Alterations in Hypothalamic KiSS-1 System in Experimental Diabetes

NEUROENDOCRINOLOGY

Alterations in Hypothalamic KiSS-1 System in Experimental Diabetes: Early Changes and Functional Consequences J. M. Castellano, V. M. Navarro, J. Roa, R. Pineda, M. A. Sańchez-Garrido, D. García-Galiano, E. Vigo, C. Dieguez, E. Aguilar, L. Pinilla, and M. Tena-Sempere Department of Cell Biology, Physiology, and 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 Co´rdoba, and CIBER Fisiopatología de la Obesidad y Nutricioń, 14004 Co´rdoba, Spain; and Department of Physiology (C.D.), University of Santiago de Compostela, 15705 Santiago de Compostela, Spain

Using long-term streptozotocin (STZ)-treated male rats, we recently proposed that defective function of hypothalamic KiSS-1 system is mechanistically relevant for central hypogonadotropism of uncontrolled diabetes. However, the temporal pattern of such defects and its potential contribution to disturbed gonadotropin secretion in the diabetic female remain so far unexplored. To cover these issues, expression analyses and hormonal tests were conducted in diabetic male (1 wk after STZ; short term) and female (4 wk after STZ; long term) rats. Short-term diabetic males had lower basal testosterone levels and decreased gonadotropin responses to orchidectomy (ORX), which associated with significantly attenuated post-ORX rises of hypothalamic KiSS-1 mRNA. Yet kisspeptin administration to diabetic males was able to acutely elicit supramaximal LH and testosterone responses and normalize post-ORX gonadotropin secretion. Long-term diabetic females showed persistent anestrus and significantly decreased basal gonadotropin levels as well as blunted LH responses to ovariectomy; changes that were linked to lowering of basal and postovariectomy expression of hypothalamic KiSS-1 mRNA. Moreover, despite prevailing gonadotropin suppression, LH responses to acute kisspeptin administration were fully preserved, and even enhanced after its repeated injection, in diabetic females. In sum, our present findings further define the temporal course and mechanistic relevance of altered hypothalamic KiSS-1 system in the hypogonadotropic state of uncontrolled diabetes. Furthermore, our data provide the basis for the potential therapeutic intervention of the KiSS-1 system as adjuvant in the management of disturbed gonadotropin secretion of type 1 diabetes in the female. (Endocrinology 150: 784 –794, 2009)

T

he secretion of pituitary gonadotropins, LH and FSH, is under the dynamic control of a plethora of regulators of central and peripheral origin that converge and integrate at the level of GnRH neurons at the forebrain (1–3), the secretion of hypothalamic GnRH being ultimately responsible for the pulsatile release of both gonadotropins (1). Whereas the mechanisms responsible for the control of the gonadotropic axis has been actively investigated and thoroughly characterized during the last decades, our understanding of the neuroendocrine pathways governing GnRH secretion has dramatically enlarged in recent years due to the identification of the reproductive roles of kisspeptins and their cognate receptor, G protein-coupled receptor 54(GPR54) (4 –7). Notably, kisspeptins, a family of peptides encoded by the

KiSS-1 gene that includes metastin and kisspeptin-10, were initially catalogued as metastasis-suppressor peptides, with potential additional roles in placental physiology (4, 6, 8 –10). However, the demonstration in late 2003 of inactivating mutations of GPR54 in patients suffering isolated or familial forms of hypogonadotropic hypogonadism unveiled the potential involvement of this ligand-receptor system in the regulation of essential aspects of reproductive maturation and function (11, 12). Indeed, during the last 4 yr, kisspeptins and GPR54, which has been now officially catalogued as KiSS-1 receptor (see http:// www.iuphar-db.org) have been proven to play pivotal roles in brain sexual differentiation, puberty onset, feedback regulation of gonadotropin secretion and/or the environmental and meta-

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2009 by The Endocrine Society doi: 10.1210/en.2008-0849 Received June 6, 2008. Accepted October 2, 2008. First Published Online October 9, 2008

Abbreviations: AUC, Area under the curve; GPR54, G protein-coupled receptor 54; i.c.v., intracerebroventricular; Kp-10, kisspeptin-10; ORX, orchidectomy; OVX, ovariectomy; STZ, streptozotocin.

784

endo.endojournals.org

Endocrinology, February 2009, 150(2):784 –794


bolic control of fertility in a diversity of species, including rodents and primates (4 – 6, 13). Overall, KiSS-1 neurons at discrete hypothalamic nuclei have been proposed to operate as nodal integrators of an array of key modulators of reproductive function, thus dictating the activation or eventual inactivation of secretion GnRH as final output pathway. Among their plethora of regulators, compelling evidence is mounting that KiSS-1 neurons are capable to sense the energy state of the organism, thereby transducing metabolic information onto the centers governing reproductive function (14 –17). This previously neglected pathway is likely to contribute to the functional coupling between the state of body energy reserves and its reproductive capacity; adverse metabolic conditions being commonly associated with defective fertility (18). Indeed, a number of experimental studies have now demonstrated that conditions of negative energy balance, such as short-term fasting in rats and mice, induce significant decreases in the hypothalamic expression of KiSS-1 gene (14, 17, 19), whereas acute administration of kisspeptin to fasted rats, despite the prevailing decrease in circulating LH levels, is able to evoke supramaximal gonadotropin responses (14, 20). Moreover, despite severe reduction of body weight due to restriction in daily food intake, repeated administration of kisspeptin was sufficient to rescue the pubertal activation of the gonadotropic axis in female rats submitted to chronic subnutrition (14). The nature of the signal(s) responsible for the metabolic regulation of the KiSS-1 system is yet to be fully uncovered. Yet convincing evidence suggests that the adipocytederived hormone, leptin, is capable to modulate KiSS-1 expression at the hypothalamus (15, 21), thereby providing a tenable mechanism for the well-known effects of leptin as gatekeeper of puberty onset and fertility. In keeping with its profound metabolic disturbances, uncontrolled diabetes is frequently associated with reproductive dysfunction, hypogonadotropic hypogonadism being a common observation in animal models of experimental diabetes (22–24). In diabetic male rats, defective function of the gonadotropic axis involves decreased basal and pulsatile LH secretion, disturbed sensitivity to negative feedback effects of androgens, reduced LH responses to gonadectomy, and defective testosterone secretion (22, 23, 25–27). Likewise, diabetic female rats display decreased basal levels of gonadotropins, disruption of the positive feedback effects of estradiol, delayed or absent preovulatory LH surges, and anovulation (28 –33). Whereas the mechanisms for the plethora of reproductive abnormalities observed in diabetes are likely to involve both peripheral and central defects, compelling evidence suggests that a failure of the neuroendocrine mechanisms governing GnRH secretion is causative for the state of hypogonadotropism detected in male and female models of uncontrolled diabetes (28, 34). Although this contention was recognized more than a decade ago, the nature of the afferent pathways altered in such conditions had remained largely unfolded. Using long-term streptozoticin (STZ)-treated adult male rats, as conventional model of type 1 diabetes, we recently documented the potential involvement of alterations of the hypothalamic KiSS-1 system in the generation of the state of impaired gonadotropic function in such model (15). Indeed, 4 wk after STZ treatment, uncontrolled diabetic males not only displayed


785

the expected decrease in circulating LH concentration but also showed a significant reduction of hypothalamic KiSS-1 mRNA levels, at both basal and postorchidectomy conditions (15). Moreover, acute and repeated administration of kisspeptin was sufficient to ameliorate the hypogonadotropic state of long-term STZ males, despite no additional metabolic intervention. Altogether these observations suggest that, at least in the male, consolidation of a state of diabetes is linked to suppression of the functionality of hypothalamic KiSS-1 system, which likely contributes to gonadotropic failure (15). Mechanistic analyses evidenced that defective leptin, but not insulin, signaling at central levels is, at least partially, responsible for such a suppression of hypothalamic KiSS-1 in uncontrolled diabetes (15). These findings are in keeping with recent in vivo and in vitro data demonstrating a positive role of leptin in the control of KiSS-1 mRNA expression at the hypothalamus (17, 21). Despite the above progress in the field, some critical features of the potential dysfunction of hypothalamic KiSS-1 system in uncontrolled diabetes remain largely unexplored. Thus, the temporal pattern of KiSS-1 defects in the diabetic male has not been assessed to date. Similarly, whether a similar disruption is detected in the diabetic female, and, if so, its potential contribution to disturbed gonadotropin secretion remain to be determined. The latter is especially relevant, given the profound impairment of gonadotropic/ovulatory function caused by diabetes in the female (28 –33). Yet previous studies had suggested the existence of sex differences in the mechanisms underlying the hypogonadal state in male and female diabetic rats (35). To cover the above issues, expression analyses and hormonal tests were conducted in short-term diabetic males (1 wk after STZ) and long-term diabetic females (4 wk after STZ). Our data document the rapid, progressive decline in the KiSS-1 system in diabetes, and strongly suggest that defective kisspeptin signaling is causative also for the state of hypogonadotropism observed in uncontrolled forms of diabetes in the female.

Materials and Methods Adult Wistar male and female rats, bred in the vivarium of the University of Co´rdoba, were used. The animals were maintained under constant conditions of light (14 h of light, from 0700 h) and temperature (22 C) and were housed in individual cages with free access to pelleted food and tap water. Experimental procedures were approved by the Co´rdoba University Ethical Committee for animal experimentation and were conducted in accordance with the European Union normative for care and use of experimental animals. Kisspeptin (110 – 119)-NH2 (termed hereafter as Kp-10) was obtained from Phoenix Pharmaceuticals Ltd. (Belmont, CA). STZ was purchased from Sigma Chemical Co. (St. Louis, MO).

Experimental designs Experiments were carried in STZ-induced diabetic rats. Two sets of studies were conducted: 1) experiments in male rats at 1 wk (short term) after STZ injections, and 2) experiments in female rats at 4 wk (long term) after STZ injections. In both settings, a protocol of two consecutive sc injections of STZ [50 mg/kg in 0.01 M citrate buffer (pH 4.5)] at a 24-h interval was used, following previously published references (15, 28, 34). After STZ injections, body weights were recorded daily and serum glucose levels were measured after 5 d and at the time of the experimental

786

Castellano et al.

KiSS-1 System and Hypogonadotropism in Diabetic Rats

procedures to check for diabetic status, animals showing severe hyperglycemia (⬎450 mg/dl) were selected for analysis (⬎95% of STZ injected males and females in the present study). For experiments using female rats, the animals were monitored for estrous cyclicity by means of vaginal cytology before initiation of experiments; only animals displaying at least two consecutive 4-d estrous cycles were selected for STZ injections and subsequent hormonal and gene expression analyses. Female rats injected with vehicle, at the diestrous-1 phase of the cycle at the time of sampling, were used as controls. In addition, for experiments involving gonadectomy, the animals were subjected to bilateral gonadectomy via the scrotal [male; orchidectomy (ORX)] or abdominal [female; ovariectomy (OVX)] route, under light ether anesthesia 1 wk before hormonal tests or tissue sampling. Sham-operated animals served as corresponding controls.

Experiments in short-term diabetic male rats


week after bilateral OVX, groups of animals (n ⫽ 10 –12) were subjected to central i.c.v. injection of Kp-10 following an experimental procedure similar to that of experiment 4, and blood samples (300 ␮l) were obtained by jugular venipuncture before (0) and at 15 and 60 min after kisspeptin administration.

Experiment 6 Besides hormonal tests, gene expression analysis were conducted in hypothalamic samples from long-term diabetic female rats, either at intact or post-OVX conditions. Protocols of STZ injections and bilateral orchidectomy were as described for experiments 4 –5. Groups of adult female rats (n ⫽ 6) were subjected to the different experimental manipulations, and at the end of the corresponding periods, the animals were killed by decapitation and the hypothalamus was immediately dissected out as described elsewhere, frozen in liquid nitrogen, and stored at ⫺80 C until RNA analysis.

Experiment 1 Basal hormonal profiles, as well as the effects of acute injection of Kp-10 on LH and testosterone secretion, were assayed in groups (n ⫽ 10 –12) of short-term diabetic male rats. A protocol of intracerebral injection of Kp-10 was conducted in STZ-injected animals and their respective controls, following previous references (15, 36). To allow delivery of kisspeptin into the lateral cerebral ventricle, the animals were implanted, under ether anesthesia, with intracerebroventricular (i.c.v.) cannulae lowered to a depth of 4 mm beneath the surface of the skull; the insert point was 1 mm posterior and 1.2 mm lateral to bregma, as described elsewhere (36). Two doses of Kp-10 were tested: 1 nmol and 10 pmol in 10 ␮l (0.9% saline). Dosage selection was based in previously published dose-response curves for LH after central administration of a range of doses of Kp-10 (37), as a means to achieve maximal and submaximal activation of GPR54 in vivo. Blood samples (300 ␮l) were obtained by jugular venipuncture under light ether anesthesia before (0) and at 15 and 60 min after Kp-10 injection.

Experiment 2 LH responses to ORX and Kp-10 administration were also evaluated in short-term diabetic males. One week after bilateral ORX, groups of animals (n ⫽ 10 –12) were subjected to central i.c.v. injection of Kp-10 after an experimental procedure similar to that of experiment 1, and blood samples (300 ␮l) were obtained by jugular venipuncture before (0) and at 15 and 60 min after kisspeptin administration.

Experiment 3 Besides hormonal tests, gene expression analysis were performed in hypothalamic samples from short-term diabetic male rats, either at intact or post-ORX conditions. Protocols of STZ injections and bilateral ORX were as described for experiments 1–2. Groups of adult male rats (n ⫽ 6) were subjected to the different experimental manipulations, and at the end of the corresponding periods, the animals were killed and the hypothalamus was immediately dissected out as described elsewhere (15, 36), frozen in liquid nitrogen, and stored at ⫺80 C for RNA analysis.

Experiments in long-term diabetic female rats Experiment 4 Basal hormonal profiles, as well as the effects of acute injection of Kp-10 on LH secretion, were evaluated in groups (n ⫽ 10 –12) of longterm diabetic female rats. For the hormonal tests, the protocol of intracerebral injection of Kp-10 was as described for experiment 1; a maximal dose of 1 nmol of Kp-10 was tested. Blood samples (300 ␮l) were obtained by jugular venipuncture under light ether anesthesia before (0) and at 15 and 60 min after Kp-10 injection.

Experiment 5 In addition to basal levels, LH responses to OVX and central Kp-10 administration were also assayed in long-term diabetic females. One

Experiment 7 Finally, the effects of repeated administration of Kp-10 in terms of LH secretory responses were monitored in long-term diabetic female rats. Groups of STZ-treated females were subjected to two different regimens of Kp-10 injections: 1) one daily i.c.v. injection of 1 nmol Kp-10 between 0900 and 1000 h; and 2) two daily i.c.v. injections of 1 nmol Kp-10 every 12 h (at 0900 and 2100 h). Treatments were continued for 7 d, and blood samples (300 ␮l) were obtained by jugular venipuncture at the morning of d 1, 3, and 7, 60 min after kisspeptin administration.

Hormone measurements Serum LH levels were determined in a volume of 50 ␮l using RIA kits supplied by the National Institutes of Health (Dr. A. F. Parlow, National Institute of Diabetes and Digestive and Kidney Diseases, National Hormone and Peptide Program, Torrance, CA). Rat LH-I-9 was labeled with 125 I using Iodo-gen tubes, following the instructions of the manufacturer (Pierce, Rockford, IL), and hormone concentrations were expressed using the reference preparation LH-RP-3 as standard. Intra- and interassay coefficients of variation were less than 8 and 10%. The sensitivity of the assay was 5 pg/tube. In addition, serum glucose concentrations were determined using an automatic glucose analyzer (Accu-Chek; Roche Diagnostics, Barcelona, Spain). Finally, in selected experimental samples, serum leptin and testosterone levels were determined using commercial kits from Linco Research (St. Charles, MO) and MP Biomedicals (Costa Mesa, CA), respectively, following the instructions of the manufacturer. The sensitivity of the assays was 0.05 ng/tube (leptin) and 0.1 ng/tube (testosterone), whereas the intraassay coefficients of variation were less than 5%.

RNA analysis by semiquantitative RT-PCR Total RNA was isolated from hypothalamic samples using the singlestep, acid guanidinium thiocyanate-phenol-chloroform extraction method. Hypothalamic expression of KiSS-1 mRNAs was assessed by RT-PCR, optimized for semiquantitative detection, using previously defined primer pairs and conditions (15). This thoroughly validated procedure allows for estimation of changes in relative amounts of the target mRNA at specific experimental conditions, as compared with the values in the corresponding control samples, measured in the same assay. As internal control for reverse transcription and reaction efficiency, amplification of a 240-bp fragment of S11 ribosomal protein mRNA was carried out in parallel in each sample. In keeping with previous optimization tests, 32 and 24 PCR cycles were chosen for semiquantitative analysis of the specific target (KiSS-1) and RP-S11 internal control, respectively (15). Specificity of PCR products was confirmed by direct sequencing (Central Sequencing Service, University of Cordoba). Quantification of intensity of RT-PCR signals was carried out by densitometric scanning using an image analysis system (1-D Manager; TDI Ltd., Madrid, Spain), and values of the specific targets were normalized to those of internal controls to express arbitrary units of relative expression. In all



787

400

100

350

75

Presentation of data and statistics

300

0

150

-25 -50

100

-75

50

-100

Glucose (mg/dL)

0

3,5

600

**

500

3,0 2,5

400

2,0

300

**

200

1,5 1,0 0,5

0

0,0

4

4

3

3

2

**

2

0

STZ-1wk

0

Control

1

STZ-1wk

1

T (ng/mL)

100

Control

Hormonal profiles and effects of kisspeptin on LH and testosterone secretion Body weight data and hormonal profiles of short-term diabetic male rats are presented in Fig. 1. One week after injection of STZ, diabetic males showed significantly lower total body weights than controls, with a negative body weight gain along the study period. As confirmation of their diabetic status, all animals injected with STZ showed serum glucose concentrations greater than 450 mg/dl, in contrast to normoglycemic controls (⬍120 mg/dl). In addition, STZ-injected males presented significantly reduced serum leptin levels. In terms of gonadotropic axis function, basal serum LH levels in 1-wk STZ rats were not significantly different from those in control animals. Conversely, shortterm diabetic males showed a clear-cut reduction in circulating testosterone levels, whose concentration was about one fourth that in control animals (Fig. 1). On this hormonal background, the gonadotropic effects of kisspeptin were tested. On the basis of our previous dose-response analyses in vivo, two doses of Kp-10 were chosen for i.c.v. injection, 1 nmol and 10 pmol, for maximal and submaximal stimulation of the gonadotropic axis, respectively. In control animals, 1 nmol and 10 pmol Kp-10 were equally effective in inducing robust LH bursts at 15 min after central injection of the peptide. However, LH levels remained elevated at 60 min after kisspeptin administration only for the high (1 nmol) dose. Likewise, in short-term diabetic animals, both doses of Kp-10 effectively induced LH secretory bursts after central injection, with persistently elevated LH levels at 60 min only in the high-dose group. Nonetheless, whereas the pattern of secretion after administration of the low dose of kisspeptin (10 pmol) was similar to that of control animals, LH responses to 1 nmol kisspeptin were significantly higher (P ⬍ 0.01) in 1-wk diabetic animals 15 min after peptide administration. Likewise, testosterone responses to the high dose of Kp-10 were not only preserved but even significantly augmented in this group. Finally, when integrated LH secretion was calculated over the 60-min study period, it was confirmed that net LH responses to low doses of kisspeptin were fully conserved, whereas those to high doses were even enhanced, in short-term diabetic male rats (Fig. 2).

25

200

LH (ng/mL)

Studies in short-term diabetic male rats

250

Leptin (ng/mL)

Results

50

**

∆BW (g)

Hormonal determinations (LH, testosterone, leptin) were conducted in duplicate, with a minimal total number of 10 samples per group. When appropriate, integrated LH secretory responses were calculated as the area under the curve (AUC), using the trapezoidal rule, over the 60-min period after administration of Kp-10 or vehicle. Semiquantitative RTPCR analyses were carried out in duplicate from at least four independent RNA samples of each experimental group. Quantitative RNA and hormonal data are presented as mean ⫾ SEM. Results were analyzed for statistically significant differences using unpaired Student t test or ANOVA followed by Student-Newman-Keuls multiple range tests (SigmaStat 2.0; Jandel Corp., San Rafael, CA). P ⱕ 0.05 was considered significant.

BW (g)

assays, liquid controls and reactions without reverse transcription resulted in negative amplification.

FIG. 1. Body weight data and hormone profiles of adult male rats 1 wk after injection of STZ (short-term diabetes). In the upper panels, total body weights (BW) and net body weight gain in control and STZ-injected animals are shown. In addition, glucose and leptin levels in short-term diabetic males and controls are presented in the middle panels. Finally, in the lower panels, serum LH and testosterone (T) levels in controls and short-term STZ animals are indicated. Data are the mean ⫾ SEM of at least 10 independent determinations per group. **, P ⬍ 0.01 vs. corresponding control values (Student t test).

Hormonal profiles and LH responses to kisspeptin in ORX diabetic male rats Hormonal profiling and functional tests were also performed in short-term STZ-injected males, at 1 wk after bilateral gonadectomy. In keeping with previous references, LH hypersecretion in response to ORX was partially blunted in diabetic animals, with an approximately 50% reduction in the post-ORX rise of serum LH levels vs. control gonadectomized animals (Fig. 3A). Central Kp-10 administration to diabetic ORX rats was able to fully reverse defective LH responses to gonadectomy. Thus, both 1 nmol and 10 pmol doses of Kp-10 induced further increases in post-ORX LH values that equaled, at 15 min after injection, the

788

Castellano et al.



CONTROL 14

LH (ng/mL)

12 10 8

**

6 4

14

**

**

**

6

**

**

4 2

0

15

30

45

15

0

15

30

0

45

60 (min)

d

AUC

500

c

**

375

10

b

5

625

a

a

250

b

125

a

LH (ng/mL·min)

T (ng/mL)

60

** ,a

20

STZ+Kp-10 1nmol

STZ+Kp-10 10pmol

STZ+Veh

C+Kp-10 1nmol

C+Kp-10 10pmol

C+Veh

STZ+Kp-10 1nmol

STZ+Veh

C+Kp-10 1nmol

C+Veh

0

10 8

2 0

12

LH (ng/mL)

Vehicle Kp-10 (10pmol) Kp-10 (1nmol)

STZ-1wk

0

FIG. 2. LH and testosterone (T) responses to central kisspeptin administration in control and short-term (STZ, 1 wk) diabetic male rats. In the upper panels, the time course for the LH-releasing effects of two doses of Kp-10 (1 nmol and 10 pmol, i.c.v.) is presented. In the lower panels, serum T levels at 60 min after injection of the high dose of Kp-10 are shown from control and 1-wk STZ animals. In addition, integrated LH secretion after central administration of Kp-10 (AUC during the 60 min study period) is presented for both doses, in control (C) and short-term diabetic animals. Data are the mean ⫾ SEM of at least 10 independent determinations per group. **, P ⬍ 0.01 vs. control values at 0-min; a, P ⬍ 0.01 vs. corresponding time-paired values in controls; for AUC data, groups with different superscript letters are statistically different (ANOVA followed by Student-Newman-Keuls multiple range test).

levels observed in ORX control animals. Yet, as was the case in intact animals, persistent increases over preinjection LH levels were only detected at 60 min after administration of the high dose of kisspeptin (Fig. 3B). Calculation of integrated LH secretory responses confirmed that net LH hypersecretion in response to ORX was significantly decreased in gonadectomized diabetic animals and that such a defective secretion could be completely rescued by exogenous administration of kisspeptin (Fig. 3C). Hypothalamic expression of KiSS-1 gene in intact and ORX diabetic male rats Expression analyses of KiSS-1 gene at the hypothalamus were conducted in short-term diabetic male rats, either intact or at 1 wk after gonadectomy. Relative KiSS-1 mRNA levels in whole hypothalamic fragments from intact short-term diabetic rats were not significantly different from those of control animals. One week after ORX, control rats presented a significant 2.5fold increase in KiSS-1 mRNA levels over those in intact males, in keeping with previous references (36). In contrast, gonadectomy of short-term diabetic rats failed to induce a significant

elevation in hypothalamic KiSS-1 mRNA expression; only a marginal rise in KiSS-1 mRNA levels over intact values was detected, but it did not reach statistical significance. Accordingly, the levels of KiSS-1 mRNA, at 1 wk after ORX, were significantly lower in short-term STZ males than in corresponding controls (Fig. 4). Studies in long-term diabetic female rats Hormonal profiles and effects of kisspeptin on LH secretion Data on body weight, glucose, and hormonal levels in longterm diabetic female rats are summarized in Fig. 5. Final body weights were significantly lower in diabetic females 4 wk after STZ injection, diabetic females showing negative body weight gain along the study period. As predicted on the basis of their diabetic status, all female rats injected with STZ showed marked hyperglycemia, with serum glucose concentrations greater than 450 mg/dl. In addition, diabetic females presented a significant reduction in serum leptin levels 4 wk after STZ injection. In terms of gonadotropic axis function, 4-wk STZ-treated female rats displayed constant anestrous at vaginal cytology (data not


12

A LH (ng/mL)

6

2

0

0

STZ

Vehicle Kp-10 (10pmol) Kp-10 (1nmol)

12

STZ-ORX

2

C-ORX

LH (ng/mL)

8

4

b

b

b

10 8

c

6

c

c

4 2

C-ORX

800

a Control

0

LH (ng/mL·min)

,a

4

14

C

12

LH (ng/mL)

**

6

16

789

10

8

Control

B

STZ-1wk

**

10


AUC

700

0 15 30 45 60 (min)

STZ-ORX b

b

b

600 500

c

400 300 200 100

a

a a STZ-ORX-K1nmol

STZ-ORX-K10pmol

STZ-ORX

STZ

C-ORX

Control

0

b

FIG. 3. In the upper panel (A), LH responses to ORX in short-term (STZ, 1 wk) diabetic male rats and their respective controls are shown. In addition, LH responses to central kisspeptin administration in short-term ORX diabetic males are presented. Thus, in the middle panel (B), the time course for the LH-releasing effects of i.c.v. injection of Kp-10 is presented from ORX diabetic males. In addition, in the lower panel (C), the integrated LH secretory responses to Kp-10 (AUC during the 60 min study period) in ORX diabetic rats are shown. For comparative purposes, the corresponding LH levels in control male rats, at 1 wk after ORX, are included in middle and lower panels. In the upper panel, **, P ⬍ 0.01 vs. corresponding controls at 0 min; a P ⬍ 0.01 vs. corresponding timepaired values in controls; in the middle and lower panels, groups with different superscript letters are statistically different (ANOVA followed by StudentNewman-Keuls multiple range test).

shown), overtly decreased serum LH concentrations, and significantly reduced uterus weight as surrogate marker of defective estradiol levels (Fig. 5). In addition, ovarian weights were also

FIG. 4. Hypothalamic expression of KiSS-1 gene in the hypothalamus of adult male rats 1 wk after injection of vehicle (control: Co) or STZ (short-term diabetes), either at intact conditions or after ORX. For each group, three representative independent samples are presented. Parallel amplification of S11 ribosomal protein mRNA served as internal control. In the lower panels, KiSS-1 mRNA levels are the mean ⫾ SEM of at least four independent determinations. Groups with different superscript letters are statistically different (P ⬍ 0.05; ANOVA followed by Student-Newman-Keuls multiple range test).

significantly decreased in 4-wk diabetic females (74.83 ⫾ 4.11 vs. 106.66 ⫾ 5.07 mg in controls; P ⬍ 0.05). In this model, injection of an effective dose of Kp-10 demonstrated that, despite significant reduction of the prevailing gonadotropin levels, LH responses to kisspeptin were grossly preserved. Thus, the profiles of LH secretion in response to 1 nmol Kp-10, with significantly increased levels at 15 min that remained elevated after 60 min, were analogous in control and long-term diabetic female rats. In good agreement, the integrated absolute LH secretory responses to Kp-10 were statistically similar in control and long-term diabetic animals. Furthermore, given the lowering of basal LH levels in diabetic females, relative responses to kisspeptin were augmented in this group (8-fold increase vs. ⬃4-fold increase in control females at diestrus-1) (Fig. 6). Hormonal profiles and LH responses to kisspeptin in OVX diabetic female rats Circulating LH levels, before and after central administration of Kp-10, were also assessed in long-term STZ-injected females at 1 wk after bilateral gonadectomy. As described previously in the female (28, 33) and in keeping with our male data (see Fig. 3), LH hypersecretion in response to OVX was significantly decreased in diabetic females, with an approximately 55% reduc-


300

100

250

75 50

**

200

25 0

150

-25

100

-50 -100

Vehicle Kp-10 (1nmol)

**

6

**

4 2

400

2,0

300

1,5

200

**

100

1,0 0,5

0

0,0

1,6

300

200 150

0,8

100

**

50

2

45

60 (min)

b

250

b

200 150 100

a

50 0

c STZ+Kp-10

Hypothalamic expression of KiSS-1 gene in intact and OVX diabetic female rats As done for the short-term diabetic male rats, relative KiSS-1 mRNA levels at the hypothalamus were assessed in 4-wk STZ diabetic females, either intact or at 1 wk after OVX. In contrast

30

STZ+Veh

tion in the post-ORX rise of serum LH levels vs. control gonadectomized animals (Fig. 7A). In this setting, intracerebral injection of 1 nmol Kp-10 was sufficient to fully reverse defective LH responses to gonadectomy. Moreover, serum LH levels at 15 and 60 min after Kp-10 injection in OVX diabetic females were significantly higher than in control OVX rats (Fig. 7B).

AUC

15

C+Kp10

FIG. 5. Body weight data and hormone profiles of adult female rats 4 wk after injection of STZ (long-term diabetes). In the upper panels, total body weights (BW) and net body weight gain in control (diestrus-1) and STZ-injected animals are shown. In addition, glucose and leptin levels in long-term diabetic females and controls are presented in the middle panels. Finally, in the lower panels, serum LH levels and uterine weights (UW) in controls and long-term STZ animals are shown. Data are the mean ⫾ SEM of at least 10 independent determinations per group. **, P ⬍ 0.01 vs. corresponding control values (Student t test).

300

0

Co+Veh

STZ-4wk

Control

STZ-4wk

Control

0

**

**

4

350

250


STZ-4wk

6

0

UW (mg)

**

1,2

LH (ng/mL)

2,5

LH (ng/mL·min)

3,0

500

0,4

8

3,5

**

600

0

Leptin (ng/mL)

Glucose (mg/dL)

0

LH (ng/mL)

Co-D1

-75

50

0,0


8

LH (ng/mL)

Castellano et al.

∆BW (g)

BW (g)

790

FIG. 6. LH responses to central kisspeptin administration in long-term (STZ, 4 wk) diabetic female rats. The time course for the LH-releasing effects of an effective dose (1 nmol) of Kp-10 is presented for control (diestrus-1: Co-D1) and diabetic females in the upper and middle panels, respectively. In the lower panels, the integrated LH secretion after central administration of Kp-10 (AUC during the 60 min study period) is presented for the different experimental groups. Data are the mean ⫾ SEM of at least 10 independent determinations per group. **, P ⬍ 0.01 vs. control values at 0 min; P ⬍ 0.01 vs. corresponding timepaired values in controls; for AUC data, groups with different superscript letters are statistically different (ANOVA followed by Student-Newman-Keuls multiple range test).

to 1-wk STZ males, long-term diabetes in the female evoked a significant reduction of greater than 50% in KiSS-1 mRNA levels in whole hypothalamic fragments. In addition, whereas 1-wk gonadectomy of control females resulted in the expected 2.5-fold increase in KiSS-1 mRNA at the hypothalamus, OVX of longterm diabetic females evoked a severely attenuated rise of hypothalamic KiSS-1 mRNA expression. These relative levels, however, were higher than those detected in intact diabetic females (Fig. 8).


A

12

12

**

6

,a

8 6

2

0

0

C-OVX

STZ-OVX

2

STZ

4

Control

4

LH (ng/mL)

LH (ng/mL)

791

10

8

STZ-4wk

B


20

LH (ng/mL)

STZ-4wk

**

10


d

d

16

b

12 8 4

c

c

a C-OVX

Control

0

c

0 15 30 45 60 (min)

STZ-OVX

FIG. 7. In the upper panel (A), LH responses to OVX in long-term (STZ, 4 wk) diabetic female rats and their respective (diestrus-1) controls are shown. In addition, LH responses to central kisspeptin administration in long-term diabetic females 1 wk after OVX are presented in the lower panel (B). For comparative purposes, the corresponding LH levels in control female rats, at 1 wk after OVX, are included. In the upper panel, **, P ⬍ 0.01 vs. corresponding controls at 0 min; ⬍ 0.01 vs. corresponding time-paired values in controls; in the lower panel, groups with different superscript letters are statistically different (ANOVA followed by Student-Newman-Keuls multiple range test).

Effects of repeated kisspeptin administration on LH secretion in diabetic female rats Finally, the effects of repeated i.c.v. administration of kisspeptin on circulating LH levels in long-term diabetic females were also explored. As experimental protocol, STZ females were submitted to daily i.c.v. injections of 1 nmol Kp-10 for 7 d at two regimens: 1) a single Kp-10 bolus at 0900 h each day of treatment; and 2) i.c.v. boluses of Kp-10 twice daily, at 0900 and 2100 h during the 7 d of treatment. Blood sampling was conducted immediately before and at 60 min after the corresponding a.m. injection of Kp-10 on d 1 (when experiment was started), d 3, and d 7. Hormonal analysis revealed that, despite significantly decreased prevailing LH levels in diabetic females, both treatment protocols evoked preserved LH responses, similar in magnitude for each regimen, throughout the study period. Of note, however, the net amplitude of LH secretion in response to Kp-10 appeared to be dependent on the regimen of administration because LH responses in STZ females injected twice daily were significantly higher than those in diabetic animals injected once a day with Kp-10 at all time points explored (Fig. 9).

FIG. 8. Hypothalamic expression of KiSS-1 gene in the hypothalamus of adult female rats 4 wk after injection of vehicle (control: Co) or STZ (long-term diabetes), either at intact conditions or after OVX. For each group, three representative independent samples are presented. Parallel amplification of S11 ribosomal protein mRNA served as internal control. In the lower panels, KiSS-1 mRNA levels are the mean ⫾ SEM of at least four independent determinations. Groups with different superscript letters are statistically different (P ⬍ 0.05; ANOVA followed by Student-Newman-Keuls multiple range test).

Discussion Whereas hypogonadotropism has been long recognized as a common complication of poorly controlled diabetes, the underlying neuroendocrine basis for such a phenomenon had remained largely unknown. Recent data from our group suggested that, in long-term STZ-treated male rats, alterations of hypothalamic KiSS-1 system are mechanistically involved in the generation of the state of central hypogonadism in diabetes (15). Our current data extend and complement those initial observations, and conclusively demonstrate that changes in expression/ function of the KiSS-1 system are readily detectable at early stages of the diabetic state in the male and that the diabetic female is also sensitive to disturbance of hypothalamic KiSS-1 system, which seems causative for defective gonadotropin secretion observed in this model, both in basal and postgonadectomy conditions. Comparison of the hormonal profiles of short-term (1 wk after STZ; present results) and long-term (4 wk after STZ) (15) diabetic male rats illustrates the progressive deterioration of gonadotropic function in the course of uncontrolled diabetes. Thus, we report herein that short-term diabetic males show significantly reduced circulating testosterone levels; however, their mean serum LH levels were roughly preserved (see Fig. 1), and serum FSH concentrations were only marginally decreased (our

792

Castellano et al.

LH (ng/mL)

8,0 6,0

STZ-Veh STZ-a.m. STZ-a.m./p.m. Control

4,0 2,0 0,0


**

d-1

**

**

** a

,b

,b

**

a

a

d-3

d-7

FIG. 9. LH responses to repeated administration of kisspeptin in long-term diabetic female rats. Diabetic females were subjected to two different protocols of intermittent administration of 1 nmol boluses of Kp-10 for 7 d: 1) a single Kp10 bolus at 0900 h each day of treatment (STZ-a.m.); and 2) i.c.v. boluses of Kp10 twice daily, at 0900 and 2100 h during the 7 d of treatment (STZ-a.m./p.m.). Hormonal levels were determined on the morning of d 1, d 3, and d 7 of treatments, before and 60 min after i.c.v. injection of kisspeptin. For comparative purposes, the corresponding LH levels in control female rats are presented for each time point as bars. **, P ⬍ 0.01 vs. corresponding preinjection values in STZ females at 0 min; a, P ⬍ 0.01 vs. corresponding time-paired values in nondiabetic controls; b, P ⬍ 0.01 vs. corresponding levels in STZ-a.m. groups (ANOVA followed by Student-Newman-Keuls multiple range test).

unpublished data). In clear contrast, long-term diabetic males were previously shown to display significantly lower basal concentrations of both LH and testosterone. These observations evidence that whereas the severity of suppression of the reproductive axis increases as function of the progression of the disease, an impact on gonadotropic function can be already detected at rather early stages of diabetes. A likely explanation for the apparent preservation of circulating LH levels in this early phase is that initial hypogonadotropism in short-term diabetes is compensated as direct consequence of a decrease in the negative feedback input of testicular testosterone. Conversely, in the long term, such compensation is no longer possible and the animals present an overt failure of gonadotropin secretion. Supporting this hypothesis, relative mRNA levels of KiSS-1 at the hypothalamus of short-term diabetic males were not overtly decreased vs. corresponding controls. This is likely to reflect a transient state of compensation due to decreased circulating testosterone because elimination of negative feedback of testicular androgen has been reported to increase hypothalamic KiSS-1 gene expression (15, 36). In contrast, long-term diabetic males displayed a significant reduction in hypothalamic KiSS-1 mRNA levels in basal conditions, in close parallelism with the marked decrease in circulating LH concentrations (15). In any event, serum LH and hypothalamic KiSS-1 levels in 1-wk STZ males must be considered as inappropriately low, given the dampening of circulating testosterone, which evidences an incipient central hypogonadism at such early phase of diabetes. The functional impact of (subtle) alterations of the hypothalamic KiSS-1 system at early stages of diabetes is indirectly documented by the observation that a single intracerebral bolus of kisspeptin was capable to evoke robust LH responses and was sufficient to normalize testosterone secretion in 1-wk STZtreated males. Of note, although different forms of kisspeptins have been described (9), in our pharmacological tests, Kp-10 was used, in line with previous studies (15, 36 –38), as a mean to achieve effective activation of GPR54 in vivo, thus proving the principle that restoration of kisspeptin signaling would be suf-


ficient to normalize the function of the gonadotropic axis despite the state of severe metabolic stress. The importance of these findings is 2-fold: 1) they document that, despite early signs of deterioration, the gonadotropic axis retains its capacity to maximally respond to kisspeptin stimulation, even at relatively low doses (10 pmol), under such altered metabolic conditions; and 2) they argue against the possibility that defective testosterone secretion in short-term diabetic males, in the presence of apparently preserved serum LH concentrations, is caused by a primary defect of testicular function. Furthermore, in short-term diabetic rats, LH secretory bursts and testosterone responses induced by the high dose of kisspeptin (1 nmol) nearly doubled those evoked in paired control animals. Taken together, these observations strongly suggest a state of enhanced responsiveness to kisspeptin in the initial phases of diabetes, which might be caused by an early, subtle decrease in the endogenous kisspeptin tone at the hypothalamus. This condition resembles the status of LH hyperresponsiveness to exogenous kisspeptin previously reported by our group in food-deprived rats, whose underlying mechanism seems to involve also the decreased expression of hypothalamic KiSS-1 (14). Anyhow, the presence of inappropriately low LH levels despite such an increased responsiveness to kisspeptin further supports the contention that a decline in expression/ secretion of kisspeptins is likely to take place at early stages of diabetes. In keeping with previous reports in long-term diabetic animals, our data demonstrate that defective LH responses to gonadectomy are already detected in short-term STZ males, documenting further their state of defective gonadotropin secretion. Notably, despite hypothalamic KiSS-1 mRNA expression was not significantly lower in short-term STZ males in basal conditions, the rise in their KiSS-1 mRNA levels after ORX was significantly attenuated (see Fig. 4). The possibility that such a blunted KiSS-1 response to gonadectomy may be causative for the defective rise of LH levels is suggested by the fact that intracerebral injection of kisspeptin in these animals was sufficient to evoke supramaximal LH secretory bursts, whose peak magnitude tended to exceed the circulating levels of LH in nondiabetic ORX males. Again, such exaggerated LH responses seem indicative of a state of LH hyperresponsiveness to kisspeptin in shortterm diabetes. From a pharmacological standpoint, it is remarkable that normalization of gonadotropic function in STZ-treated males could be achieved by solely replacing the endogenous kisspeptin tone, in both basal and post-ORX conditions. In our studies, long-term diabetic females displayed a plethora of reproductive defects manifested by persistent vaginal anestrous, decreased serum LH levels, and markedly atrophied uteri and ovaries. In addition, as was the case for STZ-injected males, the rise of LH levels after gonadectomy was severely blunted in diabetic females. Of note, defective LH secretion in these animals was invariantly coupled with decreased KiSS-1 mRNA levels in whole hypothalamic preparations, in both basal and post-OVX conditions, whereas LH responses to exogenous Kp-10 administration were roughly preserved. These observations strongly suggest that, as we previously reported in males (14), the overt state of hypogonadotropism detected in the uncontrolled, long-term diabetic female is, at least partially, due to


the suppressed function of the KiSS-1 system at the hypothalamus. This notion is functionally relevant because sex differences had been previously suggested for the mechanisms underlying the failure of gonadotropic axis linked to diabetes (35). Yet in our study some subtle differences were detected in the pattern of LH responses to kisspeptin between male and female rats because in intact STZ-treated animals, LH responses to kisspeptin were comparatively higher in males, whereas in gonadectomized animals, kisspeptin-induced LH secretion was higher in females. The basis for such apparent, as yet subtle, differences remains to be elucidated. Whereas our data document the functional consequences of altered hypothalamic expression of KiSS-1 in uncontrolled diabetes, some facets of this phenomenon await further experimental characterization. These include the identification of potential nucleus-specific changes in KiSS-1 expression, as described previously in order conditions of metabolic stress such as fasting or leptin deficiency (21, 39) as well as the translation of our current RNA data into changes in kisspeptin expression and/or release, at the protein level. The latter is indirectly evidenced by the close correlation between KiSS-1 RNA and circulating LH levels in the different models tested, yet experimental confirmation awaits further development of procedures for protein quantification of kisspeptins in hypothalamic tissue. From a therapeutic perspective, our current data provide the conceptual basis for targeting the hypothalamic KiSS-1/GPR54 system as potential adjuvant treatment of reproductive complications of diabetes in the female. In this context, it is interesting to note that, as we recently reported in cyclic female rats (40), the pattern of kisspeptin administration seems relevant to achieve maximal gonadotropic stimulation in the diabetic female. Thus, a regimen of intermittent injections of Kp-10 twice daily induced persistently higher LH secretory responses than protocols of single kisspeptin boluses every 24 h, a phenomenon whose underlying mechanism remains to be defined. Of note, desensitization of LH responses to kisspeptin has been recently documented after continuous infusion or repeated injections of high doses of kisspeptins to adult male rats and monkeys (41, 42), a phenomenon that was not observed in our experiments. Although the molecular basis for this divergence has not been established in our present study, it is tempting to speculate that the lowering of the endogenous kisspeptin tone in diabetes might have prevented the achievement of desensitizing levels, as it has been previously hypothesized in another situation of metabolic stress, such as chronic subnutrition (43). Admittedly, however, the potential translation of our current pharmacological data must be done with caution because our experiments were designed to provide a proof of principle, and thus, protocols of central injection of kisspeptin were selected. In any event, it is worth noting that diabetes has been reported to induce the suppression of preovulatory LH surges and thereby to cause anovulation (29, 31–33). Further analyses are required in our model to determine whether reduced KiSS-1 expression at the hypothalamus, and specifically at the anteroventral paraventricular nucleus, as center for the generation of the preovulatory surge (4, 6), is causative for such anovulatory state and whether repeated kisspeptin administra-


793

tion would be sufficient to restore ovulation in uncontrolled, long-term diabetic female rats. In summary, we report herein a series of hormonal and expression analyses addressing the contribution of potential changes in the hypothalamic KiSS-1 system to the alterations of the gonadotropic axis at early stages of diabetes in the male as well as their involvement in the reproductive failure observed in the diabetic female. Our data conclusively demonstrate that the diabetic state results in rapid changes in hypothalamic KiSS-1 that appear mechanistically relevant for the disturbance of reproductive function also in the female. Taken together with previous results in models of negative energy balance (16), our present observations further document the nodal role of KiSS-1 neurons as key sensors and transmitters of information concerning the metabolic state of the organism onto the centers governing the gonadotropic axis, in both males and females.

Acknowledgments Address all correspondence and requests for reprints to: Manuel TenaSempere, Physiology Section, Department of Cell Biology, Physiology, and Immunology. Faculty of Medicine, University of Co´rdoba, Avenida Meneńdez Pidal s/n, 14004 Co´rdoba, Spain. E-mail: [email protected]. This work was supported by Grants BFI 2008-00984 and BFI 200507446 from Direccioń General de Educacioń Superior e Investigcioń Cientifica (DGESIC) (Ministerio de Ciencia y Tecnología, Spain); funds from Instituto de Salud Carlos III (Red de Centros en Metabolismo y Nutricioń (RCMN) C03/08 and Project PI042082; Ministerio de Sanidad, Spain); and European Union Research Contract Developmental Effects of Environment on Reproductive Health (DEER) (FP-7). Centro de Investigacioń Biomedica en Red (CIBER) is an initiative of Instituto de Salud Carlos III (Spain). Disclosure Statement: The authors have nothing to disclose.

References 1. Herbison AE 2008 Estrogen positive feedback to gonadotropin-releasing hormone (GnRH) neurons in the rodent: the case for the rostral periventricular area of the third ventricle (RP3V). Brain Res Rev 57:277–287 2. Tena-Sempere M, Huhtaniemi I 2003 Gonadotropins and gonadotropin receptors. In: Fauser BCJM, ed. Reproductive medicine—molecular, cellular and genetic fundamentals. New York: Parthenon Publishing; 225–244 3. Schwartz NB 2000 Neuroendocrine regulation of reproductive cyclicity. In: Conn PM, Freeman ME, eds. Neuroendocrinology in physiology and medicine. Totowa, NJ: Humana Press; 135–146 4. Roa J, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M 2008 New frontiers in kisspeptin/GPR54 physiology as fundamental gatekeepers of reproductive function. Front Neuroendocrinol 29:48 – 69 5. Seminara SB 2005 Metastin and its G protein-coupled receptor, GPR54: critical pathway modulating GnRH secretion. Front Neuroendocrinol 26:131– 138 6. Popa SM, Clifton DK, Steiner RA 2008 The role of kisspeptins and GPR54 in the neuroendocrine regulation of reproduction. Annu Rev Physiol 70:213–238 7. Tena-Sempere M 2006 GPR54 and kisspeptin in reproduction. Hum Reprod Update 12:631– 639 8. Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, Terao Y, Kumano S, Takatsu Y, Masuda Y, Ishibashi Y, Watanabe T, Asada M, Yamada T, Suenaga M, Kitada C, Usuki S, Kurokawa T, Onda H, Nishimura O, Fujino M 2001 Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature 411:613– 617 9. Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden JM, Le Poul E, Brezillon S, Tyldesley R, Suarez-Huerta N, Vandeput F, Blanpain C, Schiffmann SN, Vassart G, Parmentier M 2001 The metastasis suppressor

794

10.

11.

12.

13.

14.

15.

16. 17.

18.

19. 20.

21. 22. 23. 24. 25.

26.

27.

Castellano et al.


gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G proteincoupled receptor GPR54. J Biol Chem 276:34631–34636 Bilban M, Ghaffari-Tabrizi N, Hintermann E, Bauer S, Molzer S, Zoratti C, Malli R, Sharabi A, Hiden U, Graier W, Knofler M, Andreae F, Wagner O, Quaranta V, Desoye G 2004 Kisspeptin-10, a KiSS-1/metastin-derived decapeptide, is a physiological invasion inhibitor of primary human trophoblasts. J Cell Sci 117:1319 –1328 Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno Jr JS, Shagoury JK, Bo-AbbasY,KuohungW,SchwinofKM,HendrickAG,ZahnD,DixonJ,KaiserUB, Slaugenhaupt SA, Gusella JF, O’Rahilly S, Carlton MB, Crowley Jr WF, Aparicio SA, Colledge WH 2003 The GPR54 gene as a regulator of puberty. N Engl J Med 349: 1614–1627 de Roux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E 2003 Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc Natl Acad Sci USA 100:10972–10976 Revel FG, Saboureau M, Masson-Pevet M, Pevet P, Mikkelsen JD, Simonneaux V 2006Kisspeptinmediatesthephotoperiodiccontrolofreproductioninhamsters.Curr Biol 16:1730–1735 Castellano JM, Navarro VM, Fernandez-Fernandez R, Nogueiras R, Tovar S, Roa J, Vazquez MJ, Vigo E, Casanueva FF, Aguilar E, Pinilla L, Dieguez C, Tena-Sempere M 2005 Changes in hypothalamic KiSS-1 system and restoration of pubertal activation of the reproductive axis by kisspeptin in undernutrition. Endocrinology 146:3917–3925 Castellano JM, Navarro VM, Fernandez-Fernandez R, Roa J, Vigo E, Pineda R, Dieguez C, Aguilar E, Pinilla L, Tena-Sempere M 2006 Expression of hypothalamic KiSS-1 system and rescue of defective gonadotropic responses by kisspeptin in streptozotocin-induced diabetic male rats. Diabetes 55:2602– 2610 Tena-Sempere M 2006 KiSS-1 and reproduction: focus on its role in the metabolic regulation of fertility. Neuroendocrinology 83:275–281 Luque RM, Kineman RD, Tena-Sempere M 2007 Regulation of hypothalamic expression of KiSS-1 and GPR54 genes by metabolic factors: analyses using mouse models and a cell line. Endocrinology 148:4601– 4611 Fernandez-Fernandez R, Martini AC, Navarro VM, Castellano JM, Dieguez C, Aguilar E, Pinilla L, Tena-Sempere M 2006 Novel signals for the integration of energy balance and reproduction. Mol Cell Endocrinol 254 –255:127–132 Brown RE, Imran SA, Ur E, Wilkinson M 2008 KiSS-1 mRNA in adipose tissue is regulated by sex hormones and food intake. Mol Cell Endocrinol 281:64 –72 Tovar S, Vazquez MJ, Navarro VM, Fernandez-Fernandez R, Castellano JM, Vigo E, Roa J, Casanueva FF, Aguilar E, Pinilla L, Dieguez C, Tena-Sempere M 2006 Effects of single or repeated intravenous administration of kisspeptin upon dynamic LH secretion in conscious male rats. Endocrinology 147:2696 – 2704 Smith JT, Acohido BV, Clifton DK, Steiner RA 2006 KiSS-1 neurones are direct targets for leptin in the ob/ob mouse. J Neuroendocrinol 18:298 –303 Sexton WJ, Jarow JP 1997 Effect of diabetes mellitus upon male reproductive function. Urology 49:508 –513 Jackson FL, Hutson JC 1984 Altered responses to androgen in diabetic male rats. Diabetes 33:819 – 824 Steger RW, Rabe MB 1997 The effect of diabetes mellitus on endocrine and reproductive function. Proc Soc Exp Biol Med 214:1–11 Dong Q, Lazarus RM, Wong LS, Vellios M, Handelsman DJ 1991 Pulsatile LH secretion in streptozotocin-induced diabetes in the rat. J Endocrinol 131:49 –55 Chandrashekar V, Steger RW, Bartke A, Fadden CT, Kienast SG 1991 Influence of diabetes on the gonadotropin response to the negative feedback effect of testosterone and hypothalamic neurotransmitter turnover in adult male rats. Neuroendocrinology 54:30 –35 Steger RW, Amador A, Lam E, Rathert J, Weis J, Smith MS 1989 Streptozotocin-induced deficits in sex behavior and neuroendocrine function in male rats. Endocrinology 124:1737–1743


28. Steger RW, Kienast SG, Pillai S, Rabe M 1993 Effects of streptozotocin-induced diabetes on neuroendocrine responses to ovariectomy and estrogen replacement in female rats. Neuroendocrinology 57:525–531 29. Kienast SG, Fadden C, Steger RW 1993 Streptozotocin-induced diabetes blocks the positive feedback release of luteinizing hormone in the female rat. Brain Res Bull 32:399 – 405 30. Valdes CT, Elkind-Hirsch KE, Rogers DG, Adelman JP 1991 The hypothalamic-pituitary axis of streptozotocin-induced diabetic female rats is not normalized by estradiol replacement. Endocrinology 128:433– 440 31. Bowton PA, Bryant KR, Whitehead SA 1986 Feedback effects of gonadal steroids and pituitary LH-releasing hormone receptors in the streptozotocininduced diabetic rat. Acta Endocrinol (Copenh) 111:467– 473 32. Katayama S, Brownscheidle CM, Wootten V, Lee JB, Shimaoka K 1984 Absent or delayed preovulatory luteinizing hormone surge in experimental diabetes mellitus. Diabetes 33:324 –327 33. Spindler-Vomachka M, Johnson DC 1985 Altered hypothalamic-pituitary function in the adult female rat with streptozotocin-induced diabetes. Diabetologia 28:38 – 44 34. Fraley GS, Scarlett JM, Shimada I, Teklemichael DN, Acohido BV, Clifton DK, Steiner RA 2004 Effects of diabetes and insulin on the expression of galaninlike peptide in the hypothalamus of the rat. Diabetes 53:1237–1242 35. Bestetti G, Locatelli V, Tirone F, Rossi GL, Muller EE 1985 One month of streptozotocin-diabetes induces different neuroendocrine and morphological alterations in the hypothalamo-pituitary axis of male and female rats. Endocrinology 117:208 –216 36. Navarro VM, Castellano JM, Fernandez-Fernandez R, Barreiro ML, Roa J, Sanchez-Criado JE, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M 2004 Developmental and hormonally regulated messenger ribonucleic acid expression of KiSS-1 and its putative receptor, GPR54, in rat hypothalamus and potent luteinizing hormone-releasing activity of KiSS-1 peptide. Endocrinology 145:4565– 4574 37. Navarro VM, Castellano JM, Fernandez-Fernandez R, Tovar S, Roa J, Mayen A, Nogueiras R, Vazquez MJ, Barreiro ML, Magni P, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M 2005 Characterization of the potent luteinizing hormone-releasing activity of KiSS-1 peptide, the natural ligand of GPR54. Endocrinology 146:156 –163 38. Navarro VM, Castellano JM, Fernandez-Fernandez R, Tovar S, Roa J, Mayen A, Barreiro ML, Casanueva FF, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M 2005 Effects of KiSS-1 peptide, the natural ligand of GPR54, on folliclestimulating hormone secretion in the rat. Endocrinology 146:1689 –1697 39. Kalamatianos T, Grimshaw SE, Poorun R, Hahn JD, Coen CW 2008 Fasting reduces KiSS-1 expression in the anteroventral periventricular nucleus (AVPV): effects of fasting on the expression of KiSS-1 and NPY in the AVPV or arcuate nucleus of female rats. J Neuroendocrinol 20:1089 –1097 40. Roa J, Vigo E, Garcia-Galiano D, Castellano JM, Navarro VM, Pineda R, Dieguez CM, Aguilar E, Pinilla L, Tena-Sempere M 2008 Desensitization of gonadotropin responses to kisspeptin in the female rat: analyses of LH and FSH secretion at different developmental and metabolic states. Am J Physiol Endocrinol Metab 294:E1088 –E1096 41. Thompson EL, Murphy KG, Patterson M, Bewick GA, Stamp GW, Curtis AE, Cooke JH, Jethwa PH, Todd JF, Ghatei MA, Bloom SR 2006 Chronic subcutaneous administration of kisspeptin-54 causes testicular degeneration in adult male rats. Am J Physiol Endocrinol Metab 291:E1074 –E1082 42. Ramaswamy S, Seminara SB, Pohl CR, DiPietro MJ, Crowley Jr WF, Plant TM 2007 Effect of continuous intravenous administration of human metastin 45–54 on the neuroendocrine activity of the hypothalamic-pituitary-testicular axis in the adult male rhesus monkey (Macaca mulatta). Endocrinology 148: 3364 –3370 43. Castellano JM, Roa J, Luque RM, Dieguez C, Aguilar E, Pinilla L, Tena-Sempere M, 2009 KiSS-1/kisspeptins and the metabolic control of reproduction: physiologic roles and putative physiopathological implications. Peptides 30:139–145