We have used total enteral nutrition (TEN) to moderately overfeed rats high-polyunsaturated fat diets to develop a model for nonalcoholic steatohepatitis (NASH). Male Sprague-Dawley rats were fed by TEN a 187 kcal.kg(-3/4).day(-1) diet containing 5% (total calories) corn oil or a 220 kcal.kg(-3/4).day(-1) diet in which corn oil constituted 5, 10, 25, 35, 40, or 70% of total calories for 21 or 65 days. Rats fed the 5% corn oil, 220 kcal.kg(-3/4).day(-1)diet had greater body weight gain (P < or = 0.05), fat mass (P < or = 0.05), and serum leptin and glucose levels (P < or = 0.05), but no liver pathology. A dose-dependent increase in hepatic triglyceride deposition occurred with increase in percent corn oil in the 220 kcal.kg(-3/4).day(-1) groups (P < or = 0.05). Steatosis, macrophage infiltration, apoptosis, and focal necrosis were present in the 70% corn oil group, accompanied by elevated serum alanine aminotransferase (ALT) levels (P < or = 0.05). An increase in oxidative stress (thiobarbituric acid-reactive substances) and TNF-alpha expression (P < or = 0.05) was observed in the 70% corn oil group, as well as an increase in hepatic CYP2E1 and CYP4A1 expression (P < or = 0.05). Significant positive correlations were observed between the level of dietary corn oil and the degree of pathology, ALTs, oxidative stress, and inflammation. Liver pathology was progressive with increased necrosis, accompanied by fibrosis, observed after 65 days of TEN. Increased expression of CD36 and l-fabp mRNA suggested development of steatosis was associated with increased fatty acid transport. These data suggest that intragastric infusion of a high-polyunsaturated fat diet at a caloric level of 17% excess total calories results in pathology similar to clinical NASH.
A "2-hit" model for nonalcoholic steatohepatitis (NASH) has been proposed in which steatosis constitutes the "first hit" and sensitizes the liver to potential "second hits" resulting in NASH. Oxidative stress is considered a candidate for the second hit. N-acetylcysteine (NAC), an antioxidant, has been suggested as a dietary therapy for NASH. We examined the effects of NAC in a rat total enteral nutrition (TEN) model where NASH develops as the result of overfeeding dietary polyunsaturated fat. Male Sprague-Dawley rats consumed pelleted AIN-93G diets ad libitum or were overfed a 9200 kJ.kg(-0.75).d(-1) liquid diet containing 70% corn oil with or without 2 g.kg(-1).d(-1) NAC i.g. for 65 d. Hepatic steatosis was not influenced by dietary supplementation with NAC; however, the liver pathology score was lower (P = 0.05) and NAC provided partial protection against alanine aminotransferase release (P = 0.05). NAC attenuated increased hepatic oxidative stress (TBARS; P = 0.05) and prevented increases in cytochrome P450 2E1 apoprotein and mRNA and in tumor necrosis factor-alpha (TNFalpha) mRNA. Titers of auto-antibodies against proteins adducted to lipid peroxidation products were lower in serum of the NAC group than in the 70% corn oil group (P = 0.05). NAC also decreased Picosirius red staining of collagen, a marker of fibrosis. However, markers of hepatic stellate cell activation were unaffected. Using NAC in a TEN model of NASH, we have demonstrated that NAC prevents many aspects of NASH progression by decreasing development of oxidative stress and subsequent increases in TNFalpha but does not block development of steatosis.
Metabolic syndrome is often accompanied by development of hepatic steatosis and less frequently by non-alcoholic fatty liver disease (NAFLD) leading to non-alcoholic steatohepatitis (NASH). Replacement of corn oil with medium chain triacylglycerols (MCT) in the diets of alcohol-fed rats has been shown to protect against steatosis and alcoholic liver injury. The current study was designed to determine if a similar beneficial effect of MCT occurs in a rat model of NAFLD. Groups of male rats were isocalorically overfed diets containing 10%, 35% or 70% total energy as corn oil or a 70% fat diet in which corn oil was replaced with increasing concentrations of saturated fat (18:82, beef tallow:MCT oil) from 20% to 65% for 21 days using total enteral nutrition (TEN). As dietary content of corn oil increased, hepatic steatosis and serum alanine amino transferases were elevated (P < 0.05). This was accompanied by greater expression of cytochrome P450 enzyme CYP2E1 (P < 0.05) and higher concentrations of polyunsaturated 18:2 and 20:4 fatty acids (FA) in the hepatic lipid fractions (P < 0.05). Keeping the total dietary fat at 70%, but increasing the proportion of MCT-enriched saturated fat resulted in a dose-dependent reduction in steatosis and necrosis without affecting CYP2E1 induction. There was no incorporation of C8-C10 FAs into liver lipids, but increasing the ratio of MCT to corn oil: reduced liver lipid 18:2 and 20:4 concentrations; reduced membrane susceptibility to radical attack; stimulated FA β- and ω-oxidation as a result of activation of peroxisomal proliferator activated receptor (PPAR)α, and appeared to increase mitochondrial respiration through complex III. These data suggest that replacing unsaturated fats like corn oil with MCT oil in the diet could be utilized as a potential treatment for NAFLD.
Fatty acids (FAs) are essential components of lipids and exhibit important biological functions. The analyses of FAs are routinely carried out by gas chromatography-mass spectrometry after multistep sample preparation. In this study, several key experimental factors were carefully examined, validated, and optimized to analyze free fatty acid (FFA) and FA profiles of triglycerides and phospholipids in serum or tissue samples. These factors included (1) methylation/transesterification reagents, (2) validation of internal standards, and (3) final step concentration of FA methyl esters. This new method was utilized to analyze FFAs and the FA profiles of triglycerides and phospholipids in the serum and liver from a recently established rat model of nonalcoholic steatohepatitis (NASH). In this model, rats were fed a 220 kcal kg (-3/4) day (-1) diet containing either 5 or 70% corn oil for 21 days using total enteral nutrition. FA compositions of the serum and liver were found to shift from a pattern dominated by saturated and monounsaturated FAs (C16:0/18:1) to one dominated by polyunsaturated C18:2 derived from dietary linoleic acid. Alteration of FA composition in liver after overfeeding of high polyunsaturated fat diets may contribute to the progression of pathological changes from steatosis to inflammation, necrosis, and fibrosis observed in NASH.
To assess the relative contributions of undernutrition and ethanol (EtOH) exposure to alcohol-induced hepatotoxicity, female Sprague-Dawley rats were intragastrically infused liquid diets containing 187 or 154 kcal·kg−3/4·day−1 with or without 11 g·kg−1·day−1 EtOH. EtOH clearance was impaired in the 154 kcal·kg−3/4·day−1 EtOH group ( P ≤ 0.05). A combination of undernutrition and EtOH also increased the induction of hepatic cytochrome P-450 (CYP)2E1 and CYP4A1 mRNA, apoprotein, and activities ( P ≤ 0.05). This was accompanied by increased oxidative stress ( P ≤ 0.05). The severity of liver steatosis, macrophage infiltration, and focal necrosis was comparable in both EtOH groups. Alanine aminotransferase levels were elevated ( P ≤ 0.05) but did not significantly differ between the two EtOH groups. TUNEL analysis also demonstrated a comparable increase in apoptosis in the two EtOH groups ( P ≤ 0.05). The development of alcohol-induced liver pathology was accompanied by little change in fatty acid (FA) synthesis or degradation at 187 kcal·kg−3/4·day−1 but at 154 kcal·kg−3/4·day−1 was accompanied by decreased expression of FA synthesis genes and increased expression of peroxisome proliferator-activated receptor-α (PPAR-α)-regulated FA degradation pathways ( P ≤ 0.05). In addition, 154 kcal·kg−3/4·day−1 EtOH group livers exhibited greater hepatocyte proliferation ( P ≤ 0.05). We conclude that undernutrition does not exacerbate alcoholic steatohepatitis despite additional oxidative stress produced by an increased induction of CYP2E1 and CYP4A1. However, enhanced ethanol-induced cellular proliferation, perhaps as a result of enhanced PPAR-α signaling, may contribute to an increased risk of hepatocellular carcinoma in undernourished alcoholics.
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