Intestinal-fatty acid binding protein and lipid transport in human intestinal epithelial cells

Intestinal-fatty acid binding protein (I-FABP) is a 14-15 kDa cytoplasmic molecule highly expressed in the enterocyte. Although different functions have been proposed for various FABP family members, the specific function of I-FABP in human intestine remains unclear. Here, we studied the role of I-FABP in molecularly modified normal human intestinal epithelial cells (HIEC-6). cDNA transfection resulted in 90-fold I-FABP overexpression compared to cells treated with empty pQCXIP vector. The high-resolution immunogold technique revealed labeling mainly in the cytosol and confirmed the marked phenotype abundance of I-FABP in cDNA transfected cells. I-FABP overexpression was not associated with alterations in cell proliferation and viability. Studies using these transfected cells cultured with [14C]oleic acid did not reveal higher efficiency in de novo synthesis or secretion of triglycerides, phospholipids, and cholesteryl esters compared to cells treated with empty pQCXIP vector only. Similarly, the incubation with [35S]methionine did not disclose a superiority in the biogenesis of apolipoproteins (apo) A-I, A-IV, B-48, and B-100. Finally, cells transfected with I-FABP did not exhibit an increased production of chylomicrons, VLDL, LDL, and HDL. Our observations establish that I-FABP overexpression in normal HIEC-6 is not related to cell proliferation, lipid esterification, apo synthesis, and lipoprotein assembly, and, therefore, exclude its role in intestinal fat transport.     

A peroxisome biogenesis deficiency prevents the binding of alpha-synuclein to lipid droplets in lipid-loaded yeast

Using a yeast model of Parkinson’s disease, we found that alpha-synuclein (αS) binds to lipid droplets in lipid-loaded, wild-type yeast cells but not to lipid droplets in lipid-loaded, peroxisome-deficient cells (pex3Δ). Our analysis revealed that pex3Δ cells have both fewer lipid droplets and smaller lipid droplets than wild-type cells, and that the acyl chains of the phospholipids on the surface of the lipid droplets from pex3Δ cells are on average shorter (C₁₆) than those (C₁₈) on the surface of lipid droplets from wild-type cells. We propose that the shift to shorter (C₁₈ → C₁₆) acyl chains contributes to the reduced binding of αS to lipid droplets in pex3Δ cells.     

A novel multiprotein complex is required to generate the prechylomicron transport vesicle from intestinal ER

Dietary lipid absorption is dependent on chylomicron production whose rate-limiting step across the intestinal absorptive cell is the exit of chylomicrons from the endoplasmic reticulum (ER) in its ER-to-Golgi transport vesicle, the prechylomicron transport vesicle (PCTV). This study addresses the composition of the budding complex for PCTV. Immunoprecipitation (IP) studies from rat intestinal ER solubilized in Triton X-100 suggested that vesicle-associated membrane protein 7 (VAMP7), apolipoprotein B48 (apoB48), liver fatty acid-binding protein (L-FABP), CD36, and the COPII proteins were associated on incubation of the ER with cytosol and ATP. This association was confirmed by chromatography of the solubilized ER over Sephacryl S400-HR in which these constituents cochromatographed with an apparent kDa of 630. No multiprotein complex was detected when the ER was chromatographed in the absence of PCTV budding activity (resting ER or PKCζ depletion of ER and cytosol). Treatment of the ER with anti-apoB48 or anti-VAMP7 antibodies or using gene disrupted L-FABP or CD36 mice all significantly inhibited PCTV generation. A smaller complex (no COPII proteins) was formed when only rL-FABP was used to bud PCTV. The data support the conclusion that the PCTV budding complex in intestinal ER is composed of VAMP7, apoB48, CD36, and L-FABP, plus the COPII proteins.     

Yarrowia lipolytica: A model and a tool to understand the mechanisms implicated in lipid accumulation

The oleaginous yeast Yarrowia lipolytica is known to inhabit various lipid-containing environments. One of the most striking features in this yeast is the presence of several multigene families involved in the metabolic pathways of hydrophobic substrate utilization. The complexity and the multiplicity of these genes give Y. lipolytica a wide capability range towards hydrophobic substrate (HS) utilization and storage. The combination of the increasing knowledge of this yeast's metabolism and the development of more efficient genetic tools is offering new perspectives in using Y. lipolytica as a model organism to study the mechanisms involved in lipid metabolism associated to fat uptake, storage, deposition, mobilization and regulation. Nutrient status and culture conditions seem to play a major role in obesity.     

A novel method to quantify H-ATPase-dependent Na transport across plasma membrane vesicles

To prevent sodium toxicity in plants, Na⁺ is excluded from the cytosol to the apoplast or the vacuole by Na⁺/H⁺ antiporters. The secondary active transport of Na⁺ to apoplast against its electrochemical gradient is driven by plasma membrane H⁺-ATPases that hydrolyze ATP and pump H⁺ across the plasma membrane. Current methods to determine Na⁺ flux rely either on the use of Na-isotopes (²²Na) which require special working permission or sophisticated equipment or on indirect methods estimating changes in the H⁺ gradient due to H⁺-ATPase in the presence or absence of Na⁺ by pH-sensitive probes. To date, there are no methods that can directly quantify H⁺-ATPase-dependent Na⁺ transport in plasma membrane vesicles. We developed a method to measure bidirectional H⁺-ATPase-dependent Na⁺ transport in isolated membrane vesicle systems using atomic absorption spectrometry (AAS). The experiments were performed using plasma membrane-enriched vesicles isolated by aqueous two-phase partitioning from leaves of Populus tomentosa. Since most of the plasma membrane vesicles have a sealed right-side-out orientation after repeated aqueous two-phase partitioning, the ATP-binding sites of H⁺-ATPases are exposed towards inner side. Leaky vesicles were preloaded with Na⁺ sealed for the study of H⁺-ATPase-dependent Na⁺ transport. Our data implicate that Na⁺ movement across vesicle membranes is highly dependent on H⁺-ATPase activity requiring ATP and Mg²⁺ and displays optimum rates of 2.50 μM Na⁺ mg^− 1 membrane protein min^− 1 at pH 6.5 and 25 °C. In this study, for the first time, we establish new protocols for the preparation of sealed preloaded right-side-out vesicles for the study of H⁺-ATPase-dependent Na⁺ transport. The results demonstrate that the Na⁺ content of various types of plasma membrane vesicle can be directly quantified by AAS, and the results measured using AAS method were consistent with those determined by the previous established fluorescence probe method. The method is a convenient system for the study of bidirectional H⁺-ATPase-dependent Na⁺ transport with membrane vesicles.     

Super-resolution microscopy localizes perilipin 5 at lipid droplet-mitochondria interaction sites and at lipid droplets juxtaposing to perilipin 2

Objective

Intramyocellular lipid droplets (LD) and their coat proteins PLIN2 and PLIN5 are involved in lipolysis, with a putative role for PLIN5 in mitochondrial tethering. Reportedly, these proteins co-localize and cover the surface of the LD. To provide the spatial basis for understanding how these proteins possess their distinct roles, we examined the precise location of PLIN2 and PLIN5 and explored PLIN5 presence at LD-mitochondria contact sites using Stimulated emission depletion (STED) microscopy and correlative light-electron microscopy (CLEM) in human skeletal muscle sections.

Arabidopsis acyl-CoA-binding proteins ACBP4 and ACBP5 are subcellularly localized to the cytosol and ACBP4 depletion affects membrane lipid composition

In Arabidopsis thaliana, acyl-CoA-binding proteins (ACBPs) are encoded by six genes, and they display varying affinities for acyl-CoA esters. Recombinant ACBP4 and ACBP5 have been shown to bind oleoyl-CoA esters in vitro. In this study, the subcellular localizations of ACBP4 and ACBP5 were determined by biochemical fractionation followed by western blot analyses using anti-ACBP4 and anti-ACBP5 antibodies and immuno-electron microscopy. Confocal microscopy of autofluorescence-tagged ACBP4 and ACBP5, expressed transiently in onion epidermal cells and in transgenic Arabidopsis, confirmed their expression in the cytosol. Taken together, ACBP4 and ACBP5 are available in the cytosol to bind and transfer cytosolic oleoyl-CoA esters. Lipid profile analysis further revealed that an acbp4 knockout mutant showed decreases in membrane lipids (digalactosyldiacylglycerol, monogalactosyldiacylglycerol, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol) while acbp4-complemented lines attained levels similar to wild type, suggesting that ACBP4 plays a role in the biosynthesis of membrane lipids including galactolipids and phospholipids.     

HDAC6 regulates lipid droplet turnover in response to nutrient deprivation via p62-mediated selective autophagy

Autophagy has been evolved as one of the adaptive cellular processes in response to stresses such as nutrient deprivation. Various cellular cargos such as damaged organelles and protein aggregates can be selectively degraded through autophagy. Recently, the lipid storage organelle, lipid droplet(LD), has been reported to be the cargo of starvation-induced autophagy. However, it remains largely unknown how the autophagy machinery recognizes the LDs and whether it can selectively degrade LDs. In this study, we show that Drosophila histone deacetylase 6(dHDAC6), a key regulator of selective autophagy, is required for the LD turnover in the hepatocyte-like oenocytes in response to starvation. HDAC6 regulates LD turnover via p62/SQSTM1(sequestosome 1)-mediated aggresome formation, suggesting that the selective autophagy machinery is required for LD recognition and degradation. Furthermore, our results show that the loss of dHDAC6 causes steatosis in response to starvation. Our findings suggest that there is a potential link between selective autophagy and susceptible predisposition to lipid metabolism associated diseases in stress conditions.     

The MAP1-LC3 conjugation system is involved in lipid droplet formation

Lipid droplets (LDs) are ubiquitous in eukaryotic cells, while excess free fatty acids and glucose in plasma are converted to triacylglycerol (TAG) and stored as LDs. However, the mechanism for the generation and growth of LDs in cells is largely unknown. We show here that the LC3 lipidation system essential for macroautophagy is involved in LD formation. LD formation accompanied by accumulation of TAG induced by starvation was largely suppressed in the hepatocytes that cannot execute autophagy. Under starvation conditions, LDs in addition to autophagosomes were abundantly formed in the cytoplasm of these tissue cells. Moreover, LC3 was localized on the surface of LDs and LC3-II (lipidation form) was fractionated to a perilipin (LD marker)-positive lipid fraction from the starved liver. Taken together, these results indicate that the LC3 conjugation system is critically involved in lipid metabolism via LD formation.     

Mitochondrial oxidative phosphorylation system is recruited to detergent‐resistant lipid rafts during myogenesis

Since detergent‐resistant lipid rafts play important roles in the signal transduction for myogenesis, their comprehensive proteomic analysis could provide new insights to understand their function in myotubes. Here, the detergent‐resistant lipid rafts were isolated from C2C12 myotubes and analyzed by capillary RPLC/MS/MS. Among the 327 proteins (or protein groups) identified, 28% were categorized to the plasma membrane or raft proteins, 29% to mitochondria, 20% to microsomal proteins, 10% to other proteins, and 13% to unknown proteins. The localization of oxidative phosphorylation (OXPHOS) complexes in the sarcolemma lipid rafts was further confirmed from C2C12 myotubes by cellular fractionation, surface‐biotin labeling, immunofluorescence, and lipid raft fractionation. After adding exogenous cytochrome c, the sarcolemma isolated from myotubes had an ability to consume oxygen in the presence of NADH or succinate. The generation of NADH‐dependent extracellular superoxide was increased by inhibiting or downregulating OXPHOS I, III, and IV in myotubes, indicating that OXPHOS proteins are major sources for extracellular ROS in skeletal muscle. With all these data, we can conclude that OXPHOS proteins are associated with the sarcolemma lipid rafts during C2C12 myogenesis to generate extracellular ROS.     

The heterogeneous nuclear ribonucleoprotein (hnRNP) glorund functions in the Drosophila fat body to regulate lipid storage and transport

The availability of excess nutrients in Western diets has led to the overaccumulation of these nutrients as triglycerides, a condition known as obesity. The full complement of genes important for regulating triglyceride storage is not completely understood. Genome-wide RNAi screens in Drosophila cells have identified genes involved in mRNA splicing as important lipid storage regulators. Our lab has shown that a group of splicing factors called heterogeneous nuclear ribonucleoproteins (hnRNPs) regulate lipid metabolism in the fly fat body; however, the identities of all the hnRNPs that function to control triglyceride storage are not known. Here, we used the GAL4/UAS system to induce RNAi to the hnRNP glorund (glo) in the Drosophila fat body to assess whether this hnRNP has any metabolic functions. Decreasing glo levels resulted in less triglycerides being stored throughout the fly. Interestingly, decreasing fat body glo expression resulted in increased triglyceride storage in the fat body, but blunted triglyceride storage in non-fat body tissues, suggesting a defect in lipid transport. Consistent with this hypothesis, the expression of apolipophorin (apolpp), microsomal triglyceride transfer protein (mtp), and apolipoprotein lipid transfer particle (apoltp), apolipoprotein genes important for lipid transport through the fly hemolymph, was decreased in glo-RNAi flies, suggesting that glo regulates the transport of lipids from the fly fat body to surrounding tissues. Together, these results indicate that glorund plays a role in controlling lipid transport and storage and provide additional evidence of the link between gene expression and the regulation of lipid metabolism.     

Lipoprotein-mediated lipid transport in insects: analogy to the mammalian lipid carrier system and novel concepts for the functioning of LDL receptor family members

In all animals, lipoproteins are used to transport lipids through the aqueous circulation. Lipids are delivered to mammalian cells by two different mechanisms: via endocytic uptake of the complete lipoprotein particle mediated by members of the low density lipoprotein (LDL) receptor (LDLR) family, or by selective delivery of lipoprotein-carried lipids at the cell surface, such as lipid uptake following the action of a lipoprotein lipase. Although many structural elements of the lipid transport system of insects are similar to those of mammals, insect lipoprotein-mediated lipid transport was thought to apply only to the latter concept, since the single lipoprotein acts as a reusable lipid shuttle. However, the recent identification of lipoprotein receptors of the LDLR family in insects suggests that lipid transport in these animals may also adopt the first concept. Yet, the endocytic properties of the insect LDLR homologue appear to deviate from those of the mammalian LDLR family members, resulting in the recycling of endocytosed lipoprotein in a transferrin-like manner. This indicates that a hitherto unknown as well as unexpected function can be added to the plethora of functions of LDLR family members. Analysis of the molecular mechanism of the ligand-recycling function of the insect receptor provides also new insight into the possible functioning of the mammalian family members. In the last several years, mammalian and insect lipoprotein-mediated lipid transport systems have been reviewed separately with respect to functioning and lipid delivery. This review, in which new and important developments in the insect field with respect to our understanding of lipid delivery are discussed with a particular focus on the involvement of the LDLR homologue, aims at comparing the two systems, also from an evolutionary biological perspective, and proposes that the two systems are more similar than assumed previously.     

A novel flow cytometric assay to quantify interactions between proteins and membrane lipids

A diverse set of experimental systems has been developed to probe protein-lipid interactions. These include measurements with the headgroups of membrane lipids in solution, immobilized membrane lipids, and analysis of protein binding to membrane lipids reconstituted in liposomes. Each of these methodologies has strengths but also substantial limitations. For example, measurements between proteins and lipid headgroups or with immobilized membrane lipids do not probe interactions in their natural environment, the lipid bilayer. The use of liposomes, however, was so far mostly restricted to biochemical flotation experiments that do not provide quantitative and/or kinetic data. Here, we present a fast and sensitive flow cytometric method to detect protein-lipid interactions. This technique allows for quantitative measurements of interactions between multiple fluorescently labeled proteins and membrane lipids reconstituted in lipid bilayers. The assay can be used to quantify binding efficiencies and to determine kinetic constants. The method is further characterized by a short sampling time of only a few seconds that allows for high-content screening procedures. Finally, using light scatter measurements, the described method also allows for monitoring changes of membrane curvature as well as tethering of liposomes evoked by binding of proteins.     

Perilipin 5, a lipid droplet-associated protein, provides physical and metabolic linkage to mitochondria

Maintaining cellular lipid homeostasis is crucial to oxidative tissues, and it becomes compromised in obesity. Lipid droplets (LD) play a central role in lipid homeostasis by mediating fatty acid (FA) storage in the form of triglyceride, thereby lowering intracellular levels of lipids that mediate cellular lipotoxicity. LDs and mitochondria have interconnected functions, and anecdotal evidence suggests they physically interact. However, the mechanisms of interaction have not been identified. Perilipins are LD-scaffolding proteins and potential candidates to play a role in their interaction with mitochondria. We examined the contribution of LD perilipin composition to the physical and metabolic interactions between LD and mitochondria using multiple techniques: confocal imaging, electron microscopy (EM), and lipid storage and utilization measurements. Using neonatal cardiomyocytes, reconstituted cell culture models, and rodent heart tissues, we found that perilipin 5 (Plin5) recruits mitochondria to the LD surface through a C-terminal region. Compared with control cells, Plin5-expressing cells show decreased LD hydrolysis, decreased palmitate β-oxidation, and increased palmitate incorporation into triglycerides in basal conditions, whereas in stimulated conditions, LD hydrolysis inhibition is lifted and FA released for β-oxidation. These results suggest that Plin5 regulates oxidative LD hydrolysis and controls local FA flux to protect mitochondria against excessive exposure to FA during physiological stress.     

The metabolic capacity of lipid droplet localized acyl-CoA synthetase 3 is not sufficient to support local triglyceride synthesis independent of the endoplasmic reticulum in A431 cells

ACSL3 is the only long chain fatty acyl-CoA synthetase consistently found on growing and mature lipid droplets (LDs), suggesting that this specific localization has biological relevance. Current models for LD growth propose that triglycerides are synthesized by enzymes at the LD surface, with activated fatty acids provided by LD localized ACSL3, thus allowing growth independent of the ER. Here, we tested this hypothesis by quantifying ACSL3 on LDs from human A431 cells.RNAi of ACSL3 reduced the oleoyl-CoA synthetase activity by 83%, suggesting that ACSL3 is by far the dominant enzyme of A431 cells. Molar quantification revealed that there are 1.4 million ACSL3 molecules within a single cell. Metabolic labeling indicated that each ACSL3 molecule contributed a net gain of 3.1 oleoyl-CoA/s. 3D reconstruction of confocal images demonstrated that 530 individual lipid droplets were present in an average oleate fed A431 cell. A representative single lipid droplet with a diameter of 0.66?μm contained 680 ACSL3 molecules on the surface. Subcellular fractionation showed that at least 68% of ACSL3 remain at the ER even during extensive fatty acid supplementation. High resolution single molecule microscopy confirmed the abundance of cytoplasmic ACSL3 outside of LDs. Model calculations for triglyceride synthesis using only LD localized ACSL3 gave significant slower growth of LDs as observed experimentally.In conclusion, although ACSL3 is an abundant enzyme on A431 LDs, the metabolic capacity is not sufficient to account for LD growth solely by the local synthesis of triglycerides.     

A simple and robust UPLC-SRM/MS method to quantify urinary eicosanoids

Eicosanoids are key mediators and regulators of inflammation and oxidative stress often used as biomarkers for diseases and pathological conditions such as cardiovascular and pulmonary diseases and cancer. Analytically, comprehensive and robust quantification of different eicosanoid species in a multi-method approach is problematic because most of these compounds are relatively unstable and may differ in their chemical properties. Here we describe a novel ultra-performance liquid chromatography-selected reaction monitoring mass spectroscopy (UPLC-SRM/MS) method for simultaneous quantification of key urinary eicosanoids, including the prostaglandins (PG) tetranor PGE-M, 8-iso-, and 2,3-dinor-8-iso-PGF_2α; the thromboxanes (TXs) 11-dehydro- and 2,3-dinor-TXB₂; leukotriene E₄; and 12-hydroxyeicosatetraenoic acid. In contrast to previous methods, which used time-consuming and complex solid phase extraction, we prepared samples with a simple liquid/liquid extraction procedure. Because collision-induced dissociation produced characteristic product ions for all analytes, no derivatization step for SRM/MS analysis was necessary. Analytes were separated with a short UPLC reversed-phase column (1.7 µm particles), allowing shorter run times than conventional HPLC columns. The method was validated and applied to human urine samples showing excellent precision, accuracy, detection limits, and robustness. In summary, the developed method allows robust and sensitive profiling of urinary eicosanoid species, making it a useful and valuable tool for biomarker profiling in clinical/toxicological studies.     

A hydrolase-related transport system is not required to explain the intestinal uptake of glucose liberated from phlorizin

The fate of [³H]glucose released from a wide range of [³H]phlorizin concentrations by phlorizin hydrolase has been studied under conditions where the Na⁺-dependent glucose transport system in hamster intestine is profoundly inhibited by the glucoside. At 0.2–2.0 mM phlorizin, the [³H]glucose uptake was a constant 11–12% of that generated by the enzyme and at the highest level, it was reduced to that of passive diffusion. Glucose liberated from 0.2 mM [³H]phlorizin is accumulated at a rate nearly equal to that found for 0.2 mM [¹⁴C]glucose when this free sugar uptake is measured in a medium containing 0.2 mM unlabeled phlorizin. Furthermore, without sodium, the accumulation rates of hydrolase-derived or exogenous glucose are both reduced to the rate of [¹⁴C]mannitol. Our results indicate that the glucose released from phlorizin enters the tissue via the small fraction of the Na⁺-dependent glucose carriers which escape phlorizin blockade together with a mannitol-like passive diffusion. It enjoys a kinetic advantage for tissue entry over free glucose in the medium by virtue of the position of the site where it is formed, i.e. inside the unstirred water layer and near normal entry portals. No special hydrolase-related transport system, like the one proposed for disaccharides, needs to be considered to account for our findings.