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.

Alcohol produces distinct hepatic lipidome and eicosanoid signature in lean and obese

“…Biologically active lipids are important mediators of multiple physiological and pathologic processes. Perturbed lipid homeostasis can lead to steatosis, inflammation, and fibrotic processes that are significant pathophysiological determinants of disease progression and severity in both NAFLD and ALD (16)(17)(18)(19)(20)(21)(22). Most studies have, however, focused on either NAFLD or ALD, and the interactions between obesity and alcohol consumption on lipid metabolism have not been previously described.…”

Mitochondria and Oxidative Stress in the Cardiorenal Metabolic Syndrome

“…These interrelationships may be caused by several common mechanisms of mitochondrial dysfunction and oxidative stress induced via alcohol and obesity/ diabetes. The factors contributing to mitochondrial dysfunction include decreased expression of SIRT1 [206] , decreased phosphorylation of AMPK [207,208] , altered mitochondrial permeability transition contributed by dysregulation of SIRT3 [209] , and impaired transport of glutathione across mitochondria with decreased mitochondrial glutathione levels [210] . Long-standing alcohol consumption also leads to decreased SOD2 and catalase, thereby suppressing mitochondrial antioxidant capacity [193,200,204,205,211,212] .…”

The inhibitory effect of ethanol on Sestrin3 in the pathogenesis of ethanol-induced liver injury

“…We next determined whether the effects of ethanol observed in vitro also took place in vivo using liver samples from our laboratory's previously published study from controls and pair-fed mice with Lieber Dicarli Diet (15). As shown in our previous publications, these mice developed hepatic steatosis after they were fed with ethanolcontaining diet for 4 wk (15,16). Mouse liver expressed all three Sesn family members, with Sesn3 being the most abundant Sestrin (data not shown).…”

“…Six-to eight-week-old male C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were fed the Lieber-DeCarli alcoholcontaining diet, as previously described (15,16). Mice were housed individually in a room with controlled temperature (20 -22°C), humidity (55-65%), and regular 12:12 light-dark cycle.…”

“…1, A and C). As per our laboratory's previous publications (15,16), the ethanol concentration at 50 mM was used in the subsequent experiments.…”