Establishment of a Gastric Cell-Based Assay System for Exploring Inhibitors of Octanoylated Ghrelin Production

Abstract

Ghrelin, a gastric hormone, is a growth hormone-releasing peptide. Its serine-3 acylation with octanoic acid is essential for its orexigenic activity, and therefore, inhibition of the acylation of ghrelin may help in decreasing appetite and preventing obesity. This study aimed to establish a human gastric cell-based assay system to evaluate candidate inhibitors of octanoylated ghrelin production. In human gastric carcinoma AGS cells, obligatory factors for the posttranslational modification of ghrelin, such as certain prohormone convertases responsible for processing of proghrelin to the mature ghrelin and the enzyme-catalyzing acyl-modification of ghrelin, were well expressed, but ghrelin was expressed at low levels. Accordingly, we transfected a ghrelin-expressing vector into AGS cells and isolated a stable ghrelin-expressing cell line (AGS-GHRL8). AGS-GHRL8 cells secreted octanoylated ghrelin in accordance with the concentrations of octanoic acid in the culture medium. Given that ingested heptanoic acid is used for the acyl-modification of ghrelin, we evaluated whether heptanoic acid inhibits production of octanoylated ghrelin in AGS-GHRL8 cells. Butyric acid was used as a control. Indeed, heptanoic acid predictably decreased the secretion of octanoylated ghrelin, whereas butyric acid did not. The AGS-GHRL8 line established in this study will facilitate the screening of inhibitors of octanoylated ghrelin production. Keywords AGS cells, butyric acid, des-acyl ghrelin, heptanoic acid, octanoylated ghrelin 1036 Journal of Biomolecular Screening 18(9)Ingested medium-chain fatty acids are directly used for the acylation of ghrelin.13 Therefore, inhibition of ghrelin octanoylation may suppress hyperphagia and thus contribute to the prevention and improvement of obesity. The enzyme that catalyzes acyl-modification of ghrelin is ghrelin O-acyltransferase (GOAT), 14,15 also known as membrane-bound O-acyltransferase 4. It has also been reported that a few synthetic peptides can inhibit GOAT activity 16,17 ; these peptides may therefore be useful for preventing the production of octanoylated ghrelin. However, considering that peptidic compounds generally have poor oral bioavailability, nonpeptidic inhibitors of octanoylated ghrelin production for the prevention of hyperphagia and obesity are currently being explored.To investigate the inhibitors of ghrelin activation, an in vitro system for the evaluation of octanoylated ghrelin production would be useful. A number of assay systems to determine ghrelin levels and GOAT activity have been developed. 18 As a cell-based assay system, Sf9 insect cells infected with a recombinant baculovirus encoding mouse GOAT and GOAT/proghrelin-transfected HeLa cells have been used for the evaluation of peptidic inhibitors of ghrelin-octanoylation. 16,17 Moreover, systems using gastric cells are considered useful for the evaluation of octanoylated ghrelin production because ghrelin is principally produced in the gastric cells and GOAT is largely restricted to the stomach.14,15 Howe...

Bioactivities of Eriobotrya japonica (Thunb.) Lindl. Leaf and Its Triterpenes

“…In addition, we tested other triterpenes such as asiatic acid, glycyrrhetinic acid, and betulinic acid. The triterpenes tested above have a carboxylic acid group in a molecule similar to fatty acids that we previously reported as having an inhibitory effect on octanoylated ghrelin production [41,42]. Therefore, we confirmed that the carboxylic acid group on production of octanoylated ghrelin.…”

“…AGS-GHRL8 cells expressed both GOAT and furin and produced octanoylated ghrelin in the presence of octanoic acid [41]. As shown in Figure 13, we have developed a cellbased assay system used by AGS-GHRL8 cells to screen for candidate molecules to inhibit octanoylated ghrelin production [41]. We found that fatty acids, such as heptanoic acid, stearic acid, linoleic acid, α-linolenic acid, and oleic acid, decreased octanoylated ghrelin levels [41,42].…”

“…Our previous study has established the ghrelin-expressing cell line, AGS-GHRL8, by transfecting AGS human gastric carcinoma cells with the human ghrelin gene [41]. AGS-GHRL8 cells expressed both GOAT and furin and produced octanoylated ghrelin in the presence of octanoic acid [41]. As shown in Figure 13, we have developed a cellbased assay system used by AGS-GHRL8 cells to screen for candidate molecules to inhibit octanoylated ghrelin production [41].…”

“…β-actin, COX-IV, and α-tubulin were used as the loading controls for the whole cell lysate, mitochondrial fraction, and cytosolic fraction, respectively. Our previous study has established the ghrelin-expressing cell line, AGS-GHRL8, by transfecting AGS human gastric carcinoma cells with the human ghrelin gene [41]. AGS-GHRL8 cells expressed both GOAT and furin and produced octanoylated ghrelin in the presence of octanoic acid [41].…”

“…As shown in Figure 13, we have developed a cellbased assay system used by AGS-GHRL8 cells to screen for candidate molecules to inhibit octanoylated ghrelin production [41]. We found that fatty acids, such as heptanoic acid, stearic acid, linoleic acid, α-linolenic acid, and oleic acid, decreased octanoylated ghrelin levels [41,42]. Since GOAT recognizes fatty acyl-coenzyme (Co)A molecules, such as octanoyl-, hexanoyl-, and decanoyl-CoA [43], we speculated that the carboxyl group in a molecule is an important functional group for this inhibitory effect.…”