Lipid Partitioning Research Articles

The role of mTOR in lipid homeostasis and diabetes progression.

Metabolic diseases, such as type 2 diabetes, cardiac dysfunction, hypertension, and hepatic steatosis, share one critical causative factor: abnormal lipid partitioning, that redistribution of triglycerides from adipocytes to nonadipose peripheral tissues. Lipid overload of these tissues causes a number of pathological effects collectively known as lipotoxicity. If we find the way to correct lipid partitioning, we will restrain metabolic diseases, improve life quality and life expectancy and radically reduce healthcare costs. Lipid partitioning in the body is maintained by tightly regulated and balanced rates of de novo lipogenesis, lipolysis, adipogenesis, and mitochondrial oxidation primarily in fat and liver. Recent studies highlighted in this review have established mTOR as a central regulator of lipid storage and metabolism. Increased activity of mTOR in obesity may compensate for the negative consequences of overnutrition, whereas dysregulation of mTOR may lead to abnormal lipid partitioning and metabolic disease.

Effects of acute exercise on lipid content and dietary lipid uptake in liver and skeletal muscle of lean and diabetic rats.

Insulin resistance is associated with ectopic lipid accumulation. Physical activity improves insulin sensitivity, but the impact of exercise on lipid handling in insulin-resistant tissues remains to be elucidated. The present study characterizes the effects of acute exercise on lipid content and dietary lipid partitioning in liver and skeletal muscle of lean and diabetic rats by use of magnetic resonance spectroscopy (MRS). After baseline measurements, rats were randomized to exercise or no-exercise groups. A subset of animals was subjected to MRS directly after 1 h of treadmill running for measurement of total intrahepatocellular lipid (IHCL) and intramyocellular lipid (IMCL) content (n=7 lean and diabetic rats). The other animals were administered 13C-labeled lipids orally after treadmill visit (with or without exercise) followed by MRS measurements after 4 and 24 h to determine the 13C enrichment of IHCL and IMCL (n=8 per group). Total IHCL and IMCL content were fivefold higher in diabetic vs. lean rats (P<0.001). Exercise did not significantly affect IHCL content but reduced IMCL by 25±7 and 33±4% in lean and diabetic rats (P<0.05), respectively. Uptake of dietary lipids in liver and muscle was 2.3-fold greater in diabetic vs. lean rats (P<0.05). Prior exercise did not significantly modulate dietary lipid uptake into muscle, but in liver of both lean and diabetic rats, lipid uptake was 44% reduced after acute exercise (P<0.05). In conclusion, IMCL but not IHCL represents a viable substrate source during exercise in both lean and diabetic rats, and exercise differentially affects dietary lipid uptake in muscle and liver.

Exercise and Weight Loss Improve Muscle Mitochondrial Respiration, Lipid Partitioning, and Insulin Sensitivity After Gastric Bypass Surgery.

Both Roux-en-Y gastric bypass (RYGB) surgery and exercise can improve insulin sensitivity in individuals with severe obesity. However, the impact of RYGB with or without exercise on skeletal muscle mitochondria, intramyocellular lipids, and insulin sensitivity index (SI) is unknown. We conducted a randomized exercise trial in patients (n = 101) who underwent RYGB surgery and completed either a 6-month moderate exercise (EX) or a health education control (CON) intervention. SI was determined by intravenous glucose tolerance test. Mitochondrial respiration and intramyocellular triglyceride, sphingolipid, and diacylglycerol content were measured in vastus lateralis biopsy specimens. We found that EX provided additional improvements in SI and that only EX improved cardiorespiratory fitness, mitochondrial respiration and enzyme activities, and cardiolipin profile with no change in mitochondrial content. Muscle triglycerides were reduced in type I fibers in CON, and sphingolipids decreased in both groups, with EX showing a further reduction in a number of ceramide species. In conclusion, exercise superimposed on bariatric surgery–induced weight loss enhances mitochondrial respiration, induces cardiolipin remodeling, reduces specific sphingolipids, and provides additional improvements in insulin sensitivity.

Lipid partitioning at the nuclear envelope controls membrane biogenesis.

Partitioning of lipid precursors between membranes and storage is crucial for cell growth, and its disruption underlies pathologies such as cancer, obesity, and type 2 diabetes. However, the mechanisms and signals that regulate this process are largely unknown. In yeast, lipid precursors are mainly used for phospholipid synthesis in nutrient-rich conditions in order to sustain rapid proliferation but are redirected to triacylglycerol (TAG) stored in lipid droplets during starvation. Here we investigate how cells reprogram lipid metabolism in the endoplasmic reticulum. We show that the conserved phosphatidate (PA) phosphatase Pah1, which generates diacylglycerol from PA, targets a nuclear membrane subdomain that is in contact with growing lipid droplets and mediates TAG synthesis. We find that cytosol acidification activates the master regulator of Pah1, the Nem1-Spo7 complex, thus linking Pah1 activity to cellular metabolic status. In the absence of TAG storage capacity, Pah1 still binds the nuclear membrane, but lipid precursors are redirected toward phospholipids, resulting in nuclear deformation and a proliferation of endoplasmic reticulum membrane. We propose that, in response to growth signals, activation of Pah1 at the nuclear envelope acts as a switch to control the balance between membrane biogenesis and lipid storage.

Lipid partitioning in maize (Zea mays L.) endosperm highlights relationships among starch lipids, amylose, and vitreousness.

Content and composition of maize endosperm lipids and their partition in the floury and vitreous regions were determined for a set of inbred lines. Neutral lipids, i.e., triglycerides and free fatty acids, accounted for more than 80% of endosperm lipids and are almost 2 times higher in the floury than in the vitreous regions. The composition of endosperm lipids, including their fatty acid unsaturation levels, as well as their distribution may be related to metabolic specificities of the floury and vitreous regions in carbon and nitrogen storage and to the management of stress responses during endosperm cell development. Remarkably, the highest contents of starch lipids were observed systematically within the vitreous endosperm. These high amounts of starch lipids were mainly due to lysophosphatidylcholine and were tightly linked to the highest amylose content. Consequently, the formation of amylose-lysophosphatidylcholine complexes has to be considered as an outstanding mechanism affecting endosperm vitreousness.

Protein kinase STK25 regulates hepatic lipid partitioning and progression of liver steatosis and NASH

Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease, and 10% to 20% of NAFLD patients progress to nonalcoholic steatohepatitis (NASH). The molecular pathways controlling progression to NAFLD/NASH remain poorly understood. We recently identified serine/threonine protein kinase 25 (STK25) as a regulator of whole-body insulin and glucose homeostasis. This study investigates the role of STK25 in liver lipid accumulation and NASH. Stk25 transgenic mice challenged with a high-fat diet displayed a dramatic increase in liver steatosis and hepatic insulin resistance compared to wild-type siblings. Focal fibrosis, hepatocellular damage, and inflammation were readily seen in transgenic but not wild-type livers. Transgenic livers displayed reduced β-oxidation and triacylglycerol secretion, while lipid uptake and synthesis remained unchanged. STK25 was associated with lipid droplets, colocalizing with the main hepatic lipid droplet-coating protein adipose differentiation-related protein, the level of which was increased 3.8 ± 0.7-fold in transgenic livers (P < 0.01), while a key hepatic lipase, adipose triacylglycerol lipase, was translocated from the lipid droplets surface to the cytoplasm, providing the likely mechanism underlying the effect of STK25. In summary, STK25 is a lipid droplet-associated protein that promotes NAFLD through control of lipid release from the droplets for β-oxidation and triacylglycerol secretion. STK25 also drives pathogenesis of NASH.

Seaweed enrichment of feed supplied to farm‐raised Atlantic salmon (Salmo salar) is associated with higher total fatty acid and LC n‐3 PUFA concentrations in fish flesh

AbstractSeaweeds have great potential as feed ingredients for fish farming, though their effects on the fatty acid content and profile of fish flesh are not well established. Recent studies suggest that seaweed supplementation can increase the concentration of long chain omega‐3 PUFA (LC n‐3 PUFAs) in various animal species, though this effect has never been assessed in salmon. Primarily, this study compared the fatty acid profiles of Atlantic salmon (Salmo salar) that were fed a high quality diet containing seaweed (Diet A), with those on a high quality control diet without seaweed (Diet B). In addition, fatty acid profiles of Atlantic salmon that were fed a standard industrial quality diet containing seaweed (Diet C), were compared to those on a similar diet without seaweed (Diet D). Total fatty acid concentrations were significantly higher (+30%, p&lt;0.05) in the flesh of the fish that were fed seaweed. Flesh concentrations of eicosapentaenoic acid (EPA) and DHA were 30% and 62% higher (p&lt;0.05) in Diet A and Diet C groups compared to salmon in Diet B and Diet D groups respectively. Analysis of the fish lipid revealed no significant differences in LC‐3 PUFAs between the high quality diet groups, while significantly higher (+ 45%, p&lt;0.005) concentrations EPA + DHA were found in fish that consumed Diet C compared to Diet D. These findings support the hypothesis that the inclusion of seaweed in fish farm diets alters lipid partitioning and profiles of Atlantic salmon.Practical Applications: This study reveals a possible way to substantially increase EPA and DHA concentrations in farmed salmon. Both EPA and DHA are LC n‐3 PUFAs and are essential components of the human diet. Unfortunately, consumption of such PUFAs is generally below RDI levels, which is in turn linked to several non‐communicable diseases. Moreover, EPA + DHA concentrations in farmed salmon are now declining due to the replacement of scarce fish oils (rich in LC n‐3 PUFA) by more sustainable plant‐based oils (low in EPA + DHA) in fish feeds. Since salmon is one of the world's most consumed fish and one of the few major sources of LC n‐3 PUFAs, it is of great importance to maintain or increase EPA + DHA levels in farmed salmon. The results of this study show that this could be achieved by dietary inclusion of seaweed.Seaweed can increase LC n‐3 PUFA levels in salmon.

The B Cell Receptor Dictates its Local Lipid Environment

The B cell receptor (BCR) is responsible for sensing and responding to intact antigen during the immune response and is hypothesized to nucleate a unique composition of lipids surrounding receptor clusters during antigen binding and signaling. This unique lipid composition, sometimes referred to as a “lipid raft,” is predicted to be enriched in glycosphingolipids and cholesterol. An altered lipid composition surrounding the BCR could influence the partitioning and activity of regulatory proteins and lipids, acting as a mechanism for the B cell to regulate signal transduction following stimulus by antigen. However, without direct observations of this phenomenon, theories surrounding lipid compositional heterogeneity in B cells remain controversial. In this work, we utilize two-color super-resolution localization microscopy (STORM and PALM) to directly observe the lipid composition proximal to B cell receptors, using the photoactivatable fluorescent protein mEos3.2 anchored to the plasma membrane through posttranslational lipid modifications. These probes mimic important regulatory proteins involved in BCR signaling by their membrane anchor but lack the protein interaction domains or biological activity found in the native protein. By analyzing the co-distributions of the B cell receptor and lipid probes using correlation functions, we find that the membrane surrounding B cell receptor clusters is depleted of probes bearing unsaturated and branched geranylgeranyl modifications and is enriched in probes bearing saturated palmitoyl modifications. Quantification of lipid probe distributions reveals that B cell compositional heterogeneity influences the partitioning of these lipid modifications with order 1kBT of effective potential, as expected from predications of composition fluctuations in critical systems. These findings provide definitive evidence of membrane compositional heterogeneity in an important biological signaling system. Our results suggest that compositional fluctuations contribute to cellular responses by influencing the spatial distribution of specific components in the plasma membrane.

Sticky Patches on Lipid Nanoparticles Generate Binding Geometries that Enable Effective Targeting of Otherwise Untargetable Cancers

The majority of breast cancer patients (70%) have tumors designated as ‘HER2-negative’ (<1+ HER2-expression evaluated by immunohistochemistry or < 200,000 HER2-copies per cell). For these patients there are no targeted therapeutic options utilizing the HER2 receptor. The ability of conventionally targeted nanoparticles for specific targeting stops to hold on cancer cells expressing less than 200,000 copies of HER2 per cell or less than two receptors per nanoparticle's projected area (for particles of 100 nm in-diameter). This geometry corresponds to the limit of multivalent interactions (avidity) loosely defined as multiple contacts between neighboring same-cell receptors with ligands from a single nanoparticle. An alternative therapeutic approach is needed, therefore, to enable selective targeting and effective killing of cancer cells with low or too low HER2 expression.Towards this goal we designed targeted lipid nanoparticles (vesicles) that contain HER2-targeting short peptides densely conjugated (for high local multivalency) within sticky patches. Sticky patches are phase-separated raft-like lipid-domains of high local multivalency which is induced by preferential partitioning of peptide-functionalized lipids. To enable selectivity in binding, sticky patches are exclusively triggered to form in mildly acidic environments matching the tumor interstitium. Lipid phase-separation with lowering pH is a result of the interplay of decreasing (pH-tunable) electrostatic repulsion and attractive hydrogen bonding among the domain-forming lipids.We show that lipid nanoparticles with sticky patches selectively associate with and kill HER2-negative and triple negative breast cancer cells (MCF-7 and MDA-MB-231, respectively, with 60,000 and 90,000 HER2-copies per cell) while do not affect cardiomyocytes and breast normal cells. Systematic studies of association, dissociation and internalization rates of nanoparticles by cells will be presented, and a mechanistic mathematical model will be discussed with the aim to explain the observed high avidity.

Simulation Study of Composition Fluctuations in Lipid Bilayers

Lipid bilayers constitute the basis of biological membranes. Understanding lipid mixing and phase behavior can provide important insights into membrane lateral organization (the raft hypothesis). Here we investigate model lipid bilayers below and above the miscibility transition temperatures. Molecular dynamics simulations with the MARTINI coarse-grained force field are employed to model bilayers on a length scale approaching 100 nm laterally and a time scale of tens of microseconds. We simulate lipid mixtures containing saturated and unsaturated lipids, and cholesterol at different concentrations and temperatures between 270 and 340 K. The coexistence of liquid-crystalline and gel, as well as liquid-ordered and liquid-disordered phases is reproduced. We induce a gradual transition from phase separation to mixing by raising the temperature and adding hybrid lipids (with a saturated and an unsaturated chains). The evolution of bilayer properties along this transition is analyzed. Domain size and phase boundary length, the length and time scales of composition fluctuations, and inter-leaflet coupling are quantified. The results allow characterizing partitioning of hybrid lipids between the coexisting phases, their role in composition fluctuations, and also the effect of spontaneous curvature on composition fluctuations. Curved domains are observed in both symmetric and asymmetric bilayers (with different composition of the two leaflets).

Designing lipids for selective partitioning into liquid ordered membrane domains.

Self-organization of lipid molecules into specific membrane phases is key to the development of hierarchical molecular assemblies that mimic cellular structures. While the packing interaction of the lipid tails should provide the major driving force to direct lipid partitioning to ordered or disordered membrane domains, numerous examples show that the headgroup and spacer play important but undefined roles. We report here the development of several new biotinylated lipids that examine the role of spacer chemistry and structure on membrane phase partitioning. The new lipids were prepared with varying lengths of low molecular weight polyethylene glycol (EGn) spacers to examine how spacer hydrophilicity and length influence their partitioning behavior following binding with FITC-labeled streptavidin in liquid ordered (Lo) and liquid disordered (Ld) phase coexisting membranes. Partitioning coefficients (Kp Lo/Ld) of the biotinylated lipids were determined using fluorescence measurements in studies with giant unilamellar vesicles (GUVs). Compared against DPPE-biotin, DPPE-cap-biotin, and DSPE-PEG2000-biotin lipids, the new dipalmityl-EGn-biotin lipids exhibited markedly enhanced partitioning into liquid ordered domains, achieving Kp of up to 7.3 with a decaethylene glycol spacer (DP-EG10-biotin). We further demonstrated biological relevance of the lipids with selective partitioning to lipid raft-like domains observed in giant plasma membrane vesicles (GPMVs) derived from mammalian cells. Our results found that the spacer group not only plays a pivotal role for designing lipids with phase selectivity but may also influence the structural order of the domain assemblies.

Simulations Predict 18:0-22:6 Phosphatidylserine Drives α-Synuclein into the Liquid-Disordered Phase in Synaptic Vesicle Lipid Composition

Because α-Synuclein binds to anionic lipids and synaptic vesicles consist of 12% 18:0-22:6 phosphatidylserine (SDPS), we asked where this asymmetric lipid partitions, and how it affects α-Synuclein partitioning in a lipid raft system. Our coarse-grained molecular dynamics simulations of SDPS in a lipid raft composition show that 18:0-22:6 PS partitions to the liquid disordered phase (Ld). Additional simulations of lyso-18:0 and lyso-22:6 and subsequent energy fluctuations analysis demonstrate that SDPS partitioning to Ld is due to the 22:6 acyl-chain's greater affinity for Ld than the 18:0 chain's affinity for Lo. Furthermore, we show that α-synuclein's preference for anionic lipids drives it to join SDPS in the Ld phase. Knowing that the remainder of the synaptic vesicle composition is largely uncharged, we propose that SDPS will drive α-synuclein to Ld regions of the synaptic vesicle mixture.

Characterization of M-laurdan, a versatile probe to explore order in lipid membranes

Microdomains corresponding to localized partition of lipids between ordered and less ordered environments are the subject of intensive investigations, because of their putative participation in modulating cellular responses. One popular approach in the field consists in labelling membranes with solvatochromic fluorescent probes such as laurdan and C-laurdan. In this report, we describe a high-yield procedure for the synthesis of laurdan, C-laurdan and two new fluorophores, called MoC-laurdan and M-laurdan, as well as their extensive photophysical characterization. We find that the latter probe, M-laurdan, is particularly suited to discriminate lipid phases independently of the chemical nature of the lipids, as measured by both fluorescence Generalized Polarization (GP) and anisotropy in large unilamellar vesicles made of various lipid compositions. In addition, staining of live cells with M-laurdan shows a good stability over time without any apparent toxicity, as well as a wider distribution in the various cell compartments than the other probes.

Interpreting in vitro developmental toxicity test battery results: The consideration of toxicokinetics.

In the EU collaborative project ChemScreen an alternative, in vitro assay-based test strategy was developed to screen compounds for reproductive toxicity. A toxicokinetic modeling approach was used to allow quantitative comparison between effective concentrations in the in vitro test battery and observations of developmental toxicity in vivo. This modeling strategy is based on (1) the definition of relevant observations of toxicity in vivo, (2) simulation of the corresponding systemic concentrations in vivo by toxicokinetic modeling, and (3) correction for differences in protein binding and lipid partitioning between plasma and in vitro test media. The test results of a feasibility study with a number of known reproductive toxicants has been described previously (Piersma et al. [15]). In the present paper, we take a more detailed look at the toxicokinetics of these compounds, and add the analysis of some compounds from subsequent studies. We discuss how the consideration of toxicokinetics allowed comparison between test systems with differing test medium composition, has helped to interpret the in vitro findings in light of in vivo observations, and to gain confidence in the predictive value of the test battery outcomes. The same toxicokinetic modeling strategy, in reverse order, can now be used for risk assessment purposes to predict toxic doses in vivo from effective concentrations in vitro.

Stationary phase lipophagy as a cellular mechanism to recycle sterols during quiescence

Delivery of cellular contents to yeast vacuoles/mammalian lysosomes via autophagy ensures long-term cell survival and extends life span. When cultured yeast cells are grown for a prolonged period of time to enter stationary phase, a nondividing state mimicking quiescence, vacuolar membrane proteins partition into either one of the vacuolar microdomains, liquid-ordered (Lo) or liquid-disordered (Ld). We show that during the transition to stationary phase, lipid droplets (LDs), organelles originated from the endoplasmic reticulum (ER), undergo lateral movement to reach the vacuolar surface and are confined within the specific Lo microdomain underlying the network of vacuolar quasi-symmetrical micodomains. Stationary phase lipophagy uses the autophagy machineries to modify the sterol-enriched Lo microdomain to engulf LDs and subsequently deposits the LD-containing vesicles inside the vacuole lumen, which is a pathway morphologically resembling microautophagy. Moreover, stationary phase lipophagy supplies quiescent yeast cells with sterols to sustain phase partitioning of lipids for vacuolar microdomain maintenance. A feed forward loop model was proposed to depict that the sterols boosted by LDs via stationary phase lipophagy promote the Lo microdomain maintenance that in turn stimulates lipophagy.

Native Oxy-PAHs, N-PACs, and PAHs in Historically Contaminated Soils from Sweden, Belgium, and France: Their Soil-Porewater Partitioning Behavior, Bioaccumulation in Enchytraeus crypticus, and Bioavailability

Soil quality standards are based on partitioning and toxicity data for laboratory-spiked reference soils, instead of real world, historically contaminated soils, which would be more representative. Here 21 diverse historically contaminated soils from Sweden, Belgium, and France were obtained, and the soil-porewater partitioning along with the bioaccumulation in exposed worms (Enchytraeus crypticus) of native polycyclic aromatic compounds (PACs) were quantified. The native PACs investigated were polycyclic aromatic hydrocarbons (PAHs) and, for the first time to be included in such a study, oxygenated-PAHs (oxy-PAHs) and nitrogen containing heterocyclic PACs (N-PACs). The passive sampler polyoxymethylene (POM) was used to measure the equilibrium freely dissolved porewater concentration, Cpw, of all PACs. The obtained organic carbon normalized partitioning coefficients, KTOC, show that sorption of these native PACs is much stronger than observed in laboratory-spiked soils (typically by factors 10 to 100), which has been reported previously for PAHs but here for the first time for oxy-PAHs and N-PACs. A recently developed KTOC model for historically contaminated sediments predicted the 597 unique, native KTOC values in this study within a factor 30 for 100% of the data and a factor 3 for 58% of the data, without calibration. This model assumes that TOC in pyrogenic-impacted areas sorbs similarly to coal tar, rather than octanol as typically assumed. Black carbon (BC) inclusive partitioning models exhibited substantially poorer performance. Regarding bioaccumulation, Cpw combined with liposome-water partition coefficients corresponded better with measured worm lipid concentrations, Clipid (within a factor 10 for 85% of all PACs and soils), than Cpw combined with octanol-water partition coefficients (within a factor 10 for 76% of all PACs and soils). E. crypticus mortality and reproducibility were also quantified. No enhanced mortality was observed in the 21 historically contaminated soils despite expectations from PAH spiked reference soils. Worm reproducibility weakly correlated to Clipid of PACs, though the contributing influence of metal concentrations and soil texture could not be taken into account. The good agreement of POM-derived Cpw with independent soil and lipid partitioning models further supports that soil risk assessments would improve by accounting for bioavailability. Strategies for including bioavailability in soil risk assessment are presented.

Dietary energy sources affect the partition of body lipids and the hierarchy of energy metabolic pathways in growing pigs differing in feed efficiency1,2

The use and partition of feed energy are key elements in productive efficiency of pigs. This study aimed to determine whether dietary energy sources affect the partition of body lipids and tissue biochemical pathways of energy use between pigs differing in feed efficiency. Forty-eight barrows (pure Large White) from two divergent lines selected for residual feed intake (RFI), a measure of feed efficiency, were compared. From 74 d to 132 ± 0.5 d of age, pigs (n = 12 by line and by diet) were offered diets with equal protein and ME contents. A low fat, low fiber diet (LF) based on cereals and a high fat, high fiber diet (HF) where vegetal oils and wheat straw were used to partially substitute cereals, were compared. Irrespective of diet, gain to feed was 10% better (P < 0.001), and carcass yield was greater (+2.3%; P < 0.001) in the low RFI compared with the high RFI line; the most-efficient line was also leaner (+3.2% for loin proportion in the carcass, P < 0.001). In both lines, ADFI and ADG were lower when pigs were fed the HF diet (-12.3% and -15%, respectively, relatively to LF diet; P < 0.001). Feeding the HF diet reduced the perirenal fat weight and backfat proportion in the carcass to the same extent in both lines (-27% on average; P < 0.05). Lipid contents in backfat and LM also declined (-5% and -19%, respectively; P < 0.05) in pigs offered the HF diet. The proportion of saturated fatty acids (FA) was lower, but the percentage of PUFA, especially the EFA C18:2 and C18:3, was greater (P < 0.001) in backfat of HF-fed pigs. In both lines, these changes were associated with a marked decrease (P < 0.001) in the activities of two lipogenic enzymes, the fatty acid synthase (FASN) and the malic enzyme, in backfat. For the high RFI line, the hepatic lipid content was greater (P < 0.05) in pigs fed the HF diet than in pigs fed the LF diet, despite a reduced FASN activity (-32%; P < 0.001). In both lines, the HF diet also led to lower glycogen content (-70%) and lower glucokinase activity (-15%; P < 0.05) in the liver. These results show that dietary energy sources modified the partition of energy between liver, adipose tissue, and muscle in a way that was partly dependent of the genetics for feed efficiency, and changed the activity levels of biochemical pathways involved in lipid and glucose storage in tissues.

Packing density changes of supported lipid bilayers observed by fluorescence microscopy and quartz crystal microbalance-dissipation.

Various properties of supported lipid bilayers such as diffusion and lipid partitioning are well characterized. However, little attention has been paid to their molecular packing density. In this work, the adsorption of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) vesicles on glass and silicon dioxide was investigated using fluorescence microscopy, quartz crystal microbalance-dissipation (QCM-D), and atomic force microscopy. Fluorescence recovery after photobleaching data showed that the adsorption of large unilamellar vesicles (LUVs) on glass yielded supported bilayers with full mobility under alkaline (pH 8.3) and acidic (pH 3-4) conditions. These fluid bilayers exhibited quite different diffusion constants; those at alkaline pH were ~10 times larger than those at acidic pH. The reason for this pH dependence was clarified by investigation of the rupture of giant unilamellar vesicles (GUVs) on glass. Fluorescence data revealed that the area of planar bilayer patches increased at alkaline pH. Thus, we conclude that the rapid diffusion in alkaline solution arises from the decreased molecular density. QCM-D data showed that dissipation increased in a stepwise manner during vesicle fusion on silicon dioxide at alkaline pH. We attribute this behavior to the decrease in packing density of planar bilayers.

Characterization of M-laurdan, a versatile probe to explore order in lipid membranes

Microdomains corresponding to localized partition of lipids between ordered and less ordered environments are the subject of intensive investigations, because of their putative participation in modulating cellular responses. One popular approach in the field consists in labelling membranes with solvatochromic fluorescent probes such as laurdan and C-laurdan. In this report, we describe a high-yield procedure for the synthesis of laurdan, C-laurdan and two new fluorophores, called MoC-laurdan and M-laurdan, as well as their extensive photophysical characterization. We find that the latter probe, M-laurdan, is particularly suited to discriminate lipid phases independently of the chemical nature of the lipids, as measured by both fluorescence Generalized Polarization (GP) and anisotropy in large unilamellar vesicles made of various lipid compositions. In addition, staining of live cells with M-laurdan shows a good stability over time without any apparent toxicity, as well as a wider distribution in the various cell compartments than the other probes.

Characterization of M-laurdan, a versatile probe to explore order in lipid membranes.

Microdomains corresponding to localized partition of lipids between ordered and less ordered environments are the subject of intensive investigations, because of their putative participation in modulating cellular responses. One popular approach in the field consists in labelling membranes with solvatochromic fluorescent probes such as laurdan and C-laurdan. In this report, we describe a high-yield procedure for the synthesis of laurdan, C-laurdan and two new fluorophores, called MoC-laurdan and M-laurdan, as well as their extensive photophysical characterization. We find that the latter probe, M-laurdan, is particularly suited to discriminate lipid phases independently of the chemical nature of the lipids, as measured by both fluorescence Generalized Polarization (GP) and anisotropy in large unilamellar vesicles made of various lipid compositions. In addition, staining of live cells with M-laurdan shows a good stability over time without any apparent toxicity, as well as a wider distribution in the various cell compartments than the other probes.