Rice Endoplasmic Protein-Derived Peptides, Rice-Ghretropins A and B, Stimulate Ghrelin Release in MGN3-1 Cells and Increase Plasma Acylated Ghrelin and Food Intake in Mice.

In this study, we demonstrated that novel rice-derived bioactive peptides promote the secretion of ghrelin, an endogenous orexigenic hormone secreted from the stomach. The enzymatic digest of rice endosperm protein with subtilisin, a microorganism-derived enzyme, stimulated acylated ghrelin secretion in the ghrelin-releasing cell line MGN3-1 and increased food intake after oral administration in mice. By performing a comprehensive analysis based on structure-activity relationships, we selected candidate peptides from over 30,000 peptides in the rice digest. Among them, we found that QAFEPIRSV and TNPWHSPRQGSF, corresponding to the amino acid sequence of the rice endoplasmic proteins glutelin A1 or A2(52-60) and B1 or B2(31-42), respectively, stimulated acylated ghrelin release in MGN3-1 cells. We named them rice-ghretropins A and B. Pyroglutamate formation of rice-ghretropin A, [pyr1]-rice-ghretropin A, also promoted ghrelin secretion. Furthermore, oral administration of rice-ghretropins increased food intake, plasma ghrelin concentration, and small intestinal transit in mice. In addition, the subtilisin digest of the rice protein significantly increased food intake for 4 h in 9 month-old (control: 0.61 ± 0.049 g; digest: 0.83 ± 0.059 g) and 24 month-old mice (control: 0.52 ± 0.067 g; digest: 1.01 ± 0.064 g). In summary, we found that novel bioactive peptides, namely, rice-ghretropins, from the enzymatic digest of rice endosperm stimulated acylated ghrelin secretion and increased food intake. This is the first report of rice-derived exogenous bioactive peptides that increase acylated ghrelin secretion.

Comprehensive analysis of a dipeptide library to identify ghrelin release-modulating peptides.

We performed a comprehensive analysis of ghrelin release-modulating activity of a dipeptide library using MGN3-1, a ghrelin-producing cell line. We found that most dipeptides suppress ghrelin secretion, whereas the N-terminal Ser-containing dipeptides and a few others stimulate it. N-terminal amino acid residues, but not C-terminal residues, play a dominant role in the effects of dipeptides. Among dipeptides, Leu-Ile (LI) and Ser-Val (SV) most strongly suppress and stimulate ghrelin secretion, respectively. LI activates Gi signaling and SV acts via the MAPK pathway. Orally administered LI and SV reduce and increase plasma ghrelin levels and food intake in mice, respectively. In conclusion, LI and SV, found based on the comprehensive screening of a dipeptide library, modulate ghrelin secretion in vitro and in vivo.

Identification of Rubisco anxiolytic-like peptides (rALPs) by comprehensive analysis of spinach green leaf protein digest.

Rubisco, an enzyme for photosynthetic carbon dioxide fixation, is a major green leaf protein and known as the most abundant protein on the Earth. We found that Rubisco digested mimicking gastrointestinal enzymatic conditions exhibited anxiolytic-like effects after oral administration in mice. Based on a comprehensive peptide analysis of the digest using nanoLC-Orbitrap-MS and the structure-activity relationship of known anxiolytic-like peptides, we identified SYLPPLTT, SYLPPLT and YHIEPV [termed Rubisco anxiolytic-like peptide (rALP)-1, rALP-1(1-7) and rALP-2, respectively], which exhibited potent anxiolytic-like effects after oral administration. The anxiolytic-like effects of rALP-1/rALP-1(1-7) were blocked by a serotonin 5-HT1A receptor antagonist, whereas rALP-2-induced effects were inhibited by a δ-opioid receptor antagonist. In conclusion, novel Rubisco-derived anxiolytic-like peptides, rALP-1/rALP-1(1-7) and rALP-2, act via independent neural pathways.

Orally active anti-hypertensive peptides found based on enteroendocrine cell responses to a dipeptide library.

We previously reported that an orally administered dipeptide, Arg-Phe (RF), which causes enteroendocrine cell responses, lowered blood pressure in spontaneously hypertensive rats (SHRs). In this study, we found that Phe-Trp (FW), induced the most potent enteroendocrine cell responses out of total 338 dipeptides. An FW analogue, Phe-Trp-Gly-Lys (FWGK), which was effectively produced by tryptic digestion of bovine serum albumin, decreased blood pressure after oral administration. The minimum effective dose of FWGK (50 µg/kg) was 1/300 of that of RF (15 mg/kg). FWGK stimulated cholecystokinin (CCK) secretion in the enteroendocrine cells and exhibited vasorelaxing and antihypertensive effects via the CCK1 system.

l-Ornithine and l-lysine stimulate gastrointestinal motility via transient receptor potential vanilloid 1.

SCOPE: The gastrointestinal (GI) tract senses and responds to intraluminal nutrients and these interactions often affect GI functions. We found that, among basic amino acids, l-ornithine (Orn) and l-lysine (Lys) stimulated but l-arginine (Arg) suppressed GI motility after oral administration (24 mmol/kg) in mice (Orn and Lys, 14.3 and 26.4% promotion; Arg, 7.7% suppression). We investigated the mechanism of the action of Orn and Lys on GI motility. METHODS AND RESULTS: Orn-induced promotion of small intestinal transit was significantly inhibited (p<0.05) by oral administration of capsazepine, a transient receptor potential vanilloid 1 (TRPV1) antagonist. Moreover, the stimulatory effect of Orn and Lys was abolished in TRPV1-knockout mice. In TRPV1-transfected HEK293 cells, Orn and Lys (10 mM) evoked Ca2+ influx, which was blocked by ruthenium red, a TRP channel antagonist. These results suggest that Orn and Lys promote GI motility via activation of TRPV1. The GI motility stimulation by Orn and Lys was also blocked by atropine, a muscarinic acetylcholine receptor (mAChR) antagonist, or NG -nitro-l-arginine methyl ester, a nitric oxide (NO) synthase inhibitor. CONCLUSION: Orally administered Orn and Lys stimulate GI motility via TRPV1, mAChR and NO synthase in mice.

Lacto-ghrestatin, a novel bovine milk-derived peptide, suppresses ghrelin secretion.

Ghrelin, an endogenous peptide isolated from the stomach, is known to stimulate food intake after peripheral administration. We found that the enzymatic digest of ß-lactoglobulin decreases ghrelin secretion from the ghrelin-producing cell line MGN3-1. The peptides present in the digest were comprehensively analyzed using the nanoLC-OrbitrapMS. Among them, we identified that the nonapeptide LIVTQTMKG, corresponding to ß-lactoglobulin(1-9), suppresses ghrelin secretion from MGN3-1 cells. We named LIVTQTMKG 'lacto-ghrestatin'. We found that lacto-ghrestatin decreases intracellular cAMP levels and mRNA expression levels of ghrelin production-related genes in MGN3-1 cells. Orally administered lacto-ghrestatin decreases plasma ghrelin levels and food intake in fasted mice. Lacto-ghrestatin is the first food-derived peptide to suppress ghrelin secretion in vitro and in vivo.

l-Ornithine stimulates growth hormone release in a manner dependent on the ghrelin system.

We found that intraduodenal administration of l-ornithine (l-Orn) stimulates growth hormone (GH) secretion in Wistar rats, and then investigated its mechanism. GH-releasing activity after intraduodenal administration of l-Orn was blocked by [d-Lys3]-GHRP-6, an antagonist of the ghrelin receptor; however, l-Orn (100 µM) has no affinity for the ghrelin receptor, suggesting that the GH-releasing activity of l-Orn is mediated via ghrelin release and activation of the ghrelin receptor. Intraduodenally administered l-Orn increased ghrelin mRNA expression in the duodenum but not in the stomach or hypothalamus. In addition, l-Orn-induced GH-releasing activity was inhibited by propranolol, an antagonist of ß-adrenergic receptor, which is known to be coupled to ghrelin release. In conclusion, intraduodenally administered l-Orn stimulates GH secretion through the sympathetic nervous and ghrelin systems.

Soy-ghretropin, a novel ghrelin-releasing peptide derived from soy protein.

We investigated exogenous secretagogues of ghrelin, which is an orexigenic hormone isolated from the stomach. We found that the tryptic digest of soy ß-conglycinin stimulated ghrelin secretion by the ghrelin-producing cell line, MGN3-1. We then identified a 22-amino acid peptide corresponding to the ß-conglycinin α-subunit(192-213) [ßCGα(192-213)] from an active fraction separated by HPLC. The N-terminal undecapeptide of ßCGα(192-213), NKNPFLFGSNR, exhibited ghrelin-releasing activity at a lower dose than that of ßCGα(192-213). We named NKNPFLFGSNR 'soy-ghretropin', which corresponds to ßCGα(192-202). Neither [des-N(1) K(2) ]-soy-ghretropin nor [des-R(11) ]-soy-ghretropin stimulated ghrelin secretion; hence, both the N- and C-terminal structures of soy-ghretropin were indispensable. Orally administered soy-ghretropin increased plasma ghrelin levels and food intake in vivo. Soy-ghretropin is the first exogenous ghrelin-releasing peptide derived from food protein.