Methionine adenosyltransferase 1A knockout mice are predisposed to liver injury and exhibit increased expression of genes involved in proliferation

Abstract

Liver-specific and nonliver-specific methionine adenosyltransferases (MATs) are products of two genes, MAT1A and MAT2A, respectively, that catalyze the formation of S-adenosylmethionine (AdoMet), the principal biological methyl donor. Mature liver expresses MAT1A, whereas MAT2A is expressed in extrahepatic tissues and is induced during liver growth and dedifferentiation. To examine the influence of MAT1A on hepatic growth, we studied the effects of a targeted disruption of the murine MAT1A gene. MAT1A mRNA and protein levels were absent in homozygous knockout mice. At 3 months, plasma methionine level increased 776% in knockouts. Hepatic AdoMet and glutathione levels were reduced by 74 and 40%, respectively, whereas S-adenosylhomocysteine, methylthioadenosine, and global DNA methylation were unchanged. The body weight of 3-month-old knockout mice was unchanged from wild-type littermates, but the liver weight was increased 40%. The Affymetrix genechip system and Northern and Western blot analyses were used to analyze differential expression of genes. The expression of many acute phase-response and inflammatory markers, including orosomucoid, amyloid, metallothionein, Fas antigen, and growth-related genes, including early growth response 1 and proliferating cell nuclear antigen, is increased in the knockout animal. At 3 months, knockout mice are more susceptible to choline-deficient diet-induced fatty liver. At 8 months, knockout mice developed spontaneous macrovesicular steatosis and predominantly periportal mononuclear cell infiltration. Thus, absence of MAT1A resulted in a liver that is more susceptible to injury, expresses markers of an acute phase response, and displays increased proliferation.

Figure 1

(A) Structure of the mouse MAT1A gene, targeting construct, and after homologous recombination. (B) Southern blot analysis of tail genomic DNA from offspring of a heterozygous mating.

Figure 2

(A) Northern blot analysis of MAT1A, MAT2A, and AFP in MAT1A knockout and wild-type littermates. Liver RNA (30 μg each lane) samples obtained from two wild-type and two MAT1A knockout mice were analyzed by Northern blot hybridization with a ³²P-labeled MAT1A cDNA probe, as described in Methods. The same membrane was then sequentially rehybridized with ³²P-labeled MAT2A, AFP, and 18S rRNA cDNA probes. A representative Northern blot is shown. (B) Hepatic steady-state liver-specific MAT protein level in MAT1A knockout and wild-type littermates. Liver cytosol (50 μg/lane) obtained from three wild-type and three MAT1A knockout mice was analyzed by Western blot analysis by using anti-rat liver-specific MAT antibodies, as described in Methods. Equivalent protein loading was assured by Coomassie-blue staining of gels after transblotting (Lower).

Figure 3

Expression of BHMT, CBS, PEMT2, SAHH, and GNMT in the MAT1A knockout mouse. Liver RNA (30 μg each lane) samples obtained from four wild-type and four MAT1A knockout mice were analyzed by Northern blot hybridization with specific cDNA probes, as described in Methods. The same membranes were then rehybridized with ³²P-labeled 18S rRNA cDNA probe to ensure equal loading. Representative Northern blots are shown.

Figure 4

Effect of CD diet on liver histology in the MAT1A knockout mouse. Trichrome-stained sections of representative liver samples of wild-type (A) and MAT1A knockout mice (B) fed a CD diet for 6 days. Minimal steatosis or no change was seen in the livers of the wild-type littermates (A), whereas severe macrovesicular steatosis was observed in more than 75% of hepatocytes in the knockout mice (B) (×100). Representative histologic changes are shown from four animals.

Figure 5

Liver histology from 8-month-old MAT1A knockout mice fed a normal diet. (Upper) Wild type. The portal tract demonstrates a portal vein and two small duct structures. No portal or lobular inflammation or lobular fatty change is seen. Hematoxylin and eosin × 148. (Lower) MAT1A knockout mouse. The portal tract at the left of the field (arrow) shows mild portal lymphocytic infiltrates with an unremarkable bile duct. The hepatocytes adjacent to the portal tract show macrovesicular fatty change. In addition, focal areas of inflammation are also seen involving the liver cells that contain fat (steatohepatitis); the inflammatory component is chiefly lymphocytic. Hematoxylin and eosin × 148. Representative histologic changes are shown from three knockout mice.