Citrin/Mitochondrial Glycerol-3-phosphate Dehydrogenase Double Knock-out Mice Recapitulate Features of Human Citrin Deficiency
2007; Elsevier BV; Volume: 282; Issue: 34 Linguagem: Inglês
10.1074/jbc.m702031200
ISSN1083-351X
AutoresTakeyori Saheki, Mikio Iijima, Meng Xian Li, Keiko Kobayashi, Masahisa Horiuchi, Miharu Ushikai, Fumihiko Okumura, Xiao jian Meng, Ituro Inoue, Atsushi Tajima, Mitsuaki Moriyama, Kazuhiro Eto, Takashi Kadowaki, David S. Sinasac, Lap‐Chee Tsui, Mihoko Tsuji, Akira Okano, Tsuyoshi Kobayashi,
Tópico(s)Pediatric Hepatobiliary Diseases and Treatments
ResumoCitrin is the liver-type mitochondrial aspartate-glutamate carrier that participates in urea, protein, and nucleotide biosynthetic pathways by supplying aspartate from mitochondria to the cytosol. Citrin also plays a role in transporting cytosolic NADH reducing equivalents into mitochondria as a component of the malate-aspartate shuttle. In humans, loss-of-function mutations in the SLC25A13 gene encoding citrin cause both adult-onset type II citrullinemia and neonatal intrahepatic cholestasis, collectively referred to as human citrin deficiency. Citrin knock-out mice fail to display features of human citrin deficiency. Based on the hypothesis that an enhanced glycerol phosphate shuttle activity may be compensating for the loss of citrin function in the mouse, we have generated mice with a combined disruption of the genes for citrin and mitochondrial glycerol 3-phosphate dehydrogenase. The resulting double knock-out mice demonstrated citrullinemia, hyperammonemia that was further elevated by oral sucrose administration, hypoglycemia, and a fatty liver, all features of human citrin deficiency. An increased hepatic lactate/pyruvate ratio in the double knock-out mice compared with controls was also further elevated by the oral sucrose administration, suggesting that an altered cytosolic NADH/NAD+ ratio is closely associated with the hyperammonemia observed. Microarray analyses identified over 100 genes that were differentially expressed in the double knock-out mice compared with wild-type controls, revealing genes potentially involved in compensatory or downstream effects of the combined mutations. Together, our data indicate that the more severe phenotype present in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice represents a more accurate model of human citrin deficiency than citrin knock-out mice. Citrin is the liver-type mitochondrial aspartate-glutamate carrier that participates in urea, protein, and nucleotide biosynthetic pathways by supplying aspartate from mitochondria to the cytosol. Citrin also plays a role in transporting cytosolic NADH reducing equivalents into mitochondria as a component of the malate-aspartate shuttle. In humans, loss-of-function mutations in the SLC25A13 gene encoding citrin cause both adult-onset type II citrullinemia and neonatal intrahepatic cholestasis, collectively referred to as human citrin deficiency. Citrin knock-out mice fail to display features of human citrin deficiency. Based on the hypothesis that an enhanced glycerol phosphate shuttle activity may be compensating for the loss of citrin function in the mouse, we have generated mice with a combined disruption of the genes for citrin and mitochondrial glycerol 3-phosphate dehydrogenase. The resulting double knock-out mice demonstrated citrullinemia, hyperammonemia that was further elevated by oral sucrose administration, hypoglycemia, and a fatty liver, all features of human citrin deficiency. An increased hepatic lactate/pyruvate ratio in the double knock-out mice compared with controls was also further elevated by the oral sucrose administration, suggesting that an altered cytosolic NADH/NAD+ ratio is closely associated with the hyperammonemia observed. Microarray analyses identified over 100 genes that were differentially expressed in the double knock-out mice compared with wild-type controls, revealing genes potentially involved in compensatory or downstream effects of the combined mutations. Together, our data indicate that the more severe phenotype present in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice represents a more accurate model of human citrin deficiency than citrin knock-out mice. Human citrin deficiency is a newly established disease entity (1Saheki T. Kobayashi K. J. Hum. Genet. 2002; 47: 333-341Crossref PubMed Scopus (233) Google Scholar, 2Kobayashi K. Saheki T. Seikagaku (Japanese). 2004; 76: 1543-1559PubMed Google Scholar) that encompasses both adult-onset type II citrullinemia (CTLN2 2The abbreviations used are: CTLN2adult-onset type II citrullinemiaAAAaromatic amino acidAGCaspartate-glutamate carrierBCAAbranched-chain amino acidCitcitrullineCtrncitrinCy3cyanine 3-dCTPCy5cyanine 5-dCTPFFAfree fatty acidLaclactateL/Plactate/pyruvateKOknock-outMAmalate-aspartatemGPDmitochondrial glycerol-3-phosphate dehydrogenaseNICCDneonatal intrahepatic cholestasis caused by citrin deficiencyPyrpyruvateTGtriglycerideANOVAanalysis of variance 2The abbreviations used are: CTLN2adult-onset type II citrullinemiaAAAaromatic amino acidAGCaspartate-glutamate carrierBCAAbranched-chain amino acidCitcitrullineCtrncitrinCy3cyanine 3-dCTPCy5cyanine 5-dCTPFFAfree fatty acidLaclactateL/Plactate/pyruvateKOknock-outMAmalate-aspartatemGPDmitochondrial glycerol-3-phosphate dehydrogenaseNICCDneonatal intrahepatic cholestasis caused by citrin deficiencyPyrpyruvateTGtriglycerideANOVAanalysis of variance; Online Mendelian Inheritance in Man™ number (OMIM™) 603471) and neonatal intrahepatic cholestasis (NICCD; OMIM™ number 605814), and it results from mutations in the SLC25A13 gene that encodes citrin (3Kobayashi K. Sinasac D.S. Iijima M. Boright A.P. Begum L. Lee J.R. Yasuda T. Ikeda S. Hirano R. Terazono H. Crackower M.A. Kondo I. Tsui L.C. Scherer S.W. Saheki T. Nat. Genet. 1999; 22: 159-163Crossref PubMed Scopus (361) Google Scholar). Citrin and the closely related protein aralar (encoded by SLC25A12) (4del Arco A. Satrustegui J. J. Biol. Chem. 1998; 273: 23327-23334Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) are isoforms of the aspartate (Asp)-glutamate (Glu) carrier (AGC) found within the inner mitochondrial membrane and are responsible for the electrogenic exchange of Asp for Glu and a H+ ion (5Palmieri L. Pardo B. Lasorsa F.M. del Arco A. Kobayashi K. Iijima M. Runswick M.J. Walker J.E. Saheki T. Satrustegui J. Palmieri F. EMBO J. 2001; 20: 5060-5069Crossref PubMed Scopus (390) Google Scholar). Citrin is predominantly found in liver, kidney, heart, and small intestine, whereas aralar is found in brain, skeletal muscle, kidney, and heart (3Kobayashi K. Sinasac D.S. Iijima M. Boright A.P. Begum L. Lee J.R. Yasuda T. Ikeda S. Hirano R. Terazono H. Crackower M.A. Kondo I. Tsui L.C. Scherer S.W. Saheki T. Nat. Genet. 1999; 22: 159-163Crossref PubMed Scopus (361) Google Scholar, 6Iijima M. Jalil A. Begum L. Yasuda T. Yamaguchi N. Li M.X. Kawada N. Endou H. Kobayashi K. Saheki T. Adv. Enzyme Regul. 2001; 41: 325-342Crossref PubMed Scopus (51) Google Scholar, 7Begum L. Jalil M.A. Kobayashi K. Iijima M. Li M.X. Yasuda T. Horiuchi M. del Arco A. Satrustegui J. Saheki T. Biochim. Biophys. Acta. 2002; 1574: 283-292Crossref PubMed Scopus (54) Google Scholar). Based on their partially overlapping expression patterns, citrin can be thought of as the liver-type and aralar as the brain- and muscle-type AGC. Both proteins have a characteristic bipartite structure; the C-terminal half of each protein contains the canonical mitochondrial solute carrier structure, and the N-terminal half contains EF-hand motifs that have been shown to bind calcium (8Kobayashi K. Iijima M. Yasuda T. Sinasac D.S. Yamaguchi N. Tsui L.C. Scherer S.W. Saheki T. Pochet R. Donato R. Haiech J. Heizmann C. Gerke V. Calcium: The Molecular Basis of Calcium Action in Biology and Medicine. Kluwer Academic Publishers Group, Dordrecht, Netherlands2000: 565-587Google Scholar, 9del Arco A. Agudo M. Satrustegui J. Biochem. J. 2000; 345: 725-732Crossref PubMed Scopus (79) Google Scholar), stimulating their transport activities (5Palmieri L. Pardo B. Lasorsa F.M. del Arco A. Kobayashi K. Iijima M. Runswick M.J. Walker J.E. Saheki T. Satrustegui J. Palmieri F. EMBO J. 2001; 20: 5060-5069Crossref PubMed Scopus (390) Google Scholar). The function of the AGC is to provide substrate for the synthesis of proteins, nucleotides, and urea, in addition to participating in gluconeogenesis from lactate (Lac) and transporting cytosolic NADH reducing equivalents into mitochondria as part of the malate-Asp (MA) shuttle. adult-onset type II citrullinemia aromatic amino acid aspartate-glutamate carrier branched-chain amino acid citrulline citrin cyanine 3-dCTP cyanine 5-dCTP free fatty acid lactate lactate/pyruvate knock-out malate-aspartate mitochondrial glycerol-3-phosphate dehydrogenase neonatal intrahepatic cholestasis caused by citrin deficiency pyruvate triglyceride analysis of variance adult-onset type II citrullinemia aromatic amino acid aspartate-glutamate carrier branched-chain amino acid citrulline citrin cyanine 3-dCTP cyanine 5-dCTP free fatty acid lactate lactate/pyruvate knock-out malate-aspartate mitochondrial glycerol-3-phosphate dehydrogenase neonatal intrahepatic cholestasis caused by citrin deficiency pyruvate triglyceride analysis of variance Citrullinemia is caused by a deficiency of the urea cycle enzyme argininosuccinate synthetase, which catalyzes the ligation of citrulline (Cit) and Asp to form argininosuccinate at the expense of ATP utilization. Saheki et al. (10Saheki T. Nakano K. Kobayashi K. Imamura Y. Itakura Y. Sase M. Hagihara S. Matuo S. J. Inherit. Metab. Dis. 1985; 8: 155-156Crossref PubMed Scopus (36) Google Scholar) originally described three clinical forms of citrullinemia based on argininosuccinate synthetase enzyme abnormalities; these forms have subsequently been re-classified into classical citrullinemia (CTLN1; OMIM™ number 215700) caused by mutations in the argininosuccinate synthetase gene (11Kobayashi K. Jackson M.J. Tick D.B. O'Brien W.E. Beaudet A.L. J. Biol. Chem. 1990; 265: 11361-11367Abstract Full Text PDF PubMed Google Scholar, 12Gao H.Z. Kobayashi K. Tabata A. Tsuge H. Iijima M. Yasuda T. Kalkanoglu H.S. Dursun A. Tokatli A. Coskun T. Trefz F.K. Skladal D. Mandel H. Seidel J. Kodama S. Shirane S. Ichida T. Makino S. Yoshino M. Kang J.H. Mizuguchi M. Barshop B.A. Fuchinoue S. Seneca S. Zeesman S. Knerr I. Rodes M. Wasant P. Yoshida I. De Meirleir L. Jalil M.A. Begum L. Horiuchi M. Katunuma N. Nakagawa S. Saheki T. Hum. Mutat. 2003; 22: 24-34Crossref PubMed Scopus (73) Google Scholar), and CTLN2 caused by mutations in SLC25A13 (3Kobayashi K. Sinasac D.S. Iijima M. Boright A.P. Begum L. Lee J.R. Yasuda T. Ikeda S. Hirano R. Terazono H. Crackower M.A. Kondo I. Tsui L.C. Scherer S.W. Saheki T. Nat. Genet. 1999; 22: 159-163Crossref PubMed Scopus (361) Google Scholar). CTLN2 is characterized by a liver-specific decrease in argininosuccinate synthetase protein (13Saheki T. Kobayashi K. Inoue I. Rev. Physiol. Biochem. Pharmacol. 1987; 108: 21-68Crossref PubMed Google Scholar) without any detectable abnormalities in the argininosuccinate synthetase gene or hepatic argininosuccinate synthetase mRNA levels (14Kobayashi K. Saheki T. Imamura Y. Noda T. Inoue I. Matuo S. Hagihara S. Nomiyama H. Jinno Y. Shimada K. Am. J. Hum. Genet. 1986; 38: 667-680PubMed Google Scholar, 15Kobayashi K. Shaheen N. Kumashiro R. Tanikawa K. O'Brien W.E. Beaudet A.L. Saheki T. Am. J. Hum. Genet. 1993; 53: 1024-1030PubMed Google Scholar). The causal link between citrin deficiency and the hepatic loss of argininosuccinate synthetase in CTLN2 patients still remains to be clarified (16Yasuda T. Yamaguchi N. Kobayashi K. Nishi I. Horinouchi H. Jalil M.A. Li M.X. Ushikai M. Iijima M. Kondo I. Saheki T. Hum. Genet. 2000; 107: 537-545Crossref PubMed Scopus (126) Google Scholar). Patients with CTLN2 suffer from recurring neuropsychiatric symptoms associated with hyperammonemia, including disorientation, delirium, seizures, and coma that can lead to death from brain edema (13Saheki T. Kobayashi K. Inoue I. Rev. Physiol. Biochem. Pharmacol. 1987; 108: 21-68Crossref PubMed Google Scholar). Laboratory findings of CTLN2 patients show moderately elevated plasma Cit and arginine (Arg) levels, an elevated plasma threonine to serine (Thr/Ser) ratio, a decreased Fischer ratio (branched-chain amino acids to aromatic amino acid ratio; BCAA/AAA) (17Saheki T. Kobayashi K. Miura T. Hashimoto S. Ueno Y. Yamasaki T. Araki H. Nara H. Shiozaki Y. Sameshima Y. Suzuki M. Yamauchi Y. Sakazume Y. Akiyama K. Yamamura Y. J. Clin. Biochem. Nutr. 1986; 1: 129-142Crossref Scopus (39) Google Scholar), an elevated serum level of pancreatic secretory trypsin inhibitor (18Kobayashi K. Horiuchi M. Saheki T. Hepatology. 1997; 25: 1160-1165Crossref PubMed Scopus (71) Google Scholar), and fatty liver (19Yagi Y. Saheki T. Imamura Y. Kobayashi K. Sase M. Nakano K. Matuo S. Inoue I. Hagihara S. Noda T. Am. J. Clin. Pathol. 1988; 89: 735-741Crossref PubMed Scopus (24) Google Scholar). The elevated pancreatic secretory trypsin inhibitor in CTLN2 patients results from its up-regulated transcriptional expression in the liver (20Kobayashi K. Nakata M. Terazono H. Shinsato T. Saheki T. FEBS Lett. 1995; 372: 69-73Crossref PubMed Scopus (27) Google Scholar). Finally, CTLN2 patients also have an increased incidence of hepatocellular carcinoma, pancreatitis, and hyperlipidemia (1Saheki T. 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Kobayashi K. Clin. Gastroenterol. Hepatol. 2007; 5: xxxiiAbstract Full Text Full Text PDF Scopus (16) Google Scholar). Clinically, one of the most distinct features of CTLN2 are reports of patients having a peculiar fondness for foods high in protein and fat such as beans and nuts, and a dislike of foods high in carbohydrates such as rice and sweets (1Saheki T. Kobayashi K. J. Hum. Genet. 2002; 47: 333-341Crossref PubMed Scopus (233) Google Scholar, 2Kobayashi K. Saheki T. Seikagaku (Japanese). 2004; 76: 1543-1559PubMed Google Scholar, 13Saheki T. Kobayashi K. Inoue I. Rev. Physiol. Biochem. Pharmacol. 1987; 108: 21-68Crossref PubMed Google Scholar, 27Saheki T. Kobayashi K. Iijima M. Moriyama M. Yazaki M. Takei Y. Ikeda S. Hepatol. Res. 2005; 33: 181-184Crossref PubMed Scopus (54) Google Scholar). Patients with CTLN1 or other urea cycle enzyme deficiencies typically are given restricted protein diets to minimize episodes of hyperammonemia resulting from protein degradation. In contrast, several CTLN2 patients have developed severe hyperammonemia or deterioration following an intravenous infusion of a high glucose solution (31Tamakawa S. Nakamura H. Katano T. Yoshizawa M. Ohtake K. Kubota T. J. Jpn. Soc. Intensive Care Med. (Japanese). 1994; 1: 37-41Crossref Google Scholar, 32Takahashi H. Kagawa T. Kobayashi K. Hirabayashi H. Yui M. Begum L. Mine T. Takagi S. Saheki T. Shinohara Y. Med. Sci. Monit. 2006; 12: CS13-CS15PubMed Google Scholar) or an administration of glycerol and fructose for the treatment of brain edema (33Yazaki M. Takei Y. Kobayashi K. Saheki T. Ikeda S. Intern. Med. 2005; 44: 188-195Crossref PubMed Scopus (54) Google Scholar). Furthermore, we have observed a citrin deficiency patient who exhibited an increased blood ammonia level following a typical hospital meal but showed no increase following a meal rich in protein and fat. 3T. Sakheki, unpublished data. 3T. Sakheki, unpublished data. Yajima et al. (34Yajima Y. Hirasawa T. Saheki T. Tohoku J. Exp. Med. 1982; 137: 213-220Crossref PubMed Scopus (9) Google Scholar) have reported previously that the blood ammonia levels of CTLN2 patients demonstrate diurnal fluctuations; the levels are usually higher in the evening following a meal and low prior to a meal. Together these observations suggest that CTLN2 patients may present with hyperammonemia as a result of dietary intake of carbohydrates, and not protein, following a meal. Identification of mutations in SLC25A13 in CTLN2 patients led to the discovery that a type of neonatal hepatitis was also caused by the same mutations (35Tazawa Y. Kobayashi K. Ohura T. Abukawa D. Nishinomiya F. Hosoda Y. Yamashita M. Nagata I. Kono Y. Yasuda T. Yamaguchi N. Saheki T. J. Pediatr. 2001; 138: 735-740Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 36Tomomasa T. Kobayashi K. Kaneko H. Shimura H. Fukusato T. Tabata M. Inoue Y. Ohwada S. Kasahara M. Morishita Y. Kimura M. Saheki T. Morikawa A. J. Pediatr. 2001; 138: 741-743Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 37Ohura T. Kobayashi K. Tazawa Y. Nishi I. Abukawa D. Sakamoto O. Iinuma K. Saheki T. Hum. Genet. 2001; 108: 87-90Crossref PubMed Scopus (118) Google Scholar). Because the neonatal symptoms were markedly different from those of adult CTLN2 patients, we named the neonatal presentation NICCD (neonatal intrahepatic cholestasis caused by citrin deficiency) (1Saheki T. Kobayashi K. J. Hum. Genet. 2002; 47: 333-341Crossref PubMed Scopus (233) Google Scholar, 38Yamaguchi N. Kobayashi K. Yasuda T. Nishi I. Iijima M. Nakagawa M. Osame M. Kondo I. Saheki T. Hum. Mutat. 2002; 19: 122-130Crossref PubMed Scopus (96) Google Scholar). Patients with NICCD show multiple metabolic abnormalities, including variable aminoacidemias (Cit, Thr, methionine, tyrosine, and Arg) accompanied by an increased Thr/Ser ratio, galactosemia, hypoproteinemia, hypoglycemia, cholestasis, and fatty liver (2Kobayashi K. Saheki T. Seikagaku (Japanese). 2004; 76: 1543-1559PubMed Google Scholar). Human citrin deficiency therefore results in NICCD during the first few months of life, with symptoms usually self-resolving by the 1st year in most cases. Following an apparently healthy period that can last from one to several decades, some patients with human citrin deficiency go on to develop severe CTLN2 (1Saheki T. Kobayashi K. J. Hum. Genet. 2002; 47: 333-341Crossref PubMed Scopus (233) Google Scholar, 36Tomomasa T. Kobayashi K. Kaneko H. Shimura H. Fukusato T. Tabata M. Inoue Y. Ohwada S. Kasahara M. Morishita Y. Kimura M. Saheki T. Morikawa A. J. Pediatr. 2001; 138: 741-743Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 39Kasahara M. Ohwada S. Takeichi T. Kaneko H. Tomomasa T. Morikawa A. Yonemura K. Asonuma K. Tanaka K. Kobayashi K. Saheki T. Takeyoshi I. Morishita Y. Transplantation. 2001; 71: 157-159Crossref PubMed Scopus (54) Google Scholar). There have been a few patients with NICCD that have required liver transplantation because of severe, prolonged symptoms (40Tamamori A. Okano Y. Ozaki H. Fujimoto A. Kajiwara M. Fukuda K. Kobayashi K. Saheki T. Tagami Y. Yamano T. Eur. J. Pediatr. 2002; 161: 609-613Crossref PubMed Scopus (91) Google Scholar, 41Nakabayashi H. Murakami H. Kitazawa E. Owada M. Jpn. J. Inherit. Metab. Dis. 2001; 17: 124Google Scholar), and patients presenting with CTLN2 typically continue to worsen unless also treated by liver transplantation (1Saheki T. Kobayashi K. J. Hum. Genet. 2002; 47: 333-341Crossref PubMed Scopus (233) Google Scholar, 8Kobayashi K. Iijima M. Yasuda T. Sinasac D.S. Yamaguchi N. Tsui L.C. Scherer S.W. Saheki T. Pochet R. Donato R. Haiech J. Heizmann C. Gerke V. Calcium: The Molecular Basis of Calcium Action in Biology and Medicine. Kluwer Academic Publishers Group, Dordrecht, Netherlands2000: 565-587Google Scholar, 23Ikeda S. Yazaki M. Takei Y. Ikegami T. Hashikura Y. Kawasaki S. Iwai M. Kobayashi K. Saheki T. J. Neurol. Neurosurg. Psychiatry. 2001; 71: 663-670Crossref PubMed Scopus (80) Google Scholar, 26Terada R. Yamamoto K. Kobayashi K. Sakaguchi K. Iwasaki Y. Saheki T. Shiratori Y. J. Gastroenterol. Hepatol. 2006; 21: 1634-1635Crossref PubMed Scopus (11) Google Scholar, 39Kasahara M. Ohwada S. Takeichi T. Kaneko H. Tomomasa T. Morikawa A. Yonemura K. Asonuma K. Tanaka K. Kobayashi K. Saheki T. Takeyoshi I. Morishita Y. Transplantation. 2001; 71: 157-159Crossref PubMed Scopus (54) Google Scholar). To fully understand the pathophysiology of human citrin deficiency and to aid in the development of novel therapeutics effective in treating NICCD and CTLN2 patients, it is critical to have an accurate animal model of human citrin deficiency. We have previously created a citrin (Ctrn) knock-out mouse (42Sinasac D.S. Moriyama M. Jalil M.A. Begum L. Li M.X. Iijima M. Horiuchi M. Robinson B.H. Kobayashi K. Saheki T. Tsui L.C. Mol. Cell. Biol. 2004; 24: 527-536Crossref PubMed Scopus (62) Google Scholar) by targeted disruption of the Slc25a13 gene. Although the resulting Ctrn-/- mice lacked Slc25a13 mRNA and citrin protein and demonstrated markedly reduced mitochondrial Asp transport and MA shuttle activity in vitro, there was no apparent phenotype in vivo. Nitrogen-loading experiments produced only minor changes in hepatic ammonia and amino acid levels in vivo. The mice however, did reveal deficits in ureogenesis from ammonia, gluconeogenesis from Lac, and an increase in the Lac to pyruvate (Pyr) (L/P) ratio during liver perfusion experiments. Mouse liver contains high mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) activity (43Williams M.T. Carrington H. Herrera A. Biochem. J. 1986; 233: 595-598Crossref PubMed Scopus (13) Google Scholar),3 a component of the glycerol phosphate shuttle that can similarly transport NADH reducing equivalents into mitochondria, whereas human liver is known to contain much lower activity (44Sadava D. Depper M. Gilbert M. Bernard B. McCabe E.R. Biol. Neonat. 1987; 52: 26-32Crossref Scopus (18) Google Scholar).3 These considerations led us to hypothesize that the higher glycerol phosphate shuttle activity in the liver of the Ctrn-/- mice may be sufficient to compensate for any decreases in MA shuttle activity resulting from the loss of citrin function. To test this hypothesis, we crossed mGPD-KO mice with Ctrn-KO mice. The resultant Ctrn/mGPD double knock-out (Ctrn-/-, mGPD-/-) mice revealed hyperammonemia under fed conditions, citrullinemia and hypoglycemia under fed and fasted conditions, and an altered Thr/Ser ratio and fatty liver under fasted conditions. Moreover, the hyperammonemia was enhanced by the administration of sucrose, with the rise in ammonia being closely associated with an increased L/P ratio. Finally, microarray gene expression analyses using liver RNA identified more than 100 genes that were differentially expressed in the double knock-out mice compared with wild-type controls, only a portion of which were also differentially expressed in either of the Ctrn-KO or mGPD-KO mice. These results support our contention that compensatory mechanisms in mouse are responsible for the observed differences in citrin deficiency between humans and mice, and that the more severe phenotype present in the double knock-out mice represent a more accurate model of human citrin deficiency. Animals—Mice homozygous for the targeted disruption of the Slc25a13 gene that encodes citrin (Ctrn-/- or Ctrn-KO) and for the mGPD gene that encodes mGPD (mGPD-/- or mGPD-KO) were generated as described previously (42Sinasac D.S. Moriyama M. Jalil M.A. Begum L. Li M.X. Iijima M. Horiuchi M. Robinson B.H. Kobayashi K. Saheki T. Tsui L.C. Mol. Cell. Biol. 2004; 24: 527-536Crossref PubMed Scopus (62) Google Scholar, 45Eto K. Tsubamoto Y. Terauchi Y. Sugiyama T. Kishimoto T. Takahashi N. Yamauchi N. Kubota N. Murayama S. Aizawa T. Akanuma Y. Aizawa S. Kasai H. Yazaki Y. Kadowaki T. Science. 1999; 283: 981-985Crossref PubMed Scopus (393) Google Scholar). Both KO mutations were made congenic on the C57BL/6J genetic background by backcrossing for at least nine generations. The two KO strains were intercrossed to generate mice heterozygous for both mutations (Ctrn+/-, mGPD+/-), which in turn were intercrossed to obtain mice that were heterozygous for citrin and homozygous for mGPD (Ctrn+/-, mGPD-/-). These mice were then intercrossed to generate single mGPD-KO (Ctrn+/+, mGPD-/-) and double knock-out (Ctrn-/-, mGPD-/-) littermates. Single Ctrn-KO (Ctrn-/-, mGPD+/+) and C57BL/6J wild-type mice were also generate as littermates by intercrossing mice heterozygous for the Ctrn-KO mutation. Genotyping was performed on DNA extracted from ear punch using procedures specific for each of the targeted disruptions made in the Ctrn-KO and mGPD-KO mice as described previously (42Sinasac D.S. Moriyama M. Jalil M.A. Begum L. Li M.X. Iijima M. Horiuchi M. Robinson B.H. Kobayashi K. Saheki T. Tsui L.C. Mol. Cell. Biol. 2004; 24: 527-536Crossref PubMed Scopus (62) Google Scholar, 45Eto K. Tsubamoto Y. Terauchi Y. Sugiyama T. Kishimoto T. Takahashi N. Yamauchi N. Kubota N. Murayama S. Aizawa T. Akanuma Y. Aizawa S. Kasai H. Yazaki Y. Kadowaki T. Science. 1999; 283: 981-985Crossref PubMed Scopus (393) Google Scholar). Animal Treatment—All mice were maintained at constant temperature (22 ± 1 °C) on a 12-h light/12-h dark cycle (7 a.m. to 7 p.m.) with free access to water and CE2 chow (24.9% protein, 4.6% fat, and 51.4% carbohydrate providing 346.8 kcal/100g; CLEA Japan, Tokyo, Japan). Mice used for experiments were analyzed between 80 and 140 days of age. In each experiment, both genders were analyzed, and the results were pooled within each genotype if no differences between genders were observed. A sucrose solution (50% w/v of water; 10 g/kg body weight) was administered per os using a gastric tube to fed mice between 9 and 10 a.m. For fasting experiments, food was withdrawn at 5 p.m. on the day prior to performed experiments. This study was approved by the Ethical Committee for Animal Experimentation at Kagoshima University. Determination of Plasma Parameters—Blood was drawn from the mice via cardiac puncture while under general anesthesia (intraperitoneal injection of pentobarbital, 50 mg/kg body weight) between 10 and 12 a.m. Plasma glucose and blood ammonia concentrations were assayed using glucose CII test and ammonia tests (Wako kits, Wako Pure Chemical Industries, Osaka, Japan), respectively, and plasma insulin concentration, using mouse insulin enzyme-linked immunosorbent assay (o-phenylenediamine) (AKRIN-011, Shibayagi, Gunma, Japan). Plasma amino acid concentrations were determined with a JLC-500 model amino acid analyzer (JEOL, Tokyo, Japan) after deproteinization with 3% sulfosalicylic acid. Plasma triglycerides (TG; TG-II Kainos; Kainos Laboratories, Inc., Tokyo, Japan), free fatty acids (FFA; Determiner-NEFA; Kyowa Medix Co. Ltd., Tokyo, Japan), total cholesterol (Cholesterol E-test Wako), glycerol (GLY 105; Randox Laboratories, Antrim, UK), ketone bodies (Ketone Test Sanwa; Sanwa Kagaku Kenkyusho, Nagoya, Japan), bile acid (Bile Acid Test Wako), Asp aminotransferase (TA-LN Kainos), alanine aminotransferase (TA-LN Kainos), and alkaline phosphatase (K-test Wako) were determined with commercially available kits as indicated, respectively. Determination of Urea Cycle Enzyme Activities—Liver extracts were prepared as described previously (46Saheki T. Ueda A. Hosoya M. Kusumi K. Takada S. Tsuda M. Katsunuma T. Clin. Chim. Acta. 1981; 109: 325-335Crossref PubMed Scopus (82) Google Scholar). The urea cycle enzyme activities were determined using the methods of Schimke (47Schimke R.T. J. Biol. Chem. 1962; 237: 459-468Abstract Full Text PDF PubMed Google Scholar) for carbamoyl-phosphate synthetase, Pierson et al. (48Pierson D.L. Cox S.L. Gilbert B.E. J. Biol. 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For the determination of hepatic Asp, a portion of liver was excised following blood withdrawal, quickly freeze-clamped, and homogenized with 20× volume of 2% (w/v) trichloroacetic acid after pulverization under liquid nitrogen. The resultant extract was diluted with an eluent and subjected to reversed-phase column chromatography with an ion pair reagent
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