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Lipids are highly structurally diverse molecules involved in a wide variety of biological processes. Here, we use high precision ion mobility-mass spectrometry to compile a structural database of 456 mass-resolved collision cross sections (CCS) of sphingolipid and glycerophospholipid species. Our CCS database comprises sphingomyelin, cerebroside, ceramide, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidic acid classes. Primary differences observed are between lipid categories, with sphingolipids exhibiting 2-6% larger CCSs than glycerophospholipids of similar mass, likely a result of the sphingosine backbone's restriction of the sn1 tail length, limiting gas-phase packing efficiency. Acyl tail length and degree of unsaturation are found to be the primary structural descriptors determining CCS magnitude, with degree of unsaturation being four times as influential per mass unit. The empirical CCS values and previously unmapped quantitative structural trends detailed in this work are expected to facilitate prediction of CCS in broadscale lipidomics research.
Isolevuglandins are 4-ketoaldehydes formed by peroxidation of arachidonic acid. Isolevuglandins react rapidly with primary amines including the lysyl residues of proteins to form irreversible covalent modifications. This review highlights evidence for the potential role of isolevuglandin modification in the disease processes, especially atherosclerosis, and some of the tools including small molecule dicarbonyl scavengers utilized to assess their contributions to disease.
Copyright © 2018. Published by Elsevier Inc.
Pancreatic β-cell expansion is a highly regulated metabolic adaptation to increased somatic demands, including obesity and pregnancy; adult β cells otherwise rarely proliferate. We previously showed that high-fat diet (HFD) feeding induces mouse β-cell proliferation in less than 1 wk in the absence of insulin resistance. Here we metabolically profiled tissues from a short-term HFD β-cell expansion mouse model to identify pathways and metabolite changes associated with β-cell proliferation. Mice fed HFD vs. chow diet (CD) showed a 14.3% increase in body weight after 7 days; β-cell proliferation increased 1.75-fold without insulin resistance. Plasma from 1-wk HFD-fed mice induced β-cell proliferation ex vivo. The plasma, as well as liver, skeletal muscle, and bone, were assessed by LC and GC mass-spectrometry for global metabolite changes. Of the 1,283 metabolites detected, 159 showed significant changes [false discovery rate (FDR) < 0.1]. The majority of changes were in liver and muscle. Pathway enrichment analysis revealed key metabolic changes in steroid synthesis and lipid metabolism, including free fatty acids and other bioactive lipids. Other important enrichments included changes in the citric acid cycle and 1-carbon metabolism pathways implicated in DNA methylation. Although the minority of changes were observed in bone and plasma (<20), increased p-cresol sulfate was increased >4 fold in plasma (the largest increase in all tissues), and pantothenate (vitamin B) decreased >2-fold. The results suggest that HFD-mediated β-cell expansion is associated with complex, global metabolite changes. The finding could be a significant insight into Type 2 diabetes pathogenesis and potential novel drug targets.
Cardiovascular disease risk depends on high-density lipoprotein (HDL) function, not HDL-cholesterol. Isolevuglandins (IsoLGs) are lipid dicarbonyls that react with lysine residues of proteins and phosphatidylethanolamine. IsoLG adducts are elevated in atherosclerosis. The consequences of IsoLG modification of HDL have not been studied. We hypothesized that IsoLG modification of apoA-I deleteriously alters HDL function. We determined the effect of IsoLG on HDL structure-function and whether pentylpyridoxamine (PPM), a dicarbonyl scavenger, can preserve HDL function. IsoLG adducts in HDL derived from patients with familial hypercholesterolemia ( = 10, 233.4 ± 158.3 ng/mg) were found to be significantly higher than in healthy controls ( = 7, 90.1 ± 33.4 pg/mg protein). Further, HDL exposed to myeloperoxidase had elevated IsoLG-lysine adducts (5.7 ng/mg protein) compared with unexposed HDL (0.5 ng/mg protein). Preincubation with PPM reduced IsoLG-lysine adducts by 67%, whereas its inactive analogue pentylpyridoxine did not. The addition of IsoLG produced apoA-I and apoA-II cross-links beginning at 0.3 molar eq of IsoLG/mol of apoA-I (0.3 eq), whereas succinylaldehyde and 4-hydroxynonenal required 10 and 30 eq. IsoLG increased HDL size, generating a subpopulation of 16-23 nm. 1 eq of IsoLG decreased HDL-mediated [H]cholesterol efflux from macrophages via ABCA1, which corresponded to a decrease in HDL-apoA-I exchange from 47.4% to only 24.8%. This suggests that IsoLG inhibits apoA-I from disassociating from HDL to interact with ABCA1. The addition of 0.3 eq of IsoLG ablated HDL's ability to inhibit LPS-stimulated cytokine expression by macrophages and increased IL-1β expression by 3.5-fold. The structural-functional effects were partially rescued with PPM scavenging.
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
Genome-wide association studies have identified numerous variants associated with lipid levels; yet, the majority are located in non-coding regions with unclear mechanisms. In the Insulin Resistance Atherosclerosis Family Study (IRASFS), heritability estimates suggest a strong genetic basis: low-density lipoprotein (LDL, h = 0.50), high-density lipoprotein (HDL, h = 0.57), total cholesterol (TC, h = 0.53), and triglyceride (TG, h = 0.42) levels. Exome sequencing of 1,205 Mexican Americans (90 pedigrees) from the IRASFS identified 548,889 variants and association and linkage analyses with lipid levels were performed. One genome-wide significant signal was detected in APOA5 with TG (rs651821, P = 3.67 × 10, LOD = 2.36, MAF = 14.2%). In addition, two correlated SNPs (r = 1.0) rs189547099 (P = 6.31 × 10, LOD = 3.13, MAF = 0.50%) and chr4:157997598 (P = 6.31 × 10, LOD = 3.13, MAF = 0.50%) reached exome-wide significance (P < 9.11 × 10). rs189547099 is an intronic SNP in FNIP2 and SNP chr4:157997598 is intronic in GLRB. Linkage analysis revealed 46 SNPs with a LOD > 3 with the strongest signal at rs1141070 (LOD = 4.30, P = 0.33, MAF = 21.6%) in DFFB. A total of 53 nominally associated variants (P < 5.00 × 10, MAF ≥ 1.0%) were selected for replication in six Mexican-American cohorts (N = 3,280). The strongest signal observed was a synonymous variant (rs1160983, P = 4.44 × 10, MAF = 2.7%) in TOMM40. Beyond primary findings, previously reported lipid loci were fine-mapped using exome sequencing in IRASFS. These results support that exome sequencing complements and extends insights into the genetics of lipid levels.
It has been widely recognized that individual cells that exist within a large population of cells, even if they are genetically identical, can have divergent molecular makeups resulting from a variety of factors, including local environmental factors and stochastic processes within each cell. Presently, numerous approaches have been described that permit the resolution of these single-cell expression differences for RNA and protein; however, relatively few techniques exist for the study of lipids and metabolites in this manner. This study presents a methodology for the analysis of metabolite and lipid expression at the level of a single cell through the use of imaging mass spectrometry on a high-performance Fourier transform ion cyclotron resonance mass spectrometer. This report provides a detailed description of the overall experimental approach, including sample preparation as well as the data acquisition and analysis strategy for single cells. Applying this approach to the study of cultured RAW264.7 cells, we demonstrate that this method can be used to study the variation in molecular expression with cell populations and is sensitive to alterations in that expression that occurs upon lipopolysaccharide stimulation. Graphical Abstract.
The increasing focus on lipid metabolism has revealed a need for analytical techniques capable of structurally characterizing lipids with a high degree of specificity. Lipids can exist as any one of a large number of double bond positional isomers, which are indistinguishable by single-stage mass spectrometry alone. Ozonolysis reactions coupled to mass spectrometry have previously been demonstrated as a means for localizing double bonds in unsaturated lipids. Here we describe an online, solution-phase reactor using ozone produced via a low-pressure mercury lamp, which generates aldehyde products diagnostic of cleavage at a particular double bond position. This flow-cell device is utilized in conjunction with structurally selective ion mobility-mass spectrometry. The lamp-mediated reaction was found to be effective for multiple lipid species in both positive and negative ionization modes, and the conversion efficiency from precursor to product ions was tunable across a wide range (20-95%) by varying the flow rate through the ozonolysis device. Ion mobility separation of the ozonolysis products generated additional structural information and revealed the presence of saturated species in a complex mixture. The method presented here is simple, robust, and readily coupled to existing instrument platforms with minimal modifications necessary. For these reasons, application to standard lipidomic workflows is possible and aids in more comprehensive structural characterization of a myriad of lipid species.
Stargardt disease is a juvenile onset retinal degeneration, associated with elevated levels of lipofuscin and its bis-retinoid components, such as N-retinylidene-N-retinylethanolamine (A2E). However, the pathogenesis of Stargardt is still poorly understood and targeted treatments are not available. Utilizing high spatial and high mass resolution matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS), we determined alterations of lipid profiles specifically localized to the retinal pigment epithelium (RPE) in Abca4 Stargardt model mice compared to their relevant background strain. Extensive analysis by LC-MS/MS in both positive and negative ion mode was required to accurately confirm the identity of one highly expressed lipid class, bis(monoacylgylercoro)phosphate (BMP) lipids, and to distinguish them from isobaric species. The same BMP lipids were also detected in the RPE of healthy human retina. BMP lipids have been previously associated with the endosomal/lysosomal storage diseases Niemann-Pick and neuronal ceroid lipofuscinosis and have been reported to regulate cholesterol levels in endosomes. These results suggest that perturbations in lipid metabolism associated with late endosomal/lysosomal dysfunction may play a role in the pathogenesis of Stargardt disease and is evidenced in human retinas.
We screened variants on an exome-focused genotyping array in >300,000 participants (replication in >280,000 participants) and identified 444 independent variants in 250 loci significantly associated with total cholesterol (TC), high-density-lipoprotein cholesterol (HDL-C), low-density-lipoprotein cholesterol (LDL-C), and/or triglycerides (TG). At two loci (JAK2 and A1CF), experimental analysis in mice showed lipid changes consistent with the human data. We also found that: (i) beta-thalassemia trait carriers displayed lower TC and were protected from coronary artery disease (CAD); (ii) excluding the CETP locus, there was not a predictable relationship between plasma HDL-C and risk for age-related macular degeneration; (iii) only some mechanisms of lowering LDL-C appeared to increase risk for type 2 diabetes (T2D); and (iv) TG-lowering alleles involved in hepatic production of TG-rich lipoproteins (TM6SF2 and PNPLA3) tracked with higher liver fat, higher risk for T2D, and lower risk for CAD, whereas TG-lowering alleles involved in peripheral lipolysis (LPL and ANGPTL4) had no effect on liver fat but decreased risks for both T2D and CAD.