Desaturase activities are depleted before and after weaning in liver microsomes of spontaneously hypertensive rats

In the present study, we have investigated the microsomal linoleic acid desaturation steps into arachidonic acid in 10- and 30-day-old spontaneously hypertensive rats (SHR), as compared to their normotensive control rats, Wistar Kyoto (WKY). Suckled by adoptive Wistar normotensive female, the SHR and WKY were fed the same diet. Our results show lower Delta 6 and Delta 5 desaturase activities (the limiting steps in the bioconversion of linoleic acid into arachidonic acid) in the young SHR, as compared to the WKY normotensive rats. The fatty acid composition of liver microsomal total lipids evidences a higher proportion of linoleic acid in SHR than in WKY, in agreement with the partially depleted desaturase activities. Such a loss of desaturase activities may be under the control of hormones involved in the regulation of SHR blood pressure.     

Hepatic microsomal delta 9 desaturation of stearic acid in the spontaneously diabetic female BB rat

delta 9 desaturation of stearic (1-14C) acid has been estimated from incubation of liver microsomes of adult female spontaneously diabetic BB rat, an animal model resembling the spontaneous juvenile diabetes in humans, comparatively to adult female control Wistar rat. The animals were sacrificed, when hyperglycemic, 24 hours after the last insulin injection to the BB rats. Stearic acid delta 9 desaturase activity is drastically depressed in the BB rats when fatty acid composition of liver phospholipids and microsomal total liver lipids are changed in spite of the daily injection of insulin necessary for the BB rats survival.     

Chronologic studies of the effects of a hypoproteinic diet followed by an equilibrated diet on delta-6 and delta-5 desaturations of linoleic acid in liver microsomes in the rat

A low protein diet affects amounts of linoleic and arachidonic acids in hepatic microsomal phospholipids of growing rats. Are the changes related to modifications in microsomal delta 6- and delta 5- linoleic acid desaturase activities? Two groups of Wistar rats weighing 80 +/- 5 g at the beginning of the experiment were used: Control group (T) was fed on a 16% gluten + 4% casein diet for 53 days; Experimental group (E) was fed on a 4% gluten + 1% casein diet for 26 days (MP) then Control diet for 27 days (RE). After 2, 14 and 26 days of MP and 2, 15 and 27 days of RE, rats of each group were sacrificed. Protein and water contents of liver, quantitative fatty acid, composition of total lipids in liver and hepatic microsomes were determined. delta 6- and delta 5- linoleic acid desaturase activities were estimated from incubation of liver microsomes with [1-14C] C 18: 2 n-6 or [2(14)C] C 20: 3 n-6 respectively. The low protein diet stops practically ponderal growth. The fatty-acid compositions of microsomal total lipids of E rats were affected in comparison with values of T rats. These modifications persist after 27 days of RE. The C 20: 4 n-6/C 18: 2 n-6 ratio in microsomal total lipids was slightly different between T and E rats but increased strongly during refeeding. Same modifications take place in the fatty-acid composition of hepatic total lipids. After two days of MP, delta 6- and delta 5- desaturase activities were depressed, phenomenon that not persist in the course of MP. These enzyme activities increase to higher values than those of the T after two days of RE.     

Fatty acid composition of brain hemispheres of spontaneously hypertensive rats suckled by female Wistar rats

Total lipid fatty acid composition was investigated in brain hemispheres of male Spontaneously Hypertensive Rats (SHR), compared with normotensive Wistar Kyoto rats (WKY) used as controls. Both strains were suckled by adoptive Wistar mothers, and then fed a standard diet after weaning. No difference was observed between the two hemispheres of WKY killed either at 10 or 30 days. In SHR killed at 10 days, the two hemispheres showed differences, SHR left hemispheres exhibiting greater fatty acid composition changes than those of WKY, phenomenon that toned down at 30 days. Hence, SHR pups showed a different total lipid fatty acid composition of their brain hemispheres when compared with their WKY controls, though the two strains received the same diet. Genetically programmed hypertension might be, directly or not, involved in these changes.     

Activities of fatty acid desaturases and fatty acid composition of liver microsomes in rats fed beta-carotene and 13-cis-retinoic acid

The fatty acid composition of microsomal lipids and the activities of delta 9- and delta 6-desaturases in liver microsomes of rats fed diets supplemented with beta-carotene and two levels of 13-cis-retinoic acid were studied. Four groups of male, weanling rats were fed semipurified diets containing 0 or 100 mg beta-carotene per kg diet, and 20 or 100 mg 13-cis-retinoic acid per kg diet. After 11 weeks of feeding, the rats were killed, liver microsomes were prepared and assayed for delta 9-desaturase and delta 6-desaturase activities. The activity of delta 9-desaturase was lower in liver microsomes of rats fed beta-carotene-supplemented diet or the diet supplemented with the higher level of 13-cis-retinoic acid. Microsomal delta 6-desaturase activity was, however, higher in liver of rats fed 13-cis retinoic acid; there was no effect of beta-carotene on delta 6-desaturase activity. The fatty acid compositional data on total lipids of liver microsomes were consistent with the diet-induced changes in fatty acid desaturases. Phospholipid composition of liver microsomes was also altered as a result of feeding beta-carotene or 13-cis-retinoic acid-containing diets. The proportions of phosphatidylethanolamine were generally higher, whereas those of phosphatidylcholine were lower in the experimental groups as compared with the control.     

Effects of streptozotocin and dietary fructose on delta-6 desaturation in spontaneously hypertensive rat liver

We have investigated the effects of hypertension associated with diabetes mellitus on polyunsaturated fatty acid biosynthesis. For this purpose, two rat models for these pathologies have been established: a type 1 diabetic hypertensive model obtained by streptozotocin injection to spontaneously hypertensive rat (SHR), followed or not by insulin treatment (experiment 1); a type 2 diabetic hypertensive model by feeding SHR with a fructose enriched diet (experiment 2). Liver gene expression of delta-6 desaturase (D6D), microsomal D6D activities and fatty acid composition of total lipids were estimated. In experiment 1, an increase of linoleic acid (18:2 n-6) level was observed in the streptozotocin group. D6D gene expression appeared depressed in both experimental groups. Insulin did not reverse the streptozotocin effect in SHR, as it does in insulin-dependent diabetic rats. In experiment 2, the results showed a decrease of 18:2 n-6 and of long chain products of desaturation in rats fed on fructose diet. Delta-6 n-3 desaturase activity was significantly increased, whereas gene expression tended to decrease. Feeding fructose induced a significant increase in delta-9 desaturated products, suggesting a stimulation of stearoyl-CoA desaturase. These changes in monounsaturated fatty acids strongly differ from those observed in the streptozotocin experiment, indicating that the effects on lipogenesis of hypertension linked to diabetes differ according to the type of diabetes. Then, these results indicate that the liver steatosis observed during genetic hypertension was reinforced by fructose feeding. All together, the present results showed that hypertension associated to type 1 or type 2 diabetes exacerbated the damage caused by diabetes or hypertension alone on liver lipid metabolism. The metabolic effects induced by fructose being very similar to those found in human NIDDM, SHR fed a fructose-rich diet appears to be an appropriate model for studying the consequences of the combination of hypertension and NIDDM in the metabolic syndrome diseases.     

Fatty acid desaturation and microsomal lipid fatty acid composition in experimental hypothyroidism.

We have studied the influence of experimental hypothyroidism in the rat on the synthesis of unsaturated fatty acids and on liver microsomal lipid fatty acid composition. Hypothyroid rats demonstrated an 80% decrease in delta 9 (stearate) desaturation and a 43% decrease in delta 6 (linoleate) desaturation. Liver microsomal fatty acid composition was altered in the hypothyroid animals with a significantly decreased proportion of arachidonate and increased proportions of linoleate, eicosa-8,11,14-trienoate, eicosapentaenoate and docosahexaenoate. The bulk of these changes occurred in both of the two major phospholipid components, phosphatidylcholine and phosphatidylethanolamine. All of the changes were corrected by treatment of the hypothyroid rat with 25 micrograms of tri-iodothyronine/100 g body wt. twice daily. The diminished delta 9 desaturation did not lead to any changes in fatty acid composition. The increased linoleate and decreased arachidonate levels may be due to the diminished delta 6 desaturase activity, the rate-controlling step in the conversion of linoleate into arachidonate. The increases in the proportions of the other polyunsaturated fatty acid components cannot be explained by changes in the synthesis of unsaturated fatty acids, but are probably due to diminished utilization of these fatty acids.     

Effect of long-term feeding olive and sunflower oils on fatty acid composition and desaturation activities of liver microsomes

Changes in microsomal fatty acid composition, delta 9- and delta 6-desaturase activities and cholesterol and phosphorus liver content were studied in dogs fed olive and sunflower oil diets. No changes were observed in the saturated fatty acids between dietary groups. The level of monounsaturated fatty acids was more elevated in animals fed the OO diet, because of its high relative content in this diet although the in vitro delta 9-desaturase activity was similar in microsomes from the two groups. The proportion of arachidonic acid was similar in SO and OO fed animals. This similar level occurred despite a significant increase in the level of linoleic acid in membrane lipids as a result of feeding the SO supplement. The in vitro delta 6-desaturase activity in liver microsomes showed no differences between dogs fed the two diets. Thus, the higher desaturation presented in vivo by microsomes from OO group may be related to the inhibition by linoleic acid of delta 6-desaturase in dogs fed the SO diet. The polyunsaturated fatty acids (PUFA) from the n-3 series were higher in microsomal phosphatidylcholine and phosphatidylethanolamine from animals fed the OO supplemented diet. The cholesterol/phosphorus molar ratio was higher in the SO group in which the unsaturation index was only slightly affected in phospholipids.     

Microsomal fatty acid desaturation and elongation in a human lung carcinoma grown in nude mice

Since tumor cells show abnormal fatty acid composition, it is likely that their desaturase systems were affected to some extent. Although desaturase activities in experimental tumors have been evaluated, to our knowledge, fatty acid desaturases in human neoplasms and particularly in human tumors grown in nude mice have not been assessed yet. We have therefore, chosen a rapidly growing human lung mucoepidermoid carcinoma (HLMC) grown in nude mice to study microsomal fatty acid desaturation and chain elongation activities. Tumor microsomal proteins were incubated with unlabeled malonyl-CoA and one of the following fatty acids: [1-14C]palmitic (16:0), [1-14C]linoleic (18:2), alpha-[1-14C]linolenic (alpha-18:3), and unlabeled gamma-linolenic (gamma-18:3) plus [2-14C]malonyl-CoA. Data show that HLMC microsomes were capable to desaturate 16:0, alpha-18:3, and dihomogammalinolenic acids (20:3) by delta 9, delta 6 and delta 5 desaturase, respectively; however, delta 6 desaturase activity on [14C]18:2 was not detected. The microsomal elongation system was active in all fatty acid series tested except for 18:2. These findings show that the undetectable activity for 18:2 desaturation is not exclusively found in experimental tumors.     

Site of participation of cytochrome b5 in hepatic microsomal fatty acid chain elongation. Electron input in the first reduction step

The present study provides strong evidence for the involvement of rat liver microsomal cytochrome b5 in the first reduction step of fatty acid chain elongation. The rate of reoxidation of NADH-reduced microsomal cytochrome b5 was markedly stimulated (up to 3-fold) by the addition of increasing concentrations of beta-ketohexadecanoyl-CoA (1-8 microM). A quantitative analysis of product formation, the effect of cyanide, and anaerobiosis completely exclude the possibility that desaturase activity accounted for the beta-ketohexadecanoyl-CoA-induced stimulation of the cytochrome b5 reoxidation rate. Using liver microsomes from untreated rats, the beta-keto substrate was found to stimulate the rate of reoxidation of cytochrome b5 by 30%. However, when liver microsomes from fat-free diet rats were employed the stimulation was more than 3-fold, suggesting that the beta-ketoacyl-CoA reductase is inducible by a high carbohydrate, fat-free diet. This study also provides evidence for the noninvolvement of cytochrome b5 in the terminal reaction step (second reduction step of chain elongation), which is catalyzed by the trans-2-enoyl-CoA reductase. Although trans-2-hexadecenoyl-CoA significantly stimulated the NADH-reduced cytochrome b5 reoxidation rate under aerobic conditions, it did not have any stimulatory effect under anaerobic conditions. One interpretation of these results is that the trans-2-hexadecenoyl-CoA is substrate for the microsomal delta 9 desaturase system. Consistent with this conclusion was the fact that the trans-2-hexadecenoyl-CoA inhibited the liver microsomal delta 9 desaturation of stearoyl-CoA to oleoyl-CoA.     

Spontaneous diabetes in BB rats: evidence for insulin dependent liver microsomal delta 6 and delta 5 desaturase activities

We studied linoleic acid delta 5 and dihomo-gamma-linolenic acid delta 5 desaturations, and fatty acid composition, of liver microsomes in the insulin-dependent spontaneously diabetic adult female BB rat. These desaturations were defective along the normo- and hyper-glycemic period and restored during the hypoglycemic period which followed the insulin injection to the diabetic rats. The fatty acid composition of BB rats microsomes was not consistent with the desaturase activities at the different periods of glycemia, probably because other factors than desaturation impairments were involved in the evolution of fatty acid composition.     

Effect of sodium loading (3% NaCl) on arachidonic acid biosynthesis in rat liver microsomes.

Sodium loading increases arachidonic acid (AA) metabolism by way of the prostaglandins(PGs) from series 2. Its effect on AA biosynthesis remains unknown. The purpose of the present study was to investigate the influence of sodium loading on the fatty acid composition of liver and liver microsomes, and the liver microsomal delta-6 and delta-5 desaturations of linoleic acid (LA) into AA. We found a decrease of LA and dihomo-gamma-linolenic acid (DGLA) levels in liver total lipids of Wistar rats receiving hypernatriuretic drinking water (NaCl 3%) for 60 days. At the same time AA increased. DGLA decreased and AA increased in liver microsomal total lipids. 1(14) C-LA delta-6 desaturase and 2(14) C-DGLA delta-5 desaturase activities increased in liver microsomes. These results show that, in addition to its influence on the regulation of glomerular filtration, sodium loading is involved in the regulation of liver AA biosynthesis.     

The acyl-CoA desaturases of microsomes from rat liver and the Morris 7777 hepatoma.

We have investigated the role of the microsomal oxidative desaturase in defining the aberrant phosphoglyceride fatty acid composition of hepatomas. The microsomal delta 9-stearoyl-CoA, delta 6-oleoyl(linolenoyl)-CoA, and delta 5-eicosatrienoyl-CA desaturase activities were studied in control and host liver and in the poorly differentiated Morris 7777 hepatoma. The delta 9-stearoyl-CoA desaturase of the hepatoma was significantly decreased (42%) relative to control liver, yet the hepatoma specific activity was twice that of host liver. Additionally, the specific activity of the delta 9-stearoyl-CoA desaturase of the tumor was found to decrease with increasing tumor weight. Also this desaturase was inactivated by freezing and thawing. The delta 6-oleoyl(linolenoyl)-CoA and delta 5-eicosatrienoyl-CoA desaturases of the hepatoma were 39% and 4% of control, respectively. The electron transport components involved in the desaturase system were reduced, although this did not appear to be rate-limiting. In addition, two competing metabolic reactions which could lower the observed desaturase activities, hydrolysis of the thioester and incorporation of substrate acyl-CoA molecules into glycerides, did not appear to be responsible for the lowered desaturase activities of the tumor. Thus, it appears that reduced levels of the desaturases themselves may be responsible for the observed activities. These results indicate that the capacity of the hepatoma to biosynthesize polyunsaturated fatty acids is greatly reduced and this is consistent with the decreased polyene content observed in many neoplasms.     

Effects of angiotensin II type 1 receptor blockade on the oxidative stress in spontaneously hypertensive rat tissues

The aim of this work was to investigate the production of oxidative damage in homogenized kidney, liver and brain of spontaneously hypertensive rats (SHR), as well as the involvement of angiotensin (Ang) II in this process. Groups of 12-week-old SHR and Wistar Kyoto rats (WKY) were given 10 mg/kg/day losartan in the drinking water during 14 days. Other groups of WKY and SHR without treatment were used as controls. The production of thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH) and the activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (Gpx) were determined. No significant difference in TBARS was observed between untreated SHR or WKY rats; GSH content was lower in the liver but higher in the brain of SHR compared to WKY rats. In tissues from the SHR group, SOD and Gpx activities were reduced, whereas CAT activity was slightly increased in kidney. TBARS levels did not change in WKY rats after losartan administration, but were reduced in SHR liver and brain. Losartan treatment decreased GSH content in WKY kidney, but increased GSH in SHR liver. The activity of the antioxidant enzymes was not modified by losartan in WKY rats; however, their activities increased in tissues from treated SHR. The lower activity of antioxidant enzymes in tissues from hypertensive rats compared to those detected in normotensive controls, indicates oxidative stress production. Ang II seems to play no role in this process in normotensive animals, although AT1 receptor blockade in SHR enhances the enzymatic activity indicating that Ang II is implicated in oxidative stress generation in the hypertensive animals.     

Fatty-acid desaturation and microsomal lipid fatty-acid composition in experimental hyperthyroidism.

We have studied the influence of experimental hyperthyroidism in the rat on the synthesis of unsaturated fatty acids and on liver microsomal lipid fatty-acid composition. Tri-iodothyronine treatment (25 micrograms/100 g body weight) daily for 3 weeks caused no significant changes in delta 9 (stearate) desaturation but a 24% decrease in delta 6 (linoleate) desaturation. Much larger doses of tri-iodothyronine increased delta 9 desaturation. Liver microsomal fatty-acid composition in hyperthyroidism is altered with significantly increased proportions of stearate and arachidonate and decreased proportions of palmitate, palmitoleate, linoleate (C18:2) and eicosa-8,11,14-trienoate (C20:3). These changes, other than the decreases proportion of C20:3 fatty acid, which may be due to the diminished delta 6 desaturase activity, cannot be attributed to changes in fatty-acid desaturation. Most of these changes were also found to be due not simply to the decreased weight gain or the increased food intake of the hyperthyroid animals. Only the decreased C18:2 fatty-acid proportions could be mimicked by restricting food intake of control animals and none of the changes were prevented by restricting food intake of hyperthyroid animals. Thus most of the changes in microsomal lipid fatty-acid composition are likely to be due to a thyroid hormone effect on peripheral lipid mobilization or lipid degradation.     

Fatty acid desaturase activities are modulated by phytosterol incorporation in microsomes

The effect of phytosterol-rich diets (3% beta-sitosterol + 2% campesterol) on rat liver microsomal fatty acid desaturases, membrane dynamics and lipid composition was investigated. After a 21 day period, phytosterol was incorporated into microsomes and the membrane fluidity decreased. There were no changes in either the phospholipid composition or in the total sterol content. However, the phytosterol/cholesterol ratio increased. In the animals fed phytosterols, the delta 5-, delta 6- and delta 9-fatty acid desaturases were significantly more active than in control animals. The changes in the lipid fatty acid composition were consistent with those of the desaturase activities. Hence, it is suggested that: (1) dietary phytosterol modulates desaturase activities; (2) phytosterols make the membrane more rigid but do not induce changes in the relative phospholipid composition; (3) delta 9-, delta 5- and delta 6-desaturase activities increase when the membrane becomes more rigid without changes in the phospholipid composition.     

In vitro modification of cholesterol content of rat liver microsomes. Effects upon membrane 'fluidity' and activities of glucose-6-phosphatase and fatty acid desaturation systems

The cholesterol content of rat liver microsomal membranes was modified in vitro by incubating microsomes and cytosol with liposomes prepared by sonication of microsomal lipids and cholesterol. In this way, the cholesterol to phospholipid molar ratio was increased from 0.11-0.13 in untreated microsomes to a maximal of 0.8 in treated ones. Cholesterol incorporation in microsomes produced an increase in the diphenyl-hexatriene steady-state fluorescence anisotropy and a decrease in the efficiency of pyrene-excimer formation which indicated a decrease in the rotational and translational mobility, respectively, of these probes in the membranes lipid phase. Cholesterol incorporation in microsomes did not affect significantly the glucose-6-phosphatase activity in 0.1% Triton X-100 totally disrupted microsomes, but diminished the glucose-6-phosphatase activity of 'intact' microsomes. This indicates that possibly the glucose 6-phosphate translocation across the microsomal membrane is impeded by an increase in the membrane apparent 'microviscosity'. Cholesterol incorporation in microsomes decreased NADH-cytochrome c reductase without affecting NADH-ferricyanide reductase activity. The delta 9 desaturation reaction rate was enhanced by cholesterol incorporation at low but not at high palmitic acid substrate concentration. delta 5 and delta 6 desaturase reaction-rates were increased both at low and high fatty acid substrate concentrations. These results suggest that a mechanism involving fatty acid desaturase enzymes, might exist to self-regulate the microsomal membrane lipid phase 'fluidity' in the rat liver.     

Extreme decrease of linoleic acid in renal medulla from rats after a diet supplemented with cod liver oil

Spontaneously hypertensive (SHR) and normotensive rats were fed a diet supplemented with linseed oil or cod liver oil for 22 weeks. The most remarkable finding was an extreme fall of linoleic acid in lipids from renal medulla after cod liver oil supplementation. In free fatty acids (FFA) eicosatrienoic acid (C2): 3n-9) appeared increased as a sign of essential fatty acid (EFA) deficiency.     

Incorporation into liver microsomal lipids of linoleic and stearic acids and of their respective products of delta 6 and delta 9 desaturation, gamma-linolenic and oleic acids: effect of age and of blackcurrant seed oil.

The incorporation of [1-14C]linoleic and [1-14C]stearic acid and of their delta 6 and delta 9 desaturation products (gamma-linolenic and oleic acids, respectively) into different classes of lipids was studied in liver microsomes of rats in function of the diet (blackcurrant seed oil diet, containing gamma-linolenic acid, versus control diet) and in function of age (3, 6 and 9 months). After delta 6 desaturation, total radioactivity was distributed between phospholipids, especially phosphatidylcholine, and neutral lipids. The desaturation product, gamma-linolenic acid, was totally recovered in the phospholipid fraction. Blackcurrant seed oil, which decreased the rate of delta 6 desaturation in 6- and 9-month-old rats, also decreased the incorporation of radioactivity in total phospholipids, especially in phosphatidylcholine. At 6 months of age, after delta 9 desaturation, the majority of radioactivity was recovered in neutral lipids principally as oleic acid, the desaturation product. The precursor, stearic acid, was highly incorporated into phospholipids, especially in rats on a diet of blackcurrant seed oil.     

Effect of temperature on the structure of rat liver microsomes studied by electron spin resonance, fluorescence and activity of enzymes involved in fatty acid biosynthesis

The structural properties of rat liver microsomes were studied by physical and kinetic methods. The microsomes and the lipids extracted from the microsomes were labeled with 16-doxyl-stearic acid- and N-phenyl-1-naphthylamine. The electron spin resonance spectra and the fluorescence intensities were respectively determined at different temperatures from approximately 10 to 40 C. Both methods suggested the absence of a transition temperature indicative of a phase change in the bulk of the lipids of the microsomes in the temperature range studied. The fluidity of the lipid bilayer increased smoothly with the temperature. The Arrhenius plots of the NADH-ferricyanide reductase, NADH-cyt.c reductase, delta 9 desaturase, delta 6 desaturase and palmitic elongation to stearic acid also indicated the absence of a detectable change of phase from crystalline to liquid crystalline in the boundary lipids of these enzymes from 10 C to 40 C. The transference of electrons from the NADH-cyt.b5 reductase to the cyt.b5 is the rate limiting step in the first parts of the electron transport chain. However, the delta 9 desaturase is the rate limiting step of all the series of reactions involved in the delta 9 fatty acid desaturation. Similar conclusions may be extended to the delta 6 desaturation of fatty acids. The physical state of the lipids surrounding the desaturating system would be different from boundary lipids of the cyt.P450 system.