British Journal of Nutrition

Transcription

1 British Journl of Nutrition (211), 15, q The Authors 21 doi:1.117/s Adrenoortiotrophi hormone-stimulted ortisol relese y the hed kidney inter-renl tissue from se rem (Sprus urt) fed with linseed oil nd soyen oil Rhid Gng 1 *, J. G. Bell 2, D. Montero 1, E. Atlh 1, Y. Vrskou 1, L. Tort 3, A. Fernndez 4 nd M. S. Izquierdo 1 1 Grupo de Investigión en Auiultur, Universidd de Ls Plms de Grn Cnri & Instituto Cnrio de Cienis Mrins, Trnsmontñ s/n, Arus, Ls Plms, Cnry Islnds, Spin 2 Institute of Aquulture, University of Stirling, Stirling FK9 4LA, Sotlnd, UK 3 Deprtmento of Cell Biology, Physiology nd Immunology, Universitt Autonóm de Brelon, Bellterr 8193, Spin 4 Biomr Ieri/ProAqu Nutrition, S.A., A-62, Km 99, ES-3421 Dueñs, Spin British Journl of Nutrition (Reeived 1 Ferury 21 Revised 23 June 21 Aepted 27 July 21 First pulished online 14 Septemer 21) Astrt The mode of tion of highly unsturted ftty ids (HUFA) in regulting gilthed se rem (Sprus urt) hed kidney (HK) ortisol prodution ws studied through in vitro trils using dynmi superfusion system. Fish were previously fed with different diets ontining severl inlusion levels of linseed oil (LO) or soyen oil (SO) for 26 weeks. Five diets were tested; nhovy oil ws the only lipid soure for the ontrol diet (fish oil, FO) nd two different sustitution levels (7 nd 1 %) were tested using either LO or SO (7LO, 7SO, 1LO nd 1SO). Ftty id ompositions of the HK refleted the dietry input, thus EPA, DHA, rhidoni id nd n-3 HUFA were signifintly (P, 5) redued in fish fed vegetle oils ompred with fish fed the FO diet. Feeding 7 or 1 % LO inresed signifintly (P, 5) ortisol relese in HK fter stimultion with drenoortiotrophi hormone (ACTH), while feeding SO hd no effet on this response. Cortisol stimultion ftor (SF) ws inresed in fish fed the 7LO nd 1LO diets ompred with fish fed the ontrol diet. Moreover, eiosnoid inhiition y inuting the HK tissue with indomethin (INDO) s ylo-oxygense (COX) inhiitor, or nordihydroguireti id (NDGA) s lipoxygense (LOX) inhiitor, signifintly redued (P, 5) the ortisol relese fter ACTH stimultion in the 7LO nd 1LO diets. Cortisol SF ws redued in the FO, 7LO nd 1LO diets when inuting the HK with INDO or NDGA, while it ws inresed in the 7SO diet. The present results indite tht hnging the ftty id profile of gilthed se rem HK y inluding LO nd/or SO in the fish diet ffeted the in vitro ortisol relese, nd this effet is prtly medited y COX nd/or LOX metolites. Key words: Gilthed se rem: Highly unsturted ftty ids: Linseed oil: Soyen oil: Indomethin: Nordihydroguireti id: Cortisol: Hed kidney: Superfusion Mrine teleosts hve requirements for the essentil longhin highly unsturted ftty ids (HUFA) of the n-3 series, DHA (22 : 6n-3) nd EPA (2 : 5n-3) (1 4). Other studies hve lso pointed out the importne of the long-hin n-6 HUFA, rhidoni id (ARA, 2 : 4n-6), s essentil for mrine fish (5,6). These three ftty ids, s omponents of phospholipids, onstitute ritil prt of the ell memrne of most tissues nd re responsile for mintining n dequte physiologil response of the ells (7,8), eing prtiulrly importnt to promote stress resistne nd defene ginst pthogeni hllenge (9). Stress in fish is monitored y levels of plsm ortisol, generl inditor of stressful onditions in vertertes (1), nd its relese into the irultion is ontrolled y the hypothlmus pituitry inter-renl xis. The ortisol relese is preeded y the stimultion of the inter-renl tissue y seretion of pituitry hormones, in prtiulr drenoortiotrophi hormone (ACTH) (11). Although some other hormones hve een shown to stimulte ortisol Arevitions: ACTH, drenoortiotrophi hormone; ARA, rhidoni id; COX, ylo-oxygense; EFA, essentil ftty ids; FO, fish oil; HK, hed kidney; HUFA, highly unsturted ftty ids; INDO, indomethin; LA, linolei id; LNA, -linoleni id; LO, linseed oil; LOX, lipoxygense; NDGA, nordihydroguireti id; OA, olei id; SF, stimultion ftor; SO, soyen oil; VO, vegetle oils. * Corresponding uthor: R. Gng, fx þ , emil rhid.gng@gmil.om Downloded from IP ddress: , on 1 De 218 t 21:58:56, sujet to the Cmridge Core terms of use, ville t

2 Cortisol relese y se rem 239 British Journl of Nutrition relese from inter-renl tissue, ACTH is the dominnt seretgogue (12 14). The understnding of the pthwys nd intrellulr messengers tht regulte ortisol synthesis in fish is very limited. The ortisol synthesis y ACTH hs een found to e dependent on yli AMP s n intrellulr seond messenger in the oho slmon (Onorhynhus kisuth) (15). In mmmls, the min pthwy leding to ortiosteroid synthesis y ACTH stimultion involves signlling sde integrting G-proteins, denylyl ylse, yli AMP nd protein kinse A (16). Other pthwys, involving protein kinse C vi stimultion y ngiotensin II or etylholine, oth known seretgogues of ortisol in fish (17), shre role in the regultion of ortiosteroid synthesis (18). Moreover, protein kinse A hs een suggested to e ruil stimultory omponent in the ACTH-medited signlling pthwy in fish drenl steroidogenesis (19), wheres protein kinse C hs een shown to hve n inhiitory role in the ute ortisol response in fish drenoortil ells (19). The mehnism of how lipids nd minly HUFA ould modulte ortisol relese in vertertes is not well known. In previous study, we showed tht free HUFA modulte ortisol seretion in se rem hed kidney (HK) mintined in superfusion (2). ARA, EPA nd DHA stimulted ortisol relese y inter-renl tissue, while dihomo-g-linoleni id (2 : 3n-6) inhiited relese. Besides, it ws demonstrted tht ACTH-indued ortisol relese is prtly medited y ylo-oxygense (COX) metolites (2). Moreover, PGE 2 derived from ARA hs een shown to modulte the sensitivity of the hypothlmus pituitry drenl xis, whih is responsile for the relese of ortisol in response to stress in mmmls (21,22) nd possily lso in the homologous hypothlmus pituitry inter-renl xis in fish (23). Fish oil (FO) hs een the min soure of HUFA in qufeeds, ut in reent yers, due to the onerns out FO sustinility nd ost, lterntive oil soures re frequently inluded in fish feeds. Prtil replement of FO y vegetle oils (VO) does not ffet fish growth or feed utilistion in severl speies (24 3). Nevertheless, high sustitution levels y VO hve een shown to lter fish resistne to stressful onditions y inresing their ortisol levels (31). However, little is known out the physiologil mehnisms involved in the regultion of stress y ftty ids. Sine vegetle lipids lk HUFA, its dietry inlusion my modify tissue omposition, ltering ell memrne fluidity, reeptor-medited ortisol response nd finlly leding to redued funtionlity of the trget orgn. In ddition, vegetle lipids n redue the vilility of eiosnoid preursors ARA, EPA, dihomo-g-linoleni id nd DHA leding to dysfuntionl eiosnoids signlling (32,33) whih my ffet fish response to stress. The im of the present study ws to lrify the effet of repling FO y linseed oil (LO) nd soyen oil (SO), in the gilthed se rem (Sprus urt) stress response to ACTH stimultion. For this purpose, the HK tissue ws mintined in superfusion system nd inuted with inhiitors of eiosnoid. Aordingly, five diets with different levels of FO sustitution (, 7 nd 1), either with LO or with SO, were fed to gilthed se rem juveniles. Mterils nd methods Animls The study ws rried out t Instituto Cnrio de Cienis Mrins (Cnry Islnds, Spin), nd fish were purhsed from lol fish frm (ADSA, Cnry Islnds, Spin). A totl of 75 gilthed se rem (S. urt), with n verge initil ody weight of 45 g, were rndomly distriuted in 5 litre polyethylene irulr tnks (forty-five fish/tnk, three tnks per diet). Tnks were supplied with ontinuous sewter (36 ) flow nd ertion. Fish were rered under nturl photoperiod onditions of pproximtely 12 h drk 12 h light. Wter temperture nd dissolved O 2 onentrtion during the experimentl period rnged from 2 to 24 28C nd from 5 4 to 8 7 prts per million, respetively. Diets Fish were fed the experiment diets until pprent stition (three times/d, 6 d/week), until they rehed ommeril mrket size fter 26 weeks. Five isoenergeti nd isonitrogenous experimentl diets were formulted to provide lipid ontent of 16 %. Anhovy oil ws the only dded lipid soure in the FO diet (1 % FO). All the other diets ontined VO to sustitute either 7 % of the nhovy oil y LO in 7LO diet, y SO in 7SO diet or 1 % of the nhovy oil y LO nd SO in 1LO nd 1SO diets, respetively. FO ws inluded in 7LO nd 7SO diets t level high enough to meet the essentil ftty id (EFA) requirements of this speies (34). Biohemil nlysis Lipid from the experimentl diets nd fish HK ws extrted with hloroform methnol (2:1, v/v) s desried previously (35). The ftty id methyl esters were otined y trnsesterifition with 1 % H 2 SO 4 in methnol nd purified y sorption hromtogrphy on NH 2 Sep-pk rtridges (Wters Corportion, SA, Milford, MA, USA) nd were seprted nd quntified y GLC (36). Preprtion nd stimultion of hed kidney tissue At the end of the feeding period, two fish were rndomly tken from eh tnk in less thn 1 min, immeditely nesthetised with 2-phenoxyethnol (1:1, v/v), nd lood ws olleted with hypodermi syringe from the Downloded from IP ddress: , on 1 De 218 t 21:58:56, sujet to the Cmridge Core terms of use, ville t

3 24 R. Gng et l. British Journl of Nutrition udl vein to minimise hemorrhge during the extrtion of the tissue. HK tissue ws removed from two fish in eh superfusion tril, nd in ll series of experiments eh tretment ws ssyed in qudruplites (using two fish per repli, n 8), nd ut into very smll frgments in HEPES Ringer medium, whih ws used s the superfusion medium. Afterwrds, HK homogentes were pooled nd distriuted in eight superfusion hmers (volume: 2 ml) in order to otin homogeneous liquot in eh of them. Tissues were superfused with HEPES (ph 7 4) Ringer s solution ontining 171 mm-ncl, 2 mm-kcl, 2mM-CCl 2.H 2 O, 25 % (w/v) gluose nd 3 % (w/v) ovine serum lumin (37). The system ws temperture ontrolled t 188C, nd the superfusion medium ws pumped through the hmer t rte of 75 ml/min y Msterplex L/S R multihnnel peristlti pump (Cole Prmer Intrument Compny, Vernon Hills, IL, USA). Trils were strted fter 3 h of superfusion when ortisol rehed stle seline level (38,39), in order to void devitions due to the different dispersion of interrenl ells in the perfusion preprtion nd the individul differenes or the pre-stress level of eh fish. After the stilistion period of 3 h, tissues were stimulted with ACTH t onentrtion of 5 nm-hacth 1-39 (Sigm, St Louis, MO, USA) for 2 min. Susequently, perfusion ws mintined for nother 17 min, with frtion smples eing olleted every 2 min during this period. In seond series of experiments to lrify the tion mehnisms of HUFA nd the implition of eiosnoids in this proess, the tissues were inuted with COX inhiitor (indomethin, INDO) or lipoxygense (LOX) inhiitor (nordihydroguireti id, NDGA) for 2 min t onentrtion of 25 mm diluted in superfusion medium, nd susequently the tissues were stimulted with ACTH s explined efore nd mintined for nother 17 min, with smples eing olleted every 2 min. Cortisol SF ws lulted y ompring mximum ortisol relesed long the experiment fter ACTH stimultion with seline ortisol relesed (mximum relese 2 seline relese) 1/ (seline relese) (37). Cortisol mesurements Cortisol onentrtion in the perfused fluid ws determined y RIA (37). The ntiody, Biolink, S.L. (Cost Mes, CA, USA), ws used in finl dilution of 1:6. This ntiody ross-retivity is 1 % with ortisol, 11 4 % with 21-deoxyortiosterone, 8 9 % with 11-deoxyortisol nd 1 6 % with 17-hydroxyprogesterone. The rdiotivity ws quntified using liquid sintilltion ounter. Cortisol levels re expressed s ng/g HK per h. Thiorituri id-retive sustne nlysis. Lipid peroxidtion produts were determined only in diets s thiorituri id-retive sustnes nd were nlysed ording to the method desried y Shhidi & Hong (4). Sttistil nlysis Signifine of differene (P, 5) etween dietry tretments ws determined y one-wy ANOVA followed y Dunn s multiple omprison test (41), n 8. Anlyses were performed using SPSS softwre (SPSS for windows 13; SPSS, In., Chigo, IL, USA). Results Effet of vegetle oil inlusion on ftty id ompositions Thiorituri id-retive sustnes showed no signifint differene etween the diets, rnging etween 8 56 nd 3 85 mmol of mlondildehyde/kg of wet diet (P, 5), inditing no oxidtion dmge in the different diets. Ftty id omposition of the diets refleted the inlusion of plnt oils (Tle 1). Totl SFA rnged from Tle 1. Ftty id profile of the experimentl diets (% totl identified ftty ids) Ftty ids FO 7LO 1LO 7SO 1SO 14 : : : ISO : : 1n : 1n : 2n : : 3n : 3n : 3n : 4n : : 1n : 1n : 1n : 2n : 2n : 3n : 3n : 3n : 4n : : 1n : 1n : 2n : 2n : 3n : 4n : 4n : 5n : 1n : 1n : 4n : 5n : 6n Sturtes Monoeni n n n n-3 HUFA n-3:n FO, fish oil; LO, linseed oil; SO, soyen oil; HUFA, highly unsturted ftty ids. Downloded from IP ddress: , on 1 De 218 t 21:58:56, sujet to the Cmridge Core terms of use, ville t

4 Cortisol relese y se rem 241 British Journl of Nutrition % in the 1LO diet to % in the FO diet. Totl monoenois ftty ids (minly olei id (18 : 1n-9, OA)) rnged from % in the 1LO diet to 29 9 % in the 1SO diet. -Linoleni id (18 : 3n-3, LNA) inresed with inresing inlusion of LO, from 48 % in the FO diet to % in the 1LO diet. Similrly, linolei id (18 : 2n-6, LA) inresed, prtiulrly with the inresing inlusion of SO, from 4 2 % in the FO diet to % in the 1SO diet. EPA nd DHA were redued y the inresed ontent of plnt oils in the diets rnging from 1 5 % in the FO diet to 2 6 % in the 1SO diet nd from 7 82 % in the FO diet to 2 91 % in the 1SO diet, respetively. ARA ws lso deresed y the plnt oil inlusion, rnging from 1 11 % in the FO diet to 24 % in the 1LO diet. Fish HK ftty id profile of totl lipids refleted the dietry lipid omposition (Fig. 1). n-3 ftty ids were signifintly inresed in fish fed the LO diet, while n-6 ontent ws signifintly inresed in fish fed the SO diet. n-9 ws lso inresed in fish fed the VO diet ompred with fish fed the ontrol diet, due minly to OA inrese. However, the ontent in HUFA (EPA, ARA nd DHA), minly n-3 series, ws signifintly (P, 5) redued in fish fed the two VO, ompred with fish fed the ontrol diet, whih ws signifintly redued. Then 45 % redution ws found in DHA from the HK of fish fed the 7LO diet, 55 % in fish fed the 1LO diet, 58 % in fish fed the 7SO diet nd 61 % in fish fed the 1SO diet ompred with FO-fed fish. A 64 % redution ws found in EPA from the HK of fish fed the 7LO diet, 76 % in fish fed the 1LO diet, 73 % in fish fed the 7SO diet nd 8 % in fish fed the 1SO diet ompred with FO-fed fish. Regrding ARA, 16 % redution ws found in fish fed the 7LO diet, nd 47 % redution ws found in fish fed the 1LO diet, 88 % in fish fed the 7SO diet nd 86 % in fish fed the 1SO diet. As onsequene, the ARA:EPA rtio ws lso mrkedly ffeted y the Conentrtion (g ftty id/1 g totl ftty ids) e d e d 18: 1n-9 18: 2n-6 18: 3n-3 2 : 4n-6 Ftty ids d 2: 5n-3 22 : 6n-3 Fig. 1. Conentrtion (% totl ftty ids) of 18 : 2n-6, 18 : 3n-3, 18 : 1n-9, 2 : 4n-6, 2 : 5n-3 nd 22 : 6n-3 in the hed kidney of se rem (Sprus urt) fed either fish oil (FO), linseed oil (LO) or soyen oil (SO) diet for 26 weeks. Vlues re mens.,,,d,e Men vlues with unlike letters were signifintly different (P, 5), n 8., FO;, 7LO;, 1LO;, 7SO;, 1SO. inlusion of VO nd differed depending on the dietry oil fed. Thus, the lowest ARA:EPA rtio ws found in the HK of fish fed the 7SO diet, followed y the 1SO, FO, 7LO diets nd, finlly, the 1LO diet. Effet of feeding vegetle oils on ortisol response to drenoortiotrophi hormone stimultion Feeding fish with LO inresed ortisol relese in HK tissue fter ACTH stimultion; fish fed the 1LO diet showed the highest ortisol relese t 31, 33, 37 nd 39 min fter ACTH stimultion, the ortisol response ws hrterised y first pek t 33 min followed y redution t 35 nd 37 min nd strongest rise t 39 min. Fish fed the 7LO diet showed the sme pttern with less intensity up to 37 min, nd the ortisol ontinued to derese t 39 min, inditing tht the effet of LO ws proportionl to its ontent in the diet. However, fish fed the 7SO nd 1SO diets showed slightly lower, ut NS, ortisol response fter ACTH hllenge (Fig. 2). Therefore, fish fed SO diets showed tendeny to inhiit ortisol response, wheres fish fed LO diets showed signifint enhnement of ortisol relese. The overll ortisol relese fter ACTH hllenge expressed s ortisol SF (Fig. 3) ws signifintly highest in the 1LO diet followed y the 7LO diet. No signifint differenes were found in ortisol SF of fish fed SO nd FO diets. The orreltion ftor etween SF nd ARA:EPA in HK ws 98 %, refleting the importne of oth EPA nd ARA s eiosnoid preursors on ortisol seretion. Implition of ylo-oxygense nd lipoxygense metolites in ortisol relese Cortisol relese in the HK fter ACTH hllenge ws ffeted y the inution with eiosnoid inhiitors. Control fish deresed ortisol relese fter ACTH stimultion when the HK tissue ws inuted with oth the inhiitors (INDO or NDGA) (Fig. 4), denoting the implition of metolites from oth COX nd LOX enzymes Cortisol (ngg/hk per h) ACTH Fig. 2. Asolute ortisol seretion (ng/g hed kidney per h) y hed kidney of se rem fed either fish oil (FO), linseed oil (LO) or soyen oil (SO) fter drenoortiotrophi hormone (ACTH) stimultion.,, Men vlues t the sme time point with unlike letters were signifintly different (P, 5), n 8. B- -, FO; O, 7LO; B, 1LO; - -X- -, 7SO;, 1SO. Downloded from IP ddress: , on 1 De 218 t 21:58:56, sujet to the Cmridge Core terms of use, ville t

5 242 R. Gng et l. British Journl of Nutrition SF (%) FO 7LO 1LO 7SO Diet 1SO Fig. 3. Cortisol stimultion ftor (SF) in hed kidney of se rem fed either fish oil (FO), linseed oil (LO) or soyen oil (SO) fter drenoortiotrophi hormone stimultion.,, Men vlues with unlike letters were signifintly different (P, 5), n 8. in ortisol seretion. When the HK tissue ws inuted with INDO nd NDGA, the ortisol relese ws signifintly deresed in fish fed the 7LO diet, inditing the modultion of the ortisol seretion y these two pthwys, with this derese eing more pronouned when NDGA ws used s the inhiitor ompred with INDO (Fig. 5). In fish fed the 1LO diet, inuting the tissue with INDO or NDGA delyed the ortisol relese, nd the ortisol response ws hrterised y only one highest pek t 33 min. Inuting the tissue with NDGA deresed the ortisol relese t 39 min, proving gin the role of COX nd LOX in the stress seretion. Also in this se, the response ws delyed, nd the use of NDGA ws more effetive in inhiiting the ortisol relese thn tht of INDO (Fig. 6). In fish fed the 7SO diet inuting the HK tissue with INDO inresed ortisol relese, inditing nother pthwy of tion in this fish speies. NDGA inution did not ffet ortisol relese fter ACTH stimultion (Fig. 7). HK tissue from fish fed the 1SO diet showed lso signifintly deresed ortisol relese fter ACTH hllenge when inuted with INDO, nd there ws Cortisol (ng/g hed kidney per h) , INDO/NDGA ACTH 4, Fig. 5. Asolute ortisol seretion (ng/g hed kidney per h) y hed kidney of se rem fed 7 linseed oil (7LO) diet in omprison to the ontrol diet (fish oil, FO) fter drenoortiotrophi hormone (ACTH) stimultion following inution with either indomethin (INDO) or nordihydroguireti id (NDGA)., Men vlues t the sme time point with unlike letters were signifintly different (P, 5), n 8. B, 7LO; - -X--, 7LO þ INDO; O- -, 7LO þ NDGA;..., FO. lso slightly deresed ortisol relese, ut NS when the tissue ws inuted with NDGA (Fig. 8). Cortisol SF from the experiment using INDO nd NDGA (Fig. 9) showed tht inuting HK tissue with INDO in FO-fed fish redued the ortisol SF to 26 %, nd when inuted with NDGA, the SF ws redued to 42 %. Also when the HK tissue in 7LO-fed fish ws inuted with INDO or NDGA, the ortisol SF level ws deresed y 39 nd 66 %, respetively. HK tissue from 1LO-fed fish showed pprently the sme level of derese of the ortisol SF when inuted with INDO or NDGA orresponding to 46 %. By ontrst, in fish fed the SO diet, the tion of the COX nd LOX inhiitors ws different, suh tht inuting HK tissue from fish fed the 1SO diet, with INDO, redued the ortisol SF to 2 %, nd the inution with NDGA deresed the ortisol SF to only 1 %. By omprison, the fish fed the 7SO diet showed the opposite response to the use of COX nd LOX inhiitors, s when the tissue ws inuted with INDO, the ortisol SF inresed y out 3-fold nd when it ws inuted with NDGA, the ortisol SF ws inresed y 6 %. Cortisol (ngg/hk per h) INDO/NDGA ACTH Fig. 4. Asolute ortisol seretion (ng/g hed kidney per h) y hed kidney of se rem fed fish oil (FO) fter drenoortiotrophi hormone (ACTH) stimultion following inution with either indomethin (INDO) or nordihydroguireti id (NDGA)., Men vlues t the sme time point with unlike letters were signifintly different (P, 5), n 8. B, FO; --X--, FOþ INDO; O--, FOþ NDGA. Cortisol (ng/g hed kidney per h) INDO/NDGA ACTH 1 75,, 5, 25, Fig. 6. Asolute ortisol seretion (ng/g hed kidney per h) y hed kidney of se rem fed 1 linseed oil (1LO) diet in omprison to the ontrol diet (fish oil, FO) fter drenoortiotrophi hormone (ACTH) stimultion following inution with either indomethin (INDO) or nordihydroguireti id (NDGA)., Men vlues t the sme time point with unlike letters were signifintly different (P, 5), n 8. B, 1LO; --X- -, 1LO þ INDO; O- -, 1LO þ NDGA;..., FO. Downloded from IP ddress: , on 1 De 218 t 21:58:56, sujet to the Cmridge Core terms of use, ville t

6 Cortisol relese y se rem 243 British Journl of Nutrition Cortisol (ng/g hed kidney per h) Fig. 7. Asolute ortisol seretion (ng/g hed kidney per h) y hed kidney of se rem fed 7 soyen oil (7SO) diet in omprison to the ontrol diet (fish oil, FO) fter drenoortiotrophi hormone (ACTH) stimultion following inution with either indomethin (INDO) or nordihydroguireti id (NDGA). B, 7SO; - -X- -, 7SO þ INDO; O- -, 7SO þ NDGA;..., FO. Disussion INDO/NDGA ACTH The EFA for mrine fish omprise HUFA with ron hin lengths of 2 nd 22, for oth the n-3 nd n-6 series. Beuse the HUFA nnot e synthesised de novo y the fish (42) t suffiient rte to meet the requirements, these EFA must e provided in the diet. HUFA re essentil for norml fish growth, for its ellulr struture nd funtions, inluding the mintenne of memrnes nd eiosnoid metolism (43). Repling FO, in ommeril diets, with VO tht re devoid of n-3 HUFA, resulted in redued tissue levels of ARA, EPA nd DHA (25,26,3,31,44). In this experiment, t the end of the 26-week feeding period, the ftty id profile of HK of totl lipids from gilthed se rem refleted the dietry input. Thus, fish fed the SO diet hd signifintly higher levels of LA nd OA, nd fish fed the LO diet hd signifintly higher levels of LNA nd OA. In ddition, sine LO nd SO ontin lrge quntities of C18 ftty ids, the n-6:n-3 ftty id rtio ws ltered, nd this ould ffet fish helth (3,31,45). The ontents of DHA nd EPA deresed signifintly in fish fed VO ording to the level of inlusion, for instne, Cortisol (ng/g hed kidney per h) INDO/NDGA ACTH Fig. 8. Asolute ortisol seretion (ng/g hed kidney per h) y hed kidney of se rem fed 1 soyen oil (1SO) diet in omprison to the ontrol diet (fish oil, FO) fter drenoortiotrophi hormone (ACTH) stimultion following inution with either indomethin (INDO) or nordihydroguireti id (NDGA)., Men vlues t the sme time point with unlike letters were signifintly different (P, 5), n 8. B, 1SO; - -X- -, 1SO þ INDO; O- -, 1SO þ NDGA;, FO., SF (%) ,, FO 7LO 1LO 7SO 1SO Diet EPA deresed y out 8 % in fish fed the 1SO diet nd 76 % in fish fed the 1LO diet ompred with fish fed the ontrol diet, nd DHA ws deresed y 61 % in fish fed the 1SO diet nd 55 % in fish fed the 1LO. SFA nd MUFA, prtiulrly 16 :, 18 : 1n-9, 2 : 1n-9 nd 22 : 1n-11, re esily tolised in fish to produe energy, while DHA nd EPA re less esily tolised vi -oxidtion (46). Thus, n inrese in the OA, LA nd LNA ontents in HK from fish fed VO my mke these PUFA more ville for oxidtion to produe energy neessry to del with the stress hllenges. Under stress onditions, ortisol is sereted from interrenl ells of the HK triggered y the ortiotrophinrelesing hormone nd ACTH hormonl sde of the hypothlmus pituitry inter-renl xis in fish. ACTH is well known to indue the prodution nd relese of ortisol nd is onsidered to e the mjor gluoortioid stressor in fish (47), plying n importnt regultory role in the stress response proess, espeilly in the metoli djustments to stress (48). Feeding gilthed se rem with the 7LO nd 1LO diets signifintly inresed ortisol relese from HK tissue fter ACTH stimultion, in ordne with previous results from in vivo studies (31). The levels of ortisol found in the present study for the ontrol diet-fed fish were similr to those reported in our previous study (2), wheres the results of HK from fish fed the 1LO diet were 9-fold higher thn the ontrol response, inditing n effet of inluding high levels of LO in diets for gilthed se rem on its response to stress. It is well estlished tht HUFA provided y the diet ply n importnt role in stress response in mmmls s in fish. Enhning the levels of dietry ARA promoted survivl nd resistne to stress in gilthed se rem lrve (49,5), nd feeding the ARA-supplemented diet resulted in diminution in ortisol response fter net onfinement ompred with the fish fed diet ontining low level of this ftty id (51). In the present study, the ontent of ARA in HK, Fig. 9. Cortisol stimultion ftor (SF) in hed kidney of se rem fed different diets fter drenoortiotrophi hormone (ACTH) stimultion following inution with either indomethin (INDO) or nordihydroguireti (NDGA) id., Men vlues with unlike letters for eh dietry tretment indite signifint differene due to INDO or NDGA ddition (P, 5), n 8. B, Control;, INDO;, NDGA. Downloded from IP ddress: , on 1 De 218 t 21:58:56, sujet to the Cmridge Core terms of use, ville t

7 244 R. Gng et l. British Journl of Nutrition deresed y 55 % in 7LO-fed fish, 81 % in 1LO-fed fish, 86 % in 7SO-fed fish nd 88 % in 1SO-fed fish, whih ould e ssoited prtly with the modultion of ortisol relese levels oserved in these tretments. However, numer of uthors hve lso demonstrted the importne of EPA nd DHA in stress response nd stimulting ortisol relese in fish (9,2,52 54). In this experiment, the low dietry levels of these importnt HUFA in the diet led to prllel derese in the HK using signifint modultion of ortisol relese. These results demonstrted tht feeding fish with diets poor in these EFA ffeted ortisol relese from HK fter hllenging the tissue with ACTH, lerly denoting the importne of these nutrients in the orret funtion of HK in response to stress situtions. In the literture, there is lk of informtion on the effet of PUFA (of n-3 nd or n-6 series) on ortisol relese nd stress response in fish. The pprent ontrdition in ortisol relese levels found etween fish fed the LO nd SO ould e explined y the physiologil role of different ftty ids provided y these oils. One relesed from the memrne, NEFA ould t diretly s lignds, ffeting positively or negtively the inding of steroid hormones to their speifi intrellulr reeptors (55). In ddition, ftty ids n lso o-regulte gluoortioid-dependent gene expression y modulting the tivity of protein kinses involved in the phosphorylting trnsription ftors (55). Therefore, they my e plying role in modulting the intrellulr steroid hormone-signlling pthwy to o-regulte gluoortioid-sensitive promoter (56). In humn pltelets, unsturted ftty ids suh s plmitolei id (16 : 1), OA, LA, LNA nd ARA were deteted to inhiit phospholipse A2 tivity (57), whih is the key enzyme responsile for lierting the COX nd LOX sustrte preursors from the memrne phospholipids. The derese in ortisol relese in fish fed SO ould e explined y the higher ontent of OA nd LA in their tissue. In rts, the mximl steroidogeni response to ACTH ws inhiited pproximtely to 5 % y OA, onluding tht the modultion of steroidogenesis y these undnt nturlly ourring lipids my e n importnt omponent of the ontrol mehnisms within the hypothlmus pituitry drenl xis (58). It ws lso demonstrted tht OA nd LA inhiited the tion of ACTH on the drenl glnd (59). Moreover, in humn sujets, some reports indited tht inresing dietry n-6:n-3 ftty ids rtio y inresing the rtio etween LA nd LNA up to 4:1 redued lood ortisol nd holesterol levels (6). Elsewhere, LA ws lso reported to inhiit ortisol relese in ovine drenl ells (61). By ontrst, there ws inresed ortisol relese in HK from fish fed LO rih in LNA fter ACTH stimultion. Prtiulrly, fish fed the 1LO diet registered signifintly the highest ortisol level with low-pek response, the first one t 33 min nd the mximum one t 39 min; this my suggest the prtiiption of other unknown mehnisms in suh response. These results re in ordne with previous in vivo study, whih showed tht feeding se rem with diets rih in LO inresed signifintly their plsm sl ortisol (31). Reently, the sme uthors hve demonstrted tht inuting HK from sess (Dientrrhus lrx), using the sme superfusion system nd the sme onditions, with free LNA inresed signifintly ortisol relese fter ACTH stimultion (D Montero, D Negrín- Báez, R Gng, A Nvrro, M Izquierdo nd JM Afonso, unpulished results). In ddition, it ws demonstrted tht reduing LNA in diets for postmenopusl women redued the ortisol relese during stress (62). The present results re in ordne with these reports inditing the opposite effet of LA in fish fed SO in reduing ortisol relese, while LNA inresed this response in fish fed LO. The mehnisms y whih these ftty ids ould modulte ortisol relese in fish re still sujet of mny reserh studies, nd mny hypotheses hve een suggested through different pthwys. Some reports suggested tht the effet of LNA nd LA on ortisol is medited y protein kinse C (protein kinse A) nd protein kinse A through yli AMP tivtion (59,61). Interestingly, one of the most importnt roles of these EFA is tht they provide eiosnoids preursors, well-known series of hormones produed y the tions of the COX nd LOX on these ftty ids, nd modulte mny physiologil nd immunologil proesses. The implition of COX metolites in ortisol relese hs een proved reently y in vivo studies in fish (49 51), nd in our previous in vitro study, we demonstrted tht inuting HK tissue with COX inhiitor signifintly deresed ortisol relese (2). In mmmls, there is ler evidene tht PG modulte the relese of hypothlmi ortiotrophinrelesing hormone nd/or pituitry ACTH (63 65). For exmple, it is known tht PG, nd prtiulrly PGE 2, modulte the sensitivity of the mmmlin hypothlmus pituitry drenl xis nd onsequently hnge the stress response (22,66). COX-derived PG hve een shown to inrese in vitro ortisol relese in inter-renl tissue of femle frogs during ovultion (67) s well s in humn drenl ells (68). Elsewhere, feeding se rem with diets ontining 1 % of VO s lend of linseed (58 %), rpeseed (17 %) nd plm (25 %) oils deresed signifintly plsm PGE 3 ( 69). In the present study, inuting HK tissue with INDO deresed ortisol relese in FO-, 7LO-, 1LO- nd 1SO-fed fish, proving the implition of prostnoids in ortisol relese in these fish, nd this is in ordne with our previous results (2). The implition of LOX metolites in ortisol relese ws reently investigted, ut s fr s we re wre, the present study is the first report of the implition of LOX derivtives in the modultion of ortisol seretion y HK in fish. Inuting the HK tissue with NDGA deresed the ortisol relese in the mjority of the tretments. This is in ordne with the results from other studies proposing the implition of the LOX pthwy in stimulting ACTH relese when Downloded from IP ddress: , on 1 De 218 t 21:58:56, sujet to the Cmridge Core terms of use, ville t

8 Cortisol relese y se rem 245 British Journl of Nutrition ARA ws dded to fish ells in vitro (5) nd tht LOX metolites of ARA were reported to stimulte growth hormone relese in rt nterior pituitry ells (63,7). More studies hve pointed out the role of LOX produts in ACTH seretion nd drenl steroidogenesis in mmmls (71,72). In ddition, other eiosnoids suh s epoxygense metolites ould lso e potentil modultors of the hypothlmus pituitry inter-renl xis in fish, sine they hve een demonstrted to stimulte ACTH nd endorphin seretion from rt pituitry ells (7,73,74). In onlusion, studies rried out with ftty ids require gret re in interprettion. There is ler suggestion tht hnging the rtios etween n-3 nd n-6 ftty ids in se rem diets y inluding LO nd/or SO s sustitutes to FO my lter seriously the omposition of fish HK memrnes (25 28) nd onsequently modulte their response to stress (31). It is ler tht feeding these VO ffeted ortisol relese in HK from gilthed se rem, nd this is medited prtly y the tion of COX nd LOX metolites. Moreover, the presene of higher levels of C18 ftty ids in the HK from fish fed VO ould tivte other unknown pthwys, nd more studies re needed to lrify these mehnisms responsile for ortisol relese in fish. Aknowledgements The present work ws funded y the Spnish Ministry of Sienes nd Innovtion s prt of LINOSALUD projet (AGL CO32). The experiments were designed ording to the Animl Welfre Ethis Committee guidelines of Ls Plms University. R. G. rried out the niml experiment, performed the iohemil studies nd ortisol nlysis, prtiipted in the interprettion of the results nd drfted the mnusript. J. G. B. nd D. M. prtiipted in the design of the study nd in the interprettion of the results. A. F. prtiipted in the niml experiment design nd provided the formulted niml diets. Y. V. nd E. A. prtiipted in the niml feeding nd smpling. L. T. supervised the ortisol nlysis nd the result interprettion. M. S. I. oneived the study nd its design, oordinted the work, nd prtiipted in the interprettion of the results nd the preprtion of the mnusript. All the uthors red nd pproved the finl mnusript. There re no potentil onflits of interest. Referenes 1. Wtne T, Tmiy T, Ok A, et l. (1983) Improvement of dietry vlue of live foods for fish lrve y feeding them on v3 highly unsturted ftty ids nd ft-solule vitmins. Nippon Suisn Gkki Shi 49, Wtne T, Kitjim C & Fujit S (1983) Nutritonl vlues of live orgnisms used in Jpn for mss propgtion of fish: review. Aquulture 34, Izquierdo MS, Wtne T, Tkeuhi T, et l. (1989) Requirement of lrvl red serem Pgrus mjor for essentil ftty ids. Nippon Suisn Gkki Shi 55, Izquierdo MS (1996) Essentil ftty id requirements of ultured mrine fish lrve. Aqu Nutr 2, Cstell JD, Bell JG, Toher DR, et l. (1994) Effets of purified diets ontining different omintions of rhidoni nd dooshexenoi id on survivl, growth nd ftty id omposition of juvenile turot, Sophthlmus mximus. Aquulture 128, Bessonrt M, Izquierdo MS, Slhi M, et l. (1999) Effet of dietry rhidoni id levels on growth nd survivl of gilthed se rem Sprus urt L. lrve. Aquulture 179, Gurr MI & Hrwood JL (1991) Lipid Biohemistry. London: Chpmn & Hll. 8. Srgent JR, Bell JG, Bell MV, et l. (1993) The metolism of phospholipids nd polyunsturted ftty ids in fish. In Aquulture: Fundmentl nd Applied Reserh, Costl nd Esturine Studies, 43rd ed., pp [B Lhlou nd P Vitello, editors]. Wshington, DC: Amerin Geophysil Union. 9. Montero D, Tort L, Izquierdo MS, et l. (1998) Depletion of serum lterntive omplement pthwy tivity in gilthed serem used y -toopherol nd n-3 HUFA dietry defiienies. Fish Physiol Biohem 18, Pikering AD & Pottinger TG (1989) Stress responses nd diseses response in slmonid fish: effets of hroni elevtion of plsm ortisol. 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