Inhibition of duodenal enterocyte Mg2+-ATPase by arachidonic acid is not mediated by an effect on protein kinase C

Active absorption processes in the duodenal enterocyte are driven by various ATPases. It is known that the activity of Na+,K+-ATPase, Ca2+-ATPase and Mg2+-ATPase can be modulated by polyunsaturated fatty acids of the n-6 series, for example by linoleic and gamma-linolenic acids. These effects may be achieved by protein phosphorylation via protein kinase C. The present study was undertaken to determine the effect of arachidonic acid on Mg2+-ATPase (measured colorimetrically) activity in basolateral membranes prepared from rat duodenum. It shows, for the first time, significant dose-dependent inhibition of Mg2+-ATPase (26-62%) by arachidonic acid (10-50 microg/ml) which already takes place after one minute of exposure, indicating involvement of a rapid signal transduction mechanism. Addition of the protein kinase C inhibitors bisimidolylmaleimide (2.5 microM) and calphostin (0.5 microM) did not influence the action of arachidonic acid on Mg2+-ATPase; protein kinase C involvement in this process is thus not indicated.     

Arachidonic acid stimulates TNFα production in Kupffer cells via a reactive oxygen species-pERK1/2-Egr1-dependent mechanism

Kupffer cells are a key source of mediators of alcohol-induced liver damage such as reactive oxygen species, chemokines, growth factors, and eicosanoids. Since diets rich in polyunsaturated fatty acids are a requirement for the development of alcoholic liver disease, we hypothesized that polyunsaturated fatty acids could synergize with ethanol to promote Kupffer cell activation and TNFα production, hence, contributing to liver injury. Primary Kupffer cells from control and from ethanol-fed rats incubated with arachidonic acid showed similar proliferation rates than nontreated cells; however, arachidonic acid induced phenotypic changes, lipid peroxidation, hydroperoxides, and superoxide radical generation. Similar effects occurred in human Kupffer cells. These events were greater in Kupffer cells from ethanol-fed rats, and antioxidants and inhibitors of arachidonic acid metabolism prevented them. Arachidonic acid treatment increased NADPH oxidase activity. Inhibitors of NADPH oxidase and of arachidonic acid metabolism partially prevented the increase in oxidant stress. Upon arachidonic acid stimulation, there was a rapid and sustained increase in TNFα, which was greater in Kupffer cells from ethanol-fed rats than in Kupffer cells from control rats. Arachidonic acid induced ERK1/2 phosphorylation and nuclear translocation of early growth response-1 (Egr1), and ethanol synergized with arachidonic acid to promote this effect. PD98059, a mitogen extracellular kinase 1/2 inhibitor, and curcumin, an Egr1 inhibitor, blocked the arachidonic acid-mediated upregulation of TNFα in Kupffer cells. This study unveils the mechanism whereby arachidonic acid and ethanol increase TNFα production in Kupffer cells, thus contributing to alcoholic liver disease.     

Regulation of AMP-activated protein kinase and acetyl-CoA carboxylase phosphorylation by palmitate in skeletal muscle cells

The purpose of this study was to investigate the effects of long-chain fatty acids (LCFAs) on AMP-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC) phosphorylation and beta-oxidation in skeletal muscle. L6 rat skeletal muscle cells were exposed to various concentrations of palmitate (1-800 microM). Subsequently, ACC and AMPK phosphorylation and fatty acid oxidation were measured. A 2-fold increase in both AMPK and ACC phosphorylation was observed in the presence of palmitate concentrations as low as 10 microM, which was also accompanied by a significant increase in fatty acid oxidation. The effect of palmitate on AMPK and ACC phosphorylation was dose-dependent, reaching maximum increases of 3.5- and 4.5-fold, respectively. Interestingly, ACC phosphorylation was coupled with AMPK activation at palmitate concentrations ranging from 10 to 100 microM; however, at concentrations >200 microM, ACC phosphorylation and fatty acid oxidation remained high even after AMPK phosphorylation was completely prevented by the use of a selective AMPK inhibitor. This indicates that LCFAs regulate ACC activity by AMPK-dependent and -independent mechanisms, based on their abundance in skeletal muscle cells. Here, we provide novel evidence that the AMPK/ACC pathway may operate as a mechanism to sense and respond to the lipid energy charge of skeletal muscle cells.     

食物中多不饱和脂肪酸在家蝇幼虫体内的富集与代谢及对其生长的影响

【目的】阐明家蝇 Musca domestica 幼虫对食物中各种多不饱和脂肪酸的富集能力以及代谢转化情况,并探究各种多不饱和脂肪酸对家蝇幼虫生长的影响。【方法】在基础饲料中添加不同浓度(3%, 6%和12%)的多不饱和脂肪酸(亚油酸、α-亚麻酸、花生四烯酸和二十二碳六烯酸)饲养经过脱脂传代培养的家蝇幼虫;提取家蝇幼虫的总脂肪酸,利用气相色谱仪进行检测和分析;测定统计幼虫体重,以分析多不饱和脂肪酸对家蝇幼虫生长的影响。【结果】亚油酸、α-亚麻酸和花生四烯酸在家蝇幼虫体内均能被富集,且它们的富集程度随着食物中多不饱和脂肪酸的添加浓度的升高而增加,其中亚油酸、α-亚麻酸和花生四烯酸在幼虫体内富集的最高含量(占体内总脂肪酸的比例)分别为21.93%, 16.13%和9.68%,而二十二碳六烯酸不能在家蝇幼虫体内富集,提示家蝇幼虫食物中添加的各种多不饱和脂肪酸经过代谢后并没有在其体内产生新的脂肪酸,而食物中添加的二十二碳六烯酸在家蝇幼虫体内被分解代谢后消除。饲喂α-亚麻酸及花生四烯酸后家蝇幼虫体重增长较为明显,其中6%α-亚麻酸添加组的幼虫体重显著高于对照组(取食脱脂饲料)和3%和12%α-亚麻酸添加组,3%和6%花生四烯酸添加组的幼虫体重显著高于对照组和12%花生四烯酸添加组。【结论】家蝇幼虫体内能够从食物中富集部分多不饱和脂肪酸,多不饱和脂肪酸碳链越长其富集程度越低直至不能富集,富集的多不饱和脂肪酸对家蝇幼虫生长有不同程度的影响。     

Docosahexaenoic acid stabilizes soluble amyloid-beta protofibrils and sustains amyloid-beta-induced neurotoxicity in vitro

Enrichment of diet and culture media with the polyunsaturated fatty acid docosahexaenoic acid has been found to reduce the amyloid burden in mice and lower amyloid-beta (Abeta) levels in both mice and cultured cells. However, the direct interaction of polyunsaturated fatty acids, such as docosahexaenoic acid, with Abeta, and their effect on Abeta aggregation has not been explored in detail. Therefore, we have investigated the effect of docosahexaenoic acid, arachidonic acid and the saturated fatty acid arachidic acid on monomer oligomerization into protofibrils and protofibril fibrillization into fibrils in vitro, using size exclusion chromatography. The polyunsaturated fatty acids docosahexaenoic acid and arachidonic acid at micellar concentrations stabilized soluble Abeta42 wild-type protofibrils, thereby hindering their conversion to insoluble fibrils. As a consequence, docosahexaenoic acid sustained amyloid-beta-induced toxicity in PC12 cells over time, whereas Abeta without docosahexaenoic acid stabilization resulted in reduced toxicity, as Abeta formed fibrils. Arachidic acid had no effect on Abeta aggregation, and neither of the fatty acids had any protofibril-stabilizing effect on Abeta42 harboring the Arctic mutation (AbetaE22G). Consequently, AbetaArctic-induced toxicity could not be sustained using docosahexaenoic acid. These results provide new insights into the toxicity of different Abeta aggregates and how endogenous lipids can affect Abeta aggregation.     

Choline Uptake in Cultured Human Y79 Retinoblastoma Cells: Effect of Polyunsaturated Fatty Acid Compositional Modifications

Abstract: Choline uptake in Y79 human retinoblastoma cells occurs through two kinetically distinguishable processes. The high-affinity system shows little sodium or energy dependence, and it does not appear to be linked to acetyl CoA: choline O -acetyltransferase. When the cells are grown in a culture medium containing 10% fetal bovine serum, the high-affinity system has a K' _m= 2.16 ± 0.13 μ m and V' _max= 27.0 ± 2.9 pmol min⁻¹ mg⁻¹, whereas the low-affinity system has K' _m= 20.4 ± 1.3 μ m and V' _max= 402 ± 49 pmol min⁻¹ mg⁻¹. Under these conditions, the polyunsaturated fatty acid content of the cell membranes is relatively low. When the polyunsaturated fatty acid content of the microsomal membrane fraction was increased by supplementing the culture medium with linolenic or docosahexaenoic acids (n-3 polyunsaturated fatty acids) or arachidonic acid (n-6 polyunsaturated fatty acid), the K' ^m of the high-affinity choline transport system was reduced by 40–60%. The V' _max also was reduced by 20–40%. Supplementation with oleic acid, the most prevalent monounsaturated fatty acid, did not affect either kinetic parameter. The results suggest that one functional effect of the high unsaturated fatty acid content of neural cell membranes is to facilitate the capacity of the high-affinity choline uptake system to transport low concentrations of choline. This effect appears to be specific for polyunsaturated fatty acids but not for a single type, for it is produced by members of both the n-3 and n-6 classes of polyunsaturated fatty acids.     

Postmortem brain fatty acid profile of levodopa-treated Parkinson disease patients and parkinsonian monkeys

Fatty acids play a critical role in brain function but their specific role in the pathophysiology of Parkinson disease (PD) and levodopa-induced motor complications is still unknown. From a therapeutic standpoint, it is important to determine the relation between brain fatty acids and PD because the brain fatty acid content depends on nutritional intake, a readily manipulable environmental factor. Here, we report a postmortem analysis of fatty acid profile by gas chromatography in the brain cortex of human patients (12 PD patients and nine Controls) as well as in the brain cortex of monkeys (four controls, five drug-naive MPTP monkeys and seven levodopa-treated MPTP monkeys). Brain fatty acid profile of cerebral cortex tissue was similar between PD patients and Controls and was not correlated with age of death, delay to autopsy or brain pH. Levodopa administration in MPTP monkeys increased arachidonic acid content (+7%; P < 0 .05) but decreased docosahexaenoic acid concentration (-15%; P < 0.05) and total n-3:n-6 polyunsaturated fatty acids ratio (-27%; P < 0.01) compared to drug-naive MPTP animals. Interestingly, PD patients who experienced motor complications to levodopa had higher arachidonic acid concentrations in the cortex compared to Controls (+13.6%; P < 0.05) and to levodopa-treated PD patients devoid of motor complications (+14.4%; P < 0.05). Furthermore, PD patients who took an above-median cumulative dose of levodopa had a higher relative amount of saturated fatty acids but lower monounsaturated fatty acids in their brain cortex (P < 0.01). These results suggest that changes in brain fatty acid relative concentrations are associated with levodopa treatment in PD patients and in a non-human primate model of parkinsonism.     

Antioxidant effect of conjugated linoleic acid and vitamin A during non enzymatic lipid peroxidation of rat liver microsomes and mitochondria

In the study reported here the effect of conjugated linoleic acid (CLA) and vitamin A on the polyunsaturated fatty acid composition, chemiluminescence and peroxidizability index of microsomes and mitochondria isolated from rat liver was analyzed. The effect of CLA on the polyunsaturated fatty acid composition of native microsomes was evidenced by an statistically significant p < 0.007 decrease of linoleic acid C18:2 n6, whereas in mitochondria it was observed a decrease p < 0.0001 of arachidonic acid C20:4 n6 when compared with vitamin A and control groups. Docosahexaenoic acid C22:6 n3 in mitochondria was reduced p < 0.04 in CLA and vitamin A groups when compared with control. After incubation of microsomes or mitochondria in an ascorbate (0.4 mM)-Fe⁺⁺ (2.15 M) system (120 min at 37°C) it was observed that the total cpm/mg protein originated from light emission: chemiluminescence was lower in liver microsomes or mitochondria obtained from CLA group (received orally: 12.5 mg/daily during 10 days) than in the vitamin A group (received intraperitoneal injection: daily 0.195 g/kg during 10 days). CLA reduced significantly maximal induced chemiluminescence in microsomes relative to vitamin A and control groups, whereas in mitochondria the effect was observed relative to control group The polyunsaturated fatty acid composition of liver microsomes or mitochondria changed by CLA and vitamin A treatment. The polyunsaturated fatty acids mainly affected when microsomes native and peroxidized from control group were compared were linoleic, linolenic and arachidonic acids, while in vitamin A group linoleic and arachidonic acid were mainly peroxidized, whereas in CLA group only arachidonic acid was altered. In mitochondria obtained from the three groups arachidonic acid and docosahexaenoic acid showed a significant decrease when native and peroxidized groups were compared. As a consequence the peroxidizability index, a parameter based on the maximal rate of oxidation of fatty acids, show significant changes in the CLA group compare vitamin A and control groups. The simultaneous analysis of peroxidizability index, chemiluminescence and fatty acid composition demonstrated that CLA is more effective than vitamin A protecting microsomes or mitochondria from peroxidative damage.     

Studies on polyenoic acid incorporation into human platelet lipid stores: interactions with linoleic and arachidonic acids

Several polyunsaturated fatty acids (C18-C22 acids) have been compared in their uptake by human platelets and their acylation into glycerophospholipid subclasses. This was also studied in the presence of linoleic and/or arachidonic acids, the main fatty acids of plasma free fatty acid pool. Amongst C20 fatty acids, dihomogamma linolenic acid (20:3(n-6)), 5,8,11-icosatrienoic acid (20:3(n-9)) and arachidonic acid (20:4(n-6)) were better incorporated. The uptake of 5,8,11,14,17-icosapentaenoic acid (20:5(n-3)) was significantly lower and comparable to that of C22 fatty acids (7,10,13,16-docosatetraenoic acid (22:4(n-6)) and 4,7,10,13,16,19-docosahexaenoic acid (22:6(n-3)) and linoleic acid (18:2(n-6)). In this respect, linolenic acid (18:3(n-3)) appeared the poorest substrate. The bulk of each acid was acylated into glycerophospholipids although the presence of linoleic and/or arachidonic acids diverted a part towards neutral lipids. This was prominent for 18:3(n-3) and C22 fatty acids. The glycerophospholipid distribution of each acid differed substantially and was not affected by the presence of linoleic and or arachidonic acids, except for 18:3(n-3) and 22:6(n-3) that were strongly diverted towards phosphatidylethanolamine (PE) at the expense of phosphatidylcholine (PC). The main features were an efficient acylation of 20:3(n-9) into phosphatidylinositol (PI) followed by 20:3(n-6) and 20:4(n-6), then by 20:5(n-3) and 22:4(n-6), and finally 22:6(n-3) and C18 fatty acids. This was reciprocal to the acylation into PE and to a lesser extent into PC which remained the main storage species in all cases. We conclude that human platelets may exhibit a certain specificity for taking up polyunsaturated fatty acids both in terms of total uptake and glycerophospholipid subclass distribution. Also the presence of polyunsaturated fatty acids of normal plasma, like linoleic and arachidonic acids, may interact specifically with such an uptake and distribution.     

Molecular mechanisms for biosynthesis and assembly of nutritionally important very long chain polyunsaturated fatty acids in microorganisms

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as docosahexaenoic acid (DHA, 22:6n-3), arachidonic acid (ARA, 20:4n-6) and eicosapentaenoic acid (EPA, 20:5-n3) are nutritionally important for humans and animals. De novo biosynthesis of these fatty acids mainly occurs in microorganisms and goes through either an aerobic pathway catalyzed by type I/II fatty acid synthase, desaturases and elongases or an anaerobic pathway catalyzed by a polyunsaturated fatty acid synthase. After synthesis, VLCPUFAs must be incorporated into glycerolipids for storage through acyl assembly processes. Understanding the mechanisms for the biosynthesis of VLCPUFAs and their incorporation into glycerolipids is important not only for developing a renewable, sustainable and environment-friendly source of these fatty acids in microorganisms, but also, for designing effective strategies for metabolic engineering of these fatty acids in heterologous systems. This review highlights recent findings which have increased our understanding of biosynthesis of VLCPUFAs and their incorporation into glycerolipids in microorganisms. Future directions in improving the production of VLCPUFAs in native microbial producers are also discussed along with transgenic production of these fatty acids in oleaginous microorganisms and oilseed crops for food and feed uses.     

Polyunsaturated fatty acids are potent neuroprotectors

Results reported in this work suggest a potential therapeutic value of polyunsaturated fatty acids for cerebral pathologies as previously proposed by others for cardiac diseases. We show that the polyunsaturated fatty acid linolenic acid prevents neuronal death in an animal model of transient global ischemia even when administered after the insult. Linolenic acid also protects animals treated with kainate against seizures and hippocampal lesions. The same effects have been observed in an in vitro model of seizure-like activity using glutamatergic neurons and they have been shown to be associated with blockade of glutamatergic transmission by low concentrations of distinct polyunsaturated fatty acids. Our data suggest that the opening of background K(+) channels, like TREK-1 and TRAAK, which are activated by arachidonic acid and other polyunsaturated fatty acids such as docosahexaenoic acid and linolenic acid, is a significant factor in this neuroprotective effect. These channels are abundant in the brain where they are located both pre- and post-synaptically, and are insensitive to saturated fatty acids, which offer no neuroprotection.     

Control of p70 ribosomal protein S6 kinase and acetyl-CoA carboxylase by AMP-activated protein kinase and protein phosphatases in isolated hepatocytes.

Certain amino acids, like glutamine and leucine, induce an anabolic response in liver. They activate p70 ribosomal protein S6 kinase (p70S6K) and acetyl-CoA carboxylase (ACC) involved in protein and fatty acids synthesis, respectively. In contrast, the AMP-activated protein kinase (AMPK), which senses the energy state of the cell and becomes activated under metabolic stress, inactivates by phosphorylation key enzymes in biosynthetic pathways thereby conserving ATP. In this paper, we studied the effect of AMPK activation and of protein phosphatase inhibitors, on the amino-acid-induced activation of p70S6K and ACC in hepatocytes in suspension. AMPK was activated under anoxic conditions or by incubation with 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAr) or oligomycin, an inhibitor of mitochondrial oxidative phosphorylation. Incubation of hepatocytes with amino acids activated p70S6K via multiple phosphorylation. It also activated ACC by a phosphatase-dependent mechanism but did not modify AMPK activation. Conversely, the amino-acid-induced activation of both ACC and p70S6K was blocked or reversed when AMPK was activated. This AMPK activation increased Ser79 phosphorylation in ACC but decreased Thr389 phosphorylation in p70S6K. Protein phosphatase inhibitors prevented p70S6K activation when added prior to the incubation with amino acids, whereas they enhanced p70S6K activation when added after the preincubation with amino acids. It is concluded that (a) AMPK blocks amino-acid-induced activation of ACC and p70S6K, directly by phosphorylating Ser79 in ACC, and indirectly by inhibiting p70S6K phosphorylation, and (b) both activation and inhibition of protein phosphatases are involved in the activation of p70S6K by amino acids. p70S6K adds to an increasing list of targets of AMPK in agreement with the inhibition of energy-consuming biosynthetic pathways.     

Surfactant protein A inhibits the non-enzymatic lipid peroxidation of porcine lung surfactant.

The ability of surfactant protein A (SP-A) to inhibit the ascorbate-Fe(2+) induced lipid peroxidation of polyunsaturated fatty acids found in porcine lung surfactant (surfacen) was assessed by measuring the light emission - chemiluminescence during a 180-min incubation period at 37 degrees C. The light emission (chemiluminescence) was concentration dependent. Changes in the fatty acid composition of surfacen were observed when the lung surfactant was incubated in an ascorbate-Fe(2+) system. The main polyunsaturated fatty acids C18:2 n6 and C20:4 n6 found in the lung surfactant decreased considerably after a 180-min lipid peroxidation process. Native SP-A isolated from pig lungs inhibited oxidation of surfactant long chain polyunsaturated fatty acids, mainly arachidonic acid, in a dose-dependent fashion that was half-maximal (60% inhibition) at a concentration of 2.0 microg/ml and almost complete (73.6% inhibition) at 4.0 microg/ml, as indicated by inhibition of light emission and fatty acid composition analysis. At the highest concentration of lung SP-A used a very good correlation between the protection of the most polyunsaturated fatty acids and inhibition of light emission was observed.     

Arachidonic acid potentiates exocytosis and allows neuronal SNARE complex to interact with Munc18a

Neuronal communication relies on the fusion of neurotransmitter-containing vesicles with the neuronal plasma membrane. Recent genetic studies have highlighted the critical role played by polyunsaturated fatty acids in neurotransmission, however, there is little information available about which fatty acids act on exocytosis and, more importantly, by what mechanism. We have used permeabilized chromaffin cells to screen various fatty acids of the n-3 and n-6 series for their acute effects on exocytosis. We have demonstrated that an n-6 series polyunsaturated fatty acid, arachidonic acid, potentiates secretion from intact neurosecretory cells regardless of the secretagogue used. We have shown that arachidonic acid dose dependently increases soluble NSF attachment protein receptor complex formation in chromaffin cells and bovine cortical brain extracts and that a non-hydrolysable analogue of arachidonic acid causes a similar increase in SNARE complex formation. This prompted us to examine the effect of arachidonic acid on SNARE protein interactions with Munc18a, a protein known to prevent Syntaxin1a engagement into the SNARE complex in vitro. In the presence of arachidonic acid, we show that Munc18a can interact with the neuronal SNARE complex in a dose-dependent manner. We further demonstrate that arachidonic acid directly interacts with Syntaxin1a.     

The role of marine omega-3 (n-3) fatty acids in inflammatory processes, atherosclerosis and plaque stability

Atherosclerosis has an important inflammatory component and acute cardiovascular events can be initiated by inflammatory processes occurring in advanced plaques. Fatty acids influence inflammation through a variety of mechanisms; many of these are mediated by, or associated with, the fatty acid composition of cell membranes. Human inflammatory cells are typically rich in the n-6 fatty acid arachidonic acid, but the contents of arachidonic acid and of the marine n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can be altered through oral administration of EPA and DHA. Eicosanoids produced from arachidonic acid have roles in inflammation. EPA also gives rise to eicosanoids and these are usually biologically weak. EPA and DHA give rise to resolvins which are anti-inflammatory and inflammation resolving. EPA and DHA also affect production of peptide mediators of inflammation (adhesion molecules, cytokines, etc.). Thus, the fatty acid composition of human inflammatory cells influences their function; the contents of arachidonic acid, EPA and DHA appear to be especially important. The anti-inflammatory effects of marine n-3 polyunsaturated fatty acids (PUFAs) may contribute to their protective actions towards atherosclerosis and plaque rupture.     

Changes in Arachidonic Acid Levels and Formation and in Lipid Synthesis in the Human Neuroblastoma SK-N-BE During Retinoic Acid-Induced Differentiation

Abstract: We observed that retinoic acid, which differentiates the human neuroblastoma SK-N-BE into mature neurons, induced an elevation in levels of polyunsaturated fatty acids, especially arachidonic acid (20:4 n-6). This effect was not induced by phorbol myristate acetate, another differentiating agent. We then explored the effects of retinoic acid on the formation of arachidonic acid and of docosahexaenoic acid from precursors and on the de novo lipid synthesis from acetate at various stages of differentiation, which was assessed by morphological (cell number and neurite outgrowth) and biochemical (protein content and thymidine incorporation) criteria. At 3 days of incubation with retinoic acid, in the n-6 series, total conversion of linoleic acid, especially to 20:3 n-6, was elevated, in association with preferential incorporation of acetate into phospholipids; in contrast, at 8 days, synthesis of 20-carbon polyunsaturated fatty acids declined, in association with enhanced incorporation in triglycerides. In the n-3 series, eicosapentaenoic acid was converted to docosahexaenoic acid in SK-N-BE, but the conversion was not affected by retinoic acid. During the early stage of neuronal differentiation, therefore, enhanced production of 20-carbon polyunsaturated fatty acids from their precursors occurred, and newly formed fatty acids were preferentially incorporated in phospholipids, possibly in association with membrane deposition. When differentiation was completed, arachidonic acid formation and incorporation of acetate in phospholipids and cholesterol declined with enhanced labeling of storage lipids.