Action Of Free Fatty Acids Research Articles

Dual β-oxidation pathway and transcription factor engineering for methyl ketones production in Saccharomyces cerevisiae

Methyl ketones (MK) are highly valuable fatty acid derivatives with broad applications. Microbes based biosynthesis represents an alternative route for production of these usually fossil based chemicals. In this study, we reported metabolic engineering of Saccharomyces cerevisiae to produce MK, including 2-nonanone, 2-undecanone, 2-tridecanone and 2-pentadecanone. Besides enhancing inherent peroxisomal fatty acids β-oxidation cycle, a novel heterologous cytosolic fatty acids β-oxidation pathway was constructed, and this resulted in an increased production of MK by 2-fold. To increase carbon fluxes to methyl ketones, the supply of precursors was enhanced by engineering lipid metabolism, including improving the intracellular biosynthesis of acyl-CoAs, weakening the consumption of acyl-CoAs for lipids storage, and reinforcing activation of free fatty acids to acyl-CoAs. Hereby the titer of MK was improved by 7-fold, reaching 143.72 mg/L. Finally, transcription factor engineering was employed to increase the biosynthesis of methyl ketones and it was found that overexpression of ADR1 can mimic the oleate activated biogenesis and proliferation of peroxisomes, which resulted in a further increased production of MK by 28%. With these modifications and optimization, up to 845 mg/L total MK were produced from glucose in fed-batch fermentation, which is the highest titer of methyl ketones reported produced by fungi.

The Global Regulator CcpA of Listeria monocytogenes Confers Sensitivity to Antimicrobial Fatty Acids.

Free fatty acids (FFAs) are known to exhibit antimicrobial and anti-virulent properties against bacterial pathogens. Specific FFAs, such as lauric acid (LA; C12:0), exert both effects against the foodborne pathogen Listeria monocytogenes: at low levels, LA acts to inhibit the activity of the virulence regulator PrfA, whereas at higher levels, LA inhibits bacterial growth. Deletion of prfA is known to promote tolerance toward antimicrobial FFAs, suggesting that the response of L. monocytogenes to anti-virulent and antimicrobial FFAs could be linked. In this study, we explored the response of L. monocytogenes toward antimicrobial FFAs holding an anti-virulence activity by isolating strains that can grow at high concentrations of LA. We found that LA-tolerant isolates carry mutations in the gene encoding the global regulator CcpA. Importantly, we discovered that mutation or deletion of ccpA protect L. monocytogenes against the antimicrobial activity of FFAs, whereas the ccpA mutants remain sensitive toward FFA’s PrfA inhibitory effect. A regulatory link involving CcpA, connecting the response toward the antimicrobial and anti-virulence activities of FFAs, is therefore unlikely. To further study how deletion of ccpA promotes FFA tolerance, we performed a transcriptomic analysis of the response to LA. Our data indicated that the FFA-tolerant phenotype of the ∆ccpA strain is not induced upon LA exposure but appears to be an inherent phenotypic trait of the ccpA deletion mutation. Interestingly, we found that the bacterial surface of L. monocytogenes becomes more hydrophilic upon deletion of ccpA, and we demonstrate that CcpA plays a role in the response of L. monocytogenes to other stress conditions, including low pH and antibiotics. Altogether, our study revealed that regulatory activities of CcpA lead to an increased hydrophobicity of the bacterial surface, which may confer sensitivity of L. monocytogenes against the antimicrobial activity of FFAs. Notably, CcpA is not involved in responding to the PrfA inhibitory effect of FFAs, showing that FFA-tolerant strains can still be targeted by the anti-virulent activity of FFAs.

Comparative examination of levodopa pharmacokinetics during simultaneous administration with lactoferrin in healthy subjects and the relationship between lipids and COMT inhibitory activity in vitro

ABSTRACT Background: Lactoferrin (bLF) is an iron-binding multifunctional protein that is abundant in milk. In mice, it inhibits catechol-O-methyltransferase (COMT) activity and increases blood levodopa levels. However, the clinical effects are unknown. Objective: The objective of this study was to determine the effect of bLF on the kinetics of levodopa in blood. Design: The effects of the concomitant administration of a combined formulation of levodopa and an aromatic amino acid decarboxylase inhibitor and bLF on the concentration of levodopa in blood and its metabolism were assessed in eight healthy subjects. In addition, we analyzed the association with clinical factors and evaluated whether clinical factors affected the COMT inhibitory activity of bLF in vitro. Results: Although not statistically significant, the peak plasma concentration (C max) of levodopa increased by 18.5%. From the results of the stratified analysis of total cholesterol, a relationship with ΔC max was predicted. Therefore, bLF was reacted with cholesterol in the presence of lecithin and sodium deoxycholate in vitro to evaluate COMT inhibitory activity, and an increase in inhibitory activity was observed. By contrast, the ester compound cholesteryl oleate had no effect. The inhibitory activity of free fatty acids, which are known to interact with bLF, was also enhanced. Conclusion: The COMT inhibitory activity of bLF is not effective in elevating blood levodopa levels. However, in humans with high lipid levels, such as cholesterol, interactions may enhance the inhibitory effect, resulting in the enhanced absorption of levodopa. Trial registration: ID, UMIN000026787, registered 30 March 2017; URL, https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000030749 Trial registration: UMIN Japan identifier: UMIN000026787

Antibacterial Activity of Free Fatty Acids, Potassium Soap, and Fatty Acids Methyl Esters from VCO (Virgin Coconut Oil)

Virgin coconut oil is edible oil obtained from fresh coconut meat (Cocos nucifera L.) and is widely used for health such as for alternative healthy diets, anti-bacterial, anti-virus and anti-fungal. This is because VCO contains a lot of MCFA (Medium-Chain Fatty Acids) such as lauric acid (65.84%), myristic acid (13.16%) and caprylic acid (7.08%). The antibacterial ability of MCFAs is one of the most studied, but free fatty acids from the hydrolysis of VCO have not been done. The objective of the research is to derivatize VCO and determine the potential antibacterial activity of the product. The results of the research are as follows. (1) K-soap (solid, white, melting point 46-124 °C). (2) Free fatty acids (liquid, colorless, boiling point 266-296 °C, density 0.93 g.mL−1, refractive index 1.44, viscosity 27.18 cP, acid value 184.54, saponification value 296.59 and ester value 112.05). (3) Fatty acid methyl ester (liquid, colorless, boiling point 260-262 °C, density 0.89 g.mL−1, refractive index 1.43, viscosity 3.36 cP, acid value 0.33, saponification value 268.6 and ester value 268.27). VCO and its derivatives at concentrations of 2 and 1% are active as antibacterial agents against Escherichia coli and Staphylococcus aureus.

An “Omics” Approach for Lipid Oxidation in Foods: The Case of Free Fatty Acids in Bulk Purified Olive Oil

Monitoring or preventing oxidation processes in foods, particularly heterogeneous systems or highly oxidizable lipids, has become a primary issue. Traditional approaches often fail in pursuing this aim. At the same time, the prediction in laboratory of the efficacy of antioxidant molecules and technologies often fails when challenging reality. A change in the traditional paradigm used to conceive oxidation processes and their monitoring could be a key. In the present work, an “omics” approach is suggested for the evaluation of oxidation in food lipids and of the role played by minor compounds. This holistic and comprehensive, hypothesis‐generating approach is applied to a typical research case: highly‐purified olive oil added with increasing amounts of purified free fatty acids (FFA). A comprehensive profile (“oxidome”) of oxidation products is outlined during the accelerated oxidation. The pathways considered are FFA and triacyglycerol oxidation, triacyglycerol polymerization, volatile compounds formation as well as their further oxidation. FFA affectes the overall balance of the oxidation pathways and consequently causes a drift in the evolution of the pattern of oxidation products. The balance between accelerating activity of FFA toward triacylglycerol oxidation and their high susceptibility to undergo oxidation turnes out to be dose‐dependent and time‐related, and shiftes the resulting oxidation profile of the oil. The omics approach to oxidation products profiling provides new insight into oxidation processes. “Oxidomics” could be helpful to deepen the insight into unresolved issues of oxidation, going beyond the usual single‐ or few‐marker approach.Practical Applications: This research suggests a new paradigm for approaching food lipid oxidation. Unresolved practical issues regarding food spoilage and shelf‐life, waste reduction, keeping of healthy properties are increasing along the increasing formulation of complex and dispersed systems, use of healthy highly unsaturated fatty acids and bioactive antioxidants, attention paid by consumers and industries to nutritional, sensory, health properties of foods. The new paradigm suggests going beyond the usually adopted single‐ or few‐marker approach and to consider the pattern of oxidation reactions and products as a whole, in order to better describe and predict its changes and the effects of external factors as well as added molecules.“Oxidomics” is a new paradigm for lipid oxidation, with a holistic approach to the system and evaluating a pattern of oxidation products and its changes.

Abstract P4-12-02: Serum-triglycerides among triple negative breast cancer patients as a biomarker of poor outcome

Abstract Backgound: Obesity and related metabolic imbalances, including increased activity of free fatty acids, may promote tumor growth and metastasis. Fatty acids are mainly stored as triacylglycerols. Yet, the role of serum-triglycerides on breast cancer prognosis is still undefined. Methods: A population based survival study among 575 breast cancer patients identified within the Tromsø study during 1979-2008, was conducted. Pre-diagnostic serum triglycerides, high density lipoprotein-cholesterol, total cholesterol, height and weight were measured. Histopathological and clinical data were obtained from medical records, and hormone receptor, HER2 status, and Ki-67 were re-analyzed on tissue microarray blocks. Multivariate Cox proportional Hazard regression models were used to study the associations between patient characteristics including s-triglycerides, and breast cancer survival. Results: Among 575 women with invasive breast cancer (stage 1-3), a total of 87 women were diagnosed with triple negative breast cancer (TNBC). Patients diagnosed with TNBC, compared to non-TNBC, were likely to be younger at diagnosis (55.3 vs 57.9 years, p=0.061), they had larger tumors (29.7 mm vs 22.5 mm, p=0.001), and higher Ki-67 (31.1% vs 15.9%, p<0.001). After a mean follow-up of 8.4 years, TNBC patients with above median levels of s-triglycerides (> 0.98mmol/L) compared to TNBC patients with below median levels of s-triglycerides (≤ 0.98mmol/L) had 3.0 times higher risk for breast cancer recurrence or breast cancer specific death (HR 3.02, 95% CI 1.21-7.55), and 3.4 times higher overall mortality risk (HR 3.41, 95% CI 1.38-8.45). Among the TNBC patients, women with above median s-triglycerides had 15% lower 5-year disease-free survival (76% vs 91%) and 18% lower 5-year overall survival (74% vs 92%) compared to women with below median s-triglycerides. Conclusions: Our results strongly support s-triglycerides as an important biomarker for breast cancer outcomes among triple negative breast cancer patients. Table 1: Multivariable adjusted Hazard Ratios (HRs) for incidence breast cancer recurrence or breast cancer specific death, and incidence overall mortality by pre-diagnostic serum-triglycerides among non-triple negative breast cancer (TNBC) and TNBC patients Non-TNBC, n=488 TNBC, n=87 Recurrence or breast cancer specific death (n=90)Overall mortality (n=104) Recurrence or breast cancer specific death (n=24)Overall mortality (n=33) nHR (95% CI)HR (95% CI)nHR (95% CI)HR (95% CI)s-Triglycerides Median ≤ 0.98 mmol/l2571.001.00431.001.00> 0.98 mmol/l2310.87 (0.56-1.35)1.06 (0.70-1.62)443.02 (1.21-7.55)3.41 (1.38-8.45) Tertiles ≤ 0.82 mmol/l1731.001.00261.001.000.83 – 1.22 mmol/l1660.74 (0.44-1.23)0.81 (0.49-1.36)311.37 (0.38-4.98)0.98 (0.31-3.08)≥ 1.23 mmol/l1490.88 (0.50-1.54)1.17 (0.66-1.96)306.63 (1.64-19.3)3.87 (1.52-12.0)p-trend 0.4950.357 0.0050.007 Multivariate Cox proportional Hazard regression model. Adjusted for BMI and age at attendance (continuous), age at diagnosis (continuous), breast cancer stage at diagnosis (categorical), and current smoking (categorical). Abbreviation: CI, confidence interval; n, number of cases; TNBC, triple negative breast cancer Citation Format: Lofterød T, Mortensen ES, Nalwoga H, Wilsgaard T, Frydenberg H, Risberg T, Eggen AE, McTiernan A, Aziz S, Wist EA, Reitan JB, Akslen LA, Thune I. Serum-triglycerides among triple negative breast cancer patients as a biomarker of poor outcome [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-12-02.

Enhancement of E. coli acyl-CoA synthetase FadD activity on medium chain fatty acids.

FadD catalyses the first step in E. coli beta-oxidation, the activation of free fatty acids into acyl-CoA thioesters. This activation makes fatty acids competent for catabolism and reduction into derivatives like alcohols and alkanes. Alcohols and alkanes derived from medium chain fatty acids (MCFAs, 6–12 carbons) are potential biofuels; however, FadD has low activity on MCFAs. Herein, we generate mutations in fadD that enhance its acyl-CoA synthetase activity on MCFAs. Homology modeling reveals that these mutations cluster on a face of FadD from which the co-product, AMP, is expected to exit. Using FadD homology models, we design additional FadD mutations that enhance E. coli growth rate on octanoate and provide evidence for a model wherein FadD activity on octanoate can be enhanced by aiding product exit. These studies provide FadD mutants useful for producing MCFA derivatives and a rationale to alter the substrate specificity of adenylating enzymes.

Xanthomonas campestris RpfB is a fatty Acyl-CoA ligase required to counteract the thioesterase activity of the RpfF diffusible signal factor (DSF) synthase.

In Xanthomonas campestris pv. campestris (Xcc), the proteins encoded by the rpf (regulator of pathogenicity factor) gene cluster produce and sense a fatty acid signal molecule called diffusible signalling factor (DSF, 2(Z)-11-methyldodecenoic acid). RpfB was reported to be involved in DSF processing and was predicted to encode an acyl-CoA ligase. We report that RpfB activates a wide range of fatty acids to their CoA esters in vitro. Moreover, RpfB can functionally replace the paradigm bacterial acyl-CoA ligase, Escherichia coli FadD, in the E. coli ß-oxidation pathway and deletion of RpfB from the Xcc genome results in a strain unable to utilize fatty acids as carbon sources. An essential RpfB function in the pathogenicity factor pathway was demonstrated by the properties of a strain deleted for both the rpfB and rpfC genes. The ΔrpfB ΔrpfC strain grew poorly and lysed upon entering stationary phase. Deletion of rpfF, the gene encoding the DSF synthetic enzyme, restored normal growth to this strain. RpfF is a dual function enzyme that synthesizes DSF by dehydration of a 3-hydroxyacyl-acyl carrier protein (ACP) fatty acid synthetic intermediate and also cleaves the thioester bond linking DSF to ACP. However, the RpfF thioesterase activity is of broad specificity and upon elimination of its RpfC inhibitor RpfF attains maximal activity and its thioesterase activity proceeds to block membrane lipid synthesis by cleavage of acyl-ACP intermediates. This resulted in release of the nascent acyl chains to the medium as free fatty acids. This lack of acyl chains for phospholipid synthesis results in cell lysis unless RpfB is present to counteract the RpfF thioesterase activity by catalysing uptake and activation of the free fatty acids to give acyl-CoAs that can be utilized to restore membrane lipid synthesis. Heterologous expression of a different fatty acid activating enzyme, the Vibrio harveyi acyl-ACP synthetase, replaced RpfB in counteracting the effects of high level RpfF thioesterase activity indicating that the essential role of RpfB is uptake and activation of free fatty acids.

Physiological Role of Acyl Coenzyme A Synthetase Homologs in Lipid Metabolism in Neurospora crassa

Acyl coenzyme A (CoA) synthetase (ACS) enzymes catalyze the activation of free fatty acids (FAs) to CoA esters by a two-step thioesterification reaction. Activated FAs participate in a variety of anabolic and catabolic lipid metabolic pathways, including de novo complex lipid biosynthesis, FA β-oxidation, and lipid membrane remodeling. Analysis of the genome sequence of the filamentous fungus Neurospora crassa identified seven putative fatty ACSs (ACS-1 through ACS-7). ACS-3 was found to be the major activator for exogenous FAs for anabolic lipid metabolic pathways, and consistent with this finding, ACS-3 localized to the endoplasmic reticulum, plasma membrane, and septa. Double-mutant analyses confirmed partial functional redundancy of ACS-2 and ACS-3. ACS-5 was determined to function in siderophore biosynthesis, indicating alternative functions for ACS enzymes in addition to fatty acid metabolism. The N. crassa ACSs involved in activation of FAs for catabolism were not specifically defined, presumably due to functional redundancy of several of ACSs for catabolism of exogenous FAs.

A dual action of saturated fatty acids on electrical activity in rat pancreatic β‐cells. Role of volume‐regulated anion channel and KATP channel currents

Free fatty acids (FFAs) exert complex actions on pancreatic β-cells. Typically, an initial potentiation of insulin release is followed by a gradual impairment of β-cell function, the latter effect being of possible relevance to hyperlipidaemia in type 2 diabetes mellitus. The molecular actions of FFAs are poorly understood. The present study investigated the acute effects of saturated FFAs on electrophysiological responses of rat pancreatic β-cells. Membrane potential and KATP channel activity were recorded using the perforated patch technique. Volume-regulated anion channel (VRAC) activity was assessed from conventional whole-cell recordings. Cell volume regulation was measured using a video-imaging technique. Addition of octanoate caused a transient potentiation of glucose-induced electrical activity, followed by a gradual hyper-polarisation and a prolonged inhibition of electrical activity. Octanoate caused an initial increase in VRAC activity followed by a secondary inhibition coinciding with increased KATP channel activity. Similar effects were observed with palmitate and 2-bromopalmitate whereas butyrate was virtually ineffective. Octanoate and palmitate also exerted a dual effect on electrical activity evoked by tolbutamide. Octanoate significantly attenuated cell volume regulation in hypotonic solutions, consistent with VRAC inhibition. It is concluded that medium and long chain FFAs have a dual action on glucose-induced electrical activity in rat pancreatic β-cells: an initial stimulatory effect followed by a secondary inhibition. These effects appear to be the result of reciprocal actions on VRAC and KATP channel currents, and could contribute towards the stimulatory and inhibitory actions of FFAs on pancreatic β-cell function.

Reduced bone mass in obese young rats through PPARγ suppression of Wnt/β‐catenin signaling and direct action of free fatty acids (NEFA)

The relationship of obesity to skeletal development is unclear. We utilized total enteral nutrition to feed high and low fat diets (HFD and LFD) to rats for 4 wks to produce obesity. Weight gain was matched but fat mass, serum leptin and NEFA were increased by HFD (P<0.05). HFD lowered total bone mineral content and density (P<0.05) accompanied by decreased serum osteocalcin and increases in resorption marker RatLaps. Increased bone marrow adiposity and adipogenic genes PPARγ and AP2, and suppressed osteoblastogenic genes osteocalcin and Runx2 were observed in the HFD group which exhibited down‐regulation of β‐catenin protein, but up‐regulation of PPARγ expression in bone (P<0.05). Reciprocal regulation of β‐catenin and PPARγ was observed in mesenchymal ST2 cells exposed to serum from HFD rats or treated with an artificial NEFA mixture. Transfection with PPARγ suppressed β‐catenin signaling. These data suggest NEFA directly stimulates bone marrow adipogenesis while suppressing bone formation. These effects of HFD may impair attainment of peak bone mass and increase risk of osteoporosis later in life. USDA CRIS‐6251‐51000‐007‐03S.

NTE1-encoded Phosphatidylcholine Phospholipase B Regulates Transcription of Phospholipid Biosynthetic Genes

The Saccharomyces cerevisiae NTE1 gene encodes an evolutionarily conserved phospholipase B localized to the endoplasmic reticulum (ER) that degrades phosphatidylcholine (PC) generating glycerophosphocholine and free fatty acids. We show here that the activity of NTE1-encoded phospholipase B (Nte1p) prevents the attenuation of transcription of genes encoding enzymes involved in phospholipid synthesis in response to increased rates of PC synthesis by affecting the nuclear localization of the transcriptional repressor Opi1p. Nte1p activity becomes necessary for cells growing in inositol-free media under conditions of high rates of PC synthesis elicited by the presence of choline at 37 degrees C. The specific choline transporter encoded by the HNM1 gene is necessary for the burst of PC synthesis observed at 37 degrees C as follows: (i) Nte1p is dispensable in an hnm1Delta strain under these conditions, and (ii) there is a 3-fold increase in the rate of choline transport via the Hnm1p choline transporter upon a shift to 37 degrees C. Overexpression of NTE1 alleviated the inositol auxotrophy of a plethora of mutants, including scs2Delta, scs3Delta, ire1Delta, and hac1Delta among others. Overexpression of NTE1 sustained phospholipid synthesis gene transcription under conditions that normally repress transcription. This effect was also observed in a strain defective in the activation of free fatty acids for phosphatidic acid synthesis. No changes in the levels of phosphatidic acid were detected under conditions of altered expression of NTE1. Consistent with a synthetic impairment between challenged ER function and inositol deprivation, increased expression of NTE1 improved the growth of cells exposed to tunicamycin in the absence of inositol. We describe a new role for Nte1p toward membrane homeostasis regulating phospholipid synthesis gene transcription. We propose that Nte1p activity, by controlling PC abundance at the ER, affects lateral membrane packing and that this parameter, in turn, impacts the repressing transcriptional activity of Opi1p, the main regulator of phospholipid synthesis gene transcription.

Histone H4 Is a Major Component of the Antimicrobial Action of Human Sebocytes

Antimicrobial peptides, such as cathelicidin and beta defensins, directly kill microbes and have been detected in human sebaceous glands and cell lines. Despite the presence of several such peptides, the apparent abundance of these is insufficient for direct killing of most skin pathogens. In this study, we sought to determine which molecules provide the majority of antimicrobial peptide activity in human sebocytes. Acid-soluble protein extracts of SEB-1 sebocytes were separated by reverse-phase high-performance liquid chromatography and were assayed for their capacity to inhibit the growth of Staphylococcus aureus. Antimicrobial activity was isolated in a single major fraction and identified to be histone H4 by mass spectrometry and western blot analysis. The importance of histone H4 in the antimicrobial activity of sebocytes was confirmed by a specific neutralizing antibody and by direct demonstration that recombinant histone H4 had antimicrobial activity against S. aureus and Propionibacterium acnes. In addition, histone H4 enhanced the antimicrobial action of free fatty acids in human sebum. Taken together, these results indicate that the release of histone H4 by holocrine secretion from the sebaceous gland may play an important role in innate immunity.

Prooxidant Mechanisms of Free Fatty Acids in Stripped Soybean Oil-in-Water Emulsions

The prooxidant role of free fatty acids was studied in soybean oil-in-water emulsions. Addition of oleic acid (0-5.0% of oil) to the emulsions increased lipid hydroperoxides and headspace hexanal formation and increased the negative charge of the emulsion droplet with increasing oleic acid concentration. Methyl oleate (1.0% of oil) did not increase oxidation rates. The ability of oleic acid to promote lipid oxidation in oil-in-water emulsions decreased with decreasing pH with dramatic reduction in oxidation observed when the pH was low enough so that the oleic acid was not able to increase the negative charge of the emulsion droplet. Ethylenediaminetetraacetic acid (EDTA, 200 microm) strongly inhibited lipid oxidation in emulsions with oleic acid, indicating that transition metals were responsible for accelerating oxidation. Oleic acid hydroperoxides did not increase oxidation rates, suggesting that hydroperoxides on free fatty acids are not strong prooxidants in oil-in-water emulsion. These results suggest that the prooxidant activity of free fatty acids in oil-in-water emulsions is due to their ability to attact prooxidant metals to the emulsion droplet surface.

Hypersusceptibility of neutrophil granulocytes towards lethal action of free fatty acids contained in enzyme-modified atherogenic low density lipoprotein

Objective The bulk of LDL entrapped in the arterial intima is modified by hydrolytic enzymes, leading to extensive cleavage of cholesterylesters and liberation of fatty acids. The latter induce apoptosis in endothelial cells but are far less cytotoxic towards macrophages. We have compared the cytotoxic effects of enzymatically modified LDL (E-LDL) on macrophages and polymorphonuclear granulocytes (PMN). Methods and results E-LDL displayed toxicity towards PMN at far lower concentrations than towards monocyte-derived macrophages. Native or oxidized LDL had no effect. Free fatty acids contained in E-LDL were the cause of the observed toxicity, which could be mimicked by linoleic acid, oleic acid and arachidonic acid. E-LDL provoked Ca 2+ influx and activated PMN, as witnessed by the generation of superoxide anions and peroxidase secretion. Inhibition of either oxidative burst or calcium influx did not diminish the cytotoxicity of E-LDL. Similar to free linoleic acid, E-LDL lysed red blood cells and rapidly rendered cells permeable to propidium iodide. Conclusion Possibly through their capacity to directly perturb cell membranes, free fatty acids contained in E-LDL exert potent cytotoxic effects on PMN. This may be one reason why PMN are not abundantly present in atherosclerotic lesions, and why PMN-depletion suppresses atherogenesis.

The Staphylococcus aureus Response to Unsaturated Long Chain Free Fatty Acids: Survival Mechanisms and Virulence Implications

Staphylococcus aureus is an important human commensal and opportunistic pathogen responsible for a wide range of infections. Long chain unsaturated free fatty acids represent a barrier to colonisation and infection by S. aureus and act as an antimicrobial component of the innate immune system where they are found on epithelial surfaces and in abscesses. Despite many contradictory reports, the precise anti-staphylococcal mode of action of free fatty acids remains undetermined. In this study, transcriptional (microarrays and qRT-PCR) and translational (proteomics) analyses were applied to ascertain the response of S. aureus to a range of free fatty acids. An increase in expression of the σB and CtsR stress response regulons was observed. This included increased expression of genes associated with staphyloxanthin synthesis, which has been linked to membrane stabilisation. Similarly, up-regulation of genes involved in capsule formation was recorded as were significant changes in the expression of genes associated with peptidoglycan synthesis and regulation. Overall, alterations were recorded predominantly in pathways involved in cellular energetics. In addition, sensitivity to linoleic acid of a range of defined (sigB, arcA, sasF, sarA, agr, crtM) and transposon-derived mutants (vraE, SAR2632) was determined. Taken together, these data indicate a common mode of action for long chain unsaturated fatty acids that involves disruption of the cell membrane, leading to interference with energy production within the bacterial cell. Contrary to data reported for other strains, the clinically important EMRSA-16 strain MRSA252 used in this study showed an increase in expression of the important virulence regulator RNAIII following all of the treatment conditions tested. An adaptive response by S. aureus of reducing cell surface hydrophobicity was also observed. Two fatty acid sensitive mutants created during this study were also shown to diplay altered pathogenesis as assessed by a murine arthritis model. Differences in the prevalence and clinical importance of S. aureus strains might partly be explained by their responses to antimicrobial fatty acids.

Pharmacologically Relevant Receptor Binding Characteristics and 5.ALPHA.-Reductase Inhibitory Activity of Free Fatty Acids Contained in Saw Palmetto Extract

Saw palmetto extract (SPE), used widely for the treatment of benign prostatic hyperplasia (BPH) has been shown to bind alpha(1)-adrenergic, muscarinic and 1,4-dihydropyridine (1,4-DHP) calcium channel antagonist receptors. Major constituents of SPE are lauric acid, oleic acid, myristic acid, palmitic acid and linoleic acid. The aim of this study was to investigate binding affinities of these fatty acids for pharmacologically relevant (alpha(1)-adrenergic, muscarinic and 1,4-DHP) receptors. The fatty acids inhibited specific [(3)H]prazosin binding in rat brain in a concentration-dependent manner with IC(50) values of 23.8 to 136 microg/ml, and specific (+)-[(3)H]PN 200-110 binding with IC(50) values of 24.5 to 79.5 microg/ml. Also, lauric acid, oleic acid, myristic acid and linoleic acid inhibited specific [(3)H]N-methylscopolamine ([(3)H]NMS) binding in rat brain with IC(50) values of 56.4 to 169 microg/ml. Palmitic acid had no effect on specific [(3)H]NMS binding. The affinity of oleic acid, myristic acid and linoleic acid for each receptor was greater than the affinity of SPE. Scatchard analysis revealed that oleic acid and lauric acid caused a significant decrease in the maximal number of binding sites (B(max)) for [(3)H]prazosin, [(3)H]NMS and (+)-[(3)H]PN 200-110. The results suggest that lauric acid and oleic acid bind noncompetitively to alpha(1)-adrenergic, muscarinic and 1,4-DHP calcium channel antagonist receptors. We developed a novel and convenient method of determining 5alpha-reductase activity using LC/MS. With this method, SPE was shown to inhibit 5alpha-reductase activity in rat liver with an IC(50) of 101 microg/ml. Similarly, all the fatty acids except palmitic acid inhibited 5alpha-reductase activity, with IC(50) values of 42.1 to 67.6 microg/ml. In conclusion, lauric acid, oleic acid, myristic acid, and linoleic acid, major constituents of SPE, exerted binding activities of alpha(1)-adrenergic, muscarinic and 1,4-DHP receptors and inhibited 5alpha-reductase activity.

Is Membrane Transport of FFA Mediated by Lipid, Protein, or Both?

![Figure][1] J. Patrick Kampf Torrey Pines Institute for Molecular Studies, San Diego, California ![Figure][1] Alan M. Kleinfeld Torrey Pines Institute for Molecular Studies, San Diego, California akleinfeld{at}tpims.org The mechanism of free fatty acid (FFA)

Potentiation of TRPV3 channel function by unsaturated fatty acids

Transient receptor potential vanilloid (TRPV) channels are polymodal detectors of multiple environmental factors, including temperature, pH, and pressure. Inflammatory mediators enhance TRPV function through multiple signaling pathways. The lipoxygenase and epoxygenase products of arachidonic acid (AA) metabolism have been shown to directly activate TRPV1 and TRPV4, respectively. TRPV3 is a thermosensitive channel with an intermediate temperature threshold of 31-39 degrees C. We have previously shown that TRPV3 is activated by 2-aminoethoxydiphenyl borate (2APB). Here we show that AA and other unsaturated fatty acids directly potentiate 2APB-induced responses of TRPV3 expressed in HEK293 cells, Xenopus oocytes, and mouse keratinocytes. The AA-induced potentiation is observed in intracellular Ca2+ measurement, whole-cell and two-electrode voltage clamp studies, as well as single channel recordings of excised inside-out and outside-out patches. The fatty acid-induced potentiation is not blocked by inhibitors of protein kinase C and thus differs from that induced by the kinase. The potentiation does not require AA metabolism but is rather mimicked by non-metabolizable analogs of AA. These results suggest a novel mechanism regulating the TRPV3 response to inflammation, which differs from TRPV1 and TRPV4, and involves a direct action of free fatty acids on the channel.

Establishing a Reference Interval For Measurement of Flux through the Mitochondrial Fatty Acid Oxidation Pathway in Cultured Skin Fibroblasts

Mitochondrial fatty acid oxidation (FAO) represents a normal metabolic response to increased energy demands during periods of fasting, febrile illness, or muscular exertion. FAO is a complex pathway involving activation of free fatty acids to acyl-CoA species, transport into mitochondria, and cyclic oxidation to break the long-chain fatty acids into acetyl-CoA and a two-carbon chain-shortened acyl-CoA, which is then recycled. Acetyl-CoA is used in the Krebs cycle and in liver as a substrate for ketogenesis (1). Measurement of FAO flux in cultured cells provides valuable information about the integrity of the enzymes and transporters involved in FAO at all stages. Clinical indications for the measurement of FAO flux in skin fibroblasts include fatty acid oxidation enzyme defects, unexplained myopathies, cardiomyopathies, hypoglycemia, liver disease, sudden unexplained infant death, and hypoketotic hypoglycemia. FAO flux is typically measured by monitoring the conversion of radiolabeled fatty acids to carbon dioxide and water. Labeling can be with 14C with final analysis of the rate of production of 14CO2, or with 3H and analysis of production of 3H2O (2)(3). Palmitic acid (C16) is used to detect long-chain defects, including deficiencies in very long chain acyl-CoA dehydrogenase, long-chain l-3-hydroxy acyl-CoA dehydrogenase, long-chain 3-keto acyl-CoA thiolase, carnitine/acylcarnitine translocase, carnitine pamitoyltransferase 1, and carnitine pamitoyltransferase 2. Myristic acid (C14) is used to detect defects in medium-chain FAO, such as medium-chain acyl-CoA dehydrogenase deficiency. We use tritiated fatty acids labeled in the (9,10) position. The assay will not detect defects in carnitine palmitoyl transferase 1B; short-chain FAO defects, including short-chain acyl-CoA dehydrogenase and short-chain l-3-hydroxy acyl-CoA dehydrogenase; and short-chain-3-keto acyl-CoA thiolase deficiencies. Historically, patient samples have been compared with batch controls, and a reference interval was not established for this assay. In the present study we have assigned …