Luis Fabián Canosa

Orexigenic Actions of Ghrelin in Goldfish: Feeding-Induced Changes in Brain and Gut mRNA Expression and Serum Levels, and Responses to Central and Peripheral Injections

In this study, we examined (i) the preprandial, postprandial and starvation-induced changes in the preproghrelin mRNA expression and serum ghrelin levels, and (ii) the effects of intracerebroventricular and intraperitoneal administration of ghrelin on food intake in goldfish. Slot blot analysis revealed a significant postprandial decrease in preproghrelin mRNA expression in the hypothalamus (1 and 3 h after feeding) and gut (3 h after feeding). A similar postprandial decrease (1 and 3 h after feeding) in serum ghrelin levels was also detected. In the fish that were unfed at the regular feeding time, the hypothalamic preproghrelin mRNA expression and the serum ghrelin levels remained unchanged, while the preproghrelin mRNA expression in the gut decreased 3 h after the regular feeding time. Starvation increased preproghrelin mRNA expression in the hypothalamus and gut on the 7th day. Serum ghrelin levels were significantly elevated on days 3 and 5 of starvation. Intracerebroventricular injections of n-octanoylated ghrelin-like peptides (gGRL[1–12]) (10 ng/g body weight) and human ghrelin (1 and 10 ng/g body weight) and intraperitoneal injections of n-octanoylated gGRL[1–12] (10 ng/g body weight), gGRL[1–19] (100 ng/g body weight) and human ghrelin (10 and 100 ng/g body weight) stimulated food intake in goldfish. The patterns of synthesis, secretion and actions indicate that ghrelin is an orexigen in goldfish.

Regulation of expression of somatostatin genes by sex steroid hormones in goldfish forebrain

Recently, our laboratory has identified three distinct pre-pro-somatostatin (PSS) genes in goldfish brain: PSS-I encodes for somatostatin (SRIH)-14, PSS-II encodes SRIH-28, which contains [Glu1, Tyr7, Gly10] SRIH-14 at its C-terminus, and PSS-III encodes [Pro2] SRIH-14. In goldfish, increasing levels of the sex steroid estradiol increase the plasma levels of growth hormone (GH). However, whether sex steroids act at the level of the brain to regulate GH release is unclear. In the present study, the effects of sex steroids on the expression of the three PSS genes in goldfish forebrain were examined. The results demonstrate that treatment with estradiol significantly increases the expression of PSS-I and PSS-III genes in both male and female fish. The effects of estradiol were evident after only 2.5 days of treatment. Testosterone treatment increased the expression of PSS-I and PSS-III genes in female but not male fish, and only at the highest dose used. In addition, the effects of testosterone were evident only after treatment for 5 or 10 days and were blocked by an aromatase inhibitor, suggesting that testosterone must be converted to estradiol to exhibit the effect. Neither estradiol nor testosterone treatment had effects on the expression of the PSS-II gene. These results suggest that sex steroids can act either directly or indirectly on the brain to regulate PSS-I and PSS-III gene expression, influencing in turn the regulation of GH secretion.

Changes in brain mRNA levels of gonadotropin-releasing hormone, pituitary adenylate cyclase activating polypeptide, and somatostatin during ovulatory luteinizing hormone and growth hormone surges in goldfish

In goldfish, circulating LH and growth hormone (GH) levels surge at the time of ovulation. In the present study, changes in gene expression of salmon gonadotropin-releasing hormone (sGnRH), chicken GnRH-II (cGnRH-II), somatostatin (SS) and pituitary adenylate cyclase activating polypeptide (PACAP) were analyzed during temperature- and spawning substrate-induced ovulation in goldfish. The results demonstrated that increases in PACAP gene expression during ovulation are best correlated with the GH secretion profile. These results suggest that PACAP, instead of GnRH, is involved in the control of GH secretion during ovulation. Increases of two of the SS transcripts during ovulation are interpreted as the activation of a negative feedback mechanism triggered by high GH levels. The results showed a differential regulation of sGnRH and cGnRH-II gene expression during ovulation, suggesting that sGnRH controls LH secretion, whereas cGnRH-II correlates best with spawning behavior. This conclusion is further supported by the finding that nonovulated fish induced to perform spawning behavior by prostaglandin F2alpha treatment increased cGnRH-II expression in both forebrain and midbrain, but decreased sGnRH expression in the forebrain.

The secretogranin II-derived peptide secretoneurin stimulates luteinizing hormone secretion from gonadotrophs.

Secretoneurin (SN) is a 33- to 34-amino acid neuropeptide derived from secretogranin-II, a member of the chromogranin family. We previously synthesized a putative goldfish (gf) SN and demonstrated its ability to stimulate LH release in vivo. However, it was not known whether goldfish actually produced the free SN peptide or whether SN directly stimulates LH release from isolated pituitary cells. Using a combination of reverse-phase HPLC and mass spectrometry analysis, we isolated for the first time a 34-amino acid free gfSN peptide from the whole brain. Moreover, Western blot analysis indicated the existence of this peptide in goldfish pituitary. Immunocytochemical localization studies revealed the presence of SN immunoreactivity in prolactin cells of rostral pars distalis of the anterior pituitary. Additionally, we found that magnocellular cells of the goldfish preoptic region are highly immunoreactive for SN. These neurons send heavily labeled projections that pass through the pituitary stalk and innervate the neurointermediate and anterior lobes. In static 12-h incubation of dispersed pituitary cells, application of SN antiserum reduced LH levels, whereas 1 and 10 nM gfSN, respectively, induced 2.5-fold (P < 0.001) and 1.9-fold (P < 0.01) increments of LH release into the medium, increases similar to those elicited by 100 nM concentrations of GnRH. Like GnRH, gfSN elevated intracellular Ca(2+) in identified gonadotrophs. Whereas we do not yet know the relative contribution of neural SN or pituitary SN to LH release, we propose that SN could act as a neuroendocrine and/or paracrine factor to regulate LH release from the anterior pituitary.

Regulation of the Hypothalamic Melanin-Concentrating Hormone Neurons by Sex Steroids in the Goldfish: Possible Role in the Modulation of Luteinizing Hormone Secretion

In teleost fish, melanin-concentrating hormone (MCH) is a cyclic heptadecapeptide released from the pituitary during white background adaptation. In the periphery MCH concentrates melanin granules in melanophores thus lightening the body color of fish. Evidence from mammalian studies has demonstrated the involvement of MCH in the control of energy balance and the reproductive axis. Information about the hormonal regulation of MCH neurons in non-mammalian systems is scarce and nothing is known about its role in the regulation of the reproductive axis. We here report the molecular characterization of two MCH precursors in the goldfish. Both precursors are peripherally expressed and the expression in the central nervous system is restricted to the mediobasal hypothalamus. Hypothalamic MCH-mRNA production is upregulated during white background adaptation. Both testosterone and estradiol stimulate MCH mRNA expression in the hypothalamus in a sex-dependent manner, with females showing the greatest responsiveness. In addition, in vitroexperiments demonstrated that graded doses of salmon MCH stimulate LH, but not GH, secretion from dispersed pituitary cells. Results suggest that hypothalamic MCH may participate in the steroid positive feedback loop on pituitary LH secretion.

Brain mapping of three somatostatin encoding genes in the goldfish

In the present study the brain distribution of three somatostatin (SRIF)‐encoding genes, PSS‐I, PSS‐II, and PSS‐III, was analyzed by in situ hybridization (ISH) in the goldfish. The PSS‐I mRNA showed the widest distribution throughout the brain, whereas PSS‐II transcripts were restricted to some hypothalamic nuclei. On the other hand, PSS‐III presents an intermediate distribution pattern. All SRIF encoding genes are expressed in hypophysiotropic nuclei supporting the idea that, in addition to SRIF‐14, [Pro2] SRIF‐14, and gSRIF‐28 have pituitary‐controlling functions. Moreover, each of the genes is expressed in nuclei directly associated with feeding behavior, suggesting a role for SRIF peptides in the central control of food intake and energy balance. Alternatively, they might have a role in processing sensory information related with feeding behavior, since PSS genes are expressed in the main gustatory, olfactory, and visual centers, which project to the hypothalamic feeding center in teleost fish. J. Comp. Neurol. 474:43–57, 2004.

Estradiol reduces pituitary responsiveness to somatostatin (SRIF-14) and down-regulates the expression of somatostatin sst2 receptors in female goldfish pituitary

Sex steroid hormones have been shown to regulate somatostatin (SRIF) gene expression in goldfish brain, which in turn influences the regulation of GH secretion. In this study, the influences of sex steroids on pituitary responsiveness to SRIF-14 and the pituitary expression of a type two SRIF receptor (sst(2)) were examined. Results from in vitro perifusion of pituitary fragments show that pituitaries from estradiol-primed sexually regressed female fish have significantly lower GH release responsiveness to pulse exposure to SRIF-14 than pituitaries from control or testosterone-treated sexually regressed females. Results from in vitro static culture show that pituitaries from sexually mature female fish have lower GH release responsiveness to SRIF-14 than those from sexually regressed females. In addition, the sst(2) receptor mRNA levels in pituitaries from mature and recrudescent female fish are significantly lower than in sexually regressed female fish. Our results indicate that estradiol acts at the level of the pituitary to regulate GH secretion by influencing the responsiveness to SRIF-14. The underlying mechanism includes, in part, reduction of the expression of sst(2) receptors, presumably leading to the lower number of the receptors available for SRIF binding.

Chapter 1 Neuroendocrine Systems of the Fish Brain

Publisher Summary This chapter discusses the neuroendocrine territories in fish. It describes the anatomy of the main neuroendocrine territories of the teleost brains. Teleost fish lack a canonical median eminence, and the hypothalamic neurons terminate very close to the adenohypophysial cells or make synaptoid contact upon them. This anatomical characteristic allows the study of the hypothalamo–hypophysial system by retrograde tracing experiments. Tract‐tracing techniques have corroborated early studies showing the preoptic area and tuberal hypothalamus as loci for the neuronal cell bodies whose axons reach the neuro‐ and adenohypophysis along well‐defined fiber tracts. The chapter also discusses several neuronal systems that produce hypothalamic releasing or inhibitory peptides and neurotransmitters, and innervate the pituitary. The hypothalamus–pituitary complex in teleost species shows a particular specialization in which the median eminence is greatly reduced or absent. As a consequence, the hypothalamic control of the pituitary is exerted by an important pituitary innervation that penetrates to the adenohypophysis as interdigitation of neuronal tissue. This neuronal circuitry integrates incoming information from both external and internal environments by expressing the appropriate set of hormonal receptors and through interneuronal communication.

Effects of cholecystokinin and bombesin on the expression of preprosomatostatin-encoding genes in goldfish forebrain

It was previously demonstrated that both cholecystokinin (CCK) and bombesin (BBS) stimulate growth hormone (GH) secretion in goldfish. Both peptides induce satiety and it was speculated that they integrate satiation and the postprandial increase in GH circulating levels. In the present paper we investigated the effects of CCK and BBS on the forebrain expression of the somatostatin gene family in goldfish to analyze if somatostatin peptides may be part of the effector mechanisms of CCK and BBS. We found that peripherally as well as centrally administered CCK decreases mRNA levels of preprosomatostatin (PSS)-I that encodes for SRIF-14, having no effects on PSS-II and PSS-III, which encode for gSRIF-28 and [Pro2] SRIF-14, respectively. In addition, a direct action on the pituitary to stimulate GH release, this inhibition of PSS-I expression provides a possible mechanism for CCK to increase postprandial GH levels. On the other hand, BBS inhibits the forebrain expression of PSS-I and PSS-II but does not affect PSS-III regardless of the route of administration. We conclude that this could be the most likely mechanism of action of BBS to increase GH secretion, since there are few BBS-immunoreactive (IR) fibers and BBS binding sites in the anterior pituitary of goldfish.

Effects of Sex Steroid Hormones on the Expression of Somatostatin Receptors sst1 and sst5 in Goldfish Pituitary and Forebrain

In the present paper the effects of estradiol and testosterone on the expression of the types 1 and 5 somatostatin receptors (sst1 and sst5) in the goldfish forebrain and pituitary were investigated. Estradiol increased the sst1 expression in both the forebrain and pituitary in a dose- and time-dependent manner. In addition, estradiol also increased the pituitary expression of sst5. On the other hand, testosterone had no effects on the expression of these receptor subtypes. Mature female goldfish were found to have higher sst1 and sst5 expression in the pituitary, as well as a higher expression of sst1 in the forebrain compared to sexually regressed animals. As estradiol treatment increases serum growth hormone levels in goldfish, these data suggest that sst1 and sst5 receptors are likely not directly involved in this aspect of growth hormone release.