Comparison of the Effect of trans Fatty Acid Isomers on Apolipoprotein A1 and B Secretion in HepG2 Cells

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1 Journl of Oleo Science Copyright 217 by Jpn Oil Chemists Society doi : 1.565/jos.ess1774 NOTE Comprison of the Effect of trns Ftty Acid Isomers on Apolipoprotein A1 nd B Secretion in HepG2 Cells Koji Ngo 1, Kzuki Yoshing 2, Toshihru Ngi 2, Hoyo Mizobe 2, Akihiko Yoshid 2, Fumiki Beppu 3 nd Nohiro Gotoh 3* 1 Deprtment of Applied Biochemistry nd Food Science, Sg University, 1 Honjo-mchi, Sg-shi, Sg , JAPAN 2 Tsukishim Foods Industry Co. Ltd., , Higshi Ksi, Edogw-ku, Tokyo , JAPAN 3 Deprtment of Food Science nd Technology, Tokyo University of Mrine Science nd Technology, Konn, Minto-ku, Tokyo , JAPAN Abstrct: Intke of trns ftty cid (TFA) is believed to chnge the rtio of low-density lipoprotein (LDL) to high density lipoprotein (HDL) cholesterol in blood, which leds to crdiovsculr disese. In this study, thirteen types of TFA including monoene type TFA (trns-octdecenoic ftty cid isomers, t-18:1 isomers), diene type TFA (t9,t12-18:2), nd triene type TFA (t-18:3) were dded to cultured HepG2 cells to compre the mount of polipoprotein A1 nd B (those relting to levels of HDL nd LDL cholesterol in blood, respectively) being secreted. We found tht trns-5-18:1 incresed the secretion of polipoprotein B reltive to oleic cid (cis-9-18:1, control). Secretion of polipoprotein B ws lso incresed by t-18:3; however, the mount ws not significnt compred with tht observed in the control. The secretion mount of polipoprotein B tended to increse with the number of double bonds in TFA mong trns-9-18:1, t9,t12-18:2, nd t-18:3. The secretion mount of polipoprotein A1 fter TFA tretment ws lso mesured. No significnt difference ws detected mong t-18:1 groups; however, t-18:3 incresed the mount significntly compred to tht in the control. These results suggest tht the effect of TFA isomers on the rtio of LDL to HDL cholesterol in the blood follows mechnism different from tht in cultured cells. Key words: polipoprotein, HepG2 cell, isomer, trns ftty cid, secretion 1 INTRODUCTION Trns ftty cid TFA exists in food s source of tricylglycerol, phospholipids, etc. TFA is geometricl isomer of unsturted ftty cids possessing t lest one double bond in the trns configurtion. TFA is formed by prtil hydrogention of vegetle oils nd biohydrogention of unsturted ftty cids by enteric bcteri in the rumen of ruminnt nimls 1, 2. Therefore, foods using prtilly hydrogented oil PHO, ruminnt milk, nd ruminnt met contin TFA. Mny epidemiologicl studies suggest tht excessive intke of TFA cuses multiple dverse effects tht led to crdiovsculr disese CVD, including dyslipidemi, endothelil dysfunction, nd inflmmtion 3, 4. Prticulrly, if more thn 2 of dily energy intke is from TFA, the rtio of low density lipoprotein LDL to high density lipoprotein HDL cholesterol in blood nd the risk for CVD increses drsticlly 5, 6. Severl epidemiologicl studies hve reported tht nturlly occurring TFA in ruminnt nimls is not correlted with CVD 7 1. In contrst, industrilly produced TFA is reported to be correlted with CVD 6, 8. However, the difference between TFAs in PHO nd ruminnts is mbiguous. PHO, ruminnt milk, nd ruminnt met contin monoene, diene, nd triene types of TFA with the dominnt TFA isomer being the monoene type, prticulrly octdecenoic cid 18:1 11. Distribution Abbrevitions: BSA, bovine serum lbumin; CLA, conjugted linoleic cid; CVD, crdiovsculr disese; DMEM, Dulbecco s modified Egle s medium; FBS, fetl bovine serum; HDL, high density lipoprotein; LDL, low-density lipoprotein; PHO, prtilly hydrogented oil; TFA, trns ftty cid (TFA) * Correspondence to: Nohiro Gotoh, Deprtment of Food Science nd Technology, Tokyo University of Mrine Science nd Technology, Konn, Minto-ku, Tokyo , JAPAN E-mil: ngotoh@kiyodi.c.jp Accepted June 6, 217 (received for review Mrch 16, 217) Journl of Oleo Science ISSN print / ISSN online

2 K. Ngo, K. Yoshing, T. Ngi et l. of the 18:1 TFA positionl isomer is nerly identicl between PHO nd ruminnts, nd both sources comprise 13 types of isomers trns-4-18:1 12. The predominnt trns-18:1 isomer in the ruminnts is vccenic cid trns-11-18:1, while the predominnt isomer in PHO is elidic cid trns-9-18:1, 12, 13. However, ruminnt met lso contins nd PHO lso contins. The intke mount rtio of ruminnt TFA nd industril TFA re not drsticlly different 12. In the 199s, Mensink nd Ktn demonstrted tht mong 34 women nd 25 men, monoene type TFA incresed both totl nd LDL cholesterol nd decresed HDL cholesterol 14. In the study, the mounts of diene nd triene type TFAs were not nlyzed. In contrst, Wng et l. recently reported tht the intke mount of monoene type TFA is unrelted to crdiovsculr disese nd totl mortlity, but tht of diene type TFAs strongly reltes to mortlity 15. Though the diene type TFA is not dominnt, it is present in foods. Composition of the monoene type more thn 96 ws t-18:1; however, t-14:1, 15:1, 16:1, 17:1, nd 2:1 were lso present, diene type, nd triene type TFAs in fst food such s hmburgers, pizzs, nd French fries hve been reported. For exmple, hmburger contined 4.8,.51, nd.2 of monoene, diene, nd triene type TFAs, respectively 16. Vermunt et l. demonstrted in clinicl study tht trns isomers of α-linolenic cid, which is redily formed during the deodoriztion of refined vegetle oils, significntly incresed the rtio of LDL to HDL cholesterol in plsm compred with low trns oils 17. Mny epidemiologicl studies focus on the structure of TFA, s it is relted to the rtio of LDL to HDL cholesterol in blood or overll mortlity. However, the nture of this correltion is uncler nd some of the results re in conflict. This is becuse epidemiologicl studies prohibit the use of pure TFAs. Severl studies exmined the effect of trns-18:1 positionl isomers on the rtio of LDL to HDL cholesterol in the plsm of hmsters 18, 19. Prticulrly, isomers the representtive TFA isomer in ruminnts nd the representtive TFA isomer in PHO were compred. Interestingly, the studies reveled tht did not increse the rtio of LDL to HDL cholesterol in plsm compred with tht in the control group, but rther decresed it. The TFA in PHO is believed to increse the rtio of LDL to HDL cholesterol in blood. An in vitro experiment with HepG2 cells ws conducted to compre the effect of TFA isomers on the secretion of polipoprotein B, the primry polipoprotein of LDL 2. The results indicted tht the secretion of polipoprotein B by the ddition of t8-, t9-, t11-, t12-, nd ws not significntly incresed compred with tht obtined post oleic cid cis-9-18:1, c9-18:1 tretment. Interestingly, the cells to which ws dded showed decresed secretion of polipoprotein B compred with cells treted with c9-18:1. Thus fr, no study hs reveled the specific TFA isomer tht increses the rtio of LDL to HDL cholesterol in blood. Therefore, we imed to mesure the chnges in secretion of polipoproteins A1, the min polipoprotein of HDL, nd B from HepG2 cells by dding the 13 types of t-18:1 isomer, diene type TFA, nd triene type TFA in order to elucidte the isomers responsible for CVD. 2 EXPERIMENTAL 2.1 Mterils c9-18:1 nd trns-9,trns-12-linoleic cid t9,t12-18:2 were obtined from Funkoshi Co., Ltd Tokyo, Jpn ; the purity of the ftty cids ws greter thn 98. The thirteen kinds of t-18:1 isomers were produced in-house 21. Trns α-linolenic cid t-18:3 ws lso produced in-house by the isomeriztion of purified α-linolenic cid with p-toluenesulfinte. The t-18:3 ws mixture of severl kinds of triene type TFA isomers. Humn heptom HepG2 cells nd fetl bovine serum FBS were obtined from Dinippon Phrmceuticl Co. Ltd. Osk, Jpn. Penicillin streptomycin ws purchsed from ICN Biomedicls, Inc. Auror, OH, USA. Dulbecco s modified Egle s medium DMEM nd ftty cidfree bovine serum lbumin BSA were purchsed from Sigm Chemicl St. Louis, MO, USA. The structures of TFA isomers used in this study re illustrted in Fig Cell culture HepG2 cells were mintined in DMEM contining 1 units/ml penicillin nd 1 µg/ml streptomycin; the cells were supplemented with 1 FBS t 37 in humidified tmosphere of 5 CO 2. At pproximtely 7 8 confluence, the medium ws replced with serum-free medium contining 1 BSA ftty cid-free for 24 h. To evlute the effect of trns ftty cid isomers on polipoprotein secretion, HepG2 cells were further incubted with either the control medium 1 BSA-DMEM or experimentl medium 1 BSA-DMEM with ftty cids 15 μm for 24 h, ccording to the previous nlyticl condition we reported 22. The ftty cid BSA complex ws prepred s described by Vn Hrken et l. 23 The cellulr protein concentrtion ws determined using the bicinchoninic cid method 24. To mesure cytotoxicity, wter-soluble disulfonted tetrzolium slt synthetic ctivity 25 ws determined using Cell Counting Kit-8 Dojindo Lortories, Kummoto, Jpn. 2.3 Mesurement of polipoprotein A1 nd B secretion from cells Apolipoprotein A1 nd B levels in the culture medium were quntitted using the Totl Humn Apolipoprotein A1 ELISA Assy Kit AlerCHEK, Inc., Springvle, ME, U.S.A. nd Totl Humn Apolipoprotein B ELISA Assy Kit AlerCHEK, Inc., Springvle, ME, U.S.A

3 Comprison of Apo A1 nd B secretion mong TFA isomers Monoene Diene t9,t12-18:2 Triene c9,c12,t15-18:3 (One of representtive structures of triene type TFA used in this experiment) Fig. 1 Structure of TFA isomers used in this study. 2.4 Sttisticl nlysis All vlues re expressed s mens SE. The dt were nlyzed using one-wy ANOVA test, nd ll differences were evluted using the Tukey Krmer post-hoc test Kleid Grph, Synergy Softwre, Reding, PA. Differences were considered sttisticlly significnt t p.5. 3 RESULTS 3.1 Viility of HepG2 cells The effects of TFAs on cell viility in HepG2 cells were investigted nd the results re shown in Fig. 2. c9-18:1 ws employed s control nd no cytotoxicity ws indicted by ny ftty cids. Cellulr protein concentrtions were not significntly different mong groups dt not shown. 3.2 Apolipoprotein A1 nd B secretion by the ddition of trns-18:1 isomers Thirteen types of trns-18:1 isomers were dded to HepG2 cell cultures nd the secretion mounts of polipoprotein A1 nd B were mesured. c9-18:1 ws employed s control Fig. 3. There ws no significnt difference in polipoprotein A secretion Fig. 3 A. In contrst, significntly incresed polipoprotein B secretion Fig. 3 B. nd did not significntly chnge polipoprotein B secretion compred with tht observed in the control. 3.3 Apolipoprotein A1 nd B secretion by the ddition of monoene, diene, nd triene type TFAs The secretion of polipoprotein A1 nd B from HepG2 cell culture by the ddition of two kinds of monoene type nd, diene type t9,t12-18:2, nd triene type t-18:3 TFAs were compred. c9-18:1 ws employed s control Fig. 4. The t-18:3 significntly incresed the secretion mount of polipoprotein A1 compred with tht observed in the control Fig. 4 A. significntly incresed the secretion of polipoprotein B compred with control Fig. 3 B. t-18:3 lso incresed secretion of polipoprotein B; however, the difference ws not significnt. The secretion of polipoprotein B tended to increse with the number of double bonds in TFA mong, t9,t12-18:2, nd t-18:3. 4 DISCUSSION The word Trns Ftty Acid is generic nme for ftty cids hving trns type double bonds in their structure. The definition of TFA by Codex Alimentrius defines it differently s Trns ftty cids re ll the geometricl isomers of monounsturted nd polyunsturted ftty cids hving non-conjugted, interrupted by t lest one methylene group, crbon-crbon double bonds in the trns configurtion 26. This definition removes ftty cids hving conjugted double bonds in the structure, such s conju- 1177

4 K. Ngo, K. Yoshing, T. Ngi et l. WST8 ssy (% of control) b b (A) (B) t9,12t-18:2 t-18:3 Fig. 2 Effects of TFA isomers on cell viility in HepG2 cells. A Among t-18:1 isomers, B Among monoene, diene, nd triene types TFAs Apoprotein A1 secretion (μg/mg protein) (A) Apoprotein B secretion (μg/mg protein) d c c c c c c (B) bc c c Fig. 3 Comprison on the secretion of polipoprotein A1 nd B from HepG2 cell mong thirteen kinds of t-18:1 isomers t4-18:1. A Apolipoprotein A1 secretion, B Apolipoprotein B secretion. gted linoleic cid CLA, owing to one isomer of CLA, nmely trns-1,cis-12-linoleic cid t1,c12-18:2, which functions to improve ft metolism in n niml body 27, 28. Incidentlly, the other isomer cis-9, trns-11-linoleic cid c9,t11-18:2, is not reported to hve strong helth benefits. This revels tht the position of the double bond in the unsturted ftty cid is criticl for its relevnce to the helth of n niml body. Totl TFA consists of mny different TFA isomers. For exmple, the TFA contined in ruminnts nd PHO minly consists of three TFA fmilies such s monoene, diene, nd triene types. More thn 8 of TFA in ruminnts nd PHO is monoene of type trns-18:1 11. There re thirteen potentil positionl isomers of trns-18:1. Mny reserchers hve concluded tht TFA is bd for helth ; however, no resercher hs reveled which TFA isomer is cuslly detrimentl. From the drmtic effect of the position of the double bond in CLA, it is cler tht not ll TFA is hrmful. Recently, Vhmni et l. exmined the effect of trns-18:1 isomers, nmely t6-, t9-, t1-, t11-, t13-, t14-, nd, 1178

5 Comprison of Apo A1 nd B secretion mong TFA isomers Apoprotein A1 secretion (μg/mg protein) (A) b Apoprotein B secretion (μg/mg protein) 16. c (B) bc t9,12t-18:2 t-18:3 t9,12t-18:2 t-18:3 Fig. 4 Comprison on the secretion of polipoprotein A1 nd B from HepG2 cell mong two kinds of monoene type nd, diene type t9, t12-18:2, nd triene type t-18:3 TFAs. A Apolipoprotein A1 secretion, B Apolipoprotein B secretion. on tricylglycerol nd cholesterol synthesis using HepG2 cells 29. They concluded tht t6-, t9-, nd induced lipogenic/cholesterolgenic gene expression. This is vlule study becuse the function of respective isomers, hving different double bond positions but the sme number of crbons nd double bonds, ws crefully exmined. However, the intke of TFA cuses potentil risk for coronry disese becuse TFA hs been chrcterized to increse the rtio of LDL to HDL rtio in blood 3. This study did not exmine the detiled effect on polipoprotein A1 nd B expression, relted to HDL cholesterol nd LDL cholesterol, respectively. In the current study, we exmined the effect of 13 types of trns-18:1 isomers on the secretion of polipoprotein A1 nd B from HepG2 cells. Additionlly, TFA isomers hving two nd three double bonds were lso evluted using the sme system. None of the ftty cids used in the studies ffected the viility of HepG2 cells Fig. 2. As shown in Fig. 3, significntly incresed polipoprotein B secretion. Storkson et l. lso compred the polipoprotein B secretion from HepG2 cells by the ddition of 21. The ddition of resulted in the lowest level of secretion while t13-18:1 resulted in the highest. Our results concur with their findings Fig. 3 B. The secretion mount of polipoprotein A1 ws not significntly different mong groups; however, the secretion mount of polipoprotein B significntly incresed by the ddition of. Therefore, the lso would increse the rtio of polipoprotein B to A1. The chnge in mrna expression for polipoprotein B ws mesured dt not shown. However, the mrna did not chnge significntly compred with tht of the control. Dt compring the effect of the number of double bonds on the secretion of polipoprotein A1 nd B re shown in Fig. 4. The result confirms the effect of on the secretion of polipoprotein B from HepG2 cells. Interestingly, t-18:3 incresed both polipoprotein A1 nd B compred to the control. t-18:3 ws purified using frctiontion HPLC, ensuring removl of the α-linolenic cid precursor. mrna expression for polipoprotein A1 nd B were mesured fter diene nd triene TFA tretment dt not shown. We found tht t-18:3 significntly incresed the mrna level of polipoprotein A1 compred with tht in the control; however, the mrna expression of polipoprotein B ws not significntly different. The experiment using respective t-18:3 isomers should be conducted to confirm this result. The secretion of polipoprotein B tended to increse with the number of double bonds in TFA mong monoene, diene, nd triene types of TFA. As mentioned, severl epidemiologicl studies reported tht TFA hving two or more double bonds is strongly correlted to the rtio of LDL to HDL cholesterol in blood nd on mortlity 15. Our results support these findings. Studies imed t reveling the mechnism of TFA ction on cholesterol expression re wrrnted. 5 CONCLUSION In this study, both nd t-18:3 incresed the secretion mount of polipoprotein B compred with tht in the control. The secretion of polipoprotein B tended to increse with the number of double bonds in TFA mong t9-18:1, t9,t12-18:2, nd t-18:3. These results suggest tht the effect of TFA on the rtio of LDL to HDL cholesterol vries by the isomer. As these results were obtined from cell culture, niml or humn experiments re required to elu- 1179

6 K. Ngo, K. Yoshing, T. Ngi et l. cidte the effect of individul TFA isomers. Acknowledgement This work ws supported by Science nd Technology Reserch Promotion Progrm for Agriculture, Forestry, Fisheries nd Food Industry, Jpn grnt number 279A. Author contribution K. N., F. B., nd N. G. designed reserch; K. N. conducted reserch; K. Y., T. N., H. M., nd A. Y. synthesized TFA isomers. K. N. nd N. G. wrote pper. K. N. nd N. G. hd primry responsibility for finl content. All uthors red nd pproved the finl mnuscript. Conflict of interest The uthors declre no conflicts of interest. References 1 Hunter, J.E. Dietry trns ftty cids: Review of recent humn studies nd food industry responses. Lipids 41, McKin, N.; Shingfield, K.J.; Wllce, R.J. Metolism of conjugted linoleic cids nd 18:1 ftty cids by ruminl bcteri: products nd mechnisms. Microbiology 156, Teegl, S.M.; Willett, W.C.; Mozffrin, D. Consumption nd helth effects of trns ftty cids: review. J. AOAC Int. 92, Mozffrin, D. Trns ftty cids - effects on systemic inflmmtion nd endothelil function. Atheroscler. Suppl. 7, Zock, P.L.; Ktn, M.B. Trns ftty cids, lipoproteins, nd coronry risk. Cn. J. Physiol. Phrmcol. 75, Mozffrin, D.; Ktn, M.B.; Ascherio, A.; Stmpfer, M.J.; Willett, W.C. Trns ftty cids nd crdiovsculr disese. N. Engl. J. Med. 354, Pietinen, P.; Ascherio, A.; Korhonen, P.; Hrtmn, A.M.; Willett, W.C.; Albnes, D.; Virtmo, J. Intke of ftty cids nd risk of coronry hert disese in cohort of Finnish men. The Alph-Tocopherol, Bet-Crotene Cncer Prevention Study. Am. J. Epidemiol. 145, Willett, W.C.; Stmpfer, M.J.; Mnson, J.E.; Colditz, G.A.; Speizer, F.E.; Rosner, B.A.; Smpson, L.A.; Hennekens, C.H. Intke of trns ftty cids nd risk of coronry hert disese mong women. Lncet 341, Ascherio, A.; Hennekens, C.H.; Buring, J.E.; Mster, C.; Stmpfer, M.J.; Willett, W.C. Trns-ftty cids intke nd risk of myocrdil infrction. Circultion 89, Jkobsen, M.U.; Overvd, K.; Dyerberg, J.; Heitmnn, B.L. Intke of ruminnt trns ftty cids nd risk of coronry hert disese. Int. J. Epidemiol. 37, Yoshing, K.; Asnum, M.; Mizobe, H.; Kojim, K.; Ngi, T.; Beppu, F.; Gotoh, N. Chrcteriztion of cisnd trns-octdecenoic cid positionl isomers in edible ft nd oil using gs chromtogrphy-flme ioniztion detector equipped with high polr ionic liquid cpillry column. Food Chem. 16, Wolff, R.L.; Combe, N.A.; Destillts, F.D.; Boué, C.; Precht, D.; Molkentin, J.; Entressngles, B. Follow-up the Δ4 to Δ16 trns-18:1 isomers profile nd content in French processed foods contining prtilly hydrogented vegetle oils during the period Anlyticl nd nutritionl implictions. Lipids 35, Precht, D.; Molkentin, J.; Destillts, F.; Wolff, R.L. Comprtive studies on individul isomeric 18:1 cids in cow, got, nd ewe milk fts by low-temperture high-resolution cpillry gs-liquid chromtogrphy. Lipids 36, Mensink, R.P.; Ktn, M.B. Effect of dietry trns ftty cids on high-density nd low density lipoprotein cholesterol levels in helthy subjects. N. Engl. J. Med. 323, Wng, Q.; Immur, F.; Lemitre, R.N.; Rimm, E.B.; Wng, M.; King, I.B.; Song, X.; Siscovick, D.; Mozffrin, D. Plsm phospholipid trns-ftty cids levels, crdiovsculr diseses, nd totl mortlity: The crdiovsculr helth study. J. Am. Hert Assoc. 3, e Tyburczy, C.; Delmonte, P.; Frdin-Ki, A.R.; Mossob, M.M.; Krmer, J.K.; Rder, J.I. Profile of trns ftty cids FAs including trns polyunsturted FAs in representtive fst food smples. J. Agric. Food Chem. 6, Vermunt, S.H.F.; Beufrère, B.; Riemersm, R.A.; Sébédio J.L.; Chrdigny, J.M.; Mensink, R.P.; Trns- LinE investigtors. Dietry trns α-linolenic cid from deodorised rpeseed oil nd plsm lipids nd lipoproteins in helthy men: the TrnsLinE Study. Brit. J. Nutr. 85, Meijer, G.W.; vn Tol, A.; vn Berkel, T.J.; Weststrte, J.A. Effect of dietry elidic versus vccenic cid on blood nd liver lipids in the hmster. Atherosclerosis 157, Tyburczy, C.; Mjor, C.; Lock, A.L.; Destillts, F.; Lw- 118

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