Increased 4-hydroxynonenal protein adducts in male GSTA4–4/PPAR-α double knockout mice enhance injury during early stages of alcoholic liver disease

Abstract

To test the significance of lipid peroxidation in the development of alcoholic liver injury, an ethanol (EtOH) liquid diet was fed to male 129/SvJ mice (wild-type, WT) and glutathione S-transferase A4–4-null (GSTA4−/−) mice for 40 days. GSTA4−/− mice were crossed with peroxisome proliferator-activated receptor-α-null mice (PPAR-α−/−), and the effects of EtOH in the resulting double knockout (dKO) mice were compared with the other strains. EtOH increased lipid peroxidation in all except WT mice ( P < 0.05). Increased steatosis and mRNA expression of the inflammatory markers CXCL2, tumor necrosis factor-α (TNF-α), and α-smooth muscle actin (α-SMA) were observed in EtOH GSTA4−/− compared with EtOH WT mice ( P < 0.05). EtOH PPAR-α−/− mice had increased steatosis, serum alanine aminotransferase (ALT), and hepatic CD3+ T cell populations and elevated mRNA encoding CD14, CXCL2, TNF-α, IL-6, CD138, transforming growth factor-β, platelet-derived growth factor receptor-β (PDGFR-β), matrix metalloproteinase (MMP)-9, MMP-13, α-SMA, and collagen type 1 compared with EtOH WT mice. EtOH-fed dKO mice displayed elevation of periportal hepatic 4-hydroxynonenal adducts and serum antibodies against malondialdehyde adducts compared with EtOH feeding of GSTA4−/−, PPAR-α−/−, and WT mice ( P < 0.05). ALT was higher in EtOH dKO mice compared with all other groups ( P < 0.001). EtOH-fed dKO mice displayed elevated mRNAs for TNF-α and CD14, histological evidence of fibrosis, and increased PDGFR, MMP-9, and MMP-13 mRNAs compared with the EtOH GSTA4−/− or EtOH PPAR-α−/− genotype ( P < 0.05). These findings demonstrate the central role lipid peroxidation plays in mediating progression of alcohol-induced necroinflammatory liver injury, stellate cell activation, matrix remodeling, and fibrosis.

Global Deletion of Glutathione S-Transferase A4 Exacerbates Developmental Nonalcoholic Steatohepatitis

“…We previously demonstrated that further deletion of Gsta4 in Ppara À/À mice, which are highly prone to development of steatosis, results in increased progression of liver injury to steatohepatitis and fibrosis after chronic alcohol exposure. 8 Experimental models of clinical pediatric NASH have proved difficult to produce in rodents given the slow development of this pathology relative to the short period between weaning and adulthood in rats and mice and the rapid growth rates of these animals during this period. 15,17 Because Ppara À/À mice have previously been shown to also have increased steatosis and inflammation in a high-fat liquid diet feeding model of adult NASH, we took a similar approach to develop a new model of developmental NASH by feeding Ppara À/À /Gsta4 À/À dKO mice liquid diets high in polyunsaturated fat (corn oil, rich in linoleic acid) from weaning to early adulthood.…”

“…8 In experiment 1, to establish a developmental NASH model, female dKO mice were weaned onto standard rodent chow (n Z 5), onto liquid diets containing 35% energy from polyunsaturated fat (corn oil), 18% from protein (casein), and 47% from carbohydrates (dextrose/maltodextrin, 4:1) (35% fat) (n Z 7) or liquid diets containing 70% energy from polyunsaturated fat, 18% from protein, and 12% from carbohydrate (70% fat) (n Z 7) and fed ad libitum for 12 weeks. Vitamin and mineral composition was based on National Research Council recommendations for growing mice.…”

“…Accumulation of hepatic lipid droplets was measured by quantitation of oil red O staining. 8,14 In addition, triglycerides were extracted from serum and liver homogenates with chloroform/methanol (2:1, v/v), and triglyceride concentrations were assayed using commercially available reagents (Cayman Chemical, Ann Arbor, MI). Overall hepatic oxidative stress was also assessed by measuring oxidized and reduced glutathione concentrations (reduced glutathione/ oxidized glutathione) using a kit from Cayman Chemical.…”

“…8 These data suggested the importance of lipid peroxidation products in progression of alcoholic liver disease and implicated a possible role for autoimmune responses triggered by adducted proteins in this process. Recent studies by Sutti et al 10 suggest a similar autoimmune process related to generation of reactive lipid peroxidation products may underlie development of inflammation in NASH.…”

“…7 More recently, we have made mice with a double knockout of Ppara À/À and the glutathione-S-transferase enzyme Gsta4 À/À (dKO). 8 Gsta4 is a detoxification enzyme that eliminates reactive electrophilic a,b unsaturated aldehydes, such as the lipid peroxidation product 4-hydroxynonenal (4-Hne). 9 We demonstrated that these dKO mice had enhanced hepatic Hne protein adducts, circulating autoantibodies against lipid peroxidation product-adducted proteins, necroinflammatory injury, stellate cell activation, and matrix remodeling than either wild-type mice or mice with knockout of either gene alone when fed chronically Data are expressed as means AE SEM.…”