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Title

Regulation of lipid droplet formation by group IVA phospholipase A2

AuthorsGuijas, Carlos ; Rubio, Julio M. ; Rodríguez, Juan Pablo; Balboa, María A. ; Balsinde, Jesús
Issue Date2015
CitationASBMB Annual Meeting 2015
AbstractExposure of human peripheral blood monocytes to free arachidonic acid (AA) results in the rapid induction of lipid droplet (LD) formation by these cells. LD are formed by two different routes, namely (i) the direct entry of AA into triacylglycerol and (ii) activation of intracellular signaling leading to increased neutral lipid formation utilizing fatty acids coming from the de novo biosynthetic route. The latter predominates, accounting for 60-70% of total LD formation, and can be completely inhibited by selective inhibition of the group IVA cytosolic phospholipase A2a, pointing out this enzyme as a key regulator of AA-induced signaling. LD formation in AA-treated monocytes can also be blocked by the combined inhibition of the mitogen-activated protein kinase family member p38 and JNK, which correlates with inhibition of cPLA2a activation by phosphorylation. Since cPLA2a has no effect on neutral lipid formation there must be a step in the mechanism of LD biogenesis that is critically dependent on the enzyme, e.g. generation of a positive curvature at the organelle baseline. Slide 1 – AA-
formation in human monocytes. Monocytes, treated without (left column) or with (right column) 3 μM triacsin C for 30 min, were induced LD exposed to AA, palmitic acid (16:0), oleic acid (18:1), linoleic acid (18:2), or γ-linolenic acid (γ18:3) for 2 h as indicated. All fatty acids were added at a concentration of 10 μM. After fixation and permeabilization, cells were stained with BODIPY493/503 (2 µg/ml) to visualize LD (green; right panels) and DAPI (1 g/ml) to mark the nuclei (blue; central panels). Left panels show the Nomarski images. Slide 2 – Effect of triacsin C on the incorporation of fatty acids into TAG in human monocytes. A: Effect of triacsin C on the distribution of fatty acids in TAG after treatment of the monocytes with 10 M AA for 2 h. The analysis of TAG fatty acids was carried out by GC-MS after converting the fatty acid glyceryl esters into fatty acidmethyl esters. B: Total cellular TAG values and the result of adding the masses of all fatty acids under each condition and dividing by 3. Data are given as means ± SE of three independent experiments. *Significantly different (p < 0.05) from their
respective controls. Slide 3 – Effect of various inhibitors on AA-induced LD formation. ffect of various inhibitors on AA-induced LD formation. Monocytes, preincubated with 3 μM triacsin C for 30 min, were untreated (left column) or treated with 10 M AA for 2 h (right column) in the presence of the indicated inhibitor at the following concentrations: 1 M pyrrophenone, 10 M SB203580, and 10 M SP600125. After fi xation and permeabilization, cells were stained with BODIPY493/503 (2 g/ml) to visualize LD and with DAPI (1 μg/ml) to mark the nuclei. Slide 4 – Stimulation of mitogen-activated protein kinases and cPLA2α by AA in human monocytes. A: Monocytes were treated without (Control) or with 10 μM AA for the indicated times and analyzed for expression of phosphorylated p38 and JNK by immunoblot. B: Analysis of the kinases implicated in cPLA2α phosphorylation. Monocytes were treated with 10 μM AA for 2 h as indicated. Some of the samples were preincubated with the following specific kinase inhibitors as indicated: 10 μM PD98059, 10 μM SB203580, 10μM SP600125, or 10 μM SB203580, plus 10 μM SP600125. All incubations proceeded in the presence of 3 M triacsin C. Phosphorylation of cPLA2α at Ser 505 was analyzed by immunoblot. The Western blots for phosphorylated p38, JNK, and cPLA2α were quantified from three different experiments (means ± SE). Slide 5 – LD synthesis from the cytosolic face of the smooth endoplasmic reticulum membranes. LD synthesis fromthe cytosolic face of the smooth endoplasmic reticulum membranes. The essential role of cPLA2α in inducing a positivemembrane curvature is indicated in step 3. Lysophospholipids are highlighted in pink, and phosphatidic acid in green. All other phospholipids are shown in blue. For further details see text. DGAT, diacylglycerol:acyl-CoA acyl transferase; ACAT, acyl-CoA:cholesterol acyl transferase; ER, endoplasmic reticulum; PLD, phospholipase D; LPCAT, lysophosphatidylcholine:acyl-CoA acyl transferase.
DescriptionWork presented at Experimental Biology 2015 - ASBMB Annual Meeting, held in Boston, Mass., USA, March 28 – April 1, 2015.
Publisher version (URL)https://www.asbmb.org/meeting2015/
URIhttp://hdl.handle.net/10261/116952
Appears in Collections:(IBGM) Comunicaciones congresos
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