Alpha linolenic acid in maternal diet halts the lipid disarray due to saturated fatty acids in the liver of mice offspring at weaning

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1 Shomonov-Wgner et l. Lipids in Helth nd Disese (2015) 14:14 DOI /s RESEARCH Open Access Alph linolenic cid in mternl diet hlts the lipid disrry due to sturted ftty cids in the liver of mice offspring t wening Limor Shomonov-Wgner 1,2, Amirm Rz 2 nd Alici Leikin-Frenkel 1,3* Astrct Bckground: Alph linolenic cid (ALA, 18:3) in mternl diets hs een shown to ttenute oesity ssocited insulin resistnce (IR) in dult offspring in mice. The ojective in the present study ws to detect the erly effects of mternl dietry sturted ftty cids (SFA) nd their prtil sustitution with ω-3 ALA, docos hexenoic cid (DHA,22:6) nd eicospentenoic cid 20:5 (EPA,20:5) on the HOMA index, liver lipids nd ftty cid desturses in the offspring t wening. Methods: 3 month old C57Bl6/J femle mice were fed diets contining norml mount of clories ut rich in SFA lone or prtilly replced with ALA, DHA or EPA efore mting, during pregnncy nd lcttion. Results: Pregnnt mice fed SFA produced offspring with the highest HOMA index, liver lipids nd desturse ctivities. ALA prevented SFA induced lipid increse ut DHA nd EPA only reduced it y 42% nd 31% respectively. ALA, DHA nd EPA decresed HOMA index y 84%, 75% nd 83% respectively. ALA, DHA nd EPA decresed Δ6 nd SCD1 desturse ctivities out 30%. Conclusions: SFA feeding to mothers predisposes their offspring to develop IR nd liver lipid ccumultion lredy t wening. ω3 ftty cids reduce IR, ALA hlts lipid ccumultion wheres DHA nd EPA only lunt it.ala nd DHA restore the incresed SCD1 to norml. These studies suggest tht ω-3 ftty cids hve different potencies to preclude lipid ccumultion in the offspring prtilly y ffecting pthwys ssocited to SCD1 modultion. Keywords: Mternl diet, Omeg (ω) -3 ftty cids, Sturted ftty cids, Nutrition, Liver, Lipids, Ftty cids/desturses, Progrmming, Insulin resistnce Bckground Metolic syndrome nd crdiometolic risk hve progressively ecome mjor pulic helth prolem [1]. Lifestyles fctors such s diet, incresing mternl ge, endocrine disruption, etc. were suggested [2] to ffect physiologicl nd metolic processes nd the underlying genetic networks in tissue specific mnner [3,4]. Incresed iosynthesis nd reduced oxidtion of FAs triggers oesity development nd oesity-relted complictions including insulin resistnce (IR), nd the metolic syndrome [5,6]. High consumption of dietry fts lso contriutes to these * Correspondence: lici.leikin@she.helth.gov.il 1 Lortory for Lipid Metolism in the Liver, Sckler School of Medicine, Tel Aviv 69978, Isrel 3 Bert W. Strssurger Lipid Center, She Medicl Center, Tel-Hshomer, Isrel Full list of uthor informtion is ville t the end of the rticle diseses. However, recent evidence indictes tht the qulity, eyond the quntity, of dietry FAs ffects differentilly some of these metolic prmeters with SFA hving shown to increse lipogenesis nd the expression of responsile genes in the liver nd dipose tissue [7,8]. In contrst there is evidence to suggest tht essentil FA i.e., ALA nd linoleic cid ( LA:18:2 ω-6) [9] nd the PUFA derivtives, DHA nd EPA reduce oesity- ssocited risk y promoting n increse in FA oxidtion [10,11]. The impct of dietry ftty cids on developmentl origins of metolic disese is still poorly understood. The Fetl Origins hypothesis sttes tht severl of the mjor diseses of dult life originte in impired intruterine growth nd development [12-14]. Accumulting evidence in oth humn nd niml model indictes tht ω-3 ftty cids EPA nd DHA do hve distinct cpilities 2015 Shomonov-Wgner et l.; licensee BioMed Centrl. This is n Open Access rticle distriuted under the terms of the Cretive Commons Attriution License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly credited. The Cretive Commons Pulic Domin Dediction wiver ( pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Shomonov-Wgner et l. Lipids in Helth nd Disese (2015) 14:14 Pge 2 of 11 to modulte oth cellulr metolic functions nd gene expression [15]. Thus Δ6 desturse, key enzyme in the conversion of ALA to EPA nd DHA is significntly reduced in oesity, IR, nd metolic syndrome [16,17]. On the other hnd SCD1 tht converts sturted into monounsturted ftty cids is incresed in oesity, NAFLD nd IR [18 20]. During mmmlin development, FAs consumed y the mother re trnsferred to the fetus through the plcent. Therefore, FAs present in the fetus reflect the mternl diet nd lso it s own metolic products [21-25]. Thus, the qulity nd composition of dietry FAs, in ddition to the quntity consumed, my hve criticl roles on the regultion of metolic nd/or endocrine pthwys during fetl development. This is prticulrly relevnt for the essentil ALA nd LA, which cnnot e synthesized in mmmls nd must e therefore oligtory consumed in the diet [9]. Severl studies hve shown tht supplementing the diet of mothers with ω-3 PUFA during pregnncy led to reduction in ft mss in the offspring [26,27] nd decresed inflmmtion. We hve recently reported [28] tht enriching mternl diet of mice during pregnncy-lcttion with ALA prevented oesity-ssocited insulin resistnce nd ftty liver in the dult offspring when chllenged with high ft diet (HFD).In contrst, providing mothers n SFA-enriched diet exsperted the metolic dysfunctions cused y HFD in dult offspring. In light of these findings, the effect of exposure to different ftty cids clsses qulity, rther thn quntity, during the perintl period on lipid metolism in the offspring clls for further investigtion. The hypothesis of the current study is tht mternl consumption during pregnncy of diets enriched with ALA nd, possily, other ω-3 ftty cids my prevent the erly fetl development of SFA-induced lipid derngements leding to long term disese in the offspring. We question here the differentil involvement of mternl dietry ω-3 ALA, DHA or EPA in the prevention of IR nd heptic lipid ccumultion nd the contriution of ftty cid desturses in the process in wening offspring. We show tht ALA is the most efficient ω-3 ftty cid in the prevention of liver lipids ccumultion nd tht the normliztion of desturses my contriute to the involved mechnisms. Results This study exmined the effects of feeding five groups of mothers diets contining 6 g% ft nd the sme mount of clories equivlent to those in regulr chow diet ut differing only in the ftty cid composition confirmed y GC s descried in Methods. Dms helth ws not ffected y the mternl diets nd the sme numer of pups were fed per mother during lcttion for ll dietry tretments. Mternl dietry ftty cid composition ffected differently glucose nd insulin in the offspring Plsm glucose ws slightly ut significntly lower in ALA compred to the other diets. Significntly, plsm insulin nd HOMA index were lmost 6 fold higher in SFA group thn in RD nd ALA (Figure 1A,B,C). DHA nd EPA lso lowered glucose nd HOMA index. Mternl dietry ftty cid composition differentilly ffected liver weight in the offspring In line with the ove dt, totl liver weight nd liver/ ody rtio of the offspring t wening were out 15% nd 25%, respectively, lower for ALA nimls thn for the other dietry tretments (Figure 2B nd C), while the nimls were otherwise helthy. Mternl dietry ftty cids differentilly ffected liver lipid content nd lipid clsses distriution The lower liver/ody rtio in ALA offspring suggested the possiility of lower lipid content in the liver of those nimls. Indeed, TLL were three times higher in SFA thn in RD nd ALA nd indictive of stetosis DHA nd EPA hd 50% nd 30% lower liver ft, respectively, thn SFA ut still significntly higher thn ALA nd RD (Figure 3A). The lower mount of totl ft ws due minly to decrese in TG seen y TLC (Figure 3A) nd confirmed y Oil Red O stining (Figure 3B). Other lipids clsses, mong them free ftty cids, dicylglycerides nd free cholesterol, were significntly higher in SFA thn in other tretments while they were similr in RD nd ALA. TLL higher thn 5%, ws developed in nimls fed SFA, DHA nd EPA while prevented y ALA. Interestingly, Figure 4 shows significnt correltion etween TLL nd HOMA index. It cn e seen clerly here tht in ALA nd RD oth prmeters were lower thn in other diets.sfa hd the higher prmeters, sitting high in the correltion grph. Mternl dietry ftty cids differentilly modified liver ftty cid composition nd ftty cid desturses in the offspring TLL ftty cid nlysis showed tht in the SFA offspring there were higher (n.s.) levels of SFAs nd MUFA nd lower levels of PUFA, compred with the other diets (Tle 1) except for, surprisingly, EPA. In turn DHA offspring hd the highest (n.s) mount of totl PUFAs. Ftty cid desturses re mostly ctive in the liver nd their ctivities cn e clculted from the surrogte mrkers in the ftty cid profile. Δ6 desturse index, clculted from the surrogte mrkers [GLA + AA/LA] ws significntly higher in SFA thn in other groups (Figure 5A left). SCD1 index, clculted from the surrogte mrkers in the plsm ftty cids profile [POA + OA/ PA + SA] ws decresed y the ω-3 FA diets (Figure 5A

3 Shomonov-Wgner et l. Lipids in Helth nd Disese (2015) 14:14 Pge 3 of 11 A B C Figure 1 Insulin resistnce prmeters in the offspring. A - Plsm Glucose levels, B- Plsm Insulin levels nd C-HOMA index in offspring t dy 21, fter wening. Mens (n = 7) without common letter difference p < 0.05 etween the mternl dietry tretments indicted in the x-xis, s descried in Methods. right). Similr to our previous work [28] nd with comprtive purposes, liver FADS2 mrna expression levels were clculted s fold chnge in different mternl dietry tretments relted to those in RD diet fter normliztion to ctin. ALA nd DHA hd significntly lower vlues thn other groups wheres EPA, unexpectedly, hd significnt higher vlues thn ll other diets (Figure 5B left). Liver SCD1 mrna expression levels in SFA nd EPA were significntly higher thn other diets (Figure 5B right). Discussion In this pper the role of ftty cids in mternl diets nd their effects on the liver lipid metolism nd helth sttus of the offspring ws investigted y iochemicl nd metolic pproches. We found tht SFA in mternl

4 Shomonov-Wgner et l. Lipids in Helth nd Disese (2015) 14:14 Pge 4 of 11 A B C Figure 2 Offspring ody weigh, liver weight nd liver/ody rtio. The offspring ody weight (A), liver weight (B) nd their rtio (C) t wening re shown s mens (n = 8 RD,12SFA,21ALA,7 DHA nd 15 EPA). Those without common letter differ p < 0.05 etween the mternl dietry tretments indicted in the x-xis, s descried in Methods. diets predisposed the 21 dys wening offspring to IR nd liver lipids ccumultion wheres prtil replcement with ALA hlted those chnges. Interestingly, DHA nd EPA, while preventing IR, only prtilly reduced lipid ccumultion. One of the most striking fetures of the phenotype in the model used is tht mternl SFA induced lipid ccumultion in the wening offspring in the liver in the sence of chnges in ody weight. This my point to the liver s the first sensitive orgn during development to sense the SFA detrimentl effects [3]. Moreover, this study shows the distinct eneficil effects of prtil replcement of SFA with ALA, DHA or EPA on the modultion of the induced lipid disrry. Similr to our previous study [28] nd different from other niml models previously descried [29] the dms in our study were helthy during pregnncy. Therefore, the effects of the mternl diets on the offspring cn e directly ssocited with the

5 Shomonov-Wgner et l. Lipids in Helth nd Disese (2015) 14:14 A Pge 5 of 11 Liver Lipid Clsses 10 RD SFA DHA ALA EPA mg Lipid/ mg Liver % 8,c c c,c c c 0 Totl TG FFA DG CE C PL Lipid Clsses B Figure 3 Liver lipids. A Liver lipid clsses distriution expressed s percent lipids of the totl liver mss in the offspring offspring t dy 21, fter wening. Mens (n = 7) without common letter difference p < B- Liver neutrl lipids stining with ORO nd oserved y light microscopy with 60 mgnifiction. Figure 4 Correltion etween HOMA-IR nd TLL in the offspring of mothers fed different diets, represented y different symols/ Empty squres: ALA, frmed filled squres RD, filled circles: DGA, filled lrge squres:epa, dimonds: SFA. differentil ftty cids composition of the mternl diets. HOMA index, direct indictor of insulin resistnce, ws incresed in the SFA offspring ut not in the ω3 FAs offspring. These effects were lso previously oserved in the dult offspring ut only fter HFD induced oesity [28]. They re lso in line with the oserved detrimentl effects of dietry SFA on IR when provided directly to dult nimls s previously descried y others [30]. Since in this study the offsprings were not oese t wening, the present dt strongly imply tht SFA in mternl diets hd direct per se effect on the emryionl physiologicl nd metolic pthwys involved in progrmming insulin resistnce in the offspring [31]. Our results show tht SFA in mternl diet predispose liver lipid increse in the wening offspring, independent of ody weight modifictions (Figure 3A,B). An ccumultion of sturted ftty cids in the liver my ply n

6 Shomonov-Wgner et l. Lipids in Helth nd Disese (2015) 14:14 Pge 6 of 11 Tle 1 Offspring liver ftty cid composition Ftty cid undnce (mol %) Ftty cids in liver lipids RD SFA ALA DHA EPA Sturted ftty cids 14:00 Myrisitic cid 1.0 ± ± ± ± ± :00 Plmitic cid 21.4 ± ± ± ± ± :00 Steric cid 17.7 ± ± ± ± ± 1.8 Totl SFAs 40.2 ± ± ± ± ± 3.9 Monounsturted ftty cids (MUFA) 16:1ω-7 Plmitoleic cid 0.4 ± ± ± ± ± :1 ω -9 Oleic cid 9.2 ± ± ± ± ± 0.8 Totl MUFA 9.6 ± ± ± ± ± 0.7 Polyunsturted ftty cids (PUFA) 18:2 ω6 Linoleic 27.4 ± ± ± ± ± :3 ω6 γ- Linolenic cid 0.4 ± ± ± ± ± :4 ω6 Archidonic cid 11.3 ± ± ± ± 0.8 c 8.2 ± 0.8 c Totl n-6 PUFA 39.2 ± ± ± 2.9, 23.3 ± ± :3 ω3 α-linolenic cid 1.1 ± ± ± ± ± :5 ω3 Eicospentenoic cid 0.4 ± ± ± ± ± :6 ω3 Docoshexenoic cid 9.3 ± ± ± ± ± 1.9 Totl ω n-3 PUFA 10.9 ± ± ± ± 3.1 c 26.1 ± 2.5 c Totl PUFA 50.1 ± ± ± ± ± ±4.7 Ftty cids re expressed s mole %. Ftty cids chnged mong the different mternl dietry tretments pper in old. Mens (n = 7) without common letter (, or c) difference p < 0.05 etween the mternl dietry tretments indicted in the x-xis, s descried in Methods. importnt role in the eginning of stetosis nd growing ody of literture suggests tht sturted ftty cids myinititethetoxiceffectsledingtothepthogenesis of NAFLD, impired liver function nd ultimtely liver filure [32,33]. The lower liver weight nd liver/ ody rtio in otherwise helthy ALA nimls (Figure 2B nd C) ws linked to the lck of dditionl ft in the liver of ALA offspring compred to RD nd 100% significntly lower TLL thn SFA offspring. DHA nd EPA, in turn, hd lower mount thn SFA ut still, higher thn RD, (Figure 3A,B). Hepto-stetosis my led to primry NAFLD tht, in turn, my e ssocited with IR nd my lso precede NAFLD nd its phenotypic mnifesttions [34]. In the present study oth conditions were found to e induced y mternl dietry SFA in the offspring nd decresed y the prtil replcement of SFA with ALA (Figures 3 nd 4). The significnt correltion oserved etween HOMA-IR nd TLL (Figure 4) my suggest tht ALA interferes with the mechnism/s y which SFA induce lipid increment in the offspring [35] differently thn DHA nd EPA. Recent reviews on the effects of ω-3 PUFA provided directly to dult nimls, on metolic diseses, hve focused primrily on the effects of EPA nd DHA [36,37]. Furthermore, studies on the eneficil effects of ALA in the context of NAFLD suggest tht these effects re solely due to the conversion of ALA to the long-chin products EPA nd DHA. Our results, however, support direct eneficiry effect y dietry ALA, independent of its longer chin metolic products ( the conversion of ALA to DHA nd EPA ws 68% nd 12.5% respectively). These results re in greement of others in which ALA ws fed to dult nimls [38,39]. Possile mechnisms for the eneficiry effects of ALA on preventing liver lipid ccumultion vs. the detrimentl effects of dietry SFA my e due, t lest in prt, to their differentil ilities to modulte metolic pthwys leding to higher lipid synthesis/ccumultion [40] or to promote FA oxidtion [10,11]. However, possile mechnisms involving PPARα, nd the susequent regultory pthwys, hve lredy een shown in dult mice not to ccount for the ALA eneficil effect [41]. The liver ftty cid profiles mesured in this work would indicte tht the mternl dietry ftty cids were indeed trnsferred to the offspring nd distinctly metolized. Mternl ω-3pufa led to decrese of totl MUFA nd ω-6pufa with significnt increse in totl ω-3fa compred to RD nd SFA in liver offspring. Moreover, not only ALA ws the highest in the ALA fed offspring ut it ws lso significntly converted into EPA nd DHA. DHA ws shown to e the highest in the DHA fed offspring nd represented most of the ω-3pufa. A

7 Shomonov-Wgner et l. Lipids in Helth nd Disese (2015) 14:14 Pge 7 of 11 Figure 5 Δ6 desturse nd SCD1 index. FADS2 nd SCD1 mrna levels. A - Represents the liver Δ6 desturse index clculted from the plsm ftty cids profile for linoleic cid (18:2) nd it s products of desturtion γ-linolenic (18:3) nd desturtion/elongtion rchidonic (20:4 ) cids (left) nd the SCD1 index clculted from the plsm ftty cids profile for plmitic (16:0) nd steric cids (18:0) nd their monounsturted products, plmitoleic (16:1) nd oleic (18:1) (right). B - Represents the FADS2 (left) nd SCD1 mrna (right) expression levels offspring t dy 21, fter wening in liver, clculted s fold chnge compred to the RD in the sme group. Mens (n = 7) without common letter difference p < The x-xis indictes tht the different mternl diets received y nimls, s descried in Methods nd the determintions were normlized y ctin levels nd clculted s fold chnge compred to the RD in the sme group. very low mount of EPA ws formed proly y retro conversion [42]. EPA, in turn, ws the highest in the EPA fed offspring nd ws lso significntly converted into DHA. However, neither DHA nor EPA s dietry components, chieved the potency shown y ALA to prevent lipid increse, thus highlighting the distinctiveness of ech of the ω-3fa tested, s lso ddressed y others [43]. Mternl SFA induced n increse in Δ6 desturse products γ-linolenic nd rchidonic cids (implying lso Δ5 desturse) in the offspring. This increse ws significntly lunted y ω-3fas. Mternl SFA induced n increse in SCD1 monounsturted 16:1 nd 18:1 products, compred to RD, tht ws significntly decresed y ω-3fa mternl diets. The gene expression levels of FADS2 were lower in the ALA nd DHA fed nimls thn in SFA fed ones, wheres in the EPA-fed group they were unexpectedly higher. SCD1 mrna levels were significntly incresed y SFA compred to RD nd significntly reduced for ALA nd DHA ut, surprisingly, unchnged for EPA. SCD1 deficiency hs een shown to ctivte metolic pthwys tht promote β-oxidtion nd decrese lipogenesis in liver nd skeletl muscles ndpossilylowerir[44].inturn,decreseinδ6 desturse ctivity, hs een found to e correlted with reduced inflmmtory processes [45]. The oserved lck of correltion found in this work etween the ctivity nd mrna expression for FAD2 nd SCD1 in the EPA-fed group my involve post-trnsltionl modifiction or inhiitory conditions for the enzyme ctivities. Our results hint to the potentil of dietry ftty cids ALA, DHA nd

8 Shomonov-Wgner et l. Lipids in Helth nd Disese (2015) 14:14 Pge 8 of 11 EPA, in pregnncy, to differentilly ffect FADS2 nd SCD1 expression, ctivity nd lso pthwys tht lower lipid ccumultion [20,22]. We hve found the ctive involvement of the desturses in the offspring processing of the ω-3fa consumed in the mternl diets. However, the similrities of their response do not support completely their different ility to prevent (ALA) or reduce (DHA nd EPA) liver lipid levels. This pprent contrdiction my point t different regultory pthwys [46-48] ctivted erly y ech of the mternl ftty cids in the foetuses or, mye, to genetic vritions [49,50]. Similrly, possile epigenetic modifictions like those descried for SCD1 [51] my e differentilly triggered y mternl ftty cids nd will e nlysed in the future. Even though this is still reltively unexplored re, two conclusions emerge clerly: first, tht mternl sturted ftty cids, independent of ft mount or clories, induce differentil metolic effects leding to liver lipid ccumultion tht cn e detected in the offspring phenotype s erly s 21 dys of life; nd second, tht not only lph linolenic cid nd sturted ftty cids hve divergent effects on insulin resistnce nd liver lipid levels ut, moreover, different ω-3 ftty cids ehve differently nd should e referred to nd studied individully nd not s group. Moreover, the lph linolenic cid preventive effects oserved in the present study pper to e, presently, unrelted to it s conversion to docos hexenoic cid nd eicos pentenoic cid. Steroyl CoA desturse1 decrese in lph linolenic cid orn offspring my ccount for possile mechnism of reduction of lipogenesis. However, docos hexenoic cid lso decreses Steroyl CoA desturse1 ut prtilly reduces totl liver lipids. Other mechnisms should then e serched to explin the differences. They my e found ssocited to the different interction etween the tested ftty cids nd nucler fctors involved in regulting the lnce etween ft storge nd oxidtion, nmely LXRs, PPARs nd trget genes [52]. Although the moleculr mechnism y which lph linolenic cid prevents lipid ccumultion in the liver hs not een elucidted in this work, preliminry results from our lortory my indicte tht it up regultes the expression of genes involved in lipid oxidtion. Our present findings my hve importnt clinicl implictions due to the vst numer of iologicl pthwys regulted y nutritionl ftty cids [53-55]. These results shed light on the erly time for detection nd potentil wys for the prevention of lipid-relted metolic disese in the offspring y mens of ddressing the mternl diet nd its ftty cid composition. Mterils nd methods Animls nd diets C57Bl6/J mice, 4 5 weeks old, were otined from the niml fcility of Tel-Aviv University. The studies were pproved y the Institutionl Committee for Animl Experiments t Tel Aviv University, Tel Aviv, nd Isrel. Femle mice were fed five isocloric diets.the control diet 1-RD contined 6 g% Soyen of commercil origin. The test diets contined 2-SFA 6 g% coconut oil lone: or mixture of 4.2 g% coconut oil with 1.8 g% of ω3 ftty cids 3-ALA, 4-DHA nd 5-EPA respectively. 99% pure ftty cids were generous gift of Prof. Amirm Rz (Tel Aviv University). The composition of the vrious diets is presented in Tle 2A. The dietry ftty cid (Tle 2B) nlysis showed tht the proportion of SFA diet ws 34.9% in SFA. ALA ws % in ALA diet, DHA ws 48.2% in DHA diet nd EPA ws 59.43% in EPA diet. The totl ω-3/ω-6 rtio rnged from 0.1 in RD to 3.2 in the ALA diet. C57Bl6/J femles received the experimentl diet two weeks pre-conception nd during pregnncy nd lcttion. Mles received regulr chow diet except during the mting period during which they shred their femle mtes diets (1 mle for every 2 femles). The numer of pups orn per mother for different diets ws 7 ± 1 for RD, SFA, ALA nd 6 ± 1 for DHA nd EPA. Mting ws performed more thn once. Pups were exposed to their mother s diet during lcttion. At dy 21 (wening) pups were euthnized y n overdose nesthetic (Xylzine-Ketnl), fter n overnight (12 h) fsting. Liver smples were otined under liquid nitrogen nd were kept frozen (-70 C) until further use. Plsm ws seprted from the lood nd kept frozen (-20 C) until use. At ll stges, the nimls hd free ccess to food nd wter nd were kept in ventilted rooms under light/drk cycle of 12 h/12 h. Guidelines for the use nd cre of the nimls t Tel Aviv University s Animl House were followed. Food consumption nd ody weight were monitored weekly. Biochemicl determintions PlsmglucoselevelsweremesuredyusingPrecision QID sensor MediSense (Aott Lortories Co MediSense Inc, Bedford, MA). Plsm insulin levels were determined fter overnight fst (12 h) using n insulin immunossy kit (MRC Mouse Insulin, Elis 96 T, Mercodi AB, Sweden). The HOMA index ws clculted s Plsm glucose [mg/dl] X Plsm Insulin [μu/ml]/22.5. Lipid extrction Liver smples were weighed nd homogenized with sline t rtio of 1:5 (w: v) in plstic tues on ice. Lipids were extrcted from n liquot of the liver homogente ccording to the procedure of Folch et l. [56]. The totl mount (totl liver lipids, TLL) ws clculted fter liquot evportion to constnt weight (18). TLL distriution ws nlyzed y TLC pplying 50 μg liquots. Smples were reconstituted in 50 μl of chloroform nd spotted on Silic-G thin-lyer chromtogrphy (TLC) pltes.

9 Shomonov-Wgner et l. Lipids in Helth nd Disese (2015) 14:14 Pge 9 of 11 Tle 2 Mternl diet composition A Dietry composition (g%) Mternl diet Protein 20.1 Crohydrte 53.8 Ft * 6 Other 20.1 Totl 100 Clories from Ft (%) 12.6 B Ftty cid undnce (mol%) Mternl diets RD SFA ALA DHA EPA Sturted ftty cids 14:00 Myrsitic cid :00 Plmitic cid :00 Steric cid Totl SFAs Monounsturted ftty cids (MUFA) 16:1ω-7 Plmitoleic cid :1ω-9 Oleic cid Totl MUFA Polyunsturted ftty cids (PUFA) 18:2ω-6 Linoleic :3ω-6 Gmm linolenic cid :4ω-6 Archidonic cid Totl ω-6 PUFA :3ω-3 Alph linolenic cid :5ω-3 Eicospentenoic cid :6ω-3 Docoshexenoic cid Totl ω-3 PUFA Totl PUFA ω3/ω *Ft in mternl diet ws Soyen Oil for RD, Coconut oil for SFA nd Coconut oil (4.8%) nd ALA, DHA or EPA (1.8%) for the diets under those nmes. Ft free chow diet contined fier, sh, minocides, minerls nd vitmins dded. A - All diets fed to dms during pre-conception, pregnncy nd lcttion were prepred y mixing 94 g% ft-free chow diet with 6 g% different fts/oils: RD-soyen, SFA-coconut oil, ALA-ALA + coconut, DHA-DHA + coconut, EPA-EPA + coconut. Ft-free chow diet contined fier, sh, minocides, minerls nd vitmins. B - Dietry Ftty cid composition: Ftty cid composition of the five diets provided to the dms in the present model RD, SFA, ALA, DHA nd EPA provided to the mothers efore nd during pregnncy nd lcttion. The lower row presents the rtio etween ω-3 nd ω -6 ftty cids in ech diet. The ω -3/ ω -6 rtio in ech diet is indicted in old. Stndrds for ech frction were purchsed from Sigm Aldrich (Rehovot, Isrel ) nd were spotted in seprte TLC lnes (i.e., 25 μg ofpl,tg,dg,cendffa). Pltes were then plced in 20x20 cm TLC chmer contining petroleum ether, ethyl ether, nd cetic cid (80:20:1, v/v/v) nd run for 45 min (18). PL, TG, DG, CE nd FFA nds were visulized with Iodine, scnned nd quntified (Epson V700). Neutrl lipids were lso detected in liver slices with the lysochrome ORO (C26H24N4O) nd visulized y light microscopy with n x60 mgnifiction [57]. Ftty cid nlysis Ftty cids were nlyzed s methyl ester derivtives (FAME) y gs chromtogrphy (GC) in Vrin, 3800 Series (Wlnut Creek, CA) chromtogrph (FID) with fused silic SGE cpillry column nd Vrin Str Worksttion Advnce Appliction softwre, version 6. Aliquots of food lipids were processed like the liver for the nlysis of ftty cids s follows: fter lipid extrction nd weight, liquots representing 0.5 g food or 0.25 g of liver, kept frozen t -20 C efore use, were tken into screw-cpped tue (teflon-lined) contining

10 Shomonov-Wgner et l. Lipids in Helth nd Disese (2015) 14:14 Pge 10 of 11 5 μg heptdecnoic cid s Internl Stndrd. 1 ml 5% H2SO4 in methnol ws dded. The tues were gssed with nitrogen, closed tightly nd heted t 85 C for 1.5 h with occsionl shking. After cooling, 1 ml of hexne ws dded, the tues content ws mixed nd, fter short centrifugtion, the hexne lyer ws trnsferred into new tue. Before GLC nlysis, the hexne extrcts were concentrted y evportion under nitrogen. One-twentieth of the finl re-suspension ws pplied in 1 μl hexneinto the gs chromtogrph. The ftty cid profiles were compred to tht of known mixture of ftty cids of niml source, PUFA2 (Supelco, USA) for identifiction (28). Δ6 desturse nd SCD1 ctivities Δ6 desturse nd SCD1 ctivities were estimted s the indexes [58] clculted y determining the rtio etween the finl products of the pthwy nd sustrtes of the surrogte mrkers [GLA + AA/LA] nd [POA + OA/PA + SA] respectively, tken from the liver. Δ6 desturse nd SCD1 mrna expression Totl RNA ws extrcted using TriRegent (Sigm-Aldrich, Jeruslem, Isrel) ccording to the mnufcturer s protocol. Extrcted RNA underwent reverse trnscription (RT) to form cdna y mens of the Verso RT-PCR Systems. The TqMn Gene Expression Assy-Pre-Mde for FADS2, SCD-1 s well s Bet Actin were otined from Applied Biosystems, Isrel (Agentek Ltd). Rection products were normlized ccording to expression of the Bet Actin gene nd presented s fold chnge compred to the RD dt in ech group [28]. Sttistics nlysis For compring the mens of tretment mongst the mternl diet groups, one-wy ANOVA nlysis ws implemented followed y n pproprite post hoc test: Tukey, using SSPS version 18. The numer of nimls used ws RD =8, SFA = 12, ALA = 21, DHA = 7 nd EPA = 15 for ody nd liver weight.the numer of nimls ws 7 nd representtive of different mothers for ll other determintions.sttisticlly significnt results were reported ccording to p vlue equl to or less thn All dt re shown ± SEM unless stted otherwise. For relting HOMA-IR dt with TLL dt, 2 tiled Person correltion nlysis ws performed. Arevitions Δ6 desturse:delt 6 desturse enzyme; FADS2: Delt 6 desturse gene; SCD1: Steroyl-CoA desturse enzyme nd gene 1; FAs: Ftty cids; SFA: Sturted ftty cid; MUFA: Monounsturted ftty cid; TG: Triglycerides; DG: Diglycerides; CE: Cholesterol ester; FFA: Free ftty cid; PA: Plmitic cid; POA: Plmitoleic cid; SA: Steric cid; OA: Oleic cid; GLA: Gm linolenic cid; AA: Archidonic cid; LA: Linoleic cid; GC: Gs-chromtogrphy; NAFLD: Non lcoholic ftty liver disese; HFD: High ft diet; HOMA: Homeosttic model ssessment; n.s: Not significnt; PUFA: Polyunsturted ftty cid; PL: Phospholipid; TLC: Thin lyer chromtogrphy, TLL, Totl liver lipids. Competing interests The uthors declre tht they hve no competing interests. Authors contriutions LSW, conducted the reserch; ALF conducted the lipid studies nd nlyzed dt; AR contriuted to the designed reserch nd dt nlysis. ALF, designed reserch, hd primry responsiility for finl content nd wrote the pper. All uthors red nd pproved the finl mnuscript. Acknowledgements This study ws supported y the Moshe Ishi Center for the Investigtion of Nturl Food on the Qulity of Humn Helth Grnt, the Reserch Authority of Tel Aviv University. Prt of the results in this pper ws presented y LSW s her Ms.Sc thesis. The uthors re grteful to Fred Konikoff for llowing LS the use of the fcilities t the Lortory for Lipid Metolism in the Liver, to Hn Levkovitz for her technicl ssistnce with ORO nd to Aviv Shish nd Dvid M.Mutch for their review of the mnuscript. Author detils 1 Lortory for Lipid Metolism in the Liver, Sckler School of Medicine, Tel Aviv 69978, Isrel. 2 G.S.W. Fculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Isrel. 3 Bert W. Strssurger Lipid Center, She Medicl Center, Tel-Hshomer, Isrel. Received: 6 Jnury 2015 Accepted: 13 Ferury 2015 References 1. Jmes PT, Rigi N, Lech R. The oesity epidemic, metolic syndrome nd future prevention strtegies. 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Mrkers of dietry ft qulity nd ftty cid desturtion s predictors of totl nd crdiovsculr mortlity: popultion-sed prospective study 1, 2, 3. Am J Clin Nutr. 2008;88: Sumit your next mnuscript to BioMed Centrl nd tke full dvntge of: Convenient online sumission Thorough peer review No spce constrints or color figure chrges Immedite puliction on cceptnce Inclusion in PuMed, CAS, Scopus nd Google Scholr Reserch which is freely ville for redistriution Sumit your mnuscript t