L-theanine taken into cells via solute carrier family 38 member 1 possible to by inhibits neuronal blastoma cell growth.

L-theanine taken into cells via solute carrier family 38 member 1 possible to by inhibits neuronal blastoma cell growth. | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 29 July 2025 V1 Latest version Share on L-theanine taken into cells via solute carrier family 38 member 1 possible to by inhibits neuronal blastoma cell growth. Authors : Koichi Kawada , Yuri Matsushima , Ruka Nakamura , Megumi Nishio , Yuka Watanabe , Shuhei Araki , Tamika Sudo , … Show All … , Sayuki Yoshikawa , Mitsuhiro Shibata , Toshihiko Kinjo , Kyosuke Uno , Yutaro Higashiura , Akiko Yamamuro , Yuki Ishimaru , Yasuhiro Yoshioka , Sadaaki Maeda , and Nobuyuki Kuramoto 0000-0003-2565-2067 [email protected] Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.175376195.59657581/v1 Published Neurochemical Research Version of record Peer review timeline 231 views 131 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract L-gamma-glutamylethylamide (L-theanine, theanine) is an amino acid and an umami component found in green tea. According to previous reports, theanine acts on the central nervous system by alleviating stress and maintaining natural sleep. Furthermore, theanine has been reported to have a mild cancer-suppressing effect. However, the molecular mechanism of theanine’s potential central nervous system activity remains unclear. We evaluated the inhibitory effect of theanine on the proliferation of neural cell lines and found that theanine most effectively inhibited the proliferation of NSC-34 mouse motor neuron-like hybrid cells compared to other neural cells. NSC-34 cell proliferation inhibition by theanine was completely alleviated by co-administration of α-(methylamino) isobutyric acid or leucine, a substrate of solute carrier family 38 member 1 (Slc38a1, the glutamine transporter) or Slc7a5 (the glutamine/leucine exchanger) respectively. This suggests that theanine uptake into cells occurs via Slc38a1 and excretion from cells occurs via Slc7a5. However, inhibition was observed even in the absence of glutamine and did not correlate with changes in mammalian target of rapamycin phosphorylation levels. These results suggest that theanine inhibits proliferation in a manner that is still unclear after it is taken up into cells. Based on these findings, we propose that theanine may exert an inhibitory effect on the proliferation of neural cells that have abnormally proliferating Slc38a1, namely neuroblastoma. Key words: L-theanine, Slc38a1, Slc7a5, glutamine, leucine, growth inhibition Title page: l-theanine taken into cells via solute carrier family 38 member 1 possible to by inhibits neuronal blastoma cell growth. Short Title Inhibition of cell growth by L-theanine Authors: Koichi Kawada 1 , Yuri Matsushima 1 , Ruka Nakamura 1 , Megumi Nishio 1 , Yuka Watanabe 2 , Shuhei Araki 2 , Tamika Sudo 2 , Sayuki Yoshikawa 2 , Mitsuhiro Shibata 2 , Toshihiko Kinjo 1 , Kyosuke Uno 1 , Yutaro Higashiura 1 , Akiko Yamamuro 2 , Yuki Ishimaru 2 , Yasuhiro Yoshioka 2 , Sadaaki Maeda 2 , Nobuyuki Kuramoto 1,* not-yet-known not-yet-known not-yet-known unknown Affiliations: 1Laboratory of Molecular Pharmacology and 2Laboratory of Pharmacotherapeutics, Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka, 573-0101, Japan. *Corresponding author: All correspondence should be addressed to Nobuyuki Kuramoto Ph.D., Laboratory of Molecular Pharmacology, Setsunan University Faculty of Pharmaceutical Sciences, 45-1 Nagaotoge-cho, Hirakata, Osaka, 573-0101, Japan. Tel/Fax: 81-(0)72-866-3207 E-mail: [email protected] Acknowledgements We thank the students at Setsunan University (Osaka, Japan) for their assistance in the experiments. We would like to thank Editage (www.editage.jp) for the English language editing. Abstract L-gamma-glutamylethylamide (L-theanine, theanine) is an amino acid and an umami component found in green tea. According to previous reports, theanine acts on the central nervous system by alleviating stress and maintaining natural sleep. Furthermore, theanine has been reported to have a mild cancer-suppressing effect. However, the molecular mechanism of theanine’s potential central nervous system activity remains unclear. We evaluated the inhibitory effect of theanine on the proliferation of neural cell lines and found that theanine most effectively inhibited the proliferation of NSC-34 mouse motor neuron-like hybrid cells compared to other neural cells. NSC-34 cell proliferation inhibition by theanine was completely alleviated by co-administration of α-(methylamino) isobutyric acid or leucine, a substrate of solute carrier family 38 member 1 (Slc38a1, the glutamine transporter) or Slc7a5 (the glutamine/leucine exchanger) respectively. This suggests that theanine uptake into cells occurs via Slc38a1 and excretion from cells occurs via Slc7a5. However, inhibition was observed even in the absence of glutamine and did not correlate with changes in mammalian target of rapamycin phosphorylation levels. These results suggest that theanine inhibits proliferation in a manner that is still unclear after it is taken up into cells. Based on these findings, we propose that theanine may exert an inhibitory effect on the proliferation of neural cells that have abnormally proliferating Slc38a1, namely neuroblastoma. Key words: L-thean ine, Slc38a1, Slc7a5, glutamine, leucine, growth inhibition not-yet-known not-yet-known not-yet-known unknown Running title: Theanine is taken into the cells via Slc38a1 and inhibits cell growth. not-yet-known not-yet-known not-yet-known unknown Highlights: L-Theanine inhibits glutamine-induced cell proliferation in neurons. The glutamine transporter Slc38a1 is highly expressed in neurons. L-Theanine’s inhibition of cell proliferation is reversed by leucine. Inhibition of cell proliferation by L-theanine is associated with Slc38a1, not Slc7a5. 1. Introduction l-Theanine (l-γ-glutamylethylamide; hereafter theanine) is an amino acid present in the leaves of Camellia sinensis, an evergreen shrub used for the production of tea. When consumed as part of a green tea beverage, theanine binds to the l-amino acid binding site in the venus flytrap domain of taste receptor type 1 member 1, which is a receptor present on the tongue and in the mouth that responds to the umami or savory taste stimulus (Narukawa et al. 2014). Studies have shown that orally ingested theanine is translocated to the brain and into neural cells, suggesting that it may have central nervous system (CNS) effects (Yokogishi et al. 1998; Kakuda et al. 2008). Indeed, in humans, theanine intake has been shown to reduce stress and assist in the maintenance of natural sleep (Rao et al. 2015). In rats, electroencephalographic studies have revealed that intravenous administration of theanine alleviates caffeine-induced hyperexcitability (Kakuda et al. 2000). In adult mice, theanine has been shown to alleviate behavioral abnormalities associated with severe traumatic stress (Takarada et al. 2015a). In addition to these CNS effects, theanine may also suppress the progression of cancer as well as increase insulin secretion and lower blood sugar levels (Shojaei-Zarghani et al. 2021; Yan et al. 2017). As part of efforts to verify the beneficial effects of theanine, a greater understanding of the movement of theanine at the molecular and cellular levels is needed. For example, the carriers involved in the distribution and cellular uptake of theanine remain to be clarified. It is currently understood that theanine transport from the intestinal tract to blood is mediated by sodium-dependent glutamine transporter (Kitaoka et al. 1996). Also, solute carrier family 7 member 5 (Slc7a5), a glutamine/leucine exchanger, has been shown to mediate passage of theanine across the blood–brain barrier (Yokogishi et al. 1998). Within the brain, theanine has been shown to inhibit glutamine uptake by neurons and astrocytes, suggesting that Slc38a1, a major glutamine transporter expressed in neurons and astrocytes, is involved in the transport of theanine in neural cells (Kakuda et al. 2008). In vitro experiments have shown that theanine enhances proliferation both in undifferentiated neural progenitor cells prepared from adult mouse hippocampus and in P19 pluripotent embryonic carcinoma stem cells (Takarada et al. 2015b; Ogura et al. 2012). Theanine exposure has also been shown to increase the expression of Slc38a1 in P19 cells and increase the expression of Slc1a5, another glutamine transporter, in the gastrointestinal tract of rats (Ogura et al. 2012; Yan et al. 2017). Together, these findings suggest that glutamine uptake increases cell proliferation and that theanine increases the glutamine requirement of cells. Glutamine is an important amino acid that is used not only in protein synthesis but also as a metabolic fuel (Dang et al. 2012). For example, in highly proliferative cancer cells, glucose metabolism alone is insufficient to support cell growth; therefore, glutamine is used as an alternative energy supply (Kandasamy et al. 2018; Kanai et al. 2013). Once glutamine is transported into the cytosol, it is enzymatically converted to glutamic acid and used either for the production of other amino acids, fatty acids, and nucleic acids or fed into the tricarboxylic acid cycle to produce ATP and coenzyme NADPH. Glutamic acid is also one of the three components of glutathione (l-γ-glutamyl-l-cysteinyl-glycine), which has been shown to reduce oxidative stress (Dang et al. 2012). Cellular uptake of glutamine via Slc1a5 is coupled with the exchange of glutamine and an amino acid via Slc7a5 (Kandasamy et al. 2018). In this way, glutamine is important for the uptake of essential amino acids such as leucine, which promotes cell division by activating the mechanistic target of rapamycin complex 1 (mTORC1) pathway by binding to Sestrin2, and phenylalanine, which is hydroxylated to tyrosine and is the source of catecholamines, namely dopamine, noradrenaline and adrenaline (Bhutia and Ganapathy. 2016; Wolfson et al. 2016; Brady et al. 2011). Slc7a5 is primarily a transporter of leucine, although it does transport some other neutral amino acids. Leucine uptake via Slc7a5 is inhibited by leucine itself, isoleucine, phenylalanine, methionine, tyrosine, histidine, tryptophan, and valine (Kim et al. 2006). In HEK293 human embryonic kidney cells stably expressing human Slc7a5, leucine uptake is strongly inhibited by leucine and phenylalanine (Khunweeraphong et al. 2012). Another solute carrier in the same family as Slc7a5 is Slc7a8, which is also involved in the uptake of neutral amino acids (Khunweeraphong et al. 2012; Segawa et al. 1999). Whereas Slc7a8 is widely expressed in non-cancer cells, Slc7a5 is highly expressed in tumor cells (Kanai. 2021). Both Slc7a5 and Slc7a8 are considered potential carriers of theanine (Yamamoto et al. 2012). As noted earlier, the uptake of essential amino acids by Slc7a5 depends on exchange with glutamine. Therefore, cells must first take up glutamine. Slc1a5 is a glutamine transporter found mainly in lung, colon, skeletal muscle, and adipose tissue cells, and to some degree in the cells of the nervous system (Kanai et al. 2013). In contrast, Slc38a1 is a major glutamine transporter found in neuronal cells (Schiöth et al. 2013). Therefore, in proliferative cells derived from nervous system cells, theanine may affect glutamine transport by at least three transporters: Slc1a5, Slc7a5, and Slc38a1. Here, to elucidate the effects of theanine on cells derived from the brain and which transporters are responsible for it, we examined the effect of theanine on cell growth, and the expression level of glutamine transporters in several human and mouse neural and endothelial cell lines, and the inhibitory effect of other transporter substrates and inhibitors on the effect. 2. Materials and Methods 2.1. Materials Theanine was purchased from Taiyo Kagaku Co., Ltd. (Tokyo, Japan). 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Nacalai Tesque Inc. (Kyoto, Japan). Hoechst 33342, propidium iodide, and the amino acids glutamine, phenylalanine, valine, serine, threonine, cysteine, and α-(methylamino) isobutyric acid (MeAIB) were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). JPH203 was purchased from Selleck Chemicals (Houston, TX, USA). Anti-GAPDH-antibody (016-25523), Dulbecco’s modified Eagle’s medium (DMEM) and Roswell Park Memorial Institute 1640 Medium were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Endothelial Cell Basal Medium, phenol red free was purchased from PromoCell GmbH (Heidelberg, Germany). Antibodies against Slc7a5 (sc-374232), Slc38a1 (sc-137032) or β3 tubulin (sc-80005) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and antibodies against mTOR (#4517) and p-mTOR (#2976) were products of Cell Signaling Technology (Danvers, MA, USA). All of the amino acids used were l-amino acids. All chemicals were of standard grade. 2.2. Cell cultures NSC-34 mouse motor neuron–like hybrid cells were obtained from CELLutions Biosystems, Inc. (Duluth, GA, USA). Neuro2A mouse neuroblastoma cells were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). C8-D1A mouse astrocyte type I clone cells and SH-SY5Y human neuroblastoma cells were purchased from American Type Culture Collection (Manassas, VA, USA). bEnd.3 mouse brain–derived endothelial cells (PMID: 2379237) were kindly provided by Prof. Akemichi Baba (Osaka University, Osaka, Japan). U-251MG human brain glioblastoma cells were obtained from RIKEN Bioresource Research Center (Ibaraki, Japan). Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell. All cultures were maintained under a 5% CO 2 atmosphere at 37°C. NSC-34, Neuro2a, and bEnd.3 cells were cultured in DMEM–high glucose containing 4500 mg/L of glucose and supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 0.1 mg/mL streptomycin. C8-D1A cells were cultured in DMEM containing 4500 mg/L glucose and 110 mg/L sodium pyruvate and supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 0.1 mg/mL streptomycin. U-251MG cells were cultured in DMEM–low glucose containing 1000 mg/L glucose and supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 0.1 mg/mL streptomycin. SH-SY5Y cells were cultured in Roswell Park Memorial Institute 1640 Medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 0.1 mg/mL streptomycin. HUVECs were cultured in Endothelial Cell Basal Medium, phenol red free. 2.3. MTT assay Cells were incubated with MTT at a final concentration of 0.5 mg/mL for 120 min at 37°C. The MTT-formazan produced by the cells was dissolved in 100% dimethyl sulfoxide and absorbance was measured by microplate reader model 680 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 570 nm. 2.4. Hoechst 33342/propidium iodide staining Cells were incubated with a mixture of Hoechst 33342 and propidium iodide at a final concentration of 10 μM and 2 μg/mL, respectively, for 15 min at 37°C. Photomicrographs of the cells were obtained using a phase-contrast microscope: IX71 (OLYMPUS Corporation, Tokyo, Japan) and excitation/emission wavelengths of 350/461 nm for Hoechst 33342 and 530/620 nm for propidium iodide. The numbers of cells per field positively stained with each of the stains were counted. The numerical aperture of the lens of microscope are 20x/0.40. 2.5. Quantitative polymerase chain reaction Total RNA was prepared by using an SV Total RNA Isolation System (Promega, Madison, WI, USA) and then subjected to reverse-transcription by using M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and random primers (Invitrogen). Target genes were amplified from the previously prepared cDNA templates by using THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan) and a Thermal Cycler Dice Real Time System (Takara, Ohtsu, Japan). The following primer sequences were used: Mouse ACTB-F: 5′-AGTGTGACGTTGACATCCGTA-3′ Mouse ACTB-R: 5′-GCCAGAGCAGTAATCTCCTTCT-3′ Mouse SLC38a1-F: 5′-AACGGAGCTGCAGAACATGA -3′ Mouse SLC38a1-R: 5′-CCCAAGATTCCACTGCCCAT -3′ Mouse SLC1a5-F: 5′-TGCTTTCGGGACCTCTTCTA-3′ Mouse SLC1a5-R: 5′-GGACACCCCGTTTAGTTGTG-3′ Mouse SLC7a5-F: 5′-CCACCTGCCTTCTGTCCTCT-3′ Mouse SLC7a5-R: 5′-TGAATCGGAGCCACATCATA-3′ Human ACTB-F: 5′-CTGGAACGGTGAAGGTGACA-3′ Human ACTB-R: 5′-AAGGGACTTCCTGTAACAACGCA-3′ Human SLC38a1-F: 5′-GGCAGTGGGATTTTGGGACT-3′ Human SLC38a1-R: 5′-TACGAACTTCCCTGTGGTGC-3′ Human SLC1a5-F: 5′-CATCATCCTCGAAGCAGTCA-3′ Human SLC1a5-R: 5′-GGGCAGCTCACTCTTCACTT-3′ Human SLC7a5-F: 5′-TGCCTATGGAGGATGGAATT-3′ Human SLC7a5-R: 5′-ATGACGCCCAGGTGATAGTT-3′ 2.6. Immunoblotting Cells were collected and lysed in a buffer composed of 10 mM Tris-HCl buffer (pH 7.5) containing 0.32 M sucrose, 1 mM EDTA, 1 mM EGTA, 5 mM dithiothreitol, phosphatase inhibitors (10 mM sodium β-glycerophosphate, and 10 mM sodium pyrophosphate, 50 mM sodium fluoride), and 1 µg/mL each of protease inhibitors (p-amidinophenyl-methanesulfonyl fluoride, benzamidine, leupeptin, and antipain). For sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, proteins in the lysate were solubilized at 100°C for 10 min in a buffer containing 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, and 0.01% bromophenol blue. Protein aliquots of equal weight (e.g.10 µg) were subjected to electrophoresis and migrated proteins were transferred onto polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany). After blocking unspecific protein bindings by the incubation with 5% skim milk for 1 h, the transfer membrane was incubated for the specific detections with a primary antibody for 2 h, and then with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The antibody-specific immunoreactive bands were detected on X-ray film using Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer, Waltham, MA, USA). not-yet-known not-yet-known not-yet-known unknown 2.7. Statistical analysis Data are expressed as mean ± standard error. Comparisons between groups were performed using Student’s t -test for two groups, Dunnett’s test for comparisons of multiple treatment groups with a single control group, and Bonferroni’s test for multiple comparisons. The test used is noted in each figure legend. 3. Results Since theanine suppresses the growth of cancer cells while promoting the growth of neural progenitor cells and P19 pluripotent embryonic carcinoma stem cells, it was intriguing whether theanine promotes or suppresses the proliferation in the case of proliferative neuronal cells (Shojaei-Zarghani et al. 2021; Takarada et al. 2015b; Ogura et al. 2012). Therefore, NSC-34, as a cell line derived from neurons was chosen. NSC-34 mouse motor neuron-like hybrid cells were cultured in DMEM containing 4.0 mM glutamine, and the effect of theanine treatment was examined. Theanine seemed to reduce the number of cells on the culture plate (Fig. 1a) and indeed significantly suppressed MTT reduction by the cells in both a concentration- and a time-dependent manner (Fig. 1b-c). Treatment with 10 mM theanine for 24 h significantly decreased the number of Hoechst 33342-positive cells without significantly increasing the number of propidium iodide-positive cells (Fig. 1d, e). Together, these findings suggest that theanine inhibits cell growth but does not induce cell death in NSC-34. Theanine was also observed to have significant inhibitory effects on cell growth in two other neuronal cell lines: Neuro2A mouse neuroblastoma cells and SH-SH5Y human neuroblastoma cells (Fig. 2). Cell growth was also significantly inhibited in C8-D1A mouse astrocytes. The growth inhibitory effects of theanine in SH-SH5Y and C8-D1A cells were very weak compared with that in NSC-34 or Neuro2A cells. In contrast, theanine had no effect on cell growth in U-251MG human glioblastoma cells nor in vascular endothelial bEnd.3 cells or HUVECs. Together, these findings suggest that the growth-inhibitory effects of theanine is specific to neural cells that have some sort of proliferative capacity. It is reported that theanine is taken up into cells via the glutamine transporter Slc38a1 and/or the glutamine/leucine exchanger Slc7a5 (Yokogoshi et al. 1998; Ogura et al. 2012). Among the cells investigated in the present study, the expressions of Slc38a1 and Slc7a5 were both highest in NSC-34 (Fig. 3b, d). In addition, the expression of Slc38a1 was significantly lower in Neuro2A, C8-D1A, and SH-SY5Y compared with that in NSC-34, and that in U-251MG was significantly lower than that in Neuro2A, C8-D1A, and SH-SY5Y; thus, very little expression was observed in the vascular endothelial cells, i.e. bEnd.3 and HUVEC (Fig. 3b). It is also reported that the neutral amino acid transporter Slc1a5 is little expressed in nervous system tissues (Kanai et al. 2013). As expected, the expression of Slc1a5 was markedly lower than that of Slc38a1 in all of the cell lines but the difference was particularly marked in NSC-34 cells (Fig. 3a, c). Correlated with gene expression, Slc38a1 protein expressions was higher in NSC-34 and Neuro2A than in bEnd.3, U-251MG and HUVEC (Fig. 4). However, unlike gene expression, S7a5 protein expression was nearly constant, whereas S7a5 expression in astrocyte lines C8-D1A and U251-MG was significantly lower than that in NSC-34. Considering protein expression, it was therefore suggested that the effect of theanine may depend on Slc38a1. In NSC-34 cells, in the absence of glutamine in the culture medium, cell growth was low, but it increased in a concentration-dependent manner until a glutamine concentration of 4 mM and then decreased at a glutamine concentration of 10 mM (Fig. 5a). Since 4.0 mM glutamine is a concentration contained in a commercially available medium, this concentration was used as the baseline concentration. When 10 mM theanine was added to the culture medium, cell growth was significantly reduced compared with that in the absence of theanine. Comparison of the inhibitory effects of theanine, using the MTT reduction at each glutamine concentration as 100%, revealed that the higher the glutamine concentration, the lower the inhibitory effect of theanine (Fig. 5b). In NSC-34 cells, leucine or phenylalanine (10 mM) completely ameliorated the inhibitory effect of theanine on cell growth in a concentration-dependent manner, whereas leucine or phenylalanine had no effect. (Fig. 6). Valine, a substrate of Slc7a5, and threonine, serine and cysteine, which are not substrates of Slc7a5, independently reduced cell growth by the cells (Fig. 7). However, valine significantly suppressed the inhibitory effect of theanine but threonine, serine, and cysteine did not, suggesting that substrates of Slc7a5 counteract the effect of theanine. Increasing the glutamine concentration in the medium from 0.1 to 4 mM increased the phosphorylation level of mTOR, but further increasing it to 10 mM did not increase the phosphorylation level, but rather slightly weakened it. When the glutamine concentration in the medium was 0.1 mM, the addition of 10 mM leucine significantly increased the phosphorylation level of mTOR, while combination with 10 mM theanine diminished the increase. This effect was not observed in the presence of 4 mM glutamine. Moreover, exposure to theanine alone did not change the phosphorylation level of mTOR under either condition (Fig.8). not-yet-known not-yet-known not-yet-known unknown Slc7a5 inhibitor JPH203 did not improve the inhibitory effect of theanine on the cell growth, whereas the Slc38a1 inhibitor MeAIB did (Fig. 9). Furthermore, as same as Fig. 6, leucine improved theanine-induced suppression of cell growth, while the effect of leucine was significantly disappeared by JPH203 (Fig. 10). Therefore, it was concluded that theanine is taken up via Slc38a1, and leucine expels theanine from within the cells when taken up via Slc7a5 in NSC-34 (Fig.11). 4. Discussion Although theanine is reported to enhance cell proliferation in neural progenitor cells, in the present study we found in several cell lines (i.e., NSC-34, Neuro2A, SH-SY5Y, C8-D1A, U-251MG, bEnd.3, and HUVEC cells) that theanine inhibited or had no effect on cell growth (Takarada et al. 2015b; Ogura et al. 2012). In cancer cells, it has been suggested that theanine inhibits cell growth and suppresses apoptosis, and that it has antimetastatic, antimigration, and anti-invasion effects (Shojaei-Zarghani et al. 2021). Among the cells examined in the present study, the greatest inhibition of growth was seen in NSC-34 (Fig. 1-2), which are motor nerve-like cells derived from murine spinal cord. The inhibition of cell growth by theanine in NSC-34 occurred in a concentration- and time-dependent manner but did not cause cell death (Fig. 1). From the above, the response of theanine to NSC-34 was more like cancer cells than neuronal progenitor cells. We were interested in what determines the sensitivity of theanine to suppress cell growth in NSC-34. Slc38a1 , not Slc7a5 , was predominantly expressed in neuronal cell lines NSC-34, Neuro2A, and SH-SY5Y (Fig. 3). Slc1a5 expression is limited in neuronal and cardiovascular cells, therefore protein expression of Slc1a5 was not investigated (Kanai et al. 2013). On the other hand, whereas Slc38a1 protein expression was similar to gene expression, Slc7a5 protein expression was consistent and distinct from the gene expression pattern (Fig. 4), highlighting the importance of investigating protein expression. At least in NSC-34, Slc7a5 was expressed together with Slc38a1, suggesting that glutamine uptake and the exchange of essential amino acids leucine with glutamine occurs in these cells via the combined activity of Slc38a1 and Slc7a5, as is found in cancer cells (Dang. 2012; Kandasamy et al. 2018; Kanai. 2021).The growth inhibitory effect of theanine was improved by MeAIB, strongly suggesting that Slc38a1 is the major transporter for theanine uptake (Fig.9). Addition of serine, threonine, or valine alone (10 mM each) to the culture medium significantly inhibited NSC-34 cell proliferation (Fig. 7a, b, c), likely because these amino acids are substrates for Slc38a1 Schiöth et al. 2013; Albers et al. 2001). They also inhibit Slc7a5-mediated cellular uptake of leucine (Kim et al. 2006), suggesting that serine, threonine, and valine are taken up by cells via Slc38a1 and Slc7a5, resulting in inhibition of glutamine and leucine uptake, respectively. The fact that these amino acids did not act additively with theanine to inhibit cell proliferation may hold the key to understanding these effects of theanine. The effect of cysteine on cell growth was different from the effects of the other amino acids (Fig. 7d). Cysteine is taken up into cells as cystine (a dimer of two cysteine molecules) via Slc7a11, a cystine/glutamic acid transporter, and once inside the cell it is reduced back to two cysteine molecules (Nakatsu et al. 2015). Surplus cysteine is exported from cells by neutral amino acid transporters, including Slc1a5 and Slc38a1. Cysteine has a nucleophilic side chain that is easily oxidized. Indeed, the antioxidant property of glutathione is due to the nucleophilicity of its cysteine residue (Gorrini et al. 2013). It is reported that cystine uptake is essential for cancer cells to avoid oxidative stress; however, there are contradictory reports indicating that increasing the intracellular cystine concentration instead increases oxidative stress in cancer cells, causing cell death (Gorrini et al. 2013; Shin et al. 2017). These findings suggest that high extracellular concentrations of cysteine are not always appropriate for cell survival. In the present study, the cells were exposed to a high concentration (10 mM) of cysteine, such that the above-mentioned negative effects of cysteine can be assumed to have occurred. Therefore, whether the observed inhibition of cell growth by cysteine was the result of cell death remains to be investigated. The cell growth and the phosphorylation level of mTOR were increased in a glutamine concentration-dependent manner up to 4 mM and it was decreased at 10 mM glutamine (Fig. 5a and Fig. 8). The glutamine/leucine exchange by Slc7a5 would be reversible, suggesting that if the extracellular glutamine concentration is too high, the direction of exchange by Slc7a5 is reversed, which causes leucine depletion inside the cell, resulting in suppression of cell growth.NSC-34 were able to survive and proliferate in medium containing no glutamine (Fig. 5a). This indicates that glutamine is not a critically important amino acid for these cells and that their basal growth potential depends only on a supply of glucose. The fact that theanine could still inhibit cell growth in the absence of glutamine suggests two things: first, that theanine depleted leucine from the cells by Slc7a5 exchanging, since loss of leucine would result in a failure to activate the mTORC cascade for the induction of cell proliferation, and second, that intracellularly theanine behaves as suppressor of the cell growth (Bhutia et al. 2016).High concentration of leucine (10 mM) or phenylalanine (5 or 10 mM) completely mitigated the inhibition of cell growth by theanine (Fig. 6). The cell growth inhibitory effect of theanine was improved by leucine, while this effect was abolished by the Slc7a5 inhibitor JPH203 (Fig.10), suggesting that after uptake, theanine is mainly excreted via Slc7a5. Theanine does not appear to inhibit leucine uptake but rather promotes it by turning it over.The cell growth increased in a glutamine concentration-dependent manner and that theanine partially, but not fully, inhibited the glutamine-mediated increase in the growth (Fig. 5). Increase in glutamine (up to 4 mM) and addition of 10 mM leucine to 0.1 mM glutamine enhanced the phosphorylation of mTOR (Fig. 8), while theanine never influenced the phosphorylation level of mTOR, suggesting the growth inhibitory effect of theanine is independent to mTOR cascade. In neural progenitor cells, theanine promoted cell growth concomitantly with increased mTOR phosphorylation levels, which is different from neural cell lines (Takarada et al. 2015b). Since glutamine metabolism affects not only ATP synthesis but also the metabolism of nitrogenous compounds such as nucleic acids, amino acids, proteins, and amino sugars, theanine may inhibit proliferation by inhibiting any of these metabolic pathways. Therefore, further research is needed to clarify the unknown mechanism by which theanine inhibits cell growth. 5. Conclusion Theanine is generally believed to have beneficial effects on mammals. Consequently, it will be necessary to examine the biological significance of our finding that theanine induced inhibition of cell proliferation in the brain. As previously reported, theanine has the effect of promoting the proliferation of normal neural progenitor cells and their differentiation into nerve cells. In addition, current research results have shown that theanine also has the effect of suppressing the proliferation of probably abnormally proliferating neuronal cells like neuroblastoma, and we will continue our research with this in mind. Slc38a1 is expressed in mature neurons that have completed proliferation and differentiation, and therefore theanine is taken up by mature neurons. Further investigation into the effects of theanine within cells may lead to the elucidation of therapeutic targets for CNS diseases. not-yet-known not-yet-known not-yet-known unknown The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. not-yet-known not-yet-known not-yet-known unknown Abbreviations CNS: central nervous system DMEM : Dulbecco’s modified Eagle’s medium mTOR: mechanistic target of rapamycin mTORC1 : mTOR complex 1 MTT : (4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide PI : propidium iodide Slc : solute carrier Theanine : γ-glutamylethylamide not-yet-known not-yet-known not-yet-known unknown Conflict of interest The authors declare no competing financial interests. Availability of data and material The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request. not-yet-known not-yet-known not-yet-known unknown Code availability Not applicable Author contributions: CRediT Conceptualization: SM and NK; Data curation: KK, YH, SM and NK Formal analysis: YM, RN, MN, YW, SA, TS, SY, MS and SM Funding acquisition: TK, KU, SM and NK Investigation: YM, RN, MN, YW, SA, TS, SY, MS and SM Methodology: KK, TK, KU, AY, YI, YY and NK Project administration: SM and NK Resources: YY, SM, and NK Software: N/A Supervision: KK, YI, YY, SM, and NK Validation: KK and NK Visualization: KK and NK Writing-original draft: KK and NK Writing-review and editing: KK, YH, SM and NK Initials are Koichi Kawada (KK), Yuri Matsushima (YM), Ruka Nakamura (RN), Megumi Nishio (MN), Yuka Watanabe (YW), Shuhei Araki (SA), Tamika Sudo (TS), Sayuki Yoshikawa (SY), Mitsuhiro Shibata (MS), Toshihiko Kinjo (TK), Kyosuke Uno (KU), Yutaro Higashiura (YH), Akiko Yamamuro (AY), Yuki Ishimaru (YI), Yasuhiro Yoshioka (YY), Sadaaki Maeda (SM), and Nobuyuki Kuramoto (NK). 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Authors Affiliations Koichi Kawada Setsunan University Faculty of Pharmaceutical Sciences Graduate School of Pharmaceutical Sciences View all articles by this author Yuri Matsushima Setsunan University Faculty of Pharmaceutical Sciences Graduate School of Pharmaceutical Sciences View all articles by this author Ruka Nakamura Setsunan University Faculty of Pharmaceutical Sciences Graduate School of Pharmaceutical Sciences View all articles by this author Megumi Nishio Setsunan University Faculty of Pharmaceutical Sciences Graduate School of Pharmaceutical Sciences View all articles by this author Yuka Watanabe Setsunan University Faculty of Pharmaceutical Sciences Graduate School of Pharmaceutical Sciences View all articles by this author Shuhei Araki Setsunan University Faculty of Pharmaceutical Sciences Graduate School of Pharmaceutical Sciences View all articles by this author Tamika Sudo Setsunan University Faculty of Pharmaceutical Sciences Graduate School of Pharmaceutical Sciences View all articles by this author Sayuki Yoshikawa Setsunan University Faculty of Pharmaceutical Sciences Graduate School of Pharmaceutical Sciences View all articles by this author Mitsuhiro Shibata Setsunan University Faculty of Pharmaceutical Sciences Graduate School of Pharmaceutical Sciences View all articles by this author Toshihiko Kinjo Setsunan University Faculty of Pharmaceutical Sciences Graduate School of Pharmaceutical Sciences View all articles by this author Kyosuke Uno Setsunan University Faculty of Pharmaceutical Sciences Graduate School of Pharmaceutical Sciences View all articles by this author Yutaro Higashiura Setsunan University Faculty of Pharmaceutical Sciences Graduate School of Pharmaceutical Sciences View all articles by this author Akiko Yamamuro Setsunan University Faculty of Pharmaceutical Sciences Graduate School of Pharmaceutical Sciences View all articles by this author Yuki Ishimaru Setsunan University Faculty of Pharmaceutical Sciences Graduate School of Pharmaceutical Sciences View all articles by this author Yasuhiro Yoshioka Setsunan University Faculty of Pharmaceutical Sciences Graduate School of Pharmaceutical Sciences View all articles by this author Sadaaki Maeda Setsunan University Faculty of Pharmaceutical Sciences Graduate School of Pharmaceutical Sciences View all articles by this author Nobuyuki Kuramoto 0000-0003-2565-2067 [email protected] Setsunan University Faculty of Pharmaceutical Sciences Graduate School of Pharmaceutical Sciences View all articles by this author Metrics & Citations Metrics Article Usage 231 views 131 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Koichi Kawada, Yuri Matsushima, Ruka Nakamura, et al. 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