Figure 1.
A figure showing the putative processing of NUCB2 and comparison of nesfatin-1 sequences.
Schematic outline of zebrafish NUCB2 gene and the formation of nesfatin-1 (A). In this figure, the boxes which make up the NUCB2 gene represent exons 1–13 and interspaced lines represent introns 1–12. The proposed scheme for NUCB2 prepropeptide and putative cleavage sites from the NUCB2 gene are shown. All other teleosts examined have similar, highly conserved intron:exon organization for the NUCB2A gene, except for pufferfish (Tetraodon nigroviridis) and fugu (Takifugu rubripes) which have exon 1 deleted. Figure 1B shows the amino acid sequence alignment of the nesfatin-1 region of NUCB2, NUCB2A and NUCB2B (teleost only). Figure 1C is the alignment of NUCB1 from cartilaginous fish, bony fish, amphibians, birds and mammals. The name of the species is given on the left side of the alignment and the number of amino acids in the peptide is provided above the alignment. The colored amino acids highlight the differences in conservation of the amino acids between species within the nesfatin-1 region of NUCB2 and NUCB1. In addition, the putative cleavage sites are shown in the box. Species names used in the alignment were as follows: zebrafish (Danio rerio), stickleback (Gasterosteus aculeatus), medaka (Oryzias latipes), green pufferfish (Tetraodon nigroviridis), fugu (Takifugu rubripes), Western clawed frog (Xenopus tropicalis), chicken (Gallus gallus), mouse (Mus musculus), and human (Homo sapiens).
Figure 2.
Phylogenetic analysis of nucleobindin gene sequences.
NUCB2A and NUCB2B (teleost only), and NUCB1 amino acid sequences. Each node has a bootstrap value which was obtained for 500 replicates. GenBank Accession Numbers or Ensembl identification numbers are as follows; Caenorhabditis elegans (NUCB1, NM_171763.3), Drosophila melanogaster (NUCB1, AAF49304.3), Rattus norvegicus (NUCB1, AAI00644.1), Mus musculus (NUCB1, AAH72554.1; NUCB2, AAH10459.1), Homo sapiens (NUCB1, NP_006175.2; NUCB2, NP_005004.1), Takifugu rubripes (NUCB1, ENSTRUP00000000823; NUCB2A, ENSTRUP00000044315; NUCB2B, ENSTRUP00000038477), Tetraodon nigroviridis (NUCB2A, ENSTNIP00000012254; NUCB2B, ENSTNIP00000005671), Gasterosteus aculeatus (NUCB1, ENSGACP00000016912; NUCB2A, ENSGACP00000020611; NUCB2B, ENSGACP00000008999), Oryzias latipes (NUCB2A, ENSORLP00000008372; NUCB2B, ENSORLP00000001612), Danio rerio (NUCB1, NM_001045463.1; NUCB2A, NM_201493.1; NUCB2B, NM_201479.1), Xenopus tropicalis, (NUCB2, AAH90107.1), Gallus gallus (NUCB2, NP_001006468.1), and Carassius auratus (NUCB2A, HM065567.1).
Figure 3.
Differential expression of NUCB2 mRNA in goldfish tissues.
NUCB2 and beta-actin cDNA amplicon products from the tissues of Carassius auratus by reverse transcriptase PCR (A). More precise quantitative data was obtained by using Real-Time Quantitative Reverse Transcription PCR (B). The results were normalized to β-actin, which served as a control to verify the amount and quality of samples (n = 5 fish).
Figure 4.
Feeding status affects the expression of NUCB2 mRNA within the hypothalamus and liver of goldfish.
Pre- and post-prandial changes in the expression of NUCB2 mRNA in the (A) hypothalamus and (B) liver of goldfish. The mRNA expression of nesfatin was normalized to β-actin and represented relative to the −3 hour scheduled feeding time group. Asterisks represent significant differences between groups at the same time point. Food deprivation decreased expression of NUCB2 mRNA in the goldfish hypothalamus (C) and increased its expression in the liver (D). Three and 7 day NUCB2 mRNA expression is represented as the normalized percentage of the NUCB2 mRNA expression in the 3 day food-deprived fish. One-way ANOVA, Newman-Keuls Multiple Comparison Test. Asterisks represent significant differences between groups at the same time point. * P<0.05, ** P<0.01, *** P<0.001. (n = 6 fish/group).
Figure 5.
One-day food deprivation reduces serum nesfatin-1 levels in goldfish.
Elevated serum nesfatin-1 levels can be detected up to 1 hour post-feeding but is reduced 3 hours post-feeding (A). Nesfatin-1 ELISA standard curve shows strong cross reactivity with goldfish nesfatin-1 over a range of 1–100 ng/ml (B). Western blot analysis confirmed the specificity of polyclonal pronesfatin antibody (C). Rodent and goldfish pronesfatin (NUCB2) were detected at their predicted molecular weights, ∼50 and ∼59 kDa, respectively, β-tubulin was added as a loading control and detected in both rat and goldfish tissues. No bands representing processed nesfatin-1 (∼9.5 kDa) was found in both rat and goldfish brain.
Figure 6.
Characterization of nesfatin-1 like immunoreactivity within the goldfish hypothalamus.
Immunohistochemical staining of goldfish hypothalamus for nesfatin-1-like immuunoreactivity (A, B; red), DAPI (C, D; nuclei; blue) and the merged image of nesfatin-1 and DAPI is shown in E and F. Representative cells that are immunopositive for nesfatin-1 in the NAT (A; open arrows) or in the NLTp (B; solid arrow) or in the NLTl (B; solid arrowhead) are shown. Figures G and H represents a no-primary antibody negative control, which was labeled only with secondary antibodies. Representative images were taken of 30 sections from 6 separate goldfish hypothalami.
Figure 7.
Nesfatin-1 like immunoreactivity is also present within the goldfish gut.
Nesfatin-1-like immunoreactivity found within enteroendocrine cells of the goldfish gut. Immunohistochemical localization shows nesfatin-1-like immunoreactive cells (arrows) in the intestinal villi of the gut (A). The nuclear stain DAPI (B) and merged images (C) show nesfatin-1-like immunoreactive cells along the base of the intestinal villi (solid arrow). The inset in C shows a representative nesfatin-1-like immunoreactive cell under high magnification (100x). A negative control where no primary, but only secondary antibody showed no staining (D). Representative images were taken of 18 sections from 6 separate goldfish guts.
Figure 8.
Effects of goldfish (gf) nesfatin-1 administration on food intake over 1 h post-injection.
Effects of intraperitoneal (IP) injections of relatively low doses of gfnesfatin-1 (0.5, 5, and 50 ng/g body weight [BW]) (A). Effects of IP injections of relatively high doses of gfnesfatin-1 (50, and 500 ng/g BW) (B). Effects of intracerebroventricular (ICV) injections of gfnesfatin-1 on food intake over 1 hour (C). A 0.5 and 25 ng/g BW ICV injection of the gfnesfatin-1 significantly reduced food intake over one hour by ∼43 and 50%, respectively. One-way ANOVA, Newman-Keuls Multiple Comparison Test. ** P<0.01, * P<0.05. (n = 6 fish/group).
Figure 9.
A summary on the distribution and functions of nesfatin-1 based on our findings in goldfish.
NUCB2/nesfatin-1 is abundantly expressed in the brain, liver and gut. NUCB2 mRNA was upregulated in the brain of fed fish and in the liver of fasted fish, while NUCB2 mRNA was down-regulated in the brain of fasted fish and in the liver of fed fish (solid arrows). Although the major tissue source of circulating nesfatin-1 is currently unknown, we propose that nesfatin-1 is released in a meal responsive manner from several tissues, including the brain, liver and gut to inhibit feeding in fish via central and peripheral actions. Nesfatin-1 seems to have direct effects on the liver, possibly in regulating the metabolic processes, especially during food-deprivation (eg: gluconeogenesis?). Solid lines indicate direct known effects while dashed arrows indicate potential relationships.