Susanne Sales

Galectin-3 drives glycosphingolipid-dependent biogenesis of clathrin-independent carriers.

Several cell surface molecules including signalling receptors are internalized by clathrin-independent endocytosis. How this process is initiated, how cargo proteins are sorted and membranes are bent remains unknown. Here, we found that a carbohydrate-binding protein, galectin-3 (Gal3), triggered the glycosphingolipid (GSL)-dependent biogenesis of a morphologically distinct class of endocytic structures, termed clathrin-independent carriers (CLICs). Super-resolution and reconstitution studies showed that Gal3 required GSLs for clustering and membrane bending. Gal3 interacted with a defined set of cargo proteins. Cellular uptake of the CLIC cargo CD44 was dependent on Gal3, GSLs and branched N-glycosylation. Endocytosis of β1-integrin was also reliant on Gal3. Analysis of different galectins revealed a distinct profile of cargoes and uptake structures, suggesting the existence of different CLIC populations. We conclude that Gal3 functionally integrates carbohydrate specificity on cargo proteins with the capacity of GSLs to drive clathrin-independent plasma membrane bending as a first step of CLIC biogenesis.

Gender, Contraceptives and Individual Metabolic Predisposition Shape a Healthy Plasma Lipidome.

Lipidomics of human blood plasma is an emerging biomarker discovery approach that compares lipid profiles under pathological and physiologically normal conditions, but how a healthy lipidome varies within the population is poorly understood. By quantifying 281 molecular species from 27 major lipid classes in the plasma of 71 healthy young Caucasians whose 35 clinical blood test and anthropometric indices matched the medical norm, we provided a comprehensive, expandable and clinically relevant resource of reference molar concentrations of individual lipids. We established that gender is a major lipidomic factor, whose impact is strongly enhanced by hormonal contraceptives and mediated by sex hormone-binding globulin. In lipidomics epidemiological studies should avoid mixed-gender cohorts and females taking hormonal contraceptives should be considered as a separate sub-cohort. Within a gender-restricted cohort lipidomics revealed a compositional signature that indicates the predisposition towards an early development of metabolic syndrome in ca. 25% of healthy male individuals suggesting a healthy plasma lipidome as resource for early biomarker discovery.

Harmonizing lipidomics: NIST interlaboratory comparison exercise for lipidomics using SRM 1950–Metabolites in Frozen Human Plasma

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950–Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

Regulation of Liver Metabolism by the Endosomal GTPase Rab5

The liver maintains glucose and lipid homeostasis by adapting its metabolic activity to the energy needs of the organism. Communication between hepatocytes and extracellular environment via endocytosis is key to such homeostasis. Here, we addressed the question of whether endosomes are required for gluconeogenic gene expression. We took advantage of the loss of endosomes in the mouse liver upon Rab5 silencing. Strikingly, we found hepatomegaly and severe metabolic defects such as hypoglycemia, hypercholesterolemia, hyperlipidemia, and glycogen accumulation that phenocopied those found in von Gierkes disease, a glucose-6-phosphatase (G6Pase) deficiency. G6Pase deficiency alone can account for the reduction in hepatic glucose output and glycogen accumulation as determined by mathematical modeling. Interestingly, we uncovered functional alterations in the transcription factors, which regulate G6Pase expression. Our data highlight a requirement of Rab5 and the endosomal system for the regulation of gluconeogenic gene expression that has important implications for metabolic diseases.

Enlightening discriminative network functional modules behind Principal Component Analysis separation in differential-omic science studies

Omic science is rapidly growing and one of the most employed techniques to explore differential patterns in omic datasets is principal component analysis (PCA). However, a method to enlighten the network of omic features that mostly contribute to the sample separation obtained by PCA is missing. An alternative is to build correlation networks between univariately-selected significant omic features, but this neglects the multivariate unsupervised feature compression responsible for the PCA sample segregation. Biologists and medical researchers often prefer effective methods that offer an immediate interpretation to complicated algorithms that in principle promise an improvement but in practice are difficult to be applied and interpreted. Here we present PC-corr: a simple algorithm that associates to any PCA segregation a discriminative network of features. Such network can be inspected in search of functional modules useful in the definition of combinatorial and multiscale biomarkers from multifaceted omic data in systems and precision biomedicine. We offer proofs of PC-corr efficacy on lipidomic, metagenomic, developmental genomic, population genetic, cancer promoteromic and cancer stem-cell mechanomic data. Finally, PC-corr is a general functional network inference approach that can be easily adopted for big data exploration in computer science and analysis of complex systems in physics.

Liver-restricted Repin1 deficiency improves whole-body insulin sensitivity, alters lipid metabolism, and causes secondary changes in adipose tissue in mice.

Replication initiator 1 (Repin1) is a zinc finger protein highly expressed in liver and adipose tissue and maps within a quantitative trait locus (QTL) for body weight and triglyceride (TG) levels in the rat. The QTL has further been supported as a susceptibility locus for dyslipidemia and related metabolic disorders in congenic and subcongenic rat strains. Here, we elucidated the role of Repin1 in lipid metabolism in vivo. We generated a liver-specific Repin1 knockout mouse (LRep1−/−) and systematically characterized the consequences of Repin1 deficiency in the liver on body weight, glucose and lipid metabolism, liver lipid patterns, and protein/mRNA expression. Hyperinsulinemic-euglycemic clamp studies revealed significantly improved whole-body insulin sensitivity in LRep1−/− mice, which may be due to significantly lower TG content in the liver. Repin1 deficiency causes significant changes in potential downstream target molecules including Cd36, Pparγ, Glut2 protein, Akt phosphorylation, and lipocalin2, Vamp4, and Snap23 mRNA expression. Mice with hepatic deletion of Repin1 display secondary changes in adipose tissue function, which may be mediated by altered hepatic expression of lipocalin2 or chemerin. Our findings indicate that Repin1 plays a role in insulin sensitivity and lipid metabolism by regulating key genes of glucose and lipid metabolism.

Lipidomics of Human Blood Plasma by High-Resolution Shotgun Mass Spectrometry

Clinical lipidomics is an emerging biomarker discovery approach that compares lipid profiles under pathologically and physiologically normal conditions. Here we describe a method for the absolute (molar) quantification of more than 200 molecules from 14 major lipid classes from 5 μL of human blood plasma using high-resolution top-down shotgun mass spectrometry. Because of its technical simplicity and robustness, the protocol lends itself for high-throughput clinical lipidomics screens.

The Diurnal Timing of Starvation Differently Impacts Murine Hepatic Gene Expression and Lipid Metabolism – A Systems Biology Analysis Using Self-Organizing Maps

Organisms adapt their metabolism and draw on reserves as a consequence of food deprivation. The central role of the liver in starvation response is to coordinate a sufficient energy supply for the entire organism, which has frequently been investigated. However, knowledge of how circadian rhythms impact on and alter this response is scarce. Therefore, we investigated the influence of different timings of starvation on global hepatic gene expression. Mice (n = 3 each) were challenged with 24-h food deprivation started in the morning or evening, coupled with refeeding for different lengths and compared with ad libitum fed control groups. Alterations in hepatocyte gene expression were quantified using microarrays and confirmed or complemented with qPCR, especially for lowly detectable transcription factors. Analysis was performed using self-organizing maps (SOMs), which bases on clustering genes with similar expression profiles. This provides an intuitive overview of expression trends and allows easier global comparisons between complex conditions. Transcriptome analysis revealed a strong circadian-driven response to fasting based on the diurnal expression of transcription factors (e.g., Ppara, Pparg). Starvation initiated in the morning produced known metabolic adaptations in the liver; e.g., switching from glucose storage to consumption and gluconeogenesis. However, starvation initiated in the evening produced a different expression signature that was controlled by yet unknown regulatory mechanisms. For example, the expression of genes involved in gluconeogenesis decreased and fatty acid and cholesterol synthesis genes were induced. The differential regulation after morning and evening starvation were also reflected at the lipidome level. The accumulation of hepatocellular storage lipids (triacylglycerides, cholesteryl esters) was significantly higher after the initiation of starvation in the morning compared to the evening. Concerning refeeding, the gene expression pattern after a 12 h refeeding period largely resembled that of the corresponding starvation state but approached the ad libitum control state after refeeding for 21 h. Some components of these regulatory circuits are discussed. Collectively, these data illustrate a highly time-dependent starvation response in the liver and suggest that a circadian influence cannot be neglected when starvation is the focus of research or medicine, e.g., in the case of treating victims of sudden starvation events.

Tolerogenic versus Immunogenic Lipidomic Profiles of CD11c+ Immune Cells and Control of Immunogenic Dendritic Cell Ceramide Dynamics

Lipids affect the membrane properties determining essential biological processes. Earlier studies have suggested a role of switch-activated protein 70 (SWAP-70) in lipid raft formation of dendritic cells. We used lipidomics combined with genetic and biochemical assays to analyze the role of SWAP-70 in lipid dynamics. TLR activation using LPS as a ligand represented a pathogenic immunogenic stimulus, physical disruption of cell–cell contacts a tolerogenic stimulus. Physical disruption, but not LPS, caused an increase of phosphatidylcholine ether and cholesteryl esters in CD11c+ immune cells. An increase of ceramide (Cer) was a hallmark for LPS activation. SWAP-70 was required for regulating the increase and localization of Cers in the cell membrane. SWAP-70 controls Cer accumulation through the regulation of pH-dependent acid-sphingomyelinase activity and of RhoA-dependent transport of endosomal contents to the plasma membrane. Poor accumulation of Cers in Swap70−/− cells caused decreased apoptosis. This shows that two different pathways of activation, immunogenic and tolerogenic, induce different changes in the lipid composition of cultured CD11c+ cells, and highlights the important role of SWAP-70 in Cer dynamics in dendritic cells.