Imipramine blocks ethanol-induced ASMase activation, ceramide generation, and PP2A activation, and ameliorates hepatic steatosis in ethanol-fed mice

Abstract

Our previous data showed the inhibitory effect of ethanol on AMP-activated protein kinase phosphorylation, which appears to be mediated, in part, through increased levels of hepatic ceramide and activation of protein phosphatase 2A (Liangpunsakul S, Sozio MS, Shin E, Zhao Z, Xu Y, Ross RA, Zeng Y, Crabb DW. Am J Physiol Gastrointest Liver Physiol 298: G1004-G1012, 2010). The effect of ethanol on AMP-activated protein kinase phosphorylation was reversed by imipramine, suggesting that the generation of ceramide via acid sphingomyelinase (ASMase) is stimulated by ethanol. In this study, we determined the effects of imipramine on the development of hepatic steatosis, the generation of ceramide, and downstream effects of ceramide on inflammatory, insulin, and apoptotic signaling pathways, in ethanol-fed mice. The effect of ethanol and imipramine (10 μg/g body wt ip) on ceramide levels, as well as inflammatory, insulin, and apoptotic signaling pathways, was studied in C57BL/6J mice fed the Lieber-DeCarli diet. Ethanol-fed mice developed the expected steatosis, and cotreatment with imipramine for the last 2 wk of ethanol feeding resulted in improvement in hepatic steatosis. Ethanol feeding for 4 wk induced impaired glucose tolerance compared with controls, and this was modestly improved with imipramine treatment. There was a significant decrease in total ceramide concentrations in response to imipramine in ethanol-fed mice treated with and without imipramine (287 ± 11 vs. 348 ± 12 pmol/mg tissue). The magnitude and specificity of inhibition on each ceramide species differed. A significant decrease was observed for C16 (28 ± 3 vs. 33 ± 2 pmol/mg tissue) and C24 (164 ± 9 vs. 201 ± 4 pmol/mg tissue) ceramide. Ethanol feeding increased the levels of the phosphorylated forms of ERK slightly and increased phospho-p38 and phospho-JNK substantially. The levels of phospho-p38 and phospho-JNK were reduced by treatment with imipramine. The activation of ASMase and generation of ceramide in response to ethanol feeding may underlie several effects of ethanol. ASMase inhibitors may be considered as a therapeutic target for alcohol-induced hepatic steatosis and activation of stress kinases.

Compounds of the sphingomyelin-ceramide-glycosphingolipid pathways as secondary messenger molecules: new targets for novel therapies for fatty liver disease and insulin resistance

“…Amitriptyline, an ASMase inhibitor, reduces hepatic fibrosis in mice (66,142,146). Imipramine blocks ethanol-induced ASMase activation and Cer generation, ameliorating hepatic steatosis (67,98,102). ASMase-deficient mice are resistant to alcohol-induced lipogenesis, macrosteatosis, LPS sensitization, and concanavalin A-mediated liver injury (48, 50, 68).…”

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.…”

“…Both endogenous and overexpressed Sesn3 proteins were observed between 50 and 55 kDa, indicating that GFP might be cleaved out from the recombinant protein ( Ethanol suppresses hepatic Sestrins expression in mice chronically fed with ethanol. 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).…”

“…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.…”