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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., Instituto de Investigaciones Bioquímicas de La Plata, Facultad de Ciencias Exactas

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., Instituto de Investigaciones Bioquímicas de La Plata, Facultad de Ciencias Médicas, Facultad de Ciencias Exactas

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., Instituto de Investigaciones Bioquímicas de La Plata, Facultad de Ciencias Exactas

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.

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 ≥ 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 ≥25% and linoleic 15%); approximately 40% corresponded to saturated acids and

Activity of one of the key enzymes involved in arachidonic acid (20:4 n−6) biosynthesis, the Δ5 desaturase, was found in rat liver cell nuclei. Up to now, it has been shown that the fatty acid desaturases are located exclusively in the endoplasmic reticulum. Similarly to what happens with microsomal enzyme the nuclear Δ5 desaturase enzyme was only fully active in the presence of a cytosolic factor. In this condition it reached a specific activity of 50 pmol 20:4 n−6 formed/min/mg of protein. This fact would imply that purified nuclei like purified microsomes lack a soluble cytosol factor necessary for the total desaturation reaction expression. Besides the nuclear Δ5 desaturase has an optimal pH of 7.6 and is inhibited by 1 or 10 mM KCN. Low long chain acyl-CoA synthetase activity that catalyzes the formation of 20:3 n−6-CoA, was also found in liver nuclei. This step would be essential in nuclear desaturation since when ATP and/or CoA (necessary for the acylation reaction) are omitted from the incubation mixture, the desaturation reaction does not take place.

The effect of different doses of L-triiodothyronine (T3) on the activity of Δ6 and Δ5 desaturases and lipid fatty acid composition was studied in liver microsomes of male rats. The activity of Δ6 and Δ5 desaturases was decreased 24 and 28%, respectively, in animals administered a daily intraperitoneal dose of 1000μg T3/100g body wt. for 5 days, whereas with 500μg T3/100g body wt. only Δ6 desaturase activity was decreased. On the other hand, no enzyme activity changed at a shorter period of hormone treatment. Changes in microsomal fatty acid composition did not seem to be a direct consequence of desaturation activity, since after 1 and 5 days of T3 treatment, the concentrations of 18:2 (n-6) and 20:3 (n-6) decreased and only after 1 day that of 20:4 (n-6) increased in spite of unchanged or decreased Δ6 and Δ5 desaturase activities. Other factors than desaturation activity must be involved in fatty acid composition of thyroid hormonetreated rats, such as diet, membrane lipid synthesis and degradation, fatty acid turn-over and oxidation. (Mol Cell Biochem121: 149–153, 1993)

This study was performed to determine whether fatty acids incorporated into liver cell nuclei phosphatidylcholine (PtdCho) could be remodeled in the isolated nuclear. For this reason, rat liver cell nuclei were incubated in vitro with [1- 14 C]20:4n-6-CoA. PtdCho molecular species with the highest specific activity had an unsaturated fatty acid at sn- 1 and sn- 2 positions (20:4-20:4>>18:2-20:4>18:1-20:4). 16:0-20:4 and 18:0-20:4 PtdChos showed a minor specific activity. When labeled nuclei were reincubated in the absence of labeled substrate with the addition of cytosol, ATP and CoA, the specific activity of 20:4-20:4, 18:2-20:4 and 18:1-20:4 species decreased, while that of 16:0-20:4 and 18:0-20:4 increased. In conclusion, the asymmetric fatty acid distribution of saturated fatty acids at sn- 1 position, and unsaturated fatty acids at sn- 2 position of nuclear PtdCho molecular species was re-established by an acyl-CoA-dependent remodeling process.

Liver nuclear incorporation of stearic (18∶0), linoleic (18∶2n−6), and arachidonic (20∶4n−6) acids was studied by incubation in vitro of the [1-14C] fatty acids with nuclei, with or without the cytosol fraction at different times. The [1-14C] fatty acids were incorporated into the nuclei as free fatty acids in the following order: 18∶0>20∶4n−6≫18∶2n−6, and esterified into nuclear lipids by an acyl-CoA pathway. All [1-14C] fatty acids were esterified mainly to phospholipids and triacylglycerols and in a minor proportion to diacylglycerols. Only [1-14C] 18∶2n−6-CoA was incorporated into cholesterol esters. The incorporation was not modified by cytosol addition. The incorporation of 20∶4n−6 into nuclear phosphatidylcholine (PC) pools was also studied by incubation of liver nuclei in vitro with [1-14C]20∶4n−6-CoA, and nuclear labeled PC molecular species were determined. From the 15 PC nuclear molecular species determined, five were labeled with [1-14C]20∶4n−6-CoA: 18∶0–20∶4, 16∶0–20∶4, 18∶1–20∶4, 18∶2–20∶4, and 20∶4–20∶4. The highest specific radioactivity was found in 20∶4–20∶4 PC, which is a minor species. In conclusion, liver cell nuclei possess the necessary enzymes to incorporate exogenous saturated and unsaturated fatty acids into lipids by an acyl-CoA pathway, showing specificity for each fatty acid. Liver cell nuclei also utilize exogenous 20∶4n−6-CoA to synthesize the major molecular species of PC with 20∶4n−6 at the sn-2 position. However, the most actively synthesized is 20∶4–20∶4 PC, which is a quantitatively minor component. The labeling pattern of 20∶4–20∶4 PC would indicate that this molecular species is synthesized mainly by the de novo pathway.

A long-chain fatty acyl-CoA synthetase that catalyzes the activation of long-chain fatty acids as thioesters of CoA, was described in rat liver nuclei. This is the first step for further metabolization of fatty acids in the cell. Up to now, it has been shown that long-chain fatty acyl-CoA synthetase is located in the endoplasmic reticulum, in plasma membrane, in mitochondria and in peroxisomes. The nuclear long-chain fatty acyl-CoA synthetase was assayed using palmitic (16:0), linoleic (18:2n-6) and 8,11,14-eicosatrienoic (20:3n-6) acids as substrates and was stimulated linearly with nuclear protein concentration and with incubation time The higher enzymatic activity was observed with 18:2n-6 and 20:3n-6 acids as substrates. The synthesis of palmitoyl-CoA, linoleyl-CoA and 8,11,14-eicosatrienoyl-CoA followed normal Michaelis-Menten kinetics with respect to the corresponding substrate concentrations. The acyl-CoA synthetase seems to be saturated at a substrate concentration of 12.8 microM for all the acids tested. The apparent Km values decreased in the following order 20:3n-6 > 18:2n-6 > 16:0. The lowest apparent Km for palmitic acid indicates a preference for acylation of this acid in the cell nucleus.

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. Fil: Layerenza, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; Argentina Fil: González, Pedro Horacio. Universidad Nacional de la Plata. Facultad de Cs.médicas. Cátedra de Patología Ii; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina Fil: Garcia, Margarita Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; Argentina Fil: Polo, Monica Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; Argentina Fil: Sisti, Martin Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; Argentina Fil: Ves Losada, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; Argentina

s / Chemistry and Physics of Lipids 163S (2010) S66–S68 S67 In conclusion, these results suggest that impaired geranylgeranylation of Rabs in Arabidopsis results in disturbances of the intracellular vesicular traffic. doi:10.1016/j.chemphyslip.2010.05.197

Male rats maintained on a control diet at 24 C and shifted to 12 C for five days showed no modification in delta 6 or delta 5 desaturase activity. When delta 6 and delta 5 desaturase activities were diminished by a hyperglycidic diet, shifting to cold increased both enzymatic activities. In general, modifications observed in liver microsomal fatty acid composition are consistent with those associated with the enzymatic desaturation activity.

Male rats injected with a single saturating dose of L-triiodothyronine (T3) showed, after a lag time of approximately eight hr, a sharp rise in delta 9 desaturase activity. Desaturase activity reached a plateau which was 1-1.2 times above the base line levels of rats which were not hormone-treated. The plateau was maintained for five days in animals which were kept on daily hormone-treatment. The increase in delta 9 desaturase activity by T3 required ongoing protein synthesis, because the increase in enzymatic activity due to hormone treatment was completely abolished in the presence of cycloheximide. These findings suggest that cycloheximide may block the induction of delta 9 desaturase by T3 and/or inhibit the synthesis of protein(s) essential to the desaturation-response to T3. Modifications observed in liver microsomal fatty acid composition in T3 treated rats were independent of the effect on desaturation. It is suggested that other factors, such as diet, membrane lipid synthesis and degradation, as well as fatty acid turnover and oxidation, could be involved in affecting the fatty acid composition of thyroid hormone-treated rats.

Male rats maintained at 24 C and then shifted to 5 C for 5 days increased food intake and decreased in growth rate and food conversion. No modification was observed in Δ6 desaturase activity, while Δ9 desaturase activity decreased after this period of time. These results were confirmed by liver microsomal and mitochondrial fatty acid composition. The phospholipid composition of liver microsomes was unaltered, whereas in mitochondria, phosphatidylcholine and sphingomyelin decreased and phosphatidylethanolamine increased due to the cold environment. The influence of food intake and weight changes on fatty acid metabolism was studied using (i) rats maintained at 5 C with restricted food intake to match the food intake of those kept at 24 C with food ad libitum and (ii) rats maintained at 24 C whose food intake was also restricted so that their growth rate would be the same as that of rats maintained at 5 C with food ad libitum, respectively. These results indicate that the negative metabolic balance state of these cold conditions is not an active factor modifier of Δ6 desaturase activity, whereas it decreases Δ9 desaturase activity, reflecting the lipogenic characteristics of the latter enzyme.