Intraperitoneal administration of kisspeptin-10 modulates follicle maturation, gonadal steroids, calcium and metabolites in Sterlet sturgeon, Acipenser ruthenus.

Kisspeptin is a multifunctional neurohormone, primarily involved in the regulation of reproduction. We tested whether peripheral administration of kisspeptin10 (KP-10) via intraperitoneal injection or slow release affects reproductive hormones and metabolites in Sterlet sturgeon (Acipenser ruthenus). Plasma and mucus 17ß-estradiol (E2), and testosterone (T), plasma and follicular vitellogenin (VTG) and calcium (Ca) as well as glucose and lipids were determined. Mature Sterlet sturgeon were grouped into six groups: saline i.p injection (control), human kisspeptin (hKP-10) i.p injection; acipenser kisspeptin (aKP-10) i.p injection; hKP-10 (slow release); aKP-10 (slow-release) and no treatment control. No effect for KP-10 on sturgeon body weight was found after 4 weeks of treatment. Multivariate analysis revealed a significant disparity in plasma E2 levels. It was significantly different between groups (time, P = 0.0022). E2 in epithelia mucosa showed significant difference between and within groups in the acute group (time, P = 0.0252; treatment, P = 0.0423; time × treatment, P = 0.0429). T levels were unaffected by treatments (P > 0.05). The presence of synthetic aKP-10 led to an elevation in oocyte and plasma VTG levels (P < 0.05). Prolonged exposure to this peptide resulted in an increase in plasma calcium levels. Simultaneously, there was an augmentation in the number of mature follicles. Regardless of the duration of exposure, aKP-10 significantly elevated plasma glucose levels in Sterlet (P < 0.0). Additionally, KP-10 led to an increase in plasma lipids and cholesterol in Sterlet. Overall, our data support an involvement for KP-10 in the regulation of gonadal steroid hormones, oocyte maturation and metabolite levels in sturgeon, suggesting a positive role for this peptide in the reproductive physiology of this species.

Suppressive action of nesfatin-1 and nesfatin-1-like peptide on cortisol synthesis in human adrenal cortex cells.

Nucleobindin-derived peptides, nesfatin-1 [NESF-1] and nesfatin-1-like-peptide [NLP] have diverse roles in endocrine and metabolic regulation. While both peptides showed a stimulatory effect on the synthesis of proopiomelanocortin (POMC), the adrenocorticotropic hormone (ACTH) precursor in mouse corticotrophs, whether NESF-1 and NLP have any direct effect on glucocorticoid [GC] synthesis in the adrenal cortex remains unknown. The main aim of this study was to determine if NESF-1 and/or NLP act directly on adrenal cortex cells to regulate cortisol synthesis in vitro. Whether NLP injection affects stress-hormone gene expression in the adrenal gland and pituitary in vivo in mice was also assessed. In addition, cortisol synthetic pathway in Nucb1 knockout mice was studied. Human adrenal cortical [H295R] cells showed immunoreactivity for both NUCB1/NLP and NUCB2/NESF-1. NLP and NESF-1 decreased the abundance of steroidogenic enzyme mRNAs, and cortisol synthesis and release through the AC/PKA/CREB pathway in H295R cells. Similarly, intraperitoneal injection of NLP in mice decreased the expression of enzymes involved in glucocorticoid (GC) synthesis in the adrenal gland while increasing the expression of Pomc, Pcsk1 and Crhr1 in the pituitary. Moreover, the melanocortin 2 receptor (Mc2r) mRNA level was enhanced in the adrenal gland samples of NLP injected mice. However, the global genetic disruption in Nucb1 did not affect most steroidogenic enzyme mRNAs, and Pomc, Pcsk2 and Crhr1 mRNAs in mice adrenal gland and pituitary gland, respectively. Collectively, these data provide the first evidence for a direct inhibition of cortisol synthesis and secretion by NLP and NESF-1. NUCB peptides might still elicit a net stimulatory effect on GC synthesis and secretion through their positive effects on ACTH-MC2R pathway in the pituitary.

Growth factors and female reproduction in vertebrates.

Female reproductive efficiency is influenced by the outcomes of various processes, including folliculogenesis, apoptosis, response to gonadotropin signaling, oocyte maturation, and ovulation. The role of hormones in regulating these processes and other reproductive activities has been well established. It is becoming increasingly evident that in addition to well-characterized hormones, growth factors play vital roles in regulating some of these reproductive activities. Growth factors and their receptors are widely distributed in vertebrate ovaries at different stages of ovarian development, indicating their involvement in intraovarian reproductive functions. In the ovary, cell surface receptors allow growth factors to regulate intraovarian reproductive activities. Understanding these actions in the reproductive axis would provide a tool to target growth factors and/or their receptors to yield desirable reproductive outcomes. These include enrichment of in vitro maturation and fertilization culture media, and management of infertility. This review discusses some widely characterized growth factors belonging to the TGF, EGF, IGF, FGF, and BDNF family of peptides and their role in female reproduction in vertebrates, with a focus on mammals.

Tissue-Specific Modulation of Gluco- and Growth-Regulatory Factor Abundance by Nesfatin-1 and Nesfatin-1-like Peptide in Goldfish.

Nesfatin-1 and nesfatin-1-like peptide (Nlp) are derived from precursors nucleobindin-2 and -1, two calcium and DNA binding proteins, respectively. Both peptides exhibit hormone-like actions in mammals and fish. These functions include insulinotropic effects of nesfatin-1 and Nlp seen in mice and their growth hormone suppressive actions reported in goldfish. We hypothesized that nesfatin-1 and Nlp are insulin stimulatory (in adipose tissue) and modulate growth hormone and insulin-like growth factors and glucose transporters in goldfish. To test this, goldfish were intraperitoneally injected with either nesfatin-1 or Nlp (50 ng/g BW) or saline alone (control) and sampled at one-hour post-injection (in vivo study). In a separate study, tissue samples were collected and were incubated with either nesfatin-1 or Nlp for one or six hours (in vitro study). Transcript (mRNA) abundance data from the adipose tissue suggest that both nesfatin-1 and Nlp significantly upregulate the abundance of preproinsulin, insulin receptors, and pcsk1 and pcsk2 mRNAs. Meanwhile, the abundance of preproglucagon mRNA in the adipose tissue was significantly downregulated in both in vivo and in vitro studies. These results agree with the insulinotropic and glucagonostatic roles for nesfatin-1 and Nlp reported in rodents. The transcript abundance of growth regulators (igf1, igf2a, and ghra) and glucose transporters (slc2a2 and slc5a1) were upregulated in the muscle, while an opposite effect on these mRNAs was found in the liver of goldfish following nesfatin-1 and Nlp administration. Our results suggest that both nesfatin-1 and Nlp have tissue-specific regulatory roles on growth and glucoregulatory elements in the liver and muscle of goldfish. This agrees with our previous studies that showed a suppressive action of nesfatin-1 on growth hormone in goldfish liver. The results presented here provide strong supportive/confirmatory evidence for tissue-specific insulinotropic and gluco- and growth-regulatory actions of nesfatin-1 and Nlp in goldfish.

Dietary protein:lipid ratio modulates somatic growth and expression of genes involved in somatic growth, lipid metabolism and food intake in Pejerrey fry (Odontesthes bonariensis).

Pejerrey is a freshwater fish from South America with high potential for aquaculture. This study was designed to determine the effects of different dietary protein:lipid ratio on growth rate and the expression of growth, lipid metabolism and feeding-related genes of this species during early developmental stages. Pejerrey fry were fed for 60 days with four experimental diets containing low (400 g Kg-1) or high (500 g Kg-1) protein (LP or HP, respectively) and low (120 g Kg-1) or high (200 g Kg-1) lipid (LL or HL, respectively), in the combinations: LP-LL; LP-HL; HP-LL and HP-HL. Measurements of growth, lipid and fatty acid content of fry, expression of genes from the endocrine axis (gh, ghrs, igfs), fatty acid metabolism (∆6-desaturase), and food intake behavior (nucb2/nesfatin-1) were collected. Fry fed with diets LP-LL and HP-LL showed the highest growth rate and growth hormone (gh) mRNA expression levels. The gene expression of ∆6-desaturase was high in head of fry fed with diet LP-HL. The mRNA expression of nucb2/nesfatin-1 and gh followed the same patterns in head, and the inverse pattern in body. In conclusion, diets with LL ensure a higher growth of pejerrey fry compared to those that contain HL, without altering the final lipid amount nor the fatty acid profile on fry. In LL groups, the expression of genes from the GH-IGF axis is associated with the observed promotion of somatic growth. The expression of nucb2/nesfatin-1 indicates an effect of this peptide not related to food intake regulation, e.g., a negative regulatory role on GH expression, that would warrant future research.

Liver and muscle-specific effects of phoenixin-20 on the insulin-like growth factor system mRNAs in zebrafish.

OBJECTIVE: Phoenixin-20 (Pnx-20) is a bioactive peptide with endocrine-like actions in vertebrates. Recent studies suggest Pnx-20 promotes growth hormone/insulin-like growth factors (Gh/Igf) axis, an important endocrine regulator of growth in mammals and fish. DESIGN: In this research, we determined whether Pnx-20 affects the different members of the Igf family, its binding proteins and receptors (Igf-system) in zebrafish liver and muscle. RESULTS: In vivo administration of Pnx-20 downregulated igfs, igf receptors (igfrs) and igf binding protein (igfbp) 5 mRNA expression in the liver of male and female zebrafish at both 1 and 6 h post-intraperitoneal (IP) injection. Interestingly, this effect occurred at a relatively earlier timepoint in female zebrafish suggesting sex-specific differences in Pnx-20 action. Besides, either 6 or 24 h in vitro incubations with Pnx-20 downregulated the expression of all igfs, igfrs and igfbp5 mRNAs (except igf2a) analyzed in a zebrafish liver cell (ZFL) line. Moreover, siRNA-mediated knockdown of Pnx-20 upregulated all Igf-system mRNAs analyzed in ZFL cells. Together, these results (both in vivo and in vitro) revealed a general suppressive action for both endogenous and exogenous Pnx-20 on the hepatic Igf-system of zebrafish. In contrast, a general sex-specific upregulation of the Igf-system mRNAs analyzed was found in the muscle of Pnx-20 injected fish. Future research should explore the sex- and time-differences observed in the present study. CONCLUSIONS: Collectively, this research shows that Pnx-20 is a tissue-specific regulator of the liver (suppressor) and muscle (stimulant) Igf signaling in both male and female zebrafish.

Localization of nucleobindin2/nesfatin-1-like immunoreactivity in human lungs and neutrophils.

Nucleobindin2 (NUCB2)/nesfatin-1 expression in human plasma positively correlates with the expression of pro-inflammatory cytokines in patients with chronic obstructive pulmonary disease (COPD), implicating its potential role in neutrophilic lung inflammation. There are no data on the localization of nucleobindin2 (NUCB2)/nesfatin-1 in human lungs and inflammatory cells. We examined the localization of NUCB2/nesfatin-1-immunoreactivity in normal and inflamed human lungs obtained from COPD patients and neutrophils with light and immunoelectron microscopy. Immunohistology showed localization of NUCB2/nesfatin-1-like immunoreactivity in the bronchiolar epithelium, alveolar septa, vascular endothelium and various immune cells of normal and inflamed lungs. Further, NUCB2/nesfatin-1-like immunoreactivity accumulated within 0.5 µm of the plasma membrane in human neutrophils following 90 min of 1 ng/mL LPS stimulation. NUCB2/nesfatin-1-like immunoreactivity was also found to localize in euchromatic portions of neutrophilic nuclei at five times the mean concentration compared to heterochromatin. Finally, our results indicate that NUCB2/nesfatin-1-like immunoreactivity is predominantly cytoplasmic including that in the Golgi complex and vesicles as it localizes at two times the concentration in neutrophilic cytoplasm compared to nucleus. Our study is the first to detail the localization of NUCB2/nesfatin-1-like immunoreactivity in lungs and neutrophils, and nuclear localization of NUCB2/nesfatin-1 also implicates its potential role in transcriptional regulation.

Insulin induces steroidogenesis in canine luteal cells via PI3K-MEK-MAPK.

Glucose uptake increases in canine luteal cells under insulin treatment. We hypothesize that insulin also increases luteal cell steroidogenesis. Dogs underwent elective ovariohysterectomy from days 10-60 post ovulation and their corpora lutea (CL) and blood samples were collected. Deep RNA sequencing determined differentially expressed genes in CL; those related to insulin signaling and steroidogenesis were validated in vivo by qPCR and their respective proteins by Western blotting and immunofluorescence. Next, luteal cell cultures were stimulated with insulin with or without inhibition of MAPK14, MAP2K1 and PI3K. Studied proteins except P450 aromatase showed the same expression pattern of coding genes in vivo. The expression of HSD3B and CYP19A1 was higher in insulin-treated cells (P < 0.005). Following respective pathway blockades, the culture medium had decreased concentrations of progesterone (P4) and 17b-estradiol (E2) (P < 0.01). Our results indicate that insulin increases HSD3B and CYP19A1 expression via MAPK and PI3K, and contributes to the regulation of P4 and E2 production in canine luteal cells.

Nesfatin-1-like peptide is a negative regulator of cardiovascular functions in zebrafish and goldfish.

Nucleobindins (NUCB1 and NUCB2) were originally identified as calcium and DNA binding proteins. Nesfatin-1 (NEFA/nucleobindin-2-Encoded Satiety and Fat-Influencing proteiN-1) is an 82 amino acid anorexigenic peptide encoded in the N-terminal region of NUCB2. We have shown that nesfatin-1 is a cardiosuppressor in zebrafish. Both NUCB1 and NUCB2 possess a -very highly conserved bioactive core. It was found that a nesfatin-1-like peptide (NLP) encoded in NUCB1 suppresses food intake in fish. In this research, we investigated whether NLP has nesfatin-1-like effects on cardiovascular functions. NUCB1/NLP-like immunoreactivity was found in the atrium and ventricle of the heart and skeletal muscle of zebrafish. Intraperitoneal injection (IP) of either zebrafish NLP or rat NLP suppressed cardiac functions in both zebrafish and goldfish. Irisin and RyR1b mRNA expression was downregulated by NLP in zebrafish cardiac and skeletal muscles. However, cardiac ATP2a2 mRNA expression was elevated after NLP injection. Administration of scrambled NLP did not affect irisin, RyR1b or ATP2a2 mRNA expression in zebrafish. Together, these results implicate NLP as a suppressor of cardiovascular physiology in zebrafish and goldfish.

Nesfatin-1 stimulates the hypothalamus-pituitary-interrenal axis hormones in goldfish.

Stress in vertebrates is mediated by the hypothalamus-pituitary-adrenal (in mammals)/interrenal (in fish) (HPA/I) axis, which produces the corticotropin-releasing factor (CRF), adrenocorticotropic hormone (ACTH), and corticosteroids, respectively. Nesfatin-1, a novel anorexigenic peptide encoded in the precursor nucleobindin-2 (NUCB2), is increasingly acknowledged as a peptide that influences the stress axis in mammals. The primary aim of this study was to characterize the putative effects of nesfatin-1 on the fish HPI axis, using goldfish (Carassius auratus) as an animal model. Our results demonstrated that nucb2/nesfatin-1 transcript abundance was detected in the HPI tissues of goldfish, with most abundant expression in the pituitary. NUCB2/nesfatin-1-like immunoreactivity was found in the goldfish hypothalamus, pituitary, and interrenal cells of the head kidney. GPCR12, a putative receptor for nesfatin-1, was also detected in the pituitary and interrenal cells. NUCB2/nesfatin-1-like immunoreactivity was observed in ACTH-expressing pituitary corticotrophs. Acute netting and restraint stress upregulated nucb2/nesfatin-1 mRNA levels in the forebrain, hypothalamus, and pituitary, as well as crf and crf-r1 expression in the forebrain and hypothalamus. Intraperitoneal and intracerebroventricular administration of nesfatin-1 increased cortisol release and hypothalamic crf mRNA levels, respectively. Finally, we found that nesfatin-1 significantly stimulated ACTH secretion from dispersed pituitary cells in vitro. Collectively, our data provide the first evidence showing that nesfatin-1 is a stress responsive peptide, which modulates the stress axis hormones in fish.

Nesfatin-1 is an inhibitor of the growth hormone-insulin-like growth factor axis in goldfish (Carassius auratus).

Nesfatin-1, an 82 amino acid peptide cleaved from the N-terminal of its precursor nucleobindin-2 (NUCB2), is emerging as a multifunctional peptide in fish. The present study aimed to determine whether nesfatin-1 plays a role in fish somatic growth by modulating the growth hormone (GH)/insulin-like growth factor (IGF) axis, using a representative teleost model, the goldfish (Carassius auratus). The results demonstrated that a single i.p. injection of synthetic goldfish nesfatin-1 significantly decreased the expression of hypothalamic pacap (approximately 90%) and pituitary Gh (approximately 90%) mRNAs at 15 minutes post-injection. Serum GH levels were also reduced as a result of nesfatin-1 administration, by approximately 45% and 55% at 15 and 30 minutes post-injection, respectively. Likewise, in vitro treatment of goldfish dispersed pituitary cells with nesfatin-1 reduced Gh secretion, suggesting that nesfatin-1 acts directly on pituitary somatotrophs to inhibit Gh release. Exposure of cultured liver fragments to nesfatin-1 (0.1, 1 and 10 nmol L-1 ) led to a significant reduction in igf-1 mRNA at 120 minutes and of igf-II mRNA at 30 and 60 minutes post-incubation. Collectively, these results indicate a suppressive role for nesfatin-1 on the goldfish GH/IGF axis. Immunohistochemical studies demonstrated that NUCB2/nesfatin-1-like immunoreactivity, although present in the goldfish pituitary, is not colocalised with GH in goldfish somatotrophs. Thus, nesfatin-1 does not appear to act in an autocrine manner to regulate GH secretion. Taken together, this research found that the pituitary gland is an important source of endogenous NUCB2/nesfatin-1 and also that nesfatin-1 directly suppresses the Gh/IGF axis in goldfish.

Nucleobindin-derived nesfatin-1 and nesfatin-1-like peptide stimulate pro-opiomelanocortin synthesis in murine AtT-20 corticotrophs through the cAMP/PKA/CREB signaling pathway.

Nucleobindin (NUCB)-derived peptides, nesfatin-1 (NES-1) and nesfatin-1-like peptide (NLP) have several physiological roles in vertebrates. While NES-1 is implicated in stress, whether NUCB1/NLP and NUCB2/NES-1 have any effect on proopiomelanocortin (POMC) remains unknown. The main aim of this study was to determine if NES-1 and/or NLP affect POMC synthesis in mouse corticotrophs. Immunocytochemistry was employed to target NUCB colocalization with POMC in immortalized mouse tumoral corticotrophs (AtT-20 cells). The ability of NES-1 and NLP to modulate POMC mRNA and protein in AtT-20 cells was assessed by qPCR and Western blot, respectively. Moreover, cell-signaling molecules mediating the effect of NES-1 and NLP on POMC synthesis in mouse tumoral corticotrophs were studied using pharmacological blockers. Mouse tumoral corticotrophs showed immunoreactivity for both NUCB1/NLP and NUCB2/NES-1. Both NES-1 and NLP exerted a stimulatory effect on POMC transcript abundance and protein expression in a dose- and time-dependent manner. This effect was comparable to corticotropin-releasing factor (CRF, positive control) stimulation of POMC. Incubation of mouse tumoral corticotrophs with NES-1 or NLP upregulated the phosphorylation of protein kinase A (PKA) and cAMP-response element-binding protein (CREB). The stimulatory effect of these peptides on POMC transcript abundance and protein expression was blocked by the PKA inhibitor, H89, and an adenylate cyclase inhibitor, 2',3'-dideoxyadenosine (DDA). These pharmacological studies indicate that NES-1 and NLP act through the cAMP/PKA/CREB cellular pathway to stimulate POMC synthesis. Our results provide molecular evidence to support a stimulatory role for nucleobindin-derived peptides on POMC synthesis from corticotrophs. Collectively, this research indicates that corticotrophs produce NUCBs, and the encoded peptides NES-1 and NLP could elicit a direct action to stimulate the pituitary stress hormone. This stimulatory effect is mediated by an uncharacterized G protein-coupled receptor (GPCR) that utilizes the cAMP/PKA/CREB pathway.

The sympathetic/beta-adrenergic pathway mediates irisin regulation of cardiac functions in zebrafish.

Irisin is a 23 kDa myokine encoded in its precursor, fibronectin type III domain containing 5 (FNDC5). The exercise-induced increase in the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α) promotes FNDC5 mRNA, followed by the proteolytic cleavage of FNDC5 to release irisin from the skeletal or cardiac muscle into the blood. Irisin is abundantly expressed in skeletal and cardiac muscle and plays an important role in feeding, modulates appetite regulatory peptides, and regulates cardiovascular functions in zebrafish. In order to determine the potential mechanisms of acute irisin effects, in this research, we explored whether adrenergic or muscarinic pathways mediate the cardiovascular effects of irisin. Propranolol (100 ng/g B·W) alone modulated cardiac functions, and when injected in combination with irisin (0.1 ng/g B·W) attenuated the effects of irisin in regulating cardiovascular functions in zebrafish at 15 min post-injection. Atropine (100 ng/g B·W) modulated cardiovascular physiology in the absence of irisin, while it was ineffective in influencing irisin-induced effects on cardiovascular functions in zebrafish. At 1 h post-injection, irisin downregulated PGC-1 alpha mRNA, myostatin-a and myostatin-b mRNA expression in zebrafish heart and skeletal muscle. Propranolol alone had no effect on the expression of these mRNAs in zebrafish and did not alter the irisin-induced changes in expression. At 1 h post-injection, irisin siRNA downregulated PGC-1 alpha, troponin C and troponin T2D mRNA expression, while upregulating myostatin a and b mRNA expression in zebrafish heart and skeletal muscle. Atropine alone had no effects on mRNA expression, and was unable to alter effects on mRNA expression of siRNA. Overall, this research identified a role for the sympathetic/beta-adrenergic pathway in regulating irisin effects on cardiovascular physiology and cardiac gene expression in zebrafish.

Ion-poor water and dietary salt deprivation upregulate the ghrelinergic system in the goldfish (Carassius auratus).

Because the ghrelinergic system in teleost fishes is broadly expressed in organs that regulate appetite as well as those that contribute to the regulation of salt and water balance, we hypothesized that manipulating salt and water balance in goldfish (Carassius auratus) would modulate the ghrelinergic system. Goldfish were acclimated to either freshwater (FW) or ion-poor FW (IPW) and were fed either a control diet containing 1% NaCl or low-salt diet containing 0.1% NaCl. Endpoints of salt and water balance, i.e., serum Na+ and Cl- levels, muscle moisture content and organ-specific Na+ -K+ -ATPase (NKA) activity, were examined in conjunction with brain, gill and gut mRNA abundance of preproghrelin and its receptor, growth hormone secretagogue receptor (ghs-r). Acclimation of fish to IPW reduced serum osmolality and Cl- levels and elevated kidney NKA activity, while FW fish fed a low NaCl diet exhibited a modest reduction in muscle moisture content but otherwise no apparent osmoregulatory disturbance. In contrast, a combined treatment of IPW acclimation and low dietary NaCl content reduced serum osmolality and Cl- levels, elevated muscle moisture content and increased gill, kidney and intestinal NKA activity. This intensified response to the combined effects of water and dietary ion deprivation is consistent with an increased effort to enhance ion acquisition. In association with these latter observations, a significant upregulation of preproghrelin mRNA expression in brain and gut was observed. A significant increase in ghs-r mRNAs was also observed in the gill of goldfish acclimated to IPW alone but a reduction in dietary NaCl content did not impact the ghrelinergic system of goldfish in FW. The results support the hypothesis that the ghrelinergic system is modulated in response to manipulated salt and water balance. Whether the central and peripheral ghrelinergic system contributes to ionic homeostasis in goldfish currently remains unclear and warrants further research.

Mouse gastric mucosal endocrine cells are sources and sites of action of Phoenixin-20.

Energy homeostasis is is determined by food intake and energy expenditure, which are partly regulated by the cross-talk between central and peripheral hormonal signals. Phoenixin (PNX) is a recently discovered pleiotropic neuropeptide with isoforms of 14 (PNX-14) and 20 (PNX-20) amino acids. It is a potent reproductive peptide in vertebrates, regulating the hypothalamo-pituitary-gonadal axis (HPG). It has been identified as a regulator of food intake during light phase when injected intracerebroventricularly in rats. In addition, plasma levels of PNX also increased after food intake in rats, suggesting that it might have possible roles in energy homeostasis. We hypothesized that gut is a source and site of action of PNX in mice. Immunoreactivity for PNX and its putative receptor, super-conserved receptor expressed in brain (SREB3; also known as the G-protein coupled receptor 173/GPR 173) was found in the stomach and intestine of male C57/BL6 J mice, and in MGN3-1 (mouse stomach endocrine) cells and STC-1 (mouse enteroendocrine) cells. In MGN3-1 cells, PNX-20 significantly upregulated ghrelin (10 nM) and ghrelin-O-acyl transferase (GOAT) mRNAs (1000 nM) at 6 h. In STC-1 cells, it significantly suppressed CCK (100 nM) at 2 h. No effects were found on other intestinal hormones tested (glucagon like peptide-1, glucose dependent insulinotropic polypeptide, and peptide YY). Together, these results indicate that PNX-20 is produced in the gut, and it could act directly on gut cells to regulate metabolic hormones.

Nesfatin-1 and Nesfatin-1-like peptide suppress basal and TRH-Induced expression of prolactin and prolactin regulatory element-binding protein mRNAs in rat GH3 somatolactotrophs.

Prolactin (PRL), mainly synthesized and secreted by the lactotrophs and somatolactotrophs of the anterior pituitary, is a pleiotropic hormone that regulates lactation. In the last decade, nesfatin-1 (NESF) and NESF-like peptide (NLP), encoded in nucleobindin 1 and 2 (NUCB1 and NUCB2), respectively, were characterized as metabolic factors with a potential role in the control of pituitary hormones. We hypothesized that NUCBs and their encoded peptides (NESF and NLP) suppress PRL transcription in the pituitary. The main objective of this research was to determine whether exogenous NESF and NLP, and/or endogenous NUCB1 and NUCB2 regulate the expression of prl and preb mRNAs. Using immortalized rat somatolactotrophs (GH3 cells), dose-response studies were performed to test whether NESF and NLP affect prl and preb. Moreover, the ability of these peptides to modulate the effects of the PRL stimulator thyrotropin releasing hormone (TRH) was studied. Besides, the effects of siRNA-mediated knockdown of endogenous NUCBs on prl and preb mRNAs were determined. NESF and NLP reduced the transcription of prl and preb in GH3 cells. Both NESF and NLP also prevented the stimulatory effects of TRH prl and preb expression. The knockdown of endogenous NUCB1 attenuates both basal prl and TRH-induced expression of prl and preb, while the silencing of NUCBs did not affect the actions of exogenous NESF or NLP. Overall, this work reveals that NUCBs and encoded-peptides are novel regulators of PRL. Future research should test whether the effects observed here in GH3 cells are preserved both in vivo and at the post-transcriptional level.

Loss of Nucleobindin-2/Nesfatin-1 increases lipopolysaccharide-induced murine acute lung inflammation.

NUCB2/nesfatin-1 is expressed in variety of tissues. Treatment with nesfatin-1 reduces inflammation in rat models of subarachnoid hemorrhage-induced oxidative brain damage and traumatic brain injury as well as myocardial injury. There is only one study showing anti-inflammatory actions of nesfatin-1 on acute lung inflammation. To more precisely determine the role of NUCB2/nesfatin-1 in acute lung inflammation, we conducted a study using NUCB2/nesfatin-1 knockout (NKO) mice as well as neutrophils isolated from the bone marrows of WT and NKO mice. Our findings suggest that the absence of NUCB2/nesfatin-1 significantly increases the accumulation of adherent neutrophils by approximately 3 times compared with WT within LPS-treated lungs. Integrating this with observations from both BALF and neutrophil cytokine expression, we propose that although neutrophils lacking NUCB2/nesfatin-1 individually secrete less pro-inflammatory cytokines compared with stimulated WT cells, the result of knocking out NUCB2/nesfatin-1 is net pro-inflammatory. No change was found in NUCB2/nesfatin-1 mRNA or protein expression comparing WT LPS and PBS-treated samples. Taken together, our results show that NUCB2/nesfatin-1 is constitutively expressed in mouse lungs and neutrophils and demonstrates anti-inflammatory properties in mouse lungs during acute lung injury, by inhibiting adherent neutrophil accumulation and inflammatory cytokine expression.

Nesfatin-1 and nesfatin-1-like peptide suppress growth hormone synthesis via the AC/PKA/CREB pathway in mammalian somatotrophs.

Nesfatin-1 (NESF) and NESF-like peptide (NLP), encoded in nucleobindin 2 and 1 (NUCB2 and NUCB1), respectively, are orphan ligands and metabolic factors. We hypothesized that NESF and NLP suppress growth hormone (GH) synthesis, and aimed to determine whether mammalian somatotrophs are a source and site of action of these peptides. Using immortalized rat somatotrophs (GH3 cells), NUCB expression was determined by qPCR, immunofluorescence and Western blot. NESF and NLP binding to GH3 cells was tested using fluorescence imaging. Both time- and concentration-dependent studies were performed to test whether NESF and NLP affect GH. Moreover, the ability of these peptides to modulate the effects of ghrelin, and cell-signaling pathways were studied. GH3 cells express NUCB mRNAs and protein. Labeled NESF and NLP bind to the surface of GH3 cells, and incubation with either NESF or NLP decreased GH mRNA and protein expression, downregulated pit-1 mRNA, and blocked the GH stimulatory effects of ghrelin. Pre-incubation with either of these peptides reduced CREB phosphorylation by an AC-activator, but not when PKA was directly activated by a cAMP analog. Our results indicate that rat somatotrophs are a source of NUCBs, and that NESF and NLP downregulate GH synthesis through the AC/PKA/CREB signaling pathway.

Feeding and food availability modulate brain-derived neurotrophic factor, an orexigen with metabolic roles in zebrafish.

Emerging findings point to a role for brain-derived neurotrophic factor (BDNF) on feeding in mammals. However, its role on energy balance is unclear. Moreover, whether BDNF regulates energy homeostasis in non-mammals remain unknown. This research aimed to determine whether BDNF is a metabolic peptide in zebrafish. Our results demonstrate that BDNF mRNAs and protein, as well as mRNAs encoding its receptors trkb2, p75ntra and p75ntrb, are detectable in the zebrafish brain, foregut and liver. Intraperitoneal injection of BDNF increased food intake at 1, 2 and 6 h post-administration, and caused an upregulation of brain npy, agrp and orexin, foregut ghrelin, and hepatic leptin mRNAs, and a reduction in brain nucb2. Fasting for 7 days increased bdnf and p75ntrb mRNAs in the foregut, while decreased bdnf, trkb2, p75ntra and p75ntrb mRNAs in the brain and liver. Additionally, the expression of bdnf and its receptors increased preprandially, and decreased after a meal in the foregut and liver. Finally, we observed BDNF-induced changes in the expression and/or activity of enzymes involved in glucose and lipid metabolism in the liver. Overall, present results indicate that BDNF is a novel regulator of appetite and metabolism in fish, which is modulated by energy intake and food availability.