Dietary oil composition differentially modulates intestinal endotoxin transport and postprandial endotoxemia

Transcription

1 Mni et l. Nutrition & Metolism 2013, 10:6 RESEARCH Open Access Dietry oil composition differentilly modultes intestinl endotoxin trnsport nd postprndil endotoxemi Venktesh Mni 1,2, Jmes H Hollis 3 nd Nichols K Gler 1,2* Astrct Bckground: Intestinl derived endotoxin nd the susequent endotoxemi cn e considered mjor predisposing fctors for diseses such s therosclerosis, sepsis, oesity nd dietes. Dietry ft hs een shown to increse postprndil endotoxemi. Therefore, the im of this study ws to ssess the effects of different dietry oils on intestinl endotoxin trnsport nd postprndil endotoxemi using swine s model. We hypothesized tht oils rich in sturted ftty cids (SFA) would ugment, while oils rich in n-3 polyunsturted ftty cids (PUFA) would ttenute intestinl endotoxin trnsport nd circulting concentrtions. Methods: Postprndil endotoxemi ws mesured in twenty four pigs following porridge mel mde with either wter (Control), fish oil (FO), vegetle oil (VO) or coconut oil (CO). Blood ws collected t 0, 1, 2, 3 nd 5 hours postprndil nd mesured for endotoxin. Furthermore, ex vivo ileum endotoxin trnsport ws ssessed using modified Ussing chmers nd intestines were treted with either no oil or 12.5% (v/v) VO, FO, cod liver oil (CLO), CO or olive oil (OO). Ex vivo mucosl to serosl endotoxin trnsport permeility (Ppp) ws then mesured y the ddition of fluorescent leled-lipopolyscchride. Results: Postprndil serum endotoxin concentrtions were incresed fter mel rich in sturted ftty cids nd decresed with higher n-3 PUFA intke. Compred to the no oil control, fish oil nd CLO which re rich in n-3 ftty cids reduced ex vivo endotoxin Ppp y 50% (P < 5). Contrrily, sturted ftty cids incresed the Ppp y 60% (P = 08). Olive nd vegetle oils did not lter intestinl endotoxin Ppp. Conclusion: Overll, these results indicte tht sturted nd n-3 PUFA differentilly regulte intestinl epithelil endotoxin trnsport. This my e ssocited with ftty cid regultion of intestinl memrne lipid rft medited permeility. Keywords: Dietry ft, Endotoxin, Intestine Bckground The link etween dietry ft nd endogenous lood endotoxin hs ttrcted incresed medicl nd iomedicl interest over the lst few yers. Furthermore, hyperphgi, incresed diposity nd metolic chnges ssocited with high ft feeding cn e recpitulted in mice chroniclly infused with LPS for four weeks [1]. It hs een reported tht the structure of ft consumed (emulsion vs. free oil) chnges the extent of endotoxemi * Correspondence: ngler@istte.edu 1 Deprtment of Animl Science, Iow Stte University, Ames, IA 50011, USA 2 Interdeprtmentl Toxicology Grdute Progrm, Iow Stte University, Ames, IA 50011, USA Full list of uthor informtion is ville t the end of the rticle nd tht ltering the composition, structure nd qulity of dietry fts could improve helth [2]. In helthy humns, postprndil plsm endotoxin concentrtions increse on verge 18% fter high ft mel (pproximtely 380 kcl from ft, 42% of totl energy) compred to the fsted stte [3]. These uthors concluded tht incresed postprndil LPS my contriute to the development of postprndil inflmmtion nd disese. Ghnim et l. [4,5] lso showed tht in helthy dults, high ft, high crohydrte mel (~900 kcl) incresed postprndil plsm LPS concentrtions y 70%. However, Lugerette et l. [6] recently reported tht dietry oil composition differentilly modulted murine inflmmtion nd endotoxin 2013 Mni et l.; licensee BioMed Centrl Ltd. 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 cited.

2 Mni et l. Nutrition & Metolism 2013, 10:6 Pge 2 of 9 trnsport. These uthors lso showed tht ft composition, not quntity in the diet (22 vs. 3%) ws criticl in modulting plsm endotoxemi. Collectively, these dt show tht dietry ft intke nd composition is le to modulte lood endotoxin nd tht this is ssocited with cute inflmmtion nd the metolic diseses of oesity nd dietes. Both grm positive nd grm negtive cteri re present in lrge quntities in the intestine. Interestingly, the totl quntity of endotoxin, which is the grm negtive cteril outer cell wll component, in the intestine lone could e up to one grm [7]. Even very smll quntities of endotoxin, pico-grm scle, in the systemic circultion hs the potentil to elicit n inflmmtory response in humns nd nimls [8]. Endotoxin is lso synonymously referred to s lipopolyscchride (LPS), nd oth of these compounds re mjor immunogens tht elicit n inflmmtory response in numerous tissues nd cell types vi their recognition through pthogen-ssocited moleculr ptterns (PAMPs) nd Toll like receptors in the innte immune system [9]. Endotoxin is thought to enter circultion y trnsport cross the intestinl epithelium either vi prcellulr pthwys through the openings of intestinl tight junctions etween two epithelil cells or y trnscellulr pthwy [7]. Trnscellulr trnsport nd the ssocited endocytosis of intestinl derived endotoxin my e fcilitted y intrcellulr signling processes medited y the innte immune receptor complex CD 14/Toll like receptor 4 (TLR4)/MD-2, in ssocition with the cell memrne micro domin lipid rft [10]. Furthermore, circulting endotoxin concentrtions my lso e ugmented y trnsport coupled to dietry lipids nd chylomicrons [11]. In recent yers ccumulting reserch hs investigted the link etween dietry ft nd endogenous endotoxin in reltion to metolic inflmmtion [12,13]. Current evidence suggests tht dietry ft ugments circulting endotoxin concentrtions nd the resultnt postprndil endotoxemi leds to low-grde systemic inflmmtion which hs een implicted in the development of severl metolic diseses [1,3,14]. Intestinl derived endotoxin nd the susequent cute endotoxemi re considered mjor predisposing fctors for inflmmtion ssocited diseses such s therosclerosis, sepsis, oesity, type 2 dietes nd Alzheimer's [15-17]. However, the ility of different types of oil nd ftty cids to fcilitte uptke of intestinl endotoxin hs een poorly chrcterized. Interestingly, sturted nd n-3 polyunsturted ftty cids (PUFA) hve een shown to reciproclly modulte the LPS receptor, TLR4, nd cell memrne lipid rfts [18]. This is postulted to e due to sturted ftty cids (SFA) such s luric nd myristic cid eing prt of the ftty cyl side chin composition of Lipid-A component of endotoxin nd the ility of n-3 PUFA to reduce the potency of endotoxin when sustituted in plce of sturted ftty cids in lipid-a [19,20]. Thus, there is cler linkge etween ftty cids (sturted, n-3 polyunsturted, monounsturted etc....) nd endotoxin signling. Therefore, the im of this study ws to ssess the effects of vrious dietry fts on in vivo nd ex vivo intestinl endotoxin trnsport nd circulting concen trtions using the pig s iomedicl model. We hypothesize tht oils rich in sturted ftty cids (SFA) would ugment, while the oils contining the n-3 PUFA (docoshexenoic cid [DHA] nd eicospentenoic cid [EPA]) would ttenute, intestinl endotoxin trnsport nd postprndil endotoxemi. Methods Mterils nd nimls All the chemicls used for this study were purchsed from Sigm-Aldrich (St. Louis, MO) unless otherwise stted. All niml use nd procedures were pproved y the Iow Stte University Institutionl Animl Cre nd Use Committee. Effect of dietry oil on postprndil serum endotoxin concentrtion Twenty four pigs (49 ± 7 kg BW) were rised on typicl corn-soyen diet tht met or exceeded their nutrient requirements [21] nd rndomly llocted to one of four tretments. The tretments consisted of 500 g ground corn-soyen mel dough (2,145 kcl ME) mde up with either 1) 50 ml wter (Control); 2) 50 ml fish oil (FO) (Spring Vlley Inc., UT); 3) 50 ml vegetle oil (VO) (Hy-Vee Inc., IA) ; or 4) 50 ml coconut oil (CO) (Spectrum Nturls Inc., NY). After n overnight fst, six pigs were fed one of ech porridge mel. Pigs voluntrily consumed the entire porridge mel with in ten minutes fter the mel ws offered. Blood ws collected t 0, 1, 2, 3, nd 5 hours postprndil y venipuncture using pyrogen free vccutiner tues nd sterile needles. Proper precutionry mesures were tken to prevent externl endotoxin contmintion of lood. Serum ws seprted y centrifuging t 2000 g nd 4 C. Serum ws then stored in pyrogen free tues t 80 C until further nlysis. Circulting serum endotoxin concentrtion ws mesured using the end point fluorescent ssy using the recominnt fctor C (rfc) system (Lonz,Switzerlnd). Briefly, the serum smples were diluted 1000 times nd 100 μl of the smples or stndrds were dded to 96 well plte nd incuted t 37 C for 10 min. Therefter, 100 μl of rfc enzyme, rfc ssy uffer nd rfc sustrte were dded t rtio of 1:4:5 to the plte nd n initil reding were tken followed y 1 h incution t 37 C. The reltive fluorescence unit (RFU) for ech well ws determined (excittion 380 nm nd emission 440 nm). A

3 Mni et l. Nutrition & Metolism 2013, 10:6 Pge 3 of 9 positive control stndrd from the ssy kit ws used to scertin the vlidity of the ssy nd the concentrtion of the endotoxin ws interpolted from the stndrd curve constructed from the stndrds nd corrected for smple dilution. Ex vivo intestinl integrity nd endotoxin trnsport Freshly isolted ileum segments from eleven pigs (21 28 dys old) were plced in chilled Kres-Henseleit uffer (consisting of, in mmol/l: 25 NHCO 3, 120 NCl, 1 MgSO 4, 6.3 KCl, 2 CCl 2, 0.32 NH 2 PO 4 ; ph 7.4) for trnsport to the lortory while under constnt ertion. Intestinl tissues were then stripped of their outer serosl lyer nd immeditely mounted into modified Ussing chmers (Physiologic Instruments Inc., Sn Diego, CA nd World Precision Instruments Inc. New Hven, CT). Ech chmer nd intestinl segment (0.71 cm 2 ) ws thed on its mucosl nd serosl sides with 5 ml of Kres-Henseleit uffer nd constntly gssed with 95% O 2-5% CO 2 mixture. Chmers were connected to pir of dul chnnel current nd voltge electrodes contining 3% nole gr ridges nd filled with 3 M potssium chloride to mesure electrophysiologicl prmeters of the intestinl memrnes or to mesure the mucosl to serosl trnsport of endotoxin. Trnsepithelil resistnce (TER) ws not different cross pigs, indicting no differences in prcellulr permeility or leky gut (dt not shown). To rule out ny influence tht ile cids my hve on intestinl integrity, TER nd mcromolecule permeility ws first tested on isolted ileum smples tht were incuted with porcine ile cid (0, 3, 6 nd 9 mg/ml) for thirty minutes. Therefter, FITC-leled dextrn (FITC-Dextrn, 4.4 kd) mucosl to serosl trnsport ws mesured s descried previously [22]. Briefly, the mucosl chmers were chllenged with 2.2 mg/ml FITC-Dextrn nd chmer smples from oth sides were collected every min for eighty minutes. The reltive fluorescence ws then determined using fluorescent plte reder (Bio-Tek, USA) with the excittion nd emission wvelengths of 485 nd 520 nm, respectively. Therefter, n pprent permeility coefficient (Ppp) ws clculted for ech tretment: dq Ppp ¼ ðdt A C 0 Þ Where: dq/dt = trnsport rte (μg/min); C 0 = initil concentrtion in the donor chmer (μg/ml); A = re of the memrne (cm 2 ). The effect of dietry ft on endotoxin trnsport ws studied using ex vivo permeility of fluorescein isothiocynte (FITC) leled-lps (Escherichi coli 055:B5) mounted into modified Ussing chmers. Briefly, segments of swine intestinl tissues were treted with either 12.5% (v/v) uffered sline control (CON), Fish Oil or cod liver oil (CLO) mnufctured y Spring Vlley Inc., UT) or vegetle oil, coconut oil or olive oil (OO) purchsed form Hy-Vee Supermrkets Inc., IA). All oils were commercil retil ville nd then mixed with 20 mm sodium turodeoxycholte (ile cid) for micelle formtion to simulte the intestinl milieu. Ech mucosl chmer ws then chllenged with 20 μg/ml FITC-LPS nd chmer smples were collected every min for eighty minutes. The reltive fluorescence of ech smple ws then determined using fluorescent plte reder (Bio-Tek, USA) with the excittion nd emission wvelengths of 485 nd 520 nm, respectively. The pprent permeility coefficient ws then clculted similr to tht descried ove for FITC-Dextrn. Lipid rfts, dietry oil nd ex vivo intestinl endotoxin trnsport To exmine the role of lipid rfts in intestinl endotoxin trnsport, ileum segments from 16 pigs (56 ± 4 dys of ge) were mounted in Ussing chmers s descried ove. Segments were pre-treted with or without 25 mm Methyl-β-cyclo dextrin (MβCD, synthetic lipid rft modifier) for 30 min. Therefter, the mucosl chmer ws spiked with either sline-ile cid (CON) or Coconut oil-ile cid (12.5% v/v) nd the FITC-LPS pprent permeility coefficient for ech tissue ws clculted. Ftty cid nlysis Ftty cid profiles of the dietry oils used to mke the porridge were determined nd nlyzed y GC-MS [23,24]. One ml oil ws mixed with 0.5 ml of 4:1 hexne nd 125 μg/l heptdecnoic cid ws dded to ech smple s n internl stndrd. FAME were nlyzed y GC on Hewlett-Pckrd model 6890 fitted with n Omegwx 320 (30-m 0.32-mm i.id um) cpillry column. Hydrogen ws the crrier gs. The temperture progrm rnged from 80 to 250 C with temperture rise of 5 C/min. The injector nd detector tempertures were 250 C nd 1 μl of smple ws injected nd run split. Ftty cids methyl esters were identified y their reltive retention times on the column with respect to pproprite stndrds nd heptdecnoic cid. Dt nlysis Results re presented s mens ± S.E.M nd were nlyzed with the Proc Mixed procedure of SAS (Cry, NC). In the model, repetition or dy of Ussing chmer run ws used s rndom effect. Sttisticl significnce of difference ws nlyzed y nlysis of vrince (ANOVA) followed y Tukey s rnge test for pir wise comprison of ll tretment mens. Differences were considered significnt t P 5 nd tendency t P 0.10.

4 Mni et l. Nutrition & Metolism 2013, 10:6 Pge 4 of 9 Results Dietry oil ftty cid profiles The ftty cid composition of the oils used to mke the porridge mel nd/or in the ex vivo trnsport study re reported in Tle 1. The coconut oil contined high concentrtions of sturted ftty cids (89%), prticulrly luric, myristic, plmitic cids. Olive oil contined very high content of monounsturted oleic cid nd moderte mount of plmitic cid, with sturted ft content of 29%. Vegetle oil used in this study contined high quntity (50%) of rchidonic cid (20:4n6), 32% oleic cid nd 13% plmitic cid. The fish oil used consisted of 35% docoshexenoic cid (DHA) nd 19% eicospentenoic cid (EPA), while the cod liver oil contined 32% plmitic cid, 25% rchidonic cid, 8.6% EPA nd 4.3% DHA. The n6:n3 rtio ws highest in the olive oil > vegetle oil > cod liver oil > fish oil > coconut oil. Effect of dietry oil on postprndil serum endotoxin concentrtion To ssess the effect of dietry lipids on postprndil serum endotoxin concentrtions, pigs received porridge mel contining either 50 ml of sline, CO, VO or FO. The endotoxin concentrtion of the vrious oils used did not differ (dt not shown). Chnge in postprndil serum endotoxin concentrtion due to different mel tretments re presented in Figure 1A. The overll postprndil serum endotoxin concentrtions were significntly lower in the mels constituting sline or FO, with the men overll serum endotoxin concentrtion incresing two-fold over the sturted coconut oil mel tretment (P < 5, Figure 1B). However, mels mde up with VO were not different from the sline, CO or FO tretments (P < 5). Interestingly, the CO mel significntly elevted serum endotoxin concentrtions fter 2 hours versus the sline nd FO, nd these remined elevted t 3 nd 5 hour postprndil (P < 5, Figure 1A). Effect of exogenous porcine ile cid on ex vivo intestinl integrity Bile cids hve een shown to increse the intestinl permeility in cultured Cco-2 cell lines [25]. To rule out the effect tht exogenous ile cid my reduce intestinl integrity, freshly isolted pig ileum segments were used to mesure TER (Figure 2A) nd FITC-Dextrn permeility (Figure 2B). As these segments were exposed to incresing concentrtions of porcine ile ex vivo, no differences in intestinl integrity were oserved (P > 0.10, Figure 2). This might e due to the tolernce of intestinl tissues towrds ile cid ecuse of previous exposure in vivo contrry to cell cultures where the cells re not exposed to the ile cids previously. Tle 1 Dietry oil ftty cid composition used to mke the porridge (g/100 g FA) Oil Source Compound Coconut oil 1 Fish oil 2 Olive oil 3 Vegetle oil 4 Cod liver oil 5 8: : : : : : : : :2 n :3 n :5 n :6 n Other Sturted n n n6:n Source coconut oil ws (Spectrum Nturls, NY). 2 Source fish oil ws (Spring Vlley, UT). 3 Source olive oil ws (Hy-Vee, IA). 4 Source vegetle oil ws (Hy-Vee, IA). 5 Source cod liver oil ws (Spring Vlley, UT).

5 Mni et l. Nutrition & Metolism 2013, 10:6 Pge 5 of 9 Effect of dietry oil on ex vivo intestinl endotoxin trnsport The ex vivo mucosl to serosl ileum endotoxin trnsport ws ssessed using modified Ussing chmers nd FITC- LPS permeility ssy (Figure 3). Compred to the sline no oil control tretment, the endotoxin Ppp ws significntly lower in oth the FO nd CLO tretments (P < 5). As hypothesized, the higher sturted ft content of coconut oil significntly incresed the endotoxin Ppp compred to the sline, FO nd CLO (P < 5). However, mucosl tretment with VO nd OO did not differ from the sline or n-3 tretments (P > 5), ut still ttenuted endotoxin Ppp versus the coconut oil tretment (P < 5, Figure 3). Trnsepithelil resistnce ws not different due to ex vivo oil tretment (dt not shown). Effect of lipid rft modifiction on sturted ft induced endotoxin trnsport To test the hypothesis tht destiliztion of intestinl lipid rfts would decrese sturted ft induced endotoxin A Delt Endotoxin (EU/mL) B Serum Endotoxin (EU/ml) CO VO FO Sline Hours Sline VO FO CO Figure 1 Dietry oil lters postprndil serum endotoxin concentrtions in pigs fed single dietry oil-sed mel. A) Delt chnge in serum endotoxin concentrtions. B) Men postprndil serum endotoxin concentrtion. Different letters (,) represent significnt difference t P < 5. Tretments re porridge mel mde with either no oil (sline), fish oil (FO), vegetle oil (VO) nd coconut oil (CO). n = 6 pigs/tretment. Dt re mens ± S.E.M. A Trnsepithelil Resistnce ( Ω /cm 2 ) B Dextrn Permetion Coefficient (Ppp) Bile cid (mg/ml) Bile cid (mg/ml) Figure 2 The effect of incresing porcine ile cid concentrtion on ex vivo intestinl integrity nd permeility. A) Trnsepithelil resistnce (TER) nd B) FITC-Dextrn trnsport (4.4 kd). Freshly isolted ileum smples were mounted into modified Ussing chmers nd incuted with the indicted concentrtion of ile cid for 30 minutes nd then FITC-Dextrn ws dded to mucosl side. Permetion coefficient ws clculted y tking smples from chmers every minutes nd mesuring the mount of fluorescence. Different letters represent significnt difference t P < 5. n = 11 pigs. Dt re mens ± S.E.M. permeility, ileum smples were pretreted with the lipid rft modifier methyl-β-cyclodextrin (MβCD) nd coconut oil ex vivo. FITC-LPS endotoxin trnsport permeility ws then mesured (Figure 4A). As expected, the CO tretment significntly ugmented the ileum endotoxin Ppp compred to the sline control (P < 5). However, the endotoxin Ppp ws significntly reduced with the MβCD tretment compred to the sline control (1.54 vs. 7, P = 4). In the presence of MβCD nd CO, the ileum Ppp ws ttenuted three fold from the CO lone tretment (P < 5). Importntly, ileum integrity nd permeility s mesured y trnsepithelil resistnce ws not ltered y either short term coconut oil, MβCD, or the comintion compred to the sline control (P = 0.98, Figure 4B).

6 Mni et l. Nutrition & Metolism 2013, 10:6 Pge 6 of 9 Endotoxin Permetion Coefficient (Ppp) c Sline Coconut oil Cod liver oil Fish oil Vegetle oil Olive oil Figure 3 Ex vivo endotoxin trnsport in pig ileum tissue exposed to different dietry oil tretments. Freshly isolted ileum smples were mounted into modified Ussing chmers nd mixed with the indicted oils nd 20 mm ile cid for 120 minutes nd FITC-LPS trnsport ws mesured. Different letters represent significnt difference t P < 5. n = 11 per tretment. Dt re mens ± S.E.M. c c c A Endotoxin Permetion Coefficient (Ppp) B Trnsepithelil Resistnce (Ω.cm 2 ) Sline Sline + MβCD Sline Sline + MβCD c Coconut oil Coconut oil + MβCD Coconut oil Coconut oil + MβCD Figure 4 Lipid rft modifier methyl et cyclodextrin (MβCD) decreses ex vivo endotoxin trnsport. A) Endotoxin trnsport nd B) trnsepithelil resistnce ws mesured using Ussing chmers in ileum tissues treted with either control (wter), MβCD, coconut oil, or coconut oil plus MβCD. Tissue (n = 7 /trt) were pretreted with these tretments for 30 min efore FITC-LPS trnsport ws ssessed. Different letters represent significnt difference t P < 5. Dt re mens ± S.E.M. Discussion In Western diets, vegetle, cnol nd plm oils re common components of the diet nd to lesser extent, long chin n-3 PUFA (DHA nd EPA) oils from lgl or mrine sources [26,27]. In recent yers, the development of oesity, inflmmtion, therosclerosis nd other metolic diseses hs een linked to low grde endotoxemi ssocited with high dietry ft nd energy intke [3,14,28-30]. However, these studies nd others hve rised questions on whether this diet induced endotoxemi reflects chnges in energy nd ft content of the diet, intestinl permeility or diet induced chnges in gut microiot. In the current study, we used ex vivo nd in vivo methods to exmine intestinl permeility to endotoxin s it reltes to dietry oil composition. All pigs were cliniclly helthy nd rised on typicl commercil swine corn-soyen diets. We oserved no differences in intestinl integrity due to our ex vivo tretments. Even though some cell culture experiments hve een shown to indicte ile cids ffecting the intestinl permeility, we didn t oserve ny negtive impcts from ile cids on intestinl integrity nd permeility. Further, Lng et l. reported tht ile lone my not e sufficient to induce rrier function in the Ex-vivo experiments [31]. Importntly, we only exmined the cute ctions of mel or oil olus tretment nd did not conduct prolonged feeding tril in n ttempt to chnge the pig microiot popultions or the ftty cid profiles of tissues due to diet. We hypothesized tht dietry intke of oils rich in DHA nd EPA would ttenute intestinl endotoxin trnsport nd postprndil circulting endotoxin. We found tht dietry cod liver nd fish oils ttenuted serum endotoxin concentrtions compred to the coconut oil nd the

7 Mni et l. Nutrition & Metolism 2013, 10:6 Pge 7 of 9 endotoxin levels in these pigs were similr to the control group (Figure 1). To the est of our knowledge, there re no other studies tht hve shown this effect of DHA nd EPA on endotoxin trnsport nd lood endotoxemi. Interestingly, only one pper hs exmined the effects of dietry oil composition on endotoxin uptke nd relted inflmmtion [6]. However, contrry to our results, the report y Lugerette et l. [6] sttes tht rpe seed (cnol) nd sunflower oil, with its high unsturted ftty cid content, ugmented plsm endotoxemi y 50-75%. Cni et l. [1], lso oserved similr increse in endotoxemi in mice orlly dministered corn oil with or without LPS compred to wter lone. However, we oserved no chnge in serum postprndil endotoxin concentrtion or intestinl endotoxin trnsport due to dietry vegetle oil compred to the sline control (Figures 1 nd 3). This discrepncy of these prmeters etween the studies might e prtilly explined y the use of different niml models nd lso the nture of the experimentl design. Wheres Lugerette et l. [6] performed chronic feeding study using mice for eight weeks using oils with different ftty cid composition; we used n cute mel olus model with swine to exmine endotoxin permeility. Therefore, the mice ftty cid profiles would hve mimicked their diets nd this tissue enrichment my lso modify endotoxin permeility, signling nd cute inflmmtion. Further work is needed to explin the moleculr spects of these differences etween the two studies with regrd to cute versus chronic dietry ft signling in the gstrointestinl trct. Additionlly, we oserved significnt increse in postprndil endotoxemi fter porridge mel mixed with coconut oil (Figure 1). Agin, this contrdicts dt presented y Lugerette et l. [6] in which plm oil, high in sturted ftty cids, hd no effect on plsm endotoxin concentrtions in mice. However, these uthors did report n increse in plsm LPS inding protein nd rgued tht this protein is etter mrker of endotoxemi due to the short hlf-life of circulting endotoxin. One issue is tht LPS inding protein cn e up regulted y inflmmtion nd cute stress s well s oth grm positive nd negtive infections. As the mgnitude of LBP response goes down with multiple episodes of infection [32], this could e result of the gonistic effects of sturted ftty cid on pro-inflmmtory signling nd not circulting endotoxin. Grm negtive cteri, prticulrly those found in the distl ileum nd colon, might e one of the mjor sources for circulting endotoxin [33]. It hs een estimted tht single cell of Escherichi coli contins pproximtely 10 6 Lipid A or endotoxin molecules nd typicl humn intestinl trct could hror pproximtely one grm of endotoxin [33-35]. Interestingly, the cteril popultion in the intestine is not sttic. Multiple studies hve shown tht cteril composition shifts to either grm positive mjority or grm negtive mjority sed on the composition of the diet consumed [30,36,37]. A mjority of these studies show tht consuming high sturted ft diet for longer period results in higher grm negtive cteril popultions nd high fier diets results in grm positive cteril popultions [30,38]. Lugerette et l. [6], reffirmed this nd showed tht ftty cid composition of different dietry oils cn lter intestinl microiot popultions. Moreover, these uthors demonstrted tht feeding diet high in plm oil which is rich in SFAs significntly incresed the grm negtive cteri Escherichi coli groups, which cn e significnt source of endotoxin in the cecl content of mice compred to milk ft, rpe seed nd sunflower oil fed diets. During intestinl stress, ischemi, inflmmtion nd diseses, prcellulr trnsport occurs through the tight junction, s known s leky gut [39]. Alterntively, trnscellulr or intrcellulr trnsport cn occur, prticulrly in helthy individuls [40]. Trnscellulr endotoxin trnsported cross cell memrne hs een shown to occur vi TLR4 nd solule GPI nchored receptor CD14 in lipid rft medited mechnism [41,42]. Additionlly, chylomicron ssocited LPS trnsport hs lso een suggested to ply key role in intestinl LPS trnsport from the intestinl epithelil cell [11,43,44]. Importntly, we oserved no decrese in intestinl integrity which might enhnce prcellulr permeility s ssessed y trnsepithelil resistnce or FITC-dextrn permeility due to tretment or short term rft destiliztion (Figure 4B). These dt suggest tht under helthy intestinl epithelil conditions, endotoxin is most likely trnsported vi lipid rft medited endocytosis. The signling nd trnsport process for endotoxin is initited in specilized memrne micro domins clled lipid rfts [42]. Lipid rfts re memrne regions rich in cholesterol, glycolipids, sphingolipids nd sturted ftty cids, which result in rigid memrne structure compred to the djcent fluid regions [45]. In immune cells, endotoxin triggers the recruitment of TLR4 into the lipid rft, where it intercts with CD14 nd other ssocited proteins such s MD-2 resulting in n inflmmtory signling cscde [46,47]. Thus, the two mjor consequences of preventing endotoxin recognition y dissociting the lipid rft to ttenute TLR4 recruitment include reduced inflmmtory signling nd ttenuted endotoxin trnsport. We oserved tht if intestinl lipid rfts re dissocited ex vivo with MβCD, then endotoxin permeility is ttenuted in the ileum (Figure 4). Interestingly, sturted ft induced endotoxin permeility is lso significntly reduced. Stimultion of TLR4 receptor hs een shown to result in the endotoxin trnsport cross the intestinl epithelil cells [40]. TLR4 is not only implicted in the trnscellulr trnsport of LPS ut

8 Mni et l. Nutrition & Metolism 2013, 10:6 Pge 8 of 9 lso for live cteri [48]. Since sturted ftty cids nd n-3 PUFA cn reciproclly modulte TLR4 signling [49], the ftty cid composition of oil in the diet hs the potentil to increse or decrese endotoxin trnsport. Altogether, these dt suggest tht picl endotoxin trnsport in the intestines is rguly rft medited in helthy individuls. In vitro experiments show clerly tht n-3 PUFA disrupt TLR4 signlling nd the ctivtion of NFκB y LPS in murine monocytic cell line [50]. Moreover, DHA modultes TLR4 signling in vitro in RAW mcrophges nd 293 T cells [49], humn monocytes nd dendritic cells [51] nd dipose tissue. We hve previously shown in pigs tht dietry EPA nd DHA re effective mens of influencing the inflmmtory sttus nd pthwys influenced y TLR4 signling induced y LPS [52] nd in ltering intestinl function [24,53]. Therefore, one could postulte tht ntgonizing TLR4 recruitment to lipid rfts nd it s signling y DHA nd EPA, or stimulting these processes with sturted ftty cids, would lter endotoxin trnsport nd circulting postprndil endotoxin. Another mechnism through which endotoxin cn enter the circultion is through micelles. Since the endotoxin side chins re mde up of ftty cids, endotoxins cn e incorported into the micelles nd trnsported into the intestinl epithelil cell [54]. In intestinl epithelil cells, chylomicrons trnsport the sored lipids into vrious prts of the ody. High ft dministrtion hs een shown to proportiontely increse the endotoxin content of the chylomicron indicting tht high ft consumption indeed enhnces higher endotoxin trnsport into the intestinl epithelil cell nd incorportion into chylomicron [11,28]. Furthermore, even though the mechnism is not cler, high intke of ft hs een shown to cuse internliztion of tight junction proteins nd increse in the prcellulr permeility to mcro molecules including endotoxin [30]. Even though, this mode of endotoxin trnsport cnnot e ruled out, we speculte tht the rte of incorportion of ftty cids into micelles would not vry due to oil composition. Therefore, we propose tht the difference in intestinl endotoxin trnsport we oserved is primrily trnscellulr trnsport tht involves lipid rfts nd receptor medited endocytosis [42]. In conclusion, these dt suggest tht dietry oils cn differentilly lter intestinl endotoxin trnsport. Oils rich in DHA nd EPA ttenute endotoxin trnsport, while oils high in sturted ftty cids ugment endotoxin trnsport. Furthermore, intestinl endotoxin trnsport in helthy sujects my e regulted through lipid rft medited mechnism. Sturted ftty cids my e stilizing the lipid rfts llowing for greter endotoxin trnsport. Arevitions CON: Control; VO: Vegetle oil; CLO: Cod liver oil; CO: Coconut oil; OO: Olive oil; FITC: fluorescein isothiocynte; TLR4: Toll like receptor 4; TER: Trnsepithelil resistnce; LPS: Lipopolyscchride; FA: Ftty cids; PAMPs: Pttern ssocited moleculr ptterns; PUFA: Polyunsturted ftty cids; SFA: Sturted ftty cids; Ppp: Apprent permeility co-efficient; FAME: Ftty cid methyl esters; MβCD: Methyl-et-cyclo dextrin; rfc: Recominnt fctor C; RFU: Reltive fluorescent unit; DHA: Docoshexenoic cid; EPA: Eicospentenoic cid; IEC: Intestinl epithelil cell. Competing interests The uthors declre tht they hve no competing interests. Authors contriutions VM, JH nd NKG designed nd conducted reserch presented. NKG ws the principle investigtor nd oth NKG nd JH otined funding for this work. VM ws the grdute student supervised y NKG whom conducted most of the niml nd lortory work nd wrote the mnuscript. JH nd NKG supervised nd revised the mnuscript. All uthors red nd pproved the finl mnuscript. Acknowledgements This work ws prtilly supported y funds cquired from the Iow Pork Producers Assocition nd the Iow Stte University Nutrition nd Wellness Reserch Center. The uthors would like to thnk Ms. Mrth Jeffery nd Yong Zhou for their ssistnce with the niml nd lortory work presented in this project. Author detils 1 Deprtment of Animl Science, Iow Stte University, Ames, IA 50011, USA. 2 Interdeprtmentl Toxicology Grdute Progrm, Iow Stte University, Ames, IA 50011, USA. 3 Deprtment of Food Science nd Humn Nutrition, Iow Stte University, Ames, IA, USA. Received: 3 Octoer 2012 Accepted: 21 Decemer 2012 Pulished: 10 Jnury 2013 References 1. Cni PD, Amr J, Iglesis MA, Poggi M, Knuf C, Bstelic D, Neyrinck AM, Fv F, Tuohy KM, Cho C, et l: Metolic endotoxemi initites oesity nd insulin resistnce. 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