| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 7, 2002 as doi:10.1096/fj.02-0078fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Division of Hepatology and Gene Therapy, Department of Medicine, School of Medicine, University of Navarra, Pamplona, Spain;
* Division of Gastroenterology and Liver Diseases, USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases, USC Liver Disease Research Center, USC School of Medicine, Los Angeles, California, USA; and
Department of Pathology, Rancho Los Amigos,
*, Keck School of Medicine USC, Los Angeles, California, USA
5Correspondence: Division of Hepatology & Gene Therapy, Edificio Los Castanos, Facultad de Medicina, Universidad de Navarra, 31008 Pamplona, Spain. E-mail: jmmato{at}unav.es; Division of GI & Liver Diseases, HMR Bldg., 415, Dept. of Medicine, Keck School of Medicine USC, 2011 Zonal Ave., Los Angeles, CA 90033, USA. E-mail: shellylu{at}hsc.usc.edu
SPECIFIC AIMS
In mammals, two genes, MAT1A and MAT2A, encode for methionine adenosyltransferase (MAT), which catalyzes formation of S-adenosylmethionine (AdoMet), the principal biological methyl donor and precursor for polyamines. We have shown that MAT1A knockout (referred to as MATO) mice have markedly lower hepatic AdoMet levels, hepatic hyperplasia, and develop spontaneous steatohepatitis. The aim of the current study was to examine the mechanisms and consequences of some of these changes.
PRINCIPAL FINDINGS
1. Chronic hepatic AdoMet deficiency led to an aberrant gene expression profile reminiscent of diabetes, obesity, and other conditions associated with steatohepatitis
Absence of hepatic MAT1A resulted in chronic hepatic AdoMet deficiency. To examine the consequence of this on differential hepatic gene expression profiles, we analyzed results obtained using oligonucleotide microarrays from 3-month-old wild-type (WT) and MATO mice according to the biological processes in which they participate. Most genes up-regulated in MATO mouse liver clustered into four biological processes: cell communication, cell growth and/or maintenance, cell death, and development. Most genes that were down-regulated in MATO mouse were involved in metabolism. Some of the genes implicated in hepatocyte differentiation and proliferation that were up-regulated in MATO mice include 
-fetoprotein and MAT2A, which are markers of differentiation, and proliferating cell nuclear antigen, peroxisome proliferator activator receptor 
, and early growth response 1, markers of hepatocyte proliferation. Likewise, altered gene expression was observed in a variety of genes known to be involved in acute-phase response and oxidative stress. For example, orosomucoid, metallothioneins 1 and 2, myeloperoxidase, lipopolysaccharide binding protein, CD14, and Fas antigen were up-regulated whereas mitochondrial ribosomal protein S12, CYP4A10, and CYP4A14 were down-regulated in MATO mice. The expression of numerous genes involved in lipid and carbohydrate metabolism was altered in MATO mice liver. Glucose-6-phosphate dehydrogenase, phospholipid transfer protein, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase, glycerol kinase, and phosphoglycerate dehydrogenase were up-regulated in MATO mice.
Consistent with this abnormal expression of genes involved in lipid and carbohydrate metabolism, MATO mice had elevated hepatic TGL levels (26.4±4.2 mg/g wet liver in WT mice vs. 114.5±33.1 mg/g wet liver in MATO mice, P<0.05) and hyperglycemia (8.17±0.72 mmol/L glucose in WT mice vs. 11.06±0.97 mmol/L in MATO mice, P<0.05) although the circulating levels of insulin were normal (0.99 ng/mL insulin in WT vs. 0.92 ng/mL in MATO mice).
2. Hepatic AdoMet deficiency led to spontaneous oxidative stress and increased expression of cytochrome P450 2E1 (CYP2E1) and uncoupling protein 2 (UCP2)
Since CYP2E1 is up-regulated in diabetes, obesity, and steatohepatitis, we tested the hypothesis that CYP2E1 was also increased in MATO mouse. We observed that liver CYP2E1 mRNA and activity were induced in 3-month-old MATO mice with respect to WT animals (Fig. 1
a, b) and that when knockout mice were treated with diallyl sulfide (DAS), an effective inhibitor of CYP2E1, hepatic CYP2E1 activity was decreased (Fig. 1b
). We also determined the expression of UCP2, an anion carrier that uncouples the respiratory chain from oxidative phosphorylation and hence might enhance the vulnerability of hepatocytes to necrosis. Although hepatocytes do not normally express uncoupling proteins, lipids up-regulate UCP2 expression in hepatocytes and obesity induces expression of UCP2 in hepatocytes and promotes liver ATP depletion. We observed that the hepatic content of UCP2 mRNA was markedly induced in MATO mice with respect to WT animals (Fig. 1a
).
|
Consistent with increased expression of CYP2E1 and UCP2, the serum concentration of malondialdehyde, a measure of lipid peroxidation, was higher in MATO mice than in WT animals. GSH synthetic enzymes are known to be induced under oxidative stress. Indeed, we observed that the hepatic mRNA levels of several key enzymes of the trans-sulfuration and GSH synthetic pathways—cystathione ß synthase (CBS), glutamate-cysteine ligase (GCL) subunits, and GSH synthetase—were all increased in MATO mice compared with WT animals (Fig. 1a
). Despite this increase in the expression of genes involved in GSH synthesis, hepatic content of GSH was reduced by 
40% in knockout mice vs. WT animals.
3. Three-month-old MATO mice are predisposed to carbon tetrachloride (CCl4)-induced hepatotoxicity due to increased expression and activity of CYP2E1
To examine whether the increased CYP2E1 activity in MATO mice predisposes to toxicity, we examined the effect of CCl4, a hepatotoxic molecule biotransformed by CYP2E1. Histological analysis revealed that liver injury caused by CCl4 was much more severe in MATO mice than in WT animals and that DAS administration prevented CCl4-induced liver injury in MATO mice. As previously demonstrated, DAS treatment also ameliorated CCl4-induced liver injury in WT mice. Liver injury was evaluated also by measuring blood alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Baseline blood AST and ALT levels were similar in knockout and WT mice, but the elevation of blood AST and ALT levels in response to CCl4 was much higher in MATO mice than in WT animals. DAS pretreatment markedly reduced the elevation of AST and ALT levels caused by CCl4 in knockout mice. DAS administration also protected CCl4-induced AST and ALT elevation in WT mice.
4. Chronic hepatic AdoMet deficiency led to spontaneous generation of hepatocellular carcinoma (HCC)
We examined the consequence of chronic hepatic AdoMet deficiency on hepatic tumorigenesis. By 18 months of age, five (two male and three female) of eight MATO mice (four male and four female) spontaneously developed HCC. The tumor is multifocal in two animals but unifocal in the other three. The tumor is composed of hepatocytes with round to oval nuclei and prominent nucleoli, forming thickened trabecular cords lined by endothelial cells. Some of the tumor cells contain fat (Fig. 2
). Although the other three animals did not have HCC, their livers contained cystic and inflammatory changes.
|
CONCLUSIONS AND SIGNIFICANCE
MAT1A encodes a catalytic subunit that organizes into dimers, MAT III, and tetramers, MAT I; MAT2A encodes a catalytic subunit that associates to form MAT II. Whereas the combination of MAT I/III provides the hepatocyte with a set of enzymes capable of efficiently metabolize methionine without being inhibited by the cellular accumulation of AdoMet, MAT II is strongly inhibited by µmol/L concentrations of AdoMet. Consequently, though the disruption of MAT1A in MATO mouse leads to an increased expression of MAT2A, the capacity of the knockout mice liver to metabolize methionine is markedly diminished, and this leads to hypermethioninemia and reduced hepatic AdoMet content. Thus, the MATO mouse is an excellent model with which to examine the consequence of chronic hepatic AdoMet deficiency, a situation that likely occurs in patients with advanced liver disease, where hepatic MAT activity falls and MAT1A expression is decreased.
Analysis of the differential gene expression profiles of knockout and WT mice suggests that MATO mice have abnormal lipid and carbohydrate metabolism. This was corroborated by the finding that MATO mice have elevated liver TGL levels and hyperglycemia. These alterations preceded any histological sign of steatosis in the knockout mice. CYP2E1 and UCP2 are known to be up-regulated by aberrant nutritional conditions such as diabetes and obesity. Thus, abnormal metabolism rather than a direct effect of AdoMet may be the trigger of the increased CYP2E1 and UCP2 expression in MATO mice. Consistent with this, hepatic CYP2E1 mRNA content was unchanged in 2-wk-old MATO mice; exogenous AdoMet did not affect CYP2E1 expression in primary hepatocytes from 3-month-old MATO mice but, as previously demonstrated, reduced MAT2A expression.
Consistent with this increase in CYP2E1 activity, knockout mice were more prone to develop liver injury in response to CCl4. Microarray data revealed that hepatic mRNA levels of CYP4A10 and CYP4A14 were reduced in MATO mice. These results agree with recent data indicating that the metabolic roles of CYP2E1 and CYP4A in lipid oxidation may be complementary and the regulation of their expression coordinated. Thus, in CYP2E1 null mice, the hepatic expression of CYP4A genes is up-regulated; in obese diabetic ob/ob mice and fa/fa Zucker rats, in which CYP2E1 is down-regulated, CYP4A genes are up-regulated.
We also observed an increase in total lipid peroxides in serum and a marked decrease of hepatic GSH, proof that a deficiency in AdoMet generates hepatic oxidative stress in knockout mice. Hepatic GSH levels decreased in MATO mice although the hepatic mRNA levels of several enzymes involved in cysteine and GSH synthesis were markedly increased. Whereas the up-regulation of antioxidant genes may reflect a general adaptive mechanism to dissipate oxidative stress generated by oxidant genes such as CYP2E1, UCP2, etc., the reduction in GSH content, despite the induction of its synthetic enzymes, may reflect the inability of the adaptive response to cope with the level of oxidative stress and channeling of AdoMet away from trans-sulfuration for cysteine synthesis, the rate-limiting precursor for GSH, due to its markedly depleted level.
Another important finding of this study was the observation that knockout mice spontaneously develop liver tumors. The induction in MATO mice of HCC strongly implies that MAT1A helps to prevent liver cancer through its capacity to maintain high AdoMet levels. This interpretation is consistent with our earlier work showing that in hepatocarcinoma HuH-7 cells differing only in the type of MAT gene that is expressed, MAT2A expression associates with more rapid cell growth whereas the opposite is observed for MAT1A. Cells expressing MAT2A had lower AdoMet levels than cells expressing MAT1A, and treatment of HuH-7 cells with AdoMet led to reduced cell growth. AdoMet therapy has been shown to be effective in preventing the growth of rat HCC and we have found that addition of AdoMet to hepatocytes markedly inhibits HGF mitogenic activity. AdoMet accelerated the resynthesis of I
B and blunted the activation of NF-B in cytokine-stimulated hepatocytes.
Taken together, these results suggest a broad scenario in which hepatocytes are kept in a proliferative, immature state in response to a chronic deficiency in AdoMet. These immature hepatocytes have an impaired metabolism of lipids and carbohydrates reminiscent of that found in diabetes, obesity, and other conditions associated with steatohepatitis, which leads to the development of HCC. Further studies are required to clarify the mechanism by which a chronic deficiency in AdoMet generates oxidative stress, particularly since impaired AdoMet synthesis is a common feature in human liver cirrhosis and this information may identify new targets for designing therapy of this disease. Figure 3
summarizes the main findings of this work.
|
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0078fje; to cite this article, use FASEB J. (June 7, 2002) 10.1096/fj.02-0078fje 
2 This paper is dedicated to Theo M. Konijn for his teachings and inspiration.
3 These authors contributed equally to this work.
4 These authors share senior authorship.
This article has been cited by other articles:
|
|
 |
|
  J. T. Dever and A. A. Elfarra L-Methionine Toxicity in Freshly Isolated Mouse Hepatocytes Is Gender-Dependent and Mediated in Part by Transamination J. Pharmacol. Exp. Ther., September 1, 2008; 326(3): 809 - 817. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Sun, B. Xing, Y. Sun, X. Du, M. Lu, C. Hao, Z. Lu, W. Mi, S. Wu, H. Wei, et al. Proteome Analysis of Hepatocellular Carcinoma by Two-dimensional Difference Gel Electrophoresis: Novel Protein Markers in Hepatocellular Carcinoma Tissues Mol. Cell. Proteomics, October 1, 2007; 6(10): 1798 - 1808. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Purohit, M. F Abdelmalek, S. Barve, N. J Benevenga, C. H Halsted, N. Kaplowitz, K. K Kharbanda, Q.-Y. Liu, S. C Lu, C. J McClain, et al. Role of S-adenosylmethionine, folate, and betaine in the treatment of alcoholic liver disease: summary of a symposium Am. J. Clinical Nutrition, July 1, 2007; 86(1): 14 - 24. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Petrak, D. Myslivcova, P. Man, R. Cmejla, J. Cmejlova, D. Vyoral, M. Elleder, and C. D. Vulpe Proteomic analysis of hepatic iron overload in mice suggests dysregulation of urea cycle, impairment of fatty acid oxidation, and changes in the methylation cycle Am J Physiol Gastrointest Liver Physiol, June 1, 2007; 292(6): G1490 - G1498. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Bailey, G. Robinson, A. Pinner, L. Chamlee, E. Ulasova, M. Pompilius, G. P. Page, D. Chhieng, N. Jhala, A. Landar, et al. S-adenosylmethionine prevents chronic alcohol-induced mitochondrial dysfunction in the rat liver Am J Physiol Gastrointest Liver Physiol, November 1, 2006; 291(5): G857 - G867. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Sahai, X. Pan, R. Paul, P. Malladi, R. Kohli, and P. F. Whitington Roles of phosphatidylinositol 3-kinase and osteopontin in steatosis and aminotransferase release by hepatocytes treated with methionine-choline-deficient medium Am J Physiol Gastrointest Liver Physiol, July 1, 2006; 291(1): G55 - G62. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Olivero, T. Ruggiero, S. Saviozzi, A. Rasola, N. Coltella, S. Crispi, F. Di Cunto, R. Calogero, and M. F. Di Renzo Genes regulated by hepatocyte growth factor as targets to sensitize ovarian cancer cells to cisplatin Mol. Cancer Ther., May 1, 2006; 5(5): 1126 - 1135. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Prudova, Z. Bauman, A. Braun, V. Vitvitsky, S. C. Lu, and R. Banerjee S-adenosylmethionine stabilizes cystathionine beta-synthase and modulates redox capacity PNAS, April 25, 2006; 103(17): 6489 - 6494. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Wu and A. I. Cederbaum Opposite action of S-adenosyl methionine and its metabolites on CYP2E1-mediated toxicity in pyrazole-induced rat hepatocytes and HepG2 E47 cells Am J Physiol Gastrointest Liver Physiol, April 1, 2006; 290(4): G674 - G684. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Berasain, E. R. Garcia-Trevijano, J. Castillo, E. Erroba, M. Santamaria, D. C. Lee, J. Prieto, and M. A. Avila Novel Role for Amphiregulin in Protection from Liver Injury J. Biol. Chem., May 13, 2005; 280(19): 19012 - 19020. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A M Diehl, Z P Li, H Z Lin, and S Q Yang Cytokines and the pathogenesis of non-alcoholic steatohepatitis Gut, February 1, 2005; 54(2): 303 - 306. [Full Text] [PDF]
|
|
|
|
|
|
A. Sahai, P. Malladi, X. Pan, R. Paul, H. Melin-Aldana, R. M. Green, and P. F. Whitington Obese and diabetic db/db mice develop marked liver fibrosis in a model of nonalcoholic steatohepatitis: role of short-form leptin receptors and osteopontin Am J Physiol Gastrointest Liver Physiol, November 1, 2004; 287(5): G1035 - G1043. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Gao, C. Wei, L. Chen, J. Huang, S. Yang, and A. M. Diehl Oxidative DNA damage and DNA repair enzyme expression are inversely related in murine models of fatty liver disease Am J Physiol Gastrointest Liver Physiol, November 1, 2004; 287(5): G1070 - G1077. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Sahai, P. Malladi, H. Melin-Aldana, R. M. Green, and P. F. Whitington Upregulation of osteopontin expression is involved in the development of nonalcoholic steatohepatitis in a dietary murine model Am J Physiol Gastrointest Liver Physiol, July 1, 2004; 287(1): G264 - G273. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Yang, M. R. Sadda, V. Yu, Y. Zeng, T. D. Lee, X. Ou, L. Chen, and S. C. Lu Induction of Human Methionine Adenosyltransferase 2A Expression by Tumor Necrosis Factor {alpha}: ROLE OF NF-{kappa}B AND AP-1 J. Biol. Chem., December 19, 2003; 278(51): 50887 - 50896. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Detich, S. Hamm, G. Just, J. D. Knox, and M. Szyf The Methyl Donor S-Adenosylmethionine Inhibits Active Demethylation of DNA: A CANDIDATE NOVEL MECHANISM FOR THE PHARMACOLOGICAL EFFECTS OF S-ADENOSYLMETHIONINE J. Biol. Chem., May 30, 2003; 278(23): 20812 - 20820. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. L. Martinez-Chantar, M. U. Latasa, M. Varela-Rey, S. C. Lu, E. R. Garcia-Trevijano, J. M. Mato, and M. A. Avila L-Methionine Availability Regulates Expression of the Methionine Adenosyltransferase 2A Gene in Human Hepatocarcinoma Cells: ROLE OF S-ADENOSYLMETHIONINE J. Biol. Chem., May 23, 2003; 278(22): 19885 - 19890. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Santamaria, M. A. Avila, M. U. Latasa, A. Rubio, A. Martin-Duce, S. C. Lu, J. M. Mato, and F. J. Corrales From the Cover: Functional proteomics of nonalcoholic steatohepatitis: Mitochondrial proteins as targets of S-adenosylmethionine PNAS, March 18, 2003; 100(6): 3065 - 3070. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
