In vitro lipid transfer between lipoproteins and midgut-diverticula in the spider Polybetes pythagoricus

It has been already reported that most hemolymphatic lipids in the spider Polybetes pythagoricus are transported by HDL1 and VHDL lipoproteins. We studied in vitro the lipid transfer among midgut-diverticula (M-diverticula), and either hemolymph or purified lipoproteins as well as between hemolymphatic lipoproteins. M-diverticula and hemolymph were labeled by in vivo ¹⁴C-palmitic acid injection. In vitro incubations were performed between M-diverticula and either hemolymph or isolated lipoproteins. Hemolymph lipid uptake was associated to HDL1 (67%) and VHDL (32%). Release from hemolymph towards M-diverticula showed the opposite trend, VHDL 75% and HDL1 45%. Isolated lipoproteins showed a similar behavior to that observed with whole hemolymph. Lipid transfer between lipoproteins showed that HDL1 transfer more ¹⁴C-lipids to VHDL than vice versa. Only 38% FFA and 18% TAG were transferred from M-diverticula to lipoproteins, while on the contrary 75% and 73% of these lipids, respectively, were taken up from hemolymph. A similar trend was observed regarding lipoprotein phospholipids. This study supports the hypothesis that HDL1 and hemocyanin-containing VHDL are involved in the uptake and release of FFA, phospholipids and triacylglycerols in the spider P. pythagoricus. The data support a directional flow of lipids from HDL1 and VHDL suggesting a mode of lipid transport between lipoproteins and M-diverticula.     

Dynamics of neutral lipid storage and mobilization in yeast

We make use of the yeast Saccharomyces cerevisiae as a flexible experimental system to investigate coordinate pathways of neutral lipid synthesis, storage and mobilization with special emphasis on the role of different organelles in these processes. Recently, a number of new gene products involved in triacylglycerol (TAG) and steryl ester (STE) metabolism were identified in our laboratory and by other groups. STE are synthesized by the two STE synthases Are1p and Are2p, whereas TAG are formed mainly through the action of the two TAG synthases Dga1p and Lro1p with minor contributions of Are1p and Are2p. Once formed, TAG and STE are stored in so-called lipid particles. A dga1Deltalro1Deltaare1Deltaare2Delta quadruple mutant which lacks neutral lipid synthesis and is consequently devoid of lipid particles turned out to be a valuable tool for studying the physiological role of storage lipids and lipid particles. Mobilization of neutral lipid depots occurs through catalysis of TAG lipases and STE hydrolases. Three TAG lipases named Tgl3p, Tgl4p and Tgl5p, and three STE hydrolases named Tgl1p, Yeh1p and Yeh2p were recently identified at the molecular level. Although these hydrolases exhibit overlapping function within the enzyme families, they are specific for TAG and STE, respectively. With the exception of Dga1p, whose activity is partially localized to lipid particles, TAG and STE forming enzymes are restricted to the endoplasmic reticulum. TAG lipases and STE hydrolases are components of lipid particles with the exception of Yeh2p, which is plasma membrane located. Thus, neutral lipid metabolism is not only regulated at the enzyme level but also by the distribution of the components to organelles. The fact that neutral lipid homeostasis is linked to a number of cell biological processes confirms the important role of this class of lipids as cellular modulators or effectors.     

Missense mutation in APOC3 within the C-terminal lipid binding domain of human ApoC-III results in impaired assembly and secretion of triacylglycerol-rich very low density lipoproteins: evidence that ApoC-III plays a major role in the formation of lipid precursors within the microsomal lumen

Hepatic assembly of triacylglycerol (TAG)-rich very low density lipoproteins (VLDL) is achieved through recruitment of bulk TAG (presumably in the form of lipid droplets within the microsomal lumen) into VLDL precursor containing apolipoprotein (apo) B-100. We determined protein/lipid components of lumenal lipid droplets (LLD) in cells expressing recombinant human apoC-III (C3wt) or a mutant form (K58E, C3KE) initially identified in humans that displayed hypotriglyceridemia. Although expression of C3wt markedly stimulated secretion of TAG and apoB-100 as VLDL(1), the K58E mutation (located at the C-terminal lipid binding domain) abolished the effect in transfected McA-RH7777 cells and in apoc3-null mice. Metabolic labeling studies revealed that accumulation of TAG in LLD was decreased (by 50%) in cells expressing C3KE. A Fat Western lipid protein overlay assay showed drastically reduced lipid binding of the mutant protein. Substituting Lys(58) with Arg demonstrated that the positive charge at position 58 is crucial for apoC-III binding to lipid and for promoting TAG secretion. On the other hand, substituting both Lys(58) and Lys(60) with Glu resulted in almost entire elimination of lipid binding and loss of function in promoting TAG secretion. Thus, the lipid binding domain of apoC-III plays a key role in the formation of LLD for hepatic VLDL assembly and secretion.     

A simplified approach to resonance energy transfer in membranes,lipoproteins and spatially restricted systems

A general model is developed to simulate dipole-dipole resonance energy transfer in spatially restricted systems. At low concentrations of acceptor molecule, the overall quantum yield of a donor population can be defined quantitatively in terms of transfer to multiple defined acceptor regions. Energy transfer at higher acceptor concentrations can be approximated by assuming an exponential dependence of relative quantum yield on the acceptor concentrations. Through geometrical manipulations, this algorithm has been applied using an electronic calculator to systems in which donor-acceptor interaction is limited by unique steric restriction on donor and acceptor distribution within lipid aggregates. The systems that have been analyzed include monomolecular films, bilayer membranes, small cliscoidal lipid-protein complexes and plasma lipoproteins. The observed energy transfer from N-(2-naphthyl)-23.24-dinor-5-cholen-22-amide-3β-ol to N-dansyldimyristoylphosphatidyl-ethanolamine in a dimyristoylphosphatidylcholine bilayer agrees with that predicted by this model.     

Effects of pH and diethylpyrocarbonate on the conductance states of planar lipid bilayers containing haemocyanin

The addition of haemocyanin from Megathura crenulata to the aqueous phase bathing a bilayer lipid membrane resulted in the formation of ionic channels. With an applied voltage biased negative with respect to the haemocyanin-containing side, a single conductance state was observed above pH 7.0. Below pH 7.0 several conductance states were manifested, and the maximum conductance observed for a single channel decreased with decreasing pH. Extensive treatment of the haemocyanin with diethylpyrocarbonate, which reacts primarily with histidine residues, completely prevented the formation of ionic channels; however, milder treatment produced a chemically modified haemocyanin that was capable of forming ionic channels with modified conductance properties. Each channel conductance was typically much lower than that of the channels formed from unmodified haemocyanin, and there was now substantial variation in conductance from channel to channel. Following the use of hydroxylamine to remove the carbethoxy groups from the modified haemocyanin, it formed ionic channels that were restored to the original unit channel conductance.     

Ultrastructural aspects of storage lipid mobilization in the tapetum of Lilium hybrida var. enchantment

Stages in the formation and degradation of pollenkitt in the anther of Lilium have been investigated using the electron microscope. This material, which appears to be a complex of lipid and carotenoids, is formed during the autolysis of the tapetal cells by the fusion of lipidic inclusions with globules derived from plastids. Autolysis of the tapetal cells is progressive for it commences with the disintegration of many cytoplasmic components, followed by the breakdown of storage lipids. The plasma membrane maintains its integrity during these events apparently, by proliferation, aiding in the transfer of the products of hydrolysis into the loculus. During the course of lipid breakdown, a striking vacuolar system is formed in the tapetal cytoplasm, presumably containing the products of this hydrolysis. The source of membranes for this system is clearly the lipid globules themselves. The generation of the membrane apparently involves the participation of electronopaque material, possibly enzymic, contained within the lipid globules.     

Aluminum compounds enhance lipid peroxidation in liposomes: insight into cellular damage caused by oxidative stress

Aluminum (Al) has been proposed as one of the critical environmental factors responsible for several neurodegenerative diseases such as Alzheimer's disease. However, the suggested mechanism involving the contribution of reactive oxygen species still remains controversial. We have first attempted to identify Al compounds either in its ionic or complexed forms that cause oxidative stress in biological systems. For this purpose, we examined the effect of inorganic Fe(2+)- and organic radical initiator (2,2'-azobis (2-amidinopopane) hydrochloride; AAPH)-induced lipid peroxidation by using aluminum (Al(3+)) nitrate and tris(maltolato)aluminum(III) complex (ALM) with respect to molecular oxygen (O(2)) consumption and membrane fluidity change in liposomes as biological membrane models. The following important results were obtained: (1) ALM enhanced the lipid peroxidation induced by Fe(2+) and AAPH in phosphatidylcholine liposomes; this corresponded well with the promotion of O(2) uptake in the same liposomes, (2) Al(3+) increased both lipid peroxidation and O(2) consumption in phosphatidylserine liposomes in the presence of Fe(2+), and (3) both Al(3+) and ALM affected the membrane fluidity on the inner side. It has been concluded that ALM induces higher lipid peroxidation in liposomes than Al(3+); this finding will be useful to gain an insight into the role of Al in cellular damage in relation to oxidative stress.     

Role of cholesterol in lipid raft formation: lessons from lipid model systems

Biochemical and cell-biological experiments have identified cholesterol as an important component of lipid ‘rafts’ and related structures (e.g., caveolae) in mammalian cell membranes, and membrane cholesterol levels as a key factor in determining raft stability and organization. Studies using cholesterol-containing bilayers as model systems have provided important insights into the roles that cholesterol plays in determining lipid raft behavior. This review will discuss recent progress in understanding two aspects of lipid-cholesterol interactions that are particularly relevant to understanding the formation and properties of lipid rafts. First, we will consider evidence that cholesterol interacts differentially with different membrane lipids, associating particularly strongly with saturated, high-melting phospho- and sphingolipids and particularly weakly with highly unsaturated lipid species. Second, we will review recent progress in reconstituting and directly observing segregated raft-like (liquid-ordered) domains in model membranes that mimic the lipid compositions of natural membranes incorporating raft domains.     

Locust flight activity as a model for hormonal regulation of lipid mobilization and transport

Flight activity of insects provides a fascinating yet relatively simple model system for studying the regulation of processes involved in energy metabolism. This is particularly highlighted during long-distance flight, for which the locust constitutes a long-standing favored model insect, which as one of the most infamous agricultural pests additionally has considerable economical importance. Remarkably many aspects and processes pivotal to our understanding of (neuro)hormonal regulation of lipid mobilization and transport during insect flight activity have been discovered in the locust; among which are the peptide adipokinetic hormones (AKHs), synthesized and stored by the neurosecretory cells of the corpus cardiacum, that regulate and integrate lipid (diacylglycerol) mobilization and transport, the functioning of the reversible conversions of lipoproteins (lipophorins) in the hemolymph during flight activity, revealing novel concepts for the transport of lipids in the circulatory system, and the structure and functioning of the exchangeable apolipopotein, apolipophorin III, which exhibits a dual capacity to exist in both lipid-bound and lipid-free states that is essential to these lipophorin conversions. Besides, the lipophorin receptor (LpR) was identified and characterized in the locust.In an integrative approach, this short review aims at highlighting the locust as an unrivalled model for studying (neuro)hormonal regulation of lipid mobilization and transport during insect flight activity, that additionally has offered a broad and profound research model for integrative physiology and biochemistry, and particularly focuses on recent developments in the concept of AKH-induced changes in the lipophorin system during locust flight, that deviates fundamentally from the lipoprotein-based transport of lipids in the circulation of mammals. Current studies in this field employing the locust as a model continue to attribute to its role as a favored model organism, but also reveal some disadvantages compared to model insects with a completely sequenced genome.     

Role of cholesterol in synapse formation and function

Cholesterol is a multifaceted molecule, which serves as essential membrane component, as cofactor for signaling molecules and as precursor for steroid hormones. Consequently, defects in cholesterol metabolism cause devastating diseases. So far, the role of cholesterol in the nervous system is less well understood. Recent studies showed that cultured neurons from the mammalian central nervous system (CNS) require glia-derived cholesterol to form numerous and efficient synapses. This suggests that the availability of cholesterol in neurons limits the extent of synaptogenesis. Here, I will summarize the experimental evidence for this hypothesis, describe what is known about the structural and functional role of cholesterol at synapses, and discuss how cholesterol may influence synapse development and stability.     

Phospholipid Transfer Protein Plays a Major Role in the Initiation of Apolipoprotein B-containing Lipoprotein Assembly in Mouse Primary Hepatocytes

In this study, we tested the hypothesis that phospholipid transfer protein (PLTP) is a plausible mediator of phospholipid (PL) transfer to the N-terminal 1000 residues of apoB (apoB:1000) leading to the initiation of apoB-containing lipoprotein assembly. To this end, primary hepatocytes from wild type (WT) and PLTP knock-out (KO) mice were transduced with adenovirus-apoB:1000 with or without co-transduction with adenovirus-PLTP, and the assembly and secretion of apoB:1000-containing lipoproteins were assessed. PLTP deficiency resulted in a 65 and 72% reduction in the protein and lipid content, respectively, of secreted apoB:1000-containing lipoproteins. Particles secreted by WT hepatocytes contained 69% PL, 9% diacylglycerol (DAG), and 23% triacylglycerol (TAG) with a stoichiometry of 46 PL, 6 DAG, and 15 TAG molecules per apoB:1000. PLTP absence drastically altered the lipid composition of apoB:1000 lipoproteins; these particles contained 46% PL, 13% DAG, and 41% TAG with a stoichiometry of 27 PL, 10 DAG, and 23 TAG molecules per apoB:1000. Reintroduction of Pltp gene into PLTP-KO hepatocytes stimulated the lipidation and secretion of apoB:1000-containing lipoproteins by ∼3-fold; the lipid composition and stoichiometry of these particles were identical to those secreted by WT hepatocytes. In contrast to the WT, apoB:1000 in PLTP-KO hepatocytes was susceptible to intracellular degradation predominantly in the post-endoplasmic reticulum, presecretory compartment. Reintroduction of Pltp gene into PLTP-KO hepatocytes restored the stability of apoB:1000. These results provide compelling evidence that in hepatocytes initial recruitment of PL by apoB:1000 leading to the formation of the PL-rich apoB-containing initiation complex is mediated to a large extent by PLTP.     

Autophagy in obesity and atherosclerosis: Interrelationships between cholesterol homeostasis,lipoprotein metabolism and autophagy in macrophages and other systems

The incidence of diseases characterized by a dysregulation of lipid metabolism such as obesity, diabetes and atherosclerosis is rising at alarming rates, driving research to uncover new therapies to manage dyslipidemias and resolve the metabolic syndrome conundrum. Autophagy and lipid homeostasis – both ancient cellular pathways – have seemingly co-evolved to share common regulatory elements, and autophagy has emerged as a prominent mechanism involved in the regulation of lipid metabolism. This review highlights recent findings on the role of autophagy in the regulation of cellular cholesterol homeostasis and lipoprotein metabolism, with special emphasis on macrophages. From modulation of inflammation to regulation of cellular cholesterol levels, a protective role for autophagy in atherosclerosis is emerging. The manipulation of autophagic activity represents a new possible therapeutic approach for the treatment complex metabolic disorders such as obesity and the metabolic syndrome.     

Larval nutrition affects lipid storage and growth, but not protein or carbohydrate storage in newly eclosed adults of the grasshopper Schistocerca americana

Nitrogen availability from dietary protein can have profound effects on the physiology and evolutionary ecology of insect herbivores. While many studies consider the effects of nutrition on consumption and gross body composition of protein and other important nutrients, few consider partitioning to storage for future use. I used chemically defined artificial diets to quantitatively manipulate the amount of dietary carbohydrates and proteins available to growing larvae of the grasshopper Schistocerca americana to determine how larval nutrient availability affects growth and all three classes of stored nutrients (proteins, lipids, and carbohydrates) carried over from larval feeding into adulthood. Larvae on poor diets increased consumption, but could not compensate for diet quality, eclosing small and containing no significant nutrient stores at adulthood. Individuals fed intermediate to high nutrient content diets as larvae were significantly larger and contained a significantly greater proportion of lipid stores at adult eclosion, but not protein or carbohydrate stores than individuals fed low nutrient content diets. This suggests that larvally derived lipid stores may be more important to adult fitness than carbohydrate or protein stores. This result is contrary to previous studies performed on the role of larval nutrition and allocation to protein stores, and this difference is likely due to variation in the relative availability of protein in adult diets across species.     

Structural dynamics of the cell wall precursor lipid II in the presence and absence of the lantibiotic nisin

Representing a physiological “Achilles' heel”, the cell wall precursor lipid II (L_II) is a prime target for various classes of antibiotics. Over the years L_II-binding agents have been recognized as promising candidates and templates in the search for new antibacterial compounds to complement or replace existing drugs. To elucidate the molecular structural basis underlying L_II functional mechanism and to better understand if and how lantibiotic binding alters the molecular behavior of L_II, we performed molecular dynamics (MD) simulations of phospholipid membrane-embedded L_II in the absence and presence of the L_II-binding lantibiotic nisin. In a series of 2 × 4 independent, unbiased 100 ns MD simulations we sampled the conformational dynamics of nine L_II as well as nine L_II–nisin complexes embedded in an aqueous 150 mM NaCl/POPC phospholipid membrane environment. We found that nisin binding to L_II induces a reduction of L_II mobility and flexibility, an outward shift of the L_II pentapeptide, an inward movement of the L_II disaccharide section, and an overall deeper insertion of the L_II tail group into the membrane. The latter effect might indicate an initial step in adopting a stabilizing, scaffold-like structure in the process of nisin-induced membrane leakage. At the same time nisin conformation and L_II interaction remain similar to the 1WCO L_II–nisin NMR solution structure.     

Toxicity of 1,2-dibromoethane in isolated hepatocytes: Role of lipid peroxidation

Treatment of isolated hepatocytes with 1,2-dibromoethane (DBE) caused a concentration dependent depletion of cellular glutathione (GSH) content and a parallel increase in the covalent binding of reactive intermediates to cell proteins, as a consequence of the haloalkane activation. The reduction of the hepatocyte GSH content, induced by DBE, stimulated the onset of lipid peroxidation, as measured by malondialdehyde (MDA) accumulation. N-Acetylcysteine (1 mM) was found to partially prevent GSH loss and to inhibit MDA formation, whereas equal concentrations of cysteine and methionine were ineffective on these respects. The stimulation of the peroxidative reactions appeared to be also associated with an increase in the leakage of lactate dehydrogenase (LDH) from the cells, indicative of a severe hepatocyte injury. Antioxidants such as -tocopherol, N,N′-phenyl-phenylenediamine (DPPD) and promethazine, as well as N-acetylcysteine reduced MDA formation to various extents and also protect against LDH release, yet without interfering with the covalent binding of DBE reactive intermediates to hepatocyte proteins. These results suggest the involvement of lipid peroxidation, consequent to GSH depletion, in the pathogenesis of liver cell necrosis due to DBE.     

Key enzymes for biosynthesis of neutral lipid storage compounds in prokaryotes: properties, function and occurrence of wax ester synthases/acyl-CoA: diacylglycerol acyltransferases

Triacylglycerols (TAGs) and wax esters (WEs) are beside polyhydroxyalkanoates (PHAs) important storage lipids in some groups of prokaryotes. Accumulation of these lipids occurs in cells when they are cultivated under conditions of unbalanced growth in the presence of high concentrations of a suitable carbon source, which can be used for fatty acid and storage lipid biosyntheses. The key enzymes, which mediate both WE and TAG formations from long-chain acyl-coenzyme A (CoA) as acyl donor and long-chain fatty alcohols or diacylglycerols as respective acyl acceptors in bacteria, are WE synthases/acyl-CoA:diacylglycerol acyltransferases (WS/DGATs). The WS/DGATs identified so far represent rather unspecific enzymes with broad spectra of possible substrates; this makes them interesting for many biotechnological applications. This review traces the molecular structure and biochemical properties including the probable regions responsible for acyltransferase properties, enzymatic activity and substrate specifities. The phylogenetic relationships based on amino acid sequence similarities of this unique class of enzymes were revealed. Furthermore, recent advances in understanding the physiological functions of WS/DGATs in their natural hosts including pathogenic Mycobacterium tuberculosis were discussed.     

Parameters affecting fusion between liposomes and synaptosomes. Role of proteins,lipid peroxidation,pH and temperature

We investigated the effect of several parameters, such as temperature, pH and proteins, on the fusion between synaptosomes, freshly isolated from rat brain cortex, and large unilamellar phosphatidylserine liposomes. These studies were carried out in both peroxidized and nonperoxidized synaptosomes. Mixing of membrane lipids was monitored using a fluorescence resonance energy transfer assay. Ascorbate (0.8 mm)/ Fe²⁺ (2.5 m)-induced peroxidation of synaptosomes enhanced the fusion process (twofold) which may reflect an increase in synaptosomal protein hydrophobicity and hence a facilitation of intermembrane aggregation. The fusion process was shown to be temperature sensitive, a reduction in the extent being observed (twofold) as the temperature was lowered from 37 to 25°C. This effect may be due to changes in membrane fluidity. The fusion process is pH dependent, an increase in both kinetics and extent being observed when the pH was lowered from 7.4 to 5.5. A significant inhibition (92% at pH 7.4; 35% at pH 5.5) of the interaction between synaptosomes and liposomes by trypsin pretreatment of synaptosomes was found, thus indicating that the fusion reaction is a protein-mediated process. The inhibitory effect of trypsin at pH 5.5 is not so strong as that at physiological pH. These results suggest that, in addition to the involvement of proteins, nonspecific interactions between the synaptosomal and liposomal membranes under acidic conditions may also play a role in the fusion process. The investigation of binding of synaptosomes to liposomes under several experimental conditions provided evidence for the participation of proteins in membrane aggregation, as well as for the role of electrostatic forces in this process, at mild acidic pH.This work was supported by Junta National de Investigação Científica e Tecnológica (JNICT) and the Calouste Gulbenkian Foundation, Portugal.     

Terminalia pallida fruit ethanolic extract ameliorates lipids,lipoproteins, lipid metabolism marker enzymes and paraoxonase in isoproterenol-induced myocardial infarcted rats

The present study aimed to evaluate the effect of Terminalia pallida fruit ethanolic extract (TpFE) on lipids, lipoproteins, lipid metabolism marker enzymes and paraoxonase (PON) in isoproterenol (ISO)-induced myocardial infarcted rats. PON is an excellent serum antioxidant enzyme which involves in the protection of low density lipoprotein cholesterol (LDL-C) from the process of oxidation for the prevention of cardiovascular diseases. ISO caused a significant increase in the concentration of total cholesterol, triglycerides, LDL-C, very low density lipoprotein cholesterol and lipid peroxidation whereas significant decrease in the concentration of high density lipoprotein cholesterol. ISO administration also significantly decreased the activities of lecithin cholesterol acyl transferase, PON and lipoprotein lipase whereas significantly increased the activity of 3-hydroxy-3-methylglutaryl-coenzyme-A reductase. Oral pretreatment of TpFE at doses 100, 300 and 500?mg/kg body weight (bw) and gallic acid (15?mg/kg bw) for 30?days challenged with concurrent injection of ISO (85?mg/kg bw) on 29th and 30th day significantly attenuated these alterations and restored the levels of lipids, lipoproteins and the activities of lipid metabolizing enzymes. Also TpFE significantly elevated the serum antioxidant enzyme PON. This is the first report revealed that pretreatment with TPFE ameliorated lipid metabolic marker enzymes and increased the antioxidant PON in ISO treated male albino Wistar rats.