Calcium inhibits diacylglycerol uptake by serum albumin

AbstractSerum albumin is an abundant protein in blood plasma, that is well-known for its ability to transport hydrophobic biomolecules and drugs. Recent hypotheses propose that serum albumin plays a role in the regulation of lipid metabolism in addition to its lipid transport properties. The present work explores the capacity of bovine serum albumin (BSA) to extract diacylglycerols (DAG) from phospholipid bilayers, and the inhibition of such interaction by divalent cations. Quantitative measurements using radioactive DAG and morphological evidence derived from giant unilamellar vesicles examined by confocal microscopy provide concurrent results. BSA extracts DAG from vesicles consisting of phosphatidylinositol/DAG. Long, saturated DAG species are incorporated more readily than the shorter-chain or unsaturated ones. Divalent cations hinder DAG uptake by BSA. For Ca2+, the concentration causing half-maximal inhibition is ≈10 μM; 90% inhibition is caused by 100 μM Ca2+. Sr2+ requires concentrations one order of magnitude higher, while Mg2+ has virtually no effect. As an example on how DAG uptake by BSA, and its inhibition by Ca2+, could play a regulating role in lipid metabolism, a PI-specific phospholipase C has been assayed in the presence of BSA and/or Ca2+. BSA activates the enzyme by removing the end-product DAG, but the activation is reverted by Ca2+ that inhibits DAG uptake

Liquid-crystalline, liquid-ordered, rippled and gel lipid bilayer phases as observed with nile red fluorescence

The basic matrix of cellular membranes consists of a double layer (bilayer) of phospholipids. Semisynthetic lipid bilayers are commonly used in biophysical studies of membranes. According to tem-perature and composition, lipid bilayers can exist in liquid-crystalline (or liquid-disordered), liquid -ordered, rippled, and gel phases. In the present study, the hydrophobic, solvatochromic molecule Nile red has been used as a fluorescent probe to examine the physical state of bilayers of different composi-tions in the 15-60 degrees C range. Phospholipids with saturated or unsaturated acyl chains, in the presence or absence of cholesterol have been studied. Nile red shows absorption maxima at 520-550 nm and emis-sion maxima at 580-640 nm, single photon excitation not being damaging to the system. A red/orange intensity ratio (ROIR) index has been used to normalize the results. ROIR varies clearly and reproducibly with the lipid phase, increasing in the order: liquid-ordered < gel < rippled < liquid-crystalline. It increases with temperature and decreases with cholesterol contents in the bilayers. Nile red allows an unusually clear observation of the rippled-to-liquid crystalline phase transition in saturated phospho-lipids. FLIM studies with Nile red also show differences between lamellar phases. Rotational relaxation times have been determined for Nile red in liquid-disordered (0.72 +/- 0.010 ns), gel (1.16 +/- 0.070 ns), and liquid-ordered (1.79 +/- 0.14 ns) phases, the large value of the liquid-ordered phase being an indication of the sterol hindering probe tumbling in the hydrophobic matrix.This work was supported in part by the Spanish Ministerio de Ciencia e Innovación (MCI), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) (grants No. PGC2018-099857-B-I00, PID2020-114755GB-C33 ), by the Basque Government (grants No. IT1625-22, IT1639-22,and IT1270-19), by Fundación Ramón Areces (CIVP20A6619), by Fundación Biofísica Bizkaia, and by the Basque Excellence Research Centre (BERC) program of the Basque Government

C24:0 and C24:1 sphingolipids in cholesterol-containing, five- and six-component lipid membranes

The biophysical properties of sphingolipids containing lignoceric (C24:0) or nervonic (C24:1) fatty acyl residues have been studied in multicomponent lipid bilayers containing cholesterol (Chol), by means of confocal microscopy, differential scanning calorimetry and atomic force microscopy. Lipid membranes composed of dioleoyl phosphatidylcholine and cholesterol were prepared, with the addition of different combinations of ceramides (C24:0 and/or C24:1) and sphingomyelins (C24:0 and/or C24:1). Results point to C24:0 sphingolipids, namely lignoceroyl sphingomyelin (lSM) and lignoceroyl ceramide (lCer), having higher membrane rigidifying properties than their C24:1 homologues (nervonoyl SM, nSM, or nervonoyl Cer, nCer), although with a similar strong capacity to induce segregated gel phases. In the case of the lSM-lCer multicomponent system, the segregated phases have a peculiar fibrillar or fern-like morphology. Moreover, the combination of C24:0 and C24:1 sphingolipids generates interesting events, such as a generalized bilayer dynamism/instability of supported planar bilayers. In some cases, these sphingolipids give rise to exothermic curves in thermograms. These peculiar features were not present in previous studies of C24:1 combined with C16:0 sphingolipids. Conclusions of our study point to nSM as a key factor governing the relative distribution of ceramides when both lCer and nCer are present. The data indicate that lCer could be easier to accommodate in multicomponent bilayers than its C16:0 counterpart. These results are relevant for events of membrane platform formation, in the context of sphingolipid-based signaling cascades.EGR and AGA were postdoctoral scientists supported by the University of the Basque Country (UPV/EHU). This work was supported in part by the Spanish Ministerio de Ciencia e Innovacion (MCI), Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) (Grant No. PGC2018-099857-B-I00), by the Basque Government (Grants Nos. IT1264-19, IT1270-19), by the Fundacion Biofisica Bizkaia and by the Basque Excellence Research Centre (BERC) program of the Basque Government. Dedicated to Professor J. C. Gomez-Fernandez, on occasion of his 70th birthday

Model Systems of Precursor Cellular Membranes: Long-Chain Alcohols Stabilize Spontaneously Formed Oleic Acid Vesicles

AbstractOleic acid vesicles have been used as model systems to study the properties of membranes that could be the evolutionary precursors of more complex, stable, and impermeable phospholipid biomembranes. Pure fatty acid vesicles in general show high sensitivity to ionic strength and pH variation, but there is growing evidence that this lack of stability can be counterbalanced through mixtures with other amphiphilic or surfactant compounds. Here, we present a systematic experimental analysis of the oleic acid system and explore the spontaneous formation of vesicles under different conditions, as well as the effects that alcohols and alkanes may have in the process. Our results support the hypothesis that alcohols (in particular 10- to 14-C-atom alcohols) contribute to the stability of oleic acid vesicles under a wider range of experimental conditions. Moreover, studies of mixed oleic-acid-alkane and oleic-acid-alcohol systems using infrared spectroscopy and Langmuir trough measurements indicate that precisely those alcohols that increased vesicle stability also decreased the mobility of oleic acid polar headgroups, as well as the area/molecule of lipid

Effects of a N-Maleimide-derivatized Phosphatidylethanolamine on the Architecture and Properties of Lipid Bilayers

N-maleimide-derivatized phospholipids are often used to facilitate protein anchoring to membranes. In autophagy studies, this is applied to the covalent binding of Atg8, an autophagy protein, to a phosphatidylethanolamine (PE) in the nascent autophagosome. However, the question remains on how closely the N-maleimide PE derivative (PE-mal) mimicks the native PE in the bilayer. In the present paper, spectroscopic and calorimetric techniques have been applied to vesicles containing either PE or PE-mal (together with other phospholipids) to compare the properties of the native and derivatized forms of PE. According to differential scanning calorimetry, and to infrared spectroscopy, the presence of PE-mal did not perturb the fatty acyl chains in the bilayer. Fluorescence spectroscopy and microscopy showed that PE-mal did not alter the bilayer permeability either. However, fluorescence emission polarization of the Laurdan and DPH probes indicated an increased order, or decreased fluidity, in the bilayers containing PE-mal. In addition, the infrared spectral data from the phospholipid phosphate region revealed a PE-mal-induced conformational change in the polar heads, accompanied by increased hydration. Globally considered, the results suggest that PE-mal would be a reasonable substitute for PE in model membranes containing reconstituted proteins.This work was funded in part by the Spanish Ministry of Science, Innovation, and Universities (MCIU), Agencia Estatal de Investigación (AEI), Fondo Europeo de Desarrollo Regional (FEDER) (grant No. PID2021-124461NB-I00), the Basque Government (grant No. IT1625-22), Fundación Ramón Areces (CIVP20A6619), Fundación Biofísica Bizkaia and the Basque Excellence Research Centre (BERC) program of the Basque Government. E.J.G.-R. was supported by Fundación Ramón Areces. Y.R.V. was a recipient of a pre-doctoral FPU fellowship from the Spanish Ministry of Science, Innovation, and Universities (FPU18/00799). U.B. thanks the University of the Basque Country for a pre-doctoral contract

Erythrocyte Membrane Nanomechanical Rigidity Is Decreased in Obese Patients

This work intends to describe the physical properties of red blood cell (RBC) membranes in obese adults. The hypothesis driving this research is that obesity, in addition to increasing the amount of body fat, will also modify the lipid composition of membranes in cells other than adipocytes. Forty-nine control volunteers (16 male, 33 female, BMI 21.8 ± 5.6 and 21.5 ± 4.2 kg/m2, respectively) and 52 obese subjects (16 male and 36 female, BMI 38.2± 11.0 and 40.7 ± 8.7 kg/m2, respectively) were examined. The two physical techniques applied were atomic force microscopy (AFM) in the force spectroscopy mode, which allows the micromechanical measurement of penetration forces, and fluorescence anisotropy of trimethylammonium diphenylhexatriene (TMA-DPH), which provides information on lipid order at the membrane polar–nonpolar interface. These techniques, in combination with lipidomic studies, revealed a decreased rigidity in the interfacial region of the RBC membranes of obese as compared to control patients, related to parallel changes in lipid composition. Lipidomic data show an increase in the cholesterol/phospholipid mole ratio and a decrease in sphingomyelin contents in obese membranes. ω-3 fatty acids (e.g., docosahexaenoic acid) appear to be less prevalent in obese patient RBCs, and this is the case for both the global fatty acid distribution and for the individual major lipids in the membrane phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS). Moreover, some ω-6 fatty acids (e.g., arachidonic acid) are increased in obese patient RBCs. The switch from ω-3 to ω-6 lipids in obese subjects could be a major factor explaining the higher interfacial fluidity in obese patient RBC membranes.This work was supported in part by the Basque Government Department of Economic Development, grant No. KK-2019/00028 (OBINTER); the Basque Government Department of Education, grants No. IT1264-19, IT1281-19, IT1270-19, and IT1625-22; the Basque Government Department of Health, grants No. 2019-222030, 2020-333023; Fundación Ramón Areces; and by Centre for the Development of Industrial Technology (CDTI) of the Spanish Ministry of Science and Innovation under the grant agreement: TECNOMIFOOD project (CER-20191010) and Basque Government: IT1625-22

Nanomedical research and development in Spain: improving the treatment of diseases from the nanoscale

49 p.-7 fig.-9 tab.The new and unique possibilities that nanomaterials offer have greatly impacted biomedicine, from the treatment and diagnosis of diseases, to the specific and optimized delivery of therapeutic agents. Technological advances in the synthesis, characterization, standardization, and therapeutic performance of nanoparticles have enabled the approval of several nanomedicines and novel applications. Discoveries continue to rise exponentially in all disease areas, from cancer to neurodegenerative diseases. In Spain, there is a substantial net of researchers involved in the development of nanodiagnostics and nanomedicines. In this review, we summarize the state of the art of nanotechnology, focusing on nanoparticles, for the treatment of diseases in Spain (2017–2022), and give a perspective on the future trends and direction that nanomedicine research is taking.This work has been partially supported by MCIN/AEI /10.13039/501100011033 and European Union NextGenerationEU/PRTR (PID2021-128340OA-I00, PID2020-119352RB-I00, PID2021-127033OB-C21 and RTI2018-101050-J-I00), Comunidad de Madrid (S2022/BMD-7403 RENIM-CM and Talento program 2018-T1/IND-1005), European Union’s Horizon 2020 research and innovation programme (grant agreement No 685795), la Caixa Foundation LCF/PR/HA21/52350003, Asociación Española Contra el Cáncer IDEAS21989THOM, MCIN/AEI/10.13039/501100011033 and “ESF investing in your future” (RYC2019-027489-I, RYC2020-029282-I). CTB thanks Ministerio de Educación (FPU18/06310) for the predoctoral fellowship. IMDEA Nanociencia acknowledges support from the ‘Severo Ochoa’ Programme for Centers of Excellence in R&D (MINECO, CEX2020-001039-S).Peer reviewe

Arachidyl Amido Cholanoic Acid Improves Liver Glucose and Lipid Homeostasis in Nonalcoholic Steatohepatitis Via AMPK and mTOR Regulation

BACKGROUND Arachidyl amido cholanoic acid (Aramchol) is a potent downregulator of hepatic stearoyl-CoA desaturase 1 (SCD1) protein expression that reduces liver triglycerides and fibrosis in animal models of steatohepatitis. In a phase IIb clinical trial in patients with nonalcoholic steatohepatitis (NASH), 52 wk of treatment with Aramchol reduced blood levels of glycated hemoglobin A1c, an indicator of glycemic control. AIM To assess lipid and glucose metabolism in mouse hepatocytes and in a NASH mouse model [induced with a 0.1% methionine and choline deficient diet (0.1MCD)] after treatment with Aramchol. METHODS Isolated primary mouse hepatocytes were incubated with 20 mu mol/L Aramchol or vehicle for 48 h. Subsequently, analyses were performed including Western blot, proteomics by mass spectrometry, and fluxomic analysis with(13)C-uniformly labeled glucose. For thein vivopart of the study, male C57BL/6J mice were randomly fed a control or 0.1MCD for 4 wk and received 1 or 5 mg/kg/d Aramchol or vehicle by intragastric gavage for the last 2 wk. Liver metabolomics were assessed using ultra-high-performance liquid chromatography-time of flight-MS for the determination of glucose metabolism-related metabolites. RESULTS Combination of proteomics and Western blot analyses showed increased AMPK activity while the activity of nutrient sensor mTORC1 was decreased by Aramchol in hepatocytes. This translated into changes in the content of their downstream targets including proteins involved in fatty acid (FA) synthesis and oxidation [P-ACC alpha/beta(S79), SCD1, CPT1A/B, HADHA, and HADHB], oxidative phosphorylation (NDUFA9, NDUFB11, NDUFS1, NDUFV1, ETFDH, and UQCRC2), tricarboxylic acid (TCA) cycle (MDH2, SUCLA2, and SUCLG2), and ribosome (P-p70S6K[T389] and P-S6[S235/S236]). Flux experiments with(13)C-uniformely labeled glucose showed that TCA cycle cataplerosis was reduced by Aramchol in hepatocytes, as indicated by the increase in the number of rounds that malate remained in the TCA cycle. Finally, liver metabolomic analysis showed that glucose homeostasis was improved by Aramchol in 0.1MCD fed mice in a dose-dependent manner, showing normalization of glucose, G6P, F6P, UDP-glucose, and Rbl5P/Xyl5P. CONCLUSION Aramchol exerts its effect on glucose and lipid metabolism in NASH through activation of AMPK and inhibition of mTORC1, which in turn activate FA beta-oxidation and oxidative phosphorylation.Supported by the National Institutes of Health Grant, No. R01CA172086; Plan Nacional of I+D, No. SAF2017-88041-R; Ministerio de Economia y Competitividad de Espana, No. SAF2017-87301-R; Asociacion Espanola contra el Cancer, No. AECC17/302; Ayudas Fundacion BBVA a equipos de Investigacion Cientifica 2018; Fondo Europeo de Desarrollo Regional, Ministerio de Economia y Competitividad de Espana, No. PGC2018-099857-BI00; Basque Government Grants, No. IT1264-19; Ministerio de Economia y Competitividad de Espana for the Severo Ochoa Excellence Accreditation, No. SEV2016-0644. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript