Sphingolipid Profiles Research Articles

Unique sphingolipid signature identifies luminal and triple-negative breast cancer subtypes.

Breast cancer (luminal and triple-negative breast cancer [TNBC]) is the most common cancer among women in India and worldwide. Altered sphingolipid levels have emerged as a common phenomenon during cancer progression. However, these alterations are yet to be translated into robust diagnostic and prognostic markers for cancer. Here, we present the quantified sphingolipids of tumor and adjacent-normal tissues from patients of luminal (n=70) and TNBC (n=42) subtype from an Indian cohort using targeted liquid chromatography mass spectrometry. We recorded unique sphingolipid profiles that distinguished luminal and TNBC tumors in comparison to adjacent normal tissue by six-sphingolipid signatures. Moreover, systematic comparison of the profiles of luminal and TNBC tumors provided a unique five-sphingolipid signature distinguishing the two subtypes. We further identified key sphingolipids that can stratify grade II and grade III tumors of luminal and TNBC subtype as well as their lymphovascular invasion status. Therefore, we provide the right evidence to develop these candidate sphingolipids as widely acceptable marker/s capable of diagnosing luminal vs TNBC subtype of breast cancer, and predicting the disease severity by identifying the tumor grade.

Serum sphingolipid profiling as a novel biomarker for metabolic syndrome characterization.

Sphingolipids are components of cell membrane structure, but also circulate in serum and are essential mediators of many cellular functions. While ceramides have been proposed previously as a useful biomarker for cardiometabolic disease, the involvement of other sphingolipids is still controversial. The aim of this study was to investigate the cross-sectional association between blood sphingolipidomic profiles and metabolic syndrome (MetS) as well as other atherosclerotic risk factors in a large population-based study in the U.S. Clinical data and serum sphingolipidomic profiling from 2,063 subjects who participated in the biomarker project of the Midlife in the United States (MIDUS) study were used. Consistent with previous reports, we found a positive association between most ceramide levels and obesity, atherogenic dyslipidemia, impaired glucose metabolism, and MetS prevalence. In contrast, most simple β-glycosphingolipids (i.e., hexosylceramides and lactosylceramides) were inversely associated with dysmetabolic biomarkers. However, this latter sphingolipid class showed a positive link with inflammatory and vascular damage-associated biomarkers in subjects with MetS. Through metabolic network analysis, we found that the relationship between ceramides and simple β-glycosphingolipids differed significantly not only according to MetS status, but also with respect to the participants' C-reactive protein levels. Our findings suggest that a comprehensive sphingolipid profile is more informative about MetS than ceramides alone, and it may reveal new insights into the pathophysiology and further diabetic vs. cardiovascular risk in patients with MetS.

Nanoliposomal C6-Ceramide Exhibits Synergistic Anti-Cancer Activity with a Small Molecule Agonist of PP2A in Chronic Lymphocytic Leukemia

The introduction of targeted therapies, including venetoclax (inhibitor of Bcl-2) and ibrutinib (inhibitor of Bruton's tyrosine kinase), has significantly advanced treatment of non-Hodgkin's lymphoma, particularly Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL). However, patients treated with ibrutinib, venetoclax, or the combination of ibrutinib and venetoclax are likely to exhibit treatment relapse. We have previously published that microenvironmental factors can generate anti-apoptotic resistance to combinatorial ibrutinib/venetoclax in both CLL and MCL models via NF-kB-dependent upregulation of anti-apoptotic proteins (Mcl-1 and Bcl-xL) (Jayappa et al. 2017). We have recently published the discovery of phenotypically similar drug resistant CLL cells that overexpress multiple anti-apoptotic proteins in patients de novo. These cells were enriched during treatment with venetoclax, suggesting this pool of resistant persister cells could be a source of treatment failure (Jayappa et al. 2021). We have previously formulated a non-toxic C6-ceramide nanoliposome (CNL) that greatly improves bioavailability of this bioactive sphingolipid and exerts anti-cancer activity in vivo, including CLL cell line that exhibits multidrug resistance phenotype (Lindsay et al. 2013). Thus, CNL-based novel therapies could hold promise for depleting the pool of multidrug resistant cancer cells in patients. In the current study, we screened CNL activity in CLL patient PBMC samples (N=15) using a flow cytometry-based cytotoxicity assay ex vivo. Our analysis revealed that CNL induced apoptotic/non-apoptotic cell death at overly high concentrations (EC50, 31 µM), prompting us to search for potential combinatorial therapies with enhanced toxicity profiles. To address this, we performed a combination drug screen with CNL as an anchor agent in a multidrug resistant CLL cell line (MEC1, p53-/mt) using a cell survival assay. Our analysis discovered exceptional synergistic interaction between CNL and a small molecule activator of Protein Phosphatase 2A (PP2A) (SMAP, DT061) in MEC1 cells [Bliss score (actual-Bliss predicted cytotoxicity): 56% (CNL, 6 µM; DT061, 12 µM)] and this synergistic behavior was further validated in primary CLL samples (N=5) ex vivo. Analyses using patient PBMC samples (N=3) have shown that the CNL and SMAP combination triggers apoptotic cell death with cleavage of caspase9, capase3, and PARP in CLL cells, while normal T lymphocytes in these samples were relatively less affected. At the molecular level, mutually reinforcing interactions between SMAP and CNL were observed. Using mass-spectrometric profiling of sphingolipids, we discovered that PP2A activation by SMAP modulates sphingolipid metabolism, leading to enhanced accumulation of C6-ceramide as well as synthesis of endogenous, pro-apoptotic sphingolipid molecules (C14- and C16-ceramides) in cells exposed to CNL. In addition, co-treatment with SMAP and CNL induced synergistic/additive downregulation of pBAD and cMyc, suggesting cMyc and/or Bad-dependent pathways could serve as node of convergence for synergistic interaction seen in these cells. In summary, we demonstrate that the CNL-SMAP combination displays exceptional synergy in CLL cells. At the molecular level, CNL and SMAP exhibit mutually reinforcing interactions that potentially converge on cMyc and Bad pathways, leading to synergistically increased apoptotic death in these cells. We intend to further characterize the role of cMyc and/or Bad pathway in synergistic interaction and assess the in vivo safety/efficacy of CNL-SMAP combination using a CLL xenograft mouse model. Collectively, this work provides the basis for further testing of this combination in in vivo models.

Selective Capture-Based Single-Cell Mass Spectrometry for Enhancing Sphingolipid Profiling of Neurons with Differentiation of Cell Body from Synapse.

Currently, single-cell lipidomic mass spectrometry (MS) techniques are mostly limited to detection of high-abundance phosphatidylcholines (PCs). Herein, for enhancing the coverage to low-abundance sphingolipids in single-cell analysis, in-tube solid-phase microextraction (SPME) was combined with a single-probe MS system for selective enrichment of sphingolipids during singe-cell sampling. From the results, a lab-made single probe with a 30 μm tip size proved to be able to resolve the axon from the cell body of neuron HT22 in ambient conditions. TiO2 was immobilized onto the inner wall of the transfer capillary of the single-probe device for online selective capture of sphingolipids in ammonia-acetonitrile and rapid desorption in formic acid-methanol. The results showed that the breakthrough volume of the capillary with sample loading flow rate at 500 nL/min was >14 μL. Standard experiments showed that the signals of cerebroside (CB), ceramide (Cer), and sphingomyelin (SM) were largely enhanced after selective capture in the coated capillary, while PCs were totally removed. The reusability (>10 times) and stability of the lab-made TiO2-coated capillary was verified. By introducing the coated capillary into the single-probe MS system, the new system proved to have low detection limits of SM, Cer, and CB (0.007-0.027 μg/mm2) and acceptable linearity (r > 0.98) and repeatability (RSD < 30%). Lipid coverage of the new method to SMs and CBs proved to be largely improved (SM, 21 vs 2; CB, 10 vs 0) with the new method in comparison to conventional single-probe MS without selective capture by ambient analysis of a single spot of rat cerebellum. Finally, the new system was used to perform single-neuron analysis of sphingolipids in the control and lipopolysaccharide (LPS)-treated HT22 with differentiation of the cell body from the axonal synapse. Results showed that 5 sphingolipids had significantly higher concentrations in the synapse than in the cell body, while 3 oxidized sphingolipids had significantly higher levels in the cell body than in the synapse. After LPS treatment, most of the sphingolipids largely decreased and became more accumulated in the synapse, providing new information on LPS-induced neuroinflammation.

Herpes simplex virus 1 protein pUL21 alters ceramide metabolism by activating the interorganelle transport protein CERT

Herpes simplex virus (HSV)-1 dramatically alters the architecture and protein composition of cellular membranes during infection, but its effects upon membrane lipid composition remain unclear. HSV-1 pUL21 is a virus-encoded protein phosphatase adaptor that promotes dephosphorylation of multiple cellular and virus proteins, including the cellular ceramide (Cer) transport protein CERT. CERT mediates nonvesicular Cer transport from the endoplasmic reticulum to the trans-Golgi network, whereupon Cer is converted to sphingomyelin (SM) and other sphingolipids that play important roles in cellular proliferation, signaling, and membrane trafficking. Here, we use click chemistry to profile the kinetics of sphingolipid metabolism, showing that pUL21-mediated dephosphorylation activates CERT and accelerates Cer-to-SM conversion. Purified pUL21 and full-length CERT interact with submicromolar affinity, and we solve the solution structure of the pUL21 C-terminal domain in complex with the CERT Pleckstrin homology and steroidogenic acute regulatory-related lipid transfer domains using small-angle X-ray scattering. We identify a single amino acid mutation on the surface of pUL21 that disrupts CERT binding invitro and in cultured cells. This residue is highly conserved across the genus Simplexvirus. In addition, we identify a pUL21 residue essential for binding to HSV-1 pUL16. Sphingolipid profiling demonstrates that Cer-to-SM conversion is severely diminished in the context of HSV-1 infection, a defect that is compounded when infecting with a virus encoding the mutated form of pUL21 that lacks the ability to activate CERT. However, virus replication and spread in cultured keratinocytes or epithelial cells is not significantly altered when pUL21-mediated CERT dephosphorylation is abolished. Collectively, we demonstrate that HSV-1 modifies sphingolipid metabolism via specific protein-protein interactions.

Diverse Sphingolipid Species Harbor Different Effects on Ire1 Clustering

Endoplasmic reticulum (ER) function is dedicated to multiple essential processes in eukaryotes, including the processing of secretory proteins and the biogenesis of most membrane lipids. These roles implicate a heavy burden to the organelle, and it is thus prone to fluctuations in the homeostasis of molecules which govern these processes. The unfolded protein response (UPR) is a general ER stress response tasked with maintaining the ER for optimal function, mediated by the master activator Ire1. Ire1 is an ER transmembrane protein that initiates the UPR, forming characteristic oligomers in response to irregularities in luminal protein folding and in the membrane lipid environment. The role of lipids in regulating the UPR remains relatively obscure; however, recent research has revealed a potent role for sphingolipids in its activity. Here, we identify a major role for the oxysterol-binding protein Kes1, whose activity is of consequence to the sphingolipid profile in cells resulting in an inhibition of UPR activity. Using an mCherry-tagged derivative of Ire1, we observe that this occurs due to inhibition of Ire1 to form oligomers. Furthermore, we identify that a sphingolipid presence is required for Ire1 activity, and that specific sphingolipid profiles are of major consequence to Ire1 function. In addition, we highlight cases where Ire1 oligomerization is absent despite an active UPR, revealing a potential mechanism for UPR induction where Ire1 oligomerization is not necessary. This work provides a basis for the role of sphingolipids in controlling the UPR, where their metabolism harbors a crucial role in regulating its onset.

Strong alterations in the sphingolipid profile of chickens fed a dose of fumonisins considered safe

Strong alterations in the sphingolipid profile of chickens fed a dose of fumonisins considered safe

A Comprehensive Profiling of Cellular Sphingolipids in Mammalian Endothelial and Microglial Cells Cultured in Normal and High-Glucose Conditions.

Sphingolipids (SPLs) play a diverse role in maintaining cellular homeostasis. Dysregulated SPL metabolism is associated with pathological changes in stressed and diseased cells. This study investigates differences in SPL metabolism between cultured human primary retinal endothelial (HREC) and murine microglial cells (BV2) in normal conditions (normal glucose, NG, 5 mM) and under high-glucose (HG, 25 mM)-induced stress by sphingolipidomics, immunohistochemistry, biochemical, and molecular assays. Measurable differences were observed in SPL profiles between HREC and BV2 cells. High-glucose treatment caused a >2.5-fold increase in the levels of Lactosyl-ceramide (LacCer) in HREC, but in BV2 cells, it induced Hexosyl-Ceramides (HexCer) by threefold and a significant increase in Sphingosine-1-phosphate (S1P) compared to NG. Altered SPL profiles coincided with changes in transcript levels of inflammatory and vascular permeability mediators in HREC and inflammatory mediators in BV2 cells. Differences in SPL profiles and differential responses to HG stress between endothelial and microglial cells suggest that SPL metabolism and signaling differ in mammalian cell types and, therefore, their pathological association with those cell types.

Characterization of the Sphingolipidome of the Peri-Infarct Tissue during Hemorrhagic Transformation in a Mouse Model of Cerebral Ischemia.

Cardiovascular diseases like stroke cause changes to sphingolipid mediators like sphingosine 1-phosphate (S1P) or its ceramide analogs, which bear the potential to either alleviate or exacerbate the neurological damage. Therefore, the precise identification of alterations within the sphingolipidome during ischemic stroke (IS) and hemorrhagic transformation (HT) harbors a putative therapeutic potential to orchestrate local and systemic immunomodulatory processes. Due to the scarcity of research in this field, we aimed to characterize the sphingolipidome in IS and HT. C57BL/6 mice underwent middle cerebral artery occlusion (MCAO) and specimens of the peri-infarct tissue were taken for sphingolipid profiling. Ischemic stroke resulted in reduced S1P whilst ceramides were elevated six hours post ischemia onset. However, these differences were nearly revoked at 24 hours post ischemia onset. Moreover, the topmost S1P and ceramide levels were linked to the presence of HT after MCAO. In this study we show the characterization of the sphingolipidomic landscape of the peri-infarct tissue after ischemic stroke and HT. Especially, highest values of S1P, C 18 lactosylceramide, C 18 glucosylceramide, and C 24:1 ceramide were nearly entirely expressed by mice with HT. Our results warrant further investigations into the immunomodulatory consequences of altered sphingolipid species for the development of HT after IS.

Analysis of Sphingolipids in Pediatric Patients with Cholelithiasis-A Preliminary Study.

(1) Background: Disturbances in the sphingolipid profile are observed in many diseases. There are currently no data available on the evaluation of sphingolipids and ceramides in cholelithiasis in children. The aim of this study was to evaluate the concentrations of sphingolipids in the sera of pediatric patients with gallstones. We determined their relationship with anthropometric and biochemical parameters. (2) Methods: The concentrations of sphingolipids in serum samples were evaluated using a quantitative method, ultra-high-performance liquid chromatography–tandem mass spectrometry. (3) Results: The prospective study included 48 children and adolescents diagnosed with gallstones and 38 controls. Serum concentrations of total cholesterol (TC); sphinganine (SPA); ceramides—C14:0-Cer, C16:0-Cer, C18:1-Cer, C18:0-Cer, C20:0-Cer and C24:1-Cer; and lactosylceramides—C16:0-LacCer, C18:0-LacCer, C18:1-LacCer, C24:0-LacCer and C24:1-LacCer differed significantly between patients with cholelithiasis and without cholelithiasis. After adjusting for age, gender, obesity and TC and TG levels, we found the best differentiating sphingolipids for cholelithiasis in the form of decreased SPA, C14:0-Cer, C16:0-Cer, C24:1-LacCer and C24:0-LacCer concentration and increased C20:0-Cer, C24:1-Cer, C16:0-LacCer and C18:1-LacCer. The highest area under the curve (AUC), specificity and sensitivity were determined for C16:0-Cer with cholelithiasis diagnosis. (4) Conclusions: Our results suggest that serum sphingolipids may be potential biomarkers in pediatric patients with cholelithiasis.

Ceramide Kinase (CERK) Emerges as a Common Therapeutic Target for Triple Positive and Triple Negative Breast Cancer Cells.

Simple SummaryExisting chemotherapy treatments for breast cancer patients are high on toxicity. There are very limited options available for triple-positive breast cancer (TPBC) patients, and there have not been any major breakthrough for targeted therapy for triple-negative breast cancer (TNBC) patients. Therefore, there is a need to identify common therapeutic targets for breast cancer patients. In this manuscript, we compared the sphingolipid profiles of cancer cell lines representing TPBC and TNBC, and correlated these profiles with the proliferation and migration properties the of cell types. We then associated the sphingolipid profiles for each subtype specific cell line with transcriptional and translational expression of corresponding metabolizing enzymes. Our results suggested that ceramide kinase (CERK) that catalyzes the synthesis of ceramide-1-phosphates from ceramides is dysregulated in both cell types. We also showed that the targeting of CERK at transcriptional level by siRNA therapeutics or inhibiting the CERK activity by hydrogel-mediated delivery of chemical inhibitors can be an effective strategy to slow down the tumor progression. Therefore, CERK emerges as a potential therapeutic target that can be explored further for cancer therapy.Sphingolipids are key signaling biomolecules that play a distinct role in cell proliferation, migration, invasion, drug resistance, metastasis, and apoptosis. Triple-negative (ER−PR−HER2−) and triple-positive (ER+PR+HER2+) breast cancer (called TNBC and TPBC, respectively) subtypes reveal distinct phenotypic characteristics and responses to therapy. Here, we present the sphingolipid profiles of BT-474 and MDA-MB-231 breast cancer cell lines representing the TPBC and TNBC subtypes. We correlated the level of different classes of sphingolipids and the expression of their corresponding metabolizing enzymes with the cell proliferation and cell migration properties of BT-474 and MDA-MB-231 cells. Our results showed that each cell type exhibits a unique sphingolipid profile, and common enzymes such as ceramide kinase (CERK, responsible for the synthesis of ceramide-1-phosphates) are deregulated in these cell types. We showed that siRNA/small molecule-mediated inhibition of CERK can alleviate cell proliferation in BT-474 and MDA-MB-231 cells, and cell migration in MDA-MB-231 cells. We further demonstrated that nanoparticle-mediated delivery of CERK siRNA and hydrogel-mediated sustained delivery of CERK inhibitor to the tumor site can inhibit tumor progression in BT-474 and MDA-MB-231 tumor models. In summary, distinct sphingolipid profiles of TPBC and TNBC representing cell lines provide potential therapeutic targets such as CERK, and nanoparticle/hydrogel mediated pharmacological manipulations of such targets can be explored for future cancer therapeutics.

SPTLC1 variants associated with ALS produce distinct sphingolipid signatures through impaired interaction with ORMDL proteins.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects motor neurons. Mutations in the SPTLC1 subunit of serine palmitoyltransferase (SPT), which catalyzes the first step in the de novo synthesis of sphingolipids (SLs), cause childhood-onset ALS. SPTLC1-ALS variants map to a transmembrane domain that interacts with ORMDL proteins, negative regulators of SPT activity. We show that ORMDL binding to the holoenzyme complex is impaired in cells expressing pathogenic SPTLC1-ALS alleles, resulting in increased SL synthesis and a distinct lipid signature. C-terminal SPTLC1 variants cause peripheral hereditary sensory and autonomic neuropathy type 1 (HSAN1) due to the synthesis of 1-deoxysphingolipids (1-deoxySLs) that form when SPT metabolizes L-alanine instead of L-serine. Limiting L-serine availability in SPTLC1-ALS–expressing cells increased 1-deoxySL and shifted the SL profile from an ALS to an HSAN1-like signature. This effect was corroborated in an SPTLC1-ALS pedigree in which the index patient uniquely presented with an HSAN1 phenotype, increased 1-deoxySL levels, and an L-serine deficiency. These data demonstrate how pathogenic variants in different domains of SPTLC1 give rise to distinct clinical presentations that are nonetheless modifiable by substrate availability.

Overexpression of a ceramide synthase gene,GhCS1, inhibits fiber cell initiation and elongation by promoting the synthesis of ceramides containing dihydroxy LCB and VLCFA.

Cotton is an important natural fiber crop worldwide. Cotton fiber cell is regarded as an ideal material for studying the growth and development of plant cells. Sphingolipids are important components of biomembrane and bioactive molecules which participate in many processes such as plant growth, development regulation, stimulus sensing, and stress response. However, the functions of sphingolipids in the cotton fiber development are still unclear. In the present study, we identified a cotton ceramide synthase gene, GhCS1, which is predominantly expressed in fiber cell. The GhCS1 is located in the endoplasmic reticulum and has the conserved domains of ceramide synthase. Overexpression of GhCS1 gene inhibited both vegetative and reproductive growth in cotton. Importantly, the fiber cell initiation and elongation were severely inhibited when compared with control. Comparison of the sphingolipid profile in the 0-DPA (days past anthesis) ovule (with fiber cell) between control and transgenic cotton plants showed that the content of sphingosines (Sph) decreased significantly in transgenic ovules, whereas the content of phyto-sphingosines (Phyto-Sph) had no change. Meanwhile, the content of ceramide containing Sph and very-long-chain fatty acid (VLCFA) increased significantly in transgenic ovules, while ceramide containing Phyto-Sph and long-chain fatty acids (LCFA)/VLCFA significantly decreased. These results indicated that GhCS1 was a functional ceramide synthase, which preferentially used Sph and VLCFA as substrates and was different from the Arabidopsis ceramide synthase AtLOH1/AtLOH3, which preferentially used Phyto-Sph and VLCFA as substrates, and also different from AtLOH2, which preferentially used Sph and LCFA as substrates. It is suggested that GhCS1 might be a new ceramide synthase gene in the plant, play some roles in the development of fiber cells and cotton plants.

Loss of glucosylceramide synthase impairs the growth and virulence of Fusarium oxysporum f. sp. cubense

Glucosylceramides are a class of membrane lipids that serve as vital structural and signaling molecules in eukaryotes. In this study, we explored the function of FocGCS, a glucosylceramide synthase (GCS) in Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) that causes Fusarium wilt in banana plants. FocGCS is highly expressed in germinating conidia and during early infection stage of Foc TR4. Disruption of FocGCS resulted in severely retarded vegetative growth, reduced conidiation, and production of morphologically abnormal conidia. Sphingolipid profiling revealed that the FocGCS null mutant lacks glucosylceramide. Pathogenicity assays on banana plants revealed substantial loss of virulence in the FocGCS null mutant. Moreover, biochemical analyses indicated that FocGCS is involved in cell wall integrity but is not required for oxidative and osmotic stress tolerance in Foc TR4. Transcriptome analysis suggested that disruption of FocGCS strongly affects transmembrane transport in Foc TR4. Our findings show that GCS is essential for normal fungal growth and pathogenesis in Foc TR4.

Melatonin Regulates the Daily Levels of Plasma Amino Acids, Acylcarnitines, Biogenic Amines, Sphingomyelins, and Hexoses in a Xenograft Model of Triple Negative Breast Cancer.

Metabolic dysregulation as a reflection of specific metabolite production and its utilization is a common feature of many human neoplasms. Melatonin, an indoleamine that is highly available during darkness, has a variety of metabolic functions in solid tumors. Because plasma metabolites undergo circadian changes, we investigated the role of melatonin on the profile of amino acids (AAs), biogenic amines, carnitines, sphingolipids, and hexoses present in the plasma of mice bearing xenograft triple negative breast cancer (MDA-MB-231 cells) over 24 h. Plasma concentrations of nine AAs were reduced by melatonin, especially during the light phase, with a profile closer to that of non-breast cancer (BC) animals. With respect to acylcarnitine levels, melatonin reduced 12 out of 24 molecules in BC-bearing animals compared to their controls, especially at 06:00 h and 15:00 h. Importantly, melatonin reduced the concentrations of asymmetric dimethylarginine, carnosine, histamine, kynurenine, methionine sulfoxide, putrescine, spermidine, spermine, and symmetric dimethylarginine, which are associated with the BC metabolite sets. Melatonin also led to reduced levels of sphingomyelins and hexoses, which showed distinct daily variations over 24 h. These results highlight the role of melatonin in controlling the levels of plasma metabolites in human BC xenografts, which may impact cancer bioenergetics, in addition to emphasizing the need for a more accurate examination of its metabolomic changes at different time points.

Assessing potential liver injury induced by Polygonum multiflorum using potential biomarkers via targeted sphingolipidomics

Context Polygonum multiflorum Thunb. (Polygonaceae) (PM) can cause potential liver injury which is typical in traditional Chinese medicines (TCMs)-induced hepatotoxicity. The mechanism involved are unclear and there are no sensitive evaluation indicators. Objective To assess PM-induced liver injury, identify sensitive assessment indicators, and screen for new biomarkers using sphingolipidomics. Materials and methods Male Sprague–Dawley (SD) rats were randomly divided into four groups (control, model with low-, middle- and high-dose groups, n = 6 each). Rats in the three model groups were given different doses of PM (i.g., low/middle/high dose, 2.7/8.1/16.2 g/kg) for four months. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the plasma and liver were quantitatively analyzed. Fixed liver tissue sections were stained with haematoxylin and eosin and examined under a light microscope. The targeted sphingolipidomic analysis of plasma was performed using high-performance liquid chromatography tandem mass spectrometry. Results The maximal tolerable dose (MTD) of PM administered intragastrically to mice was 51 g/kg. Sphingolipid profiling of normal and PM-induced liver injury SD rats revealed three potential biomarkers: ceramide (Cer) (d18:1/24:1), dihydroceramide (d18:1/18:0)-1-phosphate (dhCer (d18:1/18:0)-1P) and Cer (d18:1/26:1), at 867.3–1349, 383.4–1527, and 540.5–658.7 ng/mL, respectively. A criterion for the ratio of Cer (d18:1/24:1) and Cer (d18:1/26:1) was suggested and verified, with a normal range of 1.343–2.368 (with 95% confidence interval) in plasma. Conclusions Three potential biomarkers and one criterion for potential liver injury caused by PM that may be more sensitive than ALT and AST were found.

Sphingolipidome of plasma, liver, and adipose tissues and its association with insulin response to oral glucose testing in Icelandic horses.

Insulin dysregulation (ID) is a determinant of equine metabolic syndrome. Among the sphingolipids, ceramides contribute to the development of ID; however, the cross talk between the liver and adipose tissue (AT) depots and the variation among AT depots in terms of ceramide metabolism are not well understood. We aimed to characterize the sphingolipidome of plasma, liver, and AT (nuchal, NUAT; subcutaneous, SCAT; omental, OMAT; retroperitoneal, RPAT) and their associations with insulin response to oral glucose testing (OGT) in normoinsulinemic and hyperinsulinemic horses. Plasma, liver, and AT samples were collected from 12 Icelandic horses upon euthanasia and analyzed by liquid chromatography-mass spectrometry. Eighty-four targeted compounds were effectively quantified. Comparing the AT depots, greater (false discovery rate, FDR < 0.05) ceramide, dihydroceramide, and sphingomyelin concentrations and lower glucosyl- and galactosyl-ceramides were found in RPAT and OMAT than in NUAT and SCAT. Hyperinsulinemic response to OGT was associated with sphingolipidome alterations primarily in the RPAT and OMAT, whereas the NUAT sphingolipidome did not show signs of ceramide accumulation, which was inconsistent with the previously proposed role of nuchal adiposity in ID. The plasma sphingolipidome was not significantly associated with the liver or AT sphingolipidomes, indicating that plasma profiles are determined by an interplay of various organs. Furthermore, hepatic sphingolipid profiles were not correlated with the profiles of AT depots. Finally, statistically valid partial least square regression models predicting insulin response were found in the plasma (Q2 = 0.58, R2 = 0.98), liver (Q2 = 0.64, R2 = 0.74), and RPAT (Q2 = 0.68, R2 = 0.79) sphingolipidome, but not in the other adipose tissues.

Lipoprotein (a) level depending on clinical features and level of plasma sphingolipids in patients with early atherosclerosis

Background and Aims : Premature atherosclerosis is a complex multifactorial condition. The study aimed to distinguish more subgroups of the disease by measuring lipoprotein (a) (Lp(a)) and sphingolipid (SP) profile in addition to usual clinical factors.

A meal rich in palm oil or butter modifies the sphingolipid profile of postprandial triglyceride-rich lipoproteins from type 2 diabetic women

Elevated concentrations of triglyceride-rich lipoproteins (TGRL) in the fasting and postprandial states are risk factors for cardiovascular events, especially in type 2 diabetes (T2D). T2D modifies the lipid composition of plasma and lipoproteins and some sphingolipids (SP) have been validated as potent predictive biomarkers of cardiovascular disease occurrence. The main objectives of the present study were to characterize the plasma SP profile in fasting T2D patients and to determine whether SP are modified in postprandial TGRL from these patients compared to fasting TGRL. In a randomized parallel-group study, 30 T2D women ingested a breakfast including 20g lipids from either hazelnut cocoa palm oil-rich spread (Palm Nut) or Butter. Plasma was collected and TGRL were isolated by ultracentrifugation at fasting and 4h after the meal. Fasting samples of 6 control subjects from another cohort were analyzed for comparison. SP were analyzed by tandem mass spectrometry. Plasma from fasting T2D patients had higher ceramide (Cer) and ganglioside GM3 concentrations, and lower concentrations of sphingosylphosphorylcholine vs healthy subjects. In postprandial TGRL from T2D patients compared to those in the fasting state, Cer concentrations and especially C16:0, C24:1 and C24:0 molecular species, increased after the Palm Nut or Butter breakfast. A positive correlation was observed in the Palm Nut group between changes (Δ4h-fasting) of summed C16:0+C22:0+C24:1+C24:0 Cer concentrations in TGRL, and changes in plasma TG, TGRL-TG and TGRL-C16:0 concentrations. Altogether in T2D, the altered profile of plasma SP and the increased Cer concentrations in postprandial TGRL could contribute to the increased atherogenicity of TGRL.

An Altered Sphingolipid Profile as a Risk Factor for Progressive Neurodegeneration in Long-Chain 3-Hydroxyacyl-CoA Deficiency (LCHADD).

Long-chain 3-hydroxyacyl-CoA deficiency (LCHADD) and mitochondrial trifunctional protein (MTPD) belong to a group of inherited metabolic diseases affecting the degradation of long-chain chain fatty acids. During metabolic decompensation the incomplete degradation of fatty acids results in life-threatening episodes, coma and death. Despite fast identification at neonatal screening, LCHADD/MTPD present with progressive neurodegenerative symptoms originally attributed to the accumulation of toxic hydroxyl acylcarnitines and energy deficiency. Recently, it has been shown that LCHADD human fibroblasts display a disease-specific alteration of complex lipids. Accumulating fatty acids, due to defective β-oxidation, contribute to a remodeling of several lipid classes including mitochondrial cardiolipins and sphingolipids. In the last years the face of LCHADD/MTPD has changed. The reported dysregulation of complex lipids other than the simple acylcarnitines represents a novel aspect of disease development. Indeed, aberrant lipid profiles have already been associated with other neurodegenerative diseases such as Parkinson’s Disease, Alzheimer’s Disease, amyotrophic lateral sclerosis and retinopathy. Today, the physiopathology that underlies the development of the progressive neuropathic symptoms in LCHADD/MTPD is not fully understood. Here, we hypothesize an alternative disease-causing mechanism that contemplates the interaction of several factors that acting in concert contribute to the heterogeneous clinical phenotype.