The Effect of Dietary Sterol on the Activity of Fatty Acid Desaturases Isolated from Tetrahymena setosa

Tetrahymena setosa has a nutritional requirement for micro amounts of sterol, a requirement which is also satisfied by relatively large amounts of either intact phospholipids or a mixture of unsaturated fatty acids normally found in these ciliates. Three microsomal fatty acyl-CoA desaturases have been isolated from T. setosa and partially characterized. These enzymes which can account for the formation of the majority of the ciliate's unsaturated fatty acids, include: a Δ9, a Δ12 and a Δ6 desaturase which catalyze the transformation of stearoyl-CoA to oleic acid, of oleoyl-CoA to linoleic acid and of linoleoyl-CoA to ?-linolenic acid, respectively. The stearoyl CoA desaturase required NAD (or NADP), ATP and free CoA; the Δ6 and Δ12 desaturases required NADP, but not ATP or CoA. Cellular levels of the three desaturases were highest in mid-logarithmic phase cells and lowest in stationary phase cells. In order to determine if there was a relationship between the sterol requirement and the ability of the organism to desaturate, T. setosa was grown in a synthetic medium supplemented with either cholesterol or a phospholipid which permits growth in the absence of cholesterol, or with both phospholipid and cholesterol. Cells grown with phospholipid alone had only half as much stearoyl-CoA and oleoyl-CoA desaturase activity as cells of identical culture age grown either on cholesterol alone or on cholesterol plus phospholipid.     

Fatty acid desaturase activities are modulated by phytosterol incorporation in microsomes

The effect of phytosterol-rich diets (3% beta-sitosterol + 2% campesterol) on rat liver microsomal fatty acid desaturases, membrane dynamics and lipid composition was investigated. After a 21 day period, phytosterol was incorporated into microsomes and the membrane fluidity decreased. There were no changes in either the phospholipid composition or in the total sterol content. However, the phytosterol/cholesterol ratio increased. In the animals fed phytosterols, the delta 5-, delta 6- and delta 9-fatty acid desaturases were significantly more active than in control animals. The changes in the lipid fatty acid composition were consistent with those of the desaturase activities. Hence, it is suggested that: (1) dietary phytosterol modulates desaturase activities; (2) phytosterols make the membrane more rigid but do not induce changes in the relative phospholipid composition; (3) delta 9-, delta 5- and delta 6-desaturase activities increase when the membrane becomes more rigid without changes in the phospholipid composition.     

Requirements of delta 9 and delta 12 fatty acid desaturation in Neurospora

Microsomes prepared from the wild-type strain and lipid auxotrophs of Neurospora were analyzed for delta 9 - (stearoyl-CoA) and delta 12 - (oleoyl-CoA) desaturase activities. The wild-type delta 9-desaturase was found to have a 20-fold higher specific activity and 2-fold lower activation energy than the delta 12-desaturase. In addition, delta 12-desaturase had higher Km app values for oleoyl-CoA and for NADH than the equivalent values for delta 9-desaturase. These properties were correlated with a rate-limiting role of delta 12-desaturase in the production of 18:2, the major fatty acid of Neurospora. The delta 12-desaturase also exhibited a higher tolerance to pH changes and to cyanide than did the delta 9-desaturase. Both activities could be measured in the same reaction mixture using stearoyl-CoA as the substrate, indicating a coupling of the two enzymes. Enrichment of cellular membranes of the wild-type Neurospora with 18:0 and 18:1, 18:2, 18:3 fatty acids led to the conclusion that the presence of excess substrate in the membrane induces activation of the appropriate desaturase. These experiments also suggested that the membrane fluidity, as determined by the degree of unsaturation of membrane fatty acids, may influence the activities of the desaturating enzymes. Perturbation of the polar head groups of the membrane phospholipids indicated that the correct composition of anionic phospholipids is an absolute requirement for the function of both desaturases. These studies show that the activities of the delta 9-desaturase and the delta 12-desaturase are regulated by a variety of factors and that the delta 12-desaturase is subjected to less stringent controls than the delta 9-desaturase.     

A possible cyclic AMP-mediated regulation of microsomal fatty acyl-CoA desaturation system in Tetrahymena microsomes

Profound alterations in the microsomal fatty acyl-CoA desaturase activities and cyclic AMP production of a unicellular eukaryote, Tetrahymena pyriformis NT-1, originally grown in the glucose-deficient medium, were observed, following the administration of glucose or beta-adrenergic agonists such as epinephrine and isoproterenol. There was a great increase of stearoyl-CoA (delta 8) desaturase activity coincident with a 2-fold decrease of oleoyl-CoA (delta 12) desaturase activity over the first 2 h after administration of these compounds. During this period of time, it was found that the production in vivo of labeled oleic acid from [14C]acetic or [3H]palmitic acid increases 2-fold and the formation in vivo of each labeled linoleic and gamma-linolenic acids drastically decreases. Glucose or beta-adrenergic agonists caused an increase of stearoyl-CoA-stimulated reoxidation rate of NADH-reduced cytochrome b5 but depressed oleoyl-CoA-stimulated reoxidation rate of b5, indicating that both desaturase activities are controlled by the respective terminal components of the desaturase system. A significant and reproducible increase of adenylate cyclase activity and a slight decrease of cyclic AMP phosphodiesterase activity were observed to occur within the first 2 h after the addition of these compounds, when cyclic AMP content in Tetrahymena cell rose by 3-4-fold. Propranolol, a beta-adrenergic blocker, abolished the effects of glucose or beta-adrenergic agonists on the activities of fatty acyl-CoA desaturases and the terminal components as well as cyclic AMP production of cells. These results suggest that glucose and beta-adrenergic agonists may modulate the microsomal fatty acyl-CoA desaturase system in Tetrahymena by acting through the increase of intracellular cyclic AMP content.     

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.     

Isolation of a stearoyl CoA Desaturase form Tetrahymena thermophila

Cell free preparations of Tetrahymena thermophila contain an enzyme that catalyzes the direct desaturation of stearoyl CoA to octadecenoic acid. The enzyme is associated with the microsomal fraction of the ciliate. Substrate of the enzyme consists of either free stearic acid or stearoyl CoA. Both ATP and CoA are required when free stearate is the substrate and are also highly stimulatory when stearoyl CoA is the substrate. With stearoyl CoA as the substrate, either NADH or NADPH are required for desaturase activity. In presence of ATP and CoA, either NAD or NADP can replace NADH and NADPH. Desaturase activity is optimal when the enzyme is incubated at pH of 7.2 and a temperature of 30-35 degrees C. Highest levels of the stearoyl CoA desaturase are found in stationary phase ciliates grown at 35 degrees C.     

Variations in the activity of fatty ACYL-CoA desaturases and electron-transport components in Tetrahymena microsomes with temperature and age of culture

1. 1.|Alterations in the fatty acid composition of microsomes were most marked in the exponential phase of both 39.5- or 15°C- grown Tetrahymena pyriformis NT-1.

2. 2.|Activities of palmitoyl-CoA and stearoyl-CoA desaturases were lower in 15°C cells than in 39.5°C cells, while the activity of oleoyl-CoA desaturase was higher in 15°C cells.

3. 3.|Activities of the terminal component of the desaturation system as well as all three desaturases (palmitoyl-CoA, stearoyl-CoA, oleoyl-CoA) were higher in the exponential phase than in the stationary phase for cells grown at both temperatures.

4. 4.|NAD(P)H-cytochrome c reductase activity and cytochrome b₅ content were reduced whereas NADH-ferricyanide reductase activity was increased in the stationary phase at both 39.5 and 15°C.

Isolation of a Stearoyl CoA Desaturase from Tetrahymena thermophila

Cell free preparations of Tetrahymena thermophila contain an enzyme that catalyzes the direct desaturation of stearoyl CoA to octadecenoic acid. The enzyme is associated with the microsomal fraction of the ciliate. Substrate for the enzyme consists of either free stearic acid or stearoyl CoA. Both ATP and CoA are required when free stearate is the substrate and are also highly stimulatory when stearoyl CoA is the substrate. With stearoyl CoA as the substrate, either NADH or NADPH are required for desaturase activity. In the presence of ATP and CoA, either NAD or NADP can replace NADH and NADPH. Desaturase activity is optimal when the enzyme is incubated at a pH of 7.2 and a temperature of 30–35°C. Highest levels of the stearoyl CoA desaturase are found in stationary phase ciliates grown at 35°C.     

The role of Δ6-desaturase acyl-carrier specificity in the efficient synthesis of long-chain polyunsaturated fatty acids in transgenic plants

The role of acyl‐CoA‐dependent Δ6‐desaturation in the heterologous synthesis of omega‐3 long‐chain polyunsaturated fatty acids was systematically evaluated in transgenic yeast and Arabidopsis thaliana. The acyl‐CoA Δ6‐desaturase from the picoalga Ostreococcus tauri and orthologous activities from mouse (Mus musculus) and salmon (Salmo salar) were shown to generate substantial levels of Δ6‐desaturated acyl‐CoAs, in contrast to the phospholipid‐dependent Δ6‐desaturases from higher plants that failed to modify this metabolic pool. Transgenic plants expressing the acyl‐CoA Δ6‐desaturases from either O. tauri or salmon, in conjunction with the two additional activities required for the synthesis of C20 polyunsaturated fatty acids, contained higher levels of eicosapentaenoic acid compared with plants expressing the borage phospholipid‐dependent Δ6‐desaturase. The use of acyl‐CoA‐dependent Δ6‐desaturases almost completely abolished the accumulation of unwanted biosynthetic intermediates such as γ‐linolenic acid in total seed lipids. Expression of acyl‐CoA Δ6‐desaturases resulted in increased distribution of long‐chain polyunsaturated fatty acids in the polar lipids of transgenic plants, reflecting the larger substrate pool available for acylation by enzymes of the Kennedy pathway. Expression of the O. tauriΔ6‐desaturase in transgenic Camelina sativa plants also resulted in the accumulation of high levels of Δ6‐desaturated fatty acids. This study provides evidence for the efficacy of using acyl‐CoA‐dependent Δ6‐desaturases in the efficient metabolic engineering of transgenic plants with high value traits such as the synthesis of omega‐3 LC‐PUFAs.     

Thermoadaptive regulation of microsomal desaturase and electron-transport enzyme activities in lipid-manipulated Tetrahymena cells. Extent of unsaturated fatty acid production is dependent on membrane fluidity before temperature down-shift

Exposure of Tetrahymena pyriformis NT-1 to chimyl alcohol (1-O-hexadecyl glycerol) produced a reproducible enhancement in unsaturated fatty acids and a great decrease in order parameter (S), which result from the 2-fold increases of stearoyl-CoA and oleoyl-CoA desaturase activities in microsomes. When the chimyl alcohol-fed cells were shifted from 34 to 15 degrees C (down-shift), unlike the drastic increases in palmitoyl-CoA, stearoyl-CoA and oleoyl-CoA desaturase activities in the native cells, there was only a slight increase in palmitoyl-CoA desaturase activity with a parallel rise in the activity of the terminal component (cyanide-sensitive factor; CSF) of the desaturase system. During cold acclimation, the decrease of order parameter in chimyl alcohol-fed cells was smaller than that in native cells, since the order parameter had already been decreased by the addition of chimyl alcohol before the shift. These results suggest that chimyl alcohol-fed cells are easily able to accomplish temperature acclimation without requiring great modification of fatty acid composition and membrane fluidity, while the non-fed control cells have difficulty doing so.     

Characterization and solubilization of an acyl chain elongation system in microsomes of leek epidermal cells

Microsomes prepared from leek epidermal tissue readily elongate stearoyl-CoA to very long chain fatty acid with malonyl-CoA as the C2 unit. In the absence of stearoyl-CoA, but in the presence of ATP, microsomes elongate endogenous free fatty acids. Endogenous CoA is the source of CoA. Palmitoyl, stearoyl, and higher saturated acyl-CoAs are readily elongated by the microsomal system but oleoyl-CoA is ineffective; however, the higher monounsaturated acyl-CoAs can be elongated. Since the very long chain fatty acids of the leek epidermis are all saturated, it would appear that the reaction controlling the nature of the final acyl product is the inactivity of oleoyl-CoA as a substrate. There is no evidence that acyl carrier protein participates in the elongation reactions. Evidence is also presented suggesting that (a) there may be two elongation systems, one responsible for the conversion of stearoyl-CoA to arachidonyl-CoA and the second involved in the conversion of arachidonyl-CoA to very long chain fatty acids, and that (b) the elongation activities may be associated with a large polypeptide.     

Characterization of sterol-ester synthetase in Saccharomyces cerevisiae.

Cell-free extracts of Saccharomyces cerevisiae grown under aerobic as well as semi-anaerobic conditions were found to catalyze the synthesis of fatty acid ester of sterol from cholesterol, fatty acid, ATP and CoA, or from cholesterol and fatty acyl-CoA. This result indicates that the enzyme involved in the formation of the ester is acyl-CoA:sterol O-acyltransferase (EC 2.3.1.26). The enzyme had a broad substrate specificity for sterols and acyl-CoAs. The enzyme levels in the cells grown under aerobic and semi-anaerobic conditions were almost equal. The enzyme was located in the microsomal fraction of the aerobically grown cells.     

Stearoyl-CoA desaturase is involved in the control of proliferation, anchorage-independent growth, and survival in human transformed cells

Saturated and monounsaturated fatty acids are the most abundant fatty acid species in mammalian organisms, and their distribution is regulated by stearoyl-CoA desaturase, the enzyme that converts saturated into monounsaturated fatty acids. A positive correlation between high monounsaturated fatty acid levels and neoplastic transformation has been reported, but little is still known about the regulation of stearoyl-CoA desaturase in cell proliferation and apoptosis, as well as in cancer development. Here we report that simian virus 40-transformed human lung fibroblasts bearing a knockdown of human stearoyl-CoA desaturase by stable antisense cDNA transfection (hSCDas cells) showed a considerable reduction in monounsaturated fatty acids, cholesterol, and phospholipid synthesis, compared with empty vector transfected-simian virus 40 cell line (control cells). hSCDas cells also exhibited high cellular levels of saturated free fatty acids and triacylglycerol. Interestingly, stearoyl-CoA desaturase-depleted cells exhibited a dramatic decrease in proliferation rate and abolition of anchorage-independent growth. Prolonged exposure to exogenous oleic acid did not reverse either the slower proliferation or loss of anchorage-independent growth of hSCDas cells, suggesting that endogenous synthesis of monounsaturated fatty acids is essential for rapid cell replication and invasiveness, two hallmarks of neoplastic transformation. Moreover, apoptosis was increased in hSCDas cells in a ceramide-independent manner. Finally, stearoyl-CoA desaturase-deficient cells were more sensitive to palmitic acid-induced apoptosis compared with control cells. Our data suggest that, by globally regulating lipid metabolism, stearoyl-CoA desaturase activity modulates cell proliferation and survival and emphasize the important role of endogenously synthesized monounsaturated fatty acids in sustaining the neoplastic phenotype of transformed cells.     

The effect of soluble rat liver proteins on the activity of microsomal stearoyl-CoA and linoleoyl-CoA desaturase.

1. The influence of bovine serum albumin and soluble rat liver proteins on the activity of rat liver microsomal delta9 and delta6 desaturases has been studied. 2. In the absence of bovine serum albumin, the delta9 desaturase which converts stearoyl-CoA into oleoyl-CoA, shows a non-linear correlation between enzyme activity and protein concentration. 3. Optimum concentrations of bovine serum albumin have three main effects on the enzyme activity: (i) establishes a linear relationship between enzyme activity and protein concentration, (ii) stimulates the enzyme activity 2--3-fold and (iii) raises the optimum substrate concentration from 10 to 100 muM. 4. A highly purified soluble liver protein of molecular weight 24 000 also stimulated the enzyme activity and brought about a linear relationship between enzyme activity and protein concentration. 5. It was concluded that the non-linear kinetics were due to limiting amounts of substrate binding protein in the microsomal preparations. 6. The delta6 desaturase which converts linoleoyl-CoA into gamma-linolenoyl-CoA was also stimulated by bovine serum albumin and soluble liver proteins. 7. The significance of the fatty acid-binding proteins is discussed.     

Genetic regulation of unsaturated fatty acid composition in C. elegans

Delta-9 desaturases, also known as stearoyl-CoA desaturases, are lipogenic enzymes responsible for the generation of vital components of membranes and energy storage molecules. We have identified a novel nuclear hormone receptor, NHR-80, that regulates delta-9 desaturase gene expression in Caenorhabditis elegans. Here we describe fatty acid compositions, lifespans, and gene expression studies of strains carrying mutations in nhr-80 and in the three genes encoding delta-9 desaturases, fat-5, fat-6, and fat-7. The delta-9 desaturase single mutants display only subtle changes in fatty acid composition and no other visible phenotypes, yet the fat-5;fat-6;fat-7 triple mutant is lethal, revealing that endogenous production of monounsaturated fatty acids is essential for survival. In the absence of FAT-6 or FAT-7, the expression of the remaining desaturases increases, and this ability to compensate depends on NHR-80. We conclude that, like mammals, C. elegans requires adequate synthesis of unsaturated fatty acids and maintains complex regulation of the delta-9 desaturases to achieve optimal fatty acid composition.     

Modulation by dexamethasone of the fatty acyl-CoA desaturase system in Tetrahymena microsomes

Dexamethasone produced an increased activity of stearoyl-CoA desaturase through the enhancement of delta 9-terminal component activity, and a corresponding decrease of oleoyl-CoA desaturase activity via the reduced activity of delta 12-terminal component in Tetrahymena microsomes. However, the content of cytochrome b5 as well as the activities of NAD(P)H-cytochrome c and NADH-ferricyanide reductases showed no significant changes by dexamethasone. Additionally, dexamethasone evoked a 3.5-fold increase of intracellular cyclic AMP content 2 hr after administration. These results suggest that dexamethasone may modulate microsomal fatty acyl-CoA desaturase system in Tetrahymena by increasing intracellular cyclic AMP content.     

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.     

Mechanism of fatty acid desaturation: a bioorganic perspective

The desaturation of long chain fatty acids is a ubiquitous transformation which plays a critical role in the biosynthesis of lipids. Of particular interest to the bioorganic chemist is the unique ability of desaturases to oxidize unactivated hydrocarbon chains in a chemo-, regio- and stereoselective manner. The mechanism of membrane-bound desaturases has been examined using regiospecifically labelled analogues bearing deuterium, sulfur or fluorine-substituted methylene isosteres. These probes have been applied in the study of several biomedically important desaturase systems including a prototypical yeast stearoyl CoA delta(9) desaturase. In all cases, it has been found that the dehydrogenation (desaturation) process is initiated by a kinetically important hydrogen activation step at the carbon of the incipient double bond which is closest to the acyl terminus of the fatty acid chain. These results point to a common active site architecture which is highly conserved among a wide range of membranous desaturases.