Juan Pablo Layerenza

Nuclear lipid droplets: a novel nuclear domain.

We investigated nuclear neutral-lipid (NL) composition and organization, as NL may represent an alternative source for providing fatty acids and cholesterol (C) to membranes, signaling paths, and transcription factors in the nucleus. We show here that nuclear NL were organized into nonpolar domains in the form of nuclear-lipid droplets (nLD). By fluorescent confocal microscopy, representative nLD were observed in situ within the nuclei of rat hepatocytes in vivo and HepG2 cells, maintained under standard conditions in culture, and within nuclei isolated from rat liver. nLD were resistant to Triton X-100 and became stained with Sudan Red, OsO4, and BODIPY493/503. nLD and control cytosolic-lipid droplets (cLD) were isolated from rat-liver nuclei and from homogenates, respectively, by sucrose-gradient sedimentation. Lipids were extracted, separated by thin-layer chromatography, and quantified. nLD were composed of 37% lipids and 63% proteins. The nLD lipid composition was as follows: 19% triacylglycerols (TAG), 39% cholesteryl esters, 27% C, and 15% polar lipids; whereas the cLD composition contained different proportions of these same lipid classes, in particular 91% TAG. The TAG fatty acids from both lipid droplets were enriched in oleic, linoleic, and palmitic acids. The TAG from the nLD corresponded to a small pool, whereas the TAG from the cLD constituted the main cellular pool (at about 100% yield from the total homogenate). In conclusion, nLD are a domain within the nucleus where NL are stored and organized and may be involved in nuclear lipid homeostasis.

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 [1-14C]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.

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.