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

Dysregulation of lipid metabolism in individual tissues can lead to systemic disruption of insulin action and glucose metabolism. Utilizing a comprehensive lipidomic platform and mice deficient in adipose tissue lipid chaperones aP2 and mal1, we explored how metabolic alterations in adipose tissue are linked to whole-body metabolism through lipid signals. A robust increase in de novo lipogenesis rendered the adipose tissue of these mice resistant to the deleterious systemic effects of dietary lipid exposure. Systemic lipid profiling also led to identification of C16:1n7-palmitoleate as an adipose tissue-derived lipid hormone that strongly stimulates muscle insulin action and suppresses hepatosteatosis. Our data reveal a novel, lipid-mediated endocrine network and demonstrate that adipose tissue uses lipokines such as C16:1n7-palmitoleate to communicate with distant organs and regulate systemic metabolic homeostasis.

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.

Macrophages exhibit endoplasmic reticulum (ER) stress when exposed to lipotoxic signals associated with atherosclerosis, although the pathophysiological significance and the underlying mechanisms remain unknown. Here, we demonstrate that mitigation of ER stress with a chemical chaperone results in marked protection against lipotoxic death in macrophages and prevents macrophage fatty acid binding protein-4 (aP2) expression. Utilizing genetic and chemical models, we show that aP2 is the predominant regulator of lipid-induced macrophage ER stress. Lipid chaperone effects are mediated by the production of phospholipids rich in monounsaturated fatty acids and bioactive lipids that render macrophages resistant to lipid-induced ER stress. Furthermore, aP2’s impact on macrophage lipid metabolism and ER stress response is mediated by upregulation of key lipogenic enzymes by the liver X receptor. Our results demonstrate the central role for lipid chaperones in regulating ER homeostasis in macrophages in atherosclerosis and that ER responses can be modified, genetically or chemically, to protect the organism against the deleterious effects of hyperlipidemia.

Although statins are widely prescribed medications, there remains considerable variability in therapeutic response. Genetics can explain only part of this variability. Metabolomics is a global biochemical approach that provides powerful tools for mapping pathways implicated in disease and in response to treatment. Metabolomics captures net interactions between genome, microbiome and the environment. In this study, we used a targeted GC-MS metabolomics platform to measure a panel of metabolites within cholesterol synthesis, dietary sterol absorption, and bile acid formation to determine metabolite signatures that may predict variation in statin LDL-C lowering efficacy. Measurements were performed in two subsets of the total study population in the Cholesterol and Pharmacogenetics (CAP) study: Full Range of Response (FR), and Good and Poor Responders (GPR) were 100 individuals randomly selected from across the entire range of LDL-C responses in CAP. GPR were 48 individuals, 24 each from the top and bottom 10% of the LDL-C response distribution matched for body mass index, race, and gender. We identified three secondary, bacterial-derived bile acids that contribute to predicting the magnitude of statin-induced LDL-C lowering in good responders. Bile acids and statins share transporters in the liver and intestine; we observed that increased plasma concentration of simvastatin positively correlates with higher levels of several secondary bile acids. Genetic analysis of these subjects identified associations between levels of seven bile acids and a single nucleotide polymorphism (SNP), rs4149056, in the gene encoding the organic anion transporter SLCO1B1. These findings, along with recently published results that the gut microbiome plays an important role in cardiovascular disease, indicate that interactions between genome, gut microbiome and environmental influences should be considered in the study and management of cardiovascular disease. Metabolic profiles could provide valuable information about treatment outcomes and could contribute to a more personalized approach to therapy.

Statins are commonly used for reducing cardiovascular disease risk but therapeutic benefit and reductions in levels of low-density lipoprotein cholesterol (LDL-C) vary among individuals. Other effects, including reductions in C-reactive protein (CRP), also contribute to treatment response. Metabolomics provides powerful tools to map pathways implicated in variation in response to statin treatment. This could lead to mechanistic hypotheses that provide insight into the underlying basis for individual variation in drug response. Using a targeted lipidomics platform, we defined lipid changes in blood samples from the upper and lower tails of the LDL-C response distribution in the Cholesterol and Pharmacogenetics study. Metabolic changes in responders are more comprehensive than those seen in non-responders. Baseline cholesterol ester and phospholipid metabolites correlated with LDL-C response to treatment. CRP response to therapy correlated with baseline plasmalogens, lipids involved in inflammation. There was no overlap of lipids whose changes correlated with LDL-C or CRP responses to simvastatin suggesting that distinct metabolic pathways govern statin effects on these two biomarkers. Metabolic signatures could provide insights about variability in response and mechanisms of action of statins.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-010-0207-x) contains supplementary material, which is available to authorized users.

There is sparse information about specific storage and handling protocols that minimize analytical error and variability in samples evaluated by targeted metabolomics. Variance components that affect quantitative lipid analysis in a set of human serum samples were determined. The effects of freeze-thaw, extraction state, storage temperature, and freeze-thaw prior to density-based lipoprotein fractionation were quantified. The quantification of high abundance metabolites, representing the biologically relevant lipid species in humans, was highly repeatable (with coefficients of variation as low as 0.01 and 0.02) and largely unaffected by 1-3 freeze-thaw cycles (with 0-8% of metabolites affected in each lipid class). Extraction state had effects on total lipid class amounts, including decreased diacylglycerol and increased phosphatidylethanolamine in thawed compared with frozen samples. The effects of storage temperature over 1 week were minimal, with 0-4% of metabolites affected by storage at 4°C, -20°C, or -80°C in most lipid classes, and 19% of metabolites in diacylglycerol affected by storage at -20°C. Freezing prior to lipoprotein fractionation by density ultracentrifugation decreased HDL free cholesterol by 37% and VLDL free fatty acid by 36%, and increased LDL cholesterol ester by 35% compared with fresh samples. These findings suggest that density-based fractionation should preferably be undertaken in fresh serum samples because up to 37% variability in HDL and LDL cholesterol could result from a single freeze-thaw cycle. Conversely, quantitative lipid analysis within unfractionated serum is minimally affected even with repeated freeze-thaw cycles.

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