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• W2130478809 abstract "Suckling “F/A2” mice, which overexpress arginase-I in their enterocytes, develop a syndrome (hypoargininemia, reduced hair and muscle growth, impaired B-cell maturation) that resembles IGF1 deficiency. The syndrome may result from an impaired function of the GH-IGF1 axis, activation of the stress-kinase GCN2, and/or blocking of the mTORC1-signaling pathway. Arginine deficiency inhibited GH secretion and decreased liver Igf1 mRNA and plasma IGF1 concentration, but did not change muscle IGF1 concentration. GH supplementation induced Igf1 mRNA synthesis, but did not restore growth, ruling out direct involvement of the GH-IGF1 axis. In C2C12 muscle cells, arginine withdrawal activated GCN2 signaling, without impacting mTORC1 signaling. In F/A2 mice, the reduction of plasma and tissue arginine concentrations to ∼25% of wild-type values activated GCN2 signaling, but mTORC1-mediated signaling remained unaffected. Gcn2-deficient F/A2 mice suffered from hypoglycemia and died shortly after birth. Because common targets of all stress kinases (eIF2α phosphorylation, Chop mRNA expression) were not increased in these mice, the effects of arginine deficiency were solely mediated by GCN2. Suckling “F/A2” mice, which overexpress arginase-I in their enterocytes, develop a syndrome (hypoargininemia, reduced hair and muscle growth, impaired B-cell maturation) that resembles IGF1 deficiency. The syndrome may result from an impaired function of the GH-IGF1 axis, activation of the stress-kinase GCN2, and/or blocking of the mTORC1-signaling pathway. Arginine deficiency inhibited GH secretion and decreased liver Igf1 mRNA and plasma IGF1 concentration, but did not change muscle IGF1 concentration. GH supplementation induced Igf1 mRNA synthesis, but did not restore growth, ruling out direct involvement of the GH-IGF1 axis. In C2C12 muscle cells, arginine withdrawal activated GCN2 signaling, without impacting mTORC1 signaling. In F/A2 mice, the reduction of plasma and tissue arginine concentrations to ∼25% of wild-type values activated GCN2 signaling, but mTORC1-mediated signaling remained unaffected. Gcn2-deficient F/A2 mice suffered from hypoglycemia and died shortly after birth. Because common targets of all stress kinases (eIF2α phosphorylation, Chop mRNA expression) were not increased in these mice, the effects of arginine deficiency were solely mediated by GCN2. Arginine is a substrate for the synthesis of proteins, creatine, agmatine, ornithine, and nitric oxide (1Wu G. Morris Jr., S.M. Biochem. J. 1998; 336: 1-17Crossref PubMed Scopus (2157) Google Scholar). In addition, it functions as a secretagogue for hormones, such as growth hormone (GH) and insulin (2Alba-Roth J. Müller O.A. Schopohl J. von Werder K. J. Clin. Endocrinol. Metab. 1988; 67: 1186-1189Crossref PubMed Scopus (304) Google Scholar, 3Rosati B. Marchetti P. Crociani O. Lecchi M. Lupi R. Arcangeli A. Olivotto M. Wanke E. Faseb. J. 2000; 14: 2601-2610Crossref PubMed Scopus (121) Google Scholar, 4Van Haeften T.W. Van Faassen I. Van der Veen E.A. Diabetes Res. 1988; 9: 187-191PubMed Google Scholar). Under normal conditions, endogenous arginine synthesis in adult mammals suffices to sustain daily requirements (5Visek W.J. J. Nutr. 1986; 116: 36-46Crossref PubMed Scopus (196) Google Scholar), but a dietary source of arginine may become necessary when demand increases under anabolic or catabolic conditions (5Visek W.J. J. Nutr. 1986; 116: 36-46Crossref PubMed Scopus (196) Google Scholar). For this reason, arginine is considered a conditionally essential amino acid. Arginine deficiency represents a significant metabolic problem in premature infants (6Wu G. Jaeger L.A. Bazer F.W. Rhoads J.M. J. Nutr. Biochem. 2004; 15: 442-451Crossref PubMed Scopus (186) Google Scholar), but its cause remains unknown. In rapidly growing suckling rodents, endogenous arginine biosynthesis is crucial to compensate for the insufficient supply of arginine via the milk (7Davis T.A. Fiorotto M.L. Reeds P.J. J. Nutr. 1993; 123: 947-956Crossref PubMed Scopus (80) Google Scholar). The intestine rather than the kidney is primarily responsible for endogenous arginine synthesis in suckling piglets (8Wu G. Am. J. Physiol. 1997; 272: G1382-G1390Crossref PubMed Google Scholar, 9Bertolo R.F. Brunton J.A. Pencharz P.B. Ball R.O. Am. J. Physiol. Endocrinol. Metab. 2003; 284: E915-E922Crossref PubMed Scopus (81) Google Scholar). In suckling rodents, the enterocytes of the small intestine also express all enzymes necessary to synthesize arginine (10De Jonge. W.J. Dingemanse M.A. de Boer P.A. Lamers W.H. Moorman A.F. Pediatr. Res. 1998; 43: 442-451Crossref PubMed Scopus (59) Google Scholar, 11Hurwitz R. Kretchmer N. Am. J. Physiol. 1986; 251: G103-G110PubMed Google Scholar, 12Riby J.E. Hurwitz R.E. Kretchmer N. Pediatr. Res. 1990; 28: 261-265Crossref PubMed Scopus (22) Google Scholar, 13Windmueller H.G. Adv. Enzymol. Relat. Areas Mol. Biol. 1982; 53: 201-237PubMed Google Scholar), while arginase is not expressed, thus allowing efficient de novo arginine biosynthesis. To examine the function of intestinal arginine synthesis, a transgenic mouse model that overexpresses arginase-I in the enterocytes of the small intestine was produced (14de Jonge W.J. Hallemeesch M.M. Kwikkers K.L. Ruijter J.M. de Gier-de Vries C. van Roon M.A. Meijer A.J. Marescau B. de Deyn P.P. Deutz N.E. Lamers W.H. Am. J. Clin Nutr. 2002; 76: 128-140Crossref PubMed Scopus (42) Google Scholar, 15de Jonge W.J. Kwikkers K.L. te Velde A.A. van Deventer S.J. Nolte M.A. Mebius R.E. Ruijter J.M. Lamers M.C. Lamers W.H. J. Clin. Invest. 2002; 110: 1539-1548Crossref PubMed Scopus (98) Google Scholar, 16de Jonge W.J. Marescau B. D'Hooge R. De Deyn P.P. Hallemeesch M.M. Deutz N.E. Ruijter J.M. Lamers W.H. J. Nutr. 2001; 131: 2732-2740Crossref PubMed Scopus (38) Google Scholar, 17Kwikkers K.L. Ruijter J.M. Labruyère W.T. McMahon K.K. Lamers W.H. Br. J. Nutr. 2005; 93: 183-189Crossref PubMed Scopus (2) Google Scholar). During the suckling period, these “F/A2” 4The abbreviations used are: F/A2, mice overexpressing arginase-I under control of the Fabpi promoter; ATF4, activating transcription factor 4 (Atf4); Atrogin1, muscle atrophy F-box E3 ubiquitin-protein ligase or F-box-only protein 32 (Fbxo32); CAT-1, cationic amino-acid transporter-1 or Solute carrier 7A1 (Slc7A1); CHOP, C/EBP-homologous protein or growth arrest and DNA damage-inducible protein 135 or DNA-damage inducible transcript 3 (Ddit3); 4EBP1, eukaryotic translation initiation factor (eIF)4E-binding protein-1 (Eif4ebp1); GADD34, growth arrest and DNA damage-inducible protein 34 or protein phosphatase-1 regulatory subunit 15A (Ppp1r15a); GCN2, general control non-derepressible 2 kinase or eukaryotic translation initiation factor 2α kinase 4 (Eif2ak4); IGF1, insulin-like growth factor 1 (Igf1); CKM, muscle-specific creatine kinase (Ckm); mTORC1, mammalian target of rapamycin complex-1; MURF1, muscle-specific RING finger-1 or E3 ubiquitin-protein ligase (Trim63); MyHC1, myosin heavy chain-I or heavy polypeptide 7 (Myh7); MyHC2B, myosin heavy chain-IIb or heavy polypeptide 4 (Myh4); ND, neonatal day; S6K1, p70 ribosomal protein S6 kinase-1 or polypeptide 5 (Rps6ka5). mice suffer from a selective deficiency of circulating arginine (which declines to ∼25% of controls), reduced growth of hair and skeletal muscle, and inhibition of B-cell maturation (14de Jonge W.J. Hallemeesch M.M. Kwikkers K.L. Ruijter J.M. de Gier-de Vries C. van Roon M.A. Meijer A.J. Marescau B. de Deyn P.P. Deutz N.E. Lamers W.H. Am. J. Clin Nutr. 2002; 76: 128-140Crossref PubMed Scopus (42) Google Scholar). The phenotype can be rescued with arginine supplementation (14de Jonge W.J. Hallemeesch M.M. Kwikkers K.L. Ruijter J.M. de Gier-de Vries C. van Roon M.A. Meijer A.J. Marescau B. de Deyn P.P. Deutz N.E. Lamers W.H. Am. J. Clin Nutr. 2002; 76: 128-140Crossref PubMed Scopus (42) Google Scholar), but could not be ascribed to a deficiency of arginine metabolites, such as creatine or polyamines, or to arginine-dependent ADP-ribosylation of proteins (14de Jonge W.J. Hallemeesch M.M. Kwikkers K.L. Ruijter J.M. de Gier-de Vries C. van Roon M.A. Meijer A.J. Marescau B. de Deyn P.P. Deutz N.E. Lamers W.H. Am. J. Clin Nutr. 2002; 76: 128-140Crossref PubMed Scopus (42) Google Scholar, 15de Jonge W.J. Kwikkers K.L. te Velde A.A. van Deventer S.J. Nolte M.A. Mebius R.E. Ruijter J.M. Lamers M.C. Lamers W.H. J. Clin. Invest. 2002; 110: 1539-1548Crossref PubMed Scopus (98) Google Scholar, 16de Jonge W.J. Marescau B. D'Hooge R. De Deyn P.P. Hallemeesch M.M. Deutz N.E. Ruijter J.M. Lamers W.H. J. Nutr. 2001; 131: 2732-2740Crossref PubMed Scopus (38) Google Scholar, 17Kwikkers K.L. Ruijter J.M. Labruyère W.T. McMahon K.K. Lamers W.H. Br. J. Nutr. 2005; 93: 183-189Crossref PubMed Scopus (2) Google Scholar). Furthermore, the features of the “arginine-deficiency syndrome” are not seen in mice that are deficient in all three nitric-oxide synthases (18Tsutsui M. Shimokawa H. Morishita T. Nakashima Y. Yanagihara N. J. Pharmacol. Sci. 2006; 102: 147-154Crossref PubMed Scopus (72) Google Scholar). This leaves two functions of arginine as likely candidates to account for the highly characteristic phenotype of arginine deficiency in suckling mice, viz. its role as a building block in protein synthesis and as a secretagogue of hormones. Amino acids, including arginine, control translation by two well-established mechanisms. Whenever the intracellular concentration of free arginine decreases below ∼20 μm, the charging process of its corresponding tRNA slows down and the concentration of uncharged tRNA increases (19Vellekamp G. Sihag R.K. Deutscher M.P. J. Biol. Chem. 1985; 260: 9843-9847Abstract Full Text PDF PubMed Google Scholar). Uncharged tRNAs activate the ubiquitously expressed “general control non-derepressible2” (GCN2) stress kinase, which then phosphorylates the α-subunit of the eukaryotic initiation factor 2 (eIF2α) and thereby initiates the “integrated stress response” (20Ron D. J. Clin. Invest. 2002; 110: 1383-1388Crossref PubMed Scopus (730) Google Scholar, 21Wek R.C. Jiang H.Y. Anthony T.G. Biochem. Soc Trans. 2006; 34: 7-11Crossref PubMed Scopus (1008) Google Scholar, 22Zhang P. McGrath B.C. Reinert J. Olsen D.S. Lei L. Gill S. Wek S.A. Vattem K.M. Wek R.C. Kimball S.R. Jefferson L.S. Cavener D.R. Mol. Cell Biol. 2002; 22: 6681-6688Crossref PubMed Scopus (340) Google Scholar). This response blocks cap-dependent protein synthesis, facilitates the translation of specific mRNAs, such as Atf4 and Cat-1, from internal ribosome entry sites, and increases expression of specific transcription factors such as ATF2, ATF4, and CHOP (23Averous J. Bruhat A. Jousse C. Carraro V. Thiel G. Fafournoux P. J. Biol. Chem. 2004; 279: 5288-5297Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar), which, in turn, induce the transcription of genes necessary for amino acid synthesis and transport. The other mechanism senses, instead, increases in amino acid concentration, probably via the Rag GTPase complex (24Shaw R.J. Trends Biochem. Sci. 2008; 33: 565-568Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), and results in the activation of the mTORC1 kinase. mTORC1, in turn, regulates cap-dependent translation through the phosphorylation of 4EBP1 and S6K1 (25Avruch J. Long X. Ortiz-Vega S. Rapley J. Papageorgiou A. Dai N. Am. J. Physiol. Endocrinol. Metab. 2009; 296: E592-E602Crossref PubMed Scopus (305) Google Scholar, 26Ma X.M. Blenis J. Nat. Rev. Mol. Cell Biol. 2009; 10: 307-318Crossref PubMed Scopus (1897) Google Scholar). The essential amino acid leucine is the prototypic regulator of both branches of amino acid-dependent translational regulation (22Zhang P. McGrath B.C. Reinert J. Olsen D.S. Lei L. Gill S. Wek S.A. Vattem K.M. Wek R.C. Kimball S.R. Jefferson L.S. Cavener D.R. Mol. Cell Biol. 2002; 22: 6681-6688Crossref PubMed Scopus (340) Google Scholar, 27Drummond M.J. Rasmussen B.B. Curr. Opin. Clin. Nutr. Metab. Care. 2008; 11: 222-226Crossref PubMed Scopus (194) Google Scholar). However, for a non-essential amino acid, arginine has an unexpectedly strong effect on mTORC1-dependent signaling (28Yao K. Yin Y.L. Chu W. Liu Z. Deng D. Li T. Huang R. Zhang J. Tan B. Wang W. Wu G. J. Nutr. 2008; 138: 867-872Crossref PubMed Scopus (313) Google Scholar) and quantitatively rivals leucine in vitro (29Hara K. Yonezawa K. Weng Q.P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 273: 14484-14494Abstract Full Text Full Text PDF PubMed Scopus (1112) Google Scholar, 30Nakajo T. Yamatsuji T. Ban H. Shigemitsu K. Haisa M. Motoki T. Noma K. Nobuhisa T. Matsuoka J. Gunduz M. Yonezawa K. Tanaka N. Naomoto Y. Biochem. Biophys. Res. Commun. 2005; 326: 174-180Crossref PubMed Scopus (78) Google Scholar). The F/A2 phenotype could, therefore, be mediated by activation of GCN2 signaling and/or inhibition of mTORC1-mediated signaling. The more remarkable phenotypic features of F/A2 mice, namely impaired muscle and hair growth and impaired B-cell maturation, are all also affected by insulin-like growth factor-1 (IGF1) signaling (31Foulstone E.J. Huser C. Crown A.L. Holly J.M. Stewart C.E. Exp. Cell Res. 2004; 294: 223-235Crossref PubMed Scopus (60) Google Scholar, 32Kim H. Barton E. Muja N. Yakar S. Pennisi P. Leroith D. Endocrinology. 2005; 146: 1772-1779Crossref PubMed Scopus (76) Google Scholar, 33Landreth K.S. Narayanan R. Dorshkind K. Blood. 1992; 80: 1207-1212Crossref PubMed Google Scholar, 34Sumita K. Hattori N. Inagaki C. J Pharmacol. Sci. 2005; 97: 408-416Crossref PubMed Scopus (23) Google Scholar, 35Weger N. Schlake T. J. Invest. Dermatol. 2005; 125: 873-882Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Like amino acids, growth factor signals, including those arising from IGF1, stimulate protein synthesis via the mTORC1 pathway (36Soulard A. Hall M.N. Cell. 2007; 129: 434Abstract Full Text PDF PubMed Scopus (45) Google Scholar). The production of IGF1, in turn, is largely dependent on growth-hormone (GH) signaling (37Rodriguez S. Gaunt T.R. Day I.N. Hum. Genet. 2007; 122: 1-21Crossref PubMed Scopus (65) Google Scholar). Arginine, finally, is also a potent growth-hormone secretagogue (38Laron Z. Drugs. 1995; 50: 595-601Crossref PubMed Scopus (43) Google Scholar). Arginine deficiency could, therefore, also cause insufficient signaling via the mTORC1 pathway due to an inactive somatotropic axis. In the present study, we used a combination of in vivo and in vitro approaches to test the hypotheses that arginine deficiency activates GCN2 signaling or blocks mTORC1 signaling, or both. Our findings demonstrate that arginine deficiency in the neonatal and suckling period activates the GCN2 stress-kinase pathway, without affecting the mTORC1 pathway. Transgenic mice that overexpress arginase-I in their small-intestinal enterocytes (F/A2 line (14de Jonge W.J. Hallemeesch M.M. Kwikkers K.L. Ruijter J.M. de Gier-de Vries C. van Roon M.A. Meijer A.J. Marescau B. de Deyn P.P. Deutz N.E. Lamers W.H. Am. J. Clin Nutr. 2002; 76: 128-140Crossref PubMed Scopus (42) Google Scholar)) were bred hemizygously. Gcn2−/− mice (39Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2388) Google Scholar) originated from the colony of D. Ron (New York, NY). Control animals were littermates of the experimental animals. Animal studies were reviewed and approved by the committee for animal care and use of Maastricht University. Litters were limited to 5 animals on neonatal day 1 (ND1) and weighed daily. Starting on ND3, 3 mg/kg body weight of human recombinant growth hormone (hrGH; Humatrope, Lilly, Indianapolis, IN) was administered subcutaneously in 25 μl of vehicle twice daily (40Liu J.L. Yakar S. LeRoith D. Endocrinology. 2000; 141: 4436-4441Crossref PubMed Scopus (54) Google Scholar). Control animals were injected with vehicle alone. Animals were sacrificed by decapitation. Blood was collected into heparin-containing tubes and centrifuged at 2,000 × g for 5 min at 4 °C. IGF-1 protein concentrations were measured in plasma, whereas amino acids were measured in deproteinized plasma. 80 μl of plasma was added to 6.4 mg of lyophilized sulfosalicylic acid, vortexed, and stored at −20 °C. For amino acid measurements, the tissues were homogenized with silica mini-beads in 0.33 m sulfosalicylic acid. Tissues were collected at ND0, ND10, ND17, and ND21. Amino acid concentrations were determined by HPLC (41van Eijk H.M. Rooyakkers D.R. Deutz N.E. J. Chromatogr. 1993; 620: 143-148Crossref PubMed Scopus (239) Google Scholar). Tissues for protein and mRNA analysis were isolated, snap frozen in liquid nitrogen and stored at −80 °C. For histological analysis, tissues were fixed in 4% buffered formalin and embedded in paraffin. Mouse IGF-1 concentration in plasma and muscle was measured by the m/r IGF-1 E25 enzyme immunoassay, as detailed by the manufacturer (Mediagnost, Reutlingen, Germany). This assay eliminates IGFBP interference with the IGF-1 measurement. Total RNA was extracted with Trizol (Sigma). 1 μg of total RNA was reverse transcribed with the iScript cDNA synthesis kit (Bio-Rad). Primer sequences (supplemental Table S1) were optimized for an annealing temperature of 60 °C, except for the Igf1 primers that were annealed at 70 °C. cDNA samples for mRNA determination were diluted 60-fold before use and those for 18 S rRNA 200-fold. Primary fluorescent data were exported and analyzed with the Lin-Reg Analysis program (42Ruijter J.M. Ramakers C. Hoogaars W.M. Karlen Y. Bakker O. van den Hoff M.J. Moorman A.F. Nucleic Acids Res. 2009; 37: e45Crossref PubMed Scopus (2128) Google Scholar). If reverse transcriptase was omitted, no product formed. mRNA concentrations were expressed relative to 18 S rRNA content. Between session variations in replicate experiments were corrected using factor correction (43Ruijter J.M. Thygesen H.H. Schoneveld O.J. Das A.T. Berkhout B. Lamers W.H. Retrovirology. 2006; 3: 2Crossref PubMed Scopus (139) Google Scholar). For Western blot assays, 100 μg of protein were loaded per lane. Antibody (supplemental Table S2) binding was visualized using the Super Signal West Femto Maximum Sensitivity Substrate (Pierce). For detection of the phosphorylated and unphosphorylated forms of eIF2α, the Odyssey Infrared Imaging system (Li-cor Bioscience, UK) was used. For immunostaining, 5-μm sections of tissues were prepared. After deparaffinization, sections were preincubated with Teng-T (10 mm Tris (pH 7.6), 5 mm EDTA, 150 mm NaCl, 0.25% gelatin, 0.05% Tween-20)/10% normal goat serum (NGS) for 30 min, followed by an overnight incubation with the primary antibody diluted in Teng-T/10% NGS. Slides were then washed with PBS and incubated with Teng-T/10% NGS for 15 min, followed by 90 min incubation with the secondary antibody (supplemental Table S2) in Teng-T/10% NGS. After washing, the slides were incubated with alkaline phosphatase substrate (NBT/BCIP tablets; Roche) at 37 °C. Digital images of the sections were analyzed with the Leica Quantimet 500 Image Analysis System, v3.2 (44van Straaten H.W. He Y. van Duist M.M. Labruyère W.T. Vermeulen J.L. van Dijk P.J. Ruijter J.M. Lamers W.H. Hakvoort T.B. Biochem. Cell Biol. 2006; 84: 215-231Crossref PubMed Scopus (42) Google Scholar). The significant difference for one variable between two distinct groups was analyzed using an unpaired Student's t test. For more complex analyses of significance for variables with different controls, each gene was analyzed using Gaussian linear regression, including 18 S as housekeeping gene. When appropriate, additional explanatory variables such as tissue type, strain, and time were included in the model. Finally, interactions among the included variables were considered. The inference criterion used for comparing the models is their ability to predict the observed data, i.e. models are compared directly through their minimized minus log-likelihood. When the numbers of parameters in models differ, they are penalized by adding the number of estimated parameters, a form of the Akaike information criterion (45.Akaike, H., (1973) Second International Symposium on Information Theory, Akademia Kiado.Google Scholar). If the most likely model contains the treatment effect, the fold change representing the ratio between the variable of the treated group and the same variable for the control is computed, as well as its 95% confidence interval. The phenotype of F/A2 mice is compatible with a deficiency of GH and/or IGF1. We, therefore, investigated the GH content in the pituitary gland by quantitative immunohistochemistry (Fig. 1A). The somatotropes of 3-week-old hemi- and homozygous F/A2 mice contained 1.4- and 2.2-fold more GH, respectively, than those of their wild-type counterparts (Fig. 1B), indicating an inverse correlation with the severity of the F/A2 phenotype (14de Jonge W.J. Hallemeesch M.M. Kwikkers K.L. Ruijter J.M. de Gier-de Vries C. van Roon M.A. Meijer A.J. Marescau B. de Deyn P.P. Deutz N.E. Lamers W.H. Am. J. Clin Nutr. 2002; 76: 128-140Crossref PubMed Scopus (42) Google Scholar, 16de Jonge W.J. Marescau B. D'Hooge R. De Deyn P.P. Hallemeesch M.M. Deutz N.E. Ruijter J.M. Lamers W.H. J. Nutr. 2001; 131: 2732-2740Crossref PubMed Scopus (38) Google Scholar). We then used the rat pituitary cell line GH3, which produces GH (46Adrião M. Chrisman C.J. Bielavsky M. Olinto S.C. Shiraishi E.M. Nunes M.T. Neuroendocrinology. 2004; 79: 26-33Crossref PubMed Scopus (18) Google Scholar, 47Liu Y.L. Zhong Y.Q. Chi S.M. Zhu Y.L. Sheng. Li Xue Bao. 2005; 57: 254-258PubMed Google Scholar), to assess the direct effects of arginine on GH production and secretion. GH content in the GH3 cells was assessed by quantitative immunohistochemistry (supplemental Fig. S1, A and B) and ELISA (supplemental Fig. S1C). Both assays showed that, after 24 h of culture, the cellular GH content was inversely correlated with extracellular arginine concentration and decreased from ∼50 to ∼25 pg of GH per cell at 0 and 200 μm arginine in the medium, respectively (supplemental Fig. S1C; significant at ≥50 μm arginine). This decrease in cellular GH content was accounted for by a 1.5-fold increase in concentration of GH in the medium (significant at ≥25 μm arginine). Because the total GH content of the cells and the medium combined did not vary with arginine concentration in the medium, these findings reveal a pronounced concentration-dependent effect of arginine on GH secretion. The dependence of GH secretion on the ambient concentration of arginine after 72 h of culture was similar to that observed after 24 h (not shown). These data show that, below a concentration of 50 μm ambient arginine, GH secretion is reduced, with a half-maximal effect at ∼20 μm arginine. As early as 3 days after birth (ND3), F/A2 mice can be distinguished from their wild-type littermates by a lower body weight (14de Jonge W.J. Hallemeesch M.M. Kwikkers K.L. Ruijter J.M. de Gier-de Vries C. van Roon M.A. Meijer A.J. Marescau B. de Deyn P.P. Deutz N.E. Lamers W.H. Am. J. Clin Nutr. 2002; 76: 128-140Crossref PubMed Scopus (42) Google Scholar). To assess whether the typically hypomorphic tissues of F/A2 mice (muscle, hair, and B-cells (15de Jonge W.J. Kwikkers K.L. te Velde A.A. van Deventer S.J. Nolte M.A. Mebius R.E. Ruijter J.M. Lamers M.C. Lamers W.H. J. Clin. Invest. 2002; 110: 1539-1548Crossref PubMed Scopus (98) Google Scholar)) respond to GH supplementation, mice were subcutaneously injected twice daily with 3 mg/kg recombinant human (rh)GH from ND3 onwards. This dose of rhGH mediates growth in liver IGF-1-deficient mice (40Liu J.L. Yakar S. LeRoith D. Endocrinology. 2000; 141: 4436-4441Crossref PubMed Scopus (54) Google Scholar). Fig. 1C shows that the growth rate of homozygous F/A2 mice was ∼50% of that of wild-type mice and that treatment with rhGH had no effect on the growth rate of either wild-type or F/A2 mice. GH action is mediated by GH receptors (GHR) and results in the synthesis and secretion of IGF1 in target tissues. Ghr mRNA concentrations in the livers of ND1, ND10, and ND21 mice increased with age, but were not different in wild-type and F/A2 mice (supplemental Fig. S2). To assess the functionality of these GHRs and their downstream signaling pathway, Igf1 mRNA concentrations were quantified in liver and muscle of GH-treated and control mice (Fig. 1D). In both tissues, Igf1 mRNA concentrations in F/A2 mice were only 10–15% of those in wild-type mice. Although the rhGH injections increased Igf1 mRNA concentration in liver and muscle of both F/A2 and wild-type mice considerably (∼8- and ∼2.5-fold, respectively; p < 0.01), Igf1 mRNA levels in the liver and muscle of treated F/A2 mice increased to only ∼65 and ∼25%, respectively, of that in untreated wild-type mice. We then measured plasma and tissue IGF1 concentrations to assess whether F/A2 mice suffer from a low production of IGF1 (Fig. 2). In wild-type and F/A2 ND1 mice, no differences in plasma, muscle, or liver IGF1 protein were detected (Fig. 2A). However, plasma IGF1 levels increased ∼7-fold faster in wild-type than F/A2 mice during the first 5 postnatal weeks (Fig. 2B, left panel; p < 0.001)). Plasma IGF1 levels reflect hepatic IGF1 production, but liver IGF1 does not affect growth until 6 weeks postnatally (48Sjögren K. Liu J.L. Blad K. Skrtic S. Vidal O. Wallenius V. LeRoith D. Törnell J. Isaksson O.G. Jansson J.O. Ohlsson C. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 7088-7092Crossref PubMed Scopus (770) Google Scholar, 49Yakar S. Liu J.L. Stannard B. Butler A. Accili D. Sauer B. LeRoith D. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 7324-7329Crossref PubMed Scopus (1176) Google Scholar). We, therefore, measured IGF1 levels in an affected tissue (calf muscle) of the same mice (Fig. 2B, right panel). Muscular IGF1 concentration decreased gradually between 1 and 3 weeks after birth, without difference between F/A2 and wild-type mice. After weaning, the IGF1 concentration in muscle increased again, with a ∼1 week delay in F/A2 relative to wild-type mice. Although the age-dependent changes in muscle IGF1 content were significant (p < 0.01), they did not differ between F/A2 and wild-type mice. Because IGF1 in skeletal muscle affects growth only after the 3rd postnatal week (32Kim H. Barton E. Muja N. Yakar S. Pennisi P. Leroith D. Endocrinology. 2005; 146: 1772-1779Crossref PubMed Scopus (76) Google Scholar), that is, coincident with the resumption of growth in F/A2 mice (14de Jonge W.J. Hallemeesch M.M. Kwikkers K.L. Ruijter J.M. de Gier-de Vries C. van Roon M.A. Meijer A.J. Marescau B. de Deyn P.P. Deutz N.E. Lamers W.H. Am. J. Clin Nutr. 2002; 76: 128-140Crossref PubMed Scopus (42) Google Scholar), we further concluded that, although the GH-IGF-1 axis did not function properly in F/A2 mice, its disturbed function could not account for the marked hypotrophy seen in these animals. F/A2 skeletal muscles have an immature appearance (14de Jonge W.J. Hallemeesch M.M. Kwikkers K.L. Ruijter J.M. de Gier-de Vries C. van Roon M.A. Meijer A.J. Marescau B. de Deyn P.P. Deutz N.E. Lamers W.H. Am. J. Clin Nutr. 2002; 76: 128-140Crossref PubMed Scopus (42) Google Scholar). We, therefore, investigated if arginine directly affects muscle development in differentiating C2C12 myoblasts that were cultured in the presence of different concentrations of arginine. Supplemental Fig. S3A shows representative pictures after 4 days of myogenic differentiation in the presence (200 μm) or absence of arginine. Both the myogenic index (the fraction of nuclei residing in cells with three or more nuclei and considered an early differentiation parameter; supplemental Fig. S3B) and the cellular concentration of the late differentiation marker creatine kinase-M (supplemental Fig. S3C) increased with increasing arginine concentration to plateau at 25–50 μm arginine. Myogenic differentiation is, therefore, clearly dependent on arginine availability, with a half-maximal effect at 10–20 μm arginine in the medium. Amino acid deficiency can activate the GCN2 stress kinase pathway (50Gebauer F. Hentze M.W. Nat. Rev. Mol. Cell Biol. 2004; 5: 827-835Crossref PubMed Scopus (708) Google Scholar). An early step is the phosphorylation of translation factor eIF2α (51Harding H.P. Zhang Y. Zeng H. Novoa I. Lu P.D. Calfon M. Sadri N. Yun C. Popko B. Paules R. Stojdl D.F. Bell J.C. Hettmann T. Leiden J.M. Ron D. Mol. Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2335) Google Scholar). As anticipated, arginine deprivation of C2C12 cells at 80% confluence induced phosphorylation of eIF2α on serine 51 within 30 min. The protein remained phosphorylated for at least 2 h (supplemental Fig. S4A). Furthermore, arginine depletion induced the accumulation of the transcription factors ATF4 and CHOP, which are well-established downstream mRNA targets of the GCN2 stress-kinase pathway (23Averous J. Bruhat A. Jousse C. Carraro V. Thiel G. Fafournoux P. J. Biol. Chem. 2004; 279: 5288-5297Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 52Ameri K. Harris A.L. Int. J. Biochem. Cell Biol. 2008; 40: 14-21Crossref PubMed Scopus (360) Google Scholar), in a concentration-dependent way in both C2C12 and GH3 cells (supplemental Fig. S4B). In both cell types, Chop mRNA concentration increased more gradually with declining arginine concentration in the medium than Atf4 mRNA, indicating that Chop mRNA accumulation was more sensitive to arginine removal than that of Atf4 mRNA. The adaptive response of Atf4 and Chop mRNA expression in primary cultures of mouse B-lymphocytes, another hypomorphic tissue in F/A2 mice, to arginine deprivation was similar to that of C2C12 and GH3 cells (not shown). Supplemental Fig. S4C, finally, shows that ATF4 protein accumulates at arginine concentrations below 25 μm (see Ref. 52Am" @default.
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• W2130478809 title "Arginine Deficiency Causes Runting in the Suckling Period by Selectively Activating the Stress Kinase GCN2" @default.
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