Stimulation by glutamine and proline of HGF production in hepatic stellate cells

Stimulation by glutamine and proline of HGF production in hepatic stellate cells

Available online at www.sciencedirect.com Biochemical and Biophysical Research Communications 363 (2007) 978–982 www.elsevier.com/locate/ybbrc Stimu...

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Available online at www.sciencedirect.com

Biochemical and Biophysical Research Communications 363 (2007) 978–982 www.elsevier.com/locate/ybbrc

Stimulation by glutamine and proline of HGF production in hepatic stellate cells Takako Nishikawa a, Tomoaki Tomiya a,*, Natsuko Ohtomo a, Yukiko Inoue a, Hitoshi Ikeda b, Kazuaki Tejima b, Naoko Watanabe a, Yasushi Tanoue a, Masao Omata a, Kenji Fujiwara c a

Department of Gastroenterology, Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan b Department of Clinical Laboratory, University of Tokyo, Tokyo, Japan c Yokohama Rosai Hospital, Yokohama, Japan Received 6 September 2007 Available online 1 October 2007

Abstract Amino acids regulate cellular functions in a variety of cell types. Most notably, leucine stimulates protein production through the mammalian target of rapamycin (mTOR)-dependent signaling pathway. We investigated the effect of amino acids on hepatocyte growth factor (HGF) production. Treatment with glutamine and proline, as well as leucine, increased HGF levels in the culture medium of a rat hepatic stellate cell clone in a dose-dependent manner. Up-regulation of phosphorylation of 70 kDa ribosomal protein S6 kinase and eukaryotic initiation factor 4E-binding protein 1 was not apparent in the cells after treatment with glutamine or proline. When rats received injections of glutamine or proline, hepatic and circulating HGF levels increased and peaked around 12 h after treatment. Glutamine and proline may have the potential to stimulate HGF production but the mechanism underlying this stimulation seems not to be through the mTOR-dependent signaling pathway.  2007 Elsevier Inc. All rights reserved. Keywords: Hepatocyte growth factor; Hepatic stellate cells; Glutamine; Proline; Mammalian target of rapamycin; p70 S6 kinase; Eukaryotic initiation factor 4E-binding protein 1

While hepatocyte growth factor (HGF) was recognized initially as a potent stimulator of hepatocyte DNA synthesis in culture [1–4], recent advances have revealed that HGF is a pleiotropic factor that is produced by cells in various organs and influences cell growth, function, and motility [1–5]. HGF is known to have mitogenic, motogenic, morphogenic, anti-apoptotic, and tumor suppressor activities [1–5]. Exogenous administration of HGF has been Abbreviations: cHSC, hepatic stellate cell clone; 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; FCS, fetal calf serum; HBSS, Hanks’ balanced salt solution; HGF, hepatocyte growth factor; MEM, Eagle’s minimal essential medium; mTOR, mammalian target of rapamycin; p70 S6, 70 kDa ribosomal protein S6; PBS, phosphate buffered saline. * Corresponding author. Fax: +81 3 5800 8806. E-mail address: [email protected] (T. Tomiya). 0006-291X/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.09.082

shown to enhance DNA synthesis in the liver, kidney, and lung, facilitate liver function, reduce injuries of the liver, colon, kidney, lung, brain, and heart, and suppress fibrogenesis of the liver and kidney in experimental models [1–3,6–10]. These studies imply a potential application for HGF in the treatment of various diseases. In addition, increasing the levels and/or activities of HGF in vivo may offer similar benefits. Amino acids have been reported to modulate numerous cellular functions, in addition to providing the substrates for protein production [11,12]. They have been shown to exert regulatory effects on gene expression, cellular metabolism, amino acid transport, and protein turnover [1,13– 21], the so-called pharmacological actions of amino acids. Among the many amino acids, leucine is best known for its ability to stimulate protein synthesis through the

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mammalian target of rapamycin (mTOR)-dependent signaling pathway, which regulates protein production post-transcriptionally through activation of the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and the 70 kDa ribosomal protein S6 (p70 S6) kinase [23–27]. Recently, we reported that HGF production by hepatic stellate cells is up-regulated by the addition of leucine [28] through the mTOR-dependent signaling pathway [29]. We also showed that administration of leucine to rats increases circulating and hepatic HGF levels [30]. These results suggest that amino acids can up-regulate HGF production through their pharmacological actions and might be used as a therapeutic tool for various kinds of diseases. In this study, we showed that glutamine and proline, in addition to leucine, may have the potential to stimulate HGF production. The mechanism underlying the stimulation of HGF production seems to vary depending on the amino acids. Materials and methods In vitro experiments Cell culture. A hepatic stellate cell clone (cHSC) was kindly provided by Dr. Marcos Rojkind [31,32]. Cells were plated on plastic culture dishes at 7 · 104 cells/cm2 in Eagle’s minimal essential medium (MEM) (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) containing 10% (v/v) fetal calf serum (FCS) (Gibco Laboratories, Life Technologies Inc., Grand Island, New York). After a 4-h attachment period, the culture medium was replaced with MEM supplemented with 0.5% FCS. After 20 h, cells were deprived of amino acids by incubation in Hanks’ balanced salt solution (Sigma Chemical Co., St. Louis, Missouri) supplemented with a vitamin mixture (Gibco Laboratories, Life Technologies Inc., Grand Island, New York) (HBSS). After 3 h, the cells were used for the following experiments. In experiment I and II, five dishes were allocated in each group. Experiment I: The medium was changed to HBSS containing 10 mM each of 20 amino acids (isoleucine, alanine, leucine, asparagine, lysine, aspartate, methionine, cysteine, phenylalanine, glutamate, threonine, glutamine, tryptophan, glycine, valine, proline, arginine, serine, histidine, and tyrosine) which are known to make up mammalian proteins (Ajinomoto Co., Inc., Tokyo, Japan). After 36 h, HGF levels were determined in the medium. Experiment II: The medium was changed to HBSS with increasing concentrations of glutamine or proline. HGF levels in the medium were determined 36 h later. In addition, the cells were cultured in the medium containing 10 mM of glutamine, proline or leucine, and HGF levels in the medium were also determined. Experiment III: The medium was changed to HBSS with 10 mM of glutamine, proline or leucine. After an incubation period of 40 min, the cells were washed twice with ice-cold phosphate buffered saline (PBS) and used for analysis of the phosphorylation state of p70 S6 kinase and 4EBP1. Animal experiments Male Sprague–Dawley rats aged 5 weeks were obtained from Japan SLC (Shizuoka, Japan). They were housed in cages at 22 C under a 12-h light–dark cycle and fed a commercial diet, with water ad libitum. All animal study protocols adhered to the guidelines of the Faculty of Medicine, University of Tokyo, for humane care. Experiment I: Rats were starved for 12 h prior to the experiment with free access to water. They received an intraperitoneal injection of 1 g per kg body weight of glutamine or proline as a 2% solution in 10 mM PBS. They were anesthetized serially with diethyl ether and blood was collected

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through the inferior caval vein using a plastic syringe containing 3.1% sodium citrate solution (1:10 v/v) in order to prepare plasma. After collecting the blood, the liver was perfused with 25 ml of saline through the portal vein after near-total exsanguination and excised. The liver was quickly frozen in liquid nitrogen and stored at 80 C. Assays Assays for HGF levels in the culture medium, liver, and plasma. Extracts of rat liver were prepared as described previously. HGF levels were determined using a rat HGF enzyme-linked immunosorbent assay (ELISA) kit (Institute of Immunology, Tokyo, Japan) [33,34]. Examination of the phosphorylation state of p70 S6 kinase and 4E-BP1. Cells were lysed in ice-cold buffer A (50 mM Tris–HCl, pH 8.0, 1% Nonidet P-40, 120 mM NaCl, 20 mM NaF, 1 mM EDTA, 6 mM EGTA, 20 mM b-glycerophosphate, 0.5 mM dithiothreitol, 50 lM p-APMSF, 1 lg/ml aprotinin, and 1 lg/ml leupeptin) and incubated for 10 min. They were centrifuged at 10,000g for 5 min at 4 C. An aliquot of the supernatants was mixed with one-fifth volume of SDS buffer (10% SDS, 30% glycerol, 280 mM Tris–HCl, pH 6.8, and 0.6 M dithiothreitol), separated by SDS–PAGE on a 7.5% gel, and transferred to a nitrocellulose membrane. Phospho-p70 S6 kinase and total p70 S6 kinase were detected by Western blotting using anti-phospho-p70 S6 kinase antibody (#9205 Cell Signalling Technology, Inc., Danvers, MA) and anti-p70 S6 kinase antibody (#9202 Cell Signalling Technology, Inc., Danvers, MA), respectively, as reported previously [12,23,29,35]. An electrophoretic mobility was studied using anti-4E-BP1 antibody (sc-6024, Santa Cruz Biotechnology, California), as described previously [23,29]. Statistical analyses The differences between two unpaired samples were defined as significant when p-values by Student’s t test were less than 0.05. The doserelated effects were tested by one-way analysis of variance.

Results HGF levels in the medium of cHSC supplemented with amino acids Among the amino acids tested, addition of glutamine or proline, as well as leucine, to the culture medium of cHSC significantly increased HGF levels in the medium (data not shown). As shown in Fig. 1, when cHSC was cultured in medium supplemented with glutamine or proline at increasing concentrations, the HGF levels in the culture medium increased in a dose-related manner (F = 3.24, p < 0.05 and F = 2.62, p < 0.05, respectively). When cHSC was cultured in medium containing 10 mM of glutamine, proline or leucine, HGF levels in the medium were similarly increased compared to the level without amino acid treatment; means (±SEM) of the HGF levels expressed as a percentage of the control were 203 ± 34%, 194 ± 31%, and 197 ± 67% by the addition of glutamine, proline, and leucine, respectively. Effect of amino acids on phosphorylation of p70 S6 kinase and 4E-BP1 in cHSC Phosphorylation of p70 S6 kinase increased 40 min after treatment with leucine, increased slightly with proline, and did not increase with glutamine, although total p70 S6

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Fig. 3. Phosphorylation of 4E-BP1 in cHSC cultured in medium supplemented with amino acids. The electrophoretic mobility was studied using anti-4E-BP1 antibody. Faster-migrating bands (a and b bands) of 4E-BP1 indicate unphosphorylated or lower phosphorylated forms of 4EBP1, while the slower-migrating band (c band) indicates the highly phosphorylated form. AA ( ) means no addition of amino acids.

than the levels before treatment. Circulating HGF levels in these rats increased in parallel with the changes in the hepatic HGF levels, reaching a maximum at 12 h, and then decreased (Fig. 4). Fig. 1. HGF levels in the medium of cHSC cultured in HBSS supplemented with amino acids. cHSC was plated on plastic culture dishes in MEM containing 10% FCS. After 4 h, the medium was replaced with MEM supplemented with 0.5% FCS. After 20 h, the cells were deprived of amino acids by incubation in HBSS. After a further 3 h, the medium was changed to HBSS supplemented with 1–100 mM of glutamine or with 0.5–100 mM of proline. The HGF levels in the medium were determined after 36 h. Data are means ± SEM of five dishes. AA ( ) means no addition of amino acids. *p < 0.05 compared to the values without adding amino acids.

kinase protein levels did not change (Fig. 2A and B). Regarding 4E-BP1, cHSC cells exhibited a predominance of faster-migrating unphosphorylated forms of 4E-BP1 (a and b bands) before the addition of amino acids (Fig. 3). The proportion of the slower-migrating highly phosphorylated form of 4E-BP1 (c band) increased following the addition of leucine, increased slightly following the addition of proline, but did not increase following the addition of glutamine (Fig. 3).

Hepatic and circulating HGF levels in rats treated with amino acids When rats received glutamine or proline, hepatic HGF levels increased gradually, reached a plateau at 12–16 h, and decreased thereafter (Fig. 4). The levels at 4–24 h in glutamine and proline treated rats were significantly higher

Fig. 2. Phosphorylation of p70 S6 kinase in cHSC cultured in medium supplemented with amino acids. (A) Phospho-p70 S6 kinase was determined by Western blotting. (B) Total p70S6 kinase protein was also determined by Western blotting. AA ( ) means no addition of amino acids.

Discussions We have shown that glutamine and proline have the potential to stimulate HGF production. The mechanism underlying the stimulation of HGF production by glutamine and proline may differ from that by leucine. Hepatic stellate cells are one of the major sources of HGF production in the liver. However, freshly isolated hepatic stellate cells are transformed rapidly into myofibroblast-like cells during culture, resulting in a diminution of HGF production in cultured hepatic stellate cells with time. Thus, we utilized a cHSC whose phenotype remains constant during culture [31,32] and produces HGF continuously [28]. Proliferation of the cHSC does not occur in serum-free culture conditions, therefore, the number of cells remains constant [31,32]. Thus, it is reasonable to speculate that glutamine and proline stimulate HGF production by cHSC, as we previously reported for leucine. Amino acids serve as substrates for protein synthesis and are metabolized as an energy source for protein production. A simple hypothesis is that the addition of glutamine and proline provides a substrate and/or energy for HGF production in the cells, resulting in increased production of HGF. However, the stimulatory effect of addition to the medium of amino acids other than glutamine, proline, and leucine is not significant, even though the other amino acids can serve as substrates and energy sources for protein synthesis. In addition, the present results show that administration of proline or glutamine increased hepatic and circulating HGF levels promptly in rats. These observations indicate the possibility that glutamine and proline have the potential to increase HGF production through their pharmacological action. In eukaryotic cells, one of the most important intracellular signaling transducers of amino acids is considered to be the mTOR pathway, which regulates protein production post-transcriptionally through activation of 4E-BP1 and p70 S6 kinase [21,36–38]. p70 S6 kinase and 4E-BP1 control the step in initiation of translation involving the

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Fig. 4. Serial changes in hepatic and circulating HGF levels in rats treated with glutamine or proline. Rats aged 5 weeks were starved for 12 h, with free access to water, prior to the experiments. They received intraperitoneal injections of 1 g per kg body weight of glutamine (A) or proline (B) in 10 mM PBS. Plasma and hepatic HGF levels were determined serially. Data are means ± SEM of five rats. Circles and columns indicate plasma and hepatic HGF levels, respectively. *p < 0.05 and **p < 0.01 compared to the values before treatment with amino acids.

binding of mRNA to the 40S ribosomal subunit [21,36]. 4E-BP1 regulates protein synthesis through its association with eukaryotic translation initiation factor 4E. Our results showed that little or no effect was observed on the phosphorylation state of either p70 S6 kinase or 4E-BP1 in hepatic stellate cells following the addition to the medium of glutamine or proline, in contrast to leucine. The extent of the increase in HGF levels following treatment with glutamine or proline was similar to the extent of the increase in HGF levels following leucine treatment. Taken together, these observations indicate that up-regulation of HGF production by glutamine and proline seems to be independent of the activation of the mTOR-dependent signaling pathway. Previous reports indicate that glutamine influences various functions in a variety of cells [39–43]. In these examples, glutamine induced up- and down-regulation or no significant changes in the activation of the mTOR-dependent signaling pathway [39–43]. The differences in activation might depend on variation among the cells studied. As for proline, little information has been provided concerning its significance on the production of proteins [44]. Hepatic stellate cells are one of the major sources of collagen production, and proline is known to be required for collagen production, mainly as a substrate [45]. The significance of proline for hepatic stellate cells has to be elucidated further. Intracellular signaling pathways, which amino acids can modulate, have been a subject of study for many years. Although leucine is known to be one of the most potent amino acids in facilitating protein synthesis through the mTOR signaling pathway [22], leucine can affect cellular functions through an mTOR-independent pathway [46]. In addition, amino acids have been reported to have specific effects in preserving proteins by decreasing protein degradation, as well as by increasing protein synthesis [1,16,17]. Further investigations are required to elucidate the mechanism(s) of regulation of protein production by amino acids.

In clinical settings, up-regulation of HGF production may offer benefits in various disease situations. Acute and chronic hepatic failure is one of the candidate conditions to increase HGF levels or activities. However, it is not clear that administration of glutamine and proline is beneficial in patients with advanced hepatic failure considering other effects of these amino acids such as ammonia and collagen production. The combination of amino acids used in these clinical settings should be designed depending on the clinical situations. References [1] A. Ichihara, BCA, HGF, and proteasomes, Biochem. Biophys. Res. Commun. 266 (1999) 647–651. [2] R. Zarnegar, G.K. Michalopoulos, The many faces of hepatocyte growth factor: from hepatopoiesis to hematopoiesis, J. Cell. Biol. 129 (1995) 1177–1180. [3] K. Matsumoto, T. Nakamura, Hepatocyte growth factor (HGF) as a tissue organizer for organogenesis and regeneration, Biochem. Biophys. Res. Commun. 239 (1997) 639–644. [4] G.K. Michalopoulos, M.C. DeFrances, Liver regeneration, Science 276 (1997) 60–66. [5] H. Tsubouchi, Hepatocyte growth factor for liver disease, Hepatology 30 (1999) 333–334. [6] H. Funakoshi, T. Nakamura, Hepatocyte growth factor: from diagnosis to clinical applications, Clin. Chim. Acta 327 (2003) 1–23. [7] L.B. Ware, M.A. Matthay, Keratinocyte and hepatocyte growth factors in the lung: roles in lung development, inflammation, and repair, Am. J. Physiol. 282 (2002) L924–L940. [8] Y. Ishiki, H. Ohnishi, Y. Muto, K. Matsumoto, T. Nakamura, Direct evidence that hepatocyte growth factor is a hepatotrophic factor for liver regeneration and has a potent antihepatitis effect in vivo, Hepatology 16 (1992) 1227–1235. [9] K. Fujiwara, S. Nagoshi, A. Ohno, K. Hirata, Y. Ohta, S. Mochida, T. Tomiya, K. Higashio, K. Kurokawa, Stimulation of liver growth by exogenous human hepatocyte growth factor in normal and partially hepatectomized rats, Hepatology 18 (1993) 1443–1449. [10] M. Yamaoka, K. Hirata, I. Ogata, T. Tomiya, S. Nagoshi, S. Mochida, K. Fujiwara, Enhancement of albumin production by hepatocyte growth factor in rat hepatocytes: distinction in mode of action from stimulation of DNA synthesis, Liver 18 (1998) 52– 59.

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[11] P.J. Reeds, G. Biolo, Non-protein roles of amino acids: an emerging aspect of nutrient requirements, Curr. Opin. Clin. Nutr. Metab. Care 5 (2002) 43–45. [12] C.J. Nelsen, D.G. Rickheim, M.M. Tucker, T.J. McKenzie, L.K. Hansen, R.G. Pestell, J.H. Albrecht, Amino acids regulate hepatocyte proliferation through modulation of cyclin D1 expression, J. Biol. Chem. 278 (2003) 25853–25858. [13] S.M. Hutson, R.A. Harris, Symposium: Leucine as a nutritional signal: introduction, J. Nutr. 131 (2001) 839–840. [14] T.A. Gautsch, J.C. Anthony, S.R. Kimball, G.L. Paul, D.K. Layman, L.S. Jefferson, Availability of eIF4E regulates skeletal muscle protein synthesis during recovery from exercise, Am. J. Physiol. 274 (1998) C406–C414. [15] R.A. Harris, R. Kobayashi, T. Murakami, Y. Shimomura, Regulation of branched-chain a-keto acid dehydrogenase kinase expression in rat liver, J. Nutr. 131 (2001) 841S–845S.19. [16] M.E. May, M.G. Buse, Effects of branched-chain amino acids on protein turnover, Diabetes Metab. Rev. 5 (1989) 227–245. [17] R.J. Louard, E.J. Barrett, R.A. Gelfand, Overnight branched-chain amino acid infusion causes sustained suppression of muscle proteolysis, Metabolism 44 (1995) 424–429. [18] K. Peyrollier, E. Hajduch, A.S. Blair, R. Hyde, H.S. Hundal, LLeucine availability regulates phosphatidylinositol 3-kinase, p70 S6 kinase and glycogen synthase kinase-3 activity in L6 muscle cells: evidence for the involvement of the mammalian target of rapamycin (mTOR) pathway in the L-leucine-induced up-regulation of System A amino acid transport, Biochem. J. 350 (2000) 361–368. [19] K. Shigemitsu, Y. Tsujishita, H. Miyake, S. Hidayat, N. Tanaka, K. Hara, K. Yonezawa, Structural requirement of leucine for activation of p70 S6 kinase, FEBS Lett. 447 (1999) 303–306. [20] G. Xu, G. Kwon, C.A. Marshall, T.A. Lin, J.C. Lawrence Jr., M.L. McDaniel, Branched-chain amino acids are essential in the regulation of PHAS-I and p70 S6 kinase by pancreatic beta-cells. A possible role in protein translation and mitogenic signaling, J. Biol. Chem. 273 (1998) 28178–28184. [21] S.R. Kimball, L.S. Jefferson, Regulation of global and specific mRNA translation by oral administration of branched-chain amino acids, Biochem. Biophys. Res. Commun. 313 (2004) 423–427. [22] C.G. Proud, mTOR-mediated regulation of translation factors by amino acids, Biochem. Biophys. Res. Commun. 313 (2004) 429– 436. [23] C. Ijichi, T. Matsumura, T. Tsuji, Y. Eto, Branched-chain amino acids promote albumin synthesis in rat primary hepatocytes through the mTOR signal transduction system, Biochem. Biophys. Res. Commun. 303 (2003) 59–64. [24] C. Roh, J. Han, A. Tzatsos, K.V. Kandror, Nutrient-sensing mTORmediated pathway regulates leptin production in isolated rat adipocytes, Am. J. Physiol. Endocrinol. Metab. 284 (2003) E322–E330. [25] J.C. Anthony, T.G. Anthony, S.R. Kimball, L.S. Jefferson, Signaling pathways involved in translational control of protein synthesis in skeletal muscle by leucine, J. Nutr. 131 (2001) 856S–860S. [26] A.K. Reiter, T.G. Anthony, J.C. Anthony, L.S. Jefferson, S.R. Kimball, The mTOR signaling pathway mediates control of ribosomal protein mRNA translation in rat liver, Int. J. Biochem. Cell. Biol. 36 (2004) 2169–2179. [27] C.J. Lynch, B.J. Patson, J. Anthony, A. Vaval, L.S. Jefferson, T.C. Vary, Leucine is a direct-acting nutrient signal that regulates protein synthesis in adipose tissue, Am. J. Physiol. Endocrinol. Metab. 283 (2002) E503–E513. [28] T. Tomiya, Y. Inoue, M. Yanase, M. Arai, H. Ikeda, K. Tejima, K. Nagashima, T. Nishikawa, K. Fujiwara, Leucine stimulates the secretion of hepatocyte growth factor by hepatic stellate cells, Biochem. Biophys. Res. Commun. 297 (2002) 1108–1111. [29] T. Tomiya, T. Nishikawa, Y. Inoue, N. Ohtomo, H. Ikeda, K. Tejima, N. Watanabe, Y. Tanoue, M. Omata, K. Fujiwara, Leucine

[30]

[31]

[32]

[33]

[34]

[35]

[36] [37]

[38] [39]

[40]

[41]

[42]

[43]

[44] [45]

[46]

stimulates HGF production by hepatic stellate cells through mTOR pathway, Biochem. Biophys. Res. Commun. 358 (2007) 176–180. T. Tomiya, Y. Inoue, M. Yanase, M. Arai, H. Ikeda, K. Tejima, K. Nagashima, T. Nishikawa, N. Watanabe, M. Omata, K. Fujiwara, Treatment with leucine stimulates the production of hepatocyte growth factor in vivo, Biochem. Biophys. Res. Commun. 322 (2004) 772–777. P. Greenwel, M. Schwartz, M. Rosas, S. Peyrol, J.A. Grimaud, M. Rojkind, Characterization of fat-storing cell lines derived from normal and CCl4-cirrhotic livers. Differences in the production of interleukin-6, Lab. Invest. 65 (1991) 644–653. P. Greenwel, J. Rubin, M. Schwartz, E.L. Hertzberg, M. Rojkind, Liver fat-storing cell clones obtained from a CCl4-cirrhotic rat are heterogeneous with regard to proliferation, expression of extracellular matrix components, interleukin-6, and connexin 43, Lab. Invest. 69 (1993) 210–216. A. Yamada, K. Matsumoto, H. Iwanari, K. Sekiguchi, S. Kawata, Y. Matsuzawa, T. Nakamura, Rapid and sensitive enzyme-linked immunosorbent assay for measurement of HGF in rat and human tissues, Biomed. Res. 16 (1995) 105–114. T. Tomiya, I. Ogata, K. Fujiwara, Transforming growth factor alpha levels in liver and blood correlate better than hepatocyte growth factor with hepatocyte proliferation during liver regeneration, Am. J. Pathol. 153 (1998) 955–961. T. Matsumura, Y. Morinaga, S. Fujitani, K. Takehana, S. Nishitani, I. Sonaka, Oral administration of branched-chain amino acids activates the mTOR signal in cirrhotic rat liver, Hepatol. Res. 33 (2005) 27–32. C.G. Proud, Regulation of mammalian translation factors by nutrients, Eur. J. Biochem. 269 (2002) 5338–5349. D.C. Fingar, J. Blenis, Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression, Oncogene 23 (2004) 3151–3171. E.K. Rowinsky, Targeting the molecular target of rapamycin (mTOR), Curr. Opin. Oncol. 16 (2004) 564–575. C.E. Gleason, D. Lu, L.A. Witters, C.B. Newgard, M.J. Birnbaum, The role of AMPK and mTOR in nutrient sensing in pancreatic betacells, J. Biol. Chem. 282 (2007) 10341–10351. Y. Watatani, N. Kimura, Y.I. Shimizu, I. Akiyama, D. Tonaki, H. Hirose, S. Takahashi, Y. Takahashi, Amino acid limitation induces expression of ATF5 mRNA at the post-transcriptional level, Life Sci. 80 (2007) 879–885. C. Fumarola, S. La Monica, G.G. Guidotti, Amino acid signaling through the mammalian target of rapamycin (mTOR) pathway: role of glutamine and of cell shrinkage, J. Cell. Physiol. 204 (2005) 155– 165. T. Nakajo, T. Yamatsuji, H. Ban, K. Shigemitsu, M. Haisa, T. Motoki, K. Noma, T. Nobuhisa, J. Matsuoka, M. Gunduz, K. Yonezawa, N. Tanaka, Y. Naomoto, Glutamine is a key regulator for amino acid-controlled cell growth through the mTOR signaling pathway in rat intestinal epithelial cells, Biochem. Biophys. Res. Commun. 326 (2005) 174–180. Y. Xia, H.Y. Wen, M.E. Young, P.H. Guthrie, H. Taegtmeyer, R.E. Kellems, Mammalian target of rapamycin and protein kinase A signaling mediate the cardiac transcriptional response to glutamine, J. Biol. Chem. 278 (2003) 13143–13150. M. Kadowaki, T. Kanazawa, Amino acids as regulators of proteolysis, J. Nutr. 133 (2003) 2052S–2056S. H.M. Hanauske-Abel, Fibrosis: representative molecular elements, a basic concept, and a emerging targets for suppressive treatment, in: D. Zakim, T.D. Boyer (Eds.), Hepatology, a Text Book of Liver Disease, W.B. Saunders, Philadelphia, 1996, pp. 465–505. S. Nishitani, T. Matsumura, S. Fujitani, I. Sonaka, Y. Miura, K. Yagasaki, Leucine promotes glucose uptake in skeletal muscles of rats, Biochem. Biophys. Res. Commun. 299 (2002) 693–696.