Inhibitory effect of ethanol on AMPK phosphorylation is mediated in part through elevated ceramide levels

Abstract

Ethanol treatment of cultured hepatoma cells and of mice inhibited the activity of AMP-activated protein kinase (AMPK). This study shows that the inhibitory effect of ethanol on AMPK phosphorylation is exerted through the inhibition of the phosphorylation of upstream kinases and the activation of protein phosphatase 2A (PP2A).Inhibition of AMPK phosphorylation by palmitate was attributed to ceramide-dependent PP2A activation. We hypothesized that the inhibitory effect of ethanol on AMPK phosphorylation was mediated partly through the generation of ceramide. The effect of ethanol and inhibitors of ceramide synthesis on AMPK phosphorylation, ceramide levels, and PP2A activity were assessed in rat hepatoma cells (H4IIEC3). The effect of ethanol on hepatic ceramide levels was also studied in C57BL/6J mice fed the Lieber-DeCarli diet. In H4IIEC3 cells, ceramide reduced AMPK phosphorylation when they were treated for between 4 and 12 h. The basal level of AMPK phosphorylation in hepatoma cells was increased with the treatment of ceramide synthase inhibitor, fumonisin B1. Ethanol treatment significantly increased cellular ceramide content and PP2A activity by approximately 18-23%, when the cells were treated with ethanol for between 4 and 12 h. These changes in intracellular ceramide concentrations and PP2A activity correlated with the time course over which ethanol inhibited AMPK phosphorylation. The activation of PP2A and inhibition of AMPK phosphorylation caused by ethanol was attenuated by fumonisin B1 and imipramine, an acid sphingomyelinase (SMase) inhibitor. There was a significant increase in the levels of ceramide and acid SMase mRNA in the livers of ethanol-fed mice compared with controls. We concluded that the effect of ethanol on AMPK appears to be mediated in part through increased cellular levels of ceramide and activation of PP2A.

Fig. 1.

Effect of ceramide and the ceramide synthase inhibitor (fumonisin B1) treatment on AMP-activated protein kinase (AMPK) phosphorylation in rat hepatoma H4IIEC3 cells. H4IIEC3 cells were maintained in MEM supplemented with 10% FBS. Cells were then washed with PBS, refed with MEM supplemented with 10% dilapidated FBS, and, after incubation for 18 h, treated with ceramide (A) and fumonisin B1 (B) for the times indicated. Cell extracts (20 μg protein) were electrophoresed on a 10% and 6% SDS gel for AMPK and acetyl-CoA carboxylase (ACC), respectively. Phospho-AMPK (p-AMPK) and phospho-ACC (p-ACC) protein (Thr-172) were visualized by using anti-p-AMPK and anti-p-ACC antibodies. Immunoblot analysis demonstrated that treatment of ceramide (15 μM) for at least 12 h decreased the expression of phospho-AMPK and phospho-ACC compared with controls. The basal levels of phospho-AMPK and phospho-ACC were increased in the presence of ceramide synthase inhibitor (B). Values are means ± SE (the blot is a representative of 3 blots from 3 individual experiments). *Significant difference vs. control; P < 0.05.

Fig. 2.

Time course of the inhibitory effect of ethanol on AMPK phosphorylation. H4IIEC3 cells were grown as described in detailed in materials and methods. Subsequently they were incubated for 1, 2, 4, 8, 12 and 24 h with ethanol (50 mM; E1hr, etc.), then stimulated with 1 mM H₂O₂ for 10 min. Cell extracts (20 μg protein) were electrophoresed on a 10% SDS gel and blotted for p-AMPK levels. A: representative set of blots. There was no effect of ethanol pretreatment on the ability of H₂O₂ to stimulate AMPK phosphorylation until ethanol had been present for more than 8 h, at which time maximal inhibition was observed. (*P < 0.05 compared with controls, $P < 0.05 compared with H₂O₂-treated cells, B).

Fig. 3.

Effect of ethanol treatment on cellular ceramide content and protein phosphatase 2A (PP2A) activity in H4IIEC3 cells. A: H4IIEC3 cells were grown in the 10-cm plate as described in materials and methods and were treated as indicated. At the end of the experiments, cells were harvested and cellular ceramide content was measured with mass spectrometry. Intracellular ceramide content was increased when the cells were treated with ethanol for at least 12 h. Its level was increased by 18% compared with those in controls (*P < 0.05 compared with controls). B: effects of ethanol on PP2A activity were analyzed using immunoprecipitates of cell lysates. H4IIEC3 cells treated with ethanol for at least 12 h had an 18–23% increase in PP2A activity, compared with controls (*P < 0.05 compared with controls).

Fig. 4.

Effect of imipramine and fumonisin B1 on the inhibitory effect of ethanol on AMPK phosphorylation. H4IIEC3 cells were treated with ethanol (50 mM), imipramine (48 μM for 24 h), and fumonisin B1 (15 μM for 24 h) as described in materials and methods. They were then treated with H₂O₂ (1 mM 10 min), then harvested for Western blotting. The blots were quantified using a PhosphorImager and ImageQuant software analysis. A and C: imipramine and fumonisin B1 blocked the inhibitory effect of H₂O₂ induced AMPK phosphorylation. B and D: PP2A activity was measured from the supernatant as indicated. PP2A activity was significantly decreased in the presence of imipramine (B) and fumonisin B1 (D) (*P < 0.05 compared with controls, $P < 0.05 significant difference compared with pretreatment with ethanol in the absence of imipramine and fumonisin B1).

Fig. 5.

Effect of ethanol feeding on intrahepatic ceramide and sphingomyelin content. C57BL/6J mice were fed the liquid diet with or without ethanol for 4 wk and then euthanized. Ten milligrams of liver tissues were prepared for mass spectrometry analyses as described in materials and methods. Hepatic C-16 (A) and C-18 ceramide (B) contents were significantly higher in ethanol-fed mice compared with controls (n = 5 in each group, *P < 0.05). The total ceramide content (the combination of C-16 and C-18 ceramides) in the liver tissues are shown (C). There was no difference in sphingomyelin content in liver tissues from ethanol-fed mice compared with paired-fed controls (D).

Fig. 6.

Proposed mechanism on ethanol effects on AMPK phosphorylation. The inhibitory effect of ethanol on AMPK phosphorylation was attenuated by the presence of an acid sphingomyelinase (SMase) inhibitor (imipramine) or ceramide synthase inhibitor (fumonisin B1) in hepatoma cells. Ethanol also increased the mRNA levels of acid SMase in ethanol-fed mice. We propose that the inhibitory effects of ethanol on AMPK phosphorylation are secondary to increased intracellular ceramide concentrations (through increased acid SMase activity), leading to increase in PP2A activity, thus inhibiting AMPK phosphorylation. SPT, serine palmitoyl transferase.