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American Journal of Physiology - Gastrointestinal and Liver Physiology logoLink to American Journal of Physiology - Gastrointestinal and Liver Physiology
. 2010 Mar 11;298(6):G1004–G1012. doi: 10.1152/ajpgi.00482.2009

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

Suthat Liangpunsakul 1,✉, Margaret S Sozio 1, Eric Shin 1, Zhenwen Zhao 2, Yan Xu 2, Ruth A Ross 1, Yan Zeng 1, David W Crabb 1
PMCID: PMC3774334  PMID: 20224005

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 ∼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.

Keywords: protein phosphatase 2A

ampk (amp-activated protein kinase), is a key regulator of cell metabolism and homeostasis. When activated, it limits energy utilization and regulates anabolic pathways such as fatty acid and sterol synthesis. It promotes energy production by activating catabolic processes for glucose uptake and fatty acid oxidation.

We have previously shown (30) that AMPK is central to the pathogenesis of alcohol-induced hepatic steatosis. Our group has shown that ethanol treatment of cultured hepatoma cells inhibited the activity of the AMPK and reduced the amount of AMPK protein (30). This was confirmed by showing reduced AMPK phosphorylation and activity in livers of ethanol-fed mice, accompanied by decreased phosphorylation of acetyl-CoA carboxylase (ACC), increased ACC activity, and increased malonyl-CoA levels (30). The activation of sterol response element binding protein (SREBP-1) by ethanol might be mediated by the inhibition of AMPK (30). Traditionally, elevation of intracellular AMP has been considered the main activator of AMPK. However, recent studies indicated that AMPK should be considered as a stress-activated kinase. It can be activated through several upstream kinases, such as LKB1 (10, 28), calcium calmodulin-dependent protein kinase kinase (CamKK-β) (33), ataxia telangiectasia mutated (24), and transforming growth factor (TGF)-β-activated kinase 1 (TAK-1) (9), reflecting responsiveness to increased intracellular calcium, DNA damage, and engagement of cytokine receptors (for TGF-β and IL-1β), respectively.

The activity of AMPK is absolutely dependent on phosphorylation of the Thr172 of the α subunit; thus its activity is also controlled by protein phosphatases (PPs). Initially PP2C was suggested to carry out this dephosphorylation; more recently PP2A (27) was implicated. Coordinated control of kinase and phosphatase activities provides the cell with a capacity to rapidly switch AMPK from the phosphorylated to the dephosphorylated state to meet differing physiological needs. Recent studies from our group (16) found that the inhibitory effect of ethanol on AMPK phosphorylation was mediated through inhibition of AMPK upstream kinases, PKC-ζ/LKB1 and activation of PP2A. In fact, silencing of PP2A largely blocked the effect of ethanol on AMPK (16).

We are therefore interested in the control of PP2A activity. PP2A is involved in the regulation of many cellular functions and signaling pathways (11, 27). The C subunit is catalytic; the A subunit plays a role in stabilizing the C subunit; B subunits influence the substrate (target protein) specificity and cellular location of the complex. PP2A can be activated by ceramide (27), which was reported to bind to the B subunit; hence it was identified as a “ceramide-activated protein phosphatase” (17). Recently, another mechanism for ceramide activation of PP2A was suggested involving the inhibitor of PP2A, I2PP2A. This protein has the ability to bind directly with ceramide, which decreases its association with PP2A, leading to increasing PP2A activity in vitro (19).

In endothelial cells, palmitate inhibited the phosphorylation of AMPK via ceramide-dependent PP2A activation (27). We wondered whether ethanol might affect PP2A via increases in intracellular ceramide levels. Ethanol has been reported to activate acid or neutral sphingomyelinase, thereby increasing cellular ceramide in vivo (1). Acid sphingomyelinase (ASMase) knockout mice fed ethanol are resistant to hepatic steatosis (7). A recent study showed that ethanol treatment of isolated hepatocytes reduced the levels of sphingomyelin and sphingosine and increased ceramide content (25). We therefore hypothesized that the inhibitory effect of ethanol on AMPK phosphorylation was mediated partly through the generation of ceramide.

MATERIALS AND METHODS

Materials

All chemicals unless otherwise specified were purchased from Sigma Chemical (St. Louis, MO). Trypsin and tissue culture media were purchased from Life Technologies. Rat hepatoma (H4IIEC3) cell line was from the American Type Culture Collection (Manassas, VA). All antibodies were purchased from Cell Signaling Technology (Beverly, MA).

Cell Culture and Treatment

All cells were grown in MEM supplemented with 10% FBS, 100 μg/ml streptomycin, 63 μg/ml penicillin G, and 25 μg/ml of amphotericin B. Serum starvation was achieved by incubating nearly confluent cells for 18 h in medium without FBS. Then cells were exposed to ethanol or other treatments at the indicated concentrations and times. When ethanol was present, the cells were incubated in a chamber containing a 1-l beaker of water containing ethanol at the same concentration to reduce the loss of ethanol from the cultures because of evaporation.

Animals and Diets

To study the effect of ethanol on hepatic ceramides in vivo, 6- to 8-wk-old male C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were fed with Lieber-DeCarli diet as previously described (29). In brief, animals were housed individually in a room with controlled temperature (20–22°C), humidity (55–65%), and lighting (on at 6 AM and off at 6 PM). Protein content was constant at 18% of calories, and each diet had identical mineral and vitamin content. The animals were divided into two dietary groups: 1) control diet (fat comprising 10% of total calories, 6% from cocoa butter, and 4% from safflower oil, 72% of calories as carbohydrate) and 2) ethanol-containing diet [identical to the control diet but with ethanol added to account for 27.5% of total calories and the caloric equivalent of carbohydrate (maltose-dextrin) removed]. The animals were pair fed for 4 wk then euthanized. The experimental protocols were approved by the Indiana University School of Medicine Animal Care and Use Committee.

Ceramide Extraction and Analysis

Analysis of ceramide from cell lysates.

H4IIEC3 cells were grown in 10-cm plates as described above to confluence (∼8 × 106 cells) and were treated with ethanol as indicated. At the end of treatment, cell culture medium was removed and the cells were rinsed with ice-cold PBS. After the removal of PBS, 2 ml of trypsin was added directly onto the 10-cm plates. Cells were then transferred to Eppendorf tubes. Tubes were centrifuged at 14,000 rpm at 4°C for 2 min. Supernatant was discarded, and the pellets were stored at −80°C until analysis. In brief, cell pellets were diluted in PBS (1×) to 0.5 ml followed by addition of 3 ml of MeOH/chloroform (2:1) and the addition of C17–0 ceramide as an internal standard. The samples were vortexed for 1 min and incubated on ice for 10 min. One milliliter of chloroform and 1.3 ml of H2O were added to separate the phases. Then the samples were vortexed for 1 min, centrifuged (1,750 g for 10 min), and the lower phase transferred to a new glass tube. Two milliliters of chloroform were added to the remaining upper phase left in the original tube to further extract the lipids. The lower phase was then transferred to the same tube, and the solvent was evaporated under nitrogen at room temperature. After evaporation of the solvent, the dried samples were dissolved in 100 μl of methanol, and 10 μl of sample was used for mass spectrometry analyses. Mass spectrometry analyses were performed using API-4000 (Applied Biosystems/MDS SCIEX, Forster City, CA) with the Analyst data acquisition system. The instrument is equipped with a Z-spray ionization source. Both the nebulizer and desolvation gases are nitrogen, and the collision gas is argon. Typical operating parameters are as follows: nebulizing gas, 15; curtain gas, 8; collision-activated dissociation gas, 35; electrospray voltage, 5,000 with positive-ion multiple-reaction monitoring (MRM) mode, and a temperature of heater at 500°C. Precursor scan and MRM mode were used for measurement of ceramides. The ions monitored (in negative mode) were at m/z 550.2 (the parent ion)-294.3 (the product ion) for 17:0-Cer, 536.4–280.1 for 16:0-Cer, and 564.3–308.1 for 18:0-Cer. The dwell time in the MRM mode was 75 ms. Samples (10 μl) were loaded through a LC system (Agilent 1100) with an autosampler. The mobile phase was methanol/water/NH4OH (90:10:0.1, vol/vol/vol). The flow rate was 0.2 ml/min and 1.5 min/sample.

Analysis of ceramide in hepatic tissues.

At the time of euthanasia, liver tissues were harvested, as rapidly as possible, immediately freeze-clamped with Wollenberger tongs at the temperature of liquid nitrogen, powdered under liquid nitrogen with a mortar and pestle, and stored at −80°C for analysis. Ten milligrams of liver tissue powder prepared under liquid nitrogen was used for the measurement of intracellular ceramide and sphingomyelin contents with mass spectrometry.

Total RNA Isolation and qRT-PCR

Total RNA was prepared from H4IIEC3 cells and liver tissue using an Absolutely RNA RT-PCR Miniprep kit (Stratagene, Cedar Creek, TX). Reverse transcription of 1 μg total RNA to cDNA was performed using the StrataScript qPCR cDNA synthesis kit (Stratagene). qRT-PCR amplification was performed in a Stratagene MX 3005P thermal cycler (La Jolla, CA) using RT2 SYBR Green qPCR Master Mix. Primers for SYBR Green-based real-time PCR were purchased from SuperArray Bioscience (Frederick, MD). The following primers were used: Smpd2 (PPM24604A, neutral sphingomyelinase), Smpd1 (PPM25140A, acidic sphingomyelinase), Sptlc1 (PPM34459A, serine palmitoyltransferase), and GAPDH (PPM02946). The relative amount of target mRNA was calculated using the comparative cycle-threshold (Ct) method and normalizing each target gene with Ct of housekeeping gene, GAPDH.

Measurement of PP2A Activity

The activity of PP2A was measured with the PP2A immunoprecipitation phosphatase assay kit (Millipore, Temecula, CA). Threonine phosphopeptide (K-R-Pt-I-R-R) was used as the PP2A substrate. In brief, the cells were harvested in lysis buffer (0.5 M Tris·HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA), 1 mM PMSF, and protease inhibitors. Supernatants were incubated with anti-PP2A (C subunit, clone 1D6) and protein A agarose at 4°C for 2 h with constant rocking. The immunoprecipitates were then washed three times with Tris-buffered saline and diluted phosphopeptide (final concentration 750 μM), and Ser/Thr assay buffer were added. The mixtures were incubated for 10 min at 30°C in a shaking incubator, then briefly centrifuged, and 25 μl of the samples were transferred to 96-well microtiter plate. PP2A activities were determined by the addition of the Malachite Green Phosphate Detection Solution into the mixtures and measuring the absorbance at 650 nm. The absorbance values of each sample were compared with negative controls containing no PP2A enzyme activity.

Immunoblot Analysis

Immunoblot analyses were performed using 20 μg whole cell extract separated by electrophoresis in a 10% or 6% SDS-polyacrylamide gel and transferred to nitrocellulose filters. Detection of the protein bands was performed using the ECL Plus Western Blotting Detection System Kit (Amersham Biosciences, Piscataway, NJ). The intensity of the individual bands on Western blots were measured by PhosphoImager and analyzed with ImageQuant (Amersham Biosciences) software analysis.

Data Analysis

All data are presented as the means ± SE. Statistical significance was calculated with the Student's t-test or ANOVA analysis, followed by post hoc testing with least-squares difference, when appropriate. P < 0.05 was considered statistically significant.

RESULTS

Ceramide Reduces AMPK Phosphorylation in H4IIEC3 Cells

To determine the possible role of ceramide in the inhibitory effect of ethanol on AMPK phosphorylation, we first determined whether treatment of cells with a ceramide analog affected AMPK phosphorylation. To test this, H4IIEC3 cells were exposed to exogenous C2-ceramide (15 μM), a cell-permeable ceramide analog. In a time-course experiment, a reduction in AMPK phosphorylation and its downstream target, phospho-ACC (p-ACC) were observed when the cells were treated for between 4 and 12 h (Fig. 1A). We found significant toxicity of ceramide on H4IIEC3 cells at 30 μM; the cells died within 8 h of treatment. Because ceramide appeared to stimulate AMPK dephosphorylation, we next hypothesized that reduction in cellular ceramide should increase AMPK phosphorylation. We therefore examined the effect of fumonisin B1 on the basal level of AMPK phosphorylation in H4IIEC3 cells. This drug is an inhibitor of (dihydro)ceramide synthase, thus preventing ceramide formation via the de novo pathway and by the salvage pathway (whereby ceramide is degraded to sphingosine by acid ceramidase, and then sphingosine is converted back to ceramide by ceramide synthase). Indeed, we observed an increase in AMPK and ACC phosphorylation when the cells were treated with fumonisin B1 (15 μM) for between 4 and 12 h (Fig. 1B). We also found that fumonisin B1 significantly decreased the levels of cellular ceramide by 58% compared with controls. These experiments confirmed that altered levels of ceramide can influence AMPK phosphorylation. Our findings in H4IIEC3 cells were consistent with those reported by Wu et al. (27).

Fig. 1.

Fig. 1.

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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.

Ethanol Inhibits H2O2-Induced AMPK Phosphorylation and Raises Ceramide Levels and PP2A Activity in Hepatoma Cells with a Lag Phase

We have previously shown that the AMPK phosphorylation and its activity in H4IIEC3 cells were stimulated by exposure to hydrogen peroxide and that this was inhibited by pretreating the cells with ethanol for 24 h (16). To further study this effect in more detail, H4IIEC3 cells were pretreated with ethanol for different lengths of time as indicated in Fig. 2, followed by H2O2 (1 mM) treatment for short duration (10 min). The time course of the effect of ethanol is shown in Fig. 2. 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 of ∼33% was observed.

Fig. 2.

Fig. 2.

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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).

To determine the involvement of PP2A and intracellular ceramide on the time course of ethanol inhibition of AMPK phosphorylation (shown in Fig. 2), we next measured cellular ceramide levels and PP2A activity at each time point shown in Fig. 3, A and B. Hydrogen peroxide treatment alone for 10 min did not alter intracellular ceramide content. As shown in Fig. 3A, the baseline ceramide content was 202 ± 51 pmol/106 cells. Ethanol treatment significantly increased cellular ceramide content by ∼18%, when the cells were treated with ethanol for between 4 and 12 h. Interestingly, the levels of sphingomyelin were not changed with ethanol treatment (data not shown). On the basis of previous reports that ceramide activates PP2A, we next determined the effects of ethanol on PP2A activity in immunoprecipitates of cell lysates (Fig. 3B). We found that H4IIEC3 cells treated with ethanol for at least 12 h had an 18–23% increase in PP2A activity, compared with controls. The changes in ceramide levels, PP2A activity, and inhibition of AMPK phosphorylation by ethanol had similar time courses.

Fig. 3.

Fig. 3.

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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).

Inhibitory Effect of Ethanol on H2O2-Induced AMPK Phosphorylation was Attenuated by the Presence of Imipramine or Fumonisin B1, But Not by Myriocin or GW4869

To further understand the mechanism by which ethanol increased intracellular ceramide levels, several experiments were conducted with inhibitors of the key enzymes of ceramide metabolism. Ceramide can be synthesized by the hydrolysis of sphingomyelin by SMases, of which the acidic and neutral isoforms are of major relevance in the cells. Ceramide can also be synthesized in vivo in the endoplasmic reticulum through the condensation of serine and palmitoyl-CoA, catalyzed by serine palmitoyl transferase (SPT). As mentioned, ceramide can be salvaged from sphingosine by (dihydro)ceramide synthase. To differentiate which pathways might be involved in the action of ethanol, the following inhibitors were tested for their ability to block the effect of ethanol on AMPK activation: myriocin (an inhibitor of serine-palmitoyl transferase), GW4869 (an inhibitor of neutral sphingomyelinase, NSMase), fumonisin B1, and imipramine (an inhibitor of ASMase).

We found that the myriocin and GW4869 did not interfere with the ability of ethanol to inhibit H2O2-induced AMPK phosphorylation. However, the effect of ethanol on H2O2-induced AMPK phosphorylation was reversed by imipramine (Fig. 4A) and fumonisin B1 (Fig. 4C), which was accompanied by decreases in PP2A activity (Figs. 4, B and D).

Fig. 4.

Fig. 4.

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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).

Because of the slow increase in ceramide caused by ethanol and the lag in its effect on AMPK, it seemed possible that ethanol was inducing the levels of enzymes involved in ceramide generation. We therefore determined the effects of 24 h of ethanol exposure on the levels of mRNA of several genes encoding these enzymes. There were no significant changes in the levels of mRNA of ASMase, NSMase, or SPT in ethanol-treated H4IIEC3 cells compared with controls.

Effect of Ethanol on Hepatic Ceramide Levels In Vivo

To determine the effect of ethanol on hepatic ceramide metabolism in vivo, we fed mice ethanol (27.5% of the total calories) using the Lieber-DeCarli liquid diet and a pair-feeding protocol for 4 wk as previously described. Ethanol was introduced gradually into the diet; after the animals were adapted to the liquid diet, they were given alcohol at 9% of the total calories for 2 days, then 18% for 3 days, and finally 27.5%. At the end of the experiment, mice were euthanized and liver tissues were harvested for analysis. There was a 1.7-fold increase in triglyceride, as expected (data not shown). There was a 28% and 36% increase in C-16 ceramide and C-18 ceramide, respectively, in the livers of ethanol-fed mice (Fig. 5, A and B). However, there was no difference in the levels of sphingomyelin in ethanol-fed mice compared with controls (Fig. 5D).

Fig. 5.

Fig. 5.

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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).

We also examined the levels of the mRNA of the key enzyme in ceramide synthesis in livers of ethanol-fed mice. We failed to observe changes in the levels of mRNA for neutral SMase and SPT. However, the level of ASMase mRNA was increased by 1.7-fold in ethanol-fed liver compared with controls (P < 0.05).

DISCUSSION

We previously reported that ethanol inhibited AMPK phosphorylation by activation of PP2A (16). The present study provides a mechanism for this effect; ethanol simulates the generation of ceramide, a known activator of PP2A (27), through activation of ASMase. We found that 1) ethanol increased the levels of ceramide both in vitro (hepatoma cells) and in vivo (in mouse liver), 2) ceramide analogs reduce AMPK phosphorylation in cultured cells, 3) the activity of PP2A was increased in ethanol-treated cells, 4) the inhibitory effect of ethanol on AMPK phosphorylation is attenuated by imipramine, an inhibitor of ASMase and fumonisin B1, 5) fumonisin and imipramine block the increase in PP2A activity induced by ethanol treatment, and 6) ethanol increased hepatic ASMase mRNA in ethanol-fed mice. Taken together, the results from this study suggested that 1) ethanol inhibits AMPK phosphorylation in part through ceramide-induced PP2A activation and 2) ethanol increased ceramide by activating ASMase in vivo. The proposed mechanism on the effect of ethanol on AMPK is shown in Fig. 6.

Fig. 6.

Fig. 6.

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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.

The control of AMPK activity is complex. AMPK can be activated through several upstream kinases such as PKC-ζ/LKB1, CamKK, TAK-1, and ataxia-telangiectasia gene product (ataxia telangiectasia mutated) (10, 18). It is also controlled by PPs. Earlier papers indicated that PP2C was responsible for dephosphorylation of Thr172, on the basis of in vitro and in vivo experiments (26). More recent data have implicated PP2A (27). Purified PP2A catalytic subunit dephosphorylated rat liver AMPK in a fashion blocked by the presence of AMP (8). High glucose is reported to reduce phosphorylation of AMPK via activation of PP2A (21).

PP2A is a ceramide-activated PP (17). On the basis of the report that palmitate inhibited AMPK by increasing ceramide synthesis and activating PP2A (27), we examined the effect of ethanol on cell ceramide content in H4IIEC3 cells and in ethanol-fed mice. Microarrays of the H4IIEC3 cells showed that they express mRNA for all the necessary enzymes for ceramide metabolism, i.e., sphingomyelin synthases 1 and 2, serine palmitoyltransferase, long-chain sphingomyelin phosphodiesterases 1, 2, and 4 (the sphingomyelinases), and ceramide synthase 2 (Edenberg, McClintick and Liu, personal communication). We demonstrated that treatment of the cells with the C2-ceramide analog reduced, and the ceramide synthase inhibitor (fumonisin B1) increased, AMPK phosphorylation, consistent with ceramide modulation of PP2A activity. The action of C2-ceramide had a slow onset; this might reflect progressive saturation of ceramide binding sites or the need for the ceramide to accumulate in a certain cellular compartment before PP2A was activated.

Similarly, treatment of the cells with ethanol led to a gradual increase in ceramide content (Fig. 3A), which correlated reasonably well with the onset of ethanol's ability to block hydrogen peroxide-stimulated AMPK phosphorylation (Fig. 2) and increase PP2A activity (Fig. 3B). Other investigators have found that ethanol treatment of cultured hepatocytes reduced the levels of sphingomyelin and sphingosine by about 20% and increased ceramide by 17% (25). The enzymatic pathway involved in this effect was not determined, but the reduction in sphingomyelin could have resulted from activation of a SMase.

In search of the mechanism for this action of ethanol, we found that its inhibitory effect on AMPK phosphorylation was attenuated by the presence of the ASMase inhibitor, imipramine, and (dihydro)ceramide synthase inhibitor, fumonisin B, but not by inhibitors of SPT or neutral ASMase. We propose that the primary action of ethanol is activation of ASMase, which generates ceramide in the acidic endosome pool (Fig. 6). Activation of ASMase by ethanol was previously reported (6). We found an induction of the hepatic ASMase mRNA in mice fed with ethanol for 4 wk. Increased ASMase would generate ceramide by hydrolyzing sphingomyelin. However, we did not see a reduction in the total liver sphingomyelin content. A trivial explanation may be that sphingomyelin was depleted in a subcellular pool and that this is not reflected in the total sphingomyelin pool. Additional studies on the subcellular location of the activated ASMase should help clarify this. The effect of fumonisin B1 can be rationalized as follows. Because myriocin, an inhibitor of serine palmitoyl transferase (and thus the de novo pathway), did not block the inhibitory effect of ethanol on AMPK phosphorylation, our results suggested that the salvage pathway of ceramide synthesis is involved in the action of fumonisin B1. The salvage pathway involves acylation of sphingosine via ceramide synthase (12). In the presence of fumonisin B1, ceramide will be depleted because of its irreversible degradation to sphingosine (Fig. 6). We propose that the modest increase in ceramide induced by ethanol (Fig. 3) cannot be sustained when ceramide synthase is blocked by fumonisin B1.

Additional effects of ethanol on the liver in situ could affect ceramide metabolism. Ethanol feeding stimulates TNF-α secretion by Kupffer cells. TNF-α is a well-known stimulus for the generation of ceramide via activation of ASMase (14). Indeed, ASMase knockout animals were resistant to the steatotic and apoptotic effects of TNF-α on the liver (6). Another mechanism by which ethanol could raise ceramide levels is through induction of stearoyl-CoA desaturase-1 (Scd-1) (4). In Scd-1 knockout mice, ceramide levels and SPT mRNA and activity are reduced in oxidative-type muscles. This was associated with increased rates of fatty acid oxidation, reduced fatty acyl-CoA levels, and activation of AMPK (4). Ethanol feeding induces Scd-1 in mouse liver (29, 30) through its effect on SREBP-1, and this might increase SPT mRNA and ceramide. Finally, ethanol metabolism results in oxidative stress generated at the mitochondrion and in the endoplasmic reticulum. Reactive oxygen species are reported to both stimulate endoplasmic reticulum stress and to increase ceramide generation (6). Intriguingly, glutathione has been reported to inhibit NSMase 2 in liver, and thus the ethanol-induced depletion of glutathione could lead to increased activity of this enzyme (22).

In conclusion, the present study has several implications. The effect of ethanol on AMPK appears to be mediated in part through increased cellular levels of ceramide and activation of PP2A. Ceramide signaling has been linked to other pathways that are implicated with alcohol-induced liver injury. Ceramide can activate JNK and PKC-ζ, each of which participates in a network of signaling cascades. Increased levels of ceramide observed in ethanol-treated cells and mouse liver have been shown to increase hepatocyte apoptosis and mitochondrial dysfunction (5–7). Ceramide mediates the downregulation of methionine adenosyltransferase-1A, the rate-limiting enzyme responsible for the synthesis of S-adenosylmethionine, which has been shown to play a key role in alcohol-induced liver injury (5). Given that ceramide is involved in several pathways of ethanol-induced liver injury, future studies to explore pharmacological therapy that modulates ceramide synthesis on alcoholic liver diseases are warranted.

GRANTS

This study is supported by R01 AA15070 and P60 AA07611 from the NIAAA (D. Crabb), Veterans Administration Young Investigator Award/Indiana Institute for Medical Research (S. Liangpunsakul), K08 AA016570 from the NIH/NIAAA (S. Liangpunsakul), Central Society for Clinical Research Career development award (S. Liangpunsakul), and F32 AA017800 (M. Sozio).

DISCLOSURES

The authors are not aware of financial conflicts with the subject matter or materials discussed in this article with any of the authors or their academic institutions or employers.

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