British Journal of Nutrition

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1 (11), 16, q The Authors 11 doi:1.117/s Fish oil comined with SCFA synergisticlly prevent tissue ccumultion of NEFA during weight loss in oese mice Miken H. Pedersen 1,, Lotte Luritzen nd Lrs I. Hellgren 1 * 1 Deprtment of Systems Biology, Center for Biologicl Sequence Anlysis, Technicl University of Denmrk, 8 Lyngy, Denmrk Deprtment of Humn Nutrition, University of Copenhgen, 1958 Frederikserg C, Denmrk (Received 13 July 1 Revised 15 Decemer 1 Accepted 16 Ferury 11 First pulished online 3 June 11) Astrct Bsed on their proposed metolic effects, we exmined whether fish oil (FO) nd SCFA, lone or in comintion, ccelerte weight loss nd the resultnt metolic improvements. Oesity ws induced in mle C57BL/6J mice y high-energy feeding for 1 weeks. The mice were trnsferred to low-ft diet ( 5w%) for 4 weeks, the source of ft eing either FO, lrd sfflower oil mix (control), or oth types comined with SCFA. Weight, fsting insulin, tissue nd serum lipid concentrtions, s well s mrna mount of genes relted to dipose inflmmtion nd heptic ft oxidtion were determined. All groups lost weight nd showed reduced fsting insulin concentrtions nd reduced liver TAG. However, weight loss on the control-ft diet cused significnt increse in heptic nd crdic NEFA. Sustituting % of the ft with SCFA incresed weight loss y 48 % nd reduced fsting insulin 1 5-fold more thn the no-scfa diets. It furthermore significntly incresed the mount of mrna for PPAR-, nd decresed the mrna mount for NF-kB in the liver nd white dipose tissue. The FO diets enhnced improvement of tissue lipid levels. Thus, FO improved liver TAG nd NEFA levels compred with weight loss on the control diet. Comining FO nd SCFA further reduced tissue NEFA ccumultion. In conclusion, we found tht dietry SCFA hd significnt impct on gene expression in the liver nd dipose tissue, nd tht the effect of FO on tissue NEFA content ws modified y SCFA. Thus, interctions etween ftty cids should e considered when studying the effects of specific ftty cids. Key words: Acette: SCFA: Fish oil: Weight loss: Tissue NEFA Dietry ftty cid chin length nd degree of unsturtion re elieved to influence the development of insulin resistnce nd n-3 long-chin (LC)-PUFA re prticulrly interesting due to their proposed nti-inflmmtory nd lipid-modulting effects. Weight loss is lso expected to improve insulin sensitivity, restore norml function of the dipose tissue nd reduce the level of inflmmtory meditors. We therefore hypothesised tht the ddition of fish oil (FO) to weight loss diet could improve the recovery of helthy metolic function due to comined eneficil effects, including potentil increse in the rte of weight loss s indicted from studies with humn test sujects (1 3). Severl recent studies indicte tht pro-inflmmtory meditors produced in the dipose tissue promote the development of insulin resistnce (4). Ft-lden dipocytes excrete meditors tht ttrct nd ctivte mcrophges (5 7), which, in turn, excrete numer of inflmmtory molecules including TNF- (8). TNF- hs een implicted to directly ttenute the insulin response in skeletl muscle cells, dipocytes nd (9 11) heptocytes nd severl moleculr mechnisms hve een suggested to link TNF- to insulin resistnce; for exmple, direct inhiition of insulin-receptor utophosphoryltion (11). Furthermore, it hs een shown tht TNF- cn increse lipolysis, cusing n incresed flow of NEFA from dipocytes to non-dipose tissues, which is lso expected to reduce insulin sensitivity in oth skeletl muscles nd the liver (1). A eneficil effect of FO on dipose inflmmtion in d/d mice on high-ft diet hs een shown y Todoric et l. (13). Furthermore, Kres et l. (14) found dditionl improvements in metolic risk fctors in women who consumed FO while losing weight. A plusile mechnism ehind this effect could e tht n-3 LC-PUFA or their metolites ctivte PPAR, which leds to the induction of nti-inflmmtory pthwys. PPAR-g ctivtion hs een shown to reduce the production of TNF- (15). PPAR-g nd PPAR- ctivtion hs lso een shown to inhiit the ctivtion of NF-kB, which is key pro-inflmmtory regultor (16). PPAR- is lso key regultor of genes involved in lipid oxidtion nd its ctivtion Arevitions: Ctrl, control; FO, fish oil; GPR, G-protein-coupled receptor; LC-PUFA, long-chin-pufa; SREBP-1c, sterol regultory element inding protein-1c. * Corresponding uthor: Lrs I. Hellgren, fx þ , emil lih@io.dtu.dk

2 145 M. H. Pedersen et l. y dietry n-3 LC-PUFA my thus reduce the lipid lod in non-dipose tissue nd therey increse insulin sensitivity (17). The inclusion of low-energy ft in exchnge for norml ft my e nother wy to enhnce recovery during weight loss. Low-energy ft of the Sltrim type contins high mounts of SCFA (cette nd propionte), which reduces the energy content to out 1 kj/g compred with 38 kj/g in norml dietry ft (18). Furthermore, SCFA hve een shown to induce the expression of PPAR- nd reduce the expression of NF-kB in cell model (19), indicting tht SCFA might hve ntiinflmmtory ctivity. Interestingly, cette, the min orgnic cid in Sltrim, ws found to exert strong nti-inflmmtory ctivity through the ctivtion of the G-protein-coupled receptor 43 (GPR43; lso clled NEFA receptor-) (). GPR43 hs een identified s specific receptor for SCFA, nd is minly expressed in white nd rown dipose tissue, the lrge intestine, dendritic cells nd grnulocytes (,1). In the white dipose tissue, GPR43 ctivtion hs een implicted in the regultion of lipid nd glucose homeostsis nd is tody exploited s promising drug trget for the tretment of insulin resistnce nd type dietes (). Furthermore, it ws lso recently shown tht ctivtion of GPR43 in immune cells is required for norml resolution of certin inflmmtory responses (1). Thus, comining Sltrim with FO my result in synergism from the concomitnt increse in PPAR- expression nd incresed vilility of its n-3 LC-PUFA gonists, s well s the nti-inflmmtory effects nd metolic improvements in dipose tissue induced y the ctivtion of GPR43. The primry hypothesis of the present study ws tht oese mice on hypoenergetic diet enriched with FO show improved rte of weight loss nd greter improvements in metolic risk fctors compred with mice on similr diet low in n-3 LC-PUFA. We furthermore explore the hypothesis tht SCFA from Sltrim nd n-3 LC-PUFA from FO hve synergistic eneficil effects on metolic function, enhncing the rte of which it returns to len phenotype during weight loss. Metolic function ws ssessed y weight, fsting insulin, tissue lipid content nd trnscription of genes relted to inflmmtion nd lipid turnover. Methods Animls nd diets A totl of forty-eight mle C57BL/6J mice weighing 3 (SD 1 1) g (Chrles River Lortories Interntionl, Wilmington, MA, USA) were rndomised into six groups. A len reference group (len) ws kept on stndrd diet 134 from Altromin (Lge, Germny) for the entire feeding period. In the five remining groups, oesity ws induced y feeding high-energy diet for 1 weeks (6 energy% ft) (D149; Reserch Diets, New Brunswick, NJ, USA) nd y the ddition of 15 w% sucrose to drinking-wter. Following the weight gin period, one group of mice continued on the high-ft diet to serve s n oese reference group (oese), while four groups were switched to d liitum low-energy diets for 4 weeks ( 5 w% ft dded to powdered C156 diet from Altromin). There ws no difference in the verge weight of the five oese groups fter the weight gin period; the verge ws 35 g compred with 6 g in the len reference group. The weight loss diets differed in ftty cid composition (Tle 1) y the ddition of either cod liver oil (FO) or control (Ctrl) mix of lrd nd sfflower oil (Ctrl) lone or with the exchnge of 5 w% of the ft for Sltrim (FO þ Sltrim, Ctrl þ Sltrim) (the Sltrim ws gift from DANISCO, Brrnd, Denmrk). The mcronutrient composition in the four weight loss diets ws identicl with 611 g/kg crohydrtes, 5 g/kg ft, 17 g/kg protein, 14 g/kg free mino cids nd 41 g/kg fire. The mount of ft dded to the weight loss diets corresponded to the upper levels of relistic dily FO intke for humns. Thus, the 5 w% equls 6 energy%, which corresponds to dily intke of 15 g FO for person consuming 1 kj/d. The mice were housed in groups of four in wire cges t 8C, humidity etween 45 nd 65 %, nd 1 h light cycle. All hndling nd use of nimls in the present study were pproved y The Dnish Animl Experiments Inspectorte nd were crried out ccording to the guidelines of The Council of Europe Convention for the Protection of Verterte Animls used for Experimentl nd other Scientific purposes. Physiologicl nd iochemicl mesurements Weight ws recorded weekly nd fsting insulin ws mesured fter 1 nd 14 weeks. All mice were killed fter 14 weeks y nesthetistion ( 11 ml/5 g ody weight of Ketminol mixed with Nrcoxyl 1:15; Intervet Dnmrk AS, Skovlunde, Denmrk) followed y crdic puncture from where ll the lood ws drwn. The lood ws left to clot t room temperture for min nd the serum ws collected fter centrifugtion. The hert, liver nd dipose tissue were removed immeditely nd quickly frozen in liquid N efore trnsfer to 88C freezer. Smll frctions of the liver nd dipose tissue were treted with RNAlter (Invitrogen, Tstrup, Tle 1. Ftty cid composition of the four weight loss diets tht were fed to oese mice for 4 weeks* Ftty cid (g/1 g) Ctrl Ctrl þ S FO FO þ S : : : : : 1n : : 1n : 1n : n : 3n : 4n : 1n : 1n : 5n : 5n : 6n Ctrl, control diet; FO, fish oil diet; FO þ S, 5 w% of the norml ft ws exchnged for Sltrim. * The mount of ech ftty cid is given s g/1 g of totl ftty cids. The totl ft content ws 5 w%.

3 SCFA, fish oil nd metolic risk fctors 1451 Denmrk) ccording to the mnufcturer s recommendtions, prior to freezing nd lter RNA isoltion. Serum concentrtions of insulin were nlysed using ELISA kits (Mercodi AB, Uppsl, Sweden). Serum concentrtions of TAG nd NEFA were mesured using kits from Wko Chemicls (Neuss, Germny) nd Cos Mir uto-nlyzer (Roche, Bsel, Switzerlnd). Lipids were extrcted from the tissue, the serum nd the feed using the Folch procedure with the ddition of nondecnoic cid, dinondecnoylglycerophosphorylcholine nd triheptdecnoyl s internl stndrds efore extrction (3). Tissue phospholipids, NEFA nd TAG were seprted using preprtive TLC; methyl esters of the lipids were prepred from the TLC scrpings or directly from the lipid extrct of the serum nd diets nd seprted nd quntified using GLC s descried erlier (4). RNA extrction nd rel-time quntittive PCR Totl RNA ws isolted from the heptic nd dipose tissue using TRIzol (Invitrogen) nd mrna ws further isolted from the totl RNA y the use of mmacs mrna isoltion kit (Miltenyi Biotec, Bergisch Gldch, Germny). Regents for first strnd synthesis, s well s primer sets for rel-time quntittive PCR, were purchsed from Superrry (Frederick, MD, USA) in the form of commercil kits including CYBR green mster mix for rel-time detection. Act, Gpdh nd HSP9AB1 were used s reference genes. A Biometr Tpersonl thermocycler (Biometr, Goettingen, Germny) ws used for first strnd production, nd Bio-Rd icycler iq5 ws used for PCR mplifiction nd detection (Bio-Rd, Copenhgen, Denmrk). The thermocyclers were progrmmed ccording to the recommendtions from Superrry. Melting curve nlysis ws performed to check tht only one trget ws mplified. Sttistics All dt re presented s group men nd stndrd devitions. GrphPd Prism 5 (GrphPd Softwre, Inc., L Joll, CA, USA) ws used for sttisticl nlysis, nd significnce ws set t 5. A two-wy ANOVA ws performed in order to ssess the effect of ft type nd Sltrim in the four weight loss diets, compring dt from these four groups only. If the ANOVA result showed significnt interction, two-sided t-tests were computed to ssess the differences etween the individul groups. Dt from the oese nd len reference groups re included in ll grphs nd tles for comprison. Results Weight loss nd fsting insulin All weight loss groups continuously lost weight nd the oese reference group continued to gin weight etween weeks 1 nd 14 (Fig. 1(A)). Fsting insulin ws chnged ccordingly in ll groups (Fig. 1). Furthermore, there ws significnt correltion etween the chnge in weight nd chnge in fsting insulin (r 73, P, 1). Sltrim cused significnt (A) 1 Weight chnge (g) Chnge in fsting insulin (µg/l) Len, Oese Ctrl Ctrl+ S FO FO + S Len Oese Ctrl Ctrl+S FO FO+S increse in weight loss, wheres the weight loss nd susequent chnge in fsting insulin in the FO nd Ctrl groups did not differ (Fig. 1). Effects on lipid content nd composition Fig. 1. The (A) weight chnge nd chnge in fsting insulin during the weight-loss phse (weeks 1 14) in the four weight loss groups nd the len nd oese reference groups. Vlues re mens nd stndrd devitions, n 8. The len nd oese groups serve s reference groups for comprison nd were not included in the sttisticl nlyses. Sttisticl nlysis ws performed s two-wy ANOVA, using fish oil nd Sltrim s vriles. For oth weight chnge nd chnge in fsting insulin, two-wy ANOVA showed n effect of Sltrim (P, )., Men vlues with unlike letters re significntly different (P, 5). Ctrl, control diet; FO, fish oil diet; FO þ S, 5 % of the norml ft ws exchnged for Sltrim. The nlysis of EPA nd DHA mounts in liver TAG showed tht the FO groups responded to the tretment with out sixteen times more EPA nd five times more DHA incorportion compred with the Ctrl groups (Fig. (A)). Anlysis of the dipose tissue content of EPA nd DHA showed tht even though thef net flux of ftty cids during weight loss is ssumed to e wy from the dipose tissue, incorportion of the ftty cids from the FO diets hd tken plce (Fig. ). The heptic phospholipid ftty cid composition nd dipose tissue totl ftty cid composition cn e found in the online supplementry mteril ( The concentrtion of TAG in the liver ws reduced in oth FO groups with no dditionl effect of Sltrim (Tle ). The serum concentrtion of NEFA ws incresed in the FO groups,

4 145 M. H. Pedersen et l. (A) 3 lower NEFA concentrtions in the FO þ S group compred with the three other weight loss groups (Fig. 3). Totl ftty cids (%) Totl ftty cids (%) 1 1 Len Oese Ctrl Ctrl +S ut the serum concentrtion of TAG ws not ffected y ny of the tretments (Tle ). It is noteworthy tht, in oth Ctrl groups weight loss induced sustntil increse in heptic concentrtions of NEFA compred with oth the oese nd the len reference groups (Fig. 3(A) nd ). FO reduced this weight loss-induced liver ccumultion of NEFA, nd there ws pronounced interction etween FO nd Sltrim, which reduced NEFA even further (Fig. 3(A)). Anlysis of crdic NEFA ccumultion lso showed significnt interction etween FO nd Sltrim, which resulted in FO FO +S Len Oese Ctrl Ctrl + S FO FO +S Fig.. The percentge of EPA ( ) nd DHA ( ) in the (A) liver nd dipose tissue TAG s percentge of totl ftty cids in TAG. Otherwise s in Fig. 1., Men vlues with unlike letters re significntly different. Ctrl, control diet; FO, fish oil diet; FO þ S, 5 % of the norml ft ws exchnged for Sltrim. Liver nd dipose tissue mrna mounts The ddition of Sltrim to the diets resulted in significntly higher levels of PPAR- mrna nd lower levels of mrna for the P65 suunit of NF-kB in oth the liver (Fig. 4 (A) nd ) nd dipose tissue (the supplementry mteril for this rticle cn e found t org/jn) compred with the effect of the weight loss diets without Sltrim. The Sltrim groups lso hd lower levels of sterol regultory element inding protein-1c (SREBP-1c) mrna in the liver (Fig. 4(C)). TNF- mrna in the dipose tissue tended to e lower in the Sltrim groups compred with the pure FO nd Ctrl groups (the supplementry mteril for this rticle cn e found t cmridge.org/jn). Neither FO nor Sltrim significntly reduced the level of CD68 mrna, which ws used s mrker for mcrophge infiltrtion in the dipose tissue (the supplementry mteril for this rticle cn e found t No significnt group differences were found in heptic mrna mounts of the SREBP-1c-regulted enzymes; ftty cid synthse nd cetyl-coa croxylse, which together produce ftty cids from cetyl-coa (the supplementry mteril for this rticle cn e found t Nor were there ny significnt differences in the mrna mount of steroyl CoA desturse-1 key lipogenic enzyme, or in the mrna mounts of three PPAR--regulted ftty cid oxidtive enzymes; cyl-coa synthetse (ctlyses n initil step in long-chin ftty cid ctolism), the peroxisoml -oxidtion enzyme cyl-coa oxidse, nd the microsoml v-hydroxylse cytochrome P45 4A1 (the supplementry mteril for this rticle cn e found t The reltive mrna mounts of dipokines nd genes ssocited with inflmmtion nd energy metolism cn e found in the supplementry mteril t Sltrim tretment significntly reduced dipose mrna levels of TGF-, plsminogen ctivtor inhiitor-1, leptin, resistin nd the insulin receptor, nd FO tretment lso reduced dipose resistin mrna. Tle. Concentrtions of TAG nd NEFA in the liver nd serum fter 4 weeks weight loss diet (Men vlues nd stndrd devitions, n 8) Len Oese Ctrl Ctrl þ S FO FO þ S P Lipid* Men SD Men SD Men SD Men SD Men SD Men SD FO Sltrim Interction Heptic TAG (mg/g) Heptic NEFA (mg/g) Crdic TAG (mg/g) Crdic NEFA (mg/g) Serum TAG (mmol/l) Serum NEFA (mmol/l) Ctrl, control diet; FO, fish oil diet; FO þ S, 5 % of the norml ft ws exchnged for Sltrim. * Extrcted from serum nd tissue from fsted nimls. The len nd oese groups serve s reference groups for comprison nd re not included in the sttisticl nlyses. Results from the two-wy ANOVA with P, 5 nd the tretment, FO or Sltrim tht cused the effect.

5 SCFA, fish oil nd metolic risk fctors 1453 (A) 4 Heptic NEFA (mg/g) Crdic NEFA (mg/g) Discussion Len Oese Ctrl Ctrl +S In the present study we hve investigted how specific groups of dietry ftty cids nd their interctions ffect the rte of weight loss, metolic function nd risk fctors relted to the development of the metolic syndrome in mice. In our study, the inclusion of FO to weight loss diet did not ffect the rte of weight loss or fsting insulin. This is contrry to studies tht hve shown tht the inclusion of n-3 LC-PUFA to weight loss diet leds to incresed rte of weight loss in humns (1 3) nd reduced fsting insulin in rodents (5). However, nti-oesity effects re not consistent finding in humns s other studies report no effect of n-3 LC-PUFA on the rte of weight loss (6,7), wheres in rodents, the nti-oesity s well s insulin-reducing effects of n-3 LC-PUFA hve een highly consistent during weight gin (8 33). However, s our primry focus ws to study the effects of FO intke relistic for humn popultion, we hve used sustntilly lower concentrtions thn erlier studies. Nktni et l. (8) compred the effect on weight gin of different mounts of FO fed to femle C57BL/6J mice, nd found tht less thn 4 energy% FO ws insufficient in reducing weight gin significntly compred with the no FO control. We gve the mice energy% FO, which corresponds to 1 15 g/d of FO for person consuming 1 kj/d, nd higher intke might e unrelistic in humn nutritionl context. c d FO FO +S Len Oese Ctrl Ctrl+S FO FO+S Fig. 3. The (A) heptic nd crdic concentrtions of NEFA (in the four weight loss groups nd the two reference groups. As the two-wy ANOVA showed significnt interction, pir-wise t tests were performed., Men vlues with unlike letters re significntly different etween groups. The two reference groups were not included in the tests. Ctrl, control diet; FO, fish oil diet; FO þ S, 5 % of the norml ft ws exchnged for Sltrim. Furthermore, in contrst to erlier studies when FO ws given during weight gin, we cn lso expect dilution of the ftty cids of dietry origin with ftty cids relesed from the dipose tissue during weight loss in the present study. Therefore, it is noteworthy, tht the low doses used hve een sufficient to improve heptic nd crdic lipid sttus, proving tht FO intke t these reltively low levels lso improves metolic sttus during weight loss. (A) PPAR-α reltive mrna mount P65 reltive mrna mount SREBP-1c reltive mrna mount ,c, Len Oese Ctrl Ctrl+S FO FO +S,c Len Oese Ctrl Ctrl+S FO FO+S Len Oese Ctrl,, Ctrl+S FO FO+S Fig. 4. The reltive heptic mrna mount of (A) PPAR-, NF-kB suunit P65 nd (C) sterol regultory element inding protein-1c (SREBP-1c) in the four different weight loss groups nd the reference groups. The rel-time quntittive PCR dt re presented s men nd stndrd devitions reltive to the mrna level in the len reference group, n 8. The two-wy ANOVA includes only the four weight loss groups. For the reltive heptic mrna mounts of PPAR-, P65 nd SREBP-1c, two-wy ANOVA showed n effect of Sltrim of P, 4, P, nd P, 3, respectively.,,c Men vlues with unlike letters re significntly different etween individul groups. Ctrl, control diet; FO, fish oil diet; FO þ S, 5 % of the norml ft ws exchnged for Sltrim.

6 1454 M. H. Pedersen et l. Dietry n-3 LC-PUFA hve een shown to prevent the development of insulin resistnce in rodents when oesity is induced y high-energy diet (9,33 35), ut the effect of dministering n-3 LC-PUFA to oese nimls during weight loss hs, to our knowledge, not previously een investigted. A study y Rmel et l. (36) indicted tht physiologicl doses of FO (3 g/d) during weight loss would improve insulin sensitivity in humns, nd in the present study we wnted to investigte the potentil mechnisms for this effect. We hypothesised tht the proposed nti-inflmmtory effects of n-3 LC-PUFA could led to reduction of the inflmmtory stte of the dipose tissue, nd tht the ctivtion of heptic PPAR- y n-3 LC-PUFA could induce the trnscription of ft-oxidising enzymes nd thus reduce heptic lipid ccumultion. Both of these effects were expected to improve insulin sensitivity, ut no difference ws seen in fsting insulin fter 4 weeks on FO diet compred with the Ctrl group tht did not consume FO. Totl mcrophgl infiltrtion in the dipose tissue ws ssessed y the mount of mrna encoding CD68, mcrophge surfce receptor, nd this ws lso not reduced y FO tretment. Totl mcrophgl infiltrtion, however, does not give informtion on the ctivtion stte of the dipose tissue mcrophges, ut s nti-inflmmtory effects of FO on the mount of mrna encoding TNF- or NF-kB P65 were not found, n effect on the distriution etween inflmmtory M1- nd nti-inflmmtory M-tissue mcrophges is unlikely. Heptic NEFA nd TAG ccumultion ws reduced y FO reltive to the Ctrl ft, nd in oth crdic nd heptic tissue the comintion of FO nd SCFA ws prticulrly efficient in reducing NEFA ccumultion. This shows tht inclusion of FO nd SCFA in weight loss diets might e fesile strtegy to optimise the improvement in tissue lipid sttus during weight loss. The incresed lod of NEFA in tissues is strongly linked to the ccumultion of lipotoxic ftty cid derivtives, such s cyl-coa, cermide nd dicylglycerol, which hve een suggested to e prt of the etiology of oth insulin resistnce nd CVD (37,38). Hence, the incresed lod of crdic nd heptic NEFA in the weight-loss groups given Ctrl ft, constitute metolic risk tht, t lest in mice, cn e reduced through intervention with pproprite dietry ft during weight-loss progrmme. The lipid-lowering effect of FO is consistent with the expected effect of n-3 LC-PUFA on PPAR- nd SREBP-1c in the liver. However, we did not find significnt increse in the mrna mount of PPAR-regulted heptic ft-oxidising enzymes tht could explin this finding. Mori et l. (3) were le to find significnt increses in the mrna mount nd enzymtic ctivity of heptic ft-oxidising enzymes in C57BL/6J mice following weeks on FO diet. Their setup, however, differed from the present study s they fed the mice weight-incresing diet. In conclusion, we show tht (reltively) low-dose FO intervention is le to increse the rte of improvement of liver lipid levels during weight-loss progrmme without concomitnt improvement of glucose tolernce or dipose inflmmtion, compred with weight loss on the Ctrl-ft diet. We lso investigted the effect of exchnging 5 w% of norml ft with Sltrim. This exchnge ws tried in comintion with oth Ctrl ft nd FO in order to investigte the possile synergistic effects of the SCFA from Sltrim nd n-3 LC-PUFA from FO. The weight loss fter 4 weeks on low-energy diets ws evident for ll mice, ut it ws significntly greter when 5 % of the ft ws exchnged for Sltrim. The chnge in fsting insulin ws highly correlted to weight chnge nd dditionl improvements in the Sltrim groups were proly due to the difference in weight. However, Sltrim lso ffected prmeters which could not e explined y the reduced energy intke nd incresed weight loss. The mount of PPAR- mrna ws higher, nd tht of NF-kB p65 suunit ws lower in oth dipose nd heptic tissues. We were le to show in vivo the effect of SCFA on the mount of PPAR- mrna, which hd previously een shown only in in vitro experiment (19). A higher expression of PPAR- could explin the comined effect of SCFA nd n-3 LC-PUFA on heptic nd crdic NEFA levels. The trnscription of mny lipogenic enzymes is controlled y the trnscription fctor SREBP-1c, nd reduction in its ctivity could lso led to reduced ftty cid ccumultion. We found significnt reduction in the mount of mrna encoding this trnscription fctor following Sltrim tretment, ut gin without significnt reductions in the mount of mrna-encoding lipogenic enzymes under its control. The source of SCFA in the present study ws Sltrim, ut fermenttion of fire y the gut microflor provides nturl source of SCFA, which hs een mesured to e 375 mmol/l in the humn portl vein (39). The finding tht cette nd propionte in Sltrim seem to ct s systemic trnscriptionl regultors therefore hs more wide-reching implictions s it suggests tht some of the systemic effects induced y interventions with pre- or proiotic might e explined y enhnced production of SCFA. Cell studies hve shown reduced cytokine production following LPS stimultion of SCFA-treted neutrophils, nd SCFA hve furthermore een shown to inhiit the ctivity of NF-kB in vitro. The present results, showing lower heptic nd dipose trnscription of NF-kB p65 in the SCFA-treted mice, indicte tht SCFA could lso inhiit NF-kB ctivity in vivo, nd tht those nti-inflmmtory effects of SCFA could rech the systemic circultion, lthough evidence of reduced inflmmtion ws not found in the present study. Yin et l. (4) hve shown tht SCFA tretment reduced IkB (NFkB inhiitor degrdtion fter NF-kB stimultion in vitro, nd it is possile tht SCFA inhiit NF-kB ctivity y more thn one pthwy. Systemic inflmmtion is ssocited with the metolic syndrome, nd it is possile tht SCFA medite some of the eneficil effects of fire consumption y its nti-inflmmtory properties. An effect of SCFA on gene trnscription could e medited y the receptors, GPR41 nd GPR43, which re widely distriuted, lthough most commonly ssocited with dipocytes nd immune cells (1). SCFA hve previously een shown to signl through these receptors (41). In conclusion, the ddition of FO to low-ft diet during weight loss did not reduce dipose inflmmtion nd fsting insulin. However, exchnging 5 w% of the ft with Sltrim incresed weight loss, lowered fsting insulin nd ltered the mount of mrna encoding three importnt trnscription

7 SCFA, fish oil nd metolic risk fctors 1455 fctors, PPAR-, NF-kB nd SREBP-1c, which re centrl in inflmmtory response nd ftty cid metolism. However, the most importnt finding of the present study is the pronounced synergistic effect of FO nd Sltrim on tissue NEFA, since reduction in ville NEFA my ffect the concentrtion of lipotoxic ftty cid metolites tht hve een implicted in the development of insulin resistnce nd CVD. Acknowledgements We thnk Jnnie Felskov Agersten nd Pernille Wehler Güllich for technicl ssistnce nd DANISCO for finncil support nd for kindly providing us with the Sltrim used in the present study. This work ws finncilly supported y the Dnish Council for Strtegic Reserch through the Progrm Committee for Food nd Helth, nd DANISCO did not hve ny influence on the interprettion of the dt or the writing of this pper. M. H. P., L. L. nd L. I. H. hve no conflict of interests. M. H. P. conducted the experiments nd wrote the pper. L. I. 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