Ellagic acid and its metabolites urolithins A/B ameliorate most common disease phenotypes in cellular and mouse models for lysosomal storage disorders by enhancing extracellular vesicle secretion.

Niemann Pick diseases types A (NPDA) and C (NPDC) are lysosomal storage disorders (LSDs) leading to cognitive impairment, neurodegeneration, and early death. NPDA and NPDC have different genetic origins, being caused by mutations in the acid sphingomyelinase (ASM) or the cholesterol transport protein NPC1, respectively. However, they share a common pathological hallmark in the accumulation of lipids in the endolysosomal compartment. Here, we tested the hypothesis that polyphenols reduce lipid overload in NPD cells by enhancing the secretion of extracellular vesicles (ECVs). We show that among the polyphenols tested, the ellagic acid metabolites, urolithin A and B, were the safest and most efficient in increasing ECV secretion. They reduced levels of accumulating lipids and lysosomal size and permeabilization in cultured bone marrow-derived macrophages and neurons from ASMko and NPC1 mutant mice, which mimic NPDA and NPDC, respectively. Moreover, oral treatment with ellagic acid reduced lipid levels, ameliorated lysosomal alterations, and diminished microglia activation in the brain of NPD mice. These results support the therapeutic value of ECV secretion and polyphenols for NPDs, which may also help treat other LSDs characterized by intracellular lipid overload.

A normalized signal calibration with a long-term reference improves the robustness of RPLC-MRM/MS lipidomics in plasma.

Improving the reliability of quantification in lipidomic analyses is crucial for its successful application in the discovery of new biomarkers or in clinical practice. In this study, we propose a workflow to improve the accuracy and precision of lipidomic results issued by the laboratory. Lipid species from 11 classes were analyzed by a targeted RPLC-MRM/MS method. The peak areas of species were used to estimate concentrations by an internal standard calibration approach (IS-calibration) and by an alternative normalization signal calibration schema (NS-calibration). The latter uses a long-term reference plasma material as a matrix-matched external calibrator whose accuracy was compared to the NIST SRM-1950 mean consensus values reported by the Interlaboratory Lipidomics Comparison Exercise. The bias of lipid concentrations showed a good accuracy for 69 of 89 quantified lipids. The quantitation of species by the NS-calibration schema improved the within- and between-batch reproducibility in quality control samples, in comparison to the usual IS-calibration approach. Moreover, the NS-calibration workflow improved the robustness of the lipidomics measurements reducing the between-batch variability (relative standard deviation <10% for 95% of lipid species) in real conditions tested throughout the analysis of 120 plasma samples. In addition, we provide a free access web tool to obtain the concentration of lipid species by the two previously mentioned quantitative approaches, providing an easy follow-up of quality control tasks related to lipidomics.

The Antipsychotic Risperidone Alters Dihydroceramide and Ceramide Composition and Plasma Membrane Function in Leukocytes In Vitro and In Vivo.

Atypical or second-generation antipsychotics are used in the treatment of psychosis and behavioral problems in older persons with dementia. However, these pharmaceutical drugs are associated with an increased risk of stroke in such patients. In this study, we evaluated the effects of risperidone treatment on phospholipid and sphingolipid composition and lipid raft function in peripheral blood mononuclear cells (PBMCs) of older patients (mean age >88 years). The results showed that the levels of dihydroceramides, very-long-chain ceramides, and lysophosphatidylcholines decreased in PBMCs of the risperidone-treated group compared with untreated controls. These findings were confirmed by in vitro assays using human THP-1 monocytes. The reduction in the levels of very-long-chain ceramides and dihydroceramides could be due to the decrease in the expression of fatty acid elongase 3, as observed in THP-1 monocytes. Moreover, risperidone disrupted lipid raft domains in the plasma membrane of PBMCs. These results indicated that risperidone alters phospholipid and sphingolipid composition and lipid raft domains in PBMCs of older patients, potentially affecting multiple signaling pathways associated with these membrane domains.

Dihydrosphingolipids are associated with steatosis and increased fibrosis damage in non-alcoholic fatty liver disease.

Dihydrosphingolipids are lipids biosynthetically related to ceramides. An increase in ceramides is associated with enhanced fat storage in the liver, and inhibition of their synthesis is reported to prevent the appearance of steatosis in animal models. However, the precise association of dihydrosphingolipids with non-alcoholic fatty liver disease (NAFLD) is yet to be established. We employed a diet induced NAFLD mouse model to study the association between this class of compounds and disease progression. Mice fed a high-fat diet were sacrificed at 22, 30 and 40 weeks to reproduce the full spectrum of histological damage found in human disease, steatosis (NAFL) and steatohepatitis (NASH) with and without significant fibrosis. Blood and liver tissue samples were obtained from patients whose NAFLD severity was assessed histologically. To demonstrate the effect of dihydroceramides over NAFLD progression we treated mice with fenretinide an inhibitor of dihydroceramide desaturase-1 (DEGS1). Lipidomic analyses were performed using liquid chromatography-tandem mass spectrometry. Triglycerides, cholesteryl esters and dihydrosphingolipids were increased in the liver of model mice in association with the degree of steatosis and fibrosis. Dihydroceramides increased with the histological severity observed in liver samples of mice (0.024 ± 0.003 nmol/mg vs 0.049 ± 0.005 nmol/mg, non-NAFLD vs NASH-fibrosis, p < 0.0001) and patients (0.105 ± 0.011 nmol/mg vs 0.165 ± 0.021 nmol/mg, p = 0.0221). Inhibition of DEGS1 induce a four-fold increase in dihydroceramides improving steatosis but increasing the inflammatory activity and fibrosis. In conclusion, the degree of histological damage in NAFLD correlate with dihydroceramide and dihydrosphingolipid accumulation. LAY SUMMARY: Accumulation of triglyceride and cholesteryl ester lipids is the hallmark of non-alcoholic fatty liver disease. Using lipidomics, we examined the role of dihydrosphingolipids in NAFLD progression. Our results demonstrate that de novo dihydrosphingolipid synthesis is an early event in NAFLD and the concentrations of these lipids are correlated with histological severity in both mouse and human disease.

Rottlerin Stimulates Exosome/Microvesicle Release Via the Increase of Ceramide Levels Mediated by Ampk in an In Vitro Model of Intracellular Lipid Accumulation.

Exosomes/microvesicles originate from multivesicular bodies that allow the secretion of endolysosome components out of the cell. In the present work, we investigated the effects of rottlerin, a polyphenol, on exosome/microvesicle secretion in a model of intracellular lipid trafficking impairment, and elucidated the mechanism of action. In a model of lipid trafficking impairment in C6 glia cells, rottlerin increased ceramide levels, while decreasing hexosylceramide content. This was accompanied by increased exosome/microvesicle secretion, thereby reducing the concentration of lipids in the endolysosomal compartment. The reduction of hexosylceramide levels by rottlerin was attributed to the increase of ß-glucosidase (glucosylceramidase) activity, and the effects of rottlerin were abrogated by ß-glucosidase inhibitors such as isofagomine D-tartrate and AMP-deoxynojirimycin. Moreover, treatment with ML-266, a potent activator of the ß-glucosidase enzyme, recapitulated the effects of rottlerin on the sphingolipid profile and exosome/microvesicle secretion. Finally, inhibition of AMPK (AMP-activated protein kinase) using compound C prevented both exosome/microvesicle secretion and the elimination of endolysosome lipids, which were promoted by rottlerin. The results showed that the decrease in intracellular lipid deposition induced by rottlerin was mediated by ß-glucosidase activation and exosome/microvesicle release via the AMPK pathway. Rottlerin consumption could represent an additional health benefit in lysosomal deposition diseases.

Curcumin stimulates exosome/microvesicle release in an in vitro model of intracellular lipid accumulation by increasing ceramide synthesis.

Curcumin, a hydrophobic polyphenol found in the rhizome of Curcuma longa, has been shown to reduce intracellular lipid accumulation in mouse models of lysosomal storage diseases such as Niemann-Pick type C. Exosomes are small extracellular vesicles secreted by cells in response to changes in intracellular ceramide composition. Curcumin can induce exosome/microvesicle release in cellular models of lipid deposition; however, the mechanism by which curcumin stimulates this release is unknown. In a model of lipid trafficking impairment in C6 glia cells, we show that curcumin stimulated ceramide synthesis by increasing the intracellular concentration of ceramide-dihydroceramide. Ceramide overload increased exosome/microvesicle secretion 10-fold, thereby reducing the concentration of lipids in the endolysosomal compartment. These effects were blocked by inhibitors of serine palmitoyltransferase (myriocin) and ceramide synthase (fumonisin B1). It is concluded that the decrease in intracellular lipid deposition induced by curcumin is mediated by increased ceramide synthesis and exosome/microvesicle release. This action may represent an additional health benefit of curcumin.