The stimulatory effect of PYY3-36 on gonadotropin secretion is potentiated

in fasted rats

L. Pinilla1, R. Fernández-Fernández1, E. Vigo, V.M. Navarro, J. Roa, J.M. Castellano, R.

Pineda, M. Tena-Sempere and E. Aguilar

Department of Cell Biology, Physiology and Immunology. University of Córdoba, 14004.

Córdoba, Spain

Running Head: PYY3-36 and gonadotropins in adult rats

Key words: gonadotropin-releasing hormone, luteinizing hormone, follicle-stimulating

hormone, fasting, pituitary

Corresponding author: Enrique Aguilar

Physiology Section. Department of Cell Biology, Physiology and

Immunology. Faculty of Medicine, University of Córdoba

Avda. Menéndez Pidal s/n

14004 Córdoba, SPAIN

FAX: +34-957-218288

E-mail: fi1agbee@uco.es

1 Equally contributed to this work and should be considered as joint first authors

* This work was supported by grants BFI 2000-0419-CO3-03 and BFI 2002-00176 from

DGESIC (Ministerio de Ciencia y Tecnología, Spain)

Articles in PresS. Am J Physiol Endocrinol Metab (January 3, 2006). doi:10.1152/ajpendo.00469.2005

Copyright © 2006 by the American Physiological Society.

2

ABSTRACT

Development and normal function of the reproductive axis requires a precise degree of

body energy stores. Polypeptide YY3-36 (PYY3-36) is a gastrointestinal secreted molecule recently

shown to be involved in the control of food intake with agonistic activity upon neuropeptide Y

(NPY) receptor subtypes Y2 and Y5. Notably, PYY3-36 has been recently demonstrated as

putative regulator of gonadotropin secretion in the rat. However, the “reproductive” facet of this

factor remains to be fully elucidated. In this context, we report herein our analyses of the

influence of the nutritional status on the effects of PYY3-36 upon GnRH and gonadotropin

secretion. The major findings of our study are: (1) the stimulatory effect of central administration

of PYY3-36 on LH secretion was significantly enhanced after fasting, and blocked by a GnRH

antagonist; (2) besides central effects, PYY3-36 elicited LH and FSH secretion directly at the

pituitary level; a response which is also augmented by fasting; (3) PYY3-36 inhibited GnRH

secretion by hypothalamic fragments from male rats fed ad libitum, whereas a significant

stimulatory effect was observed after fasting; (4) the increase in the gonadotropin

responsiveness to PYY3-36 in fasting was not associated with changes in the expression of Y2

and Y5 receptor genes at hypothalamus and/or pituitary. In conclusion, our study extends our

previous observations suggesting a relevant, mostly stimulatory, role of PYY3-36 in the control of

gonadotropin secretion. Strikingly, such an effect was significantly enhanced by fasting.

Considering the proposed decrease in PYY3-36 levels after fasting, the possibility that reduced

PYY3-36 secretion might contribute to defective function of the gonadotropic axis after food

deprivation merits further investigation.

3

INTRODUCTION

Although it is known that conditions of negative energy balance are frequently linked to

lack of puberty onset and reproductive failure, at the moment the mechanisms involved in fitting

the reproductive function to body energy stores remain incompletely defined. Compelling

evidence has recently demonstrated that central and peripheral endocrine signals governing

energy homeostasis, such as the adipocyte-derived hormone leptin, the stomach-derived

hormone ghrelin, orexins and neuropeptide Y (NPY), are also involved in the control of

reproductive function by acting at different levels of hypothalamic-pituitary-gonadal axis.

In this context it has been demonstrated that NPY, a member of the pancreatic

polypeptide family (54), is involved in the control of food intake, reproductive function and

pituitary secretion (26, 27, 30, 33). In rats, central administration of NPY advances puberty (43),

while immunoneutralization of NPY reduced the magnitude of the LH surge during the afternoon

of first proestrus (42). A facilitatory role of NPY on the onset of puberty has been also reported

in the female rhesus monkey (23) and in chicks (18). Secretion of NPY to portal vasculature is

increased on the afternoon of proestrus and served to amplify the actions of GnRH in initiating

the preovulatory surges of LH and probably FSH (53). Acute intracerebroventricular (i.c.v.)

administration of NPY stimulated LH release in ovariectomized rats primed with ovarian steroids

(4). These excitatory effects have been shown to be the consequence of an increase in

secretion and effectiveness of GnRH (5, 14). In contrast, chronic treatment with the peptide

decreased FSH, LH and testosterone secretion (47, 9). NPY hypersecretion is observed in

genetically obese and sterile hyperphagic rodents, which demonstrated the inverse relationship

between chronic NPY secretion and reproductive function (15).

NPY exerts its actions throughout at least five receptor subtypes (16). Development of

selective agonists/antagonists for different receptors and utilization of knock-out animals have

improved our understanding about the role of different receptor subtypes for NPY in the control

of reproductive axis, although at the moment the characterization of the specific role of the

different receptor subtypes is scarce. Recent experiments have shown that the NPY Y1 receptor

inhibits the gonadotrope axis (22) and that its blockade accelerates the onset of puberty (49). In

addition, NPY Y4 receptors have been involved in the NPY effects on LH release (25) and

4

experimental evidence suggests that the inhibition of LH secretion exerted by central

administration of NPY in the rats is predominantly mediated by Y5 receptors (51).

Polypeptide YY 3-36 (PYY3-36), a hormone from gastrointestinal origin structurally

related with NPY and agonist of receptor subtypes Y2 and Y5 (36), has been recently proposed

as a putative anorexigenic signal involved in the control of food intake (3, 24, 50). Conflicting

results on the repeatability of the effects of PYY3-36 in terms of body weight control have been

also published (13, 60). Although the potential role of PYY3-36 in fitting the reproductive function

to body energy stores is still poorly characterized, recent data suggested its involvement in the

control of reproductive axis. Thus, in addition to the reported presence of PYY3-36 in placenta

(62), recent data from our laboratory indicated that PYY3-36 stimulates in vitro FSH and LH

secretion by pituitaries from prepubertal female and male rats and inhibited in vitro GnRH

secretion selectively in males (17). In addition, infusion of PYY3-36 into the lateral ventricle

rapidly inhibited estrous behavior in ovariectomized steroid-primed hamsters (35), and

decreased LH secretion in prepubertal male rats (17).

It is well known that fasting strongly inhibits gonadotropin secretion (6, 7). Considering

that PYY3-36 stimulates in vitro gonadotropin secretion (17), and that PYY3-36 secretion is

depressed during fasting (59), we hypothesized that decreased secretion of PYY3-36 during food

deprivation might be involved in the suppressive effect on pituitary-gonadal function. To test this

hypothesis, we have analyzed the in vivo and in vitro effects of PYY3-36 on GnRH and

gonadotropin secretion after fasting.

5

MATERIAL AND METHODS

Animals and drugs

Wistar rats born in our laboratory were kept under controlled conditions of light (12 h

light: 12 h darkness, lights on at 07.00 h) and temperature (22º C) with free access to pelleted

food (Pacsa Sanders, Seville, Spain) and tap water. Experiments were carried out in adult (90-

100 days) animals. The vaginal smears of adult females were monitored daily and only those

rats exhibiting two or more regular cycles were used. PYY3-36 was purchased from Bachem

(Barcelona, Spain). GnRH antagonist (GnRH-ANT) was ORG.30276 (Ac-D-p-Cl-Phe-D-p-Cl-

Phe-D-Trp-Ser-Tyr-D-Arg-Leu-Arg-Prol-D-Ala-NH2 CH3·COOH) and was purchased from

Organon International (Oss, The Netherlands). For in vivo experiments, PYY3-36 and GnRH-ANT

were dissolved in saline immediately before use, whereas for in vitro experiments PYY3-36, was

dissolved in Dulbecco’s Modified Eagle’s Medium (DMEM) (BioWhittaker; Verviers, Belgium)

immediately before use. Doses of drugs were selected on the basis of previous studies (17, 57).

Experimental designs

Experimental procedures were approved by the Córdoba University Ethical Committee

for animal experimentation and were conducted in accordance with the European Union

normative for care and use of experimental animals. The number of animals per experimental

group is provided in the figure legends. Experiments were carried out between 10.00 and 12.00

h. Special caution was taken to avoid any stressing influences upon the experimental animals

(all the animals were handled daily for a week before the experiment and humanely killed by the

same person and the different drugs were injected at random). The following experiments were

conducted:

In vivo experiments

In the first set of experiments, and to characterize the possible effects of fasting in the

effects of PYY3-36 on gonadotropin secretion, adult (90 days) males were submitted to a 4-days-

period of absolute restriction of food. Control animals were fed ad libitum. The animals were

i.c.v. injected with 3 nmol/rat of PYY3-36 or vehicle. The procedure of i.c.v. injection was as

previously described (48). Briefly, animals were implanted, two days before PYY3-36

administration, with i.c.v. cannulae under light ether anesthesia. To allow delivery of PYY3-36 into

the lateral cerebral ventricle, the cannulae were lowered to a depth of 4 mm beneath the

6

surface of the skull; the insert point was 1 mm posterior and 1.2 mm lateral to bregma. Animals

were humanely killed 15, 30 and 60 min. after injection and trunk blood samples were collected.

To analyze whether the stimulatory effect of PYY3-36 on gonadotropin secretion was

exerted throughout an increase in GnRH release, adult male rats submitted to a 4-days period

of absolute restriction of food and corresponding control groups were subcutaneously (s.c.)

injected with GnRH-ANT (5 mg/kg/rat) 48 and 24 h before i.c.v. administration of PYY3-36 (3

nmol/rat). Blood samples were obtained by decapitation 15, 30 and 60 min. after PYY3-36

injection.

In vitro experiments

Adult cyclic female and male rats were submitted to a 4-day period of absolute

restriction of food. Control animals received food ad libitum. Thereafter, the animals were

humanely killed (the control females being in metestrus) and hypothalami and pituitaries

removed to analyze the effects of PYY3-36 on GnRH and gonadotropin secretion. Hypothalamic

samples were dissected out as described in detail elsewhere (45), by a horizontal cut of about 2

mm depth with the following limits: 1 mm anteriorly from the optic chiasma, the posterior border

of mamillary bodies and the hypothalamic fisures. Hypothalamus were placed in scintillation

vials and incubated in 500 µl of DMEM in a Dubnoff shaker incubator under an atmosphere of

95% O2 and 5% CO2 at 37.5º C. After a 30-min. preincubation, the medium was removed and

hypothalamic fragments were challenged for 45 min with PYY3-36 (10-6 M) or DMEM alone. At

the end of the incubation period, medium samples were boiled for 30 min. to inactivate

endogenous protease activity and stored at -80º C until used for GnRH determinations. Anterior

pituitaries were halved and placed in scintillation vials. After 1 h. of preincubation, the medium

was replaced by fresh medium alone or medium containing PYY3-36 (10-8 and 10-6 M). Samples

were collected at 60 and 120 min. of the incubation period for LH and FSH determinations.

Finally, to analyze whether the increase in the responsiveness to gonadotropin at PYY3-

36 in fasted rats can be mediated by an increase in the NPY Y2 and Y5 receptor subtypes, adult

male were submitted to a 4-days period of absolute restriction of food. Control animals were fed

ab libitum. Thereafter, animals (9-12 animals/group) were humanely killed by decapitation and

pituitary and hypothalamus were immediately dissected (as above described), snap frozen in

liquid nitrogen and stored at -80º C until use for RNA isolation and analysis.

7

LH, FSH, GnRH and leptin measurements by specific RIAs

After centrifugation (1600 g at 4º C for 20 min.), serum was collected, frozen and stored

at –20º C until use. The concentrations of LH and FSH were measured in 5-50 µL by a double-

antibody method using RIA kits supplied by National Institute of Diabetes and Digestive and

Kidney Diseases (Bethesda, MD.USA). Rat-LH-I-10 and FSH-I-9 was labeled with 125I by the

chloramine T method and hormone concentrations were expressed using a reference

preparation LH-RP3 and FSH-RP2 as standard. Intra- and inter-assay variations were,

respectively, 8 and 10%, for LH and 6 and 9% for FSH. The sensitivities of the assay were 75

and 400 pg/mL for LH and FSH, respectively. In addition, GnRH concentrations in the

incubation media from hypothalamic explants were measured in 100-µL aliquots using a

commercial RIA kit purchased from Peninsula Laboratories Inc (Bachem group, San Carlos,

CA) following the instructions of the manufacturer. The sensitivity of the assay was 1 pg/tube.

All samples of each experiment were measured in the same assay. Serum leptin concentrations

were measured in control and fasted rats using a commercial kit from LINCO Research (St.

Charles, MI, USA), following the instructions of the manufacturer. The sensitivity of the assay

was 0.05 ng/tube, while the intra-assay coefficient of variation was below 5%.

RNA Analysis by RT-PCR

Hypothalamic and pituitary expression of NPY Y2 and NPY-Y5 receptor mRNAs was

assessed by semi-quantitative RT-PCR. Total mRNA was isolated from tissue samples using

the single-step, acid guanidinum thiocyanate-phenol-chloroform extraction method, followed by

DNase I treatment (12). For amplification of the target genes, the following primer pairs were

used: NPY-Y2-sense (nt 375-398; 5′-GGT GCC CTA TGC CCA GGG TCT GGC-3′) and NPY-

Y2-antisense (nt 530-509; 5′-GCG CTG ACA CCC CAC GCC AGG C-3′) for amplification of a

156-bp fragment of rat NPY-Y2 receptor cDNA; and NPY Y5-sense (nt 131-153; 5′-GGT CCT

GCT CCT GCC GCC ACC GC-3′) and NPY Y5-antisense (nt 274-253; 5′-CTT GTT AAA ATG

CTC CTC AAG C-3′) for amplification of a 144-bp fragment of rat NPY-Y5 receptor cDNA.

These oligo-primers were synthesized according to the published rat cDNA sequences of NPY-

Y2 and NPY-Y5 (GenBank accession nº NM-023968 and NM-012869, respectively). In addition,

to provide an appropriate internal control, parallel amplification of a 241-bp fragment of S11

ribosomal protein mRNA was carried out in each sample, using the primer pair S11-sense (nt

8

11/32; 5′-CAT TCA GAC GGA GCG TG TTA C-3′) and S11-antisense (nt 231/250; 5′-TGC ATC

TTC ATC TTC GTC AC-3′).

For amplifications of the targets, RT and PCR were run in two separate steps.

Furthermore, to enable appropriate amplification in the exponential phase for each target, PCR

amplification of specific signal and S11 ribosomal protein transcripts was carried out in separate

reactions with different number of cycles (see below), but using similar amounts of the

corresponding cDNA templates, generated in single RT reactions, as previously described (55,

58). Briefly, equal amounts of total RNA (2 µg) were heat denatured and reverse transcribed by

incubation at 42º C for 90 min with 12.5 U avian myeloblastosis virus (AMV) RT (Promega,

Madison, WI, USA), 20 U ribonuclease inhibitor RNasin (Promega), 200 mM deoxy-NTP

mixture, and 1 nM specific and internal control antisense primers in a final volume of 30 µL of 1x

AMV-RT buffer. The reactions were terminated by heating at 97º C for 5 min. and cooling on

ice, followed by dilution of the RT cDNA samples with nuclease-free H2O (final volume, 60 µL).

For semi-quantitative PCR, 10 µL-aliquots of the cDNA samples (equivalent to 650 ng total RNA

input) were amplified in 50 µL of 1x PCR buffer in the presence of 2.5 U Taq DNA polymerase

(Promega), 200 nM deoxy-NTP mixture, and the appropriate primer pairs (1 nM of each primer).

PCR reactions consisted in a first denaturing cycle at 97º C for 5 min., followed by a variable

number of cycles of amplification (n= 36 cycles for NPY-Y2 and –Y5; n= 26 cycles for RP-S11)

defined by denaturation at 96º C for 1.5 min., annealing for 1.5 min, and extension at 72º C for 3

min. A final extension cycle of 72º C for 15 min. was included. Annealing temperature was

adjusted for each target: 58º C for NPY-Y2 receptor and S11, and 61.5º C for NPY-Y5 receptor.

Different numbers of cycles were tested to optimize amplification in the exponential phase of

PCR. On this basis, the numbers of PCR cycles indicated above were chosen for further semi-

quantitative analysis targets and RP-S11 internal control.

PCR-generated DNA fragments were resolved in Tris-borate buffered 1.5% agarose

gels and visualized by ethidium bromide staining. Specificity of PCR products was confirmed by

direct sequencing (Central Sequencing Service, University of Córdoba). Quantification of

intensity of RT-PCR signals was carried out by densitometric scanning and values of the

specific targets were normalized to those of internal control to express arbitrary units of relative

9

expression. In all assays, liquid controls and reactions without RT were included, yielding

negative amplification.

Presentation of data and statistics

Values are expressed as means ± SEM. When relevant, integrated LH and FSH

secretory responses were calculated as the area under the curve (AUC), obtained following the

trapezoidal rule, over the 60-min period after administration of PYY3-36. Differences between

groups were analyzed using Student t test or two-way ANOVA followed by Student-Newman-

Keuls multiple range test.

RESULTS

Effects of PYY3-36 on in vivo gonadotropin secretion

In adult males, a 4-day period of absolute restriction of food induced a significant

decrease in body weight (282 ± 10 g vs 325 ± 5 g in controls) and in serum LH and leptin

concentrations (Table 1). I.c.v. administration of PYY3-36 (3 nmol/rat) significantly stimulated LH

and FSH secretion in controls fed ad libitum and in fasted animals (Table 1), without affecting

serum leptin concentrations. Interestingly, the LH response in fasted animals was higher than in

the control group, either when response was estimated by absolute LH levels reached after

PYY3-36 administration or by the fold-increase in serum LH concentrations (Table 1). To obtain

information about the time-course of the stimulatory effect of PYY3-36 on gonadotropin secretion,

blood samples were obtained 15, 30 and 60 min. after peptide administration in adult controls

and fasted male rats. Results obtained showed that the stimulatory effect on gonadotropin

secretion remain evident, at least, 60 min after i.c.v. administration of PYY3-36 (Figure 1).

Treatment with a GnRH-ANT abolished completely the stimulatory effect of PYY3-36 on LH and

FSH secretion in controls and fasted rats (Figure 1).

Effects of PYY3-36 on in vitro GnRH secretion in adult male rats

PYY3-36 (10-6 M) significantly inhibited GnRH secretion by hypothalamic fragments from

control males fed ad libitum (Figure 2). Food deprivation during 4 days reduced, in the limits of

statistical significance, basal GnRH secretion (11.07 ± 2.21 pg/fragment vs 16.42 ± 1.40). In

contrast with the inhibitory effect in ad libitum control animals, PYY3-36 significantly stimulated

GnRH by hypothalamic samples from fasted animals (Figure 2).

10

Effects of PYY3-36 on in vitro gonadotropin secretion

Males: In hemi-pituitaries from control rats fed ad libitum, 10-6 M PYY3-36 significantly increased

LH secretion at 60 and 120 min. of incubation period, whereas the dose of 10-8M only increased

LH secretion at 120 min. (Figure 3). After a 4-day period of absolute food restriction, both doses

of PYY3-36 significantly increased LH secretion at 60 and 120 min. of incubation period, the

responses being significantly greater than in control group (Figure 3). FSH secretion was

similarly stimulated at 60 min. of the incubation period in controls fed ad libitum and fasted rats

by both doses of PYY3-36, and the effect disappeared at 120 min of the incubation period

(Figure 3).

Females: In hemi-pituitaries from control metestrous females fed ad libitum, PYY3-36 was unable

to stimulate LH or FSH secretion, except for a significant increase in LH observed at 60 min of

the incubation period in presence of higher dose of PYY3-36 (Figure 4). However, after a 4-day

period of absolute food restriction, LH and FSH secretion was significantly stimulated by both

doses of PYY3-36 (Figure 4).

In order to compare the effectiveness of PYY3-36 in controls fed ad libitum and fasted rats,

results were expressed as changes over the concentrations measured in absence of PYY3-36.

Figure 5 clearly evidences that the overall response was greater in fasted animals than in ad

libitum control animals.

Hypothalamic and pituitary mRNA expression of NPY Y2 and Y5 receptors in fed and fasted rats

RT-PCR analysis using specific primer pairs demonstrated that the expression of the

genes encoding NPY receptors Y2 and Y5 at the hypothalamus and pituitary of adult rats was

similar in ad libitum control and fasted rats (Figure 6).

11

DISCUSSION

The relationship between energy balance, body weight and reproductive function is well

known after the pioneer works of Frish group (20, 21). Different hormonal signals such as leptin,

ghrelin and orexins seem to be involved in the fine coordination between body weight and

reproductive function (2, 52, 56).

The key signal in the regulation of reproductive function is the pulsatile secretion of

GnRH which controls gonadotropin release. GnRH release is primarily controlled by central and

peripheral signals (28, 29, 41). NPY, secreted from hypothalamus to hypothalamic-pituitary

portal system, was characterized as “an unique member of the family of gonadotropic releasing

hormones“ (19), due to their multiple effects on hypothalamic GnRH secretion and in the

pituitary responsiveness to GnRH. Selective agonists and antagonists for the different NPY

receptors and different knock-out animals have been used to elucidate the role of different NPY

receptors in the control of feeding behavior and reproductive function. Recently, it has been

demonstrated that PYY3-36, agonist of NPY Y2 and Y5 receptors, is secreted from

gastrointestinal tract depending on the nutritional status of the animal. A regulatory loop

involving this peptide has been proposed: after food intake, PYY3-36 is secreted and inhibits

feeding behavior, whereas its release is depressed in underfeeding conditions (3, 59).

In previous experiments, we demonstrated the presence of NPY Y2 and Y5 receptor

subtypes in hypothalamus and pituitary and the complex actions of PYY3-36 at hypothalamic and

pituitary level (17). PYY3-36 directly stimulated gonadotropin secretion by prepubertal pituitaries

and significantly inhibited GnRH secretion by hypothalamic explants in males. The systemic and

central administration of the peptide exerted changes in serum concentrations of gonadotropins,

the effects being sexually dimorphic (17). The present studies extend and reinforce our previous

hypothesis that PYY3-36 is involved in the control of gonadotropin secretion. Specifically, herein

we have analyzed the effects of PYY3-36 in the control of gonadotropin and GnRH secretion

during fasting.

Periods of chronic undernutrition, as well as short periods of fasting, have been shown

to adversely impact mammalian reproduction in various ways. Fasting suppresses GnRH

release and hence pituitary LH, FSH and testosterone secretion, which are reversed by pulsatile

GnRH substitution (6, 7, 31, 61). The neural pathways that relay information on insufficient

12

energy stores to GnRH neurons included many signals. Different neuropeptides and

neurotransmitters [such as NPY, adrenaline via alpha 2-adrenergic receptors, Corticotropin

releasing hormone (CRH), leptin, ciliary neurotropic factor (CNTF), gamma-aminobutyric acid

(GABA) and opioids] have been implicated in the effects of fasting on pituitary secretion (8, 32,

38, 39, 40, 44).

Acute administration of NPY stimulated GnRH/LH release (4, 5, 14), whereas

continuous NPY receptor activation results in suppression of gonadotropin secretion (9, 15, 47),

which explains that in experimental conditions that up-regulate NPY synthesis and release,

such as diabetes, the reproductive function is also impaired (31). Acute and chronic food

deprivation and undernutrition stimulate hypothalamic NPY synthesis, storage and release in

the hypothalamus (31, 46, 61). Consequently, fasting-induced up-regulation of NPY secretion

may diminish gonadotropin secretion. This hypothesis is sustained by the finding that

counteracting the fasting-induced NPY up-regulation experimentally with those naturally-

occurring compounds that inhibit the hypothalamic NPY system, such as CNTF, it is possible to

attenuate the fasting-induced suppression of pituitary LH secretion (32). In summary,

undernutrition induces an up-regulation of NPY system in arcuate nucleus which, in turn,

suppresses GnRH and gonadotropin secretion.

In addition to such phenomenon, we hypothesized that changes in PYY3-36 secretion

should also be involved in the adverse effects of fasting on reproductive function. PYY3-36

release is regulated by food intake, increasing after meal intake and decreasing during fasting

(3, 59). On the basis of this secretory profile, reduction of gonadotropin secretion during fasting

would be due, at least in part, to the decrease in PYY3-36 release. To confirm this hypothesis, we

have analyzed the effects in vivo and in vitro of PYY3-36 on gonadotropin secretion in controls

fed ad libitum and fasted rats. In adult male rats, deprivation of food during 4 days significantly

decreased serum LH concentrations (Table 1). I.c.v. administration of PYY3-36 stimulated more

efficiently LH secretion in fasted than in control animals fed ad libitum, and serum LH

concentrations reached levels higher than obtained in control males. The increased in vivo

efficiency of PYY3-36 in fasted animals was confirmed in vitro. These results support our

hypothesis that depression of gonadotropin secretion during fasting in adult animals may be

13

due, at least partially, to the decrease in the stimulatory effect carried out by PYY3-36 and that

the efficiency of this signal was increased during food deprivation.

To analyze the participation of GnRH on the stimulatory effect of i.c.v. administration of

PYY3-36 on gonadotropin release in adult fasted rats, we analyzed the effects of PYY3-36 after

pretreatment with a GnRH-ANT. Results obtained demonstrated that the blockade of GnRH

action abolished the stimulatory effect of PYY3-36 on gonadotropin secretion. In addition, we

analyzed in vitro the GnRH release in presence of PYY3-36. Two important findings were

observed: PYY 3-36 inhibited GnRH release by hypothalamus obtained from males fed ad

libitum, which agrees with data obtained in prepubertal males (17), whereas a clear stimulatory

effect was observed after fasting. The reasons for the switch from an inhibitory action of PYY3-36

on GnRH release in control males to the stimulatory effect observed after fasting are unknown

at the present. However, this finding is in strong agreement with the potentiation of PYY3-36

effectiveness on gonadotropin secretion after fasting. Overall it is evident that the control of

GnRH /gonadotropin release by PYY3-36 is critically dependent of the nutritional status. Since

the effects of PYY3-36 on GnRH are opposite in fasted and ad lib fed rats, the stimulatory effect

on LH observed in both groups of animals suggests either that PYY3-36 modulated the

hypothalamic release of different signals other than GnRH involved in the control of LH, or a

possible direct pituitary effect (via hypothalamic-pituitary portal system) of the peptide icv

delivered.

The present data also indicated that PYY3-36 directly increases in both sexes LH and

FSH secretion at the pituitary level, in accordance with data obtained in prepubertal rats (17).

The stimulatory effect is enhanced after fasting. The mechanisms of the stimulatory action of

PYY3-36 at the pituitary level and the reasons for its increase effectiveness during fasting are

unknown. It has been previously shown that the NPY Y2 and Y5 receptor mRNA expression

patterns in hypothalamus do not change during fasting, in contrast with the increase observed

for Y1 mRNA expression (11, 63), but the expression in pituitary have not been studied. To

analyze whether, in our experimental paradigm, fasting increased the expression of genes

encoding Y2 and Y5 receptors, we studied their expression levels in hypothalamus and pituitary.

The fact that similar levels of mRNA were detected after fasting argues against the possibility

that an up-regulation of NPY Y2 and Y5 receptors in hypothalamus and/or pituitary might explain

14

the increase in the effectiveness of PYY3-36 in fasted rats. Nonetheless, the possibility that

fasting-induced changes in receptor number of signaling might take place at a post-

trascriptional level cannot be ruled out on the basis of our present data.

Leptin stimulates GnRH and LH secretion (64), and serum leptin levels decreased

during fasting (1). Different experimental approaches demonstrated the cross-talk between

leptin and NPY in the control of reproductive function. For example: the inhibition of

gonadotropic axis in leptin-deficient mice is attenuated by removal of NPY Y1 receptor and

acceleration of puberty by leptin is largely facilitated in mice deficient for NPY Y1 receptors (22).

It is conceivable that suppression of leptin input to GnRH neurons and pituitary gonadotrops in

fasted rats may enhance the responsiveness to other stimulating signals such as PYY 3-36. If this

hypothesis is correct, the increased hypothalamic/pituitary responsiveness to PYY 3-36 after

fasting could be secondary to the decrease in serum leptin levels. Since underrnutrition induces,

in addition to changes observed in serum leptin concentrations, a decrease in serum levels of

insulin and an increase in those of corticosterone, and both hormones are involved in the

control of LH secretion (10, 34, 37), the possibility that these changes could be involved in the

potentiation of stimulatory effect of PYY 3-36 after fasting merits further investigation

In conclusion, present experiments demonstrated that the stimulatory effect of PYY3-36

on gonadotropin secretion is enhanced after fasting. This phenomenon included an increase in

gonadotropin responsiveness to PYY3-36 in vivo and in vitro, as well as a clear stimulatory effect

on GnRH release. We proposed that the inhibition of pituitary gonadotropin secretion which

occurs in undernutrition could be mediated, at least in part, by the decrease in PYY 3-36

secretion and is reversed by exogenous administration of the peptide.

ACKNOWLEDGMENTS

This work was supported by grants BFI 2000-0419-CO3 and BFI 2002-00176 from

DGESIC (Ministerio de Ciencia y Tecnología, Spain). The collaboration of A. Mayen is

recognized.

15

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21

LEGEND TO FIGURES

Figure 1: Serum concentrations of LH (upper panels) and FSH (lower panels) in male rats,

either fed ad libitum or at fasting, which were s.c. injected with GnRH-ANT (5 mg/kg) or vehicle

48 and 24 h before i.c.v.injection with vehicle or 3 nmol/rat of PYY3-36. Food deprivation was

maintained during 4 days. Values are expressed as means ± SEM (10-12 animals/group). **p≤

0.01 vehicle plus PYY3-36 vs other groups plus PYY3-36. (ANOVA followed by Student -Newman-

Keuls test). In the inside panels, the integrated gonadotropin secretory responses following

central administration of PYY3-36 and peripheral administration of GnRH-ANT (calculated as the

area under the curve-AUC- during the 60-min study-period) in control and fasted rats are

shown. Bars with different superscript letter were statistically differents (ANOVA followed by

Student -Newman-Keuls test)

Figure 2: GnRH released (pg/hypothalamus/45 min.) by hypothalami from adult male rats,

either fed ad libitum or at fasting, incubated in the presence of PYY3-36 (10-6M) or medium

(DMEM) alone. Food deprivation was maintained during 4 days before incubation of

hypothalami. Values are expressed as means ± SEM (10-12 samples/group). **p≤ 0.01 and *p≤

0.05 vs corresponding control (DMEM) group (ANOVA followed by Student-Newman-Keuls

test).

Figure 3: Effects of PYY3-36 (10-8 and 10-6 M) on LH (upper panels) and FSH (lower panels)

secreted by hemi-pituitaries obtained from adult controls fed ad libitum (open bars) or fasting

males (hatched bars). Food deprivation was maintained during 4 days before incubation of

hemi-pituitaries. Values are expressed as means ± SEM (10-12 samples/group). **p≤ 0.01 vs

hemi-pituitaries incubated in absence of PYY3-36; ap≤ 0.01 vs corresponding fed ad libitum

control group (ANOVA followed by Student-Newman-Keuls test).

Figure 4: Effects of PYY3-36 (10-8 and 10-6 M) on LH (upper panels) and FSH (lower panels)

secreted by hemi-pituitaries obtained from adult controls fed ad libitum (open bars) or fasting

female rats (hatched bars). Control females were studied in metestrus. Food deprivation was

maintained during 4 days before incubation of hemi-pituitaries. Values are expressed as means

± SEM (10 samples/group). **p≤ 0.01 vs hemi-pituitaries incubated in absence of PYY3-36; ap≤

22

0.01 vs corresponding fed ad libitum control group (ANOVA followed by Student-Newman-Keuls

test).

Figure 5: Comparison between the effects of PYY3-36 on LH (upper panels) and FSH (lower

panels) secreted by hemi-pituitaries obtained from adult fed ad libitum control (open bars) and

fasting (hatched bars) male and female rats. Results are presented as changes over levels

obtained in the absence of PYY3-36. **p≤ 0.01 vs corresponding fed ad libitum control group

(ANOVA followed by Student-Newman-Keuls test).

Figure 6: Expression of the genes encoding NPY receptors Y2 and Y5 at the hypothalamus

(upper panels) and pituitary (lower panels) from adult male rats fed ad libitum (open bars) or

fasting males (hatched bars). Food deprivation was maintained during 4 days before study.

Representative images of ethidium bromide-stained gel electrophoresis of the specific

amplicons are presented. Three independent samples from controls fed ad libitum (C1-C3) and

fasting rats (F1-F3) are shown. Amplification of a fragment of RP-S11 mRNA served as internal

control. Semi-quantitative values of gene expression levels in the experimental groups are

shown, which are the mean ± SEM of at least three independent determinations. Negative

controls were run in parallel with specific RT-PCR assays and yielded negative amplification

(data not shown).

Table 1 Serum LH, FSH and leptin concentrations (ng/mL) in adult males either ad libitum or submitted to 4-days of absolute restriction of food (fasting), 15

min after i.c.v. administration of PYY3-36 (3 nmol/rat).

LH (ng/mL) FSH (ng/mL) Leptin (ng/mL)

Group Vehicle PYY3-36Fold-

increase Vehicle PYY3-36Fold-

increase Vehicle PYY3-36

Fed 2.60 ± 0.44 6.63 ± 0.76 a 2.55 8.95 ± 1.13 17.33 ± 1.41 a 1.93 3.38 ± 0.33 3.02 ± 0.34

Fasting 1.72 ± 0.41* 11.73 ± 0.94**,a 6.81 8.98 ± 0.66 15.20 ± 1.32 a 1.69 0.40 ± 0.07** 0.39 ± 0.10**

Values are given as means ± SEM (n = 8-10 animals per group). * p ≤ 0.05 and ** p ≤ 0.01 vs fed ad libitum group; a p ≤ 0.01 vs corresponding

vehicle-injected group. (Student´s t test).

LH (n

g/m

L)

0

5

10

15

AUCLH

(ng/

mL.

min

)

0

200

400

600

800

b

ac c

Veh GnRH-ANT

VehPYY3-36 3 nmol

**

****

Veh + VehVeh + PYY3-36GnRH-ANT + VehGnRH-ANT + PYY3-36 LH

(ng/mL)

0

5

10

15

AUC

LH (n

g/m

L. m

in)

0

200

400

600

800

b

ac c

Veh GnRH-ANT

VehPYY3-36 3 nmol

**

**

**

Veh + VehVeh + PYY3-36GnRH-ANT + VehGnRH-ANT + PYY3-36

0 15 30 45 60

FSH

(ng/

mL)

0

10

20

30

40

50

AUC

FSH

(ng/

mL.

min

)

0

250

500

750

1000

1250

b

Time (min.)

a

c c

Veh GnRH-ANT

VehPYY3-36 3 nmol

** ** **

Veh + VehVeh + PYY3-36GnRH-ANT + VehGnRH-ANT + PYY3-36

0 15 30 45 60

FSH (ng/m

L)

0

10

20

30

40

50

AUC

FSH

(ng/

mL.

min

)

0

250

500

750

1000

1250

b

Time (min.)

a

c c

Veh GnRH-ANT

VehPYY3-36 3 nmol

**** **

Veh + VehVeh + PYY3-36GnRH-ANT + VehGnRH-ANT + PYY3-36

Fed Fasting

Pinilla et al.Figure 1

GnR

H

(pg/

hypo

thal

amus

/45

min

)

0

10

20

30DMEMPYY3-36 10-6 M

**

*

Fed Fasting

Pinilla et al.Figure 2

0

10

20

30

40

50

DMEM

****

PYY 108 M

PYY 10-6 M

DMEM

PYY 108 M

PYY 10-6 M

** **

FSH

(ng/

mL)

0

10

20

30Fed Fasting

Fed Fasting

** ****

**

a

LH (n

g/m

L)

0

10

20

30

0

10

20

30

40

5060 min.

**

DMEM

**

120 min.

PYY 108 M

PYY 10-6 M

DMEM

PYY 108 M

PYY 10-6 M

Fed Fasting

Fed Fasting

**

****

,a**

,a**

Pinilla et al.Figure 3

FSH

(ng/

mL)

0

2

4

6

8

10

0

5

10

15

20

25

DMEM

PYY 108 M

PYY 10-6 M

DMEM

PYY 108 M

PYY 10-6 M

Fed Fasting

Fed Fasting

,a** ,a**,a** ,a**

LH (n

g/m

L)

0

10

20

30

40

0

10

20

30

40

50

6060 min.

DMEM

**

120 min.

PYY 108 M

PYY 10-6 M

DMEM

PYY 108 M

PYY 10-6 M

Fed Fasting

Fed Fasting

** a

,a** ,a**

,a**

Pinilla et al.Figure 4

0

200

400

600

800

1000

LH

(% 0

f cha

nge

over

con

trol

leve

ls)

0

200

400

600

800

1000Males Females

Fed Fasting

FSH

(%

0f c

hang

e ov

er c

ontr

ol le

vels

)

-100

0

100

200

300

400

500

-100

0

100

200

300

400

500

PYY3-36 (M) PYY3-36 (M)

10-8

10-6

10-8

10-6

10-8

10-6

10-8

10-6

10-8

10-6

10-8

10-6

10-8

10-6

10-8

10-6

60 min. 120 min.

60 min. 120 min.

60 min. 120 min.

60 min. 120 min.

**

**

****

**** ** **

**

**

** **

Pinilla et al.Figure 5

NPY-Y2

NPY-Y5

RP-S11

- 290 bp

- 144 bp

- 241 bp

Hypothalamus

C1 F2C3C2 F1M F3

mR

NA

leve

ls (O

D/S

11)

0

25

50

75

100

125NPY-Y2

mR

NA

leve

ls (O

D/S

11)

0

25

50

75

100

125

Fed Fasting

NPY-Y5

Fed Fasting

- 290 bp

- 144 bp

- 241 bp

Pituitary

mR

NA

leve

ls (O

D/S

11)

0

25

50

75

100

125NPY-Y2

mR

NA

leve

ls (O

D/S

11)

0

25

50

75

100

125

Fed Fasting

NPY-Y5

Fed Fasting

NPY-Y2

NPY-Y5

RP-S11

C2 F1M F3C1 C3 F2

Pinilla et al.Figure 6