Delta6 Desaturase mRNA Research Articles

Effect of different contents of extruded linseed in the sow diet on piglet fatty acid composition and hepatic desaturase expression during the post-natal period

n-3 polyunsaturated fatty acids (n-3 PUFA) contribute to the normal growth and development of numerous organs in the piglet. The fatty acid composition of piglet tissues is linked to the fatty acid composition of sow milk and, consequently, to the composition of sow diet during the gestation and lactation period. In this study, we investigated the impact of different contents of extruded linseed in the sow diet on the fatty acid composition and desaturase gene expression of piglets. Sows received a diet containing either sunflower oil (low 18:3n-3 with 18:3n-3 representing 3% of total fatty acids) or a mixture of extruded linseed and sunflower oil (medium 18:3n-3 with 9% of 18:3n-3) or extruded linseed (high 18:3n-3 with 27% of 18:3n-3) during gestation and lactation. Fatty acid composition was evaluated on sow milk and on different piglet tissues at days 0, 7, 14, 21 and 28. The postnatal evolution of delta5 (D5D) and delta6 (D6D) desaturase mRNA expression was also measured in the liver of low 18:3n-3 and high 18:3n-3 piglets. The milk of high 18:3n-3 sows had higher proportions of n-3PUFA than that of low 18:3n-3 and medium 18:3n-3 sows. Piglets suckling the high 18:3n-3 sows had greater proportions of 18:3n-3, 20:5n-3, 22:5n-3 and 22:6n-3 in the liver, and of 22:5n-3 and 22:6n-3 in the brain than low 18:3n-3 and medium 18:3n-3 piglets. D5D and D6D mRNA expressions in piglet liver were not affected by the maternal diet at any age. In conclusion, extruded linseed in the sow diet modifies the n-3PUFA status of piglets during the postnatal period. However, a minimal content of 18:3n-3 in the sow diet is necessary to increase the n-3PUFA level in piglet liver and brain. Moreover, modifications in the n-3PUFA fatty acid composition of piglet tissue seem linked to the availability of 18:3n-3 in maternal milk and not to desaturase enzyme expression.

Differential effects of antipsychotic medications on polyunsaturated fatty acid biosynthesis in rats: Relationship with liver delta6-desaturase expression

Polyunsaturated fatty acids (PUFA), a lipid family comprised of omega-3 ( n− 3) and n− 6 fatty acids, are a critical component of cellular membranes, and recent in vitro studies have found that antipsychotic medications up-regulate genes responsible for PUFA biosynthesis. To evaluate this effect in vivo, rats were treated with risperidone (1.5, 3, 6 mg/kg/day), paliperidone (1.5, 3, 6 mg/kg/day), olanzapine (2.5, 5, 10 mg/kg/day), quetiapine (5, 10, 20 mg/kg/day), haloperidol (1, 3 mg/kg/day) or vehicle through their drinking water for 40 day. Effects on liver Fads1, Fads2, Elovl2, and Elovl5 mRNA expression , plasma indices of n− 3 (plasma 22:6/18:3 and 20:5/18:3 ratios) and n− 6 (plasma 20:4/18:2 and 20:3/18:2 ratios) biosynthesis, and peripheral (erythrocyte, heart) and central (frontal cortex) membrane PUFA composition were determined. Only risperidone and its metabolite paliperidone significantly and selectively up-regulated liver delta-6 desaturase ( Fads2) mRNA expression, and robustly increased plasma indices of n− 3 and n− 6 fatty acid biosynthesis. In risperidone- and paliperidone-treated rats, plasma indices of n− 3 and n− 6 fatty acid biosynthesis were all positively correlated with liver Fads2 mRNA expression, but not Fads1, Elovl2, or Elovl5 mRNA expression. All antipsychotics at specific doses increased erythrocyte docosahexaenoic acid (DHA, 22:6 n− 3) composition, and all except quetiapine increased arachidonic acid (AA, 20:4 n− 6) composition. Risperidone, paliperidone, and olanzapine increased heart DHA and AA composition, and no antipsychotic altered frontal cortex DHA or AA composition. These in vivo data demonstrate that augmentation of PUFA biosynthesis is not common to all antipsychotic medications, and that risperidone and paliperidone uniquely increase delta-6 desaturase ( Fads2) mRNA expression and most robustly increase PUFA biosynthesis and peripheral membrane composition.

Revisiting delta-6 desaturase regulation by C18 unsaturated fatty acids, depending on the nutritional status

Dietary polyunsaturated fatty acids (PUFA) play a key role in regulating delta-6 desaturase (D6D), the key enzyme for long-chain PUFA biosynthesis. Nevertheless, the extent of their effects on this enzyme remains controversial and difficult to assess. It has been generally admitted that C18 unsaturated fatty acids (UFAs) regulate negatively delta-6 desaturase (D6D). This inhibition has been evidenced in regard to a high glucose/fat free (HG/FF) diet used in reference. However, several nutritional investigations did not evidence any inhibition of desaturases when feeding fatty acids. Because the choice of the basal diet appeared to be of primary importance in such experiments, our goal was to reconsider the specific role of dietary UFAs on D6D regulation, depending on nutritional conditions. For that, sixteen adult Wistar rats were fed purified linoleic acid, α-linolenic acid or oleic acid, included in one of two diets at 4% by weight: an HG/FF or a high starch base (HS) where the pure UFAs replaced a mixed vegetable oil. Our results showed first that D6D specific activity was significantly greater when measured in presence of an HG/FF than with an HS/4% vegetable oil diet. Secondly, we found that linoleic and alpha-linolenic acids added to HG/FF reduced the specific activity of D6D. In contrast, when pure UFAs were added to an HS base, D6D specific activities remained unchanged or increased. Concordant results were obtained on D6D mRNA expression. Altogether, this study evidenced the importance of the nutritional status in D6D regulation by C18 UFAs: when used as control, HG/FF diet stimulates D6D compared with a standard control diet containing starch and 4% fats, leading to an overestimation of the D6D regulation by UFAs. Then, UFAs should be considered as repressors for unsaturated fatty acid biosynthesis only in very specific nutritional conditions.

The Metabolism and Distribution of Docosapentaenoic Acid (n-6) in the Liver and Testis of Growing Rats

To investigate the metabolism and distribution of docosapentaenoic acid (22:5n-6, DPA) in the liver and testis of growing rats, 22:5n-6 was administered to their dams. Newborn rats with a low hepatic arachidonic acid (20:4n-6, AA) level were generated by administrating a diet rich in docosahexaenoic acid (22:6n-3, DHA) but n-6 fatty acid (FA) free to pregnant dams. After parturition, 22:5n-6 or linoleic acid (18:2n-6, LA) was administered with a high level of 22:6n-3 to the dams until weaning. At weaning, the hepatic 20:4n-6 level was significantly highest in the DPA-DHA but not LA-DHA diet-fed animals. The hepatic delta-6 desaturase (D6D) mRNA abundance was significantly lower in both the LA-DHA and DPA-DHA diet-fed animals, connoted with the 20:4n-6 content recovered by 22:5n-6 that did not involve D6D and supporting the occurrence of retroconversion in the liver of the growing rats. The low D6D level in the 3-week-old testis was not in proportion to the elevated 22:5n-6 level, implying that early testicular 22:5n-6 accumulation might require supply from the circulation system.

Reduced active sterol regulatory element binding protein‐1 (SREBP‐1) and delta‐6 desaturase transcription are accompanied by increased membrane phosphatidyl‐ethanolamine (PE) / phosphatidyl‐choline (PC) ratio

SREBP‐1 regulates delta‐6 desaturase (D6D), the key enzyme of highly unsaturated fatty acid synthesis. While PE suppresses proteolytic activation of SREBP in Drosophila, an activation mechanism of SREBP‐1 is unknown in mammals. Dietary ethanolamine (EA) and eritadenine (ER), which increase membrane PE/PC ratio, suppress D6D activity and mRNA in rat liver. We tested the hypothesis that both EA and ER suppress D6D gene transcription by reducing active form of SREBP‐1 (nSREBP‐1). First, rats (n=6) were fed a control diet or a diet supplemented with either EA (8g/kg) or ER (50 mg/kg) for 14 days. Both EA and ER supplementation decreased D6D mRNA and nSREBP‐1 in livers (p<0.05). Second, HepG2 cells treated with EA (30μM) and ER (1mM) significantly reduced D6D mRNA and nSREBP‐1 with concomitant increase in the PE/PC ratio. Next, D6D promoter‐luciferase reporter constructs were transfected into HepG2 cells, and then treated with EA and ER. Promoter activity was suppressed in constructs with wild‐type D6D promoters by EA and ER treatments, whereas mutation of sterol regulatory element abolished or reduced the suppression by EA or ER, respectively. In conclusion, SREBP‐1 in part mediates suppression of D6D gene transcription by EA and ER with an accompanying increase in the PE/PC ratio, suggesting that like in Drosophila, SREBP‐1 activation in mammals may also be regulated by the membrane PE/PC ratio.

Impact of dietary protein on lipid metabolism in hamsters is source-dependent and associated with changes in hepatic gene expression

This study tested the hypothesis that protein source is a factor determining the impact of the diet on lipid metabolism in hamsters. Twenty-eight hamsters of similar body weight were assigned for a period of 8 weeks to one of the following four diets (seven per group) containing either 20 % (w/w) casein (CAS), beef protein (BF), wheat gluten (WG) or soya protein (SOY). The fat composition of the diet was the same (15.5 % w/w) in all groups and provided SFA, MUFA and PUFA representative of the average Canadian diet. After an overnight fast, blood and liver were collected for the measurement of serum lipids, fatty acid composition of liver phospholipids and mRNA levels of selected genes involved in lipid metabolism. WG resulted in lower total cholesterol, HDL-cholesterol and non-HDL-cholesterol but, along with SOY, in higher mRNA levels of cholesterol 7 alpha-hydroxylase and LDL receptor. Furthermore, both WG and SOY resulted in lower 18 : 3n-3, 20 : 4n-6, total n-6 PUFA, 18 : 1n-9 and total MUFA, but higher 22 : 6n-3, total n-3 PUFA, 22 : 6n-3/18 : 3n-3 and 22 : 5n-3/18 : 3n-3 ratios in liver phospholipids, and higher hepatic Delta6-desaturase mRNA levels. These results show that the impact of dietary protein on lipid metabolism is source-dependent and associated with changes in mRNA abundances of key hepatic enzymes and receptors.

Δ6 Desaturase mRNA Abundance in HepG2 Cells Is Suppressed by Unsaturated Fatty Acids

The effect of unsaturated fatty acids on the abundance of Delta6 desaturase (D6D) mRNA and the fatty acid composition of HepG2 cell membranes was examined. Supplementation of HepG2 cells with oleic acid (18:1n-9, OA), linoleic acid (18:2n-6, LA), alpha-linolenic acid (18:3n-3, ALA), arachidonic acid (20:4n-6, AA) or eicosapentaenoic acid (20:5n-3, EPA) reduced D6D mRNA abundance by 39 +/- 6.6, 40 +/- 2.2, 31 +/- 5.2, 55 +/- 4.8, and 52 +/- 5.0%, respectively, compared with control cells. Despite the reduction in D6D mRNA abundance, the level of D6D conversion products (20:3n-9, EPA and AA) in OA, ALA and LA supplemented cells, respectively, was elevated above that in control cells. Our results suggest that although unsaturated fatty acids decrease the abundance of D6D mRNA by as much as 50%, the conversion of polyunsaturated fatty acids and accumulation of long chain polyunsaturated fatty acids (LCPUFA) in HepG2 cell phospholipids continues to occur.

Δ5 desaturase mRNA levels are increased by simvastatin via SREBP-1 at early stages, not via PPARα, in THP-1 cells

In addition to inhibiting cholesterol biosynthesis, statins increase the conversion of linoleic acid to its derivatives, in particular to arachidonic acid, both in vivo and in vitro. Desaturases are the rate-limiting enzymes in this metabolic process and statins markedly enhance delta5 desaturase activity. To evaluate the delta5 desaturase gene expression and the transcription factors involved, THP-1 cells (a monocytic cell line) were incubated with 5 microM simvastatin for different time periods. The activity of the enzyme, evaluated as product/precursor ratio in the metabolic pathway (starting from [1-(14)C] linoleic acid), increased in treated cells with respect to controls after 24 h, whereas, mRNA levels of the delta5 desaturase increased after 12 h of incubation with simvastatin. Fatty acid desaturase genes are regulated by both sterol regulatory element binding proteins (SREBPs) and peroxisome proliferators activated receptors (PPARs). Both PPARalpha (WY 14643 and fenofibrate) and PPARgamma (ciglitazone) agonists did not affect linoleic acid conversion and the delta5 desaturase activity at any time considered (8-48 h), but they increased the delta5 desaturase mRNA levels, after 48 h; only fenofibrate showed a synergistic effect with simvastatin at this time, with a concomitantly increase in PPARalpha expression and beta-oxidation. Simvastatin alone increased SREBP-1 levels with respect to controls, starting from 8 h of incubation, whereas PPARalpha and linoleic acid beta-oxidation (a PPARalpha mediated process) were not affected after 48 h of incubation. These results taken together suggest that SREBP-1 is involved in the early regulation of delta5 desaturase gene by simvastatin, in THP-1 cells.

Identification and Functional Analysis of a Delta-6 Desaturase from the Marine Microalga Glossomastix chrysoplasta

A delta-6 (Delta6) desaturase gene was isolated from the marine microalga Glossomastix chrysoplasta, a stramenopile that produces large amounts of eicosapentaenoic acid (EPA). A functional analysis of this gene was performed in Saccharomyces cerevisiae. Isolation of the Delta6 fatty acid desaturase was achieved via reverse transcriptase-polymerase chain reaction (RT-PCR) with degenerate primers designed from conserved histidine motifs and 5' and 3' RACE. Two almost identical copies of Delta6 desaturase were found, differing by nine silent mutations. The existence of two such genes may be a result of a recent gene duplication event, or may have arisen from the possible diploid nature of vegetative algae. This appears to be the first instance of two Delta6 desaturase mRNA sequences existing in the same organism. The isolated mRNA sequences and their corresponding hypothetical protein, GcDES6, were found to contain features characteristic of a membrane-bound Delta6 desaturase, including membrane-spanning regions separating conserved histidine boxes and N-terminal cytochrome b5 fusion. Heterologous expression in S. cerevisiae was used to confirm Delta6 regioselectivity and the function of GcDES6. Both omega3(18:3Delta9,12,15) and omega6(18:2Delta9,12) precursors can be used by GcDES6 in vivo with similar desaturase activity. One intron site was found in the cytochrome b5 fusion region of GcDES6. Although the conservation of intron-exon junctions has been found for several desaturases in humans and in Caenorhabditis elegans, a comparison of introns in GcDES6 and other Delta6 desaturases has not revealed any strong similarities.

Peroxisome proliferator-activated receptor α is required for feedback regulation of highly unsaturated fatty acid synthesis

Delta6 desaturase (D6D), the rate-limiting enzyme for highly unsaturated fatty acid (HUFA) synthesis, is induced by essential fatty acid-deficient diets. Sterol regulatory element-binding protein-1c (SREBP-1c) in part mediates this induction. Paradoxically, D6D is also induced by ligands of peroxisome proliferator-activated receptor alpha (PPARalpha). Here, we report a novel physiological role of PPARalpha in the induction of genes specific for HUFA synthesis by essential fatty acid-deficient diets. D6D mRNA induction by essential fatty acid-deficient diets in wild-type mice was diminished in PPARalpha-null mice. This impaired D6D induction in PPARalpha-null mice was not attributable to feedback suppression by tissue HUFAs because PPARalpha-null mice had lower HUFAs in liver phospholipids than did wild-type mice. Furthermore, PPARalpha-responsive genes were induced in wild-type mice under essential fatty acid deficiency, suggesting the generation of endogenous PPARalpha ligand(s). Contrary to genes for HUFA synthesis, the induction of other lipogenic genes under essential fatty acid deficiency was higher in PPARalpha-null mice than in wild-type mice even though mature SREBP-1c protein did not differ between the genotypes. The expression of PPARgamma was markedly increased in PPARalpha-null mice and might have contributed to the induction of genes for de novo lipogenesis. Our study suggests that PPARalpha, together with SREBP-1c, senses HUFA status and confers pathway-specific induction of HUFA synthesis by essential fatty acid-deficient diets.

Desaturase activities in rat model of insulin resistance induced by a sucrose-rich diet.

A sucrose-rich diet, as compared with a similar starch diet, induces a time-dependent typical noninsulin-dependent diabetes syndrome characterized by insulin resistance in rats. Within the first 3 wk, there was glucose intolerance associated with hyperinsulinemia, hypertriglyceridemia, and high plasma FFA. In this study, we examined the effect of the sucrose-rich diet vs. the starch diet during short- (3 wk) and longterm treatment (6 mon) on hepatic delta9, delta6, and delta5 desaturases. These enzymes modulate monounsaturated FA and PUFA biosynthesis, respectively. Sucrose feeding (3 wk) caused an initial hyperinsulinemia that was normalized within 6 mon. In the early period (3 wk), stearoyl-CoA desaturase-1 (SCD-1) mRNA and activity were decreased, whereas delta6 desaturase mRNA abundance and delta6 and delta5 desaturase activities remained unchanged. After 6 mon of sucrose feeding, activities of the delta9, delta6, and delta5 desaturases were each increased. The SCD-1 and delta6 desaturase mRNA were also correspondingly higher. These increases were consistent with an increase in oleic acid, the 20:4/18:2 ratio, and 22:4n-6 and 22:5n-6 acids in liver and muscle lipids. On the other hand, the percentage of 22:6n-3 acid was decreased. In conclusion, a sucrose-rich diet after 6 mon induces an increase in rat liver SCD-1 and delta6 desaturase mRNA and enzymatic activities that are opposite to the changes reported in insulin-dependent diabetes mellitus. It appears that neither blood insulin levels nor insulin resistance is a factor affecting the delta9, delta6, and delta5 desaturase changes in mRNA and activity found with the sucrose-rich diet.

Expression and regulation of delta5-desaturase, delta6-desaturase, stearoyl-coenzyme A (CoA) desaturase 1, and stearoyl-CoA desaturase 2 in rat testis.

In mammalian cells, essential polyunsaturated fatty acids (PUFAs) are converted to longer PUFAs by alternating steps of elongation and desaturation. In contrast to other PUFA-rich tissues, the testis is continuously drained of these fatty acids as spermatozoa are transported to the epididymis. Alteration of the germ cell lipid profile from spermatogonia to condensing spermatids and mature spermatozoa has been described, but the male gonadal gene expression of the desaturases, responsible for the PUFA-metabolism, is still not established. The focus of this study was to characterize the expression and regulation of stearoyl-CoA desaturase 1 (SCD1), stearoyl-CoA desaturase 2 (SCD2), and Delta5- and Delta6-desaturase in rat testis. Desaturase gene expression was detected in testis, epididymis, and separated cells from seminiferous tubulus using Northern blot analysis. For the first time, SCD1 and SCD2 expression is demonstrated in rat testis and epididymis, both SCDs are expressed in epididymis, while testis mainly contains SCD2. Examination of the testicular distribution of Delta5- and Delta6-desaturase and SCD1 and SCD2 shows that all four desaturases seem to be localized in the Sertoli cells, with far lower expression in germ cells. In light of earlier published results showing that germ cells are richer in PUFAs than Sertoli cells, this strengthens the hypothesis of a lipid transport from the Sertoli cells to the germ cells. As opposed to what is shown in liver, Delta5- and Delta6-desaturase mRNA levels in Sertoli cells are up-regulated by dexamethasone. Furthermore, dexamethasone induces SCD2 mRNA. Insulin also up-regulates these three genes in the Sertoli cell, while SCD1 mRNA is down-regulated by both insulin and dexamethasone. Delta5- and Delta6-desaturase, SCD1, and SCD2 are all up-regulated by FSH. A similar up-regulation of the desaturases is observed when treating Sertoli cells with (Bu)2cAMP, indicating that the desaturase up-regulation observed with FSH treatment results from elevated levels of cAMP. Finally, testosterone has no influence on the desaturase gene expression. Thus, FSH seems to be a key regulator of the desaturase expression in the Sertoli cell.

Comparative effect of fenofibrate on hepatic desaturases in wild-type and peroxisome proliferator-activated receptor alpha-deficient mice.

In this study is presented the effect of fenofibrate, a prototypical peroxisome proliferator of the fibrate class, on wild-type and peroxisome proliferator-activated receptor alpha (PPARalpha)-/- mouse liver FA profile, desaturase mRNA levels, and activities. We established that, following peroxisome proliferator exposure, the hepatic FA profile was greatly modified. These modifications in hepatic FA content required the expression of PPARalpha, as they are suppressed in transgenic mice deficient in this nuclear receptor. Following peroxisome proliferator exposure, delta6- and delta5-desaturase mRNA levels and activities were increased in wild-type but not in PPARalpha-deficient mouse liver. These results suggest the involvement of PPARalpha in the control of hepatic delta6- and delta5-desaturases in mice. Their roles in minimizing long-chain PUFA depletion in the liver during peroxisome proliferator exposure are discussed.

Dietary cholesterol modulates delta6 and delta9 desaturase mRNAs and enzymatic activity in rats fed a low-eFA diet.

The effects of a 1% addition of cholesterol to a diet low in EFA on FA desaturases were examined. The administration of cholesterol markedly increased the esterified cholesterol content in microsomes and total liver lipids from the first day, whereas the proportion of free cholesterol remained unaltered throughout the treatment. An excellent homeostasis in the free cholesterol content was apparently evoked by the acyl-CoA cholesterol acyltransferase. The cholesterol esters were mainly oleate, palmitate, and stearate, and the addition of cholesterol increased the relative proportions of cholesterol palmitoleate and oleate. The addition of cholesterol to a low-EFA diet induced, as in animals fed a high-EFA diet, a marked increase in liver stearoyl-CoA desaturase-1 mRNA and enzyme activity. This increased activity apparently evoked a similar enhancement of palmitoleic and oleic acids in total and microsomal liver lipids. The cholesterol-rich diet depressed the liver A6 and delta5 desaturase activity. However, the abundance of delta6 desaturase mRNA was not modified throughout the treatment. This indicates that the depressive effect is evoked at a step beyond that controlled by the mRNA level. The depression of both enzymatic activities was consistent with the decrease in the percentages of arachidonic acid and DHA in total and microsomal liver lipids. Taken together, these results indicate that through its modulating effect on the desaturases, dietary cholesterol may lead an animal or human fed low-EFA diet to a true deficiency by the decreased synthesis of the highly polyunsaturated acids derived from linoleic and alpha-linolenic acids.

Metabolism and functions of highly unsaturated fatty acids: an update.

This review briefly examines the recent progress in knowledge about the synthesis and degradation of highly unsaturated fatty acids (HUFA) and their functions. Following the cloning of mammalian Delta6-desaturase (D6D), the D6D mRNA was found in many tissues, including adult brain, maternal organs, and fetal tissue, suggesting an active synthesis of HUFA in these tissues. The cloning also confirmed the long-postulated hypothesis that the same pathway is followed in n-6 and n-3 HUFA synthesis. Dietary n-6 and n-3 HUFA both induce fatty acid oxidation enzymes in peroxisomes when compared to their respective precursor polyunsaturated fatty acids. This suggests that peroxisomes may be the primary site of HUFA degradation when HUFA are supplied in excess from the diet. Peroxisome proliferators strongly induce the enzymes for the HUFA synthesis. The mechanism of this induction is currently unknown. Recent studies revealed new HUFA functions that are not mediated by eicosanoids. These functions include endocytosis/exocytosis, ion-channel modulation, DNA polymerase inhibition, and regulation of gene expression. These new discoveries will enable us to re-examine the underlying mechanisms for the classical symptoms of essential fatty acid deficiency as well as vitamin E deficiency. Progress has also been made in understanding the mechanism by which dietary HUFA reduce body fat deposition. One mechanism is induction of genes for fatty acid oxidation, which is mediated by peroxisome proliferator-activated receptor-alpha. Another likely mechanism is that HUFA suppress genes for fatty acid synthesis by reducing both mRNA and protein maturation of sterol regulatory element binding protein-1.

Identification of a fatty acid Δ6-desaturase deficiency in human skin fibroblasts

Polyunsaturated fatty acid (PUFA) utilization was investigated in skin fibroblasts cultured from a female patient with an inherited abnormality in lipid metabolism. These deficient human skin fibroblasts (DF) converted 85;-95% less [1-14C]linoleic acid (18:2n-6) to arachidonic acid (20:4n-6), 95% less [3-14C]tetracosatetraenoic acid (24:4n-6) to docosapentaenoic acid (22:5n-6), and 95% less [1-14C]-linolenic acid (18:3n-3) and [3-14C]tetracosapentaenoic acid (24:5n-3) to docosahexaenoic acid (22:6n-3) than did normal human skin fibroblasts (NF). The only product formed by the DF cultures from [1-14C]tetradecadienoic acid (14:2n-6) was 18:2n-6. However, they produced 50;-90% as much 20:4n-6 as the NF cultures from [1-14C]hexadecatrienoic acid (16:3n-6), [1-14C]gamma-linolenic acid (18:3n-6), and [1-14C]dihomo-gamma-linolenic acid (20:3n-6), PUFA substrates that contain Delta6 double bonds. DF also contained 80% more 18:2n-6 and 25% less 20:4n-6. These results suggested that DF are deficient in Delta6 desaturation. This was confirmed by Northern blots demonstrating an 81;-94% decrease in Delta6-desaturase mRNA content in the DF cultures, whereas the Delta5-desaturase mRNA content was reduced by only 14%. This is the first inherited abnormality in human PUFA metabolism shown to be associated with a Delta6-desaturase deficiency. Furthermore, the finding that the 18- and 24-carbon substrates are equally affected suggests that a single enzyme carries out both Delta6 desaturation reactions in human PUFA metabolism.

Effect of conjugated linoleic acid on fungal delta6-desaturase activity in a transformed yeast system.

Conjugated linoleic acid (CLA; 18:2), a group of positional and geometric isomers of linoleic acid (LA; 18:2n-6), has been shown to modulate immune function through its effect on eicosanoid synthesis. This effect has been attributed to a reduced production of n-6 polyunsaturated fatty acid (PUFA), the precursor of eicosanoids. Since delta6-desaturase is the rate-limiting enzyme of the n-6 PUFA production, it is our hypothesis that CLA, which has similar chemical structure to LA, interacts directly with delta6-desaturase. A unique and simple model, i.e., baker's yeast (Saccharomyces cerevisiae) transformed with fungal delta6-desaturase gene, previously established, was used to investigate the direct effect of CLA on delta6-desaturase. This model allows LA to be converted to y-linolenic acid (GLA; 18:3n-6) but not GLA to its metabolite(s). No metabolites of CLA were found in the lipids of the yeast transformed with delta6-desaturase. The inability to convert CLA to conjugated GLA was not due to the failure of yeast cells to take up the CLA isomers. CLA mixture and individual isomers significantly inhibited the activity of delta6-desaturase of the transformed yeast in vivo. Even though its uptake by the yeast was low, CLA c9,t11 isomer was found to be the most potent inhibitor of the four isomers tested, owing to its high inhibitory effect on delta6-desaturase. Since CLA did not cause significant changes in the level of delta6-desaturase mRNA, the inhibition of GLA production could not be attributed to suppression of delta6-desaturase gene expression at the transcriptional level.

Regulation of Hepatic Δ-6 Desaturase Expression and Its Role in the Polyunsaturated Fatty Acid Inhibition of Fatty Acid Synthase Gene Expression in Mice

Dietary polyunsaturated fatty acids (PUFA) of the (n-6) and (n-3) families uniquely suppress the expression of lipogenic genes while concomitantly inducing the expression of genes encoding proteins of fatty acid oxidation. Although considerable progress has been made toward understanding the nuclear events affected by PUFA, the intracellular mediator responsible for the regulation of hepatic lipogenic gene expression remains unclear. On the basis of earlier fatty acid composition studies, we hypothesized that the Delta-6 desaturase pathway was essential for the production of the fatty acid regulator of gene expression. To address this hypothesis, male BALB/c mice (n = 8/group) were fed for 5 d a high glucose, fat-free diet (FF) or the FF plus 50 g/kg 18:2(n-6) with and without eicosa-5, 8,11,14-tetraynoic acid (ETYA) (200 mg/kg diet), a putative inhibitor of the Delta-6 desaturase pathway. ETYA had no effect on food intake or weight gain, but it completely prevented 18:2(n-6) from suppressing the hepatic abundance of fatty acid synthase mRNA. ETYA ingestion was associated with a decrease in the hepatic content of 20:4(n-6) and an increase in the amount of 18:2(n-6). The fatty acid composition changes elicited by ETYA were accompanied by a decrease in the enzymatic activity of Delta-6 desaturase. Interestingly, the hepatic abundance of Delta-6 desaturase mRNA was actually induced by ETYA one- to twofold. When the product of Delta-6 desaturase, i.e., 18:3(n-6), was added to the ETYA plus 18:2(n-6) diet, the hepatic content of 20:4(n-6) was normalized. In addition, 18:3(n-6) consumption reduced the level of hepatic Delta-6 desaturase mRNA by 50% and completely prevented the increase in fatty acid synthase mRNA that was associated with ETYA ingestion. Apparently, Delta-6 desaturation is an essential step for the PUFA regulation of the fatty acid synthase gene transcription. Finally, the suppression of Delta-6 desaturase by PUFA and its induction by ETYA suggest that the Delta-6 desaturase gene may be regulated by two different lipid-dependent mechanisms.

Cloning, Expression, and Fatty Acid Regulation of the Human Δ-5 Desaturase

Arachidonic (20:4(n-6)), eicosapentaenoic (20:5(n-3)), and docosahexaenoic (22:6(n-3)) acids are major components of brain and retina phospholipids, substrates for eicosanoid production, and regulators of nuclear transcription factors. One of the two rate-limiting steps in the production of these polyenoic fatty acids is the desaturation of 20:3(n-6) and 20:4(n-3) by Delta-5 desaturase. This report describes the cloning and expression of the human Delta-5 desaturase, and it compares the structural characteristics and nutritional regulation of the Delta-5 and Delta-6 desaturases. The open reading frame of the human Delta-5 desaturase encodes a 444-amino acid peptide which is identical in size to the Delta-6 desaturase and which shares 61% identity with the human Delta-6 desaturase. The Delta-5 desaturase contains two membrane-spanning domains, three histidine-rich regions, and a cytochrome b(5) domain that all align perfectly with the same domains located in the Delta-6 desaturase. Expression of the open reading frame in Chinese hamster ovary cells instilled the ability to convert 20:3(n-6) to 20:4(n-6). Northern analysis revealed that many human tissues including skeletal muscle, lung, placenta, kidney, and pancreas expressed Delta-5 desaturase mRNA, but Delta-5 desaturase was most abundant in the liver, brain, and heart. However, in all tissues, the abundance of Delta-5 desaturase mRNA was much lower than that observed for the Delta-6 desaturase. When rats were fed a diet containing 10% safflower oil or menhaden fish oil, the level of hepatic mRNA for Delta-5 and Delta-6 desaturase was only 25% of that found in the liver of rats fed a fat-free diet or a diet containing triolein. Finally, a BLAST and Genemap search of the human genome revealed that the Delta-5 and Delta-6 desaturase genes reside in reverse orientation on chromosome 11 and that they are separated by <11,000 base pairs.

Cloning, Expression, and Nutritional Regulation of the Mammalian Δ-6 Desaturase

Arachidonic acid (20:4(n-6)) and docosahexaenoic acid (22:6(n-3)) have a variety of physiological functions that include being the major component of membrane phospholipid in brain and retina, substrates for eicosanoid production, and regulators of nuclear transcription factors. The rate-limiting step in the production of 20:4(n-6) and 22:6(n-3) is the desaturation of 18:2(n-6) and 18:3(n-3) by Delta-6 desaturase. In this report, we describe the cloning, characterization, and expression of a mammalian Delta-6 desaturase. The open reading frames for mouse and human Delta-6 desaturase each encode a 444-amino acid peptide, and the two peptides share an 87% amino acid homology. The amino acid sequence predicts that the peptide contains two membrane-spanning domains as well as a cytochrome b5-like domain that is characteristic of nonmammalian Delta-6 desaturases. Expression of the open reading frame in rat hepatocytes and Chinese hamster ovary cells instilled in these cells the ability to convert 18:2(n-6) and 18:3(n-3) to their respective products, 18:3(n-6) and 18:4(n-3). When mice were fed a diet containing 10% fat, hepatic enzymatic activity and mRNA abundance for hepatic Delta-6 desaturase in mice fed corn oil were 70 and 50% lower than in mice fed triolein. Finally, Northern analysis revealed that the brain contained an amount of Delta-6 desaturase mRNA that was several times greater than that found in other tissues including the liver, lung, heart, and skeletal muscle. The RNA abundance data indicate that prior conclusions regarding the low level of Delta-6 desaturase expression in nonhepatic tissues may need to be reevaluated.