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The KCNQ1 (Kv7.1) channel plays an important role in cardiovascular physiology. Cardiomyocytes co-express KCNQ1 with KCNE1-5 proteins. KCNQ1 may co-associate with multiple KCNE regulatory subunits to generate different biophysically and pharmacologically distinct channels. Increasing evidence indicates that the location and targeting of channels are important determinants of their function. In this context, the presence of K+ channels in sphingolipid-cholesterol-enriched membrane microdomains (lipid rafts) is under investigation. Lipid rafts are important for cardiovascular functioning. We aimed to determine whether KCNE subunits modify the localization and targeting of KCNQ1 channels in lipid rafts microdomains. HEK-293 cells were transiently transfected with KCNQ1 and KCNE1-5, and their traffic and presence in lipid rafts were analyzed. Only KCNQ1 and KCNE3, when expressed alone, co-localized in raft fractions. In addition, while KCNE2 and KCNE5 notably stained the cell surface, KCNQ1 and the rest of the KCNEs showed strong intracellular retention. KCNQ1 targets multiple membrane surface microdomains upon association with KCNE peptides. Thus, while KCNQ1/KCNE1 and KCNQ1/KCNE2 channels target lipid rafts, KCNQ1 associated with KCNE3-5 did not. Channel membrane dynamics, analyzed by fluorescence recovery after photobleaching (FRAP) experiments, further supported these results. In conclusion, the trafficking and targeting pattern of KCNQ1 can be influenced by its association with KCNEs. Since KCNQ1 is crucial for cardiovascular physiology, the temporal and spatial regulations that different KCNE subunits may confer to the channels could have a dramatic impact on membrane electrical activity and putative endocrine regulation. J. Cell. Physiol. 225: 692-700, 2010. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this work was to develop highly dispersible and dry formulations of insulin for use in dry powder inhalers (DPIs) using high-pressure homogenisation (HPH) and spray-drying. Several formulations were evaluated, including formulations spray-dried without excipients and formulations coated with lipids. A physiological lipid composition based on a mixture of cholesterol and phospholipids was used to form the coating film around micronised drug particles. The production technique and excipients were chosen in order to limit the degradation of the active ingredient. The resulting powders exhibited a size and shape suitable for the deep lung deposition of drugs, and good aerodynamic features were obtained for the different formulations tested, with fine particle fractions between 46% and 63% vs. 11% for raw insulin powder. The presence of a lipid coating of up to 30% (w/w) did not significantly affect the aerodynamic behaviour, and the coated formulations also exhibited a decreased residual moisture content of between 2.3% and 3.7% vs. 4.8% for raw insulin, which should improve the long-term stability of the protein formulations. No degradation of the insulin molecule occurred during the HPH/spray-drying process, as it was shown using an HPLC method (insulin content between 98.4% and 100.5%), and the content in high molecular weight proteins, assessed using a gel filtration method, stayed below 0.4%. [ABSTRACT FROM AUTHOR]

Partitioning of membrane proteins into various types of microdomains is crucial for many cellular functions. Tetraspanin-enriched microdomains (TEMs) are a unique type of protein-based microdomain, clearly distinct from membrane rafts, and important for several cellular processes such as fusion, migration and signaling. Paradoxically, HIV-1 assembly/egress occurs at TEMs, yet the viral particles also incorporate raft lipids. Using different quantitative microscopy approaches, we investigated the dynamic relationship between TEMs, membrane rafts and HIV-1 exit sites, focusing mainly on the tetraspanin CD9. Our results show that clustering of CD9 correlates with multimerization of the major viral structural component, Gag, at the plasma membrane. CD9 exhibited confined behavior and reduced lateral mobility at viral assembly sites, suggesting that Gag locally traps tetraspanins. In contrast, the raft lipid GM1 and the raft-associated protein CD55, while also recruited to assembly/budding sites, were only transiently trapped in these membrane areas. CD9 recruitment and confinement were found to be partially dependent on cholesterol, while those of CD55 were completely dependent on cholesterol. Importantly, our findings support the emerging concept that cellular and viral components, instead of clustering at preexisting microdomain platforms, direct the formation of distinct domains for the execution of specific functions. [ABSTRACT FROM AUTHOR]

The relative location of proteins and lipids in particles of medicinal leech salivary gland secretion (SGS) is revealed for the first time. Their sizes and morphology are described. Using scanning electron microscopy and transmission electron microscopy, it was determined that SGS consists of particles of different sizes and form. This picture is supported by confocal laser scanning microscopy of SGS preparations treated with fluorescein isothiocyanate. After incubation with nonionic detergents (Brij 35 and Tween 20), transmission electron microscopy revealed the dissociation of fragments composing protein-lipid particles (PLP), and in this case an increase in free protein concentration determined by a modification of the Lowry method was observed. Perylene probing of lipids in SGS preparations showed that they are concentrated mainly inside PLP and are almost absent on the surface. Cholesterol was detected during SGS probing using the cholesteryl-Bodipy (hydrophobic fluorescent analog of cholesterol) on surface sections during confocal analysis of electron microphotographs of SGS. This analysis detected PLP structures in SGS resembling caveoles full of cholesterol. SGS, preliminary frozen at −70°C, transformed into a multitude of similar small particles visualized by transmission electron microscopy, whose fixed distribution resembled water crystal structure. [ABSTRACT FROM AUTHOR]

Abstract: Lipid transfer proteins (PLTP and CETP) play roles in atherogenesis by modifying the arterial intima cholesterol content via altering the concentration and function of plasma lipoproteins and influencing inflammation. In this regard, endotoxins impair the reverse cholesterol transport (RCT) system in an endotoxemic rodent model, supporting a pro-inflammatory role of HDL reported in chronic diseases where atherosclerosis is premature. High PLTP activity related to atherosclerosis in some clinical studies, but the mechanisms involved could not be ascertained. In experimental animals the relation of elevated plasma PLTP concentration with atherosclerosis was confounded by HDL-C lowering and by unfavorable effects on several inflammatory markers. Coincidently, PLTP also increases in human experimental endotoxemia and in clinical sepsis. Human population investigations seem to favor low CETP as atheroprotective; this is supported by animal models where overexpression of huCETP is atherogenic, most likely due to increased concentration of apoB-lipoprotein-cholesterol. Thus, in spite of CETP facilitating the HDL-C-mediated RCT, the reduction of apoB-LP-cholesterol concentration is the probable antiatherogenic mechanism of CETP inhibition. On the other hand, experimental huCETP expression protects mice from the harmful effects of a bacterial polysaccharide infusion and the mortality rate of severely ill patients correlates with reduction of the plasma CETP concentration. Thus, the roles played by PLTP and CETP on atherosclerosis and acute inflammation seem contradictory. Therefore, the biological roles of PLTP and CETP must be carefully monitored when investigating drugs that inhibit their activity in the prevention of atherosclerosis. [Copyright &y& Elsevier]

We report the changes in the electrical properties of the lipid−protein film of pulmonary surfactant produced by excess cholesterol. Pulmonary surfactant (PS) is a complex lipid−protein mixture that forms a molecular film at the interface of the lung’s epithelia. The defined molecular arrangement of the lipids and proteins of the surfactant film gives rise to the locally highly variable electrical surface potential of the interface, which becomes considerably altered in the presence of cholesterol. With frequency modulation Kelvin probe force microscopy (FM-KPFM) and force measurements, complemented by theoretical analysis, we showed that excess cholesterol significantly changes the electric field around a PS film because of the presence of nanometer-sized electrostatic domains and affects the electrostatic interaction of an AFM probe with a PS film. These changes in the local electrical field would greatly alter the interaction of the surfactant film with charged species and would immediately impact the manner in which inhaled (often charged) airborne nanoparticles and fibers might interact with the lung interface. [ABSTRACT FROM AUTHOR]

Biological membranes are not structurally passive solvents of amphipathic proteins and lipids. Rather, it appears their constituents have evolved intrinsic characteristics that make homogeneous distribution of components unlikely. As a case in point, the concept of lipid rafts has received considerable attention from biologists and biophysicists since the formalization of the hypothesis more than 10 years ago. Today, it is clear that sphingolipid and cholesterol can self-associate into micron-scaled phases in model membranes and that these lipids are involved in the formation of highly dynamic nanoscale heterogeneity in the plasma membrane of living cells. However, it remains unclear whether these entities are manifestations of the same principle. A powerful means by which the molecular organization of rafts can be assessed is through analysis of their functionalized condition. Raft heterogeneity can be activated to coalesce and laterally reorganize/stabilize bioactivity in cell membranes. Evaluation of this property suggests that functional raft heterogeneity arises through principles of lipid-driven phase segregation coupled to additional chemical specificities, probably involving proteins. [ABSTRACT FROM AUTHOR]

The lipid layer of the tear film is associated with preventing evaporative loss. The ability of human Meibomian lipids to reduce evaporation in vitro was tested. Films of human or animal Meibomian lipids or mixtures of cholesterol and phosphatidylcholine were spread on the surface of either artificial buffer or on whole tears and placed on a mass balance that was enclosed in a sealed chamber. The temperature was adjusted to 37°C and gas flow was controlled. Increasing the amounts of Meibomian lipids gave a very small reduction in evaporation. It was concluded from these in vitro experiments that prevention of evaporation from the tear film is not due to the Meibomian lipids alone, but is more likely to be due to a complex interaction between components of the aqueous and the Meibomian lipids. [ABSTRACT FROM AUTHOR]

Recent experimental developments have led to a revision of the classical fluid mosaic model proposed by Singer and Nicholson more than 35 years ago. In particular, it is now well established that lipids and proteins diffuse heterogeneously in cell plasma membranes. Their complex motion patterns reflect the dynamic structure and composition of the membrane itself, as well as the presence of the underlying cytoskeleton scaffold and that of the extracellular matrix. How the structural organization of plasma membranes influences the diffusion of individual proteins remains a challenging, yet central, question for cell signaling and its regulation. Here we have developed a raft-associated glycosyl-phosphatidyl-inositol-anchored avidin test probe (Av-GPI), whose diffusion patterns indirectly report on the structure and dynamics of putative raft microdomains in the membrane of HeLa cells. Labeling with quantum dots (qdots) allowed high-resolution and long-term tracking of individual Av-GPI and the classification of their various diffusive behaviors. Using dual-color total internal reflection fluorescence (TIRF) microscopy, we studied the correlation between the diffusion of individual Av-GPI and the location of glycosphingolipid GM1-rich microdomains and caveolae. We show that Av-GPI exhibit a fast and a slow diffusion regime in different membrane regions, and that slowing down of their diffusion is correlated with entry in GM1-rich microdomains located in close proximity to, but distinct, from caveolae. We further show that Av-GPI dynamically partition in and out of these microdomains in a cholesterol-dependent manner. Our results provide direct evidence that cholesterol-/sphingolipid-rich microdomains can compartmentalize the diffusion of GPI-anchored proteins in living cells and that the dynamic partitioning raft model appropriately describes the diffusive behavior of some raft-associated proteins across the plasma membrane. [ABSTRACT FROM AUTHOR]

Abstract: Hyperlipidemia is one of the most important risk factors for atherosclerosis. This can be amplified by a localized inflammatory response mediated by macrophages. Macrophages are capable of taking up excess cholesterol, and it is well known that delivery of cholesterol to the mitochondria by steroidogenic acute regulatory (StAR) protein is the rate-limiting step for cholesterol degradation in the liver. It has also been shown that overexpression of StAR in hepatocytes dramatically increases the amount of regulatory oxysterols in the nucleus, which play an important role in the maintenance of intracellular lipid homeostasis. The goal of the present study was to determine whether StAR plays a similar role in macrophages. We have found that overexpression of StAR in human THP-1 monocyte-derived macrophages decreases intracellular lipid levels, activates liver X receptor alpha (LXRα) and proliferation peroxysome activator receptor gamma (PPARγ), and increases ABCG1 and CYP27A1 expression. Furthermore, it reduces the secretion of inflammatory factors, and prevents apoptosis. These results suggest that StAR delivers cholesterol to mitochondria where regulatory oxysterols are generated. Regulatory oxysterols can in turn activate nuclear receptors, which increase expression of cholesterol efflux transporters, and decrease secretion of inflammatory factors. These effects can prevent macrophage apoptosis. These results imply a potential role of StAR in the prevention of atherosclerosis. [Copyright &y& Elsevier]

Ca2+-triggered membrane fusion is the defining step of exocytosis. Despite realization that the fusion machinery must include lipids and proteins working in concert, only of late has work in the field focused more equally on both these components. Here we use isolated sea urchin egg cortical vesicles (CV), a stage-specific preparation of Ca2+-sensitive release-ready vesicles that enables the tight coupling of molecular and functional analyses necessary to dissect molecular mechanisms. The stalk-pore hypothesis proposes that bilayer merger proceeds rapidly via transient, high-negative curvature, intermediate membrane structures. Consistent with this, cholesterol, a major component of the CV membrane, contributes to a critical local negative curvature that supports formation of lipidic fusion intermediates. Following cholesterol depletion, structurally dissimilar lipids having intrinsic negative curvature greater than or equal to cholesterol recover the ability of CV to fuse but do not recover fusion efficiency (Ca2+ sensitivity and kinetics). Conversely, cholesterol- and sphingomyelin-enriched microdomains regulate the efficiency of the fusion mechanism, presumably by contributing spatial and functional organization of other critical lipids and proteins at the fusion site. Critical proteins are thought to participate in Ca2+ sensing, initiating membrane deformations, and facilitating fusion pore expansion. Capitalizing on a novel effect of the thiol-reactive reagent iodoacetamide (IA), potentiation of the Ca2+ sensitivity and kinetics, a fluorescently tagged IA has been used to enhance fusion efficiency and simultaneously label the proteins involved. Isolation of cholesterol-enriched CV membrane fractions, using density gradient centrifugation, is being used to narrow the list of protein candidates potentially critical to the mechanism of fast Ca2+-triggered membrane fusion. [ABSTRACT FROM AUTHOR]

The traditional Chinese formula Danning tablets exhibit wide clinical applications in liver and gallbladder diseases, and currently it is reported to be effective on fatty liver disease in clinical trials. However, the underlying mechanisms remain elusive. The purpose of the present study was to assess the effects and potential pharmacological mechanisms of Danning tablet against high fat diet (HFD)-induced obesity, fatty liver, and related metabolic disorders in mice. C57BL/6 J male mice were treated with HFD for 12 weeks to trigger obesity and fatty liver condition. Then those mice were randomly divided into 5 groups, namely HFD, Danning tablet (0.75, 1.5 or 3 g/kg bodyweight) or lovastatin (30 mg/kg bodyweight) for extra 6 weeks' treatment of HFD. Food intake and bodyweight were recorded each week. In the last week, before the mice were sacrificed, fasting blood glucose levels and insulin levels were measured. Furthermore, insulin and glucose tolerance tests were performed. Blood and hepatic lipid levels were examined, the lipid metabolism-associated gene expressions and protein levels in the liver or adipose tissues were assayed after sacrificing all mice. Our results demonstrated that a high dose of Danning tablet (3 g/kg) treatment mitigated body weight gain, reduced blood and hepatic cholesterol and triglyceride levels. The morphology analysis showed that Danning tablets could reduce lipid accumulation in both liver and brown adipose tissue. Moreover, Danning tablets could improve fasting blood glucose levels and ameliorate glucose and insulin tolerance in HFD-induced obese mice. Furthermore, qRT-PCR analysis revealed that the mRNA expressions of SREBP-1 and SREBP-2 as well as their target genes were remarkedly down-regulated in the liver and adipose tissue of diet-induced obesity (DIO) mice after treating those mice with Danning tablets. Our results indicated that Danning tablets could improve the obesity-induced metabolic associated fatty liver disease (MAFLD) and related metabolic disorders. The potential mechanism may probably involve the regulation of the SREBP pathway. [Display omitted] [ABSTRACT FROM AUTHOR]

Cholesterol, certain lipids, membrane-bound and soluble proteins, as well as viruses that are synthesized in the endoplasmic reticulum (ER), reach the plasma membrane (PM) via non-classical pathway(s) that remain poorly understood. Typical for this transport is (i) its insensitivity to brefeldin A (BFA), which dissociates selected coat complexes from membranes, resulting in the disassembly of the Golgi apparatus; (ii) its rapid kinetics as compared to the classical secretory pathway; and (iii) its role in the trafficking of lipid raft components. Based on results showing that the intermediate compartment (IC) at the ER-Golgi boundary constitutes a stable tubular network that maintains its dynamics in the presence of BFA, we propose that two bidirectional Golgi-bypass pathways to the PM exist, a direct route from early IC elements, and another, reminescent of the yeast secretory pathway, from late IC elements via the endosomal system. These pathways have implications for the organization of the secretory processes in different cell types. (Part of a Multi-author Review) [ABSTRACT FROM AUTHOR]

The article reports on the study regarding glutathione S-transferase null and cholesteryl ester transfer protein TaqI B Polymorphisms and lipid response to atorvastatin in Greek dyslipidaemic patients. A genetic interaction was detected, between polymorphisms affecting reverse cholesterol transport and the antioxidant capacity of the organism, in determining lipid response to a statin. It adds that atorvastatin treatment is of increased benefit to individuals of relatively impaired antioxidative capacity

Abstract: The aim of this review is to give a general contemporary overview of the physiologic effects of phytosterols and their role in cholesterol uptake in the intestinal tract. The mechanism of phytosterols action is based on its ability to reduce cholesterol absorption. Doses of 0.8 to 4.0 g/d of phytosterols were used to reduce low-density lipoprotein cholesterol concentrations by 10% to 15%, although most of the studies described used 2 g/d of phytosterol to achieve a reduction of 10% in low-density lipoprotein cholesterol concentrations. Although some studies point to the possibility that elevated plasma phytosterol concentrations could contribute to the development of premature coronary artery diseases, extensive safety evaluation studies have been conducted for these compounds, and they have been considered safe. [Copyright &y& Elsevier]

Membrane heterogeneity plays a significant role in regulating signal transduction and other cellular activities. We examined the protein and lipid components associated with the detergent-resistant membrane (DRM) fractions from retinal rod outer segment (ROS) disk and plasma membrane-enriched preparations. Proteomics and correlative western blot analysis revealed the presence of α and β subunits of the rod cGMP-gated ion channel and glucose transporter type 1, among other proteins. The glucose transporter was present exclusively in ROS plasma membrane (not disks) and was highly enriched in DRMs, as was the cGMP-gated channel β-subunit. In contrast, the majority of rod opsin and ATP-binding cassette transporter A4 was localized to detergent-soluble domains in disks. As expected, the cholesterol : fatty acid mole ratio was higher in DRMs than in the corresponding parent membranes (disk and plasma membranes, respectively) and was also higher in disks compared to plasma membranes. Furthermore, the ratio of saturated : polyunsaturated fatty acids was also higher in DRMs compared to their respective parent membranes (disk and plasma membranes). These results confirm that DRMs prepared from both disks and plasma membranes are enriched in cholesterol and in saturated fatty acids compared to their parent membranes. The dominant fatty acids in DRMs were 16 : 0 and 18 : 0; 22 : 6n3 and 18 : 1 levels were threefold higher and twofold lower, respectively, in disk-derived DRMs compared to plasma membrane-derived DRMs. We estimate, based on fatty acid recovery that DRMs account for only ∼ 8% of disks and ∼ 12% of ROS plasma membrane. [ABSTRACT FROM AUTHOR]

Enveloped viruses infect host cells by fusion of viral and target membranes. This fusion event is triggered by specific glycoproteins in the viral envelope. Fusion glycoproteins belong to either class I, class II or the newly described third class, depending upon their arrangement at the surface of the virion, their tri-dimensional structure and the location within the protein of a short stretch of hydrophobic amino acids called the fusion peptide, which is able to induce the initial lipid destabilization at the onset of fusion. Viral fusion occurs either with the plasma membrane for pH-independent viruses, or with the endosomal membranes for pH-dependent viruses. Although, viral fusion proteins are parted in three classes and the subcellular localization of fusion might vary, these proteins have to act, in common, on lipid assemblies. Lipids contribute to fusion through their physical, mechanical and/or chemical properties. Lipids can thus play a role as chemically defined entities, or through their preferential partitioning into membrane microdomains called “rafts”, or by modulating the curvature of the membranes involved in the fusion process. The purpose of this review is to make a state of the art on recent findings on the contribution of cholesterol, sphingolipids and glycolipids in cell entry and membrane fusion of a number of viral families, whose members bear either class I or class II fusion proteins, or fusion proteins of the recently discovered third class. [ABSTRACT FROM AUTHOR]

Objective: To investigate the role of serum cholesterol ester transfer protein (CETP) and the metabolism of various lipids including apoproteins in patients with type 2 diabetes. Materials and Methods: The relationships between serum concentrations of CETP and various lipids and apoproteins were investigated in 193 patients with type 2 diabetes mellitus and 68 age-matched healthy subjects. Serum CETP concentrations were measured by an enzyme-linked immunosorbent assay. Results: Serum CETP values were lower in diabetic patients than in healthy controls (p < 0.01). Female diabetic patients had significantly higher CETP concentrations than male patients. Serum CETP concentrations exhibited a significant positive correlation with serum concentrations of cholesterol (TC) and β-lipoproteins in diabetic patients (r = 0.485, p = 0.013). Patients with relatively high serum concentrations of high-density lipoprotein cholesterol (HDL-C) tended to have much lower CETP concentrations than patients with lower HDL-C concentrations. Serum CETP concentrations showed significant positive correlations with those of apoproteins B (Apo B; r = 0.384, p = 0.024) and E (Apo E; r = 0.341, p = 0.035). Conclusion: The data indicate that serum CETP is closely involved in the metabolism of TC, β-lipoprotein, Apo B and Apo E in type 2 diabetic patients. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Bilayer mixtures of lipids are used by many researchers as chemically simple models for biological membranes. In particular, observations on three-component bilayer mixtures containing cholesterol show rich phase behavior, including several regions of two-phase coexistence and one region of three-phase coexistence. Yet, the relationship between these simple model mixtures and biological membranes, which contain hundreds of different proteins and lipids, is not clear. Many of the model mixtures have been chosen for study because they exhibit readily observed phase separations, not because they are good mimics of cell membrane components. If the many components of cell membranes could be grouped in some way, then understanding the phase behaviors of biological membranes might be enhanced. Furthermore, if the underlying interaction energies between lipids and proteins can be determined, then it might be possible to model the distributions of lipids and proteins in a bilayer membrane, even in complex mixtures. [ABSTRACT FROM AUTHOR]

Background: Recent studies suggest that the distribution of lipoprotein subfractions is an independent predictor of vascular events. Therefore, we evaluated the effect of ezetimibe (a selective cholesterol transport inhibitor) on the concentrations of lipoprotein subfractions in patients with primary dyslipidaemia.Materials and methods: Patients (n = 50) with primary dyslipidaemias were recruited. The concentrations of the individual lipoprotein subfractions were measured using the Lipoprint system at baseline and after 16 weeks of treatment.Results: Ezetimibe reduced total, low-density lipoprotein cholesterol (LDL-C) and non-high-density lipoprotein cholesterol (HDL-C) values as well as apolipoprotein B concentrations. Subfractionation of apolipoprotein B-containing lipoproteins showed that the reduction in LDL-C values was due to a fall in the concentrations of all LDL subfractions. However, a more pronounced trend towards a decrease in the concentrations of dense LDL subfractions was observed. Patients with triglyceride values >1.7 mmol/L had significantly greater reductions in the concentrations of small, dense LDL particles compared with those with normal triglyceride levels (49 vs. 19%, respectively; p < 0.05). Ezetimibe decreased the concentrations of HDL-C mainly due to a fall in the concentration of dense HDL subfractions.Conclusion: Ezetimibe can favourably affect the distribution of LDL subfractions, especially in patients with elevated triglyceride values. Further studies are needed to clarify the significance of the ezetimibe-induced reduction in the concentrations of dense HDL particles. [ABSTRACT FROM AUTHOR]

Diverse experimental and theoretical evidence suggests that plasma membranes contain cholesterol-induced segregated domains that could play a key role in the modulation of membrane functions, including intrinsic enzyme activity. To gain insight into the role of cholesterol, we reconstituted pig kidney Na+/K+-ATPase into unilamellar vesicles of endogenous lipids mimicking the natural membrane and addressed the question of how modification of the cholesterol content could affect the ATPase activity via changes in the membrane lipid phase and in the protein structure and dynamics. We used steady-state and time-resolved fluorescence spectroscopy with the lipid phase probes DPH and Laurdan and the protein probe fluorescein and also used infrared spectroscopy using attenuated total reflectance. Upon modification of membrane cholesterol content, the ATPase activity did not change monotonically but instead exhibited abrupt changes resulting in two peaks at or close to critical cholesterol mole fractions (25 and 33.3 tool %) predicted by the superlattice or regular distribution model. Fluorescence parameters associated with the membrane probes also showed abrupt changes with peaks, coincident with the cholesterol concentrations associated with the peaks in the enzyme activity, while parameters associated with the protein probes also showed slight but abrupt changes resulting in dips at the same cholesterol concentrations. Notably, the IR amide I band maximum also showed spectral shifts, characterized by a frequency variation pattern with peaks at the same cholesterol concentrations. Overall, these results indicate that the lipid phase had slightly lower hydration, at or near the two critical cholesterol concentrations predicted by the superlattice theory. However, in the protein domains monitored there was a slight but significant hydration increase along with increased peptide backbone flexibility at these cholesterol concentrations. We propose that in the vicinity of the critical mole fractions, where superlattice formation can occur, minute changes in cholesterol concentration produce abrupt changes in the membrane organization, increasing interdomain surfaces. These changes, in turn, induce small changes in the protein's structure and dynamics, therefore acting to fine-tune the enzyme. [ABSTRACT FROM AUTHOR]

Abstract: Although the high presence of cholesterol in nerve terminals is well documented, specific roles of this lipid in transmitter release have remained elusive. Since cholesterol is a highly enriched component in the membrane microdomains known as lipid rafts, it is probable that these domains are very important in synaptic function. The extraction of lipid rafts using Brij 98 at 37°C avoids the formation of nonspecific membrane aggregates at low temperature, allowing the isolation of more physiologically relevant lipid rafts. In the present work, we examine, by means of buoyancy analysis in sucrose gradients after solubilization of the membranes with Brij 98 or with Lubrol WX, the presence of proteins involved in exocytosis in detergent-resistant membranes (DRM) using rat brain synaptosomes as a neurological model. Significant proportions of the proteins tested in the present work, which are involved in neurotransmitter release, are found in Brij 98 raft fractions, demonstrating that significant pools of synaptic proteins are segregated in specific parts of the membrane at physiological temperature. On the other hand, Lubrol WX is unable to solubilize the major fraction of the proteins tested. Treatment of synaptosomes with methyl-β-cyclodextrin (mβCD) causes alteration in the buoyancy properties of proteins initially present in Brij- as well as in Lubrol-resistant membranes, indicating the cholesterol-dependency of both kinds of microdomains. Finally, we detect the depolarization-induced enhancement of the cholesterol-dependent association of syntaxin 1 with Brij 98-rafts, under the same conditions in which prolonged neurotransmitter release is stimulated. [Copyright &y& Elsevier]

The effects of protein and fat on glycemic responses have not been studied systematically. Therefore, our aim was to determine the dose-response effects of protein and fat on the glycemic response elicited by 50 g glucose in humans and whether subjects' fasting plasma insulin (FPI) and diet influenced the results. Nondiabetic humans, 10 with FPI < [corrected] or =40 pmol/L and 10 with FPI >40 pmol/L, were studied on 18 occasions after 10 14-h overnight fasts. Subjects consumed 50 g glucose dissolved in 250 mL water plus 0, 5, 10, or 30 g fat and/or 0, 5, 10, or 30 g protein. Each level of fat was tested with each level of protein. Dietary intake was measured using a 3-d food record. Gram per gram, protein reduced glucose responses approximately 2 times more than fat (P < 0.001) with no significant fat x protein interaction (P = 0.051). The effect of protein on glycemic responses was related to waist circumference (WC) (r = -0.56, P = 0.011) and intake of dietary fiber (r = -0.60, P = 0.005) but was unrelated to FPI or other nutrient intakes. The effect of fat on glycemic responses was related to FPI (r = 0.49, P = 0.029) but was unrelated to WC or diet. We conclude that, across the range of 0-30 g, protein and fat reduced glycemic responses independently from each other in a linear, dose-dependent fashion, with protein having approximately 3-times the effect of fat. A large protein effect was associated with high WC and high dietary-fiber intake, whereas a large fat effect was associated with low FPI. These conclusions may not apply to solid meals. Further studies are needed to determine the mechanisms for these effects. [ABSTRACT FROM AUTHOR]

The accessory α2δ subunits of voltage-gated calcium channels are highly glycosylated transmembrane proteins that interact with calcium channel α1 subunits to enhance calcium currents. We compared the membrane localization and processing of native cerebellar α2δ-2 subunits with α2δ-2 stably expressed in tsA-201 cells. We identified that α2δ-2 is completely concentrated in cholesterol-rich microdomains (lipid rafts) in cerebellum, in which it substantially colocalizes with the calcium channel α1 subunit CaV2.1, although CaV2.1 is also present in the Triton X-100-soluble fraction. In tsA-201 cells, unlike cerebellum, α2δ-2 is not completely proteolytically processed into α2δ-2 and α-2. However, this processing is more complete in the lipid raft fraction of tsA-201 cells, in which α2δ-2 also colocalizes with CaV2.1. Cholesterol depletion of intact cells disrupted their lipid rafts and enhanced CaV2.1/α2δ-2/β4 currents. Furthermore, α2δ-2 coimmunoprecipitates with lipid raft-associated proteins of the stomatin family. The apparent affinity of α2δ-2 for its ligand gabapentin is increased markedly in the cholesterol-rich microdomain fractions, in both cerebellum and the stable α2δ-2 cell line. In contrast, α2δ-2 containing a point mutation (R282A) has a much lower affinity for gabapentin, and this is not enhanced in the lipid raft fraction. This R282A mutant α2δ-2 shows reduced functionality in terms of enhancement of CaV2.1/β4 calcium currents, suggesting that the integrity of the gabapentin binding site may be important for normal functioning of α2δ-2. Together, these results indicate that both α2δ-2 and CaV2.1 are normally associated with cholesterol-rich microdomains, and this influences their functionality. [ABSTRACT FROM AUTHOR]

Summary With a prevalence of 10-15% in adults in Europe and the USA, gallstones are the most common digestive disease needing admission to hospital in the West. The interplay between interprandial and postprandial physiological responses to endogenous and dietary lipids underscores the importance of coordinated hepatobiliary and gastrointestinal functions to prevent crystallisation and precipitation of excess biliary cholesterol. Indeed, identifying the metabolic and transcriptional pathways that drive the regulation of biliary lipid secretion has been a major achievement in the field. We highlight scientific advances in protein and gene regulation of cholesterol absorption, synthesis, and catabolism, and biliary lipid secretion with respect to the pathogenesis of cholesterol gallstone disease. We discuss the physical-chemical mechanisms of gallstone formation in bile and the active role of the gallbladder and the intestine. We also discuss gaps in our knowledge of the pathogenesis of gallstone formation and the potential for gene targeting in therapy. [ABSTRACT FROM AUTHOR]

Abstract: Maintaining a functional pulmonary surfactant system at depth is critical for diving mammals to ensure that inspiration is possible upon re-emergence. The lipid and protein composition of lavage extracts from three pinniped species (California sea lion, Northern elephant seal and Ringed seal) were compared to several terrestrial species. Lavage samples were purified using a NaBr discontinuous gradient. Concentrations of phospholipid classes and molecular species were measured using electrospray ionisation mass spectrometry, cholesterol was measured using high-performance liquid chromatography, surfactant protein A (SP-A) and SP-B were measured using enzyme-linked immunosorbent assays. There were small differences in phospholipid classes, with a lower level of anionic surfactant phospholipids, PG and PI, between diving and terrestrial mammals. There were no differences in PL saturation or SP-A levels between species. PC16:0/14:0, PC16:0/16:1, PC16:0/16:0, long chain PI species and the total concentrations of alkyl–acyl species of PC and PG as a ratio of diacyl species were increased in diving mammals, whereas concentrations of PC16:0/18:1, PG16:0/16:0 and PG16:0/18:1 were decreased. Cholesterol levels were very variable between species and SP-B was very low in diving mammals. These differences may explain the very poor surface activity of pinniped surfactant that we have previously described [Miller, N.J., Daniels, C.B., Schürch, S., Schoel, W.M., Orgeig, S., 2005. The surface activity of pulmonary surfactant from diving mammals. Respir. Physiol. Neurobiol. 150 (2006) 220–232], supporting the hypothesis that pinniped surfactant has primarily an anti-adhesive function to meet the challenges of regularly collapsing lungs. [Copyright &y& Elsevier]

The lipid requirements of the Torpedo californica nicotinic acetylcholine receptor (nAChR) were assessed by reconstituting purified receptors into lipid vesicles of defined composition and by using photolabeling with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) to determine functionality. Earlier studies demonstrated that nAChRs reconstituted into membranes containing phosphatidylcholine (PC), the anionic lipid phosphatidic acid (PA), and cholesterol (CH) are particularly effective at stabilizing the nAChR in the resting (closed) state that is capable of undergoing agonist-induced conformational transitions (i.e., functionality). The present studies demonstrate that (1) there is no obligatory requirement for PC, (2) increasing the CH content serves to increase the degree to which nAChRs are stabilized in the resting state, and this effect saturates at ∼35 mol % (molar lipid percentage), and (3) the effect of increasing levels of PA saturates at ∼12 mol % and in the absence of PA nAChRs are stabilized in the desensitized state (i.e., nonfunctional). Native Torpedo membranes contain ∼35 mol % CH but less than 1 mol % PA, suggesting that other anionic lipids may substitute for PA. We report that (1) phosphatidylserine (PS) and phosphatidylinositol (PI), anionic lipids that are abundant in native Torpedo membranes, also stabilize the receptor in the resting state although with reduced efficacy (∼50-60%) compared to PA, and (2) for nAChRs reconstituted into PA/CH membranes at different lipid-protein molar ratios, receptor functionality decreases rapidly below ∼65 lipids per receptor. Collectively, these results are consistent with a functional requirement of a single shell of lipids surrounding the nAChR and specific anionic lipid- and sterol (CH)-protein interactions. [ABSTRACT FROM AUTHOR]

Alzheimer's disease is the principal cause of dementia throughout the world and the fourth cause of death in developed economies. This brain disorder is characterized by the formation of brain protein aggregates, namely, the paired helical filaments and senile plaques. Oxidative stress during life, neuroinflammation, and alterations in neuron-glia interaction patterns have been also involved in the etiopathogenesis of this disease. In recent years, cumulative evidence has been gained on the involvement of alteration in neuronal lipoproteins activity, as well as on the role of cholesterol and other lipids in the pathogenesis of this neurodegenerative disorder. In this review, we analyze the links between changes in cholesterol homeostasis, and the changes of lipids of major importance for neuronal activity and Alzheimer's disease. The investigation on the fine molecular mechanisms underlying the lipids influence in the etiopathogenesis of Alzheimer's disease may shed light into its treatment and medical management. [ABSTRACT FROM AUTHOR]

Abstract: The precise mechanism by which galectin-3 and other cytosolic proteins that lack signal peptides are secreted is yet to be elucidated. In the present analyses, we determined that galectin-3, a β-galactoside binding protein, can interact directly with membrane lipids in solid phase binding assays. More interestingly, we determined by spectrophotometric methods that it can spontaneously penetrate the lipid bilayer of liposomes in either direction. These findings suggest that galectin-3 on its own has the capacity to traverse the lipid bilayer. Whereas the situation is rather simplified in liposomes, the interaction of galectin-3 with the plasma membrane may involve cholesterol-rich membrane domains where galectin-3 can be concentrated and form multimers or interact covalently with other proteins. [Copyright &y& Elsevier]

Pulmonary surfactant, a complex mixture of lipids and proteins, lowers the surface tension in terminal air spaces and is crucial for lung function. Within an animal species, surfactant composition can be influenced by development, disease, respiratory rate, and/or body temperature. Here, we analyzed the composition of surfactant in three heterothermic mammals (dunnart, bat, squirrel), displaying different torpor patterns, to determine: 1) whether increases in surfactant cholesterol (Chol) and phospholipid (PL) saturation occur during long-term torpor in squirrels, as in bats and dunnarts; 2) whether surfactant proteins change during torpor; and 3) whether PL molecular species (molsp) composition is altered. In addition, we analyzed the molsp composition of a further nine mammals (including placental/marsupial and hetero-/homeothermic contrasts) to determine whether phylogeny or thermal behavior determines molsp composition in mammals. We discovered that like bats and dunnarts, surfactant Chol increases during torpor in squirrels. However, changes in PL saturation during torpor may not be universal. Torpor was accompanied by a decrease in surfactant protein A in dunnarts and squirrels, but not in bats, whereas surfactant protein B did not change in any species. Phosphatidylcholine (PC)16:0/16:0 is highly variable between mammals and is not the major PL in the wombat, dunnart, shrew, or Tasmanian devil. An inverse relationship exists between PC16:0/16:0 and two of the major fluidizing components, PC16:0/16:1 and PC16:0114:0. The PL molsp profile of an animal species is not determined by phylogeny or thermal behavior. We conclude that there is no single PL molsp composition that functions optimally in all mammals; rather, surfactant from each animal is unique and tailored to the biology of that animal. [ABSTRACT FROM AUTHOR]

While our understanding of lipid microdomains has advanced in recent years, many aspects of their formation and dynamics are still unclear. In particular, the molecular determinants that facilitate the partitioning of integral membrane proteins into lipid raft domains are yet to be clarified. This review focuses on a family of raft-associated integral membrane proteins, termed flotillins, which belongs to a larger class of integral membrane proteins that carry an evolutionarily conserved domain called the prohibitin homology (PHB) domain. A number of studies now suggest that eucaryotic proteins carrying this domain have affinity for lipid raft domains. The PHB domain is carried by a diverse array of proteins including stomatin, podocin, the archetypal PHB protein, prohibitin, lower eucaryotic proteins such as the Dictyostelium discoideum proteins vacuolin A and vacuolin B and the Caenorhabditis elegans proteins unc-1, unc-24 and mec-2. The presence of this domain in some procaryotic proteins suggests that the PHB domain may constitute a primordial lipid recognition motif. Recent work has provided new insights into the trafficking and targeting of flotillin and other PHB domain proteins. While the function of this large family of proteins remains unclear, studies of the C. elegans PHB proteins suggest possible links to a class of volatile anaesthetics raising the possibility that these lipophilic agents could influence lipid raft domains. This review will discuss recent insights into the cell biology of flotillins and the large family of evolutionarily conserved PHB domain proteins. [ABSTRACT FROM AUTHOR]

End-product feedback inhibition of cholesterol synthesis was first demonstrated in living animals by Schoenheimer 72 years ago. Current studies define Insig proteins as essential elements of this feedback system in mouse liver. In cultured cells, Insig proteins are required for sterol-mediated inhibition of the processing of sterol regulatory element-binding proteins (SREBPs) to their nuclear forms. We produced mice with germline disruption of the Insig2 gene and Cre-mediated disruption of the Insig1 gene in liver. On a chow diet, these double-knockout mice overaccumulated cholesterol and triglycerides in liver. Despite this accumulation, levels of nuclear SREBPs and mRNAs for SREBP target genes in lipogenic pathways were not reduced. Whereas cholesterol feeding reduced nuclear SREBPs and lipogenic mRNAs in wild-type mice, this feedback response was severely blunted in the double-knockout mice, and synthesis of cholesterol and fatty acids was not repressed. The amount of HMG-CoA reductase protein was elevated out of proportion to the mRNA in the double-knockout mice, apparently owing to the failure of cholesterol to accelerate degradation of the enzyme. These studies indicate that the essential elements of the regulatory pathway for lipid synthesis function in liver as they do in cultured cells. [ABSTRACT FROM AUTHOR]

The steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain is a protein modeul of ∼210 residues that binds lipids, including sterols. Fifteen mammalian proteins, STARD1-STARD15, possess a START domain and these can be grouped into six subfamilies. Cholesterol, 25-hydroxycholesterol, phosphatidylcholine, phosphatidylethanolamine and ceramides are ligands for STARD1/STARD3/STARD5, STARD5, STARD2/STARD10, STARD10 and STARD11, respectively. The lipids or sterols bound by the remaining 9 START proteins are unknown. Recent studies show that the C-terminal end of the domain plays a fundamental role, forming a lid over a deep ligand-binding pocket that shields the ligand from the external environment. The START domain can be regarded as a lipid-exchange and/or a lipid-sending domain. Mammalian START proteins have diverse expression patterns and can be found free in the cytoplasm, attached to membranes or in the nucleus. They appear to function in a variety of distinct physiological processes, such as lipid transfer between intracellular compartments, lipid metabolism and modulation of signaling events. Mutation or misexpression of START proteins is linked to pathological processes, including genetic disorders, autoimmune disease and cancer. [ABSTRACT FROM AUTHOR]

Lipids comprise the bulk of the dry mass of the brain. In addition to providing structural integrity to membranes, insulation to cells and acting as a source of energy, lipids can be rapidly converted to mediators of inflammation or to signaling molecules that control molecular and cellular events in the brain. The advent of soft ionization procedures such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) have made it possible for compositional studies of the diverse lipid structures that are present in brain. These include phospholipids, ceramides, sphingomyelin, cerebrosides, cholesterol and their oxidized derivatives. Lipid analyses have delineated metabolic defects in disease conditions including mental retardation, Parkinson's Disease (PD), schizophrenia, Alzheimer's Disease (AD), depression, brain development, and ischemic stroke. In this review, we examine the structure of the major lipid classes in the brain, describe methods used for their characterization, and evaluate their role in neurological diseases. The potential utility of characterizing lipid markers in the brain, with specific emphasis on disease mechanisms, will be discussed. Additionally, we describe several proteomic strategies for characterizing lipid-metabolizing proteins in human cerebrospinal fluid (CSF). These proteins may be potential therapeutic targets since they transport lipids required for neuronal growth or convert lipids into molecules that control brain physiology. Combining lipidomics and proteomics will enhance existing knowledge of disease pathology and increase the likelihood of discovering specific markers and biochemical mechanisms of brain diseases. [ABSTRACT FROM AUTHOR]

Abstract: The mammalian glycolipid transfer protein, GLTP, catalyzes the transfer in vitro of glycolipids between membranes. In this study we have examined on one hand the effect of the variations in the donor vesicle composition and on the other hand the effects of variations in the acceptor vesicle composition on the GLTP-catalyzed transfer kinetics of galactosylceramide between bilayer vesicles. For this purpose a resonance energy transfer assay was used, the energy donor being anthrylvinyl-galactosylceramide and the energy acceptor DiO-C16. First, we show that the transfer of anthrylvinyl-galactosylceramide from palmitoyl-oleoyl-phosphatidylcholine donor vesicles was faster than from dipalmitoyl-phosphatidylcholine vesicles, and that there is no transfer from palmitoyl-sphingomyelin vesicles regardless of the cholesterol amount. In this setup the acceptor vesicles were always 100% palmitoyl-oleoyl-phosphatidylcholine. We also showed that the transfer in general is faster from small highly curved vesicles compared to that from larger vesicles. Secondly, by varying the acceptor vesicle composition we showed that the transfer is faster to mixtures of sphingomyelin and cholesterol compared to mixtures of phosphatidylcholines and cholesterol. Based on these experiments we conclude that the GLTP mediated transfer of anthrylvinyl-galactosylceramide is sensitive to the matrix lipid composition and membrane bending. We postulate that a tightly packed membrane environment is most effective in preventing GLTP from accessing its substrates, and cholesterol is not required to protect the glycosphingolipid in the membrane from being transferred by GLTP. On the other hand GLTP can more easily transfer glycolipids to ‘lipid raft’ like membranes, suggesting that the protein could be involved in raft assembly. [Copyright &y& Elsevier]

Recent evidence suggests that individuals with high concentrations of C-reactive protein (CRP), a marker of inflammation, are less responsive to cholesterol-lowering diets. CRP concentrations are increased by oral estrogen; however, the effect of soy phytoestrogens on inflammation has not been studied comprehensively, especially in women receiving hormone replacement therapy (HRT). This study was conducted to determine whether adding soy to a low-fat, high-fiber diet affects CRP and interleukin (IL)-6, and to examine the association between CRP levels and lipid response in moderately hypercholesterolemic adults (men = 18, postmenopausal women = 14; 6 receiving HRT). After a 3-wk run-in period with consumption of a Step I diet (27% total fat, 7% saturated fat, 275 mg cholesterol), participants were randomly assigned to diets containing 25 g/d soy protein (+ 90 mg/d isoflavones) or 25 g/d milk protein for 6 wk in a crossover design. Lipids and lipoproteins, CRP, and IL-6 were measured at the end of each diet and participants were categorized into high (>3.5 mg/L) or low CRP groups based on a median split. The addition of soy or milk protein to the Step I diet did not affect lipids or inflammatory markers. Regardless of protein source, those with low CRP exhibited significant decreases in LDL cholesterol (-3.5%) and the LDL:HDL cholesterol ratio (-4.8%), whereas those with high CRP had significant increases in LDL cholesterol (+4.8%), the LDL:HDL cholesterol ratio (+5.2%), apolipoprotein B (+3.8%), and lipoprotein(a) (+ 13.5%) compared with the run-in diet. These results suggest that inflammation may not only attenuate lipid responses, but also aggravate dyslipidemia in hypercholesterolemic subjects consuming a cholesterol-lowering diet. [ABSTRACT FROM AUTHOR]

Abstract: This review deals with four lipid transfer proteins (LTP): three are involved in cholesteryl ester (CE) synthesis or transport, the fourth deals with plasma phospholipid (PL) transfer. Experimental models of atherosclerosis, clinical and epidemiological studies provided information as to the relationship of these LTP(s) to atherosclerosis, which is the main focus of this review. Thus, inhibition of acyl-CoA:cholesterol acyltransferase (ACAT) 1 and 2 decreases cholesterol absorption, plasma cholesterol and aortic cholesterol esterification in the aorta. The discovery that tamoxifen is a potent ACAT inhibitor explained the plasma cholesterol lowering of the drug. The use of ACAT inhibition in humans is under current investigation. As low cholesteryl ester transfer protein (CETP) activity is connected with high HDL-C, several CETP inhibitors were tried in rabbits, with variable results. A new CETP inhibitor, Torcetrapib, was tested in humans and there was a 50–100% increase in HDL-C. Lecithin cholesterol acyl-transferase (LCAT) influences oxidative stress, which can be lowered by transient LCAT gene transfer in LCAT-/- mice. Phospholipid transfer protein (PLTP) deficiency reduced apo B production in apo E-/- mice, as well as oxidative stress in four models of mouse atherosclerosis. In conclusion, the ability to increase HDL-C so markedly by inhibitors of CETP introduces us into a new era in prevention and treatment of coronary heart disease (CHD). [Copyright &y& Elsevier]

Steroidogenic acute regulatory (STAR) protein facilitates import of cholesterol into adrenal and gonadal mitochondria where cholesterol is converted to pregnenolone, initiating steroidogenesis. StAR acts exclusively on the outer mitochondrial membrane (OMM) by unknown mechanisms. To identify StAR domains involved in membrane association, we reacted N-62 StAR with small unilamellar vesicles (SUVs) composed of lipids resembling the OMM. Solvent-exposed domains were digested with trypsin, Asp-N, or pepsin at different pH levels, and StAR peptides protected from proteolysis were identified by mass spectrometry. At pH 4 SUVs completely protected residues 259-282; at pH 6.5 this region was partially digested into 254-272, 254-273, and 254-274. Computer-graphic modeling of N-62 StAR indicated these peptides correspond to the C-terminal α4 helix and that residues Leu275, Thr263, and Arg272 in α4 form stabilizing interactions with Gln128, Asp150, and Asp106 in adjacent loops. CD spectroscopy of a 37-mer model of α4 (residues 247-287) indicated a random coil in aqueous buffer, but in 40% methanol the peptide was α-helical and achieved maximal α-helicity at pH 5.0 in the presence of SUVs. Reacting the 37-mer with diethyl pyrocarbamate incorporated into SUVs increased the number of modified residues. Thus the C-terminal α4 helix is critically involved in the membrane association of StAR with OMM lipids. The membrane association and the α-helical structure of the C terminus in the presence of OMM lipids are also pH-dependent. These results further support StAR undergoing a pH-dependent change in its conformation when interacting with the acidic phospholipid head groups of a membrane. [ABSTRACT FROM AUTHOR]

The two-month effects of dietary fish protein and casein on VLDL, HDL2 and HDL3 compositions and hepatic lipase (HTGL) and tissue lipoprotein lipase (LPL) activities were examined in spontaneously hypertensive rats (SHR) at 4 wk of age. After 2 mo of experiment, the fish protein diet induced lower blood pressure (-14 %) as compared to casein. Liver triacylglycerol and total cholesterol concentrations were 1.37- and 1.71-fold lower in the fish protein group than in the casein group, respectively. Total cholesterol concentration in plasma was also diminished by fish protein (-21 %) and was reflected in HDL2 fraction (-44 %). SHR fed the fish protein diet as compared with those fed casein, showed a significantly low HDL3 particle number, as measured by diminished HDL3 mass and apo A-I. The consumption of fish protein did not affect VLDL particle number, but significantly decreased VLDL-triacylglycerol (-32 %) and adipose tissue total lipid concentrations as compared to casein. This was accompanied by diminished HTGL and adipose tissue LPL activities (-10%, -91%, respectively). These data demonstrate that fish protein plays an antihypertensive role and reduces plasma and tissue lipid concentrations. Thus, a fish protein intake might be beneficial for patients with hypertension. [ABSTRACT FROM AUTHOR]

The current paper demonstrates that cholesterol and its hydroxylated derivative, 25-hydroxycholesterol (25HC), inhibit cholesterol synthesis by two different mechanisms, both involving the proteins that control sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors that activate genes encoding enzymes of lipid synthesis. Using methyl-βcyclodextrin as a delivery vehicle, we show that cholesterol enters cultured Chinese hamster ovary cells and elicits a conformational change in SREBP cleavage-activating protein (SCAP), as revealed by the appearance of a new fragment in tryptic digests. This change causes SCAP to bind to Insigs, which are endoplasmic reticulum retention proteins that abrogate movement of the SCAP·SREBP complex to the Golgi apparatus where SREBPs are normally processed to their active forms. Direct binding of cholesterol to SCAP in intact cells was demonstrated by showing that a photoactivated derivative of cholesterol cross-links to the membrane domain of SCAP. The inhibitory actions of cholesterol do not require the isooctyl side chain or the Δ5-double bond of cholesterol, but they do require the 3β-hydroxyl group. 25-HC is more potent than cholesterol in eliciting SCAP binding to Insigs, but 25-HC does not cause a detectable conformational change in SCAP. Moreover, a photoactivated derivative of 25-HC does not cross-link to SCAP. These data imply that cholesterol interacts with SCAP directly by inducing it to bind to Insigs, whereas 25-HC works indirectly through a putative 25-HC sensor protein that elicits SCAP-Insig binding. [ABSTRACT FROM AUTHOR]

Little is known about the regulation of apolipoprotein (apo) C-I production by human adipocytes. The aim of the present study, therefore, was to investigate the effect of different tissue culture conditions on the synthesis and secretion of apoC-I and apoE in human SW872 liposarcoma cells. After 3-4 d in culture (0.5 x 10(6) cells/well, DMEM/F-12 medium with 10% fetal calf serum), cells reached confluence and became growth arrested. The molar ratio of apoE:apoC-I in the cell was 8.9 +/- 0.6 and in the medium was 6.6 +/- 0.5. After 17 d in culture, SW872 cells contained significantly more cholesterol (100%) and triglyceride (3-fold) and secreted more apoC-I [4 vs. 17 d: 0.11 +/- 0.01 vs. 0.23 +/- 0.01 pmol/(10(6) cells . 24 h), P < 0.001] and apoE [0.7 +/- 0.1 vs. 3.1 +/- 0.3 pmol/(10(6) cells . 24 h), P < 0.001]. Cellular apoC-I increased 7-fold and apoE increased 16-fold. Cell maturation was associated with significantly higher levels of apoE mRNA but not apoC-I mRNA. Increases in cell lipids, apoC-I, and apoE were not dependent on the presence of extracellular lipids because similar changes occurred in cells incubated with lipoprotein-deficient serum or in cells incubated without serum. Treatment (7 d) of cells during maturation with insulin (10 or 1000 nmol/L) significantly reduced the secretion of apoC-I and apoE. These results demonstrate that in maturing SW872 cells, cholesterol and triglyceride accumulation in the presence or absence of extracellular lipids, is associated with increased apoC-I and apoE production. Furthermore, apoC-I and apoE production are differentially regulated at the transcriptional level, and long-term treatment with insulin has an inhibitory rather than stimulatory effect on apoC-I and apoE production. [ABSTRACT FROM AUTHOR]

Niemann–Pick type C, or NPC for short, is an early childhood disease exhibiting progressive neurological degeneration, associated with hepatosplenomegaly in some cases. The disease, at the cellular level, is a result of improper trafficking of lipids such as cholesterol and glycosphingolipids (GSLs) to lysosome-like storage organelles (LSOs), which become engorged with these lipids. It is believed that the initial defect in trafficking, whether of cholesterol or a GSL, results in an eventual traffic jam in these LSOs. This leads to the retention of not only other lipids, but also of transmembrane proteins that transiently associate with the late endosomes (LE) in normal cells, on their way to other cellular destinations such as the trans-Golgi network (TGN). In this review, we discuss the biophysical properties of lipids and cholesterol that might determine their intracellular itineraries, and how these itineraries are altered in NPC cells, which have defects in the proteins NPC1 or NPC2. We also discuss some potential therapeutic directions being suggested by recent research. [Copyright &y& Elsevier]

The effect of spermatogenesis and oogenesis on protein, lipid, glycogen, cholesterol and energy contents, total amino acid and fatty acid profiles of Eledone cirrhosa and Eledone moschata tissues (gonad, digestive gland and muscle) was investigated. A significant (p<0.05) increase in the amino acids and protein content of the gonad throughout sexual maturation (namely in oogenesis) was observed, but the allocation of these nitrogen compounds from the digestive gland and muscle was not evident. The major essential amino acids (EAA) in the three tissues were leucine, lysine and arginine. The major nonessential amino acids (NEAA) were glutamic acid, aspartic acid and alanine. A significant increase in lipid and fatty acid contents of gonad and digestive gland was observed. There was also little evidence of accumulated lipid storage reserves being used for egg production. It seems that for egg production Eledone species use energy directly from food, rather than from stored products. Most of saturated fatty acid (SFA) content of the three tissues was presented as 16:0 and 18:0, monounsaturated fatty acid (MUFA) content as 18:1 and 20:1 and polyunsaturated fatty acid (PUFA) content as 20:4n-6, 20:5n-3 and 22:6n-3. Cholesterol and glycogen contents significantly increased in gonad and digestive gland throughout maturation while the muscle revealed no obvious pattern. If Eledone''s component sterols are of a dietary origin, a considerable variation in the cholesterol content between species might be expected on the basis of the sterol composition of their prey. Although spermatogenesis and oogenesis had a significant effect (p<0.05) in gonad and digestive gland energy content, the biochemical composition of digestive gland and muscle may not be primarily influenced by sexual maturation, but rather by other biotic factors such as feeding activity, food availability, spawning and brooding. [Copyright &y& Elsevier]

Lipid rafts are cholesterol-rich membrane microdomains that are thought to act as coordinated signaling platforms by regulating dynamic, agonist-induced translocation of signaling proteins. They have been described to play a role in multiple prototypical cascades, among them the lipopolysaccharide pathway, and to host multiple signaling proteins, including kinases and low molecular weight G-proteins. Here we report lipopolysaccharide-induced activation of the Rho family GTPase Cdc42, and we show its activation in the human neutrophil to be mediated by a p38 mitogen-activated protein kinase-dependent mechanism. Subcellular fractionation reveals that lipopolysaccharide induces translocation of Cdc42 to lipid rafts, where it and p38 are both found to be activated. By contrast, lipopolysaccharide causes translocation of Rac from the polymorphonuclear leukocyte (PMN) rafts and does not induce its activation. With the use of methyl-β-cyclodextrin, a cholesterol-depleting agent that reversibly disrupts rafts, we confirm an important regulatory role for rafts in the activation state of p38 and Cdc42 and in the Rho GTPase-dependent functions superoxide anion production and actin polymerization. Methyl-β-cyclodextrin induces activation of p38 and Cdc42, but not Rac, in the nonstimulated PMN, yet inhibits subsequent lipopolysaccharide-induced activation of p38 and Cdc42. In parallel, methyl-β-cyclodextrin primes the human PMN for subsequent superoxide release triggered by the formylated bacterial tripeptide formyl-Met-Leu-Phe, and induces actin polymerization in a subcellular distribution distinct from that induced by lipopolysaccharide. In sum, these findings provide evidence for an important regulatory role of cholesterol in both transmission of the lipopolysaccharide signal and the inflammatory phenotype of the human neutrophil. [ABSTRACT FROM AUTHOR]

Niemann-Pick type C (NPC) 1 protein plays important roles in moving cholesterol and other lipids out of late endosomes by means of vesicular trafficking, but it is not known whether NPC1 directly interacts with cholesterol. We performed photoaffinity labeling of intact cells expressing fluorescent protein (FP)-tagged NPC1 by using [³H]7,7-azocholestanol ([³H]AC). After immunoprecipitation, ³H-labeled NPC1-GFP appeared as a single band. Including excess unlabeled sterol to the labeling reaction significantly diminished the labeling. Altering the NPC1 sterol-sensing domain (SSD) with loss-of-function mutations (P692S and Y635C) severely reduced the extent of labeling. To further demonstrate the specificity of labeling, we show that NPC2, a late endosomal/lysosomal protein that binds to cholesterol with high affinity, is labeled, whereas mutant NPC2 proteins inactive in binding cholesterol are not. Vamp7, an abundant late endosomal membrane protein without an SSD but with one transmembrane domain, cannot be labeled. Binding between [³H]AC and NPC1 does not require NPC2. Treating cells with either U-18666A, a compound that creates an NPC-like phenotype, or with bafilomycin A1, a compound that raises late endosomal pH, has no effect on labeling of NPC1-YFP, suggesting that both drugs affect processes other than NPC1 binding to cholesterol. We also developed a procedure to label the NPC1-YFP by [³H]AC in vitro and showed that cholesterol is more effective in protection against labeling than its analogs epicholesterol or 5-alpha-cholestan. Overall, the results demonstrate that there is direct binding between NPC1 and azocholestanol; the binding does not require NPC2 but requires a functional SSD within NPC1. [ABSTRACT FROM AUTHOR]

The effect of decreased fatty acid oxidation on liver lipid metabolism in HcB-19 mice, a mouse model of hyperlipidemia (Txnip(-/-)), was investigated using metabolic labeling. De novo cholesterol synthesis and de novo lipogenesis were quantified using 1-(13)C(1) acetic acid, and liver triacylglycerol (TAG) derived from dietary fatty acids was quantified using dietary glyceryl tri(hexandecanoate-d(31)). Tissue samples were analyzed for TAG, free cholesterol (FC), and cholesterol ester (CE) content. Txnip(-/-) mice had significantly elevated (P < 0.05) serum nonesterified fatty acids compared with wild-type (WT) littermates; their livers weighed more and contained more TAG and total cholesterol. Txnip(-/-) liver also contained measurable CE; CE was not detectable in WT mice. Liver CE content was elevated despite lower cholesterol fractional synthesis rates (16 vs. 31%/d in Txnip(-/-) and WT mice, respectively). FC absolute synthesis rate (ASR) in WT mice (0.28 +/- 0.0 micromol/d) was similar to the combined synthesis rates of FC (0.13 +/- 0.10 micromol/d) and CE (0.10 +/- 0.00 micromol/d) in Txnip(-/-) mice. Lipogenesis, as assessed by TAG-palmitate ASR, was significantly greater in Txnip(-/-) mice (1.47 +/- 0.08 vs. 0.49 +/- 0.06 micro mol/d) and liver fatty acid synthase activity was also higher (7.96 +/- 2.53 vs. 4.83 +/- 1.44 U/mg protein). Both elevated lipogenesis and increased fatty acid reesterification to glycerol and cholesterol contributed to fat in the livers of Txnip(-/-) mice. These data support elevated fatty acid synthesis as the primary contributor to liver TAG in Txnip(-/-) mice, although increased esterification of fatty acids also contributed to excess liver TAG. The absolute total cholesterol synthesis rate was not altered, but esterification of fatty acids to cholesterol provided an additional means to buffer physiologically the negative results of excess fatty acid availability. [ABSTRACT FROM AUTHOR]

There is growing evidence that soy protein improves the blood lipid profiles of animals and humans. We compared the effects of fish protein hydrolysate (FPH), soy protein, and casein (control) on lipid metabolism in Wistar rats and genetically obese Zucker (fa/fa) rats. In Zucker rats, FPH treatment affected the fatty acid composition in liver, plasma, and triacylglycerol-rich lipoproteins. The mRNA levels of Delta 5 and Delta 6 desaturases were reduced by FPH and soy protein feeding compared with casein feeding. In Zucker rats both FPH and soy protein treatment reduced the plasma cholesterol level. Furthermore, the HDL cholesterol:total cholesterol ratio was greater in these rats and in the Wistar rats fed FPH and soy protein compared with those fed casein. Although fecal total bile acids were greater in soy protein-fed Zucker rats than in casein-fed controls, those fed FPH did not differ from the controls. However, the acyl-CoA:cholesterol acyltransferase activity was reduced in Zucker rats fed FPH and tended to be lower (P = 0.13) in those fed soy protein compared with those fed casein. Low ratios of methionine to glycine and lysine to arginine in the FPH and soy protein diets, compared with the casein diet, may be involved in lowering the plasma cholesterol concentration. Our results indicate that the effects of FPH and soy protein on fatty acid metabolism are similar in many respects, but the hypocholesterolemic effects of FPH and soy protein appear to be due to different mechanisms. FPH may have a role as a cardioprotective nutrient. [ABSTRACT FROM AUTHOR]

Identified in the steroidogenic acute regulatory protein (STAR), is involved in the intracellular trafficking of lipids. Sixteen mammalian START domain-containing proteins have been identified to date. STAR, a protein targeted to mitochondria, stimulates the movement of cholesterol from the outer to the inner mitochondrial membranes, where it is metabolized into pregnenolone in steroidogenic cells. MLN64, the START domain protein most closely related to STAR, is localized to late endosomes along with other proteins involved in sterol trafficking, including NPC1 and NPC2, where it has been postulated to participate in sterol distribution to intracellular membranes. To investigate the role of MLN64 in sterol metabolism, we created mice with a targeted mutation in the Mln64 START domain, expecting to find a phenotype similar to that in humans and mice lacking NPC1 or NPC2 (progressive neurodegen. erative symptoms, free cholesterol accumulation in lysosomes). Unexpectedly, mice homozygous for the Mln64 mutant allele were viable, neurologically intact, and fertile. No significant alterations in plasma lipid levels, liver lipid content and distribution, and expression of genes involved in sterol metabolism were observed, except for an increase in sterol ester storage in mutant mice fed a high fat diet. Embryonic fibroblast cells transfected with the cholesterol side-chain cleavage system and primary cultures of granulosa cells from Mln64 mutant mice showed defects in sterol trafficking as reflected in reduced conversion of endogenous cholesterol to steroid hormones. These observations suggest that the Mln64 START domain is largely dispensable for sterol metabolism in mice. [ABSTRACT FROM AUTHOR]

Niemann-Pick disease type C (NPC) is characterized by the accumulation of cholesterol and sphingolipids in the late endosomal/lysosomal compartment. The mechanism by which the concentration of sphingolipids such as glucosylceramide is increased in this disease is poorly understood. We have found that, in NPC fibroblasts, the cholesterol storage affects the stability of glucosylceramidase (GCase), decreasing its mass and activity; a reduction of cholesterol raises the level of GCase to nearly normal values. GCase is activated and stabilized by saposin C (Sap C) and anionic phospholipids. Here we show by immunofluorescence microscopy that in normal fibroblasts, GCase, Sap C, and lysobisphosphatidic acid (LBPA), the most abundant anionic phospholipid in the endolysosomal system, reside in the same intracellular vesicular structures. In contrast, the colocalization of GCase, Sap C, and LBPA is markedly impaired in NPC fibroblasts but can be re-established by cholesterol depletion. These data show for the first time that the level of cholesterol modulates the interaction of GCase with its protein and lipid activators, namely Sap C and LBPA, regulating the GCase activity and stability. [ABSTRACT FROM AUTHOR]

Niemann-Pick C (NPC) disease is a fatal neurodegenerative disorder characterized by a lysosomal accumulation of cholesterol and other lipids within the cells of patients. Clinically identical forms of NPC disease are caused by defects in either of two different proteins: NPC1, a lysosomal-endosomal transmembrane protein and NPCZ, a soluble lysosomal protein with cholesterol binding properties. Although it is clear that NPC1 and NPC2 are required for the egress of lipids from the lysosome, the precise roles of these proteins in this process is unknown. To gain insight into the normal function of NPC2 and to investigate its interactions, if any, with NPC1, we have generated a murine NPC2 hypomorph that expresses 0-4% residual protein in different tissues and have examined its phenotype in the presence and absence of NPC1. The phenotypes of NPC1 and NPC2 single mutants and an NPC1;NPC2 double mutant are similar or identical in terms of disease onset and progression, pathology, neuronal storage, and biochemistry of lipid accumulation. These findings provide genetic evidence that the NPC1 and NPC2 proteins function in concert to facilitate the intracellular transport of lipids from the lysosome to other cellular sites. [ABSTRACT FROM AUTHOR]