Abstract
Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder in western countries. Despite the high prevalence of NAFLD, the underlying biology of the disease progression is not clear, and there are no approved drugs to treat non-alcoholic steatohepatitis (NASH), the most advanced form of the disease. Thus, there is an urgent need for developing advanced in vitro human cellular systems to study disease mechanisms and drug responses. We attempted to create an organoid system genetically predisposed to NAFLD and to induce steatosis and fibrosis in it by adding free fatty acids. We used multilineage 3D spheroids composed by hepatocytes (HepG2) and hepatic stellate cells (LX-2) with a physiological ratio (24:1). HepG2 and LX-2 cells are homozygotes for the PNPLA3 I148M sequence variant, the strongest genetic determinant of NAFLD. We demonstrate that hepatic stellate cells facilitate the compactness of 3D spheroids. Then, we show that the spheroids develop accumulations of fat and collagen upon exposure to free fatty acids. Finally, this accumulation was rescued by incubating spheroids with liraglutide or elafibranor, drugs that are in clinical trials for the treatment of NASH. In conclusion, we have established a simple, easy to handle, in vitro model of genetically induced NAFLD consisting of multilineage 3D spheroids. This tool may be used to understand molecular mechanisms involved in the early stages of fibrogenesis induced by lipid accumulation. Moreover, it may be used to identify new compounds to treat NASH using high-throughput drug screening.
Highlights
Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder in western countries, affecting 17–46% of adults, and is becoming the major cause of liver disease and transplantation [1]
NAFLD encompasses a wide spectrum of pathologic conditions, including hepatic steatosis and non-alcoholic steatohepatitis (NASH), which can further progress to fibrosis, cirrhosis and hepatocellular carcinoma [2]
Carriers of the patatin like phospholipase domain containing 3 (PNPLA3) I148M variant have several specific characteristics including a lower hepatic very-low density lipoprotein (VLDL) secretion [27], lower turnover of retinol in stellate cells [28,29,30,31] and a specific signature of circulating lipids [32]. They display a diverse response to statins [33], fenofibrate and omega 3 fatty acids [34,35] and present more hepatotoxicity after treatment for leukemia [36]. All these findings suggest that PNPLA3 I148M associated disease represents a separate entity within NAFLD
Summary
Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder in western countries, affecting 17–46% of adults, and is becoming the major cause of liver disease and transplantation [1]. NAFLD encompasses a wide spectrum of pathologic conditions, including hepatic steatosis and non-alcoholic steatohepatitis (NASH), which can further progress to fibrosis, cirrhosis and hepatocellular carcinoma [2]. Hallmark of this spectrum is an excessive accumulation of fat (steatosis) exceeding 5% of total liver weight [3,4,5]. To understand molecular mechanisms of NAFLD and to identify new drugs several in vitro models of fatty liver disease based on 2D cell culture and in vivo mouse models have been exploited [9,10]. New in vitro models mimicking liver complexity and better emulating pathophysiology of NAFLD and NASH are highly needed. Clinical studies show how severe fibrosis is the best predictor of mortality in patients with NAFLD [16]
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