Reproduction is sensitive to metabolic cues and the

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1 REPRODUCTION-DEVELOPMENT Obesity-Induced Hypogondism in the Mle: Premture Reproductive Neuroendocrine Senescence nd Contribution of Kiss1-Medited Mechnisms Miguel Angel Sánchez-Grrido, Frncisco Ruiz-Pino, Mri Mnfredi-Lozno, Silvi Leon, Dvid Grci-Glino, Justo P. Cstño, Rul M. Luque, Antonio Romero-Ruiz, Jun M. Cstellno, Crlos Diéguez, Leonor Pinill, Mnuel Ten-Sempere Deprtment of Cell iology, Physiology, nd Immunology; CIERobn Fisioptologí de l Obesidd y Nutrición; Deprtment of Cell iology, Physiology, nd Immunology; nd Instituto Mimonides de Investigción iomedic de Cordob (IMIIC)/Hospitl Universitrio Rein Sofi (M.A.S.-G., F.R.-P., M.M.-L., S.L., D.G.-G., J.P.C., R.M.L., A.R.-R., J.M.C., L.P., M.T.-S.), University of Córdob, Córdob, Spin; nd Deprtment of Physiology (C.D.), University of Sntigo de Compostel, Sntigo de Compostel, Spin Reproduction is sensitive to insufficient body energy reserves, especilly in femles. Metbolic regultion of the mle reproductive xis is less obvious, nd the impct of conditions of persistent energy excess hs received moderte ttention. Yet, the esclting prevlence of obesity nd the clinicl evidence of its deleterious effects on mle fertility hve rised considerble concerns. We report here phenotypic nd mechnistic studies of the reproductive impct of postntl nutritionl mnipultions (minly overnutrition) coupled to high-ft diet (HFD) fter wening. Metbolic nd hormonl nlyses in young (4 months old) nd middle-ged (10 months old) nimls reveled tht HFD cused profound metbolic perturbtions, including glucose intolernce, which were worsened by precedent postntl overfeeding; these were detectble lredy in young mles but ggrvted in 10-month-old rts. Impirment of reproductive prmeters took plce progressively, nd HFD lone ws sufficient to explin most of these ltertions, regrdless of postntl underor overnutrition. In young mles, testosterone (T) levels nd steroidogenic enzyme expression were suppressed by HFD, without compenstory increses of LH levels, which were in fct prtilly inhibited in hevier mles. In ddition, obese mles displyed suppressed hypothlmic Kiss1 expression despite low T, nd HFD inhibited LH responses to kisspeptin. Overweight nticipted some of the neuroendocrine effects of ging, such s the suppression of hypothlmic Kiss1 expression nd the decline in serum T nd LH levels. Nonetheless, HFD per se cused detectble worsening of key reproductive indices in middle-ged mles, such s bsl LH nd FSH levels s well s LH responses to kisspeptin. Our study demonstrtes tht nutritionl stress, especilly HFD, hs profound deleterious impct on metbolic nd gondotropic function s well s on the Kiss1 system nd precipittes neuroendocrine reproductive senescence in the mle. (Endocrinology 155: , 2014) Reproduction is sensitive to metbolic cues nd the mgnitude of energy reserves (1 3). This phenomenon, which llows proper coupling of body fuel stores nd fertility, is more evident in femles, where fertility is ssocited with the substntil metbolic demnds of pregnncy nd lcttion (3, 4). Notwithstnding, metbolic signls lso impct the reproductive function of mles, in which evolutionry spects of reproductive success, eg, ISSN Print ISSN Online Printed in U.S.A. Copyright 2014 by the Endocrine Society Received June 25, Accepted December 16, First Published Online Jnury 3, 2014 Abbrevitions: ARC, rcute nucleus; AUC, re under the curve; W, body weight; CD, control diet; CV, coefficient of vrition; E 2, estrdiol; HFD, high-ft diet; HPG, hypothlmic-pituitry-gondl; icv, intrcerebroventriculr; ISH, in situ hybridiztion; Kp-10, Kisspeptin ( )-NH 2 ; LL, lrge litter; NL, norml litter; OGTT, orl glucose tolernce test; PND, postntl dy; qpcr, quntittive rel-time RT-PCR; SL, smll litter. doi: /en Endocrinology, Mrch 2014, 155(3): endo.endojournls.org 1067

2 1068 Sánchez-Grrido et l Obesity-Induced Mle Hypogondism Endocrinology, Mrch 2014, 155(3): territorility nd prtner selection, re linked to dequte energy homeostsis (4, 5). The bility of metbolic cues nd fuel reserves to gte reproduction hs been well chrcterized in conditions of energy insufficiency; undernutrition, norexi, or strenuous exercise re well known to perturb puberty nd fertility (6). In ddition, forms of metbolic distress due to persistent energy excess, such s morbid obesity, lso hve cler impct on fertility (7, 8). Thus, obese men frequently disply low testosterone (T) levels nd other signs of hypogondism (7, 8). Indeed, obesity is predicted to become one of the most importnt threts for humn reproductive helth. Yet, most of the studies on the impct of obesity on reproduction provide little mechnistic insight (7), nd those ddressing its effects on mle fertility, which remin scrce, were mostly clinicl nd focused on the direct impct on testiculr T production nd sperm qulity (7, 8). Mounting evidence suggests tht conditions of severe overweight lso perturb centrl elements of the neuroendocrine reproductive (lso clled hypothlmic-pituitrygondl [HPG]) xis (7, 8). This system is governed by popultion of hypothlmic neurons, which produce the neuropeptide GnRH (6, 9). This fctor, secreted in pulstile mnner, potently stimultes pituitry gondotropin secretion nd, thereby, promotes gondl mturtion nd function (9). Hence, conditions tht perturb GnRH neurosecretion cn jeoprdize fertility. Notbly, GnRH neurons re devoid of the receptors for most of their mjor regultory signls, such s estrogen (for which they lck estrogen receptor- ), insulin, nd leptin, nd re, therefore, regulted by network of excittory nd inhibitory fferents (10), sensitive to the effects of sex steroids nd metbolic cues. Admittedly, however, GnRH neurons do express estrogen receptor-, of s yet unknown functions in this popultion, nd might be directly regulted by ndrogens, s deduced by moleculr nlyses in the GT1 7 cell line (11). Among GnRH fferents, Kiss1 neurons hve emerged s essentil gtekeepers of puberty nd fertility (9, 12 14). esides other functions, Kiss1 neurons convey, directly or indirectly, some of the regultory ctions of key metbolic signls, such s leptin nd insulin, to GnRH neurons (6, 9, 15). Yet, most of our knowledge on this front comes from studies in femles ddressing the impct of conditions of negtive energy blnce, which inhibit Kiss1 neurons (3, 9). The impct of different forms of metbolic distress on the Kiss1 system in the mle remins lrgely unexplored. Nutritionl chllenges during erly developmentl stges hve durble impct on metbolic homeostsis, so tht these dptive chnges cn permnently rewire key pthwys tht predispose to inpproprite/exggerted responses to other metbolic insults nd disese lter in life (16, 17). Frgmentry dt suggest tht the HPG xis is lso sensitive to erly nutritionl influences nd body weight chnges (18 25); gin, mjority of the studies in this re hve focused on femles. Thus, puberty nd dult reproductive function is ltered in girls with low birth weight tht subsequently develop obesity (26, 27). In ddition, mnipultions of gesttionl nd postntl feeding in rodents perturb puberty onset nd gondl mturtion nd function (25, 28, 29). Yet, the nture nd mgnitude of such chnges depend on the type nd timing of nutritionl chllenges. Although these hve been mostly studied in femles, recent dt indicte tht erly nutritionl/ metbolic ltertions, including postntl overfeeding in rts nd obesity in boys, cn perturb the timing of mle puberty (30, 31). Yet, the long-term impct of such erly metbolic unblnce on dult function of the mle HPG xis remins ill-defined. To provide novel insights into the reproductive deregultion ssocited with disrupted metbolic homeostsis in the mle, we present here series of studies tht ddress the time course nd underlying mechnisms for the impct of isolted or sequentil nutritionl (minly, obesogenic) insults on key metbolic nd reproductive prmeters in dult mle rts. Mterils nd Methods Animls, regents, nd diets Wistr rts bred in the vivrium of the University of Córdob were used. Different nutritionl mnipultions were pplied to groups of mle rts, under constnt conditions of light (14 hours) nd temperture (22 C). In ll experiments, the nimls were housed nd hndled under strictly similr conditions, llowing the integrl nlysis of dt. Experimentl procedures were pproved by Córdob University Ethicl Committee nd conducted in ccordnce with Europen Union guidelines. The experimentl nimls were fed on control diet (CD), D12450 (10% of clories from ft, 20% from protein, nd 70% from crbohydrte), or high-ft diet (HFD), D12451 (45% of clories from ft, 20% from protein, nd 35% from crbohydrte; Reserch Diets Inc). Kisspeptin ( )-NH 2 (Kp-10) ws obtined from Phoenix Phrmceuticls (elmont, CA). GnRH (L7134) ws purchsed from Sigm-Aldrich. Glucose ws from Schrlu Chemie SA. Experimentl design Different rt models of erly nutritionl mnipultion were used. Pregnnt dms were generted by mting with dult mles of proven fertility. On postntl dy (PND)-1, pups were crossfostered nd rered in 3 different litter sizes, s described elsewhere (18, 19, 32, 33): smll litter (SL) (4 pups per litter; postntl overnutrition), norml litter (NL) (12 pups per litter), nd lrge litter (LL) (20 pups per litter; postntl undernutriton).

3 doi: /en endo.endojournls.org 1069 Subgroups of SL, NL, nd LL mles were fed from PND-23 onwrd on HFD or CD d libitum. Anlyses were pplied t 2 ges (4 nd 10 months), representtive of young dult nd middle-ged rts, in keeping with previous studies (34 36). These included phenotypic indices, biochemicl/hormonl prmeters in serum, nd expression nlyses in brin/hypothlmic nd testiculr smples. Whenever possible, nmely, for noninvsive or minimlly invsive prmeters, the nimls were followed in longitudinl mnner, so tht experimentl procedures nd smpling were pplied sequentilly in the sme nimls. lood/ tissue smpling ws conducted between 9:00 AM nd noon to void the potentil interference of circdin vritions (28, 37 40). lood smpling ws conducted independent of killing by mens of jugulr venipuncture using procedures extensively vlidted in our lbortory (31, 37, 41 46). An overview of the experimentl design nd groups, s well s of the group sizes, is provided in Figure 1. Mjor vribles nd nlyticl procedures Phenotypic nlyses ody weight (W) gin, terminl W, nd dily energy intke were monitored. Dily energy intke ws clculted in 4-monthold mles from men food ingestion per dy (during 1 week), using the kiloclories per grm index provided by the mnufcturer (3.85 kcl/g for CD; 4.73 kcl/g for HFD). A irth Mle rts ody weight (g) Control (NL) Over-feeding (SL) Under-feeding (LL) Wening 1 23 Lcttion Age (dys) CD or HFD d libitum Age (dys) Scrifice Post-wening Group iochemicl nd hormonl nlyses Cholesterol nd glucose were mesured in blood smples, tken from the experimentl nimls t the 2 ges indicted bove. Serum levels of LH, FSH, T, estrdiol (E 2 ), leptin, nd insulin were ssyed by specific RIAs. Insulin nd glucose levels were mesured in specific serum smples obtined fter n overnight fst. Orl glucose tolernce tests (OGTT) Four-month-old mle rts were weighed nd fsted overnight. sl glucose levels were recorded t 8:00 to 9:00 AM, 1 hour before glucose overlod. A bolus of glucose (1 g/kg W) ws pplied by gstric gvge, nd glucose concentrtions were mesured t 20 nd 60 minutes. Integrl glucose chnges were estimted s re under the curve (AUC) during the 60 minutes, clculted by the trpezoidl method. Due to the limited volume of blood drwn during the tests, simultneous mesurements of insulin levels could not be conducted in the sme smples. Group Size (n) Scrifice Hormonl determintions (n) NL/CD NL/HFD S L/CD S L/HFD LL/CD LL/HFD Figure 1. A, Schemtic representtion of the vrious nutritionl mnipultions nd experimentl groups included in this study. Different metbolic insults during lcttion nd fter wening re represented in chronologicl mnner long the timeline of postntl mturtion., W curves over the first 4 months (120 dys) postprtum of the different experimentl groups; W dt were clculted using vlues from ll the experimentl nimls included in the different experimentl groups (C). Note tht the gry zone in the W plots represents the initil 50 dys of postntl life; W dt from similr groups hve been included in our recent study ddressing the impct of vrious pre- nd postntl nutritionl mnipultions in the timing of mle nd femle puberty (31). C, Numbers of nimls included in ech experimentl group, nd the miniml numbers of independent smples for hormonl determintions in the study re included. C Kisspeptin stimultion tests The LH-relesing bility of low dose of Kp-10 ws monitored. To llow delivery of Kp-10 into the lterl cerebrl ventricle, the rts were implnted with intrcerebroventriculr (icv) cnnule, s described previously (45 47). A dose of 50 pmol/rt ws used to evoke detectble but submximl stimultion of the gondotropic xis (45, 47), thus llowing discrimintion of subtle chnges in sensitivity. lood smples were obtined before (0 minutes) nd t 15, 60, nd 120 minutes fter Kp-10 by jugulr venipuncture, following well-vlidted procedures t our lbortory. esides individul hormonl levels, AUC responses were clculted over the 120-minute period. Kp-10 tests were implemented in ll the experimentl groups, t the 2 ges under nlysis (4-month nd 10-monthold mles), with totl of 12 tests. To limit the number of nimls used, prllel tests with vehicle were not included, nd comprisons were mde of net secretory LH responses to Kp-10 mong the different experimentl groups for ech ge. GnRH stimultion tests The LH-relesing bility of GnRH ws lso tested. Given the primry site of ction of GnRH (ie, pituitry), systemic dministrtion of GnRH ws pplied. Rts were subjected to ip injection of 1 g/rt GnRH, nd blood smples were obtined before (0 minutes) nd t 15 nd 60 minutes fter GnRH, following procedures similr to those of Kp-10 tests. AUC responses were clculted over the 60-minute period. GnRH tests were selectively implemented in middle-ged mles from ll the experi-

4 1070 Sánchez-Grrido et l Obesity-Induced Mle Hypogondism Endocrinology, Mrch 2014, 155(3): mentl groups, with totl of 6 tests. To limit the number of nimls used, prllel tests with vehicle were not included, nd comprisons were mde of net secretory LH responses to GnRH mong the different groups. Expression nlyses rin nd/or testiculr smples were dissected immeditely upon euthnsi of subsets of nimls from the different experimentl groups t the 2 ges under nlysis, nd tissues were processed for quntittive rel-time RT-PCR (qpcr) or in situ hybridiztion (ISH). For qpcr nlyses, tissues were snp-frozen in liquid N 2 nd stored t 80 C until used for RNA isoltion. Hypothlmic blocks were excised from the nimls s described in previous references (39, 46). For ISH, whole-brin specimens were excised nd llowed to grdully freeze by plcing upside-down on dry ice. Tissues were stored t 80 C until used. Hormonl nd metbolic mesurements Serum LH nd FSH levels were mesured using RIA kits from the Ntionl Hormone nd Peptide Progrm. Rt LH- I-10 nd FSH-I-9 were lbeled with 125 I using Iodo-gen tubes (Pierce). Hormone concentrtions were expressed using reference preprtions LH-RP-3 nd FSH-RP-2. For both hormones, the intr- nd interssy coefficients of vrition (CV) were 8% nd 10%, respectively. Serum T levels were ssyed using RIA kits from ICN iomedicls, Inc. The sensitivity of the ssy ws 0.1 ng/ml, nd the intr- nd interssy CV were 5% nd 8%, respectively. Circulting estrdiol (E 2 ) levels were determined using commercil ultrsensitive RIA kit from eckmn Coulter. The sensitivity of the ssy ws 2.2 pg/ml, nd the intr- nd interssy CV were 8.9% A C ody weight (g) Leptin (ng/ml),,b D E F,,b E nergy intke (K c l/rt/dy) Ins ulin (ng/ml) Figure 2. A F, W (A), dily energy intke (), nd serum cholesterol (C), leptin (D), insulin (E), nd bsl glucose (F) levels in young dult (4-month-old) mle rts, submitted or not to postntl overnutrition during lcttion (SL vs NL) nd/or HFD fter wening (HFD vs CD) in 4 different experimentl groups: NL/CD, NL/HFD, SL/CD, nd SL/HFD. Note tht insulin nd glucose levels were mesured in specific serum smples obtined fter n overnight fst. Sttisticlly significnt differences were ssessed by two-wy ANOVA to nlyze the effects of litter size nd diet nd their interctions. When significnt differences were found, the dt were further nlyzed using Newmn-Keuls tests to identify simple effects., P.01, effect of HFD;, P.05, effect of litter size; b, P.05, interction of HFD/litter size., C holes terol (mg/dl ) sl glucose (mg/dl) nd 12.2%, respectively. Serum leptin nd insulin levels were mesured using RIA kits from Linco Reserch; the sensitivity of the ssys were 0.5 (leptin) nd 0.1 (insulin) ng/ml. Cholesterol levels were mesured using colorimetric ssy from Químic Clínic Aplicd SA; intr-ssy CV ws 2%. Glucose levels were ssyed using glucometer (ACCU- CHEK) form Aviv (Roche Dignostics). RNA mesurements by qpcr Hypothlmic nd testiculr smples were processed for totl RNA isoltion using TRIsure (IOLINE). Two-microgrm RNA smples were used for the reverse trnscriptse rection (io-rd Lbortories). Rel-time RT-PCR ws performed using the icycler iq rel-time PCR system (io-rd). Hypothlmic Kiss1 s well s testiculr StAR, P450ssc, nd 17-HSD expression ws mesured using specific primer pirs (Supplementl Tble 1, published on The Endocrine Society s Journls Online website t mplifiction of 240-bp frgment of S11 ribosoml protein mrna ws used s internl control. Clcultion of reltive expression levels of the trgets ws conducted bsed on the cycle threshold (C T ) method, s described in detil elsewhere (37). In situ hybridiztion ISH ws pplied to brin specimens to quntittively ssess the levels of Kiss1 mrna within the hypothlmus, with ntomicl resolution; specil focus ws mde in the nlysis of the rcute nucleus (ARC), s the mjor reported site for expression of Kiss1 in the mle brin (48). To comply with the technicl demnds of the procedure nd to llow simultneous nlysis of ll the sections, brins were collected from representtive individuls of the groups of extreme metbolic conditions: NL/CD vs SL/HFD t the 2 ges (4 nd 10 months old) under nlysis,, resulting in totl of 4 groups. Individuls (n 5 per group) were rndomly ssigned mong the members of ech group. From ech brin, 5 sets of coronl plne sections 20 m thick were generted nd thw mounted in SuperFrost Plus slides (VWR Scientific). Stndrd procedures of tissue collection were pplied, strting in fixed coordinte in the rostrl hypothlmic re up to the ARC to encompss equivlent res including nterior nd medil portions of this nucleus, where Kiss1 neurons re bundntly locted (9, 12, 13). A rdiolbeled Kiss1 ntisense riboprobe ws generted using T7 RNA polymerse (io-rd) nd Kiss1 cdna templte contining the T7 polymerse region, s described elsewhere (37). Rdiolbeled RNA probes were synthesized by dding the following ingredients in volume of 25 L: 250 Ci [ 33 P]UTP (Perkin-Elmer), 1 g of templte product; 0.5mM ratp, rctp, nd rgtp; nd 40 U T7 RNA polymerse. Residul DNA ws di-

5 doi: /en endo.endojournls.org 1071 gested with 4 U deoxyribonuclese (io-rd), nd the rection ws terminted by ddition of 2 L 0.5M EDTA. The tissue sections were hybridized nd wshed, s described previously (37, 40, 49). Hybridiztion ws performed during 16 hours t 55 C. Hybridiztion buffer contined 0.03 pmol/ml of rdiolbel. After hybridiztion nd wshing, slides were dipped in Kodk utordiogrphy emulsion-type NT. The sections were developed nd fixed following the mnufcturer instructions (Kodk). For nlysis, 35 to 40 sections from ech niml (10 slides; 4 sections per slide) were evluted, with 5 nimls per group. Presenttion of dt nd sttistics Metbolic nd hormonl determintions were conducted in duplicte, with miniml totl number of 10 individul determintions per group (see Figure 1); the miniml number of smples per group ws set on the bsis of our previous dt, using similr experimentl procedures (31) nd priori power clcultions, to ensure robust estimtions of significnt differences mong the experimentl groups. RNA nlyses were performed in duplicte from t lest 5 independent smples per group. All dt re expressed s the men SEM. Results were nlyzed using Student s t test (when 2 groups were compred) or two-wy ANOVA, for the nlysis of the effects of the 2 mjor vribles under study (litter size nd diet) nd their interctions. For ANOVA, when significnt differences were found (P.05), the dt were further nlyzed through A Glucose (mg/dl) Time (min) C AUC - Glucose Glucose (mg/dl),,b post hoc comprison, using Newmn-Keuls tests, to identify simple effects, in keeping with previous studies (50 52). Significnce level ws set t P.05 (Prism GrphPd version5.0 softwre). Results Impct of nutritionl mnipultions on metbolic nd reproductive prmeters in young mles Postntl overfeeding (SL) cused persistently elevted W vlues (vs control NL groups) from PND-10, wheres postntl underfeeding (LL) decresed W from the infntile period onwrd. HFD fter wening cused n increse of W in SL (from PND-70) nd NL (from PND-50) mles, wheres LL mles fed on n HFD did not disply higher W thn the respective CD mles, except for modest elevtion t PND-110. W curves in SL/CD mles were similr to those of NL/HFD mles from PND-70 onwrd (Figure 1). Note tht W curves between PND-1 nd PND-50 from similr experimentl groups re lso included in lrger study of our group, focusing on the impct of different forms of erly nutritionl stress on mle nd femle puberty (31). Anlyses in 4-month-old nimls confirmed the bove trends; postntl overfeeding resulted in n increse in W nd dily energy intke t PND-120, which were 10% higher thn in corresponding NL controls. In ddition, HFD cused significnt elevtion of W nd dily clorie intke, with n incrementl Time (min) Figure 3. OGGTs in young dult mle rts, submitted or not to postntl overnutrition during lcttion (SL vs NL) nd/or HFD fter wening (HFD vs CD) in 4 different experimentl groups: NL/CD, NL/HFD, SL/CD, nd SL/HFD. The nimls received n orl bolus of glucose (1 g/kg W), nd blood glucose levels were mesured t 0 minutes (before) nd 20 nd 60 minutes fter glucose dministrtion. oth time-course dt (A nd ) nd integrl glucose levels, estimted s AUC by the trpezoidl rule (C), re presented. For AUC dt, sttisticlly significnt differences were ssessed by two-wy ANOVA to nlyze the effects of litter size nd diet nd their interctions. When significnt differences were found, the dt were further nlyzed using Newmn-Keuls tests to identify simple effects., P.01, effect of HFD;, P.05, effect of litter size; b, P.05, interction of HFD/litter size. interction of both nutritionl insults, so tht SL/HFD mles displyed 25% higher W nd dily clorie intke thn corresponding NL/CD controls (Figure 2). Postntl overnutrition per se did not cuse detectble ltertions in bsl cholesterol, leptin, or insulin levels but cused sustined elevtion in bsl glucose. In contrst, HFD significntly elevted cholesterol, leptin, nd glucose levels, with synergistic interction with postntl feeding in the cse of leptin. Likewise, glucose concentrtions were significntly higher in SL/HFD nimls thn in the other groups. Interestingly, despite elevtion of bsl glucose, HFD tended to decrese insulin concentrtions, regrdless of the pttern of

6 1072 Sánchez-Grrido et l Obesity-Induced Mle Hypogondism Endocrinology, Mrch 2014, 155(3): postntl feeding, lthough the decline in SL nimls ws not sttisticlly significnt (Figure 2). Glucose homeostsis ws further explored in young dult mles by OGTT. Despite the elevtion of bsl glycemi, postntl overnutrition did not cuse detectble ltertion in the tolernce to orl glucose overlod. In contrst, HFD-treted groups displyed impired glucose tolernce, s reflected by n increse in AUC glucose levels fter n orl overlod. However, lthough HFD cused 15% increse in AUC glucose levels in control NL mles during n OGTT, these were 45% higher in SL mles fed n HFD; nmely, 3-fold higher glucose intolernce thn in NL/HFD mles, therefore pointing out n interction between postntl overfeeding nd HFD to mrkedly worsen the tolernce to glucose overlod (Figure 3). Erly undernutrition (LL) cused sustined 10% to 15% decrese in W nd energy intke, nd n HFD fter wening modestly but significntly incresed both prmeters. Although W in LL/HFD mles remined lower thn in the corresponding control (NL/CD) group, dily clorie intke tended to be higher thn in controls. Postntl undernutrition per se did not lter ny of the bsl metbolic prmeters t PND-120 (cholesterol, leptin, insulin, or glucose). However, HFD significntly worsened A LH (ng/ml) FSH (ng/ml) C LH (ng/ml) Time (min) AUC - LH ll metbolic indices in LL mles, except for decrese in bsl insulin levels, despite the persistently elevted glycemi. OGTT in these groups reveled tht postntl undernutrition prevented the impct of HFD on glucose intolernce (Supplementl Figure 1). Postntl overfeeding ws ssocited with modertely higher bsl LH levels vs NL controls t PND-120, without vritions in FSH concentrtions. LH responses to Kp-10, estimted by the time-course profiles nd integrl (AUC) secretory mss over 120 minutes fter icv injection of the peptide, were not ffected by postntl overnutrition. In contrst, HFD significntly suppressed bsl LH levels in SL mles nd cused n 25% drop in globl (AUC) LH responses to Kp-10 in control (NL) nimls (Figure 4). In ddition, lthough serum T concentrtions were not ffected by postntl overfeeding, T levels were significntly suppressed by HFD in both NL nd SL rts. A similr profile ws detected for testiculr expression of key steroidogenic fctors, so tht HFD significntly decresed the expression of StAR, P450scc, nd 17 -HSD genes in NL mles, wheres SL rts hd lower StAR nd P450scc expression, lthough the ltter did not rech sttisticl significnce (Figure 5). Serum E 2 levels were not ltered by postntl overfeeding or HFD lone, but cir- Responses to Kp-10 * LH (ng/ml) Time (min) Figure 4. A nd, Serum LH nd FSH levels in bsl blood smples from young dult (4-month-old) mle rts, submitted or not to postntl overnutrition during lcttion (SL vs NL) nd/or HFD fter wening (HFD vs CD) in 4 different experimentl groups: NL/CD, NL/HFD, SL/CD, nd SL/ HFD. C, LH responses to n icv bolus of Kp-10 in the sme experimentl groups. Animls were icv injected with single dose of Kp-10 (50 pmol/ rt), nd blood smples were obtined t 0 minutes (before) nd 20, 60, nd 120 minutes fter Kp-10 dministrtion. oth time-course dt nd integrl LH levels, estimted s AUC by the trpezoidl rule, re shown. For bsl levels nd AUC dt, sttisticlly significnt differences were ssessed by two-wy ANOVA to nlyze the effects of litter size nd diet nd their interctions. When significnt differences were found, the dt were further nlyzed using Newmn-Keuls tests to identify simple effects. *, P.05;, P.01, effect of HFD;, P.05, effect of litter size.

7 doi: /en endo.endojournls.org 1073 A Tes tos terone (ng/ml) E s trdiol (pg/ml ) * C D E % Δ StAR /S11 culting E 2 concentrtions were prtilly suppressed in SL/HFD mles; ie, fter combintion of both obesogenic insults (Figure 5). Postntl undernutrition cused sustined elevtion of LH nd FSH levels t PND-120, wheres HFD significntly decresed bsl LH concentrtions in LL mles. Postntl underfeeding persistently reduced bsl T, but not E 2, levels, lthough ddition of HFD did not cuse further decreses in T or E 2 t PND-120 (Supplementl Figure 2). Likewise, testiculr gene expression of P450scc nd, to lesser extent, StAR, were lower in LL mles; yet, in contrst to controls, HFD filed to ffect the expression of those trgets in LL nimls (dt not shown). Integrl AUC LH responses to Kp-10 were not ffected by postntl undernutrition, but HFD significntly dmpened net LH responses to Kp-10 in LL mles (Supplementl Figure 2). Finlly, ISH nd qpcr were implemented t PND-120 to detect chnges in hypothlmic Kiss1 mrna levels cused by extreme W/metbolic conditions, nmely, postntl overfeeding coupled with HFD fter wening (SL/HFD). In keeping with previous studies (9, 48), prominent popultion of Kiss1-expressing neurons ws detected in the ARC, regrdless of the nutritionl mnipultion, wheres expression in the rostrl re ws negligible (48). ISH nlyses reveled 50% drop in Kiss1 levels in the ARC of SL/HFD vs control NL/CD vlues, % Δ P450s cc/s 11 Figure 5. A E, Serum T (A) nd estrdiol E 2 () levels s well s testiculr expression of the mrnas encoding key steroidogenic fctors, nmely StAR, P450scc, nd 17 -HSD (C E), in young dult (4-month-old) mle rts, submitted or not to postntl overnutrition during lcttion (SL vs NL) nd/or HFD fter wening (HFD vs CD) in 4 different experimentl groups: NL/CD, NL/HFD, SL/CD, nd SL/HFD. Sttisticlly significnt differences were ssessed by two-wy ANOVA to nlyze the effects of litter size nd diet nd their interctions. When significnt differences were found, the dt were further nlyzed using Newmn-Keuls tests to identify simple effects. *, P.05;, P.01, effect of HFD. % Δ 17β-HSD/S11 reduction tht ws confirmed by qpcr in hypothlmic frgments (encompssing the ARC) of young dult SL/ HFD mles (Figure 6). Impct of nutritionl mnipultions on metbolic nd reproductive prmeters in middle-ged mles Middle-ged (10-month-old) mles were on verge 15% hevier thn 4-month-old nimls. At this ge, terminl W tended to be higher ( 18% increse) in SL rts thn in NL/CD mles. HFD per se cused significnt 11% increse in W, which esclted to 27% rise by the combintion of postntl overnutrition nd HFD. Tenmonth-old SL rts displyed nonsignificnt 25% elevtion of bsl leptin levels without chnges in bsl insulin, wheres HFD ws unble to lter bsl leptin or insulin, except for detectble increse in leptin levels when combined with postntl overnutrition (Figure 7). Of note, control middle-ged mles showed 300% increse in leptin levels vs young dult nimls, wheres insulin levels were not ffected by ge. Yet, bsl glucose levels were significntly incresed in ll groups vs corresponding 4-month-old vlues, regrdless of the diet (dt not shown). On the other hnd, erly undernutrition cused sustined 10% decrese in W in 10-month-old mles, wheres HFD significntly incresed W vlues in LL mles. In ddition, middle-ged LL/CD mles displyed lower bsl levels of leptin nd insulin vs NL/CD nimls,

8 1074 Sánchez-Grrido et l Obesity-Induced Mle Hypogondism Endocrinology, Mrch 2014, 155(3): A 3V 3V NL/CD 4-mo NL/CD 10-mo wheres HFD significntly elevted leptin concentrtions nd tended to increse bsl insulin levels in LL rts (Supplementl Figure 3). Reproductive nlyses in middle-ged mles demonstrted tht, wheres control NL/CD mles hd LH levels similr to young dult nimls, postntl overfeeding tended to decrese LH concentrtions. In turn, FSH levels incresed with ge nd were not ffected by postntl overfeeding. HFD hd substntil impct on gondotropin levels in 10-month-old mles; LH nd FSH concentrtions were reduced by HFD regrdless of the postntl diet. In ddition, bsl T levels were suppressed by ge in ll groups, nd HFD further decresed circulting T concentrtions in control middle-ged mles (Figure 7). Hypothlmic expression of Kiss1 declined with ge, so tht Kiss1 mrna levels in the ARC of 10-month-old NL/CD rts were 40% of those of young dult controls. However, HFD did not further decrese Kiss1 expression in middle-ged mles (Figure 6). Finlly, postntl underfeeding per se lowered LH nd T levels in 10-month-old rts without chnges in FSH, wheres HFD further decresed LH concentrtions in middle-ged LL mles (Supplementl Figure 3). Net (AUC) LH responses to icv injection of Kp-10 were lso explored in middle-ged mles. LH responses to Kp-10 were not ffected by postntl overfeeding, wheres HFD significntly decresed net secretory responses in NL nd SL groups. However, lthough the drop 3V 3V SL/HFD 4-mo S L/HFD 10-mo Figure 6. A, Representtive ISH photomicrogrphs of Kiss1 mrna-expressing neurons in the nterior region of the ARC in young dult (4-month-old) nd middle-ged (10-month-old) mle rts, submitted or not to postntl overnutrition during lcttion (SL vs NL) nd HFD fter wening (HFD vs CD). Anlyses were specificlly conducted in groups with extreme W differences t the 2 ges, nmely NL/CD vs SL/HFD., Quntifiction of Kiss1 mrna expression, estimted s grin density in the ARC region subjected to nlysis, for the 2 ges. Sttisticlly significnt differences were ssessed by two-wy ANOVA to nlyze the effects of overweight (SL/HFD) nd ge nd their interctions. When significnt differences were found, the dt were further nlyzed using Newmn-Keuls tests to identify simple effects., P.01, effect of SL/ HFD;, P.05, effect of ge. C, Confirmtion of quntittive chnges detected by ISH by mens of qpcr of Kiss1 mrna levels in the ARC of young dult NL/CD vs SL/HFD mles., P.01 vs control NL/CD young mles (Student s t test). C Kiss1 mr NA (% controls) % Δ Kiss1/S11 4-mo 10-mo in LH responses to Kp-10 ws 55% in NL mles, the decrese in integrl LH responses to Kp-10 ws 35% in SL rts. These profiles were compred with responses to GnRH in the sme groups. LH responsiveness to GnRH ws not ltered in SL mles, wheres HFD modertely inhibited LH responses to GnRH only in the NL group. However, the mgnitude of such decline ( 25% in NL/HFD rts) ws much more modest thn the drop induced in Kp-10 responses. In fct, SL/HFD mles did not show ny decline in LH responses to GnRH, despite the substntil reduction in LH responsiveness to Kp-10 (Figure 8). On the other hnd, wheres postntl underfeeding did not lter LH responses to Kp-10 in middle-ged mles, HFD evoked profound ( 55%) suppression of LH secretory mss fter Kp-10, which lrgely exceeded tht induced by HFD on GnRH responsiveness (only 25% drop; Supplementl Figure 3). Discussion Prevlence of obesity nd other metbolic disorders, such s type 2 dibetes, hs esclted drmticlly during the lst decdes, obesity being one of the most importnt threts for popultion helth (8). Compelling evidence hs documented tht obese ptients disply reproductive bnormlities. However, reproductive-metbolic interctions hve been more exhustively studied in femles, even though evidence suggests tht the mle reproductive xis is lso sensitive to metbolic hormones nd nutritionl cues (4, 5, 53). Indeed, reproductive impirment nd decresed testiculr function hve been reported in obese men (7, 8). Yet, little ttention hs been pid to elucidte the contribution of centrl mechnisms to this phenomenon. Notwithstnding, it ws recently suggested tht the risk of developing centrl hypogondism in men is 8-fold higher with MI 30 kg/m 2 (54). In ddition, most of the studies published to dte were cross-sectionl nd hence filed to provide insights into the time course for insturtion of reproductive deficits nd the influence of erly vs lte metbolic insults, relevnt issue given the growing concerns on the erly origins of dult metbolic diseses (55) nd the suspected role of erly developmentl

9 doi: /en endo.endojournls.org 1075 A C, ody weight (g) Leptin (ng/ml) Ins ulin (ng/ml) LH (ng/ml) D E F events in the pthogenesis of vrious reproductive disorders (56 58). However, lthough metbolic stress in utero hs been linked to perturbtions of the timing of femle FSH (ng/ml) Figure 7. A F, W (A) nd serum leptin (), insulin (C), LH (D), FSH (E), nd T (F) levels in middle-ged (10-month-old) mle rts, submitted or not to postntl overnutrition during lcttion (SL vs NL) nd/or HFD fter wening (HFD vs CD) in 4 different experimentl groups: NL/CD, NL/HFD, SL/CD, nd SL/HFD. Control vlues from young dult, 4-month-old NL/CD mles re denoted for reference purposes s dotted lines in ech grph. Sttisticlly significnt differences were ssessed by two-wy ANOVA to nlyze the effects of litter size nd diet nd their interctions. When significnt differences were found, the dt were further nlyzed using Newmn-Keuls tests to identify simple effects., P.01, effect of HFD;, P.05, effect of litter size. A AUC - LH Responses to Kp-10 AUC - LH Responses to GnRH Figure 8. A, LH responses to n icv bolus of Kp-10 from middle-ged (10-month-old) mle rts, submitted or not to postntl overnutrition during lcttion (SL vs NL) nd/or HFD fter wening (HFD vs CD) in 4 different experimentl groups: NL/CD, NL/HFD, SL/CD, nd SL/HFD. Animls were icv injected with single dose of Kp-10 (50 pmol/rt), nd blood smples were obtined t 0 minutes (before) nd 20, 60, nd 120 minutes fter Kp-10 dministrtion. Integrl LH levels, estimted s AUC by the trpezoidl rule, re shown., For comprtive purposes, LH responses in the sme experimentl groups to n ip bolus of GnRH. The only difference is tht blood smpling ws conducted t 0, 20, nd 60 minutes fter GnRH dministrtion. Sttisticlly significnt differences of AUC dt were ssessed by twowy ANOVA to nlyze the effects of litter size nd diet nd their interctions. When significnt differences were found, the dt were further nlyzed using Newmn-Keuls tests to identify simple effects., P.01, effect of HFD. Tes tos terone (ng/ml ) puberty nd ovrin disorders (27, 56, 59), the impct of erly nutritionl insults on metbolic nd reproductive helth in mles remins lrgely unexplored. Our study discloses the prtilly divergent impct of postntl overfeeding nd HFD on metbolic nd reproductive indices in the mle. These insults were both considered obesogenic, becuse they cused overweight, vribly coupled to hyperleptinemi nd/or indices of perturbed glucose homeostsis. Indeed, these nutritionl mnipultions hd durble effects per se on W nd dily energy intke, wheres they synergisticlly intercted or summted to worsen key metbolic prmeters, such s bsl leptin nd glucose levels nd glucose tolernce. These effects somewht nticipted the deteriortion of metbolic sttus observed with ging, when W nd bsl glucose nd leptin levels were incresed in ll groups. Of note, lthough postntl overnutrition lone did not impir vrious metbolic indices, it predisposed to exggerted dverse responses, such s perturbed glucose homeostsis, to lter nutritionl chllenges. Admittedly, full interprettion of some of the dverse metbolic responses, such s glucose intolernce to n orl overlod, would require simultneous mesurement of insulin levels, which ws not possible in our study due to limited smple vilbility during the OGTT. In ny event, our findings em-

10 1076 Sánchez-Grrido et l Obesity-Induced Mle Hypogondism Endocrinology, Mrch 2014, 155(3): phsize the importnce of erly nutritionl hbits s mjor determinnt of dult metbolic helth nd stress the importnce of preventing ctions of child obesity. Substntil deteriortion of different reproductive indices, such s T nd LH levels, s well s hypothlmic Kiss1 expression nd LH responses to Kp-10, were observed in overweight groups, but, t lest for hormonl levels, this ws mostly ttributble to HFD, wheres postntl overnutrition lone tended to lower LH levels only in middle-ged SL mles. Recent studies from our group hve disclosed tht postntl overfeeding dvnces mle puberty, wheres HFD hd no effect (31). In contrst, exposure to HFD cused significnt decline in T levels lredy in young dult mles (present results), response tht ws ssocited with lower expression levels of key limiting fctors of testiculr steroidogenesis (60), such s StAR nd P450scc. The decrese in circulting T levels ws not ssocited with compenstory increses in circulting gondotropins, which would be expected in cse of primry testiculr impct of HFD; this suggests centrl inhibitory component. In fct, HFD lowered bsl LH levels in SL mles nd decresed LH responsiveness to Kp-10 in control NL mles. Furthermore, young obese SL/HFD mles displyed significntly decresed Kiss1 mrna levels in the ARC, despite the lower T levels, which should hve brought bout compenstory increse in Kiss1 nd LH levels (48). Although dditionl prmeters of gondl function, such s inhibin- levels, might hve provided dditionl vluble informtion (61), the bove observtions strongly suggest tht overweight hs n inhibitory impct on centrl elements of the mle HPG xis even t young ge. The possibility tht the observed suppression in ARC Kiss1 expression might be cused by rise in circulting E 2 levels ppers unlikely, becuse E 2 concentrtions were ctully decresed in young SL/HFD mles, finding tht reinforces the hypothesis of primry centrl inhibitory impct of overweight on the hypothlmic Kiss1 system. Intriguingly, we filed to detect stte of hyperestrogenism in young dult mles subjected to obesogenic insults, despite the proven cpcity of ft tissue to convert drenl nd gondl ndrogens into E 2 (62), nd present evidence for incresed diposity in our models. One tenble explntion is tht given the centrl impct of obesogenic mnipultions, especilly HFD, on the HPG xis, the persistent reduction of circulting T levels might cuse depletion of the substrte for dipose romtiztion, thus preventing, t lest in young mles, n overt increse in serum E 2 levels. Due to limited smple vilbility, whether E 2 levels ctully incresed in our overweight middle-ged mles could not be evluted in the present study. The HFD nticipted the decline in reproductive prmeters observed with (incipient) ging. Thus, middleged rts displyed signs of neuroendocrine reproductive senescence lredy t 10 months of ge, s evidenced by lower circulting levels of T nd hypothlmic expression of Kiss1, even in the bsence of obesogenic insults. The bove ltertions were recpitulted by overweight in young mles. Moreover, HFD hd discernible negtive impct on specific reproductive prmeters, such s bsl LH nd FSH levels nd LH responses to Kp-10, lso in middle-ged mles. These observtions further document the deleterious effects of HFD, which ppers not only to nticipte but lso to ggrvte mle reproductive senescence, t lest from neuroendocrine perspective. Admittedly, other (eg, behviorl) spects of reproductive ging were not directly ddressed in our study. Our study revels the potentil involvement of perturbed function of the hypothlmic Kiss1 system in the pthogenesis of the gondotropic ltertions of obese mles. The contribution of centrl defects in this phenomenon remined scrcely ddressed, nd to our knowledge, studies in this re hd been conducted only in femles (63, 64). Moreover, mouse strins prone to develop obesityinduced infertility, such s the DA/2J mouse, were previously used (63, 64), thus limiting the pthophysiologicl trnsltion of those studies. Our work, using strin of robust reproductive cpcity, such s Wistr rts, conclusively demonstrtes centrl impct of obesity on the reproductive brin, which is conducted, t lest prtilly, by trgeting the kisspeptin system t 2 mjor sites: 1) expression of Kiss1 gene in the ARC nd 2) LH responsiveness to kisspeptin, likely t the level of GnRH neurons. Regrding the former, our study extends nd complements one previous report in DA/2J femle mice showing tht HFDinduced obesity decreses hypothlmic Kiss1/kisspeptin expression (64). Notbly, we previously documented tht overfeeding during lcttion increses Kiss1 expression in the hypothlmus of femle rts during puberty (29). In ddition, previous qpcr nlyses demonstrted tht shorter ( 3 weeks) protocols of HFD resulted in enhnced hypothlmic Kiss1 levels in femle rts (65). Although sex differences in responses to overfeeding/hfd re lso possible, ltogether, the bove observtions suggest dul effect of overweight on Kiss1 expression, nmely, n initil increse followed by suppression in the long term, which would explin the complex effects of obesity on the reproductive xis long the lifespn. Admittedly, the underlying mechnisms for overweight-induced suppression of hypothlmic Kiss1 expression re yet to be exposed. In this context, the contribution of peripherl metbolic hormones nd centrl inflmmtory meditors, trgeting

11 doi: /en endo.endojournls.org 1077 neurons nd/or glil cells, in this phenomenon is currently under investigtion in our lbortory. Our study is lso the first to document the deleterious impct of HFD on the mgnitude of the LH response to Kp-10. This inhibitory effect ws much higher thn on GnRH responsiveness. This observtion suggests tht significnt component of the decline in gondotropic responses to kisspeptin in overweight mles is due to primry ltertion upstrem of the pituitry nd rgues in fvor of direct impirment of GnRH neurons s mjor trgets of kisspeptins (9). In fct, very recent study suggested tht the metbolic syndrome cn impir GnRH neurons in rbbits (66). On the other hnd, previous studies in rodents demonstrted tht shorter protocols of nutritionl mnipultion, such s cute fsting, did not suppress, but rther ugmented, LH responsiveness to Kp-10 (67, 68). Likewise, recent studies in young obese, type 2 dibetic ptients documented robust LH responses to Kp- 10, despite the previling hypogondotropic stte (69). Thus, the responsiveness to kisspeptins my be lso vribly ffected by obesity, depending on its durtion nd mgnitude. In this context, it will be interesting to explore whether obese/dibetic ptients would disply decresed responses to kisspeptins in the long term. Our study lso ddressed the impct of erly undernutrition on different reproductive indices in mles nd its potentil interply with HFD. This llowed us to ddress the potentil protective effect of persistent lowering of W on the reproductive impct of HFD nd to evlute whether erly dverse metbolic conditions my worsen metbolic nd reproductive helth lter in life. To our knowledge, lthough the impct of gesttionl subnutrition on vrious indices of ovrin function hd been previously evluted (25), nlyses on the consequences of erly undernutrition on the dult mle HPG xis hd not been reported. In our study, postntl undernutrition persistently lowered W, energy intke, nd leptin levels nd pprently protected from HFD-induced glucose intolernce. Nonetheless, HFD hd discernible impct on different metbolic prmeters lso in LL mles, including bsl leptin nd glucose levels. Additionlly, postntl underfeeding ffected different reproductive prmeters, lthough some of the effects were dependent on ge. For instnce, LL mles displyed persistently reduced T levels, yet bsl LH concentrtions were incresed in young LL mles, suggesting compenstory dpttion to low T cused by primry ltertion of testiculr development nd/or function. Nonetheless, LL mles developed stte of hypogondotropic hypogondism (low LH/low T) with ging, which is comptible with puttive impct lso t centrl levels. In ddition, HFD lso hd mrked effect on different reproductive indices in LL mles, s it decresed bsl LH levels nd LH responses to Kp-10. These observtions stress the impct of HFD on the centrl components of the gondotropic xis. In fct, comprison of our dt on LH responsiveness with Kp-10 nd GnRH strongly suggest hypothlmic component for the impct of HFD in LL mles tht is likely to include the perturbtion of kisspeptin signling or ctions in GnRH neurons. esides its obvious reproductive effects, T hs recently emerged s key metbolic hormone (70). Accordingly, fctors ffecting T levels my hve n importnt deleterious impct not only in reproductive but lso in metbolic nd crdiovsculr helth (70). Our study, whose mjor results re summrized in Supplementl Tble 2, thoroughly document the metbolic nd reproductive consequences, in young dulthood nd middle ge, of isolted or sequentil nutritionl chllenges in mle rts. Our nlyses not only chrcterize the biochemicl nd hormonl signture of obesity-induced mle hypogondism but lso dissect out the differentil contribution of vrious forms of nutritionl stress tking plce t different developmentl windows. In ddition, our dt re the first to provide mechnistic insight for the centrl impct of HFD, in combintion with postntl overfeeding, on the hypothlmic Kiss1 system, s mjor physiologic regultor of the mle gondotropic xis (9). Our study will help to explin differences in susceptibility nd to define puttive phrmcologicl interventions in the mngement of obesity-induced hypogondism of the mle. Acknowledgments We re indebted to Dr Enrique Aguilr, Jun Ro, nd Frncisco Gytn, from the University of Cordob (Spin), for helpful comments nd suggestions during preprtion of this mnuscript. The superb technicl ssistnce of the members of our tem t the University of Cordob nd IMIIC, An elén Rodríguez nd Antoni Sánchez-Arroyo, is cordilly pprecited. Address ll correspondence nd requests for reprints to: Mnuel Ten-Sempere, Physiology Section, Deprtment of Cell iology, Physiology, nd Immunology, Fculty of Medicine, University of Córdob, Avenid Menéndez Pidl, Córdob, Spin. E-mil: fi1tesem@uco.es. This work ws supported by Grnts FU nd FU (Ministerio de Economí y Competitividd, Spin; cofunded with EU funds from the FEDER Progrm); Project P08-CVI (Junt de Andlucí, Spin); EU Reserch Contrct DEER FP7-ENV CIER Fisioptologí de l Obesidd y Nutrición is n inititive of Instituto de Slud Crlos III, Ministerio de Snidd, Spin. M.A.S.-G. conducted the experimentl studies, evluted the dt, nd prtilly wrote the mnuscript; F.R.-P., M.M.L., nd

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