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.

Brain and hippocampus fatty acid composition in phospholipid classes of aged-relative cognitive deficit rats.

The aim of this work was to study the composition of long chain fatty acids and the n-6 and n-3 fatty acid ratios in aged and young Wistar rats in brain and hippocampus, related to relative cognitive deficits. The aged animals showed cognitive deficits during acquisition of a memory task (delayed alternation). In brain, results showed a decrease in palmitoleic and palmitic acid percentages in all the studied phospholipid classes and in the phosphatidylserine and phosphatidylcholine classes, respectively, in old rats, compared to the young ones. There was also an increase in oleic and stearic acid amounts in the sphingomyelin, phosphatidylserine and phosphatidylinositol classes and in the phosphatidylserine and phosphatidylcholine classes, respectively. Arachidonic acid amount was decreased in old rats, compared to the young ones, in the phosphatidylserine and phosphatidylinositol classes. Total n-6 and n-3 fatty acid amounts were both decreased in all phospholipid classes, with a stable n-6/n-3 ratio. Our results confirm that arachidonic acid concentration is decreased in aged rats and that this reduction, more significant in phosphatidylserine and phosphatidylinositol classes, should be related to the fact that low concentrations of arachidonic acid are observed during activation of glutamate receptor.

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.

5-HT3 receptor-channels coupled with Na+ influx in human T cells: role in T cell activation.

The study was conducted on a human (Jurkat) T cell line, loaded with a Na+ fluorescent probe, SBFI/AM. Serotonin and an agonist of 5-HT3 receptor-channels, 2-methyl-5HT, evoked Na+ influx, whereas the agonists of other serotonergic receptor subtypes, i.e., 5-HT1A and 5-HT1B receptors, failed to induce Na+ influx in these cells. By using 3H-BRL43694, an agonist of 5-HT3 receptor-channels, we characterized 5-HT3 lymphocyte receptors which exhibited a density (Bmax) of 300 +/- 20 fmol/10(6) cells and a Kd of 30 nM in Jurkat T cells. The T-cell 5-HT3 receptor-channel is not regulated either by the protein kinase C or by the free intracellular calcium concentrations as the agents known to activate the PKC and to induce increases in intracellular free calcium concentrations failed to influence the free intracellular Na+ concentrations, [Na+]i, in these cells. Furthermore, an increase in [Na+]i, induced by 2-methyl-5HT, via 5-HT3 receptor-channels seems to stimulate T-cell activation by facilitating the progression of T cells from S to G2/M phase of the cell cycle.

Fatty acid desaturase activities and polyunsaturated fatty acid composition in human liver between the seventeenth and thirty-sixth gestational weeks.

OBJECTIVE: The aim of the study was to characterize n-3 and n-6 fatty acid delta5- and delta6-desaturase activities and their time course variations in human fetal liver between the 17th and 36th gestational week. STUDY DESIGN: Twenty-one biologic samples were obtained after legally approved medical abortion, according to French law. The desaturase activities were measured in the 21 liver samples by a radiochemical method by means of reverse-phase high-performance liquid chromatography. The fatty acid composition (percentage by weight) of liver phospholipids was assessed in 16 samples by gas-liquid chromatographic analysis. RESULTS: Both delta5- and delta6-desaturase activities were significantly expressed between the 17th and 36th gestational weeks. During the second trimester n-6 fatty acid delta5- and delta6-desaturase activities showed opposite patterns of variation; both then remained stable between the 25th and 36th weeks. Delta6-desaturation was higher in n-3 than n-6 fatty acids and peaked at the 18th gestational week. The percentages of linoleic and docosahexaenoic acids in liver microsomes were positively correlated with the gestation age (P < .01), whereas arachidonic acid remained stable. CONCLUSION: Significant n-3 and n-6 delta5- and delta6-desaturase activities are expressed in human fetal liver as early as the 17th gestational week and are stable throughout the third trimester. Their theoretic capacity evaluated from in vitro measurements appears lower than polyunsaturated fatty acid requirements and is not directly related to liver microsomal membrane fatty acid composition.

Changes in tissue polyunsaturated fatty acids with age, in spontaneously hypertensive rats.

The relationship between the biosynthesis of long-chain fatty acids and their distribution in the key organs of hypertension is of considerable interest because of their role in the production of vasoactive eicosanoids and their effects on membrane properties. The present study analyzed the fatty acid compositions of the total lipids in the kidney, aorta, heart, and hepatocytes of 1-, 3-, and 6-mon-old spontaneously hypertensive rats (SHR) and their normotensive controls, Wistar Kyoto rats (WKY) by capillary gas chromatography . The major changes concerned the polyunsaturated fatty acids (PUFA). The percentage of arachidonic acid (AA) was significantly greater in the 1-mon-old SHR kidney than in the WKY kidney, but it was lower at 3 and 6 mon. The percentage of eicosapentaenoic acid was very low in the SHR kidney. The results for the aorta were similar, with marked decreases in 18:2n-6 and 18:3n-3 in SHR aged 1 and 6 mon. Despite a higher proportion of 18:2n-6 and AA at 6 mon, there was no major change in the SHR heart lipids. The fatty acid spectrum in the liver provides additional evidence for the previously reported inhibition of desaturase activities in SHR. Thus, this study shows that the PUFA composition is modified differently in different tissues in SHR, and this may be related to the pathogenesis of hypertension in these animals.

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.

Liver microsomal membrane fluidity and microsomal desaturase activities in adult spontaneously hypertensive rats.

OBJECTIVE: The purpose of the present study was to investigate liver microsomal membrane fluidity simultaneously with membrane fatty acid composition and desaturase activities in spontaneously hypertensive rats (SHR). DESIGN AND METHODS: The membrane fluidity was determined, after electron spin resonance (ESR) measurement, in SHR compared with normotensive Wistar-Kyoto (WKY) rats, by calculating the order parameter S from ESR spectra of 5-nitroxide stearate and 10-nitroxide stearate, used as spin-labelled fatty acids. Desaturase activities were measured by incubating SHR and WKY rat liver microsomes with [14C]-radiolabeled fatty acids as substrates for desaturation reactions. The fatty acid composition of liver microsomal membranes was determined by gas-liquid chromatography. RESULTS: Whereas no significant difference between S of 5-nitroxide stearate was observed for SHR and WKY rats, S of 10-nitroxide stearate was significantly lower in SHR than it was in WKY rat microsomal membrane, indicating that the core microsomal membrane fluidity was higher in SHR. Significant differences between fatty acid compositions were observed for SHR and WKY rat microsomal membranes. Delta9 and n-6 delta6 microsomal desaturase activities were significantly lower in SHR. CONCLUSION: These results suggest that the higher liver core microsomal membrane fluidity observed in SHR might be dependent on the increased proportion of mono-unsaturated fatty acids. Such observed modifications and the alterations in delta9 and n-6 delta6 desaturase activities suggest that an impaired polyunsaturated fatty acid biosynthesis is related to changes in microsomal membrane fluidity in hypertension.