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.     

Evidence for the different responses of delta9-, delta6- and delta5-fatty acyl-CoA desaturases to cytoplasmic proteins.

Microsomes prepared from the livers of 4-week-old rats were, after extraction with 0.1 M potassium phosphate buffer, pH 7.4, unable to catalyse either the delta6 desaturation of alpha-linolenic acid (9c.12c.15c., 18 : 3) into 6c.9c.12c.15c., 18 : 4 or the delta5 desaturation of eicosatrienoic acid (8c.11c.14c., 20 : 3) into arachidonic acid (5c.8c.11c.14c., 20 : 4). Both these enzymes only showed full activity after incubation of the microsomes with either the 100 000 X g supernatant fraction or with purified bovine catalase. Bovine serum albumin, while capable of restoring 50% of the delta5 desaturase activity has no effect on the delta6 desaturase. In contrast the delta9 desaturase activity of microsomes was never completely lost after extraction with buffer but could be stimulated by optimum concentrations of both bovine serum albumin and catalase. The significance of the different responses of the three desaturases to the cytoplasmic components is discussed.     

Immunochemical evidence for the enzymatic difference of delta 6-desaturase from delta 9- and delta 5-desaturase in rat liver microsomes

The enzymatic properties of the three types of microsomal acyl-CoA desaturases, delta 6-, delta 9- and delta 5-desaturases, were immunologically compared using a monospecific antibody raised against the purified linoleoyl-CoA desaturase (delta 6-desaturase). By the double immunodiffusion technique, the anti-delta 6-desaturase antibody showed a single precipitin line to the purified delta 6-desaturase and microsomes treated with Triton X-100, but no line was observed with the partially purified delta 9-desaturase. The antibody even inhibited definitely delta 6-desaturase activity in microsomes, but neither stearoyl-CoA (delta 9-) nor eicosatrienoic acid (delta 5-) desaturations were inhibited. By these immunological investigations it was confirmed that terminal delta 6-desaturase is different enzyme from desaturases delta 9- and delta 5.     

A palmitoyl-CoA-specific delta9 fatty acid desaturase from Caenorhabditis elegans

Biosynthesis of polyunsaturated fatty acids in C. elegans is initiated by the introduction of a double bond at the delta9 position of a saturated fatty acid. We identified three C. elegans fatty acid desaturase genes related to the yeast delta9 desaturase OLE1 and the rat stearoyl-CoA desaturase SCD1. Heterologous expression of all three genes rescues the fatty acid auxotrophy of the yeast delta9 desaturase mutant ole1. Examination of the fatty acid composition of the transgenic yeast reveals striking differences in the substrate specificities of these desaturases. Two desaturases, FAT-6 and FAT-7, readily desaturate stearic acid (18:0) and show less activity on palmitic acid (16:0). In contrast, the other desaturase, FAT-5, readily desaturates palmitic acid (16:0), but shows nearly undetectable activity on the common delta9 substrate stearic acid. This is the first report of a palmitoyl-CoA-specific membrane fatty acid desaturase.     

Modulation of delta 6 and delta 5 rat liver microsomal desaturase activities by dexamethasone-induced factor

This report supports evidence for the existence of a dexamethasone-induced factor that modulates fatty acid desaturase activities. Dexamethasone at a dose of 1 mg/rat produced a significant decrease in microsomal delta 6 and delta 5 desaturation activity 12 h after the injection. Both desaturase activities were depressed by a soluble factor present in the cytosolic fraction of cells, since the supernatant of microsomes separated at 110,000 X g from hormonal-treated rat liver homogenates, added to crude or washed control microsomes, was able to inhibit in vitro linoleic and homo-gamma-linolenic conversion to gamma-linolenic and arachidonic acids, respectively. The inhibitory factor was loosely bound to microsomes, since it was also present in a soluble fraction obtained after washing crude microsomes from dexamethasone-treated rats with a low-ionic-strength solution. Besides, trypsin digestion deactivates the dexamethasone-induced factor. Therefore, the depressing effect of glucocorticoids on delta 6 and delta 5 desaturation capacity depends on an unchanged protein structure present in the cytosolic fraction of the cell and whose biosynthesis is brought about by hormonal induction.     

The effect of insulin on delta5 desaturation in hepG2 human hepatoma cells and L6 rat muscle myoblasts.

In humans there is a correlation between the ratio of arachidonic acid (20:4n-6) to cis 8,11,14 eicosatrienoic acid (20:3n-6) in skeletal muscle phospholipids and insulin sensitivity. This has been interpreted as indicating a link between the activity of the delta5 desaturase enzyme and muscle insulin sensitivity. The present study addressed the possibility that insulin regulates delta5 desaturase activity using L6 rat myoblasts and hepG2 human hepatoma cells. Both cell lines responded to insulin by increasing the amount of D-[U-14C] glucose incorporated into glycogen. In L6 cells, insulin stimulated cis 8,11,14 eicosatrienoic acid uptake and arachidonic acid production but had no effect on the percentage conversion of cis 8,11,14 eicosatrienoic acid to arachidonic acid. In hepG2 cells, insulin had no effect on cis 8,11,14 eicosatrienoic acid uptake or arachidonic acid production. These results suggest that insulin has no direct effect on delta5 desaturase activity in the liver but can alter arachidonic acid production in muscle by altering substrate availability.     

Identification of a novel bifunctional delta12/delta15 fatty acid desaturase from a basidiomycete, Coprinus cinereus TD#822-2

A new gene encoding a delta12 fatty acid desaturase-related protein was cloned from a multicellular basidiomycete Coprinus cinereus TD#822-2. The 1326 bp full-length gene, designated as Cop-odeA, codes for a putative protein of 442 amino acids with a MW of 49224. The Cop-odeA yeast transformant accumulated four new fatty acids identified as 9,12-hexadecadienoic acid, 9,12,15-hexadecatrienoic acid, linoleic acid, and alpha-linolenic acid, which comprised 8.8%, 1.0%, 29.0%, and 0.6% of the total fatty acids, respectively. The Cop-odeA protein was confirmed to be a novel bifunctional fatty acid desaturase with both high delta12 desaturase activity and unusual delta15 desaturase activity.     

Effect of clofibrate on fatty acid desaturation of rats treated with ethanol

The effect of clofibrate and ethanol in the rat was studied on the following aspects of lipid composition and metabolism: liver delta 5, delta 6 and delta 9 fatty acid desaturases, fatty acid synthetase and fatty acid desaturase microsomal electron transport chain activity and serum cholesterol, triacylglycerols and high (HDL), low (LDL) and very low density lipoprotein (VLDL) levels. Clofibrate administered for 9 days (0.3% W/W) did not modify the relative composition of liver phospholipids and cholesterol, but did diminish triacylglycerol levels increased by ethanol. This effect could be explained by the possible beta-adrenergic blocking properties of clofibrate or by an increased activity of peroxisomal beta-oxidation. Clofibrate also promoted a decrease in serum cholesterol and triacylglycerol levels, delta 6 desaturase activity and a suppression of the electron transport chain as measured by NADH cytochrome b5 reductase and NADH cytochrome c reductase. The drug increased delta 9 desaturase activity and fatty acid synthetase, while no effect could be found in delta 5 desaturase activity. The hypocholesterolenic effect of clofibrate can not be explained through the delta 6 desaturase inhibition, or the fatty acid synthetase enhancement. Ethanol increased the HDL and VLDL and lowered LDL serum concentrations, while clofibrate reversed these results. Considering that clofibrate could have antiatherosclerotic effect in the rat, it is difficult to explain it through these changes in lipoprotein levels, since according to Miller and Miller low HDL levels are predictive of coronary heart disease.     

小眼虫藻Δ8-脂肪酸脱氢酶基因的克隆及其在酿酒酵母中的表达

Δ8途径是合成多不饱和脂肪酸的替代途径,Δ8-脂肪酸脱氢酶是该途径的关键酶之一。根据已报道的Δ8-脂肪酸脱氢酶基因设计引物,分别从小眼虫藻基因组DNA和cDNA中扩增得到该基因片段,序列分析表明：结构基因长1 266 bp,编码421个氨基酸;该基因没有内含子,比已经报道的Δ8-脂肪酸脱氢酶基因长6 bp,并且N末端序列也有所不同。利用酿酒酵母的载体pYES2.0构建Δ8-脂肪酸脱氢酶表达载体pYEFD,并转化到营养缺陷型酿酒酵母菌株INVSc1中,在选择培养基中筛选得到酿酒酵母转化菌株YD8。YD8在合适的培养条件下,添加外源底物二十碳二烯酸和二十碳三烯酸并诱导基因表达。脂肪酸甲酯气相色谱分析表明小眼虫藻Δ8-脂肪酸脱氢酶基因在酿酒酵母中获得了高效表达,将二十碳二烯酸和二十碳三烯酸分别转化成二高-γ-亚麻酸和二十碳四烯酸,其底物转化率分别达到了31.2％和46.3％。     

Accelerated essential fatty acid deficiency by delta 9 desaturase induction: dissociation between the effects on liver and other tissues

Essential fatty acid (EFA) deficiency is an important tool in probing the role of arachidonic acid (20:4(n-6] in pathophysiologic processes, but requires stringent and prolonged deprivation of (n-6) fatty acids. The present study investigated whether induction of the delta 9 desaturase, which is responsible for the synthesis of oleate, the precursor of 20:3(n-9) which uniquely accumulates in the deficiency state, might serve to accelerate the biochemical and biological effects of EFA deficiency. By alternately fasting and feeding animals a fat-free diet, it was possible to induce markedly the delta 9 desaturase selectively in liver. This dietary manipulation in consequence led to dramatic and rapid changes in hepatic phospholipid fatty acid composition. Within 2 weeks, 20:3(n-9) to 20:4(n-6) ratios in liver phospholipids were several fold greater than those seen in animals fed a fat-free diet alone. These changes, however, contrasted with those seen in the serum and other tissues. The mol% of 20:3(n-9) in serum was not increased by delta 9 desaturase induction and the 20:3(n-9) to 20:4(n-6) ratio was only modestly increased. The effects of delta 9 desaturase induction were even more attenuated in tissues other than the liver. Desaturase induction led to a doubling in the 20:3(n-9) to 20:4(n-6) ratio in phosphatidylcholine in renal cortex and heart, although the ratio in the other phospholipids was unaffected. The 20:3(n-9) to 20:4(n-6) ratio in peritoneal macrophage phospholipids was unaffected by desaturase induction. Thus, delta 9 desaturase induction greatly augments the synthesis of (n-9) fatty acids within the liver and leads to the rapid and substantial accumulation of the abnormal fatty acid, 20:3(n-9). This markedly augmented synthesis of hepatic 20:3(n-9), however, is not reflected in increased plasma levels of 20:3(n-9), and thus the effects of delta 9 desaturase induction are attenuated in tissues other than the liver. These data underscore the notable ability of the liver to maintain polyunsaturated fatty acid homeostasis.     

Polyunsaturated fatty acid metabolism in fish cells: differential metabolism of (n-3) and (n-6) series acids by cultured cells originating from a freshwater teleost fish and from a marine teleost fish

1. The incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids (PUFA) supplemented to growing cultures were studied in rainbow trout (RTG-2) and turbot (TF) cell lines. 2. A fatty acid concentration of 20 microM considerably altered the fatty acid composition of the cells without affecting lipid class composition or the appearance of cytoplasmic lipid droplets. 3. Both cell lines exhibited considerable delta 6 desaturase activities. 4. Whereas delta 5 desaturase activity was expressed in RTG-2 cells, delta 4 desaturase activity was absent and, conversely, delta 4 desaturase activity was expressed in TF cells, but there was an apparent deficiency in the C18 to C20 elongase multi-enzyme complex. 5. The delta 6 desaturase activity in both cell lines showed little preference between 18:2(n-6) and 18:3(n-3) but the delta 5 desaturase activity of RTG-2 cells and the delta 4 desaturase activity of TF cells showed a preference for (n-3)PUFA. 6. Two fish oil concentrates were assessed for their ability to generate fatty acid compositions in the cell lines more closely resembling those of intact fish tissues.     

Functional identification of a delta8-sphingolipid desaturase from Borago officinalis

The similarities between delta12- and delta5-fatty acyl desaturase sequences were used to construct degenerate primers for PCR experiments with cDNA transcribed from mRNA of developing borage seeds. Screening of a borage seed cDNA library with an amplified DNA fragment resulted in the isolation of a full-length cDNA corresponding to a deduced open-reading frame of 446 amino acids. The protein showed high similarity to plant delta8-sphingolipid desaturases as well as to the delta6-fatty acyl desaturase from Borago officinalis. The sequence is characterized by the presence of a N-terminal cytochrome b5 domain. Expression of this open-reading frame in Saccharomyces cerevisiae resulted in the formation of delta8-trans/cis-phytosphingenines not present in wild-type cells, as shown by HPLC analysis of sphingoid bases as their dinitrophenyl derivatives. GLC-MS analysis of the methylated di-O-trimethylsilyl ether derivatives confirmed the presence of delta8-stereoisomers of C18- and C20-phytosphingenine. Furthermore, Northern blotting showed that the gene encoding a stereo-unselective delta8-sphingolipid desaturase is primarily expressed in young borage leaves.     

Hormonal modulation of delta6 and delta5 desaturases: case of diabetes

Animal biosynthesis of high polyunsaturated fatty acids from linoleic, alpha-linolenic and oleic acids is mainly modulated by the delta6 and delta5 desaturases through dietary and hormonal stimulated mechanisms. From hormones, only insulin activates both enzymes. In experimental diabetes mellitus type-1, the depressed delta6 desaturase is restored by insulin stimulation of the gene expression of its mRNA. However, cAMP or cycloheximide injection prevents this effect. The depression of delta6 and delta5 desaturases in diabetes is rapidly correlated by lower contents of arachidonic acid and higher contents of linoleic in almost all the tissues except brain. However, docosahexaenoic n-3 acid enhancement, mainly in liver phospholipids, is not explained yet. In experimental non-insulin dependent diabetes, the effect upon the delta6 and delta5 desaturases is not clear. From all other hormones glucagon, adrenaline, glucocorticoids, mineralocorticoids, oestriol, oestradiol, testosterone and ACTH depress both desaturases, and a few hormones: progesterone, cortexolone and pregnanediol are inactive.     

Chronologic studies of the effects of a hypoproteinic diet followed by an equilibrated diet on delta-6 and delta-5 desaturations of linoleic acid in liver microsomes in the rat

A low protein diet affects amounts of linoleic and arachidonic acids in hepatic microsomal phospholipids of growing rats. Are the changes related to modifications in microsomal delta 6- and delta 5- linoleic acid desaturase activities? Two groups of Wistar rats weighing 80 +/- 5 g at the beginning of the experiment were used: Control group (T) was fed on a 16% gluten + 4% casein diet for 53 days; Experimental group (E) was fed on a 4% gluten + 1% casein diet for 26 days (MP) then Control diet for 27 days (RE). After 2, 14 and 26 days of MP and 2, 15 and 27 days of RE, rats of each group were sacrificed. Protein and water contents of liver, quantitative fatty acid, composition of total lipids in liver and hepatic microsomes were determined. delta 6- and delta 5- linoleic acid desaturase activities were estimated from incubation of liver microsomes with [1-14C] C 18: 2 n-6 or [2(14)C] C 20: 3 n-6 respectively. The low protein diet stops practically ponderal growth. The fatty-acid compositions of microsomal total lipids of E rats were affected in comparison with values of T rats. These modifications persist after 27 days of RE. The C 20: 4 n-6/C 18: 2 n-6 ratio in microsomal total lipids was slightly different between T and E rats but increased strongly during refeeding. Same modifications take place in the fatty-acid composition of hepatic total lipids. After two days of MP, delta 6- and delta 5- desaturase activities were depressed, phenomenon that not persist in the course of MP. These enzyme activities increase to higher values than those of the T after two days of RE.     

The acyl-CoA desaturases of microsomes from rat liver and the Morris 7777 hepatoma.

We have investigated the role of the microsomal oxidative desaturase in defining the aberrant phosphoglyceride fatty acid composition of hepatomas. The microsomal delta 9-stearoyl-CoA, delta 6-oleoyl(linolenoyl)-CoA, and delta 5-eicosatrienoyl-CA desaturase activities were studied in control and host liver and in the poorly differentiated Morris 7777 hepatoma. The delta 9-stearoyl-CoA desaturase of the hepatoma was significantly decreased (42%) relative to control liver, yet the hepatoma specific activity was twice that of host liver. Additionally, the specific activity of the delta 9-stearoyl-CoA desaturase of the tumor was found to decrease with increasing tumor weight. Also this desaturase was inactivated by freezing and thawing. The delta 6-oleoyl(linolenoyl)-CoA and delta 5-eicosatrienoyl-CoA desaturases of the hepatoma were 39% and 4% of control, respectively. The electron transport components involved in the desaturase system were reduced, although this did not appear to be rate-limiting. In addition, two competing metabolic reactions which could lower the observed desaturase activities, hydrolysis of the thioester and incorporation of substrate acyl-CoA molecules into glycerides, did not appear to be responsible for the lowered desaturase activities of the tumor. Thus, it appears that reduced levels of the desaturases themselves may be responsible for the observed activities. These results indicate that the capacity of the hepatoma to biosynthesize polyunsaturated fatty acids is greatly reduced and this is consistent with the decreased polyene content observed in many neoplasms.     

少根根霉Δ^6-脂肪酸脱氢酶基因在毕赤酵母中的表达

Δ^6-脂肪酸脱氢酶是一种膜整合蛋白,也是多不饱和脂肪酸合成途径中的限速酶。在前期工作中,通过RT-PCR和RACE技术,从少根根霉NK300037中克隆到一个潜在编码Δ^6-脂肪酸脱氢酶的序列,序列和功能分析结果表明该序列具有一个长度为1377bp、编码由458个氨基酸组成、大小为52kD的新的Δ^6-肪酸脱氢酶基因。把少根根霉Δ^6-脂肪酸脱氢酶基因（RAD6）亚克隆到表达载体pPIC3．5K,构建重组表达载体pPICRAD6,并转化到毕赤酵母菌株GS115进行表达。提取酵母细胞总脂肪酸和进行甲酯化,经气相色谱和气相色谱-质谱连用分析表明,目的基因的编码产物能将C16：1、C17：1、C18：1、亚油酸和α-亚麻酸在△6和7位间特异性脱氢而引入一个新的双键,生成更高不饱和的脂肪酸,该催化反应没有链长特异性,只有键位特异性。此外,按Kozak序列特点,改变目的基因转译起始密码子周边序列结构,并把改变后序列导入毕赤酵母GS115中进行功能表达分析,结果表明在毕赤酵母中这种改变同样能提高目的基因的表达水平。综合所有分析结果表明,巴斯德毕赤酵母更适合用来综合分析Δ^6-脂肪酸脱氢酶基因的功能。     

Increased response of liver microsomal delta 6-desaturase activity to dietary methionine in rats

The effects of dietary casein level (5-40%) on the liver microsomal phospholipid profile, delta 6-desaturase activity and related variables were investigated in rats to examine whether the dietary protein level affected the delta 6-desaturase activity through an alteration of the liver microsomal phospholipid profile. The effects of supplementing a 10% casein diet with certain amino acids were also investigated. The concentration of hepatic S-adenosylmethionine (SAM), the ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) and the delta 6-desaturase activity in liver microsomes, and the ratio of arachidonate to linoleate of microsomal PC increased with increasing dietary casein level. There were significant correlations between the dietary methionine content and hepatic SAM concentration, hepatic SAM concentration and microsomal PE concentration, and microsomal PE concentration and delta 6-desaturase activity. Supplementation of the 10% casein diet with methionine significantly increased the hepatic SAM concentration, PC/PE ratio, delta 6-desaturase activity, and arachidonate/linoleate ratio, whereas cystine supplementation had no or little effect on these variables. These increases induced by methionine were significantly suppressed by additional glycine. The results obtained here, together with those in our previous report, suggest that quantity and type of dietary protein might affect the delta 6-desaturase activity through an alteration of the liver microsomal profile of phospholipids, especially PE, and that the alteration of phospholipid profile might be mediated by a hepatic SAM concentration that reflects the dietary methionine level.     

Evidence of isozymes for delta6 fatty acid desaturase in rat hepatocytes

The expression of delta6 fatty acid desaturase, previously identified, was suppressed almost completely by hyper expression of the corresponding antisense gene in a transformant of the rat hepatic cell line BRL-3A. Conversion rates of [1-14C] linoleic acid, alpha-linolenic acid, and tetracosapentaenoic acid into the respective delta6 fatty acids were equivalent to those in control cells. This finding suggested that all of these reactions were catalyzed by at least two delta6 desaturase isozymes in rat hepatocytes.     

Studies of the delta 12 desaturase of Carthamus tinctorius L

The delta 12 desaturase of developing safflower seeds responsible for the conversion of an oleoyl moiety to the linoleoyl moiety of phospholipids was further characterized. The protein concentration of the microsomal preparation, the oleoyl-CoA concentration (the primary substrate), short incubation periods, and the addition of lysophospholipids must be controlled to obtain optimal desaturation. No evidence could be obtained to implicate cytochrome b5 as the intermediate electron carrier. Attempts to solubilize the desaturase with a variety of detergents and chaotropic reagents were not successful. Brief exposure of the microsomal preparation to trypsin resulted in rapid loss of activity. The overall evidence would suggest that the delta 12 desaturase requires a reductant (NADPH), a NADPH:electron carrier reductase, an electron carrier, a specific desaturase, and an acyltransferase with oleoyl-CoA as the substrate to acylate lysophospholipid to the active oleoyl phospholipids (presumably phosphatidylcholine or phosphatidylethanolamine). The complexity of this system suggests that purification of the components and a reassembling of the purified components will be difficult.