Fatty Acid Synthetase Activity Research Articles

Chicken liver fatty acid synthetase: Proteolysis by subtilisin and isolation and properties of palmitoyl thioesterase

Subtilisin hydrolysis of chicken liver fatty acid syntheiase yields polypeptides of molecular weights 220,000, 160,000 and 35,000. The larger peptides are further degraded to proteins of molecular weights 122,000 and 105,000. When 50% and 80% of the synthetase subunits are cleaved, there is a loss of 10% and 40% of fatty acid synthetase activity, respectively, indicating that proteolysis of the 240,000-mol. wt. subunit does not substantially affect palmitate synthesis provided that the component polypeptides remain associated with each other. Ammonium sulfate fractionation yields a fraction containing the palmitoyl thioesterase activity. Polyacrylamide gel electrophoresis of this fraction under both nondenaturing and denaturing conditions yields one band with an estimated molecular weight of 35,000. The isolated thioesterase is specific for palmitoyl and stearoyl thioesters (myristoyl-CoA is hydrolyzed at 15% the rate of palmitoyl-CoA). The enzyme is inhibited byN-ethylmaleimide and diisopropylfluorophosphate, suggesting that both an active -SH and -OH are involved in catalysis. However, preincubation of the thioesterase with decanoly-CoA protected the enzyme against inhibition by diisopropylfluorophosphate but not byN-ethylmaleimide, suggesting that an active OH (seryl or threonyl) is involved in the hydrolysis of the palmitoyl group. This active hydroxyl group is uniquely inhibited by diisopropylfluorophosphate, as evidenced by the incorporation of 2 mol of [32P]diisopropylfluorophosphate per mole of synthetase (M r = 480,000) and the fact that all the radioactivity was associated with the isolated thioesterase. These results indicate that there are two copies of the thioesterase per mole of synthetase or one copy of the enzyme per 240,000-mol. wt. subunit.

Isolation and partial purification of elastase-released peptide domains that contain the partial activities of pigeon liver fatty acid synthetase

Proteolytic cleavage of pigeon liver fatty acid synthetase with elastase (4% w/w) for 5 h yields two peptides, designated II and IV, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of peptides II and IV are 130,000 and 86,000, respectively. These peptides are separated by dye-ligand chromatography on an Affi-Gel Blue column. Peptide II is found to contain the domains of β-ketoacyl and enoyl reductases and β-hydroxyacyl dehydrase, whereas peptide IV contains the domains of acetyl- and malonyl-CoA:pantetheine transacylases.

The effects of experimental inflammation on turnover of fatty acid synthetase and levels of fatty acid synthetase messenger RNA in rat liver.

Suppression, by experimental inflammation, induced by subcutaneous injection of oil of turpentine, of the usual increase in liver fatty acid synthetase (FAS) activity resulting from fat-free feeding following starvation (adaptive synthesis) was shown to result entirely from lowered hepatic content of FAS protein. Comparison of changes in the relative rate of synthesis of FAS, determined radioimmunochemically during adaptive synthesis with and without inflammation, with concomitant changes in FAS activity, revealed that inflammation partically suppressed the increased rate of synthesis characteristic of adaptive synthesis, but insufficiently to account entirely for the suppression of enzyme activity. Inflammation accelerated the relative rate of degradation of FAS, causing a 50% decrease in enzyme half-life and a corresponding increase in kd and turnover index. Levels of translatable FAS mRNA rose only fivefold after 24 h of adaptive synthesis, while the relative rate of FAS synthesis increased 12-fold indicating the operation of both transcriptional and translational control. Inflammation, induced at the start of adaptive synthesis, caused a 65% lowering of the relative rate of FAS synthesis after 24 h and a 60% decrease in mRNA translatable as FAS, but was without effect on the total translational activity of the mRNA although alterations in the size distribution of RNA species in the mRNA fraction were noted.

Suppression of Rat Liver Fatty Acid Synthesis by Eicosa-5,8,11,14-tetraynoic Acid without a Reduction in Lipogenic Enzymes

In meal-fed rats supplementation of safflower oil (5 g per 100 g diet) to a fat-free basal diet resulted in the characteristic suppression of liver fatty acid synthetase and acetyl-CoA carboxylase activities, which was accompanied by a 60% decrease in the rate of hepatic fatty acid synthesis. The decline in activity of these lipogenic enzymes was completely prevented by adding 0.05% eicosa-5,8,11,14-tetraynoic acid (TYA) to the safflower oil diet. Fatty acid analysis of the livers indicated that TYA significantly impaired the conversion of linoleate to arachidonate. Apparently the selective suppression of lipogenic enzymes by dietary linoleate is not caused by linoleate per se but requires its conversion to longer-chain fatty acids and/or prostaglandins. In spite of high activities of fatty acid synthetase and acetyl-CoA carboxylase, liver fatty acid synthesis continued to be inhibited by the safflower oil + TYA dietary regimen. This continued inhibition of lipogenesis was due to the TYA, because addition of TYA to the fat-free diet precipitated a significant decline in liver fatty acid synthesis without a drop in lipogenic enzymes. Inhibition of fatty acid synthesis by TYA could not be attributed to a decrease in liver glucose utilization based on hepatic glycogen concentration, nor was it due to a reduction in the fraction of catalytically active polymeric acetyl-CoA carboxylase based on sensitivity of the enzyme activity to avidin.lipogenesis eicosatetraynoic acid polyunsaturated fats

Early effect of myo-inositol deficiency on fatty acid synthetic enzymes of rat liver

Young rats (100 g) were fed either a purified myo-inositol-deficient balanced diet or a control diet containing 0.5% by weight myo-inositol, ad libitum, for up to 2 weeks following a 48 h fast. Weight gain was the same for animals in both groups. Liver triacylglycerol levels in the deficient animals were 1.8–, 3.5– and 3.0-fold higher than the corresponding levels in the control animals after 4, 8 and 14 days of feeding, respectively. In the myo-inositol-deficient group the specific activities of liver fatty acid synthetase and acetyl-CoA carboxylase were elevated 1.5–2.0-fold over controls, reaching a maximum after 3–4 days of feeding. Subsequently, activities declined to control levels. Rates of fatty acid synthetase synthesis in the deficient group, as measured by [ 3H]leucine incorporation into immunoprecipitable fatty acid synthetase polypeptide, were significantly higher (1.5–2.0-fold) than controls after 12–18 h of feeding and then declined to control levels by 1 day. No difference was noted between groups in either the rate of total, soluble liver protein synthesis or the half-life of fatty acid synthetase over this time period. These results suggest that the liver lipodystrophy observed during myo-inositol deficiency in rats may be due in part to elevated levels of lipogenic enzymes in this tissue in the early stage of the deficiency.

Lipase-induced alterations of fatty acid synthesis by subcellular fractions from germinating pea (Pisum sativum L.).

1. The effect of exogenous lipases on fatty acid synthesis from [14C]malonyl-CoA by the microsomal and soluble fractions from germinating peas was studied. 2. Addition of phospholipase A2 or the lipase from Rhizopus arrhizus had no effect on total fatty acid synthesis by the soluble fraction but caused severe inhibition of that by the microsomal fraction. 3. The addition of enzymes with phospholipase activity particularly inhibited the microsomal stearate elongase. 4. Control studies indicated that the phospholipase-induced inhibition of fatty acid synthesis was due to the location of fatty acid synthetase, palmitate elongase and stearate elongase on the outside of the microsomal vesicles. 5. Experiments with a trypsin-like proteinase showed that approximately half the microsomal fatty acid synthesis was resistant to proteolysis. 6. Although addition of exogenous phospholipases had no effect on total fatty acid synthesis by the soluble fraction, it did increase alpha-hydroxylation of newly-formed palmitate and stearate. 7. The results provide further evidence for differences between the soluble and particulate fatty acid synthetase and palmitate elongase activities of germinating pea.

Effect of dietary polyunsaturated fatty acids on the activity and content of fatty acid synthetase in mouse liver

When mice, previously fed a standard laboratory mouse chow diet, were fed a high carbohydrate (50% glucose) diet containing 15% (w/w) hydrogenated cottonseed oil, the activity of hepatic fatty acid synthetase per mg cytosolic protein increased approximately 3-fold over an 11-day period. However, when mice were placed on an isocaloric diet containing 15% (w/w) corn oil, the specific activity of the enzyme did not increase above the chow-fed levels. Using antibody prepared against pure mouse liver fatty acid synthetase, we showed that the increase in the specific activity of fatty acid synthetase in the hydrogenated cottonseed oil-fed animals resulted from an elevation in the hepatic content of the enzyme. This increase was a result of (a) an increase in the rate of synthesis of the enzyme relative to that of total protein and (b) a decrease in the enzyme's degradative rate, when compared to these parameters measured in the livers of the corn oil-fed animals. Furthermore, these dietary-induced changes in enzyme specific activity were not accompanied by changes in the catalytic efficiency of fatty acid synthetase; since both hydrogenated cottonseed oil-fed and corn oil-fed animals showed identical immunoequivalences and contained similar amounts of immunoprecipitable 3H-labeled enzyme protein per unit enzyme activity (in mice pulse-labeled with [ 3H]leucine). The results of experiments in which we administered pure fatty acids (oleic (cis- Δ 9-18:1), ricinoleic (12-hydroxy-cis- Δ 9-18:1), Iinoleic (cis, cis- Δ 9,12-18:2), α-linoleic ( cis, cis, cis- Δ 9,12,15-18:3), columbinic ( trans, cis, cis- Δ 5,9,12-18:3) and arachidonic (all-cis- Δ 5,8,11,14-20:4)) to mice maintained on a 50% glucose diet suggested that the ability of a fatty acid to inhibit hepatic fatty acid synthetase activity and to prevent an increase in hepatic fatty acid synthetase protein was related to the degree and position of unsaturation of the fatty acid administered and not to the ability of the fatty acid to act as prostaglandin precursor. Those 18-carbon fatty acids which possessed a double bond at positions Δ 9,12 (linoleic, columbinic and α-linolenic) were the most effective at inhibiting hepatic fatty acid synthetase activity and in preventing an increase in enzyme content.

Metabolic alterations induced by voluntary exercise and discontinuation of exercise in hamsters

A study was conducted to determine the possible biochemical mechanisms responsible for the rapid increase in body weight and body fat during the early phase of exercise-retirement. Thirty-two adult female hamsters were allotted to four groups of eight each. One group served as sedentary controls. Other groups had access to voluntary disc-running during a 35-day period of the 76-day experimental period and were retired from running for 0, 8, and 41 days, respectively, before the termination of the experiment. Disc-running reduced body fat and body weight. Discontinuation of running led to a fast gain of body fat and body weight. Disc-running decreased serum triacylglycerol levels but had no effect on serum cholesterol levels. Serum high-density lipoprotein-cholesterol concentration was significantly elevated at 8 days after retirement but was not changed during exercise or at 41 days after retirement. Exercise or discontinuation of exercise had no effect on serum T-3 or T-4 levels. The rate of fatty acid synthesis and the activities of malic enzyme and fatty acid synthetase in the liver were increased during running and during the early phase of retirement. Liver glucose-6-phosphate dehydrogenase activity was increased at 8 days after discontinuation of running. Disc-running enhanced food intake which was not reduced to the sedentary level until about 10 days after retirement, suggesting a delay in adaptation to reduced energy expenditure. Results of the present study suggest that both the delay in food intake adaptation and the enhanced lipogenic activity play a role in the rapid gain of body weight and body fat during the early phase of exercise-retirement.

Limited elastase digestion of pigeon liver fatty acid synthetase with retention of all partial enzyme activities.

Pigeon liver fatty acid synthetase which contains two subunits of 240,000 daltons each has been treated with elastase. This treatment yields four protein fragments which can be separated on sodium dodecyl sulfate (SDS)-gel electrophoresis. After the subunit protein has been treated with elastase, all of the partial enzyme activities catalyzed by the complex are present, but enzyme activity for fatty acid synthesis is lost. The formation of protein fragments during proteolysis has been followed by densitometric scanning of the SDS gels. The results of these scans have suggested that (a) there are two peptide components present in the highest molecular weight band, (b) both are rapidly digested to yield the second and third largest peptides, and (c) a further cleavage of the third largest peptide gives rise to the smallest of the four major peptides. Crossed-rocket immunoelectrophoretic analysis of the four protein fragments has confirmed these conclusions and established also that the three smallest peptides are homogeneous. Each of the four peptides has been isolated by preparative SDS-gel electrophoresis, and antibody to one has been prepared. This antibody fraction immunotitrates overall fatty acid synthetase activity and immunoprecipitates the native enzyme. Immunoelectrophoresis of the four elastase-digested synthetase products against this antibody showed some cross-reactivity with a peptide that was neither the precursor nor the product of the immunogen. This cross-reacting antibody was removed by reaction with the nonrelated protein to yield antibody specific for one region of the fatty acid synthetase complex.

Partial separation of individual enzyme activities of an ACP-dependent fatty acid synthetase from barley chloroplasts

An acyl carrier protein (ACP) dependent fatty acid synthetase (fas) from barley chloroplast stroma was purified five-fold by ammonium sulphate precipitation and gel filtration on Sephacryl S-300. The β-ketoacyl-ACP reductase, β-ketoacyl-ACP synthetase, acetyl-CoA:ACP transacylase and malonyl-CoA:ACP transacylase activities were resolved on the Sephacryl S-300 column with apparent molecular weights of respectively 125, 92, 82 and 41 kilodalton. The fas activity exhibited an apparent molecular weight of 87 kilodalton resulting from the overlapping portions of the component activities. A fifth component of the active fas, ACP, was separated completely from the other four individual enzyme activities by the ammonium sulphate precipitation. When the fas purified by gel filtration was applied to a Mātrex Gel Blue B column, the component activities were separated into two groups. A bound fraction contained all the malonyl-CoA:ACP transacylase whereas the β-ketoacyl synthetase activity was exclusively present in the non-bound fraction. Neither the bound nor the non-bound fraction showed any fas activity alone, but complete reconstitution of fas activity was obtained when both protein fractions were combined. The barley chloroplast fas is therefore not a multifunctional protein but consists of at least five separable components. Characterization with respect to cofactor requirements was also performed. Variation of certain cofactor concentrations markedly altered the pattern of fatty acid synthesis.

Effect of thyroid hormones and cyclic AMP on some lipogenic enzymes of the fat cell.

Thyroid hormones exert two types of effect on the lipogenic pathway of the fat cell of the young rat: one which is independent of cyclic AMP, and another which is under cyclic nucleotide control: Thyroidectomy leads to an enhanced conversion of glucose to glucose 6‐phosphate by the hexokinase system. The activity of hexokinase I is increased twofold in the fat cell from hypothyroid rats. The oxidation of glucose 6‐phosphate by the pentose phosphate pathway is also increased after thyroidectomy. The V of both glucose 6‐phosphate and gluconate‐6‐phosphate dehydrogenases is doubled (56 mU × min−1× mg−1 to 114 mU × min−1× mg−1) whereas the Km, is unchanged (19 μM). The activities of ATP citrate lyase and of acetyl‐CoA carboxylase are higher in the adipocytes from hypothyroid rats. The activity of fatty acid synthetase is also stimulated, but to a lesser extent. The apparent Km of acetyl‐CoA carboxylase is 0.14 mM and 0.06 mM and the V is 17.8 nmol xmin−1 xmg‐′ and 27.8 nmolxmin−1 xmg protein−1 in the adipocytes from normal and hypothyroid rats respectively. In contrast, for ATP citrate lyase the V only is doubled (62 mU × min−1× mg−1 to 111 mUxmin−1xmg−1). When fat cells from normal or hypothyroid rat were incubated in the presence of methylxanthines, dibutyryladenosine 3′,5′‐monophosphate, isoproterenol or epinephrine the activities of the hexokinase system and of glucose‐6‐phosphate and gluconate‐6‐phosphate dehydrogenases remained unchanged. In contrast, dibutyryl‐ adenosine 3′,5′‐monophosphate inhibited, by 60 ‐ 80%, the activities of ATP citrate lyase and acetyl‐CoA carboxylase in both cell types. In adipocytes prepared from normal rats, isoproterenol and epinephrine inhibited the activities of these two latter enzymes by 30 ‐ 60% while isobutylmethylxanthine potentiated their effect. In contrast, with fat cells from hypothyroid rats, isoproterenol and epinephrine had no effect upon the activities of ATP citrate lyase and acetyl‐CoA carboxylase, whereas a combination of epinephrine and ibobutylmethylxanthine was clearly inhibitory. In conclusion, thyroid hormones regulate lipogenesis in the fat cell of the young rat firstly because they control the accumulation of the substrates of this pathway, glucose 6‐phosphate and NADPH, by changing the concentrations of the hexokinase system and of the rate‐limiting enzymes of the pentose phosphate pathway. Secondly, because they have a specific effect on the amount of the enzymes (ATP citrate lyase and acetyl‐CoA carboxylase) which channel acetyl‐CoA towards long chain fatty acid synthesis; and thirdly because they allow, in response to the lipolytic hormones, the accumulation of cyclic AMP which is responsible for the inactivation of these two latter enzymes.

Alterations in lipogenic enzymes and lipoprotein lipase activity during gram-negative sepsis in the rat.

The effects of sepsis on lipid metabolism have not been clearly defined. This study was designed to observe the changes in adipose tissue lipoprotein lipase (LPL) and fatty acid synthetase (FAS) after administration of Escherichia coli bacteria. Male Lewis rats, weighing 245 to 270 g, were assigned to two groups and fed a powdered chow diet for 14 days. On day 14, one group was inoculated with E coli. Twenty-four hours later, both groups were killed by decapitation. Serum triglyceride levels were significantly elevated in the E coli-treated rats. Adipose tissue LPL and FAS activity was significantly decreased by 50% in E coli-treated rats compared with the control rats. These results suggest that the elevated serum triglyceride levels associated with sepsis maybe caused by a decreased rate of clearance of lipids from the blood and an increased rate of hepatic lipid synthesis.

Bio-inorganic regulation of pathways of carbohydrate and lipid metabolism. II. The effect of iron-deficiency on the profile of enzymes in the developing rat adrenal gland.

The effect of iron-deficiency anaemia and iron supplementation on the enzyme profile and conversion of specifically labelled glucose to 14CO2 and 14C-lipid was measured in rat adrenal glands. Enzymes involved in lipogenesis were severely depressed in iron deficiency, addition of Fe2+ to the diet resulted in increased activity of fatty acid synthetase (+75%), NADP isocitrate dehydrogenase (+466%), glucose-6-phosphate dehydrogenase (+27%) and malic enzyme (+46%). Parallel increases occurred in the flux of glucose into lipid. The effects of iron-deficiency anaemia on adrenal and thyroid function in relation to developmental processes are discussed.

The effect of dietary methyl branched-chain fatty acids on aspects of hepatic lipid metabolism in the rat.

1. Rats were fed to appetite on a stock laboratory diet or on diets consisting of the stock diet and in addition 50 or 200 g triolein/kg, 50 g palmitic acid/kg or 50 g/kg of a concentrate mixture of methyl branched-chain fatty acids (Me-BCFA) which had been prepared from sheep adipose triacylglycerols. 2. No differences could be detected in the delta 9-desaturase activity or fatty acid synthetase activity of liver preparations from rats which had been fed on either the stock diet, the 50 g palmitic acid/kg or the 50 and 200 g triolein/kg diet; the palmitic acid diet was therefore taken as the control diet in subsequent experiments. 3. Rats consuming the 50 g Me-BCFA/kg diet exhibited a marked reduction in the capacity of their liver microsomes for delta 9-desaturation when compared with animals receiving the control diet. The delta 6-desaturase activity also showed an inhibitory trend with the Me-BCFA diet. 4. Microsomal omega-oxidation of fatty acids, mitochondrial succinate oxidation and the activity of cytosolic fatty acid synthetase (FAS) were unaffected by the ingestion of the Me-BCFA mixture compared with the diet which included palmitic acid. 5. There were no differences in the plasma concentration of thyroxin, insulin and glucagon between animals fed on the diets containing palmitic acid or the Me-BCFA. 6. For a given concentration of fatty acids the Me-BCFA had a greater inhibitory effect when added to incubations of liver microsomes from rats fed on the standard diet than did the addition of palmitic acid. 7. The observations in vivo and in vitro strongly suggested that the Me-BCFA were having a specific inhibitory effect on the desaturation reaction.

The effects of dietary lipid and phenobarbitone on the production and utilization of NADPH in the liver. A combined biochemical and quantitative cytochemical study.

The validity of the concept that cellular NADPH utilization in the cytoplasm can, by quantitative cytochemical procedures, be classified into two pathways (Pathway I, in which NADPH is oxidized via the microsomal electron-transport system, and Pathway II, in which NADPH supplies reducing equivalents for biosynthetic processes) was tested. The amount of NADPH, generated by glucose 6-phosphate dehydrogenase, entering Pathways I and II in the centrilobular and periportal regions was measured by quantitative cytochemistry, and the values obtained were compared with biochemical measurements of mixed-function oxidase and fatty acid synthetase activity after the administration of sodium phenobarbitone or by altering the quantity and nature of the dietary lipid. Phenobarbitone stimulates hepatic mixed-function oxidation measured biochemically and Pathway I, but not Pathway II. Variation in the type and quantity of dietary lipid can also regulate the activity in mixed-function oxidation and alter the amount of NADPH entering Pathways I and II. It is concluded that, in general, the concept of two main pathways of NADPH utilization in the liver is valid, but that the ratios of NADPH utilization in the two pathways gives a better indication of the use of NADPH in vivo than is obtained for absolute values for the two pathways. Moreover, the centrilobular and periportal hepatocytes showed different patterns in their response to changes in dietary lipid and the administration of phenobarbitone. These results indicate the different metabolic roles that these two groups of cells may play in the metabolism of foreign compounds.

Kinetic studies of the fatty acid synthetase multienzyme complex from Euglena gracilis variety bacillaris.

A fatty acid synthetase multienzyme complex was purified from Euglena gracilis variety bacillaris. The fatty acid synthetase activity is specifically inhibited by antibodies against Escherichia coli acyl-carrier protein. The Euglena enzyme system requires both NADPH and NADH for maximal activity. An analysis was done of the steady-state kinetics of the reaction catalysed by the fatty acid synthetase multienzyme complex. Initial-velocity studies were done in which the concentrations of the following pairs of substrates were varied: malonyl-CoA and acetyl-CoA, NADPH and acetyl-CoA, malonyl-CoA and NADPH. In all three cases patterns of the Ping Pong type were obtained. Product-inhibition studies were done with NADP+ and CoA. NADP+ is a competitive inhibitor with respect to NADPH, and uncompetitive with respect to malonyl-CoA and acetyl-CoA. CoA is uncompetitive with respect to NADPH and competitive with respect to malonyl-CoA and acetyl-CoA. When the concentrations of acetyl-CoA and malonyl-CoA were varied over a wide range, mutual competitive substrate inhibition was observed. When the fatty acid synthetase was incubated with radiolabelled acetyl-CoA or malonyl-CoA, labelled acyl-enzyme was isolated. The results are consistent with the idea that fatty acid synthesis proceeds by a multisite substituted-enzyme mechanism involving Ping Pong reactions at the following enzyme sites: acetyl transacylase, malonyl transacylase, beta-oxo acyl-enzyme synthetase and fatty acyl transacylase.

Comparative Studies of Adaptive Responses of Fatty Acid Synthetase Activities in Rat Liver and Adipose Tissue

The specific activities of the fatty acid synthetases in the cytosolic fractions of livers and epididymal fat pads from fed, fasted or refed rats were determined. Refed rats received diets which provided, as the primary energy sources, sucrose or starch (75%) or both (59%) with beef tallow or safflower oil (16%) or the monosaccharide components (75%). From these determinations of fatty acid synthetase activity, the relative contribution of each tissue to the rat’s overall lipogenic capacity was estimated. In rats fed a cereal-based stock diet ad libitum the adipose tissue accounted for 58% of total activity. During a 2-day fast the hepatic activity decreased from 14 units/100 g body weight to 2.7 units whereas the adipose tissue activity fell from 19 to 13.4 units/100 g body weight. At this time, 82.8% of the activity was in the latter tissue. After refeeding the sucrose diet for 2 days, the hepatic activity had increased 45-fold to 122.3 units/100 g body weight while the adipose tissue activity increased only 2.2-fold to 29.1 units, 19% of the total activity. We estimate that these adaptive responses deteriorate slowly (12.3 days, adipose tissue; 15.4 days, liver) with continued refeeding to the levels present in rats fed the cereal-based stock diet. Activation of residual or constituative enzyme was the major factor in the adipose tissue response whereas activation and induction played roles in the hepatic response. Responses to the refeeding of the other diets were of lesser magnitude. Generally our estimates based on fatty acid synthetase activities are consistent with other estimates based on radiolabel incorporation, NADPH generation and rate-limiting enzyme activities.fatty acid synthetase fat fasting carbohydrate

Polyoxyethylated cholesterol derivatives Organic synthesis, cellular uptake and effect on lipid metabolism in cultured skin fibroblasts

Polyoxyethylated derivatives of cholesterol were synthesized by adduction of ethylene oxide to the 3-β-hydroxy position of cholesterol. Derivatives with the methoxy terminal were prepared by condensing methoxypoly(oxyethylene)methane sulfonate with cholesterol. Derivatives with nine ethoxy groups were isolated by the combined use of high pressure liquid chromatography and thin-layer chromatography and identified by proton magnetic resonance. In cultured human skin fibroblasts, uptake of polyoxyethylated [ 3H]cholesterol was linear up to a concentration of 60 μM. Uptake at 10 μM concentration was linear for 5 h and amounted to 2.5% of the total added label per h. All of the cellular radioactivity was recovered in polyoxyethylated cholesterol and no label appeared in other lipid fractions. Polyoxyethylated cholesterol (10 μM) inhibited the incorporation of acetate into cholesterol and the activity of 3-hydroxy-3-methylglutaryl-CoA reductase, 50% inhibition occurring at 1 and 3 h, respectively. After 18 h incubation, 50% inhibition of both activities occurred at 5 μM and near total inhibition at 25 μM. The incorporation of leucine into protein, uridine into RNA and thymidine into DNA were not affected significantly. Methoxypolyoxyethylated cholesterol gave similar results. There were no differences between normal and homozygous familial hypercholesterolemic fibroblasts which lack membrane receptors for low density lipoproteins. Incorporation of acetate into fatty acids was suppressed by polyoxyethylated cholesterol but not by 25-hydroxycholesterol. Polyoxyethylated cholesterol had no effect on the activity of fatty acid synthetase. Efflux of polyoxyethylated cholesterol from cells had a rapid phase amounting to 55% in the first 2 h, followed by a slow leakage of 15% in the next 20 h. Recovery of the activity of 3-hydroxy-3-methylglutaryl-CoA reductase was faster than that of incorporation of acetate to cholesterol and fatty acids but all these activities remained below the control levels, suggesting that the polyoxyethylated cholesterol retained within the cell continues to exert its inhibitory effect on lipid synthesis.

Infradian cycles in glucose utilization and lipogenic enzyme activity in dormouse (Glis glis) adipocytes.

This study investigated glucose utilization and lipogenic enzyme activities in adipocytes of dormice ( Glis glis ) at different phases of their infradian body weight cycle. Basal glucose utilization and the activities of fatty acid synthetase, glucose-6-phosphate dehydrogenase, and citrate cleavage enzyme were all markedly increased in animals with higg food intakes (>20 g/day) as compared to animals with low food intakes (<5 g/day). Values for dormice with stable weights and food intakes were intermediate. These results indicate that changes in glucose utilization by dormouse adipocytes are likely to be an important part of insulin-dependent effector mechanisms for energy storage in this species.

Demonstration of the occurrence of inactive fatty acid synthetase in rat liver by immunotitration and its in vitro partial activation.

Direct immunotitrations of rat liver fatty acid synthetase in crude tissue homogenates with monospecific rabbit anti-rat liver fatty acid synthetase antibody enabled us to make a comparison of fatty acid synthetase protein and activity (percentage of maximal activity) as a function of the nutritional state in normal, diabetic, and insulin- and glucagon-insulin treated animals. Previous results, in which large changes in fatty acid synthetase activity were related to protein synthesis and degradation rather than to enzyme activation, were confirmed. It was also shown that fatty acid synthetase activation does not occur immediately on synthesis but follows the synthesis of fatty acid synthetase protein. In order to characterize the enzymatically inactive protein found on immunotitration and to develop an in vitro system for fatty acid synthetase activation, conditions were sought to obtain large amounts of fatty acid synthetase protein free from, or low in, activity. It was found that treatment of hypophysectomized rats with triiodothyronine meets these requirements, yielding milligram quantities of inactive fatty acid synthetase protein with less than 2% of maximal activity. A part of the inactive fatty acid synthetase was found to be the apoenzyme as indicated by beta-ketoreductase and thioesterase activities, by its ability to incorporate label from [G3H]CoA, and by its partial in vitro activation, which led to an increase in overall synthetase activity in crude and partially purified cell-free systems. The components required for activation include magnesium ion and a transferase fraction prepared from livers of 48-h fasted, 12-h refed rats.