Amino Acids DOI 10.1007/s00726-017-2478-2
ORIGINAL ARTICLE
Expression of cationic amino acid transporters in pig skeletal muscles during postnatal development Aiko Ishida1 · Akane Ashihara1 · Kazuki Nakashima1 · Masaya Katsumata1,2
Received: 23 November 2016 / Accepted: 1 August 2017 © Springer-Verlag GmbH Austria 2017
Abstract The cationic amino acid transporter (CAT) protein family transports lysine and arginine in cellular amino acid pools. We hypothesized that CAT expression changes in pig skeletal muscles during rapid pig postnatal development. We aimed to investigate the tissue distribution and changes in the ontogenic expression of CATs in pig skeletal muscles during postnatal development. Six piglets at 1, 12, 26, 45, and 75 days old were selected from six litters, and their longissimus dorsi (LD), biceps femoris (BF), and rhomboideus (RH) muscles, and their stomach, duodenum, jejunum, ileum, colon, liver, kidney, heart, and cerebrum were collected. CAT-1 was expressed in all the 12 tissues investigated. CAT-2 (CAT-2A isoform) expression was highest in the skeletal muscle and liver and lowest in the jejunum, ileum, kidney, and heart. CAT-3 was expressed mainly in the colon and detected in the jejunum, ileum, and cerebrum. The CAT-1 expression was higher in the skeletal muscle of day 1 pigs than in that of older pigs (P < 0.05). The CAT-2 mRNA level was lowest at day 1, but increased with postnatal development (P < 0.05). There was no significant change in CAT-1 expression among the LD, BF, and RH during postnatal development (P > 0.05); however, there Handling Editor: E. I. Closs. Electronic supplementary material The online version of this article (doi:10.1007/s00726-017-2478-2) contains supplementary material, which is available to authorized users. * Aiko Ishida
[email protected] 1
Institute of Livestock and Grassland Science, NARO, Tsukuba, Ibaraki 305‑0901, Japan
2
Present Address: School of Veterinary Science, Azabu University, Sagamihara, Kanagawa 252‑5201, Japan
was a change in CAT-2 expression. The CAT-2 expression was highest in the LD of 12-, 26-, 45-, and 75-day-old pigs, followed by the BF and RH (P < 0.05). These results suggest that CAT-1 and CAT-2 play different roles in pig skeletal muscles during postnatal development. Keywords Cationic amino acid transporter · Pig · Postnatal development · Skeletal muscle
Introduction Cationic amino acids (CAAs) such as arginine, lysine, and histidine are essential for optimal growth of pigs, with lysine frequently being the major limiting amino acids (AA) in typical pig diets. Lysine is an important building block of proteins; hence, its deficiency results in an AA imbalance, which inhibits protein synthesis (Ishida et al. 2011). The AAs absorbed by the intestine are transported to cells by AA transporters (AATs), which differ in their substrate specificity and driving force (Christensen 1990). The CAAs are transported at high affinity by two gene families: the system y+ cationic amino transporter (CAT) and system b0+ (Verrey et al. 2003). The CATs are the primary AAT system for the accumulation of lysine and arginine in cellular AA pools for use in nitrogen metabolism (Verrey et al. 2003). Skeletal muscle comprises the largest organ of the body, playing vital roles in locomotion and representing approximately 40% of the body mass; therefore, the muscle protein pool represents a very large reservoir of AAs in the body. In response to nutritional and physiological status, AAs are released from skeletal muscle during muscle atrophy and are incorporated into skeletal muscle during muscle growth or hypertrophy. The AATs play a crucial role in maintaining AA homeostasis and supply AAs for growth of skeletal
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muscle; however, the mechanisms behind these processes are less clear. The neonatal stage of animal growth shows a more rapid growth than any other stage, with a high rate of protein turnover. The increase of protein mass is greater in skeletal muscle than in the entire body during postnatal growth. The fractional rate of growth and protein synthesis decreases during neonatal development (Davis et al. 1989; Goldspink and Kelly 1984). Dramatic changes occur in skeletal muscle during postnatal growth, including changes in the rate of protein accretion, changes in myosin heavy chain proportion (Suzuki and Cassens 1980), and an increase in glycolytic metabolism (Davies 1972), as well as the induction of myofiber hypertrophy (Harrison et al. 1997). We hypothesized that the expression of CATs changes in pig skeletal muscle during postnatal development. However, there is little information about the distribution of CATs in different tissues and no information on the expression of CATs in pig skeletal muscle. To test the above hypothesis, we investigated the expression of CAT-1, -2, and -3 in different skeletal muscles and several tissues of pig at five time points during the first 10 weeks after birth.
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Quantitative real‑time PCR Total RNA was extracted from the tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), in accordance with the manufacturer’s instructions. The quantity and quality of RNA were measured using a spectrophotometer (NanoVue; GE Healthcare, Tokyo, Japan). Complementary DNA (cDNA) was synthesized from total RNA (0.5 μg) using a random primer (Takara, Kusatsu, Japan) and M-MLV reverse transcriptase (TOYOBO, Osaka, Japan). Quantitative real-time PCR analysis of CAT-1, -2, and -3 and six candidate housekeeping genes was performed using a Roche LightCycler (Roche Diagnostics, Indianapolis, IN, USA) using the QuantiTect SYBR Green PCR kit (Qiagen, Hilden, Germany). We also measured AA transporter B0,+ (ATB0,+), B0,+-type amino acid transporter 1 ( B0,+AT), and y+LAT1, which also transport CAAs. Table 1 and Supplemental Table 1 list the primers used for quantitative realtime PCR. To identify stable endogenous control in skeletal muscles during postnatal development, six housekeeping genes, namely, 18S ribosomal RNA (18S rRNA), β-actin Table 1 Primer sequences for real-time PCR
Materials and methods Animals and experimental design All experimental procedures were conducted in accordance with the guidelines of the Animal Research Committee of the Institute of Livestock and Grassland Science, NARO. Thirty male Duroc × (Large White × Landrace) pigs were selected from six litters (five pigs/litter, birth weight, 1.52 ± 0.17 kg). The pigs were housed with littermates under standard conditions and fed commercial diets, i.e., starting creep feeding [crude protein (CP) 23%, total digestible nutrients (TDN) 90%; JA Zenn-noh, Tokyo, Japan] on day 14, weaning on day 28, starting the transition diet (CP 21%, TDN 85%; JA Zenn-noh) on day 31, and starting grower meal (CP 16%, TDN 78%; Shimizukou Shiryo, Shizuoka, Japan) on day 63. The piglets were castrated at 21 days of age. One pig from each litter was sacrificed by electrical stunning, followed by exsanguination at five different time points (i.e., 1, 12, 26, 45, and 75 days), without direct stress resulting from castration, weaning, or a change in diet. Longissimus dorsi (LD), rhomboideus (RH), and biceps femoris (BF) muscles were collected from six pigs at each slaughter time point. For the tissue distribution studies, the LD, BF, and RH muscles, and stomach, duodenum, jejunum, ileum, colon, liver, kidney, heart, and cerebrum were collected from piglets at each time point. The samples were removed immediately, frozen in liquid nitrogen, and stored at −80 °C.
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Primer sequence (5′–3′)b
CAT-1
F: catcaaaaactggcagctca R: tggtagcgatgcagtcaaag CAT-2 F: ggtcctgccaagtatgtcgt R: aaatgacccctgcagtcaag CAT-3 F: accgaaccctatccgagact R: ccacacctagggccactaaa RPL4 F: aggaggctgttctgcttctg R: tccagggatgtttctgaagg HPRT1 F: aagcttgctggtgaaaagga R: gggactccagatgtttccaa HMBS F: ggcctgcagtttgaaatcat R: ggggtgaaagacaacagcat F: aacagttcagtagttatgagcTBPc caga R: agatgttctcaaacgcttcg ACTB F: tccagccctccttcctgggc R: agcaccgtgttggcgtagag 18S rRNA F: agtcggcatcgtttatggtc R: cgcggttctattttgttggt
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NM_001012613
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a CAT-1, CAT-2 and CAT-3 cationic amino acid transporter 1, 2, and 3, respectively, RPL4 ribosomal protein L4, HPRT1 hypoxanthine phosphoribosyltransferase 1; HMBS, hydroxymethyl-bilane synthase, TBP TATA box binding protein, ACTB β-actin, 18S rRNA 18S ribosomal RNA b c
F forward primer, R reverse primer
Nygard et al. (2007)
Expression of cationic amino acid transporters in pig skeletal muscles during postnatal…
(ACTB), ribosomal protein L4 (RPL4), hypoxanthine phosphoribosyltransferase 1 (HPRT1), TATA box binding protein (TBP), and hydroxymethyl-bilane synthase (HMBS), were selected from commonly used reference genes. The stability of the genes was measured using NormFinder (http://www. mdl.dk/publicationsnormfinder.htm) software. To examine the effects of postnatal growth, the data were normalized using the geometric means of RPL4, HPRT1, and HMBS as reference genes for skeletal muscles (Uddin et al. 2011; Vandesompele et al. 2002). The LD muscle was used as a calibrator for making relative comparisons among the different skeletal muscles. The fold change was calculated using the 2−ΔΔCt method (Livak and Schmittgen 2001). Statistical analysis Statistical analyses were performed using analysis of variance according to the MIXED model procedure (SAS Inst. Inc., Cary, NC, USA). For the analysis of ontogenic expression, the model included the fixed effect of age in days and the random effect of the dam. For the skeletal muscle comparison, the model included the fixed effects of the skeletal muscle and the random effect of the individual animal. Differences between the fixed effects were assessed using the Tukey–Kramer honestly significant difference test within SAS software. P < 0.05 was considered significant.
Results and discussion Tissue distribution The CAT family in rodents and humans comprises CAT-1, -2, -3, and -4 which are encoded by the Slc7A1, 2, 3, and 4 genes, respectively. The cDNA sequences for porcine CAT-1 (Cui et al. 2005), -2 (Zou et al. 2009), and -3 (Zuo et al. 2013) have been reported. In rodents and humans, differential splicing of Slc7A2 (CAT-2) results in two isoforms, CAT-2A and CAT-2B, and the cDNA encoding them were cloned. CAT-2A and CAT-2B differ only in a stretch of 42 AAs. That difference determines the transport properties of CAT-2 proteins, namely, a 10–30-fold lower substrate affinity of CAT-2A (Closs et al. 1993, 1997). Meanwhile, porcine CAT-2 mRNA measured in this study was cloned in 2009 by Zou et al. The cDNA (NM_001110420.1) shows significant homology with human and mouse CAT-2A and less with CAT-2B. There are two predicted porcine CAT-2 sequences: transcript variant X1 (XM_021077148) encoding CAT-2A and transcript variant X2 (XM_021077148) encoding CAT-2B. The primer used in this study for CAT-2 matched with the predicted sequence of CAT-2A. We began the present study by quantifying CAT expression in different tissues, although there have been other
limited tissue distribution studies focusing on individual CATs separately. Here the expression of CAT was quantified in 12 tissues, including different skeletal muscles (LD, BF, and RH muscles) and the stomach, duodenum, jejunum, ileum, colon, liver, kidney, heart, and cerebrum of 45-dayold pigs (n = 6 per tissue; Fig. 1). Although previous studies reported ubiquitous expression of CAT-1 in humans, rodents, and chickens (Ito and Groudine 1997; MacLeod and Kakuda 1996), little information on CAT-1 expression in porcine tissues is available. In agreement with previous reports, CAT-1 expression was detected in all tissues investigated, including the skeletal muscles (Fig. 1a). By contrast, the expression of CAT-2 and -3 was restricted to specific tissues. The CAT-2 expression was highest in the skeletal muscles and liver in agreement with an earlier study (Ito and Groudine 1997), with low but significant expression detected in the jejunum, ileum, liver, kidney, and heart (Fig. 1b). In support of this, Zou et al. (2009) reported that CAT-2 mRNA was detected in the brain, kidney, intestines, lung, liver, heart, and muscle of 6-day-old Landrace pigs, and that the highest abundance of CAT-2 mRNA occurred in the heart. CAT-3 was expressed mainly in the colon, and low expression was detected in the jejunum, ileum, and cerebrum (Fig. 1c). Although we also investigated CAT-3 expression in different skeletal muscles, mRNA levels were below the detection limit in these cases. Zuo et al. (2013) reported moderate CAT-3 expression in pig muscles, with the highest expressions in the brain and heart. In other adult mammals, CAT-3 expression has been found to be restricted to the brain and kidneys (Hosokawa et al. 1997). CAT-3 also plays a major role during embryogenesis (Verrey et al. 2003). We also measured the expression of A TB0,+, b0,+AT, and y+LAT1 which transport CAAs. Although the expression of ATB0,+ and b0,+AT was restricted to specific tissues and the mRNA of A TB0,+ and b 0,+AT was undetectable in the skeletal muscles, y+LAT1 expression was detected in all tissues investigated (Supplemental Figs. 1 and 2). A TB0,+ was expressed in the colon, and low expression was detected in the jejunum, ileum, and cerebrum (Supplemental Fig. 1a). The b0,+AT was expressed mainly in the intestines and kidney, and low expression was detected in the colon, liver, and cerebrum (Supplement Fig. 1b). Regarding the distribution in the intestines, the ileum had the highest b 0,+AT mRNA level, whereas the colon had the lowest, in agreement with an earlier study (Zhi et al. 2008). Matching previous reports, the levels of y +LAT1 expression in the intestine and kidney were high and lower levels of expression were detected in the skeletal muscles, heart, and cerebrum (Supplemental Fig. 2) (Pfeiffer et al. 1999). Although these results suggest that CAT-1 and -2 play important roles in transporting CAAs to skeletal muscles, our results are limited to the mRNA level, with the protein level and kinetic parameters, Km and Vmax, of transporters
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not being determined. Further experiments on the affinity and maximum velocity of porcine CATs are clearly needed to identify precisely the degree of contribution of each CAT to the transport of AA in skeletal muscle.
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Fig. 1 Expression of cationic amino acid transporter (CAT)-1 (a), CAT-2 (b), and CAT-3 (c) in different tissues of pig on postnatal day 45. The LD muscle was used for CAT-1 and CAT-2, and the jejunum was used for CAT-3 as a calibrator for making relative comparisons among the different tissues. The results are presented as mean ± standard error of the mean (SEM) (n = 6). ND no detectable expression, LD longissimus dorsi muscle, RH rhomboideus muscle, BF biceps femoris muscle
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weights of pigs on days 1, 12, 26, 45, and 75 are shown in Fig. 2. These weights ranged from 1.2–1.7 kg at birth to 33.8–47.6 kg on day 75. The mean daily weight gains were similar for all six litters examined (mean ± SEM = 0.37 ± 0.02 kg day−1). Humphrey et al. (2004) reported the ontogenic expressions of CAT-1 and CAT-2 in the skeletal muscles of broilers, whereas Zou et al. (2009) investigated the ontogenic expression of CAT-2 in the intestine of pigs. However, no reports have been published on the ontogenic expression of CAT-1 and -2 in skeletal muscles of growing pigs and
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other mammals. Compared with 12-, 26-, 45-, and 75-dayold pigs, the CAT-1 mRNA level was highest in the skeletal muscle of 1-day-old pigs (P < 0.05, Fig. 3). Furthermore, there was no change in CAT-1 expression on postnatal days 12, 26, 45, and 75 (Fig. 3). The ontogenic expression of CAT-1 in the skeletal muscles was similar to that in the heart, stomach, liver, and kidney (Fig. 4a, b, g, h). Although CAT-2 expression was lowest in the skeletal muscles of day 1 pigs, it increased with development (P < 0.05, Fig. 5). There was no change in the CAT-2 mRNA level in RH muscle on days 12, 26, 45, and 75. However, the CAT-2 mRNA level in the LD muscle was higher on day 75 than on day 12. It was also higher in the BF muscle on day 75 than on days 12 and 26. Although the ontogenic expression of CAT-2 in the heart was similar to that in the skeletal muscle, it was higher in the ileum of day 1 pigs than in the ileum of older pigs (P < 0.05, Fig. 6a, c). The CAT-3 expression was detected only in the colon, jejunum, ileum, and cerebrum. The changes in ontogenic expression were only detected in the jejunum and ileum (P < 0.05, Fig. 7). CAT-1 and -2
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had different patterns of the ontogenic expression, and there was no significant difference in the patterns among the LD, BF, and RH muscles. These results suggest that CAT-1 and -2 play different roles in the development of skeletal muscle. Humphrey et al. (2004) reported that CAT-1 expression increased in the gastrocnemius and pectoralis muscles in broilers, whereas the CAT-2 mRNA level decreased during the first week posthatch. Fasting and surgical trauma such as partial hepatectomy or splenectomy has been found to increase CAT-2 but not that of CAT-1 in the skeletal muscles of mice (Kakuda et al. 1998). CAT-1 expression was highest in LD, RH, and BF muscles on day 1, with levels decreasing and remaining constant thereafter. CAT-1 expression in the liver of rats was induced following treatment with dexamethasone (Liu and Hatzoglou 1998). Due to the increase in plasma cortisol in fetal pigs during the last 2 weeks of gestation, the plasma cortisol concentration in pigs on day 1 postnatally was fiveto tenfold higher than that in fetal pigs (Fowden et al. 1995; Heo et al. 2003). A rapid decline in cortisol concentration was observed by day 3, with the total cortisol concentration remaining unchanged thereafter (Kattesh et al. 1990). Thus, it is possible that changes in the cortisol concentration may affect CAT-1 expression shortly after birth. CAT-2 expression increased in skeletal muscles during growth. Nitric oxide (NO), a multifunctional signaling molecule that plays a key role in various physiological processes, is synthesized from l-arginine by NO synthases (NOS). During biological processes, CAT-2 supplies arginine to NOS; thus, NO production is dependent upon CAT-2 expression and CAT-2-mediated arginine uptake (Stathopulos et al. 2001). Although the expression of CAT-2 changes with the expression of NOS in the aorta of rats, CAT-1 expression remains unchanged (Stathopulos et al. 2001). Three NOS isoforms have been identified (nNOS, iNOS, and eNOS), and the skeletal muscle predominantly expresses nNOS (McConell et al. 2007). The nNOS levels in the skeletal
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Fig. 4 Changes in the ontogenic expression of cationic amino acid transporter (CAT)-1 in the heart (a), stomach (b), duodenum (c), jejunum (d), ileum (e), colon (f), liver (g), kidney (h), and cerebrum (i)
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Fig. 6 Changes in the ontogenic expression of cationic amino acid transporter (CAT)-2 in the heart (a), jejunum (b), ileum (c), liver (d), and kidney (e) of pig on postnatal days 1, 12, 26, 45 (n = 6 per time point), and 75 (n = 5). The results are presented as mean ± standard error of the mean (SEM)
Fig. 7 Changes in the ontogenic expression of cationic amino acid transporter (CAT)-3 in the jejunum (a), ileum (b), colon (c), and cerebrum (d) of pig on postnatal days 1, 12, 26, 45 (n = 6 per time point), and 75 (n = 5). The results are presented as mean ± standard error of the mean (SEM)
muscles of rat increase dramatically during development (Capanni et al. 1998; Chang et al. 1996). Therefore, the increase in CAT-2 expression during postnatal development may contribute to NO production in pig skeletal muscles during postnatal development.
(Christova et al. 1997). Although the LD muscle has large proportion of a fast-twitch glycolytic muscle (Karlsson et al. 1993; Lefaucheur et al. 2002), the RH and BF muscles are mixtures of slow- and fast-twitch oxido-glycolytic muscles. Compared with the RH muscle, the BF muscle contains a greater amount of fast-twitch glycolytic muscle (Harrison et al. 1997). Due to differences in their myofiber type composition, these three skeletal muscles have different characteristics related to metabolism (Lefaucheur et al. 2011). In the fiber type composition, the differences of the characteristics of each of the skeletal muscles become distinct during postnatal growth. Harrison et al. (1997) reported an increase in the proportion of type II fibers, but a decrease in the proportion of type I fibers in the LD muscle between birth and day 2 postnatal in pigs. In addition, Suzuki and Cassens (1980) and Lefaucheur and Vigneron (1986) reported that the proportion of fiber type change from birth
Expression of CAT in different skeletal muscles There was no significant change in CAT-1 expression among the LD, BF, and RH muscles during postnatal development (P > 0.05, Fig. 8); however, there was a change in CAT-2 expression. CAT-2 expression was highest in LD muscle of pigs on days 12, 26, 45, and 75, followed by the BF and RH muscles (P < 0.05, Fig. 9). The proportion of type IIA/X muscle fiber was found to be positively correlated with the content of nNOS in pigs (Liu et al. 2015) as well as the activity of NOS in rats (Kobzik et al. 1994) and mice
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Fig. 8 Expression level of cationic amino acid transporter (CAT)-1 in the longissimus dorsi (LD), rhomboideus (RH), and biceps femoris (BF) muscles of pig on days 1 (a), 12 (b), 26 (c), 45 (d) (n = 6 per time point), and 75 (e) (n = 5). The results are presented as mean ± standard error of the mean (SEM)
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Fig. 9 Expression level of cationic amino acid transporter (CAT)-2 in the longissimus dorsi (LD), rhomboideus (RH), and biceps femoris (BF) muscles of pig on days 1 (a), 12 (b), 26 (c), 45 (d) (n = 6 per time point), and 75 (e) (n = 5). The results are presented as mean ± standard error of mean (SEM)
Expression of cationic amino acid transporters in pig skeletal muscles during postnatal…
up to 2 months of age, with no significant change thereafter. We also previously reported that the expression of MHC IIA/X greatly increased in the LD and the BF muscles during postnatal development, whereas the age in days had no effect on its level in the RH muscle (Katsumata et al. 2017). The changes in CAT-2 expression in skeletal muscles from 12-day-old pigs may be associated with changes in NOS expression caused by their fiber type composition. Further studies are required to understand whether CAT-2 expression contributes to fiber type and proportion, as well as to establish the relation between CAT-2 expression and NOS activity. Moreover, as there are two isoforms of CAT-2, further investigation of each isoform is needed. Regarding the CATs of swine, their protein expression and kinetics parameters have yet to be elucidated, as in this study we investigated only their mRNA expression. Further experiments on the affinity and maximum velocity of porcine CATs are, thus, needed for adequate evaluation of the physiological significance of the changes detected. If these results are combined, it will provide important information about the AA requirements and AA metabolism at each organization during growth.
Conclusions We found differences in CAT-1 and -2 expression in several tissues during postnatal development in pigs. Although there was no difference in CAT-1 expression among the three types of skeletal muscles during postnatal development in pigs, there was a striking difference in CAT-2 expression. These results suggest that CAT-1 and -2 play different roles in pig skeletal muscles during postnatal development. Acknowledgements The authors are grateful to the animal care team of the Pig Unit of NARO for the care of the pigs and support for the sample collections. Part of this work was previously published as an abstract and presented as a poster at the 4th EAAP International Symposium on Energy and Protein Metabolism and Nutrition, 9–12 September, California, USA (Ishida et al. 2013). In this research, we used the supercomputer of the Agriculture, Forestry and Fisheries Research IT (AFFRIT), Ministry of Agriculture, Forestry, and Fisheries (MAFF), Japan. Compliance with ethical standards Conflict of interest The authors declare that they have no conflicts of interest. Ethical approval All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. Funding This work was partially supported by a Grant-in-Aid for Young Scientists (B) (Grant No. 26850170) from the Ministry of Education, Science and Culture, Japan.
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