Xinwei Lin

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.

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.

Evolution of neuroendocrine peptide systems: gonadotropin-releasing hormone and somatostatin.

Nine vertebrate and two protochordate gonadotropin-releasing hormone (GnRH) decapeptides have been identified and sequenced. Multiple molecular forms of GnRH peptide were present in the brain of most species examined, and cGnRH-II generally coexists with one or more GnRH forms in all the major vertebrate groups. The presence of multiple GnRH forms has been further confirmed by the deduced GnRH peptide structure from cDNA and/or gene sequences in several teleost species and tree shrew. High conservation of the primary structure of GnRH decapeptides and the overall structure of GnRH genes and precursors suggests that they are derived from a common ancestor. Somatostatin (SRIF) is a phylogenetically ancient, multigene family of peptides. A tetradecapeptide, SRIF (SRIF14) has been conserved, with the same amino acid sequence, in representative species of all classes of vertebrate. Four molecular variants of SRIF14 have been identified. SRIF14 is processed from preprosomatostatin-I, which contains SRIF14 at its C-terminus; preprosomatostatin-I is also processed to SRIF28 in mammals and SRIF26 in bowfin. Teleost fish possess a second somatostatin precursor, preprosomatostatin-II, containing [Tyr7, Gly10]-SRIF14 at the C-terminus, that is mainly processed into large forms of SRIF.

Somatostatins and their receptors in fish.

Somatostatin (SRIF) is a multigene family of peptides. SRIF-14 is conserved with identical primary structure in species across the vertebrates. The presence of multiple SRIF genes has been demonstrated in a number of fish species. Notably, three distinct SRIF genes have been identified in goldfish. One of these genes, which encodes [Pro(2)]SRIF-14, has also been identified in sturgeon and African lungfish, and is closely associated with the amphibian [Pro(2),Met(13)]SRIF-14 gene and mammalian cortistatin gene. The main neuroendocrine role of SRIF-14 peptide that has been determined in fish is the inhibition of pituitary growth hormone secretion. The functions of SRIF-14 variant or larger forms of SRIF peptide and the regulation of SRIF gene expression remain to be explored. Type one and two SRIF receptors have been identified from goldfish and type three SRIF receptor from an electric fish. Fish SRIF receptors display considerable homology to mammalian counterparts in terms of primary structure and negative coupling to adenylate cyclase. The identification of the multiple gene family of SRIF peptides and multiple types of SRIF receptors in fish opens a new avenue for the study of physiological roles of SRIF, and the molecular and cellular mechanisms of SRIF actions in fish.

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.

Molecular cloning and expression of two type one somatostatin receptors in goldfish brain

Somatostatin (SRIF or SS) exerts diverse inhibitory actions through binding to specific receptors. In this study, two SRIF receptor complementary DNAs (cDNAs) were cloned and sequenced from goldfish brain using PCR and cDNA library screening. The two cDNAs share 92% similarity in nucleotide sequence and 98% similarity in the deduced amino acid sequences and are presumably derived from duplicate genes, as goldfish are tetraploid. Two cDNAs encode two 367-amino acid goldfish type one SRIF receptors (designated as sst1A and sst1B, respectively), with seven putative transmembrane domains (TMD) and YANSCANP motif in the 7th TMD, a signature sequence for mammalian SRIF receptor (sst) family. In addition, the amino acid sequences of two receptors have 76% and 75% similarity to human or rat sst1, respectively, and 39-55% similarities to other mammalian sst subtypes (sst2-5), suggesting that the two receptors could be the goldfish homologs of mammalian sst1. The difference between goldfish and mammalian sst1 is mainly reflected by the extreme divergence in their extracellular N termini. Both SRIF-14 and [Pro2]SRIF-14, two of the native goldfish SRIF forms, significantly inhibited forskolin-stimulated cAMP release in COS-7 cells transiently expressing goldfish sSt1A or sst1B, suggesting functional coupling of the two receptors to adenylate cyclase. Northern blot and RT-PCR showed that messenger RNAs (mRNAs) for both receptors are widely distributed throughout goldfish brain, whereas only one receptor mRNA is expressed in the pituitary. RT-PCR analysis also detected sst1 receptor mRNAs in several peripheral tissues. These findings provide fundamental information for studying the mechanism of SRIF actions in vertebrates and structural analysis of mammalian sst receptors.

Somatostatin-like receptors in goldfish: cloning of four new receptors.

In this study, four somatostatin-like receptor (Sst) cDNAs were identified from goldfish pituitary, using RT-PCR screening and rapid amplification of cDNA ends (RACE) strategies. These include two type-five like Sst (Sst(5B) and Sst(5C)) and two type-three like Sst receptors (Sst(3A) and Sst(3B)), designated based on their amino acid sequence similarities to the known mammalian and fish Sst(5) and Sst(3). Both Sst(5C) and Sst(3A) mRNAs are widely expressed in all brain regions and pituitary; however, Sst(3B) expression is restricted to forebrain and Sst(5B) expression is mainly detected in pituitary and spinal cord.

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.

Goldfish γ -Preprotachykinin mRNA Encodes the Neuropeptides Substance P, Carassin, and Neurokinin A 1

Abstract Lin, X.-W. and R. E. Peter. Goldfish γ -preprotachykinin mRna encodes the neuropeptides substance P, carassin, and neurokinin A. Peptides 18(6) 817–824, 1997.—Two cDNAs, size 969 bp and 1146 bp respectively, encoding goldfish γ -preprotachykinin ( γ -PPT) were identified. Both cDNAs contain the same 345 bp open reading frame. The deduced 114-amino acid γ -PPT contains the sequence of substance P, carassin and neurokinin A. Sequence analysis of the two cDNA 5′-untranslated regions shows that the two cDNAs may represent different PPT-A gene transcripts resulting from the alternative transcriptional start sites. Expression of γ -PPT mRNA was detected in a wide range of brain areas from the olfactory bulbs to the posterior brain region, as well as in the intestine, testis and pituitary.