Distribution of Cortical Endoplasmic Reticulum Determines Positioning of Endocytic Events in Yeast Plasma Membrane

In many eukaryotes, a significant part of the plasma membrane is closely associated with the dynamic meshwork of cortical endoplasmic reticulum (cortical ER). We mapped temporal variations in the local coverage of the yeast plasma membrane with cortical ER pattern and identified micron-sized plasma membrane domains clearly different in cortical ER persistence. We show that clathrin-mediated endocytosis is initiated outside the cortical ER-covered plasma membrane zones. These cortical ER-covered zones are highly dynamic but do not overlap with the immobile and also endocytosis-inactive membrane compartment of Can1 (MCC) and the subjacent eisosomes. The eisosomal component Pil1 is shown to regulate the distribution of cortical ER and thus the accessibility of the plasma membrane for endocytosis

Combined intervention with pioglitazone and n-3 fatty acids in metformin-treated type 2 diabetic patients: improvement of lipid metabolism

Background: The marine n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) exert numerous beneficial effects on health, but their potency to improve treatment of type 2 diabetic (T2D) patients remains poorly characterized. We aimed to evaluate the effect of a combination intervention using EPA?+?DHA and the insulin-sensitizing drug pioglitazone in overweight/obese T2D patients already treated with metformin.Methods: In a parallel-group, four-arm, randomized trial, 69 patients (66 % men) were assigned to 24-week-intervention using: (i) corn oil (5 g/day; Placebo), (ii) pioglitazone (15 mg/day; Pio), (iii) EPA?+?DHA concentrate (5 g/day, containing ~2.8 g EPA?+?DHA; Omega-3), or (iv) pioglitazone and EPA?+?DHA concentrate (Pio&amp; Omega-3). Data from 60 patients were used for the final evaluation. At baseline and after intervention, various metabolic markers, adiponectin and cytokines were evaluated in serum using standard procedures, EPA?+?DHA content in serum phospholipids was evaluated using shotgun lipidomics and mass spectrometry, and hyperinsulinemic-euglycemic clamp and meal test were also performed. Indirect calorimetry was conducted after the intervention. Primary endpoints were changes from baseline in insulin sensitivity evaluated using hyperinsulinemic-euglycemic clamp and in serum triacylglycerol concentrations in fasting state. Secondary endpoints included changes in fasting glycemia and glycated hemoglobin (HbA1c), changes in postprandial glucose, free fatty acid and triacylglycerol concentrations, metabolic flexibility assessed by indirect calorimetry, and inflammatory markers.Results: Omega-3 and Pio&amp; Omega-3 increased EPA?+?DHA content in serum phospholipids. Pio and Pio&amp; Omega-3 increased body weight and adiponectin levels. Both fasting glycemia and HbA1c were increased by Omega-3, but were unchanged by Pio&amp; Omega-3. Insulin sensitivity was not affected by Omega-3, while it was improved by Pio&amp; Omega-3. Fasting triacylglycerol concentrations and inflammatory markers were not significantly affected by any of the interventions. Lipid metabolism in the meal test and metabolic flexibility were additively improved by Pio&amp; Omega-3.Conclusion: Besides preventing a modest negative effect of n-3 fatty acids on glycemic control, the combination of pioglitazone and EPA?+?DHA can be used to improve lipid metabolism in T2D patients on stable metformin therapy.Trial registration: EudraCT number 2009-011106-42.<br/

Activation of H+-ATPase of the Plasma Membrane of Saccharomyces cerevisiae by Glucose: The Role of Sphingolipid and Lateral Enzyme Mobility

Activation of the plasma membrane H+-ATPase of the yeast Saccharomyces cerevisiae by glucose is a complex process that has not yet been completely elucidated. This study aimed to shed light on the role of lipids and the lateral mobility of the enzyme complex during its activation by glucose. The significance of H+-ATPase oligomerization for the activation of H+-ATPase by glucose was shown using the strains lcb1-100 and erg6, with the disturbed synthesis of sphyngolipid and ergosterol, respectively. Experiments with GFP-fused H+-ATPase showed a decrease in fluorescence anisotropy during the course of glucose activation, suggesting structural reorganization of the molecular domains. An immunogold assay showed that the incubation with glucose results in the spatial redistribution of ATPase complexes in the plasma membrane. The data suggest that (1) to be activated by glucose, H+-ATPase is supposed to be in an oligomeric state, and (2) glucose activation is accompanied by the spatial movements of H+-ATPase clusters in the PM

ABC Transporter Pdr10 Regulates the Membrane Microenvironment of Pdr12 in Saccharomyces cerevisiae

The eukaryotic plasma membrane exhibits both asymmetric distribution of lipids between the inner and the outer leaflet and lateral segregation of membrane components within the plane of the bilayer. In budding yeast (Saccharomyces cerevisiae), maintenance of leaflet asymmetry requires P-type ATPases, which are proposed to act as inward-directed lipid translocases (Dnf1, Dnf2, and the associated protein Lem3), and ATP-binding cassette (ABC) transporters, which are proposed to act as outward-directed lipid translocases (Pdr5 and Yor1). The S. cerevisiae genome encodes two other Pdr5-related ABC transporters: Pdr10 (67% identity) and Pdr15 (75% identity). We report the first analysis of Pdr10 localization and function. A Pdr10-GFP chimera was located in discrete puncta in the plasma membrane and was found in the detergent-resistant membrane fraction. Compared to control cells, a pdr10∆ mutant was resistant to sorbate but hypersensitive to the chitin-binding agent Calcofluor White. Calcofluor sensitivity was attributable to a partial defect in endocytosis of the chitin synthase Chs3, while sorbate resistance was attributable to accumulation of a higher than normal level of the sorbate exporter Pdr12. Epistasis analysis indicated that Pdr10 function requires Pdr5, Pdr12, Lem3, and mature sphingolipids. Strikingly, Pdr12 was shifted to the detergent-resistant membrane fraction in pdr10∆ cells. Pdr10 therefore acts as a negative regulator for incorporation of Pdr12 into detergent-resistant membranes, a novel role for members of the ABC transporter superfamily

Proteomic Analysis of Rta2p-Dependent Raft-Association of Detergent-Resistant Membranes in Candida albicans

In Candida albicans, lipid rafts (also called detergent-resistant membranes, DRMs) are involved in many cellular processes and contain many important proteins. In our previous study, we demonstrated that Rta2p was required for calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Here, we found that Rta2p was co-localized with raft-constituted ergosterol on the plasma membrane of C. albicans. Furthermore, this membrane expression pattern was totally disturbed by inhibitors of either ergosterol or sphingolipid synthesis. Biochemical fractionation of DRMs together with immunoblot uncovered that Rta2p, along with well-known DRM-associated proteins (Pma1p and Gas1p homologue), was associated with DRMs and their associations were blocked by inhibitors of either ergosterol or sphingolipid synthesis. Finally, we used the proteomic analysis together with immunoblot and identified that Rta2p was required for the association of 10 proteins with DRMs. These 5 proteins (Pma1p, Gas1p homologue, Erg11p, Pmt2p and Ali1p) have been reported to be DRM-associated and also that Erg11p is a well-known target of azoles in C. albicans. In conclusion, our results showed that Rta2p was predominantly localized in lipid rafts and was required for the association of certain membrane proteins with lipid rafts in C. albicans

Metformin strongly affects transcriptome of peripheral blood cells in healthy individuals

Funding Information: The study was supported by the European Regional Development Fund under the project ?Investigation of interplay between multiple determinants influencing response to metformin: search for reliable predictors for efficacy of type 2 diabetes therapy? (Project No.: 1.1.1.1/16/A/091, https://ec.europa.eu/regional_policy/en/funding/ erdf/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors would like to thank all the volunteers for their participation and acknowledge the Genome Database of the Latvian Population for providing biological material and data. Publisher Copyright: © 2019 Ustinova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Metformin is a commonly used antihyperglycaemic agent for the treatment of type 2 diabetes mellitus. Nevertheless, the exact mechanisms of action, underlying the various therapeutic effects of metformin, remain elusive. The goal of this study was to evaluate the alterations in longitudinal whole-blood transcriptome profiles of healthy individuals after a one-week metformin intervention in order to identify the novel molecular targets and further prompt the discovery of predictive biomarkers of metformin response. Next generation sequencing-based transcriptome analysis revealed metformin-induced differential expression of genes involved in intestinal immune network for IgA production and cytokine-cytokine receptor interaction pathways. Significantly elevated faecal sIgA levels during administration of metformin, and its correlation with the expression of genes associated with immune response (CXCR4, HLA-DQA1, MAP3K14, TNFRSF21, CCL4, ACVR1B, PF4, EPOR, CXCL8) supports a novel hypothesis of strong association between metformin and intestinal immune system, and for the first time provide evidence for altered RNA expression as a contributing mechanism of metformin’s action. In addition to universal effects, 4 clusters of functionally related genes with a subject-specific differential expression were distinguished, including genes relevant to insulin production (HNF1B, HNF1A, HNF4A, GCK, INS, NEUROD1, PAX4, PDX1, ABCC8, KCNJ11) and cholesterol homeostasis (APOB, LDLR, PCSK9). This inter-individual variation of the metformin effect on the transcriptional regulation goes in line with well-known variability of the therapeutic response to the drug.publishersversionPeer reviewe