A Novel Lipidomics Workflow for Improved Human Plasma Identification and Quantification Using RPLC-MSn Methods and Isotope Dilution Strategies.

Lipid identification and quantification are essential objectives in comprehensive lipidomics studies challenged by the high number of lipids, their chemical diversity, and their dynamic range. In this work, we developed a tailored method for profiling and quantification combining (1) isotope dilution, (2) enhanced isomer separation by C30 fused-core reversed-phase material, and (3) parallel Orbitrap and ion trap detection by the Orbitrap Fusion Lumos Tribid mass spectrometer. The combination of parallelizable ion analysis without time loss together with different fragmentation techniques (HCD/CID) and an inclusion list led to higher quality in lipid identifications exemplified in human plasma and yeast samples. Moreover, we used lipidome isotope-labeling of yeast (LILY)-a fast and efficient in vivo labeling strategy in Pichia pastoris-to produce (nonradioactive) isotopically labeled eukaryotic lipid standards in yeast. We integrated the 13C lipids in the LC-MS workflow to enable relative and absolute compound-specific quantification in yeast and human plasma samples by isotope dilution. Label-free and compound-specific quantification was validated by comparison against a recent international interlaboratory study on human plasma SRM 1950. In this way, we were able to prove that LILY enabled quantification leads to accurate results, even in complex matrices. Excellent analytical figures of merit with enhanced trueness, precision and linearity over 4-5 orders of magnitude were observed applying compound-specific quantification with 13C-labeled lipids. We strongly believe that lipidomics studies will benefit from incorporating isotope dilution and LC-MSn strategies.

A relative quantitative positive/negative ion switching method for untargeted lipidomics via high resolution LC-MS/MS from any biological source.

INTRODUCTION: Advances in high-resolution mass spectrometry have created renewed interest for studying global lipid biochemistry in disease and biological systems. OBJECTIVES: Here, we present an untargeted 30 min. LC-MS/MS platform that utilizes positive/negative polarity switching to perform unbiased data dependent acquisitions (DDA) via higher energy collisional dissociation (HCD) fragmentation to profile more than 1000-1500 lipid ions mainly from methyl-tert-butyl ether (MTBE) or chloroform:methanol extractions. METHODS: The platform uses C18 reversed-phase chromatography coupled to a hybrid QExactive Plus/HF Orbitrap mass spectrometer and the entire procedure takes ~10 h from lipid extraction to identification/quantification for a data set containing 12 samples (~4 h for a single sample). Lipids are identified by both accurate precursor ion mass and fragmentation features and quantified using Lipid-Search and Elements software. RESULTS: Using this approach, we are able to profile intact lipid ions from up to 18 different main lipid classes and 66 subclasses. We show several studies from different biological sources, including cultured cancer cells, resected tissues from mice such as lung and breast tumors and biological fluids such as plasma and urine. CONCLUSIONS: Using mouse embryonic fibroblasts, we showed that TSC2-/- KD significantly abrogates lipid biosynthesis and that rapamycin can rescue triglyceride (TG) lipids and we show that SREBP-/- shuts down lipid biosynthesis significantly via mTORC1 signaling pathways. We show that in mouse EGFR driven lung tumors, a large number of TGs and phosphatidylmethanol (PMe) lipids are elevated while some phospholipids (PLs) show some of the largest decrease in lipid levels from ~ 2000 identified lipid ions. In addition, we identified more than 1500 unique lipid species from human blood plasma.

Perilipin 5 regulates islet lipid metabolism and insulin secretion in a cAMP-dependent manner: implication of its role in the postprandial insulin secretion.

Elevation of circulating fatty acids (FA) during fasting supports postprandial (PP) insulin secretion that is critical for glucose homeostasis and is impaired in diabetes. We tested our hypothesis that lipid droplet (LD) protein perilipin 5 (PLIN5) in ß-cells aids PP insulin secretion by regulating intracellular lipid metabolism. We demonstrated that PLIN5 serves as an LD protein in human islets. In vivo, Plin5 and triglycerides were increased by fasting in mouse islets. MIN6 cells expressing PLIN5 (adenovirus [Ad]-PLIN5) and those expressing perilipin 2 (PLIN2) (Ad-PLIN2) had higher [(3)H]FA incorporation into triglycerides than Ad-GFP control, which support their roles as LD proteins. However, Ad-PLIN5 cells had higher lipolysis than Ad-PLIN2 cells, which increased further by 8-Br-cAMP, indicating that PLIN5 facilitates FA mobilization upon cAMP stimulation as seen postprandially. Ad-PLIN5 in islets enhanced the augmentation of glucose-stimulated insulin secretion by FA and 8-Br-cAMP in G-protein-coupled receptor 40 (GPR40)- and cAMP-activated protein kinase-dependent manners, respectively. When PLIN5 was increased in mouse ß-cells in vivo, glucose tolerance after an acute exenatide challenge was improved. Therefore, the elevation of islet PLIN5 during fasting allows partitioning of FA into LD that is released upon refeeding to support PP insulin secretion in cAMP- and GPR40-dependent manners.

Reducing plasma membrane sphingomyelin increases insulin sensitivity.

It has been shown that inhibition of de novo sphingolipid synthesis increases insulin sensitivity. For further exploration of the mechanism involved, we utilized two models: heterozygous serine palmitoyltransferase (SPT) subunit 2 (Sptlc2) gene knockout mice and sphingomyelin synthase 2 (Sms2) gene knockout mice. SPT is the key enzyme in sphingolipid biosynthesis, and Sptlc2 is one of its subunits. Homozygous Sptlc2-deficient mice are embryonic lethal. However, heterozygous Sptlc2-deficient mice that were viable and without major developmental defects demonstrated decreased ceramide and sphingomyelin levels in the cell plasma membranes, as well as heightened sensitivity to insulin. Moreover, these mutant mice were protected from high-fat diet-induced obesity and insulin resistance. SMS is the last enzyme for sphingomyelin biosynthesis, and SMS2 is one of its isoforms. Sms2 deficiency increased cell membrane ceramide but decreased SM levels. Sms2 deficiency also increased insulin sensitivity and ameliorated high-fat diet-induced obesity. We have concluded that Sptlc2 heterozygous deficiency- or Sms2 deficiency-mediated reduction of SM in the plasma membranes leads to an improvement in tissue and whole-body insulin sensitivity.

Selective reduction in the sphingomyelin content of atherogenic lipoproteins inhibits their retention in murine aortas and the subsequent development of atherosclerosis.

OBJECTIVE: We used the sphingomyelin (SM) synthase 2 (Sms2) gene knockout (KO) approach to test our hypothesis that selectively decreasing plasma lipoprotein SM can play an important role in preventing atherosclerosis. METHODS AND RESULTS: The sphingolipid de novo synthesis pathway is considered a promising target for pharmacological intervention in atherosclerosis. However, its potential is hampered because the substance's atherogenic mechanism is not completely understood. We prepared Sms2 and apolipoprotein E (Apoe) double-KO mice. They showed a significant decrease in plasma lipoprotein SM levels (35%, P<0.01) and a significant increase in ceramide and dihydroceramide levels (87.5% and 27%, respectively; P<0.01) but no significant changes in other tested sphingolipids, cholesterol, and triglyceride. Non-high-density lipoproteins from the double-KO mice showed a reduction of SM, but not cholesterol, and displayed less tendency toward aortic sphingomyelinase-mediated lipoprotein aggregation in vitro and retention in aortas in vivo when compared with controls. More important, at the age of 19 weeks, Sms2 KO/Apoe KO mice showed a significant reduction in atherosclerotic lesions of the aortic arch and root (52%, P<0.01) compared with controls. The Sms2 KO/Apoe KO brachiocephalic artery contained significantly less SM, ceramide, free cholesterol, and cholesteryl ester (35%, 32%, 58%, and 60%, respectively; P<0.01) than that of the Apoe KO brachiocephalic artery. CONCLUSIONS: Decreasing plasma SM levels through decreasing SMS2 activity could become a promising treatment for atherosclerosis.

Impaired de novo choline synthesis explains why phosphatidylethanolamine N-methyltransferase-deficient mice are protected from diet-induced obesity.

Phosphatidylcholine (PC) is synthesized from choline via the CDP-choline pathway. Liver cells can also synthesize PC via the sequential methylation of phosphatidylethanolamine, catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). The current study investigates whether or not hepatic PC biosynthesis is linked to diet-induced obesity. Pemt(+/+) mice fed a high fat diet for 10 weeks increased in body mass by 60% and displayed insulin resistance, whereas Pemt(-/-) mice did not. Compared with Pemt(+/+) mice, Pemt(-/-) mice had increased energy expenditure and maintained normal peripheral insulin sensitivity; however, they developed hepatomegaly and steatosis. In contrast, mice with impaired biosynthesis of PC via the CDP-choline pathway in liver became obese when fed a high fat diet. We, therefore, hypothesized that insufficient choline, rather than decreased hepatic phosphatidylcholine, was responsible for the lack of weight gain in Pemt(-/-) mice despite the presence of 1.3 g of choline/kg high fat diet. Supplementation with an additional 2.7 g of choline (but not betaine)/kg of diet normalized energy metabolism, weight gain, and insulin resistance in high fat diet-fed Pemt(-/-) mice. Furthermore, Pemt(+/+) mice that were fed a choline-deficient diet had increased oxygen consumption, had improved glucose tolerance, and gained less weight. Thus, de novo synthesis of choline via PEMT has a previously unappreciated role in regulating whole body energy metabolism.

Liver-specific deficiency of serine palmitoyltransferase subunit 2 decreases plasma sphingomyelin and increases apolipoprotein E levels.

Sphingomyelin (SM) is one of the major lipid components of plasma lipoproteins. Serine palmitoyltransferase (SPT) is the key enzyme in SM biosynthesis. Mice totally lacking in SPT are embryonic lethal. The liver is the major site for plasma lipoprotein biosynthesis, secretion, and degradation, and in this study we utilized a liver-specific knock-out approach for evaluating liver SPT activity and also its role in plasma SM and lipoprotein metabolism. We found that a deficiency of liver-specific Sptlc2 (a subunit of SPT) decreased liver SPT protein mass and activity by 95 and 92%, respectively, but had no effect on other tissues. Liver Sptlc2 deficiency decreased plasma SM levels (in both high density lipoprotein and non-high density lipoprotein fractions) by 36 and 35% (p < 0.01), respectively, and increased phosphatidylcholine levels by 19% (p < 0.05), thus increasing the phosphatidylcholine/SM ratio by 77% (p < 0.001), compared with controls. This deficiency also decreased SM levels in the liver by 38% (p < 0.01) and in the hepatocyte plasma membranes (based on a lysenin-mediated cell lysis assay). Liver-specific Sptlc2 deficiency significantly increased hepatocyte apoE secretion and thus increased plasma apoE levels 3.5-fold (p < 0.0001). Furthermore, plasma from Sptlc2 knock-out mice had a significantly stronger potential for promoting cholesterol efflux from macrophages than from wild-type mice (p < 0.01) because of a greater amount of apoE in the circulation. As a result of these findings, we believe that the ability to control liver SPT activity could result in regulation of lipoprotein metabolism and might have an impact on the development of atherosclerosis.

Sphingomyelin synthase 2 is one of the determinants for plasma and liver sphingomyelin levels in mice.

BACKGROUND: It has been proposed that plasma sphingomyelin (SM) plays a very important role in plasma lipoprotein metabolism and atherosclerosis. Sphingomyelin synthase (SMS) is the last enzyme for SM de novo biosynthesis. Two SMS genes, SMS1 and SMS2, have been cloned and characterized. METHODS AND RESULTS: To evaluate the in vivo role of SMS2 in SM metabolism, we prepared SMS2 knockout (KO) and SMS2 liver-specific transgenic (LTg) mice and studied their plasma SM and lipoprotein metabolism. On a chow diet, SMS2 KO mice showed a significant decrease in plasma SM levels (25%, P<0.05), but no significant changes in total cholesterol, total phospholipids, or triglyceride, compared with wild-type (WT) littermates. On a high-fat diet, SMS2 KO mice showed a decrease in plasma SM levels (28%, P<0.01), whereas SMS2LTg mice showed a significant increase in those levels (29%, P<0.05), but no significant changes in other lipids, compared with WT littermates. Atherogenic lipoproteins from SMS2LTg mice displayed a significantly stronger tendency toward aggregation after mammalian sphingomyelinase treatment, compared with controls. Moreover, SMS2 deficiency significantly increased plasma apoE levels (2.0-fold, P<0.001), whereas liver-specific SMS2 overexpression significantly decreased those levels (1.8-fold, P<0.01). Finally, SMS2 KO mouse plasma promoted cholesterol efflux from macrophages, whereas SMS2LTg mouse plasma prevented it. CONCLUSIONS: We therefore believe that regulation of liver SMS2 activity could become a promising treatment for atherosclerosis.

Sphingomyelin synthase 2 deficiency attenuates NFkappaB activation.

BACKGROUND: NFkappaB has long been regarded as a proatherogenic factor, mainly because of its regulation of many of the proinflammatory genes linked to atherosclerosis. Metabolism of sphingomyelin (SM) has been suggested to affect NFkappaB activation, but the mechanism is largely unknown. SMS2 regulates SM levels in cell plasma membrane and lipid rafts and has a potential to regulate NFkappaB activation. METHODS AND RESULTS: To investigate the role of SMS2 in NFkappaB activation we used macrophages from SMS2 knockout (KO) mice and SMS2 siRNA-treated HEK 293 cells. We found that NFkappaB activation and its target gene expression are attenuated in macrophages from SMS2 KO mice in response to lipopolysaccharide (LPS) stimulation and in SMS2 siRNA- treated HEK 293 cells after tumor necrosis factor (TNF)-alpha simulation. In line with attenuated NFkappaB activation, we found that SMS2 deficiency substantially diminished the abundance of toll like receptor 4 (TLR4)-MD2 complex levels on the surface of macrophages after LPS stimulation, and SMS2 siRNA treatment reduced TNF-alpha-stimulated lipid raft recruitment of TNF receptor-1 (TNFR1) in HEK293 cells. SMS2 deficiency decreased the relative amounts of SM and diacylglycerol (DAG) and increased ceramide, suggesting multiple mechanisms for the decrease in NFkappaB activation. CONCLUSIONS: SMS2 is a modulator of NFkappaB activation, and thus it could play an important role in NFkappaB-mediated proatherogenic process.

AGPAT6 is a novel microsomal glycerol-3-phosphate acyltransferase.

AGPAT6 is a member of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family that appears to be important in triglyceride biosynthesis in several tissues, but the precise biochemical function of the enzyme is unknown. In the current study, we show that AGPAT6 is a microsomal glycerol-3-phosphate acyltransferase (GPAT). Membranes from HEK293 cells overexpressing human AGPAT6 had higher levels of GPAT activity. Substrate specificity studies suggested that AGPAT6 was active against both saturated and unsaturated long-chain fatty acyl-CoAs. Both glycerol 3-phosphate and fatty acyl-CoA increased the GPAT activity, and the activity was sensitive to N-ethylmaleimide, a sulfhydryl-modifying reagent. Purified AGPAT6 protein possessed GPAT activity but not AGPAT activity. Using [(13)C(7)]oleic acid labeling and mass spectrometry, we found that overexpression of AGPAT6 increased both lysophosphatidic acid and phosphatidic acid levels in cells. In these studies, total triglyceride and phosphatidylcholine levels were not significantly altered, although there were significant changes in the abundance of specific phosphatidylcholine species. Human AGPAT6 is localized to endoplasmic reticulum and is broadly distributed in tissues. Membranes of mammary epithelial cells from Agpat6-deficient mice exhibited markedly reduced GPAT activity compared with membranes from wild-type mice. Reducing AGPAT6 expression in HEK293 cells through small interfering RNA knockdown suggested that AGPAT6 significantly contributed to HEK293 cellular GPAT activity. Our data indicate that AGPAT6 is a microsomal GPAT, and we propose renaming this enzyme GPAT4.

Inhibition of sphingomyelin synthase (SMS) affects intracellular sphingomyelin accumulation and plasma membrane lipid organization.

Sphingomyelin plays a very important role both in cell membrane formation that may well have an impact on the development of diseases like atherosclerosis and diabetes. However, the molecular mechanism that governs intracellular and plasma membrane SM levels is largely unknown. Recently, two isoforms of sphingomyelin synthase (SMS1 and SMS2), the last enzyme for SM de novo synthesis, have been cloned. We have hypothesized that SMS1 and SMS2 are the two most likely candidates responsible for the SM levels in the cells and on the plasma membrane. To test this hypothesis, cultured cells were treated with tricyclodecan-9-yl-xanthogenate (D609), an inhibitor of SMS, or with SMS1 and SMS2 siRNAs. Cells were then pulsed with [14C]-L-serine (a precursor of all sphingolipids). SMS activity and [14C]-SM in the cells were monitored. We found that SMS activity was significantly decreased in cells after D609 or SMS siRNA treatment, compared with controls. SMS inhibition by D609 or SMS siRNAs significantly decreased intracellular [14C]-SM levels. We measured cellular lipid levels, including SM, ceramide, phosphatidylcholine, and diacylglycerol and found that SMS1 and SMS2 siRNA treatment caused a significant decrease of SM levels (20% and 11%, respectively), compared to control siRNA treatment; SMS1 but not SMS2 siRNA treatment caused a significant increase of ceramide levels (10%). There was a decreasing tendency for diacylglycerol levels after both SMS1 and SMS2 siRNA treatment, however, it was not statistical significant. As shown by lipid rafts isolation and lipid determination, SMS1 and SMS2 siRNA treatment led to a decrease of SM content in detergent-resistant lipid rafts on the cell membrane. Furthermore, SMS1 and SMS2 siRNA-treated cells had a stronger resistance than did control siRNA-treated cells to lysenin (a protein that causes cell lysis due to its affinity for plasma membrane SM). These results indicate that both SMS1 and SMS2 contribute to SM de novo synthesis and control SM levels in the cells and on the cell membrane including plasma membrane, implying an important relationship between SMS activity and cell functions.