Triterpenes Suppress Octanoylated Ghrelin Production in Ghrelin-Expressing Human Gastric Carcinoma Cells

Biomedical Research (Tokyo) 37 (6) 343–349, 2016

Triterpenes suppress octanoylated ghrelin production in ghrelin-expressing human gastric carcinoma cells

1 1 1 2 1 Kensuke NAKAJIMA , Shigeru OISO , Takuhiro UTO , Osamu MORINAGA , Yukihiro SHOYAMA , and Hiroko 1 KARIYAZONO 1 Graduate School of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298, Japan and 2 Department of Natural Medicines, Daiichi University of Pharmacy, 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan (Received 3 October 2016; and accepted 11 October 2016)

ABSTRACT Ghrelin is an appetite-stimulating peptide hormone with an octanoyl modification at serine 3 that is essential for its orexigenic effect. Ghrelin O-acyltransferase (GOAT) is the enzyme that catalyzes ghrelin acylation using fatty acyl-coenzyme A as a substrate. We previously developed an assay system based on the AGS-GHRL8 cell line that produces octanoylated ghrelin in the presence of octanoic acid, and demonstrated that some fatty acids suppressed octanoylated ghrelin production. Recent studies have reported that triterpenes have anti-obesity effect. Since such triterpenes, like fatty acids, have a carboxyl group, we speculated that they can suppress octanoylated ghrelin production. To test this hypothesis, we investigated the effect of triterpenes on octanoylated ghrelin production. Asiatic acid, corosolic acid, glycyrrhetinic acid, oleanolic acid and ursolic acid suppressed octanoylated ghrelin levels in AGS-GHRL8 cells without decreasing transcript expression of GOAT or furin, a protease required for ghrelin maturation. β-amyrin had no effect on octanoylated ghrelin level, which was only slightly inhibited by uvaol; the fact that both these triterpenes lack a carboxyl group indicates that this group is important for suppressing octanoylated ghrelin production. These results suggest that triterpenes may have the potential as obesity-pre- venting agents with suppressive effect on octanoylated ghrelin production.

Ghrelin is a 28-amino acid peptide that was isolated mone convertases including furin are required to from rat stomach (11) and induces body weight gain generate mature ghrelin from the proghrelin precur- by increasing food intake (12). Ghrelin exists as two sor (21, 27). isoforms in vivo: octanoylated ghrelin, which has an We previously established the ghrelin-expressing octanoyl modification at serine 3, and des-acyl ghre- cell line, AGS-GHRL8, by transfecting AGS human lin without acyl modification (9). Only the former gastric carcinoma cells with the human ghrelin gene stimulates appetite (13). Suppressing octanoylated (16). AGS-GHRL8 cells expressed both GOAT and ghrelin production is considered as a promising furin, and produced octanoylated ghrelin in the pres- therapeutic strategy for the treatment of obesity. ence of octanoic acid (16). We used AGS-GHRL8 Ghrelin O-acyltransferase (GOAT) catalyzes ghrelin cells to develop a cell-based assay system to screen octanoylation (1, 7, 26), and one or more prohor- for molecules that inhibit octanoylated ghrelin production (16) and found that fatty acids such as hepta- noic acid, stearic acid, linoleic acid, α-linolenic acid Address correspondence to: Hiroko Kariyazono, Ph.D., Graduate School of Pharmaceutical Sciences, Nagasaki and oleic acid decreased octanoylated ghrelin levels International University, 2825-7 Huis Ten Bosch, Sasebo, (16, 17). Since GOAT recognizes fatty acyl-CoA such Nagasaki 859-3298, Japan as octanoyl-, hexanoyl-, and decanoyl-CoA (14), we Tel: +81-956-20-5697, Fax: +81-956-20-5697 speculated that the carboxyl group is an important E-mail: [email protected] structure for this inhibitory effect. 344 K. Nakajima et al.

Triterpenes are phytochemicals consisting of six culture medium. The optical density was measured isoprene units that have anti-oxidant, anti-inflamma- at 570 nm using a Multiscan FC instrument (Thermo tory, and anti-proliferative effects (2, 22, 24). Cer- Fisher Scientific, Waltham, MA, USA). Values are tain triterpenes such as asiatic acid, betulinic acid, expressed as the ratio of absorbance of treated cells corosolic acid, glycyrrhetinic acid, oleanolic acid, to that of untreated control cells. and ursolic acid have shown anti-obesity effects in mice fed a high-fat-diet (4, 5, 18, 19, 23, 25). Like Evaluation of effects of triterpenes on octanoylated fatty acids, these triterpenes have a carboxyl group ghrelin production in AGS-GHRL8 cells. AGS-GHRL8 in their structure, suggesting that they could also cells produce octanoylated ghrelin in the presence of suppress octanoylated ghrelin production. octanoic acid (16). To determine the effect of triter- We investigated this possibility in the present penes on octanoylated ghrelin production, we com- study by examining the inhibitory effect of triter- pared the levels of octanoylated ghrelin secreted penes on octanoylated ghrelin production in AGS- from AGS-GHRL8 cells after adding octanoic acid GHRL8 cells. We also evaluated GOAT and furin with or without triterpenes (16, 17). mRNA expression in cells treated with triterpenes AGS-GHRL8 cells were seeded in 12-well plates and the significance of the triterpene carboxyl group at 4 × 105 cells/well in DMEM and cultured for 24 h. in suppressing octanoylated ghrelin production. After two washes with phosphate-buffered saline (PBS), fresh DMEM containing octanoic acid with or without the test triterpenes was added to each MATERIALS AND METHODS well. After 24 h, the culture medium was collected Reagents. Asiatic acid, betulinic acid, corosolic acid, in a microtube, and 1/10 volume of 1 mol/L hydro- oleanolic acid, ursolic acid, and uvaol were purchased chloric acid was added to each sample. Cells were from Tokyo Chemical Industry Co. (Tokyo, Japan). washed twice with PBS, and harvested using 0.05% Glycyrrhetinic acid and β-amyrin were purchased trypsin and 0.02% EDTA solution. Cell number was from Sigma-Aldrich (Tokyo, Japan). Octanoic acid quantified using an EVE Automatic Cell Counter and 3-(4,5-dimethylthiazol-2-yl) 5-diphenyltetrazoli- (AR Brown, Tokyo, Japan). Octanoylated ghrelin um bromide (MTT) were obtained from Wako Pure concentration in the culture medium was measured Chemical Industries (Osaka, Japan). Dulbecco’s with enzyme-linked immunosorbent assay (ELISA) modified Eagle’s medium (DMEM), RNAlater, Fast kits, which specifically detect octanoylated ghrelin SYBR Green Master Mix, and TRIzol reagent were but not des-acyl ghrelin. ELISA kits for measuring purchased from Life Technologies (Carlsbad, CA, octanoylated ghrelin levels were purchased from USA), and ReverTra Ace was purchased from Toyobo SCETI K.K. (Tokyo, Japan) and Bertin Pharma (Osaka, Japan). Primers targeting GOAT, furin and (Montigny le Bretonneux, France). The concentra- 18S ribosomal RNA were synthesized by Genenet tion of octanoylated ghrelin was adjusted for the (Fukuoka, Japan) and Greiner Japan (Kanagawa, Ja- number of cells in the well. pan). Other reagents were obtained from Wako Pure Chemical Industries unless otherwise indicated. Quantitative reverse transcriptase (qRT)-PCR analysis. Relative mRNA levels of GOAT and furin in Cell culture. Ghrelin-expressing AGS-GHRL8 cells AGS-GHRL8 cells treated with triterpenes were were cultured in DMEM containing 10% fetal bo- evaluated by qRT-PCR. Total RNA was extracted vine serum at 37°C in a humidified atmosphere of from AGS-GHRL8 cells treated with test triterpenes

5% CO2. using TRIzol reagent and reverse-transcribed into cDNA with ReverTra Ace. PCR was carried out on MTT assay. The MTT assay was performed as de- a 7900HT Fast Real-Time PCR System (Applied scribed in our previous studies (15–17). Briefly, Biosystems/Life Technologies, Carlsbad, CA, USA). AGS-GHRL8 cells were seeded in 96-well plates at The reaction mixture containing 10 μL of 2 × Fast 3 × 103 cells/well and cultured for 24 h in DMEM. SYBR Green Master Mix, 0.4 pmol sense and anti- Triterpenes were added to the culture medium at sense primers, and 2 μL diluted cDNA (17) was concentrations ranging from 0 to 100 μmol/L; 24 h loaded onto a 386-well plate; amplification was car- later, 200 μg/mL MTT (final concentration) was ried out under the following conditions: 95°C for added to each well and cells were cultured for an 20 s, followed by 40 cycles of 95°C for 1 s and additional 4 h. The resultant formazan crystals were 60°C for 20 s. The following sense and antisense dissolved in dimethyl sulfoxide after removing the primers were used: human GOAT, 5′-ACAGCTCGA Triterpenes suppress ghrelin 345

Fig. 1 Chemical structures of test triterpenes.

TGGCTCCGACG-3′ and 5′-AGCTTCCACCATC 12.5 μmol/L, respectively (Fig. 2). ACGGCCC-3′; human furin, 5′-GAAGTGCACGGA We then examined the effects of triterpenes on GTCTCACA-3′ and 5′-CCGCCATGTGAGGTTCTT octanoylated ghrelin production in AGS-GHRL8 AT-3′; and 18S ribosomal RNA, 5′-GTAACCCGTT cells using nontoxic levels of each triterpene. Treat- GAACCCCATT-3′ and 5′-CCATCCAATCGGTAGT ment with 25 and 50 μmol/L asiatic acid, 12.5 and AGCG-3′. Transcript levels were estimated from the 25 μmol/L corosolic acid, 100 μmol/L glycyrrhetinic respective standard curves and normalized to 18S ri- acid, 50 and 100 μmol/L oleanolic acid, and 6.25 bosomal RNA (internal control). and 12.5 μmol/L ursolic acid decreased the levels of octanoylated ghrelin in the culture medium, while Statistical analysis. Values are presented as mean ± none of the tested concentrations of betulinic acid SD. Differences between groups were analyzed with affected octanoylated ghrelin production (Fig. 3). a two sample t-test or Tukey’s test for multiple com- parisons. Differences were considered significant at Triterpenes do not suppress GOAT and furin mRNA P < 0.05. levels GOAT and furin are involved in the processing of proghrelin to the octanoylated form of the peptide (1, RESULTS 7, 21, 26, 27) and are expressed in AGS-GHRL8 Triterpenes suppress octanoylated ghrelin produc- cells (16). We investigated whether the suppression tion in AGS-GHRL8 cells of octanoylated ghrelin production by triterpenes We evaluated the effects of the triterpenes such as was due to decreased gene expression. GOAT and asiatic acid, betulinic acid, corosolic acid, glycyrrhe- furin mRNA levels were upregulated by asiatic acid tinic acid, oleanolic acid, and ursolic acid on oc- (Fig. 4). On the other hand, corosolic acid, glycyr- tanoylated ghrelin production (Fig. 1). To first rhetinic acid, oleanolic acid, and ursolic acid had no determine the appropriate test concentrations, we as- effect on GOAT and furin expression (Fig. 4). sessed the viability of triterpene-treated AGS-GHRL8 cells with the MTT assay. Glycyrrhetinic acid and Carboxyl groups in oleanolic acid and ursolic acid oleanolic acid showed no toxicity to cells in the have an influence on octanoylated ghrelin levels in range of 0 to 100 μmol/L, whereas asiatic acid, bet- AGS-GHRL8 cells ulinic acid, corosolic acid, and ursolic acid were Triterpenes that were found to reduce octanoylated non-toxic up to concentrations of 50, 6.25, 25, and ghrelin levels in this study have carboxyl group in 346 K. Nakajima et al.

Fig. 2 Viability of AGS-GHRL8 cells treated with test triterpenes. Asiatic acid decreased cell viability at 100 μmol/L; betulinic acid at ≥ 12.5 μmol/L; corosolic acid at ≥ 50 μmol/L; and ursolic acid at ≥ 25 μmol/L. Glycyrrhetinic acid and oleanolic acid had no effect on cell viability. Data are shown relative to the viability of control cells (100%) and represent mean ± SD (n = 6). *P < 0.01 vs. untreated control (0 μmol/L).

Fig. 3 Effect of test triterpenes on octanoylated ghrelin production in AGS-GHRL8 cells. Octanoic acid was used at 100 μmol/L. Asiatic acid, corosolic acid, glycyrrhetinic acid, oleanolic acid, and ursolic acid but not betulinic acid suppressed octanoylated ghrelin production. Data are shown as octanoylated ghrelin concentrations relative to the control (0 μmol/L). Each value represents mean ± SD (n = 6). *P < 0.01 vs. untreated control (0 μmol/L). their structures similar to fatty acids that we previ- group is substituted with methyl and hydroxyl groups, ously reported as having an inhibitory effect on oc- respectively (Fig. 5A). The maximum concentrations tanoylated ghrelin production (16, 17). We therefore of β-amyrin and uvaol that were non-toxic to AGS- investigated the significance of the triterpene car- GHRL8 cells were 6.25 and 25 μmol/L, respectively, boxyl group on production of octanoylated ghrelin. as determined by the MTT assay (data not shown). As a negative control we used β-amyrin and uvaol, We compared the effects of oleanolic acid and which have similar structures to oleanolic acid and β-amyrin at the same concentration (6.25 μmol/L). ursolic acid, respectively, except that the carboxyl The effect of uvaol was evaluated at a concentration Triterpenes suppress ghrelin 347

Fig. 4 Effect of test triterpenes on mRNA expression of (A) GOAT and (B) furin in AGS-GHRL8 cells. Asiatic acid increased GOAT and furin mRNA levels, whereas other triterpenes had no effect on the expression of either gene. Data are shown as mRNA levels in triterpene-treated as compared to untreated control cells (0 μmol/L). Each value represents mean ± SD (n = 4). *P < 0.01 vs. untreated control (0 μmol/L). of 12.5 μmol/L, which was the highest concentration tanoylated ghrelin production to determine whether of ursolic acid that was tested. Oleanolic acid but these molecules can be used to prevent or treat obe- not β-amyrin suppressed octanoylated ghrelin pro- sity. Our results showed that asiatic acid, corosolic duction at 6.25 μmol/L (Fig. 5B). Uvaol had a slight acid, glycyrrhetinic acid, oleanolic acid, and ursolic suppressive effect at 12.5 μmol/L (Fig. 5C), which acid but not betulinic acid inhibited production of reduced octanoylated ghrelin levels by 29% vs. 89% octanoylated ghrelin in AGS-GHRL8 cells. for ursolic acid at the same concentration (Fig. 3). Betulinic acid has a five-membered ring at position 17 whereas the other test triterpenes have a six- membered ring. In addition, the side chain of DISCUSSION betulinic acid at position 19 is bulkier than those of Octanoylated ghrelin stimulates appetite and induces other triterpenes. These structural features may ac- body weight gain (12, 13). The present study evalu- count for its lack of effect on octanoylated ghrelin ated the inhibitory effects of triterpenes on oc- production. Nonetheless, betulinic acid has been re- 348 K. Nakajima et al.

ported to reduce plasma ghrelin levels in mice (4), which could be due to decreased ghrelin mRNA expression. However, it is not possible to validate changes in ghrelin mRNA expression in betulinic acid-treated AGS-GHRL8 cells since these were established by transfecting AGS human gastric carcinoma cells with the human ghrelin gene. Some triterpenes are known to reduce mRNA expression of various proteins, including GREB1 (growth regulation by estrogen in breast cancer 1) (10), estrogen receptor α (10) and monocyte chemoattractant protein-1 (3). We therefore examined the effect of triterpenes on GOAT and furin mRNA expression. Asiatic acid, corosolic acid, glycyrrhetinic acid, oleanolic acid, and ursolic acid did not de- crease GOAT and furin transcript levels, implying that their expression is not related to the inhibitory effects on octanoylated ghrelin production. Although asiatic acid increased mRNA levels of GOAT and furin, the consequent increase in octanoylated ghrelin production would likely be masked by the stronger inhibitory effect of octanoylated ghrelin production via other mechanisms. We also found that the triterpene carboxyl group may contribute to suppression of octanoylated ghrelin production. Octanoyl-, hexanoyl-, and decanoyl-CoA are acyl group donors for GOAT (14); complexes are formed by dehydrocondensation of fatty acid carboxyl groups and CoA thiol groups. It is thus possible that triterpenes with carboxyl groups form a complex with CoA and compete with octanoyl- CoA as a substrate of GOAT, thereby inhibiting octanoylated ghrelin production. Our previous study demonstrated that oleic acid, which has a carboxyl group, decreased otanoylated ghrelin production in AGS-GHRL8 cells as well as plasma levels of octanoylated ghrelin in mice (17). Therefore, triterpenes that decreased octanoylated ghrelin production in this study may also have similar effects in vivo. Although it is difficult to extract a large amount of pure triterpene from plants (6), Fig. 5 Contribution of the triterpene carboxyl group to the inhibitory effect on octanoylated ghrelin production in AGS- the genes encoding some of the enzymes involved GHRL8 cells. (A) Structures of β-amyrin and uvaol. These in triterpene synthesis have been identified and iso- molecules have the same structures as oleanolic acid and lated (6, 8, 20), which can facilitate large-scale pro- ursolic acid, respectively, except that the carboxyl groups duction of these molecules. The results of our study are substituted by methyl and hydroxyl groups, respectively. suggest that triterpenes can be effective prophylactic (B, C) Effect of oleanolic acid and β-amyrin (B) and uvaol (C) on octanoylated ghrelin production in AGS-GHRL8 or therapeutic agents in the treatment of obesity by cells. Oleanolic acid but not β-amyrin suppressed oc- suppressing octanoylated ghrelin production. tanoylated ghrelin levels. Uvaol showed an inhibitory effect but to a lesser degree than ursolic acid (refer to Fig. 3). Oc- tanoic acid was used at 100 μmol/L. Data are shown as Acknowledgements relative concentrations of octanoylated ghrelin. Each value represents mean ± SD (n = 6). *P < 0.01 vs. untreated con- This work was supported by JSPS KAKENHI Grant trol (0 μmol/L). Numbers 25350169 and 15H06805. Triterpenes suppress ghrelin 349

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