PPARalpha expression protects male mice from high fat-induced nonalcoholic fatty liver

Abstract

Emerging evidence suggests that the lack of PPARα enhances hepatic steatosis and inflammation in Ppara-null mice when fed a high-fat diet (HFD). Thus, the aim of this study was to determine whether Ppara-null mice are more susceptible to nonalcoholic steatohepatitis (NASH) than their wild-type (WT) counterparts following short-term feeding with a HFD. Age-matched male WT and Ppara-null mice were randomly assigned to consume ad libitum a standard Lieber-DeCarli liquid diet (STD) (35% energy from fat) or a HFD (71% energy from fat) for 3 wk. Liver histology, plasma transaminase levels, and indicators of oxidative/nitrosative stress and inflammatory cytokines were evaluated in all groups. Levels of lobular inflammation and the NASH activity score were greater in HFD-exposed Ppara-null mice than in the other 3 groups. Biochemical analysis revealed elevated levels of ethanol-inducible cytochrome P450 2E1 and TNFα accompanied by increased levels of malondialdehyde as well as oxidized and nitrated proteins in Ppara-null mice. Elevated oxidative stress and inflammation were associated with activation of c-Jun-N-terminal kinase and p38 kinase, resulting in increased hepatocyte apoptosis in Ppara-null mice fed a HFD. These results, with increased steatosis, oxidative stress, and inflammation observed in Ppara-null mice fed a HFD, demonstrate that inhibition of PPARα functions may increase susceptibility to high fat-induced NASH.

FIGURE 1

Liver histology of WT and Ppara-null mice fed STD or HFD for 3 wk (A–D). Hematoxylin and eosin stain shows intracellular lipid accumulation and inflammation (200×). The enclosed box in each panel shows the enlarged images (400×) and black arrows (D) indicate inflammatory foci. (E) Values are means ± SEM, n = 4 or 3 (WT STD). *Different from corresponding WT group, P = 0.05; +different from Null-STD, P = 0.05. A color version of this figure is available online as Supplemental Figure 2.

FIGURE 2

Mitochondrial thioase protein (A), densitometric levels (B), and catalytic activities (C) in livers of male WT and Ppara-null mice fed STD or HFD for 3 wk. The mitochondrial complex II protein was used to show equal protein loading. Values are means ± SEM, n = 4 or 3 (WT-STD). G, genotype.

FIGURE 3

Levels of CYP2E1 protein in cytoplasm (A) or mitochondria (C) in livers of male WT and Ppara-null mice fed STD or HFD for 3 wk. The densities measured by immunoblot analysis were normalized to GAPDH (B) and complex II subunit (D), respectively, and were plotted as a percentage of the value in WT-STD mice. Values are means ± SEM, n = 4 or 3 (WT-STD). G, genotype; D, diet.

FIGURE 4

Activation of JNK (A) or p38 kinase (C) in livers of WT and Ppara-null mice fed STD or HFD for 3 wk. Cytosolic proteins (100 μg/lane) were used to determine the activation of JNK and p38 kinase. The densities of immunoreactive bands were normalized to GAPDH (B,D) and plotted as a percentage of the levels in WT-STD mice. Significance levels are indicated for differences between genotypes (G). Values are means ± SEM, n = 4 or 3 (WT-STD). (D) Labeled means without a common letter differ, P < 0.05.

FIGURE 5

Levels of apoptotic hepatocytes (A-D) in livers of WT and Ppara-null mice fed STD or HFD for 3 wk. TUNEL-positive hepatocytes were identified by black arrows (D) and quantified (E) in 20 high-power fields (×400). Values are means ± SEM, n = 4 or 3 (WT-STD). *Different from the corresponding WT group, P = 0.05; +different from Null-STD, P = 0.05. A color version of this figure is available online as Supplemental Figure 3.

FIGURE 6

A proposed role of PPARα in the development of NAFLD/NASH. The early accumulation of intracellular lipids (first hit) followed by the increased oxidative stress and inflammatory cytokines (second hit), which can activate JNK and p38 kinase, contribute to NAFLD/NASH, and ultimately lead to apoptosis of hepatocytes.