Sex Dimorphism of Nonalcoholic Fatty Liver Disease (NAFLD) in Pparg-Null Mice.

Men with nonalcoholic fatty liver disease (NAFLD) are more exposed to nonalcoholic steatohepatitis (NASH) and liver fibrosis than women. However, the underlying molecular mechanisms of NALFD sex dimorphism are unclear. We combined gene expression, histological and lipidomic analyses to systematically compare male and female liver steatosis. We characterized hepatosteatosis in three independent mouse models of NAFLD, ob/ob and lipodystrophic fat-specific (PpargF <sup>Δ/Δ</sup> ) and whole-body PPARγ-null (Pparg <sup>Δ/Δ</sup> ) mice. We identified a clear sex dimorphism occurring only in Pparg <sup>Δ/Δ</sup> mice, with females showing macro- and microvesicular hepatosteatosis throughout their entire life, while males had fewer lipid droplets starting from 20 weeks. This sex dimorphism in hepatosteatosis was lost in gonadectomized Pparg <sup>Δ/Δ</sup> mice. Lipidomics revealed hepatic accumulation of short and highly saturated TGs in females, while TGs were enriched in long and unsaturated hydrocarbon chains in males. Strikingly, sex-biased genes were particularly perturbed in both sexes, affecting lipid metabolism, drug metabolism, inflammatory and cellular stress response pathways. Most importantly, we found that the expression of key sex-biased genes was severely affected in all the NAFLD models we tested. Thus, hepatosteatosis strongly affects hepatic sex-biased gene expression. With NAFLD increasing in prevalence, this emphasizes the urgent need to specifically address the consequences of this deregulation in humans

Characterisation of adipocyte-derived extracellular vesicle subtypes identifies distinct protein and lipid signatures for large and small extracellular vesicles

Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues

Deciphering lipid structures based on platform-independent decision rule sets

We developed decision rule sets for Lipid Data Analyzer (LDA; http://genome.tugraz.at/lda2), enabling automated and reliable annotation of lipid species and their molecular structures in high-throughput data from chromatography-coupled tandem mass spectrometry. Platform independence was proven in various mass spectrometric experiments, comprising low- and high-resolution instruments and several collision energies. We propose that this independence and the capability to identify novel lipid molecular species render current state-of-the-art lipid libraries now obsolete

Quantitative analysis of N-acylphosphatidylethanolamine molecular species in rat brain using solid-phase extraction combined with reversed-phase chromatography and tandem mass spectrometry

10.1002/jssc.201600172Journal of Separation Science39132474-248

Balanced mTORC1 activity in oligodendrocytes is required for accurate CNS myelination

The mammalian target of rapamycin (mTOR) pathway integrates multiple signals and regulates crucial cell functions via the molecular complexes mTORC1 and mTORC2. These complexes are functionally dependent on their raptor (mTORC1) or rictor (mTORC2) subunits. mTOR has been associated with oligodendrocyte differentiation and myelination downstream of the PI3K/Akt pathway, but the functional contributions of individual complexes are largely unknown. We show, by oligodendrocyte-specific genetic deletion of Rptor and/or Rictor in the mouse, that CNS myelination is mainly dependent on mTORC1 function, with minor mTORC2 contributions. Myelin-associated lipogenesis and protein gene regulation are strongly reliant on mTORC1. We found that also oligodendrocyte-specific overactivation of mTORC1, via ablation of tuberous sclerosis complex 1 (TSC1), causes hypomyelination characterized by downregulation of Akt signaling and lipogenic pathways. Our data demonstrate that a delicately balanced regulation of mTORC1 activation and action in oligodendrocytes is essential for CNS myelination, which has practical overtones for understanding CNS myelin disorders

Proteome profiling of extracellular vesicles derived from adipocytes reveal the presence of key metabolic proteins involved in obesity-associated metabolic dysfunctions

International audienc

Proteome profiling of extracellular vesicles derived from adipocytes reveal the presence of key metabolic proteins involved in obesity-associated metabolic dysfunctions

International audienc

Omics approach to characterize milk-derived extracellular vesicles/exosomes isolated from goats expressing or not alphas1-CN

International audienceBackgroundGoats homozygous for a null allele (O/O) at the CSN1S1 locus encoding αs1-casein display a chronic endoplasmic reticulum (ER) stress (Unfolded Protein Response) due to an accumulation of the other caseins in this compartment, thus triggering a general MEC dysfunction with a strong impact on milk composition. Milk-derived extracellular vesicles (MEVs) contain molecular information, which are thus recognized as mediators of intercellular communication. We made the assumption that the absence of CSN1S1 expression may influence MEVs cargos including miRNA, proteins, lipids and metabolites. The objective of this study was to assess the impact of αs1-casein deficit on MEVs cargos and to compare the biological material they convey.Material & MethodsWe have developed an improved method based on a density gradient ultracentrifugation to isolate MEVs. The quality of MEVs was analyzed morphologically by transmission electron microscopy (TEM) with negative staining (uranyl acetate), the specific “exosome” protein markers were detected by Western blot and ELISA and the size distribution and particle concentration were measured by NTA. The MEV’s proteome was acquired by LC-MS/MS and nucleic acid content (mRNA and miRNA) by NGS and qPCR. EV lipid content was extracted with the MTBE method. Data acquisition was performed by an Orbitrap-MS and analyzed using Lipid Data Analyzer. MEV metabolites were extracted using MPLEx protocol, data acquired by LC coupled with HRMS and analyzed using Metaboanalyst online workbenches. Differential statistical and bioinformatic analyses were performed using appropriate softwares.ResultsThe novel purification method gives MEV populations, free of contamination by other EVs and milk components, at sufficient concentrations to perform subsequent analyses. Nearly 280 proteins involved in the biogenesis of exosomes and MVB formation, their adhesion and internalization as well as proteins associated with membrane transport and enzymes involved in cellular metabolism were identified, among which 41 exosomal proteins differed between CSN1S1 O/O (null) and A/A (wildtype) genotypes. Ongoing profiling of RNA from MEVs has already identified over 230 miRNA and confirmed MEC origin due to the presence of mRNA encoding specific major milk proteins. The comparison of exosomal miRNomes of goat homozygous for A and O alleles at the CSN1S1 locus pointed out 15 miRNAs differentially abundant, potentially related to the MEC phenotype. Sphingomyelin and phosphatidylcholin were the major phospholipids observed in MEV populations. We have totally identified ca. 4,000 compounds using pHILIC and RPLC, 79 of which were significantly up or down-regulated in studied genotypes.ConclusionSeveral differences distinguishing goats according to the genotype at the CSN1S1 locus were found at each level of the omic analysis of MEVs. Differentially abundant miRNAs and transcriptome analyses are in agreement with UPR phenotype and confirmed their involvement in post-transcriptional regulatory mechanisms