The secretion of pituitary gonadotropins, LH and FSH, is under

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1 NEUROENDOCRINOLOGY Altertions in Hypothlmic KiSS-1 System in Experimentl Dietes: Erly Chnges nd Functionl Consequences J. M. Cstellno, V. M. Nvrro, J. Ro, R. Pined, M. A. Sánchez-Grrido, D. Grcí-Glino, E. Vigo, C. Dieguez, E. Aguilr, L. Pinill, nd M. Ten-Sempere Deprtment of Cell Biology, Physiology, nd Immunology (J.M.C., V.M.N., J.R., R.P., M.A.S.-G., D.G.-G., E.V., E.A., L.P., M.T.-S.), University of Córdo, nd CIBER Fisioptologí de l Oesidd y Nutrición, 1 Córdo, Spin; nd Deprtment of Physiology (C.D.), University of Sntigo de Compostel, 1575 Sntigo de Compostel, Spin Using long-term streptozotocin (STZ)-treted mle rts, we recently proposed tht defective function of hypothlmic KiSS-1 system is mechnisticlly relevnt for centrl hypogondotropism of uncontrolled dietes. However, the temporl pttern of such defects nd its potentil contriution to distured gondotropin secretion in the dietic femle remin so fr unexplored. To cover these issues, expression nlyses nd hormonl tests were conducted in dietic mle (1 wk fter STZ; short term) nd femle ( wk fter STZ; long term) rts. Short-term dietic mles hd lower sl testosterone levels nd decresed gondotropin responses to orchidectomy (ORX), which ssocited with significntly ttenuted post-orx rises of hypothlmic KiSS-1 mrna. Yet kisspeptin dministrtion to dietic mles ws le to cutely elicit suprmximl LH nd testosterone responses nd normlize post-orx gondotropin secretion. Long-term dietic femles showed persistent nestrus nd significntly decresed sl gondotropin levels s well s lunted LH responses to ovriectomy; chnges tht were linked to lowering of sl nd postovriectomy expression of hypothlmic KiSS-1 mrna. Moreover, despite previling gondotropin suppression, LH responses to cute kisspeptin dministrtion were fully preserved, nd even enhnced fter its repeted injection, in dietic femles. In sum, our present findings further define the temporl course nd mechnistic relevnce of ltered hypothlmic KiSS-1 system in the hypogondotropic stte of uncontrolled dietes. Furthermore, our dt provide the sis for the potentil therpeutic intervention of the KiSS-1 system s djuvnt in the mngement of distured gondotropin secretion of type 1 dietes in the femle. (Endocrinology 15: 7 79, 9) The secretion of pituitry gondotropins, LH nd FSH, is under the dynmic control of plethor of regultors of centrl nd peripherl origin tht converge nd integrte t the level of GnRH neurons t the forerin (1 3), the secretion of hypothlmic GnRH eing ultimtely responsile for the pulstile relese of oth gondotropins (1). Wheres the mechnisms responsile for the control of the gondotropic xis hs een ctively investigted nd thoroughly chrcterized during the lst decdes, our understnding of the neuroendocrine pthwys governing GnRH secretion hs drmticlly enlrged in recent yers due to the identifiction of the reproductive roles of kisspeptins nd their cognte receptor, G protein-coupled receptor 5(GPR5) ( 7). Notly, kisspeptins, fmily of peptides encoded y the ISSN Print ISSN Online Printed in U.S.A. Copyright 9 y The Endocrine Society doi: 1.11/en.-9 Received June,. Accepted Octoer,. First Pulished Online Octoer 9, KiSS-1 gene tht includes metstin nd kisspeptin-1, were initilly ctlogued s metstsis-suppressor peptides, with potentil dditionl roles in plcentl physiology (,, 1). However, the demonstrtion in lte 3 of inctivting muttions of GPR5 in ptients suffering isolted or fmilil forms of hypogondotropic hypogondism unveiled the potentil involvement of this lignd-receptor system in the regultion of essentil spects of reproductive mturtion nd function (11, 1). Indeed, during the lst yr, kisspeptins nd GPR5, which hs een now officilly ctlogued s KiSS-1 receptor (see hve een proven to ply pivotl roles in rin sexul differentition, puerty onset, feedck regultion of gondotropin secretion nd/or the environmentl nd met- Arevitions: AUC, Are under the curve; GPR5, G protein-coupled receptor 5; i.c.v., intrcereroventriculr; Kp-1, kisspeptin-1; ORX, orchidectomy; OVX, ovriectomy; STZ, streptozotocin. 7 endo.endojournls.org Endocrinology, Ferury 9, 15():7 79 Downloded from y guest on July 1

2 Endocrinology, Ferury 9, 15():7 79 endo.endojournls.org 75 olic control of fertility in diversity of species, including rodents nd primtes (, 13). Overll, KiSS-1 neurons t discrete hypothlmic nuclei hve een proposed to operte s nodl integrtors of n rry of key modultors of reproductive function, thus dictting the ctivtion or eventul inctivtion of secretion GnRH s finl output pthwy. Among their plethor of regultors, compelling evidence is mounting tht KiSS-1 neurons re cple to sense the energy stte of the orgnism, therey trnsducing metolic informtion onto the centers governing reproductive function (1 17). This previously neglected pthwy is likely to contriute to the functionl coupling etween the stte of ody energy reserves nd its reproductive cpcity; dverse metolic conditions eing commonly ssocited with defective fertility (1). Indeed, numer of experimentl studies hve now demonstrted tht conditions of negtive energy lnce, such s short-term fsting in rts nd mice, induce significnt decreses in the hypothlmic expression of KiSS-1 gene (1, 17, 19), wheres cute dministrtion of kisspeptin to fsted rts, despite the previling decrese in circulting LH levels, is le to evoke suprmximl gondotropin responses (1, ). Moreover, despite severe reduction of ody weight due to restriction in dily food intke, repeted dministrtion of kisspeptin ws sufficient to rescue the puertl ctivtion of the gondotropic xis in femle rts sumitted to chronic sunutrition (1). The nture of the signl(s) responsile for the metolic regultion of the KiSS-1 system is yet to e fully uncovered. Yet convincing evidence suggests tht the dipocytederived hormone, leptin, is cple to modulte KiSS-1 expression t the hypothlmus (15, 1), therey providing tenle mechnism for the well-known effects of leptin s gtekeeper of puerty onset nd fertility. In keeping with its profound metolic disturnces, uncontrolled dietes is frequently ssocited with reproductive dysfunction, hypogondotropic hypogondism eing common oservtion in niml models of experimentl dietes ( ). In dietic mle rts, defective function of the gondotropic xis involves decresed sl nd pulstile LH secretion, distured sensitivity to negtive feedck effects of ndrogens, reduced LH responses to gondectomy, nd defective testosterone secretion (, 3, 5 7). Likewise, dietic femle rts disply decresed sl levels of gondotropins, disruption of the positive feedck effects of estrdiol, delyed or sent preovultory LH surges, nd novultion ( 33). Wheres the mechnisms for the plethor of reproductive normlities oserved in dietes re likely to involve oth peripherl nd centrl defects, compelling evidence suggests tht filure of the neuroendocrine mechnisms governing GnRH secretion is custive for the stte of hypogondotropism detected in mle nd femle models of uncontrolled dietes (, 3). Although this contention ws recognized more thn decde go, the nture of the fferent pthwys ltered in such conditions hd remined lrgely unfolded. Using long-term streptozoticin (STZ)-treted dult mle rts, s conventionl model of type 1 dietes, we recently documented the potentil involvement of ltertions of the hypothlmic KiSS-1 system in the genertion of the stte of impired gondotropic function in such model (15). Indeed, wk fter STZ tretment, uncontrolled dietic mles not only displyed the expected decrese in circulting LH concentrtion ut lso showed significnt reduction of hypothlmic KiSS-1 mrna levels, t oth sl nd postorchidectomy conditions (15). Moreover, cute nd repeted dministrtion of kisspeptin ws sufficient to meliorte the hypogondotropic stte of long-term STZ mles, despite no dditionl metolic intervention. Altogether these oservtions suggest tht, t lest in the mle, consolidtion of stte of dietes is linked to suppression of the functionlity of hypothlmic KiSS-1 system, which likely contriutes to gondotropic filure (15). Mechnistic nlyses evidenced tht defective leptin, ut not insulin, signling t centrl levels is, t lest prtilly, responsile for such suppression of hypothlmic KiSS-1 in uncontrolled dietes (15). These findings re in keeping with recent in vivo nd in vitro dt demonstrting positive role of leptin in the control of KiSS-1 mrna expression t the hypothlmus (17, 1). Despite the ove progress in the field, some criticl fetures of the potentil dysfunction of hypothlmic KiSS-1 system in uncontrolled dietes remin lrgely unexplored. Thus, the temporl pttern of KiSS-1 defects in the dietic mle hs not een ssessed to dte. Similrly, whether similr disruption is detected in the dietic femle, nd, if so, its potentil contriution to distured gondotropin secretion remin to e determined. The ltter is especilly relevnt, given the profound impirment of gondotropic/ovultory function cused y dietes in the femle ( 33). Yet previous studies hd suggested the existence of sex differences in the mechnisms underlying the hypogondl stte in mle nd femle dietic rts (35). To cover the ove issues, expression nlyses nd hormonl tests were conducted in short-term dietic mles (1 wk fter STZ) nd long-term dietic femles ( wk fter STZ). Our dt document the rpid, progressive decline in the KiSS-1 system in dietes, nd strongly suggest tht defective kisspeptin signling is custive lso for the stte of hypogondotropism oserved in uncontrolled forms of dietes in the femle. Mterils nd Methods Adult Wistr mle nd femle rts, red in the vivrium of the University of Córdo, were used. The nimls were mintined under constnt conditions of light (1 h of light, from 7 h) nd temperture ( C) nd were housed in individul cges with free ccess to pelleted food nd tp wter. Experimentl procedures were pproved y the Córdo University Ethicl Committee for niml experimenttion nd were conducted in ccordnce with the Europen Union normtive for cre nd use of experimentl nimls. Kisspeptin (11 119)-NH (termed herefter s Kp-1) ws otined from Phoenix Phrmceuticls Ltd. (Belmont, CA). STZ ws purchsed from Sigm Chemicl Co. (St. Louis, MO). Experimentl designs Experiments were crried in STZ-induced dietic rts. Two sets of studies were conducted: 1) experiments in mle rts t 1 wk (short term) fter STZ injections, nd ) experiments in femle rts t wk (long term) fter STZ injections. In oth settings, protocol of two consecutive sc injections of STZ [5 mg/kg in.1 M citrte uffer (ph.5)] t -h intervl ws used, following previously pulished references (15,, 3). After STZ injections, ody weights were recorded dily nd serum glucose levels were mesured fter 5 d nd t the time of the experimentl Downloded from y guest on July 1

3 7 Cstellno et l. KiSS-1 System nd Hypogondotropism in Dietic Rts Endocrinology, Ferury 9, 15():7 79 procedures to check for dietic sttus, nimls showing severe hyperglycemi ( 5 mg/dl) were selected for nlysis ( 95% of STZ injected mles nd femles in the present study). For experiments using femle rts, the nimls were monitored for estrous cyclicity y mens of vginl cytology efore initition of experiments; only nimls displying t lest two consecutive -d estrous cycles were selected for STZ injections nd susequent hormonl nd gene expression nlyses. Femle rts injected with vehicle, t the diestrous-1 phse of the cycle t the time of smpling, were used s controls. In ddition, for experiments involving gondectomy, the nimls were sujected to ilterl gondectomy vi the scrotl [mle; orchidectomy (ORX)] or dominl [femle; ovriectomy (OVX)] route, under light ether nesthesi 1 wk efore hormonl tests or tissue smpling. Shm-operted nimls served s corresponding controls. Experiments in short-term dietic mle rts Experiment 1 Bsl hormonl profiles, s well s the effects of cute injection of Kp-1 on LH nd testosterone secretion, were ssyed in groups (n 1 1) of short-term dietic mle rts. A protocol of intrcererl injection of Kp-1 ws conducted in STZ-injected nimls nd their respective controls, following previous references (15, 3). To llow delivery of kisspeptin into the lterl cererl ventricle, the nimls were implnted, under ether nesthesi, with intrcereroventriculr (i.c.v.) cnnule lowered to depth of mm eneth the surfce of the skull; the insert point ws 1 mm posterior nd 1. mm lterl to regm, s descried elsewhere (3). Two doses of Kp-1 were tested: 1 nmol nd 1 pmol in 1 l (.9% sline). Dosge selection ws sed in previously pulished dose-response curves for LH fter centrl dministrtion of rnge of doses of Kp-1 (37), s mens to chieve mximl nd sumximl ctivtion of GPR5 in vivo. Blood smples (3 l) were otined y jugulr venipuncture under light ether nesthesi efore () nd t 15 nd min fter Kp-1 injection. Experiment LH responses to ORX nd Kp-1 dministrtion were lso evluted in short-term dietic mles. One week fter ilterl ORX, groups of nimls (n 1 1) were sujected to centrl i.c.v. injection of Kp-1 fter n experimentl procedure similr to tht of experiment 1, nd lood smples (3 l) were otined y jugulr venipuncture efore () nd t 15 nd min fter kisspeptin dministrtion. Experiment 3 Besides hormonl tests, gene expression nlysis were performed in hypothlmic smples from short-term dietic mle rts, either t intct or post-orx conditions. Protocols of STZ injections nd ilterl ORX were s descried for experiments 1. Groups of dult mle rts (n ) were sujected to the different experimentl mnipultions, nd t the end of the corresponding periods, the nimls were killed nd the hypothlmus ws immeditely dissected out s descried elsewhere (15, 3), frozen in liquid nitrogen, nd stored t C for RNA nlysis. Experiments in long-term dietic femle rts Experiment Bsl hormonl profiles, s well s the effects of cute injection of Kp-1 on LH secretion, were evluted in groups (n 1 1) of longterm dietic femle rts. For the hormonl tests, the protocol of intrcererl injection of Kp-1 ws s descried for experiment 1; mximl dose of 1 nmol of Kp-1 ws tested. Blood smples (3 l) were otined y jugulr venipuncture under light ether nesthesi efore () nd t 15 nd min fter Kp-1 injection. Experiment 5 In ddition to sl levels, LH responses to OVX nd centrl Kp-1 dministrtion were lso ssyed in long-term dietic femles. One week fter ilterl OVX, groups of nimls (n 1 1) were sujected to centrl i.c.v. injection of Kp-1 following n experimentl procedure similr to tht of experiment, nd lood smples (3 l) were otined y jugulr venipuncture efore () nd t 15 nd min fter kisspeptin dministrtion. Experiment Besides hormonl tests, gene expression nlysis were conducted in hypothlmic smples from long-term dietic femle rts, either t intct or post-ovx conditions. Protocols of STZ injections nd ilterl orchidectomy were s descried for experiments 5. Groups of dult femle rts (n ) were sujected to the different experimentl mnipultions, nd t the end of the corresponding periods, the nimls were killed y decpittion nd the hypothlmus ws immeditely dissected out s descried elsewhere, frozen in liquid nitrogen, nd stored t C until RNA nlysis. Experiment 7 Finlly, the effects of repeted dministrtion of Kp-1 in terms of LH secretory responses were monitored in long-term dietic femle rts. Groups of STZ-treted femles were sujected to two different regimens of Kp-1 injections: 1) one dily i.c.v. injection of 1 nmol Kp-1 etween 9 nd 1 h; nd ) two dily i.c.v. injections of 1 nmol Kp-1 every 1 h (t 9 nd 1 h). Tretments were continued for 7 d, nd lood smples (3 l) were otined y jugulr venipuncture t the morning of d 1, 3, nd 7, min fter kisspeptin dministrtion. Hormone mesurements Serum LH levels were determined in volume of 5 l using RIA kits supplied y the Ntionl Institutes of Helth (Dr. A. F. Prlow, Ntionl Institute of Dietes nd Digestive nd Kidney Diseses, Ntionl Hormone nd Peptide Progrm, Torrnce, CA). Rt LH-I-9 ws leled with 15 I using Iodo-gen tues, following the instructions of the mnufcturer (Pierce, Rockford, IL), nd hormone concentrtions were expressed using the reference preprtion LH-RP-3 s stndrd. Intr- nd interssy coefficients of vrition were less thn nd 1%. The sensitivity of the ssy ws 5 pg/tue. In ddition, serum glucose concentrtions were determined using n utomtic glucose nlyzer (Accu-Chek; Roche Dignostics, Brcelon, Spin). Finlly, in selected experimentl smples, serum leptin nd testosterone levels were determined using commercil kits from Linco Reserch (St. Chrles, MO) nd MP Biomedicls (Cost Mes, CA), respectively, following the instructions of the mnufcturer. The sensitivity of the ssys ws.5 ng/tue (leptin) nd.1 ng/tue (testosterone), wheres the intrssy coefficients of vrition were less thn 5%. RNA nlysis y semiquntittive RT-PCR Totl RNA ws isolted from hypothlmic smples using the singlestep, cid gunidinium thiocynte-phenol-chloroform extrction method. Hypothlmic expression of KiSS-1 mrnas ws ssessed y RT-PCR, optimized for semiquntittive detection, using previously defined primer pirs nd conditions (15). This thoroughly vlidted procedure llows for estimtion of chnges in reltive mounts of the trget mrna t specific experimentl conditions, s compred with the vlues in the corresponding control smples, mesured in the sme ssy. As internl control for reverse trnscription nd rection efficiency, mplifiction of -p frgment of S11 riosoml protein mrna ws crried out in prllel in ech smple. In keeping with previous optimiztion tests, 3 nd PCR cycles were chosen for semiquntittive nlysis of the specific trget (KiSS-1) nd RP-S11 internl control, respectively (15). Specificity of PCR products ws confirmed y direct sequencing (Centrl Sequencing Service, University of Cordo). Quntifiction of intensity of RT-PCR signls ws crried out y densitometric scnning using n imge nlysis system (1-D Mnger; TDI Ltd., Mdrid, Spin), nd vlues of the specific trgets were normlized to those of internl controls to express ritrry units of reltive expression. In ll Downloded from y guest on July 1

4 Endocrinology, Ferury 9, 15():7 79 endo.endojournls.org 77 ssys, liquid controls nd rections without reverse trnscription resulted in negtive mplifiction. Presenttion of dt nd sttistics Hormonl determintions (LH, testosterone, leptin) were conducted in duplicte, with miniml totl numer of 1 smples per group. When pproprite, integrted LH secretory responses were clculted s the re under the curve (AUC), using the trpezoidl rule, over the -min period fter dministrtion of Kp-1 or vehicle. Semiquntittive RT- PCR nlyses were crried out in duplicte from t lest four independent RNA smples of ech experimentl group. Quntittive RNA nd hormonl dt re presented s men SEM. Results were nlyzed for sttisticlly significnt differences using unpired Student t test or ANOVA followed y Student-Newmn-Keuls multiple rnge tests (SigmStt.; Jndel Corp., Sn Rfel, CA). P.5 ws considered significnt. Results Studies in short-term dietic mle rts Hormonl profiles nd effects of kisspeptin on LH nd testosterone secretion Body weight dt nd hormonl profiles of short-term dietic mle rts re presented in Fig. 1. One week fter injection of STZ, dietic mles showed significntly lower totl ody weights thn controls, with negtive ody weight gin long the study period. As confirmtion of their dietic sttus, ll nimls injected with STZ showed serum glucose concentrtions greter thn 5 mg/dl, in contrst to normoglycemic controls ( 1 mg/dl). In ddition, STZ-injected mles presented significntly reduced serum leptin levels. In terms of gondotropic xis function, sl serum LH levels in 1-wk STZ rts were not significntly different from those in control nimls. Conversely, shortterm dietic mles showed cler-cut reduction in circulting testosterone levels, whose concentrtion ws out one fourth tht in control nimls (Fig. 1). On this hormonl ckground, the gondotropic effects of kisspeptin were tested. On the sis of our previous dose-response nlyses in vivo, two doses of Kp-1 were chosen for i.c.v. injection, 1 nmol nd 1 pmol, for mximl nd sumximl stimultion of the gondotropic xis, respectively. In control nimls, 1 nmol nd 1 pmol Kp-1 were eqully effective in inducing roust LH ursts t 15 min fter centrl injection of the peptide. However, LH levels remined elevted t min fter kisspeptin dministrtion only for the high (1 nmol) dose. Likewise, in short-term dietic nimls, oth doses of Kp-1 effectively induced LH secretory ursts fter centrl injection, with persistently elevted LH levels t min only in the high-dose group. Nonetheless, wheres the pttern of secretion fter dministrtion of the low dose of kisspeptin (1 pmol) ws similr to tht of control nimls, LH responses to 1 nmol kisspeptin were significntly higher (P.1) in 1-wk dietic nimls 15 min fter peptide dministrtion. Likewise, testosterone responses to the high dose of Kp-1 were not only preserved ut even significntly ugmented in this group. Finlly, when integrted LH secretion ws clculted over the -min study period, it ws confirmed tht net LH responses to low doses of kisspeptin were fully conserved, wheres those to high doses were even enhnced, in short-term dietic mle rts (Fig. ). BW (g) Glucose (mg/dl) STZ-1wk STZ-1wk -1 T (ng/ml) Hormonl profiles nd LH responses to kisspeptin in ORX dietic mle rts Hormonl profiling nd functionl tests were lso performed in short-term STZ-injected mles, t 1 wk fter ilterl gondectomy. In keeping with previous references, LH hypersecretion in response to ORX ws prtilly lunted in dietic nimls, with n pproximtely 5% reduction in the post-orx rise of serum LH levels vs. control gondectomized nimls (Fig. 3A). Centrl Kp-1 dministrtion to dietic ORX rts ws le to fully reverse defective LH responses to gondectomy. Thus, oth 1 nmol nd 1 pmol doses of Kp-1 induced further increses in post-orx LH vlues tht equled, t 15 min fter injection, the ,5 3,,5, 1,5 1,,5, BW (g) Leptin (ng/ml) FIG. 1. Body weight dt nd hormone profiles of dult mle rts 1 wk fter injection of STZ (short-term dietes). In the upper pnels, totl ody weights (BW) nd net ody weight gin in control nd STZ-injected nimls re shown. In ddition, glucose nd leptin levels in short-term dietic mles nd controls re presented in the middle pnels. Finlly, in the lower pnels, serum LH nd testosterone (T) levels in controls nd short-term STZ nimls re indicted. Dt re the men SEM of t lest 1 independent determintions per group., P.1 vs. corresponding control vlues (Student t test). Downloded from y guest on July 1

5 7 Cstellno et l. KiSS-1 System nd Hypogondotropism in Dietic Rts Endocrinology, Ferury 9, 15(): CONTROL Vehicle Kp-1 (1pmol) Kp-1 (1nmol) STZ-1wk (min) T (ng/ml) , AUC c d LH (ng/ml min) STZ+Kp-1 1nmol STZ+Kp-1 1pmol STZ+Veh C+Kp-1 1nmol C+Kp-1 1pmol C+Veh STZ+Kp-1 1nmol STZ+Veh C+Kp-1 1nmol C+Veh FIG.. LH nd testosterone (T) responses to centrl kisspeptin dministrtion in control nd short-term (STZ, 1 wk) dietic mle rts. In the upper pnels, the time course for the LH-relesing effects of two doses of Kp-1 (1 nmol nd 1 pmol, i.c.v.) is presented. In the lower pnels, serum T levels t min fter injection of the high dose of Kp-1 re shown from control nd 1-wk STZ nimls. In ddition, integrted LH secretion fter centrl dministrtion of Kp-1 (AUC during the min study period) is presented for oth doses, in control (C) nd short-term dietic nimls. Dt re the men SEM of t lest 1 independent determintions per group., P.1 vs. control vlues t -min;, P.1 vs. corresponding time-pired vlues in controls; for AUC dt, groups with different superscript letters re sttisticlly different (ANOVA followed y Student-Newmn-Keuls multiple rnge test). levels oserved in ORX control nimls. Yet, s ws the cse in intct nimls, persistent increses over preinjection LH levels were only detected t min fter dministrtion of the high dose of kisspeptin (Fig. 3B). Clcultion of integrted LH secretory responses confirmed tht net LH hypersecretion in response to ORX ws significntly decresed in gondectomized dietic nimls nd tht such defective secretion could e completely rescued y exogenous dministrtion of kisspeptin (Fig. 3C). Hypothlmic expression of KiSS-1 gene in intct nd ORX dietic mle rts Expression nlyses of KiSS-1 gene t the hypothlmus were conducted in short-term dietic mle rts, either intct or t 1 wk fter gondectomy. Reltive KiSS-1 mrna levels in whole hypothlmic frgments from intct short-term dietic rts were not significntly different from those of control nimls. One week fter ORX, control rts presented significnt.5- fold increse in KiSS-1 mrna levels over those in intct mles, in keeping with previous references (3). In contrst, gondectomy of short-term dietic rts filed to induce significnt elevtion in hypothlmic KiSS-1 mrna expression; only mrginl rise in KiSS-1 mrna levels over intct vlues ws detected, ut it did not rech sttisticl significnce. Accordingly, the levels of KiSS-1 mrna, t 1 wk fter ORX, were significntly lower in short-term STZ mles thn in corresponding controls (Fig. ). Studies in long-term dietic femle rts Hormonl profiles nd effects of kisspeptin on LH secretion Dt on ody weight, glucose, nd hormonl levels in longterm dietic femle rts re summrized in Fig. 5. Finl ody weights were significntly lower in dietic femles wk fter STZ injection, dietic femles showing negtive ody weight gin long the study period. As predicted on the sis of their dietic sttus, ll femle rts injected with STZ showed mrked hyperglycemi, with serum glucose concentrtions greter thn 5 mg/dl. In ddition, dietic femles presented significnt reduction in serum leptin levels wk fter STZ injection. In terms of gondotropic xis function, -wk STZ-treted femle rts displyed constnt nestrous t vginl cytology (dt not Downloded from y guest on July 1

6 Endocrinology, Ferury 9, 15():7 79 endo.endojournls.org 79 A 1 1 STZ-1wk 1 1, B C LH (ng/ml min) 1 Vehicle Kp-1 (1pmol) 1 Kp-1 (1nmol) AUC C-ORX C-ORX C-ORX shown), overtly decresed serum LH concentrtions, nd significntly reduced uterus weight s surrogte mrker of defective estrdiol levels (Fig. 5). In ddition, ovrin weights were lso c c c (min) STZ-ORX STZ c STZ-ORX STZ STZ-ORX-K1pmol STZ-ORX STZ-ORX-K1nmol FIG. 3. In the upper pnel (A), LH responses to ORX in short-term (STZ, 1 wk) dietic mle rts nd their respective controls re shown. In ddition, LH responses to centrl kisspeptin dministrtion in short-term ORX dietic mles re presented. Thus, in the middle pnel (B), the time course for the LH-relesing effects of i.c.v. injection of Kp-1 is presented from ORX dietic mles. In ddition, in the lower pnel (C), the integrted LH secretory responses to Kp-1 (AUC during the min study period) in ORX dietic rts re shown. For comprtive purposes, the corresponding LH levels in control mle rts, t 1 wk fter ORX, re included in middle nd lower pnels. Intheupper pnel,, P.1 vs. corresponding controls t min; P.1 vs. corresponding timepired vlues in controls; in the middle nd lower pnels, groups with different superscript letters re sttisticlly different (ANOVA followed y Student- Newmn-Keuls multiple rnge test). FIG.. Hypothlmic expression of KiSS-1 gene in the hypothlmus of dult mle rts 1 wk fter injection of vehicle (control: Co) or STZ (short-term dietes), either t intct conditions or fter ORX. For ech group, three representtive independent smples re presented. Prllel mplifiction of S11 riosoml protein mrna served s internl control. In the lower pnels, KiSS-1 mrna levels re the men SEM of t lest four independent determintions. Groups with different superscript letters re sttisticlly different (P.5; ANOVA followed y Student-Newmn-Keuls multiple rnge test). significntly decresed in -wk dietic femles ( vs mg in controls; P.5). In this model, injection of n effective dose of Kp-1 demonstrted tht, despite significnt reduction of the previling gondotropin levels, LH responses to kisspeptin were grossly preserved. Thus, the profiles of LH secretion in response to 1 nmol Kp-1, with significntly incresed levels t 15 min tht remined elevted fter min, were nlogous in control nd long-term dietic femle rts. In good greement, the integrted solute LH secretory responses to Kp-1 were sttisticlly similr in control nd long-term dietic nimls. Furthermore, given the lowering of sl LH levels in dietic femles, reltive responses to kisspeptin were ugmented in this group (-fold increse vs. -fold increse in control femles t diestrus-1) (Fig. ). Hormonl profiles nd LH responses to kisspeptin in OVX dietic femle rts Circulting LH levels, efore nd fter centrl dministrtion of Kp-1, were lso ssessed in long-term STZ-injected femles t 1 wk fter ilterl gondectomy. As descried previously in the femle (, 33) nd in keeping with our mle dt (see Fig. 3), LH hypersecretion in response to OVX ws significntly decresed in dietic femles, with n pproximtely 55% reduc- Downloded from y guest on July 1

7 79 Cstellno et l. KiSS-1 System nd Hypogondotropism in Dietic Rts Endocrinology, Ferury 9, 15():7 79 BW (g) Glucose (mg/dl) , 1,,,, STZ-wk STZ-wk 1-1 BW (g) tion in the post-orx rise of serum LH levels vs. control gondectomized nimls (Fig. 7A). In this setting, intrcererl injection of 1 nmol Kp-1 ws sufficient to fully reverse defective LH responses to gondectomy. Moreover, serum LH levels t 15 nd min fter Kp-1 injection in OVX dietic femles were significntly higher thn in control OVX rts (Fig. 7B). Hypothlmic expression of KiSS-1 gene in intct nd OVX dietic femle rts As done for the short-term dietic mle rts, reltive KiSS-1 mrna levels t the hypothlmus were ssessed in -wk STZ dietic femles, either intct or t 1 wk fter OVX. In contrst ,5 3,,5, 1,5 1,,5, Leptin (ng/ml) UW (mg) FIG. 5. Body weight dt nd hormone profiles of dult femle rts wk fter injection of STZ (long-term dietes). In the upper pnels, totl ody weights (BW) nd net ody weight gin in control (diestrus-1) nd STZ-injected nimls re shown. In ddition, glucose nd leptin levels in long-term dietic femles nd controls re presented in the middle pnels. Finlly, in the lower pnels, serum LH levels nd uterine weights (UW) in controls nd long-term STZ nimls re shown. Dt re the men SEM of t lest 1 independent determintions per group., P.1 vs. corresponding control vlues (Student t test). LH (ng/ml min) Co-D1 STZ-wk Vehicle Kp-1 (1nmol) Vehicle Kp-1 (1nmol) (min) AUC Co+Veh C+Kp1 to 1-wk STZ mles, long-term dietes in the femle evoked significnt reduction of greter thn 5% in KiSS-1 mrna levels in whole hypothlmic frgments. In ddition, wheres 1-wk gondectomy of control femles resulted in the expected.5-fold increse in KiSS-1 mrna t the hypothlmus, OVX of longterm dietic femles evoked severely ttenuted rise of hypothlmic KiSS-1 mrna expression. These reltive levels, however, were higher thn those detected in intct dietic femles (Fig. ). c STZ+Veh STZ+Kp-1 FIG.. LH responses to centrl kisspeptin dministrtion in long-term (STZ, wk) dietic femle rts. The time course for the LH-relesing effects of n effective dose (1 nmol) of Kp-1 is presented for control (diestrus-1: Co-D1) nd dietic femles in the upper nd middle pnels, respectively. In the lower pnels, the integrted LH secretion fter centrl dministrtion of Kp-1 (AUC during the min study period) is presented for the different experimentl groups. Dt re the men SEM of t lest 1 independent determintions per group., P.1 vs. control vlues t min; P.1 vs. corresponding timepired vlues in controls; for AUC dt, groups with different superscript letters re sttisticlly different (ANOVA followed y Student-Newmn-Keuls multiple rnge test). Downloded from y guest on July 1

8 Endocrinology, Ferury 9, 15():7 79 endo.endojournls.org 791 A 1 1 STZ-wk 1 1, B C-OVX Vehicle Kp-1 (1nmol) 1 1 C-OVX STZ-wk STZ STZ-OVX Effects of repeted kisspeptin dministrtion on LH secretion in dietic femle rts Finlly, the effects of repeted i.c.v. dministrtion of kisspeptin on circulting LH levels in long-term dietic femles were lso explored. As experimentl protocol, STZ femles were sumitted to dily i.c.v. injections of 1 nmol Kp-1 for 7 d t two regimens: 1) single Kp-1 olus t 9 h ech dy of tretment; nd ) i.c.v. oluses of Kp-1 twice dily, t 9 nd 1 h during the 7 d of tretment. Blood smpling ws conducted immeditely efore nd t min fter the corresponding.m. injection of Kp-1 on d 1 (when experiment ws strted), d 3, nd d 7. Hormonl nlysis reveled tht, despite significntly decresed previling LH levels in dietic femles, oth tretment protocols evoked preserved LH responses, similr in mgnitude for ech regimen, throughout the study period. Of note, however, the net mplitude of LH secretion in response to Kp-1 ppered to e dependent on the regimen of dministrtion ecuse LH responses in STZ femles injected twice dily were significntly higher thn those in dietic nimls injected once dy with Kp-1 t ll time points explored (Fig. 9). d d c c c (min) STZ-OVX FIG. 7. In the upper pnel (A), LH responses to OVX in long-term (STZ, wk) dietic femle rts nd their respective (diestrus-1) controls re shown. In ddition, LH responses to centrl kisspeptin dministrtion in long-term dietic femles 1 wk fter OVX re presented in the lower pnel (B). For comprtive purposes, the corresponding LH levels in control femle rts, t 1 wk fter OVX, re included. In the upper pnel,, P.1 vs. corresponding controls t min;.1 vs. corresponding time-pired vlues in controls; in the lower pnel, groups with different superscript letters re sttisticlly different (ANOVA followed y Student-Newmn-Keuls multiple rnge test). FIG.. Hypothlmic expression of KiSS-1 gene in the hypothlmus of dult femle rts wk fter injection of vehicle (control: Co) or STZ (long-term dietes), either t intct conditions or fter OVX. For ech group, three representtive independent smples re presented. Prllel mplifiction of S11 riosoml protein mrna served s internl control. In the lower pnels, KiSS-1 mrna levels re the men SEM of t lest four independent determintions. Groups with different superscript letters re sttisticlly different (P.5; ANOVA followed y Student-Newmn-Keuls multiple rnge test). Discussion Wheres hypogondotropism hs een long recognized s common compliction of poorly controlled dietes, the underlying neuroendocrine sis for such phenomenon hd remined lrgely unknown. Recent dt from our group suggested tht, in long-term STZ-treted mle rts, ltertions of hypothlmic KiSS-1 system re mechnisticlly involved in the genertion of the stte of centrl hypogondism in dietes (15). Our current dt extend nd complement those initil oservtions, nd conclusively demonstrte tht chnges in expression/ function of the KiSS-1 system re redily detectle t erly stges of the dietic stte in the mle nd tht the dietic femle is lso sensitive to disturnce of hypothlmic KiSS-1 system, which seems custive for defective gondotropin secretion oserved in this model, oth in sl nd postgondectomy conditions. Comprison of the hormonl profiles of short-term (1 wk fter STZ; present results) nd long-term ( wk fter STZ) (15) dietic mle rts illustrtes the progressive deteriortion of gondotropic function in the course of uncontrolled dietes. Thus, we report herein tht short-term dietic mles show significntly reduced circulting testosterone levels; however, their men serum LH levels were roughly preserved (see Fig. 1), nd serum FSH concentrtions were only mrginlly decresed (our Downloded from y guest on July 1

9 79 Cstellno et l. KiSS-1 System nd Hypogondotropism in Dietic Rts Endocrinology, Ferury 9, 15():7 79,,,,, STZ-Veh STZ-.m. STZ-.m./p.m.,, d-1 d-3 d-7 FIG. 9. LH responses to repeted dministrtion of kisspeptin in long-term dietic femle rts. Dietic femles were sujected to two different protocols of intermittent dministrtion of 1 nmol oluses of Kp-1 for 7 d: 1) single Kp- 1 olus t 9 h ech dy of tretment (STZ-.m.); nd ) i.c.v. oluses of Kp- 1 twice dily, t 9 nd 1 h during the 7 d of tretment (STZ-.m./p.m.). Hormonl levels were determined on the morning of d 1, d 3, nd d7of tretments, efore nd min fter i.c.v. injection of kisspeptin. For comprtive purposes, the corresponding LH levels in control femle rts re presented for ech time point s rs., P.1 vs. corresponding preinjection vlues in STZ femles t min;, P.1 vs. corresponding time-pired vlues in nondietic controls;, P.1 vs. corresponding levels in STZ-.m. groups (ANOVA followed y Student-Newmn-Keuls multiple rnge test). unpulished dt). In cler contrst, long-term dietic mles were previously shown to disply significntly lower sl concentrtions of oth LH nd testosterone. These oservtions evidence tht wheres the severity of suppression of the reproductive xis increses s function of the progression of the disese, n impct on gondotropic function cn e lredy detected t rther erly stges of dietes. A likely explntion for the pprent preservtion of circulting LH levels in this erly phse is tht initil hypogondotropism in short-term dietes is compensted s direct consequence of decrese in the negtive feedck input of testiculr testosterone. Conversely, in the long term, such compenstion is no longer possile nd the nimls present n overt filure of gondotropin secretion. Supporting this hypothesis, reltive mrna levels of KiSS-1 t the hypothlmus of short-term dietic mles were not overtly decresed vs. corresponding controls. This is likely to reflect trnsient stte of compenstion due to decresed circulting testosterone ecuse elimintion of negtive feedck of testiculr ndrogen hs een reported to increse hypothlmic KiSS-1 gene expression (15, 3). In contrst, long-term dietic mles displyed significnt reduction in hypothlmic KiSS-1 mrna levels in sl conditions, in close prllelism with the mrked decrese in circulting LH concentrtions (15). In ny event, serum LH nd hypothlmic KiSS-1 levels in 1-wk STZ mles must e considered s inppropritely low, given the dmpening of circulting testosterone, which evidences n incipient centrl hypogondism t such erly phse of dietes. The functionl impct of (sutle) ltertions of the hypothlmic KiSS-1 system t erly stges of dietes is indirectly documented y the oservtion tht single intrcererl olus of kisspeptin ws cple to evoke roust LH responses nd ws sufficient to normlize testosterone secretion in 1-wk STZtreted mles. Of note, lthough different forms of kisspeptins hve een descried (9), in our phrmcologicl tests, Kp-1 ws used, in line with previous studies (15, 3 3), s men to chieve effective ctivtion of GPR5 in vivo, thus proving the principle tht restortion of kisspeptin signling would e sufficient to normlize the function of the gondotropic xis despite the stte of severe metolic stress. The importnce of these findings is -fold: 1) they document tht, despite erly signs of deteriortion, the gondotropic xis retins its cpcity to mximlly respond to kisspeptin stimultion, even t reltively low doses (1 pmol), under such ltered metolic conditions; nd ) they rgue ginst the possiility tht defective testosterone secretion in short-term dietic mles, in the presence of pprently preserved serum LH concentrtions, is cused y primry defect of testiculr function. Furthermore, in short-term dietic rts, LH secretory ursts nd testosterone responses induced y the high dose of kisspeptin (1 nmol) nerly douled those evoked in pired control nimls. Tken together, these oservtions strongly suggest stte of enhnced responsiveness to kisspeptin in the initil phses of dietes, which might e cused y n erly, sutle decrese in the endogenous kisspeptin tone t the hypothlmus. This condition resemles the sttus of LH hyperresponsiveness to exogenous kisspeptin previously reported y our group in food-deprived rts, whose underlying mechnism seems to involve lso the decresed expression of hypothlmic KiSS-1 (1). Anyhow, the presence of inppropritely low LH levels despite such n incresed responsiveness to kisspeptin further supports the contention tht decline in expression/ secretion of kisspeptins is likely to tke plce t erly stges of dietes. In keeping with previous reports in long-term dietic nimls, our dt demonstrte tht defective LH responses to gondectomy re lredy detected in short-term STZ mles, documenting further their stte of defective gondotropin secretion. Notly, despite hypothlmic KiSS-1 mrna expression ws not significntly lower in short-term STZ mles in sl conditions, the rise in their KiSS-1 mrna levels fter ORX ws significntly ttenuted (see Fig. ). The possiility tht such lunted KiSS-1 response to gondectomy my e custive for the defective rise of LH levels is suggested y the fct tht intrcererl injection of kisspeptin in these nimls ws sufficient to evoke suprmximl LH secretory ursts, whose pek mgnitude tended to exceed the circulting levels of LH in nondietic ORX mles. Agin, such exggerted LH responses seem indictive of stte of LH hyperresponsiveness to kisspeptin in shortterm dietes. From phrmcologicl stndpoint, it is remrkle tht normliztion of gondotropic function in STZ-treted mles could e chieved y solely replcing the endogenous kisspeptin tone, in oth sl nd post-orx conditions. In our studies, long-term dietic femles displyed plethor of reproductive defects mnifested y persistent vginl nestrous, decresed serum LH levels, nd mrkedly trophied uteri nd ovries. In ddition, s ws the cse for STZ-injected mles, the rise of LH levels fter gondectomy ws severely lunted in dietic femles. Of note, defective LH secretion in these nimls ws invrintly coupled with decresed KiSS-1 mrna levels in whole hypothlmic preprtions, in oth sl nd post-ovx conditions, wheres LH responses to exogenous Kp-1 dministrtion were roughly preserved. These oservtions strongly suggest tht, s we previously reported in mles (1), the overt stte of hypogondotropism detected in the uncontrolled, long-term dietic femle is, t lest prtilly, due to Downloded from y guest on July 1

10 Endocrinology, Ferury 9, 15():7 79 endo.endojournls.org 793 the suppressed function of the KiSS-1 system t the hypothlmus. This notion is functionlly relevnt ecuse sex differences hd een previously suggested for the mechnisms underlying the filure of gondotropic xis linked to dietes (35). Yet in our study some sutle differences were detected in the pttern of LH responses to kisspeptin etween mle nd femle rts ecuse in intct STZ-treted nimls, LH responses to kisspeptin were comprtively higher in mles, wheres in gondectomized nimls, kisspeptin-induced LH secretion ws higher in femles. The sis for such pprent, s yet sutle, differences remins to e elucidted. Wheres our dt document the functionl consequences of ltered hypothlmic expression of KiSS-1 in uncontrolled dietes, some fcets of this phenomenon wit further experimentl chrcteriztion. These include the identifiction of potentil nucleus-specific chnges in KiSS-1 expression, s descried previously in order conditions of metolic stress such s fsting or leptin deficiency (1, 39) s well s the trnsltion of our current RNA dt into chnges in kisspeptin expression nd/or relese, t the protein level. The ltter is indirectly evidenced y the close correltion etween KiSS-1 RNA nd circulting LH levels in the different models tested, yet experimentl confirmtion wits further development of procedures for protein quntifiction of kisspeptins in hypothlmic tissue. From therpeutic perspective, our current dt provide the conceptul sis for trgeting the hypothlmic KiSS-1/GPR5 system s potentil djuvnt tretment of reproductive complictions of dietes in the femle. In this context, it is interesting to note tht, s we recently reported in cyclic femle rts (), the pttern of kisspeptin dministrtion seems relevnt to chieve mximl gondotropic stimultion in the dietic femle. Thus, regimen of intermittent injections of Kp-1 twice dily induced persistently higher LH secretory responses thn protocols of single kisspeptin oluses every h, phenomenon whose underlying mechnism remins to e defined. Of note, desensitiztion of LH responses to kisspeptin hs een recently documented fter continuous infusion or repeted injections of high doses of kisspeptins to dult mle rts nd monkeys (1, ), phenomenon tht ws not oserved in our experiments. Although the moleculr sis for this divergence hs not een estlished in our present study, it is tempting to speculte tht the lowering of the endogenous kisspeptin tone in dietes might hve prevented the chievement of desensitizing levels, s it hs een previously hypothesized in nother sitution of metolic stress, such s chronic sunutrition (3). Admittedly, however, the potentil trnsltion of our current phrmcologicl dt must e done with cution ecuse our experiments were designed to provide proof of principle, nd thus, protocols of centrl injection of kisspeptin were selected. In ny event, it is worth noting tht dietes hs een reported to induce the suppression of preovultory LH surges nd therey to cuse novultion (9, 31 33). Further nlyses re required in our model to determine whether reduced KiSS-1 expression t the hypothlmus, nd specificlly t the nteroventrl prventriculr nucleus, s center for the genertion of the preovultory surge (, ), is custive for such novultory stte nd whether repeted kisspeptin dministrtion would e sufficient to restore ovultion in uncontrolled, long-term dietic femle rts. In summry, we report herein series of hormonl nd expression nlyses ddressing the contriution of potentil chnges in the hypothlmic KiSS-1 system to the ltertions of the gondotropic xis t erly stges of dietes in the mle s well s their involvement in the reproductive filure oserved in the dietic femle. Our dt conclusively demonstrte tht the dietic stte results in rpid chnges in hypothlmic KiSS-1 tht pper mechnisticlly relevnt for the disturnce of reproductive function lso in the femle. Tken together with previous results in models of negtive energy lnce (1), our present oservtions further document the nodl role of KiSS-1 neurons s key sensors nd trnsmitters of informtion concerning the metolic stte of the orgnism onto the centers governing the gondotropic xis, in oth mles nd femles. Acknowledgments Address ll correspondence nd requests for reprints to: Mnuel Ten- Sempere, Physiology Section, Deprtment of Cell Biology, Physiology, nd Immunology. Fculty of Medicine, University of Córdo, Avenid Menéndez Pidl s/n, 1 Córdo, Spin. E-mil: fi1tesem@uco.es. This work ws supported y Grnts BFI -9 nd BFI 5-7 from Dirección Generl de Educción Superior e Investigción Cientific (DGESIC) (Ministerio de Cienci y Tecnologí, Spin); funds from Instituto de Slud Crlos III (Red de Centros en Metolismo y Nutrición (RCMN) C3/ nd Project PI; Ministerio de Snidd, Spin); nd Europen Union Reserch Contrct Developmentl Effects of Environment on Reproductive Helth (DEER) (FP-7). Centro de Investigción Biomedic en Red (CIBER) is n inititive of Instituto de Slud Crlos III (Spin). Disclosure Sttement: The uthors hve nothing to disclose. References 1. Herison AE Estrogen positive feedck to gondotropin-relesing hormone (GnRH) neurons in the rodent: the cse for the rostrl periventriculr re of the third ventricle (RP3V). Brin Res Rev 57:77 7. Ten-Sempere M, Huhtniemi I 3 Gondotropins nd gondotropin receptors. In: Fuser BCJM, ed. 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