Stearoyl-coenzyme A desaturase activity in novikoff hepatoma

The activities of microsomal stearoyl-CoA desaturation, NADH-cytochrome b₅ reductase, NADH-cytochrome c reductase, and the content of cytochrome b₅ were similar in livers of normal and host rats. On the other hand, stearoyl-CoA desaturation activity was absent in Novikoff hepatoma. The activities of NADH-cytochrome b₅ and NADH-cytochrome c reductases in the hepatoma microsomes were 4.8% and 2.2%, respectively, of those in normal liver. Furthermore, in hepatoma microsomes, cytochrome b₅ was absent. An active stearoyl-CoA desaturation was reconstituted only on addition of both cytochrome b₅ and the terminal desaturase enzyme to the hepatoma microsomes. These results indicated that a complete absence of cytochrome b₅ and terminal desaturase is responsible for the lack of stearoyl-CoA desaturation in Novikoff hepatoma microsomes.     

Membrane-bound phospholipid desaturases

This review covers studies on membrane-bound phospholipid desaturases in yeast and rat liver carried out in this laboratory. In yeast the desaturase system was shown to effect the direct desaturation of dioleoyl-lecithin to dilinoleoyl-lecithin. In rat liver the desaturase was capable of converting 2-eicosatrienoyl-lecithin to 2-arachidonoyl-lecithin. Both systems required reduced pyridine nucleotides, O₂ and cytochrome b₅. Eicosatrienoyl-lecithin desaturase along with eicosatrienoyl-CoA desaturase of rat liver microsomes was solubilized with detergents and purified 7–8-fold from the microsomal pellets. Both activities were reconstituted in the presence of deoxycholate on addition of the other components of the cytochrome b₅-electron transport chain (cytochrome b₅ and NADH-cytochrome b₅ reductase) to the solubilized desaturase; addition of lecithin further stimulated the activities. The demonstration of desaturation of eicosatrienoyl-lecithin by a solubilized and partially purified desaturase provides strong evidence for the direct desaturation of the lecithin substrate without prior conversion to the acyl-CoA thiolester.     

Analysis of the stearoyl-CoA desaturase system in the Morris hepatoma 7288C and 7288CTC

The microsomal stearoyl-CoA desaturase system was examined in both the Morris hepatoma 7288CTC, maintained in the host Buffalo strain rat, and the Morris hepatoma 7288C, maintained in tissue culture. In vitro examination shows the stearoyl-CoA desaturase system to be similar in the 2 tissues. Both show extremely low overall stearoyl-CoA desaturase activity, having 4% and 8% of normal liver values respectively. Examination of the electron transport system showed both tissues have decreased electron transport components cytochrome b₅ and cytochrome b₅ reductase. Particularly noticeable were the extremely low levels of cytochrome b₅ (2% compared with normal liver). Microsomes from both tissues showed a decreased ability to reduce an artificial electron acceptor, cytochrome c. With the low levels of cytochrome b₅ observed in these tissues, the low levels of overall desaturase activity may be caused by lack of terminal enzyme, lack of sufficient cytochrome b₅, or both. Analysis of the stearoyl-CoA desaturase system in cultured hepatoma cells suggests that these cells are similar to the host-grown tumor in this respect and may be used as a model in further examinations of the stearoyl-CoA desaturase system.     

Effect of ethanol administration on fatty acid desaturation

The effect of ethanol on the fatty acid desaturation by rat liver has been studied using liquid diets of different composition. Acute ethanol administration increased triacylglycerols of total liver lipids, but did not modify significantly the lipidic composition of microsomes. The Δ6 and Δ5 desaturases were inhibited by ethanol whereas the Δ9 desaturase and fatty acid synthetase were apparently modified only by diet composition. NADH-cytochrome (cyt.) c reductase was partially inhibited, whereas NADH-cyt. b₅ reductase remained practically unaltered and NADPH-cyt. c reductase activity was enhanced. Decreased electrons supplied by the microsomal cyt. b₅ electron transport chain would not be the reason for the inhibition of Δ6 and Δ5 desaturases by ethanol.     

Stearoyl-CoA desaturase activity in mammary adenocarcinomas carried by C3H mice

Transplantable mammary adenocarcinomas and livers of C3H mice fed a stock diet or a linoleate rich diet (15% corn oil) contain similar amounts of oleate (ca 3 mg/gm tissue). On feeding either a high carbohydrate, fat free or a high carbohydrate, saturated fat-containing (15% hydrogenated coconut or cottonseed oil) diet for 6 weeks, oleate levels increased 2-fold in tumor and 5-fold in liver. The specific activity of stearoyl-CoA desaturase in liver microsomes was similar to that in the corresponding fractions of mammary glands of lactating mice. In liver, this activity was enhanced 2- to 3-fold by feeding a high carbohydrate, fat free or a high carbohydrate, saturated fat diet. The desaturase activity in mammary tumor microsomes, while only 10% of that in hepatic microsomes, remained unaltered regardless of the type of diet fed. These observations suggest that (a) a major portion of the oleate in the mammary tumor is not produced within the tissue, (b) dietary adaptation is not a general characteristic of stearoyl-CoA desaturase in neoplastic tissues, and (c) enhanced desaturase activity in liver is directly related to the absence of linoleate or oleate, or to a large decrease in oleate in the diet.     

Modification of microsomal lipid composition and electron transport enzyme activities in isovalerate-supplemented cells of novelTetrahymena ISO

Tetrahymena ISO cells, which have an unusually high level of iso odd-numbered fatty acids, were grown medium supplemented with various concentrations of isovalerate. There was a marked increase in the total proportion of iso odd-numbered fatty acids in supplemented whole cells (28.9 leads to 70.3%) and microsomes (37.7 leads to 84%), with a corresponding decrease in normal fatty acids, although no significant alteration of phospholipid composition was observed during 11 hr isovalerate-supplementation. Microsomal palmitoyl-CoA and stearoyl-CoA desaturase activities in isovalerate-supplemented cells decreased by 45.7% and 30.6% during 11 hr, respectively. NADH-cytochrome c reductase and NADH-ferricyanide reductase activities as well as the content of cytochrome b560ms, which is similar to mammalian microsomal cytochrome b5, were reduced in microsomes from 11 hr-supplemented cells, whereas NADPH-cytochrome c reductase activity was constant. It is suggested that the alteration of the cross-sectional area of lipid molecules in the bilayer, which results from the replacement of normal fatty acids with iso- 15:0 and iso- 17:1, would result in the decline of palmitoyl- and stearoyl-CoA desaturation in the isovalerate-supplemented cells, in order to maintain membrane fluidity at a functional level.     

The dietary regulation of acyltransferase and desaturase activities in microsomal membranes of rat liver

Dietary manipulation produces marked alterations in desaturase activities of rat liver microsomes with no concomitant changes in acyltransferase activities. Desaturation of stearoyl-CoA (Δ9-desaturase), linoleoyl-CoA (Δ6-desaturase), eicosatrienoyl-CoA (Δ5-desaturase) and eicosatrienoyl-phosphatidylcholine (Δ5-desaturase) was elevated in animals fed a corn oil diet and lowered in those fed a coconut oil diet compared to control animals. The Δ5-desaturase activities were also lowered in starved animals and elevated in starved animals refed a fat-free diet. However, no changes in acyl-CoA:1-acylsn-glycero-3-phosphocholine acyltransferase activity were observed in the membranes of animals maintained on any of the dietary regimens studied. These observations suggest that the desaturases of rat liver microsomes are regulated independently of the acyltransferases and that desaturation of eicosatrienoyl-phosphatidylcholine is regulated at the level of the desaturase itself and not by availability of the phospholipid substrate.     

Stearyl CoA as a precursor of oleic acid and glycerolipids in mammary microsomes from lactating bovine: Possible regulatory step in milk triglyceride synthesis

The stearyl desaturase of lactating bovine mammary tissue is located in the microsomes and requires activated fatty acid and NADH for activity. Other enzymes, acyl-transferase(s) and deacylase which apparently compete with the desaturase for substrate are also present. Both the substrate 1-¹⁴C-stearyl CoA and the oleic acid produced by desaturase are esterified into the various lipid classes. The oleic acid is preferentially acylated into positionsn-3 of the triglycerides andsn-2 of the phosphatidylcholine. Experimental conditions causing reduced desaturase activity depressed triglyceride synthesis, and stimulation of desaturation by NADH L^−α GP, acidic pH, 5.6, was accompanied by increased incorporation of radioactive fatty acids into the triglycerides. These data indicated that desaturase and glyceride acyl transferase were located contiguously within the microsomal membranes. The possibility that desaturase activity might control triglyceride synthesis in vivo is discussed. It was observed that mammary tissue from nonlactating cows 1–2 weeks and 2 days prior to calving lacked or possessed very low stearyl desaturase activity.     

Effect of ATP on the microsomal desaturation of unsaturated fatty acids

The effect of ATP on the microsomal desaturation of linoleic acid to γ-linolenic acid was studied in a system in vitro with the following results: (1) preincubation of rat liver microsomes with ATP alone in N₂ or in the presence of CoA and Mg⁺⁺ followed by subsequent incubation with 1-¹⁴C-linoleic acid plus NADH in O₂ resulted in enhancement of 1-¹⁴C-linoleic acid desaturation when compared with control samples in which no preincubation was performed; (2) the preincubation of the microsomes with ATP, Mg⁺⁺ and CoA in the presence of 1-¹⁴C-linoleic acid decreased the desaturation of the labeled acid to γ-linolenic acid upon subsequent incubation with NADH, as a consequence of incorporation of the acid into the microsomal lipids; (3) the increase of linoleic acid desaturation depended on the ATP concentration during preincubation and followed a sigmoidal curve. It was specific for ATP, and neither GTP, CTP, ADP nor AMP produced a similar effec. However, GTP or CTP could replace ATP as a cofactor in the microsomal desaturation of free linoleic acid to γ-linolenic, suggesting that directly or indirectly they may activate conversion of the free acid to linoleyl-CoA; (4) preincubation of microsomes with ATP activated the acylation of CoA. However, this activation showed no quantitative correlation with enhancement of the desaturation reaction; (5) addition of ATP also stimulated conversion of linoleyl-CoA to γ-linolenic acid. This enhancement was not related to inhibition of the linoleyl-CoA hydrolase; (6) however, in spite of these results, preincubation with ATP did not increase the initial velocity of linoleic acid or linoleyl-CoA desaturation; (7) preincubation of microsomes with ATP also increased the 6-desaturation of oleic acid and α-linolenic acid but did not increase the 9-desaturation of plamitic and stearic acid.     

Effect of epinephrine on the oxidative desaturation of fatty acids in the rat adrenal gland

Delta-6 and Δ5 desaturation activity of rat adrenal gland microsomes was studied to determine the effect of microsomal protein and the substrate saturation curves. This tissue has a very active Δ6 desaturase for linoleic and α-linoleic acids and a Δ5 desaturase for eicosa-8,11,14-trienoic acid. The administration of epinephrine (1 mg/kg body weight) 12 hr before killing, produced approximately a 50% decrease in desaturation of [1-¹⁴C]linoleic acid to γ-linolenic acid, [1-¹⁴C]α-linolenic acid to octadeca-6,9,12,15-tetraenoic acid and [1-¹⁴C]eicosa-8,11,14-trienoic acid to arachidonic acid. A 30% decrease in Δ5 desaturation activity was also shown after 7 hr of epinephrine treatment. The changes on the oxidative desaturation of the same fatty acids in liver microsomes were similar. No changes were observed in the total fatty acid composition of adrenal microsomes 12 hr after epinephrine treatment. Mechanisms of action of the hormone on the biosynthesis of polyunsaturated fatty acids in the adrenal gland are discussed.     

Effect of environmental temperature changes on rat liver fatty acid desaturases

Female rats warm-adapted at 30–32 C for 20–25 days and then shifted to 13–15 C for 12, 24, 48, 72 and 120 hr showed that Δ9 desaturase and fatty acid synthetase activity decay after 24 hr of cold exposure, while Δ6 and Δ5 desaturases were increased after this period of time. These results were confirmed by an increase of arachidonic acid of heart and liver microsomes phosphatidylcholine and a decrease of oleic acid. Neither NADH-cyt b₅ reductase nor NADH-cyt c reductase activity of liver microsomes were significantly affected. Male rats warm-adapted under the same conditions and then shifted to 13–15 C for 120 hr did not show significant changes in fatty acid synthetase, Δ9 and Δ6 desaturases and enzymes of the microsomal electron transport chain. Therefore, the desaturase response to environmental temperature changes could be plausibly linked to female hormones.     

Δ9 desaturase activity in normal mouse liver and hepatoma SS1K

The activity of Δ9 desaturase was determined in the microsomal fraction of normal mouse liver and hepatoma SS1K in the presence of the 105,000 × g supernatant. Neither hepatic nor hepatoma soluble fractions were able to modify the low desaturating capacity. Two enzymes from the microsomal electron transport chain associated with Δ9 desaturase, namely NADH-cytochrom b₅ reductase and NADH-cytochrome C reductase were also measured. The results indicate that the low Δ9 desaturase activity in hepatoma SS1K could be related to the reduced amount of desaturase.     

Δ6 and Δ5 desaturase activities in liver from obese zucker rats at different ages

Δ6 Desaturation of linoleic acid (18∶2 n−6) and Δ5 desaturation of dihomo-γ-linolenic acid (20∶3 n−6) were measured in liver microsomes from genetically obese Zucker rats (fa/fa) and from their lean littermates (Fa/−). Both groups were fed a balanced commercial diet. The rats were 6, 9 and 12 weeks old, which corresponded to stages in their active growth period. The content of total fatty acids and n−6 polyunsaturated fatty acids in whole liver and liver microsomes was also determined in order to ascertain how the desaturase activities measuredin vitro reflected regulation of essential fatty acid metabolismin vivo. Contrary to values obtained for Δ6 desaturation, Δ5 desaturation at nonsaturating substrate levels were lower in obese rats than in lean controls. In contrast, at saturating substrate level, the maximal Δ5 desaturase activities were the same in both phenotypes and they increased with age. Study of Δ5 desaturation kinetics (1/V vs 1/S) showed that V_m did not differ between 12-week-old obese and lean rats, whereas K_M in obese rats was much lower than in controls, expressing the very low affinity of the enzyme for the substrate in obese animals. The fatty acid composition of liver lipids reflected the results of desaturase activitiesin vitro. In particular, the ratios 20∶4 n−6/20∶3 n−6 were lower in obese rats than in lean rats, which can be explained by the lower conversion of 20∶3 n−6 into 20∶4 n−6 by Δ5 desaturation. However, the total amount of 20∶4 n−6 in the whole liver did not differ between phenotypes, whatever their age. This work presents evidence for a relationship between the changes in fatty acid compositional data in hepatic total lipids, total lipids of liver microsomes and modifications of fatty acid desaturase activities in the genetically obese Zucker rat.     

Involvement of cytochrome b5 in the oxidative desaturation of linoleic acid to γ-linolenic acid in rat liver microsomes

The effects of antibodies against microsomal electron-transport components on the in vitro activity of Δ⁶-desaturation of linoleic acid to γ-linolenic acid have been studied in intact microsomal membranes of rat liver. Reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) (0.87 mM) served as electron donors, and effectively prompted the Δ⁶-desaturase activities with yields of about 1.1 to 1.3 nmol per mg of protein in 10 min. Of the two antibodies studied under the same in vitro conditions, i.e., rabbit antisera preparations against rat liver microsomal hydrophilic parts of cytochrome b₅ and NADPH-cytochrome c reductase, only the antibody against cytochrome b₅ demonstrated a marked ability to inhibit the Δ⁶-desaturase activity. This evidence supports a participation of cytochrome b₅ in the Δ⁶-desaturation of linoleic acid and suggests a pathway analogous to the Δ⁹-desaturation of stearyl-CoA.     

Effect of dietary fatty acids on Δ5 desaturase activity and biosynthesis of arachidonic acid in rat liver microsomes

The effect of different fatty acids supplemented to a fat-free diet on the activity of Δ5 desaturase was studied. Fat-free diet produces a reduction in the conversion of eicosa-8,11,14-trienoic acid to arachidonic acid. The addition of thecis-ω6 acids, linoleic, γ-linolenic or arachidonic to the diet produces an increase of eicosatrienoic acid desaturation, shifting Δ5 desaturase activity towards the controls on a balanced diet. This reactivation is apparently produced by induction of enzyme biosynthesis since linoleate effect was suppressed by simultaneous cycloheximide injection. On the contrary, no changes in Δ5 desaturation activity were found when the diet was supplemented with palmitic or 9-trans,12-trans-linoleic acid. The changes on the activity of Δ5 desaturase were compared with the fatty acid composition of plasma and liver microsomes.     

Rosiglitazone,a PPAR-γ Agonist,Fails to Attenuate CLA-Induced Milk Fat Depression and Hepatic Lipid Accumulation in Lactating Mice

Our objective was to investigate the combination of rosiglitazone (ROSI) and conjugated linoleic acid (CLA) on mammary and hepatic lipogenesis in lactating C57Bl/6 J mice. Twenty-four lactating mice were randomly assigned to one of four treatments applied from postpartum day 6 to day 10. Treatments included: (1) control diet, (2) control plus 1.5 % dietary CLA (CLA) substituted for soybean oil, (3) control plus daily intra-peritoneal (IP) rosiglitazone injections (10 mg/kg body weight) (ROSI), and (4) CLA plus ROSI (CLA-ROSI). Dam food intake and milk fat concentration were depressed with CLA. However, no effects were observed with ROSI. The CLA-induced milk fat depression was due to reduced expression for mammary lipogenic genes involved in de-novo fatty acid (FA) synthesis, FA uptake and desaturation, and triacyglycerol synthesis. Liver weight (g/100 g body weight) was increased by CLA due to an increase in lipid accumulation triggering a compensatory reduction in mRNA abundance of hepatic lipogenic enzymes, including acetyl-CoA carboxylase I and stearoyl-CoA desaturase I. On the contrary, no effects were observed with ROSI on hepatic and mammary lipogenic gene and enzyme expression. Overall, feeding CLA to lactating mice induced milk fat depression and increased hepatic lipid accumulation, probably due to the presence of trans-10, cis-12 CLA isomer, while ROSI failed to significantly attenuate both hepatic steatosis and reduction in milk fat content.     

The Δ5 and Δ6 desaturation of fatty acids of varying chain length by rat liver: A preliminary report

The Δ6 desaturase of rat liver can accommodate substrates with a wide range of chain lengths. Δ9-cis,12-cis-Dienoic acids of chain lengths 14–22 carbon atoms were all desaturated at the Δ6 position by microsomal preparations from rat liver. By contrast, the Δ5 desaturase appeared much more chain-length sensitive. The percentage Δ5 desaturation of a series of Δ8-cis- and Δ9-trans-monoenoic acids increased with increasing chain length (from C₁₆ to C₂₀).     

Effect of different acids with Δ9,12-dienoic structures on Δ9 desaturation activity in rat liver microsomes

The effect of oral administration, for 24 or 48 hr, of different octadeca fatty acids containing a 9,12-dienoic structure on the fatty acid composition and Δ9 desaturation activity of liver microsomes of rat fed a fat-free diet was studied. The ethyl esters of linoelaidic and γ-linolenic acids, the methyl ester of linoleic acid and free columbinic acid were administered to rats maintained on a fat-free diet. The supplementation of the fat-free diet with linoelaidate produced no relevant changes in the fatty acid composition pattern of liver microsomes and did not modify the percentage of conversion of palmitic to palmitoleic acid. The addition of linoleate or γ-linolenate to the fat-free diet returned liver microsome Δ9 desaturation activity toward the control and partially restored the liver microsome fatty acid spectrum found in the fat-free diet. Columbinic acid (5-trans-9-cis,12-cis-18∶3), which cannot be transformed into arachidonic acid, also decreased the Δ9 desaturation activity enhanced by the fat-free diet and evoked changes in the microsomal fatty acid composition similar to those produced by the ω6 fatty acids. These results suggest that the modulation of Δ9 desaturase activity evoked by dietary administration of unsaturated acids of ω6 series would depend on thecis double bond configuration of these acids.     

Highly unsaturated fatty acid synthesis in vertebrates: New insights with the cloning and characterization of a Δ6 desaturase of Atlantic salmon

Fish are an important source of the n−3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher vertebrates. The synthesis of HUFA involves enzyme-mediated desaturation, and a Δ5 fatty acyl desaturase cDNA has been cloned from Atlantic salmon (Salmo salar) and functionally characterization of a Δ6 fatty acyl desaturase of Atlantic salmon and describe its genomic structure, tissue expression, and nutritional regulation. A salmon genomic library was screened with a salmon Δ5 desaturase cDNA and positive recombinant phage isolated and subcloned. The full-length cDNA for the putative fatty acyl desaturase was shown to comprise 2106 bp containing an open reading frame of 1365 bp specifying a protein of 454 amino acids (GenBank accession no. AY458652). The protein sequence included three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b₅ domain containing the heme-binding motif HPGG, all of which are characteristic of microsomal fatty acid desaturases. Functional expression showed that this gene possessed predominantly Δ6 desaturase activity. Screening and sequence analysis of the genomic DNA of a single fish revealed that the Δ6 desaturase gene constituted 13 exons in 7965 bp of genomic DNA. Quantitative real-time PCR assay of gene expression in Atlantic salmon showed that both Δ6 and Δ5 fatty acyl desaturase genes, and a fatty acyl elongase gene, were highly expressed in intestine, liver, and brain, and less so in kidney, heart, gill, adipose tissue, muscle, and spleen. Furthermore, expression of both Δ6 and Δ5 fatty acyl desaturase genes in intestine, liver, red muscle, and adipose tissue was higher in salmon fed a diet containing vegetable oil than in fish fed a diet containing fish oil.     

Desaturases fused to their electron donor

Fatty acid desaturations in the carboxy‐terminal segment from C1—C10 are catalyzed in many, but not in all cases, by desaturase enzymes which are fused to their electron donor cytochrome b₅. Several of these enzymes (“front‐end desaturases”) from a wide variety of organisms have been cloned and functionally expressed for proof of regio‐, stereo‐ and chain length‐selectivity. In most cases the actual status of the substrate chain, whether coenzyme A thioester or component of a membrane lipid, is not known. The cytochrome b₅ domain is located N‐terminally, internally or C‐terminally. Compared to the free cytochrome b₅ , the fused domains show a significant reduction of acidic amino acid residues on the surface of the four helices enclosing the heme group. It is discussed how this may contribute to hydrophobic domain pairing required for interdomain electron transport. This is in contrast to the mode of interaction of free cytochrome b₅ with its partners, which is governed by electrostatic charge pairing. A look at crystallized or computer‐simulated models involving fused or free cytochrome b₅ helps to outline the problems encountered by optimizing the docking of partners and the exchange of electrons between domains of different degrees of mobility.