The spectrum of nonalcoholic fatty liver disease (NAFLD) includes a nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). The specific types and amounts of lipids that accumulate in NAFLD are not fully defined. The free fatty acid (FFA), diacylglycerol (DAG), triacylglycerol (TAG), free cholesterol (FC), cholesterol ester, and phospholipid contents in normal livers were quantified and compared to those of NAFL and NASH, and the distribution of fatty acids within these classes was compared across these groups. Hepatic lipids were quantified by capillary gas chromatography. N onalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease in North America. 1 NAFLD is associated with insulin resistance and the metabolic syndrome. 2,3 The clinical-histologic spectrum of NAFLD extends from a nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH). 4 Although NASH is distinguished from NAFL by the presence of cytologic ballooning and inflammation, both conditions are characterized by a fatty liver. 4,5 Thus, hepatic fat accumulation is the hallmark of NAFLD.The compositions of the lipids that accumulate in the livers of subjects with NAFLD are not well characterized. Most of the published literature has focused on triglyceride accumulation as the key defect in NAFLD. 6,7 However, it is not known whether there are substantial changes in other lipid classes, such as cholesterol and specific phospholipids (PLs). Although an increase in the n-6:n-3 fatty acid ratio in total lipids in NAFLD has been described recently, 8,9 the distribution of these fatty acids within specific lipid classes has not been extensively characterized. Given the important biological activities of many lipids, such information could provide potential insights into the pathophysiology of NAFLD and the metabolic syndrome.A lipidomic approach was taken to quantify the major lipid classes and the distribution of fatty acids within these classes in the liver. The specific aims of the study were to (1) quantify the absolute and relative amounts of free fatty acids (FFAs), diacylglycerol (DAG), triacylglycerol

The expression of microRNA in nonalcoholic steatohepatitis (NASH) and their role in the genesis of NASH are not known. The aims of this study were to: (1) identify differentially expressed microRNAs in human NASH, (2) tabulate their potential targets, and (3) define the effect of a specific differentially expressed microRNA, miR-122, on its targets and compare these effects with the pattern of expression of these targets in human NASH. The expression of 474 human microRNAs was compared in subjects with the metabolic syndrome and NASH versus controls with normal liver histology. Differentially expressed microRNAs were identified by the Paraflo microRNA microarray assay and validated using quantitative real-time polymerase chain reaction (PCR). The effects of a specific differentially expressed miRNA (miR-122) on its predicted targets were assessed by silencing and overexpressing miR-122 in vitro. A total of 23 microRNAs were underexpressed or overexpressed. The predicted targets of these microRNAs are known to affect cell proliferation, protein translation, apoptosis, inflammation, oxidative stress, and metabolism. The miR-122 level was significantly decreased in subjects with NASH (63% by real-time PCR, P < 0.00001). Silencing miR-122 led to an initial increase in mRNA levels of these targets (P < 0.05 for all) followed by a decrease by 48 hours. This was accompanied by an increase in protein levels of these targets (P < 0.05 for all). Overexpression of miR-122 led to a significant decrease in protein levels of these targets. Conclusions: NASH is associated with altered hepatic microRNA expression. Underexpression of miR-122 potentially contributes to altered lipid metabolism implicated in the pathogenesis of NASH. (HEPATOLOGY 2008;48: 1810-1820.)

Specific alterations in hepatic lipid composition characterize the spectrum of nonalcoholic fatty liver disease (NAFLD), which extends from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH). However, the plasma lipidome of NAFLD and whether NASH has a distinct plasma lipidomic signature are unknown. A comprehensive analysis of plasma lipids and eicosanoid metabolites quantified by mass spectrometry was performed in NAFL (n = 25) and NASH (n = 50) subjects and compared with lean normal controls (n = 50). The key findings include significantly increased total plasma monounsaturated fatty acids driven by palmitoleic (16:1 n7) and oleic (18:1 n9) acids content (P < 0.01 for both acids in both NAFL and NASH). The levels of palmitoleic acid, oleic acid, and palmitoleic acid to palmitic acid (16:0) ratio were significantly increased in NAFLD across multiple lipid classes. Linoleic acid (8:2n6) was decreased (P < 0.05), with a concomitant increase in γ-linolenic (18:3n6) and dihomo γ-linolenic (20:3n6) acids in both NAFL and NASH (P < 0.001 for most lipid classes). The docosahexanoic acid (22:6 n3) to docosapentenoic acid (22:5n3) ratio was significantly decreased within phosphatidylcholine (PC), and phosphatidylethanolamine (PE) pools, which was most marked in NASH subjects (P < 0.01 for PC and P < 0.001 for PE). The total plasmalogen levels were significantly decreased in NASH compared with controls (P < 0.05). A stepwise increase in lipoxygenase (LOX) metabolites 5(S)-hydroxyeicosatetraenoic acid (5-HETE), 8-HETE, and 15-HETE characterized progression from normal to NAFL to NASH. The level of 11-HETE, a nonenzymatic oxidation product of arachidonic (20:4) acid, was significantly increased in NASH only. Conclusions: Although increased lipogenesis, desaturases, and LOX activities characterize NAFL and NASH, impaired peroxisomal polyunsaturated fatty acid (PUFA) metabolism and nonenzymatic oxidation is associated with progression to NASH.

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