Effect of dietary n - 3 polyunsaturated fatty acids on oxidant/antioxidant status in macrosomic offspring of diabetic rats.

The aim of this work was to determine the effect of dietary n - 3 PUFA on oxidant/antioxidant status, in vitro very low and low density lipoprotein (VLDL-LDL), and VLDL-LDL-fatty acid composition in macrosomic pups of diabetic mothers. We hypothesized that n - 3 PUFA would improve oxidative stress in macrosomia. Diabetes was induced in female Wistar rats fed with the ISIO diet (control) or with the EPAX diet (enriched in n - 3 PUFAs), by streptozotocin. The macrosomic pups were killed at birth (day 0) and at adulthood (day 90). Lipid parameters and VLDL-LDL-fatty acid composition were investigated. The oxidant/antioxidant status was determined by measuring plasma oxygen radical absorbance capacity (ORAC), hydroperoxides, carbonyl proteins, and VLDL-LDL oxidation. Macrosomic rats of ISIO fed diabetic mothers showed an increase in plasma and VLDL-LDL-triglycerides and VLDL-LDL-cholesterol levels and altered VLDL-LDL-fatty acid composition. Plasma ORAC was low with high hydroperoxide and carbonyl protein levels. The in vitro oxidizability of VLDL-LDL was enhanced in these macrosomic rats. The EPAX diet corrected lipid parameters and improved oxidant/antioxidant status but increased VLDL-LDL susceptibility to oxidation. Macrosomia is associated with lipid abnormalities and oxidative stress. n - 3 PUFA exerts favorable effects on lipid metabolism and on the oxidant/antioxidant status of macrosomic rats. However, there are no evident effects on VLDL-LDL oxidation.

Fat absorption and metabolism in bile duct ligated rats.

BACKGROUND: Bile excretion is obstructed in children with extrahepatic bile duct atresia (EHBA) resulting in fat malabsorption and disturbed lipid metabolism. AIM: Investigate if the bile duct ligated rat exhibits similar deviations as patients with EHBA under different feeding conditions. METHODS: 6 bile duct ligated Wistar rats and 12 matched paired controls were randomised over 3 feeding groups. Rats were killed 16 or 30 days postsurgery. Faeces, blood and livers were collected. Fat absorption was evaluated, markers for cholestasis and the fatty acid composition of serum phospholipids (PL) and cholesterol esters (CE) were determined. Fatty acid desaturation activities in liver microsomes were measured. RESULTS: Cholestatic bile duct ligated rats have a lower fat absorption coefficient and a lower fraction of 18:2n-6 and 18:3n-3 in serum triglycerides than their controls. This demonstrates that bile duct ligated rats suffer from fat malabsorption. In contrast to the observations in serum triglycerides, 18:2n-6 and 18:3n-3 were not reduced in serum PL and CE of cholestatic rats. Overflow of 18:2n-6 rich biliary PL in the general circulation could contribute to this observation. In agreement with what was found in man, serum PL of cholestatic rats have a higher 16:0/18:0 ratio, increased monoenes and reduced unsaturated fatty acids. However, no differences were observed in microsomal desaturation activities. CONCLUSION: Cholestatic bile duct ligated rats exhibit similar deviations in serum fatty acid composition as found in patients with EHBA, therefore they can be used as a model for this human disease.

Effect of ethanol on polyunsaturated fatty acid biosynthesis in hepatocytes from spontaneously hypertensive rats.

BACKGROUND: Polyunsaturated fatty acids (PUFA) play a major role in membrane structures that are modified during alcoholism. PUFA are also precursors of second messengers-eicosanoids-involved in the regulation of blood pressure. Alcohol has been related to hypertension and to alterations in liver PUFA metabolism. We investigated the effects of ethanol on PUFA biogenesis in hepatocytes of Wistar Kyoto (WKY) rats and Spontaneously Hypertensive Rats (SHR). The effects of a diet enriched with n-3 PUFA, which is known to modulate hypertension, were also studied. METHODS: Isolated hepatocytes from male normotensive Wistar Kyoto (WKY) rats and SHR were incubated for 60 min in the presence of labeled linoleic acid and DGLA, which are precursors of the limiting desaturation steps of PUFA biosynthesis, into a medium containing different concentrations of ethanol. Hepatocytes from SHR that were fed a diet supplemented with n-3 PUFA were incubated with the same precursors. RESULTS: First, the hepatic biogenesis of PUFA is dependent on the level of ethanol in the incubation medium. Second, Delta5 desaturase was more sensitive than Delta6 desaturase to changes in alcohol concentration. Third, in SHR, a tremendous decrease of arachidonic acid biosynthesis was evidenced in alcohol-intoxicated hepatocytes; the effect was reinforced when ethanol concentration was high, mainly for Delta5 desaturase. Fourth, in the presence of ethanol, the biogenesis of PUFA was altered in isolated hepatocytes from SHR that were fed the diet supplemented with n-3 PUFA, particularly via an inhibition of Delta5 desaturation. CONCLUSIONS: Our study showed that hepatocyte PUFA biogenesis is dependent on ethanol concentration. Ethanol strongly inhibits the synthesis of PUFA in hepatocytes from SHR, which can explain the deficit of prostaglandin precursors observed in cardiovascular diseases linked to ethanol intoxication. n-3 PUFA supplemented diet reinforces the inhibition of arachidonic acid synthesis, likely by a substrate competition toward Delta5 desaturation. This in vitro approach provides a better understanding of the effects of ethanol on fatty acid metabolism in relation to hypertension.

Depletion of delta 9 desaturase (EC 1.14.99.5) activity in lactating rat during protein restriction.

The effects of protein restriction on the activity of delta9 desaturase (EC 1.14.99.5) were investigated in lactating rats. A control group was fed a balanced diet (20% casein) for 14 days, whereas the experimental groups were fed a low-protein diet (8% casein), supplemented with or without L-methionine (0.4%), for 14 days. The enzyme activity was measured by incubations of hepatic microsomal pellets with (1-14C) stearic acid. Results showed a decreased delta9 desaturase activity, after 2,7 and 14 days of depleted diet, of -50, -40 and -33% respectively, compared with control. The supplementation of the low-protein diet with 0.4% methionine, which favours food consumption as well as growth, did not improve the altered delta9 desaturase activity. Our data evidenced that delta9 desaturase activity is depleted by protein restriction during lactation, when the protein needs are high for the biosynthesis of animal tissues. This change has to be considered as a sign of depressed delta9 desaturase biosynthesis or modifications of enzymatic properties, or both.

Membrane fluidity and fatty acid metabolism in kidney cells from rats fed purified eicosapentaenoic acid or purified docosahexaenoic acid.

Rats were given a supplement (1.5 ml/day) of purified eicosapentaenoic acid (EPA, 20:5,n-3), purified docosahexaenoic acid (DHA, 22:6,n-3)), or corn oil for 10 days. Membrane fluidity, measured as the steady-state fluorescence polarization of diphenylhexatriene (DPH), was approximately 20% lower in kidney cells from rats fed purified EPA than in cells from the DHA-fed or corn-oil fed animals. The level of 20:5(n-3) in kidney phospholipids was 18 times higher in rats fed EPA, and four times higher in those fed DHA as compared to the corn-oil group. The level of arachidonic acid (20:4,n-6) was concomitantly decreased, while linoleic acid (18:2,n-6) was increased in kidney-phospholipids in the n-3 fatty acid fed rats. The proportion of 22:6(n-3) in kidney phospholipids was not affected by EPA supplementation, while the DHA diet slightly increased the level of this fatty acid. The distribution of phospholipid subclasses was significantly altered in that phosphatidylcholine was increased and phosphatidylethanolamine was concomitantly decreased. It is suggested that the decrease in 20:4(n-6) is relatively more important in the regulation of fluidity than a concomitant increase in 20:5(n-3). It is also suggested that the compensatory modifications of the phospholipid subclass distribution as a response to decreased 20:4(n-6)/20:5(n-3) ratio was not sufficient to maintain fluidity when the ratio was as low as in the present study. The incorporation of labelled linolenic acid (18:3,n-3) in phospholipids was decreased in cells from the n-3 supplemented rats. Since endogenous 22:5(n-3) in phospholipids was only increased in the EPA group, 22:6(n-3) only in the DHA group, and 20:5(n-3) in both, it is suggested that the decreased incorporation of labelled 18:3(n-3) into phospholipids of the DHA-fed rats in particular is correlated to the increased level of 22:6(n-3) in the membrane phospholipids. The incorporation of fatty acids into phopholipids may thus show substrate specificity, in that 22:6(n-3) is less exchangable with labelled 18:3(n-3) than is 20:5(n-3). These results demonstrate that increasing levels of n-3 fatty acids in membranes affect the uptake and intracellular metabolism of fatty acids as well as membrane fluidity in the kidney.

Psychosocial stress, catecholamines, and essential fatty acid metabolism in rats.

To examine the effects of psychosocial stress and the "stress hormone," epinephrine, on essential fatty acid metabolism in rats, two studies were conducted. In the first, the effects of four weeks of (i) social isolation and (ii) group housing (control) on liver microsomal delta 6 and delta 5 n-6 desaturase activity were studied in group-reared male normotensive (Wistar Kyoto) and spontaneously hypertensive (SHR) rats (n = 5/group). The second study examined the effects of acute ip epinephrine (0.0, 1.0, 2.0, and 4.0 mg/kg) 6 hr prior to and following an ig dose (4 g/kg) of safflower oil (rich in 18:2n-6, LA) on plasma and liver LA, 20:4n-6 (AA), and LA/AA ratios in adult essential fatty acid deficient Sprague-Dawley rats (n = 6/group). In the first experiment, isolation stress significantly inhibited the activity of delta 6 (P < 0.05) and delta 5 (P < 0.01) desaturase in the normotensive rats and of delta 5 desaturase in the SHR (P < 0.05). In the second study, epinephrine increased plasma and liver LA at doses 1.0 and 2.0 mg/kg in most of the fractions examined, and suppressed AA levels. The response of the LA/AA ratio to epinephrine varied between tissues and among lipid fractions, but increased this ratio at the moderate doses (2.0-4.0 mg/kg) of epinephrine in most cases. These data suggest that psychosocial stressors are capable of inhibiting the rate limiting steps of essential fatty acid metabolism and that this response is more pronounced in the SHR than in the Wistar Kyoto. They also suggest that epinephrine is capable of altering the in vivo metabolism of essential fatty acids in the rat.

Time-course effects of protein malnutrition on hepatic fatty acids delta 6 and delta 5 desaturation in the growing rat.

1. In growing rats, the time-course effects of giving a normal-protein diet (200 g casein/kg; NP) for 52 d, a low-protein diet (20 g casein/kg; LP) for 52 d and a LP diet for 26 d followed by balanced refeeding (200 g casein/kg; BR) for 26 d, on the fatty acid composition of liver total lipids and microsomal phospholipids were investigated together with delta 6- and delta 5-microsomal desaturase activities. 2. The oleic acid content (mg/g tissue) of liver total lipids increased progressively with the LP diet, while linoleic acid was increased only at days 7 and 52. 20:3 omega 6, 20:4 omega 6, 22:5 omega 6 and 22:6 omega 3 fatty acids decreased during the period on the LP diet. BR for 7 d was sufficient to restore the fatty acid composition of total lipids to control values. Changes in the fatty acid composition of liver microsomal L-alpha-phosphatidylcholines were observed only after 52 d on the LP diet; the proportions (% w/w total fatty acids) of 18:0, 20:3 omega 6 and 20:4 omega 6 fatty acids decreased while oleic acid increased. The fatty acid composition of L-alpha-phosphatidylethanolamines was less affected. 3. delta 6- and delta 5-desaturase activities decreased to 20-30% of their original values after 2 d on the LP diet; a smaller deficit prevailed after 14 d but disappeared after 25 d, to appear again after 52 d. As early as day 2 of BR, desaturase activities were greatly recovered and returned to control values at day 13. 4. The present work shows that modifications in microsomal delta 6- and delta 5-desaturase activities are not strictly paralleled by the changes in the composition of fatty acids of liver total lipids and microsomal phospholipids.