Fatty acid-related modulations of membrane fluidity in cells: detection and implications

Metabolic homeostasis of fatty acids is complex and well-regulated in all organisms. The biosynthesis of saturated fatty acids (SFA) in mammals provides substrates for ?-oxidation and ATP production. Monounsaturated fatty acids (MUFA) are products of desaturases that introduce a methylene group in cis geometry in SFA. Polyunsaturated fatty acids (n-6 and n-3 PUFA) are products of elongation and desaturation of the essential linoleic acid and ?-linolenic acid, respectively. The liver processes dietary fatty acids and exports them in lipoproteins for distribution and storage in peripheral tissues. The three types of fatty acids are integrated in membrane phospholipids and determine their biophysical properties and functions. This study was aimed at investigating effects of fatty acids on membrane biophysical properties under varying nutritional and pathological conditions, by integrating lipidomic analysis of membrane phospholipids with functional two-photon microscopy (fTPM) of cellular membranes. This approach was applied to two case studies: first, pancreatic beta-cells, to investigate hormetic and detrimental effects of lipids. Second, red blood cells extracted from a genetic mouse model defective in lipoproteins, to understand the role of lipids in hepatic diseases and metabolic syndrome and their effect on circulating cells

Membrane manipulation by free fatty acids improves microbial plant polyphenol synthesis

Microbial synthesis of nutraceutically and pharmaceutically interesting plant polyphenols represents a more environmentally friendly alternative to chemical synthesis or plant extraction. However, most polyphenols are cytotoxic for microorganisms as they are believed to negatively affect cell integrity and transport processes. To increase the production performance of engineered cell factories, strategies have to be developed to mitigate these detrimental effects. Here, we examine the accumulation of the stilbenoid resveratrol in the cell membrane and cell wall during its production using Corynebacterium glutamicum and uncover the membrane rigidifying effect of this stilbenoid experimentally and with molecular dynamics simulations. A screen of free fatty acid supplements identifies palmitelaidic acid and linoleic acid as suitable additives to attenuate resveratrol’s cytotoxic effects resulting in a three-fold higher product titer. This cost-effective approach to counteract membrane-damaging effects of product accumulation is transferable to the microbial production of other polyphenols and may represent an engineering target for other membrane-active bioproducts

Main fatty acid changes in NB100 cell membranes after fatty acids supplementation.

<p>(<b>A</b>) In the left panel, the main fatty acid variations in NB100 cell membranes after 150 µM PA supplementation are reported. In the right panel, fluctuations of the corresponding fatty acid families are evidenced. (<b>B</b>) In the left panel, the main fatty acid variations in NB100 cell membranes after 150 µM PA +50 µM OA +50 µM AA supplementation are reported. The right panel evidences the fatty acid family changes after this supplementation. (<b>C</b>) In the left panel, the main fatty acid variations and the corresponding fatty acid families are evidenced in NB100 cell membranes after 50 µM PA supplementation. In the right panel, the same fatty acid variations are reported for cells incubated with 150 µM PA for 8 hours and 24 hours. Data are obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055537#pone-0055537-t001" target="_blank">Tables 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055537#pone-0055537-t002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055537#pone-0055537-t003" target="_blank">3</a>. Values are means ± SD. Statistical significances are as reported in the notes to the tables.</p

Evaluation of apoptosis in NB100 cells supplemented with fatty acids.

<p>(<b>A</b>) Caspase activation in NB100 cells exposed to PA 150 µM (▪) or PA 150 µM+OA 50 µM+AA 50 µM (•). Caspase-2, -8, -9 and -3/7 activation was determined at 3, 8, 16, 24 hours as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055537#s2" target="_blank">Materials and Methods</a>. Caspase activity is expressed as percentage of control values obtained form cultures grown in the absence of FA supplementation. Mean results ± SD are reported. (<b>B</b>) Cell viability was evaluated at 24 hours on NB100 cells pretreated with 30 µM of the irreversible tetrapeptide pan-caspase inhibitor Z-VAD-fmk, added to the culture 3 hours before the 150 µM PA supplementation.</p

Effect of PA on NB100 cell line and cell morphology.

<p>(<b>A</b>) Effect of FA supplementation on NB100 cell viability. Cell viability was determined by MTS assay. Values are means ± SD of four determinations. Left graph: cells were incubated in complete medium supplemented with 50 µM PA (□), 150 µM PA (▪), 150 µM PA +50 µM OA (♦), 150 µM PA +50 µM AA (▴), 150 µM PA +50 µM OA +50 µM AA (○). Right graph: cells were incubated in complete medium supplemented with PA at various concentrations for 1 hour (•) or for 2 hours (○), and then incubated in complete medium for 48 hours after wash. (<b>B</b>) NB100 cells morphology assessed by phase contrast microscopy. Control cultures grown in the absence of FA supplementation are shown in comparison with cells treated for 24 hours with 150 µM PA or 150 µM PA +50 µM OA +50 µM AA. Magnification 200×. (<b>C</b>) Nuclei of NB100 cells stained with DAPI and assessed by fluorescence microscopy (×600 magnification objective). Cells were incubated in complete medium supplemented with 150 µM PA (4, 8, 16, 24 hours) or 150 µM PA +50 µM OA +50 µM AA (24 hours). Control cultures grown in the absence of FA supplementation (24 hours) are also shown.</p

Membrane phospholipid fatty acids of NB100 cells treated for the indicated times with 50 µM and 150 µM PA and compared to controls grown in the same conditions without PA supplementation at each time intervals.

<p> <b>The values are reported as % rel of the total fatty acid peak areas detected in the GC analysis. They are mean values ± SD of the n repetitions of the same experiment.</b></p>a<p>FAME are obtained from total lipid extraction, derivatization, and GC analysis.</p>b<p>The identification of the peaks have been performed by authentic samples and the identified peaks accounted for >98% of the total peaks.</p>c<p>Values higher than untreated control (***P = 0.0001).</p>d<p>Values lower than untreated control (***P = 0.0001).</p>e<p>Values higher than untreated control (**P<0.001).</p>f<p>Values lower than untreated control (**P<0.001).</p>g<p>Values higher than untreated control (*P<0.01).</p>h<p>Values lower than untreated control (*P<0.01).</p>*<p>Evaluated with standard compounds (mono-trans arachidonic acid isomers) obtained following references <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055537#pone.0055537-Ferreri1" target="_blank">[12]</a>.</p>#<p>This value includes EPA and DHA.</p