(The) mGluR5-mediated endocannabinoid production of hepatic stellate cells causes alcoholic fatty liver

Abstract

Part Ⅰ: The mGluR5-Mediated Endocannabinoid Production of Hepatic Stellate Cells Causes Alcoholic Fatty Liver 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. Here, we found that chronic ethanol consumption significantly increased glutamate levels both in mice and in human patients. RNA-Seq 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. 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.

Part Ⅱ. Experimental Applications of in situ Liver Perfusion Machinery for the Study of Liver Disease The liver is involved in a wide range of activities in vertebrates and some other animals, including metabolism, protein synthesis, detoxification, and the immune system. Until now, various methods have been devised to study liver diseases

however, each method has its own limitations. In situ liver perfusion machinery, originally developed in rats, has been successfully adapted to mice, enabling the study of liver diseases. Here we describe the protocol, which is a simple but widely applicable method for investigating the liver diseases. The liver is perfused in situ by cannulation of the portal vein and suprahepatic inferior vena cava (IVC), with antegrade closed circuit circulation completed by clamping the infrahepatic IVC. In situ liver perfusion can be utilized to evaluate immune cell migration and function, hemodynamics and related cellular reactions in each type of hepatic cells, and the metabolism of toxic or other compounds by changing the composition of the circulating media. In situ liver perfusion method maintains liver function and cell viability for up to 2 h. This study also describes an optional protocol using density-gradient centrifugation for the separation of different types of hepatic cells, allowing the determination of changes in each cell type. In summary, this method of in situ liver perfusion will be useful for studying liver diseases as a complement to other established methods.