The transition from fatty liver to NASH associates with SAMe depletion in db/db mice fed a methionine choline-deficient diet

Abstract

Nonalcoholic fatty liver disease (NAFLD) is highly prevalent in the Western population. By mechanisms that are not completely understood, this disease may progress to nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). db/db mice spontaneously develop hepatic steatosis, which progresses to NASH when these mice are fed a methionine choline-deficient (MCD) diet. The goal of our studies was to identify lipid and methionine metabolism pathways affected by MCD feeding to determine potential causal events leading to the development of NASH from benign steatosis. db/db mice fed the MCD diet for 2 weeks exhibited signs of incipient NASH development such as upregulated cytokines and chemokines. At this time point, MCD diet feeding caused S-adenosylmethionine (SAMe) depletion in db/db mice, while wild-type mice on the same diet retained hepatic SAMe levels. SAMe depletion exerts pleiotropic effects upon liver homeostasis and is commonly associated with a variety of liver insults such as thioacetamide, CCL4, and alcohol treatment; thus, SAMe depletion may serve as the second hit in NASH development. It is possible that differences in hepatic lipid and/or methionine metabolism between wild-type and db/db mice underlay the differential maintenance of SAMe levels during methionine and choline restriction. Indeed, db/db mice exhibited inhibited lipid oxidation pathways, which may be a priming factor for NASH development, and db/db mice fed the MCD diet had differential methionine adenosyltransferase (MAT) expression. The occurrence of SAMe depletion at this early, benign stage of NASH development in db/db mice with fatty liver suggests that SAMe supplementation may be (A) targeted to individuals susceptible to NASH (i.e., NAFLD patients) and (B) preventative of NASH before substantial liver injury has occurred.

Fig. 1

Liver histology of wild-type and db/db mice fed the MCD diet. Wild-type (a and b) or db/db (c and d) mice were fed either control diets (a and c) or the MCD diet for 2 weeks (b and d). Liver sections were stained with hematoxylin-eosin to assess morphology. Micrographs are representative of five mice of each group

Fig. 2

Hepatic TNFα, IL-1β, and MIP-2 mRNA levels in wild-type and db/db mice fed the MCD diet. Wild-type (□) or db/db (■) mice were fed either a control diet or the MCD diet for 2 weeks. mRNA levels of (a) TNFα, (b) IL-1β, and (c) MIP-2 was quantified relative to GAPDH as described in the Materials and Methods section. Data represent mean ± SD (n = 5). ^*P<0.05 compared to wild-type mice fed the same diet, #P<0.05 compared to control diet-fed mice of the corresponding genotype

Fig. 3

Hepatic TGFβ, α-SMA, and collagen 1α mRNA levels in wild-type and db/db mice fed the MCD diet. Wild-type (□) or db/db (■) mice were fed either a control diet or the MCD diet for 2 weeks. mRNA levels of (a) TGFβ, (b) α-SMA, and (c) collagen 1α was quantified relative to GAPDH as described in the Materials and Methods section. Data represent mean ± SD (n = 5). ^*P<0.05 compared to wild-type mice fed the same diet, ^#P<0.05 compared to control-diet-fed mice of the corresponding genotype

Fig. 4

Hepatic FAS and SCD-1 mRNA levels in wild-type and db/db mice fed the MCD diet. Wild-type (□) or db/db (■) mice were fed either a control diet or the MCD diet for 2 weeks. mRNA levels of (a) FAS and (b) SCD-1 was quantified relative to GAPDH as described in the Materials and Methods section. Data represent mean ± SD (n = 5). ^*P<0.05 compared to wild-type mice fed the same diet, ^#P<0.05 compared to control-diet-fed mice of the corresponding genotype

Fig. 5

Hepatic PGC-1α, PGC-1β, CPT-1, and Cyp4a14 mRNA levels in wild-type and db/db mice fed the MCD diet. Wild-type (□) or db/db (■) mice were fed either a control diet or the MCD diet for 2 weeks. mRNA levels of (a) PGC-1α, (b) PGC-1β, (c) CPT-1, and (d) Cyp4a14 expression was quantified relative to GAPDH as described in the Materials and Methods section. Data represent mean ± SD (n = 5). ^*P<0.05 compared to wild-type mice fed the same diet, ^#P<0.05 compared to control-diet-fed mice of the corresponding genotype

Fig. 6

Hepatic SAMe and GSH metabolism of wild-type and db/db mice fed the MCD diet. Wild-type (□) or db/db (■) mice were fed either a control diet or the MCD diet for 2 weeks. Hepatic (a) SAMe concentrations, (b) SAH concentrations, (c) SAMe:SAH ratios, (d) GSH concentrations, (e) GSSG concentrations, and (f) GSH:GSSG ratios were quantified as described in the Materials and Methods section. Data represent mean ± SD (a, b, and c, n = 3; d, e, and f, n = 5). ^*P<0.05 compared to wild-type mice fed the same diet, ^#P<0.005 compared to control-diet-fed mice of the corresponding genotype

Fig. 7

Hepatic MATI/III and MATII mRNA and protein levels of wild-type and db/db mice fed the MCD diet. Wild-type (□) or db/db (■) mice were fed either a control diet or the MCD diet for 2 weeks. mRNA levels of (a) MAT1A and (b) MAT2A were quantified relative to GAPDH as described in the Materials and Methods section. (c) MATI/III and (d) MATII protein levels and (e) MATII:MATI/III ratio was quantified relative to GAPDH using Western blotting as described in the Materials and Methods section. Densitometry graphs (c) and (d) are accompanied with representative blots chosen from three samples per group. Data represent mean ± SD (a and b, n = 5; c, d, and e, n = 3). ^*P<0.05 compared to wild-type mice fed the same diet, ^#P<0.05 compared to control-diet-fed mice of the corresponding genotype