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Acorbine, a Corbicula japonica–derived tripeptide containing non-proteinogenic amino acids, suppresses ethanol-induced liver injury
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Acorbine, a Corbicula japonicaederived tripeptide containing nonproteinogenic amino acids, suppresses ethanol-induced liver injury Miho Tamai a, b, c, Hidemitsu Uchisawa d, Yukari Saito c, Hajime Matsue e, Masaya Kawase f, Tetsushi Naraoka d, Yoh-ichi Tagawa b, c, * a
Faculty of Dental Medicine, Hokkaido University, Kita 13-jo, Nishi 7-chome, Kita-ku, Sapporo, 060-8586, Japan School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa, 226-8501, Japan Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa, 226-8501, Japan d Industrial Research Institute, Aomori Prefectural Industrial Technology Research Center, 221-10 Yamaguchi, Nogi, Aomori, 030-0142, Japan e Aomori University of Health and Welfare, 58-1 Mase, Hamadate, Aomori, 030-8505, Japan f Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama-shi, Shiga, 526-0829, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 November 2019 Accepted 20 November 2019 Available online xxx
Since ancient times, Corbicula extract has been believed in Japan to have hepatoprotective effects, but it remains unclear whether these claims are true, and if so, which component is responsible for hepatoprotection. In this study, we showed that Corbicula extract exerted a protective effect against liver damage. Recent work identified acorbine (b-alanyl-ornithyl-ornithine), a novel tripeptide containing non-proteinogenic amino acids, in the extract of Corbicula japonica. Synthesized acorbine cured alcoholinduced liver damage in mice. In addition, acorbine purified from Corbicula extract exerted a protective effect against alcohol-induced hepatotoxicity in a culture liver model derived from mouse ES/iPS cells. Thus, acorbine is one of the components of Corbicula extract that protects hepatocytes against ethanolinduced death. © 2019 Elsevier Inc. All rights reserved.
Keywords: Acorbine Corbicula Hepatitis Hepatoprotection Liver tissue
1. Introduction Orally ingested alcohol is delivered to the liver from the intestine, primarily through the hepatic portal vein, and is subsequently oxidized to finally acetic acid by a two-step reaction in hepatocytes. In the first step, ethanol is oxidized to acetaldehyde by alcohol dehydrogenase, concomitant with reduction of NADþ to NADH. Second, the resultant acetaldehyde is oxidized to acetic acid by acetaldehyde dehydrogenase in a reaction that also produces NADH. Ethanol is oxidized also by other enzymes, including catalase and microsomal CYP2E1. Ethanol and acetaldehyde can induce apoptosis in hepatocytes [1]. Excess NADH accumulation from these ethanol oxidation reactions also affects a variety of metabolic pathways [2]. Alcohol-related chronic hepatitis is a worldwide public health problem.
* Corresponding author. School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa, 226-8501, Japan. E-mail address: [email protected] (Y.-i. Tagawa).
Recent studies have focused on improving hepatic functions in patients with liver failure using local food products, such as curcumin [3] and polyphenolic extracts [4]. Although these substances have been commercialized as nutraceuticals worldwide, it remains unclear how they protect against liver injury and prevent clinically important diseases. For example, extracts of brackish-water bivalves, such as the freshwater clam Corbicula japonica, are thought to be beneficial for the liver after consumption of alcohol; however, it is not clear whether this claim is true, and if so, which component exerts the hepatoprotective effect. Bivalves of genus Corbicula are very popular as ingredients of soup in north Asian countries, especially miso soup in Japan. Recently, acorbine (b-alanylornithyl-ornithine), a novel tripeptide containing nonproteinogenic amino acids, was discovered in extract from Corbicula japonica (Corbicula extract) [5]. To date, acorbine has been identified only in Corbicula, and in no other type of organism. It is difficult to identify molecules that protect against liver injury because the liver regenerates itself after the injury; consequently, it has been challenging to develop useful liver-alternative culture system for investigating the functions of this organ. To address this issue, we established a murine ES cellderived in vitro
https://doi.org/10.1016/j.bbrc.2019.11.131 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: M. Tamai et al., Acorbine, a Corbicula japonicaederived tripeptide containing non-proteinogenic amino acids, suppresses ethanol-induced liver injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.131
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M. Tamai et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
liver model, termed IVLmES, which includes both the hepatocyte layer and the accompanying sinusoid vascularlike network [6], and exhibits cytochrome P450 activity [7] and enhancement of urea production by excess ornithine [8]. Using IVLmES, we demonstrated the hepatoprotective effect of L-ornithine, which was not evident in monolayer single culture of mouse primary hepatocytes. In addition to being a major component of Corbicula extract, L-ornithine is abundant in hepatocytes as a component of the urea cycle. In this study, we found that acorbine could cure alcohol-induced liver damage in mice, and that acorbine exerts a protective effect against alcohol-induced hepatotoxicity in IVLmES/iPS.
diet containing 1e5% ethanol, as shown in Fig. 1A. The ethanolcontaining liquid diet was freshly prepared every day. The body weights of all mice were measured every day, and the blood was collected from the retroorbital plexus under isoflurane anesthesia once per week. Anesthesia was induced by placing each mouse in a regulated inhalator with 3% isoflurane. Blood samples were deposited in 1.5 mL tubes and centrifuged (1,200 g, 30 min, 4 C) to separate serum. Mouse livers were isolated as follows: after the mice were killed by cervical fracture, the livers were sliced and washed to remove hemoglobin, and then fixed in 4% paraformaldehyde/PBS. The study protocol was approved by the Animal Experimentation Committees of Tokyo Institute of Technology.
2. Materials and methods 2.3. Histological staining 2.1. Corbicula extract and acorbine Corbicula extract was prepared from the brackish-water bivalve Corbicula japonica obtained in Lake Jusanko, Aomori prefecture, Japan by Uchisawa et al. [5]. Corbicula extract was provided from Fukushimashoten Co., LTD. Its composition was analyzed by common methods: moisture level was determined by heating/drying at atmospheric pressure; protein by the Kjeldahl method, lipid by dimethyl ether extraction; carbohydrate by difference; ash by the direct ashing method; glycogen by the sodium thiosulfate method; calcium, sodium, and iron by atomic absorption; vitamin B2 by fluorometry; vitamin B6 and vitamin B12 by microbial assay; alanine and ornithine using an amino acid analyzer; and acorbine by high-performance liquid chromatography (HPLC), as shown in Table 1. Acorbine was isolated and purified as described previously [5]. Briefly, gel permeation HPLC was performed on an HPLC system (Hitachi, Japan) using a column (10 mm 30 cm) of Superdex Peptide HR 10/30 (Amersham Biosciences, Sweden) with a mobile phase of 250 mM NaCl in 20 mM phosphate buffer (pH 7.2) at room temperature (flow rate, 0.25 ml/min). Elution was monitored by absorbance at 205 nm. The fraction containing acorbine was desalted by dialysis using a Spectra/Por with an MWCO of 500, lyophilized, and then used for structural analyses. Acorbine with purity of 80.9%, as estimated by HPLC analysis, was used in this study.
Paraffin-embedded blocks and sections were performed by Genostaff (Tokyo, Japan). Tissues were fixed in 4% paraformaldehyde/PBS, embedded in paraffin, and sectioned at a thickness of 4 mm. Sections were stained with hematoxylin and eosin for histological examination. 2.4. Quantification of released liver enzyme activities Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) activities in the medium or serum were assessed using SPOTCHEM™ EZ SP-4430 and
2.2. Alcohol-induced chronic liver injury in mice The LiebereDeCarli diet (Oriental Yeast, Tokyo, Japan), a liquid diet, was naturally fed to female C57BL/6 J mice (6 weeks old at the start of the experiment) for 3 days, and then replaced with liquid
Table 1 Proximate Composition and Maine Ingredients of the Brackish-water Bivalve Extract followed by HPLC analysis. Unit
Components
Value (mg/100 g)
g/100 g g/100 g g/100 g g/100 g g/100 g g/100 g g/100 g g/100 g mg/100 g/100 g mg/100 mg/100 mg/100 g/100 g g/100 g g/100 g
Moisture Protein Lipid Carbohydrate Ash Glycogen Calcium Sodium Iron Succinic acid Vitamin B2 Vitamin B6 Vitamin B12 Alanine Ornithine Acorbine
5.3 34.0 0.1 48.9 11.7 41.0 0.95 3.0 9.3 7.1 3.01 0.43 0.26 1.29 0.17 0.84
g g g g
Fig. 1. Effect of Corbicula extract or synthesized acorbine on alcohol-induced chronic liver injury in mice. A: Alcohol administration schedule for the induction of alcoholinduced chronic liver injury. AST (B) and ALT (C) levels in the serum of ethanolreceiving (white column, N ¼ 24) or control (black column, N ¼ 6) BALB/cA mice. AST (D) and ALT (E) levels in the serum of mice receiving ethanol and Corbicula extract (CE), synthesized acorbine (Ac), or PBS (P), on 42 (white column), 49 (cross column), and 63 (black column) days after the start of ethanol administration.
Please cite this article as: M. Tamai et al., Acorbine, a Corbicula japonicaederived tripeptide containing non-proteinogenic amino acids, suppresses ethanol-induced liver injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.131
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SPOTCHEM™ II LDH (ARKRAY, Kyoto, Japan). 2.5. Culture and differentiation of murine ES cells and murine iPS cells
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All mice were sacrificed 63 days after initiation of ethanol intake, and their livers were histologically investigated. These experiments had to be carried out within 63 days, because many lipid droplets were present in the livers of control mice not receiving ethanol (Fig. 2A), indicating that the control mice had steatosis. On
The murine ES cell lines ST1 (originally established from the BALB/cA strain [9]) and Bruce 4 (derived from C57BL/6, OZ gene) [10,11] and the murine iPS cell line iPS-MEF-Ng-20D-17 (derived from 75% DBA, 12.5% C57BL/6, and 12.5% 129S4) [11,12] were obtained from RIKEN Bioresource Center (Ibaraki, Japan). The cells were grown on feeder layers of mitomycin Ctreated murine embryonic fibroblasts in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Tokyo, Japan) containing 15% fetal bovine serum (FBS) (Nichirei Biosciences, Tokyo, Japan), 1 mM sodium pyruvate (Invitrogen), 100 mM nonessential amino acids (Invitrogen), 100 mM 2mercaptoethanol (Sigma-Aldrich Japan, Tokyo, Japan), and 103 U/ mL leukemia inhibitory factor (LIF) (Chemicon International, Temecula, CA, USA). The IVLmES/iPS was generated by differentiation from murine ES cells as previously described [6,8,11]. Briefly, mES/iPS cells were dissociated with trypsin and re-suspended in Iscove’s modified Dulbecco’s medium (IMDM) (Invitrogen) containing 15% FBS, 1 mM sodium pyruvate, 100 mM nonessential amino acids, and 100 mM 2mercaptoethanol without LIF, and then cultured for 5 days in a hanging drop at a concentration of 1,000 cells per 50 mL in order to form embryoid bodies (EBs). Fifty well-formed EBs were collected and plated in each well of a gelatin-coated 6-well plate; the day of plating was denoted as day 0 (A0). In this study, IVLmES/iPS EBs were used 18 days after the plating (A18). 2.6. Quantification of urea concentrations Urea in the culture medium after centrifugation at 300 g for 5 min was quantified using a QuantiChrom™ Urea Assay Kit (BioAssay Systems, Hayward, CA, USA). 2.7. Statistical evaluation Values are expressed as means ± SEM. Calculation of descriptive statistics and paired t-test were performed using the R software [13]. Differences between groups were considered significant when P < 0.05. 3. Results 3.1. Acorbine has a beneficial therapeutic effect in the alcoholinduced chronic hepatitis in mice Most ingredients of Corbicula extract are present in other bivalves, with the exception of acorbine, a tripeptide containing nonproteinogenic amino acids (Table 1). Because acorbine is a unique ingredient in C. japonica, we investigated whether it was responsible for the hepatoprotective effect of Corbicula extract. To generate an alcohol-induced liver injury mouse model, a liquid diet containing ethanol was naturally administered to C57BL/6 J female mice following the schedule shown in Fig.1A. The activity levels of AST and ALT in the serum gradually increased after the intake of ethanol, and were higher than the control levels 28 days after ethanol administration (Fig. 1B and C). Corbicula extract at 1 g/kg of body weight or synthesized acorbine at 9 mg/kg of body weight had been orally administrated once a day using feeding needles since 42 days after initiation of ethanol intake (Fig. 1A). The release of AST and ALT in serum was completely suppressed in mice receiving Corbicula extract or synthesized acorbineeadministrated mice for at least 3 weeks during ethanol natural intake (Fig. 1D and E).
Fig. 2. Histological examination of hematoxylin and eosin staining of the liver. A: control, B: ethanol, C: ethanol and Corbicula extract, D: ethanol and acorbine administrated mice on 63 days after the start of ethanol administration.
Please cite this article as: M. Tamai et al., Acorbine, a Corbicula japonicaederived tripeptide containing non-proteinogenic amino acids, suppresses ethanol-induced liver injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.131
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the other hand, many ballooning hepatocytes were present in the livers of ethanol-receiving mice (Fig. 2B), indicative of typical ethanol-induced chronic hepatitis. Ballooning hepatocytes were much less abundant in the livers of mice receiving Corbicula extract or acorbine (Fig. 2C and D). In particular, some lipid droplets, but no ballooning hepatocytes, were detected in the livers of acorbinereceiving mice (Fig. 2D).
that acorbine could suppress ethanol-induced hepatocyte death in the IVLmES/iPS. To investigate hepatic activity of surviving hepatocytes in the IVLmES/iPS, we measured urea production in the medium. The results revealed that urea production levels in the medium in the presence of ethanol could also be maintained by the addition of acorbine (Fig. 4AeC).
3.2. Acorbine protects against alcohol-induced cell death of the hepatocytes in murine ES- or iPS-derived hepatic tissue models
4. Discussion
The murine ES/iPS cellderived in vitro liver model, IVLmES/iPS, was prepared from the murine embryonic stem cell lines ST1 (BALB/cA background) and Bruce 4 (C57BL/6) and the murine iPS cell line iPS-MEF-Ng-20D-17 (derived from 75% DBA, 12.5% C57BL/6 and 12.5% 129S4) by in vitro liver organogenesis. Corbicula extract at a final concentration of 2% (w/v), or purified acorbine chloride at a final concentration of 40 mM, was added into the medium of the IVLmES/iPS along with ethanol at a final concentration of 3% (w/v), and the activity of released LDH in the medium was measured 24 h later. Release of LDH by addition of ethanol was suppressed in the medium of the IVLmES/iPS containing Corbicula extract or purified acorbine, in comparison with PBS controls (Fig. 3AeC). In particular, purified acorbine significantly suppressed the increase of LDH in the medium. This result suggests
In Japan, Corbicula extract has been believed since ancient times to exert hepatoprotective effects. In this study, we found that Corbicula extract protected against alcohol-induced hepatocyte death in mouse (Figs. 1 and 2). Corbicula extract contains several potentially hepatoprotective substances, e.g., L-ornithine [8] and taurine [14]. Given that L-ornithine mediates protective effects on hepatocytes in the IVLmES culture system [8], this molecule is a strong candidate for a downstream factor in the hepatoprotective effect associated with Corbicula extract. However L-ornithine is present in other bivalves, such as scallop. Acorbine (b-Ala-OrnOrn), a novel tripeptide containing non-proteinogenic amino acids, is present only in C. japonica, but not in scallop [5]. In this study, we showed that chemically synthesized acorbine could cure alcohol-induced liver damage in mice and we demonstrated that acorbine purified from Corbicula extract exerts a
Fig. 3. Cell death in the IVLmES/iPS stimulated by ethanol. The each IVL was induced cell death by the addition of 3% ethanol in the medium and simultaneous addition of Corbicula extract (CE), acorbine (Ac), or PBS (P). AeC: Released LDH levels in the medium of the IVLmES/iPS. The IVL was derived from ES ST1 (A), ES Bruce4 (B), and iPS iPSMEF-Ng-20D-17 (C).
Fig. 4. Hepatic function in the IVLmES/iPS stimulated by ethanol. The each IVL was induced cell death by the addition of 3% ethanol in the medium and simultaneous addition of Corbicula extract (CE), acorbine (Ac), or PBS (P). AeC: Urea production of the IVLmES/iPS. The IVL was derived from ES ST1 (A), ES Bruce4 (B), and iPS iPS-MEF-Ng20D-17 (C).
Please cite this article as: M. Tamai et al., Acorbine, a Corbicula japonicaederived tripeptide containing non-proteinogenic amino acids, suppresses ethanol-induced liver injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.131
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protective effect against alcohol-induced hepatotoxicity in a culture model. We found that acorbine is one of the compounds in Corbicula extract that protects against ethanol-induced hepatocyte death. Acorbine can be precipitated by either acetate or chloride; we used acorbine chloride in the assay of IVLmES/iPS. Initially, we tried to investigate the hepatoprotective effect in the IVLmES/iPS using acorbine acetate, but the acetate strongly induced cell death. By changing the acetate to chloride, we succeeded in protecting the hepatocyte in the IVLmES/iPS after exposure to alcohol. L-ornithine protects against alcohol-induced hepatocyte death [8]. Therefore, because acorbine is hydrolyzed to L-ornithine and balanine in mouse, it is possible that acorbine-derived L-ornithine acts on hepatocytes to reduce the level of ammonium ion. The production of urea was significantly higher in the IVLmES/iPS in the presence of Corbicula extract or acorbine than in the absence of these supplements; however, acorbine might not be hydrolyzed under the culture conditions we used. Non-hydrolyzed acorbine could be detected in the liver by HPLC analysis, indicating that acorbine was delivered directly to the liver through the portal vein. The net dose of acorbine in Corbicula extract administered to mice was lower than the dose of singly administered acorbine, suggesting that other substances in Corbicula extract exert a synergistic hepatoprotective effect. We are still looking for such additional active substances in Corbicula extract. In summary, we found that Corbicula extract exerted a protective effect against liver damage. Acorbine (b-alanyl-ornithyl-ornithine), a tripeptide containing non-proteinogenic amino acids, could cure alcohol-induced liver damage in mice. Acorbine purified from Corbicula extract also protected against alcohol-induced hepatotoxicity in a liver culture model derived from mouse ES/iPS cells. Thus, acorbine is one of the components of Corbicula extract that protects against ethanol-induced hepatocyte death. Declaration of competing interest The authors declare no conflicts of interest. Acknowledgements This study was supported by a Grant-in-Aid for Challenging Exploratory Research [No. 24650254](to Y. Tagawa), for Scientific Research (A) [No. 25242040] (to Y. Tagawa), for Young Scientists (B) [No. 26750145] (to M. Tamai) from the Japan Society for the Promotion of Science (JSPS) and a Grant-in-Aid for Scientific Research on Innovative Areas [No. 231190003] (to Y. Tagawa) from the Ministry of Education, Culture, Sports, Science and Technology
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(MEXT) of Japan and by Building of Consortia for the Development of Human Resources in Science and Technology, Ministry of Education, Culture, Sports, Science and Technology, Japan. The mouse embryonic fibroblast-derived iPS cell line, iPS-MEFNg-20D-17 (APS0001), was provided by RIKEN Bio-Resource Center (Ibaraki, Japan) in agreement with Dr. Shinya Yamanaka (Center for iPS Cell Research and Application, Kyoto University).
Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.131.
References [1] J.B. Minana, L. Gomez-Cambronero, A. Lloret, et al., Mitochondrial oxidative stress and CD95 ligand: a dual mechanism for hepatocyte apoptosis in chronic alcoholism, Hepatology 35 (2002) 1205e1214. [2] A.I. Cederbaum, Alcohol metabolism, Clin. Liver Dis. 16 (2012) 667e685. [3] C. Wang, H.H. Nie, K. Li, et al., Curcumin inhibits HMGB1 releasing and attenuates concanavalin A-induced hepatitis in mice, Eur. J. Pharmacol. 697 (2012) 152e157. [4] C. Kumarappan, M. Vijayakumar, E. Thilagam, et al., Protective and curative effects of polyphenolic extracts from Ichnocarpus frutescense leaves on experimental hepatotoxicity by carbon tretrachloride and tamoxifen, Ann. Hepatol. 10 (2011) 63e72. [5] H. Uchisawa, T. Naraoka, T. Ono, A novel ornithine-containing tripeptide isolated from the extract of the brackish-water bivalve Corbicula japonica, Biochim. Biophys. Acta 1770 (2007) 790e796. [6] S. Ogawa, Y. Tagawa, A. Kamiyoshi, et al., Crucial roles of mesodermal cell lineages in a murine embryonic stem cell-derived in vitro liver organogenesis system, Stem Cells 23 (2005) 903e913. [7] M. Tsutsui, S. Ogawa, Y. Inada, et al., Characterization of cytochrome P450 expression in murine embryonic stem cell-derived hepatic tissue system, Drug Metab. Dispos. 34 (2006) 696e701. [8] M. Tamai, M. Aoki, A. Nishimura, et al., In vitro recapitulation of the urea cycle using murine embryonic stem cell-derived in vitro liver model, Amino Acids 45 (2013) 1343e1351. [9] H. Aikawa, M. Tamai, K. Mitamura, et al., Innate immunity in an in vitro murine blastocyst model using embryonic and trophoblast stem cells, J. Biosci. Bioeng. 117 (2014) 358e365. [10] F. Kontgen, G. Suss, C. Stewart, et al., Targeted disruption of the MHC class II Aa gene in C57BL/6 mice, Int. Immunol. 5 (1993) 957e964. [11] M. Tamai, A. Yamashita, Y. Tagawa, Mitochondrial development of the in vitro hepatic organogenesis model with simultaneous cardiac mesoderm differentiation from murine induced pluripotent stem cells, J. Biosci. Bioeng. 112 (2011) 495e500. [12] K. Okita, T. Ichisaka, S. Yamanaka, Generation of germline-competent induced pluripotent stem cells, Nature 448 (2007) 313e317. [13] R Development Core Team R, A Language and Environment for Statistical Computing, R Foundation for Statistical Computing, Vienna, Austria, 2012, ISBN 3-900051-07-0. http://www.R-project.org/. [14] M.D. Kerai, C.J. Waterfield, S.H. Kenyon, et al., Taurine: protective properties against ethanol-induced hepatic steatosis and lipid peroxidation during chronic ethanol consumption in rats, Amino Acids 15 (1998) 53e76.
Please cite this article as: M. Tamai et al., Acorbine, a Corbicula japonicaederived tripeptide containing non-proteinogenic amino acids, suppresses ethanol-induced liver injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.131
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Acorbine, a Corbicula japonicaederived tripeptide containing nonproteinogenic amino acids, suppresses ethanol-induced liver injury Miho Tamai a, b, c, Hidemitsu Uchisawa d, Yukari Saito c, Hajime Matsue e, Masaya Kawase f, Tetsushi Naraoka d, Yoh-ichi Tagawa b, c, * a
Faculty of Dental Medicine, Hokkaido University, Kita 13-jo, Nishi 7-chome, Kita-ku, Sapporo, 060-8586, Japan School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa, 226-8501, Japan Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa, 226-8501, Japan d Industrial Research Institute, Aomori Prefectural Industrial Technology Research Center, 221-10 Yamaguchi, Nogi, Aomori, 030-0142, Japan e Aomori University of Health and Welfare, 58-1 Mase, Hamadate, Aomori, 030-8505, Japan f Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama-shi, Shiga, 526-0829, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 November 2019 Accepted 20 November 2019 Available online xxx
Since ancient times, Corbicula extract has been believed in Japan to have hepatoprotective effects, but it remains unclear whether these claims are true, and if so, which component is responsible for hepatoprotection. In this study, we showed that Corbicula extract exerted a protective effect against liver damage. Recent work identified acorbine (b-alanyl-ornithyl-ornithine), a novel tripeptide containing non-proteinogenic amino acids, in the extract of Corbicula japonica. Synthesized acorbine cured alcoholinduced liver damage in mice. In addition, acorbine purified from Corbicula extract exerted a protective effect against alcohol-induced hepatotoxicity in a culture liver model derived from mouse ES/iPS cells. Thus, acorbine is one of the components of Corbicula extract that protects hepatocytes against ethanolinduced death. © 2019 Elsevier Inc. All rights reserved.
Keywords: Acorbine Corbicula Hepatitis Hepatoprotection Liver tissue
1. Introduction Orally ingested alcohol is delivered to the liver from the intestine, primarily through the hepatic portal vein, and is subsequently oxidized to finally acetic acid by a two-step reaction in hepatocytes. In the first step, ethanol is oxidized to acetaldehyde by alcohol dehydrogenase, concomitant with reduction of NADþ to NADH. Second, the resultant acetaldehyde is oxidized to acetic acid by acetaldehyde dehydrogenase in a reaction that also produces NADH. Ethanol is oxidized also by other enzymes, including catalase and microsomal CYP2E1. Ethanol and acetaldehyde can induce apoptosis in hepatocytes [1]. Excess NADH accumulation from these ethanol oxidation reactions also affects a variety of metabolic pathways [2]. Alcohol-related chronic hepatitis is a worldwide public health problem.
* Corresponding author. School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa, 226-8501, Japan. E-mail address: [email protected] (Y.-i. Tagawa).
Recent studies have focused on improving hepatic functions in patients with liver failure using local food products, such as curcumin [3] and polyphenolic extracts [4]. Although these substances have been commercialized as nutraceuticals worldwide, it remains unclear how they protect against liver injury and prevent clinically important diseases. For example, extracts of brackish-water bivalves, such as the freshwater clam Corbicula japonica, are thought to be beneficial for the liver after consumption of alcohol; however, it is not clear whether this claim is true, and if so, which component exerts the hepatoprotective effect. Bivalves of genus Corbicula are very popular as ingredients of soup in north Asian countries, especially miso soup in Japan. Recently, acorbine (b-alanylornithyl-ornithine), a novel tripeptide containing nonproteinogenic amino acids, was discovered in extract from Corbicula japonica (Corbicula extract) [5]. To date, acorbine has been identified only in Corbicula, and in no other type of organism. It is difficult to identify molecules that protect against liver injury because the liver regenerates itself after the injury; consequently, it has been challenging to develop useful liver-alternative culture system for investigating the functions of this organ. To address this issue, we established a murine ES cellderived in vitro
https://doi.org/10.1016/j.bbrc.2019.11.131 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: M. Tamai et al., Acorbine, a Corbicula japonicaederived tripeptide containing non-proteinogenic amino acids, suppresses ethanol-induced liver injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.131
2
M. Tamai et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
liver model, termed IVLmES, which includes both the hepatocyte layer and the accompanying sinusoid vascularlike network [6], and exhibits cytochrome P450 activity [7] and enhancement of urea production by excess ornithine [8]. Using IVLmES, we demonstrated the hepatoprotective effect of L-ornithine, which was not evident in monolayer single culture of mouse primary hepatocytes. In addition to being a major component of Corbicula extract, L-ornithine is abundant in hepatocytes as a component of the urea cycle. In this study, we found that acorbine could cure alcohol-induced liver damage in mice, and that acorbine exerts a protective effect against alcohol-induced hepatotoxicity in IVLmES/iPS.
diet containing 1e5% ethanol, as shown in Fig. 1A. The ethanolcontaining liquid diet was freshly prepared every day. The body weights of all mice were measured every day, and the blood was collected from the retroorbital plexus under isoflurane anesthesia once per week. Anesthesia was induced by placing each mouse in a regulated inhalator with 3% isoflurane. Blood samples were deposited in 1.5 mL tubes and centrifuged (1,200 g, 30 min, 4 C) to separate serum. Mouse livers were isolated as follows: after the mice were killed by cervical fracture, the livers were sliced and washed to remove hemoglobin, and then fixed in 4% paraformaldehyde/PBS. The study protocol was approved by the Animal Experimentation Committees of Tokyo Institute of Technology.
2. Materials and methods 2.3. Histological staining 2.1. Corbicula extract and acorbine Corbicula extract was prepared from the brackish-water bivalve Corbicula japonica obtained in Lake Jusanko, Aomori prefecture, Japan by Uchisawa et al. [5]. Corbicula extract was provided from Fukushimashoten Co., LTD. Its composition was analyzed by common methods: moisture level was determined by heating/drying at atmospheric pressure; protein by the Kjeldahl method, lipid by dimethyl ether extraction; carbohydrate by difference; ash by the direct ashing method; glycogen by the sodium thiosulfate method; calcium, sodium, and iron by atomic absorption; vitamin B2 by fluorometry; vitamin B6 and vitamin B12 by microbial assay; alanine and ornithine using an amino acid analyzer; and acorbine by high-performance liquid chromatography (HPLC), as shown in Table 1. Acorbine was isolated and purified as described previously [5]. Briefly, gel permeation HPLC was performed on an HPLC system (Hitachi, Japan) using a column (10 mm 30 cm) of Superdex Peptide HR 10/30 (Amersham Biosciences, Sweden) with a mobile phase of 250 mM NaCl in 20 mM phosphate buffer (pH 7.2) at room temperature (flow rate, 0.25 ml/min). Elution was monitored by absorbance at 205 nm. The fraction containing acorbine was desalted by dialysis using a Spectra/Por with an MWCO of 500, lyophilized, and then used for structural analyses. Acorbine with purity of 80.9%, as estimated by HPLC analysis, was used in this study.
Paraffin-embedded blocks and sections were performed by Genostaff (Tokyo, Japan). Tissues were fixed in 4% paraformaldehyde/PBS, embedded in paraffin, and sectioned at a thickness of 4 mm. Sections were stained with hematoxylin and eosin for histological examination. 2.4. Quantification of released liver enzyme activities Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) activities in the medium or serum were assessed using SPOTCHEM™ EZ SP-4430 and
2.2. Alcohol-induced chronic liver injury in mice The LiebereDeCarli diet (Oriental Yeast, Tokyo, Japan), a liquid diet, was naturally fed to female C57BL/6 J mice (6 weeks old at the start of the experiment) for 3 days, and then replaced with liquid
Table 1 Proximate Composition and Maine Ingredients of the Brackish-water Bivalve Extract followed by HPLC analysis. Unit
Components
Value (mg/100 g)
g/100 g g/100 g g/100 g g/100 g g/100 g g/100 g g/100 g g/100 g mg/100 g/100 g mg/100 mg/100 mg/100 g/100 g g/100 g g/100 g
Moisture Protein Lipid Carbohydrate Ash Glycogen Calcium Sodium Iron Succinic acid Vitamin B2 Vitamin B6 Vitamin B12 Alanine Ornithine Acorbine
5.3 34.0 0.1 48.9 11.7 41.0 0.95 3.0 9.3 7.1 3.01 0.43 0.26 1.29 0.17 0.84
g g g g
Fig. 1. Effect of Corbicula extract or synthesized acorbine on alcohol-induced chronic liver injury in mice. A: Alcohol administration schedule for the induction of alcoholinduced chronic liver injury. AST (B) and ALT (C) levels in the serum of ethanolreceiving (white column, N ¼ 24) or control (black column, N ¼ 6) BALB/cA mice. AST (D) and ALT (E) levels in the serum of mice receiving ethanol and Corbicula extract (CE), synthesized acorbine (Ac), or PBS (P), on 42 (white column), 49 (cross column), and 63 (black column) days after the start of ethanol administration.
Please cite this article as: M. Tamai et al., Acorbine, a Corbicula japonicaederived tripeptide containing non-proteinogenic amino acids, suppresses ethanol-induced liver injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.131
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SPOTCHEM™ II LDH (ARKRAY, Kyoto, Japan). 2.5. Culture and differentiation of murine ES cells and murine iPS cells
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All mice were sacrificed 63 days after initiation of ethanol intake, and their livers were histologically investigated. These experiments had to be carried out within 63 days, because many lipid droplets were present in the livers of control mice not receiving ethanol (Fig. 2A), indicating that the control mice had steatosis. On
The murine ES cell lines ST1 (originally established from the BALB/cA strain [9]) and Bruce 4 (derived from C57BL/6, OZ gene) [10,11] and the murine iPS cell line iPS-MEF-Ng-20D-17 (derived from 75% DBA, 12.5% C57BL/6, and 12.5% 129S4) [11,12] were obtained from RIKEN Bioresource Center (Ibaraki, Japan). The cells were grown on feeder layers of mitomycin Ctreated murine embryonic fibroblasts in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Tokyo, Japan) containing 15% fetal bovine serum (FBS) (Nichirei Biosciences, Tokyo, Japan), 1 mM sodium pyruvate (Invitrogen), 100 mM nonessential amino acids (Invitrogen), 100 mM 2mercaptoethanol (Sigma-Aldrich Japan, Tokyo, Japan), and 103 U/ mL leukemia inhibitory factor (LIF) (Chemicon International, Temecula, CA, USA). The IVLmES/iPS was generated by differentiation from murine ES cells as previously described [6,8,11]. Briefly, mES/iPS cells were dissociated with trypsin and re-suspended in Iscove’s modified Dulbecco’s medium (IMDM) (Invitrogen) containing 15% FBS, 1 mM sodium pyruvate, 100 mM nonessential amino acids, and 100 mM 2mercaptoethanol without LIF, and then cultured for 5 days in a hanging drop at a concentration of 1,000 cells per 50 mL in order to form embryoid bodies (EBs). Fifty well-formed EBs were collected and plated in each well of a gelatin-coated 6-well plate; the day of plating was denoted as day 0 (A0). In this study, IVLmES/iPS EBs were used 18 days after the plating (A18). 2.6. Quantification of urea concentrations Urea in the culture medium after centrifugation at 300 g for 5 min was quantified using a QuantiChrom™ Urea Assay Kit (BioAssay Systems, Hayward, CA, USA). 2.7. Statistical evaluation Values are expressed as means ± SEM. Calculation of descriptive statistics and paired t-test were performed using the R software [13]. Differences between groups were considered significant when P < 0.05. 3. Results 3.1. Acorbine has a beneficial therapeutic effect in the alcoholinduced chronic hepatitis in mice Most ingredients of Corbicula extract are present in other bivalves, with the exception of acorbine, a tripeptide containing nonproteinogenic amino acids (Table 1). Because acorbine is a unique ingredient in C. japonica, we investigated whether it was responsible for the hepatoprotective effect of Corbicula extract. To generate an alcohol-induced liver injury mouse model, a liquid diet containing ethanol was naturally administered to C57BL/6 J female mice following the schedule shown in Fig.1A. The activity levels of AST and ALT in the serum gradually increased after the intake of ethanol, and were higher than the control levels 28 days after ethanol administration (Fig. 1B and C). Corbicula extract at 1 g/kg of body weight or synthesized acorbine at 9 mg/kg of body weight had been orally administrated once a day using feeding needles since 42 days after initiation of ethanol intake (Fig. 1A). The release of AST and ALT in serum was completely suppressed in mice receiving Corbicula extract or synthesized acorbineeadministrated mice for at least 3 weeks during ethanol natural intake (Fig. 1D and E).
Fig. 2. Histological examination of hematoxylin and eosin staining of the liver. A: control, B: ethanol, C: ethanol and Corbicula extract, D: ethanol and acorbine administrated mice on 63 days after the start of ethanol administration.
Please cite this article as: M. Tamai et al., Acorbine, a Corbicula japonicaederived tripeptide containing non-proteinogenic amino acids, suppresses ethanol-induced liver injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.131
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the other hand, many ballooning hepatocytes were present in the livers of ethanol-receiving mice (Fig. 2B), indicative of typical ethanol-induced chronic hepatitis. Ballooning hepatocytes were much less abundant in the livers of mice receiving Corbicula extract or acorbine (Fig. 2C and D). In particular, some lipid droplets, but no ballooning hepatocytes, were detected in the livers of acorbinereceiving mice (Fig. 2D).
that acorbine could suppress ethanol-induced hepatocyte death in the IVLmES/iPS. To investigate hepatic activity of surviving hepatocytes in the IVLmES/iPS, we measured urea production in the medium. The results revealed that urea production levels in the medium in the presence of ethanol could also be maintained by the addition of acorbine (Fig. 4AeC).
3.2. Acorbine protects against alcohol-induced cell death of the hepatocytes in murine ES- or iPS-derived hepatic tissue models
4. Discussion
The murine ES/iPS cellderived in vitro liver model, IVLmES/iPS, was prepared from the murine embryonic stem cell lines ST1 (BALB/cA background) and Bruce 4 (C57BL/6) and the murine iPS cell line iPS-MEF-Ng-20D-17 (derived from 75% DBA, 12.5% C57BL/6 and 12.5% 129S4) by in vitro liver organogenesis. Corbicula extract at a final concentration of 2% (w/v), or purified acorbine chloride at a final concentration of 40 mM, was added into the medium of the IVLmES/iPS along with ethanol at a final concentration of 3% (w/v), and the activity of released LDH in the medium was measured 24 h later. Release of LDH by addition of ethanol was suppressed in the medium of the IVLmES/iPS containing Corbicula extract or purified acorbine, in comparison with PBS controls (Fig. 3AeC). In particular, purified acorbine significantly suppressed the increase of LDH in the medium. This result suggests
In Japan, Corbicula extract has been believed since ancient times to exert hepatoprotective effects. In this study, we found that Corbicula extract protected against alcohol-induced hepatocyte death in mouse (Figs. 1 and 2). Corbicula extract contains several potentially hepatoprotective substances, e.g., L-ornithine [8] and taurine [14]. Given that L-ornithine mediates protective effects on hepatocytes in the IVLmES culture system [8], this molecule is a strong candidate for a downstream factor in the hepatoprotective effect associated with Corbicula extract. However L-ornithine is present in other bivalves, such as scallop. Acorbine (b-Ala-OrnOrn), a novel tripeptide containing non-proteinogenic amino acids, is present only in C. japonica, but not in scallop [5]. In this study, we showed that chemically synthesized acorbine could cure alcohol-induced liver damage in mice and we demonstrated that acorbine purified from Corbicula extract exerts a
Fig. 3. Cell death in the IVLmES/iPS stimulated by ethanol. The each IVL was induced cell death by the addition of 3% ethanol in the medium and simultaneous addition of Corbicula extract (CE), acorbine (Ac), or PBS (P). AeC: Released LDH levels in the medium of the IVLmES/iPS. The IVL was derived from ES ST1 (A), ES Bruce4 (B), and iPS iPSMEF-Ng-20D-17 (C).
Fig. 4. Hepatic function in the IVLmES/iPS stimulated by ethanol. The each IVL was induced cell death by the addition of 3% ethanol in the medium and simultaneous addition of Corbicula extract (CE), acorbine (Ac), or PBS (P). AeC: Urea production of the IVLmES/iPS. The IVL was derived from ES ST1 (A), ES Bruce4 (B), and iPS iPS-MEF-Ng20D-17 (C).
Please cite this article as: M. Tamai et al., Acorbine, a Corbicula japonicaederived tripeptide containing non-proteinogenic amino acids, suppresses ethanol-induced liver injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.131
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protective effect against alcohol-induced hepatotoxicity in a culture model. We found that acorbine is one of the compounds in Corbicula extract that protects against ethanol-induced hepatocyte death. Acorbine can be precipitated by either acetate or chloride; we used acorbine chloride in the assay of IVLmES/iPS. Initially, we tried to investigate the hepatoprotective effect in the IVLmES/iPS using acorbine acetate, but the acetate strongly induced cell death. By changing the acetate to chloride, we succeeded in protecting the hepatocyte in the IVLmES/iPS after exposure to alcohol. L-ornithine protects against alcohol-induced hepatocyte death [8]. Therefore, because acorbine is hydrolyzed to L-ornithine and balanine in mouse, it is possible that acorbine-derived L-ornithine acts on hepatocytes to reduce the level of ammonium ion. The production of urea was significantly higher in the IVLmES/iPS in the presence of Corbicula extract or acorbine than in the absence of these supplements; however, acorbine might not be hydrolyzed under the culture conditions we used. Non-hydrolyzed acorbine could be detected in the liver by HPLC analysis, indicating that acorbine was delivered directly to the liver through the portal vein. The net dose of acorbine in Corbicula extract administered to mice was lower than the dose of singly administered acorbine, suggesting that other substances in Corbicula extract exert a synergistic hepatoprotective effect. We are still looking for such additional active substances in Corbicula extract. In summary, we found that Corbicula extract exerted a protective effect against liver damage. Acorbine (b-alanyl-ornithyl-ornithine), a tripeptide containing non-proteinogenic amino acids, could cure alcohol-induced liver damage in mice. Acorbine purified from Corbicula extract also protected against alcohol-induced hepatotoxicity in a liver culture model derived from mouse ES/iPS cells. Thus, acorbine is one of the components of Corbicula extract that protects against ethanol-induced hepatocyte death. Declaration of competing interest The authors declare no conflicts of interest. Acknowledgements This study was supported by a Grant-in-Aid for Challenging Exploratory Research [No. 24650254](to Y. Tagawa), for Scientific Research (A) [No. 25242040] (to Y. Tagawa), for Young Scientists (B) [No. 26750145] (to M. Tamai) from the Japan Society for the Promotion of Science (JSPS) and a Grant-in-Aid for Scientific Research on Innovative Areas [No. 231190003] (to Y. Tagawa) from the Ministry of Education, Culture, Sports, Science and Technology
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(MEXT) of Japan and by Building of Consortia for the Development of Human Resources in Science and Technology, Ministry of Education, Culture, Sports, Science and Technology, Japan. The mouse embryonic fibroblast-derived iPS cell line, iPS-MEFNg-20D-17 (APS0001), was provided by RIKEN Bio-Resource Center (Ibaraki, Japan) in agreement with Dr. Shinya Yamanaka (Center for iPS Cell Research and Application, Kyoto University).
Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.131.
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Please cite this article as: M. Tamai et al., Acorbine, a Corbicula japonicaederived tripeptide containing non-proteinogenic amino acids, suppresses ethanol-induced liver injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.131