Galanin-like peptide as a link in the integration of metabolism and reproduction

The arcuate nucleus is a hypothalamic center that couples energetics and reproduction. Peptide-releasing neurons in the arcuate nucleus receive and process humoral signals from the periphery and relay this information to other nuclei in the hypothalamus and preoptic area. Galanin-like peptide (GALP) is expressed in the arcuate nucleus, and GALP-containing neurons are targets for the action of leptin. GALP-containing neurons are closely apposed to gonadotropin-releasing hormone (GnRH) neurons in the preoptic area, and CNS injections of GALP stimulate GnRH-mediated secretion of luteinizing hormone. These observations indicate that GALP is a molecular signal that couples circulating indices of metabolism to the neuroendocrine reproductive system and, thus, regulates reproductive activity as a function of the energy state. In this article, we describe the involvement of GALP in metabolism and reproduction, and in the coupling between these two processes.

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title: Leptin and insulin action in the central nervous system.
abstract: Body adiposity is known to be carefully regulated and to remain relatively stable for long periods of time in most mammalian species. This review summarizes old and recent data implicating insulin and leptin as key circulating signals to the central nervous system, particularly the ventral hypothalamus, in communicating the size and the distribution of body fat stores. This input ultimately alters food intake and energy expenditure to maintain constancy of the adipose depot. The key primary neurons in the arcuate nucleus containing NPY/AgRP and POMC/CART appear be critical constituents of the CNS regulating system, and are shown to contribute to anabolic and catabolic signaling systems to complete the feedback loop. New data to indicate shared intracellular signaling from leptin and insulin is provided. The satiety system for meals, consisting of neural afferents to the hindbrain from the gastrointestinal tract, is described and its effectiveness is shown to vary with the strength of the insulin and leptin signals. This provides an efferent mechanism that plays a key role in a complex feedback system that allows intermittent meals to vary from day to day, but provides appropriate long-term adjustment to need. Recently described contributions of this system to obesity are described and potential therapeutic implications are discussed.

title: The hypothalamus and the control of energy homeostasis Different circuits, different purposes
abstract: The hypothalamus regulates many aspects of energy homeostasis, adjusting both the drive to eat and the expenditure of energy in response to a wide range of nutritional and other signals. It is becoming clear that various neural circuits operate to different degrees and probably serve specific functions under particular conditions of altered feeding behaviour. This review will discuss this functional diversity by illustrating hypothalamic neurones that express neuropeptide Y (NPY), the melanocortin-4 receptor (MC4-R) and the orexins. NPY neurones in the arcuate nucleus (ARC) release NPY, a powerful inducer of feeding and obesity, in the paraventricular nucleus (PVN) and the lateral hypothalamic area (LHA). ARC-NPY neurones are inhibited by leptin and insulin and become overactive when levels of these hormones fall during undernutrition. They may function physiologically to protect against starvation. With disruption of the inhibitory leptin signals due to gene mutations, the NPY neurones are overactive, which contributes to hyperphagia and obesity in the ob/ob and db/db mice and fa/fa Zucker rat. The MC4-R is activated by alpha-melanocyte-stimulating hormone [alpha-MSH; a cleavage product of pro-opiomelanocortin (POMC), which is expressed in the other ARC neurones] and inhibits feeding. This effect is antagonised by agouti gene-related peptide (AGRP), which is coexpressed by the ARC-NPY neurones only. Activation of MC4-R, possibly mediated by blockade of AGRP release, appears to restrain overeating of a palatable diet. This response may be programmed by a transient rise in leptin soon after presentation of palatable food, and rats that fail to do this will overeat and become obese. Orexin-A and -B (corresponding to hypocretins 1 and 2) are expressed in specific LHA neurones. These have extensive reciprocal connections with many areas involved in appetite control, including the nucleus of the solitary tracts (NTS), which relays vagal afferent satiety signals from the viscera. Orexin neurones also have close anatomical connections with LHA glucose-sensitive neurones. Orexin-A induces acute feeding but does not cause obesity. Orexin neurones are stimulated by hypoglycaemia partly via the NTS and inhibited by food ingestion. These neurones may therefore be involved in the severe hyperphagia of hypoglycaemia and short-term control of feeding.

title: The arcuate nucleus as a conduit for diverse signals relevant to energy homeostasis
abstract: Arcuate nucleus neurons are known to be responsive to a wide array of hormones and nutrients, including leptin, insulin, gonadal steroids and glucose. In addition to potential transport mechanisms, peripheral substances may access these neurons via arcuate cell bodies in and projections to the median eminence, a region considered to be a circumventricular organ. The arcuate is a potent site of leptin action, probably mediating a component of leptin's effects via arcuate neuropeptide Y/agouti-related peptide (NPY/AgRP) and pro-opiomelanocortin (POMC) neurons, and implicating this structure in the long-term control of energy stores. However, ghrelin, the endogenous ligand of the growth hormone secretagogue receptor, may also stimulate feeding and weight gain, in part through action on receptors in arcuate NPY neurons. Since ghrelin is secreted by the stomach upon content depletion, with a half-life of no more than an hour, the arcuate nucleus may also be important in sensing and responding to acute changes in nutrients. We have developed a system for recording from arcuate POMC neurons using a mouse containing a transgene in which the POMC promoter is driving expression of the green fluorescent protein (GFP). In these mice, 99% of the β-endorphin positive neurons express GFP, making whole cell patch clamp recordings from the sparsely distributed POMC neurons facile. All of the POMC neurons appear to be activated by leptin, via two different mechanisms, while approximately 30–50% of the neurons appear to be inhibited by a gamma-melanocyte stimulating hormone (MSH) specific agonist. The latter result suggests that the melanocortin-3 receptor (MC3-R) may act as an autoinhibitory receptor on some POMC neurons. This hypothalamic slice preparation also confirms the responsiveness of arcuate POMC neurons to a wide variety of nutrients and hormones. Thus the arcuate melanocortin system is best described as a conduit of many diverse signals involved in energy homeostasis, with leptin acting tonically to regulate the responsiveness of the circuit to a wide variety of hormones and nutrients.

title: Neuroendocrine mechanisms regulating food intake and body weight
abstract: In the field of obesity research, two separate lines of study have emerged which explore the mechanism by which food intake is regulated: short‐term control of food intake, and the central regulation of energy balance. The former studies the satiety response during consumption of meals, whereby satiety signalling originating in the gut is transduced into a neural signal that modulates satiety pathways in the brainstem. This review describes a neuroanatomically based model in which leptin and insulin signalling in the hypothalamus governs long‐term regulation of energy balance via mechanisms that are integrated with satiety hormone signalling in the brainstem. The functional outcome of this integration is a cumulative meal‐to‐meal regulation of food intake, that over relatively long intervals serves to maintain stable adipose stores. Our model provides a context within which continued investigation of neuroendocrine mechanisms that control food intake and body weight can be explored, and has potential application to our current understanding of clinical obesity and its treatment.

title: Central regulators of food intake.
abstract: Obesity is a major public health problem and is associated with significant morbidity and mortality from coronary heart disease and type 2 diabetes. The population is becoming increasingly obese, yet for individuals weight is regulated within a narrow range. This regulation depends on the balance between energy intake (in the form of food) and energy expenditure. Thus in order to regulate weight and energy stores, the body must be able to control food intake accurately. Recently there has been a remarkable increase in our understanding of the molecular mechanisms regulating food intake and energy homeostasis.



There is increased understanding of the central pathways by which known neurotransmitters affect food intake. These include peptides expressed in hypothalamic nuclei, but other brain regions including brain stem, amygdala and nucleus accumbens, are also important. Further central transmitters have been identified, such as that encoded by the gene identified in mice with the mahoganoid mutation, and roles for known transmitters have been elucidated, such as endocannabinoids and acetylcholine. There is new evidence regarding peripheral modulation of these pathways including the role of ghrelin as an initiator of feeding, and peptide YY as a medium-term satiety signal.



Complex central circuitry controls food intake and energy expenditure. Circulating factors that modulate these pathways have appetite stimulation or satiety effects. These are potentially important targets for therapy in obesity. Single-gene models of obesity in animals are important in understanding these pathways, however human clinical correlates for genetically determined obesity are uncommon.

title: Hypothalamic pathways underlying the endocrine, autonomic, and behavioral effects of leptin
abstract: Leptin affects feeding, metabolism, and neuroendocrine status. It is now clearly established that the hypothalamus coordinates these responses, though the specific brain regions engaged by leptin remain unclear. We have used combinations of neuroanatomic techniques to identify candidate pathways in the central nervous system underlying leptin action. Leptin decreases body weight in part by activating the sympathetic nervous system, resulting in increased thermogenesis and energy expenditure. We investigated hypothalamic pathways underlying leptin's effects on stimulating the sympathetic nervous system. We found that leptin activates neurons in the retrochiasmatic area (RCA) and lateral arcuate nucleus (Arc) that innervate the sympathetic preganglionic neurons in the thoracic spinal cord and also contain cocaine- and amphetamine-regulated transcript (CART). We also found that CART neurons in the RCA and the Arc coexpress pro-opiomelanocortin (POMC) mRNA. Recent studies have reinforced the view that the lateral hypothalamic area (LHA) regulates food intake and body weight. Using retrograde tracing with leptin administration, we found retrogradely labeled cells in the Arc contained neuropeptide Y (NPY) mRNA or POMC mRNA. Following leptin administration, NPY cells in the Arc did not express Fos but expressed suppressor of cytokine signaling-3 (SOCS-3) mRNA. In contrast, leptin induced both Fos and SOCS-3 expression in POMC neurons, many of which also innervated the LHA. We suggest that leptin directly activates POMC/CART neurons that project to the LHA, the paraventricular hypothalamic nucleus (PVH), and spinal sympathetic preganglionic neurons. These projections link circulating leptin and neurons that regulate feeding behavior, energy expenditure, and body weight homeostasis.

title: Neuroendocrine Interactions between Galanin, Opioids, and Neuropeptide Y in the Control of Reproduction and Appetite a
abstract: Galanin is a pleiotropic neuroendocrine signal produced in discrete subpopulations of neurons distributed in several sites in the hypothalamus. Neuropeptide Y and β‐endorphin also display pleiotropism, but they are produced by subpopulations of neurons located only in the arcuate nucleus of the hypothalamus. Each of these neuropeptides exerts a regulatory influence on reproduction and appetitive behavior. Experimental and morphologic evidence from our laboratory show direct contacts and interplay among these diverse signals. Seemingly, an interconnected network composed of these three neuropeptide‐producing neurons provides precision and site specificity in the relay of information necessary to govern reproduction and appetite. Disruptions in this interplay are likely to manifest in untoward consequences such as infertility and obesity.

title: Neuroendocrine control processes. Pubertal onset and progression.
abstract: This discussion has outlined current concepts in neuroendocrinologic control of pubertal onset and progression. Central nervous system regulation of the arcuate nucleus (ventromedial hypothalamus) pulse generator that subsequently controls pituitary gonadotropin synthesis and secretion has been highlighted. Significant investigative issues that deserve assessment in the next several years include the following: 1. Systematic neuropharmacologic, electrophysiologic, and anatomic assessment of the hypothalamic arcuate nucleus. These assessments would include the use of recombinant DNA technology to probe cellular regulation of GnRH production. 2. Physiologically oriented examination of hypothalamic GnRH synthesis and secretion, along with function in the remaining reproductive endocrine system, during situations of nutritional impairment and excessive energy utilization and psychologic stress. 3. Further assessment of the neurophysiologic inhibition of GnRH production during childhood and the late prepubertal reactivation of the arcuate nucleus pulse generator. Roles of opioids, dopamine, other neurotransmitters, and metabolic signals remain to be clarified. 4. Exploration of regulators of hypothalamic, pituitary, and gonadal function when pulsatile GnRH administration has replaced the usual hypothalamic mechanisms. Pituitary-gonadal interactions may be independently assessed. 5. Assessment of pubertal growth, endocrine function, and neuropharmacologic control mechanisms in circumstances of chemical removal of pituitary gonadotrope function by GnRH agonists or antagonists. 6. Concordance and discordance of potency estimates of gonadotropins made by bioassay and immunoassay. The biologic basis for qualitative changes in bioassayable levels of LH and FSH, often related to carbohydrate content of the glycoprotein, may help to explain changes of gonadal function during the pubertal process. The potential for significant molecular heterogeneity of the gonadotropins is recognized and suggests substantial posttranslational changes of LH and FSH. 7. A cogent delineation of the hormonal, nutritional, and energy regulators of the pubertal growth spurt, though not discussed in this manuscript, remains to be accomplished. The relationship between pituitary gonadotropins and growth hormone, sex steroids, and the various peptide growth factors, especially the relationship between the growth factors and intragonadal steroidogenesis and germ-cell production, remain to be resolved. The importance of local production and action of peptide-growth factors in diverse tissues, skeletal and other, is being increasingly recognized.(ABSTRACT TRUNCATED AT 400

title: Acute administration of alpha-melanotropin exerts a stimulatory control on puberty.
abstract: The present experiments were undertaken to throw light on the physiological role played by alpha-MSH in the advent of female puberty. The serum and pituitary alpha-MSH concentrations were determined in prepubertal rats 5, 15, 25, 30, 33 days old and at vaginal opening. The serum levels showed a significant increase on day 30 and the pituitary alpha-MSH levels increased steadily from day 5 to day 33. To determine if a relation exists between the increase in alpha-MSH levels and the peak of LH prior to vaginal opening, prepubertal rats of the same age range as above received an injection of alpha-MSH or saline solution and were sacrificed 30 min later. The peptide increased LH serum levels only at 30 days of age. To examine the hypothesis that alpha-MSH was involved in determining the timing of puberty, prepubertal rats pretreated with estradiol benzoate received an injection of alpha-MSH or saline solution on day 28. This treatment advanced the time of vaginal opening by 2 days in the experimental animals. With the same experimental procedure, an increase in serum levels of LH and progesterone was also observed at 29 days of age. The action of alpha-MSH on GnRH was analysed by incubating median eminence from prepubertal rats 20, 25 and 30 days of age, with the peptide, alpha-MSH increased GnRH release only in 25-day-old rats. The above results show that acute treatment with alpha-MSH induces modifications in the hormones related to puberty and hence allows us to include alpha-MSH in the chain of neuroendocrine events involved in reproductive maturation.

title: Beta-endorphin regulation of luteinizing hormone-releasing hormone release at the median eminence in ewes: immunocytochemical and physiological evidence.
abstract: Beta-endorphin (beta-END) is an inhibitory factor in the neuroendocrine control of luteinizing hormone (LH) release and thus, presumably also of hypophysiotropic luteinizing hormone-releasing hormone (LHRH) release. In order to address if the median eminence (ME) is a site of beta-END action, we studied its functional role in ewes by assessing: (a) the hypothalamic distribution of beta-END using immunolabeling and by comparing this distribution with our data on the localization of LHRH; (b) the ME in vivo release of LHRH and beta-END during the luteal (day 12) and the follicular (day 15) phases of the estrous cycle; (c) the in vivo release of LHRH from the posterior-lateral ME, as assessed by push-pull cannula (PPC) sampling, before, during, and after infusion of increasing doses of beta-END or naloxone through the PPC, during the follicular phase; and (d) the in vivo release of ME-LHRH and serum LH, before, during, and after infusion of beta-END or naloxone in luteal and follicular ewes. In the ewe, beta-END-containing perikarya are located in and around the arcuate nucleus. Their processes are also present in the diagonal band, medial septal nucleus, and medial and lateral hypothalamic areas, including the preoptic region and posterior ME. Perikarya containing LHRH are located in the preoptic area and project also to the ME, providing opportunities for synaptic interactions between beta-END and LHRH-containing perikarya and processes at these levels. ME in vivo release of LHRH and beta-END increase from the luteal (low LH/high progesterone, P4) to the follicular phase (high LH/low P4). In follicular ewes, in vivo LHRH and LH release is decreased, in a dose-dependent manner, by beta-END infused through the PPC probe into the posterior-lateral ME. In contrast, infusion of naloxone under similar conditions increases LHRH and LH release, also in a dose-dependent fashion. The inhibitory effect of beta-END on LHRH and LH, as well as the stimulatory effect of naloxone on LHRH and LH, were only marginally apparent in luteal ewes. These results suggest that the ME is a major control site where beta-END exerts its influence on hypophysiotropic LHRH release. The strength of this inhibitory effect apparently increases throughout the follicular phase, and might prevent the premature onset of the preovulatory surge of LHRH and LH.

title: Hypothalamic neuropeptide-Y gene expression increases before the onset of the ovarian steroid-induced luteinizing hormone surge.
abstract: Neuropeptide-Y (NPY), a hypothalamic peptide, is involved in stimulation of LHRH and LH surges during proestrus and those induced by ovarian steroids in ovariectomized (ovx) rats. The NPY neurons that reside in the arcuate nucleus of the medial basal hypothalamus (MBH) and accumulate 17 beta-estradiol participate in the initiation of LHRH and LH surges. To determine whether NPY synthesis is altered in conjunction with the LH surge, we studied the dynamic changes in prepro-NPY mRNA levels in the MBH in association with the LH surge elicited by estradiol benzoate (EB) alone or by progesterone (P) in EB-primed ovx rats. Five days after ovariectomy, rats received oil or EB (30 micrograms/rat) at 1000 h on day 0. On day 2, these rats were injected with either oil or P (2 mg/rat) at 1000 h. Rats were killed before (1000 h) and at 2-h intervals after oil or P injection. The MBHs were dissected out and processed for determination of prepro-NPY mRNA levels by solution hybridization/RNase protection assay using a cRNA probe. Although in control ovx rats, prepro-NPY mRNA levels remained unchanged between 1000-1600 h, prepro-NPY mRNA levels showed dynamic changes in steroid-primed rats. In the EB-primed rats, prepro-NPY mRNA levels rose significantly (100%) at 1200 and 1400 h before the LH rise at 1600 h, and the levels remained elevated up to 1800 h. After P injection to the EB-primed rats, this response was further augmented, with a slightly different temporal pattern. Prepro-NPY mRNA levels rose at 1400 h (600%) before the onset of the LH rise at 1600 h and declined steadily to significantly lower values at 1800 h, coincident with the highest rate of LH secretion. These studies demonstrate dynamic shifts in hypothalamic NPY gene expression in association with the LH (LHRH) surge, and that maximal increases occur before the onset of the LH rise, but thereafter, NPY gene expression diverged in the two ovarian-steroid treatment models. These findings along with previous evidence of similar antecedent increases in NPY content in the median eminence, followed by release, suggest that augmented NPY synthesis and release are two temporally dissociable neural events for the LHRH and LH surges.

title: Galanin — a novel biologically active peptide from porcine intestine
abstract: The isolation of a novel biologically active peptide, designated galanin, is described. The peptide was discovered by the detection of its C‐terminal amide structure in porcine intestinal extract using a chemical method. It was found that galanin consists of 29 amino acids and the complete amino acid sequence is: contract smooth muscle preparations from the rat and to cause a mild and sustained hyperglycemia in dog.

title: Galanin: a biologically active peptide.
abstract: Galanin is a biologically active neuropeptide, widely distributed in the central and peripheral nervous systems and the endocrine system. The amino acid sequence of galanin is very conserved (almost 90% among species), indicating the importance of the molecule. Galanin has multiple biological effects. In the central nervous system, galanin alters the release of several neurotransmitters. In particular the ability of galanin to inhibit acetylcholine release, along with the observation of hyperinervation of galanin fibres in the human basal forebrain of Alzheimer's disease patients, suggest a possible role for galanin in the cholinergic dysfunction, characteristic of the disease. Moreover, galanin has been suggested to be involved in other neuronal functions, such as learning and memory, epileptic activity, nociception, spinal reflexes and feeding. Galanin has also been shown to increase the levels of growth hormone, prolactin and luteinizing hormone, to inhibit glucose induced insulin release and to affect gastrointestinal motility. The expression of galanin (mRNA and peptide levels) is elevated following estrogen administration, neuronal activation, denervation and/or nerve injury, as well as during development. The spectrum of galanin's activities indicates that galanin is an important messenger for intercellular communication within the nervous system and the neuroendocrine axis. Galanin acts at specific membrane receptors to exert its effects; so far three human and rodent galanin receptor subtypes have been cloned. Galanin agonists have been shown to have therapeutic application in treatment of chronic pain; galanin antagonists have therapeutic potential in treatment of Alzheimer's disease, depression, and feeding disorders.

title: Galanin--10 years with a neuroendocrine peptide.
abstract: Galanin is a 29/30 amino acids long neuropeptide which does not belong to any known peptide family. The N-terminal first 16 amino acids of the molecule are both necessary and sufficient for receptor recognition and receptor activation. The main pharmacophores of galanin in its central and pancreatic actions are Gly1, Trp2, Asn5 and Tyr9, respectively. The neuropeptide galanin has multiple effects in both the central and peripheral nervous systems. Centrally, galanin potently stimulates fat intake and impairs cognitive performance. Anoxic glutamate release in the hippocampus is inhibited by galanin and the noradrenergic tonus in the brain is influenced by a hyperpolarizing action of galanin in the locus coeruleus. In the spinal cord galanin inhibits spinal excitability and potentiates the analgesic effect of morphine. In the neuroendocrine system galanin acts in a stimulatory manner on the release of growth hormone and prolactin, and peripherally galanin inhibits glucose induced insulin release. Galanin also causes contraction of the jejunum. The galanin receptor is a Gi-protein-coupled, membrane-bound glycoprotein with an estimated molecular mass of 53 kDa. Several putative tissue specific galanin receptor subtypes have been proposed on a pharmacological basis. The distribution of galanin receptors and of galanin like immunoreactivity are overlapping in the CNS, both being high in areas such as the locus coeruleus, raphe nucleus and hypothalamus. Galanin receptor activation leads to a reduced intracellular Ca(2+)-concentration, either by direct action on voltage sensitive Ca(2+)-channels or indirectly via opening of K(+)-channels or via inhibition of adenylyl cyclase activity. The lowered intracellular Ca2+ level subsequently leads to a reduced PLC activity. Galanin also inhibits cGMP synthesis induced by depolarization. A number of synthetic high affinity galanin receptor antagonists of the peptide type were developed recently, which have enabled the elucidation of functional roles of endogenous galanin in several systems. Furthermore, putative subtypes of galanin receptors can be distinguished by the use of these new galanin receptor ligands.

title: Galanin receptor subtypes.
abstract: The neuropeptide galanin, which is widely expressed in brain and peripheral tissues, exerts a broad range of physiological effects. Pharmacological studies using peptide analogues have led to speculation about multiple galanin receptor subtypes. Since 1994, a total of three G-protein-coupled receptor (GPCR) subtypes for galanin have been cloned (GAL1, gal2 and gal3). Potent, selective antagonists are yet to be found for any of the cloned receptors. Major challenges in this field include linking the receptor clones with each of the known physiological actions of galanin and evaluating the evidence for additional galanin receptor subtypes.

title: Galanin overexpressing transgenic mice
abstract: Galanin overexpressing transgenic mice (GAL-tg) were generated on two different promoters. Both lines of GAL-tg displayed high levels of galanin in the hippocampus and reduced sensitivity to seizures, as compared to their respective wildtype littermate controls (WT). Performance deficits on learning and memory tasks, impaired long-term potentiation, reduced hippocampal excitability, lower evoked glutamate release, and reduced numbers of choline acetyltransferase immunoreactive neurons in the horizontal limb of the diagonal band were detected in GAL-tg as compared to WT. Changes in sensitivity to nociceptive stimuli were demonstrated in one line. GAL-tg represent a new model for investigating the biological actions of endogenous galanin, and for testing novel therapeutics based on galanin receptor ligands.

title: Nutritional Infertility: The Role of the Interconnected Hypothalamic Neuropeptide Y–Galanin–Opioid Network
abstract: Neuropeptide Y (NPY) has been implicated in the hypothalamic regulation of reproduction and energy homeostasis. The perikarya located primarily in the arcuate nucleus (ARC) of the hypothalamus constitute a common source of NPY. Projections from these neurons along two distinct pathways, namely, the reproductive axis of the ARC--median eminence--medial preoptic area and the orexigenic axis of the ARC--paraventricular nucleus and neighboring regions, participate in regulation of these two neuroendocrine functions. Additionally, the NPY neuronal system within the basal hypothalamus is morphologically and functionally linked with galanin and the opioid networks which also play roles in the control of reproduction and ingestive behavior. Recent experimental evidence indicates that genetic, hormonal, and environmental factors that cause nutritional imbalance and metabolic disturbances, along with depressed pituitary gonadal function, produce alterations in the synthesis, storage, and release of NPY and in other connected peptidergic systems in the hypothalamus. The current findings summarized in this review support our concept that NPY may-be one of the essential messenger molecules that serve as a communication bridge between the neural processes that regulate reproduction and those that maintain energy homeostasis. Modification in information flow within this peptidergic network due to nutritional imbalance may adversely impact hypothalamo-pituitary-gonadal function.

title: Galanin An unassuming neuropeptide moves to center stage in reproduction
abstract: Galanin is a neuropeptide widely distributed throughout the reproductive system, including the brain, pituitary, testes, ovaries, and accessory reproductive tracts of both sexes. Within the hypothalamus, a subset of GnRH-producing neurons also synthesizes galanin. Galanin gene expression in these GnRH neurons is highly inducible by estrogen. Galanin is thought to play an important role in the generation of the preovulatory LH surge, and may serve other functions yet to be discovered.

title: Galanin/GALP and galanin receptors: role in central control of feeding, body weight/obesity and reproduction?
abstract: Scientific and commercial pharmacological interest in the role of galanin and galanin receptors in the regulation of food intake, energy balance, and obesity has waned recently, following initial enthusiasm during the 1980-1990s. It has been replaced by efforts to understand the role of newly discovered peptide systems such as the hypocretin/orexins, melanocortins and cocaine- and amphetamine-regulated transcript (CART) and their relationship to the important hormones, leptin and insulin. Thus, while numerous studies have revealed the ability of galanin to stimulate food intake via actions at sites within the hypothalamus, and shown reliable changes in hypothalamic galanin synthesis in response to food ingestion; findings including the lack of a 'body weight/obesity' phenotype in galanin transgenic mouse strains and a lack of agonists/antagonists for galanin receptor subtypes have probably served to reduce enthusiasm. However, as more is learnt about the general and galanin-related neurochemistry of brain pathways involved in feeding, metabolism and body weight control, the potential importance of galanin systems is again in focus. Studies of the newly discovered galanin family peptide, 'galanin-like peptide' (GALP), highlight the likely role of galanin peptides and receptors in the physiological coupling of body weight, adiposity and reproductive function. GALP is produced by a discrete population of neurons within the basomedial arcuate nucleus (and median eminence) that send projections to the anterior paraventricular nucleus and that make close contacts with leutinizing hormone-releasing hormone (LHRH) neurons in basal forebrain. Furthermore, GALP neurons express leptin receptors and respond to leptin treatment by increasing their expression of GALP mRNA. Centrally administered GALP activates LHRH-immunoreactive neurons and increases plasma LH levels. These findings suggest a direct stimulatory action of endogenous GALP on gonadotropin secretion via actions within the hypothalamus/basal forebrain, with leptin actions linking this system to body adipose levels.

title: Isolation and cDNA Cloning of a Novel Galanin-like Peptide (GALP) from Porcine Hypothalamus*
abstract: Galanin is a widely distributed neuropeptide with a variety of physiological functions. Three galanin receptor subtypes, GALR1, GALR2, and GALR3, have been reported. We isolated a novel galanin-like peptide (GALP) from porcine hypothalamus by observing its activity for increasing [35S]GTPγS binding to a membrane preparation of GALR2-transfected cells. The peptide had 60 amino acid residues and a non-amidated C terminus. The amino acid sequence of GALP-(9–21) was completely identical to that of galanin-(1–13). A cloned porcine GALP cDNA indicated that GALP was processed from a 120-amino acid GALP precursor protein. The structures of rat and human GALP-(1–60) were deduced from cloned cDNA, which indicated that the amino acid sequences 1–24 and 41–53 were highly conserved between humans, rats, and pigs. Receptor binding studies revealed that porcine GALP-(1–60) had a high affinity for the GALR2 receptor (IC50 = 0.24 nm) and a lower affinity for the GALR1 receptor (IC50 = 4.3 nm). In contrast, galanin showed high affinity for the GALR1 (IC50= 0.097 nm) and GALR2 receptors (IC50 = 0.48 nm). GALP is therefore an endogenous ligand that preferentially binds the GALR2 receptor, whereas galanin is relatively non-selective.

title: Genomic organization and localization of the gene encoding human preprogalanin.
abstract: An approximately 35-kb region of genomic DNA encoding the human preprogalanin gene including 5' and 3' flanking sequences has been cloned and characterized. Exons and flanking introns were sequenced to determine the structural organization of the gene. The gene spans 6.5 kb, with the first exon encoding only the 5' untranslated sequence. The coding region of preprogalanin and the 3' untranslated sequence is divided into five exons. Using high-resolution fluorescence in situ hybridization, the position of the single human preprogalanin gene was localized to chromosome 11q13.3-q13.5. Several oncogenes have been mapped to this region, which is also the breakpoint for the translocation t(11;14)(q13;q32) in chronic lymphocytic leukemia and diffuse B-cell lymphoma.

title: Regulation of Galanin‐Like Peptide Gene Expression By Pituitary Hormones and Their Downstream Targets
abstract: Galanin‐like peptide (GALP) mRNA is expressed in neurones of the hypothalamic arcuate nucleus and within pituicytes in the neurohypophysis. Several neuropeptides that are expressed in the arcuate nucleus participate in the neuroendocrine regulation of pituitary hormone secretion. Our objective was to determine the extent to which GALP might be a target for regulation by pituitary hormones or their downstream targets in the rat. The expression of GALP mRNA in the arcuate nucleus was reduced by hypophysectomy as determined by in situ hybridization. However, this did not appear to be attributable to the loss of either gonadal or adrenal steroids because castrated, ovariectomized and adrenalectomized rats had GALP mRNA expression that was indistinguishable from their respective controls. Next, we investigated the effects of growth hormone deficiency on GALP mRNA expression by studying dwarf rats and found that GALP gene expression was not different between dwarf rats and controls. We found that thyroidectomy led to a significant reduction in GALP mRNA expression compared to intact controls, and thyroidectomized rats implanted with thyroxine pellets had GALP mRNA expression that was similar to intact controls. Thus, the reduction of GALP mRNA expression seen in hypophysectomized animals may reflect, in part, a selective loss of thyroid hormone. We also found that the expression of GALP mRNA was increased in the neurohypophysis of lactating rats compared to nonlactating rats, whereas GALP mRNA expression in the arcuate nucleus was unaffected by lactation. This suggests that the induction of GALP gene expression in pituicytes is physiologically associated with activation of oxytocin and vasopressin secretion during lactation.

title: Galanin-like peptide (GALP) is a target for regulation by leptin in the hypothalamus of the rat.
abstract: Galanin-like peptide (GALP), which was recently isolated from the porcine hypothalamus, shares sequence homology with galanin and binds with high affinity to galanin receptors. To study the distribution and regulation of GALP-expressing cells in the brain, we cloned a 120 base-pair cDNA fragment of rat GALP and produced an antisense riboprobe. In situ hybridization for GALP mRNA was then performed on tissue sections throughout the forebrain of adult ovariectomized female rats. We found GALP mRNA-containing cells in the arcuate nucleus (Arc), caudal dorsomedial nucleus, median eminence and the pituitary. Because GALP mRNA in the Arc appeared to overlap with the known distribution of leptin receptor mRNA, we tested the hypothesis that GALP expression is regulated by leptin. Using in situ hybridization, we compared the number of GALP mRNA-containing cells among groups of rats that were fed ad lib or fasted for 48 h and treated with either leptin or vehicle. Fasting reduced the number of identifiable cells containing GALP mRNA in the Arc, whereas the treatment of fasted animals with leptin produced a 4-fold increase in the number of cells expressing GALP message. The presence of GALP mRNA in the hypothalamus and pituitary and its regulation by leptin suggests that GALP may have important neuroendocrine functions, including the physiological regulation of feeding, metabolism, and reproduction.

title: Distribution and Regulation of Galanin-Like Peptide (GALP) in the Hypothalamus of the Mouse.
abstract: Galanin-like peptide (GALP) is a newly discovered molecule whose expression in the brain is confined to the arcuate nucleus and median eminence. In the rat, cellular levels of GALP mRNA are reduced by fasting and reversed by peripheral administration of leptin. The purpose of this investigation was 1) to clone and map the distribution of GALP mRNA in the brain of the mouse; 2) to compare the pattern and magnitude of GALP mRNA expression in the leptin-deficient obese (ob/ob) mouse with that of wild-type controls; and 3) to examine the effects of leptin delivered into the brain on the expression of GALP mRNA in the ob/ob mouse. We report the sequence of a mouse GALP cDNA and show that GALP mRNA is expressed in the arcuate nucleus, median eminence, infundibular stalk, and the neurohypophysis of this species. The expression of GALP mRNA in the brain was markedly reduced in the ob/ob mice, compared with wild-type animals. Intracerebroventricular infusion of leptin to ob/ob mice increased both the number of GALP mRNA-expressing neurons and their content of GALP mRNA, compared with vehicle-treated controls. These observations demonstrate that GALP mRNA is induced by leptin through a direct action on the brain.

title: Galanin-Like Peptide (GALP) mRNA Expression Is Restricted to Arcuate Nucleus of Hypothalamus in Adult Male Rat Brain
abstract: Galanin-like peptide (GALP) is a novel 60-amino acid neuropeptide, isolated from porcine hypothalamus and subsequently identified in rats and humans, which has reported selectivity for the Gal-R2 galanin receptor [Ohtaki T et al: J Biol Chem 1999; ]. In the current study, the regional and cellular distribution of GALP mRNA in rat brain has been investigated by in situ hybridization of [35S]-labelled oligonucleotide probes. In a thorough screening of adult male rat brain, GALP mRNA expression was detected only throughout the rostrocaudal extent of the arcuate nucleus (ARC) with the most abundant hybridization signal in the posterior, periventricular zones. GALP mRNA-positive neurons were mostly localized in the ventromedial division of the ARC, with many closely adjacent to the wall of the third ventricle. Smaller numbers of labelled neurons were also found in ventrolateral areas. The distribution of GALP mRNA was somewhat complementary to that for galanin (GAL) mRNA in the ARC, but contrasted with the broad distribution of this transcript throughout the hypothalamus. GAL mRNA was also distributed along the rostrocaudal extent of the ARC, but was most abundant in the anterior to middle levels and in ventrolateral regions. Interestingly, somatostatin mRNA expression appeared to overlap the distribution of GALP mRNA in posterior, ventromedial regions of the ARC. Thus, in adult rat brain GALP mRNA expression was restricted to a discrete subpopulation of neurons in the ARC, with a unique localization pattern unlike GAL or many other known peptide- and transmitter-containing cells in this region. GALP could, however, be co-expressed in sub-populations of other neuronal phenotypes (e.g. somatostatin neurons) or within cells that express Gal-R2 receptors. In view of the established anatomy and function of the ARC and the restricted localization of GALP mRNA, this novel peptide is likely to play a role in regulation of anterior pituitary hormone secretion, or in regulation of other hypothalamic peptide and transmitter systems.

title: Cloning and distribution of galanin-like peptide mRNA in the hypothalamus and pituitary of the macaque.
abstract: Galanin-like peptide (GALP) is a newly discovered hypothalamic neuropeptide, which is regulated by leptin and implicated in the regulation of GnRH secretion in the rodent. We searched the human genome database and determined that the human GALP gene comprises six exons, as has been shown for human galanin. We used rapid amplification of cDNA ends to clone a full-length cDNA (802 bp) of the macaque homologue of GALP and found it to be highly conserved between human and macaque at both the nucleotide (93%) and peptide (94%) levels. Mature GALP is predicted to be 60 amino acids in the macaque as in other species, and the region of GALP (9-21) that shows homology to the N-terminal 13 amino acids of galanin is perfectly conserved. We mapped the distribution of GALP mRNA in the hypothalamus and pituitary of the macaque by in situ hybridization and observed that, as in rodent species, the expression of GALP mRNA is confined to the arcuate nucleus, median eminence, and neurohypophysis. Using double-label in situ hybridization, we found that nearly all (98%) GALP mRNA-expressing cells in the arcuate nucleus also express mRNA for the long form of the leptin receptor. These findings suggest that a leptin-GALP signaling pathway exists in a primate species.

title: Galanin and galanin receptors in human gliomas
abstract: Galanin-like immunoreactivity (GAL-LI) and specific GAL binding sites have been shown to be widely distributed in the central nervous system (CNS) and in CNS tumors. GAL and its receptors have also been shown to be present in glial cells, but to date it is still unknown whether human gliomas produce GAL and express GAL receptors. In this study 20 brain tumors consisting of 15 glioblastomas, 4 meningiomas and 1 gliosarcoma were investigated for the presence of GAL-LI and GAL receptors. Immunofluorescence analysis revealed a dense network of GAL-LI positive cellular processes and cell bodies in 18 of the 20 tumors. In contrast, in vitro 125I-labeled GAL receptor autoradiography showed substantial GAL binding in only 6 glioblastoma tissues. Reverse transcription-PCR analysis detected mRNA of all three known galanin receptors in the tumor tissues, with most tumors expressing multiple receptor subtypes. Pharmacological analysis of tumor membrane homogenates with GAL and the specific GAL receptor GalR2 agonist, AR-M1896, revealed that the GAL receptor GalR1 is most likely the receptor responsible for the observed GAL binding in the glioblastomas. No correlation could be found between GAL-LI, the level of GAL binding and proliferative activity as determined by immunostaining with the cell proliferation marker Ki-67.

title: Galanin‐like peptide (GALP) is expressed in rat hypothalamus and pituitary, but not in DRG
abstract: Galanin-like peptide (GALP) was recently purified on the basis of its preferential activation of galanin receptor subtype 2 (GALR2) compared with galanin receptor subtype 1 (GALR1). Using in situ hybridization of adult rat brain, pituitary and dorsal root ganglia (DRG) we demonstrate that GALP mRNA expression is restricted to the arcuate nucleus and median eminence of the hypothalamus, and to the posterior lobe of the pituitary. No expression was detected elsewhere in brain, or in the DRG. In adult mouse, no expression was detected in brain or in DRG either before or after axotomy, suggesting that GALP has no apparent role in the axotomy response of DRG.

title: Galanin-like peptide is co-localized with α-melanocyte stimulating hormone but not with neuropeptide Y in the rat brain
abstract: Galanin-like peptide (GALP), recently isolated from the hypothalamus, is a novel peptide of 60 amino acid residues. GALP is an endogenous ligand of the orphan receptor and shows a high affinity to its specific receptor GalR2. GALP mRNA was shown to be expressed predominantly in the arcuate nucleus (ARC) of the rat hypothalamus, a region considered to be one of the most important feeding-regulating centers in the brain. According to recent reports of morphological and physiological experiments, GALP-containing neurons express leptin receptors and respond to leptin treatment by increasing mRNA expression. However, the relationships between GALP and other feeding-regulating neurons have not yet been proven. In this study, we examined the relationships between GALP- and neuropeptide Y (NPY)- or alpha-melanocyte stimulating hormone ( MSH)-containing neurons by using a dual immunostaining technique. We found that many NPY-immunoreactive fibers were in close apposition with GALP-immunoreactive cell bodies. Furthermore, immunoreactivity for GALP and alpha-MSH was detectable in the same neurons (3.3-11.8%) in the ARC. However, the co-existence of GALP and NPY was never demonstrated. These findings strongly suggest that GALP may participate in the regulation of feeding behavior in harmony with alpha-MSH.

title: Intracellular signalling: Key enzyme in leptin-induced anorexia
abstract: Leptin is a key hormonal regulator of energy balance that acts upon hypothalamic neurons to reduce food intake, but the intracellular mechanisms involved are incompletely understood. Here we show that systemic administration of leptin in rats activates the enzyme phosphatidylinositol-3-OH kinase (PI(3)K) in the hypothalamus and that intracerebro-ventricular (i.c.v.) infusion of inhibitors of this enzyme prevents leptin-induced anorexia. Our results indicate that PI(3)K is a crucial enzyme in the signal-transduction pathway that links hypothalamic leptin to reduced food intake.

title: Insulin activation of phosphatidylinositol 3-kinase in the hypothalamic arcuate nucleus: a key mediator of insulin-induced anorexia.
abstract: In peripheral tissues, insulin signaling involves activation of the insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase (PI3K) enzyme system. In the hypothalamus, insulin functions with leptin as an afferent adiposity signal important for the regulation of body fat stores and hepatic glucose metabolism. To test the hypothesis that hypothalamic insulin action involves intracellular PI3K signaling, we used histochemical and biochemical methods to determine the effect of insulin on hypothalamic IRS-PI3K activity. Here, we report that insulin induces tyrosine phosphorylation of the insulin receptor and IRS-1 and -2, increases binding of activated IRS-1 and -2 to the regulatory subunit of PI3K, and activates protein kinase B/Akt, a downstream target of PI3K. Using an immunohistochemical technique to detect PI 3,4,5-triphosphate, the main product of PI3K activity, we further demonstrate that in the arcuate nucleus, insulin-induced PI3K activity occurs preferentially within cells that contain IRS-2. Finally, we show that the food intake- lowering effects of insulin are reversed by intracerebroventricular infusion of either of two PI3K inhibitors at doses that have no independent feeding effects. These findings support the hypothesis that the IRS-PI3K pathway is a mediator of insulin action in the arcuate nucleus and, combined with recent evidence that leptin activates PI3K signaling in the hypothalamus, provide a plausible mechanism for neuronal cross-talk between insulin and leptin signaling.

title: Galanin-like peptide stimulates the release of gonadotropin-releasing hormone in vitro and may mediate the effects of leptin on the hypothalamo-pituitary-gonadal axis.
abstract: Leptin regulates the hypothalamo-pituitary-gonadal axis in relation to nutritional status. The mechanism through which leptin mediates its effects on neuroendocrine reproductive circuits remains unclear. Galanin-like peptide (GALP) is a recently identified hypothalamic peptide, localized in the arcuate nucleus, which seems to be regulated by leptin and stimulates LH when administered centrally. Here, we demonstrate that leptin stimulates the release of GALP and GnRH in vitro from hypothalamic explants harvested from male rats. In addition, we show that GALP stimulates the release of GnRH from hypothalamic explants and GT1-7 cells. Furthermore, we demonstrate that GALP antiserum blocks the stimulatory action of leptin on GnRH release from hypothalamic explants. GALP is a ligand of the galanin receptors. We therefore investigated whether the effect of GALP on GnRH release may be mediated via a known galanin receptor. GALP-stimulated GnRH release from hypothalamic explants was attenuated (but not abolished) by the galanin receptor antagonist galantide. However, GALP-stimulated GnRH release from GT1-7 cells was not diminished by the coadministration of galantide. In addition, none of the cloned galanin receptors were expressed in GT1-7 cells by RT-PCR. These observations suggest that GALP may stimulate GnRH release through an indirect pathway involving a galanin receptor and via a direct action on GnRH neurons, possibly through a novel receptor. These findings suggest that GALP may mediate the actions of leptin on the reproductive axis and provide a link between nutrition and fertility.

title: Galanin-like peptide stimulates food intake in the rat
abstract: We have isolated a novel hypothalamic peptide, Galanin-like peptide (GALP), as a ligand for galanin receptor subtype GalR2. To investigate the physiological role of GALP, we examined the effect of the intracerebroventricular administration of GALP and found that GALP induced food intakes. GALP had ten-fold the orexigenic activity of galanin. We also observed the anxiogenic-like behavior after the administration of 1 nmol GALP. These results suggest that GALP is a novel orexigenic and anxiogenic peptide.

title: Centrally Administered Galanin‐Like Peptide Modifies Food Intake in the Rat: A Comparison with Galanin
abstract: Galanin‐like peptide (GALP) is a recently identified neuropeptide that shares sequence homology with the orexigenic neuropeptide, galanin. In contrast to galanin, GALP is reported to bind preferentially to the galanin receptor 2 subtype (GalR2) compared to GalR1. The aim of this study was to determine the effect of GALP on feeding, body weight and core body temperature after central administration in rats compared to the effects of galanin. Intracerebroventricular (i.c.v.) injection of GALP (1 µg−10 µg) significantly stimulated feeding at 1 h in both satiated and fasted Sprague‐Dawley rats. However, 24 h after GALP injection, body weight gain was significantly reduced and food intake was also usually decreased. In addition, i.c.v. GALP caused a dose‐related increase in core body temperature, which lasted until 6–8 h after injection, and was reduced by peripheral administration of the cyclooxygenase inhibitor, flurbiprofen (1 mg/kg). Similar to GALP, i.c.v. injection of galanin (5 µg) significantly increased feeding at 1 h in satiated rats. However, there was no difference in food intake and body weight at 24 h, and galanin only caused a transient rise in body temperature. Thus, similar to galanin, GALP has an acute orexigenic effect on feeding. However, GALP also has an anorectic action, which is apparent at a later time. Therefore, GALP has complex opposing actions on energy homeostasis.

title: Effect of galanin on food intake in rats: involvement of lateral and ventromedial hypothalamic sites.
abstract: Galanin has previously been reported to elicit feeding in satiated animals when injected into the hypothalamic paraventricular nucleus. It is not known, however, 1) whether this action is due to activation of feeding signals or suppression of satiety signals or both or 2) whether other hypothalamic regions such as the lateral hypothalamus (LH) or the ventromedial hypothalamus (VMH) are involved in this action. The effects of galanin on food intake were therefore examined in satiated and in fasted rats both after intracerebroventricular injection (0.1, 1, and 10 micrograms/10 microliters) and after microinjection (1 and 5 micrograms/0.5 microliters) into the LH and VMH. Twenty minutes after intracerebroventricular injection, galanin significantly and dose dependently augmented food intake by up to sevenfold in freely feeding rats and by up to 79% in fasted animals. The galanin-induced augmentation of cumulative food intake up to 2 h after injection was due to the initial increase in food consumption during the 0 to 20-min interval. This suggests that galanin acts by activation of feeding behavior and not by suppression of satiety signals in these fasted animals, in which satiety signals are presumably not initially operative. Twenty minutes after intrahypothalamic injections into both the LH and VMH, galanin (5 micrograms) significantly increased food consumption, fivefold in freely feeding rats and 30-35% in fasted rats. Thus stimulation of feeding by centrally injected galanin also involves loci within the LH and VMH.

title: Enkephalin contributes to the locomotor stimulating effects of 3,4‐methylenedioxy‐N‐methylamphetamine
abstract: 3,4‐Methylenedioxy‐N‐methylamphetamine (MDMA, ‘Ecstasy’) is a potent inhibitor of serotonin uptake, which induces both an increase in locomotion and a decrease in exploratory activity in rodents. Serotonin 5‐HT1B receptors, located on the terminals of striatal efferent neurons, have been suggested to mediate these motor effects of MDMA. Striatal neurons projecting to the globus pallidus contain met‐enkephalin, whilst those projecting to the substantia nigra contain substance P. We therefore analysed the levels of both peptides using radioimmunocytochemistry after MDMA administration (10 mg/kg, 3 h) in wild‐type and 5‐HT1B receptor knockout mice. Our results demonstrate that MDMA induces a decrease in pallidal met‐enkephalin immunolabelling in wild‐type, but not in 5‐HT1B receptor knockout mice. Similar results were obtained following treatment with the 5‐HT1A/1B agonist RU24969 (5 mg/kg, 3 h), suggesting that activation of 5‐HT1B receptors leads to a reduction in met‐enkephalin levels in the globus pallidus. In contrast, MDMA had no effect on the nigral substance P levels. We have previously shown that both MDMA and RU24969 fail to stimulate locomotor activity in 5‐HT1B receptor knockout mice. Our present data indicate that the opioid antagonist naloxone suppressed the locomotor effects of MDMA. This study is the first to demonstrate that Enk contributes to MDMA‐induced increases in locomotor activity. Such an effect may be related to the 5‐HT control of pallidal met‐enkephalin levels via the 5‐HT1B receptors.

title: Galanin-like peptide as a possible link between metabolism and reproduction in the macaque.
abstract: Galanin-like peptide (GALP) is a hypothalamic neuropeptide that has been implicated in the control of feeding, metabolism, and reproduction. The goal of this study was to examine the effects of central infusions of GALP on GnRH and LH secretion and to identify physiological factors that influence the expression of GALP mRNA in the brain of a primate species. Infusions of GALP into the lateral cerebroventricle of the macaque caused a significant increase in LH secretion, which was blocked by administration of the GnRH receptor antagonist acyline. However, the expression of GALP mRNA in the arcuate nucleus, as determined by in situ hybridization, was not regulated by either estradiol or progesterone. Compared with feeding ad libitum, fasting for 48 h produced a significant reduction in the hypothalamic expression of GALP mRNA. GALP neurons were found to express both neuropeptide Y Y1 receptor and serotonin 2C receptor by double-label in situ hybridization. Taken together, these results suggest that GALP neurons play a role of integrating metabolic signals, which are relayed to circuits controlling GnRH release in the macaque.

title: Galanin-like peptide is target for regulation by orexin in the rat hypothalamus
abstract: Galanin-like peptide (GALP) is a newly discovered 60 amino acid peptide from the porcine hypothalamus. GALP has been shown to be expressed predominantly in the arcuate nucleus (ARC) of the rat hypothalamus, a region considered to be one of the most important feeding-regulating centers in the brain. GALP-containing neurons in the ARC express leptin receptors, but relationships between GALP and other feeding-regulating neurons have not yet been fully elucidated. Given that Orexin (OX)-containing neurons make synaptic inputs to the ARC, we thus examined the relationship between GALP and OX in the ARC by use of a dual immunostaining technique. OX-immunoreactive fibers appeared to be closely apposed to GALP-immunoreactive cell bodies and their processes. We also examined whether the OX receptor, OX(1)-R was expressed in the GALP-containing neurons. Immunoreactivity for both OX(1)-R and GALP was detectable in 9.6 % neurons (range 4.2-14.6%) in the ARC. These findings strongly suggest that GALP may participate in the regulation of feeding behavior under the influence of leptin and OX.

title: Leptin is a metabolic signal to the reproductive system.
abstract: Leptin, a newly-discovered hormonal product of the obese (ob) gene, is expressed by adipocytes and thought to play a role in the regulation of food intake and metabolism. We tested the hypothesis that leptin signals metabolic information to the reproductive system by examining its effects on the reproductive system of ob/ob mice, which have a congenital deficiency in leptin and are infertile. We treated pair-fed males and females with leptin (50 microg twice daily, ip) or vehicle (n=10/group) for 14 days, after which the animals were bled and killed. Leptin-treated females had significantly elevated serum levels of LH, increased ovarian and uterine weights, and stimulated aspects of ovarian and uterine histology compared to controls. Leptin-treated males had significantly elevated serum levels of FSH, increased testicular and seminal vesicle weights, greater seminal vesicle epithelial cell height, and elevated sperm counts compared to controls. These results demonstrate that leptin stimulates the reproductive endocrine system in both sexes of ob/ob mice and suggest that leptin may serve as a permissive signal to the reproductive system of normal animals.

title: Leptin regulation of reproductive function and fertility.
abstract: Leptin, a 16-KD protein secreted primarily by adipose tissue, was first discovered in the search for a satiety signal. When administered into the brain, leptin depresses appetite. Interestingly, hyperphagic, obese, transgenic mice with leptin deficiency were noted to be reproductively incompetent, and administration of leptin restored their fertility. These pivotal observations led to numerous studies on the site of action of leptin within the hypothalamo-hypophyseal-gonadal axis, and a variety of models have been used ranging from the prepubertal condition to fasting suppression of reproductive hormones. The preponderance of studies thus far has focused on how leptin serves as a metabolic signal of energy balance within the neuroendocrine system, particularly as a regulator of GnRH/LH secretion. Less research has been conducted with other components of the reproductive system, but local effects of leptin have been demonstrated in the gonads where hyperleptinemia suppresses steroidogenesis and potentially affects gamete maturation. This presentation will review the major concepts for the role of leptin in the modulation of fertility and will consider the potential use of leptin in assisted reproductive technology and embryo transfer.

title: IRS-2 pathways integrate female reproduction and energy homeostasis
abstract: Severe dietary restriction, catabolic states and even short-term caloric deprivation impair fertility in mammals. Likewise, obesity is associated with infertile conditions such as polycystic ovary syndrome. The reproductive status of lower organisms such as Caenorhabditis elegans is also modulated by availability of nutrients. Thus, fertility requires the integration of reproductive and metabolic signals. Here we show that deletion of insulin receptor substrate-2 (IRS-2), a component of the insulin/insulin-like growth factor-1 signalling cascade, causes female infertility. Mice lacking IRS-2 have small, anovulatory ovaries with reduced numbers of follicles. Plasma concentrations of luteinizing hormone, prolactin and sex steroids are low in these animals. Pituitaries are decreased in size and contain reduced numbers of gonadotrophs. Females lacking IRS-2 have increased food intake and obesity, despite elevated levels of leptin. Our findings indicate that insulin, together with leptin and other neuropeptides, may modulate hypothalamic control of appetite and reproductive endocrinology. Coupled with findings on the role of insulin-signalling pathways in the regulation of fertility, metabolism and longevity in C. elegans and Drosophila, we have identified an evolutionarily conserved mechanism in mammals that regulates both reproduction and energy homeostasis.

title: Role of Brain Insulin Receptor in Control of Body Weight and Reproduction
abstract: Insulin receptors (IRs) and insulin signaling proteins are widely distributed throughout the central nervous system (CNS). To study the physiological role of insulin signaling in the brain, we created mice with a neuron-specific disruption of the IR gene (NIRKO mice). Inactivation of the IR had no impact on brain development or neuronal survival. However, female NIRKO mice showed increased food intake, and both male and female mice developed diet-sensitive obesity with increases in body fat and plasma leptin levels, mild insulin resistance, elevated plasma insulin levels, and hypertriglyceridemia. NIRKO mice also exhibited impaired spermatogenesis and ovarian follicle maturation because of hypothalamic dysregulation of luteinizing hormone. Thus, IR signaling in the CNS plays an important role in regulation of energy disposal, fuel metabolism, and reproduction.

title: Thyroid disease and male reproductive function
abstract: Despite the high prevalence of thyroid diseases in the general population, male reproductive function in patients with thyroid disease has been the subject of only a few studies. Hyperthyroidism appears to cause sperm abnormalities (mainly reduction in motility), which reverse after restoration of euthyroidism. Radioiodine therapy for hyperthyroidism or thyroid cancer may cause transient reductions in sperm count and motility, but there appears to be little risk of permanent effects provided that the cumulative dose is less than 14 MBq. The effects of hypothyroidism on male reproduction appear to be more subtle than those of hyperthyroidism and reversible. Severe, prolonged hypothyroidism in childhood may be associated with permanent abnormalities in gonadal function.

title: Thyroid function and puberty.
abstract: Thyroid hormones are essential for normal growth, sexual development and reproductive function. During puberty, changes in thyroid functions and an increase in thyroid volume occur as an adaptation to body and sexual development. Hypothyroidism diagnosed late in prepubertal years, usually due to Hashimoto's thyroiditis, can cause a delay of puberty or incomplete isosexual precocity (development of breast and internal genitalia in girls and increased testis volume in boys without adrenarche). In contrast, normal pubertal development and adequate menarche have been documented in congenital hypothyroidism detected by neonatal screening and treated early. The effect of hyperthyroidism on pubertal development is not well known, but a short period of hyperthyroidism seems not to have major negative effects. In adolescence or young adulthood, menstrual dysfunction, infertility, and stillbirth or premature birth are associated with thyroid dysfunction.