Vaccenic acid suppresses intestinal inflammation by increasing the endocannabinoid anandamide

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1 Vccenic cid suppresses intestinl inflmmtion by incresing the endocnnbinoid nndmide nd non-cnnbinoid signling molecules in rt model of the metbolic syndrome Author(s): Mirim Jcome-Sos 1, Cludi Vcc 2, Rbbn Mngt 1, Abdoulye Dine 1, Rndy C. Nelson 1, Mrtin J. Reney 3, Jinheng Shen 3, Jonthn M. Curtis 4, Donn F. Vine 1, Ctherine J. Field 4, Miki Igrshi 5, Dniele Piomelli 6, Sebstino Bnni 2 nd Spencer D. Proctor 1. Affilitions: 1 Metbolic nd Crdiovsculr Disese Lbortory, Group on Moleculr nd Cell Biology of Lipids, Albert Dibetes nd Mznkowski Hert Institutes, University of Albert, Edmonton, AB, Cnd. 2 Deprtment of Biomedicl Sciences, University of Cgliri, Cittdell Universitri, Monserrto, Cgliri, Itly. 3 Deprtment of Plnt Science, University of Ssktchewn, Ssktoon, SK, Cnd. 4 Deprtment of Agriculturl Food nd Nutritionl Science, University of Albert, Edmonton, AB, Cnd. 5 Lbortory for Medicl Homeostsis, RIKEN center for Integrtive Medicl Sciences, Kngw, Jpn. 6 Deprtment of Antomy nd Neurobiology, Phrmcology nd Biologicl Chemistry, University of Cliforni, Irvine, CA, USA. Corresponding uthor: Dr. Spencer D. Proctor Address: 4-2J Li K Shing Centre for Helth Reserch Innovtion, University of Albert, Edmonton, AB, T6G 2E1, Cnd Emil: spencer.proctor@ulbert.c Phone: , Fx: Running title: Vccenic cid regultes intestinl endocnnbinoids 1

2 Abbrevitions: AA: rchidonic cid; AEA: nndmide; 2-AG: 2-rchidonoylglycerol; CB: cnnbinoid; CD: control diet; CLA: conjugted linoleic cid; EC: endocnnbinoids; ECS: endocccnbinoid system; FAAH: ftty cid mide hydrolse; FAS: ftty cid synthse; IL-1β: interleukin 1 bet; LC-ESI-MS: liquid chromtogrphy/electrospry ioniztion mss spectrometry; OEA: N-oleoylethnolmide (OEA); PL: phospholipids; PEA: N-plmitoylethnolmide; SREBP1: sterol response element binding protein 1; rtfa: ruminnt trns ftty cids; VA: vccenic cid; VAT: viscerl dipose tissue 2

3 Abstrct Vccenic cid (VA), the predominnt ruminnt-derived trns ft in the food chin, meliortes hyperlipidemi yet mechnisms remin elusive. We investigted whether VA could influence tissue endocnnbinoids (EC) by ltering the vilbility of their biosynthetic precursor, rchidonic cid (AA) in membrne phospholipids (PL). JCR:LA-cp rts were ssigned to control diet (CD) with/without VA (1% w/w), cis-9, trns-11 conjugted linoleic cid (CLA) (1% w/w) or VA+CLA (1%+.5% w/w) for 8 weeks. VA reduced the EC 2-rchidonoylglycerol (2-AG) in the liver nd viscerl dipose tissue (VAT) reltive to CD (P<.1) but did not chnge AA in tissue PL. There ws no dditive effect of combining VA+CLA on 2-AG reltive to VA lone (P>.5). Interestingly, VA incresed jejunl concentrtions of nndmide nd those of the non-cnnbinoid signling molecules, oleoylethnolmide nd plmitoylethnolmide, reltive to CD (P<.5). This ws consistent with lower jejunl protein bundnce (but not ctivity) of their degrding enzyme, ftty cid mide hydrolse nd mrna expression TNFα nd IL-1β (P<.5). The bility of VA to reduce 2-AG in the liver nd VAT provides potentil mechnistic explntion to llevite ectopic lipid ccumultion. The opposing regultion of EC nd other non-cnnbinoid lipid signling molecules by VA suggests n ctivtion of benefit vi the EC system in the intestine. Key words: Vccenic cid, endocnnbinoids, N-cylethnolmines, metbolic syndrome, intestinl inflmmtory diseses. 3

4 Introduction Trns-11 18:1 (vccenic cid or VA) is the most bundnt ruminnt-derived trns ftty cid (rtfa) in the food chin nd hs sprked mjor interest due to mndtory lbeling of ll trns ft on foods in North Americ. This interest hs lso been simulted by the recent nnouncement by the FDA to retrct the GRAS (generlly recognized s sfe) sttus of rtificil trns ftty cids (1). VA is lso the precursor to endogenous synthesis of conjugted linoleic cid (CLA), the first rtfa to be recognized s hving numerous helth-relted effects. Interestingly, growing body of evidence from studies in niml models hs suggested bioctivity for VA independent of its conversion to CLA. More specificlly, VA hs been shown to ttenute complictions observed in the metbolic syndrome, including dyslipidemi, ftty liver disese nd low-grde inflmmtion (2-5). It hs been proposed tht the lipid-lowering nd ntiinflmmtory effects of VA my be prtilly ssocited with its bility to lignd ctivte PPAR-γ regulted pthwys (6, 7), by cting directly in the intestine (6) nd dipose tissue (5). In generl, bioctive long chin ftty cids lso ct by modifying the composition of membrne phospholipids (PL) nd potentilly replcing or interfering with the synthesis of PL-derived lipid signling molecules including endocnnbinoids (8, 9). However, the incorportion of VA into membrne phospholipids nd potentil effects on endocnnbinoid pthwys remins unknown. Endocnnbinoids (EC), the endogenous lignds for cnnbinoid (CB) receptors, re lipid-derived messengers tht hve emerged s key regultors of ppetite behvior, energy metbolism nd intestinl inflmmtion. The most common EC include N-rchidonoylethnolmide (nndmide; AEA) nd 2- rchidonoylglycerol (2-AG); nd re derivtives of the polyunsturted ftty cid (PUFA) rchidonic cid (AA; C2:4, n-6) in PL nd cn be modulted in response to dietry PUFA intke (1-13). Incresed plsm endocnnbinoid concentrtions, due to ltertions in the ctivity/expression of enzymes regulting their biosynthesis nd degrdtion, re ssocited with bdominl obesity, dyslipidemi nd insulin resistnce in humns (14-16). Intriguingly, CB receptor over-ctivity cn result in tissue-specific metbolic effects. For instnce, centrl nd heptic CB receptor ctivtion leds to hyperphgi (17), 4

5 heptic de novo lipogenesis nd insulin resistnce (18-2). In contrst, in niml models of experimentl colitis incresed CB receptor signling hs been shown to meliorte smooth musculr irrittion (21) nd the T cell-medited berrnt immune response (22); thus, countercting the excessive inflmmtory responses/signs in intestinl disese conditions. Therefore, nutritionl interventions tht trget tissuespecific EC pthwys could be used s potentil therpeutic strtegies for tretment of obesity-ssocited metbolic diseses. A recent study in hypercholesterolemic subjects provided the first evidence tht diry product nturlly enriched with VA nd CLA, decreses plsm concentrtions of nndmide in dose-dependent mnner (23). However, the direct effect of VA on EC regultion ws not ble to be determined in this study. Furthermore, given the bioctive properties of VA to fvorbly modulte whole body energy metbolism nd low-grde inflmmtion (24, 5) we proposed to explore novel regultory effects of VA on tissue EC s potentil mechnism of ction for these metbolic effects. In order to ddress the specific role of dietry VA lone or in combintion with CLA in EC metbolism we supplemented the diets of n estblished rodent model of metbolic syndrome (the JCR:LA-cp rt) with these bioctive long chin ftty cids, nd exmined tissue concentrtions of CB receptor lignds nndmide nd 2-AG nd the biosynthetic precursor AA in membrne PL. We lso nlyzed two non-cnnbinoid lipid signling molecules, N-oleoylethnolmide (OEA) nd N-plmitoylethnolmide (PEA), which only differ from nndmide by their cyl chin nd hve been shown to induce stiety (25, 26) nd exert ntiinflmmtory effects through ctivtion of the PPARα receptor (27). 5

6 Mterils nd methods Animls nd diets Rts of the JCR:LA-cp strin tht re homozygous for the corpulent trit (cp/cp) hve complete bsence of the leptin receptor in the plsm membrne nd spontneously develop symptoms ssocited with the metbolic syndrome nd the pre-dibetic stte typiclly observed in humns; including obesity, insulin resistnce nd dyslipidemi (28). Mle JCR:LA-cp rts were rised in our estblished breeding colony t the University of Albert s previously described (29). At 8 weeks (wk) of ge, rts (n=5) were rndomized nd ssigned to one of four diets (control nd experimentl diets) for 8 wk nd hd free ccess to food nd wter. The control diet ws prepred by dding 1% cholesterol nd 15% w/w of ft to n 85% bsl mix diet (Hrln lbortories, TD.626) nd contined 42% of energy from crbohydrte, 23.7% of energy from protein nd 34% of energy from ft, s previously described (5). Experimentl diets were prepred by djusting the ftty cid composition (replcing oleic cid by either VA, CLA or VA+CLA) of the control diet to provide pproximtely 1% w/w of VA (VA), 1% w/w of cis-9, trns-11 CLA (CLA) or both 1% w/w of VA +.5% w/w of cis-9, trns-11 CLA (VA+CLA). The mount of VA in the diet (~2% of totl energy from VA) ws chosen bsed on previous published studies (2-4) nd ws intended to be compred with helth effects of moderte doses of rtfa previously exmined in humn clinicl trils (3). The ft composition of control nd experimentl diets is shown in supplementl tble 1. Control nd experimentl diets were iso-cloric nd hd constnt PUFA to SFA rtio of.4 nd constnt n6 to n3 PUFA rtio of 8. Purified VA ws synthesized by chemicl lkli isomeriztion from linoleic cid-rich vegetble oil (31). Semi-purified cis-9, trns-11 CLA (G-c9t11 8:2) contining 59.8% of cis-9, trns-11 CLA nd 14.4% of trns-1, cis-12 CLA ws kindly provided by Lipid Nutrition. Ftty cid composition of diets ws confirmed by gs chromtogrph nlysis (32) of the ft blend smples (Tble 1). After scrifice, smples of the hypothlmus, skeletl muscle, viscerl dipose tissue (VAT), liver, nd jejunl mucos segments of the intestine were excised nd snp frozen t -8 C until nlysis. Animl cre nd 6

7 experimentl procedures were conducted in ccordnce with the Cndin Council on Animl cre nd pproved by the University of Albert Animl Cre nd Use Committee-Livestock. Tissue lipid extrction Tissues (.2-.3g) were homogenized nd totl lipids extrcted with chloroform/methnol (2:1, v/v) contining internl deuterted stndrds for nndmide (AEA), 2-AG, OEA nd PEA to quntify for recovery efficiency ([ 2 H] 8-AEA, 2 ng/ml), ([ 2 H] 5-2-AG, 2 ng/ml), ([ 2 H] 2-OEA, 2 ng/ml) nd ([ 2 H] 4-PEA, 2 ng/ml) (Cymn Chemicl, MI, USA). This mixture ws wshed with.25 volume of.9% KCl ccording to the Folch procedure (33) to seprte the phses. Smples were centrifuged nd the lipid contining lower phse ws trnsferred to clen tubes nd evported to dryness under strem of nitrogen t room temperture. After lipid extrction from tissue smples, lipid clsses were seprted by solid phse extrction using commercil silic crtridges Strt SI-1 (Phenomenex, Torrnce, USA). Smples were reconstituted in 5 µl of chloroform, vortexed nd loded to the column following by wshing with 1 ml of chloroform to elute neutrl lipids. The frctions contining endocnnbinoids were then eluted with 1 ml chloroform/methnol (9:1, v/v), evported to dryness under nitrogen nd reconstituted in methnol until nlysis by LC/MS. The frctions contining phospholipids (PL) were eluted with 1 ml methnol nd stored t -2 ºC until further preprtion for ftty cid nlysis. Recovery of endocnnbinoids in the chloroform/methnol (9:1, v/v) elutes ws confirmed by LC/MS nd estimted to be higher thn 9%. Purity of the PL frction ws confirmed by thin lyer chromtogrphy (TLC) using heptne/isopropyl ether/cetic cid (6:4:4, by vol) s previously described (34). Anlysis of endocnnbinoids nd nndmide nlogues Smples were nlyzed by liquid chromtogrphy/electrospry ioniztion mss spectrometry (LC-ESI- MS) using n Agilent 12 series HPLC coupled to 32 QTRAP mss spectrometer (AB SCIEX; Concord, ON, Cnd). LC seprtion ws performed through n Ascentis Express C18 column (7.5 cm x 2.1 mm, 2.7 m prticle size) t flow rte of.3 ml/min. Two mobile phses were used: A) methnol 7

8 with.2% formic cid nd B) 5 mm mmonium formte, ph 3. The grdient elution method strted t 85% A from -.1 min; then the mobile phse A linerly incresed to 95% from.1-2 min nd held for 2 dditionl min (2.1-4 min). Then, the mobile phse returned to 85% A nd held t this composition for 6 min equilibrium time prior to the next injection. The mss spectrometer ws operted in the multiple rection monitoring (MRM) scn mode under positive ioniztion. Nitrogen ws used s curtin gs, drying nd nebulizing gs. Anndmide, 2-AG nd the two N-cylethnolmines (OEA nd PEA) in their protonted forms [M+H] + were identified s peks with the pproprite m/z vlues nd quntified by comprison with its externl synthetic stndrds rn under the sme conditions. The MRM trnsitions monitored were s follows: AEA m/z (35eV); AEA-d8 m/z (35eV); 2-AG m/z (18eV); 2-AG-d5 m/z (18eV); PEA m/z 3 62 (3eV); PEA-d4 m/z (3eV); OEA m/z (3eV); OEA-d2 m/z (3eV). The liner rnge for the clibrtion (stndrd) curves for AEA, PEA nd OEA ws 5 to 5 ng/ml nd for 2-AG ws.1 to 1 µg/ml. Since 2-AG nd 1-AG undergo rpid isomeriztion (35), results for 2-AG were reported s the sum of the individul peks of 2-AG + 1-AG. Ftty cid nlysis in PL PL frctions were trnsesterified using.5 N methnolic bse (metllic sodium in methnol), (Sigm- Aldrich) t 8 C for 15 min forming ftty cid methyl esters (FAME). The FAME were flushed with N 2 nd stored t 35 C until nlysis nd ftty cids were seprted by GC/FID (Vrin 39, Mississug, ON) using 1-m CP-Sil 88 fused-silic cpillry column (1 m x.25 mm i.d. x.2 μm film thickness; Vrin Inc., Mississug, ON) s previously described (32). The FAME were identified by comprison with retention times of commercil GC reference FAME stndrds (FAME mix #463 nd CLA FAME #UC-59M) from Nu-Chek Prep Inc. 8

9 Mesurement of ftty cid mide hydrolse (FAAH) protein expression in the jejunum Smples of the jejunum were wshed with phosphte-buffered sline, nd 2 cm-segment ws cut from 1 cm below the pyloric sphincter. Mucosl smples were then scrpped from jejunl segments. Proteins from jejunl mucos homogentes were seprted by SDS-PAGE electrophoresis on Tris-cette polycrylmide gels (3 8%, InVitrogen), trnsferred to PVDF membrne nd incubted with nti- FAAH1 rbbit polyclonl (1:1; ctlogue no. 9179; Cell Signling, Technology ) nd nti-β-ctin mouse polyclonl (1:5; ctlogue no. b8226; Abcm, St Louis, MO, USA) ntibodies s previously described (36). Detection ws chieved using nti-rbbit nd nti-murine secondry ntibodies nd the ECL dvnce detection system (Amershm Biosciences). Results re expressed s rtio of trget protein:β-ctin protein. Enzyme ctivity ssys The ctivity of FAAH nd NAPE-PLD in jejunl mucos ws mesured by stndrd ssys s previously described using [ 3 H]-nndmide nd 1,2-diplmitoyl-sn-glycero-3-phosphoethnolmine-Nheptdecenoyl s substrtes, respectively (37). Mesurement of pro-inflmmtory genes in the jejunum Totl RNA ws isolted from frozen segments of jejunl mucos using TRIzol (Invitrogen, Cnd) s described in the mnufcturer s protocol nd reversed trnscribed into complementry DNA using MultiScribee Reverse trnscriptse (High-Cpcity cdna Reverse Trnscription Kit; Applied Biosystems, Foster City, CA, USA). The expression of CB 1, FAAH, nd the pro-inflmmtory cytokines; TNF-α nd interleukin 1 bet (IL-1β) reltive to the housekeeping gene Actb (β-ctin) ws ssessed by quntittive rel-time PCR, using the StepOne Plus Rel-Time PCR system (Applied Biosystems) nd StepOne Softwre (version 2). PCR contined complementry DNA templte, 1 nm of commercilly vilble, pre-mixed trget-specific primers nd Tqmn FAM -lbelled probe (Applied Biosystems) for CB 1, FAAH, TNF-α, IL-1β nd Actb. Therml cycling conditions were s follows: 95 C for 2 s, 9

10 followed by forty cycles of 95 C for 1 s nd 6 C for 2 s. Reltive mrna expression for ech trget gene ws normlized to Actb mrna nd quntified using the comprtive cycle threshold (Ct) method. Dt ws expressed s rtio of trget mrna expression reltive to β-ctin. All ssys were performed in triplicte. Cco-2 cell culture nd mesurement of FAAH inhibition on inflmmtory cytokines Cco-2 cells (ATCC) were cultured in MEM (M4655, Sigm) with 1% fetl bovine serum nd 1% penicillin/streptomycin nd kept t 37 C in 5% CO 2 nd 95% humidity. Cells were grown in 6 well pltes nd seeded t 1 6 cells per insert (24 mm dimeter,.4 m pore polycrbonte inserts). Cells t pssges were grown for minimum of 18 dys nd used for experimenttion between dys. Cells were mintined for 12 hours in 1% ftty cid free BSA fter which they were treted with vehicle control or VA (1 M), VA (1 M) + URB597 (FAAH inhibitor, Cedrlne) (1 M) in the presence or bsence of OEA (1 M) for 24 hours. Following this, the cells were chllenged with LPS (1 g/ L) for 24 hours. During this time fresh vehicle control or VA (1 M), URB597 (1 M), OEA (1 M) in.5% BSA ws dded. Totl RNA ws isolted from Cco-2 cells nd reversed trnscribed into complementry DNA. The expression of TNF-α reltive to the housekeeping gene GAPDH ws ssessed by quntittive rel-time polymerse chin rection (PCR) using SYBR Green (Applied Biosystems). Sttisticl nlysis All results re expressed s men ± SEM. Sttisticl comprisons between dietry groups were nlyzed using one-wy ANOVA followed by Tukey s post hoc test. The level of significnce ws set t P<.5 (Grph Pd Prism 5., USA). 1

11 Results Supplementtion with VA lone or in combintion with CLA does not ffect endocnnbinoid concentrtions in plsm, brin or skeletl muscle Contrry to our hypothesis, dietry supplementtion with VA, CLA or VA+CLA for 8 wk did not ffect endocnnbinoid concentrtions in plsm (Tble 2), the hypothlmus (dt not shown) or skeletl muscle (Figure 1) reltive to control diet (P>.5). All dietry tretments (VA, CLA nd VA+CLA) significntly reduced (P<.1) the concentrtions of OEA (-2%, -19% nd -17%, respectively) while only VA reduced the concentrtion of PEA (21%, P<.5) in the skeletl muscle reltive to control (Figure 1). Dietry supplementtion with VA lone or in combintion with CLA decreses the concentrtion of 2-AG nd nndmide nlogues (OEA nd PEA) in the liver nd VAT Supplementtion with VA, CLA or VA+CLA significntly reduced 2-AG concentrtions in the liver (83%, P<.1; 47.6%, P<.5 nd 74%, P<.1, respectively) reltive to control diet. Interestingly, the VA diet (but not the VA+CLA diet) lowered liver 2-AG concentrtions by 68% s compred to the CLA fed group (P<.5). In ddition, VA or VA+CLA supplementtion (but not CLA) resulted in decresed (P<.1) concentrtions of the N-cylethnolmines, OEA (to not detectble levels) nd PEA (-57% nd -56%, respectively) in this tissue s compred to control (Figure 2). Anndmide ws not detected in the liver of JCR:LA-cp rts. VA nd VA+CLA (but not CLA) significntly (P<.5) reduced the concentrtions of 2-AG (-86% nd - 87%, respectively) nd OEA (-59%, P<.5 nd -74%, P<.1, respectively) in VAT s compred to control (Figure 3). Collectively, results from this study suggest tht there were no dditive effects of combining VA with CLA on reducing EC concentrtions in liver nd VAT. 11

12 Supplementtion with VA increses the concentrtion of nndmide nd nndmide nlogues (OEA nd PEA) in the jejunum Unexpectedly, dietry supplementtion with VA (but not CLA or VA+CLA) significntly incresed jejunl concentrtions of the endocnnbinoid nndmide (3.8 fold, P<.5) nd its nlogues OEA (1.7 fold, P<.5) nd PEA (1.9 fold, P<.1) (Figure 4). It is importnt to note tht VA did not lter food intke in the present study (dt no shown), nor in previous studies using different niml models (2, 3, 38, 39). Therefore, it is unlikely tht incresed nndmide concentrtions in the jejunum (following VA tretment) would result in n ppetite stimultory effect s result of CB receptor ctivtion. Effects of dietry supplementtion with VA, CLA or their combintion on tissue PL ftty cid composition We nlyzed tissue PL ftty cid composition to determine whether chnges in tissue endocnnbinoid nd N-cylethnolmine concentrtions were due to ltertions in the vilbility of their biosynthetic precursors in membrne PL (Tble 3). Interestingly, we found tht VA supplementtion incresed the endocnnbinoid precursor, AA in liver (3%, P<.1) nd skeletl muscle (11%, P<.5) PL compred to control diet. VA+CLA fed-rts lso hd incresed mounts of AA (2%) in liver PL reltive to control rts (P<.1). The incorportion of AA in VAT, jejunl nd hypothlmus PL ws not ffected by ny experimentl diet (VA, CLA or VA+CLA) reltive to control (P>.5). VA nd VA+CLA fed rts hd lower concentrtions of the OEA precursor, oleic cid (-35% nd -4%, respectively) while VA-fed rts only hd lower mounts of the PEA precursor, plmitic cid (-21%) in liver PL reltive to control rts (P<.1). CLA nd VA+CLA fed rts hd lower (P<.1) mounts of oleic cid (-15% nd -17%, respectively) in VAT PL, while only VA+CLA rts hd lower concentrtions of oleic cid (-33%, P<.1) in jejunl PL reltive to control rts. Collectively, findings from ftty cid nlysis in tissue PL suggest tht the regultory effect of VA on tissue EC could not be explined by chnges in their biosynthetic membrne PL precursor (AA). 12

13 We lso ssessed the PL incorportion of VA nd CLA in ll tissues nlyzed. As expected, VA nd VA+CLA fed-rts hd higher (P<.1) concentrtions of VA in liver (8 fold), VAT (4.5 fold), skeletl muscle (1 fold) nd jejunl (3.8 fold) PL compred with rts fed the control diet. The incorportion of VA into the hypothlmus of JCR:LA-cp rts ws found to be t trce mounts (.1 g/1 g totl ftty cids). CLA ws not detected in tissues of VA-fed rts, suggesting limited incorportion of CLA (produced from VA desturtion) into tissue PL. Furthermore, CLA ws only incorported in the liver, VAT, hypothlmus nd jejunl PL of CLA fed-rts (<1 g/1 g totl ftty cids) nd to lesser extent in tissue PL of rts fed the VA+CLA diet (P<.5). The mgnitude of incorportion of VA in membrne PL is tissue-dependent To ssocite the tissue-specific effects of VA with its mgnitude of incorportion, we conducted comprison of VA in tissue membrne PL (Figure 5). VA ws incorported in VAT (1.8 g/1 g ftty cids), followed by the liver (1.6 g/1 g ftty cids), jejunum (1.5 g/1 g ftty cids), skeletl muscle (1. g/1 g ftty cids) nd hypothlmus (.1 g/1 g ftty cids). Dietry supplementtion with VA lters the mrna nd protein expression of FAAH but does not ffect the mrna expression of CB 1 receptor in the jejunum To determine whether selective increse of nndmide, OEA nd PEA in the jejunl mucos by VA could be ssocited with synthetic or degrdtive pthwys, we first mesured the mrna expression nd protein ctivity of jejunl NAPE-PLD (N-cylphosphtidylethnolmine phospholipse D type), one of the key enzymes responsible for the synthesis of these N-cylethnolmines but no differences were found between groups (dt no shown, P>.5). We then mesured the expression of FAAH, their primry hydrolyzing enzyme. Interestingly, while VA nd VA+CLA fed rts hd higher jejunl mrna expression of FAAH compred to the control group (1.4 fold, P<.5 nd 1.6 fold, P<.1, respectively), the protein bundnce of this enzyme ws reduced in VA fed-rts only (-34%, P<.5) (Figure 6A, B nd 13

14 C). However, the protein ctivity of jejunl FAAH did not differ between groups (Figure 6E, P>.5). We lso found tht VA tended to lower jejunl mrna expression of CB1 reltive to the other diets; however, this did not rech sttisticl significnce (P>.5) (Figure 6D). Dietry supplementtion with VA reduced the mrna expression of pro-inflmmtory cytokines in the jejunum The endocnnbinoid system (ECS) is upregulted in humn inflmmtory bowel diseses nd experimentl models of colitis nd colorectl cncer growth (21, 4-42). During these conditions, n overctive ECS is proposed to be n dptive response to counterct the consequences of inflmmtion, such s T cell-medited berrnt immune response (41). Therefore, explored whether the increse in jejunl nndmide, OEA nd PEA we observed ws ssocited with intestinl inflmmtion. Indeed, the expression of pro-inflmmtory cytokines (TNFα (Figure 7A) nd IL-1β (Figure 7B) in the intestine ws significntly lower in rts fed the VA supplemented diet compred with the control rts (-8% nd -64%, respectively; P<.5). Cco2 cell culture to verify VA ction on nti-inflmmtory pthwys We tested the nti-inflmmtory properties of VA lone or in combintion with the FAAH inhibitor URB597 in the presence or bsence of the FAAH substrte, OEA in the Cco2 cell model of humn intestinl epithelil cells. Tretment with VA lone reduced the LPS-induced mrna expression of TNFα by 64% while ddition of URB597 did not result in further nti-inflmmtory properties in the Cco2 cells (Figure 8). The mrna expression TNF-α ws not reduced by VA in the bsence of OEA (P>.5), suggesting OEA is required for the observed nti-inflmmtory properties of VA in this cell model. 14

15 Discussion Dietry supplementtion with VA reduces liver nd VAT 2-AG without ltering the vilbility of PL biosynthetic precursor Incresed plsm EC concentrtions hve been found to be positively correlted with viscerl ft mss nd wist circumference in humns (14-16). Consequently, the ECS hs been proposed s criticl trget for the tretment of bdominl obesity nd ssocited metbolic bnormlities in the metbolic syndrome. While phrmcologicl blockde of the CB1 receptor with Rimonbnt hs shown some clinicl success, the dverse psychitric side effects ssocited with this drug hve led to its withdrwl s tretment option (43). Given tht the ECS cn be modulted in response to dietry ft (9), nutritionl interventions with tissue-specific effects could be n ttrctive lterntive pproch to cliniclly trget the systemic ECS during metboliclly bnorml conditions. To our knowledge this is the first report to demonstrte tht despite lck of direct effect on either the plsm or the brin ECs, dietry supplementtion with VA cn effectively decrese liver nd VAT 2-AG concentrtions in rt model of metbolic syndrome. Our findings resemble those reported for n3-long chin (LC)-PUFA (in the form of krill oil) in the Zucker f/ft rt (44) nd my lso suggest puttive mechnism of ction for the bility of VA to decrese ectopic lipid ccumultion nd heptic TG secretion (5). The rtionle for this hypothesis is tht CB receptor ctivtion in the liver nd VAT cn led to incresed de novo lipogenesis nd viscerl diposity (18, 19, 45), both of which re ttenuted by VA (3, 5). Interestingly, effects of VA on liver nd VAT 2-AG concentrtions cnnot be explined by chnges in AA levels in membrne PL but rther due to incorportion of VA into PL. Thus, it is plusible tht n incresed incorportion of VA (reltive to AA) into the lipid precursor of 2-AG (i.e dicylglycerol) my occur during PL remodeling. This modifiction would result in the synthesis of vccenic cid-derived glycerol compound insted of synthesis of 2-AG, thereby leding to decresed concentrtions of 2-AG but this requires further investigtion. 15

16 Effects of VA on tissue EC concentrtions re ssocited with its incorportion into tissue PL Previous studies in the JCR:LA-cp rt hve shown tht the lipid-lowering effects of CLA re enhnced by the ddition of VA when compred to dietry supplementtion with CLA lone (24). In this study we provide evidence tht VA per se cn independently reduce tissue 2-AG concentrtions corresponding with its mgnitude of incorportion into tissue PL when compred to CLA (Tble 3). Our findings lso revel tht effects of VA on EC concentrtions mybe tissue-specific nd prllel the extent of VA incorportion into respective tissue membrne PL (Figure 5). We note tht while the incorportion of VA in peripherl tissues (VAT, liver nd jejunl mucos) is within the sme generl level, the lower hypothlmic incorportion of VA suggests decresed ctive trnsport of VA cross the blood brin brrier but this requires further investigtion. Further studies re lso needed to determine the mechnism of how the incorportion of VA into tissue PL medites lowering of EC concentrtions in liver nd VAT. Incresed jejunl nndmide, OEA nd PEA by VA is ssocited with down-regultion of FAAH protein expression nd my explin nti-inflmmtory properties of VA We hve observed tht VA re-equilibrtes intestinl nd heptic lipid homeostsis while exerting differentil trnscriptionl regultion in both orgns, s reflected in mrna levels of SREBP1 nd FAS (5). In this study we found consistent effect of VA to decrese 2-AG concentrtions in the liver nd VAT. In contrst, jejunl concentrtions of nndmide nd its nlogues (OEA nd PEA) were selectively incresed following VA tretment lone. This selective increse of jejunl N- cylethnolmines by VA could not be explined by chnges in their biosynthetic PL precursors, but ssocited with reduction in protein expression of the enzyme FAAH (known to hydrolyze nndmide, OEA nd PEA). Notbly, feeding n-3 LCPUFA is ssocited with n increse in the FAAH inhibitor, rchidonoyl-serotonin (AA-5-HT) nd other jejunl LCPUFA-serotonins (lso cpble of inhibiting FAAH ctivity in vitro) in mice (46). Interestingly, in the present study we found discordnce between incresed mrna levels nd reduced protein bundnce of FAAH in the jejunl mucos upon VA 16

17 supplementtion which suggests compenstory mechnism for the reduced protein nd infers nd ctive feedbck pthwy for the enzyme. Furthermore, the protein ctivity of FAAH ws not different between groups (Figure 6E). Although it is plusible tht VA supplementtion my stimulte the formtion of lipid meditors tht regulte FAAH bundnce, the fctors tht regulte FAAH bundnce nd/or ctivity re complex. For exmple, the literture describes complex interction of FAAH with ppetite nd sex hormones (47, 48), s well s preferentil membrne stbiliztion of FAAH by sterols (49) tht could potentilly be involved. Trgeting the ECS (vi inhibition of FAAH) hs therpeutic potentil in the tretment of intestinl inflmmtory diseses (5-52). Anndmide is found to be incresed in humn intestinl inflmmtory diseses nd is proposed to be n dptive response to counterct the inflmmtory milieu during these conditions (4-42). In contrst to the pthologicl role of the ECS in the liver nd VAT (13, 18, 53), ctivtion of CB1 or CB2 receptors (vi exogenous gonists or FAAH inhibition) hs been shown to reduce mcroscopic dmge scores of colonic inflmmtion nd to ttenute the ctivtion of T-cells nd the infiltrtion of inflmmtory cells in niml models of colitis (21, 22, 5, 51). Additionlly, the N- cylethnolmine PEA hs lso been shown to exert nti-inflmmtory effects through ctivtion of PPARα, including reduction or pro-inflmmtory cytokine production (54). We therefore exmined the mrna expression of two key inflmmtory mrkers, TNF-α nd IL-1β in the intestine to explore potentil ssocition with the incresed nndmide nd PEA concentrtions observed in the jejunum. Indeed, the reduction in mrna expression of these pro-inflmmtory cytokines by VA is consistent with previous observtions in this rt model. VA tretment hs been shown to normlize the production of IL-2 nd TNFα in mesenteric gut-ssocited lymph nodes fter mitogen stimultion in this rodent model of metbolic syndrome (4). Results from the present study provide further evidence for potentil ntiinflmmtory effect of VA vi ctivtion of the ECS in the intestine. We wish to note tht both CB nd non-cnnbinoid receptor (e.g. PPAR) mechnisms my contribute to the nti-inflmmtory effects of FAAH inhibition (52). Further, we hve shown previously tht VA feeding increses the expression of 17

18 PPAR nd PPAR in jejunl mucos (6). Therefore, incresing the vilbility of endogenous lignds to CB nd PPAR receptors (e.g. nndmide nd PEA, respectively), my offer mechnistic explntion for the nti-inflmmtory effects of VA in this orgn. Results from this study lso suggest potentil synergistic effect of VA nd OEA on reducing the LPSinduced mrna expression of TNFα in the Cco2 cell model (Figure 8). The mechnisms for this interction re not cler, but previous studies suggest nti-inflmmtory effects of OEA re ssocited with reduced ctivtion of the trnscriptionl cytokine ctivtor NF-κB (55, 56). Unlike industrillyderived TFA, VA hs been shown to protect ginst NF-κB ctivtion (57) nd inflmmtion s mesured by reduced T-helper cell cytokine production in PPARy-dependent mnner (7). Therefore, it is plusible tht OEA nd VA my ct synergisticlly to reduce the LPS-induced pro-inflmmtory response vi PPAR ctivtion. Alterntively, VA nd OEA my lter cell receptor function (e.g. toll-like receptor 4) nd receptor-induced ctivtion of NF-κB indirectly by ltering membrne nd lipid rft function but this wrrnts further investigtion. In conclusion, we demonstrte tht dietry supplementtion with VA exerts tissue-specific regultion of endocnnbinoids tht could be used s n ttrctive lterntive pproch to trget the ECS during conditions of metbolic syndrome nd intestinl inflmmtory diseses. We hve shown tht VA effectively reduces liver nd VAT 2-AG concentrtions corresponding with its previously observed properties to beneficilly modulte lipid storge comprtments. We hve lso provided evidence tht VA cn ct independently of CLA, which seems to be ssocited with its incorportion into tissue PL. Additionlly, the present findings delinete unique opposing regultion of VA on nndmide nd its N- cylethnolmine nlogues tht cnnot be explined by chnges in their biosynthetic PL precursors. Rther, our results suggest n inhibitory effect of VA on the protein expression of FAAH in the intestine tht my result in ctivtion of protective pthwys of the ECS in this orgn. Collectively, findings from this study hve provided potentil novel mechnism of ction for the helth benefits of VA nd 18

19 highlight the need for further investigtion to explore the efficcy of VA on intestinl inflmmtory diseses. 19

20 Acknowledgements: We thnk Shron Sokolik nd Sndr Kelly for their excellent technicl ssistnce ssocited with this project. The reserch ws supported in prt by Diry Frmers of Cnd, Cndin Institutes of Helth Reserch, Albert Livestock Industry Development Fund nd Albert Livestock nd Met Agency. MMJS ws supported by scholrship from the Ntionl Council of Mexico for Science nd Technology (CONACyT). SDP ws supported by New Investigtor Awrd from Hert nd Stroke Foundtion of Cnd nd the Nturl Science nd Engineering Reserch Council of Cnd. All uthors hve declred no conflict of interest. 2

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26 Figure Legends Figure 1 Endocnnbinoid nd N-cylethnolmine concentrtions in the skeletl muscle of JCR:LA-cp rts following dietry supplementtion with VA, CLA or VA+CLA. Vlues re mens +/- SEM, represented by verticl brs (n=5). Mens without common letter differ (p<.5). Figure 2 Endocnnbinoid nd N-cylethnolmine concentrtions in the liver of JCR:LA-cp rts following dietry supplementtion with VA, CLA or VA+CLA. Vlues re mens +/- SEM, represented by verticl brs (n=5). Mens without common letter differ (p<.5). ND, not detectble Figure 3 Endocnnbinoid nd N-cylethnolmine concentrtions in VAT of JCR:LA-cp rts following dietry supplementtion with VA, CLA or VA+CLA. Vlues re mens +/- SEM, represented by verticl brs (n=5). Mens without common letter differ (p<.5). Figure 4 Endocnnbinoid nd N-cylethnolmine concentrtions in the jejunl mucos of JCR:LA-cp rts following dietry supplementtion with VA, CLA or VA+CLA. Vlues re mens +/- SEM, represented by verticl brs (n=5). Mens without common letter differ (p<.5). Figure 5 Incorportion of VA in tissue PL in JCR:LA-cp rts supplemented with VA for 8 wk. Vlues re mens +/- SEM, represented by verticl brs (n=5). Mens without common letter differ, P<.5. Figure 6 Jejunl mucosl mrna expression of FAAH nd CB 1 nd protein bundnce nd ctivity of FAAH in JCR:LA-cp rts fed control or experimentl diets for 8 wk. The mrna expression for FAAH (A) nd CB 1 (D) is reltive to the housekeeping gene β-ctin. A representtive blot (B) nd grph (C) for FAAH reltive protein bundnce re shown. FAAH ctivity ws not significnt within groups. Vlues re mens +/- SEM, represented by verticl brs (n=5). Mens without common letter differ, P<.5. Figure 7 Jejunl mucos mrna expression of pro-inflmmtory cytokines in JCR:LA-cp rts fed control or experimentl diets for 8 wk. The expression of TNF-α (A) nd IL-1β (B) is reltive to the 26

27 housekeeping gene β-ctin. Vlues re mens +/- SEM, represented by verticl brs (n=5). Mens without common letter differ, P<.5. Figure 8 Effect of VA in the presence or bsence of OEA nd the FAAH inhibitor URB597 in the intestinl Cco2 cell model of inflmmtion. The expression of TNF-α is reltive to the housekeeping gene GAPDH. Vlues re mens +/- SEM, represented by verticl brs (n=5). Mens without common letter differ, P<.5. 27

28 Tbles nd Figures Tble 1 Ftty cid composition of control nd experimentl diets 1 Ftty cid Control diet VA diet CLA diet VA+CLA diet g/1 FAME* C12: C14: C14: C16: C16: C18: C18:1 trns C18:1 trns-11 (VA) C18:1 cis-9 (oleic cid) C18:1 cis C18:2 n C18:3 n cis9, trns11 CLA Summries SFA C12:, C14:, C16: PUFA cismufa trnsmufa n n n6:n3 rtio PUFA:SFA rtio Vlues re expressed s percentge of ftty cids *FAME, ftty cid methyl esters 28