Novel Regulators of Feeding and Cardiovascular Physiology in Fish

Nesfatin-1, an 82 amino acid anorexigen is encoded in a secreted precursor, nucleobindin-2 (NUCB2). NUCB2 was named so due to its high sequence similarity with nucleobindin-1 (NUCB1). It was recently reported that NUCB1 encodes an insulinotropic nesfatin-1-like peptide (NLP) in mice. Irisin, a muscle protein is encoded in its precursor fibronectin type III domain containing 5 (FNDC5) and released into blood from skeletal muscle. Here we aimed to characterize NLP and irisin in fish, and to study whether these are novel regulators of feeding and cardiovascular functions in zebrafish and goldfish. Western blot analysis and immunohistochemical studies determined the expression of NUCB1/NLP in central and peripheral tissues of goldfish. Administration of rat and goldfish NLP at 10 and 100 ng/g body weight doses caused potent inhibition of food intake in goldfish. NLP also downregulated the expression of preproghrelin and orexin-A mRNA, and upregulated cocaine and amphetamine regulated transcript (CART) mRNA in goldfish brain. Intraperitoneal (I.P) administration of NLP reduced cardiac functions in zebrafish and goldfish, downregulated irisin, and RyR1b mRNA expression in zebrafish. Irisin was detected in zebrafish heart and skeletal muscle. Single I.P. injection of irisin did not affect feeding, but its knockdown using siRNA caused a significant reduction in food intake. Knockdown of irisin reduced ghrelin and orexin-A mRNA expression, and increased CART mRNA expression in zebrafish brain and gut. Meanwhile, injection of irisin (0.1 and 1 ng/g B.W) increased cardiac functions, while knockdown of irisin resulted in reverse effects on cardiovascular physiology. Administration of propranolol attenuated the effects of irisin on cardiac physiology. Collectively, my research discovered that NLP and irisin modulate food intake and cardiac physiology in fish. Future studies should focus on the mechanisms of action of NLP and irisin in regulating metabolism and cardiovascular biology in fish

Effect of extrusion cooking on the nutritional properties of amaranth, quinoa, kañiwa and lupine

Amaranth, quinoa, kañiwa and lupine are good sources of protein, fat, dietary fibre and bioactive compounds. The literature review deals with the nutritional properties and the stability of bioactive compounds and the effect of extrusion cooking on amaranth, quinoa, kañiwa and lupine. The main aim of this study was to (1) chemically characterize amaranth, quinoa, kañiwa and lupine, and (2) to determine the effect of extrusion cooking on the nutritional properties and the stability of bioactive compounds. Extrudates were processed using twin screw extruder at two different extrusion temperatures (140 and 160 °C) containing two different contents of tested flour mixtures (20 and 50%). The raw materials and the extrudates were stored at -18 °C and chemically characterized to determine fatty acid composition, tocopherol composition and total phenolic acid content. Fatty acid composition was determined using GC while tocopherol composition was detected using HPLC. The total phenolic acid content was analyzed using Folin-Ciocalteu method. The protein and dietary fibre content in lupine accounted for 29 and 50 g/100 g d.m., respectively. The extrudates containing 50% lupine and processed at 140 °C possessed higher content of oleic, linoleic and linolenic fatty acids. At higher content of tested flours, extrusion cooking at 160 °C resulted in better retention of unsaturated fatty acids in the extrudates of amaranth, kañiwa and quinoa. Higher extrusion temperatures resulted in lower retention of tocopherols in all the extrudates. The total phenolic acid resulted in higher contents in the extrudates of kañiwa when compared to other extrudates. At higher seed contents of tested flours (%), higher retention of total phenolic acid was achieved during extrusion cooking at 140 °C in the extrudates of amaranth, quinoa and kañiwa. This study showed that extrusion conditions could be optimized in order to obtain lesser effects on the nutritional properties and better retention of bioactive compounds. The research study provides supportive information for obtaining gluten-free cereal snack products with lower glycemic index

Why goldfish? Merits and challenges in employing goldfish as a model organism in comparative endocrinology research

Goldfish has been used as an unconventional model organism to study a number of biological processes. For example, goldfish is a well-characterized and widely used model in comparative endocrinology, especially in neuroendocrinology. Several decades of research has established and validated an array of tools to study hormones in goldfish. The detailed brain atlas of goldfish, together with the stereotaxic apparatus, are invaluable tools for the neuroanatomic localization and central administration of endocrine factors. In vitro techniques, such as organ and primary cell cultures, have been developed using goldfish. In vivo approaches using goldfish were used to measure endogenous hormonal milieu, feeding, behaviour and stress. While there are many benefits in using goldfish as a model organism in research, there are also challenges associated with it. One example is its tetraploid genome that results in the existence of multiple isoforms of endocrine factors. The presence of extra endogenous forms of peptides and its receptors adds further complexity to the already redundant multifactorial endocrine milieu. This review will attempt to discuss the importance of goldfish as a model organism in comparative endocrinology. It will highlight some of the merits and challenges in employing goldfish as an animal model for hormone research in the post-genomic era.Fil: Blanco, Ayelén Melisa. University of Saskatchewan; Canadá. Universidad Complutense de Madrid; EspañaFil: Sundarrajan, Lakshminarasimhan. University of Saskatchewan; CanadáFil: Bertucci, Juan Ignacio. University of Saskatchewan; Canadá. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Unniappan, Suraj. University of Saskatchewan; Canad

Irisin regulates cardiac physiology in zebrafish.

Irisin is a myokine encoded in its precursor fibronectin type III domain containing 5 (FNDC5). It is abundantly expressed in cardiac and skeletal muscle, and is secreted upon the activation of peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1 alpha). We aimed to study the role of irisin on cardiac function and muscle protein regulation in zebrafish. Western blot analyses detected the presence of irisin protein (23 kDa) in zebrafish heart and skeletal muscle, and irisin immunoreactivity was detected in both tissues. Irisin siRNA treated samples did not show bands corresponding to irisin in zebrafish. In vitro studies found that treatment with irisin (0.1 nM) downregulated the expression of PGC-1 alpha, myostatin a, and b, while upregulating troponin C mRNA expression in zebrafish heart and skeletal muscle. Exogenous irisin (0.1 and 1 ng/g B.W) increased diastolic volume, heart rate and cardiac output, while knockdown of irisin (10 ng/g B.W) showed opposing effects on cardiovascular function. Irisin (1 and 10 ng/g B.W) downregulated PGC-1 alpha, myostatin a and b, and upregulated troponin C and troponin T2D mRNA expression. Meanwhile, knockdown of irisin showed opposing effects on troponin C, troponin T2D and myostatin a and b mRNAs in zebrafish heart and skeletal muscle. Collectively, these results identified muscle proteins as novel targets of irisin, and added irisin to the list of peptide modulators of cardiovascular physiology in zebrafish

Xenin is a novel anorexigen in goldfish (Carassius auratus).

Xenin, a highly conserved 25 amino acid peptide cleaved from the N-terminus of the coatomer protein alpha (COPA), is emerging as a food intake regulator in mammals and birds. To date, no research has been conducted on xenin biology in fish. This study aims to identify the copa mRNA encoding xenin in goldfish (Carassius auratus) as a model, to elucidate its regulation by feeding, and to describe the role of xenin on appetite. First, a partial sequence of copa cDNA, a region encoding xenin, was identified from goldfish brain. This sequence is highly conserved among both vertebrates and invertebrates. RT-qPCR revealed that copa mRNAs are widely distributed in goldfish tissues, with the highest levels detected in the brain, gill, pituitary and J-loop. Immunohistochemistry confirmed also the presence of COPA peptide in the hypothalamus and enteroendocrine cells on the J-loop mucosa. In line with its anorexigenic effects, we found important periprandial fluctuations in copa mRNA expression in the hypothalamus, which were mainly characterized by a gradually decrease in copa mRNA levels as the feeding time was approached, and a gradual increase after feeding. Additionally, fasting differently modulated the expression of copa mRNA in a tissue-dependent manner. Peripheral and central injections of xenin reduce food intake in goldfish. This research provides the first report of xenin in fish, and shows that this peptide is a novel anorexigen in goldfish

Xenin-like immunoreactivity in the goldfish brain.

<p>(<b>A</b>) Sagittal view of a goldfish brain stained with DAPI (blue). Arrows indicate the region of the posterior periventricular nucleus (NPP<i>v</i>), nucleus anterior tuberis (NAT), and posterior nucleus lateralis tuberis (NLT<i>p</i>). Scale bar = 500 μm. (<b>B–D</b>) Immunohistochemical staining of goldfish NAT, NLT<i>p</i> and NPP<i>v</i> neurons for xenin-like immunoreactivity (red). All images are merged with DAPI showing nuclei in blue. Arrows point to immunopositive cells. In C, open arrows point to xenin-like immunoreactive neurons along the ventricle, and closed arrows to positive neurons lateral to the ventricle. Scale bars = 50 μm (B), 100 μm (C, D), 20 μm (inset in D). (<b>E</b>) Image of a negative control slide stained with secondary antibody alone. Scale bar = 50 μm. (<b>F, G</b>) Transversal representative sections of goldfish brain treated with specific primary anti-xenin antibody pre-absorbed in xenin. Scale bars = 200 μm.</p

Nesfatin-1-Like Peptide Encoded in Nucleobindin-1 in Goldfish is a Novel Anorexigen Modulated by Sex Steroids, Macronutrients and Daily Rhythm

Nesfatin-1 is an 82 amino acid anorexigen encoded in a secreted precursor nucleobindin-2 (NUCB2). NUCB2 was named so due to its high sequence similarity with nucleobindin-1 (NUCB1). It was recently reported that NUCB1 encodes an insulinotropic nesfatin-1-like peptide (NLP) in mice. Here, we aimed to characterize NLP in fish. RT-qPCR showed NUCB1 expression in both central and peripheral tissues. Western blot analysis and/or fluorescence immunohistochemistry determined NUCB1/NLP in the brain, pituitary, testis, ovary and gut of goldfish. NUCB1 mRNA expression in goldfish pituitary and gut displayed a daily rhythmic pattern of expression. Pituitary NUCB1 mRNA expression was downregulated by estradiol, while testosterone upregulated its expression in female goldfish brain. High carbohydrate and fat suppressed NUCB1 mRNA expression in the brain and gut. Intraperitoneal injection of synthetic rat NLP and goldfish NLP at 10 and 100 ng/g body weight doses caused potent inhibition of food intake in goldfish. NLP injection also downregulated the expression of mRNAs encoding orexigens, preproghrelin and orexin-A, and upregulated anorexigen cocaine and amphetamine regulated transcript mRNA in goldfish brain. Collectively, these results provide the first set of results supporting the anorectic action of NLP, and the regulation of tissue specific expression of goldfish NUCB1.Fil: Sundarrajan, Lakshminarasimhan. University of Saskatchewan; CanadáFil: Blanco, Ayelén Melisa. Universidad Complutense de Madrid; EspañaFil: Bertucci, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Ramesh, Naresh. University of Saskatchewan; CanadáFil: Canosa, Luis Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Unniappan, Suraj. University of Saskatchewan; Canad

Phylogenetic analysis of the partial COPA sequence obtained from goldfish.

<p><b>(A)</b> Phylogenetic tree showing the evolutionary relationships of the obtained nucleotide sequence of goldfish <i>copa</i> with those of other species. Tree was inferred by the neighbor-joining method using the online tool <a href="http://www.phylogeny.fr" target="_blank">www.phylogeny.fr</a>. The scale bar indicates the average number of substitutions per position (a relative measure of evolutionary distance). The names of the species used for the alignment are provided in the figure. GenBank accession numbers of the sequences used are as follows: <i>Bombyx mori</i>, NM_001172721.1; <i>Bos taurus</i>, NM_001105645.1; <i>Carassius auratus</i>, JQ929912.1; <i>Cavia porcellus</i>, XM_003466569.3; <i>Ciona intestinalis</i>, XM_002131228.4; <i>Cricetulus griseus</i>, XM_007629307.2; <i>Danio rerio</i>, NM_001001941.2; <i>Equus caballus</i>, XM_023640888.1; <i>Gallus gallus</i>, NM_001031405.2; <i>Homo sapiens</i>, NM_001098398.1; <i>Loxodonta africana</i>, XM_023554162.1; <i>Macaca mulatta</i>, XM_015113467.1; <i>Meleagris gallopavo</i>, XM_003213951.3; <i>Monodelphis domestica</i>, XM_016430274.1; <i>Mus musculus</i>, NM_009938.4; <i>Nomascus leucogenys</i>, XM_012510889.1; <i>Pan troglodytes</i>, XM_001171563.4; <i>Rattus norvegicus</i>, NM_001134540.1; <i>Saccoglossus kowalevskii</i>, XM_002731243.2; <i>Salmo salar</i>, XM_014140665.1; <i>Sus scrofa</i>, XM_001928697.6; <i>Thalassiosira pseudonana</i>, XM_002291058.1; <i>Xenopus laevis</i>, NM_001093019.2; <i>Xenopus tropicalis</i>, NM_001127994.1. <b>(B)</b> Alignment of the first 61 aa of COPA from an algal species, invertebrate species, teleost fishes, amphibians, avians, and mammals. The species names are provided on the left-hand side of the alignment and the number of aa is present on the right-hand side of the alignment. The coloured aa highlights the differences in conservation between species. The first 25 aa (which correspond to the xenin region) are boxed.</p

Pre- and post-prandial changes of <i>copa</i> mRNA expression in the goldfish hypothalamus (A), J-loop (B), and liver (C).

<p>The mRNA expression of <i>copa</i> was normalized to <i>β-actin</i> and represented relative to the -3 h scheduled feeding group. Data are presented as mean + SEM (n = 4 fish). Columns sharing a same letter are not statistically different (p < 0.05, one-way ANOVA and Student-Newman-Keuls tests).</p

Fasting-induced changes of <i>copa</i> mRNA expression in goldfish central and peripheral tissues.

<p>(<b>A–C</b>) Expression of <i>copa</i> mRNAs after a 3-day fasting period in the goldfish hypothalamus (A), J-loop (C), and liver (E). The mRNA expression of <i>copa</i> was normalized to <i>β-actin</i> and represented relative to the unfed group. Data are presented as mean + SEM (n = 4 fish). Columns with a different letter are statistically different (p < 0.05, t-test). (<b>D–F</b>) Expression of <i>copa</i> mRNAs after a 7-day fasting period and refeeding in the goldfish hypothalamus (D), J-loop (E), and liver (F). The mRNA expression of <i>copa</i> was normalized to <i>β-actin</i> and represented relative to the unfed group. Data are presented as mean + SEM (n = 4 fish). Columns with a different letter are statistically different (p < 0.05, one-way ANOVA and Student-Newman-Keuls tests).</p