Molecular species selectivity of lipid transport creates a mitochondrial sink for di-unsaturated phospholipids.

Mitochondria depend on the import of phospholipid precursors for the biosynthesis of phosphatidylethanolamine (PE) and cardiolipin, yet the mechanism of their transport remains elusive. A dynamic lipidomics approach revealed that mitochondria preferentially import di-unsaturated phosphatidylserine (PS) for subsequent conversion to PE by the mitochondrial PS decarboxylase Psd1p. Several protein complexes tethering mitochondria to the endomembrane system have been implicated in lipid transport in yeast, including the endoplasmic reticulum (ER)-mitochondrial encounter structure (ERMES), ER-membrane complex (EMC), and the vacuole and mitochondria patch (vCLAMP). By limiting the availability of unsaturated phospholipids, we created conditions to investigate the mechanism of lipid transfer and the contributions of the tethering complexes in vivo. Under these conditions, inactivation of ERMES components or of the vCLAMP component Vps39p exacerbated accumulation of saturated lipid acyl chains, indicating that ERMES and Vps39p contribute to the mitochondrial sink for unsaturated acyl chains by mediating transfer of di-unsaturated phospholipids. These results support the concept that intermembrane lipid flow is rate-limited by molecular species-dependent lipid efflux from the donor membrane and driven by the lipid species' concentration gradient between donor and acceptor membrane.

Silencing of ceramide synthase 2 in hepatocytes modulates plasma ceramide biomarkers predictive of cardiovascular death.

Emerging clinical data show that three ceramide molecules, Cer d18:1/16:0, Cer d18:1/24:1, and Cer d18:1/24:0, are biomarkers of a fatal outcome in patients with cardiovascular disease. This finding raises basic questions about their metabolic origin, their contribution to disease pathogenesis, and the utility of targeting the underlying enzymatic machinery for treatment of cardiometabolic disorders. Here, we outline the development of a potent N-acetylgalactosamine-conjugated antisense oligonucleotide engineered to silence ceramide synthase 2 specifically in hepatocytes in vivo. We demonstrate that this compound reduces the ceramide synthase 2 mRNA level and that this translates into efficient lowering of protein expression and activity as well as Cer d18:1/24:1 and Cer d18:1/24:0 levels in liver. Intriguingly, we discover that the hepatocyte-specific antisense oligonucleotide also triggers a parallel modulation of blood plasma ceramides, revealing that the biomarkers predictive of cardiovascular death are governed by ceramide biosynthesis in hepatocytes. Our work showcases a generic therapeutic framework for targeting components of the ceramide enzymatic machinery to disentangle their roles in disease causality and to explore their utility for treatment of cardiometabolic disorders.

Organic matter processing by microbial communities throughout the Atlantic water column as revealed by metaproteomics.

The phylogenetic composition of the heterotrophic microbial community is depth stratified in the oceanic water column down to abyssopelagic layers. In the layers below the euphotic zone, it has been suggested that heterotrophic microbes rely largely on solubilized particulate organic matter as a carbon and energy source rather than on dissolved organic matter. To decipher whether changes in the phylogenetic composition with depth are reflected in changes in the bacterial and archaeal transporter proteins, we generated an extensive metaproteomic and metagenomic dataset of microbial communities collected from 100- to 5,000-m depth in the Atlantic Ocean. By identifying which compounds of the organic matter pool are absorbed, transported, and incorporated into microbial cells, intriguing insights into organic matter transformation in the deep ocean emerged. On average, solute transporters accounted for 23% of identified protein sequences in the lower euphotic and ∼39% in the bathypelagic layer, indicating the central role of heterotrophy in the dark ocean. In the bathypelagic layer, substrate affinities of expressed transporters suggest that, in addition to amino acids, peptides and carbohydrates, carboxylic acids and compatible solutes may be essential substrates for the microbial community. Key players with highest expression of solute transporters were Alphaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria, accounting for 40%, 11%, and 10%, respectively, of relative protein abundances. The in situ expression of solute transporters indicates that the heterotrophic prokaryotic community is geared toward the utilization of similar organic compounds throughout the water column, with yet higher abundances of transporters targeting aromatic compounds in the bathypelagic realm.

Increasing jojoba-like wax ester production in Saccharomyces cerevisiae by enhancing very long-chain, monounsaturated fatty acid synthesis.

BACKGROUND: Fatty acids (FAs) with a chain length of more than 18 carbon atoms (> C18) are interesting for the production of specialty compounds derived from these FAs. These compounds include free FAs, like erucic acid (C22:1-Δ13), primary fatty alcohols (FOHs), like docosanol (C22:0-FOH), as well as jojoba-like wax esters (WEs) (C38-WE to C44-WE), which are esters of (very) long-chain FAs and (very) long-chain FOHs. In particular, FAs, FOHs and WEs are used in the production of chemicals, pharmaceuticals and cosmetic products. Jojoba seed oil is highly enriched in diunsaturated WEs with over 70 mol% being composed of C18:1-C24:1 monounsaturated FOH and monounsaturated FA moieties. In this study, we aim for the production of jojoba-like WEs in the yeast Saccharomyces cerevisiae by increasing the amount of very long-chain, monounsaturated FAs and simultaneously expressing enzymes required for WE synthesis. RESULTS: We show that the combined expression of a plant-derived fatty acid elongase (FAE/KCS) from Crambe abyssinica (CaKCS) together with the yeast intrinsic fatty acid desaturase (FAD) Ole1p leads to an increase in C20:1 and C22:1 FAs in S. cerevisiae. We also demonstrate that the best enzyme candidate for C24:1 FA production in S. cerevisiae is a FAE derived from Lunaria annua (LaKCS). The combined overexpression of CaKCS and Ole1p together with a fatty acyl reductase (FAR/FAldhR) from Marinobacter aquaeolei VT8 (MaFAldhR) and a wax synthase (WS) from Simmondsia chinensis (SciWS) in a S. cerevisiae strain, overexpressing a range of other enzymes involved in FA synthesis and elongation, leads to a yeast strain capable of producing high amounts of monounsaturated FOHs (up to C22:1-FOH) as well as diunsaturated WEs (up to C46:2-WE). CONCLUSIONS: Changing the FA profile of the yeast S. cerevisiae towards very long-chain monounsaturated FAs is possible by combined overexpression of endogenous and heterologous enzymes derived from various sources (e.g. a marine copepod or plants). This strategy was used to produce jojoba-like WEs in S. cerevisiae and can potentially be extended towards other commercially interesting products derived from very long-chain FAs.

Quantitative lipidomics reveals age-dependent perturbations of whole-body lipid metabolism in ACBP deficient mice.

The acyl-CoA binding protein (ACBP) plays a key role in chaperoning long-chain acyl-CoAs into lipid metabolic processes and acts as an important regulatory hub in mammalian physiology. This is highlighted by the recent finding that mice devoid of ACBP suffer from a compromised epidermal barrier and delayed weaning, the physiological process where newborns transit from a fat-based milk diet to a carbohydrate-rich diet. To gain insights into how ACBP impinges on weaning and the concomitant remodeling of whole-body lipid metabolism we performed a comparative lipidomics analysis charting the absolute abundance of 613 lipid molecules in liver, muscle and plasma from weaning and adult Acbp knockout and wild type mice. Our results reveal that ACBP deficiency affects primarily lipid metabolism of liver and plasma during weaning. Specifically, we show that ACBP deficient mice have elevated levels of hepatic cholesteryl esters, and that lipids featuring an 18:1 fatty acid moiety are increased in Acbp depleted mice across all tissues investigated. Our results also show that the perturbation of systemic lipid metabolism in Acbp knockout mice is transient and becomes normalized and similar to that of wild type as mice grow older. These findings demonstrate that ACBP serves crucial functions in maintaining lipid metabolic homeostasis in mice during weaning.

Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis.

The majority of mass spectrometry-based protein quantification studies uses peptide-centric analytical methods and thus strongly relies on efficient and unbiased protein digestion protocols for sample preparation. We present a novel objective approach to assess protein digestion efficiency using a combination of qualitative and quantitative liquid chromatography-tandem MS methods and statistical data analysis. In contrast to previous studies we employed both standard qualitative as well as data-independent quantitative workflows to systematically assess trypsin digestion efficiency and bias using mitochondrial protein fractions. We evaluated nine trypsin-based digestion protocols, based on standard in-solution or on spin filter-aided digestion, including new optimized protocols. We investigated various reagents for protein solubilization and denaturation (dodecyl sulfate, deoxycholate, urea), several trypsin digestion conditions (buffer, RapiGest, deoxycholate, urea), and two methods for removal of detergents before analysis of peptides (acid precipitation or phase separation with ethyl acetate). Our data-independent quantitative liquid chromatography-tandem MS workflow quantified over 3700 distinct peptides with 96% completeness between all protocols and replicates, with an average 40% protein sequence coverage and an average of 11 peptides identified per protein. Systematic quantitative and statistical analysis of physicochemical parameters demonstrated that deoxycholate-assisted in-solution digestion combined with phase transfer allows for efficient, unbiased generation and recovery of peptides from all protein classes, including membrane proteins. This deoxycholate-assisted protocol was also optimal for spin filter-aided digestions as compared with existing methods.

Dietary intake of a MFGM/EV-rich concentrate promotes accretion of very long odd-chain sphingolipids and increases lipid metabolic turnover at the whole-body level.

Lipids from cow milk fat globule membranes (MFGMs) and extracellular vesicles (EVs) are considered beneficial for neurodevelopment, cognitive maintenance and human health in general. Nevertheless, it is largely unknown whether intake of infant formulas and medical nutrition products rich in these particles promote accretion of specific lipids and whether this affects metabolic homeostasis. To address this, we carried out a 16-week dietary intervention study where mice were supplemented with a MFGM/EV-rich concentrate, a control diet supplemented with a whey protein concentrate and devoid of milk lipids, or regular chow. Assessment of commonly used markers of metabolic health, including body weight, glucose intolerance and liver microanatomy, demonstrated no differences across the dietary regimes. In contrast, in-depth lipidomic analysis revealed accretion of milk-derived very long odd-chain sphingomyelins and ceramides in blood plasma and multiple tissues of mice fed the MFGM/EV diet. Furthermore, lipidomic flux analysis uncovered that mice fed the MFGM/EV diet have increased lipid metabolic turnover at the whole-body level. These findings help fill a long-lasting knowledge gap between the intake of MFGM/EV-containing foods and the health-promoting effects of their lipid constituents. In addition, the findings suggest that dietary sphingomyelins or ceramide-breakdown products with very long-chains can be used as structural components of cellular membranes, lipoprotein particles and signaling molecules that modulate metabolic homeostasis and health.

Lipotype acquisition during neural development is not recapitulated in stem cell-derived neurons.

During development, different tissues acquire distinct lipotypes that are coupled to tissue function and homeostasis. In the brain, where complex membrane trafficking systems are required for neural function, specific glycerophospholipids, sphingolipids, and cholesterol are highly abundant, and defective lipid metabolism is associated with abnormal neural development and neurodegenerative disease. Notably, the production of specific lipotypes requires appropriate programming of the underlying lipid metabolic machinery during development, but when and how this occurs is unclear. To address this, we used high-resolution MSALL lipidomics to generate an extensive time-resolved resource of mouse brain development covering early embryonic and postnatal stages. This revealed a distinct bifurcation in the establishment of the neural lipotype, whereby the canonical lipid biomarkers 22:6-glycerophospholipids and 18:0-sphingolipids begin to be produced in utero, whereas cholesterol attains its characteristic high levels after birth. Using the resource as a reference, we next examined to which extent this can be recapitulated by commonly used protocols for in vitro neuronal differentiation of stem cells. Here, we found that the programming of the lipid metabolic machinery is incomplete and that stem cell-derived cells can only partially acquire a neural lipotype when the cell culture media is supplemented with brain-specific lipid precursors. Altogether, our work provides an extensive lipidomic resource for early mouse brain development and highlights a potential caveat when using stem cell-derived neuronal progenitors for mechanistic studies of lipid biochemistry, membrane biology and biophysics, which nonetheless can be mitigated by further optimizing in vitro differentiation protocols.

Deconvolution of mixture spectra and increased throughput of peptide identification by utilization of intensified complementary ions formed in tandem mass spectrometry.

A cornerstone of mass spectrometry based proteomics is to relate with high statistical significance experimentally obtained tandem mass spectrometry (MS/MS) data to peptide sequences from a protein database. Most sequence specific fragment ions in MS/MS spectra are represented by a subset of complementary ion pairs. Here, we investigated the reliabilities of complementary ion pairs formed in CAD and CAD/ETD MS/MS and developed a reliability-based approach of intensification of ion signals of complementary pairs prior to database searching. In a large-scale proteomics experiment using high-resolution orbitrap mass spectrometry, an increase in the number of peptide identifications was obtained relative to the original CAD MS/MS spectra when intensified golden complementary (+18.6%) and CAD complementary pairs (+17.2%) were submitted to the Mascot search engine. This also exceeded the results obtained by deisotoping/deconvolution of CAD MS/MS spectra. A novel approach for extracting sequence-specific fragment ions of co-isolated peptides was developed based on the complementarity rules. This technique demonstrated an impressive gain of 42.4% more peptide identifications as compared with the use of the initial data set.

Precision mapping of coexisting modifications in histone H3 tails from embryonic stem cells by ETD-MS/MS.

Post-translational modifications (PTMs) of histones play a major role in regulating chromatin dynamics and influence processes such as transcription and DNA replication. Here, we report 114 distinct combinations of coexisting PTMs of histone H3 obtained from mouse embryonic stem (ES) cells. Histone H3 N-terminal tail peptides (amino acids 1-50, 5-6 kDa) were separated by optimized weak cation exchange/hydrophilic interaction liquid chromatography (WCX/HILIC) and sequenced online by electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS). High mass accuracy and near complete sequence coverage allowed unambiguous mapping of the major histone marks and discrimination between isobaric and nearly isobaric PTMs such as trimethylation and acetylation. Hierarchical data analysis identified H3K27me2-H3K36me2 as the most frequently observed PTMs in H3. Modifications at H3 residues K27 and K36 often coexist with the abundant mark K23ac, and we identified two frequently occurring quadruplet marks 'K9me1K23acK27me2K36me2' and 'K9me3K23acK27me2K36me', which might indicate a role in crosstalk. Co-occurrence frequency analysis revealed also an interplay between methylations of K9, K27, and K36, suggesting interdependence between histone methylation marks. We hypothesize that the most abundant coexisting PTMs may provide a signature for the permissive state of mouse ES cells.

Lipidomic Characterization of Whey Concentrates Rich in Milk Fat Globule Membranes and Extracellular Vesicles.

Lipids from milk fat globule membranes (MFGMs) and extracellular vesicles (EVs) are considered beneficial for cognitive development and human health. Milk-derived whey concentrates rich in these lipids are therefore used as ingredients in infant formulas to mimic human milk and in medical nutrition products to improve the metabolic fitness of adults and elderly people. In spite of this, there is no consensus resource detailing the multitude of lipid molecules in whey concentrates. To bridge this knowledge gap, we report a comprehensive and quantitative lipidomic resource of different whey concentrates. In-depth lipidomic analysis of acid, sweet, and buttermilk whey concentrates identified 5714 lipid molecules belonging to 23 lipid classes. The data show that the buttermilk whey concentrate has the highest level of fat globule-derived triacylglycerols and that the acid and sweet whey concentrates have the highest proportions of MFGM- and EV-derived membrane lipids. Interestingly, the acid whey concentrate has a higher level of cholesterol whereas sweet whey concentrate has higher levels of lactosylceramides. Altogether, we report a detailed lipid molecular compendium of whey concentrates and lay the groundwork for using in-depth lipidomic technology to profile the nutritional value of milk products and functional foods containing dairy-based concentrates.

Beta2-glycoprotein I can exist in 2 conformations: implications for our understanding of the antiphospholipid syndrome.

The antiphospholipid syndrome is defined by the presence of antiphospholipid antibodies in blood of patients with thrombosis or fetal loss. There is ample evidence that beta(2)-glycoprotein I (beta(2)GPI) is the major antigen for antiphospholipid antibodies. The autoantibodies recognize beta(2)GPI when bound to anionic surfaces and not in solution. We showed that beta(2)GPI can exist in at least 2 different conformations: a circular plasma conformation and an "activated" open conformation. We also showed that the closed, circular conformation is maintained by interaction between the first and fifth domain of beta(2)GPI. By changing pH and salt concentration, we were able to convert the conformation of beta(2)GPI from the closed to the open conformation and back. In the activated open conformation, a cryptic epitope in the first domain becomes exposed that enables patient antibodies to bind and form an antibody-beta(2)GPI complex. We also demonstrate that the open conformation of beta(2)GPI prolonged the activated partial thromboplastin time when added to normal plasma, whereas the activated partial thromboplastin time is further prolonged by addition of anti-beta(2)GPI antibodies. The conformational change of beta(2)GPI, and the influence of the autoantibodies may have important consequences for our understanding of the antiphospholipid syndrome.

Proteome-wide alterations in Escherichia coli translation rates upon anaerobiosis.

Enzyme reprofiling in bacteria during adaptation from one environmental condition to another may be regulated by both transcription and translation. However, little is known about the contribution of translational regulation. Recently, we have developed a pulse labeling method using the methionine analog azidohomoalanine to determine the relative amounts of proteins synthesized by Escherichia coli in a brief time frame upon a change in environmental conditions. Here we present an extension of our analytical strategy, which entails measuring changes in total protein levels on the same time scale as new protein synthesis. This allows identification of stable and labile proteins and demonstrates that altered levels of most newly synthesized proteins are the result of a change in translation rate rather than degradation rate. With this extended strategy, average relative translation rates for 10 min immediately after a switch from aerobiosis to anaerobiosis were determined. The majority of proteins with increased synthesis rates upon an anaerobic switch are involved in glycolysis and pathways aimed at preventing glycolysis grinding to a halt by a cellular redox imbalance. Our method can be used to compare relative translation rates with relative mRNA levels at the same time. Discrepancies between these parameters may reveal genes whose expression is regulated by translation rather than by transcription. This may help unravel molecular mechanism underlying changes in translation rates, e.g. mediated by small regulatory RNAs.

DDA-imaging with structural identification of lipid molecules on an Orbitrap Velos Pro mass spectrometer.

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a useful technique for visualizing the spatial distribution of lipid molecules in tissues. Nevertheless, the use of MSI to investigate local lipid metabolic hallmarks has until recently been hampered by a lack of adequate technology that supports confident lipid identification. This limitation was recently mitigated by the development of DDA-imaging technology where high-resolution MSI is combined with parallel acquisition of lipid tandem MS2 spectra on a hybrid ion trap-Orbitrap Elite mass spectrometer featuring a resolving power of 240,000 and a scan time of 1 s. Here, we report the key tenets related to successful transfer of the DDA-imaging technology onto an Orbitrap Velos Pro instrument featuring a resolving power of 120,000 and a scan time of 2 s. Through meticulous performance assessments and method optimization, we tuned the DDA-imaging method to be able to confidently identify 73 molecular lipid species in mouse brain sections and demonstrate that the performance of the technology is comparable with DDA-imaging on the Orbitrap Elite. Altogether, our work shows that DDA-imaging on the Orbitrap Velos Pro instrument can serve as a robust workhorse for lipid imaging in routine applications.

Cholestasis alters brain lipid and bile acid composition and compromises motor function in neonatal piglets.

Infants with neonatal cholestasis are prone to neurodevelopmental deficits, however, the underlying pathogenesis is unclear. Lipid malabsorption and accumulation of potentially neurotoxic molecules in the blood such as bile acids are important yet relatively unexplored pathways. Here, we developed a translational piglet model to understand how the molecular bile acid and lipid composition of the brain is affected by this disease and relates to motor function. Piglets (8-days old) had bile duct ligation or sham surgery and were fed a formula diet for 3 weeks. Alongside sensory-motor deficits observed in bile duct-ligated animals, we found a shift toward a more hydrophilic and conjugated bile acid profile in the brain. Additionally, comprehensive lipidomics of the cerebellum revealed a decrease in total lipids including phosphatidylinositols and phosphatidylserines and increases in lysophospholipid species. This was paralleled by elevated cerebellar expression of genes related to inflammation and tissue damage albeit without significant impact on the brain transcriptome. This study offers new insights into the developing brain's molecular response to neonatal cholestasis indicating that bile acids and lipids may contribute in mediating motor deficits.

Brain lipidomics and neurodevelopmental outcomes in intrauterine growth restricted piglets fed dairy or vegetable fat diets.

Breast milk has neurodevelopmental advantages compared to infant formula, especially in low-birth-weight infants, which may in part relate to the fat source. This study compared neurodevelopmental outcomes in three-day-old normal birth weight (NBW) and intrauterine growth restricted (IUGR) piglets fed a formula diet with either vegetable oil (VEG) or bovine milk fat sources (MILK) for three weeks in a 2 × 2 factorial design. Behavioural tests, lipidomics, MRI and RNA sequencing analyses of plasma and brain tissue were conducted. The absolute levels of 82% and 11% of lipid molecules were different between dietary groups in plasma and hippocampus, respectively. Of the lipid molecules with differential abundance in the hippocampus, the majority were upregulated in MILK versus VEG, and they mainly belonged to the group of glycerophospholipids. Lower absolute brain weights, absolute grey and white matter volumes and behaviour and motor function scores, and higher relative total brain weights were present in IUGR compared to NBW with minor influence of diet. Cognitive function and cerebellar gene expression profiles were similar for dietary and weight groups, and overall only minor interactive effects between diet and birth weight were observed. Overall, we show that the dietary fat source influences the plasma and to a lesser degree the hippocampal lipidome and is unable to improve on IUGR-induced brain structural and functional impairments.

Determination of the binding sites for oxaliplatin on insulin using mass spectrometry-based approaches.

Using insulin as a model protein for binding of oxaliplatin to proteins, various mass spectrometric approaches and techniques were compared. Several different platinum adducts were observed, e.g. addition of one or two diaminocyclohexane platinum(II) (Pt(dach)) molecules. By top-down analysis and fragmentation of the intact insulin-oxaliplatin adduct using nano-electrospray ionisation quadrupole time-of-flight mass spectrometry (nESI-Q-ToF-MS), the major binding site was assigned to histidine5 on the insulin B chain. In order to simplify the interpretation of the mass spectrum, the disulphide bridges were reduced. This led to the additional identification of cysteine6 on the A chain as a binding site along with histidine5 on the B chain. Digestion of insulin-oxaliplatin with endoproteinase Glu-C (GluC) followed by reduction led to the formation of five peptides with Pt(dach) attached. Identification of several of the binding sites was obtained using matrix-assisted laser desorption/ionization (MALDI)-ToF-ToF-MS and liquid chromatography-nESI-Q-ToF-MS. Upon comparing the top-down and bottom-up approaches, the suitability of the bottom-up approach for determining binding sites was questioned, as the release and possible re-association of Pt(dach) were demonstrated upon enzymatic digestion. The associated advantages and disadvantages of ESI and MALDI were also pointed out.

Identification and quantitation of newly synthesized proteins in Escherichia coli by enrichment of azidohomoalanine-labeled peptides with diagonal chromatography.

A method is presented to identify and quantify several hundreds of newly synthesized proteins in Escherichia coli upon pulse labeling cells with the methionine analogue azidohomoalanine (azhal). For the first 30 min after inoculation, a methionine-auxotrophic strain grows equally well on azhal as on methionine. Upon a pulse of 15 min and digestion of total protein, azhal-labeled peptides are isolated by a retention time shift between two reversed phase chromatographic runs. The retention time shift is induced by a reaction selective for the azido group in labeled peptides using tris(2-carboxyethyl)phosphine. Selectively modified peptides are identified by reversed phase liquid chromatography and on-line tandem mass spectrometry. We identified 527 proteins representative of all major Gene Ontology categories. Comparing the relative amounts of 344 proteins synthesized in 15 min upon a switch of growth temperature from 37 to 44 degrees C showed that nearly 20% increased or decreased more than 2-fold. Among the most up-regulated proteins many were chaperones and proteases in accordance with the cells response to unfolded proteins due to heat stress. Comparison of our data with results from previous microarray experiments revealed the importance of regulation of gene expression at the level of transcription of the most elevated proteins under heat shock conditions and enabled identification of several candidate genes whose expression may predominantly be regulated at the level of translation. This work demonstrates for the first time the use of a bioorthogonal amino acid for proteome-wide detection of changes in the amounts of proteins synthesized during a brief period upon variations in cellular growth conditions. Comparison of such data with relative mRNA levels enables assessment of the separate contributions of transcription and translation to the regulation of gene expression.

Lipid molecular timeline profiling reveals diurnal crosstalk between the liver and circulation.

Diurnal regulation of whole-body lipid metabolism plays a vital role in metabolic health. Although changes in lipid levels across the diurnal cycle have been investigated, the system-wide molecular responses to both short-acting fasting-feeding transitions and longer-timescale circadian rhythms have not been explored in parallel. Here, we perform time-series multi-omics analyses of liver and plasma revealing that the majority of molecular oscillations are entrained by adaptations to fasting, food intake, and the postprandial state. By developing algorithms for lipid structure enrichment analysis and lipid molecular crosstalk between tissues, we find that the hepatic phosphatidylethanolamine (PE) methylation pathway is diurnally regulated, giving rise to two pools of oscillating phosphatidylcholine (PC) molecules in the circulation, which are coupled to secretion of either very low-density lipoprotein (VLDL) or high-density lipoprotein (HDL) particles. Our work demonstrates that lipid molecular timeline profiling across tissues is key to disentangling complex metabolic processes and provides a critical resource for the study of whole-body lipid metabolism.

Dairy-Derived Emulsifiers in Infant Formula Show Marginal Effects on the Plasma Lipid Profile and Brain Structure in Preterm Piglets Relative to Soy Lecithin.

Breastfed infants have higher intestinal lipid absorption and neurodevelopmental outcomes compared to formula-fed infants, which may relate to a different surface layer structure of fat globules in infant formula. This study investigated if dairy-derived emulsifiers increased lipid absorption and neurodevelopment relative to soy lecithin in newborn preterm piglets. Piglets received a formula diet containing soy lecithin (SL) or whey protein concentrate enriched in extracellular vesicles (WPC-A-EV) or phospholipids (WPC-PL) for 19 days. Both WPC-A-EV and WPC-PL emulsions, but not the intact diets, increased in vitro lipolysis compared to SL. The main differences of plasma lipidomics analysis were increased levels of some sphingolipids, and lipid molecules with odd-chain (17:1, 19:1, 19:3) as well as mono- and polyunsaturated fatty acyl chains (16:1, 20:1, 20:3) in the WPC-A-EV and WPC-PL groups and increased 18:2 fatty acyls in the SL group. Indirect monitoring of intestinal triacylglycerol absorption showed no differences between groups. Diffusor tensor imaging measurements of mean diffusivity in the hippocampus were lower for WPC-A-EV and WPC-PL groups compared to SL indicating improved hippocampal maturation. No differences in hippocampal lipid composition or short-term memory were observed between groups. In conclusion, emulsification of fat globules in infant formula with dairy-derived emulsifiers altered the plasma lipid profile and hippocampal tissue diffusivity but had limited effects on other absorptive and learning abilities relative to SL in preterm piglets.