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

Emilio J Vélez,Suraj Unniappan

doi:10.1038/s41598-020-73840-4

Abstract

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.

Highlights

In recent years, it has been observed that two DNA and calcium-binding secreted peptides named nucleobindins (NUCBs, designated as NUCB1 and NUCB2) are involved in many processes, including the activation of G protein s ignaling[1]
NUCB1/ nesfatin-1-like peptide (NLP) immunoreactivity was mainly located in the cytoplasm of GH3 cells (Fig. 1c)
GH3 cells stained positive for growth hormone (GH) within the cytoplasm were immunopositive for both NUCB1 and NUCB2 (Fig. 2a), and similar results were observed with RC-4B/C cells

Summary

Introduction

It has been observed that two DNA and calcium-binding secreted peptides named nucleobindins (NUCBs, designated as NUCB1 and NUCB2) are involved in many processes, including the activation of G protein s ignaling[1]. NUCBs are present within the pituitary in some species, and NESF regulates pituitary gonadotropin levels[17,18,19,20] These results suggest that pituitary is a site of synthesis of NUCBs and its action. We hypothesized that NUCB-encoded peptides, NESF and NLP, suppress GH synthesis in somatotrophs. This main objective of this research was to determine whether mammalian somatotrophs are a source and site of action of NESF and NLP. An in vitro model (GH3 and RC-4B/C cell lines) was employed to test if NESF and NLP regulate GH gene and protein expression and determine the main signaling pathways (GH3 cells) mediating their action on somatotrophs

Objectives

Methods

Results

Conclusion