Plasma Metabonomics of Human Adenovirus-infected Patients with Pneumonia and Upper Respiratory Tract Infection.

OBJECTIVE: Human adenovirus (HAdV) infection is common and can develop to serious conditions with high mortality, yet the mechanism of HAdV infection remains unclear. In the present study, the serum metabolite profiles of HAdV-7-infected patients with pneumonia or upper respiratory tract infection (URTI) were explored. METHODS: In total, 35 patients were enrolled in the study following an outbreak of HAdV-7 in the army, of whom 14 had pneumonia and 21 had URTI. Blood samples were collected at the acute stage and at the recovery stage and were analyzed by untargeted metabolomics. RESULTS: Over 90% of the differential metabolites identified between the pneumonia patients and URTI patients were lipids and lipid-like molecules, including glycerophospholipids, fatty acyls, and sphingolipids. The metabolic pathways that were significantly enriched were primarily the lipid metabolism pathways, including sphingolipid metabolism, glycerophospholipid metabolism, and linoleic acid metabolism. The sphingolipid metabolism was identified as a significantly differential pathway between the pneumonia patients and URTI patients and between the acute and recovery stages for the pneumonia patients, but not between the acute and recovery stages for the URTI patients. Ceramide and lactosylceramide, involved in sphingolipid metabolism, were significantly higher in the pneumonia patients than in the URTI patients with good discrimination abilities [area under curve (AUC) 0.742 and 0.716, respectively; combination AUC 0.801]. CONCLUSION: Our results suggested that HAdV modulated lipid metabolism for both the patients with URTI and pneumonia, especially the sphingolipid metabolism involving ceramide and lactosylceramide, which might thus be a potential intervention target in the treatment of HAdV infection.

Convenient and Sensitive Measurement of Lactosylceramide Synthase Activity Using Deuterated Glucosylceramide and Mass Spectrometry.

Lactosylceramide is necessary for the biosynthesis of almost all classes of glycosphingolipids and plays a relevant role in pathways involved in neuroinflammation. It is synthesized by the action of galactosyltransferases B4GALT5 and B4GALT6, which transfer galactose from UDP-galactose to glucosylceramide. Lactosylceramide synthase activity was classically determined in vitro by a method based on the incorporation of radiolabeled galactose followed by the chromatographic separation and quantitation of the product by liquid scintillation counting. Here, we used deuterated glucosylceramide as the acceptor substrate and quantitated the deuterated lactosylceramide product by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We compared this method with the classical radiochemical method and found that the reactions have similar requirements and provide comparable results in the presence of high synthase activity. Conversely, when the biological source lacked lactosylceramide synthase activity, as in the case of a crude homogenate of human dermal fibroblasts, the radiochemical method failed, while the other provided a reliable measurement. In addition to being very accurate and sensitive, the proposed use of deuterated glucosylceramide and LC-MS/MS for the detection of lactosylceramide synthase in vitro has the relevant advantage of avoiding the costs and discomforts of managing radiochemicals.

A Biomarker Study in Patients with GBA1-Parkinson's Disease and Healthy Controls.

BACKGROUND: Molecules related to glucocerebrosidase (GCase) are potential biomarkers for development of compounds targeting GBA1-associated Parkinson's disease (GBA-PD). OBJECTIVES: Assessing variability of various glycosphingolipids (GSLs) in plasma, peripheral blood mononuclear cells (PBMCs), and cerebrospinal fluid (CSF) across GBA-PD, idiopathic PD (iPD), and healthy volunteers (HVs). METHODS: Data from five studies were combined. Variability was assessed of glucosylceramide (various isoforms), lactosylceramide (various isoforms), glucosylsphingosine, galactosylsphingosine, GCase activity (using fluorescent 4-methylumbeliferryl-ß-glucoside), and GCase protein (using enzyme-linked immunosorbent assay) in plasma, PBMCs, and CSF if available, in GBA-PD, iPD, and HVs. GSLs in leukocyte subtypes were compared in HVs. Principal component analysis was used to explore global patterns in GSLs, clinical characteristics (Movement Disorder Society - Unified Parkinson's Disease Rating Scale Part 3 [MDS-UPDRS-3], Mini-Mental State Examination [MMSE], GBA1 mutation type), and participant status (GBA-PD, iPD, HVs). RESULTS: Within-subject between-day variability ranged from 5.8% to 44.5% and was generally lower in plasma than in PBMCs. Extracellular glucosylceramide levels (plasma) were slightly higher in GBA-PD compared with both iPD and HVs, while intracellular levels were comparable. GSLs in the different matrices (plasma, PBMCs, CSF) did not correlate. Both lactosylceramide and glucosylsphingosine were more abundant in granulocytes compared with monocytes and lymphocytes. Absolute levels of GSL isoforms differed greatly. GBA1 mutation types could not be differentiated based on GSL data. CONCLUSIONS: Glucosylceramide can stably be measured over days in both plasma and PBMCs and may be used as a biomarker in clinical trials targeting GBA-PD. Glucosylsphingosine and lactosylceramide are stable in plasma but are strongly affected by leukocyte subtypes in PBMCs. GBA-PD could be differentiated from iPD and HVs, primarily based on glucosylceramide levels in plasma. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Glucosylceramide is essential for Heartland and Dabie bandavirus glycoprotein-induced membrane fusion.

Due to climate changes, there has been a large expansion of emerging tick-borne zoonotic viruses, including Heartland bandavirus (HRTV) and Dabie bandavirus (DBV). As etiologic agents of hemorrhagic fever with high fatality, HRTV and DBV have been recognized as dangerous viral pathogens that likely cause future wide epidemics. Despite serious health concerns, the mechanisms underlying viral infection are largely unknown. HRTV and DBV Gn and Gc are viral surface glycoproteins required for early entry events during infection. Glycosphingolipids, including galactosylceramide (GalCer), glucosylceramide (GlcCer) and lactosylceramide (LacCer), are a class of membrane lipids that play essential roles in membrane structure and viral lifecycle. Here, our genome-wide CRISPR/Cas9 knockout screen identifies that glycosphingolipid biosynthesis pathway is essential for HRTV and DBV infection. The deficiency of UDP-glucose ceramide glucosyltransferase (UGCG) that produces GlcCer resulted in the loss of infectivity of recombinant viruses pseudotyped with HRTV or DBV Gn/Gc glycoproteins. Conversely, exogenous supplement of GlcCer, but not GalCer or LacCer, recovered viral entry of UGCG-deficient cells in a dose-dependent manner. Biophysical analyses showed that GlcCer targeted the lipid-head-group binding pocket of Gc to form a stable protein-lipid complex, which allowed the insertion of Gc protein into host lysosomal membrane lipid bilayers for viral fusion. Mutagenesis showed that D841 residue at the Gc lipid binding pocket was critical for GlcCer interaction and thereby, viral entry. These findings reveal detailed mechanism of GlcCer glycosphingolipid in HRTV and DBV Gc-mediated membrane fusion and provide a potential therapeutic target for tickborne virus infection.

Molecular Dynamics of Glycolipids in Liposomes.

Glycosphingolipids (GSLs) in the mammalian plasma membrane are essential for various biological events as they form glycolipid-rich membrane domains, such as lipid rafts. GSLs consist of a certain oligosaccharide head group and a ceramide tail with various lengths of acyl chains. The structure of the head group as well as the carbon number and degree of the unsaturation of the acyl chain are known to regulate the membrane distributions and interleaflet couplings of GSLs by altering physicochemical properties, such as dynamics, interactions, and cluster sizes. This chapter provides the detailed use of time-resolved fluorescence measurement for investigating the membrane properties of lactosylceramide (LacCer)-enriched domains in bilayer membranes. LacCer belongs to the neutral GSLs and is believed in forming a highly ordered phase in model membranes and biological membranes, while the details of the domain remain unclear. Here, we suggest using trans-parinaric acid (tPA) and tPA-LacCer fluorescent probes to reveal the dynamics and size of the GSL domains since they prefer to be distributed in the GSL-rich ordered phase. The fluorescence lifetime in the nanosecond timescale reveals the difference in the surrounding membrane environments, which relates to hydrocarbon chain ordering, membrane hydration, and submicrometer domain size. The fluorescence lifetime of these probes can thus provide important information on submicron- to nano-scale small GSL domains not only in model membranes but also in biological membranes.

Functional Impairment of the Nervous System with Glycolipid Deficiencies.

Patients with nervous system disorders suffer from impaired cognitive, sensory and motor functions that greatly inconvenience their daily life and usually burdens their family and society. It is difficult to achieve functional recovery for the damaged central nervous system (CNS) because of its limited ability to regenerate. Glycosphingolipids (GSLs) are abundant in the CNS and are known to play essential roles in cell-cell recognition, adhesion, signal transduction, and cellular migration, that are crucial in all phases of neurogenesis. Despite intense investigation of CNS regeneration, the roles of GSLs in neural regeneration remain unclear. Here we focus on the respective potentials of glycolipids to promote regeneration and repair of the CNS. Mice lacking glucosylceramide, lactosylceramide or gangliosides show lethal phenotypes. More importantly, patients with ganglioside deficiencies exhibit severe clinical phenotypes. Further, neurodegenerative diseases and mental health disorders are associated with altered GSL expression. Accumulating studies demonstrate that GSLs not only delimit physical regions but also play central roles in the maintenance of the biological functions of neurons and glia. We anticipate that the ability of GSLs to modulate behavior of a variety of molecules will enable them to ameliorate biochemical and neurobiological defects in patients. The use of GSLs to treat such defects in the human CNS will be a paradigm-shift in approach since GSL-replacement therapy has not yet been achieved in this manner clinically.

Glycosphingolipids in Diabetes, Oxidative Stress, and Cardiovascular Disease: Prevention in Experimental Animal Models.

Diabetes contributes to about 30% morbidity and mortality world-wide and has tidal wave increases in several countries in Asia. Diabetes is a multi-factorial disease compounded by inflammation, dyslipidemia, atherosclerosis, and is sometimes accompanied with gains in body weight. Sphingolipid pathways that interplay in the enhancement of the pathology of this disease may be potential therapeutic targets. Thus, the application of advanced sphingolipidomics may help predict the progression of this disease and therapeutic outcomes in man. Pre-clinical studies using various experimental animal models of diabetes provide valuable information on the role of sphingolipid signaling networks in diabetes and the efficacy of drugs to determine the translatability of innovative discoveries to man. In this review, we discuss three major concepts regarding sphingolipids and diabetes. First, we discuss a possible involvement of a monosialodihexosylceramide (GM3) in insulin-insulin receptor interactions. Second, a potential role for ceramide (Cer) and lactosylceramide (LacCer) in apoptosis and mitochondrial dysfunction is proposed. Third, a larger role of LacCer in antioxidant status and inflammation is discussed. We also discuss how inhibitors of glycosphingolipid synthesis can ameliorate diabetes in experimental animal models.

Sphingolipids in Atherosclerosis: Chimeras in Structure and Function.

Atherosclerosis-a systemic inflammatory disease-is the number one cause of mortality and morbidity worldwide. As such, the prevention of disease progression is of global interest in order to reduce annual deaths at a significant scale. Atherosclerosis is characterized by plaque formation in the arteries, resulting in vascular events such as ischemic stroke or myocardial infarction. A better understanding of the underlying pathophysiological processes at the cellular and molecular level is indispensable to identify novel therapeutic targets that may alleviate disease initiation or progression. Sphingolipids-a lipid class named after the chimeric creature sphinx-are considered to play a critical and, metaphorically, equally chimeric regulatory role in atherogenesis. Previous studies identified six common sphingolipids, namely dihydroceramide (DhCer), ceramide (Cer), sphingosine-1-phosphate (S1P), sphingomyelin (SM), lactosylceramide (LacCer), and glucosylceramide (GluCer) in carotid plaques, and demonstrated their potential as inducers of plaque inflammation. In this review, we point out their specific roles in atherosclerosis by focusing on different cell types, carrier molecules, enzymes, and receptors involved in atherogenesis. Whereas we assume mainly atheroprotective effects for GluCer and LacCer, the sphingolipids DhCer, Cer, SM and S1P mediate chimeric functions. Initial studies demonstrate the successful use of interventions in the sphingolipid pathway to prevent atherosclerosis. However, as atherosclerosis is a multifactorial disease with a variety of underlying cellular processes, it is imperative for future research to emphasize the circumstances in which sphingolipids exert protective or progressive functions and to evaluate their therapeutic benefits in a spatiotemporal manner.

Aberrant Ganglioside Functions to Underpin Dysregulated Myelination, Insulin Signalling, and Cytokine Expression: Is There a Link and a Room for Therapy?

Gangliosides are molecules widely present in the plasma membranes of mammalian cells, participating in a variety of processes, including protein organization, transmembrane signalling and cell adhesion. Gangliosides are abundant in the grey matter of the brain, where they are critically involved in postnatal neural development and function. The common precursor of the majority of brain gangliosides, GM3, is formed by the sialylation of lactosylceramide, and four derivatives of its a- and b-series, GM1, GD1a, GD1b and GT1b, constitute 95% of all the brain gangliosides. Impairments in ganglioside metabolism due to genetic abnormalities of GM-synthases are associated with severe neurological disorders. Apart from that, the latest genome-wide association and translational studies suggest a role of genes involved in brain ganglioside synthesis in less pervasive psychiatric disorders. Remarkably, the most recent animal studies showed that abnormal ganglioside functions result in dysregulated neuroinflammation, aberrant myelination and altered insulin receptor signalling. At the same time, these molecular features are well established as accompanying developmental psychiatric disorders such as attention-deficit hyperactivity disorder (ADHD) and autism spectrum disorders (ASD). This led us to hypothesize a role of deficient ganglioside function in developmental neuropsychiatric disorders and warrants further gene association clinical studies addressing this question. Here, we critically review the literature to discuss this hypothesis and focus on the recent studies on ST3GAL5-deficient mice. In addition, we elaborate on the therapeutic potential of various anti-inflammatory remedies for treatment of developmental neuropsychiatric conditions related to aberrant ganglioside functions.

Metabolome-wide association study on ABCA7 indicates a role of ceramide metabolism in Alzheimer's disease.

Genome-wide association studies (GWASs) have identified genetic loci associated with the risk of Alzheimer's disease (AD), but the molecular mechanisms by which they confer risk are largely unknown. We conducted a metabolome-wide association study (MWAS) of AD-associated loci from GWASs using untargeted metabolic profiling (metabolomics) by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS). We identified an association of lactosylceramides (LacCer) with AD-related single-nucleotide polymorphisms (SNPs) in ABCA7 (P = 5.0 × 10-5 to 1.3 × 10-44). We showed that plasma LacCer concentrations are associated with cognitive performance and genetically modified levels of LacCer are associated with AD risk. We then showed that concentrations of sphingomyelins, ceramides, and hexosylceramides were altered in brain tissue from Abca7 knockout mice, compared with wild type (WT) (P = 0.049-1.4 × 10-5), but not in a mouse model of amyloidosis. Furthermore, activation of microglia increases intracellular concentrations of hexosylceramides in part through induction in the expression of sphingosine kinase, an enzyme with a high control coefficient for sphingolipid and ceramide synthesis. Our work suggests that the risk for AD arising from functional variations in ABCA7 is mediated at least in part through ceramides. Modulation of their metabolism or downstream signaling may offer new therapeutic opportunities for AD.

Neurodegenerative Lysosomal Storage Disorders: TPC2 Comes to the Rescue!

Lysosomal storage diseases (LSDs) resulting from inherited gene mutations constitute a family of disorders that disturb lysosomal degradative function leading to abnormal storage of macromolecular substrates. In most LSDs, central nervous system (CNS) involvement is common and leads to the progressive appearance of neurodegeneration and early death. A growing amount of evidence suggests that ion channels in the endolysosomal system play a crucial role in the pathology of neurodegenerative LSDs. One of the main basic mechanisms through which the endolysosomal ion channels regulate the function of the endolysosomal system is Ca2+ release, which is thought to be essential for intracellular compartment fusion, fission, trafficking and lysosomal exocytosis. The intracellular TRPML (transient receptor potential mucolipin) and TPC (two-pore channel) ion channel families constitute the main essential Ca2+-permeable channels expressed on endolysosomal membranes, and they are considered potential drug targets for the prevention and treatment of LSDs. Although TRPML1 activation has shown rescue effects on LSD phenotypes, its activity is pH dependent, and it is blocked by sphingomyelin accumulation, which is characteristic of some LSDs. In contrast, TPC2 activation is pH-independent and not blocked by sphingomyelin, potentially representing an advantage over TRPML1. Here, we discuss the rescue of cellular phenotypes associated with LSDs such as cholesterol and lactosylceramide (LacCer) accumulation or ultrastructural changes seen by electron microscopy, mediated by the small molecule agonist of TPC2, TPC2-A1-P, which promotes lysosomal exocytosis and autophagy. In summary, new data suggest that TPC2 is a promising target for the treatment of different types of LSDs such as MLIV, NPC1, and Batten disease, both in vitro and in vivo.

Enhancing the accuracy of surgical wound excision following burns trauma via application of Rapid Evaporative IonisationMass Spectrometry (REIMS).

BACKGROUND: Surgical wound excision is a necessary procedure for burn patients that require the removal of eschar. The extent of excision is currently guided by clinical judgement, with excessinto healthy tissue potentially leading to excessive scar, or inadequate debridement increasing risk of infection. Thus, an objective real-time measure to facilitate accurate excision could support clinical judgement and improve this surgical procedure. This study was designed to investigate the potential use of Rapid evaporative ionisation mass spectrometry (REIMS) as a tool to support data-driven objective tissue excision. METHODS: Data were acquired using a multi-platform approach that consisted of both Rapid Evaporative Ionisation Mass Spectrometry (REIMS) performed on intact skin, and comprehensive liquid chromatography-mass spectrometry (LC-MS/MS) lipidomics performed on homogenised skin tissue extracts. Data were analysed using principal components analysis (PCA) and multivariate orthogonal projections to latent squares discriminant analysis (OPLS-DA) and logistic regression to determine the predictability of the models. RESULTS: PCA and OPLS-DA models of the REIMS and LC-MS/MS lipidomics data reported separation of excised and healthy tissue. Molecular fingerprints generated from REIMS analysis of healthy skin tissue revealed a high degree of heterogeneity, however, intra-individual variance was smaller than inter-individual variance. Both platforms indicated high levels of skin classification accuracy. In addition, OPLS-DA of the LC-MS/MS lipidomic data revealed significant differences in specific lipid classes between healthy control and excised skin samples; including lower free fatty acids (FFA), monoacylglycerols (MAG), lysophosphatidylglycerol (LPG) and lysophosphatidylethanolamines (LPE) in excised tissue and higher lactosylceramides (LCER) and cholesterol esters (CE) compared to healthy control tissue. CONCLUSIONS: Having established the heterogeneity in the biochemical composition of healthy skin using REIMS and LC-MS/MS, our data show that REIMS has the potential to distinguish between excied and healthy skin tissue samples. This pilot study suggests that REIMS may be an effective tool to support accurate tissue excision during burn surgery.

Comprehensive metabolomics study on the pathogenesis of anaplastic astrocytoma via UPLC-Q/TOF-MS.

Anaplastic astrocytoma (AA) is a malignant carcinoma whose pathogenesis remains to be fully elucidated. System biology techniques have been widely used to clarify the mechanism of diseases from a systematic perspective. The present study aimed to explore the pathogenesis and novel potential biomarkers for the diagnosis of AA according to metabolic differences. Patients with AA (n = 12) and healthy controls (n = 15) were recruited. Serum was assayed with untargeted ultraperformance liquid chromatography-quadrupole/time-of-flight-mass spectrometry (UPLC-Q/TOF-MS) metabolomic techniques. The data were further evaluated using multivariate analysis and bioinformatic methods based on the KEGG database to determine the distinct metabolites and perturbed pathways. Principal component analysis and orthogonal projections to latent structures-discriminant analysis (OPLS-DA) identified the significance of the distinct metabolic pattern between patients with AA and healthy controls (P < .001) in both ESI modes. Permutation testing confirmed the validity of the OPLS-DA model (permutation = 200, Q2 < 0.5). In total, 24 differentiated metabolites and 5 metabolic pathways, including sphingolipid, glycerophospholipid, caffeine, linoleic acid, and porphyrin metabolism, were identified based on the OPLS-DA model. 3-Methylxanthine, sphinganine, LysoPC(18:1), and lactosylceramide were recognized as potential biomarkers with excellent sensitivity and specificity (area under the curve > 98%). These findings indicate that the perturbed metabolic pattern related to immune regulation and cellular signal transduction is associated with the pathogenesis of AA. 3-Methylxanthine, sphinganine, LysoPC(18:1), and lactosylceramide could be used as biomarkers of AA in future clinical practice. This study provides a therapeutic basis for further studies on the mechanism and precise clinical diagnosis of AA.

Lactosylceramide-enriched microdomains mediate human neutrophil immunological functions via carbohydrate-carbohydrate interaction.

The innate immune system of mammalian cells is the first line of defense against pathogenic microorganisms. Phagocytes, which play the central role in this system, engulf microorganisms by a mechanism that involves pattern recognition receptors on their own surface and pathogen-associated molecular patterns (PAMPs) expressed by the microorganism. Components of PAMPs include glycans (polysaccharides) and glycoconjugates (carbohydrates covalently linked to other biological molecules). Pathogenic microorganisms display specific binding affinity to various types of glycosphingolipids (sphingosine-containing glycolipids; GSLs), and GSLs are involved in host-pathogen interactions. We observed that lactosylceramide (LacCer), a neutral GSL, binds directly to certain pathogen-specific molecules (e.g., Candida albicans-derived ß-glucans, mycobacterial lipoarabinomannan) via carbohydrate-carbohydrate interaction. LacCer is expressed highly on human neutrophils, and forms membrane microdomains. Such LacCer-enriched microdomains mediate several important neutrophil functions, including chemotaxis, phagocytosis, and superoxide generation. Human neutrophils phagocytose pathogenic mycobacteria (including Mycobacterium tuberculosis) through carbohydrate-carbohydrate interaction between LacCer on their own surface and mannose-capped lipoarabinomannan on the bacterium. During recognition of pathogen-specific glycans, direct association of LacCer-containing C24 fatty acid chain with Lyn (a Src family kinase) is necessary for signal transduction from the neutrophil exterior to interior. Pathogenic mycobacteria utilize a similar interaction to avoid killing by neutrophils. We describe here the mechanisms whereby LacCer mediates neutrophil immune systems via carbohydrate-carbohydrate interaction.

Galactocerebrosidase deficiency induces an increase in lactosylceramide content: A new hallmark of Krabbe disease?

Galactocerebrosidase (GALC) hydrolyses galactose residues from various substrates, including galactosylceramide, psychosine (galactosylsphingosine), and lactosylceramide. Its severe deficiency has been associated with the accumulation of psychosine, a toxic molecule with detergent-like features, which alters membrane structures and signalling pathways, inducing the death of oligodendrocytes and a sequence of events in the nervous system that explain the appearance of many clinical signs typical of Krabbe disease. Nevertheless, new evidence suggests the existence of other possible links among GALC action, myelination, and myelin stability, apart from psychosine release. In this study, we demonstrated that lactosylceramide metabolism is impaired in fibroblasts isolated from patients with Krabbe disease in the absence of psychosine accumulation. This event is responsible for the aberrant and constitutive activation of the AKT/prolin-rich AKT substrate of 40 kDa (PRAS40) signalling axis, inducing B cell lymphoma 2 (BCL2) overexpression and glycogen synthase kinase 3 beta (GSK-3ß) inhibition. In addition, nuclear factor E2-related factor 2 (NRF2) showed increased nuclear translocation. Due to the relevance of these molecular alterations in neurodegeneration, lactosylceramide increase should be evaluated as a novel marker of Krabbe disease, and because of its significant connections with signalling pathways.

Diabetes and kidney dysfunction markedly alter the content of sphingolipids carried by circulating lipoproteins.

BACKGROUND: We have previously shown that very long ceramides/lactosylceramides predicted the development of macroalbuminuria (MA) in type 1 diabetes and expanded our studies into type 2 diabetes. OBJECTIVE: This study proposes comparing the levels of plasma sphingolipids and their distribution in circulating lipoproteins (VLDL/IDL, LDL, HDL2 and HDL3) between a healthy control group and two groups of subjects with type 2 diabetes, one with and other without MA. METHODS: Plasma and lipoprotein sphingolipids/glycosphingolipids were measured using HPLC-MS/MS in 114 subjects (40 controls; 74 type 2 diabetes, 40 without MA; and 34 with MA) and the levels were compared between controls and the two groups of diabetes. Group effect sizes were calculated using Cohen's d. RESULTS: Sphingomyelin species carried by LDL are significantly higher in diabetic patients with MA than in those with normal albumin excretion rate (AER). Compared to controls, significant decreases in the levels of sphingolipids carried by HDL in patients with diabetes with normal AER or MA were observed for all sphingolipid classes except for hexosylceramide, which was normal in diabetic patients without MA. Although lower than in controls, the levels of lactosylceramides carried by HDL2/HDL3 were significantly higher in diabetes with MA. CONCLUSIONS: Considering the critical role sphingolipids play in major cell biological responses and cell signaling pathways, the consequences for disease development of changes in the distribution of sphingolipids/glycosphingolipids carried by lipoproteins could be considerable. Our work is just a first step to address a considerable gap in our present knowledge in this important field.

Effect of cholesterol on the lactosylceramide domains in phospholipid bilayers.

Lactosylceramide (LacCer) in the plasma membranes of immune cells is an important lipid for signaling in innate immunity through the formation of LacCer-rich domains together with cholesterol (Cho). However, the properties of the LacCer domains formed in multicomponent membranes remain unclear. In this study, we examined the properties of the LacCer domains formed in Cho-containing 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) membranes by deuterium solid-state NMR and fluorescence lifetimes. The potent affinity of LacCer-LacCer (homophilic interaction) is known to induce a thermally stable gel phase in the unitary LacCer bilayer. In LacCer/Cho binary membranes, Cho gradually destabilized the LacCer gel phase to form the liquid-ordered phase by its potent order effect. In the LacCer/POPC binary systems without Cho, the 2H NMR spectra of 10',10'-d2-LacCer and 18',18',18'-d3-LacCer probes revealed that LacCer was poorly miscible with POPC in the membranes and formed stable gel phases without being distributed in the liquid crystalline domain. The lamellar structure of the LacCer/POPC membrane was gradually disrupted at around 60°C, whereas the addition of Cho increased the thermal stability of the lamellarity. Furthermore, the area of the LacCer gel phase and its chain order were decreased in the LacCer/POPC/Cho ternary membranes, whereas the liquid-ordered domain, which was observed in the LacCer/Cho binary membrane, was not observed. Cho surrounding the LacCer gel domain liberated LacCer and facilitated forming the submicron to nano-scale small domains in the liquid crystalline domain of the LacCer/POPC/Cho membranes, as revealed by the fluorescence lifetimes of trans-parinaric acid and trans-parinaric acid-LacCer. Our findings on the membrane properties of the LacCer domains, particularly in the presence of Cho, would help elucidate the properties of the LacCer domains in biological membranes.

Empagliflozin Improves Metabolic and Hepatic Outcomes in a Non-Diabetic Obese Biopsy-Proven Mouse Model of Advanced NASH.

Empagliflozin, an established treatment for type 2 diabetes (T2DM), has shown beneficial effects on liver steatosis and fibrosis in animals and in humans with T2DM, non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH). However, little is known about the effects of empagliflozin on liver function in advanced NASH with liver fibrosis and without diabetes. This study aimed to assess the effects of empagliflozin on hepatic and metabolic outcomes in a diet-induced obese (DIO) and insulin-resistant but non-diabetic biopsy-confirmed mouse model of advanced NASH. Male C57BL/6JRj mice with a biopsy-confirmed steatosis and fibrosis on AMLN diet (high fat, fructose and cholesterol) for 36-weeks were randomized to receive for 12 weeks: (a) Empagliflozin (10 mg/kg/d p.o.), or (b) vehicle. Metabolic outcomes, liver pathology, markers of Kupffer and stellate cell activation and lipidomics were assessed at the treatment completion. Empagliflozin did not affect the body weight, body composition or insulin sensitivity (assessed by intraperitoneal insulin tolerance test), but significantly improved glucose homeostasis as assessed by oral glucose tolerance test in DIO-NASH mice. Empagliflozin improved modestly the NAFLD activity score compared with the vehicle, mainly by improving inflammation and without affecting steatosis, the fibrosis stage and markers of Kupffer and stellate cell activation. Empagliflozin reduced the hepatic concentrations of pro-inflammatory lactosylceramides and increased the concentrations of anti-inflammatory polyunsaturated triglycerides. Empagliflozin exerts beneficial metabolic and hepatic (mainly anti-inflammatory) effects in non-diabetic DIO-NASH mice and thus may be effective against NASH even in non-diabetic conditions.

Host lactosylceramide enhances Edwardsiella tarda infection.

Edwardsiella tarda is a Gram-negative bacterium causing economic damage in aquaculture. The interaction of E. tarda with microdomains is an important step in the invasion, but the target molecules in microdomains remain undefined. Here, we found that intraperitoneal injection of E. tarda altered splenic glycosphingolipid patterns in the model host medaka (Oryzias latipes) accompanied by alteration of glycosphingolipid metabolism-related gene expressions, suggesting that glycosphingolipid levels are involved in E. tarda infection. To ascertain the significance of glycosphingolipids in the infection, fish cell lines, DIT29 cells with a high amount of lactosylceramide (LacCer) and glucosylceramide (GlcCer), and GAKS cells with a low amount of these lipids, were treated with methyl-ß-cyclodextrin to disrupt the microdomain. E. tarda infection was suppressed in DIT29 cells, but not in GAKS cells, suggesting the involvement of microdomain LacCer and GlcCer in the infection. DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, an inhibitor of glycosphingolipid-synthesis, attenuated the infection in DIT29 cells, while Neu3-overexpressing GAKS cells, which accumulated LacCer, enhanced the infection. E. tarda possessed binding ability towards LacCer, but not GlcCer, and LacCer preincubation declined the infection towards fish cells, possibly due to the masking of binding sites. The present study suggests that LacCer may be a positive regulator of E. tarda invasion.