Transfer of phospholipids from fat body to lipophorin in Rhodnius prolixus

32P-Labeled fat bodies (32P-fat bodies) of Rhodnius prolixus females were incubated in the presence of non radioactive purified lipophorin and the release of radioactivity to the medium was analysed to answer the question of whether lipophorin is a reusable shuttle for phospholipids. The radioactivity found in the medium was associated with lipophorin phospholipids. When the 32P-fat bodies were incubated in the absence of lipophorin, only a small amount of radioactivity was released and it was not associated with lipophorin, indicating that there was no release of pre-labeled 32P-lipophorin by the tissue. Analysis of 32P-phospholipids transferred from fat bodies to the lipophorin particles by thin-layer chromatography revealed a predominance of phosphatidylethanolamine and phosphatidylcholine, with minor amounts of phosphatidylserine, phosphatidylinositol, and sphingomyelin. The transfer of phospholipids to lipophorin was linear with time up to 45 min and the process was inhibited at low temperature and by the metabolic inhibitors azide and fluoride. The transfer of phospholipids from the fat bodies to lipophorin was saturable with respect to the concentration of lipophorin, which was half-maximal at about 8 mg/ml. A directional movement of phospholipids from the fat body to lipophorin was observed. The net gain of phospholipids in 2 h of incubation with fat body was 8.54 nmol per insect, which corresponds to 6.69% of increase in the lipophorin phospholipid content. The rate of 32P-phospholipid transfer from fat body to lipophorin particles varied during the days after a blood meal increasing up to day 10 and then decreasing in parallel with the process of oogenesis.     

Domain-specific lipid distribution in macrophage plasma membranes

Lipid rafts, defined as cholesterol- and sphingolipid-rich domains, provide specialized lipid environments understood to regulate the organization and function of many plasma membrane proteins. Growing evidence of their existence, protein cargo, and regulation is based largely on the study of isolated lipid rafts; however, the consistency and validity of common isolation methods is controversial. Here, we provide a detailed and direct comparison of the lipid and protein composition of plasma membrane "rafts" prepared from human macrophages by different methods, including several detergent-based isolations and a detergent-free method. We find that detergent-based and detergent-free methods can generate raft fractions with similar lipid contents and a biophysical structure close to that previously found on living cells, even in cells not expressing caveolin-1, such as primary human macrophages. However, important differences between isolation methods are demonstrated. Triton X-100-resistant rafts are less sensitive to cholesterol or sphingomyelin depletion than those prepared by detergent-free methods. Moreover, we show that detergent-based methods can scramble membrane lipids during the isolation process, reorganizing lipids previously in sonication-derived nonraft domains to generate new detergent-resistant rafts. The role of rafts in regulating the biological activities of macrophage plasma membrane proteins may require careful reevaluation using multiple isolation procedures, analyses of lipids, and microscopic techniques.     

Methodological concerns in measuring the lipid fraction of carbon fixation

Carbon fixation into lipid (lipid production) by phytoplankton was measured in 3 lakes on the edge of the Canadian Shield by two different extraction methods. The amount of lipid detected in the plankton samples was generally 11% lower when extracted by a Folch-like lipid solvent (dichloromethane:methanol (2:1)) than with the lipid solvent (80% ethanol and 80% ethanol-diethyl ether) used in a sequential extraction method. The difference between methods was not due to losses of fixed carbon during extraction since the sum of the extraction fractions from both methods were not different from the amount of carbon fixed on a replicate acidified filter. Although more carbon was detected in the lipid fraction of the sequential extraction method, an additional 5% of total carbon fixed was found in the lipid extract of another fraction from the sequential method, the low molecular weight fraction. Our results suggest that accurate comparisons of lipid production data can only occur after compensating for differences in extraction methods while comparing the LFCF determined by different lipid extraction should be avoided.     

Overexpression of full-length cholesteryl ester transfer protein in SW872 cells reduces lipid accumulation

Cells produce two cholesteryl ester transfer protein (CETP) isoforms, full-length and a shorter variant produced by alternative splicing. Blocking synthesis of both isoforms disrupts lipid metabolism and storage. To further define the role of CETP in cellular lipid metabolism, we stably overexpressed full-length CETP in SW872 cells. These CETP⁺ cells had several-fold higher intracellular CETP and accumulated 50% less TG due to a 26% decrease in TG synthesis and 2.5-fold higher TG turnover rate. Reduced TG synthesis was due to decreased fatty acid uptake and impaired conversion of diglyceride to TG even though diacylglycerol acyltransferase activity was normal. Sterol-regulatory element binding protein 1 mRNA levels were normal, and although PPARγ expression was reduced, the expression of several of its target genes including adipocyte triglyceride lipase, FASN, and APOE was normal. CETP⁺ cells contained smaller lipid droplets, consistent with their higher levels of perilipin protein family (PLIN) 3 compared with PLIN1 and PLIN2. Intracellular CETP was mostly associated with the endoplasmic reticulum, although CETP near lipid droplets poorly colocalized with this membrane. A small pool of CETP resided in the cytoplasm, and a subfraction coisolated with lipid droplets. These data show that overexpression of full-length CETP disrupts lipid homeostasis resulting in the formation of smaller, more metabolically active lipid droplets.     

The effect of headgroup class on the conformation of membrane lipids in Acholeplasma Laidlawii: A 2H-NMR study

[2-²H₂]Oleic, [2-²H₂]palmitic, [2-²H₂]dihydrosterculic and [3-²H₂]oleic acids were biosynthetically incorporated into the membrane lipids of Acholeplasma laidlawii B. ²H-NMR spectroscopy and spectral ‘de-Parking” (M. Bloom, J.H. Davis and M.I. Valic, Can. J. Phys., 58 (1980) 1510) were used to study the effect of lipid headgroup class on the conformational order in the vicinity of the C-2 position of the acyl chains of lipids in the liquid crystalline phase. The results indicate that although the orientation and conformations of the membrane lipids in the region of the C-2 position of the chains are qualitatively very similar among the various lipid classes, quantitatively there are some differences, particularly between the glycolipids and the phospholipids. These differences do not exted to the C-3 position. Unlike the headgroup class, the membrane proteins appear to have little if any effect on the molecular ordering of the lipids.     

Brij detergents reveal new aspects of membrane microdomain in erythrocytes

Membrane microdomains enriched in cholesterol, sphingolipids (rafts), and specific proteins are involved in important physiological functions. However their structure, size and stability are still controversial. Given that detergent-resistant membranes (DRMs) are in the liquid-ordered state and are rich in raft-like components, they might correspond to rafts at least to some extent. Here we monitor the lateral order of biological membranes by characterizing DRMs from erythrocytes obtained with Brij-98, Brij-58, and TX-100 at 4?°C and 37?°C. All DRMs were enriched in cholesterol and contained the raft markers flotillin-2 and stomatin. However, sphingomyelin (SM) was only found to be enriched in TX-100-DRMs – a detergent that preferentially solubilizes the membrane inner leaflet – while Band 3 was present solely in Brij-DRMs. Electron paramagnetic resonance spectra showed that the acyl chain packing of Brij-DRMs was lower than TX-100-DRMs, providing evidence of their diverse lipid composition. Fatty acid analysis revealed that the SM fraction of the DRMs was enriched in lignoceric acid, which should specifically contribute to the resistance of SM to detergents. These results indicate that lipids from the outer leaflet, particularly SM, are essential for the formation of the liquid-ordered phase of DRMs. At last, the differential solubilization process induced by Brij-98 and TX-100 was monitored using giant unilamellar vesicles. This study suggests that Brij and TX-100-DRMs reflect different degrees of lateral order of the membrane microdomains. Additionally, Brij DRMs are composed by both inner and outer leaflet components, making them more physiologically relevant than TX-100-DRMs to the studies of membrane rafts.     

Import and fate of fluorescent analogs of oxidized phospholipids in vascular smooth muscle cells

Lipid oxidation is now thought to be an initiating and sustaining event in atherogenesis. Oxidatively fragmented phospholipids, namely 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), present in minimally modified LDL and atherosclerotic lesions, have been reported to elicit a wide range of pathophysiological responses in the cells of the vascular wall. Nevertheless, the question of their potential sites of action and their primary molecular targets remains open. To address this issue, a series of fluorescently labeled analogs, which differ with regard to structure and binding site of the fluorophore, were synthesized and used as tools for studying the uptake, intracellular stability, and distribution of PGPC and POVPC in vascular smooth muscle cells (VSMCs). We demonstrate that in accordance with their lysophospholipid-like structure, these highly similar molecules transferred rapidly either from aqueous phospholipid dispersions or preloaded native LDL into VSMCs, producing disparate fluorescence patterns irrespective of the attached fluorophore. PGPC derivatives were translocated to the lysosomes. In sharp contrast, POVPC analogs were initially captured in the plasma membrane, most likely in consequence of the formation of covalent adducts with free amino and sulfhydryl groups of proteins and phospholipids. LDL internalization is not required for cellular lipid uptake. Collectively, our data provide evidence that oxidized phospholipids, owing to their high exchangeability between lipoproteins and cell membranes, may act within a short time on different cellular sites in VSMCs and affect various lipid and protein components through physical or chemical interactions, which might then serve as starting points for intracellular signaling.     

Butyrylcholinesterase in lipid metabolism: A new outlook

Cholinesterase enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are traditionally associated with the termination of acetylcholine mediated neural signaling. The fact that these ubiquitous enzymes are also found in tissues not involved in neurotransmission has led to search for alternative functions for these enzymes. Cholinesterases are reported to be involved in many lipid related disease states. Taking into view that lipases and cholinesterases belong to the same enzyme class and by comparing the catalytic sites, we propose a new outlook on the link between BChE and lipid metabolism. The lipogenic substrates of BChE that have recently emerged in contrast to traditional cholinesterase substrates are explained through the hydrolytic capacity of BChE for ghrelin, 4-methyumbelliferyl (4-mu) palmitate, and arachidonoylcholine and through endogenous lipid mediators such as cannabinoids like anandamide and essential fatty acids. The abundance of BChE in brain, intestine, liver, and plasma, tissues with active lipid metabolism, supports the idea that BChE may be involved in lipid hydrolysis. BChE is also regulated by various lipids such as linoleic acid, alpha-linolenic acid or dioctanoylglycerol, whereas AChE is inhibited. The finding that BChE is able to hydrolyze 4-mu palmitate at a pH where lipases are less efficient points to its role as a backup in lipolysis. In diseases such as Alzheimer, in which elevated BChE and impaired lipid levels are observed, the lipolytic activity of BChE might be involved. It is possible to suggest that fatty acids such as 4-mu palmitate, ghrelin, arachidonoylcholine, essential fatty acids, and other related lipid mediators regulate cholinesterases, which could lead to some sort of compensatory mechanism at high lipid concentrations.

     

Complicated trafficking behaviors involved in paradoxical regulation of sortilin in lipid metabolism

This review aims to summarize and discuss the most recent advances in our understanding of the underlying mechanisms of the paradoxical effects of sortilin on lipid metabolism. The vacuolar protein sorting 10 protein (Vps10p) domain in the sortilin protein is responsible for substrate binding. Its cytoplasmic tail interacts with adaptor molecules, and modifications can determine whether sortilin trafficking occurs via the anterograde or retrograde pathway. The complicated trafficking behaviors likely contribute to the paradoxical roles of sortilin in lipid metabolism. The anterograde pathway of sortilin trafficking in hepatocytes, enterocytes, and peripheral cells likely causes an increase in plasma lipid levels, while the retrograde pathway leads to the opposite effect. Hepatocyte sortilin functions via the anterograde or retrograde pathway in a complicated and paradoxical manner to regulate apoB-containing lipoprotein metabolism. Clarifying the regulatory mechanisms underlying the trafficking behaviors of sortilin is necessary and may lead to artificial sortilin intervention as a potential therapeutic strategy for lipid disorder diseases. Conclusively, the paradoxical regulation of sortilin in lipid metabolism is likely due to its complicated trafficking behaviors.     

Unsaturated diether phospholipids in the Antartic methanogen Methanococcoides burtonii

Abstract The Antarctic methanogen Methanococcoides burtonii contained only diether phospholipids. These membrane components were analysed by gas chromatography and gas chromatography mass spectrometry. Of particular interest was the occurrence of unsaturated diether lipids in M. burtonii ; unsaturated ether lipids accounted for 57% of the diether phospholipids. To our knowledge, unsaturated ether lipids have not been previously reported in a methanogen. The presence of the unsaturated ether lipids in M. burtonii is probably the result of temperature adaptation by the bacterium. It may be possible to use these components as a chemical signature for methanogens in Antarctic and Southern Ocean environments.     

Lipid polymorphism and the roles of lipids in membranes

The reasons for lipid diversity in membranes are not understood. Here we review evidence supporting the proposal that factors related to the polymorphic capabilities of lipids provide a rationale for lipid diversity. In particular, the ability of lipids to adopt different polymorphic phases appears to be related to a generalized shape property, where lipids with a cylindrical geometry preferentially adopt the bilayer phase whereas ‘cone’ shaped lipids adopt the hexagonal H_II phase. Lipid diversity may then be considered to satisfy three demands. The first is obviously a need for bilayer forming lipids to provide the basic permeability barrier, whereas the second concerns a need for non-bilayer lipids and associated structures for fusion and related membrane contact phenomena to proceed. A third, and less obvious demand satisfied by non-bilayer lipids concerns the ability of lipids of different shapes to modulate the order in the hydrocarbon region when constrained to a bilayer organization. These possibilities are summarized in a metamorphic mosaic model of membranes.     

sn-Glycerol-3-phosphate acyltransferases in plants

sn-Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the acylation at sn-1 position of glycerol-3-phosphate to produce lysophosphatidic acid (LPA). LPA is an important intermediate for the formation of different types of acyl-lipids, such as extracellular lipid polyesters, storage and membrane lipids. Three types of GPAT have been found in plants, localizing to the plastid, endoplasmic reticulum, and mitochondria. These GPATs are involved in several lipid biosynthetic pathways and play important biological roles in plant development. In the present review, we will focus on the recent progress in studying the physiological functions of GPATs and their metabolic roles in glycerolipid biosynthesis.     

Contrasting effects of anoxia on rhizome lipids in Iris species

A 14-day period of anoxia resulted in losses of polar lipids, particularly their saturated fatty acid components, from the anoxia-tolerant species Iris pseudacorus. By complete contrast, the anoxia-intolerant, closely related species I. germanica, although possessing a highly similar lipid profile, exhibited no changes in lipid composition in response to anoxia. The consequences of the lipid alterations in I. pseudacorus for membrane function and their possible role in adaptation to anoxia are discussed.     

牛磺酸抗小鼠动脉粥样硬化的研究

为了探讨牛磺酸抗小鼠动脉粥样硬化的影响及可能机制,将C57BL/6J小鼠分成五组,正常对照组给予基础饲料喂养,高脂组给予高脂饲料,牛磺酸组分别给予含1.0%、3.0%和5.0%牛磺酸的高脂饲料,实验时间为90d。结果表明,高脂组小鼠主动脉内膜和肝脏发生了粥样硬化病变和脂肪变性,而牛磺酸组病变程度随剂量增大而减轻。高脂组较正常对照组血清及肝脏甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-c)、动脉硬化指数(AI)显著升高,高密度脂蛋白胆固醇(HDL-c)显著降低;牛磺酸组较高脂组显著改善。可见牛磺酸可通过改善脂质代谢紊乱发挥抗动脉粥样硬化的作用。     

Sterol carrier protein-2 (SCP-2) involvement in cholesterol hydroperoxide cytotoxicity as revealed by SCP-2 inhibitor effects

Sterol carrier protein-2 (SCP-2) plays an important role in cholesterol trafficking and metabolism in mammalian cells. The purpose of this study was to determine whether SCP-2, under oxidative stress conditions, might also traffic hydroperoxides of cholesterol, thereby disseminating their cytotoxic effects. Two inhibitors, SCPI-1 and SCPI-3, known to block cholesterol binding by an insect SCP-2, were used to investigate this. A mouse fibroblast transfectant clone (SC2F) overexpressing SCP-2 was found to be substantially more sensitive to apoptotic killing induced by liposomal 7α-hydroperoxycholesterol (7α-OOH) than a wild-type control. 7α-OOH uptake by SC2F cells and resulting apoptosis were both inhibited by SCPI-1 or SCPI-3 at a subtoxic concentration. Preceding cell death, reactive oxidant accumulation and loss of mitochondrial membrane potential were also strongly inhibited. Similar SCPI protection against 7α-OOH was observed with two other types of SCP-2-expressing mammalian cells. In striking contrast, neither inhibitor had any effect on H₂O₂-induced cell killing. To learn whether 7α-OOH cytotoxicity is due to uptake/transport by SCP-2, we used a fluorescence-based competitive binding assay involving recombinant SCP-2, NBD-cholesterol, and SCPI-1/SCPI-3 or 7α-OOH. The results clearly showed that 7α-OOH binds to SCP-2 in SCPI-inhibitable fashion. Our findings suggest that cellular SCP-2 not only binds and translocates cholesterol but also cholesterol hydroperoxides, thus expanding their redox toxicity and signaling ranges under oxidative stress conditions.     

Polymorphism of 1-O-alkylglycerols and general lipid polymorphic behaviour

The polymorphic behaviour of a series of l-O-alkylglycerols have been studied; mainly by DTA and X-ray powder diffraction techniques. The behaviour of these ether lipids is quite similar to that of the closely related l-acylglycerols. The background to the polymorphic behaviour of these lipids is discussed. In the α-phase, the alkyl chains are non-crystalline but still partly ordered in a lattice. The main reason for the formation of the sub-α-phase is a ‘chain crystallization’. The polar group region in the sub-α-phase is crystallized in a state with higher energy, relative to the state of the polar group region in the β-phase. This can account for the difference in stability the β - and sub-ga-phases.     

Spatial variation in density, mean size and physiological condition of the holarctic amphipod Diporeia spp. in Lake Michigan

• 1 We examined spatial patterns in population characteristics (density, biomass, mean body length) and physiological condition (lipid content, length‐weight) of the amphipod Diporeia spp. in Lake Michigan by collecting samples at up to 85 sites in late summer 1994 and 1995. Variables were examined relative to water depth and three lake regions: south, central and north. Most major river systems are found in the south, and this region is more nutrient‐enriched compared to the north.
• 2 Over all sites, mean density was 5240^‐2, biomass was 4.1g dry wt m^‐2, and mean body length was 5 mm. While maximum densities were related to depth, with a peak at 30–70 m, greatest densities occurred on the west side of the lake, and low densities were found in the south‐east, north‐east and lower Green Bay. High densities in the west probably resulted from upwelling, and reduced densities in the south‐east may reflect food competition with Dreissena polymorpha (zebra mussel).
• 3 Lipid content, weight per unit length, and mean length declined with increased water depth, but depth‐related trends were most evident in the south. Overall, mean lipid content and weight per unit length were significantly lower in the south (16.6% dry wt, 0.59 mg at 5 mm body length) compared to the north (23.7% dry wt, 0.78 mg at 5 mm body length). These regional differences may have resulted from greater diatom availability in the north and competition from D. polymorpha in the south. Triacylglycerols and phospholipids were the dominant lipid classes in all three regions. Although the mean proportion of triacylglycerols, the energy‐storage lipid, was lower in the south than in the north, regional differences in proportions of lipid classes were not significant.
• 4 Mean lipid content and weight per unit length of Diporeia in the south were lower than values found in the late 1980s prior to the establishment of Dreissena. Mean lipid content of mature individuals is now at levels considered a minimum for successful reproduction.

     

Effects of a Longitudinal Training Program on Responses to Exercise in Overweight Men

Objective: The aim of this study was to determine how training modifies metabolic responses and lipid oxidation in overweight young male subjects. Research Methods and Procedures: Eleven overweight subjects were selected for a 4‐month endurance training program. Before and after the training period, they cycled for 60 minutes at 50% of their Vo ₂max after an overnight fast or 3 hours after eating a standardized meal. Various metabolic and endocrine parameters, and respiratory exchange ratio values were evaluated. Results: Exercise‐induced plasma norepinephrine concentration increases were similar before and after training in fasted or fed conditions. After food intake, exercise promoted a decrease in plasma glucose and a higher increase in epinephrine than in fasting conditions. The increase in epinephrine after the meal was more marked after training (264 ± 32 vs. 195 ± 35 pg/mL). Training lowered the resting plasma nonesterified fatty acids. During exercise, changes in glycerol were similar to those found before training. Lipid oxidation during exercise was higher in fasting than in fed conditions (15.5 ± 1.4 vs. 22.3 ± 1.7 g/h). Training did not significantly increase fat oxidation when exercise was performed in fed conditions, but it did in fasting conditions (18.6 ± 1.4 vs. 27.2 ± 1.8 g/h). Discussion: Endurance training decreased plasma nonesterified fatty acids, cholesterol, and insulin concentrations. Training increased lipid oxidation during exercise, in fasting conditions, and not when exercise was performed after the meal. During exercise in overweight subjects, the fasting condition seems more suited to oxidizing fat and maintaining glucose homeostasis than a 3‐hour wait after a standard meal.