultion of dietry AGEs modultes liver injury, inflmmtion, nd liver fibrosis vi RAGE dependent pthwy. This suggests tht phrmologil nd dietry strtegies trgeting the AGE/RAGE pthwy ould slow the progression of NAFLD. Key words: Advned glytion end-produts; Frutose; Stetoheptitis; Non-loholi ftty liver disese; Hepti fibrosis; Oxidtive stress The Author(s) Published by Bishideng Publishing Group In. All rights reserved. Core tip: A novel high frutose, high holesterol diet produes hepti non-loholi stetoheptitis (NASH) with fibrosis in 33 wk nd inresing the Advned glytion end produts (AGEs), ontent of this diet vi bking inreses hepti fibrosis whilst vinegr mrintion dereses dietry AGE levels, brogting the hrmful effets of AGEs. RAGE -/- nimls ppered to be proteted from the dditionl hrmful effets of high AGE ontining diet suggesting the entrl role of RAGE in progression of NASH. Inresed ell prolifertion nd oxidtive stress in isolted primry Kupffer ells with the ddition of AGEs suggests they re n importnt mehnism in whih AGEs ontribute to liver injury. Leung C, Herth CB, Ji Z, Andrikopoulos S, Brown BE, Dvies MJ, River LR, Furness JB, Forbes JM, Angus PW. Dietry dvned glytion end-produts ggrvte non-loholi ftty liver disese. World J Gstroenterol 2016; 22(35): Avilble from: URL: v22/i35/8026.htm DOI: i INTRODUCTION Non-loholi ftty liver disese (NAFLD) is the most ommon liver disese in the world nd is strongly linked to the burgeoning rtes of dibetes nd obesity [1]. Although most ptients with NAFLD re symptomti nd do not develop linilly signifint liver injury, mny progress to non-loholi stetoheptitis (NASH), irrhosis nd liver ner. However, the host nd/or environmentl risk ftors tht determine whether ptients with simple hepti stetosis go on to develop NASH nd its omplitions remin unler [2]. Advned glytion end produts (AGEs), lso known s glyotoxins, re omplex group of ompounds tht re formed when tive sugr moieties beome bound to proteins using browning nd other irreversible modifitions [3]. Foods tht re highly proessed or dry heted t high tempertures, suh s broiled foods, hve prtiulrly high AGE ontent [4]. However, they re lso formed endogenously nd this ours t n inresed rte in dibetes, most likely beuse of hyperglyemi. There is now onsiderble evidene tht umultion of AGEs re implited in the pthogenesis of dibeti renl, neurologil, retinl nd vsulr omplitions [5]. AGEs t on severl reeptors but the reeptor for dvned glytion end produts (RAGE), member of the immunoglobulin superfmily of ellsurfe moleules, is the best hrterised of these reeptors [6]. Enggement with RAGE, whih in turn inreses inflmmtion nd oxidtive stress is thought to be the min wy in whih AGEs imprt these pthogeni effets [7]. This reeptor is expressed in number of ell types, inluding endothelil ells, vsulr smooth musle ells, peripherl blood mononuler ells, mrophges (inluding Kupffer ells) nd HSCs [8]. A number of lines of evidene to suggest tht, in NAFLD, AGEs my be seond hit tht ontributes to the progression from simple stetosis to NASH nd liver fibrosis [9]. Severl studies hve shown tht RAGE plys role in ute liver injury nd tht blokde of RAGE n meliorte toxi, ishemi nd holestti liver dmge [10-12]. In hroni liver injury, hepti expression of RAGE is signifintly inresed [13], nd in NAFLD AGE levels orrelte with the severity 8027 September 21, 2016 Volume 22 Issue 35

3 Leung C et l. Progression of NAFLD of fibrosis, leding to speultion tht AGEs ply primry role in disese pthogenesis [14]. At ellulr level, AGEs indues ROS vi oxidtive stress [15] nd this is signifint mehnism underlying the pthogenesis of NASH [16]. Furthermore, dibetes, whih inreses AGE formtion RAGE expression nd oxidtive stress, worsens the progression of fibrosis in number of humn liver diseses, inluding NAFLD [17] nd heptitis C [18]. In previous study, we showed tht in short term methionine holine defiient (MCD) model of NAFLD, high AGE diet inresed hepti AGE ontent nd exerbted liver injury, oxidtive stress nd liver fibrosis nd tht AGEs produed RAGE dependent profibroti effets in tivted hepti stellte ells (HSCs) [19]. However, the relevne of the MCD model to humn disese is limited sine the metboli profile of MCD fed nimls is generlly the onverse of humn NAFLD [20]. In ontrst, longterm murine models bsed on high-lorie diet high in ft, frutose nd holesterol [high frutose, high holesterol () diet], yield NASH ssoited with mny of the metboli fetures of humn NASH [21]. The im of the urrent study ws to explore whether inresing dietry AGE onsumption ould preipitte the development of NASH in novel model of NAFLD. We lso performed omplementry studies in RAGE KO nimls to determine the role of RAGE signlling in our liver disese model. MATERIALS AND METHODS Experimentl design Experiments were pproved by the Austin Helth Animl Ethis Committee nd performed ording to the Ntionl Helth nd Medil Reserh Counil (NHMRC) of Austrli Guidelines for niml experimenttion. Mle C57Bl6 mie (n = 10/group) were fed high ft, high frutose, high holesterol () diet [Speilty Feeds SF (21% sturted ft, 10% frutose, 2% holesterol)] for 33 wk nd ompred with nimls on norml how (Speilty Feeds AIN93G). This is novel dietry model ombining mounts of sturted ft (espeilly niml fts suh s ghee) nd frutose to mimi unhelthy Western diets [22]. This model is different to other high ft diets in tht we gve mounts of ft tully onsumed by ptients for longer period to indue fibrosis [23]. Two perent holesterol ws inorported s this hs been shown to be ritil driver for fibrosis in murine dietry models of NASH [22] nd 10% frutose ws dded, both due to its prevlene in Western diet s well s its potent bility to indue AGE formtion [24] (See Supplementry mterils 2). In two further groups, the effets of further inresing dietry AGE ontent by bking (desribed below), were studied in nimls fed norml how nd in nimls fed the diet. A finl group ws fed bked diet tht ws mrinted in eti id (vinegr 4% w/v) prior to heting to prevent the formtion of AGEs, s is seen in Mediterrnen style diets [25]. In seond experiment, RAGE KO nimls were fed diet or high AGE diet for 33 wk nd ompred with WT ontrols. At the ompletion of eh experiment, insulin resistne ws mesured vi HOMA-IR nd orl gluose tolerne testing (OGTT) performed s previously desribed [26]. Livers were hrvested for ssessment of liver injury nd serum lnine minotrnsminse (ALT) levels were mesured by utonlyser (Bekmn Instruments, Fullerton, CA). Prodution of AGEs Dietry AGE ontent ws inresed by bking t 160 for 1 h whih we hve previously shown produes n pproximtely 4 fold inrese in AGE levels [12]. CML is the predominnt AGE in food nd the extent of dvned glytion in the diet ws ssessed by mesuring CML ontent using high performne liquid hromtogrphy with fluoresene detetion ginst uthenti stndrds of CML [27]. For ell studies, AGEs were prepred in vitro by inubting bovine serum lbumin (BSA, 50 mg/ml) with 0.5 mol/l gluose in 100 mmol/l sodium phosphte buffer, ph 7.4. This ws inubted t 37 for 6 wk, followed by dilysis ginst phosphte buffered sline (PBS) for 48 h t 4 to remove ny free gluose. AGE-BSA nd BSA were then pssed through n endotoxin olumn (Detoxigel, Piere, Rokford, IL, United Sttes) to remove ny possible endotoxin ontminnts. The extent of dvned glytion ws ssessed by CML levels with ELISA nd by isotope dilution, seleted ion monitoring gs hromtogrphymss spetrometry [12,27]. Liver histology Prffin embedded, prformldehyde fixed setions of liver (4 µm) were stined with hemtoxylin nd eosin nd pirosirius red (Polysienes In. Wrrington, PA) for ssessment of liver fibrosis nd stetoheptitis by n independent pthologist. The NAFLD Ativity Sore (NAS) system ws used to quntify stetoheptitis using ten 200 light mirosopi fields in blinded fshion s previously desribed [28]. Eh field ws sored using the following riteri: For hepti stetosis: grde 0, no ft; grde 1, stetosis oupying less thn 33% of the hepti prenhym; grde 2, 34%-66% of the hepti prenhym; grde 3, more thn 66% of the hepti prenhym; for inflmmtory ell infiltrtion: grde 0:none; grde 1, 1-2 foi/field; grde 2, 3-4 foi/field; grde 3, more thn 4 foi/field (stetosis 0-3, lobulr inflmmtion 0-2, heptoellulr bllooning 0-3 nd fibrosis 0-4) [28]. Collgen ontent of the liver ws quntified histologilly using omputerized quntifition of pirosirius red stining, s desribed previously [12]. This ws ssessed t 100 mgnifition in totl of ten 8028 September 21, 2016 Volume 22 Issue 35

4 Leung C et l. Progression of NAFLD Tble 1 Primer nd probe sequenes used for rel time qpcr Gene nme Probe/ primer Sequene IL-6 Probe 5-FAM ATTGCCATTGCACAACT-3 Forwrd 5-GGGAAATCGTGGAAATGAGAAA-3 Reverse 5-AAGTGCATCATCGTTGTTCATACA-3 MCP1 Probe 5-FAM-AATGGGTCCAGACATAC-3 Forwrd 5-GTCTGTGCTGACCCCAAGAAG-3 Reverse 5-TGGTTCCGATCCAGGTTTTTA-3 TNF Probe 5-FAM-TCACCCACACCGTCAG-3 Forwrd 5-GGCTGCCCCGACTACGT-3 Reverse 5-TTTCTCCTGGTATGAGATAGCAAATC-3 RAGE Probe 5-FAM-CACAGCCCGGATTG-3 Forwrd 5-GCTGTAGCTGGTGGTCAGAACA-3 Reverse 5-CCCCTTACAGCTTAGCACAAGTG-3 lphsma Probe 5-FAM-TGCCAGATCTTTTCC-3 Forwrd 5-GACGCTGAAGTATCCGATAGAACA-3 Reverse 5-GGCCACACGAAGCTCGTTAT-3 COL1A1 Probe 5-FAM-ATCGACCCTAACCAAG-3 Forwrd 5-GACTGGAAGAGCGGAGAGTACTG-3 Reverse 5-CCTTGATGGCGTCCAGGTT-3 CTGF Probe 5-FAM-C ACTGCCTGGTCCAGAC-3 Forwrd 5-GCTGCCTACCGACTGGAAGA-3 Reverse 5-CTTAGAACAGGCGCTCCACTCT-3 fields per setion (per niml), using omputerized quntifition nd results were expressed s proportion of pirosirius red stining per field. Assessment of hepti oxidtive stress Superoxide nion nd ROS re key meditors of liver injury nd ellulr dysfuntion ssoited with the progression of ftty liver disese. Immunohistohemistry for HNE, mrker of lipid peroxidtion, ws performed on 4 µm setions of prffin embedded liver s previously desribed [12]. The primry HNE ntibody (1:500 Alph Dignosti Interntionl, Sn Antonio, Texs, United Sttes) in 0.1% norml got serum with PBS ws pplied nd then inubted with biotinylted seondry got nti-mouse ntibody (1/100) followed by inubtion with vidin-biotin horserdish peroxidse. Peroxidse onjugtes were subsequently lolized using diminobenzidine tetrhydrohloride hromogen (Sigm-Aldrih, Sydney, Austrli). The reltive positive stining in eh group ws determined by omputerized quntifition (MCID; Imging Reserh, Ontrio, Cnd) t 100 mgnifition (10 fields per niml). Quntittive rel time PCR Totl RNA ws extrted using TRI regent (Sigm- Aldrih) nd reverse trnsribed to DNA using protool previously desribed [29]. Gene expression ws normlized to the expression of the endogenous ontrol, ribosoml 18S. Eh smple ws run nd nlysed in duplite. The normlized vlues from norml how livers were used s the librtor with given vlue of 1 nd the other groups ompred with this librtor. The gene probe, forwrd nd reverse primer sequenes re detiled in Tble 1. Kupffer ell experiments Kupffer ells (KCs) were isolted from rt livers s desribed previously [30,31]. Further detils n be found in Supplementry mterils 1. KCs were ultured in M199 medium (Invitrogen), supplemented with 10% (v/v) FCS (foetl lf serum) ontining 100 units/ml peniillin nd 100 µg/ml streptomyin (Invitrogen), t 37 in n oxygented, humidified binet ontining 5% CO2. Twenty thousnd KCs per well were plted in blk 96 well pltes t 37. The ells were inubted with 2,7 -dihlorodihydrofluoresein diette (DCFDA) (10 µmol/l, Sigm-Aldrih, St Louis, MO, United Sttes) for 30 min, nd wshed twie with PBS. The ells were then treted with either AGEs or vehile BSA t physiologil onentrtions of 100 µg/ml. Mesurement of intrellulr ROS genertion ws performed using 2,7 -dihlorodihydrofluoresein diette (DCFDA) s desribed previously [32]. Briefly, fluoresene mesurements were tken with exittion nd emission wvelengths of 485 nm nd 520 nm respetively on n Optim Miroplte Reder (BMG Lbteh, Mornington, Austrli). KCs were ssyed for their prolifertive response to AGEs using BrdU ell prolifertion ssy (Rohe Applied Siene, IN, United Sttes) s per mnufturer s instrutions. KCs were ultured in 96 well pltes, with ells per well nd treted with BSA nd AGEs t 100 µg/ml. Sttistil nlysis Results re expressed s men ± SE nd nlysed by nlysis of vrine (ANOVA) nd Student s twotil, unpired t-test where pproprite with Prism 5 softwre (GrphPd Softwre, Sn Diego, United Sttes). Dt tht were not normlly distributed with equl vrine were log trnsformed prior to nlysis. P < 0.05 ws onsidered signifint. RESULTS Novel 33 week high ft, dietry model of NAFLD produed both stetoheptitis nd signifint fibrosis The diet ws well tolerted nd the initil body weights of the experimentl groups were similr. All the groups (, bked nd bked + eti id) gined more weight, s expeted, over the 33 wk ompred with norml how mie, nd there ws no finl differene in weight gin, liver weight or liver to body weight rtio mong the groups (Tble 2). The dily weight of how onsumed eh dy ws the sme in ll the groups The diet produed fetures similr to humn NASH with bllooning of heptoytes nd oxidtive stress s ssessed by HNE umultion nd stetosis grde > 3 (Figure 1A-C), with signifint inreses in ALT nd lobulr inflmmtion (Figure 1D nd E). The diet lso indued hepti fibrosis with the typil river delt tendrils of lobulr fibrosis seen in fibroti humn NASH 8029 September 21, 2016 Volume 22 Issue 35

5 Leung C et l. Progression of NAFLD Tble 2 Animl hrteristis nd metboli findings Body weight (g) Epididyml ft (g) Liver weight (g) Blood gluose (mmol/l) OGTT (AUC) Norml how 35.4 ± ± ± ± ± ± ± ± ± ± 65.2 bked 45.5 ± ± ± ± ± 70.2 bked nd eti id 44.6 ± ± ± 0.2 Dt re men ± SEM t 33 wk of norml how or high ft high holesterol feeding. P < 0.01 vs groups. AUC: Are under the urve; OGTT: Orl gluose tolerne test. (Figure 2A). This ws ssoited with n elevtion in profibroti nd proinflmmtory ytokine expression in the liver (Figure 2B nd C, Supplementry mterils 3). Thus using ombintion of physiologil mounts of ft, frutose nd prolonged durtion of feeding, relible model of NASH with fibrosis ws generted. Importntly this diet did not result in insulin resistne in ny of the groups s mesured by blood gluose OGTT (Tble 2). Diet high in AGEs did not use stetosis or stetoheptitis in the norml liver (norml how nimls) Both norml how nd norml how bked groups hd similr food intke nd there ws no differene in weight gin, liver weight or liver to body weight rtio between the two groups. In line with previous studies [19], bking the norml how diet inresed AGE ontent by over 6 fold (Figure 3) but feeding with bked how did not hnge liver biohemistry, produe stetosis, oxidtive stress or fibrosis (Figures 1 nd 2). Chnges in dietry AGE ontent modulte oxidtive stress nd heptoyte bllooning in indued stetoheptitis Bking the diet mrkedly inresed CML ontent (Figure 3). Other studies hve shown tht premrintion of food in vinegr dereses AGE levels to muh lower levels omprble to rw food or boiled food. Importntly, the pre-mrintion of the bked diet with eti id redued CML levels in the bked diet to non-bked levels (Figure 3). These AGE levels in the bked diet re similr to the CML levels found in moderte to high AGE typil Western diet [33]. Stetosis ws signifintly inresed in ll groups but not further inresed by bking to inrese its AGE ontent (Figure 1C). However, the bllooning produed by feeding ws further inresed by the high AGE/ diet nd this effet ws inhibited by the redution in dietry AGE ontent hieved by vinegr pre-mrintion (Figure 1A). Oxidtive stress is strongly implited in liver injury nd bllooning degenertion in NASH. Higher levels of HNE were deteted in the livers of the high AGE group ompred to those fed the diet lone. Furthermore, hepti HNE ontent ws redued to the levels observed in the group when AGE ontent of the diet ws redued by eti id (Figure 1B). These findings implite inresed genertion of oxidtive stress in the pthogenesis of AGE medited injury in this model. We previously showed tht AGEs inrese tivtion nd prolifertion of hepti stellte ells nd led to the prodution of ROS by these ells [19]. However KCs re responsible for hepti AGE uptke nd ply entrl role in NASH pthogenesis. We therefore isolted primry KCs to determine the effets of AGEs on these key drivers of inflmmtion nd injury in NAFLD. As shown in Figure 4A, the genertion of ROS by isolted Kupffer ells, s mesured by 2,3-DCFDA, ws signifintly inresed by the ddition of AGEs to the medium ompred to vehile. Moreover, Kupffer ell prolifertion s ssessed by BrDU inorportion ws lso signifintly inresed in Kupffer ells exposed to AGEs ompred to BSA vehile lone (Figure 4B). Lobulr inflmmtion, ALT levels nd proinflmmtory ytokine expression were ll signifintly inresed in the model (Figure 1D nd E, supplement 3) but were not ffeted by the dietry AGE ontent. Modultion of dietry AGE ontent inresed mrkers of liver fibrosis in the model As outlined bove, in this novel model, fibrosis ws lrgely onfined to zone 3 nd produed sinusoidl periellulr fine river delt pttern. This is in keeping with humn NASH where fibrosis tends to follow entrizonl pttern [34]. The proportionl re stined ws further inresed when the diet hd been bked to inrese AGE ontent (Figure 2A). In ddition to more pronouned zone 3 fibrosis, periportl fibrosis ws lso observed in high AGE fed nimls following the pttern of fibrosis progression in humn NAFLD [34]. However, the portl pttern of inflmmtion ws not ffeted by hnging dietry AGE ontent. In keeping with the results of pirosirius red stining, COL1A gene expression ws signifintly inresed in both groups ompred with norml how ontrols nd levels were higher in the mie with high orl intke of AGEs ompred with lone (Figure 2B). Ativtion of myofibroblsts, s ssessed by hepti α-sma gene expression ws lso signifintly inresed in the model nd further 8030 September 21, 2016 Volume 22 Issue 35

6 Leung C et l. Progression of NAFLD A Norml how bked bked + eti id 1.5 Bllooning Bllooning sore Norml how Norml how bked bked bked + eti id B Norml how bked bked + eti id 8031 September 21, 2016 Volume 22 Issue 35

7 Leung C et l. Progression of NAFLD 0.15 Hepti 4-hydroxynonenl ontent b b Proportionl re stined b b 0.00 Norml how Norml how bked bked bked + eti id C D Stetosis Grde ALT (U/L) Norml how Norml how bked bked bked + eti id 0 Norml how Norml how bked bked bked + eti id E 1.5 Lobulr inflmmtion Norml how Norml how bked bked bked + eti id Figure 1 Novel 33 wk high frutose, high holesterol dietry model indued hnges of non-loholi stetoheptitis. A: Hemtoxylin nd eosin stined setions showing bllooning fter high frutose, high holesterol () diets (exmples of bllooning irled). At right, bllooning sores determined morphometrilly; B: Oxidtive stress reveled by the immunohistohemil lolistion of 4-hydroxynonenl; C: Stetosis grde (grde 0, no ft; grde 1, stetosis oupying less thn 33% of the hepti prenhym; grde 2, 34%-66% of the hepti prenhym; grde 3, more thn 66% of the hepti prenhym); D: Plsm levels of lnine trnsminse (ALT) in the different tretment groups; nd E: Lobulr inflmmtion (grde 0:none; grde 1, 1-2 foi/field; grde 2, 3-4 foi/field; grde 3, more thn 4 foi/field). Bllooning nd oxidtive stress were signifintly inresed by rising the AGE ontent of the diet. These hnges were ttenuted when dietry AGE ontent ws redued by eti id premrintion. P < 0.05, b P < 0.01, P < September 21, 2016 Volume 22 Issue 35

8 Leung C et l. Progression of NAFLD A Norml how Norml how bked b bked d bked + eti id Pirosirius red ontent 0.05 b e Proportionl re stined b 0.00 Norml how Norml how bked bked bked + eti id B C 25 5 COL1 gene expression (fold indution normlised to 1) SMA gene expression (fold indution normlised to 1) Norml how Norml how bked bked bked + eti id 0 Norml how Norml how bked bked bked + eti id 8033 September 21, 2016 Volume 22 Issue 35

9 Leung C et l. Progression of NAFLD D 0.08 RAGE immunohistohemistry proportionl re stined Norml how Norml how bked bked bked + eti id Figure 2 Elevtion in profibroti nd proinflmmtory ytokine expression in the liver. A: Pirosirius red stining of ollgen showing entrilobulr sinusoidl fibrosis indued by the 33 wk high frutose, high holesterol () dietry model (). In nimls reeiving the diet, fibrosis ws further inresed by rising the dvned glytion end produts (AGEs) ontent of the diet (d) nd ttenuted when dietry AGE ontent ws redued by eti id premrintion prior to bking (e). However inresing the AGE ontent of norml how did not use inresed fibrosis (ompre, b). Hepti gene expression of COL1A (B) nd SMA (C) were lso signifintly inresed by rising the AGE ontent of the diet. Expression of RAGE, key reeptor for AGEs, ws elevted in the model but this ws not ffeted by vrying the AGE ontent of the diet, P < 0.05, b P < 0.01 (D). Crboxymethyl lysine ontent (nmol/mol of serine) AGE ontent of how b b b A ROS genertion fluoresene (RFU) B BrDU (rtio inrese over sline) Norml how Norml how bked bked bked + eti id Figure 3 Levels of rboxymethyl lysine in the different diets. Bking the high frutose, high holesterol () diet nd norml how inresed CML ontent of feed by > 6 fold. Levels of CML were mrkedly redued by premrinting with vinegr prior to bking, b P < inresed (P < 0.05) with high orl intke of AGEs (Figure 2C). The role of AGEs in mediting inresed liver fibrosis ws further supported by the finding tht pirosirius stining, COL 1A gene expression nd hepti α-sma gene expression were redued to those observed in nimls fed the diet lone, with vinegr pre-mrintion to redue AGE ontent during bking (Figure 2B nd C). 0 BSA BSA-AGE Figure 4 Effets of dvned glytion end produts on retive oxygen speies nd prolifertion in isolted Kupffer ells exposed to physiologil mounts of dietry dvned glytion end produts ompred with bovine serum lbumin vehile. A: Advned glytion end produts (AGEs) inresed ROS genertion s mesured by 2,7 -dihlorodihydrofluoresein diette. B: AGEs inresed ell prolifertion s ssessed by BrDU inorportion, P Pthologil effets of dietry AGEs re RAGE dependent In dibetes, mny of the hrmful effets of AGEs re thought to be medited vi AGE/RAGE signlling [35]. RAGE expression, s mesured by immunohistohemistry [19], ws miniml in helthy liver from nimls fed norml how however RAGE expression ws inresed in ll groups ompred to nimls fed stndrd how (Figure 2D). Given the puttive role of RAGE in mediting the BSA BSA-AGE 8034 September 21, 2016 Volume 22 Issue 35

10 Leung C et l. Progression of NAFLD hrmful effets of AGEs nd the finding tht its expression ws inresed in our dietry model, we performed further study in whih we exmined whether RAGE deletion brogted liver injury nd the hrmful effet of AGEs in the model. As in the initil study, in this experiment, diet indued liver fibrosis nd 4HNE stining were inresed by rising dietry AGE ontent nd the re of fibrosis ws expnded to inlude the periportl region (Figure 5A-C). RAGE KO nimls fed the diet hd similr levels of fibrosis to wild type nimls (5A). However RAGE KO nimls ppered to be proteted from the inresed liver fibrosis indued by AGEs. Compred to wild type nimls fed the high AGE/ diet, there ws less pirosirius stining nd COL 1A gene expression in the RAGE deleted nimls (Figure 5A-B). The inresed oxidtive stress in nimls fed bked diet ws lso brogted signifintly in orresponding RAGE KO nimls (Figure 5C). However, HNE levels did not return bk to norml how levels. These findings suggest tht AGEs t vi RAGE to inrese liver fibrosis in this model of experimentl NASH. DISCUSSION Our experiments show tht AGEs my be importnt modifible dietry oftors whih ontribute to the development of liver fibrosis in NAFLD. The urrent findings re in keeping with previous studies whih showed tht AGEs dministered intrperitonelly or in the diet exerbte liver injury nd fibrosis [12,19]. However these previous experiments were onduted in short term models of liver disese of limited relevne to humn NAFLD [36]. For the present studies, therefore, novel model of NAFLD ws developed using physiologil mounts of ft, holesterol nd frutose tht our in humn diets nd it relibly produed slowly progressive stetosis, bllooning, oxidtive stress nd predominntly perientrl sinusoidl fibrosis with tempo of disese onsistent with the long progressive nturl history of humn NASH. In this study, ll of these key fetures of high ft diet indued NASH were exerbted by inresing dietry AGE ontent The two-hit hypothesis of NASH pthogenesis suggests tht seond injury or oftor is required for progression from simple benign stetosis to hrmful stetoheptitis or fibrosis, irrhosis nd heptoellulr rinom [37]. There hs been onsiderble interest in ftors whih ould serve s this seond hit. In line with our previous study in MCD nimls [19], we found tht high AGE ontining diet hs no effet on liver biohemistry or histology in nimls without hepti stetosis. However, we showed they t s o-ftor to inrese injury in disesed livers. AGEs hve been shown to exert their effets through severl reeptors, the best studied of whih is RAGE. Ativtion of RAGE stimultes multiple signl trnsdution pthwys [38], ultimtely leding to the genertion of ROS [39]. This ulmintes in the tivtion of NF-κB, redox sensitive trnsription ftor, whih in turn trnslotes into the nuleus [40]. The promoter region of the RAGE gene ontins n NF-κB binding site nd therefore, one of the importnt onsequenes of NF-κB tivtion nd trnslotion is upregultion of RAGE itself. This sets up positive feedbk loop nd ensures mintenne of RAGE signlling. Furthermore, the genertion of ROS triggered by RAGE tivtion uses inresed AGE formtion nd ontributes to viious yle of AGE formtion, genertion of oxidtive stress nd further RAGE tivtion [7]. Our study showed tht RAGE expression ws miniml in helthy livers but upregulted in non-dibeti model of ftty liver disese. These differenes in RAGE expression my explin why the high AGE diet hd no hrmful effets in helthy ontrols but exerbted liver injury in nimls with NAFLD. However, sine the dietry AGE ontent of norml mouse how is so high it is lso fesible tht the dietry AGE ontent of norml mouse how is so high tht mie beome preonditioned ginst the effets of further inreses. In keeping with these findings, lthough RAGE KO nimls developed NASH in response to the diet they were proteted from the dditionl hrmful effets of the high AGE diet. Oxidtive stress nd the umultion of superoxide is key meditor of bllooning, ellulr dysfuntion nd fibrosis in non-loholi stetoheptitis [7,41]. HNE, n end-produt of peroxidtion of membrne N-6- polyunsturted ftty ids, is prtiulrly good mrker of lipid oxidtion during liver injury nd is relted to the intensity of neroinflmmtion [42]. Given the long- term nture of our model, ssessment of oxidtive stress in the liver ws therefore performed by mesuring HNE dduts whih reflet umultion of oxidtive stress over time. This showed tht HNE ontent ws signifintly elevted in ll the groups (Figure 1C). Consistent with the known effets of AGEs, we found tht diet high in AGEs signifintly inresed oxidtive stress in indued NASH. This ws ssoited with inresed bllooning, stellte ell tivtion s ssessed by SMA expression nd inresed fibrosis. These findings re onsistent with our previous work whih showed tht in tivted primry murine hepti stellte ells whih express RAGE, ROS prodution, ell tivtion nd prolifertion were mrkedly inresed in the presene of AGEs. These effets were inhibited by RAGE blokde or NADPH oxidse inhibition. However, Kupffer ells (KCS) express RAGE [43] nd uptke of AGEs in the liver ours primrily vi these ells [11]. It is known tht KCs ply key role in promoting hepti stetosis, stetoheptitis nd bllooning in NASH [44] ; nd genertion of ROS in KCs rther thn by HSCs hs been ssoited with NAFLD disese progression [45]. Given the importnt role of KCs in NAFLD progression nd their key role in 8035 September 21, 2016 Volume 22 Issue 35

11 Leung C et l. Progression of NAFLD A WT norml how RAGE KO norml how WT RAGE KO WT bked RAGE KO bked * 0.05 Pirosirius red ontent Proportionl re stined Norml how RAGE KO norml how RAGE KO bked RAGE KO bked 8036 September 21, 2016 Volume 22 Issue 35

12 Leung C et l. Progression of NAFLD B 25 COL1A gene expression C 0.15 Hepti 4-Hydroxynonenl ontent COL1A gene expression (fold indution normlised to 1) Proportionl re stined Norml how RAGE KO norml how RAGE KO bked RAGE KO bked 0.00 Norml how RAGE KO norml how RAGE KO bked RAGE KO bked Figure 5 Effets of the high frutose, high holesterol diet nd dietry dvned glytion end produts mnipultion on fibrosis in the reeptor for dvned glytion end produts KO nimls. A: The bsene of reeptor for dvned glytion end (RAGE) ttenuted the effets of inresing the dvned glytion end produts (AGEs) ontent of the high frutose, high holesterol () diet s evidened by redution in pirosirius red stining to levels observed in nimls on the diet lone; B: There ws ommensurte redution in COL1A gene expression; C: A redution in hepti 4-hydroxynonenl ontent ( P < 0.05). svenging AGEs [46], we explored the effet of AGEs on primry KCs nd showed tht AGEs inrese ell prolifertion nd the genertion of ROS by these ells. As expeted, in our model there ws mrked upregultion of number of key proinflmmtory nd fibrogeni ytokines. It is unler why, despite showing tht AGEs inrese oxidtive stress, heptoyte bllooning nd fibrosis, we did not find they inresed inflmmtory infiltrtion of the liver or in inflmmtory ytokines expression. In keeping with this finding, in very similr dietry model of NASH, Lo et l [47] found tht lthough dibetes worsened stetosis indued liver fibrosis this ws not ssoited with mesurble inreses in liver inflmmtory infiltrtion or proinflmmtory ytokine levels. These finding suggest tht profibroti effets of AGEs nd dibetes my not be medited primrily through inresing hepti inflmmtion. The formtion of AGEs in foods involves the ondenstion of n mino group with the rbonyl group of reduing rbohydrte (gluose, frutose, mltose, ltose or ribulose) to form intermedite Amdori produts. Oxidtion of Amdori produts leds to more stble ompound, CML, used in studies s n inditor of the AGE ontent of foods [4]. A ommon method of high dietry AGE formtion is vi heting t high tempertures in non-queous environments (e.g., bking or broiling) [4]. High levels of AGEs re thus found in mny ommon foods suh s bked breds nd bisuits/ookies, tosted brekfst erels, grilled stek, brewed beer, nd rosted offee bens. For exmple, tosting white bred inreses its AGE ontent by more thn 3 times [48]. Interestingly, idifying suh foods prior to ooking by mrinting them in eti id (vinegr) or lemon juie, redues their CML ontent substntilly without ompromising pltbility [49]. In this study, we hve found reduing AGEs prodution by vinegr mrintion prior to bking n brogte the hrmful effets of high AGE diet in n niml model of NAFLD. This my be one mehnism by whih the Mediterrnen diet whih inludes the use of vinegr nd lemon juie mrintion is benefiil for metboli helth nd in NAFLD [50]. It hs been shown tht ptients with NASH hve higher levels of irulting AGEs thn those with simple stetosis [46]. However whether this reflets n inresed dietry exposure to AGEs or greter endogenous AGE prodution in ptients who hve both ftty liver nd gluose intolerne is unler. Studies exmining the reltionship between dietry exposure to AGEs nd the histologil severity of liver injury in non-dibeti ptients will help lrify this issue. In onlusion, we show tht high dietry AGE exposure worsens liver pthology in experimentl NASH, impliting AGE/RAGE signlling in ftty liver disese progression. Deresing dietry AGEs by ltering food seletion nd ooking methods my thus offset the possible hrmful effets fforded by high AGE diet. If onfirmed in humn studies, our findings hve brod implitions for the wy we proess foods nd the dietry dvie given to ptients with NAFLD. They lso suggest possible role for therpies trgeting the AGE/RAGE pthwy in the tretment nd prevention of NASH. ACKNOWLEDGMENTS Mny thnks to A/Prof Trishe Leong from the Deprtment of Antomil Pthology, Austin Helth, for her expert histopthologil ssistne September 21, 2016 Volume 22 Issue 35

13 Leung C et l. Progression of NAFLD COMMENTS Bkground There is onsiderble interest in identifying the ftors whih drive progression of unomplited non-loholi ftty liver disese (NAFLD) to non-loholi stetoheptitis (NASH), irrhosis nd liver ner. Advned glytion end produts (AGEs) re omplex group of ompounds formed in foods tht re highly proessed or dry heted t high tempertures whih hve been implited in the pthogenesis of dibeti omplitions. Our previous work in methionine holine defiient model of NASH hs shown tht AGEs my be n importnt environmentl risk ftor tht serves s seond hit driving simple stetosis to stetoheptitis nd irrhosis. The ims of the urrent study were to determine whether AGEs drive NAFLD progression in long term high ft, high holesterol, high frutose dietry model of NASH tht mimis mny of the fetures of humn NASH. We lso imed to exmine the role of the reeptor for AGEs (RAGE) nd Kupffer ells in this proess. Reserh frontiers There is mjor interest in identifying so-lled seond hits whih drive liver inflmmtion nd fibrosis in NAFLD. AGEs hve been implited in progression of number of disese proesses, inluding dibeti nephropthy, retinopthy, vsulopthy nd neuropthy. This study investigtes whether AGEs ould represent n importnt potentil dietry nd phrmologil trget in the mngement of the burgeoning problem of ftty liver disese. Innovtions nd brekthroughs This is the first study whih exmines the impt of ltering dietry AGE ontent in long term physiologil model of NASH tht utilises proportions of fts nd frutose found typilly in Western diets. We hve found tht hnges in AGE ontent influene liver inflmmtion nd fibrosis. This is lso the first study to show tht in RAGE knok out nimls, these deleterious effets of AGEs re brogted, suggesting RAGE dependent pthwy. Studies by Leung et l in isolted primry Kupffer ells lso show AGEs inrese ell prolifertion nd oxidtive stress, suggesting likely mehnism by whih these ompounds ontribute to liver injury. Applitions Food souring nd preprtion methods tht redue AGE ontent ould influene the progression of NAFLD. This study lso suggests tht phrmologil therpies whih trget the AGE/RAGE pthwy my hve role in tretment of NAFLD. Terminology AGEs, lso known s glyotoxins, re omplex group of ompounds tht re formed when tive sugr moieties beome bound to proteins using browning nd other irreversible modifitions. Foods tht re highly proessed or dry heted t high tempertures, suh s broiled foods, hve prtiulrly high AGE ontent. However, they re lso formed endogenously nd this ours t n inresed rte in dibetes, most likely beuse of hyperglyemi. There is now onsiderble evidene tht umultion of AGEs re implited in the pthogenesis of dibeti renl, neurologil, retinl nd vsulr omplitions. AGEs t on severl reeptors but the reeptor for dvned glytion end produts (RAGE), member of the immunoglobulin superfmily of ell-surfe moleules, is the best hrterised of these reeptors. Enggement with RAGE, whih in turn inreses inflmmtion nd oxidtive stress, is thought to be the min wy in whih AGEs imprt these pthogeni effets. This reeptor is expressed in number of ell types, inluding endothelil ells, vsulr smooth musle ells, peripherl blood mononuler ells, mrophges (inluding Kupffer ells) nd HSCs. Peer-review A well designed nd orgnized study. REFERENCES 1 Willims CD, Stengel J, Asike MI, Torres DM, Shw J, Contrers M, Lndt CL, Hrrison SA. Prevlene of nonloholi ftty liver disese nd nonloholi stetoheptitis mong lrgely middleged popultion utilizing ultrsound nd liver biopsy: prospetive study. Gstroenterology 2011; 140: [PMID: DOI: /j.gstro ] 2 Arteel GE. Beyond resonble doubt: who is the ulprit in lipotoxiity in NAFLD/NASH? Heptology 2012; 55: [PMID: DOI: /hep.25721] 3 Henle T. Protein-bound dvned glytion endproduts (AGEs) s biotive mino id derivtives in foods. Amino Aids 2005; 29: [PMID: DOI: /s ] 4 Goldberg T, Ci W, Pepp M, Drdine V, Blig BS, Uribrri J, Vlssr H. Advned glyoxidtion end produts in ommonly onsumed foods. J Am Diet Asso 2004; 104: [PMID: DOI: /j.jd ] 5 Aso Y, Inuki T, Tym K, Tkemur Y. Serum onentrtions of dvned glytion endproduts re ssoited with the development of theroslerosis s well s dibeti mirongiopthy in ptients with type 2 dibetes. At Dibetol 2000; 37: [PMID: ] 6 Stitt AW, Bul R, Vlssr H. Atherogenesis nd dvned glytion: promotion, progression, nd prevention. Ann N Y Ad Si 1997; 811: ; disussion [PMID: ] 7 Miyt T, Hori O, Zhng J, Yn SD, Ferrn L, Iid Y, Shmidt AM. The reeptor for dvned glytion end produts (RAGE) is entrl meditor of the intertion of AGE-bet2miroglobulin with humn mononuler phgoytes vi n oxidnt-sensitive pthwy. Implitions for the pthogenesis of dilysis-relted myloidosis. 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14 Leung C et l. Progression of NAFLD Si 2013; 14: [PMID: DOI: / ijms ] 17 Adms LA, Snderson S, Lindor KD, Angulo P. The histologil ourse of nonloholi ftty liver disese: longitudinl study of 103 ptients with sequentil liver biopsies. J Heptol 2005; 42: [PMID: DOI: /j.jhep ] 18 Rtziu V, Muntenu M, Chrlotte F, Bonyhy L, Poynrd T. Fibrogeni impt of high serum gluose in hroni heptitis C. J Heptol 2003; 39: [PMID: DOI: / S (03) ] 19 Leung C, Herth CB, Ji Z, Goodwin M, Mk KY, Wtt MJ, Forbes JM, Angus PW. Dietry glyotoxins exerbte progression of experimentl ftty liver disese. J Heptol 2014; 60: [PMID: DOI: /j.jhep ] 20 Lrter CZ, Yeh MM. Animl models of NASH: getting both pthology nd metboli ontext right. J Gstroenterol Heptol 2008; 23: [PMID: DOI: / j x] 21 Kohli R, Kirby M, Xnthkos SA, Softi S, Feldstein AE, Sxen V, Tng PH, Miles L, Miles MV, Blistreri WF, Woods SC, Seeley RJ. High-frutose, medium hin trns ft diet indues liver fibrosis nd elevtes plsm oenzyme Q9 in novel murine model of obesity nd nonloholi stetoheptitis. Heptology 2010; 52: [PMID: DOI: /hep.23797] 22 Vn Rooyen DM, Gn LT, Yeh MM, High WG, Lrter CZ, Ionnou G, Teoh NC, Frrell GC. Phrmologil holesterol lowering reverses fibroti NASH in obese, dibeti mie with metboli syndrome. J Heptol 2013; 59: [PMID: DOI: /j.jhep ] 23 Xu ZJ, Fn JG, Ding XD, Qio L, Wng GL. Chrteriztion of high-ft, diet-indued, non-loholi stetoheptitis with fibrosis in rts. Dig Dis Si 2010; 55: [PMID: DOI: /s ] 24 Tkgi Y, Kshiwgi A, Tnk Y, Ashin T, Kikkw R, Shiget Y. Signifine of frutose-indued protein oxidtion nd formtion of dvned glytion end produt. J Dibetes Complitions 1995; 9: [PMID: DOI: / (94) G] 25 Dvid E. A Book of Mediterrnen Food (New York Review Books Clssis). New York City: New York Review Books, Andrikopoulos S, Blir AR, Delu N, Fm BC, Proietto J. Evluting the gluose tolerne test in mie. 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15 Leung C et l. Progression of NAFLD progression ftor for hepti fibrosis in high ft fed mouse obesity model of non-loholi stetoheptitis. J Heptol 2011; 55: [PMID: DOI: /j.jhep ] 48 Vlssr H, Striker G. Glyotoxins in the diet promote dibetes nd dibeti omplitions. Curr Dib Rep 2007; 7: [PMID: DOI: /s z] 49 Uribrri J, Woodruff S, Goodmn S, Ci W, Chen X, Pyzik R, Yong A, Striker GE, Vlssr H. Advned glytion end produts in foods nd prtil guide to their redution in the diet. J Am Diet Asso 2010; 110: e12 [PMID: DOI: /j.jd ] 50 Hoffmn R, Gerber M. Evluting nd dpting the Mediterrnen diet for non-mediterrnen popultions: ritil pprisl. Nutr Rev 2013; 71: [PMID: DOI: /nure.12040] P- Reviewer: Busuttil RW, Mendez-Snhez N, Tnoglu A S- Editor: Qi Y L- Editor: A E- Editor: M S 8040 September 21, 2016 Volume 22 Issue 35

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