Changes in lipid composition of liver microsomes and fatty acyl-CoA desaturase activities induced by medium chain triglyceride feeding

Changes in fatty acid composition, microsomal Δ⁹- and Δ⁶-desaturase activities and liver contents of cholesterol and phospholipids were studied in rats fed medium chain triglyceride-supplemented diets. Weanling rats were divided into four groups and fed for three weeks a basal diet with different 10%-fat supplements: corn oil, medium chain triglyceride-corn oil, olive oil and medium chain triglyceride-olive oil. The highest relative content of saturated fatty acids corresponded to corn oil-fed animals. Both monounsaturated fatty acid content and Δ⁹-desaturase activity were higher in the animals fed olive oil diets than in corn oil-fed rats. The long chain polyunsaturated fatty acids of the n−3 series were increased in the olive oil and medium chain triglyceride-olive oil-fed groups probably due to the lower linoleic/α-linolenic ratios found in these two diets. The cholesterol/phospholipid molar ratio was unaffected by diet and the unsaturation index was only slightly changed in the four groups. Thus, some mechanism may be operative under these conditions to maintain the homeostasis of the membrane.     

Effect of magnesium deficiency on Δ6 desaturase activity and fatty acid composition of rat liver microsomes

Experimental Mg²⁺ deficiency was induced in a group of rats by feeding them a Mg²⁺-deficient diet for 23 days. They were pair-fed to compare with a control group of rats fed a Mg²⁺-sufficient diet. In the Mg²⁺-deficient group the plasma total cholesterol and triglyceride levels were increased while HDL-cholesterol was decreased. In the Mg²⁺-deficient group the plasma level of thiobarbituric acid reacting substances (TBARS) used as a measure for lipid peroxidation was increased. The increase was attributed to the increased cytosolic Ca²⁺ in Mg²⁺-deficiency which can cause: 1) increase of hydro and endoperoxide levels as a consequence of the increase of arachidonic acid release and eicosanoid synthesis in Mg²⁺-deficiency, and 2) inhibition of the mitochondrial respiratory activity and activation of Ca²⁺-dependent proteases which may activate the conversion of xanthine dehydrogenase to xanthine oxidase which generates active O₂ species. In the Mg²⁺-deficient group, the fatty acid composition of the liver microsomes indicated a slower rate of conversion of linoleic acid to arachidonic acid which was consistent with the decrease of Δ6 desaturase activity in liver microsomes of Mg²⁺-deficient rats as measuredin vitro. The decrease of Δ6 desaturase activity was attributed to the lower concentration of actual enzyme molecules as a result of the decreased rate of protein synthesis in Mg²⁺-deficiency. The possible effects of the increased catecholamine release in Mg²⁺-deficiency are discussed.     

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.     

The effects of dietary protein on the fatty acid composition and Δ6 desaturase activity of rat hepatic microsomes

Recently, significant differences between rats fed a casein diet and rats fed a soybean protein diet have been observed in hepatic phospholipid fatty acid patterns (Sjöblom, L., and Eklund, A.,Biochim. Biophys. Acta 1004, 187–192, 1990). The influence of these two diets on the Δ6 desaturase activity was investigated in the present study because the hepatic desaturase system is a source of unsaturated fatty acids. The rats fed a casein diet showed higher desaturase activity than those fed soybean protein when using either linoleic acid (P<0.005) or oleic acid (P<0.05) as substrates. The phosphatidylcholine fraction of hepatic microsomes showed increases in oleic acid (P<0.005) and 20∶3ω9 (P<0.001) levels as well as decreases in stearic acid (P<0.001), linoleic acid (P<0.005) and arachidonic acid (P<0.005) levels in rats which were fed casein rather than soybean protein. Similar differences between the two groups were also observed in the phosphatidylethanolamine and phosphatidylinositol fractions. These data indicate that the qualitative properties of the dietary protein source may influence the fatty acid pattern of rat hepatic microsomes by interfering with Δ6 desaturase activity.     

Age-related changes in Δ6 and Δ5 desaturase activities in rat liver microsomes

Age-related changes in Δ6 desaturation of [1-¹⁴C]α-linolenic acid and [1-¹⁴C]linoleic acid and in Δ5 desaturation of [2-¹⁴C]dihomo-γ-linolenic acid were studied in liver microsomes from Wistar male rats at various ages ranging from 1.5 to 24 mon. Desaturase activities were expressed both as specific activity of liver microsomes and as the capacity of whole liver to desaturate by taking into account the total amount of liver microsomal protein. Δ6 Desaturation of α-linolenic acid increased from 1.5 to 3 mon and then decreased linearly up to 24 mon to reach the same desaturation capacity of liver measured at 1.5 mon. The capacity of liver to desaturate linoleic acid increased up to 6 mon and then remained constant, whereas microsomal specific activity was equal at 1.5 and 24 mon of age. The capacity of liver to convert dihomo-γ-linolenic acid to arachidonic acid by Δ5 desaturation decreased markedly from 1.5 to 3 mon. It then increased to reach, at 24 mon, the same level as that observed at 1.5 mon. Age-related changes in the fatty acid composition of liver microsomal phospholipids at the seven time points studied and of erythrocyte lipids at 1.5 and 24 mon were consistent with the variations in desaturation capacity of liver. In particular, arachidonic acid content in old rats was slightly higher than in young rats whereas contents in linoleic and docosahexaenoic acids varied little throughout the life span. The results suggest that, in liver, the activity of desaturases may be regulated in the course of aging to maintain a constant level of polyunsaturated fatty acids in cellular membranes.     

Effect of dietary fats on desaturase activities and the biosynthesis of fatty acids in rat-liver microsomes

Four groups of rats were fed diets containing 15% (w/w) high-oleic safflower oil (SFO, rich incis-18∶1 acids), a mixture of 80% partially hydrogenated soybean oil plus 20% corn oil (H+CO, rich intrans-18∶1 acids), lard (L, rich in saturated fatty acids) and corn oil (Co, rich in 18∶2ω6). Fatty acid composition of liver microsomes and activities of the Δ⁵, Δ⁶ and Δ⁹ desaturases were determined. Microsomal Δ⁶ desaturase activity and arachidonic acid were lower in the H+CO group compared with SFO of L. No difference was found in the Δ⁵ or Δ⁶ desaturase activity of CO and SFO groups. Thus, the oleic-acid level of the SFO diet had no effect on the metabolism of 18∶2ω6. Fluorescent polarization studies, usingtrans-parinaric acid as a probe, showed no differences between the physical states of phospholipid vesicles made from lipids isolated from each group. We concluded that thetrans-18∶1 acids in partially hydrogenated soybean oil have a more inhibitory effect than saturated acids on EFA metabolism, even in the presence of adequate amounts of essential fatty acid.     

Effect of L-triiodothyronine on Δ9 desaturase activity in liver microsomes of male rats

Male rats injected with a single saturating dose of L-triiodothyronine (T₃) showed, after a lag time of approximately eight hr, a sharp rise in Δ9 desaturase activity. Desaturase activity reached a plateau which was 1–1.2 times above the base line levels of rats which were not hormone-treated. The plateau was maintained for five days in animals which were kept on daily hormone-treatment. The increase in Δ9 desaturase activity by T₃ required ongoing protein synthesis, because the increase in enzymatic activity due to hormone treatment was completely abolished in the presence of cycloheximide. These findings suggest that cycloheximide may block the induction of Δ9 desaturase by T₃ and/or inhibit the synthesis of protein(s) essential to the desaturation-response to T₃. Modifications observed in liver microsomal fatty acid composition in T₃ treated rats were independent of the effect on desaturation. It is suggested that other factors, such as diet, membrane lipid synthesis and degradation, as well as fatty acid turnover and oxidation, could be involved in affecting the fatty acid composition of thyroid hormone-treated rats.     

Adaptive changes in Δ9 desaturase activity in rat liver

The Δ9 desaturase activity and the¹⁴C radioactivity of the de novo synthesized fatty acids incorporated into microsomal lipids and serum triglycerides were measured under different nutritional conditions. The results obtained indicate a correlation between the values of the three parameters studied after starvation or after refeeding Purina chow or either a high carbohydrate or a high protein diet. These data suggest that liver lipogenesis and Δ9 desaturase activities respond to the same regulatory factors.     

Change of substrate specificity of rat liver microsomal fatty acyl-CoA synthetase activity by triton X-100

The effect of Triton X-100 on the activities and apparent molecular size of fatty acyl-CoA synthetase, solubilized and partially purified from rat liver microsomes, was studied. In the presence of Triton X-100, the activity for lignoceroyl-CoA synthesis was decreased, but activity was restored when the detergent was removed. The appearance and disappearance of lignoceroyl-CoA synthesis appeared related to the size, of the aggregated from of the enzyme. On the other hand, activity for palmitoyl-CoA synthesis was not significantly affected by the detergent. Because available evidence suggests that both fatty acids are converted to CoA esters by the same enzyme, it seems likely that the substrate specificity of the enzyme is influenced by changes in the aggregation state branes may determine the substrate specificity of acyl-CoA synthetase.     

Do rat kidney cortex microsomes possess the enzymatic machinery to desaturate and chain elongate fatty acyl-CoA derivatives?

Rat kidney cortex microsomal preparations were unable to catalyze Δ9, Δ6, and Δ5 desaturation of stearoylcoenzyme A (CoA), linoleoyl-CoA and dihomo-γ-linolenoyl-CoA, respectively. The kidney cortex microsomal fraction, however, did catalyze the malonyl-CoA dependent fatty acyl-CoA elongation. The biochemical properties of palmitoyl-CoA elongation were studied as a function of protein concentration, time, reduced nicotinamide adenine dinucleotide phosphate (NADPH), malonyl-CoA and substrate concentrations; of the substrates investigated, Δ6.9.12–18∶3 was the most active. Unlike what was observed in the hepatic system, a high-carbohydrate, fat-free diet did not induced kidney fatty acid chain elongation. All intermediate kidney cortex microsomal reactions,i.e., β-ketoacyl-CoA reductase, β-hydroxyacyl-CoA dehydrase andtrans-2-enoyl-CoA reductase activities, were significantly higher (greater than one order of magnitude) than the condensing enzyme activity, suggesting that the rate-limiting step in total elongation is the initial condensation reaction. Contrary to other reports, the results suggest that the kidney cannot synthesize arachidonic acid needed for eicosanoid production.     

Desaturation of saturated fatty acids by rat liver microsomes

A method was developed for the rapid determination of the initial velocity of the desaturation of saturated fatty acids. In the reaction, DPNH was a more efficient electron donor than TPNH. Fatdeficient rats have a 2.5-fold greater level of acyl desaturase per milligram of liver microsomal protein than did animals fed lab chow. Increasing the chain length of the acyl substrate from 10∶0 to 18∶0 increases the rate of monoene formation, but 19∶0 is desaturated at a rate lower than that for 15∶0. The energy of activation (Ea) for the overall desaturation reaction has been determined for 12∶0 through 19∶0. The Ea values for desaturation of 13∶0 and 16∶0 are markedly lowr than for the other acids. An interaction between the alkyl chain of the substrate and polyunsaturated acids of the microsomal membrane-bound phospholipids is postulated to explain the recurring 3-carbon pattern of the relative reaction rates of the various acyl substrates.     

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.     

Effects of estradiol and environmental temperature changes on rat liver Δ6 microsomal desaturase activity

The regulation of Δ6 desaturase activity by environmental temperature changes was studied in the microsomal membranes from female and ovariectomized female rat liver. Female rats adapted at 30–32 C for 20–25 days and then shifted to 13–15 C for 5 days showed an increased Δ6 desaturase system. Ovariectomized rats adapted under the same conditions did not show significant changes in this enzyme. The fatty acid compositions of microsomal phosphatidylcholine showed a decrease in arachidonic acid in female rats at 30 C compared to females at 15 C and ovariectomized rats at both temperatures. These results suggest that a modification of ovaric sex hormone levels might be responsible for the different Δ6 desaturase activity in female rats acclimated at both temperatures. In this regard, serum estradiol radioimmunoassay yielded slight differences between the two groups of female rats, suggesting that estradiol could play a role in the regulation of the Δ6 desaturase. The administration of a pharmacological dose of 17-β estradiol to female and ovariectomized rats kept at 30 and 15 C decreased the Δ6 microsomal desaturase activity. These data suggest that estradiol levels are involved in the regulation of the Δ6 desaturase during cold adaptation.     

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.     

Fatty acid desaturase systems of hen liver and their inhibition by cyclopropene fatty acids

Hen liver preparations which desaturate stearic acid at the 9,10 position to form oleic acid have been found to desaturate other saturated fatty acids of carbon chain length from 12 to 20 and 22. The 9,10-monoenoic fatty acid of the same chain length as the substrate fatty acid is the major product formed. Minor amounts of the 10,11- and 11, 12-monoenoic acids are also formed. Maximum desaturation occurred with the C₁₄ fatty acid substrate and with the fatty acids C₁₇ and C₁₈, suggesting the presence of at least two desaturating systems. The cyclopropene fatty acids, sterculic and malvalic acids, inhibited the desaturation of all thefatty acids at the 9,10 position but desaturation at the 10,11 and 11, 12 positions was affected only slightly. The effect is not due to inhibition of the primary activating enzyme, the long chain acyl CoA synthetase. Sterculic acid is a more effective inhibitor than either malvalic acid or sterculyl alcohol, probably because these cyclopropene compounds do not block the desaturating site of the enzyme as completely as sterculic acid.     

Interrelationship between the dietary regulation of fatty acid synthesis and the fatty acyl-CoA desaturases

In this paper we present further evidence for the close control of fatty acid synthetase and stearoyl-CoA desaturase. Furthermore, we have established that whereas dietary palmitic acid may influence the activity of this desaturase but not of fatty acid synthetase, dietary linoleic acid appears to control both these enzymes. Finally, we have studied the influence of dietary fat and carbohydrate on the activities of the delta6 and delta5 desaturases. The former is only slightly affected by these dietary components. The delta5 desaturase activity is stimulated as the dietary fat content rises but is unaffected by dietary carbohydrate. The control of these enzymes is therefore independent of the control of fatty acid synthetase and stearoyl-CoA desaturase. From the data presented, the magnitude of the controlling effect of polyunsaturated fatty acids on fatty acid synthetase and stearoyl-CoA desaturase activity is determined and its relevance to lipogenesis in man based on daily intake of carbohydrate and linoleic acid is discussed.     

Determination of individual long-chain fatty acyl-CoA esters in heart and skeletal muscle

A method has been developed for determination of individual long-chain fatty acyl-CoA esters from heart and skeletal muscle using high performance liquid chromatography (HPLC). The esters were extracted from freezeclamped tissue of pig and rat hearts and rat skeletal muscle for analysis on a radially compressed C₁₈ 5μ reversephase column. Nine peaks in the extract with carbon chain lengths from C₁₂ to C₂₀ that subsequently disappeared on alkaline hydrolysis were identified. The major acyl-CoA peaks were 14∶1, 18∶2, 16∶0 and 18∶1 and additionally in rat heart 18∶0. Total long-chain acyl-CoA esters obtained by summation of the individual molecular species was 11.34±1.48 nmol/g wet wt. pig heart; 14.51±2.11 nmol/g wet wt. in rat heart, and 4.35±0.71 nmol/g wet wt. in rat skeletal muscle. These values were approximately 132% of those obtained using a separate procedure that measured total CoA by HPLC after alkaline hydrolysis of the esters. The described method demonstrates the quantitation of individual acyl-CoA species in muscle tissue. Therefore, it has a number of advantages in that it permits information to be obtained on the individual molecular species under various nutritional and metabolic conditions.     

Chain length specificity of the saturated fatty acyl desaturase in rat liver preparations

Chain length specificity of the saturated fatty acyl desaturase was studied in rat liver preparations using both de novo synthesized long chain fatty acids and equimolar mixtures of preformed fatty acids. Both experiments show that myristic acid is least desaturated and palmitic and stearic acids are desaturated in about equal amounts.     

Suppression by carotenoids of microtenoids of microcystin-induced morphological changes in mouse hepatocytes

Microcystin-LR is a liver tumor promoter in the okadaic acid class, a group of potent inhibitors of protein phosphatases 1 and 2A. Because of inhibition of protein phosphatases, microcystin-LR induces hyperphosphorylation of cellular proteins, including cytoskeletal proteins—cytokeratins 8 and 18—and causes morphological changes in mouse hepatocytes in primary culture. We studied the effects of carotenoids to antagonize microcystin-LR-induced morphological changes in hepatocytes. β-Carotene (100 nM to 100 μM), suppressed the morphological changes induced by 100 nM microcystin-LR in a dose-dependent manner. Other carotenoids tested exerted similar suppressive effects, although retinoids, such as all-trans retinol, all-trans retinoic acid, and 9-cis retinoic acid, were only weakly suppressive. The relative potency of the suppression correlated significantly with the number of conjugated double bonds in thetrans configuration. β-Carotene strongly suppressed the hyperphosphorylation of cellular proteins induced by microcystin-LR without significant changes in the basal phosphorylation level. Other antioxidants, such as α-tocopherol, did not protect the cells against microcystin-LR. Taken together, the antagonistic effects of carotenoids against microcystin-LR are difficult to explain by their antioxidant or provitamin A activities. Suppression of the hyperphosphorylation of cellular proteins may be a novel mechanism by which carotenoids inhibit tumor promotion.