Pavlina T. Ivanova

Adiponutrin Functions as a Nutritionally Regulated Lysophosphatidic Acid Acyltransferase

Numerous studies in humans link a nonsynonymous genetic polymorphism (I148M) in adiponutrin (ADPN) to various forms of fatty liver disease and liver cirrhosis. Despite its high clinical relevance, the molecular function of ADPN and the mechanism by which I148M variant affects hepatic metabolism are unclear. Here we show that ADPN promotes cellular lipid synthesis by converting lysophosphatidic acid (LPA) into phosphatidic acid. The ADPN-catalyzed LPA acyltransferase (LPAAT) reaction is specific for LPA and long-chain acyl-CoAs. Wild-type mice receiving a high-sucrose diet exhibit substantial upregulation of Adpn in the liver and a concomitant increase in LPAAT activity. In Adpn-deficient mice, this diet-induced increase in hepatic LPAAT activity is reduced. Notably, the I148M variant of human ADPN exhibits increased LPAAT activity leading to increased cellular lipid accumulation. This gain of function provides a plausible biochemical mechanism for the development of liver steatosis in subjects carrying the I148M variant.

Chronic overexpression of PNPLA3 I148M in mouse liver causes hepatic steatosis

A genetic variant in PNPLA3 (PNPLA3(I148M)), a triacylglycerol (TAG) hydrolase, is a major risk factor for nonalcoholic fatty liver disease (NAFLD); however, the mechanism underlying this association is not known. To develop an animal model of PNPLA3-induced fatty liver disease, we generated transgenic mice that overexpress similar amounts of wild-type PNPLA3 (PNPLA3(WT)) or mutant PNPLA3 (PNPLA3(I148M)) either in liver or adipose tissue. Overexpression of the transgenes in adipose tissue did not affect liver fat content. Expression of PNPLA3(I148M), but not PNPLA3(WT), in liver recapitulated the fatty liver phenotype as well as other metabolic features associated with this allele in humans. Metabolic studies provided evidence for 3 distinct alterations in hepatic TAG metabolism in PNPLA3(I148M) transgenic mice: increased formation of fatty acids and TAG, impaired hydrolysis of TAG, and relative depletion of TAG long-chain polyunsaturated fatty acids. These findings suggest that PNPLA3 plays a role in remodeling TAG in lipid droplets, as they accumulate in response to food intake, and that the increase in hepatic TAG levels associated with the I148M substitution results from multiple changes in hepatic TAG metabolism. The development of an animal model that recapitulates the metabolic phenotype of the allele in humans provides a new platform in which to elucidate the role of PNLPA3(I148M) in NAFLD.

Overview of the Alliance for Cellular Signaling

The Alliance for Cellular Signaling is a large-scale collaboration designed to answer global questions about signalling networks. Pathways will be studied intensively in two cells — B lymphocytes (the cells of the immune system) and cardiac myocytes — to facilitate quantitative modelling. One goal is to catalyse complementary research in individual laboratories; to facilitate this, all alliance data are freely available for use by the entire research community.The Alliance for Cellular Signaling is a large-scale collaboration designed to answer global questions about signalling networks. Pathways will be studied intensively in two cells — B lymphocytes (the cells of the immune system) and cardiac myocytes — to facilitate quantitative modelling. One goal is to catalyse complementary research in individual laboratories; to facilitate this, all alliance data are freely available for use by the entire research community.

The TMAO-Generating Enzyme Flavin Monooxygenase 3 Is a Central Regulator of Cholesterol Balance

Circulating levels of the gut microbe-derived metabolite trimethylamine-N-oxide (TMAO) have recently been linked to cardiovascular disease (CVD) risk. Here, we performed transcriptional profiling in mouse models of altered reverse cholesterol transport (RCT) and serendipitously identified the TMAO-generating enzyme flavin monooxygenase 3 (FMO3) as a powerful modifier of cholesterol metabolism and RCT. Knockdown of FMO3 in cholesterol-fed mice alters biliary lipid secretion, blunts intestinal cholesterol absorption, and limits the production of hepatic oxysterols and cholesteryl esters. Furthermore, FMO3 knockdown stimulates basal and liver X receptor (LXR)-stimulated macrophage RCT, thereby improving cholesterol balance. Conversely, FMO3 knockdown exacerbates hepatic endoplasmic reticulum (ER) stress and inflammation in part by decreasing hepatic oxysterol levels and subsequent LXR activation. FMO3 is thus identified as a central integrator of hepatic cholesterol and triacylglycerol metabolism, inflammation, and ER stress. These studies suggest that the gut microbiota-driven TMA/FMO3/TMAO pathway is a key regulator of lipid metabolism and inflammation.

Lipidomics: a mass spectrometry based systems level analysis of cellular lipids.

Lipidomics is a logical outcome of the history and traditions of lipid biochemistry and advances in mass spectrometry are at the heart of a renaissance in understanding the roles of lipids in cellular functions. Our desire to understand the complexity of lipids in biology has led to new techniques that allow us to identify over 1000 phospholipids in mammalian cell types and tissues. Improvements in chromatographic separation and mass spectrometry have positioned us to determine not only the lipid composition (i.e. parts list) of cells and tissues, but also address questions regarding lipid substrates and products that previously overwhelmed traditional analytical technologies. In the decade since lipidomics was conceived much of the efforts have been on new methodologies, development of computer programs to decipher the gigabytes of raw data, and struggling with the highly variable nature of biological systems where absolute quantities of a given metabolite may be less important than its relative change in concentration. It is clear that the technology is now sufficiently developed to address fundamental questions about the roles of lipids in cellular signaling and metabolic pathways.

Electrospray ionization mass spectrometry analysis of changes in phospholipids in RBL-2H3 mastocytoma cells during degranulation.

Biological membranes contain an extraordinary diversity of lipids. Phospholipids function as major structural elements of cellular membranes, and analysis of changes in the highly heterogeneous mixtures of lipids found in eukaryotic cells is central to understanding the complex functions in which lipids participate. Phospholipase-catalyzed hydrolysis of phospholipids often follows cell surface receptor activation. Recently, we demonstrated that granule fusion is initiated by addition of exogenous, nonmammalian phospholipases to permeabilized mast cells. To pursue this finding, we use positive and negative mode Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) to measure changes in the glycerophospholipid composition of total lipid extracts of intact and permeabilized RBL-2H3 (mucosal mast cell line) cells. The low energy of the electrospray ionization results in efficient production of molecular ions of phospholipids uncomplicated by further fragmentation, and changes were observed that eluded conventional detection methods. From these analyses we have spectrally resolved more than 130 glycerophospholipids and determined changes initiated by introduction of exogenous phospholipase C, phospholipase D, or phospholipase A2. These exogenous phospholipases have a preference for phosphatidylcholine with long polyunsaturated alkyl chains as substrates and, when added to permeabilized mast cells, produce multiple species of mono- and polyunsaturated diacylglycerols, phosphatidic acids, and lysophosphatidylcholines, respectively. The patterns of changes of these lipids provide an extraordinarily rich source of data for evaluating the effects of specific lipid species generated during cellular processes, such as exocytosis.

Increased diacylglycerols characterize hepatic lipid changes in progression of human nonalcoholic fatty liver disease; comparison to a murine model.

Background and Aims The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and progression to cirrhosis. While differences in liver lipids between disease states have been reported, precise composition of phospholipids and diacylglycerols (DAG) at a lipid species level has not been previously described. The goal of this study was to characterize changes in lipid species through progression of human NAFLD using advanced lipidomic technology and compare this with a murine model of early and advanced NAFLD. Methods Utilizing mass spectrometry lipidomics, over 250 phospholipid and diacylglycerol species (DAGs) were identified in normal and diseased human and murine liver extracts. Results Significant differences between phospholipid composition of normal and diseased livers were demonstrated, notably among DAG species, consistent with previous reports that DAG transferases are involved in the progression of NAFLD and liver fibrosis. In addition, a novel phospholipid species (ether linked phosphatidylinositol) was identified in human cirrhotic liver extracts. Conclusions Using parallel lipidomics analysis of murine and human liver tissues it was determined that mice maintained on a high-fat diet provide a reproducible model of NAFLD in regards to specificity of lipid species in the liver. These studies demonstrated that novel lipid species may serve as markers of advanced liver disease and importantly, marked increases in DAG species are a hallmark of NAFLD. Elevated DAGs may contribute to altered triglyceride, phosphatidylcholine (PC), and phosphatidylethanolamine (PE) levels characteristic of the disease and specific DAG species might be important lipid signaling molecules in the progression of NAFLD.

Biomarkers of NAFLD progression: a lipidomics approach to an epidemic

The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and cirrhosis. Recognition and timely diagnosis of these different stages, particularly NASH, is important for both potential reversibility and limitation of complications. Liver biopsy remains the clinical standard for definitive diagnosis. Diagnostic tools minimizing the need for invasive procedures or that add information to histologic data are important in novel management strategies for the growing epidemic of NAFLD. We describe an “omics” approach to detecting a reproducible signature of lipid metabolites, aqueous intracellular metabolites, SNPs, and mRNA transcripts in a double-blinded study of patients with different stages of NAFLD that involves profiling liver biopsies, plasma, and urine samples. Using linear discriminant analysis, a panel of 20 plasma metabolites that includes glycerophospholipids, sphingolipids, sterols, and various aqueous small molecular weight components involved in cellular metabolic pathways, can be used to differentiate between NASH and steatosis. This identification of differential biomolecular signatures has the potential to improve clinical diagnosis and facilitate therapeutic intervention of NAFLD.

Synaptic vesicle-like lipidome of human cytomegalovirus virions reveals a role for SNARE machinery in virion egress

Human cytomegalovirus induces and requires fatty acid synthesis. This suggests an essential role for lipidome remodeling in viral replication. We used mass spectrometry to quantify glycerophospholipids in mock-infected and virus-infected fibroblasts, as well as in virions. Although the lipid composition of mock-infected and virus-infected fibroblasts was similar, virions were markedly different. The virion envelope contained twofold more phosphatidylethanolamines and threefold less phosphatidylserines than the host cell. This indicates that the virus buds from a membrane with a different lipid composition from the host cell as a whole. Compared with published datasets, the virion envelope showed the greatest similarity to the synaptic vesicle lipidome. Synaptosome-associated protein of 25 kDa (SNAP-25) is a component of the complex that mediates exocytosis of synaptic vesicles in neurons; and its homolog, SNAP-23, functions in exocytosis in many other cell types. Infection induced the relocation of SNAP-23 to the cytoplasmic viral assembly zone, and knockdown of SNAP-23 inhibited the production of virus. We propose that cytomegalovirus capsids acquire their envelope by budding into vesicles with a lipid composition similar to that of synaptic vesicles, which subsequently fuse with the plasma membrane to release virions from the cell.

Quantitative analysis of glycerophospholipids by LC-MS: acquisition, data handling, and interpretation

As technology expands what it is possible to accurately measure, so too the challenges faced by modern mass spectrometry applications expand. A high level of accuracy in lipid quantitation across thousands of chemical species simultaneously is demanded. While relative changes in lipid amounts with varying conditions may provide initial insights or point to novel targets, there are many questions that require determination of lipid analyte absolute quantitation. Glycerophospholipids present a significant challenge in this regard, given the headgroup diversity, large number of possible acyl chain combinations, and vast range of ionization efficiency of species. Lipidomic output is being used more often not just for profiling of the masses of species, but also for highly-targeted flux-based measurements which put additional burdens on the quantitation pipeline. These first two challenges bring into sharp focus the need for a robust lipidomics workflow including deisotoping, differentiation from background noise, use of multiple internal standards per lipid class, and the use of a scriptable environment in order to create maximum user flexibility and maintain metadata on the parameters of the data analysis as it occurs. As lipidomics technology develops and delivers more output on a larger number of analytes, so must the sophistication of statistical post-processing also continue to advance. High-dimensional data analysis methods involving clustering, lipid pathway analysis, and false discovery rate limitation are becoming standard practices in a maturing field.