Suraj Unniappan

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.

Pancreatic beta cells colocalize insulin and pronesfatin immunoreactivity in rodents.

Nesfatin-1 is a recently discovered feeding inhibitory peptide encoded in the precursor protein, nucleobindin 2 (pronesfatin). Previous studies have shown pronesfatin expression in the brain, stomach and pancreas. However, the identity of cells that express nesfatin in the pancreas remain unknown. The objective of this study was to determine which cells in the pancreas of mice and rats express pronesfatin immunoreactivity. We found pronesfatin immunopositive cells exclusively in the pancreatic islets of both CD1 mice and Fischer 344 rats. Our novel results indicate that the insulin producing beta cells colocalize pronesfatin in the islets of both mice and rats. No colocalization of glucagon and pronesfatin was found in mice, while some glucagon positive cells were positive for pronesfatin in rat islets. The abundant presence of pronesfatin immunoreactivity and its colocalization with insulin suggests a potential role for pronesfatin-derived peptides in islet biology and glucose homeostasis in rodents.

Nesfatin-1 exerts a direct, glucose-dependent insulinotropic action on mouse islet β- and MIN6 cells

Nesfatin-1 is a recently discovered multifunctional metabolic hormone abundantly expressed in the pancreatic islets. The main objective of this study is to characterize the direct effects of nesfatin-1 on insulin secretion in vitro using MIN6 cells and islets isolated from C57BL/6 mice. We also examined the expression of the nesfatin-1 precursor protein, nucleobindin 2 (NUCB2) mRNA, and nesfatin-1 immunoreactivity (ir) in the islets of normal mice and in the islets from mice with streptozotocin-induced type 1 diabetes and diet-induced obese (DIO) mice with type 2 diabetes. Nesfatin-1 stimulated glucose-induced insulin release in vitro from mouse islets and MIN6 cells in a dose-dependent manner. No such stimulation in insulin secretion was found when MIN6 cells/islets were incubated with nesfatin-1 in low glucose. In addition, a fourfold increase in nesfatin-1 release from MIN6 cells was observed following incubation in high glucose (16.7  mM) compared to low glucose (2  mM). Furthermore, we observed a significant reduction in both NUCB2 mRNA expression and nesfatin-1-ir in the pancreatic islets of mice with type 1 diabetes, while a significant increase was observed in the islets of DIO mice. Together, our findings indicate that nesfatin-1 is a novel insulinotropic peptide and that the endogenous pancreatic islet NUCB2/nesfatin is altered in diabetes and diet-induced obesity.

Nutrient Responsive Nesfatin-1 Regulates Energy Balance and Induces Glucose-Stimulated Insulin Secretion in Rats

Nesfatin-1 is a recently discovered anorexigen, and we first reported nesfatin-like immunoreactivity in the pancreatic β-cells. The aim of this study was to characterize the effects of nesfatin-1 on whole-body energy homeostasis, insulin secretion, and glycemia. The in vivo effects of continuous peripheral delivery of nesfatin-1 using osmotic minipumps on food intake and substrate partitioning were examined in ad libitum-fed male Fischer 344 rats. The effects of nesfatin-1 on glucose-stimulated insulin secretion (GSIS) were examined in isolated pancreatic islets. L6 skeletal muscle cells and isolated rat adipocytes were used to assess the effects of nesfatin-1 on basal and insulin-mediated glucose uptake as well as on major steps of insulin signaling in these cells. Nesfatin-1 reduced cumulative food intake and increased spontaneous physical activity, whole-body fat oxidation, and carnitine palmitoyltransferase I mRNA expression in brown adipose tissue but did not affect uncoupling protein 1 mRNA in the brown adipose tissue. Nesfatin-1 significantly enhanced GSIS in vivo during an oral glucose tolerance test and improved insulin sensitivity. Although insulin-stimulated glucose uptake in L6 muscle cells was inhibited by nesfatin-1 pretreatment, basal and insulin-induced glucose uptake in adipocytes from nesfatin-1-treated rats was significantly increased. In agreement with our in vivo results, nesfatin-1 enhanced GSIS from isolated pancreatic islets at both normal (5.6 mM) and high (16.7 mM), but not at low (2 mM), glucose concentrations. Furthermore, nesfatin-1/nucleobindin 2 release from rat pancreatic islets was stimulated by glucose. Collectively, our data indicate that glucose-responsive nesfatin-1 regulates insulin secretion, glucose homeostasis, and whole-body energy balance in rats.

Fasting induces preproghrelin mRNA expression in the brain and gut of zebrafish, Danio rerio

Ghrelin is a gut/brain hormone with a unique acyl modification and various biological functions in fish and mammals. In addition to its possible role as a circulating orexigenic factor, ghrelin has been shown to regulate several other physiological processes in fish in a species-specific manner. The objectives of this project were to identify the zebrafish ghrelin gene organization, study tissue specific preproghrelin mRNA expression and investigate the fasting induced changes in the expression of preproghrelin mRNA in zebrafish. Our reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed the predicted ghrelin sequence available in the GenBank and identified preproghrelin mRNA expression in several tissues including the brain, gut, ovary, testis, heart and gill. PCR using genomic DNA identified that the ghrelin gene in zebrafish is comprised of four exons and three introns. Quantitative (real-time) PCR studies indicate that there is a significant increase in preproghrelin mRNA expression in the brain and gut of zebrafish fasted for 3, 5 and 7 days when compared to the expression in ad libitum fed fish. Refeeding after a 7 day fast caused a significant and dramatic decrease in preproghrelin mRNA expression in the gut and brain of zebrafish. An increase in the expression of preproghrelin mRNA during fasting, and its decrease following refeeding suggests an orexigenic role for ghrelin in zebrafish. Overall, our results provide evidence for a highly conserved structure and biological actions of ghrelin in zebrafish. Further studies are required to identify the tissue specific functions of ghrelin in zebrafish.

Molecular, cellular and physiological evidences for the anorexigenic actions of nesfatin-1 in goldfish.

Background Nesfatin-1 is a recently discovered anorexigen encoded in the precursor peptide, nucleobindin-2 (NUCB2) in mammals. To date, nesfatin-1 has not been described in any non-mammalian species, although some information is available in the sequenced genomes of several species. Our objective was to characterize nesfatin-1 in fish. Methodology/Principal Findings In the present study, we employed molecular, immunohistochemical, and physiological studies to characterize the structure, distribution, and appetite regulatory effects of nesfatin-1 in a non-mammalian vertebrate. A very high conservation in NUCB2 sequences, especially in the nesfatin-1 region was found in lower vertebrates. Abundant expression of NUCB2 mRNA was detected in several tissues including the brain and liver of goldfish. Nesfatin-1-like immunoreactive cells are present in the feeding regulatory nucleus of the hypothalamus and in the gastrointestinal tract of goldfish. Approximately 6-fold increase in NUCB2 mRNA levels was found in the liver after 7-day food-deprivation, and a similar increase was also found after short-term fasting. This points toward a possible liver specific role for NUCB2 in the control of metabolism during food-deprivation. Meanwhile, ∼2-fold increase at 1 and 3 h post-feeding and an ∼3-fold reduction after a 7-day food-deprivation was observed in NUCB2 mRNA in the goldfish hypothalamus. In vivo, a single intraperitoneal injection of the full-length native (goldfish; gf) nesfatin-1 at a dose of 50 ng/g body weight induced a 23% reduction of food intake one hour post-injection in goldfish. Furthermore, intracerebroventricular injection of gfnesfatin-1 at a dose of 5 ng/g body weight resulted in ∼50% reduction in food intake. Conclusions/Significance Our results provide molecular, anatomical and functional evidences to support potential anorectic and metabolic roles for endogenous nesfatin-1 in goldfish. Collectively, we provide novel information on NUCB2 in non-mammals and an anorexigenic role for nesfatin-1 in goldfish.

Molecular characterization, appetite regulatory effects and feeding related changes of peptide YY in goldfish

Peptide YY (PYY) is a 36 amino acid multifunctional gut-brain hormone in mammals. PYY has recently raised great interest as it was shown to reduce food intake and body weight of mammals. While PYY and its receptors have been sequenced from many non-mammalian vertebrates, its functional role, especially in the regulation of food intake in lower vertebrates remain unknown. In this study, we identified the gene organization of goldfish PYY (gfPYY) and found abundant expression of PYY mRNA in the brain and digestive tract of goldfish. A 2.5-fold increase at 3h post-feeding and a 1.5-fold decrease in fasted animals was observed of PYY mRNA expression in the brain, suggesting an anorectic role for PYY in goldfish. A single intraperitoneal injection of 10 ng/g body weight gfPYY(1-36) or an intracerebroventricular injection of 5 ng/g body weight gfPYY(1-36) caused a 27% or 30% reduction in food intake in goldfish, respectively. Overall, our results, for the first time provide molecular and functional evidence for anorectic actions of PYY in goldfish.

Nucleobindins: Bioactive precursor proteins encoding putative endocrine factors?

The nucleobindins, nucleobindin 1 (NUCB1) and nucleobindin 2 (NUCB2), are homologous multidomain calcium and DNA binding proteins. NUCB1 is a well-characterized Golgi protein found within the rat pituitary, liver and kidney with functions related to immunity, calcium homeostasis and G protein signaling. NUCB2 is found both in the hypothalamus and brain stem centers, as well as peripherally in the digestive tract. Renewed interest in the nucleobindins has been sparked by the recent discovery of nesfatin-1, an endocrine factor post-translationally processed from the N-terminal of NUCB2. Nesfatin-1 has quickly established itself as a novel regulator of appetite, insulin secretion, energy homeostasis and reproduction with important consequences to the etiology of metabolic diseases including diabetes and obesity. The discovery of nesfatin-1 and it endocrine functions attracted more attention to the nucleobindins that are already known to have important intracellular functions. From the sequence information available, it is possible that nucelobindins itself or nesfatin-1 like peptides within the NUCB1 could also elicit nesfatin-1-like biological functions. The research on nesfatin-1 in last 5years further adds to the importance of nucleobindins as potential endocrine precursors. This review aims to summarize some of the most recent findings on the functional significance of NUCB1, NUCB2, as well as encoded proteins and highlights the questions that remain unanswered.

Nesfatin-1 Regulates the Hypothalamo-Pituitary-Ovarian Axis of Fish

ABSTRACT Nesfatin-1 is an anorexigen in goldfish. In the present study, we provide novel data indicating the presence and regulatory effects of nesfatin-1 on the hypothalamo-pituitary-ovarian (HPO) axis of goldfish. Nucleobindin-2 (NUCB2)/nesfatin-1-like immunoreactive (ir) cells are present in the hypothalamus and in the pituitary, suggesting a hypophysiotropic role for nesfatin-1. NUCB2/nesfatin-1-like ir cells colocalize gonadotropin-releasing hormone (GnRH) in the nucleus lateralis tuberis posterioris and the nucleus anterior tuberis of the goldfish hypothalamus. The presence of nesfatin-1 with GnRH in these two nuclei implicated in pituitary hormone release suggests a role for nesfatin-1 on gonadotropin secretion. A single i.p. injection of synthetic goldfish nesfatin-1 (50 ng/g body wt) resulted in an acute decrease (∼75%) in the expression of hypothalamic chicken GnRH-II and salmon GnRH mRNAs at 15 min postinjection in goldfish. Meanwhile, pituitary luteinizing hormone (LH) beta and follicle-stimulating hormone beta mRNAs were also inhibited (∼80%), but only at 60 min postinjection. Nesfatin-1 administration also resulted in a significant reduction (∼60%) in serum LH levels at 60 min postadministration. Nesfatin-1-like immunoreactivity was also found in the follicle cells, but not the oocytes, in zebrafish and goldfish ovaries. Incubation of zebrafish follicles with nesfatin-1 resulted in a significant reduction in basal germinal vesicle breakdown (∼50%) during the oocyte maturation. In addition, nesfatin-1 also attenuated the stimulatory effects of maturation-inducing hormone on germinal vesicle breakdown. Together, the current results indicate that nesfatin-1 is a metabolic hormone with an inhibitory tone on fish reproduction. Nesfatin-1 appears to elicit this suppressive effect through actions on all three tissues in the fish HPO axis.

Ontogenic pattern of nucleobindin-2/nesfatin-1 expression in the gastroenteropancreatic tissues and serum of Sprague Dawley rats.

Nesfatin-1 is a novel metabolic hormone that has glucose-responsive insulinotropic actions. Islet β-cells and gastrointestinal tissues have been reported as abundant sources of nesfatin-1 and its precursor hormone nucleobindin-2 (NUCB2). While nesfatin-1 is emerging as a multifunctional hormone, there are no reports on the developmental expression of NUCB2/nesfatin-1. The main objective of this study was to examine the ontogenic expression of NUCB2 mRNA, and NUCB2/nesfatin-1 immunoreactivity in the pancreas, stomach and duodenum, and the circulating levels NUCB2/nesfatin-1 in Sprague Dawley rats. In addition, we also determined the co-localization of NUCB2/nesfatin-1 and insulin immunoreactivity during development. NUCB2/nesfatin-1 immunoreactivity was found in the rat stomach from postnatal days 13-27. Furthermore, NUCB2/nesfatin-1 immunoreactivity was also detected in the enteroendocrine cells of the duodenum at postnatal days 13 and 27. Duodenal NUCB2 mRNA expression at postnatal day 27 was highest. Serum NUCB2/nesfatin-1 levels on embryonic day 21 and postnatal day 1 were lower than serum NUCB2/nesfatin-1 levels of adults and neonates at postnatal days 13, 20 and 27, gradually increasing with growth, suggesting an increase in its production and secretion from tissues including the gastrointestinal tract and pancreas. Our findings indicate that NUCB2/nesfatin-1 colocalizes with insulin in the islet β-cells at all developmental stages, but the percentage of colocalization varies in an age-dependent manner. These findings suggest that NUCB2/nesfatin-1 has potential age- and tissue-specific role in the developmental physiology of rats during growth.