Sphingolipids (SLs) are essential lipids with important functions in membrane formation and cell signaling. The presence of a long chain base (LCB) structure is common to all SLs. De novo SL synthesis is initiated by the enzyme serine-palmitoyltransferase (SPT), which forms an LCB by the conjugation from serine and fatty acyl-CoAs. SPT can metabolize a variety of acyl-CoA substrates, which form diverse LCB structures within and across species. The LCB then undergoes further metabolic modifications resulting in an extraordinarily diverse spectrum of sphingolipids formed. SL analysis, using liquid chromatography-mass spectrometry (LC-MS)-based methods, poses challenges due to the diverse range of frequently isobaric species. This complexity complicates the identification of underlying LCB structures using standard lipidomics approaches. Here, we describe a simplified method to analyze the LCB profile in cells, tissue, and blood. The procedure involves chemical hydrolysis to remove the conjugated headgroups and N-acyl chains, allowing to specifically resolve the underlying LCB structures by LC-MS. This method can also be combined with an isotope labeling approach to determine in vivo SPT activity and total SL de novo synthesis over time.

In this community effort, we compare measurements between 34 laboratories from 19 countries, utilizing mixtures of labelled authentic synthetic standards, to quantify by mass spectrometry four clinically used ceramide species in the NIST (National Institute of Standards and Technology) human blood plasma Standard Reference Material (SRM) 1950, as well as a set of candidate plasma reference materials (RM 8231). Participants either utilized a provided validated method and/or their method of choice. Mean concentration values, and intra- and inter-laboratory coefficients of variation (CV) were calculated using single-point and multi-point calibrations, respectively. These results are the most precise (intra-laboratory CVs ≤ 4.2%) and concordant (inter-laboratory CVs < 14%) community-derived absolute concentration values reported to date for four clinically used ceramides in the commonly analyzed SRM 1950. We demonstrate that calibration using authentic labelled standards dramatically reduces data variability. Furthermore, we show how the use of shared RM can correct systematic quantitative biases and help in harmonizing lipidomics. Collectively, the results from the present study provide a significant knowledge base for translation of lipidomic technologies to future clinical applications that might require the determination of reference intervals (RIs) in various human populations or might need to estimate reference change values (RCV), when analytical variability is a key factor for recall during multiple testing of individuals.

ABSTRACT Introduction Along with recent advances in analytical technologies, TCA-cycle intermediates are increasingly identified as promising makers for cellular ischemia and mitochondrial dysfunction during hemorrhagic shock (HS). For traumatized patients, the knowledge of the role of lipid oxidation substrates is sparse. In this study, we aimed to analyze the dynamics of systemic acylcarnitine (AcCa) release in a standardized polytrauma model with HS. Methods 52 male pigs (50 ± 5 kg) were randomized into two groups: Group IF (isolated fracture) was subject to a standardized femur shaft fracture. Group PT (polytrauma) was subject to a femur fracture, followed by blunt chest trauma, liver laceration and a pressure controlled hemorrhagic shock for 60 min. Resuscitation was performed with crystalloids. Fractures were stabilized by intramedullary nailing. Venous samples were collected at 6 timepoints (baseline, trauma, resuscitation, 2 h, 4 h and 6 h). Lipidomic analysis was performed via liquid chromatography coupled mass spectrometry. Measurements were collated with clinical markers and near-infrared spectrometry measurements (NIRS) of tissue perfusion. Longitudinal analyses were performed with linear mixed models and spearman’s correlations were calculated. A p-value of 0.05 was defined as threshold for statistical significance. Results From a total of 303 distinct lipids, we identified two species of long-chain AcCas. Both showed a highly significant (p < 0.001) two-fold increase after HS in Group PT that promptly normalized after resuscitation. This increase was associated with a significant decrease of the base excess (p = 0.005) but recovery after resuscitation was faster. For both AcCas, there were significant correlations with decreased muscle tissue oxygen delivery (p = 0.008, p = 0.003) and significant time-lagged correlations with the increase of creatine kinase (p < 0.001, p < 0.001). Conclusion Our results point to plasma AcCas as a possible indicator for mitochondrial dysfunction and cellular ischemia in HS. The more rapid normalization after resuscitation in comparison to acid base changes may warrant further investigation. Study Type Experimental Animal Model Level of Evidence N/A

Metabolic dysfunction-associated steatohepatitis (MASH) is a leading cause of chronic liver disease with few therapeutic options. To narrow the translational gap in the development of pharmacological MASH treatments, a 3D liver model from primary human hepatocytes and non-parenchymal cells derived from patients with histologically confirmed MASH was established. The model closely mirrors disease-relevant endpoints, such as steatosis, inflammation and fibrosis, and multi-omics analyses show excellent alignment with biopsy data from 306 MASH patients and 77 controls. By combining high-content imaging with scalable biochemical assays and chemogenomic screening, multiple novel targets with anti-steatotic, anti-inflammatory, and anti-fibrotic effects are identified. Among these, activation of the muscarinic M1 receptor (CHRM1) and inhibition of the TRPM8 cation channel result in strong anti-fibrotic effects, which are confirmed using orthogonal genetic assays. Strikingly, using biosensors based on bioluminescence resonance energy transfer, a functional interaction along a novel MASH signaling axis in which CHRM1 inhibits TRPM8 via Gq/11 and phospholipase C-mediated depletion of phosphatidylinositol 4,5-bisphosphate can be demonstrated. Combined, this study presents the first patient-derived 3D MASH model, identifies a novel signaling module with anti-fibrotic effects, and highlights the potential of organotypic culture systems for phenotype-based chemogenomic drug target identification at scale.

Background Metabolic cardiomyopathy (MCM), characterized by intramyocardial lipid accumulation, drives the progression to heart failure with preserved ejection fraction (HFpEF). Although evidence suggests that the mammalian silent information regulator 1 (Sirt1) orchestrates myocardial lipid metabolism, it is unknown whether its exogenous administration could avoid MCM onset. We investigated whether chronic treatment with recombinant Sirt1 (rSirt1) could halt MCM progression. Methods db/db mice, an established model of MCM, were supplemented with intraperitoneal rSirt1 or vehicle for 4 weeks and compared with their db/ + heterozygous littermates. At the end of treatment, cardiac function was assessed by cardiac ultrasound and left ventricular samples were collected and processed for molecular analysis. Transcriptional changes were evaluated using a custom PCR array. Lipidomic analysis was performed by mass spectrometry. H9c2 cardiomyocytes exposed to hyperglycaemia and treated with rSirt1 were used as in vitro model of MCM to investigate the ability of rSirt1 to directly target cardiomyocytes and modulate malondialdehyde levels and caspase 3 activity. Myocardial samples from diabetic and nondiabetic patients were analysed to explore Sirt1 expression levels and signaling pathways. Results rSirt1 treatment restored cardiac Sirt1 levels and preserved cardiac performance by improving left ventricular ejection fraction, fractional shortening and diastolic function (E/A ratio). In left ventricular samples from rSirt1-treated db/db mice, rSirt1 modulated the cardiac lipidome: medium and long-chain triacylglycerols, long-chain triacylglycerols, and triacylglycerols containing only saturated fatty acids were reduced, while those containing docosahexaenoic acid were increased. Mechanistically, several genes involved in lipid trafficking, metabolism and inflammation, such as Cd36 , Acox3 , Pparg , Ncoa3 , and Ppara were downregulated by rSirt1 both in vitro and in vivo. In humans, reduced cardiac expression levels of Sirt1 were associated with higher intramyocardial triacylglycerols and PPARG-related genes. Conclusions In the db/db mouse model of MCM, chronic exogenous rSirt1 supplementation rescued cardiac function. This was associated with a modulation of the myocardial lipidome and a downregulation of genes involved in lipid metabolism, trafficking, inflammation, and PPARG signaling. These findings were confirmed in the human diabetic myocardium. Treatments that increase Sirt1 levels may represent a promising strategy to prevent myocardial lipid abnormalities and MCM development. Graphical Abstract

Sphingolipids (SL) represent a structurally diverse class of lipids that are central to cellular physiology and neuronal development and function. Defects in the sphingolipid metabolism are typically associated with nervous system disorders. The C4-dihydroceramide desaturase (DEGS1) catalyzes the conversion of dihydroceramide to ceramide, the final step in the SL de-novo synthesis. Loss of function mutations in DEGS1 cause a hypomyelinating leukodystrophy, which is associated with increased plasma dihydrosphingolipids (dhSL) and with the formation of an atypical SPB 18:1(14Z);O2 metabolite. Here, we characterize two novel DEGS1 variants of unknown significance (VUS), provide a structural model with a predicted substrate binding site, and propose a regulatory link between DEGS1 and fatty acid desaturase 3 (FADS3). Both VUS involve single amino acid substitutions near the C-terminus within conserved regions of the enzyme. Patient 1 (p.R311K variant) shows severe progressive tetraspasticity, intellectual disability, and epilepsy in combination with brain magnetic resonance imaging (MRI) findings, typical for DEGS1-related leukodystrophy. Patient 2 (p.G270E variant) presents with delayed psychomotor development, oculomotor apraxia, and a normal brain MRI. Plasma from the p.R311K carrier showed a significantly elevated dhSL species and the presence of SPB 18:1(14Z);O2, while the plasma SL profile for the p.G270E variant was not altered. This suggests the p.R331K variant is pathogenic, while the p.G270E appears benign. As an increase in dihydroSL species is also seen in other pathological disorders of the SL metabolism, the SPB 18:1(14Z);O2 seems to be a more specific biomarker to discriminate between pathogenic and benign DEGS1 variants.

Background Occult hypoperfusion describes the absence of sufficient microcirculation despite normal vital signs. It is known to be associated with prolonged elevation of serum lactate and later complications in severely injured patients. We hypothesized that changes in circulating lipids are related to responsiveness to resuscitation. The purpose of this study is investigating the relation between responsiveness to resuscitation and lipidomic course after poly trauma. Methods Twenty-five male pigs were exposed a combined injury of blunt chest trauma, liver laceration, controlled haemorrhagic shock, and femoral shaft fracture. After 1 h, animals received resuscitation and fracture stabilization. Venous blood was taken regularly and 233 specific lipids were analysed. Animals were divided into two groups based on serum lactate level at the end point as an indicator of responsiveness to resuscitation (<2 mmol/L: responder group (R group), ≧2 mmol/L: occult hypoperfusion group (OH group)). Results Eighteen animals met criteria for the R group, four animals for the OH group, and three animals died. Acylcarnitines showed a significant increase at 1 h compared to baseline in both groups. Six lipid subgroups showed a significant increase only in R group at 2 h. There was no significant change at other time points. Conclusions Six lipid groups increased significantly only in the R group at 2 h, which may support the idea that they could serve as potential biomarkers to help us to detect the presence of occult hypoperfusion and insufficient resuscitation. We feel that further study is required to confirm the role and mechanism of lipid changes after trauma.

Objective: ApoM enriches S1P (sphingosine-1-phosphate) within HDL (high-density lipoproteins) and facilitates the activation of the S1P1 (S1P receptor type 1) by S1P, thereby preserving endothelial barrier function. Many protective functions exerted by HDL in extravascular tissues raise the question of how S1P regulates transendothelial HDL transport.Approach and Results: HDL were isolated from plasma of wild-type mice, Apom knockout mice, human apoM transgenic mice or humans and radioiodinated to trace its binding, association, and transport by bovine or human aortic endothelial cells. We also compared the transport of fluorescently-labeled HDL or Evans Blue, which labels albumin, from the tail vein into the peritoneal cavity of apoE-haploinsufficient mice with (apoE-haploinsufficient mice with endothelium-specific knockin of S1P1) or without (control mice, ie, apoE-haploinsufficient mice without endothelium-specific knockin of S1P1) endothelium-specific knockin of S1P1. The binding, association, and transport of HDL from Apom knockout mice and human apoM-depleted HDL by bovine aortic endothelial cells was significantly lower than that of HDL from wild-type mice and human apoM-containing HDL, respectively. The binding, uptake, and transport of 125I-HDL by human aortic endothelial cells was increased by an S1P1 agonist but decreased by an S1P1 inhibitor. Silencing of SR-BI (scavenger receptor BI) abrogated the stimulation of 125I-HDL transport by the S1P1 agonist. Compared with control mice, that is, apoE-haploinsufficient mice without endothelium-specific knockin of S1P1, apoE-haploinsufficient mice with endothelium-specific knockin of S1P1 showed decreased transport of Evans Blue but increased transport of HDL from blood into the peritoneal cavity and SR-BI expression in the aortal endothelium.Conclusions: ApoM and S1P1 promote transendothelial HDL transport. Their opposite effect on transendothelial transport of albumin and HDL indicates that HDL passes endothelial barriers by specific mechanisms rather than passive filtration.

Primary cilia are key sensory organelles whose dysfunction leads to ciliopathy disorders such as Bardet-Biedl syndrome (BBS). Retinal degeneration is common in ciliopathies, since the outer segments (OSs) of photoreceptors are highly specialized primary cilia. BBS1, encoded by the most commonly mutated BBS-associated gene, is part of the BBSome protein complex. Using a new bbs1 zebrafish mutant, we show that retinal development and photoreceptor differentiation are unaffected by Bbs1-loss, supported by an initially unaffected transcriptome. Quantitative proteomics and lipidomics on isolated OSs show that Bbs1 is required for BBSome-entry into OSs and that Bbs1-loss leads to accumulation of membrane-associated proteins in OSs, with enrichment in proteins involved in lipid homeostasis. Disruption of the tightly regulated OS lipid composition with increased OS cholesterol content are paralleled by early functional visual deficits, which precede progressive OS morphological anomalies. Our findings identify a new role for Bbs1/BBSome in OS lipid homeostasis and suggest a new pathomechanism underlying retinal degeneration in BBS.

OBJECTIVE:Debate continues regarding the influence of dietary fats and sugars on the risk of developing metabolic diseases, including insulin resistance and nonalcoholic fatty liver disease (NAFLD). We investigated the effect of two eucaloric diets, one enriched with saturated fat (SFA) and the other enriched with free sugars (SUGAR), on intrahepatic triacylglycerol (IHTAG) content, hepatic de novo lipogenesis (DNL), and whole-body postprandial metabolism in overweight males.RESEARCH DESIGN AND METHODS:Sixteen overweight males were randomized to consume the SFA or SUGAR diet for 4 weeks before consuming the alternate diet after a 7-week washout period. The metabolic effects of the respective diets on IHTAG content, hepatic DNL, and whole-body metabolism were investigated using imaging techniques and metabolic substrates labeled with stable-isotope tracers.RESULTS:Consumption of the SFA diet significantly increased IHTAG by mean ± SEM 39.0 ± 10.0%, while after the SUGAR diet IHTAG was virtually unchanged. Consumption of the SFA diet induced an exaggerated postprandial glucose and insulin response to a standardized test meal compared with SUGAR. Although whole-body fat oxidation, lipolysis, and DNL were similar following the two diets, consumption of the SUGAR diet resulted in significant (P < 0.05) decreases in plasma total, HDL, and non-HDL cholesterol and fasting β-hydroxybutyrate plasma concentrations.CONCLUSIONS:Consumption of an SFA diet had a potent effect, increasing IHTAG together with exaggerating postprandial glycemia. The SUGAR diet did not influence IHTAG and induced minor metabolic changes. Our findings indicate that a diet enriched in SFA is more harmful to metabolic health than a diet enriched in free sugars.

Sphingolipids (SLs) are chemically diverse lipids that have important structural and signaling functions within mammalian cells. SLs are commonly defined by the presence of a long-chain base (LCB) that is normally formed by the conjugation of l-serine and palmitoyl-CoA. This pyridoxal 5-phosphate (PLP)-dependent reaction is mediated by the enzyme serine-palmitoyltransferase (SPT). However, SPT can also metabolize other acyl-CoAs, in the range of C14 to C18, forming a variety of LCBs that differ by structure and function. Mammalian SPT consists of three core subunits: SPTLC1, SPTLC2, and SPTLC3. Whereas SPTLC1 and SPTLC2 are ubiquitously expressed, SPTLC3 expression is restricted to certain tissues only. The influence of the individual subunits on enzyme activity is not clear. Using cell models deficient in SPTLC1, SPTLC2, and SPTLC3, we investigated the role of each subunit on enzyme activity and the LCB product spectrum. We showed that SPTLC1 is essential for activity, whereas SPTLC2 and SPTLC3 are partly redundant but differ in their enzymatic properties. SPTLC1 in combination with SPTLC2 specifically formed C18, C19, and C20 LCBs while the combination of SPTLC1 and SPTLC3 yielded a broader product spectrum. We identified anteiso-branched-C18 SO (meC18SO) as the primary product of the SPTLC3 reaction. The meC18SO was synthesized from anteiso-methyl-palmitate, in turn synthesized from a precursor metabolite generated in the isoleucine catabolic pathway. The meC18SO is metabolized to ceramides and complex SLs and is a constituent of human low- and high-density lipoproteins.

Glycosylation is an integral part in health and disease, as emphasized by the growing number of identified glycosylation defects. In humans, proteins are modified with a diverse range of glycoforms synthesized in complex biosynthetic pathways. Glycosylation disorders have been described in congenital disorders of glycosylation (CDG) as well as in acquired disease conditions such and non-alcoholic fatty liver disease (NAFLD). A hallmark in a subset of CDG cases is the reduced glycosylation site occupancy of asparagine-linked glycans. Using an optimized method protocol, we determined the glycosylation site occupancy from four proteins of hepatic and lymphatic origin from CDG and NAFLD patients. We found variable degrees of site occupancy, depending on the tissue of origin and the disease condition. In CDG glycosylation sites of IgG2 and IgA1 were occupied to normal levels. In NAFLD haptoglobin and transferrin glycosylation sites were hyper-glycosylated, a property qualifying for its use as a potential biomarker. Furthermore, we observed, that glycosylation sites of liver-originating transferrin and haptoglobin are differentially occupied under physiological conditions, a further instance not noticed in serum proteins to date. Our findings suggest the use of serum protein hyperglycosylation as a biomarker for early stages of NAFLD.

Acanthamoeba polyphaga mimivirus is a giant virus encoding 1262 genes among which many were previously thought to be exclusive to cellular life. For example, mimivirus genes encode enzymes involved in the biosynthesis of nucleotide sugars and putative glycosyltransferases. We identified in mimivirus a glycogenin-1 homologous gene encoded by the open reading frame R707. The R707 protein was found to be active as a polymerizing glucosyltransferase enzyme. Like glycogenin-1, R707 activity was divalent-metal-ion-dependent and relied on an intact DXD motif. In contrast with glycogenin-1, R707 was, however, not self-glucosylating. Interestingly, the product of R707 catalysis featured α1-6, β1-6 and α1-4 glycosidic linkages. Mimivirus R707 is the first reported glycosyltransferase able to catalyse the formation of both α and β linkages. Mimivirus-encoded glycans play a role in the infection of host amoebae. Co-infection of Acanthamoeba with mimivirus and amylose and chitin hydrolysate reduced the number of infected amoebae, thus supporting the importance of polysaccharide chains in the uptake of mimivirus by amoebae. The identification of a glycosyltransferase capable of forming α and β linkages underlines the peculiarity of mimivirus and enforces the concept of a host-independent glycosylation machinery in mimivirus.

Acanthamoeba polyphaga mimivirus is a member of the giant nucleocytoplasmic large DNA viruses, infecting various Acanthamoeba spp. The genomes of giant viruses encode components previously thought to be exclusive to cellular life, such as proteins involved in nucleic acid and protein synthesis. Recent work on enzymes involved in carbohydrate biosynthesis and metabolism show that instead of utilizing host cell resources, Mimivirus produces its own glycosylation machinery. To obtain a more detailed view of glycosylation in Mimivirus, we developed a periodate oxidation-based method to selectively enrich Mimivirus surface glycoproteins. O-Glycosylation in Mimivirus glycoproteins was identified by permethylation and matrix-assisted laser desorption/ionization-mass spectrometry analyses of beta-eliminated glycans. We sequenced 26 previously undescribed O-glycans, most of which contain glucose as their reducing end saccharide. These data will facilitate future studies on the functional significance of glycosylation in Mimivirus.

Deficiency in N-linked protein glycosylation is a long-known characteristic of alcoholic liver disease and congenital disorders of glycosylation. Previous investigations of ethanol-induced glycosylation deficiency demonstrated perturbations in the early steps of substrate synthesis and in the final steps of capping N-linked glycans in the Golgi. The significance of the biosynthesis of N-glycan precursors in the endoplasmic reticulum, however, has not yet been addressed in alcoholic liver disease. Ethanol-metabolizing hepatoma cells were treated with increasing concentrations of ethanol. Transcript analysis of genes involved in the biosynthesis of N-glycans, activity assays of related enzymes, dolichol-phosphate quantification, and analysis of dolichol-linked oligosaccharides were performed. Upon treatment of cells with ethanol, we found a decrease in the final N-glycan precursor Dol-PP-GlcNAc(2) Man(9) Glc(3) and in C95- and C100-dolichol-phosphate levels. Transcript analysis of genes involved in N-glycosylation showed a 17% decrease in expression levels of DPM1, a subunit of the dolichol-phosphate-mannose synthase, and an 8% increase in RPN2, a subunit of the oligosaccharyl transferase. Ethanol treatment decreases the biosynthesis of dolichol-phosphate. Consequently, the formation of N-glycan precursors is affected, resulting in an aberrant precursor assembly. Messenger RNA levels of genes involved in N-glycan biosynthesis are slightly affected by ethanol treatment, indicating that the assembly of N-glycan precursors is not regulated at the transcriptional level. This study confirms that ethanol impairs N-linked glycosylation by affecting dolichol biosynthesis leading to impaired dolichol-linked oligosaccharide assembly. Together our data help to explain the underglycosylation phenotype observed in alcoholic liver disease and congenital disorders of glycosylation.

ABSTRACT Collagens, the most abundant proteins in animals, also occur in some recently described nucleocytoplasmic large DNA viruses such as Mimiviridae , which replicate in amoebae. To clarify the impact of viral collagens on the immune response of animals exposed to Mimiviridae , we have investigated the localization of collagens in Acanthamoeba polyphaga mimivirus particles and the response of mice to immunization with mimivirus particles. Using protein biotinylation, we have first shown that viral collagen encoded by open reading frame L71 is present at the surface of mimivirus particles. Exposure to mimivirus collagens elicited the production of anti-collagen antibodies in DBA/1 mice immunized intradermally with mimivirus protein extracts. This antibody response also targeted mouse collagen type II and was accompanied by T-cell reactivity to collagen and joint inflammation, as observed in collagen-induced arthritis following immunization of mice with bovine collagen type II. The broad distribution of nucleocytoplasmic large DNA viruses in the environment suggests that humans are constantly exposed to such large virus particles. A survey of blood sera from healthy human subjects and from rheumatoid arthritis patients indeed demonstrated that 30% of healthy-subject and 36% of rheumatoid arthritis sera recognized the major mimivirus capsid protein L425. Moreover, whereas 6% of healthy-subject sera recognized the mimivirus collagen protein L71, 22% of rheumatoid arthritis sera were positive for mimivirus L71. Accordingly, our study shows that environmental exposure to mimivirus represents a risk factor in triggering autoimmunity to collagens.

Time 8:30 Glycobiology of Human Pluripotent Stem Cells; Steve Dalton, University of Georgia #1 9:00 Siglec-Sialoglycan Binding Regulates Cell-Cell Interactions; Ronald Schnaar, Johns Hopkins School of Medicine 9:30 Stem Cell Therapies in Epidermolysis Bullosa; Angela Christiano, Columbia University Medical Center 10:00 – 10:30 am Coffee Break (Grande Foyer and Grande Ballroom A) 10:30 am – 12:00 pm Plenary II: Biomaterials and Matrix Engineering (Grande Ballroom B & C) Chair: Adam Engler, University of California, San Diego 10:30 Hydrogels as Synthetic Extracellular Matrices; Kristi Anseth, University of Colorado at Boulder 11:00 Mechanical Regulation of Cell Adhesion and Function; Christopher Chen, University of Pennsylvania Joint Meeting of the Society for Glycobiology & American Society for Matrix Biology Conference Program by A rm en Peosyan on N ovem er 2, 2012 http://glycfordjournals.org/ D ow nladed from