Metabolomic changes in fatty liver can be modified by dietary protein and calcium during energy restriction

Transcription

1 Online Sumissions: wjg.wjgnet.om World J Gstroenterol 28 July 28; 14(28): [email protected] World Journl of Gstroenterology ISSN doi:1.3748/wjg The WJG Press. All rights reserved. BASIC RESEARCH Metolomi hnges in ftty liver n e modified y dietry protein nd lium during energy restrition Tru K Pilvi, Tuulikki Seppänen-Lkso, Helen Simolin, Piet Finkenerg, Anne Huotri, Krl-Heinz Herzig, Riitt Korpel, Mtej Orešič, Eero M Mervl Tru K Pilvi, Piet Finkenerg, Riitt Korpel, Eero M Mervl, Institute of Biomediine, Phrmology, Biomedium Helsinki, PO Box 63, FI-14 University of Helsinki, Helsinki FI-39, Finlnd Tru K Pilvi, Riitt Korpel, Foundtion for Nutrition Reserh, PO Box 3, Helsinki FI-39, Finlnd; Vlio Ltd, Reserh Center, PO Box 3, Vlio, Helsinki FI-39, Finlnd Tuulikki Seppänen-Lkso, Helen Simolin, Mtej Orešič, VTT Tehnil Reserh Centre of Finlnd, Tietotie 2, VTT FI-244, Finlnd Anne Huotri, Krl-Heinz Herzig, A. I. Virtnen Institute for Moleulr Sienes, PO Box 1627, University of Kuopio, Kuopio FI-7211, Finlnd Krl-Heinz Herzig, Deprtment of Internl Mediine, Kuopio University Hospitl, PO Box 1777, Kuopio FI-7211, Finlnd; Institute of Biomediine, Deprtment of Physiology, nd Bioenter of Oulu PO Box 5, University of Oulu, Oulu FI-914, Finlnd Supported y Foundtion for Nutrition Reserh, Ademy of Finlnd, Sigrid Juselius Foundtion nd Vlio Ltd., Helsinki, Finlnd Author ontriutions: Pilvi TK designed nd onduted the study, nlysed the dt nd prepred the mnusript. Seppänen- Lkso T, Simolin H nd Orešič M did the metolomi nlyses. Huotri A nd Herzig KH onduted the metoli performne nd lorimetry tests. Korpel R nd Mervl EM were involved in designing the study, reviewing nd interpreting the results nd ll uthors were involved in drfting the mnusript. Correspondene to: Eero M Mervl, MD, Professor, Institute of Biomediine, Phrmology, Biomedium Helsinki, PO Box 63, University of Helsinki, Helsinki FI-39, Finlnd. [email protected] Telephone: Fx: Reeived: April 16, 28 Revised: June 23, 28 Aepted: June 3, 28 Pulished online: July 28, 28 Astrt AIM: To hrterise the effet of energy restrition (ER) on liver lipid nd primry metolite profile y using metolomi pproh. We lso investigted whether the effet of energy restrition n e further enhned y modifition of dietry protein soure nd lium. METHODS: Liver metolomi profile of len nd oese C57Bl/6J mie (n = 1/group) were ompred with two groups of weight-redued mie. ER ws performed on ontrol diet nd whey protein-sed high-lium diet (whey + C). The metolomi nlyses were performed using the UPLC/MS sed lipidomi pltform nd the HPLC/MS/MS sed primry metolite pltform. RESULTS: ER on oth diets signifintly redued hepti lipid umultion nd lipid droplet size, while only whey + C diet signifintly deresed lood gluose (P <.1) nd serum insulin (P <.1). In hepti lipid speies the iggest redution ws in the level of triylglyerols nd ermides while the level of holesterol esters ws signifintly inresed during ER. Interestingly, diylglyerol to phospholipid rtio, n inditor of reltive mount of dietogeni diglyeride speies, ws inresed in the ontrol ER group, ut deresed in the whey + C ER group (P <.1, vs oese). ER on whey + C diet lso totlly reversed the oesity indued inrese in the reltive level of lipotoxi ermides (P <.1, vs oese; P >.5, vs len). These hnges were ompnied with up-regulted TCA yle nd pentose phosphte pthwy metolites. CONCLUSION: ER-indued hnges on hepti metolomi profile n e signifintly ffeted y dietry protein soure. The therpeuti potentil of whey protein nd lium should e further studied. 28 The WJG Press. All rights reserved. Key words: Ftty liver; Metolomis; Energy restrition; Whey protein; Dietry lium Peer reviewer: Dr. Philip Arhm, Professor, Consultnt Gstroenterologist & Heptologist, PD Hinduj Ntionl Hospitl & Medil Reserh Centre, Veer Svrkr Mrg, Mhim, Mumi 416, Indi Pilvi TK, Seppänen-Lkso T, Simolin H, Finkenerg P, Huotri A, Herzig KH, Korpel R, Orešič M, Mervl EM. Metolomi hnges in ftty liver n e modified y dietry protein nd lium during energy restrition. World J Gstroenterol 28; 14(28): Aville from: URL: DOI: dx.doi.org/1.3748/wjg INTRODUCTION Oesity is losely ssoited with different omponents of metoli syndrome [1]. However, not ll oese

2 Pilvi TK et l. Energy restrition nd ftty liver 4463 individuls develop metoli syndrome nd not ll individuls with metoli syndrome re oese. It hs reently een suggested tht ft umultion in the liver is the key feture distinguishing those individuls who develop metoli syndrome from those who do not [2]. The mehnisms leding to hepti ft umultion re not fully understood nd, hene, the mens of preventing nd treting this ondition re limited. One ft hs umulted, the liver is insulin resistnt nd overprodues mjor rdiovsulr risk ftors, suh s C-retive protein, very low density lipoprotein nd plsminogen tivtor inhiitor-1 [3]. At the moment, improving insulin resistne through energy restrition nd susequent weight loss remins the ornerstone of therpy for non-loholi ftty liver disese [4]. Lipids re highly diverse lss of moleules, whih hve importnt roles s signlling nd struturl moleules in ddition to serving s energy storge. Therefore, it is ruil to identify the vriety of lipid speies umulting in the liver in order to understnd the omplex proess of hepti insulin resistne. The level of triylglyerides (TAG) nd diylglyerides (DAG) hs een shown to e inresed in non-loholi ftty liver disese in humns, while totl mount of phosphtidylholines is deresed [5]. Similr hnges in liver lipids hve een deteted in o/o mie with upregultion of TAG nd DAG, diylphosphoglyerols nd speifi ermide speies nd down-regultion of sphingomyelins [6]. Interestingly, reent humn study reveled tht high level of liver ft is lso ssoited with hnges in the lipidomi profile of suutneous dipose tissue [7]. Inresed dipose tissue ermides, SM, ether phospholipids nd long-hin TAG were ssoited with higher liver ft level. Hene, the umultion of ermides nd TAG lso in the suutneous dipose tissue seems to reflet the development of ftty liver. However, more studies re needed to support the findings of ftty liver lipidomis. Even though weight loss is the min therpeuti wy to redue liver ft, the informtion on the effet of energy restrition on liver lipidomi profile is sre. Also the enefiil effet of different dietry omponents on liver ft speies is nerly n unexplored re [8]. High intke of diry produts is relted to lower risk of insulin resistne [9], type two dietes [1,11] nd metoli syndrome [12-15], ut the mehnism of tion hs not een estlished. The inresed intke of diry produts or lium hs lso een shown to ugment weight loss oth in humns nd mie [16-19]. Although prt of the effet of diry produts on weight loss n e ttriuted to lium, it hs een repetedly demonstrted tht the nti-oesity effet of diry is superior to tht of lium lone [18,2]. It hs een suggested tht the whey protein frtion of milk is soure of iotive peptides or other ompounds ple of regulting dipose tissue metolism, energy expenditure or stiety signls [21]. In our previous study, we showed tht whey protein in omintion with lium ttenutes weight gin [22], ut the effets of whey protein during energy restrition hve not een reported. Also, the effet of whey protein ontining high-lium diet on hepti lipid profile hs not een previously desried. The im of this study ws to hrterise the effet of high-ft diet-indued oesity nd the susequent ER on liver lipidomi nd primry metolite profile in C57Bl/6 mie, widely studied model of diet indued oesity. In ddition we investigted whether the effet of ER my e signifintly improved y modulting the protein soure nd lium ontent of the weight loss diet. MATERIALS AND METHODS Animls nd diets Eight-week old mle C57Bl/6J mie (n = 4) were purhsed from Hrln (Horst, The Netherlnds). The mie were housed five in ge in stndrd experimentl niml lortory, illuminted from 6:3-18:3, temperture 22 ± 1. The protools were pproved y the Animl Experimenttion Committee of the University of Helsinki, Finlnd nd the priniples of lortory niml re (NIH pulition no , revised 1985) were followed. The mie hd free ess to feed nd tp wter during the experiment. After one-week limtistion period on norml how diet (Hrln Teklnd 218, Hrln Holding, In, Wilmington, DE, USA) thirty mie (25.5 g ±.3 g) were put on high-ft diet (6% of energy from ft, D53111M, Reserh Diets In., New Brunswik, NJ, USA) for 14 wk. Ten remining mie ontinued on norml how diet (d liitum) throughout the study nd served s len ontrol group. After the weight gin period on high-ft diet one group of mie (oese group, n = 1) ws srified, nd the remining mie were put on lorie restrition diet for 7 wk. During the lorie restrition period, the mie were given 7% of the energy they te during the d liitum feeding. In the eginning of the lorie restrition period, the ody weight mthed mie were divided into two groups: whey + C group nd ontrol group. Whey + C group reeived high-ft diet (D53114M, Reserh Diets In., New Brunswik, NJ, USA) with 1.8% CCO 3 nd ll protein (18% of energy) from whey protein isolte (Alen 895, NZMP, Auklnd, New Zelnd). The ontrol group ontinued with the sme high-ft diet (D53111M) s during the weight gin period. The powdered diets were moistened with tp wter (2 ml/kg in whey + C, 11 ml/kg in ontrol nd 7 ml/kg in norml how diet) using industril dough mixer, pked in one-dy portions nd stored t -2. The ody weight ws monitored weekly during the weight gin period, nd twie per week during the lorie restrition period using stndrd tle sle (Ohus Sout Pro, SP41, Nänikon, Switzerlnd). The onsumption of feed ws monitored dily using the sme tle sle. The ody ft ontent ws nlysed y dul-energy X-ry sorptiometry (DEXA, Lunr PIXImus, GE Helthre, Chlfont St. Giles, UK) t the end of the weight gin nd lorie restrition periods.

3 4464 ISSN CN /R World J Gstroenterol July 28, 28 Volume 14 Numer 28 Clorimetry nd metoli performne The dietry protein-indued differenes in metoli performne, energy expenditure, physil tivity nd drinking nd feeding ehviour were nlysed y housing n dditionl group of nimls (n = 4/whey group nd n = 3/sein group) in home ge sed monitoring system for lortory nimls (LMster, Bd Homurg, Germny). The instrument onsists of omintion of highly sensitive feeding nd drinking sensors for utomted online mesurement. The lorimetry system is n open-iruit system tht determines O 2 onsumption, CO 2 prodution, nd respirtory quotient (RQ = VCO 2 /VO 2, where V is volume), respirtory exhnge rte nd het. A photoem-sed tivity monitoring system detets nd reords every multory movement, inluding rering nd liming movements in every ge. The sensors for detetion of movement operte effiiently in oth light nd drk phses, llowing ontinuous reording. All of the prmeters were mesured ontinuously nd simultneously in ll nimls over the susequent 7 d fter 5 d of dpttion in identil trining ges. Fel ft exretion For the olletion of fees, the mie were housed in metoli ges for 72 h t the end of the weight gin nd weight redution periods. The intke of feed nd drink ws monitored dily nd fees olleted t the end of the 72 h period. The fees were weighed nd stored in -7 until ssyed. The ft ontent of the fel smples ws determined y SBR (Shmid-Bondzynski-Rtzlff) method modified for fel smple nlysis [23]. The pprent ft sorption ws lulted from the mount of feed onsumed nd the mount of ft exreted during the housing in metoli ges. Apprent ft sorption (%) ws determined s 1 (ft intke-fel ft)/(ft intke). To estimte the effet of ft exretion on energy sorption during the whole study period, we lulted the pprent umultive energy sorption from ft using the umultive energy intke dt (pprent ft sorption % umultive energy intke from ft) s desried previously [24]. Blood gluose nd serum insulin Blood gluose nd ws nlysed from the lood smples tken t the termintion of the nimls. Blood gluose ws determined y gluometer (Super Gluord II, GT-163, Arkry Ftory In., Shig, Jpn). Serum insulin ws nlysed from frozen serum smples y ELISA kit for mouse insulin (Ultr sensitive Mouse Insulin ELISA kit 98, Crystl Chem In., IL, USA). The smple preprtion At the end of the tretment period, the mie were rendered unonsious with CO 2 /O 2 (95%/5%; AGA, Riihimäki, Finlnd) nd depitted. The lood smples were tken into hilled plsti tues, nd the serum ws seprted y entrifugtion t 4 for 15 min. The livers nd suutneous, epididyml, dominl nd perirenl ft pds were removed, wshed with sline, lotted dry nd weighted. The tissue smples for lipidomi nd primry metolite nlysis were snp-frozen in liquid nitrogen nd stored t -8 until ssyed. The smples for oil red O stining were frozen in isopentne (-38 ) nd stored t -8 until further proessed. The smples for histology were fixed in 4 g/l formldehyde nd emedded in prffin with routine tehniques. Liver histology nd Oil Red O stining For histologil evlution of the liver smples 4 mm setions of the prffin emedded smples were ut with mirotome, stined with H&E nd exmined with light mirosopy. The severity of the oserved lesions ws grded s previously desried [25]. In order to determine the reltive mount of lipids in the liver smples, frozen setions (4 mm) were stined with Oil Red O, mounted nd photogrphed. From the otined mirosopi imges, the mount of Oil Red O-positive stining ws determined with AnlySIS Prosoftwre (Soft Imging System, Münster, Germny). Lipidomis The lipidomi nlysis of liver tissue smples (n = 1/group) ws performed s desried previously desried [26]. Liver tissue lipid extrts were exmined y Q-Tof Premier mss spetrometer y introduing the smple through n Aquity UPLCTM system equipped with n Aquity UPLCTM BEH C18 1 mm 5 mm olumn with 1.7 mm prtiles. The ompounds were deteted y using eletrospry ioniztion in positive ion mode (ESI+). Dt ws olleted t m/z 3-12 with sn durtion of.2 s. Dt ws proessed using MZmine softwre version.6 [27,28], nd metolites were identified using internl spetrl lirry or with tndem mss spetrometry s previously desried [6,29]. Primry metolites Twenty mg of frozen liver tissue (n = 1/group) ws weighed into Eppendorf tues nd 2 ml of methnol (-8 ) nd 1 ml of 13 C leled internl stndrd ws dded. Smple ws homogenized with Miro Dismemrtor S (Srtorius, Germny) y using glss eds ( mm) nd 3 r/min for 3 min. Homogenized smples were oiled immeditely in 8 for 3 min nd t 1 r/min for 5 min. Superntnt ws olleted nd evported to dryness under strem of nitrogen. Smples were reonstituted in 1 ml of ultr pure wter. The liver extrts were nlyzed with HPLC-MS/MS method for quntittive nlysis of phosphorous nd TCA-yle ompounds. The system onsisted of HT- Alline HPLC (Wters, Milford, MA, USA) working t high ph. The nlytes were resolved y nion exhnge hromtogrphy omined with post olumn ASRS Ultr Ⅱ 2 mm ion suppressor (Dionex, Sunnyvle, CA) nd deteted with Quttro Miro triple qudruple mss spetrometry (Wters, Milford, MA, USA) operting in eletrospry negtive ion mode. The nlytil olumn

4 Pilvi TK et l. Energy restrition nd ftty liver 4465 A 45 B 45 Finl ody weight (g) 4 35 Body ft (%) ER ontrol. ER ontrol C 1 D 5 Blood gluose (mmol/l) Serum insulin (ng/ml) ER ontrol ER ontrol Figure 1 A: The ody weight of C57Bl/6J mie t the end of the study; B: The ody ft ontent of C57Bl/6J mie mesured y DEXA t the end of the study; C: The lood gluose of C57Bl/6J mie t the end of the study; D: The serum insulin of C57Bl/6J mie t the end of the study. Dt is presented s men ± SE. The letters denote signifint differene etween the groups ( P <.5; P <.1, P <.1; n = 1/group). ws IonP AS11 (2 mm 25 mm, Dionex, Sunnyvle, CA) nd gurd olumn IonP AG11 (2 mm 5 mm, Dionex, Dionex Sunnyvle, CA). Flow rte ws 25 ml/ min nd injetion volume 5 ml. The temperture of the olumn ws 35 nd utosmpler 1. The ompounds were deteted in Multiple Retion Monitoring mode for optiml sensitivity nd seletivity. Mss spetrometri prmeters, one voltge nd ollision energy were optimized for eh omponent. A smll liquot of 13 C-lelled metolites from yestfed th ultivtion ws used s n internl stndrd for oth lirtion stndrds nd smples. Hexose phosphtes (gluose-6-phosphte, frutose-6-phosphte, mnnose-6-phosphte nd 6-gluose-1-phosphte), pentose phosphtes (riose-5-phosphte nd riulose-5- phosphte), frutose isphosphte, glyerte-2-phosphte nd glyerte-3-phosphte, phosphoenolpyruvte, 6-phosphogluonte, suinte, mlte, -ketoglutrte, oxloette, itrte, iso-itrte, glyoxylte nd pyruvte were quntittively mesured with this method. Dt ws proessed with MssLynx 4.1 softwre nd internl lirtion urves were lulted sed on response of 12 C nlyte nd 13 C lelled nlogue. Sttistil nlysis Dt re presented s men ± SEM. Sttistilly signifint differene in men vlues were tested y ANOVA followed y Tukey s test. The dt were nlysed using GrphPd Prism, version 4.2 (GrphPd Softwre, In., Sn Diego, CA, USA). Sttistil nlyses of metolomis dt were performed using R sttistil softwre ( RESULTS Weight nd ft loss nd ft sorption during ER The ody weight of the high-ft fed mie inresed signifintly during the 14 wk d liitum feeding. At the end of the weight gin period the high-ft fed mie weighed signifintly more thn the how fed ontrol mie (Figure 1A). The oese mie lso hd signifintly more ft tissue thn the len ontrols (Figure 1B). The 7-week ER redued the ody weight in the whey + C group, to the level of len ontrols, ut the derese in ody weight ws not sttistilly signifint in the ontrol group. Whey + C lso redued the ft pd weights more thn the weight loss on ontrol diet

5 4466 ISSN CN /R World J Gstroenterol July 28, 28 Volume 14 Numer 28 Tle 1 Ft pd weights (g) ER ANOVA Control Whey + C P vlue Suutneous ft.4 ± ± ±.1 d,e 1. ±.1,d <.1 Epididyml ft 1.4 ± ± ±.1 f 1.3 ±.5.6 Perirenl ft.7 ± ± ±.1 d,f.9 ±.1 <.1 Dt is presented s men ± SE (n = 1/group). P <.5, P <.1, P <.1, vs oese, respetively; d P <.1 vs len; e P <.5, f P <.1 vs whey + C, respetively. Tle 2 Inidenes of the oserved histopthologil lesions in liver smples ER Control Whey + C Numer of smples No normlities deteted 8 Mrovesiulr ftty hnge (totl) Severe 1 Mrked 1 Moderte Slight Miniml 1 Infiltrtion of inflmmtory ells, miniml Fol heptoyte nerosis, 2 2 totl Slight 1 Miniml 1 2 A B Oil red O positive re (%) Ft droplet size (ritrry units) ER ontrol (Tle 1). Apprent ft sorption ws redued in the whey + C group in omprison with the ontrol diet (96.9% ±.3% vs 98.4% ±.1% in whey + C nd ontrol diet, respetively; P =.4). Blood gluose, serum insulin nd liver histology ER on oth diets redued the lood gluose to the level of len ontrols, ut the derese ws sttistilly signifint only in the whey + C group (Figure 1C). Also the serum insulin ws signifintly deresed only in the whey + C group (Figure 1D). In the oese group, the liver histology showed n evident mrovesiulr ftty hnge of diffuse pttern, with severity rnging from slight to severe (Tle 2). In the ER groups, the oserved ftty hnge ws less severe. The ft droplets were smller nd minly present in the perivenulr regions. Miniml foi of inflmmtory ells nd neroti heptoytes were osionlly noted, ut firosis ws sent. Oil Red O stining demonstrted tht ER on ontrol nd whey + C diet signifintly redued hepti lipid umultion nd lipid droplet size (Figure 2), ut the mount of ft did not reh the level oserved in the len mie. The effet of protein soure on metoli performne In order to investigte whether the more pronouned weight loss effet in the whey protein fed mie ws result of differenes in drinking nd feeding ehviour, inresed tivity or hnges in metoli performne, ER ontrol Figure 2 A: The Oil Red O positive re of prffin emedded liver smples of C57Bl/6J mie t the end of the study; B: The men ft droplet re (ritrry units) of prffin emedded liver smples of C57Bl/6J mie t the end of the study. Dt is presented s men ± SE. The letters denote the signifint differene etween the groups ( P <.5; P <.1, P <.1; n = 1/group). n dditionl group of mie were housed in home ge sed monitoring system. A 7-dy monitoring did not revel differenes in umultive feed or wter intke, respirtory exhnge rte, het prodution, O 2 onsumption, CO 2 prodution, totl or rering tivity or multory movements during the oservtion period (Figure 3). The effet of ER on liver lipid profile Of the totl 2498 hepti lipid peks deteted, 391 mjor peks were identified nd inluded in further nlysis. The redution of lipids ws minly seen in the level of triylglyerols (TAG) nd ermides nd ER on whey + C diet even restored the level of ermides to the level of len mie (Figure 4). The mount of holesterol esters ws signifintly inresed in oth ER groups. The TAG to phospholipid rtio, whih reflets the reltion of memrne lipids to storge lipids, ws signifintly redued only in whey + C group, ut it ws still higher thn in the len ontrols (Figure 5A). Interestingly, diylglyerol (DAG) to phospholipid rtio ws inresed in the ontrol ER group, ut deresed in

6 Pilvi TK et l. Energy restrition nd ftty liver 4467 Food intke (g) Cumultive food intke RER Respirtory exhnge rte Kl/h per kg Het prodution Counts Totl tivity Figure 3 A: Cumultive food intke over 7 d nlyzed y LMster system. The umultive totl food intke ws similr etween the groups; B: LMster nlysis of respirtory exhnge rte (RER); C: Het prodution mesured y LMster; D: Totl multory movement did not differ etween the groups. In ll figures dt is presented s men ± SE, n = 4 in sein group (red) nd n = 3 in whey group (green). the whey + C ER group (Figure 5B). ER on whey + C diet lso totlly reversed the oesity-indued inrese in the ermide to sphingomyelin rtio (Figure 5C). Phosphtidi id (GPA) nd phosphtidylglyerol (GPGro) peks ould not e uniquely distinguished y our method. The level of GPA/GPGro, phosphtidylholines nd lysophosphtidylethnolmines ws not ffeted y oesity or ER. The most signifintly hnged lipids re presented in Figure 6. Interestingly TAG (58:3) nd TAG (58:2) were t higher level in the ontrol weight loss group even though they were lredy inresed s result of oesity, nd so ws the level of TAG (5:8) in the whey + C group. Weight loss further inresed the level of GPCho (32:2) even though its level ws lredy over 1 times higher in the oese group thn in the len mie. On the other hnd, the level of TAG (52:) deresed during ER even though its level ws lredy lower in oese thn in the len nimls. The most distint fetures of whey + C ER group were the signifint inreses in the level of totl phosphtidylserines, phosphtidylethnolmines nd sphingomyelins (Figure 6). It is lso of note tht the level of ertin phosphtidylholine speies ws signifintly deresed during ER on ontrol diet wheres there ws no hnge in whey + C group. Whey + C speifilly ffeted ertin ermide speies [Cer (d18:/22:5), Cer (d18:/22:6), Cer (d18:1/23:3), Cer (d18:1/23:5), Cer (d18:1/26:4)], whose level ws redued to the level of len mie, wheres their level ws unffeted y ER on ontrol diet. Cer (d18:1/25:4) ws even inresed y ER on ontrol diet while its level did not differ etween len, oese nd whey + C group. The effet of ER on primry metolites The primry metolite nlysis led to identifition of 13 metolites (gluose-6-phosphte, frutose- 6-phosphte, mnnose-6-phosphte, fr utose isphosphte, glyerte- 3- phosphte, riose- 5- phosphte, suinte, mlte, itrte, pyruvte, phosphoenolpyruvte, 6-phosphogluonte nd fumrte). The high-ft diet feeding nd susequent oesity led to redution of glyolyti metolites, suh s gluose-6-phosphte, frutose-6-phosphte nd pyruvte (Tle 3, Figure 7). ER with whey + C diet ws ssoited with signifint inreses of suinte, whih elongs to the TCA yle nd of riose-5- phosphte, whih is produt of pentose phosphte pthwy. Whey + C diet lso deresed the level of glyolyti metolites gluose-6-phosphte, frutose-6- phosphte nd frutose isphosphte in ontrst with ER on ontrol diet, whih did not ffet the level of these metolites. DISCUSSION In this study, we showed tht deresing liver ft y ER signifintly modultes the overll profile of liver

7 4468 ISSN CN /R World J Gstroenterol July 28, 28 Volume 14 Numer 28 TAG DAG MAG Cermides Cholesterol esters GPA/GPGro A TAG/Phospholipid rtio ER ontrol Phosphtidylholines B.2 Phosphtidylethnolmines Phosphtidylserines Lysophosphtidyl holines Lysophosphtidylethnolmines DAG/Phospholipid rtio Sphingomyelins. ER ontrol Fold hnge vs oese Figure 4 Men fold hnges in lipid lsses in len (white), ER ontrol (lk) nd (grey) groups in reltion to the oese group (n = 1/group). The letters denote signifint differene in omprison with the oese group ( P <.5, P <.1, P <.1). lipid speies. The min finding of the study ws tht the protein soure nd lium ontent of the diet hd signifint effet on the ER-indued hepti lipid hnges. Even though the histologil nlysis did not revel signifint differenes in the mount of liver ft etween the ER groups, the metolomi dt demonstrted tht ER on whey + C diet ws le to redue the reltive level of potentilly dietogeni ermides nd diylglyerols to the level oserved in len nimls. This finding is in ordne with the deresed level of serum insulin in this group. These hnges were ompnied y derese in glyolyti metolites while the metolites from the pentose phosphte pthwy nd TCA yle were inresed together with shift towrds gluoneogenesis. The UPLC/MS sed lipidomis pltform nd the HPLC/MS/MS sed primry metolite pltform tehniques were used to hrterise the hepti lipid nd primry metolite hnges in this study. These tehniques provide n overview of key metolites involved in energy metolism, inluding rod profile overing ll mjor lipid lsses present in liver s well s key metolites of the entrl ron metolism. Trditionl nlyses of lipids hve een generlly limited C Cermide/Sphingomyelin rtio ER ontrol Figure 5 A: The liver TAG/Phospholipid rtio; B: The liver DAG/Phospholipid rtio; C: The liver Cermide/Sphingomyelin rtio. Lipids mesured y UPLC/ MS. Dt is presented s men ± SE. The letters denote signifint differene etween the groups ( P <.5; P <.1, P <.1; n = 1/group). to investigtions of lipid lss-or ftty id-speifi hnges [3]. These new nlytil methods omined with informtion tehnology provide extremely sensitive tools to mesure the extended metolome, nd my help to explore the mehnisms of mny omplex diseses [31,32]. However, one evident shortoming of the method is tht mjor prt of the spetrl peks re still unidentified. To our knowledge this is the first study to demonstrte the effet of ER on ftty liver lipidomi nd primry metolite profile in diet indued oese mie. In

8 Pilvi TK et l. Energy restrition nd ftty liver 4469 LysoGPCho (18:2e) DAG (38:4) TAG (54:5) GPCho (36:5) SM (d18:1/21:) SM (d18:1/22:1) GPCho (4:2) GPCho (38:2) GPCho (3:1) GPCho (42:5) GPCho (4:4) GPCho (42:7) GPCho(32:) TAG (61:12) GPEtn (34:) TAG (52:) GPCho (38:8) GPCho (36:5)_v2 GPEtn (36:5) TAG (54:3) TAG (52:8) GPCho (32:2) TAG (56:1) TAG (5:8) TAG (58:11) TAG (56:2) GPCho (42:9) TAG (52:6) TAG (5:5) TAG (62:14) TAG (58:2) TAG (58:3) TAG (58:1) TAG (56:9) TAG (6:12) DAG (4:6) Cer (d18:/22:6) TAG (54:8) TAG (58:9) TAG (54:3)_v2 ER ontrol ER ontrol ER ontrol ER ontrol ER ontrol ER ontrol ER ontrol ER ontrol ER ontrol ER ontrol Fold hnge (log2) Figure 6 Twenty most signifintly up- nd down-regulted lipids etween oese nd len group. Fold hnge for eh individul mouse within eh group s log2 rtio etween the lipid onentrtion in individul smple nd the medin lipid onentrtion in the oese group. Hierrhil lustering using Wrd linkge ws pplied. Tle 3 Conentrtions of primry metolites in liver smples (mmol/g tissue) ER ANOVA P vlue Control Whey + C Gluose-6-phosphte 28.6 ± ± ± 1.7 h 11.2 ± 3.2 f <.1 Frutose-6-phosphte 7. ± ± ±.7 h 3.2 ±.9 e.1 Mnnose-6-phosphte 1.7 ± ± ±.1 g.8 ±.2,f.3 Frutose isphosphte 3.2 ± ± ± 2.4 d,h.5 ±.1.5 Glyerte-3-phosphte 26.2 ± ± ± 1.2 d 13.1 ± 1.4 f.8 Riose-5-phosphte.3 ±.2.7 ±.1.7 ±.1 d,i 1.3 ±.1,f <.1 Suinte 24.5 ± ± ± 2.4 f,i 24. ± 4. <.1 Mlte 42. ± ± ± 4.7 d,g 4.5 ± Citrte 4.9 ±.7 2. ± ±.2 f 1.8 ±.3 f <.1 Pyruvte 5.6 ±.8.9 ± ±.4 e 1.5 ±.3 f <.1 Phosphoenolpyruvte 2.8 ± ± ± ± phosphogluonte 3.5 ± ± ±.2 e,g 2.6 ±.4.23 Fumrte 9.6 ± ± ± 1.7,f,g 11.9 ± Dt is presented s men ± SE (n = 1/group). P <.5, P <.1, P <.1 vs oese, respetively; d P <.5, e P <.1, f P <.1 vs len, respetively; g P <.5, h P <.1, i P <.1 vs whey + C, respetively. ordne with previous studies on lipidomi profile of non-loholi ftty liver disese, we lso found inresed levels of TAG, DAG nd speifi ermide speies nd down-regultion of sphingomyelins in the oese group [6]. Interestingly, only ermides were signifintly deresed y ER on ontrol diet, while the level of DAG inresed non-signifintly nd sphingomyelins styed un-hnged. However, ER on whey + C diet signifintly inresed the level of sphingomyelins nd deresed the level of DAG hnging the ermide/sphingomyelin nd DAG/ phospholipid rtios to the level of len nimls. The umultion of oth ermides nd DAG in peripherl tissues ontriute to insulin resistne [33-35] nd, therefore, the derese of these lipids n e onsidered

9 447 ISSN CN /R World J Gstroenterol July 28, 28 Volume 14 Numer 28 3-Phosphoglyerte Mnnose-6-phosphte Phosphoenolpyruvte Citrte Fumrte 6-Phosphoglyerte Frutose-6-phosphte Mlte Gluose-6-phosphte Suinte Frutose-1,6-isphosphte Pyruvte Riose-5-phosphte ER ontrol ER ontrol ER ontrol ER ontrol ER ontrol ER ontrol ER ontrol ER ontrol ER ontrol ER ontrol Fold hnge (log2) Figure 7 Primry metolite profiles for eh individul mouse s log2 rtio etween the metolite onentrtion in individul smples nd the medin metolite onentrtion in the oese group. Hierrhil lustering using Wrd linkge ws pplied. prtiulrly enefiil. Additionlly n inrese in liver holesterol ester level, whih ws seen in oth of the ER groups, hs een demonstrted to our lso s result of ute 24 h food deprivtion [36]. One of the min funtions of holesterol is mintining of memrne fluidity y interting with other memrne lipid omponents suh s phosphtidylholines nd sphingomyelin [37]. However, only holesterol esters were found to e inresed s result of ER in the ontrol group. The level of primry metolites ws prtiulrly ffeted in the whey + C group. Energy restrition in norml weight, helthy mie, is known to enhne hepti gluoneogenesis [38,39] nd suppress glyolysis [4]. This effet ws prtiulrly pronouned in the whey + C group s indited y the striking derese of frutose isphosphte, the key regultor of gluoneogenesis, nd signifint derese of glyolyti intermedites gluose-6- phosphte nd frutose-6-phosphte. An inresed level of riose-5-phosphte in the whey + C group indites enhned flux through pentose phosphte pthwy, whih is known to e triggered y low onentrtions of frutose-2,6-isphosphte [41]. ER on whey + C diet lso deresed the level of suinte to the level of len nimls, wheres the level of suinte did not hnge in the ER ontrol group. The signifine of the derese of mnnose-6-phosphte in whey + C groups remins to e eluidted. One of the few dietry omponents whih re known to influene the liver ft profile during ER is the type nd mount of dietry ft [8]. In this study there were no differenes in either the type or mount of dietry ft etween the ER groups. However, the pprent ft sorption ws deresed in the whey + C group. Clium preferentilly inds sturted ftty ids in the intestine [42], nd therefore, lso the qulity of the sored ft might hve een influened in the whey + C group. Even though these findings my help to understnd why inresed diry lium intke my lower the risk of metoli syndrome, the moleulr mehnism y whih whey protein nd lium modulte the liver lipid profile remin unnswered. Whey protein onsists of severl smll protein types, inluding lf-ltlumin, etltogloulin, ovine serum lumin, ltoferrin nd other minor peptides [43]. In order to investigte the possile effets of whey protein on energy expenditure nd food intke, we mesured the metoli performne of mie fed either sein or whey sed diet in lorimetry system, ut did not see ny differenes etween the proteins. The prinipl question regrding the mehnism is whether the enefiil effet is derived only from the mino ids or if iotive peptides re formed during the digestion nd sorption of the protein. The present study demonstrtes tht ER-indued hnges in ftty liver re signifintly ffeted y dietry protein soure nd lium. Reduing liver ft y ER is urrently the min tretment for non-loholi ftty liver disese nd therefore, it is ruil to understnd whih dietry ftors hve signifint effets on the outome of ER in liver. These results indite tht whey protein nd lium ould e enefiil in the dietry tretment of ftty liver, nd re likely ontriute to the inverse reltionship etween diry intke nd the risk of insulin resistne. The therpeuti potentil of whey protein nd lium in linil setting, nd the mehnism of tion remin to e eluidted. ACKNOWLEDGMENTS We re grteful to MS Mrjut Louhelinen, MS Sr Mersto, Ms Sri Lkkonen, Mrs Anneli von Behr for expert tehnil ssistne. COMMENTS Bkground Ftty liver is onsidered to e n importnt link etween oesity nd the development of metoli syndrome nd insulin resistne. Liver ft n e redued y weight loss, ut the effet of weight loss on the qulity of hepti lipid profile is urrently not well estlished. Reserh frontiers Some dietry ftors, like the quntity nd qulity of ft during weight loss hve een shown to hve n effet on liver ft. However, the importne of dietry protein soure nd lium ontent hs not een investigted previously. Innovtions nd rekthroughs This study hrterises the hnges in hepti lipid profile during energy

10 Pilvi TK et l. Energy restrition nd ftty liver 4471 restrition in mouse model of diet indued oesity. The effet of protein soure nd lium ontent of the weight loss diet is lso studied. This study demonstrtes for the first time tht dietry protein soure my enefiilly modulte the lipid profile of ftty liver during energy restrition. Applitions Weight loss y life style hnges is the min therpeuti pproh to redue liver ft. Therefore, it is ruil to identify dietry omponents whih my improve the outome of weight loss in the level of hepti lipid profile. These results indite tht whey protein nd lium enefiilly modulte the hepti lipid profile trgeting speifilly the lipotoxi diylglyerol nd ermide speies. Peer review The study is well onduted nd hs importnt informtion out the pthophysiology of ftty liver. REFERENCES 1 Mison P, Byrne CD, Hles CN, Dy NE, Wrehm NJ. Do different dimensions of the metoli syndrome hnge together over time? Evidene supporting oesity s the entrl feture. 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