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.

Evidence for insulin dependent hepatic microsomal gamma-linolenic acid chain elongation in spontaneously diabetic Wistar BB rats.

We studied hepatic microsomal gamma-linolenoyl-CoA elongation and fatty acid composition of liver microsomes in spontaneously diabetic Wistar BB rats. The liver microsomal gamma-linolenoyl-CoA elongation was decreased in diabetic Wistar BB rats during both normo- and hyperglycemic periods and restored during the hypoglycemic period following insulin treatment. These results are in agreement with our previously reported data on linoleic acid delta 6 and delta 5 desaturations and support the non-parallel relationship between the chain elongation system and the glycemia. The fatty acid composition of BB rat liver microsomes was only partially consistent with the gamma-linolenoyl-CoA elongation activity at the different periods of glycemia, probably because factors other than elongation impairments were involved in the evolution of fatty acid composition.

The effects of alkylthioacetic acids (3-thia fatty acids) on fatty acid metabolism in isolated hepatocytes.

Long-chain alkylthioacetic acids (3-thia fatty acids) inhibit fatty acid synthesis from [1-14C]acetate in isolated hepatocytes, while fatty acid oxidation is nearly unaffected or even stimulated. Desaturation of [1-14C]stearate (delta 9-desaturase) is also unaffected. [1-14C]Dodecylthioacetic acid (a 3-thia fatty acid) is incorporated in triacylglycerol and in phospholipids more efficiently than [1-14C]palmitate in isolated hepatocytes. The metabolism of [1-14C]dodecylthioacetic acid to acid-soluble products (by omega-oxidation) is slow compared to the oxidation of [1-14C]palmitate. In hepatocytes from adapted rats (rats fed tetradecylthioacetic acid for 4 days) the rate of [1-14C]palmitate oxidation is increased and its rate of esterification is decreased. Stearate desaturation is also decreased. The rate of cyanide-insensitive peroxisomal fatty acid beta-oxidation is several-fold increased. The metabolic effects of long-chain 3-thia fatty acids are discussed and it is concluded that they behave essentially like normal fatty acids except for their slow breakdown due to the sulfur atom in the 3 position, which blocks normal beta-oxidation.

The effect of dietary alpha-bromopalmitate on blood lipids in the rat.

When alpha-bromopalmitate was fed to rats for 9-30 days, the level of serum triacylglycerol increased up to 2-fold over the concentration of controls. alpha-Bromopalmitate treatment had no effect on concentration of complex lipids in liver, while the triacylglycerol level in heart was significantly enhanced. From metabolic studies using isolated hepatocytes and liver microsomes, it is suggested that the increased serum triacylglycerol level after alpha-bromopalmitate feeding is mainly due to reduced fatty acid oxidation in both liver and peripheral tissues, and to a lesser extent, to inhibited fatty acid uptake and esterification.

Evidence that liver microsomes of human neonates desaturate essential fatty acids.

delta 6- and delta 5-Desaturation of essential fatty acids of n-6 and n-3 series are required for the biosynthesis of polyunsaturated fatty acids (PUFAs), which are precursors of eicosanoids and constituents of membrane phospholipids. This pathway could be of special importance during the perinatal period, when PUFAs accretion in the central nervous system is very active. However, experimental evidence of delta 6- and delta 5-desaturase activities in man is very scarce, and no data are available for newborns. We report the delta 6- and delta 5-desaturase activities detected in human liver microsomes from three neonates who died from associated malformations. Radiochemical assays of delta 6- and delta 5-desaturase activities performed with reverse phase HPLC analysis of the products in the n-6 series ranged from 4.8-13.6 to 3.2-16.4 pmol substrate converted.min-1.mg-1 microsomal proteins, respectively. In the n-3 series delta 6-desaturase activity ranged from 5.3 to 12.8 pmol.min-1.mg-1. The relationships between enzyme activities and substrate concentrations suggest excess substrate inhibition for n-6 and not for n-3 fatty acids. These results demonstrate significant delta 6- and delta 5-desaturase activities in human liver of neonates, but this activity was lower than previously reported in adult humans and in mammals, especially rodents.

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.

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 metabolism, pharmacological nutrients and hypertension.

The purpose of the present study was to investigate the effect of a concentrated preparation (EPA 30) containing eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) on the limiting desaturation steps of the polyunsaturated fatty acid biosynthesis in spontaneously hypertensive rats (SHR). Adult SHR were divided into two groups: one group received a standard diet, and the experimental group the standard diet including 0.8% of EPA30 for 9 weeks. Blood pressure was measured at the end of the diets. The desaturase activities and fatty acid composition were determined in isolated hepatocytes. The blood pressure did not decrease in the experimental group. The desaturated products of the n-6 family (gamma-linolenic acid, 18:3 n-6 and arachidonic acid, 20:4 n-6) were lowered in the EPA30 group, when their respective substrates (18:2 n-6 and 20:3 n-6) were increased. EPA and DHA were higher in the experimental group delta 6 n-3, delta 6 n-6 and delta 5 n-6 desaturase activities were depressed approximately 20% in the EPA30 group. EPA30 being an active nutrient on the EFAs cascade, increasing the level of PG3 precursors and decreasing the level of PG2 precursors, favourable conditions have been established to reduce hypertension. The underlying mechanism related to the regulation of desaturase activities by these fatty nutrients remains to be elucidated.

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.

Age-related depletion of linoleic acid desaturation in liver microsomes from young spontaneously hypertensive rats.

The present study was designed to investigate the microsomal interconversion of linoleic acid (LA) into arachidonic acid (AA) in young spontaneously hypertensive rats (SHR), in relation to the pathogenesis of hypertension. Our results show lower delta 6 and delta 5 desaturase activities (the limiting steps in the bioconversion of LA into AA) in young SHR, as compared to Wistar Kyoto normotensive rats. This impairment of desaturase activities is raised when the blood pressure increases and is related to the age of animals. The fatty acid composition of liver lipids shows a lower proportion of AA and a higher proportion of LA in SHR than in normotensive rats, confirming the depletion of the enzymatic system studied. Such a loss of desaturase activity may be under the control of hormones involved in the regulation of SHR blood pressure.

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.

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.

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.

Adrenic acid delta 4 desaturation and fatty acid composition in liver microsomes of spontaneously diabetic Wistar BB rats.

We examined the delta 4 (n-6) desaturation and the fatty acid composition of liver microsomes in the insulin-dependent spontaneously diabetic Wistar Bio-Breeding (BB) rat. The desaturation of adrenic acid to n-6 docosapentaenoic acid was decreased in the normo- and hyperglycemic diabetic rats. Insulin treatment with 1.0 IU. 100 g body weight-1 twice a day for 2 days restored the reduced activity during the hypoglycemic period. The pattern of responses was similar to that of linoleic acid delta 6 and dihomo-gamma-linolenic acid delta 5 desaturases, with a non-parallel relationship between the desaturation system and the glycemia. The microsomal fatty acid composition of BB rat liver reflected only partially to the delta 4 desaturation at different states of glycemia. Factors other than impaired desaturation system are involved in the fatty acid metabolism of spontaneously diabetic rats.

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.

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.

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.