Control of leptin by metabolic state and its regulatory interactions with pituitary growth hormone and hepatic growth hormone receptors and insulin like growth factors in the tilapia (Oreochromis mossambicus).

Leptin is an important cytokine for regulating energy homeostasis, however, relatively little is known about its function and control in teleost fishes or other ectotherms, particularly with regard to interactions with the growth hormone (GH)/insulin-like growth factors (IGFs) growth regulatory axis. Here we assessed the regulation of LepA, the dominant paralog in tilapia (Oreochromis mossambicus) and other teleosts under altered nutritional state, and evaluated how LepA might alter pituitary growth hormone (GH) and hepatic insulin-like growth factors (IGFs) that are known to be disparately regulated by metabolic state. Circulating LepA, and lepa and lepr gene expression increased after 3-weeks fasting and declined to control levels 10days following refeeding. This pattern of leptin regulation by metabolic state is similar to that previously observed for pituitary GH and opposite that of hepatic GHR and/or IGF dynamics in tilapia and other fishes. We therefore evaluated if LepA might differentially regulate pituitary GH, and hepatic GH receptors (GHRs) and IGFs. Recombinant tilapia LepA (rtLepA) increased hepatic gene expression of igf-1, igf-2, ghr-1, and ghr-2 from isolated hepatocytes following 24h incubation. Intraperitoneal rtLepA injection, on the other hand, stimulated hepatic igf-1, but had little effect on hepatic igf-2, ghr1, or ghr2 mRNA abundance. LepA suppressed GH accumulation and gh mRNA in pituitaries in vitro, but had no effect on GH release. We next sought to test if abolition of pituitary GH via hypophysectomy (Hx) affects the expression of hepatic lepa and lepr. Hypophysectomy significantly increases hepatic lepa mRNA abundance, while GH replacement in Hx fish restores lepa mRNA levels to that of sham controls. Leptin receptor (lepr) mRNA was unchanged by Hx. In in vitro hepatocyte incubations, GH inhibits lepa and lepr mRNA expression at low concentrations, while higher concentration stimulates lepa expression. Taken together, these findings indicate LepA gene expression and secretion increases with fasting, consistent with the hormones function in promoting energy expenditure during catabolic stress. It would also appear that LepA might play an important role in stimulating GHR and IGFs to potentially spare declines in these factors during catabolism. Evidence also suggests for the first time in teleosts that GH may exert important regulatory effects on hepatic LepA production, insofar as physiological levels (0.05-1 nM) suppresse lepa mRNA accumulation. Leptin A, may in turn exert negative feedback effects on basal GH mRNA abundance, but not secretion.

Phylogenetic Analysis of Molluscan Metallothioneins: Evolutionary Insight from Crassostrea virginica.

Mechanisms by which organisms genetically adapt to environmental conditions are of fundamental importance to studies of evolutionary biology and environmental physiology. Natural selection acts on existing genetic variation leading to adaptation through selection of new mutations that confer beneficial advantages to populations. The American oyster, Crassostrea virginica, is an excellent model to investigate interactions between environmental and ecological factors as driving forces for natural selection. A great example of this is represented by the diversity of C. virginica metallothioneins (CvMT), metal-binding proteins involved in homeostasis and tolerance, that have resulted from a series of duplication events to produce the greatest structural diversity of MT proteins found in a single species. We present phylogenetic evidence of two distinct ancestral ß-domain MTs that gave rise to a variety of ßß and αß CvMT proteins, as well as CvMT-II proteins consisting solely of one to four α-domains. Furthermore, we annotate the complete locus containing the paralogous CvMT-I, -II, and -IV genes, providing supporting evidence of a hypothesized series of exon and gene duplication events that gave rise to the various CvMT-I and -II isoforms. We also highlight unique MT expression profiles from four separate C. virginica populations to demonstrate differences in gene diversity and copy number which appear to be enriched in southeastern U.S. oyster populations. These observations contribute to a better understanding of the molecular mechanisms leading to adaptation in organisms that experience substantial environmental stress, with a specific focus on evolutionary adaptations of gene structure.

Lrp13 is a novel vertebrate lipoprotein receptor that binds vitellogenins in teleost fishes.

Transcripts encoding a novel member of the lipoprotein receptor superfamily, termed LDL receptor-related protein (Lrp)13, were sequenced from striped bass (Morone saxatilis) and white perch (Morone americana) ovaries. Receptor proteins were purified from perch ovary membranes by protein-affinity chromatography employing an immobilized mixture of vitellogenins Aa and Ab. RT-PCR revealed lrp13 to be predominantly expressed in striped bass ovary, and in situ hybridization detected lrp13 transcripts in the ooplasm of early secondary growth oocytes. Quantitative RT-PCR confirmed peak lrp13 expression in the ovary during early secondary growth. Quantitative mass spectrometry revealed peak Lrp13 protein levels in striped bass ovary during late-vitellogenesis, and immunohistochemistry localized Lrp13 to the oolemma and zona radiata of vitellogenic oocytes. Previously unreported orthologs of lrp13 were identified in genome sequences of fishes, chicken (Gallus gallus), mouse (Mus musculus), and dog (Canis lupus familiaris). Zebrafish (Danio rerio) and Nile tilapia (Oreochromis niloticus) lrp13 loci are discrete and share genomic synteny. The Lrp13 appears to function as a vitellogenin receptor and may be an important mediator of yolk formation in fishes and other oviparous vertebrates. The presence of lrp13 orthologs in mammals suggests that this lipoprotein receptor is widely distributed among vertebrates, where it may generally play a role in lipoprotein metabolism.

Prolactin is a major inhibitor of hepatic Leptin A synthesis and secretion: studies utilizing a homologous Leptin A ELISA in the tilapia.

The present study identifies regulatory interactions between leptin A (LepA) and the pituitary hormone prolactin (PRL). In order to measure tilapia (Oreochromis mossambicus) LepA, an enzyme-linked immunosorbent assay (ELISA) utilizing a rabbit polyclonal antibody specific to tilapia LepA was first developed. The antibody shows strong cross reactivity to recombinant tilapia LepA (rtLepA), and a corresponding 16kDa protein in both tilapia and striped bass plasma, but not to recombinant human leptin (rhLep). The assay has a linear detection range of 0.25-1000nM, with intra- and interassay variability of 9% and 16%, respectively. Plasma LepA levels measured in tilapia ranged from 0.8 to 3.9nM, similar to that found for other vertebrates. Hypophysectomy (Hx) increased circulating LepA and lepa mRNA levels in the liver, the dominant source of hormone production. Adminstration of ovine PRL (oPRL, 5µg/g BW) to Hx fish restored circulating LepA and hepatic lepa mRNA levels to those of control fish. Additionally, oPRL reduced lepa mRNA levels in a dose-dependent fashion in cultured hepatocytes following an 18h incubation. Previous work in our lab indicates that rhLep stimulates PRL release in vitro from tilapia pituitaries. Here, both rtLepA and rhLep (0.5µg/g BW) increased mRNA expression of tilapia prolactin mRNAs (prl1, prl2) in the pituitary in vivo. These results demonstrate that LepA enhances pituitary prolactin synthesis and release, while PRL in turn inhibits hepatic leptin secretion and synthesis in teleosts. We postulate this regulatory interaction may be necessary for mobilizing energy reserves during acute hyperosmotic adaptation.

Cloning and characterization of leptin in a Perciform fish, the striped bass (Morone saxatilis): control of feeding and regulation by nutritional state.

In mammals, leptin is an anorexigenic peptide hormone that regulates energy homeostasis. It is produced predominantly by white adipose tissue and circulates as an endocrine indicator of energy reserves. Teleost leptin has been characterized in a few fish species, but its regulation is not well understood, particularly in response to nutritional status. In this study, we cloned a putative leptin in striped bass (Morone saxatilis) and report the first characterization of leptin in a Perciforme, the largest and most diverse order of fish. The striped bass leptin coding sequence was 65% homologous with pufferfish, 52% with Atlantic salmon, and 46% with human. PCR showed that leptin mRNA was exclusively expressed in the liver, and not adipose or other tissues. The leptin coding sequence of striped bass and the more widely cultured hybrid striped bass variety (HSB; Morone chrysops, white bass×M. saxatilis) were identical. We then evaluated whether the metabolic status of HSB might alter leptin gene expression. Juvenile HSB were subjected to 3weeks feed deprivation followed by 3weeks of refeeding. Quantitative PCR showed that fasting for 3weeks reduced hepatic leptin mRNA levels relative to fed controls. Leptin mRNA levels then increased upon refeeding, albeit levels were not completely restored to those seen in control fish fed throughout the experiment. Intraperitoneal injection of human leptin suppressed appetite in HSB. In as much as hepatic HSB leptin mRNA is regulated by nutritional state and has a corresponding anorexigenic effect, our results suggest that leptin may play a role in energy homeostasis in these advanced Perciformes.

Enhanced biodiversity of gut flora and feed efficiency in pond cultured tilapia under reduced frequency feeding strategies.

Feed constitutes 50-70% of total production costs of tilapia, one of the most widely cultured finfishes in the world. We evaluated reduced-feeding strategies for improving production efficiency of Nile tilapia (Oreochromis niloticus). In a 12-week pond trial, fish were fed daily, every other day, every third day, or not at all. Ponds were fertilized to enhance natural foods. In a fifth group fish were fed daily without pond fertilization. Fish fed daily with or without pond fertilization and fish fed every other day had higher specific growth rates, survivability, and net production than the other two treatments. Fish feed efficiency and benefit to cost ratio was highest for treatments fed in a pulsatile manner (i.e. fed every other day or every third day) with fish fed on alternate days providing the best net return among all groups. Fish fed on alternate days had more moderate gene expression levels of intestinal nutrient transporters which may allow for a more balanced and efficient nutrient uptake. Fecal microbe analyses identified 145 families of prokaryotic and 132 genera of eukaryotic organisms in tilapia. The highest diversity of prokaryotes was found in fish fed either every other day or daily in fertilized ponds and the highest diversity of eukaryotes was found in fish fed every other day. These studies indicate feeding Nile tilapia on alternate days along with weekly pond fertilization has no deleterious effects on growth, survivability, or production versus daily feeding regimes, but enhances feed efficiency by 76% and provides the greatest net return on investments. Our studies also suggest for the first time that combining alternate-day feeding with pond fertilization produces the greatest microbial biodiversity in the intestine that could contribute to enhanced feed efficiency and overall health of tilapia.

Transcriptional regulation of prolactin in a euryhaline teleost: Characterisation of gene promoters through in silico and transcriptome analyses.

The sensitivity of prolactin (Prl) cells of the Mozambique tilapia (Oreochromis mossambicus) pituitary to variations in extracellular osmolality enables investigations into how osmoreception underlies patterns of hormone secretion. Through the actions of their main secretory products, Prl cells play a key role in supporting hydromineral balance of fishes by controlling the major osmoregulatory organs (ie, gill, intestine and kidney). The release of Prl from isolated cells of the rostral pars distalis (RPD) occurs in direct response to physiologically relevant reductions in extracellular osmolality. Although the particular signal transduction pathways that link osmotic conditions to Prl secretion have been identified, the processes that underlie hyposmotic induction of prl gene expression remain unknown. In this short review, we describe two distinct tilapia gene loci that encode Prl177 and Prl188 . From our in silico analyses of prl177 and prl188 promoter regions (approximately 1000 bp) and a transcriptome analysis of RPDs from fresh water (FW)- and seawater (SW)-acclimated tilapia, we propose a working model for how multiple transcription factors link osmoreceptive processes with adaptive patterns of prl177 and prl188 gene expression. We confirmed via RNA-sequencing and a quantitative polymerase chain reaction that multiple transcription factors emerging as predicted regulators of prl gene expression are expressed in the RPD of tilapia. In particular, gene transcripts encoding pou1f1, stat3, creb3l1, pbxip1a and stat1a were highly expressed; creb3l1, pbxip1a and stat1a were elevated in fish acclimated to SW vs FW. Combined, our in silico and transcriptome analyses set a path for resolving how adaptive patterns of Prl secretion are achieved via the integration of osmoreceptive processes with the control of prl gene transcription.

Role of the Campylobacter jejuni Cj1461 DNA methyltransferase in regulating virulence characteristics.

Mutation of the cj1461 predicted methyltransferase gene reduced the motility of Campylobacter jejuni 81-176. Electron microscopy revealed that the mutant strain had flagella but with aberrant structure. The Deltacj1461 mutant was sevenfold more adherent to but 50-fold less invasive of INT-407 human epithelial cells than the wild type.

Leptin Stimulates Cellular Glycolysis Through a STAT3 Dependent Mechanism in Tilapia.

We assessed if leptin, a cytokine hormone known to enhance energy expenditure by promoting lipid and carbohydrate catabolism in response to physiologic stress, might directly regulate cellular glycolysis. A transcriptomic analysis of prolactin cells in the tilapia (Oreochromis mossambicus) pituitary rostral pars distalis (RPD) revealed that recombinant leptin (rtLep) differentially regulates 1,995 genes, in vitro. Machine learning algorithms and clustering analyses show leptin influences numerous cellular gene networks including metabolism; protein processing, transport, and metabolism; cell cycle and the hypoxia response. Leptin stimulates transcript abundance of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (gapdh) in a covariate manner to the hypoxic stress gene network. Orthogonal tests confirm that rtLepA dose-dependently increases gapdh gene expression in the RPD along with transcript abundance of 6-phosphofructo-1-kinase (pfk1), the rate limiting glycolytic enzyme. Functional testing demonstrated that leptin stimulates PFK activity and glycolytic output, while Stattic (a STAT3 blocker) was sufficient to suppress these responses, indicating leptin stimulates glycolysis through a STAT3-dependent mechanism. Leptin also stimulated pfk1 gene expression and lactate production in primary hepatocyte incubations in a similar manner to those shown for the pituitary RPD. This work characterizes a critical metabolic action of leptin to directly stimulate glycolysis across tissue types in a teleost model system, and suggest that leptin may promote energy expenditure, in part, by stimulating glycolysis. These data in a teleost fish, suggest that one of leptin's ancient, highly-conserved functions among vertebrates may be stimulation of glycolysis to facilitate the energetic needs associated with various stressors.

Ecology of sleeping: the microbial and arthropod associates of chimpanzee beds.

The indoor environment created by the construction of homes and other buildings is often considered to be uniquely different from other environments. It is composed of organisms that are less diverse than those of the outdoors and strongly sourced by, or dependent upon, human bodies. Yet, no one has ever compared the composition of species found in contemporary human homes to that of other structures built by mammals, including those of non-human primates. Here we consider the microbes and arthropods found in chimpanzee beds, relative to the surrounding environment (n = 41 and 15 beds, respectively). Based on the study of human homes, we hypothesized that the microbes found in chimpanzee beds would be less diverse than those on nearby branches and leaves and that their beds would be primarily composed of body-associated organisms. However, we found that differences between wet and dry seasons and elevation above sea level explained nearly all of the observed variation in microbial diversity and community structure. While we can identify the presence of a chimpanzee based on the assemblage of bacteria, the dominant signal is that of environmental microbes. We found just four ectoparasitic arthropod specimens, none of which appears to be specialized on chimpanzees or their structures. These results suggest that the life to which chimpanzees are exposed while in their beds is predominately the same as that of the surrounding environment.

Regulation of metallothionein genes in the American oyster (Crassostrea virginica): ontogeny and differential expression in response to different stressors.

Metallothioneins (MTs) are typically low molecular weight (6-7 kDa), metal-binding proteins with characteristic repeating cysteine motifs (Cys-X-Cys or Cys-Xn-Cys) and a prolate ellipsoid shape containing single alpha- and beta-domains. While functionally diverse, they play important roles in metals homeostasis, detoxification and the stress response. The present study, combined with previous observations (e.g., Jenny et al., Eur. J. Biochem. 2005; 271:1702-1712) defines an unprecedented diversity of MT primary structure and domain organization in the American oyster, Crassostrea virginica. Two novel molluscan MT families are described. One of these (CvMT-III) is characterized by the presence of two beta-domains and the absence of alpha-domains. This family exhibits constitutive expression during larval development and is the dominant CvMT isoform expressed in larvae. CvMT-III displays low basal levels of expression in adult tissues and only moderate responsiveness to metal challenges in both larvae and adults. A second novel MT isoform (CvMT-IV) was isolated from hemocytes by subtractive hybridization techniques following a 4-hour immune challenge with heat-killed bacteria (Vibrio, Bacillus, Micrococcus spp. mixture). Based on conservation of the cysteine motifs, this isoform appears to be a sub-family related to the molluscan alphabeta-domain MTs. A series of amino acid substitutions has resulted in four additional cysteines which give rise to a Cys-Cys motif and three Cys-Cys-Cys motifs. Northern blot analyses demonstrate that CvMT-IV is down-regulated upon sterile wounding and immune challenge, displays moderate expression in larvae and adults and differential gene induction in response to metals exposure.

Role for leptin in promoting glucose mobilization during acute hyperosmotic stress in teleost fishes.

Osmoregulation is critical for survival in all vertebrates, yet the endocrine regulation of this metabolically expensive process is not fully understood. Specifically, the function of leptin in the regulation of energy expenditure in fishes, and among ectotherms, in general, remains unresolved. In this study, we examined the effects of acute salinity transfer (72  h) and the effects of leptin and cortisol on plasma metabolites and hepatic energy reserves in the euryhaline fish, the tilapia (Oreochromis mossambicus). Transfer to 2/3 seawater (23  ppt) significantly increased plasma glucose, amino acid, and lactate levels relative to those in the control fish. Plasma glucose levels were positively correlated with amino acid levels (R2=0.614), but not with lactate levels. The mRNA expression of liver leptin A (lepa), leptin receptor (lepr), and hormone-sensitive and lipoprotein lipases (hsl and lpl) as well as triglyceride content increased during salinity transfer, but plasma free fatty acid and triglyceride levels remained unchanged. Both leptin and cortisol significantly increased plasma glucose levels in vivo, but only leptin decreased liver glycogen levels. Leptin decreased the expression of liver hsl and lpl mRNAs, whereas cortisol significantly increased the expression of these lipases. These findings suggest that hepatic glucose mobilization into the blood following an acute salinity challenge involves both glycogenolysis, induced by leptin, and subsequent gluconeogenesis of free amino acids. This is the first study to report that teleost leptin A has actions that are functionally distinct from those described in mammals acting as a potent hyperglycemic factor during osmotic stress, possibly in synergism with cortisol. These results suggest that the function of leptin may have diverged during the evolution of vertebrates, possibly reflecting differences in metabolic regulation between poikilotherms and homeotherms.

Phylogenetic revision of the claudin gene family.

Claudins are four-transmembrane proteins acting to collectively regulate paracellular movement of water and ions across cellular tight junctions in vertebrate tissues. Despite the prominence of zebrafish (Danio rerio) as a developmental model and the existence of an annotated genome, the diversity and evolutionary history of these claudins, with respect to other vertebrate groups, is poorly described. In this study, we identify 54 zebrafish claudins, including 24 that were previously unreported, and infer homology of the encoded polypeptide sequences with other vertebrate claudin groups using Bayesian phylogenetic analysis. In this analysis, 197 vertebrate claudin and claudin-like proteins were classified into discrete 'superclades' of related proteins. Based on these groupings, an interim reclassification is proposed, which will resolve ambiguity in the present nomenclature of several vertebrate models. Fifty-two of the 54 identified claudins were detected in cDNA preparations from whole, adult zebrafish, and 43 exhibited distinct tissue expression profiles. Despite prolific expansion of the claudin gene family in teleost genomes, these claudins can still be broadly separated into two functional groups: (1) "classic" claudins that characteristically contain an equal number of opposing, charged residues in the first extracellular loop (ECL1) and (2) "non-classic" claudins that typically have an ECL1 containing a variable number of charged residues. Functional analysis of these groups indicates that 'classic' claudins may act to reduce overall paracellular permeability to water and dissolved ions, whereas 'non-classic' claudins may constitute pores that facilitate selective ion permeability.

Salinity regulates claudin mRNA and protein expression in the teleost gill.

The teleost gill carries out NaCl uptake in freshwater (FW) and NaCl excretion in seawater (SW). This transformation with salinity requires close regulation of ion transporter capacity and epithelial permeability. This study investigates the regulation of tight-junctional claudins during salinity acclimation in fish. We identified claudin 3- and claudin 4-like immunoreactive proteins and examined their expression and that of select ion transporters by performing Western blot in tilapia (Oreochromis mossambicus) gill during FW and SW acclimation. Transfer of FW tilapia to SW increased plasma osmolality, which was corrected after 4 days, coinciding with increased gill Na+-K+-ATPase and Na+-K+-2Cl(-) cotransporter expression. Gill claudin 3- and claudin 4-like proteins were reduced with exposure to SW. Transfer to FW increased both claudin-like proteins. Immunohistochemistry shows that claudin 3-like protein was localized deep in the FW gill filament, whereas staining was found apically in SW gill. Claudin 4-like proteins are localized predominantly in the filament outer epithelial layer, and staining appears more intense in the gill of FW versus SW fish. In addition, tilapia claudin 28a and 30 genes were characterized, and mRNA expression was found to increase during FW acclimation. These studies are the first to detect putative claudin proteins in teleosts and show their localization and regulation with salinity in gill epithelium. The data indicate that claudins may be important in permeability changes associated with salinity acclimation and possibly the formation of deeper tight junctions in FW gill. This may reduce ion permeability, which is a critical facet of FW osmoregulation.