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.

Oxidative stress and maternal obesity: feto-placental unit interaction.

OBJECTIVE: To determine oxidative stress markers in maternal obesity during pregnancy and to evaluate feto-placental unit interaction, especially predictors of fetal metabolic alterations. PATIENTS AND METHODS: 40 obese pregnant women (prepregnancy BMI > 30 kg/m²) were compared to 50 control pregnant women. Maternal, cord blood and placenta samples were collected at delivery. Biochemical parameters (total cholesterol and triglycerides) and oxidative stress markers (malondialdehyde, carbonyl proteins, superoxide anion expressed as reduced Nitroblue Tetrazolium, nitric oxide expressed as nitrite, reduced glutathione, catalase, superoxide dismutase) were assayed by biochemical methods. RESULTS: Maternal, fetal and placental triglyceride levels were increased in obese group compared to control. Maternal malondialdehyde, carbonyl proteins, nitric oxide and superoxide anion levels were high while reduced glutathione concentrations and superoxide dismutase activity were low in obesity. In the placenta and in newborns of these obese mothers, variations of redox balance were also observed indicating high oxidative stress. Maternal and placental interaction constituted a strong predictor of fetal redox variations in obese pregnancies. DISCUSSION: Maternal obesity compromised placental metabolism and antioxidant status which strongly impacted fetal redox balance. Oxidative stress may be one of the key downstream mediators that initiate programming of the offspring. CONCLUSION: Maternal obesity is associated with metabolic alterations and dysregulation of redox balance in the mother-placenta - fetus unit. These perturbations could lead to maternal and fetal complications and should be carefully considered.

Distinct regulation of stearoyl-CoA desaturase 1 gene expression by cis and trans C18:1 fatty acids in human aortic smooth muscle cells.

Consumption of trans fatty acids is positively correlated with cardiovascular diseases and with atherogenic risk factors. Trans fatty acids might play their atherogenic effects through lipid metabolism alteration of vascular cells. Accumulation of lipids in vascular smooth muscle cells is a feature of atherosclerosis and a consequence of lipid metabolism alteration. Stearoyl-CoA desaturase 1 (scd1) catalyses the production of monounsaturated fatty acids (e.g. oleic acid) and its expression is associated with lipogenesis induction and with atherosclerosis development. We were interested in analysing the regulation of delta-9 desaturation rate and scd1 expression in human aortic smooth muscle cells (HASMC) exposed to cis and trans C18:1 fatty acid isomers (cis-9 oleic acid, trans-11 vaccenic acid or trans-9 elaidic acid) for 48 h at 100 µM. Treatment of HASMC with these C18:1 fatty acid isomers led to differential effects on delta-9 desaturation; oleic acid repressed the desaturation rate more potently than trans-11 vaccenic acid, whereas trans-9 elaidic acid increased the delta-9 desaturation rate. We then correlated the delta-9 desaturation rate with the expression of scd1 protein and mRNA. We showed that C18:1 fatty acids controlled the expression of scd1 at the transcriptional level in HASMC, leading to an increase in scd1 mRNA content by trans-9 elaidic acid treatment, whereas a decrease in scd1 mRNA content was observed with cis-9 oleic acid and trans-11 vaccenic acid treatments. Altogether, this work highlights a differential capability of C18:1 fatty acid isomers to control scd1 gene expression, which presumes of different consequent effects on cell functions.

5,6-dihydro-2H-pyranones and 5,6-dihydro-2H-pyridones and their derivatives modulate in vitro human T lymphocyte function.

The aim of this work was to study the in vitro effects of δ-lactone 1, δ-lactam 3 and their enaminone derivatives 2 and 4, synthesized in our laboratory, on the proliferative responses of human lymphocytes, Th1 and Th2 cytokine secretion and intracellular redox status. Peripheral blood lymphocytes were isolated using differential centrifugation on a density gradient of Histopaque. They were cultured with mitogen concanavalin A (Con A) and with different concentrations of the compounds 1, 2, 3 and 4 (0.1-10 µM). Proliferation (MTT assay), IL-2, INFγ and IL-4 (Elisa kits), oxidative markers (intracellular glutathione, hydroperoxide and carbonyl protein contents) and cytotoxic effect (micronucleus test) were determined. The compounds 1 and 2 are immunosuppressive and decrease IL-2, INFγ and IL-4 secretion with a shift away from Th2 response to Th1 phenotype. The compounds 3 and 4 were immunostimulant and increased cytokine secretion with a shift away from Th1 response to Th2. The introduction of an enamine group to 1 and 3 to provide 2 and 4 seemed to attenuate their immunological properties. These immunomodulatory properties were, however, accompanied by an increase in lymphocyte intracellular oxidative stress, especially with 1 and 2 at high concentrations. In conclusion, the compounds 1, 2, 3 and 4 could be used to provide cell-mediated immune responses for novel therapies in T-cell mediated immune disorders.

Effects of the association of aging and obesity on lipids, lipoproteins and oxidative stress biomarkers: a comparison of older with young men.

In this study, plasma lipids, lipoproteins and markers of oxidant/antioxidant status were investigated in young (n = 45) and older (n = 40) obese men and compared to those in young (n = 65) and older (n = 55) normal weight controls. The purpose was to determine whether obesity exacerbates or not lipid, lipoprotein abnormalities and oxidative stress in older men. Our findings showed that all obese patients had increased plasma triglyceride, cholesterol, LDL-cholesterol, -triglyceride and HDL-triglyceride levels concentrations compared to controls (P < 0.01). However, the younger obese men had relatively larger and accentuated changes in plasma lipids and lipoproteins than the older patients. Additionally, total antioxidant capacity (ORAC), vitamins C and E were lower while hydroperoxides and carbonyl proteins were higher in young and older obese patients compared to their respective controls (P < 0.001). Erythrocyte antioxidant SOD and catalase activities were enhanced in obese young patients, but reduced in obese older men. Glutathione peroxidase activity was low in obesity irrespective of age. In multiple regression analysis, BMI significantly predicted total cholesterol, LDL-C, LDL-TG and HDL-TG (P < 0.0001). These relationships were not modified by age. BMI alone was a not a significant predictor for ORAC, vitamins C, E, catalase and Glutathione peroxidase. However, the interaction BMI-age significantly predicted these parameters and explained 28-45% of their changes. BMI was a significant predictor of SOD, carbonyl proteins and hydroperoxides. This effect became more significant (P < 0.0001) and worsened with BMI-age interaction. In conclusion, lipoprotein metabolism and oxidant/antioxidant status are altered in obesity irrespective of age. However, obesity-related lipid and lipoprotein alterations were attenuated while oxidative stress was aggravated in older adults.

Dehydroepiandrosterone up-regulates the Adrenoleukodystrophy-related gene (ABCD2) independently of PPARalpha in rodents.

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disease caused by mutations in the ABCD1 gene, which encodes a peroxisomal ABC transporter, ALDP, supposed to participate in the transport of very long chain fatty acids (VLCFA). The adrenoleukodystrophy-related protein (ALDRP), which is encoded by the ABCD2 gene, is the closest homolog of ALDP and is considered as a potential therapeutic target since functional redundancy has been demonstrated between the two proteins. Pharmacological induction of Abcd2 by fibrates through the activation of PPARalpha has been demonstrated in rodent liver. DHEA, the most abundant steroid in human, is described as a PPARalpha activator and also as a prohormone able to mediate induction of several genes. Here, we explored the in vitro and in vivo effects of DHEA on the expression of peroxisomal ABC transporters. We show that Abcd2 and Abcd3 but not Abcd4 are induced in primary culture of rat hepatocytes by DHEA-S. We also demonstrate that Abcd2 and Abcd3 but not Abcd4 are inducible by an 11-day treatment with DHEA in the liver of male rodents but not in brain, testes and adrenals. Finally and contrary to Abcd3, we show that the mechanism of induction of Abcd2 is independent of PPARalpha.

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.

A new hypotensive polyunsaturated fatty acid dietary combination regulates oleic acid accumulation by suppression of stearoyl CoA desaturase 1 gene expression in the SHR model of genetic hypertension.

Polyunsaturated fatty acids (PUFA) are known to repress SCD-1 gene expression, key enzyme of monounsaturated fatty acid biosynthesis. Alterations of the monounsaturated/saturated fatty acids ratio have been implicated in various diseases related to the metabolic syndrome, including hypertension. We previously evidenced that lipogenesis end-products accumulated in spontaneously hypertensive rats (SHR), and that a dietary combination of n-6/n-3 PUFA had hypotensive effects. Our present objective was to test the hypothesis that these SHR liver lipid disorders might be modulated, in response to this hypotensive combination, by changes in SCD-1 expression and activity. So we studied, in hepatocytes, SCD-1 transcription by Northern blotting, as well as plasma and liver fatty acid composition by gas-liquid chromatography. Liver SCD-1 gene expression was suppressed by 50%, and in different lipid classes, relative abundance of stearic and oleic acids decreased. Consequently, the Delta9 desaturation index, calculated from the ratio of oleic vs. stearic acids, decreased. In addition, the level of circulating saturated fatty acids decreased when one of oleic acids increased. These data provided evidence that the tested hypotensive PUFA combination reverses the high monounsaturated/saturated fatty acids ratio associated to hypertension in SHR, via a regulation monounsaturated fatty acid relative abundance by repression of SCD-1 gene.

Liver oleic acid biogenesis is impaired during the prehypertensive period in the spontaneously hypertensive rat.

In the present study, we have investigated the liver microsomal stearic acid delta9 desaturation, and the fatty acid composition of liver microsomal total lipids in 10- and 30-day-old spontaneously hypertensive rats (SHRs), compared to the normotensive Wistar Kyoto (WKY) control rats. So as to avoid any influence related to the diet, the composition of the milk being different in SHR and WKY strains, the pups were suckled by adoptive normotensive female Wistar. After weaning, the 30-day-old rats were fed a standard commercial diet and then killed. Our results show lower liver microsomal delta9 desaturase activities in the 10- and 30-day-old SHR versus the WKY of the same age. The fatty acid composition of the SHR liver microsomal total lipids are not in agreement with the changes in the delta9 desaturase activities at the two studied ages. This phenomenon depends not only on desaturation/elongation but also on other interacting aspects of lipid metabolism including oxidation, substrate availability, acyl exchange, and eicosanoid synthesis, as well as hormonal status.

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.

Antihypertensive effects of a dietary unsaturated FA mixture in spontaneously hypertensive rats.

The aim of the present study was to investigate whether a mixture of dietary n-6 and n-3 PUFA could lower blood pressure in spontaneously hypertensive rats (SHR) of different ages. In addition, we studied how such a treatment could normalize the FA composition of plasma TAG and cholesterol esters (CE), and of red blood cell (RBC) total lipids. SHR (ages 4, 19, and 50 wk) were fed a normal diet (control groups) or a semisynthetic diet containing a mixture of gamma-linolenic acid (GLA), EPA, and DHA (experimental groups). Systolic blood pressure was measured at regular intervals. After 11 wk of consuming this diet, plasma TAG and CE were separated by TLC and analyzed for their FA composition. Total FA composition of RBC was also determined. The degree to which blood pressure was elevated was reduced in SHR after 11 wk of diet. The largest decrease was obtained with the oldest animals. In RBC, EPA and DHA contents increased. In plasma TAG and CE, EPA, DHA, and GLA increased whereas arachidonic acid decreased. The n-6 and n-3 unsaturated FA mix slowed the development of hypertension in young SHR and decreased blood pressure in adult and aged SHR. In addition, the present treatment altered the n-3 and n-6 PUFA content of SHR lipids to that seen in normotensive rats.

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.

Desaturation and esterification of fatty acids in kidney cells from spontaneously hypertensive rats.

In previous studies, several alterations in lipid metabolism have been related to hypertension, but the mechanisms explaining this relationship have not been elucidated. None of the previous works has focused on the lipid metabolism in kidney, which is a key organ in the overall regulation of blood pressure. The aim of the present work was to study the metabolism of polyunsaturated fatty acids, and the possible compositional changes in kidney from hypertensive rats. Radiolabelled linoleic acid (18:2,n-6) and dihomogammalinolenic acid (20:3, n-6) were incubated with isolated kidney cells from spontaneously hypertensive rats (SHR) or the parent normotensive strain (Wistar Kyoto, WKY). The rats were divided into groups of age 9 (young) and 17 (adult) weeks. Cellular uptake, desaturation, chain-elongation, oxidation and distribution into phospholipids and triacylglycerols were measured. Additionally, the lipid composition of kidney was characterized. With each of the labelled fatty acid substrates the uptake in cells from the SHR rats, compared to the WKY rats, was numerically lower in the young group and higher in the adult group. The incorporation of labelled fatty acids into phospholipids was increased and concomitantly decreased in triacylglycerols in cells from adult SHR rats. The delta6-desaturation, measured as the conversion of labelled 18:2(n-6) to 18:3(n-6) was between two and three times increased in cells from the adult rats compared to the young ones, while no difference was found in hypertensives compared to normotensives. Concomitantly, no difference in conversion of labelled 20:3(n-6) to 20:4(n-6) was observed in relation to blood pressure, but, different from delta6-desaturation, the delta5-desaturation was significantly decreased by age. Taken together, this study demonstrates for the first time desaturation of long-chain polyunsaturated fatty acids in isolated kidney cells in suspension and that, contrary to what has been observed in liver, the desaturase activity is unaffected by hypertension. Also different from what has been observed in liver, no blood-pressure-related changes in lipid composition of kidney were found.

Dietary docosahexaenoic acid affects stearic acid desaturation in spontaneously hypertensive rats.

Docosahexaenoic acid (DHA, 22:6n-3) is an n-3 polyunsaturated fatty acid which attenuates the development of hypertension in spontaneously hypertensive rats (SHR). The effects of DHA on delta-9-desaturase activity in hepatic microsomes and fatty acid composition were examined in young SHR. Two groups of SHR were fed either a DHA-enriched diet or a control diet for 6 wk. Desaturase activity and fatty acid composition were determined in hepatic microsomes following the dietary treatments. Delta-9-desaturase activity was decreased by 53% in DHA-fed SHR and was accompanied by an increase in 16:0 and a reduction in 16:1n-7 content in hepatic microsomes. The DHA diet also increased the levels of eicosapentaenoic acid (20:5n-3) and DHA. The n-6 fatty acid content was also affected in DHA-fed SHR as reflected by a decrease in gamma-linolenic acid (18:3n-6), arachidonic acid (20:4n-6), adrenic acid (22:4n-6), and docosapentaenoic acid (22:5n-6). A higher proportion of dihomo-gamma-linolenic acid (20:3n-6) and a lower proportion of 20:4n-6 is indicative of impaired delta-5-desaturase activity. The alterations in fatty acid composition and metabolism may contribute to the antihypertensive effect of DHA previously reported.

Stearoyl-CoA desaturase 1 coding sequences and antisense RNA affect lipid secretion in transfected chicken LMH hepatoma cells.

Hepatic stearoyl CoA desaturase (SCD) activity in chickens from a fat line is higher than that of chickens from a lean line and correlates with plasma triacylglycerol concentrations. Furthermore, in these lines, the hepatic SCD1 mRNA level is positively correlated with the adipose tissue weight. To analyze the contribution of the SCD1 gene in the regulation of adiposity in the early stages of triacylglycerol secretion, SCD1 coding sequence and antisense RNA expression vectors were transfected in LMH cells. After selection, these cells were analyzed with regard to SCD1 expression and lipid secretion. The amounts of secreted triacylglycerols and phospholipids were shown to be higher in LMH cells transfected with the SCD1 gene, but reduced in those transfected with the SCD1 antisense sequences when compared to cells transfected with the vector alone (without SCD1 sequences). These results provide direct evidence that the expression of the SCD1 gene plays a major role in the triacylglycerol and phospholipid secretion process.

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.