Nesfatin-1 stimulates the hypothalamus-pituitary-interrenal axis hormones in goldfish.

Stress in vertebrates is mediated by the hypothalamus-pituitary-adrenal (in mammals)/interrenal (in fish) (HPA/I) axis, which produces the corticotropin-releasing factor (CRF), adrenocorticotropic hormone (ACTH), and corticosteroids, respectively. Nesfatin-1, a novel anorexigenic peptide encoded in the precursor nucleobindin-2 (NUCB2), is increasingly acknowledged as a peptide that influences the stress axis in mammals. The primary aim of this study was to characterize the putative effects of nesfatin-1 on the fish HPI axis, using goldfish (Carassius auratus) as an animal model. Our results demonstrated that nucb2/nesfatin-1 transcript abundance was detected in the HPI tissues of goldfish, with most abundant expression in the pituitary. NUCB2/nesfatin-1-like immunoreactivity was found in the goldfish hypothalamus, pituitary, and interrenal cells of the head kidney. GPCR12, a putative receptor for nesfatin-1, was also detected in the pituitary and interrenal cells. NUCB2/nesfatin-1-like immunoreactivity was observed in ACTH-expressing pituitary corticotrophs. Acute netting and restraint stress upregulated nucb2/nesfatin-1 mRNA levels in the forebrain, hypothalamus, and pituitary, as well as crf and crf-r1 expression in the forebrain and hypothalamus. Intraperitoneal and intracerebroventricular administration of nesfatin-1 increased cortisol release and hypothalamic crf mRNA levels, respectively. Finally, we found that nesfatin-1 significantly stimulated ACTH secretion from dispersed pituitary cells in vitro. Collectively, our data provide the first evidence showing that nesfatin-1 is a stress responsive peptide, which modulates the stress axis hormones in fish.

Nesfatin-1 regulates the hypothalamo-pituitary-ovarian axis of fish.

Nesfatin-1 is an anorexigen in goldfish. In the present study, we provide novel data indicating the presence and regulatory effects of nesfatin-1 on the hypothalamo-pituitary-ovarian (HPO) axis of goldfish. Nucleobindin-2 (NUCB2)/nesfatin-1-like immunoreactive (ir) cells are present in the hypothalamus and in the pituitary, suggesting a hypophysiotropic role for nesfatin-1. NUCB2/nesfatin-1-like ir cells colocalize gonadotropin-releasing hormone (GnRH) in the nucleus lateralis tuberis posterioris and the nucleus anterior tuberis of the goldfish hypothalamus. The presence of nesfatin-1 with GnRH in these two nuclei implicated in pituitary hormone release suggests a role for nesfatin-1 on gonadotropin secretion. A single i.p. injection of synthetic goldfish nesfatin-1 (50 ng/g body wt) resulted in an acute decrease (∼75%) in the expression of hypothalamic chicken GnRH-II and salmon GnRH mRNAs at 15 min postinjection in goldfish. Meanwhile, pituitary luteinizing hormone (LH) beta and follicle-stimulating hormone beta mRNAs were also inhibited (∼80%), but only at 60 min postinjection. Nesfatin-1 administration also resulted in a significant reduction (∼60%) in serum LH levels at 60 min postadministration. Nesfatin-1-like immunoreactivity was also found in the follicle cells, but not the oocytes, in zebrafish and goldfish ovaries. Incubation of zebrafish follicles with nesfatin-1 resulted in a significant reduction in basal germinal vesicle breakdown (∼50%) during the oocyte maturation. In addition, nesfatin-1 also attenuated the stimulatory effects of maturation-inducing hormone on germinal vesicle breakdown. Together, the current results indicate that nesfatin-1 is a metabolic hormone with an inhibitory tone on fish reproduction. Nesfatin-1 appears to elicit this suppressive effect through actions on all three tissues in the fish HPO axis.

Calcium and other signalling pathways in neuroendocrine regulation of somatotroph functions.

Relative to mammals, the neuroendocrine control of pituitary growth hormone (GH) secretion and synthesis in teleost fish involves numerous stimulatory and inhibitory regulators, many of which are delivered to the somatotrophs via direct innervation. Among teleosts, how multifactorial regulation of somatotroph functions are mediated at the level of post-receptor signalling is best characterized in goldfish. Supplemented with recent findings, this review focuses on the known intracellular signal transduction mechanisms mediating the ligand- and function-specific actions in multifactorial control of GH release and synthesis, as well as basal GH secretion, in goldfish somatotrophs. These include membrane voltage-sensitive ion channels, Na(+)/H(+) antiport, Ca(2+) signalling, multiple pharmacologically distinct intracellular Ca(2+) stores, cAMP/PKA, PKC, nitric oxide, cGMP, MEK/ERK and PI3K. Signalling pathways mediating the major neuroendocrine regulators of mammalian somatotrophs, as well as those in other major teleost study model systems are also briefly highlighted. Interestingly, unlike mammals, spontaneous action potential firings are not observed in goldfish somatotrophs in culture. Furthermore, three goldfish brain somatostatin forms directly affect pituitary GH secretion via ligand-specific actions on membrane ion channels and intracellular Ca(2+) levels, as well as exert isoform-specific action on basal and stimulated GH mRNA expression, suggesting the importance of somatostatins other than somatostatin-14.

Fluoxetine affects weight gain and expression of feeding peptides in the female goldfish brain.

Serotonin has been implicated in the regulation of feeding and growth in vertebrates. However, the mechanisms through which serotonin mediates its anorectic effects are only partially understood. In this study we measured food intake and difference in weight gain in sexually regressed female goldfish intraperitionally injected with fluoxetine, a selective serotonin reuptake inhibitor (SSRI). The experiment was conducted in July, a period in which female goldfish show maximum body growth rates. After an acclimation period of one week, goldfish were injected every 3 d with 5 microg/g body weight fluoxetine for 13 d. Fluoxetine injections resulted in a significant decrease in food intake, as well as a significant decrease in weight gain. To investigate potential mechanisms, neuropeptide gene expression in the hypothalamus and telencephalon was determined using real-time RT-PCR. We found a 2.3-fold up-regulation of both CRF1 (p<0.03) and NPY mRNAs (p<0.04) in the hypothalamus. In the telencephalon there was a 2.3-fold decrease (p<0.02) of NPY mRNA and a 3.2-fold increase (p<0.02) in CART-1 mRNA. No changes in tachykinin mRNA were observed in either hypothalamus or telencephalon. In contrast, brain somatostatin-2 and serum GH levels were unaffected by fluoxetine. These results indicate that alteration of central serotoninergic tone reduces food intake and weight gain and increases the expression of potent inhibitory feeding neuropeptides. However, expression of the orexigenic neuropeptide NPY was increased in the hypothalamus. The results are discussed in the context of fluoxetine as a pharmaceutical of concern in the aquatic environment.

Endogenous hypothalamic somatostatins differentially regulate growth hormone secretion from goldfish pituitary somatotropes in vitro.

Using Southern blot analysis of RT-PCR products, mRNA for three different somatostatin (SS) precursors (PSS-I, -II, and -III), which encode for SS(14), goldfish brain (gb)SS(28), and [Pro(2)]SS(14), respectively, were detected in goldfish hypothalamus. PSS-I and -II mRNA, but not PSS-III mRNA, were also detected in cultured pituitary cells. We subsequently examined the effects of the mature peptides, SS(14), gbSS(28), and [Pro(2)]SS(14), on somatotrope signaling and GH secretion. The gbSS(28) was more potent than either SS(14) or [Pro(2)]SS(14) in reducing basal GH release but was the least effective in reducing basal cellular cAMP. The ability of SS(14), [Pro(2)]SS(14), and gbSS(28) to attenuate GH responses to GnRH were comparable. However, gbSS(28) was less effective than SS(14) and [Pro(2)]SS(14) in diminishing dopamine- and pituitary adenylate cyclase-activating polypeptide-stimulated GH release, as well as GH release resulting from the activation of their underlying signaling cascades. In contrast, the actions of a different 28-amino-acid SS, mammalian SS(28), were more similar to those of SS(14) and [Pro(2)]SS(14). We conclude that, in goldfish, SSs differentially couple to the intracellular cascades regulating GH secretion from pituitary somatotropes. This raises the possibility that such differences may allow for the selective regulation of various aspects of somatotrope function by different SS peptides.