Regulated accumulation of desmosterol integrates macrophage lipid metabolism and inflammatory responses.

Inflammation and macrophage foam cells are characteristic features of atherosclerotic lesions, but the mechanisms linking cholesterol accumulation to inflammation and LXR-dependent response pathways are poorly understood. To investigate this relationship, we utilized lipidomic and transcriptomic methods to evaluate the effect of diet and LDL receptor genotype on macrophage foam cell formation within the peritoneal cavities of mice. Foam cell formation was associated with significant changes in hundreds of lipid species and unexpected suppression, rather than activation, of inflammatory gene expression. We provide evidence that regulated accumulation of desmosterol underlies many of the homeostatic responses, including activation of LXR target genes, inhibition of SREBP target genes, selective reprogramming of fatty acid metabolism, and suppression of inflammatory-response genes, observed in macrophage foam cells. These observations suggest that macrophage activation in atherosclerotic lesions results from extrinsic, proinflammatory signals generated within the artery wall that suppress homeostatic and anti-inflammatory functions of desmosterol.

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.

Enhanced synthesis of saturated phospholipids is associated with ER stress and lipotoxicity in palmitate treated hepatic cells.

High levels of saturated FAs (SFAs) are acutely toxic to a variety of cell types, including hepatocytes, and have been associated with diseases such as type 2 diabetes and nonalcoholic fatty liver disease. SFA accumulation has been previously shown to degrade endoplasmic reticulum (ER) function leading to other manifestations of the lipoapoptotic cascade. We hypothesized that dysfunctional phospholipid (PL) metabolism is an initiating factor in this ER stress response. Treatment of either primary hepatocytes or H4IIEC3 cells with the SFA palmitate resulted in dramatic dilation of the ER membrane, coinciding with other markers of organelle dysfunction. This was accompanied by increased de novo glycerolipid synthesis, significant elevation of dipalmitoyl phosphatidic acid, diacylglycerol, and total PL content in H4IIEC3 cells. Supplementation with oleate (OA) reversed these markers of palmitate (PA)-induced lipotoxicity. OA/PA cotreatment modulated the distribution of PA between lipid classes, increasing the flux toward triacylglycerols while reducing its incorporation into PLs. Similar trends were demonstrated in both primary hepatocytes and the H4IIEC3 hepatoma cell line. Overall, these findings suggest that modifying the FA composition of structural PLs can protect hepatocytes from PA-induced ER stress and associated lipotoxicity.

Regulation of phospholipase D activity and phosphatidic acid production after purinergic (P2Y6) receptor stimulation.

Phosphatidic acid (PA) is a lipid second messenger located at the intersection of several lipid metabolism and cell signaling events including membrane trafficking, survival, and proliferation. Generation of signaling PA has long been primarily attributed to the activation of phospholipase D (PLD). PLD catalyzes the hydrolysis of phosphatidylcholine into PA. A variety of both receptor-tyrosine kinase and G-protein-coupled receptor stimulations have been shown to lead to PLD activation and PA generation. This study focuses on profiling the PA pool upon P2Y6 receptor signaling manipulation to determine the major PA producing enzymes. Here we show that PLD, although highly active, is not responsible for the majority of stable PA being produced upon UDP stimulation of the P2Y6 receptor and that PA levels are tightly regulated. By following PA flux in the cell we show that PLD is involved in an initial increase in PA upon receptor stimulation; however, when PLD is blocked, the cell compensates by increasing PA production from other sources. We further delineate the P2Y6 signaling pathway showing that phospholipase Cß3 (PLCß3), PLCδ1, DGKζ and PLD are all downstream of receptor activation. We also show that DGKζ is a novel negative regulator of PLD activity in this system that occurs through an inhibitory mechanism with PKCα. These results further define the downstream events resulting in PA production in the P2Y6 receptor signaling pathway.

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.

Sum of the parts: mass spectrometry-based metabolomics.

Metabolomics is a rapidly growing field of research used in the identification and quantification of the small molecule metabolites within an organism, thereby providing insights into cell metabolism and bioenergetics as well as processes important in clinical medicine, such as disposition of pharmaceutical compounds. It offers comprehensive information about thousands of low-molecular mass compounds (<1500 Da) that represent a wide range of pathways and intermediary metabolism. Because of its vast expansion in the past two decades, mass spectrometry has become an indispensable tool in "omic" analyses. The use of different ionization techniques such as the more traditional electrospray and matrix-assisted laser desorption, as well as recently popular desorption electrospray ionization, has allowed the analysis of a wide range of biomolecules (e.g., peptides, proteins, lipids, and sugars), and their imaging and analysis in the original sample environment in a workup free fashion. An overview of the current state of the methodology is given, as well as examples of application.

Diacylglycerol kinase delta promotes lipogenesis.

We have studied the relationship between diacylglycerol kinase delta (DGKδ) and lipogenesis. There is a marked increase in the expression of DGKδ during the differentiation of 3T3-L1 cells to adipocytes, as well as in the synthesis of neutral and polar lipids. When 3T3-L1 undifferentiated fibroblasts are transfected to express DGKδ, there is increased triglyceride synthesis without differentiation to adipocytes. Hence, expression of DGKδ promotes lipogenesis. Lipid synthesis is decreased in DGKδ knockout mouse embryo fibroblasts, especially for lipids with shorter acyl chains and limited unsaturation. This reduction occurs for both neutral and polar lipids. These findings suggest reduced de novo lipid synthesis. This is confirmed by measuring the incorporation of glycerol into polar and neutral lipids, which is higher in the wild type cells than in the DGKδ knockouts. In comparison, there was no change in lipid synthesis in DGKε knockout mouse embryo fibroblasts. We also demonstrate that the DGKδ knockout cells had a lower expression of acetyl-CoA carboxylase and fatty acid synthase as well as a lower degree of activation by phosphorylation of ATP citrate lyase. These three enzymes are involved in the synthesis of long chain fatty acids. Our results demonstrate that DGKδ markedly increases lipid synthesis, at least in part as a result of promoting the de novo synthesis of fatty acids.

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.

Regional skeletal muscle remodeling and mitochondrial dysfunction in right ventricular heart failure.

Exercise intolerance is a cardinal symptom of right ventricular heart failure (RV HF) and skeletal muscle adaptations play a role in this limitation. We determined regional remodeling of muscle structure and mitochondrial function in a rat model of RV HF induced by monocrotaline injection (MCT; 60 mg·kg(-1); n = 11). Serial sections of the plantaris were stained for fiber type, succinate dehydrogenase (SDH) activity and capillaries. Mitochondrial function was assessed in permeabilized fibers using respirometry, and isolated complex activity by blue native gel electrophoresis (BN PAGE). All measurements were compared with saline-injected control animals (CON; n = 12). Overall fiber cross-sectional area was smaller in MCT than CON: 1,843 ± 114 vs. 2,322 ± 120 µm(2) (P = 0.009). Capillary-to-fiber ratio was lower in MCT in the oxidative plantaris region (1.65 ± 0.09 vs. 1.93 ± 0.07; P = 0.03), but not in the glycolytic region. SDH activity (P = 0.048) and maximal respiratory rate (P = 0.012) were each ∼15% lower in all fibers in MCT. ADP sensitivity was reduced in both skeletal muscle regions in MCT (P = 0.032), but normalized by rotenone. A 20% lower complex I/IV activity in MCT was confirmed by BN PAGE. MCT-treatment was associated with lower mitochondrial volume density (lower SDH activity), quality (lower complex I activity), and fewer capillaries per fiber area in oxidative skeletal muscle. These features are consistent with structural and functional remodeling of the determinants of oxygen supply potential and utilization that may contribute to exercise intolerance and reduced quality of life in patients with RV HF.

A mouse macrophage lipidome.

We report the lipidomic response of the murine macrophage RAW cell line to Kdo(2)-lipid A, the active component of an inflammatory lipopolysaccharide functioning as a selective TLR4 agonist and compactin, a statin inhibitor of cholesterol biosynthesis. Analyses of lipid molecular species by dynamic quantitative mass spectrometry and concomitant transcriptomic measurements define the lipidome and demonstrate immediate responses in fatty acid metabolism represented by increases in eicosanoid synthesis and delayed responses characterized by sphingolipid and sterol biosynthesis. Lipid remodeling of glycerolipids, glycerophospholipids, and prenols also take place, indicating that activation of the innate immune system by inflammatory mediators leads to alterations in a majority of mammalian lipid categories, including unanticipated effects of a statin drug. Our studies provide a systems-level view of lipid metabolism and reveal significant connections between lipid and cell signaling and biochemical pathways that contribute to innate immune responses and to pharmacological perturbations.

Extensive sphingolipid depletion does not affect lipid raft integrity or lipid raft localization and efflux function of the ABC transporter MRP1.

We show that highly efficient depletion of sphingolipids in two different cell lines does not abrogate the ability to isolate Lubrol-based DRMs (detergent-resistant membranes) or detergent-free lipid rafts from these cells. Compared with control, DRM/detergent-free lipid raft fractions contain equal amounts of protein, cholesterol and phospholipid, whereas the classical DRM/lipid raft markers Src, caveolin-1 and flotillin display the same gradient distribution. DRMs/detergent-free lipid rafts themselves are severely depleted of sphingolipids. The fatty acid profile of the remaining sphingolipids as well as that of the glycerophospholipids shows several differences compared with control, most prominently an increase in highly saturated C(16) species. The glycerophospholipid headgroup composition is unchanged in sphingolipid-depleted cells and cell-derived detergent-free lipid rafts. Sphingolipid depletion does not alter the localization of MRP1 (multidrug-resistance-related protein 1) in DRMs/detergent-free lipid rafts or MRP1-mediated efflux of carboxyfluorescein. We conclude that extensive sphingolipid depletion does not affect lipid raft integrity in two cell lines and does not affect the function of the lipid-raft-associated protein MRP1.

Molecular species of phosphatidylinositol-cycle intermediates in the endoplasmic reticulum and plasma membrane.

Phosphatidylinositol (PI) turnover is a process requiring both the plasma and ER membranes. We have determined the distribution of phosphatidic acid (PA) and PI and their acyl chain compositions in these two subcellular membranes using mass spectrometry. We assessed the role of PI cycling in determining the molecular species and quantity of these lipids by comparing the compositions of the two membranes isolated from embryonic fibroblasts obtained from diacylglycerol kinase epsilon (DGKepsilon) knockout (KO) and wild-type (WT) mice. In the KO cells, the conversion of arachidonoyl-rich DAG to PA is blocked by the absence of DGKepsilon, resulting in a reduction in the rate of PI cycling. The acyl chain composition is very similar for PI and PA in the endoplasmic reticulum (ER) versus plasma membrane (PM) and for WT versus KO. However, the acyl chain profile for PI is very different from that for PA. This indicates that DGKepsilon is not facilitating the direct transfer of a specific species of PA between the PM and the ER. Approximately 20% of the PA in the ER membrane has one short acyl chain of 14 or fewer carbons. These species of PA are not converted into PI but may play a role in stabilizing regions of high positive curvature in the ER. There are also PI species in both the ER and PM for which there is no detectable PA precursor, indicating that these species of PI are unlikely to arise via the PI cycle. We find that in the PM of KO cells the levels of PI and of PA are decreased approximately 3-fold in comparison with those in either the PM of WT cells or the ER of KO cells. The PI cycle is slowed in the KO cells; hence, the lipid intermediates of the PI cycle can no longer be interconverted and are depleted from the PI cycle by conversion to other species. There is less of an effect of the depletion in the ER where de novo synthesis of PA occurs in comparison with the PM.

Lipidomics reveals a remarkable diversity of lipids in human plasma.

The focus of the present study was to define the human plasma lipidome and to establish novel analytical methodologies to quantify the large spectrum of plasma lipids. Partial lipid analysis is now a regular part of every patient's blood test and physicians readily and regularly prescribe drugs that alter the levels of major plasma lipids such as cholesterol and triglycerides. Plasma contains many thousands of distinct lipid molecular species that fall into six main categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. The physiological contributions of these diverse lipids and how their levels change in response to therapy remain largely unknown. As a first step toward answering these questions, we provide herein an in-depth lipidomics analysis of a pooled human plasma obtained from healthy individuals after overnight fasting and with a gender balance and an ethnic distribution that is representative of the US population. In total, we quantitatively assessed the levels of over 500 distinct molecular species distributed among the main lipid categories. As more information is obtained regarding the roles of individual lipids in health and disease, it seems likely that future blood tests will include an ever increasing number of these lipid molecules.

Diacylglycerol kinase epsilon is selective for both acyl chains of phosphatidic acid or diacylglycerol.

The phosphatidylinositol (PI) cycle mediates many cellular events by controlling the metabolism of many lipid second messengers. Diacylglycerol kinase epsilon (DGK epsilon) has an important role in this cycle. DGK epsilon is the only DGK isoform to show inhibition by its product phosphatidic acid (PA) as well as substrate specificity for sn-2 arachidonoyl-diacylglycerol (DAG). Here, we show that this inhibition and substrate specificity are both determined by selectivity for a combination of the sn-1 and sn-2 acyl chains of PA or DAG, respectively, preferring the most prevalent acyl chain composition of lipids involved specifically in the PI cycle, 1-stearoyl-2-arachidonoyl. Although the difference in rate for closely related lipid species is small, there is a significant enrichment of 1-stearoyl-2-arachidonoyl PI because of the cyclical nature of PI turnover. We also show that the inhibition of DGK epsilon by PA is competitive and that the deletion of the hydrophobic segment and cationic cluster of DGK epsilon does not affect its selectivity for the acyl chains of PA or DAG. Thus, this active site not only recognizes the lipid headgroup but also a combination of the two acyl chains in PA or DAG. We propose a mechanism of DGK epsilon regulation where its dual acyl chain selectivity is used to negatively regulate its enzymatic activity in a manner that ensures DGK epsilon remains committed to the PI turnover cycle. This novel mechanism of enzyme regulation within a signaling pathway could serve as a template for the regulation of enzymes in other pathways in the cell.

Spatial and temporal alterations of phospholipids determined by mass spectrometry during mouse embryo implantation.

Molecular events involved in successful embryo implantation are not well understood. In this study, we used MALDI imaging mass spectrometry (IMS) technologies to characterize the spatial and temporal distribution of phospholipid species associated with mouse embryo implantation. Molecular images showing phospholipid distribution within implantation sites changed markedly between distinct cellular areas during days 4-8 of pregnancy. For example, by day 8, linoleate- and docosahexaenoate-containing phospholipids localized to regions destined to undergo cell death, whereas oleate-containing phospholipids localized to angiogenic regions. Arachidonate-containing phospholipids showed different segregation patterns depending on the lipid class, revealing a strong correlation of phosphatidylethanolamines and phosphatidylinositols with cytosolic phospholipase A(2alpha) and cyclooxygenase-2 during embryo implantation. LC-ESI-MS/MS was used to validate MALDI IMS phospholipid distribution patterns. Overall, molecular images revealed the dynamic complexity of lipid distributions in early pregnancy, signifying the importance of complex interplay of lipid molecules in uterine biology and implantation.

Lipidomic profiling in mouse brain reveals differences between ages and genders, with smaller changes associated with alpha-synuclein genotype.

Advances in lipidomics technology have facilitated the precise detection, identification and profiling of lipid species within tissues. Mass spectrometry allows for identification of lipids as a function of the total number of carbons and double bonds in their acyl chains. Such detailed descriptions of lipid composition can provide a basis for further investigation of cell signaling and metabolic pathways, both physiological and pathological. Here, we applied phospholipid profiling to mouse models relevant to Parkinson's disease, using mice that were transgenic for human alpha-synuclein (alphaSyn) or deleted of endogenous alphaSyn. Proposed functions of alphaSyn include phospholipid binding, regulation of membrane composition, and regulation of vesicular pools. We investigated whether alphaSyn gene dosage interacts with differences in phospholipid composition across brain regions or with age-related changes in brain phospholipid composition. The most dramatic phospholipid changes were observed in alphaSyn wild-type animals as a function of age and gender. alphaSyn genotype-specific changes were also observed in aged, but not young, mice. Our results provide a detailed and systematic characterization of brain phospholipid composition in mice and identify age-related changes relevant both to Parkinson's disease and to normal aging.

Dramatic differences in the roles in lipid metabolism of two isoforms of diacylglycerol kinase.

Lipid species changes for SV40-transformed fibroblasts from wild-type or from diacylglycerol kinase-epsilon (DGKepsilon) or diacylglycerol kinase-alpha (DGKalpha) knockout mice were determined for glycerophospholipids, polyphosphatidylinositides (GPInsP n ) and diacylglycerol (DAG) using direct infusion mass spectrometry. Dramatic differences in arachidonate (20:4 fatty acid)-containing lipids were observed for multiple classes of glycerophospholipids and polyphosphatidylinositides between wild-type and DGKepsilon knockout cells. However, no difference was observed in either the amount or the acyl chain composition of DAG between DGKepsilon knockout and wild-type cells, suggesting that DGKepsilon catalyzed the phosphorylation of a minor fraction of the DAG in these cells. The differences in arachidonate content between the two cell lines were greatest for the GPInsP n lipids and lowest for DAG. These findings indicate that DGKepsilon plays a significant role in determining the enrichment of GPInsP n with 20:4 and that there is a pathway for the selective translocation of arachidonoyl phosphatidic acid from the plasma membrane to the endoplasmic reticulum. In contrast, no substantial difference was observed in the acyl chain composition of any class of glycerophospholipid or diacylglycerol between lipid extracts from fibroblasts from wild-type mice or from DGKalpha knockout mice. However, the cells from the DGKalpha knockout mice had a higher concentration of DAG, consistent with the lack of downregulation of the major fraction of DAG by DGKalpha, in contrast with DGKepsilon that is primarily responsible for enrichment of GPInsP n with arachidonoyl acyl chains.

Effects of dietary salt loading on the responses of isolated rat mesenteric arteries to leptin.

BACKGROUND: Leptin induces relaxation of vascular smooth muscle through an endothelium-dependent release of nitric oxide (EDNO) and administration of a high-salt diet reduces the relaxation of vessels to EDNO. We would, therefore, predict that salt loading would reduce the leptin-induced dilatation. However, in salt-loaded animals the relaxation to acetylcholine is maintained through an endothelial-dependent hypopolarizing factor instead of EDNO. These experiments were, therefore, designed to examine whether in salt-loaded animals the response to leptin would be reduced or whether, as for acetylcholine, an alternative mechanism would be substituted. METHODS: Weanling rats were given diets containing either 0.4% (n = 10) or 8% (n = 9) sodium chloride for 4 weeks. They were then killed and a length of second order mesenteric artery was mounted in a perfusion myograph with diameter changes measured using a microscope-video tracking system. The vessels were preconstricted with norepinephrine and then the effects of graded concentrations of leptin were determined. RESULTS: In vessels from the low salt animals leptin caused a dose-dependent dilatation (maximum change 31.4% +/- 5.8% of the initial norepinephrine-induced constriction) but in the high salt animals the change was only 3.4% +/- 1.1%. The nitric oxide synthase blocker Nomega-nitro-L-arginine methyl ester (L-NAME) abolished the responses, although responses could still be obtained in vessels from both groups to the NO donor, sodium nitroprusside. CONCLUSIONS: These results indicate that salt loading to rats almost completely abolishes the vasodilatation to leptin. This implies endothelial disruption and, unlike the response to acetylcholine, no other vasodilator mechanism is implicated. This could provide a link between high salt intake and hypertension because the known increase in sympathetic activity caused by leptin would not be countered by a direct vasorelaxation.

Lipid composition of viral envelope of three strains of influenza virus - not all viruses are created equal.

While differences in the rate of virus fusion and budding from the host cell membrane have been correlated with pathogenicity, no systematic study of the contribution of differences in viral envelope composition has previously been attempted. Using rigorous virus purification, marked differences between virions and host were observed. Over 125 phospholipid species have been quantitated for three strains of influenza (HKx31- H3N2, PR8- H1N1, and VN1203- H5N1) grown in eggs. The glycerophospholipid composition of purified virions differs from that of the host or that of typical mammalian cells. Phosphatidylcholine is the major component in most mammalian cell membranes, while in purified virions phosphatidylethanolamine dominates. Due to its effects on membrane curvature, it is likely that the variations in its content are important to viral processing during infection. This integrated method of virion isolation with systematic analysis of glycerophospholipids provides a tool for the assessment of species specific biomarkers of viral pathogenicity.