Nesfatin-1-Like Peptide (mouse)

Nucleobindin-1 has high sequence similarity to nucleobindin-2, which encodes the anorectic and metabolic peptide, nesfatin-1. We previously reported a nesfatin-1-like peptide (NLP), anorectic in fish and insulinotropic in mice islet beta-like cells. The main objective of this research was to determine whether NLP is a metabolic regulator in male Wistar rats. A single intraperitoneal (IP) injection of NLP (100 μg/kg BW) decreased food intake and increased ambulatory movement, without causing any change in total activity or energy expenditure when compared to saline-treated rats. Continuous subcutaneous infusion of NLP (100 μg/kg BW) using osmotic mini-pumps for 7 days caused a reduction in food intake on days 3 and 4. Similarly, water intake was also reduced for two days (days 3 and 4) with the effect being observed during the dark phase. This was accompanied by an increased RER and energy expenditure. However, decreased whole-body fat oxidation, and total activity were observed during the long-term treatment (7 days). Body weight gain was not significantly different between control and NLP infused rats. The expression of mRNAs encoding adiponectin, resistin, ghrelin, cholecystokinin and uncoupling protein 1 (UCP1) were significantly upregulated, while leptin and peptide YY mRNA expression was downregulated in NLP-treated rats. These findings indicate that administration of NLP at 100 μg/kg BW reduces food intake and modulates whole body energy balance. In summary, NLP is a novel metabolic peptide in rats.

Nesfatin-1 (82 amino acid) is an anorexigenic and insulinotropic peptide encoded in a secreted precursor, nucleobindin-2 (NUCB2). Nucleobindin-1 (NUCB1) is a protein with very high sequence similarity to NUCB2. We hypothesized that a nesfatin-1 like peptide (NLP) is encoded in NUCB1, and this peptide is biologically active. In silico analysis found a signal peptide cleavage site at position 25 (Arginine) and 26 (Valine) preceding the NLP region in NUCB1 sequence, and potential proprotein convertase cleavage sites at Lys-Arg (KR), forming a 77 amino acid NLP. RT-PCR studies found NUCB1 mRNA in both pancreas and MIN6 cells. NUCB1-like immunoreactivity was detected in mouse insulinoma (MIN6) cells, and pancreatic islet beta cells of mice. In order to determine the biological activity of NLP, MIN6 cells were incubated with synthetic rat NLP. NLP (10nM and 100nM) upregulated preproinsulin mRNA expression and insulin secretion at 1h post-incubation. In identical experiments using MIN6 cells, a scrambled peptide based on the NLP sequence did not elicit any effects on preproinsulin mRNA expression or insulin secretion. From this result, it is clear that an intact NLP sequence is required for its biological activity. NLP appears as another endogenous insulinotropic peptide encoded in NUCB1.

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.