Nonalcoholic steatosis (NAFLD) and steatohepatitis (NASH) may accompany a variety of clinical disorders including parenteral nutrition, nutritional protein deficiency, congenital metabolic disorders, jejuno-ileal bypass, chronic inflammatory bowel disease, and chronic chemical intoxication [10
]. A number of animal models of NAFLD and NASH have been described. Many of these involve abnormalities of the hepatic transmethylation and transsulfuration pathways including mice deficient for the enzymes methionine adenosyltransferase 1A (MAT1A), methylene tetrahydrofolate reductase (MTHFR), and phosphatidylethanolamine N-methyltransferase (PEMT) [36
]. Methionine (Meth), a sulfur amino acid, serves as a precursor for the biosynthesis of cysteine and GSH through the trans-sulfuration pathway; however, the reverse reaction does not occur, making Meth of critical importance [37
]. Consumption of Meth choline deficient (MCD) diet provokes steatohepatitis. This model mimics nutritional deficiency that produces lesions similar to the histological and biochemical lesion caused by NASH.
In the present study, we explored the mechanistic action for Meth deficiency in the development of hepatic injury in this model. Young rats fed amino-acid-based MCD for two (MCS/MCD) to five consecutive weeks (MCD) lost, respectively, 30% and 65% of their bodyweight, but appeared otherwise normal. This weight loss was attributed predominately to the choline deficiency, since Meth replacement only partially improved this abnormality. Weight loss has been reported previously in rats fed a high-fat Meth-restricted choline-deficient (MCD) diet (not amino acid) after 12 weeks [38
]. In the current study, rats developed severe hepatic lesions after two weeks as evidenced by fatty degeneration (steatosis) and inflammation (MCS/MCD score: 2.8 ± 0.5) that progressed to necrosis after five weeks on the deficient diet (MCD score: 3.6 ± 0.5, P
< 0.001). MCD rats showed significant hepatic injury without exogenous lipopolysaccharide (LPS) challenge as purportedly required in previous studies [35
]. These changes were accompanied by a marked elevation in serum aspartate transaminase (AST, 5.8-fold), and alanine transaminase (ALT, 3.2-fold) enzyme activity as early as after two weeks on deficient diet. Hepatic enzymes normalized and histological damage partially but significantly improved with Meth supplementation (Meth/MCD versus MCD, P
In the current study, there was a significant overexpression of inflammatory genes, such as TNF-α (7.7- fold), IL-6 (17-fold), IL-1β (7-fold), TGF-β (3-fold), and SOCS1 (2.4-fold) in hepatic tissue from rats on deficient diets. Similarly, genes involved in the structure of extracellular matrix, MMP-9 (gelatinase-B, 8.5-fold), MMP-13 (4-fold), collagen-α1 (9.2-fold), were upregulated. Meth administration significantly attenuated the upregulation of these deleterious genes.
Meth is readily oxidized by the reactive oxygen species (ROS) [39
]. Meth deficiency results in GSH depletion, which in turn promotes oxidative damage by cellular constituents, including amino acids, lipids, and nucleic acids, and contributes to tissue destruction in a wide variety of diseases, including steatohepatitis [40
]. GSH is a crucial cellular antioxidant that neutralizes free radicals, and adequate protein nutrition is necessary for the maintenance of GSH homeostasis. In addition, oral administration of methionine and 2(RS)-n-propylthiazoli-dine-4(R)-carboxylic acid (PTCA) are effective precursors of cysteine for tissue GSH synthesis in animal models for inflammatory complications such as overdose acetaminophen hepatotoxicity [42
], dextran sodium sulfate (DSS)-induced colitis [43
], and steatohepatitis [45
]. GSH is depleted during illness and, as illustrated in the present investigation, animals fed the MCD diet developed marked depletion of hepatic and blood reduced GSH (35%) when compared to those fed the sufficient diet (MCS). However, Meth supplementation and cysteine GSH pro-drug treatments circumvented this deficiency. SAMe deficiency is often associated with GSH deficiency, [35
] since SAMe is a precursor for hepatic GSH. Endogenous liver SAMe is also a substrate for de novo biosynthesis of choline. In this study, animals fed an MCD diet developed GSH depletion that accompanied by a reduction in endogenous hepatic SAMe concentrations. Meth replacement normalized reduced blood and hepatic GSH storages caused by MCD diet, and partially improved the hepatic SAMe concentrations.
Previous reports have also demonstrated significantly lower hepatic concentrations of both SAMe and choline after two weeks on Meth-restricted choline-deficient diet [35
]. Daily IM injection with SAMe restored hepatic concentrations of SAMe but did not affect hepatic choline levels [35
], suggesting that dietary supplementation of choline is necessary to normalize hepatic choline concentrations in these animals. We recently reported that oral administration of SAMe for two consecutive weeks partially but significantly restored hepatic SAMe after induction of hepatic injury in MCD rats [45
]. Hepatic SAMe deficiency is associated with increased susceptibility to LPS-induced hepatotoxicity and increased serum TNF-α
]. Similarly, acetaminophen toxicity is associated with hepatic SAMe deficiency and increased serum TNF-α
levels. In addition, oral administration of SAMe has proven beneficial in acetaminophen-induced hepatotoxicity in mice [42
] as well as in alcohol-induced liver injury [46
], by improving GSH levels and decreasing TNF-α
The importance of steatosis as a sensitizer to a drug was also demonstrated in a recent work [48
]. Male Sprague-Dawley rats were fed a methionine- and choline-deficient diet for 31 days to induce steatosis. On day 32, administration of a nonlethal dose of carbon tetrachloride (CCl4—2 ml/kg, intraperitoneally) yielded 70% mortality in steatotic rats 12–72 h after CCl4 administration, whereas all nonsteatotic rats survived.
Miele et al. clearly established the role of dietary deficiencies in the formation and progression of liver damage in non-alcoholic steatohepatitis in humans [49
]. The present study demonstrates that Meth deficiency in a dietary model causes major hepatic pathology and inflammatory/fibrotic gene overexpression as early as two weeks after consumption of an MCD diet. The abnormalities were largely reversible by Meth replacement therapy. These data support the pivotal role played by Meth in the pathogenesis and expression of inflammatory/fibrogenic genes as early as two weeks in this dietary model for steatohepatitis.