Inhibition of p53 attenuates steatosis and liver injury in a mouse model of non-alcoholic fatty liver disease

Background & Aims

p53 and its transcriptional target miRNA34a have been implicated in the pathogenesis of fatty liver. We tested the efficacy of a p53 inhibitor, pifithrin-α p-nitro (PFT) in attenuating steatosis, associated oxidative stress and apoptosis in a murine model of non-alcoholic fatty liver disease (NAFLD).

Methods

C57BL/6 mice were fed a high-fat (HFD) or control diet for 8 weeks; PFT or DMSO (vehicle) was administered three times per week. Markers of oxidative stress and apoptosis as well as mediators of hepatic fatty acid metabolism were assessed by immunohistochemistry, Western blot, real-time PCR, and biochemical assays.

Results

PFT administration suppressed HFD-induced weight gain, ALT elevation, steatosis, oxidative stress, and apoptosis. PFT treatment blunted the HFD-induced upregulation of miRNA34a and increased SIRT1 expression. In the livers of HFD-fed, PFT-treated mice, activation of the SIRT1/PGC1α/PPARα axis increased the expression of malonyl-CoA decarboxylase (MLYCD), an enzyme responsible for malonyl-CoA (mCoA) degradation. Additionally, the SIRT1/LKB1/AMPK pathway (upstream activator of MLYCD) was promoted by PFT. Thus, induction of these two pathways by PFT diminished the hepatic mCoA content by enhancing MLYCD expression and function. Since mCoA inhibits carnitine palmitoyltransferase 1 (CPT1), the decrease of hepatic mCoA in the PFT-treated, HFD-fed mice increased CPT1 activity, favored fatty acid oxidation, and decreased steatosis. Additionally, we demonstrated that PFT abrogated steatosis and promoted MLYCD expression in palmitoleic acid-treated human HepaRG cells.

Conclusions

The p53 inhibitor PFT diminished hepatic triglyceride accumulation and lipotoxicity in mice fed a HFD, by depleting mCoA and favoring the β-oxidation of fatty acids.

Introduction

There is accumulating evidence that p53 plays a central role in the pathogenesis of alcoholic [1], [2], [3], [4] and non-alcoholic fatty liver disease (NAFLD) [5], [6], [7]. For example, chronic ethanol feeding in rats has been shown to increase the hepatic mRNA abundance, acetylation, and transcriptional activity of p53 [1], [3], [4], [8]. The importance of p53 and its pro-apoptotic and pro-oxidant downstream target p66^shc in alcoholic liver disease (ALD) is highlighted by the observations that genetic ablation of p53 or p66^shc significantly reduced ethanol-induced hepatocellular injury and oxidative stress [2], [9]. The pivotal role of p53 in the pathogenesis of ALD has been further elucidated by a recent study, where rat strain-specific susceptibility to ethanol-induced liver injury was associated with differences in hepatic p53 activation and corresponding downstream changes in signaling cascades regulating apoptosis, oxidative stress, insulin signaling, and fatty acid metabolism [4].

Similar to ALD, p53 was found to be upregulated in the livers of various murine models of NAFLD [7]. Hepatocyte apoptosis was linked to p53 activation in mice fed a HFD [5]. In addition, there was a positive correlation between the level of steatosis and p53 expression in human liver samples [6]. Based on these findings, p53 activation may be a broader metabolic event important in the pathogenesis of fatty liver, irrespective of etiology, that not only facilitates apoptosis and oxidative stress, but also generates hepatic abnormalities such as steatosis and insulin resistance, which contribute to injury. Regarding the role of p53 in steatosis, it has recently been observed that the regulatory control of p53 appears to fail in fatty liver disease, since activated p53 upregulates the transcription of miRNA34a [10] as a direct downstream target of p53 [11]. In this regard, miRNA34a has been shown to be upregulated in the livers of mice fed a HFD as well as in patients with metabolic syndrome and non-alcoholic steatohepatitis (NASH) [12], [13]. Its expression level also correlated with the susceptibility to NASH development in various murine strains [13]. It has been demonstrated that silent mating type information regulation 2 homolog 1 (SIRT1) expression in the liver is suppressed by miRNA34a [10]. SIRT1 has a central role in regulating hepatic fatty acid metabolism; it abrogates ectopic fat accumulation by facilitating fatty acid oxidation and curbing the de novo fatty acid synthesis. Accordingly, hepatocyte-specific ablation of SIRT1 results in hepatic steatosis and inflammation upon HFD feeding in mice [14].

The mechanisms, by which SIRT1 activation may attenuate steatosis, include the deacetylation and inactivation [15] of sterol regulatory element-binding protein 1c (SREBP1c, as a master transcriptional regulator of de novo fatty acid synthesis) as well as the promotion of deacetylation and activation [16] of peroxisome proliferator-activated receptor-γ co-activator 1α (PGC1α). This latter event may increase the PPARα-mediated gene expression (e.g., fatty acid oxidizing enzymes and malonyl-CoA decarboxylase (MLYCD)) [14], [17]. Additionally, the interaction between SIRT1 and PPARα was shown to be necessary for efficient PGC1α activation [14]. Finally, SIRT1 can deacetylate and promote the cytosolic translocation [18] of liver kinase B1 (LKB1) that initiates the LKB1/5′ adenosine monophosphate-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) signaling cascade, ultimately leading to decreased intrahepatic mCoA [19]. The mechanisms of AMPK-mediated malonyl-CoA decrease have been described and include the phosphorylation and subsequent inactivation of ACC [20] (involved in mCoA synthesis) as well as the phosphorylation and activation of MLYCD [21] (responsible for mCoA degradation). Malonyl-CoA is a key physiological regulator of cellular fatty acid oxidation and lipid partitioning by allosterically inhibiting carnitine palmitoyltransferase 1 (CPT1), which regulates the mitochondrial uptake of long-chain fatty-acyl CoA molecules for oxidation. The transcriptional and post-translational activation of MLYCD via the SIRT1/PGC1α/PPARα and SIRT1/LKB1/AMPK signaling cascades, respectively, in concert with the inactivation of ACC, may decrease the hepatic mCoA content. This biochemical event, associated with SIRT1 activation, favors fatty acid oxidation and decreases the likelihood of excess lipid accumulation.

Given these observations, we hypothesized that dysregulation of p53 in the liver during diet-induced obesity may favor excess accumulation of lipids by activating miRNA34a and altering SIRT1 expression. Steatosis promotes oxidative stress and increases the vulnerability of hepatocytes to acute injury [22]. Additionally, increased oxidative stress may further facilitate p53 stabilization in a feed-forward regulatory mechanism, activating downstream genes that are involved in apoptosis, oxidative stress and insulin resistance [4], [23]. Here we demonstrate that pharmacologic inhibition of p53 with a specific transcriptional inhibitor, pifithrin-α p-nitro [24], attenuates hepatic steatosis and liver injury in mice fed a HFD.