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Summary: Endocrine cells employ regulated exocytosis of secretory granules to secrete hormones and neurotransmitters. Secretory granule exocytosis depends on spatiotemporal variables such as proximity to the plasma membrane and age, with newly generated granules being preferentially released. Despite recent advances, we lack a comprehensive view of the molecular composition of insulin granules and associated changes over their lifetime. Here, we report a strategy for the purification of insulin secretory granules of distinct age from insulinoma INS-1 cells. Tagging the granule-resident protein phogrin with a cleavable CLIP tag, we obtain intact fractions of age-distinct granules for proteomic and lipidomic analyses. We find that the lipid composition changes over time, along with the physical properties of the membrane, and that kinesin-1 heavy chain (KIF5b) as well as Ras-related protein 3a (RAB3a) associate preferentially with younger granules. Further, we identify the Rho GTPase-activating protein (ARHGAP1) as a cytosolic factor associated with insulin granules.

Objective: The liver regulates the availability of insulin to other tissues and is the first line insulin response organ physiologically exposed to higher insulin concentrations than the periphery. Basal insulin during fasting inhibits hepatic gluconeogenesis and glycogenolysis, whereas postprandial insulin peaks stimulate glycogen synthesis. The molecular consequences of chronic insulin deficiency for the liver have not been studied systematically. Methods: We analyzed liver samples of a genetically diabetic pig model (MIDY) and of wild-type (WT) littermate controls by RNA sequencing, proteomics, and targeted metabolomics/lipidomics. Results: Cross-omics analyses revealed increased activities in amino acid metabolism, oxidation of fatty acids, ketogenesis, and gluconeogenesis in the MIDY samples. In particular, the concentrations of the ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) and of retinol dehydrogenase 16 (RDH16), which catalyzes the first step in retinoic acid biogenesis, were highly increased. Accordingly, elevated levels of retinoic acid, which stimulates the expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK1), were measured in the MIDY samples. In contrast, pathways related to extracellular matrix and inflammation/pathogen defense response were less active than in the WT samples. Conclusions: The first multi-omics study of a clinically relevant diabetic large animal model revealed molecular signatures and key drivers of functional alterations of the liver in insulin-deficient diabetes mellitus. The multi-omics data set provides a valuable resource for comparative analyses with other experimental or clinical data sets. Keywords: Liver, Insulin deficiency, Transcriptome, Proteome, Metabolome, Lipidome

Objective: Shotgun lipidomics enables an extensive analysis of lipids from tissues and fluids. Each specimen requires appropriate extraction and processing procedures to ensure good coverage and reproducible quantification of the lipidome. Adipose tissue (AT) has become a research focus with regard to its involvement in obesity-related pathologies. However, the quantification of the AT lipidome is particularly challenging due to the predominance of triacylglycerides, which elicit high ion suppression of the remaining lipid classes. Methods: We present a new and validated method for shotgun lipidomics of AT, which tailors the lipid extraction procedure to the target specimen and features high reproducibility with a linear dynamic range of at least 4 orders of magnitude for all lipid classes. Results: Utilizing this method, we observed tissue-specific and diet-related differences in three AT types (brown, gonadal, inguinal subcutaneous) from lean and obese mice. Brown AT exhibited a distinct lipidomic profile with the greatest lipid class diversity and responded to high-fat diet by altering its lipid composition, which shifted towards that of white AT. Moreover, diet-induced obesity promoted an overall remodeling of the lipidome, where all three AT types featured a significant increase in longer and more unsaturated triacylglyceride and phospholipid species. Conclusions: The here presented method facilitates reproducible systematic lipidomic profiling of AT and could be integrated with further –omics approaches used in (pre-) clinical research, in order to advance the understanding of the molecular metabolic dynamics involved in the pathogenesis of obesity-associated disorders. Keywords: Shotgun mass spectrometry, Lipidomics, Adipose tissue, Method validation, Lipid extraction, Mouse, Chow and high-fat diet, Lipid remodeling

Objective: The prevalence of diabetes mellitus and associated complications is steadily increasing. As a resource for studying systemic consequences of chronic insulin insufficiency and hyperglycemia, we established a comprehensive biobank of long-term diabetic INSC94Y transgenic pigs, a model of mutant INS gene-induced diabetes of youth (MIDY), and of wild-type (WT) littermates. Methods: Female MIDY pigs (n = 4) were maintained with suboptimal insulin treatment for 2 years, together with female WT littermates (n = 5). Plasma insulin, C-peptide and glucagon levels were regularly determined using specific immunoassays. In addition, clinical chemical, targeted metabolomics, and lipidomics analyses were performed. At age 2 years, all pigs were euthanized, necropsied, and a broad spectrum of tissues was taken by systematic uniform random sampling procedures. Total beta cell volume was determined by stereological methods. A pilot proteome analysis of pancreas, liver, and kidney cortex was performed by label free proteomics. Results: MIDY pigs had elevated fasting plasma glucose and fructosamine concentrations, C-peptide levels that decreased with age and were undetectable at 2 years, and an 82% reduced total beta cell volume compared to WT. Plasma glucagon and beta hydroxybutyrate levels of MIDY pigs were chronically elevated, reflecting hallmarks of poorly controlled diabetes in humans. In total, ∼1900 samples of different body fluids (blood, serum, plasma, urine, cerebrospinal fluid, and synovial fluid) as well as ∼17,000 samples from ∼50 different tissues and organs were preserved to facilitate a plethora of morphological and molecular analyses. Principal component analyses of plasma targeted metabolomics and lipidomics data and of proteome profiles from pancreas, liver, and kidney cortex clearly separated MIDY and WT samples. Conclusions: The broad spectrum of well-defined biosamples in the Munich MIDY Pig Biobank that will be available to the scientific community provides a unique resource for systematic studies of organ crosstalk in diabetes in a multi-organ, multi-omics dimension.

A calorie-rich diet is one reason for the continuous spread of metabolic syndromes in western societies. Smart food design is one powerful tool to prevent metabolic stress, and the search for suitable bioactive additives is a continuous task. The nutrient-sensing insulin pathway is an evolutionary conserved mechanism that plays an important role in metabolism, growth and development. Recently, lipid cues capable to stimulate insulin signaling were identified. However, the mechanistic base of their activity remains obscure to date. We show that specific Akt/Protein-kinase B isoforms are responsive to different calorie-rich diets, and potentiate the activity of the cellular insulin cascade. Our data add a new dimension to existing models and position Drosophila as a powerful tool to study the relation between dietary lipid cues and the insulin-induced cellular signal pathway.

Blood is arguably the most important bodily fluid and its analysis provides crucial health status information. A first routine measure to narrow down diagnosis in clinical practice is the differential blood count, determining the frequency of all major blood cells. What is lacking to advance initial blood diagnostics is an unbiased and quick functional assessment of blood that can narrow down the diagnosis and generate specific hypotheses. To address this need, we introduce the continuous, cell-by-cell morpho-rheological (MORE) analysis of diluted whole blood, without labeling, enrichment or separation, at rates of 1000 cells/sec. In a drop of blood we can identify all major blood cells and characterize their pathological changes in several disease conditions in vitro and in patient samples. This approach takes previous results of mechanical studies on specifically isolated blood cells to the level of application directly in blood and adds a functional dimension to conventional blood analysis.

Driven by interactions between lipids and proteins, biological membranes display lateral heterogeneity that manifests itself in a mosaic of liquid-ordered (Lo) or raft, and liquid-disordered (Ld) or non-raft domains with a wide range of different properties and compositions. In giant plasma membrane vesicles and giant unilamellar vesicles, specific binding of Cholera Toxin (CTxB) to GM1 glycolipids is a commonly used strategy to label raft domains or Lo membrane environments. However, these studies often use acyl-chain labeled bodipy-GM1 (bdGM1), whose headgroup accessibility and membrane order or phase partitioning may differ from those of GM1, rendering the interpretation of CTxB binding data quite problematic. To unravel the molecular basis of CTxB binding to GM1 and bdGM1, we explored the partitioning and the headgroup presentation of these gangliosides in the Lo and Ld phases using atomistic molecular dynamics simulations complemented by CTxB binding experiments. The conformation of both GM1 and bdGM1 was shown to be largely similar in the Lo and Ld phases. However, bdGM1 showed reduction in receptor availability when reconstituted into synthetic bilayer mixtures, highlighting that membrane phase partitioning of the gangliosides plays a considerable role in CTxB binding. Our results suggest that the CTxB binding is predominately modulated by the partitioning of the receptor to an appropriate membrane phase. Further, given that the Lo and Ld partitioning of bdGM1 differs from those of GM1, usage of bdGM1 for studying GM1 behavior in cells can lead to invalid interpretation of experimental data.

The pathogenesis and progression of many tumors, including hematologic malignancies is highly dependent on enhanced lipogenesis. De novo fatty-acid synthesis permits accelerated proliferation of tumor cells by providing membrane components but these may also alter physicochemical properties of lipid bilayers, which can impact signaling or even increase drug resistance in cancer cells. Cancer type-specific lipid profiles would permit us to monitor and interpret actual effects of lipid changes, potential fingerprints of individual tumors to be explored as diagnostic markers. We have used the shotgun MS approach to identify lipid patterns in different types of acute myeloid leukemia (AML) patients that either show no karyotype change or belong to t(8;21) or inv16 types. Differences in lipidomes of t(8;21) and inv(16) patients, as compared to AML patients without karyotype change, presented mostly as substantial modulation of ceramide/sphingolipid synthesis. Furthermore, between the t(8;21) and all other patients we observed significant changes in physicochemical membrane properties. These were related to a marked alteration in lipid saturation levels. The discovered differences in lipid profiles of various AML types improve our understanding of the pathobiochemical pathways involved and may serve in the development of diagnostic tools.

Lateral compositional and physicochemical heterogeneity is a ubiquitous feature of cellular membranes on various length scales, from molecular assemblies to micrometric domains. Segregated lipid domains of increased local order, referred to as rafts, are believed to be prominent features in eukaryotic plasma membranes; however, their exact nature (i.e. size, lifetime, composition, homogeneity) in live cells remains difficult to define. Here we present evidence that both synthetic and natural plasma membranes assume a wide range of lipid packing states with varying levels of molecular order. These states may be adapted and specifically tuned by cells during active cellular processes, as we show for stimulated insulin secretion. Most importantly, these states regulate both the partitioning of molecules between coexisting domains and the bioactivity of their constituent molecules, which we demonstrate for the ligand binding activity of the glycosphingolipid receptor GM1. These results confirm the complexity and flexibility of lipid-mediated membrane organization and reveal mechanisms by which this flexibility could be functionalized by cells.

BACKGROUND: Specific interactions between sphingomyelin (SM) and cholesterol (Ch) are commonly believed to play a key role in the formation of rafts in the biological membranes. A weakness of this model is the implication that these microdomains are confined to the outer bilayer leaflet. The cytoplasmic leaflet, which contains the bulk of phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI), is thought also to harbour half of the membrane cholesterol. Moreover, SLPE (1-stearoyl-2-linoleoyl-sn-glycero-3-phosphatidyl-ethanolamine) has recently been shown to be enriched in isolated detergent-resistant membranes (DRM), and this enrichment was independent of the method of isolation of DRM. METHODOLOGY/PRINCIPAL FINDINGS: Here we present quantitative evidence coming from Langmuir-Blodgett monolayer experiments that SLPE forms complex with Ch similar to that between SM and Ch. The energies of these interactions as calculated form the monolayer studies are highly negative. FRAP analysis showed that NBD-Ch recovery was similar in liposomes composed of DOPC/Ch SM or SLPE but not DPPE, providing further evidence that SLPE may form an l(o) phase in the presence of high Ch concentration. Experiments on the solubility of DOPC liposomes containing DPPE/Ch (1ratio1), SM/Ch (1ratio1) or SLPE/Ch (1ratio1) showed the presence of Triton X-100 insoluble floating fraction (TIFF) in the case of SM/Ch or SLPE/Ch but not in DPPE/Ch containing liposomes. Quantitative determination of particular lipid species in the TIFF fraction confirms the conclusion that SLPE (or similar PE species) could be an important constituent of the inner leaflet raft. CONCLUSION: Such interactions suggest a possible existence of inner-leaflet nanoscale assemblies composed of cholesterol complexes with SLPE or similar unsaturated PE species.

Although evidences that cell membrane contains microdomains are accumulating, the exact properties, diversity and levels of organization of small lipid patches built mainly of cholesterol and sphingomyelin, termed rafts, remain to be elucidated. Our understanding of the cell membrane is increasing with each new raft feature discovered. Nowadays rafts are suggested to act as sites of cell signaling events, to be a part of protein sorting machinery but also they are used by several pathogens as gates into the cells. It is still unclear how rafts are connected to the membrane skeleton and cytoskeleton and with how many different types of rafts are we actually dealing with. This review summarizes some of the most recent discoveries trying to make a view of the complex raft properties.

The review is focused on the domain structure and function of protein 4.1, one of the proteins belonging to the membrane skeleton. The protein 4.1 of the red blood cells (4.1R) is a multifunctional protein that localizes to the membrane skeleton and stabilizes erythrocyte shape and membrane mechanical properties, such as deformability and stability, via lateral interactions with spectrin, actin, glycophorin C and protein p55. Protein 4.1 binding is modulated through the action of kinases and/or calmodulin-Ca2+. Non-erythroid cells express the 4.1R homologues: 4.1G (general type), 4.1B (brain type), and 4.1N (neuron type), and the whole group belongs to the protein 4.1 superfamily, which is characterized by the presence of a highly conserved FERM domain at the N-terminus of the molecule. Proteins 4.1R, 4.1G, 4.1N and 4.1B are encoded by different genes. Most of the 4.1 superfamily proteins also contain an actin-binding domain. To date, more than 40 members have been identified. They can be divided into five groups: protein 4.1 molecules, ERM proteins, talin-related molecules, protein tyrosine phosphatase (PTPH) proteins and NBL4 proteins. We have focused our attention on the main, well known representatives of 4.1 superfamily and tried to choose the proteins which are close to 4.1R or which have distinct functions. 4.1 family proteins are not just linkers between the plasma membrane and membrane skeleton; they also play an important role in various processes. Some, such as focal adhesion kinase (FAK), non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells, play the role in cell adhesion. The other members control or take part in tumor suppression, regulation of cell cycle progression, inhibition of cell proliferation, downstream signaling of the glutamate receptors, and establishment of cell polarity; some are also involved in cell proliferation, cell motility, and/or cell-to-cell communication.

It was shown previously that an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine (PE/PC) binding site maps to the N-terminal part of the ankyrin-binding domain of β-spectrin (ankBDn). Here we have identified the amino acid residues within this domain which are responsible for recognizing monolayers and bilayers composed of PE/PC mixtures. In vitro binding studies revealed that a quadruple mutant with substituted hydrophobic residues W1771, L1775, M1778 and W1779 not only failed to effectively bind PE/PC, but its residual PE/PC-binding activity was insensitive to inhibition with ankyrin. Structure prediction and analysis, supported by in vitro experiments, suggests that "opening" of the coiled-coil structure underlies the mechanism of this interaction. Experiments on red blood cells and HeLa cells supported the conclusions derived from the model and in vitro lipid-protein interaction results, and showed the potential physiological role of this binding. We postulate that direct interactions between spectrin ankBDn and PE-rich domains play an important role in stabilizing the structure of the spectrin-based membrane skeleton.