Attenuated progression of diet-induced steatohepatitis in glutathione-deficient mice

Abstract

In nonalcoholic fatty liver disease (NAFLD), depletion of hepatic antioxidants may contribute to the progression of steatosis to nonalcoholic steatohepatitis (NASH) by increasing oxidative stress that produces lipid peroxidation, inflammation, and fibrosis. We investigated whether depletion of glutathione (GSH) increases NASH-associated hepatic pathology in mice fed a diet deficient in methionine and choline (MCD diet). Wild-type (wt) mice and genetically GSH-deficient mice lacking the modifier subunit of glutamate cysteine ligase (Gclm null mice), the rate-limiting enzyme for de novo synthesis of GSH, were fed the MCD diet, a methionine/choline-sufficient diet, or standard chow for 21 days. We assessed NASH-associated hepatic pathology, including steatosis, fibrosis, inflammation, and hepatocyte ballooning, and used the NAFLD Scoring System to evaluate the extent of changes. We measured triglyceride levels, determined the level of lipid peroxidation products, and measured by qPCR the expression of mRNAs for several proteins associated with lipid metabolism, oxidative stress, and fibrosis. MCD-fed GSH-deficient Gclm null mice were to a large extent protected from MCD diet-induced excessive fat accumulation, hepatocyte injury, inflammation, and fibrosis. Compared with wt animals, MCD-fed Gclm null mice had much lower levels of F₂-isoprostanes, lower expression of acyl-CoA oxidase, carnitine palmitoyltransferase 1a, uncoupling protein-2, stearoyl-coenzyme A desaturase-1, transforming growth factor-β, and plasminogen activator inhibitor-1 mRNAs, and higher activity of catalase, indicative of low oxidative stress, inhibition of triglyceride synthesis, and lower expression of profibrotic proteins. Global gene analysis of hepatic RNA showed that compared with wt mice, the livers of Gclm null mice have a high capacity to metabolize endogenous and exogenous compounds, have lower levels of lipogenic proteins, and increased antioxidant activity. Thus, metabolic adaptations resulting from severe GSH deficiency seem to protect against the development of steatohepatitis.

Figure 1

Total hepatic GSH concentrations measured in wild-type (wt) or Gclm null (ko) mice on standard mouse chow or after 21 days on the MCD diet. *P <0.01 vs wt MCD; # <0.01 vs wt chow. For all groups, n = 4–6 animals.

Figure 2

Hematoxylin and eosin-stained formalin-fixed sections showing representative hepatic pathology in wild-type (wt) and Gclm null mice after 21 days on the MCD diet. Arrows point to macrovesicular steatosis. Arrowheads highlight areas of infiltrating neutrophils.

Figure 3

Hepatic steatosis in wild-type (wt) and Gclm null (ko) mice. Oil red-stained section of MCD-fed wild-type liver (a) shows increased oil red staining (arrows) typical of hepatic lipid accumulation. Decreased oil red staining in MCD-fed Gclm null liver (b) indicates decreased hepatic steatosis. Hepatic triglycerides (c) in wt and Gclm null mice on standard mouse chow (panel c), methionine-choline-sufficient diet (MCS), or methionine-choline-deficient (MCD) diet. *P <0.05, compared with wt MCD; ^#P <0.05, compared with null C.

Figure 4

Expression of hepatic stearoyl-Co enzyme A desaturase-1 (SCD-1) (a), and fatty acid synthase (FAS) (b) mRNA by qPCR in wt and Gclm null (ko) mice fed methionine-choline-sufficient (MCS) or methionine-choline-deficient (MCD) diet. *P <0.05 compared with wt MCS; ^#P <0.05 compared with ko MCS; ^$P <0.05 compared with wt MCD, for all groups, n = 4–6 animals. Relative expression was determined by the ΔΔCt method as described in the Materials and Methods section, in which hepatic expression in wt, chow-fed, age-matched, male mice is 1.0.

Figure 5

Gclm null mice are resistant to MCD diet-induced fibrosis. Sirius red-stained section of MCD-fed liver of wt mouse (a) shows pericellular Sirius red staining (arrow), indicating collagen deposition; little staining is detected in MCD-fed Gclm (ko) null liver (b). Morphometric analysis of hepatic Sirius red staining (c). Staining was significantly increased in MCD-fed wt liver (wt MCD) compared with MCS-fed wt mice (wt MCS), and MCS or MCD-fed Gclm null mice (ko MCS; ko MCD). *P <0.05 compared with wt MCD, for all groups, n = 4–6 animals.

Figure 6

Low expression of αSMA in Gclm null mice fed the MCD diet. αSMA-stained sections of the livers of MCS-fed wt (a) and Gclm null (b) and of MCD-fed wt (c, d) and Gclm null (e, f) mice. The pericellular αSMA staining in MCD-fed wt (panels c, d) compared with that of MCD-fed Gclm null mice (panels e, f) or MCS-fed animals (panels a, b) must be noted. Magnifications, for panels a, b, c, e, × 10; for panels d, f, × 40).

Figure 7

Expression of hepatic fibrogenic genes in wt and Gclm (ko) null mice. TIMP-1 (a), PAI-1 (b), TGF-β (c), and Col1A1 (d) mRNAs from the livers of wt or Gclm null (ko) mice fed the MCS or MCD diet for 21 days. Relative expression was determined by the ΔΔCt method in which hepatic expression in wt, chow-fed, age-matched, male mice is 1.0. For panel a, ^##P <0.01 compared with wt MCS; ^ΦP <0.05 compared with ko MCS. For panel b, *P <0.05 compared with wt MCD. For panel d, ^#P <0.05 compared with wt MCS.

Figure 8

(a) Concentration of hepatic F₂-isoprostanes in wt and Gclm null mice fed the MCS or MCD diet. *P <0.05 compared with wt MCD. (b) Catalase activity in MCD-fed Gclm null mice. *P <0.05 compared with wt MCD; ^#P <0.05 compared with wt chow; ^ΦP <0.05 compared with Gclm null chow.

Figure 9

Hepatic expression of Acyl-CoA oxidase (ACO) (a) carnitine palmitoyltransferase 1-a (CPT-1) (b), uncoupling protein-2 (UCP-2) (c), or PPARα (d), mRNA in wt or Gclm null (ko) mice after 21 days on either a methionine-choline-sufficient (MCS), or methionine-choline-deficient (MCD) diet. ^ΦP <0.05 compared with wt MCD; *P <0.05 compared with wt MCS; ^#P <0.05 compared with ko MCS. Relative expression was determined by the ΔΔCt method as described in the Materials and Methods section, in which hepatic expression in wt, chow-fed, age-matched, male mice is 1.0.

Figure 10

Correlation of gene expression data obtained by cDNA microarray and qPCR. Differences in gene expression between wt and gclm null liver RNA from analyses by cDNA microarray (expressed as fold changes) and qPCR (expressed as ΔΔCt). (r²) = 0.913. Gene symbols: Cbr3 (carbonyl reductase 3), Cbr1 (carbonyl reductase 1), HO-1 (heme oxygenase 1), gclc (glutamate-cysteine ligase, catalytic subunit), PAI-1 ( plasminogen activator inhibitor 1), RRM2 (ribonucleotide reductase M2), EGFR (epidermal growth factor receptor), scd1 (stearoyl-Co enzyme A desatuase-1).