Action of leptin on in vitro luteinizing hormone release in the European sea bass (Dicentrarchus labrax).

The discovery of leptin has sparked a rapidly growing number of publications concerning the role of leptin in the regulation of body adiposity, feeding, and reproductive system in mammals. To date, there have been no reports on the presence of leptin-related peptide, and functional studies on the role of leptin remain limited in fishes. We investigated the effect of mouse recombinant leptin on basal and sea bream (sb) GnRH-induced LH release from dispersed pituitary cells obtained from male European sea bass (Dicentrarchus labrax) at different stages of sexual development. The potential interaction of leptin with the porcine neuropeptide Y (pNPY), known to play a dual role in feeding and reproduction in vertebrates, was also investigated. High doses of leptin (10(-8)-10(-6) M) and/or pNPY (0.1 and 1 nM) had different effects on LH release at various stages of sexual development. Porcine NPY alone was weakly effective on basal LH release, but it enhanced LH release induced by leptin (10(-6) M) in late prepuberty but not in early postpuberty. Additive or inhibitory effects of leptin were observed on sbGnRH-induced LH release depending on sbGnRH dose and stage of sexual development. The direct action of leptin on LH release at the pituitary level in sea bass suggests that leptin is a regulator of the reproductive system in fishes.

Brain regulation of feeding behavior and food intake in fish.

In mammals, the orexigenic and anorexigenic neuronal systems are morphologically and functionally connected, forming an interconnected network in the hypothalamus to govern food intake and body weight. However, there are relatively few studies on the brain control of feeding behavior in fish. Recent studies using mammalian neuropeptides or fish homologs of mammalian neuropeptides indicate that brain orexigenic signal molecules include neuropeptide Y, orexins, galanin and beta-endorphin, whereas brain anorexigenic signal molecules include cholecystokinin, bombesin, corticotropin-releasing factor, cocaine- and amphetamine-regulated transcript, and serotonin. Tachykinins may also have an anorectic action in fish. The brain hypothalamic area is associated with regulation of food intake, while sites outside the hypothalamus are also involved in this function. There is correlation between short-term changes in serum growth hormone levels and feeding behavior, although possible mechanisms integrating these functions remain to be defined.

Regulation of food intake by neuropeptide Y in goldfish.

In mammals, neuropeptide Y (NPY) is a potent orexigenic factor. In the present study, third brain ventricle (intracerebroventricular) injection of goldfish NPY (gNPY) caused a dose-dependent increase in food intake in goldfish, and intracerebroventricular administration of NPY Y1-receptor antagonist BIBP-3226 decreased food intake; the actions of gNPY were blocked by simultaneous injection of BIBP-3226. Goldfish maintained on a daily scheduled feeding regimen display an increase in NPY mRNA levels in the telencephalon-preoptic area and hypothalamus shortly before feeding; however, a decrease occured in optic tectum-thalamus. In both fed and unfed fish, brain NPY mRNA levels decreased after scheduled feeding. Restriction in daily food ration intake for 1 wk or food deprivation for 72 h resulted in increased brain NPY mRNA levels. Results from these studies demonstrate that NPY is a physiological brain signal involved in feeding behavior in goldfish, mediating its effects, at least in part, through Y1-like receptors in the brain.

Molecular cloning and expression of cDNA encoding a brain bombesin/gastrin-releasing peptide-like peptide in goldfish.

A complementary DNA (cDNA) of 928 bp encoding a bombesin (BBS)/gastrin-releasing peptide (GRP) precursor was identified from goldfish brain. Goldfish BBS/GRP messenger RNA (mRNA) encodes a 157 amino acid precursor, which contains a signal peptide sequence, the 22 amino acid putative BBS/GRP-like peptide, and a carboxy-terminal extension peptide. Reverse transcription-polymerase chain reaction (PCR) (RT-PCR) demonstrated that the mRNA for this precursor has a widespread distribution in goldfish brain, and is also present in skin, gastrointestinal tract, gonad, and gill. Phylogenetic analysis of BBS/GRP-like peptide precursors in vertebrates shows that goldfish BBS/GRP is more closely related to the known GRP precursors than to BBS precursors.

Preprotachykinin gene expression in goldfish brain: sexual, seasonal, and postprandial variations.

Recently, we described the complete nucleotide sequence of gamma-preprotachykinin (gamma-PPT) mRNA and the deduced amino acid sequence of the precursor on the basis of molecular cloning and sequence analysis of cDNA from goldfish brain. In the present study, gamma-PPT gene expression in the brain of goldfish was examined using quantitative Northern blot analysis. The results showed that the gamma-PPT gene is highly but differentially expressed in the olfactory bulbs, hypothalamus, and posterior brain regions. There are sexual dimorphism and seasonal variations in gamma-PPT gene expression. In addition, the postprandial changes in gamma-PPT gene expression in the olfactory bulbs and hypothalamus suggest that tachykinin peptides are involved in regulation of feeding behavior in goldfish.

Postprandial, seasonal and sexual variations in cholecystokinin gene expression in goldfish brain.

Recently, we described the complete nucleotide sequence of cholecystokinin (CCK) mRNA and the deduced amino acid sequence of the precursor on the basis of molecular cloning and sequence analysis of cDNA from goldfish brain. In this study, we investigated the hypothesis that CCK has a role in feeding behavior by examining CCK gene expression in the brain of goldfish using Northern blot. We showed that CCK gene is widely but differentially expressed in broad areas of the goldfish brain, including the olfactory bulbs, telencephalon and preoptic region, hypothalamus, optic tectum-thalamus and posterior brain regions, with highest levels in hypothalamus. We found that CCK mRNA levels in goldfish olfactory bulbs, telencephalon-preoptic region, optic tectum-thalamus, and posterior brain were influenced by sex at least sometime of the seasonal gonadal cycle, with female fish having higher levels than males during at least one of the four seasonal sampling times. We also observed a transient and acute increase in the CCK mRNA levels in the olfactory bulbs, telencephalon-preoptic region, hypothalamus, and posterior brain at 120 min after a meal. These widespread postprandial changes in CCK gene expression in goldfish brain indicate that CCK peptides have multiple roles in regulation of feeding behavior in goldfish. This supports the idea that CCK plays a role as a satiety factor in goldfish.

Molecular cloning and expression of cDNA encoding brain preprocholecystokinin in goldfish.

A cholecystokinin (CCK) precursor cDNA of 782 bp was identified from goldfish brain. The open reading frame (369 bp) encodes the 123 amino acid precursor which contains mono- and di-basic amino acid endoproteolytic cleavage, C-terminal alpha-amidation and tyrosyl sulfation sites. Expression studies revealed the presence of preproCCK mRNA in the gastrointestinal tract, pituitary and a wide range of brain areas from the olfactory bulbs to the posterior brain region. We have also confirmed the presence of CCK mRNA in the posterior ventrolateral hypothalamus by in situ hybridization, supporting a role of CCK in feeding behavior and regulation of pituitary hormone secretion.

Metabolic studies on eel (Anguilla anguilla L.) hepatocytes in primary culture: effect of 17 beta-estradiol and growth hormone.

Previous studies demonstrated that native and recombinant growth hormone from mammalian and fish species potentiate the estrogenic induction of vitellogenin synthesis by cultured eel hepatocytes. In the present study, the metabolic competence (respiratory activity and estradiol catabolism) of cultured hepatocytes and their functional capacity to synthesize a specific protein, vitellogenin, in the presence of estradiol and/or bovine growth hormone was investigated. In addition, we examined the possible role of insulin-like growth factors as mediators of growth hormone. Hepatocytes retain a high level of metabolic activity under the primary culture conditions applied. Estradiol has a half life of several hours in the hepatocyte culture, and is metabolized into conjugated forms. Estradiol and/or growth hormone had no effects on respiratory activity of the cultured hepatocytes. Moreover, the estradiol catabolic parameters were not affected by growth hormone. Finally, human and trout recombinant insulin-like growth factors do not potentiate vitellogenin synthesis induced by estradiol.

Potentiating effect of growth hormone on vitellogenin synthesis induced by 17 beta-estradiol in primary culture of female silver eel (Anguilla anguilla L.) hepatocytes.

Previous in vivo experiments have indicated a potentiating role of growth hormone (GH) during experimentally induced vitellogenesis by 17-beta-estradiol (E2) in the female silver eel (Anguilla anguilla L.). To investigate whether GH has direct hepatic actions, the effects of hypophysial-purified and recombinant QH on vitellogenin (Vg) synthesis in response to E2 were tested on primary cultures of hepatocytes. Hepatocytes were prepared from control or E2-primed eels. Addition of E2 alone into the culture medium induced both Vg synthesis and secretion in a dose- and time-related fashion. Bovine growth hormone (bGH) alone had no effect on the induction of Vg synthesis or secretion. Bovine GH enhanced the in vitro effects of F2 on both Vg synthesis and secretion, an effect attenuated by an in vivo E2 priming which was dose-dependent with an ED(50) of 5 ng/ml. To investigate the specificity of GH action, purified eel and salmon GH and salmon, trout, and tilapia prolactins (PRL), as well as recombinant trout and tilapia GH, were tested, and the responses were compared to bGH. Purified salmon and homologous eel GH potentiated the vitellogenic response to F2. Recombinant GH were highly efficacious, excluding the presence of active contaminants in the potentiating effect of GH preparations. The potentiating effect of recombinant trout GH on the vitellogenic response was reduced at high doses (above 20 ng/ml), suggesting a down-regulation of GH binding sites by GH itself. Salmon PRL has minimal activity, but not trout and tilapia PRL, indicating that PRL is not an important potentiating factor on Vg synthesis in our model. It is concluded that GH acts directly on the liver to potentiate E2 induction of eel hepatic Vg synthesis. The potentiating effect of GH appears to be time- and dose-dependent and modulated as a function of hormonal status (E2 priming) of the eel.

Synthesis of vitellogenin by eel (Anguilla anguilla L.) hepatocytes in primary culture: requirement of 17 beta-estradiol-priming.

Control and 17 beta-estradiol-primed eels were used to investigate the hormonal requirement of vitellogenesis in an immature fish, the eel. A primary culture of isolated liver cells from female silver eels was developed. The hepatocytes were maintained as monolayers on poly-L-lysine coated dishes for up to 12 days in a defined medium alone or supplemented with 17 beta-estradiol (E2, from 10(-8) to 10(-5) M). The amounts of vitellogenin (Vg) in the cells and secreted into the medium were measured at 2-day intervals using a homologous vitellogenin ELISA. Different E2-priming conditions were determined before hepatocyte isolation (one injection of 250 micrograms of E2 21 days, 17 days, or 24 hr). The vitellogenic response of hepatocytes to E2 stimulation was studied in relation to the duration of the E2-priming. After 8 days of culture, when hepatocytes from control eels were used, Vg was undetectable both in cells and in culture media, even if the culture was performed in the presence of E2 10(-5) M. However, Vg was detectable both in cells and in culture media of hepatocytes from E2-primed eels. If the priming was performed 24 hr before the culture, the Vg synthesis significantly increased (P < 0.001) in the presence of E2 10(-5) M after 10 days of culture but remained low. When the culture was performed 17 or 21 days after the priming, the level of the vitellogenic response was higher than after a short priming. In particular, with hepatocytes from 21-day E2-primed eel, the concentration of secreted Vg was 1.5 times higher than in control dishes (P < 0.01), in the presence of E2 10(-8) M after 12 days of culture. Higher doses of E2 (10(-5) M) increased Vg 2.7-fold over control values (P < 0.01) after 4 days of culture. In control dishes, cultured without steroid, the amounts of secreted and intracellular Vg remained unchanged over 12 days of culture (respectively, 72.8 +/- 2.7 ng/10(6) cells/48 hr and 28.7 +/- 2.7 ng/10(6) cells). These results show that cultured hepatocytes retain their functional capacity by synthesizing a specific protein, Vg, in the presence of E2 and there are dose- and time-related effects of E2 on in vitro Vg synthesis. The induction of hepatic vitellogenesis in vitro requires a preliminary in vivo E2-priming.