Gregory S. Fraley

KISSPEPTIN ACTIVATION OF GONADOTROPIN RELEASING HORMONE NEURONS AND REGULATION OF KISS-1 MRNA IN THE MALE RAT

The KiSS-1 gene codes for a family of neuropeptides called kisspeptins which bind to the G-protein-coupled receptor GPR54. To assess the possible effects of kisspeptins on gonadotropin secretion, we injected kisspeptin-52 into the lateral cerebral ventricles of adult male rats and found that kisspeptin-52 increased the serum levels of luteinizing hormone (p < 0.05). To determine whether the kisspeptin-52-induced stimulation of luteinizing hormone secretion was mediated by gonadotropin-releasing hormone (GnRH), we pretreated adult male rats with a GnRH antagonist (acyline), then challenged the animals with intracerebroventricularly administered kisspeptin-52. The GnRH antagonist blocked the kisspeptin-52-induced increase in luteinizing hormone. To examine whether kisspeptins stimulate transcriptional activity in GnRH neurons, we administered kisspeptin-52 intracerebroventricularly and found by immunocytochemistry that 86% of the GnRH neurons coexpressed Fos 2 h after the kisspeptin-52 challenge, whereas fewer than 1% of the GnRH neurons expressed Fos following injection of the vehicle alone (p < 0.001). To assess whether kisspeptins can directly act on GnRH neurons, we used double-label in situ hybridization and found that 77% of the GnRH neurons coexpress GPR54 mRNA. Finally, to determine whether KiSS-1 gene expression is regulated by gonadal hormones, we measured KiSS-1 mRNA levels by single-label in situ hybridization in intact and castrated males and found significantly higher levels in the arcuate nucleus of castrates. These results demonstrate that GnRH neurons are direct targets for regulation by kisspeptins and that KiSS-1 mRNA is regulated by gonadal hormones, suggesting that KiSS-1 neurons play an important role in the feedback regulation of gonadotropin secretion.

Rat RFamide-related peptide-3 stimulates GH secretion, inhibits LH secretion, and has variable effects on sex behavior in the adult male rat

A recently described avian neuropeptide, gonadotropin inhibitory hormone (GnIH), has been shown to have seasonal regulatory effects on the hypothalamic-pituitary-gonadotropin axis (HPG) in several avian species. In the bird, GnIH expression is increased during the photorefractory period and has inhibitory effects on the HPG. A recently described mammalian neuropeptide, RF-amide-related peptide-3 (RFRP-3), may be genetically related and functionally similar to this avian neuropeptide. The purposes of this study were to first see if rat RFRP-3 is expressed in the male rat brain and second to determine if ICV injections of RFRP-3 will have effects on feeding and sex behaviors, as well as hormone release from the anterior pituitary. Results confirm other studies in that immunoreactive cell bodies and fibers are observable in areas of the male rat brain known to control the HPG and feeding and sex behaviors. RFRP-3 fibers are also observed in close proximity to GnRH immunoreactive cell bodies. Behavioral tests indicate that high but not low ICV RFRP-3 (500 vs. 100 ng, respectively) significantly (p<0.05) suppressed all facets of male sex behavior while not having any observable effects on their ability to ambulate. Sex behavior was later exhibited when those same male rats received the ICV vehicle. While suppressing sex behavior, ICV RFRP-3 significantly (p<0.05) increased food intake compared to controls. ICV RFRP-3 also significantly reduced plasma levels of luteinizing hormone but increased growth hormone regardless of the time of day; however, at no time did RFRP-3 alter plasma levels of FSH, thyroid hormone, or cortisol. These results indicate that although RFRP-3 has similar effects on LH as observed with GnIH in avian species, in the rat RFRP-3 has additional roles in regulating feeding and growth.

Gonadotropin-inhibitory hormone is a hypothalamic peptide that provides a molecular switch between reproduction and feeding

Objective: Gonadotropin-inhibitory hormone (GnIH)-3 is a neuropeptide that plays a major role in the regulation of reproduction and feeding in mammals. Materials and Methods: We measured endocrine and behavioural parameters of reproduction in sheep, and sexual behaviour in sheep, mice and cynomolgus monkeys. In addition, GnIH gene expression (in situ hybridization) was examined in ewes, and effects of GnIH-3 on food intake and energy expenditure were measured in various species. GnIH-3 was infused (i.v.) into ewes after an i.m. injection of estradiol benzoate to determine whether the peptide blocks the surge in luteinizing hormone (LH) secretion. Results: GnIH gene expression was reduced in the preovulatory period in ewes. Infusion (i.v.) of GnIH-3 blocked the estrogen-induced LH surge (in ewes). Intracerebroventricular infusion had no effect on female or male sexual behaviour in each of the three species, but increased food intake. There were no effects on energy expenditure in sheep or rats. GnIH increased fos protein (immunohistochemistry) was seen in orexigenic neurons (in sheep and rats), but also in anorexigenic neurons (in sheep). Conclusions: GnIH-3 reduces reproductive hormone levels and increases food intake in mammals without reducing energy expenditure. There is minimal effect on reproductive behaviour. The dual effect on reproduction and feeding suggests that GnIH-3 provides a molecular switch between these two functions. Blockade of the positive feedback effect of estrogen with parenteral infusion indicates that this peptide may have utility as a blocker of reproductive function in mammals.

Effects of galanin-like peptide (GALP) on locomotion, reproduction, and body weight in female and male mice.

Galanin-like peptide (GALP) has been implicated in the neuroendocrine regulation of both feeding and reproduction. In male rodents and primates, intracerebroventricular (icv) infusions of GALP stimulate luteinizing hormone (LH) release, induce Fos expression in brain areas implicated in feeding and reproduction, and affect food intake and body weight in rodents. In gonad-intact and castrated male rats, icv administration of GALP also stimulates male sexual behavior. While the effects of GALP on male physiology and behavior are well documented, no studies have addressed such a role of GALP in females. We tested the effects of icv GALP infusions on LH release, locomotor activity, motor control, and body weight regulation in adult ovariectomized female mice hormonally primed with estradiol benzoate and progesterone. In addition, sexually-experienced male and female mice were treated with GALP and tested for sexual behavior. In females, GALP reduced open-field locomotor activity, the ability to maintain grip on an accelerating rotarod, and 24-h body weight in a dose-dependent manner. GALP also increased LH secretion in female mice, an effect that was blocked by pre-treatment with Antide, a gonadotropin-releasing hormone (GnRH) type-1 receptor antagonist. GALP infusions slightly decreased the occurrence of lordosis behavior in female mice and significantly increased the latencies with which females displayed receptivity. Unlike previous reports in male rats, GALP inhibited male sexual behavior in mice. Our data indicate that in female mice, GALP stimulates LH release via GnRH, and decreases body weight, motor control, and locomotor activity via GnRH-independent pathways. Furthermore, our sexual behavior and locomotor findings suggest species-specific differences in the mechanism and/or location of GALP action in the brains of rats and mice.

Rat RFRP-3 alters hypothalamic GHRH expression and growth hormone secretion but does not affect KiSS-1 gene expression or the onset of puberty in male rats.

Background/Aims: RFRP-3 is a recently described neuropeptide that influences a variety of physiologic factors, including the inhibition of gonadotropin secretion and reproductive behaviors. We hypothesized that endogenous RFRP-3 could function to inhibit the onset of puberty in young male rats. Methods: To test this hypothesis, we first placed cannulas into the third ventricle of 24-day-old male rat pups. The cannulas were attached to osmotic minipumps that infused antisense oligonucleotides (ODNs) against RFRP-3. Second, cannulas were placed in the ventricle of 35-day-old pups and infused with RFRP-3, NPFF (putatively, an alternative transcript of the RFRP gene), or vehicle. Results: No treatment altered the onset of puberty compared to controls. RFRP-3 ODN rats had significantly larger testes compared to control rats. Similarly, the RFRP-3 ODN-treated rats had a significant increase in plasma LH, but not FSH, compared to control rats. Rats infused with RFRP-3 exhibited significantly smaller testes compared to control rats. RFRP-3 rats also had a significant decrease in plasma LH levels. RFRP-3 infusion elicited a significant increase in growth hormone-releasing hormone mRNA and plasma growth hormone levels compared to control rats. Neither peptide had an effect on KiSS-1 mRNA expression. Conclusion: These data suggest that endogenous rat RFRP-3 does not affect the timing of puberty in male rats but may be associated with peripubertal rise in growth hormone secretion.

Central Insulin-Like Growth Factor 1 Receptors Play Distinct Roles in the Control of Reproduction, Food Intake, and Body Weight in Female Rats

Abstract Estradiol and progesterone induction of the LH surge in ovariectomized female rats requires concurrent activation of brain insulin-like growth factor 1 (IGF1) receptors. The present study determined whether brain IGF1 receptor signaling is required for estrous cyclicity in gonadally intact female rats. A selective IGF1 receptor antagonist (JB-1) or vehicle was continuously administered into the third ventricle by osmotic minipumps. Following surgical placement of the minipumps, all rats temporarily reduced food intake, lost weight, and suspended estrous cycles. Control rats resumed cycles within a few days and exhibited compensatory hyperphagia until they returned to presurgical body weight. Animals receiving JB-1 had severely delayed or absent estrous cycles, failed to show rebound feeding, and regained body weight more slowly. Vehicle-infused animals pair fed to JB-1-treated rats had even lower body weights but resumed estrous cycles sooner than those given drug alone. Chronic infusion of IGF1 alone had no effect on any of these parameters, but coinfusion of IGF1 with the antagonist completely reversed JB-1 effects on food intake and estrous cyclicity and partially reversed the effects on body weight. There were no significant differences in the expression of galanin-like peptide (Galp) or Kiss1 mRNA in the arcuate or periventricular hypothalamic area of control and JB-1-treated animals at a time point when food intake and estrous cycles were different between controls and JB-1-treated rats. These data suggest that brain IGF1 signaling is necessary for normal estrous cycles as well as compensatory hyperphagia and that IGF1 modulation of the reproductive axis is not secondary to reduced food intake.

Alarin stimulates food intake in male rats and LH secretion in castrated male rats

Alarin is a newly identified member of the galanin family of neuropeptides that includes galanin-like peptide (GALP) and galanin. Alarin was discovered as an alternate transcript of the GALP gene in neuroblastoma cells, and subsequently alarin mRNA was detected in the brain of rodents. GALP and galanin are important central regulators of both feeding and reproductive behavior. We hypothesized, that, as a member of the galanin family of peptides, alarin would also have central effects on feeding and reproduction. To test this hypothesis, we treated male rats with alarin intracerebroventricularly (i.c.v.) and measured its effects on food intake and energy homeostasis as well as sexual behavior and luteinizing hormone (LH) secretion. We observed that i.c.v. injection of 1.0 nmol alarin significantly increased food intake (p<0.01) and body weight (p<0.05). Alarin did not affect sexual behavior in male rats; however, alarin did significantly (p<0.01) increase LH levels in castrated, but not intact, male rats. Alarin immunoreactive cell bodies were detected within the locus coeruleus and locus subcoeruleus of the midbrain, which is a brainstem nucleus involved in coordinating many physiological activities, including food intake and reproduction. Lastly, alarin stimulated Fos induction in hypothalamic nuclei, such as the paraventricular nucleus and the nucleus of the tractus solitarious. Our studies demonstrate that alarin, like other members of the galanin family, is a neuromediator of food intake and body weight.

Distribution and sequence of gonadotropin-inhibitory hormone and its potential role as a molecular link between feeding and reproductive systems in the Pekin duck (Anas platyrhynchos domestica)

The reproductive status of adult Pekin drakes is very sensitive to nutritional status. Thus, the purpose of this study was to increase our understanding of the neurobiology underlying the depressive effect of fasting on the secretion of reproductive hormones. It was hypothesized that this effect was mediated by gonadotropin-inhibitory hormone (GnIH). Networks of GnIH fibers were present throughout the diencephalon, and cell bodies were present primarily, in the hypothalamic paraventricular nucleus (PVN). The duck GnIH gene was cloned and sequenced and found to encode GnIH and two GnIH-related peptides (GnIH-RP1, GnIH-RP2) which have a similar identity to those found in other avian species. Intracerebroventricular injection of GnIH, but not of GnIH-RP1, depressed plasma LH and stimulated feeding. Fasting for 48h depressed plasma LH and induced fos expression in about half the population of GnIH-ir neurons. These data suggest that GnIH neurons are mediators between feeding and reproductive systems in Pekin drakes.

Galanin-like Peptide (GALP) is a Hypothalamic Regulator of Energy Homeostasis and Reproduction

Galanin-like peptide (GALP) was discovered in 1999 in the porcine hypothalamus and was found to be a 60 amino acid neuropeptide. GALP shares sequence homology to galanin (1-13) in position 9-21 and can bind to, as well as activate, the three galanin receptor subtypes (GalR1-3). GALP-expressing cells are limited, and are mainly found in the arcuate nucleus of the hypothalamus (ARC) and the posterior pituitary. GALP-positive neurons in the ARC project to several brain regions where they appear to make contact with multiple neuromodulators. These neuromodulators are involved in the regulation of energy homeostasis and reproduction, anatomical evidence that suggests a role for GALP in these physiological functions. In support of this idea, GALP gene expression is regulated by several factors that reflect metabolic state including the metabolic hormones leptin and insulin, thyroid hormones, and blood glucose. Considerable evidence now exists to support the hypothesis that GALP has a role in the regulation of energy homeostasis and reproduction; and, that GALPs role may be independent of the known galanin receptors. In this review, we (1) provide an overview of the distribution of GALP, and discuss the potential relationship between GALP and other neuromodulators of energy homeostasis and reproduction, (2) discuss the metabolic factors that regulate GALP expression, (3) review the evidence for the role of GALP in energy homeostasis and reproduction, (4) discuss the potential downstream mediators and mechanisms underlying GALPs effects, and (5) discuss the possibility that GALP may mediate its effects via an as yet unidentified GALP-specific receptor.

Altered response to metabolic challenges in mice with genetically targeted deletions of galanin-like peptide

Galanin-like peptide (GALP) is expressed in the arcuate nucleus and is implicated in the neuroendocrine regulation of metabolism and reproduction. To investigate the physiological significance of GALP, we generated and characterized a strain of mice with a genetically targeted deletion in the GALP gene [GALP knockout (KO) mice]. We report that GALP KO mice have a subtle, but notable, metabolic phenotype that becomes apparent during adaptation to changes in nutrition. GALP KO mice are indistinguishable from wild-type (WT) controls in virtually all aspects of growth, sexual development, body weight, food and water consumption, and motor behaviors, when they are allowed unlimited access to standard rodent chow. However, GALP KO mice have an altered response to changes in diet. 1) Male GALP KO mice consumed less food during refeeding after a fast than WT controls (P < 0.01). 2) GALP KO mice of both sexes gained less weight on a high-fat diet than WT controls (P < 0.01), despite both genotypes having consumed equal amounts of food. We conclude that although GALP signaling may not be essential for the maintenance of energy homeostasis under steady-state nutritional conditions, GALP may play a role in readjusting energy balance under changing nutritional circumstances.