Lipidome Unsaturation Affects the Morphology and Proteome of the Drosophila Eye.

Organisms respond to dietary and environmental challenges by altering the molecular composition of their glycerolipids and glycerophospholipids (GPLs), which may favorably adjust the physicochemical properties of lipid membranes. However, how lipidome changes affect the membrane proteome and, eventually, the physiology of specific organs is an open question. We addressed this issue in Drosophila melanogaster, which is not able to synthesize sterols and polyunsaturated fatty acids but can acquire them from food. We developed a series of semisynthetic foods to manipulate the length and unsaturation of fatty acid moieties in GPLs and singled out proteins whose abundance is specifically affected by membrane lipid unsaturation in the Drosophila eye. Unexpectedly, we identified a group of proteins that have muscle-related functions and increased their abundances under unsaturated eye lipidome conditions. In contrast, the abundance of two stress response proteins, Turandot A and Smg5, is decreased by lipid unsaturation. Our findings could guide the genetic dissection of homeostatic mechanisms that maintain visual function when the eye is exposed to environmental and dietary challenges.

Eye proteome of Drosophila melanogaster.

Drosophila melanogaster is a popular model organism to elucidate the molecular mechanisms that underlie the structure and function of the eye as well as the causes of retinopathies, aging, light-induced damage, or dietary deficiencies. Large-scale screens have isolated genes whose mutation causes morphological and functional ocular defects, which led to the discovery of key components of the phototransduction cascade. However, the proteome of the Drosophila eye is poorly characterized. Here, we used GeLC-MS/MS to quantify 3516 proteins, including the absolute (molar) quantities of 43 proteins in the eye of adult male Drosophila reared on standard laboratory food. This work provides a generic and expandable resource for further genetic, pharmacological, and dietary studies.

Lipidome unsaturation affects the morphology and proteome of the Drosophila eye.

While the proteome of an organism is largely determined by the genome, the lipidome is shaped by a poorly understood interplay of environmental factors and metabolic processes. To gain insights into the underlying mechanisms, we analyzed the impacts of dietary lipid manipulations on the ocular proteome of Drosophila melanogaster . We manipulated the lipidome with synthetic food media that differed in the supplementation of an equal amount of saturated or polyunsaturated triacylglycerols. This allowed us to generate flies whose eyes had a highly contrasting length and unsaturation of glycerophospholipids, the major lipid class of biological membranes, while the abundance of other membrane lipid classes remained unchanged. By bioinformatically comparing the resulting ocular proteomic trends and contrasting them with the impacts of vitamin A deficiency, we identified ocular proteins whose abundances are differentially affected by lipid saturation and unsaturation. For instance, we unexpectedly identified a group of proteins that have muscle-related functions and increase their abundances in the eye upon lipidome unsaturation but are unaffected by lipidome saturation. Moreover, we identified two differentially lipid-responsive proteins involved in stress responses, Turandot A and Smg5, whose abundances decrease with lipid unsaturation. Lastly, we discovered that the ocular lipid class composition is robust to dietary changes and propose that this may be a general homeostatic feature of the organization of eukaryotic tissues, while the length and unsaturation of fatty acid moieties is more variable to compensate environmental challenges. We anticipate that these insights into the molecular responses of the Drosophila eye proteome to specific lipid manipulations will guide the genetic dissection of the mechanisms that maintain visual function when the eye is exposed to dietary challenges.

Eye proteome of Drosophila melanogaster.

The Drosophila melanogaster eye is a popular model to elucidate the molecular mechanisms that underlie the structure and function of the eye as well as the causes of retinopathies. For instance, the Drosophila eye has been used to investigate the impacts of ageing and environmental stresses such as light-induced damage or dietary deficiencies. Moreover, large-scale screens have isolated genes whose mutation causes morphological and functional ocular defects, which includes key components of the phototransduction cascade. However, the proteome of the Drosophila eye is poorly characterized. Here, we used GeLC-MS/MS to quantify 3516 proteins he adult Drosophila melanogaster eye and provide a generic and expandable resource for further genetic, pharmacological, and dietary studies.

Vitamin A Deficiency Alters the Phototransduction Machinery and Distinct Non-Vision-Specific Pathways in the Drosophila Eye Proteome.

The requirement of vitamin A for the synthesis of the visual chromophore and the light-sensing pigments has been studied in vertebrate and invertebrate model organisms. To identify the molecular mechanisms that orchestrate the ocular response to vitamin A deprivation, we took advantage of the fact that Drosophila melanogaster predominantly requires vitamin A for vision, but not for development or survival. We analyzed the impacts of vitamin A deficiency on the morphology, the lipidome, and the proteome of the Drosophila eye. We found that chronic vitamin A deprivation damaged the light-sensing compartments and caused a dramatic loss of visual pigments, but also decreased the molar abundance of most phototransduction proteins that amplify and transduce the visual signal. Unexpectedly, vitamin A deficiency also decreased the abundances of specific subunits of mitochondrial TCA cycle and respiratory chain components but increased the levels of cuticle- and lens-related proteins. In contrast, we found no apparent effects of vitamin A deficiency on the ocular lipidome. In summary, chronic vitamin A deficiency decreases the levels of most components of the visual signaling pathway, but also affects molecular pathways that are not vision-specific and whose mechanistic connection to vitamin A remains to be elucidated.

Nonalcoholic fatty liver disease stratification by liver lipidomics.

Nonalcoholic fatty liver disease (NAFLD) is a common metabolic dysfunction leading to hepatic steatosis. However, NAFLD's global impact on the liver lipidome is poorly understood. Using high-resolution shotgun mass spectrometry, we quantified the molar abundance of 316 species from 22 major lipid classes in liver biopsies of 365 patients, including nonsteatotic patients with normal or excessive weight, patients diagnosed with NAFL (nonalcoholic fatty liver) or NASH (nonalcoholic steatohepatitis), and patients bearing common mutations of NAFLD-related protein factors. We confirmed the progressive accumulation of di- and triacylglycerols and cholesteryl esters in the liver of NAFL and NASH patients, while the bulk composition of glycerophospho- and sphingolipids remained unchanged. Further stratification by biclustering analysis identified sphingomyelin species comprising n24:2 fatty acid moieties as membrane lipid markers of NAFLD. Normalized relative abundance of sphingomyelins SM 43:3;2 and SM 43:1;2 containing n24:2 and n24:0 fatty acid moieties, respectively, showed opposite trends during NAFLD progression and distinguished NAFL and NASH lipidomes from the lipidome of nonsteatotic livers. Together with several glycerophospholipids containing a C22:6 fatty acid moiety, these lipids serve as markers of early and advanced stages of NAFL.

Hydroxylated sphingolipid biosynthesis regulates photoreceptor apical domain morphogenesis.

Apical domains of epithelial cells often undergo dramatic changes during morphogenesis to form specialized structures, such as microvilli. Here, we addressed the role of lipids during morphogenesis of the rhabdomere, the microvilli-based photosensitive organelle of Drosophila photoreceptor cells. Shotgun lipidomics analysis performed on mutant alleles of the polarity regulator crumbs, exhibiting varying rhabdomeric growth defects, revealed a correlation between increased abundance of hydroxylated sphingolipids and abnormal rhabdomeric growth. This could be attributed to an up-regulation of fatty acid hydroxylase transcription. Indeed, direct genetic perturbation of the hydroxylated sphingolipid metabolism modulated rhabdomere growth in a crumbs mutant background. One of the pathways targeted by sphingolipid metabolism turned out to be the secretory route of newly synthesized Rhodopsin, a major rhabdomeric protein. In particular, altered biosynthesis of hydroxylated sphingolipids impaired apical trafficking via Rab11, and thus apical membrane growth. The intersection of lipid metabolic pathways with apical domain growth provides a new facet to our understanding of apical growth during morphogenesis.

Phosphoinositide Profile of the Mouse Retina.

Phosphoinositides are known to play multiple roles in eukaryotic cells. Although dysregulation of phosphoinositide metabolism in the retina has been reported to cause visual dysfunction in animal models and human patients, our understanding of the phosphoinositide composition of the retina is limited. Here, we report a characterization of the phosphoinositide profile of the mouse retina and an analysis of the subcellular localization of major phosphorylated phosphoinositide forms in light-sensitive photoreceptor neurons. Using chromatography of deacylated phosphatidylinositol headgroups, we established PI(4,5)P2 and PI(4)P as two major phosphorylated phosphoinositides in the retina. Using high-resolution mass spectrometry, we revealed 18:0/20:4 and 16:0/20:4 as major fatty-acyl chains of retinal phosphoinositides. Finally, analysis of fluorescent phosphoinositide sensors in rod photoreceptors demonstrated distinct subcellular distribution patterns of major phosphoinositides. The PI(4,5)P2 reporter was enriched in the inner segments and synapses, but was barely detected in the light-sensitive outer segments. The PI(4)P reporter was mostly found in the outer and inner segments and the areas around nuclei, but to a lesser degree in the synaptic region. These findings provide support for future mechanistic studies defining the biological significance of major mono- (PI(4)P) and bisphosphate (PI(4,5)P2) phosphatidylinositols in photoreceptor biology and retinal health.

Disrupted Blood-Retina Lysophosphatidylcholine Transport Impairs Photoreceptor Health But Not Visual Signal Transduction.

Retinal photoreceptor cells contain the highest concentration of docosahexaenoic acid (DHA) in our bodies, and it has been long assumed that this is critical for supporting normal vision. Indeed, early studies using DHA dietary restriction documented reduced light sensitivity by DHA-deprived retinas. Recently, it has been demonstrated that a major route of DHA entry in the retina is the delivery across the blood-retina barrier by the sodium-dependent lipid transporter, Mfsd2a. This discovery opened a unique opportunity to analyze photoreceptor health and function in DHA-deprived retinas using the Mfsd2a knock-out mouse as animal model. Our lipidome analyses of Mfsd2a-/- retinas and outer segment membranes corroborated the previously reported decrease in the fraction of DHA-containing phospholipids and a compensatory increase in phospholipids containing arachidonic acid. We also revealed an increase in the retinal content of monounsaturated fatty acids and a reduction in very long chain fatty acids. These changes could be explained by a combination of reduced DHA supply to the retina and a concomitant upregulation of several fatty acid desaturases controlled by sterol regulatory element-binding transcription factors, which are upregulated in Mfsd2a-/- retinas. Mfsd2a-/- retinas undergo slow progressive degeneration, with ∼30% of photoreceptor cells lost by the age of 6 months. Despite this pathology, the ultrastructure Mfsd2a-/- photoreceptors and their ability to produce light responses were essentially normal. These data demonstrate that, whereas maintaining the lysophosphatidylcholine route of DHA supply to the retina is essential for long-term photoreceptor survival, it is not important for supporting normal phototransduction.SIGNIFICANCE STATEMENT Phospholipids containing docosahexaenoic acid (DHA) are greatly enriched in the nervous system, with the highest concentration found in the light-sensitive membranes of photoreceptor cells. In this study, we analyzed the consequences of impaired DHA transport across the blood-retina barrier. We have found that, in addition to a predictable reduction in the DHA level, the affected retinas undergo a complex, transcriptionally-driven rebuilding of their membrane lipidome in a pattern preserving the overall saturation/desaturation balance of retinal phospholipids. Remarkably, these changes do not affect the ability of photoreceptors to produce responses to light but are detrimental for the long-term survival of these cells.

Quantitative Fragmentation Model for Bottom-Up Shotgun Lipidomics.

Quantitative bottom-up shotgun lipidomics relies on molecular species-specific "signature" fragments consistently detectable in tandem mass spectra of analytes and standards. Molecular species of glycerophospholipids are typically quantified using carboxylate fragments of their fatty acid moieties produced by higher-energy collisional dissociation of their molecular anions. However, employing standards whose fatty acids moieties are similar, yet not identical, to the target lipids could severely compromise their quantification. We developed a generic and portable fragmentation model implemented in the open-source LipidXte software that harmonizes the abundances of carboxylate anion fragments originating from fatty acid moieties having different sn-1/2 positions at the glycerol backbone, length of the hydrocarbon chain, and number and location of double bonds. The postacquisition adjustment enables unbiased absolute (molar) quantification of glycerophospholipid species independent of instrument settings, collision energy, and employed internal standards.

Monitoring Membrane Lipidome Turnover by Metabolic 15N Labeling and Shotgun Ultra-High-Resolution Orbitrap Fourier Transform Mass Spectrometry.

Lipidomes undergo permanent extensive remodeling, but how the turnover rate differs between lipid classes and molecular species is poorly understood. We employed metabolic 15N labeling and shotgun ultra-high-resolution mass spectrometry (sUHR) to quantify the absolute (molar) abundance and determine the turnover rate of glycerophospholipids and sphingolipids by direct analysis of total lipid extracts. sUHR performed on a commercial Orbitrap Elite instrument at the mass resolution of 1.35 × 106 (m/z 200) baseline resolved peaks of 13C isotopes of unlabeled and monoisotopic peaks of 15N labeled lipids (Δm = 0.0063 Da). Therefore, the rate of metabolic 15N labeling of individual lipid species could be determined without compromising the scope, accuracy, and dynamic range of full-lipidome quantitative shotgun profiling. As a proof of concept, we employed sUHR to determine the lipidome composition and fluxes of 62 nitrogen-containing membrane lipids in human hepatoma HepG2 cells.

Spatiotemporal Control of Lipid Conversion, Actin-Based Mechanical Forces, and Curvature Sensors during Clathrin/AP-1-Coated Vesicle Biogenesis.

Clathrin/adaptor protein-1-coated carriers connect the secretory and the endocytic pathways. Carrier biogenesis relies on distinct protein networks changing membrane shape at the trans-Golgi network, each regulating coat assembly, F-actin-based mechanical forces, or the biophysical properties of lipid bilayers. How these different hubs are spatiotemporally coordinated remains largely unknown. Using in vitro reconstitution systems, quantitative proteomics, and lipidomics, as well as in vivo cell-based assays, we characterize the protein networks controlling membrane lipid composition, membrane shape, and carrier scission. These include PIP5K1A and phospholipase C-beta 3 controlling the conversion of PI[4]P into diacylglycerol. PIP5K1A binding to RAC1 provides a link to F-actin-based mechanical forces needed to tubulate membranes. Tubular membranes then recruit the BAR-domain-containing arfaptin-1/2 guiding carrier scission. These findings provide a framework for synchronizing the chemical/biophysical properties of lipid bilayers, F-actin-based mechanical forces, and the activity of proteins sensing membrane shape during clathrin/adaptor protein-1-coated carrier biogenesis.

Intensity-Independent Noise Filtering in FT MS and FT MS/MS Spectra for Shotgun Lipidomics.

Shotgun lipidomics relies on the direct infusion of total lipid extracts into a high resolution tandem mass spectrometer. A single shotgun analysis produces several hundred of densely populated FT MS and FT MS/MS spectra, each of which might comprise thousands of peaks although a very small percentage of those belong to lipids. Eliminating noise by adjusting a minimal peak intensity threshold is biased and inefficient since lipid species and classes vary in their natural abundance and ionization capacity. We developed a method of peak intensity-independent noise filtering in shotgun FT MS and FT MS/MS spectra that capitalizes on a stable composition of the infused analyte leading to consistent time-independent detection of its bona fide components. Repetition rate filtering relies on a single quantitative measure of peaks detection reproducibility irrespectively of their absolute intensities, masses, or assumed elemental compositions. In comparative experiments, it removed more than 95% of signals detectable in shotgun spectra without compromising the accuracy and scope of lipid identification and quantification. It also accelerated spectra processing by 15-fold and increased the number of simultaneously processed spectra by ∼500-fold hence eliminating the major bottleneck in high-throughput bottom-up shotgun lipidomics.

Detection of Independent Associations of Plasma Lipidomic Parameters with Insulin Sensitivity Indices Using Data Mining Methodology.

OBJECTIVE: Glucolipotoxicity is a major pathophysiological mechanism in the development of insulin resistance and type 2 diabetes mellitus (T2D). We aimed to detect subtle changes in the circulating lipid profile by shotgun lipidomics analyses and to associate them with four different insulin sensitivity indices. METHODS: The cross-sectional study comprised 90 men with a broad range of insulin sensitivity including normal glucose tolerance (NGT, n = 33), impaired glucose tolerance (IGT, n = 32) and newly detected T2D (n = 25). Prior to oral glucose challenge plasma was obtained and quantitatively analyzed for 198 lipid molecular species from 13 different lipid classes including triacylglycerls (TAGs), phosphatidylcholine plasmalogen/ether (PC O-s), sphingomyelins (SMs), and lysophosphatidylcholines (LPCs). To identify a lipidomic signature of individual insulin sensitivity we applied three data mining approaches, namely least absolute shrinkage and selection operator (LASSO), Support Vector Regression (SVR) and Random Forests (RF) for the following insulin sensitivity indices: homeostasis model of insulin resistance (HOMA-IR), glucose insulin sensitivity index (GSI), insulin sensitivity index (ISI), and disposition index (DI). The LASSO procedure offers a high prediction accuracy and and an easier interpretability than SVR and RF. RESULTS: After LASSO selection, the plasma lipidome explained 3% (DI) to maximal 53% (HOMA-IR) variability of the sensitivity indexes. Among the lipid species with the highest positive LASSO regression coefficient were TAG 54:2 (HOMA-IR), PC O- 32:0 (GSI), and SM 40:3:1 (ISI). The highest negative regression coefficient was obtained for LPC 22:5 (HOMA-IR), TAG 51:1 (GSI), and TAG 58:6 (ISI). CONCLUSION: Although a substantial part of lipid molecular species showed a significant correlation with insulin sensitivity indices we were able to identify a limited number of lipid metabolites of particular importance based on the LASSO approach. These few selected lipids with the closest connection to sensitivity indices may help to further improve disease risk prediction and disease and therapy monitoring.

Gender, Contraceptives and Individual Metabolic Predisposition Shape a Healthy Plasma Lipidome.

Lipidomics of human blood plasma is an emerging biomarker discovery approach that compares lipid profiles under pathological and physiologically normal conditions, but how a healthy lipidome varies within the population is poorly understood. By quantifying 281 molecular species from 27 major lipid classes in the plasma of 71 healthy young Caucasians whose 35 clinical blood test and anthropometric indices matched the medical norm, we provided a comprehensive, expandable and clinically relevant resource of reference molar concentrations of individual lipids. We established that gender is a major lipidomic factor, whose impact is strongly enhanced by hormonal contraceptives and mediated by sex hormone-binding globulin. In lipidomics epidemiological studies should avoid mixed-gender cohorts and females taking hormonal contraceptives should be considered as a separate sub-cohort. Within a gender-restricted cohort lipidomics revealed a compositional signature that indicates the predisposition towards an early development of metabolic syndrome in ca. 25% of healthy male individuals suggesting a healthy plasma lipidome as resource for early biomarker discovery.

Increasing plasma lysophosphatidylcholine levels in patients with regular dextran sulfate lipoprotein apheresis.

OBJECTIVES: Previously we found a highly significant increase of phosphatidylethanolamines (PE) in response to acute lipoprotein apheresis (LA) with whole blood dextran sulfate adsorption (DSA) in contrast to the overall tendency of reduction of lipid metabolites of all lipid classes in post-apheresis plasma. Therefore, the aim of the present study was to analyze long-term modifications of the plasma lipidomic profile in patients with repeated DSA apheresis. METHODS: Nine patients weekly treated with DSA were followed for 40 weeks. Pre- and post-apheresis levels of routine lipid parameters and lipidomic profiles of five apheresis sessions were assessed. RESULTS: The main finding of the present study was a progressive increase of pre- and post-apheresis plasma lysophosphatidylcholine (LPC) levels, which doubled in concentration at the end of the 40 week observation period. LPC metabolites which mainly contributed to this increase were LPC 20:4 > 18:0 > 18:1 > 16:0 > 20:3 > 18:2. CONCLUSION: These data indicate that long-term application of DSA technology may be associated with a continuous increase in LPC levels. Possible pro- or anti-atherogenic consequences should be elucidated in further studies.

Shotgun lipidomics on a LTQ Orbitrap mass spectrometer by successive switching between acquisition polarity modes.

Top-down shotgun lipidomics relies on direct infusion of total lipid extracts into a high-resolution tandem mass spectrometer and implies that individual lipids are recognized by their accurately determined m/z. Lipid ionization efficiency and detection specificity strongly depend on the acquisition polarity, and therefore it is beneficial to analyze lipid mixtures in both positive and negative modes. Hybrid LTQ Orbitrap mass spectrometers are widely applied in top-down lipidomics; however, rapid polarity switching was previously unfeasible because of the severe and immediate degradation of mass accuracy. Here, we report on a method to rapidly acquire high-resolution spectra in both polarity modes with sub-ppm mass accuracy and demonstrate that it not only simplifies and accelerates shotgun lipidomics analyses but also improves the lipidome coverage because more lipid classes and more individual species within each class are recognized. In this way, shotgun analysis of total lipid extracts of human blood plasma enabled to quantify 222 species from 15 major lipid classes within 7 min acquisition cycle.

LipidXplorer: a software for consensual cross-platform lipidomics.

LipidXplorer is the open source software that supports the quantitative characterization of complex lipidomes by interpreting large datasets of shotgun mass spectra. LipidXplorer processes spectra acquired on any type of tandem mass spectrometers; it identifies and quantifies molecular species of any ionizable lipid class by considering any known or assumed molecular fragmentation pathway independently of any resource of reference mass spectra. It also supports any shotgun profiling routine, from high throughput top-down screening for molecular diagnostic and biomarker discovery to the targeted absolute quantification of low abundant lipid species. Full documentation on installation and operation of LipidXplorer, including tutorial, collection of spectra interpretation scripts, FAQ and user forum are available through the wiki site at: https://wiki.mpi-cbg.de/wiki/lipidx/index.php/Main_Page.

Altered desaturation and elongation of fatty acids in hormone-sensitive lipase null mice.

BACKGROUND: Hormone-sensitive lipase (HSL) is expressed predominantly in adipose tissue, where it plays an important role in catecholamine-stimulated hydrolysis of stored lipids, thus mobilizing fatty acids. HSL exhibits broad substrate specificity and besides acylglycerides it hydrolyzes cholesteryl esters, retinyl esters and lipoidal esters. Despite its role in fatty acid mobilization, HSL null mice have been shown to be resistant to diet-induced obesity. The aim of this study was to define lipid profiles in plasma, white adipose tissue (WAT) and liver of HSL null mice, in order to better understand the role of this multifunctional enzyme. METHODOLOGY/PRINCIPAL FINDINGS: This study used global and targeted lipidomics and expression profiling to reveal changed lipid profiles in WAT, liver and plasma as well as altered expression of desaturases and elongases in WAT and liver of HSL null mice on high fat diet. Decreased mRNA levels of stearoyl-CoA desaturase 1 and 2 in WAT were consistent with a lowered ratio of 16:1n7/16:0 and 18:1n9/18:0 in WAT and plasma. In WAT, increased ratio of 18:0/16:0 could be linked to elevated mRNA levels of the Elovl1 elongase. CONCLUSIONS: This study illustrates the importance of HSL for normal lipid metabolism in response to a high fat diet. HSL deficiency greatly influences the expression of elongases and desaturases, resulting in altered lipid profiles in WAT, liver and plasma. Finally, altered proportions of palmitoleate, a recently-suggested lipokine, in tissue and plasma of HSL null mice, could be an important factor mediating and contributing to the changed lipid profile, and possibly also to the decreased insulin sensitivity seen in HSL null mice.

Bottom-up shotgun lipidomics by higher energy collisional dissociation on LTQ Orbitrap mass spectrometers.

Higher energy collision dissociation (HCD) is a complementary fragmentation tool that has recently become available on mass spectrometers of the LTQ Orbitrap family. We report on a shotgun bottom-up lipidomics approach that relies on HCD of the isolated lipid precursors. HCD, together with the high mass resolution and mass accuracy of the Orbitrap analyzer, improved the confidence of molecular species assignment and accuracy of their quantification in total lipid extracts. These capabilities were particularly important for accounting for biologically interesting lipid species comprising polyunsaturated and odd numbered fatty acid moieties. We argue that now both bottom-up and top-down shotgun lipidomics could be performed on the same instrumentation platform.