Anterior gradient 2 increases long-chain fatty acid uptake via stabilizing FABP1 and facilitates lipid accumulation

Anterior gradient 2 (AGR2), a protein disulfide isomerase (PDI), is a well-established oncogene. Here, we found that Agr2^-/- mice had a decreased fat mass and hepatic and serum lipid levels compared with their wild-type littermates after fasting, and exhibited reduced high-fat diet (HFD)-induced fat accumulation. Transgenic mice overexpressing AGR2 (Agr2/Tg) readily gained fat weight on a HFD but not a normal diet. Proteomic analysis of hepatic samples from Agr2^-/- mice revealed that depletion of AGR2 impaired long-chain fatty acid uptake and activation but did not affect de novo hepatic lipogenesis. Further investigations led to the identification of several effector substrates, particularly fatty acid binding protein-1 (FABP1) as essential for the AGR2-mediated effects. AGR2 was coexpressed with FABP1, and knockdown of AGR2 resulted in a reduction in FABP1 stability. Physical interactions of AGR2 and FABP1 depended on the PDI motif in AGR2 and the formation of a disulfide bond between these two proteins. Overexpression of AGR2 but not a mutant AGR2 protein lacking PDI activity suppressed lipid accumulation in cells lacking FABP1. Moreover, AGR2 deficiency significantly reduced fatty acid absorption in the intestine, which might be resulted from decreased fatty acid transporter CD36 in mice. These findings demonstrated a novel role of AGR2 in fatty-acid uptake and activation in both the liver and intestine, which contributed to the AGR2-mediated lipid accumulation, suggesting that AGR2 is an important regulator of whole-body lipid metabolism and down-regulation of AGR2 may antagonize the development of obesity.     

Alterations of polyunsaturated fatty acid metabolism in ovarian tissues of polycystic ovary syndrome rats

The metabolism of polyunsaturated fatty acids (PUFAs) remains poorly characterized in ovarian tissues of patients with polycystic ovary syndrome (PCOS). This study aimed to explore alterations in the levels of PUFAs and their metabolites in serum and ovarian tissues in a PCOS rat model treated with a high‐fat diet and andronate. Levels of PUFAs and their metabolites were measured using gas/liquid chromatography‐mass spectrometry after the establishment of a PCOS rat model. Only 3 kinds of PUFAs [linoleic acid, arachidonic acid (AA) and docosahexaenoic acid] were detected in both the circulation and ovarian tissues of the rats, and their concentrations were lower in ovarian tissues than in serum. Moreover, significant differences in the ovarian levels of AA were observed between control, high‐fat diet‐fed and PCOS rats. The levels of prostaglandins, AA metabolites via the cyclooxygenase (COX) pathway, in ovarian tissues of the PCOS group were significantly increased compared to those in the controls. Further studies on the mechanism underlying this phenomenon showed a correlation between decreased expression of phosphorylated cytosolic phospholipase A2 (p‐cPLA2) and increased mRNA and protein expression of COX2, potentially leading to a deeper understanding of altered AA and prostaglandin levels in ovarian tissues of PCOS rats.     

Specific long-chain fatty acids promote optimal growth of Frankia: accumulation and intracellular distribution of palmitic and propionic acid

Frankia isolates from nodules of the genera Casuarina (BR, S21, Thr), Allocasuarina (Allo2), and Gymnostoma (G80) were found to grow exponentially with high biomass yield and minimal sporangia formation in stirred propionate mineral medium when supplemented either with 2.4 μM palmitic acid (C16:0), pentadecanoic (C15:0), heptadecanoic (C17:0), or linoleic (C18:2, cis 9, 12) fatty acids. Strains also grew with lauric (C12:0) or myristic (C14:0) acids, but gave lower biomass yield. Stearic acid (C18:0) produced a good biomass yield, but cultures slowly accumulated sporangia; oleic acid (C18:1, cis-9) was detrimental to growth. Caprylic (C8:0) or capric (C10:0) acids proved to be prejudicial for long-term storage of Frankia strains. In experiments using labeled 1,2-dipalmitoyl phosphatidylcholine and palmitic acid, radioactivity bound rapidly to the insoluble, but solvent-extractable fraction of Frankia cells. In contrast, label from propionic acid accumulated in the cytosolic fraction. Therefore, the beneficial effect of some specific phospatidylcholines or free fatty acids on Frankia growth appears to result from their utilization as building blocks for the membrane, suggesting that membrane biosynthesis may be the limiting step for Frankia growth in unamended propionate mineral medium. Received: 9 October 1995 / Accepted: 24 February 1996     

Lysosomal release of cathepsins causes ischemic damage in the rat hippocampal slice and depends on NMDA-mediated calcium influx, arachidonic acid metabolism, and free radical production

NMDA-mediated calcium entry and reactive oxygen species (ROS) production are well-recognized perpetrators of ischemic neuronal damage. The current studies show that these events lead to the release of the protein hydrolase, cathepsin B, from lysosomes 2 h following 5-min oxygen–glucose deprivation in the rat hippocampal slice. This release reflects a lysosomal membrane permeabilization (LMP) and was measured as the appearance of diffuse immunolabeled cathepsin B in the cytosol of CA1 pyramidal neurons. Necrotic neuronal damage begins after the release of cathepsins and is prevented by inhibitors of either cathepsin B or D indicating that the release of cathepsins is an important mediator of severe damage. There was an increase in superoxide levels, measured by dihydroethidium fluorescence, at the same time as LMP and reducing ROS levels with antioxidants, Trolox or N -tert-butyl-α-phenyl nitrone, blocked LMP. Both LMP and ROS production were blocked by an NMDA channel blocker (MK-801) and by inhibitors of mitogen-activated protein kinase kinase (U0126), calcium-dependent/independent phospholipases A2 (methyl arachidonyl fluorophosphonate) but not calcium-independent phospholipases A2 (bromoenol lactone) and cyclooxygenase-2 (NS398). A cell-permeant specific inhibitor of calpain (PD150606) prevented LMP, but not ROS production. It is concluded that LMP results in part from calcium-initiated and extracellular signal-regulated kinase-initiated arachidonic acid metabolism, which produces free radicals; it also requires the action of calpain.     

Phosphatidic acid accumulation is an early response in the Cf-4/Avr4 interaction

The Cladosporium fulvum (Cf)-4 gene of tomato confers resistance to the fungus C. fulvum, expressing the corresponding avirulence (Avr)4 gene, which codes for an elicitor protein. Little is known about how such mechanisms work, but previous studies have shown that elicitor recognition activates Ca(2+) signalling and protein kinases, such as mitogen-activated protein kinase (MAPK) and calcium-dependent protein kinase (CDPK). Here, we provide evidence that a new signalling component, the lipid second messenger phosphatidic acid (PA), is produced within a few minutes of AVR4/Cf-4 interaction. Using transgenic tobacco cells expressing the tomato Cf-4-resistance gene as a model system, phospholipid signalling pathways were studied by pre-labelling the cells with (32)P(i) and assaying for the formation of lipid signals after challenge with the fungal elicitor AVR4. A dramatic rapid response was an increase in (32)P-PA, together with its metabolic product diacylglycerol pyrophosphate (DGPP). AVR4 increased the levels of PA and DGPP in a Cf-4(+)-, time- and dose-dependent manner, while the non-matching elicitor AVR9 did not trigger any response. In general, PA signalling can be triggered by two different pathways: via phospholipase D (PLD), which generates PA directly by hydrolysing structural phospholipids like phosphatidylcholine (PC), or via PLC, which generates diacylglycerol (DAG) that is subsequently phosphorylated to PA by DAG kinase (DGK). To determine the origin of the AVR4-induced PA formation, a PLD-specific transphosphatidylation assay and a differential (32)P-labelling protocol were used. The results clearly demonstrated that most PA was produced via the phosphorylation of DAG. Neomycin and U73122, inhibitors of PLC activity, inhibited AVR4-induced PA accumulation, suggesting that the increase in DGK activity was because of increased PLC activity producing DAG. Lastly, evidence is provided that PLC signalling and, in particular, PA production could play a role in triggering responses, such as the AVR4-induced oxidative burst. For example, PLC inhibitors inhibited the oxidative burst, and when PA was added to cells, an oxidative burst was induced.     

Hyperosmotic stress rapidly generates lyso-phosphatidic acid in Chlamydomonas

Plant cells are continuously exposed to environmental stresses such as hyper-osmolarity, and have to respond in order to survive. When 32P-labelled Chlamydomonas moewusii cells were challenged with NaCl, the formation of a new radiolabelled phospholipid was stimulated, which was barely detectable before stimulation. The phospholipid was identified as lyso-phosphatidic acid (LPA), and was the only lyso-phospholipid to be accumulated. The increase in LPA was dose- and time-dependent. When other osmotically active compounds were used, the formation of LPA was also induced with similar kinetics, although salts were better inducers than non-salts. At least part of the LPA was generated by phospholipase A2 (PLA2) hydrolysing phosphatidic acid (PA). This claim is based on PA formation preceding LPA production, and PLA2 inhibitors decreasing the accumulation of LPA and promoting the conversion of PA to diacylglycerol pyrophosphate. The latter is another metabolic derivative of PA that is implicated in cell signalling. The involvement of multiple lipid-signalling pathways in hyperosmotic stress responses is discussed.     

Induction of secretory phospholipase A2 in reactive astrocytes in response to transient focal cerebral ischemia in the rat brain

Although mRNA expression of group IIA secretory phospholipase A2 (sPLA2-IIA) has been implicated in responses to injury in the CNS, information on protein expression remains unclear. In this study, we investigated temporal and spatial expression of sPLA2-IIA mRNA and immunoreactivity in transient focal cerebral ischemia induced in rats by occlusion of the middle cerebral artery. Northern blot analysis showed a biphasic increase in sPLA2-IIA mRNA expression following 60-min of ischemia-reperfusion: an early phase at 30 min and a second increase at a late phase ranging from 12 h to 14 days. In situ hybridization localized the early-phase increase in sPLA2-IIA mRNA to the affected ischemic cortex and the late-phase increase to the penumbral area. Besides sPLA2-IIA mRNA, glial fibrillary acidic protein (GFAP) and cyclo-oxygenase-2 mRNAs, but not cytosolic PLA2, also showed an increase in the penumbral area at 3 days after ischemia-reperfusion. Immunohistochemistry of sPLA2-IIA indicated positive cells in the penumbral area similar to the GFAP-positive astrocytes but different from the isolectin B4-positive microglial cells. Confocal microscopy further confirmed immunoreactivity of sPLA2-IIA in reactive astrocytes but not in microglial cells. Taken together, these results demonstrate for the first time an up-regulation of the inflammatory sPLA2-IIA in reactive astrocytes in response to cerebral ischemia-reperfusion.     

Fat mass and obesity-associated protein regulates lipogenesis via m6A modification in fatty acid synthase mRNA

As the first identified N⁶-methyladenosine (m⁶A) demethylase, fat mass and obesity-associated (FTO) protein is associated with fatty acid synthase (FASN) and lipid accumulation. However, little is known about the regulatory role of FTO in the expression of FASN and de novo lipogenesis through m⁶A modification. In this study, we used FTO small interfering RNA to explore the effects of FTO knockdown on hepatic lipogenesis and its underlying epigenetic mechanism in HepG2 cells. We found that knockdown of FTO increased m⁶A levels in total RNA and enhanced the expression of YTH domain family member 2 which serves as the m⁶A-binding protein. The de novo lipogenic enzymes and intracellular lipid content were significantly decreased under FTO knockdown. Mechanistically, knockdown of FTO dramatically enhanced m⁶A levels in FASN messenger RNA (mRNA), leading to the reduced expression of FASN mRNA through m⁶A-mediated mRNA decay. The protein expressions of FASN along with acetyl CoA carboxylase and ATP-citrate lyase were further decreased, which inhibited de novo lipogenesis, thereby resulting in the deficiency of lipid accumulation in HepG2 cells and the induction of cellular apoptosis. The results reveal that FTO regulates hepatic lipogenesis via FTO-dependent m⁶A demethylation in FASN mRNA and indicate the critical role of FTO-mediated lipid metabolism in the survival of HepG2 cells. This study provides novel insights into a unique RNA epigenetic mechanism by which FTO mediates hepatic lipid accumulation through m⁶A modification and indicates that FTO could be a potential target for obesity-related diseases and cancer.     

Spatio-temporal expression of patatin-like lipid acyl hydrolases and accumulation of jasmonates in elicitor-treated tobacco leaves are not affected by endogenous levels of salicylic acid

We have previously isolated three tobacco genes (NtPat) encoding patatin-like proteins, getting rapidly induced during the hypersensitive response (HR) to tobacco mosaic virus, in advance to jasmonate accumulation. NtPAT enzymes are lipid acyl hydrolases that display high phospholipase A2 (PLA2) activity and may mobilize fatty acid precursors of oxylipins. Here, we performed a detailed study of NtPat gene regulation under various biotic and abiotic stresses. PLA2 activity was poorly induced in response to drought, wounding, reactive oxygen intermediates, salicylic acid (SA) or methyl-jasmonate (MJ) whereas the ethylene (ET) precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), provoked a moderate induction. In contrast, PLA2 activity was strongly induced when ACC was combined with MJ, and in response to the bacterium Erwinia carotovora or to the fungus Botrytis cinerea, as well as to treatment with beta-megaspermin, a cell death-inducing protein elicitor. A simplified system based on the infiltration of beta-megaspermin into leaves was used to dissect the spatio-temporal activation of PLA2 activity with regards to the accumulation of jasmonates and to the influence of endogenous SA. NtPat-encoded PLA2 activity was rapidly induced in the infiltrated zone before the appearance of cell death and with some delay in the surrounding living cells. A massive accumulation of 12-oxo-phytodienoic and jasmonic acids occurred in the elicitor-infiltrated zone, but only low levels were detectable outside this area. A similar picture was found in SA-deficient plants, showing that in tobacco, accumulation of jasmonates is not affected by the concomitant HR-induced build-up of endogenous SA. Finally, ET-insensitive plants showed a weakened induction of PLA2 activity outside the elicitor-infiltrated tissue.     

Regulation of free arachidonic acid levels in isolated salivary glands from the lone star tick: A role for dopamine

An important regulatory step for prostaglandin synthesis is the availability of the precursor, free arachidonic acid (AA). In isolated salivary glands of the lone star tick, Amblyomma americanum (L.), the level of free AA appears to depend on higher phospholipase A₂ (PLA₂) activity rather than decreased rates of re-esterification by lysophosphatide acyl transferase (LAT). This conclusion is supported by experiments where inhibition of LAT with merthiolate was without effect, while the calcium ionophore A23187, a PLA₂ stimulant, increased levels of free AA. The PLA₂ activity in A. americanum was reduced by the substrate analog, PLA₂ inhibitor, oleyloxyethyl phosphorylcholine in a dose-dependent manner, but was insensitive to the other mammalian PLA₂ inhibitors mepacrine (20μM), aristolochic acid (45μM), and dexamethasone (50μM). No substrate preference was observed for the functional group of the phospholipid, with phosphatidylcholine and phosphatidylethanolamine being equal sources of AA in A23187-stimulated glands. Compared to phospholipids containing other fatty acids, only arachidonyl-phospholipid (arachidonyl-PL) was significantly hydrolyzed by PLA₂ activity in A23187-stimulated glands. Dopamine was as effective as A23187 as a stimulant of PLA₂ activity in isolated glands, but this effect was abolished in the presence of the calcium channel blocking agent verapamil. It is concluded that free AA levels in tick salivary glands are increased through activation of a Type IV-like PLA₂ following an increase of intracellular calcium caused by the opening of voltage-dependent calcium channels due to dopamine stimulation. © 1995 Wiley-Liss, Inc.     

Okadaic acid causes breakdown of self-incompatibility in Brassica oleracea: evidence for the involvement of protein phosphatases in the incompatible response

Summary Detached pistils from inbred lines of Brassica oleracea L. var alboglabra were fed with okadaic acid (OA), an inhibitor of serine/threonine protein phosphatases, via the transpiration stream. Following self-pollination, pollen tubes were observed to have grown into or through the styles of pistils treated with OA, but not those of untreated controls. Treatment with 1 M OA was sufficient to completely overcome self-incompatibility (SI) in an inbred line homozygous for the S63 allele, though an OA concentration of 5 M was required to cause breakdown of SI in an inbred line homozygous for the S29 allele. At the higher concentration used, pollen tube growth was arrested before the pollen tubes reached the ovary, but this effect was also noted in cross-pollinated styles treated in the same manner. These data provide evidence for the involvement of type 1 and/or type 2A protein phosphatases in the Brassica SI signal transduction mechanism. Present address after November 1993: Department of Biology, Colorado State University, Fort Collins, Colorado, USA     

Citrus abscission and Arabidopsis plant decline in response to 5-chloro-3-methyl-4-nitro-1H-pyrazole are mediated by lipid signalling

The compound 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMNP) is a pyrazole-derivative that induces abscission selectively in mature citrus (Citrus sinensis) fruit when applied to the canopy and has herbicidal activity on plants when applied to roots. Despite the favourable efficacy of this compound, the mode of action remains unknown. To gain information about the mode of action of CMNP, the effect of application to mature citrus fruit and Arabidopsis thaliana roots was explored. Peel contact was essential for mature fruit abscission in citrus, whereas root drenching was essential for symptom development and plant decline in Arabidopsis. CMNP was identified as an uncoupler in isolated soybean (Glycine max) mitochondria and pea (Pisum sativum) chloroplasts and an inhibitor of alcohol dehydrogenase in citrus peel, but not an inhibitor of protoporphyrinogen IX oxidase. CMNP treatment reduced ATP content in citrus peel and Arabidopsis leaves. Phospholipase A₂ (PLA₂) and lipoxygenase (LOX) activities, and lipid hydroperoxide (LPO) levels increased in flavedo of citrus fruit peel and leaves of Arabidopsis plants treated with CMNP. An inhibitor of PLA₂ activity, aristolochic acid (AT), reduced CMNP-induced increases in PLA₂ and LOX activities and LPO levels in citrus flavedo and Arabidopsis leaves and greatly reduced abscission in citrus and delayed symptoms of plant decline in Arabidopsis. However, AT treatment failed to halt the reduction in ATP content. Reduction in ATP content preceded the increase in PLA₂ and LOX activities, LPO content and the biological response. The results indicate a link between lipid signalling, abscission in citrus and herbicidal damage in Arabidopsis.     

Calcium-calmodulin plays a major role in bradykinin-induced arachidonic acid release by bovine aortic endothelial cells

We provided evidence that calcium-calmodulin plays a major role in bradykinin-induced arachidonic acid release by bovine aortic endothelial cells. In cells labeled for 16 hr with ³H-arachidonic acid, ionomycin and Ca²⁺-mobilizing hormones such as bradykinin, thrombin and platelet activating factor induced arachidonic acid release. However, arachidonic acid release was not induced by agents known to increase cyclic AMP (forskolin, isoproterenol) or cyclic GMP (sodium nitroprusside). Bradykinin induced the release of arachidonic acid in a dose-dependent manner (EC₅₀ = 1.6 ± 0.7 nM). This increase was rapid, reaching a maximal value of fourfold above basal level in 15 min. In a Ca²⁺-free medium, bradykinin was still able to release arachidonic acid but with a lower efficiency. Quinacrine (300 μM), a blocker of PLA₂, completely inhibited bradykinin-induced arachidonic acid release. The B₂ bradykinin receptor antagonist HOE-140 completely inhibited bradykinin-induced arachidonic acid release. The B₁-selective agonist DesArg⁹-bradykinin was inactive and the B₁-selective antagonist [Leu⁸]DesArg⁹-bradykinin had no significant effect on bradykinin-induced arachidonic acid release. The phospholipase C inhibitor U-73122 (100 μM) decreased bradykinin-induced arachidonic acid release. The calmodulin inhibitor W-7 (50 μM) drastically reduced the bradykinin- and ionomycin-induced arachidonic acid release. Also, forskolin decreased bradykinin-induced arachidonic acid release. These results suggest that the activation of PLA₂ by bradykinin in BAEC is a direct consequence of phospholipase C activation. Ca²⁺-calmodulin appears to be the prominent activator of PLA₂ in this system. © 1996 Wiley-Liss, Inc.     

Dietary eicosapentaenoic acid supplementation accentuates hepatic triglyceride accumulation in mice with impaired fatty acid oxidation capacity

Reduced mitochondrial fatty acid (FA) β-oxidation can cause accumulation of triglyceride in liver, while intake of eicosapentaenoic acid (EPA) has been recommended as a promising novel therapy to decrease hepatic triglyceride content. However, reduced mitochondrial FA β-oxidation also facilitates accumulation of EPA. To investigate the interplay between EPA administration, mitochondrial activity and hepatic triglyceride accumulation, we investigated the effects of EPA administration to carnitine-deficient mice with impaired mitochondrial FA β-oxidation. C57BL/6J mice received a high-fat diet supplemented or not with 3% EPA in the presence or absence of 500 mg mildronate/kg/day for 10 days. Liver mitochondrial and peroxisomal oxidation, lipid classes and FA composition were determined. Histological staining was performed and mRNA level of genes related to lipid metabolism and inflammation in liver and adipose tissue was determined. Levels of pro-inflammatory eicosanoids and cytokines were measured in plasma. The results showed that mildronate treatment decreased hepatic carnitine concentration and mitochondrial FA β-oxidation and induced severe triglyceride accumulation accompanied by elevated systemic inflammation. Surprisingly, inclusion of EPA in the diet exacerbated the mildronate-induced triglyceride accumulation. This was accompanied by a considerable increase of EPA accumulation while decreased total n-3/n-6 ratio in liver. However, inclusion of EPA in the diet attenuated the mildronate-induced mRNA expression of inflammatory genes in adipose tissue. Taken together, dietary supplementation with EPA exacerbated the triglyceride accumulation induced by impaired mitochondrial FA β-oxidation. Thus, further thorough evaluation of the potential risk of EPA supplementation as a therapy for NAFLD associated with impaired mitochondrial FA oxidation is warranted.     

Protein kinase C-independent activation of a novel nonspecific phospholipase C pathway by phorbol myristate acetate releases arachidonic acid for prostaglandin synthesis in MC3T3-E1 osteoblasts

The effects of phorbol myristate acetate, an activator of protein kinase C, on the release of [³H]arachidonic acid and prostaglandin synthesis were studied in an osteoblast cell line (MC3T3-E1). Phorbol myristate acetate (20 uM) liberated 16 and 55% of the [³H]arachidonate in prelabeled phosphatidylinositol and phosphatidylethanolamine, respectively, and evoked a 19-fold stimulation in the synthesis of prostaglandin E₂. Phorbol myristate acetate doubled the cellular mass of 1,2-diacylglycerol and stimulated the liberation of [³H]arachidonate from the diacylglycerol pool in prelabeled cells. The diacylglycerol lipase inhibitor RHC 80267 blocked 75–80% of the phorbol ester-promoted (total) cellular liberation of [³H]arachidonic acid and production of prostaglandin E₂. In comparison, the release of [³H]arachidonate from phosphatidylethanolamine (but not phosphatidylinositol) was only partially antagonized (to the same degree) by the PLA₂ inhibitor p-bromophenacylbromide and the protein kinase C inhibitor Et-18-OMe. PMA-induced formation of diacylglycerol or synthesis of PGE₂ was not affected by the prior inhibition of protein kinase C. Therefore, we have shown a novel pathway for the liberation of arachidonic acid in osteoblasts involving the nonspecific hydrolysis of phosphatidylinositol and phosphatidylethanolamine by phospholipase C followed by the deesterification of diacylgycerol. This pathway can be activated by a phorbol ester through a protein kinase C-independent mechanism.     

Vitamin D3 inhibits fatty acid synthase expression by stimulating the expression of long-chain fatty-acid-CoA ligase 3 in prostate cancer cells

FAS and FACL3 are enzymes of fatty acid metabolism. In our previous studies, we found that FAS and FACL3 genes were vitamin D3-regulated and involved in the antiproliferative effect of 1alpha,25(OH)2D3 in the human prostate cancer LNCaP cells. Here, we elucidated the mechanism behind the downregulation of FAS expression by vitamin D3. Triacsin C, an inhibitor of FACL3 activity, completely abolished the downregulation of FAS expression by vitamin D3, whereas an inhibitor of FAS activity, cerulenin, had no significant effect on the upregulation of FACL3 expression by vitamin D3 in LNCaP cells. In human prostate cancer PC3 cells, in which FACL3 expression is not regulated by vitamin D3, no regulation of FAS expression was seen. This suggests that the downregulation of FAS expression by vitamin D3 is mediated by vitamin D3 upregulation of FACL3 expression. Myristic acid, one of the substrates preferential for FACL3, enhanced the repression of FAS expression by vitamin D3. The action of myristic acid was abrogated by inhibition of FACL3 activity, suggesting that the enhancement in the downregulation of FAS expression by vitamin D3 is due to the formation of myristoyl-CoA. The data suggest that vitamin D3-repression of FAS mRNA expression is the consequence of feedback inhibition of FAS expression by long chain fatty acyl-CoAs, which are formed by FACL3 during its upregulation by vitamin D3 in human prostate cancer LNCaP cells.     

Alterations in phospholipid and fatty acid lipid profiles in primary neocortical cells during oxidant-induced cell injury

Specific phospholipids and fatty acids altered during oxidant-induced neuronal cell injury were determined using electrospray ionization mass spectrometry (ESI-MS) and ion trapping. The oxidants hydrogen peroxide (H(2)O(2), 0-1000 microM) and tert-butylhydroperoxide (TBHP, 0-400 microM) induced time- and concentration-dependent increases in reactive oxygen species in primary cultures of mouse neocortical cells as determined by 2',7'-dichlorofluorescein diacetate staining and thiobarbituric acid formation. ESI-MS analysis of 26 m/z values, representing 42 different phospholipids, demonstrated that H(2)O(2) and TBHP increased the abundance of phospholipids containing polyunsaturated fatty acids, but had minimal affect on those containing mono- or di-unsaturated fatty acids. These increases correlated to time-dependent increase in 16:1-20:4, 16:0-20:4, 18:1-20:4 and 18:0-20:4 phosphatidylcholine. Oxidant exposure also increased mystric (14:0), palmitic (16:0), and stearic (18:0) acid twofold, oleic acid (18:1) two- to threefold, and arachidonic acid (20:4) fourfold, compared to controls. Increases in arachidonic acid levels occurred prior to increases in the phospholipids, but after increases in ROS, and correlated to increases in oxidized arachidonic acid species, specifically [20:4-OOH]-H(2)O-, 20:4-OH-, and Tri-OH-20:4-arachidonic acid. Treatment of cells with methyl arachidonyl flourophosphonate an inhibitor of Group IV and VI PLA(2), decreased oxidant-induced arachidonic acid release, while bromoenol lactone, an inhibitor of Group VI PLA(2), did not. Collectively, these data identify phospholipids and fatty acids altered during oxidant treatment of neurons and suggest differential roles for Group IV and VI PLA(2) in oxidant-induced neural cell injury.     

Bicuculline induces free fatty acid release from phospholipids in Neuro-2A cells in culture

This study characterizes free fatty acid release in a neuroblastoma cell line (Neuro-2A), a potential model system for the study of factors that control phospholipase A₂ in neurons. Two compounds, bicuculline (an antagonist at -aminobutyric acid receptors), and A23187 (a Ca²⁺ ionophore), were examined. The release of endogenous fatty acids and the turnover of radiolabeled arachidonic and docosahexaenoic acids were measured. The cells actively incorporated radiolabeled fatty acids into various glycerolipid pools. Both endogenous fatty acids and radiolabeled fatty acids were released from glycerolipids in a time-dependent manner. Phosphatidylcholine was a major source of released fatty acids. Release of free fatty acids was markedly stimulated by both bicuculline and A23187. We conclude that the Neuro-2A cell contain phospholipase activity that is sensitive to Ca²⁺ ionophore and bicuculline, and may provide a good system for further studies on the regulation of phospholipase A₂ in neurons.Abbteviations 160 palmitic acid - 180 stearic acid - 181 oleic acid - 182 linoleic acid - 183 linolenic acid - 204 arachidonic acid - 226 docosahexaenoic acid - DG diacylglycerol - FAME fatty acid methyl ester - FFA free fatty acid - GABA -aminobutyric acid - PA phosphatidic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - TG triacylglycerol     

Chronic lithium administration attenuates up-regulated brain arachidonic acid metabolism in a rat model of neuroinflammation

Neuroinflammation, caused by a 6-day intracerebroventricular infusion of lipopolysaccharide (LPS) in rats, is associated with the up-regulation of brain arachidonic acid (AA) metabolism markers. Because chronic LiCl down-regulates markers of brain AA metabolism, we hypothesized that it would attenuate increments of these markers in LPS-infused rats. Incorporation coefficients k* of AA from plasma into brain, and other brain AA metabolic markers, were measured in rats that had been fed a LiCl or control diet for 6 weeks, and subjected in the last 6 days on the diet to intracerebroventricular infusion of artificial CSF or of LPS. In rats on the control diet, LPS compared with CSF infusion increased k* significantly in 28 regions, whereas the LiCl diet prevented k* increments in 18 of these regions. LiCl in CSF infused rats increased k* in 14 regions, largely belonging to auditory and visual systems. Brain cytoplasmic phospholipase A(2) activity, and prostaglandin E(2) and thromboxane B(2) concentrations, were increased significantly by LPS infusion in rats fed the control but not the LiCl diet. Chronic LiCl administration attenuates LPS-induced up-regulation of a number of brain AA metabolism markers. To the extent that this up-regulation has neuropathological consequences, lithium might be considered for treating human brain diseases accompanied by neuroinflammation.