Mus musculus strain:C57BL/6J Transcriptome or Gene expression

Background: Paracrine activation of hepatic cannabinoid receptor 1 by hepatic stellate cell (HSC)-derived 2-arachidnoylglycerol (2-AG) is one of the critical mechanisms mediating alcoholic steatosis by stimulating de novo lipogenesis in hepatocytes. However, the precise mechanism of 2-AG production in HSCs is unknown.Methods: To examine alcohol-induced glutamate excretion, endocannabinoid production and consequent steatosis, C57BL/6 wild type, global metabotropic glutamate receptor 5 (mGluR5) knockout and hepatocyte-specific xCT knockout mice were fed liquid ethanol diet for 8 weeks. RNA sequencing, histology, immunoblots, quantitative polymerase chain reaction, and metabolite measurements were performed.Results: We found that chronic ethanol consumption significantly increased glutamate levels in blood and liver of ethanol-fed mice and blood of patients with alcoholic liver disease compared to controls. To find out the mechanism, RNA sequencing analysis exhibited the upregulation of xCT (a cystine-glutamate antiporter) through the antioxidant transcription factor Nrf2 in the liver of ethanol-fed mice. Mechanistically, cysteine deficiency and subsequent glutathione depletion was induced by impaired transsulfuration pathway in ethanol-fed mice, which leads to the glutamate excretion for cystine uptake (immediately reduced to cysteine) via upregulation of xCT in hepatocytes. Intriguingly, comparing with other hepatic cells, the mGluR5 was highly expressed in HSCs and 2-AG production in HSCs was remarkably increased by mGluR5 activation. Consistently, genetic or pharmacologic inhibition of mGluR5 or xCT significantly attenuated alcoholic steatosis in ethanol-fed mice, followed by suppression of 2-AG production and de novo lipogenesis.Conclusion: Taken together, our findings demonstrate that increased excretion of hepatic glutamate by xCT as a lipogenic mediator promotes alcoholic steatosis through mGluR5-mediated 2-AG production in HSCs, which could be a potential therapeutic target for alcoholic liver disease.

背景：肝星状细胞（hepatic stellate cell, HSC）分泌的2-花生四烯酰甘油（2-arachidonoylglycerol, 2-AG）旁激活肝大麻素受体1（hepatic cannabinoid receptor 1），是通过刺激肝细胞内的从头脂肪生成（de novo lipogenesis）介导酒精性脂肪变性的关键机制之一。然而，肝星状细胞中2-AG的具体生成机制尚不明确。
方法：为探究酒精诱导的谷氨酸排泄、内源性大麻素生成及由此引发的脂肪变性，本研究将C57BL/6野生型小鼠、全局代谢型谷氨酸受体5（metabotropic glutamate receptor 5, mGluR5）敲除小鼠及肝细胞特异性xCT敲除小鼠饲喂液体乙醇饲料8周。随后开展了RNA测序、组织学分析、免疫印迹实验、定量聚合酶链式反应（quantitative polymerase chain reaction, qPCR）及代谢物检测。
结果：本研究发现，与对照组相比，慢性乙醇摄入可显著升高乙醇饲喂小鼠血液及肝脏中的谷氨酸水平，同时升高酒精性肝病患者血液中的谷氨酸水平。为明确其机制，RNA测序分析显示，乙醇饲喂小鼠肝脏中，胱氨酸-谷氨酸逆向转运蛋白（cystine-glutamate antiporter, xCT）通过抗氧化转录因子Nrf2发生上调。机制层面，乙醇饲喂小鼠的转硫通路受损，引发半胱氨酸缺乏及后续谷胱甘肽耗竭，进而通过上调肝细胞内xCT，促进谷氨酸排泄以摄取胱氨酸（胱氨酸可快速还原为半胱氨酸）。有趣的是，与其他肝细胞相比，代谢型谷氨酸受体5在肝星状细胞中高表达，且肝星状细胞内的2-AG生成可通过mGluR5激活显著增加。与之一致的是，对mGluR5或xCT进行遗传或药理学抑制，可显著减轻乙醇饲喂小鼠的酒精性脂肪变性，同时抑制2-AG生成与从头脂肪生成。
结论：综上，本研究结果表明，xCT介导的肝脏谷氨酸排泄作为成脂介质，通过肝星状细胞中mGluR5介导的2-AG生成促进酒精性脂肪变性，该通路或可成为酒精性肝病的潜在治疗靶点。