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.

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.

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.

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.

Nesfatin-1-like peptide suppresses hypothalamo-pituitary-gonadal mRNAs, gonadal steroidogenesis, and oocyte maturation in fish†.

Nucleobindin (Nucb)-1 and Nucb2 are DNA and Ca2+ binding proteins with multiple functions in vertebrates. Prohormone convertase-mediated processing of Nucb2 results in the production of biologically active nesfatin-1. Nesfatin-1 is involved in the regulation of reproduction in many vertebrates, including fish. Our lab originally reported a nesfatin-1-like peptide (Nlp) encoded in Nucb1 that exhibits nesfatin-1-like metabolic effects. We hypothesized that Nlp has a suppressive role in the reproductive physiology of fish. In this research, whether Nlp regulates reproductive hormones and oocyte maturation in fish were determined. Single intraperitoneal (IP) injection of goldfish Nlp (50 ng/g body weight) suppressed salmon and chicken gonadotropin-releasing hormone (sgnrh and cgnrh2), gonadotropin-inhibiting hormone (gnih) and its receptor (gnihr), and kisspeptin and brain aromatase mRNA expression in the hypothalamus of both male and female goldfish. In the pituitary, Nlp decreased mRNAs encoding lhb, fshb and kisspeptin and its receptor, while a significant increase in gnih and gnihr was observed. In the gonads, lh (only in male fish) and fsh receptor mRNAs were also significantly downregulated in Nlp-injected fish. Sex-specific modulation of gnih, gnihr, and kisspeptin system in the gonads was also observed. Nlp decreased sex steroidogenic enzyme encoding mRNAs and circulating levels of testosterone and estradiol. In addition, incubation of zebrafish ovarian follicles with Nlp resulted in a reduction in oocyte maturation. These results provide evidence for a robust role for Nlp in regulating reproductive hormones in goldfish and oocyte maturation in zebrafish, and these effects resemble that of nesfatin-1.

Nucleobindins and encoded peptides: From cell signaling to physiology.

Nucleobindins (NUCBs) are DNA and calcium binding, secreted proteins with various signaling functions. Two NUCBs, nucleobindin-1 (NUCB1) and nucleobindin-2 (NUCB2), were discovered during the 1990s. These two peptides are shown to have diverse functions, including the regulation of inflammation and bone formation, among others. In 2006, Oh-I and colleagues discovered that three peptides encoded within the NUCB2 could be processed by prohormone convertases. These peptides were named nesfatin-1, 2 and 3, mainly due to the satiety and fat influencing properties of nesfatin-1. However, it was found that nesfatin-2 and -3 have no such effects. Nesfatin-1, especially its mid-segment, is very highly conserved across vertebrates. Although the receptor(s) that mediate nesfatin-1 effects are currently unknown, it is now considered an endogenous peptide with multiple functions, affecting central and peripheral tissues to regulate metabolism, reproduction, endocrine and other functions. We recently identified a nesfatin-1-like peptide (NLP) encoded within the NUCB1. Like nesfatin-1, NLP suppressed feed intake in mice and fish, and stimulated insulin secretion from pancreatic beta cells. There is considerable evidence available to indicate that nucleobindins and its encoded peptides are multifunctional regulators of cell biology and whole animal physiology. This review aims to briefly discuss the structure, distribution, functions and mechanism of action nucleobindins and encoded peptides.