Ana M. Bernasconi

Dietary cholesterol modulates Δ6 and Δ9 desaturase mRNAs and enzymatic activity in rats fed a low-EFA diet

The effects of a 1% addition of cholesterol to a diet low in EFA on FA desaturases were examined. The administration of cholesterol markedly increased the esterified cholesterol content in microsomes and total liver lipids from the first day, whereas the proportion of free cholesterol remained unaltered throughout the treatment. An excellent homeostasis in the free cholesterol content was apparently evoked by the acyl-CoA cholesterol acyltransferase. The cholesterol esters were mainly oleate, palmitate, and stearate, and the addition of cholesterol increased the relative proportions of cholesterol palmitoleate and oleate. The addition of cholesterol to a low-EFA diet induced, as in animals fed a high-FFA diet, a marked increase in liver stearoyl-CoA desaturase-1 mRNA and enzyme activity. This increased activity apparently evoked a similar enhancement of palmitoleic and oleic acids in total and microsomal liver lipids. The cholesterol-rich diet depressed the liver Δ6 and Δ5 desaturase activity. However, the abundance of Δ6 desaturase mRNA was not modified throughout the treatment. This indicates that the depressive effect is evoked at a step beyond that controlled by the mRNA level. The depression of both enzymatic activities was consistent with the decrease in the percentages of arachidonic acid and DHA in total and microsomal liver lipids. Taken together, these results indicate that through its modulating effect on the desaturases, dietary cholesterol may lead an animal or humaan fed low-EFA diet to a true deficiency by the decreased synthesis of the highly polyunsaturated acids derived from linoleic and α-linolenic acids.

Effect of polyunsaturated fatty acid deficiency on dipole relaxation in the membrane interface of rat liver microsomes

The influence of a fat-free diet on the lipid dynamics of rat liver microsomes and liposomes of microsomal lipids was studied by using different fluorescence methods. Lifetime distribution and rotational diffusion of probes with different localization in the lipid bilayer were measured using multifrequency fluorometry. Lateral mobility was studied by measuring excimer formation of pyrenedodecanoic acid. Dipolar relaxation in the interfacial region was studied using 2-dimethyl-amino-6-lauroylnaphthalene (Laurdan). In spite of large changes in the fatty acid composition of microsomal lipids, polyunsaturated fatty acid deficiency showed no effect on the lifetime distribution and rotational mobility of 1,6-diphenyl-1,3,5-hexatriene (DPH). l-(4-(trimethylamino)phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), 2- 7- and 12-(9-anthroiloxy)stearic acids. The treatment did not change the lateral diffusion of pyrenedodecanoic acid, either. However, generalized polarization of Laurdan fluorescence was higher in polyunsaturated fatty acid deficient microsomes as compared to the polyunsaturated fatty acid sufficient ones. This effect was also observed in liposomes of the total microsomal lipids, indicating that the changes in fatty acid composition resulting from polyunsaturated fatty acid deficiency produced a small but significant decrease in the rate of dipolar relaxation in the region of the lipid polar groups of the bilayer. The absence of lipid gel phase domains in rat liver microsomes was also indicated by Laurdan fluorescence features.

Prostaglandin biosynthesis in the gonads of the hematophagus insect Triatoma infestans

1. 1. Dietary arachidonic acid accumulated in male gonad lipids of the blood sucking bug Triatoma infectans. Fair amounts of arachidonic acid were found in testicles, male accessory glands and spermatophores whereas only traces of eicosa-8,11,14-trienoic acid were detected. 2. 2. Smaller amounts of arachidonic acid were found in the virgin female tissues: bursa copulatrix, spermathecae and ducts. 3. 3. In the spermatophores arachidonic acid was specially high in phosphatidyl inositol molecules (27.8%) followed by phosphatidyl ethanolamine (10.9%), phosphatidyl choline (3.4%) and phosphatidyl serine (1.1%). 4. 4. Prostaglandin PGE2 was synthesized from 14C arachidonic acid in testicles and spermatophores and the biosynthesis of PGE1 was also tested and recognized in spermatophores after the incubation with 14C eicosa-8,11,14-trienoic acid. 5. 5. No PGE2 synthetase activity could be detected in spermathecae of virgin females but it was recognized in the spermathecae 6 hr after copulation. 6. 6. Results indicate that males transfer to females during copulation by means of the spermatophores not only significant amounts of arachidonic acid in phospholipid molecules but also the PGE2 synthesizing enzymes.

Dietary cholesterol induces changes in molecular species of hepatic microsomal phosphatidylcholine.

After 21 days on a diet containing 1g% cholesterol and 0.5g% cholic acid, rats had an increased content of cholesterol in liver microsomal lipids. In liver, both cholesterol content and δ9 desaturase activity increased, whereas δ6 and δ5 desaturase activities decreased. These changes correlated with increases in oleic, palmitoleic, and linoleic acids and decreases in arachidonic and docosahexenoic acids in total microsomal lipids. Similar fatty acid changes were found in phosphatidylcholine (PC), the principal lipid of the microsomal membrane. In PC the predominant molecular fatty acid species (67% of the total) in the control rats were 18:0/20:4, 16:0/20:4, and 16:0/18:2; and they mainly determined the contribution of PC to the biophysical and biochemical properties of the phospholipid bilayer. The cholesterol diet decreased specifically the 18:0/20:4 species, and to a lesser extent, 16:0/20:4 and 18:0/22:6. The 18:1-containing species, especially 18:1/18:2 and less so 16:0/18:1 and 18:1/20:4, were increased. A new 18:1/18:1 species appeared. The independent effects of the presence of cholesterol and change of the fatty acid composition of the phospholipid bilayer of liver microsomes on the packing were studied by fluorescence methods using 6-lauroyl-2,4-dimethylaminonaphthalene, 1,6-diphenyl-1,3,5-hexatriene and 1-(4-trimethylammonium phenyl)-6-phenyl-1,3,5-hexatriene, which test different parameters and depths of the bilayer. Data showed that the increase of cholesterol in the membrane, and not the change of the fatty acid composition of phospholipids, was the main determinant of the increased bulk packing of the bilayer. The increase of fluid oleic- and linoleic-containing species almost compensated for the drop in 20:4- and 22:6-containing molecules. But the most important effect was that the general drop in essential n-6 and n-3 polyunsaturated fatty acids meant that this endogenous source for the needs of the animal decreased.

The occurrence of arachidonic acid in Triatoma infestans

1. Gas-liquid chromatography and mass spectrometry were used to verify the presence of arachidonic acid (20:4 omega 6) in adult male and female organs of the blood-suckling bug Triatoma infestans. Fat body, gonad and head lipids were analyzed. 2. Male gonads contained the highest percentage of phospholipids and the highest percentage of arachidonic acid. 3. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and sphingomyelin were isolated from gonads and heads of sexed insects and the fatty acid composition analyzed. 4. All the phospholipids analyzed contained relevant percentages of linoleic acid and fair percentages of arachidonic acid, except for sphingomyelin where no 20:4 omega 6 acid was found. 5. However, arachidonic acid was specifically incorporated in phosphatidylinositol since this phospholipid contained very large percentages of the acid. 6. In male gonads, phosphatidylcholine also contained a similar high percentage of the acid. Arachidonic acid is apparently incorporated, selectively, into these lipids from the blood of the host.

Molecular species of phosphoglycerides in liver microsomes of rats fed a fat-free diet.

The influence of a fat-free diet on the molecular species composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) of rat liver microsomes was studied by using reversed-phase high-pressure liquid chromatography. In the three phosphoglyceride classes analyzed, the fat-free diet produced a large decrease in the 18:0/20:4n−6 species but less important changes were found in the 16:0/20:4n−6 species. In PC, the most abundant phosphoglyceride class of rat liver microsomes, the fall in the 18:0/20:4n−6 species was counterbalanced mainly by an enhancement in the 16:0/18:1n−9 species although it was not evident in PE. In PI, the decrease in the 18:0/20:4n−6 species was counterbalanced by an increase in the 18:0/20:3n−9 species. Fluorescence polarization measurements of 1,6-diphenyl-1,3,5-hexatriene in liposomes of 16:0/18:1n−9-, 18:0/18:1n−9-, 16:0/20:4n−6-, and 18:0/20:4n−6-PC indicated that the change in the saturated fatty acid in the sn-1 position accompanying the replacement of 20:4n−6 by 18:1n−9 could be very important for a homeoviscous compensation, maintaining the membrane physical properties without large alterations in spite of the essential fatty acid deficiency due to the fat-free diet.