Nuclear-lipid-droplet proteome: carboxylesterase as a nuclear lipase involved in lipid-droplet homeostasis.

Nuclear-lipid droplets (nLD)-a dynamic cellular organelle that stores neutral lipids, within the nucleus of eukaryotic cells-consists of a hydrophobic triacylglycerol -cholesterol-ester core enriched in oleic acid (OA) surrounded by a monolayer of polar lipids, cholesterol, and proteins. nLD are probably involved in nuclear-lipid homeostasis serving as an endonuclear buffer that provides or incorporates lipids and proteins participating in signaling pathways, as transcription factors and enzymes of lipid metabolism and nuclear processes. In the present work, we analyzed the nLD proteome and hypothesized that nLD-monolayer proteins could be involved in processes similar as the ones occurring in the cLD including lipid metabolism and other cellular functions. We evaluated the rat-liver-nLD proteome under physiological and nonpathological conditions by GeLC-MS2. Since isolated nLD are highly diluted, a protein-concentrating isolation protocol was designed. Thirty-five proteins were identified within the functional categories: cytoskeleton and structural, transcription and translation, histones, protein-folding and posttranslational modification, cellular proliferation and/or cancer, lipid metabolism, and transport. Purified nLD contained an enzyme from the lipid-metabolism pathway, carboxylesterase 1d (Ces1d/Ces3). Nuclear Carboxylesterase localization was confirmed by Western blotting. By in-silico analyses rat Ces1d/Ces3 secondary and tertiary structure predicted would be equivalent to human CES1. These results-the first nLD proteome-demonstrate that a tandem-GeLC-MS2-analysis protocol facilitates studies like these on rat-liver nuclei. A diversity of cellular-protein function was identified indicating the direct or indirect nLD participation and involving Ces1d/Ces3 in the LD-population homeostasis.

Reversible Nuclear-Lipid-Droplet Morphology Induced by Oleic Acid: A Link to Cellular-Lipid Metabolism.

Neutral lipids-involved in many cellular processes-are stored as lipid droplets (LD), those mainly cytosolic (cLD) along with a small nuclear population (nLD). nLD could be involved in nuclear-lipid homeostasis serving as an endonuclear buffering system that would provide or incorporate lipids and proteins involved in signalling pathways as transcription factors and as enzymes of lipid metabolism and nuclear processes. Our aim was to determine if nLD constituted a dynamic domain. Oleic-acid (OA) added to rat hepatocytes or HepG2 cells in culture produced cellular-phenotypic LD modifications: increases in TAG, CE, C, and PL content and in cLD and nLD numbers and sizes. LD increments were reversed on exclusion of OA and were prevented by inhibition of acyl-CoA synthetase (with Triacsin C) and thus lipid biosynthesis. Under all conditions, nLD corresponded to a small population (2-10%) of total cellular LD. The anabolism triggered by OA, involving morphologic and size changes within the cLD and nLD populations, was reversed by a net balance of catabolism, upon eliminating OA. These catabolic processes included lipolysis and the mobilization of hydrolyzed FA from the LD to cytosolic-oxidation sites. These results would imply that nLD are actively involved in nuclear processes that include lipids. In conclusion, nLD are a dynamic nuclear domain since they are modified by OA through a reversible mechanism in combination with cLD; this process involves acyl-CoA-synthetase activity; ongoing TAG, CE, and PL biosynthesis. Thus, liver nLD and cLD are both dynamic cellular organelles.

Inhibition of Mevalonate Pathway and Synthesis of the Storage Lipids in Human Liver-Derived and Non-liver Cell Lines by Lippia alba Essential Oils.

The essential oils (EOs) of Lippia alba, an herb extensively used as a folk medicine in Latin America, are today promoted as an effective means of eliminating problems caused by hyperlipemia. We hypothesized that L.alba EOs inhibited cholesterol and triacylglycerols synthesis and decreased the intracellular depots of those lipids (lipid droplets), mechanisms involving the induction of a hypolipidemic response. Our aim was, therefore, to evaluate the hypolipogenic capability of the EOs of four L. alba chemotypes on liver-derived (HepG2) and non-liver (A549) human cell lines and to identify the potential biochemical targets of those chemotypes, particularly within the mevalonate pathway (MP). [14C]Acetate was used as radioactive precursor for assays. Lipid analyses were performed by thin-layer and capillary gas chromatography, lipid droplets analyzed by fluorescence microscopy, and HMGCR levels determined by Western blot. In both cell lines, all four chemotypes exerted hypocholesterogenic effects within a concentration range of 3.2-32 µg/mL. Nonsaponifiable lipids manifested a decrease in incorporation of [14C]acetate into squalene, lanosterol, lathosterol, and cholesterol, but not into ubiquinone, thus suggesting an inhibition of enzymes in the MP downstream from farnesyl pyrophosphate. The tagetenone chemotype, the most efficacious hypocholesterogenic L. alba EO, lowered HMGCR protein levels; inhibited triacylglycerols, cholesteryl esters, and phospholipids synthesis; and diminished lipid droplets in size and volume. These results revealed that L. alba EOs inhibited different lipogenic pathways and such lipid-lowering effects could prove essential to prevent cardiovascular diseases.

Arachidonic acid pools of rat kidney cell nuclei.

We have assessed that nuclear lipids from rat kidney cells are not only membrane components, but they are also found within the nucleus. The most abundant nuclear and endonuclear lipids have a high proportion of unsaturated fatty acids (n-6 series: arachidonic > linoleic), mainly esterified to PtdCho. Nuclear most abundant molecular species are 16:0-20:4, 16:0-18:2, 18:0-20:4, 18:0-18:2, and 16:0-18:1. Arachidonic acid is esterified at the sn-2 position of PtdCho: 16:0-20:4(25%), 18:0-20:4(15%), 18:2-20:4(3%), 18:1-20:4(2%). Exogenous [1-(14)C]20:4n-6-CoA is esterified in vitro in GP (glycerophospholipids) > > TAG and DAG. Five PtdCho molecular species were labeled: 16:0-20:4, 18:0-20:4, 18:1-20:4, 18:2-20:4, and 20:4-20:4. In conclusion, these results demonstrated that: (1) there is an important lipid pool within kidney cell nuclei; (2) main nuclear and endonuclear lipid pools were PtdCho molecular species which contained a high proportion of unsaturated fatty acids (20:4n-6 and 18:2n-6) esterified at sn-2 position and 16:0 esterified at sn-1 position; (3) kidney cell nuclei also contained the necessary enzymes to esterify exogenous 20:4n-6-CoA to glycerolipids and to GP; (4) exogenous 20:4n-6-CoA was esterified in five PtdCho molecular species with 20:4n-6 at the sn-2 position, although the most actively synthesized PtdCho contained 20:4n-6 at both the sn-1 and sn-2 positions of the molecule; (5) we can infer that by a remodeling process, the unsaturated fatty acids at the sn-1 position of PtdCho molecular species could be replaced by 16:0 and 18:0, and thus PtdCho would achieve the physiological profile characteristic of the organ.

Incorporation of arachidonic and stearic acids bound to L-FABP into nuclear and endonuclear lipids from rat liver cells.

The incorporation of exogenous fatty acids bound to L-FABP into nuclei was studied. Rat liver cell nuclei and nuclear matrices (membrane depleted nuclei) were incubated in vitro with [1-(14)C]18:0 and 20:4n-6 either free or bound to L-FABP, ATP and CoA. FA esterification in whole nuclei and endonuclear lipids was ATP-CoA-dependent, and with specificity regarding fatty acid type and lipid class. 18:0 and 20:4n-6, free or L-FABP bound, showed the same incorporation and esterification pattern in lipids of whole nuclei. Only 20:4n-6 L-FABP bound was less incorporated into TAG with respect to free 20:4n-6. In the nuclear matrix, 18:0 free or L-FABP bound was esterified with a higher specific activity (SA) into: PtdEtn > PtdIns, PtdSer > PtdCho. 20:4n-6 free or L-FABP bound was esterified into: PtdIns > PtdEtn > PtdCho. 20:4n-6:L-FABP was esterified in endonuclear total-PL and PtdIns with a greater SA with respect to free 20:4n-6 and with a minor one as FFA. To summarize, trafficking of FA to nuclei includes esterification of 18:0 and 20:4n-6 either free or L-FABP-bound, into nuclear and endonuclear lipids by an ATP-CoA-dependent pathway. Endonuclear fatty acid esterification was more active than that in whole nuclei, and independent of the nuclear membrane. Esterification patterns of fatty acids L-FABP-bound or free into whole nuclear lipids were the same whereas in the nuclear matrix, L-FABP could play an important role in the mobilization of 20:4n-6 into specific sites of utilization such as the PtdIns pools.

Endonuclear lipids in liver cells.

Lipids are not only components of cell nucleus membranes, but are also found in the membrane-depleted nuclei where they fulfill special functions. We have investigated the lipid composition of membrane-depleted rat liver nuclei obtained by incubation with low Triton X-100 concentrations of 0.04% and 0.08%, which rendered them unaltered or hardly altered. Under these conditions, 26% of proteins and 22% of phospholipids were recovered. The main phospholipids were phosphatidylcholine > phosphatidylethanolamine > phosphatidylinositol = or > phosphatidylserine and sphingomyelin (in decreasing concentrations). The fatty acid components of total lipids and phosphatidylcholine were mainly unsaturated. Over 40% belonged to the n-6 series (arachidonic > or = 25% and linoleic 15%); approximately 40% corresponded to saturated acids and <10% were monoenoic. Endonuclear phosphatidylcholine was built up by 16 molecular species, the most abundant being 18:0-20:4 (32%), 16:0-20:4 (19%), 16:0-18:2 (13%), and 18:0-18:2 (11%). The fatty acid composition and phosphatidylcholine molecular species distribution in the membrane-depleted nucleus of rat liver showed patterns similar to the whole nucleus, mitochondria, microsomes, and homogenate of the parent liver cells, suggesting that endonuclear lipid pool composition is mainly determined by a liver organ profile.