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...
