Stephanie M. Krasnow

Distribution and Regulation of Galanin-Like Peptide (GALP) in the Hypothalamus of the Mouse

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.

Neuroendocrine Profiles in Galanin-Overexpressing and Knockout Mice

The peptide galanin has been implicated in the neuroendocrine regulation of reproduction and energy balance. To gain more insight into the functional significance of galanin in these processes, we studied the phenotype of mice that either overexpress galanin in the brain under the control of the dopamine β-hydroxylase promoter (GALTG) or have a complete absence of galanin expression (GALKO). Both GALTGs and GALKOs had body weights and feeding patterns that were indistinguishable from wild-type (WT) control animals, and both genotypes were reproductively competent. Serum levels of follicle-stimulating hormone were significantly higher in GALKOs and slightly lower in GALTGs than in their respective WT controls. Both GALTGs and GALKOs showed a normal response to fasting, but when GALKO mice were treated with leptin during fasting, levels of corticosterone and testosterone were altered compared to WT mice. In addition, GALKOs were more sensitive than WT controls to the effects of chronic leptin treatment on body weight and fat pad mass, whereas GALTGs showed responses to this metabolic challenge that were indistinguishable from their controls. When galanin was administered centrally, GALKOs had lower testosterone and corticosterone levels than did WT mice. These results suggest that the complete loss of galanin leads to significant alterations in neuroendocrine homeostasis, whereas targeted overexpression of galanin in the brain does not interfere with normal neuroendocrine function.

Analysis of the contribution of galanin receptors 1 and 2 to the central actions of galanin-like peptide

Galanin-like peptide (GALP) shares partial sequence identity with galanin and exhibits agonistic activity at two of the galanin receptor subtypes (GALR1 and GALR2) in vitro. The goal of these experiments was to determine whether galanin receptors mediate the effects of central GALP administration on food intake, body weight, and luteinizing hormone (LH) secretion in the mouse. We first evaluated the effects of intracerebroventricular injections of GALP or its vehicle alone in GALR1 knockout mice, GALR2 knockout mice, and their respective wild-type controls. GALP reduced food intake and body weight after 24 h to a similar degree in wild-type, GALR1 knockout, and GALR2 knockout mice. The wild-type, GALR1 knockout, and GALR2 knockout mice also exhibited significant increases in serum levels of LH following the GALP injections. To help delineate the biologically active moiety of the GALP molecule, we injected wild-type mice with shorter fragments of the full-length GALP peptide. Neither GALP(1–21) (the fragment containing the galanin-homologous sequence) nor GALP(22–60) (the C-terminal portion of the GALP molecule lacking sequence identity with galanin) had any discernable effect on food intake, body weight or circulating LH. These observations demonstrate that neither GALR1 nor GALR2 are essential for mediating the effects of GALP on feeding, body weight or LH secretion. Furthermore, the galanin-homologous region of the GALP molecule is not sufficient to mimic the effects of full-length GALP. Together, these findings argue against the hypothesis that GALP signals solely through galanin receptors in vivoand suggest the existence of a yet-to-be-identified GALP-specific receptor.

Regulation of Galanin-Like Peptide Gene Expression By Pituitary Hormones and Their Downstream Targets

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.

Physiological mechanisms integrating metabolism and reproduction

This chapter provides an overview of the physiological mechanisms integrating metabolism and reproduction. The reproductive system of some mammals is more sensitive than others to reductions in energy availability. Of the various factors that dictate an animals sensitivity to energetic challenges, three are significant—sex, body size, and domestication. The reproductive function is influenced by short-term alterations in metabolic status. The rapidity with which the hypothalamic–pituitary–gonadal (HPG) axis responds to an improved metabolic state is illustrated by multiple observations that pulsatile luteinizing hormone (LH) secretion commences or resumes within minutes to hours after food-restricted prepubertal or adult animals are given unlimited access to food. In addition to the metabolic factors, a variety of other hormones has been implicated in relaying metabolic cues to the neuroendocrine reproductive axis, including growth hormone, insulin-like growth factor I, and glucocorticoids. Based on their anatomical distribution pattern, gonadotropin-releasing hormone (GnRH) neurons appear to be ideally situated to directly detect metabolic fuels and hormones that enter the brain from the systemic circulation.