Liver-specific ceramide reduction alleviates steatosis and insulin resistance in alcohol-fed mice. Liver-specific ceramide reduction alleviates steatosis and insulin resistance in alcohol-fed mice

Alcohol’s impairment of both hepatic lipid metabolism and insulin resistance (IR) are key drivers of alcoholic steatosis, the initial stage of alcoholic liver disease (ALD). Pharmacologic reduction of lipotoxic ceramide prevents alcoholic steatosis and glucose intolerance in mice, but potential off-target effects limit its strategic utility. Here, we employed a hepatic-specific acid-ceramidase (ASAH) overexpression model to reduce hepatic ceramides in a Lieber-DeCarli model of experimental alcoholic steatosis. We examined effects of alcohol on hepatic lipid metabolism, body composition, energy homeostasis and insulin sensitivity as measured by hyperinsulinemic-euglycemic clamp. Our results demonstrate that hepatic ceramide reduction ameliorates the effects of alcohol on hepatic lipid droplet accumulation by promoting VLDL secretion and lipophagy, the latter of which involves ceramide cross-talk between the lysosomal and lipid droplet compartments. We additionally demonstrate that hepatic ceramide reduction prevents alcohol’s inhibition of hepatic insulin signaling. These effects on the liver are associated with a reduction in oxidative stress markers and are relevant to humans, as we observe peri-lipid droplet ASAH expression in human ALD. Together, our results suggest a potential role for hepatic ceramide inhibition in preventing ALD. Overall design: Liver RNA from mice fed an alcohol diet mice was analyzed by RNAseq (N=4/group). Hepatic ceramides were reduced by inducing the transgene ASAH1, a ceramidase, in a liver-specific, dox-inducible fashion. To assess the optimal control for transgene induction, we performed RNAseq to compare strategies varying either genetics (transgene- or transgene+ fed Etoh-dox) or diet (transgene+ mice fed an Etoh or Etoh-dox diet).

酒精对肝脏脂质代谢与胰岛素抵抗（insulin resistance, IR）的损伤作用，是酒精性肝病（alcoholic liver disease, ALD）初始阶段——酒精性脂肪变性的核心驱动因素。通过药物手段降低脂毒性神经酰胺，可在小鼠模型中缓解酒精性脂肪变性与葡萄糖耐量异常，但潜在的脱靶效应限制了其应用前景。本研究构建了肝脏特异性过表达酸性神经酰胺酶（acid-ceramidase, ASAH）的模型，以在李贝尔-德卡里（Lieber-DeCarli）实验性酒精性脂肪变性模型中降低肝脏神经酰胺水平。我们通过高胰岛素-正常血糖钳夹试验（hyperinsulinemic-euglycemic clamp）评估了酒精对肝脏脂质代谢、身体成分、能量稳态与胰岛素敏感性的影响。研究结果显示，肝脏神经酰胺降低可通过促进极低密度脂蛋白（very low-density lipoprotein, VLDL）分泌与脂噬作用（lipophagy），改善酒精诱导的肝脏脂滴蓄积；其中脂噬作用涉及溶酶体与脂滴区室之间的神经酰胺信号串扰。此外，肝脏神经酰胺降低可阻断酒精对肝脏胰岛素信号通路的抑制作用。上述肝脏保护效应与氧化应激标志物水平降低相关，且该结果适用于人类：我们在人类酒精性肝病患者的脂滴周围观察到了ASAH的表达。综上，本研究结果提示肝脏神经酰胺抑制在预防酒精性肝病中具有潜在应用价值。实验整体设计：通过肝脏特异性多西环素诱导型（dox-inducible）转基因系统，诱导神经酰胺酶ASAH1表达以降低肝脏神经酰胺水平。我们对喂食酒精饲料的小鼠肝脏组织进行RNA测序（RNAseq, 每组n=4）。为优化转基因诱导的对照策略，我们开展了RNA测序以对比两种分组方案：一是遗传学分组（携带转基因与否，以及喂食酒精+多西环素饲料），二是饮食分组（携带转基因的小鼠分别喂食酒精饲料或酒精+多西环素饲料）。