Monika SBupecka, Katarzyna Romanowicz, and JarosBaw WoliNski. 1. Introduction

Transcription

1 Interntionl Journl of Endocrinology Volume 216, Article ID , 9 pges Reserch Article Mternl High-Ft Diet during Pregnncy nd Lcttion Influences Obesttin nd Ghrelin Concentrtions in Milk nd Plsm of Wistr Rt Dms nd Their Offspring Monik SBupeck, Ktrzyn Romnowicz, nd JrosBw WoliNski Deprtment of Endocrinology, The Kielnowski Institute of Animl Physiology nd Nutrition, Polish Acdemy of Sciences, 5-11 Jbłonn, Polnd Correspondence should be ddressed to Monik Słupeck; m.slupeck@ifzz.pl Received 22 October 215; Revised 18 Februry 216; Accepted 6 Mrch 216 Acdemic Editor: Ichiro Skt Copyright 216 Monik Słupeck et l. This is n open ccess rticle distributed under the Cretive Commons Attribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. The study ims to estblish the effect of mternl high-ft diet on obesttin concentrtion, totl ghrelin, nd ghrelin/obesttin rtio during pregnncy nd lcttion of Wistr rts nd their offspring in the first 21 dys of life. On the mting dy, femles were rndomly llocted nd fed either high-ft diet (3% of ft; HF) or breeding diet (5% ft; BD) till the of lcttion. Hormones were nlyzed in the blood plsm nd milk of rt dms s well s in the blood plsm of their offspring. HF resulted in significnt decrese in obesttin level on the of lcttion nd elevtion on the. Plsm obesttin in HFD offspring ws significntly higher thn in BD ones. HF diet did not significntly ffect dm plsm ghrelin until the of lcttion. The ghrelin concentrtions in milk fter both diets were significntly lower thn in blood plsm. Milk ghrelin in HF dms ws significntly higher thn in the BD ones. Plsm ghrelin from HF offspring ws significntly higher thn tht from BD dms. Our results demonstrte tht mternl HF diet during pregnncy nd lcttion influences ghrelin nd obesttin level in both dms nd their offspring. 1. Introduction Development nd growth during the erly life period re influenced by mternl helth nd diet composition [1]. Evidence from both epidemiologicl nd niml model studies indictes tht mternl diet influences metbolic sttus nd plys crucil role in the development of metbolic functions in offspring nd their susceptibility to metbolic diseses in dulthood. Incresed ft-consumption, n ttribute of the Western diet, is considered mjor triggering fctor of metbolic impirments such s obesity, type II dibetes, insulin resistnce, dyslipidemi, nd hypertension. Studies in rts show tht high-ft diet during pregnncy nd lcttion hs mrked impct on offspring body composition, risk of metbolic syndrome [2], nd the development of obesity in both erly life nd dulthood [3, 4]. Moreover, it ws shown tht even mild mternl overnutrition cused by high-ft dietledtoincreseddiposity[5],glucoseintolernce[5],nd ltered brin ppetite regultors in offspring [6, 7]. Ghrelin nd obesttin re gstrointestinl peptides involved in the regultion of metbolic functions in rodents nd humns. Although both peptides derive from common preprohormone, different posttrnsltion processing determines their functions which re reported to be opposite to severl physiologicl functions. Moreover, severl findings on obesttin re controversil s subsequent studies hve provided contrdictory results on its role in the regultion of food intke nd body weight, energy expenditure control, nd growth hormone secretion [7 9]. The nture of the obesttin receptor hs lso remined n open question. The initilly proposed G-protein-coupled receptor GPR39 hs been questioned due to series of studies, which filed to demonstrte the bility of obesttin to bind to nd ctivte this receptor [1 12]. Although obesttin s role in the control of metbolism is still uncler, the previous studies showed tht the ghrelin/obesttin blnce is essentil for modulting energy homeostsis nd dpting the orgnism to nutritionl chllenges. For exmple, deregultion in the

2 2 Interntionl Journl of Endocrinology Amount per kg Tble1:Mincomponentsofdietsgiventorts. Stndrd breeding diet (BD, 5% ft) High-ft diet (HF, 3% ft) Dry mtter 885 g 918 g Crude protein 222 g 19 g Crude lipids 5g 297g Crbohydrtes 572 g 41 g Crude fiber 46g 32g Crude sh 41 g 3 g Metbolic energy 12,8MJ/357kcl 19MJ/4658kcl ghrelin/obesttin rtio ws observed in norexi nervos [13] nd obesity, in both childhood [14] nd dulthood [15]. Substntil mounts of ghrelin nd obesttin were previously reported in the colostrum nd humn brest milk [16]. Obesttin nd ghrelin s milk-contined bioctive peptides which cn modulte energy homeostsis my ply n importnt role in metbolism progrmming of newborns. However, the reltionship between different mternl diets nd obesttin nd ghrelin blnce during gesttion nd lcttion of rts hs not been exmined yet. The present study ims both to explin how the concentrtion of stted hormones chnges when diets vrying in ft content will be given to rts during pregnncy nd lcttion nd to lern whether they influence the plsm level of these two hormones in offspring until wening time. 2. Mterils nd Methods 2.1. Animls. The experiments nd tretments were conducted in complince with Europen Union regultions concerning the protection of experimentl nimls. The Locl Animl Ethics Committee locted in Wrsw pproved the study protocol. Sixteen dult femle nd sixteen mle Wistr Hn rts (11 weeks old) were obtined from the Center of Experimentl Medicine t the Medicl University of Bilystok, Polnd. After two weeks of cclimtiztion, the femles were exmined with vginl impednce checker (Muromchi Kiki Co., Ltd.) for the precise determintion of the stge of estrus for mting time. Bsed upon these mesurements, femle rts t the pproprite point in the estrous cycle were selected for breeding. The mting dy ws lso dy when the nimls were rndomly llocted for either high-ft diet (HF, 3% ft; 4.7 kcl/g; n = 6)or stndrd breeding diet (BD; 5% ft; 3.1 kcl/g; n=6) (diets were purchsed from Wytwórni Psz Morwski (Polnd)). Min components of diets given to rts were presented in Tble 1. Porcine lrd ws used s the source of ft in the HF diet. The following morning, mted femles were exmined for the presence of vginl plug, nd the dy the plug ws observed ws considered s dy 1 of gesttion. On dy 14 of gesttion, the femles were smpled for blood, weighed, nd seprted from the mles. The dy when femles gve birth ws considered dy 1 of lcttion. Only femles who gve birth 21 ± 1 dys fter mting were chosen for the experiment (n =6for ech diet). On the first dy of lcttion, the litters were stndrdized to ten pups. The supernumerry pups were smpledforbloodndeuthnized(dy1,hfon=7;bdo n = 5). On dy 14 of lcttion, two pups from ech litter weresmpledforbloodndthenscrificed(n=12for ech mternl diet). The rest of the pups styed with their dms nd continued to be used in the experiment till the of lcttion when they were blood-smpled. Then ll pups nd rt dms (n = 6 for ech diet) were scrificed. Two pups from ech litter were tken for further blood smpling (n = 12 for ech mternl diet) while the rest were tken for nother experiment. Strting from dy 1 till dy 21 of lcttion ll pups were blood-smpled vi crdic puncture under isoflurne or CO 2 inhltion nd then euthnized by overdosing of isoflurne (Bxter, USA) (dy 1) or CO 2 (dys 14, 21 nd rt dms) Smpling Blood nd Milk. Blood smpling ws done on dy just before mting, on 14th-15th dy of gesttion, nd on 14th nd s of lcttion during the light phse between 11. nd 11.3.m. (resting phse for rodents), but not during the drk period (ctive feeding phse), s the plsm ghrelin levelhsbeenshowntobelteredbyfoodintke[17]. Blood ws smpled from the pups on the 1st (within 24 h fter birth), 14th, nd s of life. Milk ws smpled on 14th nd s of lcttion before the blood smpling. In brief, 3 min before smpling, rt dms were seprted from their pups nd n intrmusculr injection of 5 IU of oxytocin (Oxytocinum synt. 1 IU/mL, Biowet Pułwy, Polnd) ws performed. Around ml smples of milk were obtined from different rndomly selected tets from ech rt. Blood smples from the dms were collected from til vein. Just before the collection, the tils were heted in wter bth (4 C) to increse blood flow. Blood from the neontes nd dms on the of lcttion ws collected vi crdic puncture. The blood glucose levels were detected from til vein using Contour Link (Byer, USA). Blood nd milk smples were withdrwn on EDTA nd protinin (.6 TIU/mL of blood) nd immeditely centrifuged t 1.6 g for15minutest4 C. Blood plsm ws hrvested, distributed into Eppendorf tubes, deep frozen ( 8 C), nd stored until nlysis. Before the milk smples were distributed into Eppendorf tubes nd deep frozen ( 8 C), the ft lyer on the top ws discrded. Concentrtion of blood plsm triglycerides ws determined spectrophotometriclly (MAXMAT PL multidisciplinry dignostic pltform, Erb Dignostics Frnce SARL, Frnce) using ELITech redy-to-use regents (ELITech Group, Frnce) ccording to method described previously [18] Obesttin nd Ghrelin Rdioimmunossy. Rt milk nd plsm smples were ssyed for obesttin nd totl ghrelin (cyl nd noncyl) concentrtion using commercilly vilble rdioimmunossy (RIA) kits: Ghrelin (Rt, Mouse) RIA Kit nd Obesttin (Rt, Mouse) Ultr-Sensitive RIA Kit (Phoenix Phrmceuticls, Inc., USA) ccording to the mnufcturer s instructions. Spike nd recovery of obesttin ndtotlghrelinweremesuredinthreedifferentplsmor

3 Interntionl Journl of Endocrinology 3 Tble 2: Body weight, blood glucose, nd plsm triglycerides in rt dms () nd neontes. () Mting Pregnncy Lcttion First d 14th d 14th d 21st d BD HF BD HF BD HF BD HF Body weight (g) 246 ± ± 6 A 28 ± ± 8 B 285 ± ± 6 B 283 ± ± 6 B Blood glucose (mmol/l) 7.38 ± ±.12 A 6.88 ± ±.11 A ±.61 b 8.61 ±.61 B ± 1.33 b 9.88 ±.72 AB Plsm triglycerides (mmol/l) 1.1 ±.1.8 ±.1 A 2.7 ±.3 b 3.2 ±.8 B 1. ± ±.5 B 1.3 ±.1 b 2.8 ±.5 B BD: femles fed with stndrd breeding diet during pregnncy nd lcttion; HF: femles fed with high-ft diet during pregnncy nd lcttion. Dt re expressed s mens ± SEM.,b Sttisticl differences between dys within BD tretment. A,B Sttisticl differences between dys within HF tretment. Sttisticl differences between BD nd HF group ( p <.5, p <.1, p <.1). First d 14th d 21st d BDO HFO BDO HFO BDO HFO Body weight (g) 6.2 ± ±.25 A 22 ±.3 b 27 ±.3 B 37 ± 1.3 c 45 ± 1.1 C Blood glucose (mmol/l) 4.3 ± ±.4 A 7.8 ±.4 b 7.4 ±.6 AB 9.4 ± 1.2 b 9.9 ± 2. B Plsm triglycerides (mmol/l) 1.7 ±.2 1. ±.3 A.6 ±.1 b 2.1 ±.2 AB 2.3 ± ±.8 B BDO: rt neontes from femles fed with stndrd breeding diet during pregnncy nd lcttion; HFO: rt neontes from femles fed with high-ft diet during pregnncy nd lcttion. Dt re expressed s mens ± SEM.,b,c Sttisticl differences between dys within BDO tretment. A,B,C Sttisticl differencesbetweendyswithinhfotretment. Sttisticl differences between BDO nd HFO group ( p <.5, p <.1, p <.1). milk smples. The ssys were performed in one run. The men percent recovery for obesttin nd ghrelin in plsm ws 9% nd 95%, respectively. The interssy coefficient of vrition (CV) for obesttin nd ghrelin in plsm ws 6.6% nd 6.%, respectively. Before the ssys, the milk smples were sonified for 15 min. Men percent recovery ws 9% for both obesttin nd ghrelin in milk. The interssy CV for obesttin nd ghrelin in milk ws 7.6% nd 5.6%, respectively Sttisticl Anlysis. The dt re expressed s mens ± SEM. One-wy nlysis of vrince (ANOVA) followed by post hoc Tukey-Krmer or Kruskl-Wllis test followed by post hoc Dunn s Multiple Comprison Test or regulr twowy ANOVA ws used to determine sttisticlly significnt differences between the groups tested time points or/nd differentsmples(prism5formcosx,version5.d,grph Pd Softwre, Sn Diego, CA, USA). The Person test ws used for correltion nlysis. In ll sttisticl nlyses, p <.5 ws tken s the level of significnce. 3. Results 3.1. Body Weights, Blood Glucose, nd Plsm Triglycerides. Bodyweightsofrtfemlesfrommtinguntil21stdyof lcttion, the blood glucose level, nd plsm triglycerides during tested time points were presented in Tble 2(). No differences in body weight between BD dms nd HF dms were observed. The level of plsm triglycerides ws unchnged in pregnncy but during lcttion (14th nd 21st dy) incresed significntly in HF dms. Feeding dm rts with high-ft diet during pregnncy nd lcttion resulted in significnt increse in body weight nd plsm triglycerides of their offspring (HFO) strting from the of life s compred to control pups (BDO) (Tble 2). Mternl diet hd no effect on the litter size (dt not shown) nd the level of blood glucose in rt neontes (Tble 2) Obesttin in Blood Plsm of Rt Dms nd Pups nd in Mternl Milk. In comprison with tht in nonpregnnt femle rts (dy ), the mternl obesttin concentrtion in plsm of BD rts ws unchnged in pregnncy ( of gesttion)ndintestedtimepointsduringlcttion(14thnd ). In contrst, the HF diet during pregnncy nd lcttion resulted in significnt decrese in the obesttin level on of lcttion nd significnt elevtion on in comprison with both nonpregnnt femles nd lctting BD femles (Figure 1()). The obesttin concentrtion in milk in 14th nd of lcttion ws significntly higher thn in the blood plsm of rt dms. In control rt dms (BD group), concentrtion of obesttin in milk did not differ significntly in tested time points during lcttion, wheres the HF diet resulted in significnt elevtion in milk obesttin s compred to the BD diet, where peptide concentrtion rising significntly from dy 14 till of lcttion (Figure 1()). A reltionship between the obesttin concentrtion in milk nd mternl blood in of lcttion ws observed in both diets (Tble 3()). In the BD group, we observed negtive correltion (r =.838, p =.38), wheres in the HF rt dms we found strong positive correltion (r = 1., p <.1). Significnt negtive correltions were observed between mternl plsm obesttin nd ghrelin for BD rts in 14th nd of lcttion nd for HF rts in of pregnncy nd lcttion (Tble 3()). Interestingly, positive reltionships between these hormones in milk were observed in the of lcttion for BD dms (r =.859, p =.14) nd in the 21st of lcttion for HF dms (r =.72, p =.14) (Tble 4()). Also reltionship between the body weight of dms nd the obesttin concentrtion in blood plsm in 21st

4 4 Interntionl Journl of Endocrinology Concentrtion of obesttin (pg/ml) Pregnncy Lcttion b Ctrl BD HF BD HF BD HF Dy () Concentrtion of obesttin (pg/ml) fff f BD HF BD HF Concentrtion of obesttin (pg/ml) A b B A c C BDO HFO BDO HFO BDO HFO Dy 1 (c) Figure 1: Obesttin concentrtion (pg/ml) in blood plsm () nd milk of femle rts fed with stndrd breeding diet (BD) or high-ft diet (HF) in pregnncy nd lcttion during different reproductive stges (before mting, dy, of pregnncy, nd 14th nd 21st dyoflcttion)ndbloodplsmofpupsfrombddms(bdo)orhfdms(hfo)inthefirst21dysoflife(c).resultsrepresenteds mens ± SEM.,b,c Sttisticl differences in obesttin plsm concentrtion between dys within control tretment (BD or BDO). A,B Sttisticl differences in obesttin plsm concentrtion between dys within HF tretment nd HFO tretment. + Sttisticl differences between BD nd HF group nd between BDO nd HFO group (p <.5). + Sttisticl differences in obesttin milk concentrtion between dys within tretment (p <.5). f indictes sttisticl differences in milk obesttin concentrtion between BD nd HF group ( f p <.5, fff p <.1). dy of lcttion ws observed on both diets. The positive correltion ws observed between the body weight of BD dms nd plsm blood obesttin concentrtion in the 21st dy of lcttion (r =.976, p =.2), wheres in the HF dms we found negtive correltion (r =.881, p =.1) (Tble 3()). No correltion ws found between the glucose concentrtion in mternl blood nd the obesttin level in either blood or mternl milk (Tble 3()). The highest obesttin concentrtion in the blood plsm of offspring from control rt dms (group BDO) ws observed on dy 1 of lcttion nd then the significnt decrese ws observed in the following dys of lcttion (Figure 1(c)). In pups from HF rt dms (HFO), the obesttin concentrtion ws higher in tested time points (significnt difference in the of life, p =.32) but the sme dependence ws observed in the form of significnt decrese in hormone level in the successive dys of life. No correltions were found between plsm obesttin nd ghrelin in offspring on either tretment (Tble 4) Ghrelin in Blood Plsm of Rt Dms nd Pups nd in Mternl Milk. In the BD rt dms, the ghrelin concentrtion in blood plsm decresed in pregnncy nd lcttion period in comprison to nonpregnnt femles (dy ) (Figure 2()). On the of lcttion, significnt increse in ghrelin concentrtion in HF dms ws found (p =.5) in comprison to BD ones. The observed level of ghrelin in this time point did not differ (p =.75)fromthebsllevelfor ghrelin in rt dms before mting (Figure 2()). The ghrelin concentrtions in mternl milk from both studied groups of rt dms were significntly lower thn in blood plsm nd grew significntly in the subsequent dys of lcttion. The milk ghrelin concentrtion in HF dms ws significntly higher thn tht in the BD nimls (Figure 2). Negtive correltions were found between blood plsm nd milk ghrelininthecontrolrtdmsonthe21stdyoflcttion (r =.737, p =.29) nd between body weight nd the plsm ghrelin concentrtion on the of pregnncy in the HF rt dms (r =.93, p =.48). By contrst, positive correltion ws found between blood plsm ghrelin nd glucose on the of lcttion (r =.852, p =.33) (Tble 3). The ghrelin blood plsm concentrtion in neontes incresed significntly in the subsequent dys of life. On the of lcttion, the ghrelin concentrtion in the blood plsm of neontes from the HF dms ws significntly

5 Interntionl Journl of Endocrinology 5 Tble 3: Correltions for obesttin () nd ghrelin in milk nd blood plsm of rt dms nd their offspring. Plsm obesttin versus body weight Plsm obesttin versus milk obesttin Milk obesttin versus newborns body weight () Pregnncy Lcttion 14th d 14th d 21st d BD HF BD HF BD HF r =.319 p =.299 r =.547 p =.234 r =.531 p =.139 r =.267 p =.312 r =.158 p =.367 r =.625 p =.187 r =.838 r = 1. r =.31 p =.38 p <.1 p =.36 r =.653 p =.56 r =.858 r =.185 p =.7 p =.317 r =.976 r =.881 p =.2 p =.1 r =.527 p =.141 r =.271 p =.21 No correltions were observed for following prmeters: plsm obesttin versus blood glucose; plsm obesttin versus newborn plsm obesttin; milk obesttin versus body weight; milk obesttin versus blood glucose. Plsm ghrelin versus body weight Plsm ghrelin versus milk ghrelin Plsm ghrelin versus blood glucose Milk ghrelin versus newborns plsm ghrelin Pregnncy Lcttion 14th d 14th d 21st d BD HF BD HF BD HF r =.351 p =.325 r =.319 p =.311 r =.567 p =.219 r =.93 r =.23 p =.48 p =.371 r =.228 p =.374 r =.14 p =.512 r =.54 p =.23 r =.113 p =.412 r =.463 p =.148 r =.738 p =.236 r =.978 p =.67 r =.478 p =.196 r =.517 p =.117 r =.451 p =.223 r =.737 r =.21 p =.29 p =.484 r =.342 p =.254 r =.98 r =.45 p =.1 p =.462 r =.852 p =.33 r =.332 p =.174 No correltions were observed for following prmeters: plsm ghrelin versus newborn plsm ghrelin, milk ghrelin versus body weight, milk ghrelin versus blood glucose, nd milk ghrelin versus newborn plsm ghrelin. BD: femles fed with stndrd breeding diet during pregnncy nd lcttion; HF: femle rts fed with high-ft diet during pregnncy nd lcttion; p <.5, p <.5, p <.1. higher thn in neontes from the control (BD) rt dms (Figure 2(c)) Ghrelin/Obesttin Rtio in Blood Plsm of Rt Dms nd Pups nd in Mternl Milk. The ghrelin/obesttin rtio for the control mternl plsm ws stble during the tested time points except for pek on the of lcttion when we observed both significnt elevtion in this prmeter nd difference with HF dms. In the HF dms, the rtio ws unchnged except on the of lcttion when the rtio wentupwhichresultedinsignificntdifferencewithbd dms (Figure 3()). On the of lcttion, the ghrelin/obesttin rtio significntly decresed in milk from experimentl dms (HF) in comprison to control (BD) rt dms (Figure 3). In plsm neontes from the control dms (BDO), the rtio ws stbleduringthefirstfourteendysoflifendthenincresed significntly on the of life (p <.1). The sme pttern ws observed in neontes from the experimentl dms (HFO), but the vlues for the ghrelin/obesttin rtio in thefirstfourteendysoflifeweresignificntlylower(p =.6; p =.1,resp.)thninthoseinthecontrolneontes (Figure 3(c)). 4. Discussion Our study reports on the concentrtions of ghrelin nd obesttin in the blood of rt dms, fed diets differing in ft content during pregnncy nd lcttion, together with their concentrtions in the milk during lcttion nd blood of offspring. We reported tht high-ft diet during pregnncy nd lcttion did not chnge the ghrelin plsm pttern on the of pregnncy nd lcttion. However, on the of lcttion the ghrelin concentrtion in HF dms wselevtedincomprisontothtinthebdnimls.the effect of HF diet consumption on ghrelin concentrtion is not conclusive s both no chnges [19, 2] nd the significnt decrese [21, 22] of this hormone hve been observed in rodents. In our study, the ghrelin concentrtion in HF dm rts could be dditionlly ssocited with specific metbolic chnges tht occur during pregnncy nd lcttion. Previous studies reveled tht the energy demnds for lcttion in rodents cn exceed 6% resulting in threefold increse in food intke [23]. However, reduced food intke nd greter weight loss were shown in obese rts in the first dys of lcttion [23, 24]. Keesey nd Hirvonen [25] proposed the existence of body weight set point in rodents nd humns so tht body weight decrese or increse is corrected by ltering food intke nd energy expenditure to mintin the trget body weight. Although this weight-control mechnism ws not investigted in the present study, it could explin the lck of effects on dm body weight depending on the diet which ws reported in our study s well s in other studies on rodents fed with HF diet during gesttion nd lcttion [26 28]. This phenomenon my be lso explined

6 6 Interntionl Journl of Endocrinology Tble 4: Correltions between obesttin nd ghrelin in blood plsm nd milk of rt dms () nd plsm of neontes. Dm plsm obesttin versus ghrelin Milk obesttin versus ghrelin () Pregnncy Lcttion 14th d 14th d 21st d BD HF BD HF BD HF r =.66 p =.44 r =.995 r =.97 r =.84 r = 1. r =.634 p =.2 p =.47 p =.37 p =.1 p =.88 r =.859 p =.14 r =.87 p =.445 r =.35 p =.473 r =.72 p =.14 BD: femles fed with stndrd breeding diet during pregnncy nd lcttion; HF: femle rts fed with high-ft diet during pregnncy nd lcttion; p <.5, p <.1. Newborns plsm obesttin versus ghrelin 1st d 14th d 21st d BDO HFO BDO HFO BDO HFO r =.461 p =.217 r =.614 p =.97 r =.72 p =.53 r =.51 p =.19 r =.421 p =.87 r =.252 p =.173 BDO: pups from femles fed with stndrd breeding diet during pregnncy nd lcttion; HFO: pups from femles fed with high-ft diet during pregnncy nd lcttion. Concentrtion of ghrelin (pg/ml) Pregnncy Lcttion A A b b B b B Ctrl BD HF BD HF BD HF Dy () Concentrtion of ghrelin (pg/ml) f f ++ BD HF BD HF Concentrtion of ghrelin (pg/ml) C c b B A BDO HFO BDO HFO BDO HFO Dy 1 (c) Figure 2: Ghrelin concentrtion (pg/ml) in blood plsm () nd milk of femle rts fed with stndrd breeding diet (BD) or high-ft diet (HF) in pregnncy nd lcttion during different reproductive stges (before mting, dy, of pregnncy, nd 14th nd 21st dyoflcttion)ndbloodplsmofpupsfrombddms(bdo)orhfdms(hfo)inthefirst21dysoflife(c).resultsrepresenteds mens ± SEM.,b,c Sttisticl differences in ghrelin plsm concentrtion between dys within control tretment (BD or BDO). A,B,C Sttisticl differences in ghrelin plsm concentrtion between dys within HF tretment nd HFO group. Sttisticl differences between BD nd HF groupndbetweenbdondhfogroup p <.1. + Sttisticl differences in ghrelin milk concentrtion between dys within tretment ( ++ p <.1). f indictes sttisticl differences in ghrelin milk concentrtion between BD nd HF group ( f p <.5).

7 Interntionl Journl of Endocrinology 7 5 Pregnncy Lcttion Ghrelin/obesttin rtio A A B B b A Ctrl BD HF BD HF BD HF Dy () 5 Ghrelin/obesttin rtio ff BD HF BD HF Ghrelin/obesttin rtio b B A A BDO HFO BDO HFO BDO HFO Dy 1 Figure 3: Ghrelin/obesttin rtio in blood plsm () nd milk of femle rts fed with stndrd breeding diet (BD) or high-ft diet (HF) in pregnncy nd lcttion during different reproductive stges (before mting, dy, of pregnncy, nd 14th nd of lcttion) nd blood plsm of pups from BD dms (BDO) or HF dms (HFO) in the first 21 dys of life (c). Results re presented s mens ± SEM.,b Sttisticl differences in ghrelin/obesttin plsm concentrtion between dys within control tretment (BD or BDO). A,B Sttisticl differences in ghrelin/obesttin plsm concentrtion between dys within HF tretment nd HFO group. Sttisticl differences between BDndHFgroup( p <.1, p <.1). + Sttisticl differences in ghrelin/obesttin milk concentrtion between dys within tretment ( + p <.5). f indictes sttisticl differences in ghrelin/obesttin milk concentrtion between BD nd HF group ( ff p <.1). (c) by results showing tht ft mobiliztion slightly contributes to totlmilkenergyoutputndthtlrgermilkftproduction in obese rts is chieved by the mobiliztion of triglycerides from dipose tissue nd lrger plsm lipid extrction rther thn incresed food intke. In our study we did not mesure the dily food intke; however we observed the increse in milk ft content on the 14th nd of lcttion (dt not shown) together with significntly higher concentrtion of plsm triglycerides of HF rts strting from the of gesttion till the of lcttion. Incresed lipolysis during lcttion in HF rts my be ssocited with n observed decrese in the concentrtion of plsm obesttin on the 14th dy of lcttion nd then its significnt increse t the end of lcttion () lthough recent studies by Pruszyńsk- Oszmłek et l. [29] hve suggested tht obesttin my inhibit lipogenesis. It should lso be mentioned tht, in our study, rts were fed HF diet for reltively short period of six weeks. Moreover, in our model we compred high-ft diet (HF) together with stndrd breeding diet (BD), which ws formulted to fulfill higher energetic demnds of rt dms during gesttion nd lcttion, so it is more energetic thn stndrd rodent chow. We hve lso found tht the ghrelin/obesttin rtio incresed in the plsm of HF dms on the of lcttion nd then significntly decresed on the 21st dy. Previous studies on humns showed tht the ghrelin/obesttin rtio ws lower in obese subjects nd negtively correlted with BMI nd indices of bdominl ft distribution [15]. This finding seems to support the concept tht, during lcttion, HF diet leds to negtive energy blnce (e.g., incresedlipolysis)ndthenwhenlcttionends(onthe21st dy) the HF diet strts to develop overweight or/nd obesity. Although we did not observe ny chnges in the body weight of dms depending on the diet, we reported chnges in the hormones concentrtion to occur in the milk nd plsm of neontes. Both tested hormones were significntly elevted in the milk of HF dms nd resulted in tendency to increse their plsm concentrtions in neontes. On the of lcttion, this increse ws significnt for obesttin concentrtion in the plsm of offspring from HF dms (HFO), nd on the of lcttion we observed significnt elevtionintheghrelinlevelinhfo.theseresultssuggest tht in rts the crucil metbolic chnges occur from the 14th dy of life. A higher concentrtion of ghrelin, n orexigenic hormone [3], in blood t wening suggests tht offspring from HF dms my be progrmmed by ghrelin to tke in more food from n erly ge. Moreover, mothers milk my be n importnt fctor in this progrmming. The intestine of

8 8 Interntionl Journl of Endocrinology rtreminsopentotheuptkeofmternlmilk-derived mcromolecules until the ge of 21 dys, when intestinl permebility decreses to the dult level [31]. However, in our studies the reltionships between mternl hormones in milk nd their concentrtion in the blood plsm of their offspring were observed only for ghrelin in HFO on the of lcttion.ourstudyshowsthttheconcentrtionsofboth studied hormones incresed due to the mternl high-ft diet, which rises the question of its possible effect on neonte development. It ws previously shown tht gut peptides my be involved in the pthwys ffecting both ppetite nd other complex signling pthwys within the brin which my potentilly ffect behviorl nd homeosttic processes beyond ppetite regultion. Further studies re needed. We lso reported tht the mternl high-ft diet resulted in chnges in the ghrelin/obesttin rtio in both rt dms nd their offspring. Vicennti et l. [15] observed lower ghrelin/obesttin rtio in the blood plsm of obese women nd suggested tht disprte chnges in circulting ghrelin nd obesttin level represent dptive modifictions to obesity development rther thn primry defects nd tht their ltertion in circulting blood levels reflects n imblnce of regultoryfctorsormechnismsresponsible,inturn,for their metbolic processes nd ction. This hypothesis is in greement with our findings s we observed significnt decrese in the bovementioned rtio in HF dms when lcttion is ending nd in their pups in the first 14 dys of life. Our results confirm studies in humns showing tht the obesttin concentrtion in brest milk is over twofold higher thn in mternl plsm [16]. These observtions suggest tht the mmmry glnd is source of obesttin. Interestingly, the pttern of obesttin concentrtion in rt milk during lcttion is opposite to its concentrtion in the blood plsm of neontes. Our results show tht the highest plsm concentrtion of obesttin is observed in newborns compred to tht during postntl development decreses suggesting tht milk-born obesttin could be n importnt source of this hormone in the very erly stge of postntl development. Although ghrelin nd obesttin originte from the sme precursor peptide, their developmentl blood ptterns in rt pups differ. A plsm concentrtion of ghrelin in neontes incresing with ge s well s peptide concentrtion in the blood plsm of dm rts during pregnncy nd lcttion hd been previously reported [32, 33]. Similr to studies by Tylor et l. [32] we observed no chnges in BD rt dms in ghrelin concentrtion between pregnncy nd lcttion. However, similr to results shown by Shibt et l. [33] we observed significnt decrese in plsm ghrelin concentrtion in pregnnt ( of gesttion) rts in comprison to rts in the proestrus stge of the estrous cycle, wheres Tylor et l. [32] did not report ny significnt chnges. In ddition, studies on pregnnt women hve shown significnt decrese in ghrelin concentrtion during the third trimester s compred with nonpregnnt women [19]. This suggests tht reduced plsm ghrelin level my hve importnt implictions for the course of pregnncy. Shibt et l. [33] reported tht ghrelin relese by the hypothlmus ffected ghrelin plsm concentrtion during pregnncy insted of ghrelin production in the stomch. 5. Conclusions In conclusion, this study shows tht mternl high-ft diet during pregnncy nd lcttion influences ghrelin nd obesttinplsmlevelsinbothdmsndtheiroffspring. Moreover, mother milk is n importnt fctor for the trnsmission of informtion between mother nd infnt nd the mternl dietry ft content influences its ghrelin nd obesttin concentrtion. Competing Interests The uthors declre tht they hve no competing interests. Acknowledgments This reserch ws supported by Ntionl Science Center Grnt no. 211/3/D/NZ9/3697. References [1] S. C. Lngley-Evns, Nutrition in erly life nd the progrmming of dult disese: review, JournlofHumnNutritionnd Dietetics,vol.28,no.1,pp.1 14,215. [2]M.Desi,J.K.Jellymn,G.Hn,M.Bell,R.H.Lne,nd M. G. Ross, Mternl obesity nd high-ft diet progrm offspring metbolic syndrome, Americn Journl of Obstetrics nd Gynecology, vol. 211, no. 3, pp. 237.e1 237.e13, 214. [3] H. Chen, D. Simr, K. Lmbert, J. Mercier, nd M. J. Morris, Mternl nd postntl overnutrition differentilly impct ppetite regultors nd fuel metbolism, Endocrinology, vol. 149, no. 11, pp , 28. [4]C.L.White,M.N.Purper,ndC.D.Morrison, Mternl obesity is necessry for progrmming effect of high-ft diet on offspring, Americn Journl of Physiology Regultory Integrtive nd Comprtive Physiology, vol.296,no.5,pp.r1464 R1472, 29. [5]S.Rji,H.Chen,ndM.J.Morris, Mternlovernutrition impcts offspring diposity nd brin ppetite mrkersmodultion by postwening diet, Journl of Neuroendocrinology,vol.22,no.8,pp ,21. [6]M.J.MorrisndH.Chen, Estblishedmternlobesityin the rt reprogrms hypothlmic ppetite regultors nd leptin signling t birth, Interntionl Journl of Obesity, vol.33,no. 1, pp , 29. [7] R. Nogueirs, P. Pfluger, S. Tovr et l., Effects of obesttin on energy blnce nd growth hormone secretion in rodents, Endocrinology,vol.148,no.1,pp.21 26,27. [8]L.M.Seone,O.Al-Mssdi,Y.Pzos,U.Pgotto,ndF.F. Csnuev, Centrl obesttin dministrtion does not modify either spontneous or ghrelin-induced food intke in rts, Journl of Endocrinologicl Investigtion, vol.29,no.8,pp. RC13 RC15, 26. [9] P.Zizzri,R.Longchmps,J.Epelbum,ndM.T.Bluet-Pjot, Obesttin prtilly ffects ghrelin stimultion of food intke ndgrowthhormonesecretioninrodents, Endocrinology,vol. 148, no. 4, pp , 27.

9 Interntionl Journl of Endocrinology 9 [1] M. Chrtrel, R. Alver-Perez, J. Leprince et l., Comment on Obesttin, peptide encoded by the ghrelin gene, opposes ghrelin seffectsonfoodintke, Science, vol. 315, no. 5813, p. 766, 27. [11] B. Holst, K. L. Egerod, E. Schild et l., GPR39 signling is stimulted by zinc ions but not by obesttin, Endocrinology,vol. 148, no. 1, pp. 13 2, 27. [12] C.-M. Zho, M. W. Furnes, B. Stenström, B. Kulseng, nd D. Chen, Chrcteriztion of obesttin- nd ghrelin-producing cells in the gstrointestinl trct nd pncres of rts: n immunohistochemicl nd electron-microscopic study, Cell nd Tissue Reserch,vol.331,no.3,pp ,28. [13] P. Monteleone, C. Serritell, V. Mrtidis, P. Scognmiglio, nd M. Mj, Plsm obesttin, ghrelin, nd ghrelin/obesttin rtio re incresed in underweight ptients with norexi nervos but not in symptomtic ptients with bulimi nervos, The Journl of Clinicl Endocrinology & Metbolism,vol.93,no.11,pp , 28. [14] C.Shen,T.Yu,Z.H.Tng,ndK.M.Wu, Chngesinghrelin nd obesttin levels before nd fter mel in children with simple obesity nd norexi, Hormone Reserch in Peditrics, vol. 79, no. 6, pp , 213. [15] V. Vicennti, S. Genghini, R. De Isio, F. Psqui, U. Pgotto, nd R. Psquli, Circulting obesttin levels nd the ghrelin/obesttin rtio in obese women, Europen Journl of Endocrinology,vol.157,no.3,pp ,27. [16] S. Aydin, Y. Ozkn, F. Ermn et l., Presence of obesttin in brest milk: reltionship mong obesttin, ghrelin, nd leptin in lctting women, Nutrition,vol.24,no.7-8,pp ,28. [17] D. E. Cummings, J. Q. Purnell, R. S. Fryo, K. Schmidov, B. E. Wisse, nd D. S. Weigle, A preprndil rise in plsm ghrelin levels suggests role in mel initition in humns, Dibetes,vol. 5, no. 8, pp , 21. [18] P. Fossti nd L. Prencipe, Serum triglycerides determined colorimetriclly with n enzyme tht produces hydrogen peroxide, Clinicl Chemistry,vol. 28, no.1, pp , [19] Y.Mkino,H.Hosod,K.Shibtetl., Altertionofplsm ghrelin levels ssocited with the blood pressure in pregnncy, Hypertension,vol.39,no.3,pp ,22. [2] A. Sugiishi, M. Kimur, R. Kmiy et l., Derngement of ghrelin secretion fter long-term high-ft diet feeding in rts, Heptology Reserch, vol. 43, no. 1, pp , 213. [21] S. J. Kentish, G. A. Wittert, L. A. Blckshw, nd A. J. Pge, A chronic high ft diet lters the homologous nd heterologous control of ppetite regulting peptide receptor expression, Peptides,vol.46,pp ,213. [22] G. Gomez, S. Hn, E. W. Englnder, nd G. H. Greeley, Influence of long-term high-ft diet on ghrelin secretion nd ghrelin-induced food intke in rts, Regultory Peptides, vol. 173, no. 1 3, pp. 6 63, 212. [23] D. J. Flint, M. T. Trvers, M. C. Brber, N. Binrt, nd P. A. Kelly, Diet-induced obesity impirs mmmry development nd lctogenesis in murine mmmry glnd, Americn Journl of Physiology-Endocrinology nd Metbolism,vol.288,no.6,pp. E1179 E1187, 25. [24] B. J. Rolls, P. M. vn Duijvenvoorde, nd E. A. Rowe, Effects of diet nd obesity on body weight regultion during pregnncy nd lcttion in the rt, Physiology & Behvior, vol.32,no.2, pp , [25] R. E. Keesey nd M. D. Hirvonen, Body weight set-points: determintion nd djustment, Journl of Nutrition, vol. 127, supplement 9, pp. S1875 S1878, [26] P. D. Tylor, I. Y. Khn, L. Lksing et l., Uterine rtery function in pregnnt rts fed diet supplemented with niml lrd, Experimentl Physiology, vol. 88, no. 3, pp , 23. [27] C. Mendes-d-Silv, C. Á. Giriko, L. V. Mennitti, L. F. Hosoume, T. D. S. Souto, nd A. V. d Silv, Mternl high-ft diet during pregnncy or lcttion chnges the somtic nd neurologicl development of the offspring, Arquivos de Neuro-Psiquitri, vol.72,no.2,pp ,214. [28] M. E. Cerf nd E. Herrer, High ft diet dministrtion during specific periods of pregnncy lters mternl ftty cid profiles in the ner-term rt, Nutrients,vol.8,no.1,rticle25,216. [29] E. Pruszyńsk-Oszmłek, D. Szczepnkiewicz, I. Hertig et l., Obesttin inhibits lipogenesis nd glucose uptke in isolted primry rt dipocytes, Journl of Biologicl Regultors nd Homeosttic Agents,vol.27,no.1,pp.23 33,213. [3] K. G. Murphy nd S. R. Bloom, Gut hormones nd the regultion of energy homeostsis, Nture, vol. 444, no. 7121, pp , 26. [31]T.Arvol,I.Rntl,A.Mrttinen,ndE.Isoluri, Erly dietry ntigens dely the development of gut mucosl brrier in prewening rts, Peditric Reserch, vol.32,no.3,pp.31 35, [32] V. J. Tylor, M. Ptterson, M. A. Ghtei, S. R. Bloom, nd C. A. Wilson, Ghrelin nd peptide YY (PYY) profiles in gstrointestinl tissues nd the circultion of the rt during pregnncy nd lcttion, Peptides,vol.3,no.12,pp , 29. [33] K.Shibt,H.Hosod,M.Kojimetl., Regultionofghrelin secretion during pregnncy nd lcttion in the rt: possible involvement of hypothlmus, Peptides, vol. 25, no. 2, pp , 24.

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