Effect of starvation and pesticide exposure on neutral lipid composition of the digestive gland of males of the apple snails Pomacea canaliculata.

Pollutants as well as starvation usually modify homeostasis of neutral lipids in aquatic organisms. However, studies on the simultaneous effects of both stressors are scarce. The aim of this study was to evaluate the effect of toxicant exposure under starvation conditions on neutral lipids of the freshwater snail Pomacea canaliculata, selected as the model organism. Starved adult male snails were exposed to sublethal concentration of the pesticide cypermethrin (100 µg/L) during 4 and 10 days. Fed snails were sacrificed at the onset of the experiment (T0), along with starved snails exposed to the pesticide vehicle (ethanol) and another group without solvent served as controls. Total lipid content, neutral lipid classes, fatty acid composition, and pesticide accumulation were determined in the digestive gland of snails. The ethanol concentration used was not an additional stressful agent. As expected, starvation caused a decrease in neutral lipid content in the digestive gland of snails with respect to T0 snails. Pesticide exposure caused, on the other hand, an increase in triacylglycerol content compared to ethanol exposure at day 10 of the bioassay. This increment correlated with the bioconcentration of cypermethrin, which was 47% higher by day 10 than by day 4. The fatty acid profile of triacylglycerols in the digestive gland was significantly altered under starvation and pesticide exposure. Stressed male snails showed the ability to preserve polyunsaturated fatty acids, as evidenced by their significant increase with respect to T0 snails. These results suggest that the alteration of lipid homeostasis could be involved in an adaptive mechanism of aquatic organisms to lipophilic and obesogenic pollutants.

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.

Phosphatidyl choline fatty acid remodeling in the hepatic cell nuclei.

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 [1-14C]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.

Incorporation and distribution of saturated and unsaturated fatty acids into nuclear lipids of hepatic cells.

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.

Incorporation of delta 5 desaturase substrate (dihomogammalinolenic acid, 20:3 n-6) and product (arachidonic acid 20:4 n-6) into rat liver cell nuclei.

The incorporation of [1-(14)C]20:3 n-6 and its desaturation product, [1-(14)C]20:4 n-6 into nuclear lipids from rat liver cells were investigated during in vitro delta5 desaturation. [1-(14)C]20:3 n-6 activated as 20:3 n-6-CoA by nuclear long chain acyl-CoA synthetase was: (1) incorporated into nuclear lipids mainly esterified to phospholipids and in a lesser proportion, to triglycerides and diglycerides; and (2) desaturated to 20:4 n-6-CoA by the nuclear delta5 desaturase. The amount of [1-(14)C]20:4 n-6 acid synthesized in cell nuclei increased along with time and was stimulated by the cytosol fraction. The major proportion of 20:4 n-6 was found in phospholipids and in a lesser proportion it remained as free fatty acids and was esterified to triglycerides and diglycerides. 20:4 n-6-CoA was incorporated into nuclear lipids and hydrolyzed to free fatty acid. These results indicate that liver cell nuclei possess the necessary enzymes to incorporate the delta5 desaturase substrate (20:3 n-6) as well as the product of desaturation (20:4 n-6) into nuclear TG, DG and PL following an acyl-CoA dependent pathway.

Long-chain fatty Acyl-CoA synthetase enzymatic activity in rat liver cell nuclei.

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.

Fatty acid delta 5 desaturation in rat liver cell nuclei.

Activity of one of the key enzymes involved in arachidonic acid (20:4 n-6) biosynthesis, the delta 5 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 delta 5 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 delta 5 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.

Effect of L-triiodothyronine on liver microsomal delta 6 and delta 5 desaturase activity of male rats.

The effect of different doses of L-triiodothyronine (T3) on the activity of delta 6 and delta 5 desaturases and lipid fatty acid composition was studied in liver microsomes of male rats. The activity of delta 6 and delta 5 desaturases was decreased 24 and 28%, respectively, in animals administered a daily intraperitoneal dose of 1000 micrograms T3/100 g body wt. for 5 days, whereas with 500 micrograms T3/100 g body wt. only delta 6 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 delta 6 and delta 5 desaturase activities. Other factors than desaturation activity must be involved in fatty acid composition of thyroid hormone-treated rats, such as diet, membrane lipid synthesis and degradation, fatty acid turn-over and oxidation.

Effect of L-triiodothyronine on delta 9 desaturase activity in liver microsomes of male rats.

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.

Effect of cold environment on hepatic microsomal delta 6 and delta 9 desaturase activity of male 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 delta 6 desaturase activity, while delta 9 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 delta 6 desaturase activity, whereas it decreases delta 9 desaturase activity, reflecting the lipogenic characteristics of the latter enzyme.