Increase of docosahexaenoic acid production by Schizochytrium sp. through mutagenesis and enzyme assay.

The present study focused on improving docosahexaenoic acid (DHA) production by Schizochytrium sp. through N-methyl-N-nitro-N-nitrisiguanidine treatment coupled with ultraviolet radiation based on the metabolic pathway analysis. The activity of glucose-6-phosphate dehydrogenase of the mutant was higher than the parent strain, which indicated that the hexose monophosphate pathway of the mutant was strengthened, and more NADPH was thus produced. Also, the activities of malic enzyme and ATP-citrate lyase in the cell extract of the mutant were higher than the parent strain, which indicated that the screening method increased NADPH and acetyl-CoA supply in vivo effectively. Finally, in the batch culturing of the mutant, 34.84% higher lipid was accumulated with the cell dry weight at the same level compared with the parent strain. Moreover, the DHA percentage of the total fatty acids up to 56.22% was achieved using the mutant, which was 38.88% higher than the parent strain. When the cultures were maintained under appropriate conditions, the final DHA yield was 0.20 and 0.11 g/g dry biomass, for the mutant and parent, respectively.

Improving estimates of trophic shift in Nile tilapia, Oreochromis niloticus (L.), using measurements of lipogenic enzyme activities in the liver.

To test whether the measurement of selected enzyme activities could be used to estimate more precisely the trophic shift of C isotopes, Nile tilapia (Oreochromis niloticus) were fed semi-synthetic diets differing in their lipid contents (1.7%, 5.0%, 10.8% and 20.0%). The diets were formulated to contain the same amount of nitrogen and metabolizable energy and were made from casein, wheat starch, corn germ oil supplemented with vitamins, minerals and L-arginine. The influence of the different diets on the activity of two lipogenic enzymes, ATP-citrate lyase and malic enzyme, on delta13C values in the whole fish, the liver and their correlation was investigated. There was a strong positive correlation between delta13C values in the lipids of whole fish and those of their livers. The activities of lipogenic enzymes increased significantly with increasing trophic shift of C isotopes (Deltadelta13Cdiet-fish values) in the lipids. If the relationship between trophic shift and enzyme activity can be confirmed in situations where feed quantity and quality are not known, the determination of enzyme activities would enable better estimates of the trophic shift to be made thus significantly improving back-calculation of diets from stable isotope data.

Nickel deficiency alters liver lipid metabolism in rats.

The present investigation was designed to examine the effect of nickel deficiency on lipid metabolism in liver and serum lipoproteins of rats. Therefore, a study over two generations was conducted feeding a nickel-deficient diet containing 13 microg/kg nickel or a nickel-adequate diet supplemented with 1 mg/kg nickel. Male 7-wk-old pups from the second offspring were studied. Pups fed a diet poor in nickel tended to have lower weight gains (P < 0.15), nickel concentrations in liver (P < or = 0.1) and iron levels in serum (P < 0.1) than nickel-adequate rats. They were classified as nickel-deficient on the basis of significantly lower erythrocyte counts, hemoglobin concentrations, hematocrits and nickel concentrations in kidney compared with nickel-adequate rats. Nickel deficiency caused a significant triacylglycerol accumulation in liver, with greater concentrations of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids than nickel-adequate rats. Nickel deficiency had slight but significant effects on the fatty acid composition of liver total lipids and phosphatidylcholine and phosphatidylethanolamine. Moreover, nickel-deficient rats had significantly lower activities of the lipogenic enzymes glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, malic enzyme and fatty acid synthase than nickel-adequate rats. Nickel-depleted pups had significantly higher concentrations of triacylglycerols and phospholipids in serum VLDL, and cholesterol in serum LDL than nickel-adequate pups. Most of these alterations in lipid metabolism are similar to those obtained in several iron-deficiency studies. Because nickel deficiency also slightly compromised iron status, it is possible that at least some of the observed alterations are due to the moderate iron deficiency.

Soybean protein suppresses hepatic lipogenic enzyme gene expression in Wistar fatty rats.

The effects of dietary soybean protein on lipogenic enzyme gene expression in livers of genetically fatty rats (Wistar fatty) have been investigated. When Wistar fatty rats and their lean littermates (7-8-wk old) were fed a casein or soybean protein isolate diet containing hydrogenated fat (4% hydrogenated fat plus 1% corn oil) or corn oil (5%) for 3 wk, the hepatic messenger RNA concentrations and activities of lipogenic enzymes were significantly lower in rats fed soybean protein than in those fed casein, regardless of genotype or dietary fat. The conversion rates of thyroxine to triiodothyronine by liver microsomes and plasma triiodothyronine concentrations were lower in the fatty rats than in the lean rats and were significantly greater in rats fed soybean protein than in those fed casein. Conversely, plasma and liver triacylglycerol concentrations were lower in soybean protein-fed fatty and lean rats than in those fed casein. The body weight was less in the fatty rats fed soybean protein than in those fed casein after 3 wk of feeding. Moreover, dietary polyunsaturated fatty acids suppressed lipogenic enzyme gene expression in the lean rats but did not in the fatty rats. Dietary soybean protein appeared to be useful for the reduction of obesity.

Vanadate induces normolipidemia and a reduction in the levels of hepatic lipogenic enzymes in obese Zucker rat.

The effects of vanadate administration on the plasma lipids and hepatic lipogenic enzymes were investigated in Zucker (fa/fa) rat, a model for obesity and non insulin-dependent diabetes. These animals were administered sodium orthovanadate through drinking water for a period of four months. The plasma levels of insulin, triacylglycerols and total cholesterol were significantly (p < 0.001) elevated in untreated obese control rats as compared to the lean animals. In the livers of obese rats, the number of insulin receptors decreased by 60% and the activities of lipogenic enzymes acetyl-CoA carboxylase and ATP-citrate lyase increased by 4.7- and 5.6-folds, respectively. The messenger RNA for ATP-citrate lyase as measured by Northern blot analysis showed a parallel increase in obese control rats. Treatment of these rats with vanadate caused 56-77% decreases in the plasma levels of insulin, triacylglycerols and total cholesterol. The insulin receptor numbers in vanadate-treated obese rats increased (119%) compared to levels in untreated obese animals. The elevated activities of acetyl-CoA carboxylase and ATP-citrate lyase observed in livers of obese rats were significantly reduced by vanadate. The messenger RNA for ATP-citrate lyase also decreased in vanadate-treated obese rats back to the lean control levels. This study demonstrates that vanadate exerts potent actions on lipid metabolism in diabetic animals in addition to the recognized effects on glucose homeostasis.

Polyunsaturated fatty acid-mediated suppression of insulin-dependent gene expression of lipogenic enzymes in rat liver.

The effects of dietary polyunsaturated fat on insulin-dependent gene expression of lipogenic enzymes and a possible mechanism for PUFA-mediated suppression of the gene expression have been investigated in rat livers. When diabetic rats were injected with insulin, the insulin dose-dependent induction of lipogenic enzyme mRNAs were markedly reduced with increasing dietary corn oil. On the other hand, the PUFA-mediated suppression of the mRNA concentrations was partially restored by treatment with pioglitazone, a candidate for increasing insulin receptor phosphorylation. Moreover, insulin binding to receptors of liver, receptor autophosphorylation, and kinase activity toward exogenous substrate were lower in the corn oil diet group than in the hydrogenated fat group. The PUFA-mediated suppression of insulin binding was somewhat restored by pioglitazone, and the suppression of insulin receptor phosphorylation was significantly restored. It is suggested that the PUFA-mediated suppression of insulin-dependent gene expression of lipogenic enzymes can be ascribed to a decrease in insulin receptor binding primarily and also to receptor phosphorylation. Thus, PUFA appears to suppress the lipogenic enzyme gene expression stimulated by insulin.

Lipids of selected molds grown for production of n-3 and n-6 polyunsaturated fatty acids.

The lipid classes and component fatty acids of seven fungi were examined. Three marine fungi, Thraustochytrium aureum, Thraustochytrium roseum and Schizochytrium aggregatum (grown at 30, 25 and 25 degrees C, respectively), produced less than 10% lipid but contained docosahexaenoic acid (DHA) up to 30% and eicosapentaenoic acid (EPA) up to 11% of the total fatty acids. Mortierella alpinapeyron produced 38% oil containing solely n-6 polyunsaturated fatty acids (PUFA) with arachidonic acid (AA) at 11% of the total fatty acids. Conidiobolus nanodes and Entomorphthora exitalis produced 25% oil and contained both n-3 and n-6 PUFA, with AA at 16% and 18%, respectively. Saprolegnia parasitica produced 10% oil and contained AA and EPA, respectively, at 19% and 18%. The triacylglycerol fraction always represented the major component at between 44% and 68% of the total lipid. Each fungus, except T. aureum, had the greatest degree of fatty acid unsaturation in the phospholipid fraction. The triacylglycerol fraction of T. aureum was the most unsaturated with DHA representing 29% (w/w) of all fatty acids present. The presence of the enzyme ATP:citrate lyase correlated with the ability of molds to accumulate more than 10% (w/w) lipid when the fungi were grown in nitrogen-limiting media. In those molds that failed to accumulate more than 10% lipid, the enzyme was absent.

Zonation of hepatic lipogenic enzymes identified by dual-digitonin-pulse perfusion.

The zonal distribution within rat liver of acetyl-CoA carboxylase, ATP citrate-lyase and fatty acid synthase, the principal enzymes of fatty acid synthesis, was investigated by using dual-digitonin-pulse perfusion. Analysis of enzyme mass by immunoblotting revealed that, in normally feeding male rats, the periportal/perivenous ratio of acetyl-CoA carboxylase mass was 1.9. The periportal/perivenous ratio of ATP citrate-lyase mass was 1.4, and fatty acid synthase exhibited the largest periportal/perivenous mass gradient, having a ratio of 3.1. This pattern of enzyme distribution was observed in male rats only; in females, the periportal/perivenous ratio of enzyme mass was nearly equal. The periportal/perivenous gradients for acetyl-CoA carboxylase, ATP citrate-lyase and fatty acid synthase observed in fed (and fasted) males were abolished when animals were fasted (48 h) and refed (30 h) with a high-carbohydrate/low-fat diet. As determined by enzyme assay of eluates obtained from the livers of normally feeding male rats, there is also periportal zonation of acetyl-CoA carboxylase activity, expressed either as units per mg of eluted protein or units per mg of acetyl-CoA carboxylase protein, suggesting the existence of gradients in both enzyme mass and specific activity. From these results, we conclude that the enzymes of fatty acid synthesis are zonated periportally in the liver of the normally feeding male rat.

Halothane-induced alterations of glucose and pyruvate metabolism in rat cerebra synaptosomes.

Synaptosomes isolated from rat cerebra were used to study the effects of the inhalational anesthetic, halothane, on cholinergic processes. To identify possible mechanisms responsible for the depression of acetylcholine synthesis, we examined the effects of halothane on precursor metabolite metabolism involved with supplying the cytosol with acetyl-CoA for acetylcholine synthesis. Three percent halothane/air (vol/vol) depressed 14CO2 evolution from labeled pyruvate and glucose. Steady-state 14CO2 evolution from [1-14C]glucose was depressed 84% by halothane, while 14CO2 evolution from [6-14C]glucose and [3,4-14C]glucose was decreased 67 and 52%, respectively, when compared with control conditions. Halothane inhibited the activities of both pyruvate dehydrogenase (14% depression) and ATP-citrate lyase (32% depression). Total synaptosomal acetyl-CoA concentrations were unaffected by halothane. Three percent halothane/air (vol/vol) caused a 77% increase in medium glucose depletion rate from 1.38 nmol (mg protein)-1 min-1 to 2.44 nmol (mg protein)-1 min-1. Production of lactate by the synaptosomes in the presence of halothane increased by 231% from a control rate of 1.44 nmol (mg protein)-1 min-1 to 4.77 nmol (mg protein)-1 min-1. Lactate production rate from pyruvate was also enhanced by 56% in the presence of halothane. These data lend support to the concept that the NAD+/NADH potential may be involved in the halothane-induced depression of acetylcholine synthesis.

Electrophoretic elution of proteins from polyacrylamide gel slices.

A method for electrophoretic elution of proteins from polyacrylamide gel slices is described. Eluted proteins were retained by a discontinuous conductivity gradient (M. Otto and M. Snejdárková, Anal. Biochem. 111, 111-114 (1981)). The method has been adapted to slices from slab gels and gels that have been stained and destained. Proteins were eluted as their sodium dodecyl sulfate complexes. Minute amounts of proteins (0.1 microgram) were recovered in high yield (85-95%) in 2 h in less than 0.1 ml volume.

Effect of litter size on glucose metabolism in rat pup adipose tissue during the suckling period.

The effect of milk intake on the glucose tolerance capacity, lipogenesis, ATP citrate lyase and acetyl CoA carboxylase activities in rat adipose tissues during the suckling period was studied. The glucose tolerance capacity in the suckling rats was far lower than that in rats at the age of 5 weeks. However, glucose tolerance capacity in the suckling animals of 4-membered litters at the age of 2 weeks was higher than that in control animals of 8-12 membered litters at the same age. Lipid synthesis, fatty acid synthesis and ATP citrate lyase activity in subcutaneous adipose tissues of animals of 4-membered litters in the suckling period were higher than those of control animals. It was observed that ATP citrate lyase activity in subcutaneous adipose tissues of animals of 4-membered litters in the suckling period were higher than those of control animals. It was observed that ATP citrate lyase activity in adipose tissues during the suckling period was increased by glucose injection alone. These results suggest that the development of the glucose tolerance capacity and the lipogenesis in adipose tissues during the suckling period were influenced by nutritional status as well as chronological age.

Properties and organ distribution of ATP citrate (pro-3S)-lyase.

1. The lipogenic enzyme ATP citrate lyase (ATP:citrate oxaloacetate-lyase (pro-3S-CH2COO-acetyl-CoA; ATP-dephosphorylating), EC 4.1.3.8) is partially purified from human liver by ammonium sulfate fractionation and anionexchange chromatography. 2. Km values for the substrates are 1.1 x 10(-5) 1.3 x 10(-3), and 1.2 x 10(-4) M for CoASH, ATP and citrate, respectively. The hypolipidemic drug L(-)-hydroxycitrate is a competitive inhibitor with respect to citrate (Ki = 3 x 10(-4) M). 3. Specific activities measured in liver, adipose tissue and intestinal mucosa (autopsic and biopsic material) are in the range of 1 mU/mg protein suggesting that the citrate pathway does not significantly contribute to human lipogenesis. No stimulation is found after a 3-day carbohydrate-rich diet. 4. Specific activities of other key-enzymes of the acetyl-CoA production from carbohydrates (pyruvate dehydrogenase, cytosolic acetyl-CoA synthetase) are of the same low magnitude.

Fat cell protein phosphorylation. Identification of phosphoprotein-2 as ATP-citrate lyase.

We have purified to apparent homogeneity a phosphoprotein from rat adipose tissue which is rapidly phosphorylated in vitro by ATP. The native phosphoprotein has an approximate sedimentation coefficient of 14.8 S. On sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, the protein dissociated into identical subunits of Mr = 128,000. A phosphoprotein with similar properties was also isolated from liver. Purified phosphoproteins from fat cells and liver had ATP-citrate lyase activity and co-migrated on sodium dodecyl sulfate gels with fat cell phosphoprotein-2, the phosphorylation of which is increased by incubating fat cells with insulin. The phosphoamino acid residue of the cells with insulin. The phosphoamino acid residue of the phosphoprotein was identified as tau-phosphohistidine. These evidences suggest that fat cell phosphoprotein-2 is ATP-citrate lyase.

Effect of sucrose on lipogenesis of rats chronically treated with ethanol.

The effect of chronic ethanol administration with and without sucrose on the lipogenic enzymes of liver and adipose tissue of rats was studied. Ethanol markedly influenced the adipose lipogenic enzymes at 28 days. Sucrose caused a 2-10fold increase in lipogenic enzymes of both adipose and liver.