Oleic acid induces endothelin-1 expression through activation of protein kinase C and NF-kappa B

This study investigated the effect of oleic acid on the expression levels of endothelin-1 (ET-1) and on the signaling pathways mediating it in human aortic endothelial cells (HAECs). ET-1 mRNA expression was significantly increased by oleic acid in a dose- and time-dependent manner. Elevation of ET-1 expression in response to oleic acid was inhibited by the protein kinase C (PKC) inhibitor, GF109203X, or the NF-kappa B inhibitor, pyrrolidine dithiocarbamate. In addition, both PKC and NF-kappa B activities were significantly increased by oleic acid. Immunoblot analysis revealed that conventional PKCs (PKC-alpha and -beta II isoforms) were significantly increased in the membranous fractions of HAECs treated with oleic acid. PKC inhibitor completely abolished oleic acid-induced NF-kappa B activation, suggesting that PKC activation is upstream of NF-kappa B activation in oleic acid-induced ET-1 expression. These data suggest that elevated plasma oleic acid levels observed in obese, insulin-resistant subjects result in endothelial dysfunction, at least in part, through an increase in ET-1 expression.     

Oleic acid-induced PKC isozyme translocation in RAW 264.7 macrophages

Fatty acids are important second messengers that mediate various cellular functions, but their role in the formation of macrophage foam cells is not known. High plasma levels of oleic acid (OA) in obese patients are often associated with a high risk for atherosclerosis. In this study, we investigated the protein kinase C (PKC) isozymes involved in OA-induced lipid accumulation in RAW 264.7 macrophages. The results show that OA induces translocation of PKC alpha, beta1, and delta from the cytosol to the cell membrane 5 min after the treatment. After 16 h incubation with OA, PKC delta was found to be colocalized with adipose differentiation-related protein (ADRP) on the surface of lipid droplets, but immunoprecipitation experiments showed that PKC delta was not biochemically associated with ADRP. After 16 h incubation with OA plus phorbol 12-myristate 13-acetate (PMA), PKC delta staining on the lipid droplet surface was not seen, whereas the accumulation of lipid droplets was unaffected. Furthermore, downregulation of PKC delta was confirmed by immunoblotting. These results demonstrate possible involvement of specific PKC isozymes in the early phase of lipid accumulation, possibly during the uptake of OA.     

Fatty acyl esters potentiate fatty acid induced activation of protein kinase C

Purified rat pancreas protein kinase C (PKC) is activated by unsaturated free fatty acids (oleic and arachidonic). The ethyl esters of these fatty acids are ineffective as enzyme activators. However, when the ethyl esters are added in combination with a free fatty acid, there is significant enhancement of enzyme activation. Nearly optimal PKC activation was obtained when non-activating ethyl oleate or ethyl arachidonate was added to sub-optimally activating concentrations of oleic or arachidonic acids. In addition to the ethyl esters, 1-monooleylglycerol also had a potentiating effect on PKC activation by oleic acid. However, the degree of activation observed in the presence of a free fatty acid and an acyl ester of the fatty acid quantitatively never surpassed that produced by sn-1,2-dioleylglycerol. Our findings indicate that significant PKC activation can be achieved by presenting the enzyme with an environment which we believe approximates the structural characteristics of the endogenous activator, sn-1,2-diacylglycerol.     

Dual modulation of protein kinase C activity by sphingosine.

Sphingosine is one of a number of cationic amphiphiles that inhibit the activity of protein kinase C (PKC) in commonly used assay conditions. This inhibition occurs only at high concentrations of this amphiphile. In the presence of excess negative charge from oleic acid, the addition of sphingosine surprisingly leads to activation of PKC. The results are explicable in terms of the dual role of charge and lipid phase propensity. When the positive charge on sphingosine is compensated by the negative charge on oleic acid, sphingosine, a hexagonal phase promoting amphiphile, becomes an activator of PKC. This does not occur with a bilayer stabilizing cationic amphiphile, N,N,N-Trimethyl-N'-cholesteryl amido-ethyl ammonium which is an inhibitor of PKC at all mol fractions, as well as in the presence of oleic acid. The results indicate that effects of sphingosine on more complex biological systems should be interpreted with caution because of this dual role of the amphiphile.     

Activation of rat brain protein kinase C by lipid oxidation products

The unsaturated fatty acid components of membrane lipids are susceptible to oxidation in vitro and in vivo. The initial oxidation products are hydroperoxy fatty acids that are converted spontaneously or enzymatically to a variety of products. Hydroperoxy derivatives of oleic, linoleic, or arachidonic acids stimulate the activity of protein kinase C (PKC) purified from rat brain. The hydroperoxy acids satisfy the requirement of PKC for phospholipid (e.g., phosphatidylserine). Activation is observed in the presence or absence of 1 mM Ca2+. Reduction of the hydroperoxides to alcohols or dehydration of the hydroperoxides to ketones increases the Ka for activation three- to fourfold but does not significantly reduce the maximal extent of PKC activation. The Ka's for activation by hydroperoxy acids are approximately half the values exhibited by the unoxidized fatty acids. Since oxidation of unsaturated fatty acids to hydroperoxides is the first event in lipid peroxidation, activation of PKC by hydroperoxy fatty acids may be an early cellular response to oxidative stress.     

Cytoplasmic lipid bodies of neutrophils: formation induced by cis- unsaturated fatty acids and mediated by protein kinase C

Lipid bodies, nonmembrane-bound cytoplasmic inclusions, serve as repositories of esterified arachidonate and are increased in cells associated with inflammatory reactions. We have evaluated stimuli and mechanisms responsible for lipid body formation within human polymorphonuclear leukocytes (PMNs). Arachidonic acid and oleic acid stimulated dose-dependent formation of lipid bodies over 0.5-1 h. Other C20 and C18 fatty acids were less active and demonstrated rank orders as follows: cis-unsaturated fatty acids were much more active than trans-fatty acids, and activity diminished with decreasing numbers of double bonds. Lipid bodies elicited in vitro with cis-fatty acids were ultrastructurally identical to lipid bodies present in PMNs in vivo. Lipid body induction was not because of fatty acid-elicited oxidants or fatty acid-induced ATP depletion. Cis-fatty acid-induced activation of protein kinase C (PKC) was involved in lipid body formation as evidenced by the capacity of other PKC activators, 1-oleoyl-2-acetyl-glycerol and two active phorbol esters, phorbol myristate acetate, and phorbol 12,13 dibutyrate, but not an inactive phorbol, to induce lipid body formation. The PKC inhibitor, 1-O-hexadecyl-2-O-methyl-glycerol, inhibited PMN lipid body formation induced by oleic and arachidonic acids and by 1-oleoyl-2-acetyl-glycerol and phorbol myristate acetate. Other PKC inhibitors (staurosporine, H-7) also inhibited lipid body formation. Formation of lipid bodies in PMNs is a specific cellular response, stimulated by cis-fatty acids and diglycerides and apparently mediated by PKC, which results in the mobilization and deposition of lipids within discrete, ultrastructurally defined cytoplasmic domains.     

Enhanced activity of Candida rugosa lipase modified by polyethylene glycol derivatives

The derivatives of polyethylene glycol (PEG) were prepared by reacting PEG with propylene oxide to enhance its hydrophobicity and introduce a branched structure. The PEG derivatives were activated with cyanuric chloride and used to modify the lipase fromCandida rugosa. The maximum specific activity of lipase modified with the PEG derivatives was about 2-fold of that modified with PEG for the esterification of oleic acid and lauryl alcohol in hexane.     

Biotransformation of oleic acid by alcaligenes sp. 5–18, a bacterium tolerant to high concentrations of oleic acid

We isolated from soil 160 bacterial strains which are tolerant to high concentrations of oleic acid, and examined their ability to transform oleic acid to new fatty acid derivatives. One of the isolated strains, Alcaligenes sp. 5–18, produced several compounds from oleic acid: 3-hydroxyoleic acid, 3-hydroxyhexadecenoic acid, octadecadienoic acid, hexadecadienoic acid, and tetradecadienoic acid. These compounds are intermediates in the β-oxidation pathway of oleic acid, and their accumulation is probably due to defective β-oxidation of oleic acid in the microorganism. Neither hydroxy nor enoic derivatives of fatty acids with a carbon chain length shorter than 14 were produced.     

Brownian dynamics simulation of the unsaturated lipidic molecules oleic and docosahexaenoic acid confined in a cellular membrane

A Brownian dynamics (BD) simulation of two unsaturated molecules, oleic and docosahexaenoic acid, in an environment that reproduces a cellular membrane, is presented. The results of the simulations, performed using mean-field potentials, were calibrated with experimental results obtained for oleic acid in a cellular membrane. The agreement between simulation and experimental results is excellent which validates subsequent simulation outcome for docosahexaenoic acid. This molecule is a major component of several cellular membranes thought to be involved in specific biological functions that require conformational changes of membrane components. The results for docosahexaenoic acid indicate that it is minimally influenced by temperature changes and that it presents great conformational variability.     

Brownian dynamics simulation of the unsaturated lipidic molecules oleic and docosahexaenoic acid confined in a cellular membrane

A Brownian dynamics (BD) simulation of two unsaturated molecules, oleic and docosahexaenoic acid, in an environment that reproduces a cellular membrane, is presented. The results of the simulations, performed using mean-field potentials, were calibrated with experimental results obtained for oleic acid in a cellular membrane. The agreement between simulation and experimental results is excellent which validates subsequent simulation outcome for docosahexaenoic acid. This molecule is a major component of several cellular membranes thought to be involved in specific biological functions that require conformational changes of membrane components. The results for docosahexaenoic acid indicate that it is minimally influenced by temperature changes and that it presents great conformational variability.     

Binding of isoxazole and pyrazole derivatives of curcumin with the activator binding domain of novel protein kinase C

The protein kinase C (PKC) family of serine/threonine kinases is an attractive drug target because of its involvement in the regulation of various cellular functions, including cell growth, differentiation, metabolism, and apoptosis. The endogenous PKC activator diacylglycerol contains two long carbon chains, which are attached to the glycerol moiety via ester linkage. Natural product curcumin (1), the active constituent of Curcuma L., contains two carbonyl and two hydroxyl groups. It modulates PKC activity and binds to the activator binding site (Majhi et al., Bioorg. Med. Chem.2010, 18, 1591). To investigate the role of the carbonyl and hydroxyl groups of curcumin in PKC binding and to develop curcumin derivatives as effective PKC modulators, we synthesized several isoxazole and pyrazole derivatives of curcumin (2-6), characterized their absorption and fluorescence properties, and studied their interaction with the activator-binding second cysteine-rich C1B subdomain of PKCδ, PKCε and PKCθ. The EC(50)s of the curcumin derivatives for protein fluorescence quenching varied in the range of 3-25 μM. All the derivatives showed higher binding with the PKCθC1B compared with PKCδC1B and PKCεC1B. Fluorescence emission maxima of 2-5 were blue shifted in the presence of the C1B domains, confirming their binding to the protein. Molecular docking revealed that hydroxyl, carbonyl and pyrazole ring of curcumin (1), pyrazole (2), and isoxazole (4) derivatives form hydrogen bonds with the protein residues. The present result shows that isoxazole and pyrazole derivatives bind to the activator binding site of novel PKCs and both carbonyl and hydroxy groups of curcumin play roles in the binding process, depending on the nature of curcumin derivative and the PKC isotype used.     

Arachidonic acid-induced IL-6 expression is mediated by PKC alpha activation in osteoblastic cells

There are several pieces of evidence supporting the important role that essential fatty acids (EFAs) and their metabolites play in regulating calcium and bone metabolism, and their relevance to the pathobiology of bone disease, with particular reference to modulating effects on cytokines. We found that arachidonic acid (AA) triggers a cell signal in osteoblasts and leads to the expression of IL-6. To explore the biochemical pathways involved in AA induction of cytokine gene expression, we evaluated the potential protein kinase C (PKC) dependent mechanism accounting for the AA effect on IL-6 gene expression. The osteoblast-like cell line MG-63 was pretreated with calphostin C, a PKC inhibitor, or phorbol 12-myristate 13-acetate (PMA) for an extended period, a condition which causes PKC downregulation, and subsequently with AA. After these treatments, IL-6 gene expression was no longer evident. We also showed that PKC and, in particular, PKC alpha, which are both recruited to the particulate fraction, undergo proteolysis and autophosphorylation; all of these steps are required for PKC activation and, subsequently, for AA-induced signaling. It is interesting that other unsaturated fatty acids, such as oleic acid (OA) or eicosapentaenoic acid (EPA), are unable to induce either PKC activation or IL-6 gene expression.     

Formation of monohydroxy derivatives of arachidonic acid, linoleic acid, and oleic acid during oxidation of low density lipoprotein by copper ions and endothelial cells.

An important event in the formation of atherosclerotic lesions is the uptake of modified low density lipoprotein (LDL) by macrophages via scavenger receptors. Modification of LDL, which results in its recognition by these receptors, can be initiated by peroxidation of LDL lipids. The first step in this process is the formation of monohydroperoxy derivatives of fatty acids, which are subsequently degraded to the corresponding monohydroxy compounds, or to a variety of secondary oxidation products. In order to understand this process more completely, we have developed a mass spectrometric procedure to measure the amounts of specific hydroperoxy/hydroxy fatty acids formed by oxidation of the major unsaturated fatty acids in human LDL, oleic acid, linoleic acid, and arachidonic acid. Oxidation of human LDL in the presence of a relatively strong stimulus (20 microM CuSO4) resulted in very large increases in the amounts of the major monohydroxy derivatives of linoleic acid (9- and 13-hydroxy derivatives) and arachidonic acid (5-, 8-, 9-, 11-, 12-, and 15-hydroxy derivatives) in LDL lipids in the early stages of the reaction. After 20 h, the amounts of these products declined due to substrate depletion, but large amounts of monohydroxy derivatives of oleic acid (8-, 10-, and 11-hydroxy derivatives) were detected. Although thiobarbituric acid-reactive substances clearly increased under these conditions, the changes were not nearly so dramatic as those observed for monohydroxy fatty acids. Oxidation of LDL in the presence of endothelial cells, a much milder stimulus, resulted in 2.5- to 3-fold increases in the amounts of monohydroxy derivatives of linoleic and arachidonic acids, as well as thiobarbituric acid-reactive substances, with more modest increases in the amounts of hydroxylated derivatives of oleic acid. There was little positional specificity in the oxidation of the above fatty acids in the presence of either stimulus, suggesting that the formation of these products proceeds primarily by lipid peroxidation, rather than by catalysis by lipoxygenases. However, an important role for lipoxygenases in the initiation of these reactions cannot be excluded. In conclusion, oxidation of LDL in the presence of copper ions or endothelial cells results in the formation of a large number of monohydroxy derivatives of oleic, linoleic, and arachidonic acids. The relative amounts of products formed from each of these fatty acids depends on the strength of the stimulus as well as the incubation time.     

Regulation of distinct pools of protein kinase C δ in beta cells

Previous studies from our laboratory have demonstrated the presence of several isoforms of protein kinase C (PKC), Ca²⁺-independent and Ca²⁺-dependent, in both whole islets and tumor-derived beta cells. In the basal state, a major proportion of the isoform was found in the crude membrane fraction with smaller amounts found in both the cytosolic and cytoskeletal fractions. Whole islets showed a similar distribution of the isoform. These studies were done to analyze the effects of insulin secretagogues on the distribution of PKC δ to different cellular pools in isolated insulinoma beta cells. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), produced a transient association of PKC δ with the beta cell cytoskeleton along with sustained decreases in cytosolic enzyme and transient increases in membrane enzyme. Neither glucose nor carbachol could acutely affect the subcellular distribution of PKC δ. Oleic acid decreased the amount of the enzyme associated with the cytoskeleton and led to a sustained decrease of cytosolic enzyme and a transient increase in membrane enzyme. Oleic acid was also able to prevent the increase in cytoskeletal enzyme induced by PMA. Both oleic acid and PMA potentiated glucose-induced insulin release but oleic acid, in contrast to PMA, was unable to initiate insulin release in the presence of substimulatory concentrations of glucose. These data demonstrate that different activators of PKC may have different effects on localization of the enzyme within the cells and suggest that there are at least three apparently distinct pools of PKC δ within the beta cell which may be important in insulin secretion or other aspects of beta cell function. © 1996 Wiley-Liss, Inc.     

Evidence for participation of cytochrome b5 in microsomal delta-6 desaturation of fatty acids.

The delta-6 desaturation of linoleic acid to gamma-linolenic acid and oleic acid to 6,9-octadecadienoic acid by rat liver microsomes was investigated. Using a specific antibody prepared against purified rat liver cytochrome b5, we demonstrated that cytochrome b5 participated in delta-6 desaturation of both fatty acids. The reaction products were identified as their methyl ester derivatives by argentation thin-layer chromatography, gas-liquid chromatography, and reductive ozonolysis followed by gas-liquid chromatography.     

Influence of cytokinins on growth of Phycomyces blakesleeanus and on the activities of the glyoxylate cycle enzymes isocitrate lyase and malate synthase

Treatment of the 1 + strain of Phycomyces blakesleeanus Bgff. with various cytokinins resulted in a stimulation of growth. The magnitude of growth stimulation depended on both the structure of the hormone used and the carbon source in the culture medium. Most of the cytokinin derivatives were active effect in glucose and oleic acid cultures. Benzyladenine (BA) and benzyladenosine stimulated the fungal growth only when oleic acid was the sole carbon source, while they had no effect in glucose cultures within the tested range of concentrations. [¹⁴C]-BA was accumulated by the mycelium of oleic acid cultures. Therefore, differences in BA uptake between glucose and oleic acid cultures could account mainly for the specific growth-promoting effect of BA. In oleic acid cultures isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2) activities were enhanced by 40 and 34%, respectively, in the presence of BA. A time course of the hormone effect suggests that BA is not involved in induction, but in the regulation of the mentioned enzymes in Phycocmyces. In contrast, acetate when presented as the sole carbon source or after addition to a glucose culture medium, induced isocitrate lyase activity. This enzyme induction was prevented by simultaneous addition of cycloheximide.     

Small‐angle X‐ray scattering of BAMLET at pH 12: A complex of α‐lactalbumin and oleic acid

BAMLET (Bovine Alpha‐lactalbumin Made LEthal to Tumors) is a member of the family of the HAMLET‐like complexes, a novel class of protein‐based anti‐cancer complexes that incorporate oleic acid and deliver it to cancer cells. Small angle X‐ray scattering (SAXS) was performed on the complex at pH 12, examining the high pH structure as a function of oleic acid added. The SAXS data for BAMLET species prepared with a range of oleic acid concentrations indicate extended, irregular, partially unfolded protein conformations that vary with the oleic acid concentration. Increases in oleic acid concentration correlate with increasing radius of gyration without an increase in maximum particle dimension, indicating decreasing protein density. The models for the highest oleic acid content BAMLET indicate an unusual coiled elongated structure that contrasts with apo‐α‐lactalbumin at pH 12, which is an elongated globular molecule, suggesting that oleic acid inhibits the folding or collapse of the protein component of BAMLET to the globular form. Circular dichroism of BAMLET and apo‐α‐lactalbumin was performed and the results suggest that α‐lactalbumin and BAMLET unfold in a continuum of increasing degree of unfolded states. Taken together, these results support a model in which BAMLET retains oleic acid by non‐specific association in the core of partially unfolded protein, and represent a new type of lipoprotein structure. Proteins 2014; 82:1400–1408. © 2014 Wiley Periodicals, Inc.     

Nitrated fatty acids: Endogenous anti-inflammatory signaling mediators

Nitroalkene derivatives of linoleic acid (LNO2) and oleic acid (OA-NO2) are present; however, their biological functions remain to be fully defined. Herein, we report that LNO2 and OA-NO2 inhibit lipopolysaccharide-induced secretion of proinflammatory cytokines in macrophages independent of nitric oxide formation, peroxisome proliferator-activated receptor-gamma activation, or induction of heme oxygenase-1 expression. The electrophilic nature of fatty acid nitroalkene derivatives resulted in alkylation of recombinant NF-kappaB p65 protein in vitro and a similar reaction with p65 in intact macrophages. The nitroalkylation of p65 by fatty acid nitroalkene derivatives inhibited DNA binding activity and repressed NF-kappaB-dependent target gene expression. Moreover, nitroalkenes inhibited endothelial tumor necrosis factor-alpha-induced vascular cell adhesion molecule 1 expression and monocyte rolling and adhesion. These observations indicate that nitroalkenes such as LNO2 and OA-NO2, derived from reactions of unsaturated fatty acids and oxides of nitrogen, are a class of endogenous anti-inflammatory mediators.