Activation of p53 enhances apoptosis and insulin resistance in a rat model of alcoholic liver disease

Abstract

Background & aims: Chronic ethanol consumption in the Long-Evans (LE) rat has been associated with hepatic p53 activation, and inhibition of the insulin/PI3K/AKT signal transduction cascade due to increased expression of PTEN. We hypothesize that p53 activation and altered insulin signaling may influence the susceptibility of rats to ethanol-induced liver damage. Furthermore, p53 not only activates programmed cell death pathways and suppresses hepatocellular survival signals, but also promotes gluconeogenesis to increase systemic insulin resistance due to a novel metabolic function.

Methods: Fischer (F), Sprague-Dawley (SD) and LE rats were fed ethanol-containing or control liquid diet for 8 weeks. Histopathological and biochemical changes were assessed.

Results: Here, we demonstrate that chronic ethanol feeding in rats promotes p53 activation, hepatic steatosis, oxidative stress, PUMA, and PTEN expression, which contribute to hepatocellular death and diminished insulin signaling in the liver. Such changes are pronounced in the LE, less prominent in SD, and virtually absent in the F rat strain. More importantly, there is activation of Tp53-induced glycolysis and apoptosis regulator (TIGAR) in the ethanol-fed LE rat. This event generates low hepatic fructose-2,6-bisphosphate (Fru-2,6-P₂) levels, reduced lactate/pyruvate ratio and may contribute to increased basal glucose turnover and high residual hepatic glucose production during euglycemic hyperinsulinemic clamp.

Conclusions: p53 activation correlates with the susceptibility to ethanol-induced liver damage in different rat strains. p53 not only orchestrates apoptosis and suppresses cell survival, but by activating TIGAR and decreasing hepatic Fru-2,6-P₂) levels it promotes insulin resistance and therefore, contributes to the metabolic abnormalities associated with hepatic steatosis.

Figure 1. Chronic alcohol-fed LE rats exhibit steatosis and ALT elevations

Hematoxylineosin staining revealed micro- and macrovesicular steatosis, cell dropout and disorganized liver architecture in the LE rat. These changes were less pronounced in the other two (F, SD) ethanol-fed strains. (panels F&I vs A-H in Fig. 1A). Enhanced steatosis was confirmed by a liver triglyceride assay (Fig. 1B). Histopathological changes in the LE model were associated with ALT elevation (Fig. 1C). (*): LE control vs LE ethanol, (#): LE ethanol vs SD ethanol, p<0.05

Figure 2. Oxidative stress, mtDNA damage and p53 activation

Oxidative stress was measured in isolated hepatocytes (Fig. 2A). Mitochondrial DNA damage was detected in the liver of LE rats (Fig. 2B). Nuclear abundance of p53 was assessed by Western blot analysis and quantified by densitometry (Fig. 2C). In vitro binding activity of p53 to DNA target sites was assessed by EMSA. The results of densitometry are shown in Fig. 2D. (*): LE control vs LE ethanol, (#): LE ethanol vs SD ethanol, (¶): LE ethanol vs F ethanol, p<0.05.

Figure 3. p53 activation by chronic ethanol promotes PUMA-mediated apoptosis, caspase-3 and PARP-cleavage

Cytosolic PUMA-α accumulation was assessed by Western-blot analysis (Fig. 3A). Caspase-3 activity was measured using liver lysates (Fig. 3B). PARP cleavage was assessed by Western-blot analysis (Fig. 3C). Results for apoptosis were expressed as number of apoptotic cells per square millimeter (Fig. 3D). (*): LE control vs LE ethanol (#): LE vs SD ethanol, (¶): LE ethanol vs F ethanol, p<0.05.

Figure 4. Chronic ethanol consumption activates p53, induces PTEN and alters AKT phosphorylation

Elements of AKT signaling were assessed by Western-blot analysis using whole liver lysates (Fig. 4A). The result of PTEN densitometry is shown (Fig. 4B) (*): LE control vs LE ethanol, p<0.05.

Figure 5. The p53 downstream target gene TIGAR reduces hepatic Fru-2,6-P₂ content and alters glucose metabolism. Chronic ethanol consumption produces hepatic insulin resistance in alcohol-fed rats

TIGAR expression was assessed by Western-blot analysis in whole liver lysates (Fig. 5A). Hepatic fructose-2,6-bisphosphate (Fig. 5B), lactate and pyruvate levels were determined (Fig. 5C). A euglycemic hyperinsulinemic clamp was performed on catheterized conscious unrestrained control and alcohol-fed rats (n=8–9). The rate of 25% D-glucose infused exogenously to maintain euglycemia during the hyperinsulinemic clamp is illustrated (Fig. 5D). The absolute rate of HGP was quantitated as the difference between the ³H-glucose determined rate of total glucose Rd and the exogenous glucose infusion rate (Fig. 5E). The percent suppression of HGP during the clamp was calculated as described (Fig. 5F). (*): control vs ethanol (#): LE ethanol vs SD ethanol, (¶): LE ethanol vs F ethanol (&) SD ethanol vs F ethanol, p<0.05

Figure 6. Diagram illustrating a role for p53 in ALD

Activation of p53 is linked to hepatic steatosis and oxidative stress. Enhanced p53 expression promotes apoptosis, suppresses insulin signaling and cell survival, as well as shifts energy metabolism towards ROS-prone oxidative phosphorylation, decreases glycolysis and favors gluconeogenesis. In this context, increased HGP enhances systemic insulin resistance and further promotes steatosis by activating SREBP1c. Grey boxes represent the results of signaling abnormalities during chronic ethanol consumption.