Maternal essential fatty acid deficiency depresses serum leptin levels in suckling rat pups

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1 Mternl essentil ftty cid deficiency depresses serum leptin levels in suckling rt pups M. Korotkov, 1, * B. Grielsson, L. Å. Hnson, nd B. Strndvik* Deprtments of Peditrics* nd Clinicl Immunology, Reserch Center for Endocrinology nd Metolism in Deprtment of Internl Medicine, Göteorg University, SE Göteorg, Sweden Astrct Dietry lipid quntity nd qulity hve recently een shown to ffect serum leptin levels in dult rts. Moreover, suckling pups from dms fed high ft diet hd incresed serum leptin levels. The im of the present study ws to nlyze the influence of essentil ftty cid (EFA) deficiency on serum leptin levels in dms nd their pups during the suckling period. For the lst 10 dys of gesttion nd throughout lcttion, pregnnt rts were fed control or n EFA-deficient (EFAD) diet. The levels of leptin nd EFA in the serum of the dms nd pups were nlyzed 1, 2, nd 3 weeks fter delivery. In prllel, serum levels of glucose nd corticosterone were nlyzed in the pups. Low serum leptin levels were found in the control lctting dms during the entire lcttion period compred with the gemtched nonlctting nimls. The leptin concentrtions in the lctting dms fed the EFAD diet were lower compred with those fed the control diet. The serum leptin levels of suckling pups from dms on the EFAD diet were mrkedly decresed compred with controls (P 0.05). The reduced serum leptin levels could not e explined y nutritionl restriction s evluted y serum levels of glucose nd corticosterone. These results indicte the importnce of the EFA composition of the mternl diet for serum leptin levels in oth dms nd pups. EFA deficiency in lctting dms my cuse long-term effects on the pups through dysregultion of leptin nd leptin-dependent functions. Korotkov, M., B. Grielsson, L. Å. Hnson, nd B. Strndvik. Mternl essentil ftty cid deficiency depresses serum leptin levels in suckling rt pups. J. Lipid Res : Supplementry key words lcttion linoleic cid rchidonic cid diet corticosterone glucose which cn ct s second messengers nd regulte gene expression (6). EFA re lso precursors of eicosnoids, which modulte cell ehvior, intercellulr interctions, nd intrcellulr signl responses (7). It hs een shown in severl recent studies tht in ddition to the regultion of food intke nd energy expenditure (8), the o gene product leptin plys importnt roles in vriety of physiologicl nd pthologicl processes. Leptin my e involved in the development of the centrl nervous system (9), sexul mturtion (10), regultion of the hypothlmus-pituitry-drenl xis (11), insulin homeostsis (12), nd immune responses (13). The effect of leptin my e especilly striking during intruterine nd neontl growth for the development of the neuroendocrine nd immune systems. It hs een shown tht dietry ft quntity (14, 15) nd qulity (16) ffect serum leptin levels. Incresed mternl ft intke rises plsm leptin concentrtions in neontl rts nd ffects hypothlmus-pituitry-drenl responsiveness in neontes nd prepuertl rts (14). A diet rich in polyunsturted ftty cids (PUFA) increses leptin levels in diet-induced oese dult rts (16). Therefore, vrition in the type of diet during pregnncy nd lcttion might significntly modulte fetl nd neontl growth nd development y leptin-ssocited mechnisms. The im of the present study ws to estimte the influence of EFA deficiency on the serum leptin concentrtion in dms nd their pups during the suckling period. Nutritionl fctors such s n dequte intke of essentil ftty cids (EFA) during pregnncy nd lcttion re importnt for optiml fetl nd postntl development. Dietry deficiency of EFA in erly life hs long-term effects on development of the neuroendocrine (1) nd immune systems (2). Dietry ftty cids (FA) my indirectly modify mny cell functions y influencing memrne fluidity nd permeility (3, 4). The memrne sttus cn ffect vrious receptor nd enzyme functions, memrne trnsport, s well s signl trnsduction (5). In ddition, EFA re precursors of long-chin polyunsturted ftty cids (LCPUFA), MATERIALS AND METHODS Animls Pregnnt Sprgue-Dwley rts (BK Universl, Stockholm, Sweden) were received on dy 7 of gesttion nd kept in our niml Arevitions: EFA, essentil ftty cid; EFAD, essentil ftty cid deficient; LCPUFA, long-chin polyunsturted ftty cid; PL, phospholipid; PUFA, polyunsturted ftty cid; SGA, smll for gesttionl ge. 1 To whom correspondence should e ddressed. e-mil: Mrin.Korotkov@shlgrensk.se Journl of Lipid Reserch Volume 42,

2 fcility under constnt conditions of humidity (70 80%), temperture (22 25 C), nd light (12-h light nd drk cycle). The rts were housed individully in plstic cges, with food nd wter ville d liitum. Ten dys efore delivery, the femles were ssigned to one of two groups (n 6 in ech group) receiving either control or n EFA-deficient (EFAD) diet. Agemtched nonpregnnt femle rts fed the control or the EFAD diet (n 6 in ech group) were strted simultneously with the pregnnt rts. Becuse the EFAD diet resulted in too mny ded pups when strting efore conception, nd n EFAD diet t irth did not result in n EFA deficiency in the pups for severl weeks (unpulished dt), it ws necessry to strt the diet 10 dys efore irth, then continue the diet fter irth nd during the lcttion period. In this study, therefore, the effect of EFAD on the pups is developed minly during the lcttion period. The pups were kept with their mothers until wening on dy 21. Suckling pups (n 6) rndomized from ech litter were used for ech time point. Body weights of dms nd pups were recorded every week. Diets The dms were fed one of two experimentl powdered diets (AnlyCen, Lidköping, Sweden) for the lst 10 dys of gesttion nd throughout lcttion. The diets differed only y lipid composition: 7% soyen oil for the control diet nd 7% hydrogented lrd for the EFAD diet. The composition of the two diets is given in Tle 1. The dt on mjor components, slt, nd vitmins hve een otined from the mnufcturer. The FA composition ws determined in our lortory with the method descried elow. The totl metolizle energy for the diets ws 13.9 MJ/kg. TABLE 1. Composition of the control nd EFAD diets Diet Control EFAD wt% Csein Potto strch Glucose Cellulosl flour Minerl mix Vitmin mix Soyen oil 7.0 Hydrogented lrd 7.0 Ftty cids 12: : : : : : : : : : : : Slt mixture contining (wt %) KH 2 PO 4 (34.1), CCO 3 (35.9), KCl (2.5), NCl (18), MgSO 4 H 2 O (5.1), FeC 6 H 5 O 7 5H 2 O (3.3), MnO (0.27), Cu 2 C 6 H 4 O 7 2.5H 2 O (0.06), Zn 3 (C 6 H 5 O 7 )2 2H 2 O (0.04), CoCl 2 6H 2 O (0.002), KAl(SO 4 )2 2H 2 O (0.008), NF (0.025), KIO 3 (0.009), N 2 B 4 O 7 10H 2 O (0.002), N 2 SeO 3 5H 2 O (0.001), nd N 2 MoO 4 2H 2 O (0.001). Vitmin mixture contining vitmin A (11.9 IE/g), vitmin D 3 (1.5 IE/g), vitmin B 1 (4 mcg/g), vitmin B 2 (12 mcg/g), vitmin B 6 (5 mcg/g), C-pntotente 45% (11 mcg/g), nicin (40 mcg/g), vitmin B 12 (0.02 mcg/g), vitmin K 3 (7.75 mcg/g), iotin 2% (3 mcg/g), vitmin C (500 mcg/g), inositol (30 mcg/g), vitmin E (42 mcg/g), choline chloride 50% (1 mg/g), nd folic cid (0.5 mcg/g). Determintion of the FA composition of serum phospholipids Blood smples were collected from dms nd pups t 1, 2, nd 3 weeks (w1, w2, nd w3, respectively) of lcttion etween 9:00 m nd 11:00 m. Blood smples were collected from gemtched rts t the sme time. Truncl lood ws collected from the pups, nd lood smples were tken from the tip of the til of the dult rts. Ser were kept frozen ( 20 C) until nlyses of leptin, glucose, nd corticosterone levels nd of FA composition of serum phospholipids (PL; the comprtment reflecting the EFA sttus of tissue lipids). Lipids were extrcted from the serum with chloroform methnol 2:1 (v/v) contining 0.01% utylted hydroxytoluene (17). The lipids were frctionted on single SEP-PAK minopropyl crtridge (Wters Corp., Milford, MA, USA) y the method descried y Kluzny et l. (18) nd modified y Pietsch nd Lorenz (19). The frction of PL ws trnsmethylted in methnolic-hcl-3n t 90 C over 4 h. The FA methyl esters were extrcted with n-hexne nd, therefter, wshed with wter until neutrl, dried with MgSO 4, nd then dried with nitrogen. The FA methyl esters were seprted y cpillry gs-liquid chromtogrphy in Hewlett-Pckrd 6890 gs chromtogrph equipped with 30 m 0.25-mm SP-2380 column, film thickness 20 m. Helium t 2.0 ml/min ws used s crrier gs, nd splitless injection ws used. The injector nd detector tempertures were 300 C nd 250 C, respectively. The column oven temperture ws progrmmed from 50 C to 230 C t heting rte of 20 C/min up to 180 C nd, therefter, 2 C/min. The seprtion ws recorded with HP GC Chem Sttion softwre (HP GC, Wilmington, DE). C21:1 ws used s internl stndrd nd the FA methyl esters identified y comprison with retention times of pure reference sustnces (Sigm Aldrich Sweden AB, Stockholm, Sweden). The rtio of 20:3(n-9) to rchidonte ws used to define the deficiency stte: rtio of greter thn 0.4 is the iochemicl criterion of EFA deficiency (20). Anlysis of leptin, glucose, nd corticosterone levels in serum Leptin concentrtions were mesured y rt leptin rdioimmunossy (RIA; Linco Reserch Ltd., St. Chrles, MO, USA) nd ll smples from one experiment were nlyzed in duplictes in the sme ssy. The intr-ssy coefficient of vrition (CV) t 0.25 ng/ml nd 20 ng/ml ws 2.4% nd 1.6%, respectively. Serum glucose levels were mesured y the glucose Trinder method (Sigm Aldrich Sweden AB), nd serum corticosterone levels were mesured y BIOTRAK rt corticosterone RIA (Amershm Phrmci Biotech, Bucks, Englnd) following the mnufcturer s protocol. Before strting the ssy, serum smples for corticosterone mesurements were heted t 60 C for 30 min to displce corticosterone from cortisol-inding gloulin. The intr-ssy CV for the corticosterone ssy ws 5%. Sttisticl nlysis The dt were nlyzed using Mnn-Whitney s U test nd Friedmn s test. The EFAD-fed nimls were compred with the control-fed nimls t ech stge of the tretment. A vlue of P 0.05 ws considered sttisticlly significnt. RESULTS FA composition of serum PL in nonlctting rts Tle 2 shows chnges in FA composition of serum PL in dult nonlctting rts receiving the EFAD diet compred with nimls fed the control diet. Strting t w1, significnt decreses in the contents of linoleic 18:2(n-6) 360 Journl of Lipid Reserch Volume 42, 2001

3 TABLE 2. Serum PL ftty cid composition in the control nd EFAD nonlctting rts mtched y time with lctting rts t weeks 1, 2, nd 3 Control Diet (n 6) EFAD Diet (n 6) Ftty Acids 1 Week 2 Weeks 3 Weeks 1 Week 2 Weeks 3 Weeks 12: : : :1(n-7) : :1(n-9) :2(n-6) : :3(n-3) :2(n-6) :3(n-9) : :4(n-6) : :1(n-9) :6(n-3) :1/18: :3/20: Vlues given s mens SD. P 0.05 from control. P from control. nd -linolenic 18:3(n-3) cids were oserved, s well s compenstory rise in the levels of oleic 18:1(n-9), eicostrienoic 20:3(n-9), nd plmitoleic 16:1(n-7) cids. Those chnges were chrcteristic for chnged lipid metolism resulting from the EFAD diet (21, 22). However, there ws no chnge in the content of rchidonic 20:4(n-6) cid in the EFAD group. The iochemicl index of the EFA nutritionl deficit, the rtio of 20:3(n-9) to 20:4(n-6) in the nonlctting EFAD group, ws elow the empiric vlue of 0.4 t w3 (20, 21). At w1 nd w2, the proportion of docoshexoenoic 22:6(n-3) cid incresed significntly in the EFAD group compred with the control group. FA composition of serum PL in rts during lcttion During lcttion, the FA composition of the serum PL in rts fed the control diet differed significntly from tht in nonlctting rts receiving the sme diet (Tle 2 nd Tle 3). In the serum PL of the control dms during lcttion, elevted levels of 18:1(n-9), 18:2(n-6), nd 18:3(n-3) cids were oserved compred with the nonlctting gemtched group of rts. Simultneously, the levels of 20:4(n-6), 22:6(n-3), steric 18:0, ehenic 22:0, lignoceric 24:0, nd nervonic 24:1 cids were decresed in the serum of the dms compred with the nonlctting group of rts. At w2 to w3 of lcttion, the sturted luric 12:0 nd myristic 14:0 cid levels were decresed. Three weeks fter wening, the levels of FA in the serum PL of control dms reched the levels of those in the nonlctting group of rts (dt not shown). Feeding the EFAD diet resulted in drmtic chnges of the FA composition of serum PL in the lctting dms compred with controls (Tle 3). A significnt decrese in the level of 18:2(n-6) nd 18:3(n-3) cids ws oserved long with n ccumultion of 16:1(n-7), 18:1(n-9), 20:3(n-9), nd 24:1(n-9) cids. At w1, the proportion of 20:4(n-6) nd 22:6(n-3) incresed significntly in the EFAD group compred with the control group. Despite this increse, the iochemicl index of the EFA nutritionl deficit, the triene/tetrene rtio, ws ove the empiricl upper norml vlue of 0.4 in the EFAD group from w2. FA composition of the serum PL in the rt pups The FA composition of PL in the serum during suckling ws, in generl, similr in the pups nd the mothers of the control rts (Tle 3 nd Tle 4), lthough the proportions of the sturted FA 14:0, 16:0, 22:0, nd 24:0, nd the levels of the LCPUFA 20:4(n-6), 20:2(n-6), nd 22:6(n-3) were incresed in the pups t w1. There were significnt chnges in the EFA composition in the serum of the pups of the EFAD group 1 week fter delivery compred with the control group, followed y n even more drmtic difference 2 weeks lter (Tle 4). The levels of 18:0, 18:2(n-6), 18:3(n-3), nd 20:4(n-6) cids were decresed long with n ccumultion of 14:0, 16:0, 18:1(n-9), 20:3(n-9), 16:1(n-7), nd 24:1(n-9) cids. The rtio of 20:3(n-9) to 20:4(n-6) in the EFAD group ws ove 0.4 from w3 on. The deficiency ws lso expressed in the incresed 18:1/18:2 rtio. At w1, the proportion of 22:6(n-3) incresed significntly in the EFAD group compred with the control group. The deficiency in the pups during the first nd second week of life ws less pronounced thn in the dms, ut ecme similr to tht of the dms fter 3 weeks of ge. Body weights The men ody weight of the dult nonlctting rts receiving the EFAD diet did not differ from tht of the control nonlctting group. Similr men ody weights were found in the lctting dms during the whole lcttion pe- Korotkov et l. Perintl EFAD depresses leptin 361

4 TABLE 3. Serum PL ftty cid composition in the control nd EFAD lctting rts t 1, 2, nd 3 weeks of lcttion Control Diet (n 6) EFAD Diet (n 6) Ftty Acids 1 Week 2 Weeks 3 Weeks 1 Week 2 Weeks 3 Weeks 12: : : :1(n-7) : :1(n-9) :2(n-6) : :3(n-3) :2(n-6) :3(n-9) : :4(n-6) : :1(n-9) :6(n-3) :1/18: :3/20: Vlues given s mens SD. P 0.05 from control. P from control. riod, unrelted to their diets (dt not shown). The men weight of the pups from the dms receiving the control diet grdully incresed from g t w1 to g efore wening. The rte of weight gin in the suckling pups from dms on the EFAD diet ws significntly reduced, eing g t w1 nd g efore wening (P 0.05). TABLE 4. Serum leptin levels in the rts nd their pups Low serum leptin levels were found in the control lctting dms during the whole lcttion period. The concentrtions of leptin during the feeding with the control diet vried from ng/ml t w1 of lcttion, with significnt increse to ng/ml t w2 (P 0.05) nd ng/ml just efore wening (Fig. 1B). Nonlctting nimls fed the control diet nd tested for the sme period of time showed significntly higher concentrtions of leptin, rnging from 1.91 to 2.74 ng/ml (Fig. 1A). The serum leptin levels in the pups from the dms with the control diet incresed slightly from ng/ml t the end of the first week to ng/ ml efore wening (not significnt; Fig. 1C). Serum PL ftty cids in the control nd EFAD rt pups t 1, 2, nd 3 weeks of ge Control Diet (n 6) EFAD Diet (n 6) Ftty Acids 1 Week 2 Weeks 3 Weeks 1 Week 2 Weeks 3 Weeks 12: : : :1(n-7) : :1(n-9) :2(n-6) : :3(n-3) :2(n-6) :3(n-9) : :4(n-6) : : :6(n-3) :1/18: :3/20: Vlues given s mens SD. P 0.05 from control. P from control. 362 Journl of Lipid Reserch Volume 42, 2001

5 Fig. 1. Effect of the EFAD diet on serum leptin levels (men SE) in (A) nonlctting rts, (B) lctting rts, nd (C) pups during suckling. Ech point represents six nimls. Filled symols represent nimls on control diet nd open symols represent nimls on EFAD diet. * P 0.05 from control. Feeding dms with the EFAD diet ffected the leptin levels in the suckling pups from the first week of lcttion nd up to wening. The leptin concentrtions in the control group during this period of time were 3 to 4 times higher thn those in the EFAD group (Fig. 1C). Lctting dms lso hd lower serum leptin levels when fed the EFAD diet compred with controls, ut this only occurred during the 2nd nd 3rd weeks (P 0.05; Fig. 1B). In the nonlctting rts, the serum leptin levels did not differ significntly etween the control nd the EFAD groups (Fig. 1A). Serum glucose nd corticosterone levels in the rt pups Serum glucose levels of the suckling pups from dms fed the control diet were similr to those of the offspring from dms fed the EFAD diet during the suckling period (men SE): versus mg/dl t w1, versus mg/dl t w2, nd versus mg/dl t w3 in control nd EFAD pups, respectively. The men serum corticosterone levels in the suckling pups were similr in the control nd EFAD groups (men SE): versus ng/ml t w1, versus ng/ml t w2, nd versus ng/ml t w3, respectively. DISCUSSION Our study shows tht deficiency of dietry EFA during pregnncy nd lcttion cused mrked suppression of serum leptin levels in neontl rts. The EFAD diet lso decresed the serum leptin levels in the lctting dms. The involvement of the peptide hormone leptin in different physiologicl processes in the orgnism rises the question of the reltion etween the dietry ft qulity nd the development of disese. It ws shown recently tht vritions in the leptin levels of dult nimls might ffect the development of the immune response (13) nd glucose homeostsis (23). The qulity of dietry ft cn regulte the serum leptin levels in dult rts irrespective of dipose tissue mss (16). A high ft diet oviously influences the serum leptin levels nd the responsiveness of the hypothlmic-pituitry-drenl xis in rt pups during the suckling period (14). Following this, n indequte dietry ft intke during the pregnncy nd lctting period resulting in low leptin levels in the offspring my hve significnt negtive effects on postntl development of the niml. Feeding the EFAD diet to the nonlctting nimls chnged the serum lipid profile, ut not drmticlly. On the other hnd, EFA deficiency induced mrked chnges in the serum PL FA composition of lctting rts. In ddition, during lcttion, the FA composition of the serum PL of the dms receiving the control diet differed from those of the nonlctting nimls. The serum PL levels of LCPUFA, 20:4(n-6) nd 22:6(n-3), were significntly decresed in the lctting rts; this ws ccompnied y elevted serum PL levels of the precursors of LCPUFA (linoleic cid nd -linolenic cids) nd oleic cid compred with the nonlctting nimls. Our results showed tht even when the mternl diet contined dequte levels of linoleic cid, the level of rchidonic cid in the serum of these nimls remined reltively lower thn in the nonlctting nimls. This is in greement with dt in humns showing reltive deficiencies of LCPUFA during the lst trimester nd lcttion (24). The chnges seen in plsm PL profiles suggested significnt trnsfer of n-3 nd n-6 PUFA from the mother to the fetus nd neonte (25). Deficiency of EFA in the diet during pregnncy nd lcttion of the dms resulted in fster development of EFA deficiency in the lctting nimls. EFA deficiency lso developed in the pups of the nimls on the deficient diet, lthough this effect ws not so drmtic during their first 2 weeks of life. This delyed development of EFA deficiency could e explined t lest prtly y the pups eing compensted y the mother vi the milk. Becuse the dms did not receive n dequte mount of EFA from the food, the EFA in the milk my hve een produced with involvement of EFA from mternl tissues. A grdul depletion of the mothers resources during lcttion might chnge the composition of the milk to ecome more deficient in EFA. This my susequently influence the EFA composition of the serum PL in the pups, incresing the normlity during the third week fter delivery. The norml EFA levels might then cuse the chnge in the leptin levels of the pups. It is well estlished tht the min source of serum leptin is mture dipocytes. The murine 3T3-L1 predipocyte cell line cn e induced to differentite into mture dipocytes y humn milk (26). The possile ctive fctor(s) ws isolted from the lipid frction of humn milk nd ws suggested to e ftty cids. The importnce of LCPUFA precursors for dipocyte function is indicted y their specific ccumultion in rt dipose tissue (27). We oserved tht in the lctting dms, the serum leptin levels were significntly higher during the second nd third weeks of lcttion in the control group compred with the EFAD group. Other reports indicted tht during lcttion, the serum leptin levels in rts re down regulted (28, 29). We lso found low serum leptin levels in the control lctting dms during the entire lcttion pe- Korotkov et l. Perintl EFAD depresses leptin 363

6 riod nd in the EFAD group during the second nd third weeks of lcttion compred with nonlctting nimls. The mechnism responsile for the diminished leptin secretion during lcttion is uncler, ut oth neuronl nd hormonl fctors could contriute. The lredy downregulted levels of leptin during lcttion were further diminished y the EFAD diet. We oserved tht the pups from the dms on the EFAD diet hd significntly lower leptin levels in the serum compred with the control pups. This difference ws found during the entire period of suckling nd could not e explined y ltered nutritionl sttus in the EFAD pups, s evluted y glucose nd corticosterone levels. As EFAD hs een shown to e one fctor contriuting to infnts orn smll for gesttionl ge (SGA) (30), nd it hs een suggested tht some of these infnts my hve prolems with overweight lter in their life (31), it is of interest tht SGA neworns hve decresed leptin levels, compred to oth preterm infnts nd infnts orn t term (32). There my e severl mechnisms involved in the down-regultion of the leptin levels y EFA deficiency oth in the dms nd the pups. It hs een shown tht leptin concentrtion flls during fsting (33). Indeed, we found considerle difference in the men weight of the EFAD pups compred with the control group, ut we did not see ny difference in the serum glucose nd corticosterone levels etween these groups. It hs een shown tht serum glucose nd corticosterone levels were diminished in growth-retrded undernourished rt pups (34). We ssume tht the low serum leptin levels in the pups fed the EFAD diet re not due to nutritionl restriction. The growth retrdtion oserved in the EFAD pups my e cused y dysfunction of growth hormone regultion tht hs een demonstrted in EFAD nimls (35). In this context, it is interesting to note tht low serum leptin level in children is negtive predictor for successful growth hormone therpy (36). It hs een shown tht humn fetuses might produce prt of the circulting leptin in their own dipocytes (37), nd the o gene is expressed nd leptin is produced erly in postntl life in rts (38). A diet rich in PUFA is shown to increse serum leptin levels in dult rts y using mechnism tht is unrelted to chnges in dipose tissue mss (16). On the other hnd, pronounced EFA deficiency induced in rts fed n EFAD diet for 16 weeks fter 3 weeks of ge cused significntly reduced weight gin y 20%, ut no effect on ft pd weight (39). PUFA nd their metolites regulte dipocyte differentition (40) nd dipocyte gene expression (6) y ffecting the nucler peroxisome prolifertor ctivted receptor. During the development of the EFA deficiency in the dms nd the pups, we found significnt decrese of the reltive concentrtion of linoleic, linolenic, nd rchidonic cids in the serum PL of the EFAD nimls compred with the controls. The EFA deficiency might ffect dipocyte cpcity to produce leptin. The significntly higher level of leptin in the control pups my lso e explined y consumption of leptin vi the milk. It hs recently een shown tht leptin is present in oth humn nd mouse milk (41, 42). Moreover, the mouse milk leptin levels clerly incresed during the lte lcttion period (42). Further studies re in progress to investigte the effects of EFA deficiency on leptin content in milk. In conclusion, we found significnt decrese of serum leptin levels in the pups of dms on n EFAD diet. Our dt might hve relevnce lso for some of the humn SGA neworns. This suggests the importnce of n dequte diet during pregnncy nd lcttion to ttin norml levels of leptin in the offspring. Such levels my e importnt for norml development of the neuroendocrine nd immunologicl systems. The long-term effects of neontl disturnces in leptin homeostsis need to e investigted. The uthors wish to express their pprecition to Ms. Helen Khu for excellent technicl ssistnce. This study ws supported y grnts from the Swedish Medicl Reserch Council (4995), Göteorg Msonic Order, nd the Royl Acdemy of Science. Mnuscript received 21 Mrch 2000, in revised form 20 Septemer 2000, nd in re-revised form 31 Octoer REFERENCES 1. McKenn, M. C., nd A. T. Cmpgnoni Effect of pre- nd postntl essentil ftty cid deficiency on rin development nd myelintion. J. Nutr. 109: Dvork, B., nd R. Stepnkov Effects of dietry essentil ftty cid deficiency on the development of the rt thymus nd immune system. Prostglnd. Leukotr. Ess. Ftty Acids. 46: Tppi, P. S., S. Ldh, D. C. Clrk, nd R. F. Grimle The influence of memrne fluidity, TNF receptor inding, camp production nd GTPse ctivity on mcrophge cytokine production in rts fed vriety of ft diets. Mol. Cell. Biochem. 166: Crtwright-Shmoon, J. M., J. A. Dodge, nd C. McMster A complex iochemicl modultion of intestinl ion trnsport in rts fed on high-ft diets. J. Peditr. Gstroenterol. Nutr. 20: Grer, R., C. Sumid, nd E. A. 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