Diacylglycerol Formation Research Articles

Nonylphenol releases arachidonic acid in rat Sertoli cells via activation of PKA and PLA2.

The endocrine disruptor, nonylphenol (NP) increases 20:4n-6 release in Sertoli cells via PKA/cPLA2 activation. Our data show that lipid metabolism could be a target of NP-induced abnormal reproductive outcomes. Nonylphenol (NP), an endocrine-disrupting chemical, is an environmental contaminant, and many notorious effects on male fertility have been reported in animal models and wild-type species. Here, we evaluated the effects of NP in follicle-stimulating hormone (FSH) signal transduction pathways and lipid metabolism using an in vitro model of rat Sertoli cell (SC) primary culture. Results show that an acute (1 h) SC exposure to NP (10 µM) increased the intra- and extra-cellular concentrations of free fatty acids (FFAs), mainly arachidonic acid (20:4n-6). Phosphatidylinositol seemed to be the major phospholipid source of this 20:4n-6 release by activation of the protein kinase A (PKA)/cytoplasmic phospholipase A2 (cPLA2) pathway. NP also increased diacylglycerols (DAG) levels and the expression (mRNA) of cyclooxygenase 2 (Cox2) and prostaglandin E2 (PGE2) levels. It is noteworthy that accumulation of lipid droplets took place after 24 h NP exposition, which was prevented by both a PKA inhibitor and a PLA2 inhibitor. Like FSH, NP triggers the release of 20:4n-6, which is a substrate for PGE2 synthesis via PKA/PLA2 activation. In addition, NP induces the formation of DAG, which could be required as a cofactor of the PKC-mediated activation of the COX2 inflammatory pathway. Our findings suggest that NP alters lipid homeostasis in SCs by inducing the activation of pro-inflammatory pathways that may trigger adverse effects in testis physiology over time. Concomitantly, the SC enhances the acylation of surplus FFAs (including 20:4n-6) in neutral lipids as a protective mechanism to shield itself from lipotoxicity and pro-inflammatory signals.

Effects of Heterogeneous Sulfated Acid Photocatalysts and Irradiation of Ultraviolet Light on the Chemical Conversion and Characteristics of Antifreeze from Bioglycerol

The purity of crude glycerol, a by-product of biodiesel production, may be as low as 50%. Thus, it has relatively low economic value without previously applying adequate physical purification or chemical conversion processes. A solid-state sulfated acid photocatalyst, TiO2/SO42− was prepared in this study to catalyze the chemical conversion of bioglycerol with acetic acid to produce an antifreeze of glycerine acetate to improve the low-temperature fluidity of liquid fuel. The experimental results show that similar X-ray intensity structures appeared between the catalysts of TiO2/SO42− and SO42−. An infrared spectra analysis using a Fourier transform infrared (FTIR) spectrometer confirmed the successful sintering of SO42− and ligating with TiO2 for preparing TiO2/SO42−. The effects of the photocatalyst were further excited by the irradiation of ultraviolet light. The highest weight percentage of glycerine acetate was obtained under a reaction time and reaction temperature of 10 h and 120 °C, respectively. In addition, it was observed that the glycerol conversion ratio reached 98.65% and the triacylglycerols compound amounted to 40.41 wt.% when the reacting molar ratio was 8. Moreover, the freezing point of the product mixture of glycerine acetate under the same molar ratio reached as low as −46.36 °C; the lowest among the products made using various molar ratios of acetic acid/glycerol. The UV light irradiation rendered higher triacylglycerols and diacylglycerols with lower diacylglycerol formation ratios than those without light irradiation.

Enzymatic synthesis and purification of diacylglycerols from mutton tallow and hemp seed oil

Abstract: Production of preparations based on natural ingredients has become a priority in the cosmetic and pharmaceutical industries. The novelty of the study was to present a possible method for obtaining pure diacylglycerols (DAG) from natural raw materials like fats (hemp seed oil and mutton tallow). We have determined the optimum water content for enzymatic interesterification to shift the reaction equilibrium towards hydrolysis and obtain the highest DAG formation yield. Then, DAG were separated and purified by column chromatography. Raw fats were characterized by fatty acids composition and their positional distribution using gas chromatography. Triacylglycerols, diacylglycerols, monoacylglycerols and free fatty acid contents were determined using gel permeation chromatography. The main fatty acids found in sn-2 MAG of hemp seed oil was linoleic acid, which was followed by oleic acid. Saturated fatty acids in hemp seed oil’s TAG showed preference for the outer sn-1,3 positions. For mutton tallow, the majority of oleic acid were found at sn-2 position. The optimum water content applied during enzymatic interesterification for DAG formation was estimated to be 2.0 % w/w, using the following conditions: 5 % w/w of catalyst, 200 rpm, 60 °C, 6h. DAG yield was 27 %. Purification of the mutton tallow-based and hemp seed oil-based DAG yielded with 83 % w/w and 82 % w/w, respectively. High purity DAG could be applied as effective emulsifiers in food, cosmetic or pharmaceutical emulsions.

The role of the endocannabinoid 2-arachidonoylglycerol in the in vivo spinal oxytocin-induced antinociception in male rats

Oxytocin receptor (OTR) activation at the spinal level produces antinociception. Some data suggest that central OTR activation enhances social interaction via an increase of endocannabinoids (eCB), but we do not know if this could occur at the spinal level, modulating pain transmission. Considering that oxytocin via OTR stimulates diacylglycerol formation, a key intermediate in synthesizing 2-arachidonylglycerol (2-AG), an eCB molecule, we sought to test the role of the eCB system on the spinal oxytocin-induced antinociception. Behavioral and electrophysiological experiments were conducted in naïve and formalin-treated (to induce long-term mechanical hypersensitivity) male Wistar rats. Intrathecal RHC 80267 injections, an inhibitor of the enzyme diacylglycerol lipase (thus, decreasing 2-AG formation), produces transient mechanical hypersensitivity, an effect unaltered by oxytocin but reversed by gabapentin. Similarly, in in vivo extracellular recordings of naïve spinal wide dynamic range cells, juxtacellular picoinjection of RHC 80267 increases the firing of nociceptive Aδ-, C-fibers, and post-discharge, an effect unaltered by oxytocin. Interestingly, in sensitized rats, oxytocin picoinjection reverses the RHC 80627-induced hyperactivity of Aδ-fibers (but not C- or post-discharge activity). In contrast, a sub-effective dose of JZL184 (a monoacylglycerol lipase inhibitor, thus favoring 2-AG levels), which does not have per se an antinociceptive effect in the formalin-induced hypernociception, the oxytocin-induced antinociception is boosted. Similarly, electrophysiological experiments suggest that juxtacellular JZL184 diminishes the neuronal firing of nociceptive fibers, and co-injection with oxytocin prolongs and enhances the antinociceptive effect. These data may imply that 2-AG formation may play a role in the spinal antinociception induced by oxytocin.

The Proteobacterial Methanotroph Methylosinus trichosporium OB3b Remodels Membrane Lipids in Response to Phosphate Limitation.

ABSTRACTMethane is a potent greenhouse gas in the atmosphere, and its concentration has continued to increase in recent decades. Aerobic methanotrophs, bacteria that use methane as the sole carbon source, are an important biological sink for methane, and they are widely distributed in the natural environment. However, relatively little is known on how methanotroph activity is regulated by nutrients, particularly phosphorus (P). P is the principal nutrient constraining plant and microbial productivity in many ecosystems, ranging from agricultural land to the open ocean. Using a model methanotrophic bacterium, Methylosinus trichosporium OB3b, we demonstrate here that this bacterium can produce P-free glycolipids to replace membrane phospholipids in response to P limitation. The formation of the glycolipid monoglucuronic acid diacylglycerol requires plcP-agt genes since the plcP-agt mutant is unable to produce this glycolipid. This plcP-agt-mediated lipid remodeling pathway appears to be important for M. trichosporium OB3b to cope with P stress, and the mutant grew significantly slower under P limitation. Interestingly, comparative genomics analysis shows that the ability to perform lipid remodeling appears to be a conserved trait in proteobacterial methanotrophs; indeed, plcP is found in all proteobacterial methanotroph genomes, and plcP transcripts from methanotrophs are readily detectable in metatranscriptomics data sets. Together, our study provides new insights into the adaptation to P limitation in this ecologically important group of bacteria.

Диабетическая кардиомиопатия: эпидемиология, этиология и патогенез

В обзоре детально проанализированы современные взгляды на эпидемиологию, этиологию и патогенез диабетической кардиомиопатии. Сахарный диабет вызывает различные структурные и функциональные модификации ткани миокарда. Эти патофизиологические изменения возникают в результате метаболических нарушений, вызванных гипергликемией, инсулинорезистентностью и дислипидемией. Свободные жирные кислоты могут стимулировать окисление и накапливаться в цитозоле, что приводит к липотоксическим эффектам путем образования церамидов, диацилглицерола и реактивных форм кислорода. Гипергликемия также вызывает увеличение содержания реактивных форм кислорода и образования конечных продуктов гликирования, что сопровождается развитием сердечной глюкотоксичности. Сочетание этих патофизиологических процессов, дефицит АТФ и липо-/глюкотоксичность являются промоторами дисбаланса Ca2+, стресса митохондриального/эндоплазматического ретикулума и развития апоптоза, активации сигнальных путей протеинкиназы С, митоген-активируемой протеинкиназы, убиквитин-протеасомной системы, протеотоксического стресса, активации циклического модулятора аденозиновых 5’-монофосфатов, ренин-ангиотензиновой системы, вызывают хроническое воспаление низкой интенсивности, диастолическую, а в дальнейшем систолическую дисфункцию, фиброз миокарда. Хроническая гипергликемия, инсулиновая резистентность и гиперинсулинемия вызывают резистентность кардиомиоцитов к инсулину и метаболические расстройства, которые усиливают дисфункцию митохондрий, оксидативный стресс, выработку конечных продуктов гликирования, нарушение обмена Ca2+ в митохондриях, хроническое воспаление низкой интенсивности, активацию системы «ренин — ангиотензин — альдостерон», стресс эндоплазматического ретикулума, гибель кардиомиоцитов, а также микрососудистую дисфункцию. Эти патофизиологические нарушения способствуют жесткости сердца, гипертрофии и фиброзу, развитию диастолической и систолической дисфункции миокарда и сердечной недостаточности.

The small molecule SERCA activator CDN1163 increases energy metabolism in human skeletal muscle cells

Background and objectiveA number of studies have highlighted muscle-specific mechanisms of thermogenesis involving futile cycling of Ca2+ driven by sarco (endo)plasmic reticulum Ca2+-ATPase (SERCA) and generating heat from ATP hydrolysis to be a promising strategy to counteract obesity and metabolic dysfunction. However, to the best of our knowledge, no experimental studies concerning the metabolic effects of pharmacologically targeting SERCA in human skeletal muscle cells have been reported. Thus, in the present study, we aimed to explore the effects of SERCA-activating compound, CDN1163, on energy metabolism in differentiated human skeletal muscle cells (myotubes). MethodsIn this study, we used primary myotube cultures derived from muscle biopsies of the musculus vastus lateralis and musculi interspinales from lean, healthy male donors. Energy metabolism in myotubes was studied using radioactive substrates. Oxygen consumption rate was assessed with the Seahorse XF24 bioanalyzer, whereas metabolic genes and protein expressions were determined by qPCR and immunoblotting, respectively. ResultsBoth acute (4 ​h) and chronic (5 days) treatment of myotubes with CDN1163 showed increased uptake and oxidation of glucose, as well as complete fatty acid oxidation in the presence of carbonyl cyanide 4-(trifluromethoxy)phenylhydrazone (FCCP). These effects were supported by measurement of oxygen consumption rate, in which the oxidative spare capacity and maximal respiration were enhanced after CDN1163-treatment. In addition, chronic treatment with CDN1163 improved cellular uptake of oleic acid (OA) and fatty acid β-oxidation. The increased OA metabolism was accompanied by enhanced mRNA-expression of carnitine palmitoyl transferase (CPT) 1B, pyruvate dehydrogenase kinase (PDK) 4, as well as increased AMP-activated protein kinase (AMPK)Thr172 phosphorylation. Moreover, following chronic CDN1163 treatment, the expression levels of stearoyl-CoA desaturase (SCD) 1 was decreased together with de novo lipogenesis from acetic acid and formation of diacylglycerol (DAG) from OA. ConclusionAltogether, these results suggest that SERCA activation by CDN1163 enhances energy metabolism in human myotubes, which might be favourable in relation to disorders that are related to metabolic dysfunction such as obesity and type 2 diabetes mellitus.

Miltefosine inhibits the membrane remodeling caused by phospholipase action by changing membrane physical properties

Miltefosine (hexadecylphosphocholine or HePC) is an alkylphosphocholine approved for the treatment of visceral and cutaneous Leishmaniasis. HePC exerts its effect by interacting with lipid membranes and affecting membrane-dependent processes. The molecular geometry of HePC suggests that the pharmacological function of HePC is to alter membrane curvature. As a model system, we studied the enzyme production in model membranes of diacylglycerol (DAG) or ceramide (CER), lipids involved in cell signaling which alter the structure of membranes. Here, we studied the effect of HePC on changes in phospholipase activity and on the effect that the lipid products have on the curvature and fusogenicity of membranes where they accumulate. Our results indicate that HePC inhibits the long-time restructuring of membranes, characteristic of the DAG and CER enzyme formation processes. In addition, the drug also reduces the fusogenicity of phospholipase-derived products. We postulate that the effect of HePC is due to a non-specific geometric compensation of HePC to the inverted cone-shape of DAG and CER products, acting as a relaxation agent of membrane curvature stress. These data are important for understanding the mechanism of action by which HePC regulates the lipid metabolism and signal transduction pathways in which these enzymes are involved.

Diacylglycerol kinases regulate TRPV1 channel activity

The transient receptor potential vanilloid 1 (TRPV1) channel is activated by heat and by capsaicin, the pungent compound in chili peppers. Calcium influx through TRPV1 has been shown to activate a calcium-sensitive phospholipase C (PLC) enzyme and to lead to a robust decrease in phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] levels, which is a major contributor to channel desensitization. Diacylglycerol (DAG), the product of the PLC-catalyzed PI(4,5)P2 hydrolysis, activates protein kinase C (PKC). PKC is known to potentiate TRPV1 activity during activation of G protein-coupled receptors, but it is not known whether DAG modulates TRPV1 during desensitization. We found here that inhibition of diacylglycerol kinase (DAGK) enzymes reduces desensitization of native TRPV1 in dorsal root ganglion neurons as well as of recombinant TRPV1 expressed in HEK293 cells. The effect of DAGK inhibition was eliminated by mutating two PKC-targeted phosphorylation sites, Ser-502 and Ser-800, indicating involvement of PKC. TRPV1 activation induced only a small and transient increase in DAG levels, unlike the robust and more sustained increase induced by muscarinic receptor activation. DAGK inhibition substantially increased the DAG signal evoked by TRPV1 activation but not that evoked by M1 muscarinic receptor activation. Our results show that Ca2+ influx through TRPV1 activates PLC and DAGK enzymes and that the latter limits formation of DAG and negatively regulates TRPV1 channel activity. Our findings uncover a role of DAGK in ion channel regulation.

Enhanced surfactant production using glycerol-based deep eutectic solvent as a novel reaction medium for enzymatic glycerolysis of soybean oil

Diacylglycerols (DAG) is one biosurfactant from oil, which can be prepared by the enzymatic glycerolysis of triacylglycerols (TAG). However, the poor miscibility of glycerol and TAG results in the low glycerolysis efficiency. In this work, glycerol-based eutectic solvent (DES) was firstly used as reaction solvent for the enzymatic glycerolysis of TAG. Five DESs (ChCl:Gly, ChCl:2Gly, ChCl:3Gly Bt:2Gly and Et:2Gly) were compared and used as reaction media. Three immobilized lipases (Lipozyme RMIM, Novozym 435 and Lipozyme TLIM) were screened and compared. Effects of DES load (0–50%), temperature (60 °C–120 °C), substrate ratio (2:1, 1:2, 1:4 and 1:6), enzyme load (3–12%) and water load added (0–5%) on the glycerolysis and the reusability of Novozym 435 in DES were evaluated. Results indicated that, among the tested immobilized lipases and DES, Novozym 435 showed the best capability of catalysis, and ChCl:2Gly as reaction medium was the best choice for the glycerolysis. The maximum DAG yield (49.83 ± 1.26%) and TAG conversion (75.80 ± 0.21%) were obtained under the following conditions: ChCl:2Gly dosage 10%, TAG/glycerol molar ratio 1:4, 80 °C, enzyme load 8%, 8 h and no water added. The selectivity for DAG formation in ChCl:2Gly system was 0.64–0.78, and the immobilized Novozym 435 can be used 5 times without obvious activity loss. Activation energy of DAG formation was 20.30 kJ/mol. Furthermore, kinetic values for K’m and Vmax were 1.35 mol/L and 7.9 × 10−2 mol/(L min), respectively.

Receptor-specific regulation of atrial GIRK channel activity by different Ca2+-dependent PKC isoforms

G Protein-activated K+ channels (GIRK) channels are inhibited by depletion of PtdIns(4,5)P2(PIP2), and/or channel phosphorylation by proteinkinase C (PKC). By using FRET-based biosensors, expressed in HEK293 cells or in atrial myocytes, we quantified receptor-specific Gq-coupled receptor (GqPCR) signalling on the level of phospholipase C (PLC) activation by monitoring PIP2-depletion and diacylglycerol (DAG) formation. Simultaneous voltage-clamp experiments on GIRK channel activity were performed as a functional readout for Gq-coupled α1B- and ET-receptor-induced signalling. GqPCR-induced fast inhibition of GIRK channel activity is mediated by depletion of PIP2, whereas phosphorylation of GIRK channels results in delayed, but effective GIRK current inhibition.We demonstrate a receptor-induced inhibitory component on GIRK activity that is independent of PIP2-depletion, but attributed to the activation of Ca2+-dependent PKC isoforms. As a novel finding, we demonstrate receptor-dependent differences in GIRK inhibition according to receptor-specific activation of the Ca2+-dependent PKC isoforms PKCα and PKCβ. Pharmacological inhibition of PKCα, but not of PKCβ, abolishes GIRK inhibition induced by stimulation of α1B-receptors. In contrast, ET-R-induced reduction of GIRK activity is sensitive to pharmacological block of PKCβ, but not of PKCα. Coexpression of α1B-receptors (or ETB-R) and PKCα (or PKCβ) in HEK 293 cells increased homologous receptor desensitization as indicated by a rapid decline of the CKAR FRET signal monitoring receptor activity. These data suggest that receptor-species dependent differences in PKC isoform activation regulate both GIRK channel activity and the strength of the receptor signal via a negative feedback mechanism.

Feedback regulation of insulin secretion by extended synaptotagmin-1.

Endoplasmic reticulum (ER)-plasma membrane (PM) contacts are dynamic structures with important roles in the regulation of calcium (Ca2+) and lipid homeostasis. The extended synaptotagmins (E-Syts) are ER-localized lipid transport proteins that interact with PM phosphatidylinositol 4,5-bisphosphate in a Ca2+-dependent manner. E-Syts bidirectionally transfer glycerolipids, including diacylglycerol (DAG), between the 2 juxtaposed membranes, but the biologic significance of this transport is still unclear. Using insulin-secreting cells and live-cell imaging, we now show that Ca2+-triggered exocytosis of insulin granules is followed, in sequence, by PM DAG formation and E-Syt1 recruitment. E-Syt1 counteracted the depolarization-induced DAG formation through a mechanism that required both voltage-dependent Ca2+ influx and Ca2+ release from the ER. E-Syt1 knockdown resulted in prolonged accumulation of DAG in the PM, resulting in increased glucose-stimulated insulin secretion. We conclude that Ca2+-triggered exocytosis is temporally coupled to Ca2+-triggered E-Syt1 PM recruitment and removal of DAG to negatively regulate the same process.-Xie, B., Nguyen, P. M., Idevall-Hagren, O. Feedback regulation of insulin secretion by extended synaptotagmin-1.

TAG synthesis and storage under osmotic stress. A requirement for preserving membrane homeostasis in renal cells

Hyperosmolarity is a controversial signal for renal cells. It can induce cell stress or differentiation and both require an active lipid metabolism. We showed that hyperosmolarity upregulates phospholipid (PL) de novo synthesis in renal cells. PL synthesis requires fatty acids (FA), usually stored as triglycerides (TAG). PL and TAG de novo synthesis utilize the same initial biosynthetic route: sn-glycerol 3P (G3P) → phosphatidic acid (PA) → diacylglycerol (DAG). In the present work, we evaluate how such pathway contributes to PL and TAG synthesis in renal cells subjected to hyperosmolarity. Our results show an increase in PA and DAG formation under hyperosmotic conditions; augmented DAG production, due to lipin enzyme activity, lead to the increase of both TAG and PL. However, at early stages (24 and 48 h), most of the de novo synthesized DAG was directed to PL synthesis; longer treatments downregulated PL synthesis and the DAG formed was mainly driven to TAG synthesis. Hyperosmolarity induced ACC and FASN transcription which mediated FA de novo synthesis. New FA molecules were stored in TAG. Silencing experiments revealed that hyperosmotic-induction of lipin-1 and -2 was mediated by SREBP1. Interestingly, SREBP1 knockdown also dropped SREBP2, indicating a modulatory action between both isoforms. Impairing SREBP activity leads to a decline in TAG levels but not PL. Membrane homeostasis is maintained through the adequate PL synthesis and renewal and constitute a protective mechanism against hyperosmolarity. The present data reveal the relevance of TAG synthesis and storage for PL synthesis in renal cells.

Control of neuronal excitability by Group I metabotropic glutamate receptors.

Metabotropic glutamate (mGlu) receptors couple through G proteins to regulate a large number of cell functions. Eight mGlu receptor isoforms have been cloned and classified into three Groups based on sequence, signal transduction mechanisms and pharmacology. This review will focus on Group I mGlu receptors, comprising the isoforms mGlu1 and mGlu5. Activation of these receptors initiates both G protein-dependent and -independent signal transduction pathways. The G-protein-dependent pathway involves mainly Gαq, which can activate PLCβ, leading initially to the formation of IP3 and diacylglycerol. IP3 can release Ca2+ from cellular stores resulting in activation of Ca2+-dependent ion channels. Intracellular Ca2+, together with diacylglycerol, activates PKC, which has many protein targets, including ion channels. Thus, activation of the G-protein-dependent pathway affects cellular excitability though several different effectors. In parallel, G protein-independent pathways lead to activation of non-selective cationic currents and metabotropic synaptic currents and potentials. Here, we provide a survey of the membrane transport proteins responsible for these electrical effects of Group I metabotropic glutamate receptors.

Receptor Species-dependent Desensitization Controls KCNQ1/KCNE1 K+ Channels as Downstream Effectors of Gq Protein-coupled Receptors

Activation of Gq protein-coupled receptors (GqPCRs) might induce divergent cellular responses, related to receptor-specific activation of different branches of the Gq signaling pathway. Receptor-specific desensitization provides a mechanism of effector modulation by restricting the spatiotemporal activation of signaling components downstream of Gq We quantified signaling events downstream of GqPCR activation with FRET-based biosensors in CHO and HEK 293 cells. KCNQ1/KCNE1 channels (IKs) were measured as a functional readout of receptor-specific activation. Activation of muscarinic M1 receptors (M1-Rs) caused robust and reversible inhibition of IKs. In contrast, activation of α1B-adrenergic receptors (α1B-ARs) induced transient inhibition of IKs, which turned into delayed facilitation after agonist withdrawal. As a novel finding, we demonstrate that GqPCR-specific kinetics of IKs modulation are determined by receptor-specific desensitization, evident at the level of Gαq activation, phosphatidylinositol 4,5-bisphosphate (PIP2) depletion, and diacylglycerol production. Sustained IKs inhibition during M1-R stimulation is attributed to robust membrane PIP2 depletion, whereas the rapid desensitization of α1B-AR delimits PIP2 reduction and augments current activation by protein kinase C (PKC). Overexpression of Ca2+-independent PKCδ did not affect the time course of α1B-AR-induced diacylglycerol formation, excluding a contribution of PKCδ to α1B-AR desensitization. Pharmacological inhibition of Ca2+-dependent PKC isoforms abolished fast α1B receptor desensitization and augmented IKs reduction, but did not affect IKs facilitation. These data indicate a contribution of Ca2+-dependent PKCs to α1B-AR desensitization, whereas IKs facilitation is induced by Ca2+-independent PKC isoforms. In contrast, neither inhibition of Ca2+-dependent/Ca2+-independent isoforms nor overexpression of PKCδ induced M1 receptor desensitization, excluding a contribution of PKC to M1-R-induced IKs modulation.

Effect of intermittent frying on fatty acids, vitamin E, lipid oxidation and acrylamide in oils and plantain chips collected from small- scale producers in Cameroon

Deep-fat frying is susceptible to induce the formation of undesirable products as lipid oxidation products and acrylamide in fried foods. Plantain chips produced by small-scale producers are sold to consumers without any control. The objective of this study was to evaluate the quality of plantain chips from local producers in relation to production process parameters and oils, and to identify the limiting factors for the production of acrylamide in plantain chips. Samples of frying oils and plantain chips prepared with either palm olein or soybean oil were collected from 10 producers in Yaounde. Quality parameters determined in this study were: fatty acid composition of the oils, determined by gas chromatography (GC) of free acid methyl ester; trans fatty acids, determined by Fourier transform infra-red spectroscopy; Tocopherols and tocotrienols as markers of nutritional quality were analyzed by High Performance Liquid Chromatography in isocratic mode. Free fatty acids and acylglycerols as markers of lipid hydrolysis were analyzed by GC of trimethylsilyl derivatives of glycerides. Conjugated dienes, Anisidine value and viscosity as markers of lipid oxidation and thermal decomposition of the oils; acrylamide which is formed through Maillard reaction and identified as a toxic compound in various fried products. Asparagine content of the raw fresh plantain powder was also determined. Fatty acid composition of palm oleins was stable within a day of intermittent frying. In soybean oils, about 57% and 62.5% of linoleic and linolenic acids were lost but trans fatty acids were not detected. Soybean oils were partly hydrolysed leading to the formation of free fatty acids, monoacylglycerols and diacylglycerols. In both oils, tocopherols and tocotrienols contents decreased significantly by about 50%. Anisidine value (AV) and polymers contents increased slightly in fried palm oleins while conjugated hydroperoxides, AV and polymers greatly increased in soybean oils. Acrylamide was not detected in the chips. This is explained by the absence of asparagine in the raw plantains, the other acrylamide precursors being present. This study shows that the plantain chips prepared at the small-scale level in Yaounde with palm olein are of good quality regarding oxidation and hydrolysis parameters and the absence of acrylamide. In contrast, oxidation developed with soybean oil whose usage for frying should be questioned. Considering that asparagine is the limiting factor for the formation of acrylamide in plantain chips, its content depending on several factors such as production parameters and maturity stage should be explored. Keywords: frying, plantain, producers, palm olein, soybean oil, oxidation, acrylamide, asparagine

Characterization of monoacylglycerol acyltransferase 2 inhibitors by a novel probe in binding assays

Monoacylglycerol acyltransferase 2 (MGAT2) is a membrane-bound lipid acyltransferase that catalyzes the formation of diacylglycerol using monoacylglycerol and fatty acyl CoA as substrates. MGAT2 is important for intestinal lipid absorption and is an emerging target for the treatment of metabolic diseases. In the current study, we identified and characterized four classes of novel MGAT2 inhibitors. We established both steady state and kinetic binding assay protocols using a novel radioligand, [3H]compound A. Diverse chemotypes of MGAT2 inhibitors were found to compete binding of [3H]compound A to MGAT2, indicating the broad utility of [3H]compound A for testing various classes of MGAT2 inhibitors. In the dynamic binding assays, the kinetic values of MGAT2 inhibitors such as Kon, Koff, and T1/2 were systematically defined. Of particular value, the residence times of inhibitors on MGAT2 enzyme were derived. We believe that the identification of novel classes of MGAT2 inhibitors and the detailed kinetic characterization provide valuable information for the identification of superior candidates for in vivo animal and clinical studies. The current work using a chemical probe to define inhibitory kinetics can be broadly applied to other membrane-bound acyltransferases.

Role of cyclic AMP and cyclic GMP as modulators of platelet cytosolic calcium

Studies from our laboratory at the University of Minnesota as well as that of others have demonstrated that agonist-mediated receptor stimulation leads to the activation of phospholipase C and formation of second messengers, diacyl glycerol and inositol trisphosphate, and mobilization of cytosolic free calcium. Elevation in intracellular calcium activates phospholipase A2 and release of arachidonic acid. This fatty acid is further converted to active metabolites, prostaglandin endoperoxides, and thromboxanes (TXA 2 ). TXA 2 promote fibrinogen binding, irreversible aggregation, and the release of granule contents, including serotonin and ADP. Agents that promote irreversible aggregation, facilitate fibrinogen binding, and those drugs that dissociate this process do so by lowering the cytosolic calcium levels. Endogenous antagonists such as PGE 1 , PGI 2 , and NO exert their inhibitory effect on platelet function by the action of cyclic nucleotides, cyclic AMP and cyclic GMP. Epinephrine-induced modulation of alpha-adrenergic receptor restores the action of agonists in drug-induced refractory platelets. As far as the role of cyclic nucleotides is considered, the observations are consistent with the concept that cyclic nucleotides participation in the biologic regulation may be modulated by cytoplasmic cations, and that a change in intracellular cyclic nucleotides and appropriate cations serves to promote the participation of both regulatory effectors. Anti-platelet drugs such as aspirin, indomethacin, and PGE 1 do not inhibit platelet interactions with the vessel wall. Based on this collective knowledge, novel antiplatelet drugs should be developed for the prevention of acute vascular events.

Membrane-Binding Mechanism of Clostridium perfringens Alpha-Toxin.

Clostridium perfringens alpha-toxin is a key mediator of gas gangrene, which is a life-threatening infection that manifests as fever, pain, edema, myonecrosis, and gas production. Alpha-toxin possesses phospholipase C and sphingomyelinase activities. The toxin is composed of an N-terminal domain (1–250 aa, N-domain), which is the catalytic site, and a C-terminal domain (251–370 aa, C-domain), which is the membrane-binding site. Immunization of mice with the C-domain of alpha-toxin prevents the gas gangrene caused by C. perfringens, whereas immunization with the N-domain has no effect. The central loop domain (55–93 aa), especially H….SW84Y85….G, plays an important role in the interaction with ganglioside GM1a. The toxin binds to lipid rafts in the presence of a GM1a/TrkA complex, and metabolites from phosphatidylcholine to diacylglycerol through the enzymatic activity of alpha-toxin itself. These membrane dynamics leads to the activation of endogenous PLCγ-1 via TrkA. In addition, treatment with alpha-toxin leads to the formation of diacylglycerol at membrane rafts in ganglioside-deficient DonQ cells; this in turn triggers endocytosis and cell death. This article summarizes the current the membrane-binding mechanism of alpha-toxin in detail.

Synthesis of Cocoa Butter Equivalent from Formulated Hard Palm Oil Mid-Fraction and Canola Oil Blends

Cocoa butter (CB) is an important major constituent of chocolate and other confectionary products. Several factors such as premium price, uncertainty in supply and variability in quality, have led the search for an alternative such as cocoa butter equivalent (CBE) from available and cheap commercial oils or fats. The aim of this research was to produce CBEs which contain omega-3 and omega-6 by blending hard palm oil mid-fraction (PMF) with canola oil. The reaction was performed by using Lipozyme RM IM as the biocatalyst. It aims to retain omega-3 and omega-6 content in CBE after interesterification. The effect of lipase load (LL), time reaction (TR) and stearic acid (ST) on CBE properties were studied to produce nearly similar CBE properties to CB. The best reaction conditions for maximizing POS (palmitic-oleic-stearic), SOS (stearic-oleic-stearic), and SMP (slip melting point) value while minimizing POP (palmitic-oleic-palmitic) and the levels of diacylglycerol (DAG) formation were; LL, 7.5% (w/w); TR, 8 hours; ST, with 44% stearic acid addition. Omega-3 (5.35%) and omega-6 (1.97%) content in CBE (after interesterification) were not significantly different (p > 0.05) to omega-3 (5.71%) and omega-6 (2.16%) content in initial mixture (before interesterification). The properties of CBE which include POP, POS, SOS, DAG and SMP values were 30.33%, 17.53%, 3.26%, 6.75%, and 46.45°C, respectively under these conditions.