Richard E. Peter

Role of leptin in the control of feeding of goldfish Carassius auratus: interactions with cholecystokinin, neuropeptide Y and orexin A, and modulation by fasting

To assess the role of leptin on food intake regulation in goldfish, we examined the effects of central (intracerebroventricular, ICV) and peripheral (intraperitoneal, IP) injections of recombinant murine leptin on feeding behavior. Centrally (100 ng/g) and peripherally (300 ng/g) injected leptin both caused a significant decrease in food intake, compared to the saline-treated controls. To test the hypothesis that leptin influenced orexigenic neuropeptide systems in goldfish, fish were co-injected with neuropeptide Y (NPY) or orexin A and leptin. Both NPY (5 ng/g) and orexin A (10 ng/g) significantly increased food intake. Fish co-injected ICV with NPY (5 ng/g) or orexin A (10 ng/g) and leptin (1 or 10 ng/g) had a food intake lower than that of fish treated with NPY or orexin A alone. NPY mRNA expression in goldfish brain was reduced 2 and 6 h following central injection of leptin. To test the hypothesis that the cholecystokinin (CCK) mediates the effects of leptin in goldfish, fish were simultaneously injected ICV with an ineffective dose of leptin (10 ng/g) and either ICV or IP with an ineffective doses of CCK (1 ng/g ICV or 25 ng/g IP). These fish had a food intake lower than vehicle-treated fish, suggesting that leptin potentiates the satiety actions of CCK. CCK hypothalamic mRNA expression was increased 2 h following central treatment with leptin. The CCK receptor antagonist proglumide blocked both central and peripheral CCK satiety effects. Blockade of CCK brain receptors by proglumide resulted in an inhibition of the leptin-induced decrease in food intake and an attenuation of the inhibiting action of leptin on both NPY- and orexin A-induced feeding. These data suggests that CCK has a role in mediating the effects of leptin on food intake. Fasting potentiated the actions of leptin and attenuated the effects of CCK. Whereas fasting had no effects on the brain mRNA expression of CCK, it increased the brain mRNA expression of NPY and decreased the expression of CART. These changes in neuropeptide expression were partially reversed when fish were treated ICV with leptin. These results provide strong evidence that, in goldfish, leptin influences food intake, in part by modulating the orexigenic effects of NPY and orexin and that its actions are mediated, at least in part, by CCK.

Differential distribution of two molecular forms of gonadotropin-releasing hormone in discrete brain areas of goldfish (Carassius auratus)

Two molecular forms of gonadotropin-releasing hormone (GnRH) were identified in the extracts of various brain areas, spinal cord and pituitary in female and male goldfish and had chromatographic and immunological properties similar to [His5, Trp7, Tyr8]-GnRH (cGnRH-II) and [Trp7,Leu8]-GnRH (sGnRH). Radioimmunoassay using different GnRH antisera after high pressure liquid chromatography did not reveal significant peaks of mammalian GnRH, [Gln8]-GnRH and [Tyr3,Leu5,Glu6,Trp7,Lys8]-GnRH in the brain extracts. The proportion of cGnRH-II-like immunoactivity to sGnRH-like immunoactivity was higher in the caudal brain areas compared to the rostral areas. The differential distribution of two GnRH forms suggest that the different GnRH forms may have different physiological functions.

Induced ovulation and spawning of cultured freshwater fish in China: Advances in application of GnRH analogues and dopamine antagonists

Abstract The Linpe method of induced ovulations and/or spawning of cultured fish consists of treatment with an analogue of gonadotropin-releasing hormone (GnRH) plus a dopamine antagonist. Initial experiments on common carp, silver carp, mud carp, bream, grass carp, bighead carp, black carp and loach confirm that ovulation can be induced in each of these species by injection of pimozide or domperidone plus [D-Ala 6 , Pro 9 NEt]-luteinizing hormone-releasing hormone (LHRH-A) or [D-Arg 6 , Trp 7 , Leu 8 , Pro 9 NEt-]-LHRH (sGnRH-A). Domperidone and sGnRH-A are preferred because of higher potencies in several species, allowing for their use at low dosages. For most species one set of simultaneous injections of the two drugs has proven sufficient to induce a high rate of ovulation within a predictable period of time. Ovulations are complete and ovulated eggs have been shown to be fertile. Hatching rates and fry survival are as high as high as with traditional methods of induced spawning. Subsequent reproductive cycles appear not to be altered by induced ovulation with the Linpe method. Although some refinement of dosages of drugs is required, the Linpe method has proven to be a highly successful procedure for induced ovulation and/or spawning of cultured freshwater fish in China.

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.

Properties of common carp gonadotropin I and gonadotropin II

Two gonadotropins, GtH I and GtH II, were extracted with 35% ethanol-10% ammonium acetate, pH 6.1, from female common carp pituitary glands and purified by ion-exchange chromatography on a DE-52 column followed by gel filtration on a Sephadex G-75 column. Molecular weights of GtH I and GtH II as determined by SDS-PAGE were 45,000 and 35,000, respectively. Both GtHs dissociate into two subunits following reduction with beta-mercaptoethanol. These subunits contain different N-terminal amino acids (Tyr and Gly for GtH I; Tyr and Ser for GtH II). GtH I was acid stable and did not dissociate into subunits following treatment with 0.1% trifluoroacetic acid; GtH II readily dissociated into subunits by this treatment. GtH I and GtH II have distinct elution profiles on reverse-phase HPLC. The N-terminal amino acid sequence of the beta-subunit of GtH II was identical to that of common carp maturational GtH described by other workers suggesting that GtH I is a newly identified molecule. This was supported by radioimmunoassay analysis. GtH II and a common carp maturational GtH preparation (F11 cGtH; Peter et al., 1982, J. Interdiscipl. Cycle Res. 13, 229-239) had similar immunological activity in tests with antisera to the beta-subunit of maturational GtH whereas GtH I had low (less than 6%) cross-reactivity. GtH I, GtH II, and F11 cGtH were equipotent in tests with antisera to the alpha-subunit of maturational GtH suggesting these molecules contain a similar alpha-subunit. In vitro bioassays using goldfish revealed that GtH I and GtH II share the same spectrum of biological activities causing stimulation of ovarian and testicular steroidogenesis and induction of oocyte final maturation. The demonstration of two chemically distinct GtHs in common carp is similar to what has been described for chum and coho salmon.

Ghrelin : A multifunctional hormone in non-mammalian vertebrates

In mammals, ghrelin is a non-amidated peptide hormone, existing in both acylated and non-acylated forms, produced mainly from the X/A or ghrelin cells present in the mucosal layer of the stomach. Ghrelin is a natural ligand of the growth hormone (GH) secretagogue-receptor (GHS-R), and functions primarily as a GH-releasing hormone and an orexigen, as well as having several other biological actions. Among non-mammalian vertebrates, amino acid sequence of ghrelin has been reported in two species of cartilaginous fish, seven species of teleosts, two species of amphibians, one species of reptile and six species of birds. The structure and functions of ghrelin are highly conserved among vertebrates. This review presents a concise overview of ghrelin biology in non-mammalian vertebrates.

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.

A reinvestigation of the Gn-RH (gonadotrophin-releasing hormone) systems in the goldfish brain using antibodies to salmon Gn-RH

SummaryThe organization of Gn-RH systems in the brain of teleosts has been investigated previously by immunohistochemistry using antibodies against the mammalian decapeptide which differs from the teleostean factor. Here, we report the distribution of immunoreactive Gn-RH in the brain of goldfish using antibodies against synthetic teleost peptide.Immunoreactive structures are found along a column extending from the rostral olfactory bulbs to the pituitary stalk. Cell bodies are observed within the olfactory nerves and bulbs, along the ventromedial telencephalon, the ventrolateral preoptic area and the latero-basal hypothalamus. Large perikarya are detected in the dorsal midbrain tegmentum, immediately caudal to the posterior commissure. A prominent pathway was traced from the cells located in the olfactory nerves through the medial olfactory tract and along all the perikarya described above to the pituitary stalk. In the pituitary, projections are restricted to the proximal pars distalis. A second immunoreactive pathway ascends more dorsally in the telencephalon and arches to the periventricular regions of the diencephalon. Part of this pathway forms a periventricular network in the dorsal and posterior hypothalamus, whereas other projections continue caudally to the medulla oblongata and the spinal cord. Lesions of the ventral preoptic area demonstrate that most of the fibers detected in the pituitary originate from the preoptic region.

Use of a pituitary cell dispersion method and primary culture system for the studies of gonadotropin-releasing hormone action in the goldfish, Carassius auratus. I. Initial morphological, static, and cell column perifusion studies.

Two cell dispersion methods for excised goldfish pituitary glands were tested, and a cultured dispersed cell system based on trypsin enzymatic tissue digestion was developed and characterized. Controlled trypsin/DNase treatment of goldfish pituitary gland yielded dispersed cells of high viability (trypsin blue exclusion test) that responded to gonadotropin (GTH)-releasing hormone (GnRH) challenges with GTH secretion in a time- and dose-dependent manner following overnight culture. Electron microscopy revealed that cell preparations produced by the trypsin dispersion were free of cell debris and nerve terminals. The dispersed pituitary cells also retained distinct morphological and immunological identities. Under static incubation conditions, 2-hr treatments with 0.1 nM to 1 microM [Trp7,Leu8]-GnRH (sGnRH) and [D-Arg6,Pro9-N-ethylamide]-sGnRH (sGnRHa) stimulated GTH release with similar efficacy, but with ED50S of 1.92 +/- 0.48 and 0.19 +/- 0.08 nM, respectively. [His5,Trp7,Tyr8]-GnRH (cGnRH-II) stimulated GTH release in a nonsigmoidal, but dose-dependent manner, and with a higher efficacy than sGnRH. In contrast, sGnRH, sGnRHa, and cGnRH-II were equipotent in inducing growth hormone (GH) secretion in static culture studies and with ED50S of 0.29 +/- 0.13, 0.18 +/- 0.11, and 0.19 +/- 0.17 nM, respectively. When trypsin/DNase-dispersed cells cultured overnight with cytodex beads were tested in a cell column perifusion system, dose-related increase in GTH secretion, as well as GH release, were also observed with 0.5 to 50 nM sGnRH. These results suggest that trypsin-dispersed goldfish pituitary cells can be used effectively to study the actions of GnRH on teleost pituitary either in short-term static incubation or column perifusion studies. Differences in the GTH and GH responses to the two native GnRH forms, sGnRH and cGnRH-II, are also indicated.

Effects of Dopamine on Gonadotropin Release in Female Goldfish, Carassius auratus

Intraperitoneal injections of dopamine (DA) or its agonist, apomorphine decreased and pimozide, a DA antagonist, increased serum gonadotropin (GtH) levels in normal female goldfish. Injection of DA into the third cranial ventricular did not alter serum GtH concentrations. Intraperitoneal injections of DA or apomorphine each reduced the highly elevated serum GtH levels caused by preoptic lesions which abolish an inhibitory hypothalamic influence on GtH release allowing prolonged spontaneous release of GtH. Intraperitoneal injections of DA or apomorphine blocked the stimulation of GtH release induced by injections of a luteinizing hormone-releasing hormone analogue. These results indicate that DA has GtH release-inhibitory activity by actions directly on gonadotrophs to inhibit spontaneous secretion of GtH, and by blocking the actions of the GtH-releasing hormone.