Regulation Of Fatty Acid Biosynthesis Research Articles

Acetoacetyl-acyl carrier protein synthase, a potential regulator of fatty acid biosynthesis in bacteria.

The first condensation reaction in the fatty acid biosynthetic pathway in Escherichia coli was rate-limiting as judged by analysis of the relative pool sizes of acyl carrier protein (ACP) thioester intermediates in vivo. Comparable concentrations of acetyl-ACP, malonyl-ACP, and nonesterified ACP were present during logarithmic growth, whereas long-chain acyl-ACP comprised a minor fraction of the total ACP pool. The antibiotic cerulenin was used to irreversibly inhibit both beta-ketoacyl-ACP synthases I and II. However, acyl-ACP formation in vivo was not blocked by this antibiotic, and short-chain (4-8-carbon) acyl-ACPs increased to 60% of the total ACP pool in cerulenin-treated cells. These data suggested that existence of a cerulenin-resistant condensing enzyme that was capable of catalyzing the initial steps in chain elongation. A unique enzymatic activity, acetoacetyl-ACP synthase, that specifically catalyzed the condensation of malonyl-ACP and acetyl-ACP was detected in E. coli cell extracts. Acetoacetyl-ACP synthase activity was not inhibited by cerulenin and was present in extracts prepared from a double mutant harboring genetic lesions in beta-ketoacyl-ACP synthases I and II (fabB20 fabF3). These data point to the condensation of malonyl-ACP and acetyl-ACP as the rate-controlling reaction in fatty acid biosynthesis and implicate acetoacetyl-ACP synthase as the pacemaker of fatty acid production in organisms and organelles that possess dissociated (Type II) fatty acid synthase systems.

Regulation of fatty acid biosynthesis by hydrocarbon substrates in Mycobacterium convolutum.

When Mycobacterium convolutum R22 was grown on the n-alkanes C13 through C16, the predominant fatty acids were of the same chain length as the growth substrate. Cells grown on C13 through C16 n-alkanes incorporated between 15 and 85 pmol of acetate per microgram of lipid into the fatty acids, whereas acetate- or propane-grown cells incorporated 280 and 255 pmol of acetate per microgram of lipid, respectively. In vivo experiments demonstrated that hexadecane, hexadecanoic acid, and hexadecanoylcoenzyme A (CoA) all inhibited de novo fatty acid synthesis. Hexadecanoyl-CoA was the most potent inhibitor. Hexadecane and hexadecanoic acid inhibited acetyl-CoA carboxylase by up to 37 and 39%, respectively, at 1 mM. Hexadecanoyl-CoA inhibited the enzyme activity by 65% at 50 micrometer. Cells that were grown on C14 through C16 n-alkanes had about 25 times less acetyl-CoA carboxylase activity than did cells grown on acetate or propane, suggesting repressed levels of the enzyme. Hexadecane- or pentadecane-grown cells were found to have 5 to 10 times more intracellular free fatty acid than cells grown on acetate, propane, or ethane.

The regulation of fatty acid biosynthesis: Simple procedure for the purification of acetyl CoA carboxylase from lactating rabbit mammary gland, and its phosphorylation by endogenous cyclic AMP-dependent and -independent protein kinase activities

A number of key regulatory enzymes in eukaryotic cells are now known to be controlled by phosphorylation-dephosphorylation mechanisms, and there is increasing evidence that these interconversion reactions underlie the short term regulation of metabolism by external physiological stimuli (reviewed [I] ). The rate at which cytoplasmic acetyl CoA is converted to long chain fatty acids is stimulated by insulin in fat cells [2] and inhibited by glucagon in hepatocytes [3] . Acetyl CoA carboxylase has been implicated as the enzyme at which these hormonal effects are exerted, but the mechanisms are not understood in molecular terms. Acetyl CoA carboxylase is completely dependent on the allosteric activator citrate for activity, and it is inhibited by very low concentrations of palmityl CoA [4] . There is also preliminary evidence which indicates that acetyl CoA carboxylase may be regulated by a phosphorylation-dephosphorylation mechanism. Relatively crude preparations from rat liver [S] or bovine mammary gland [6] undergo a time-dependent inactivation if they are incubated with magnesium ions and [Y-~~P] ATP [7-91 and 32P-radioactivity is incorporated into a protein(s) which is precipitated by antiserum raised to a proteolytic fragment of rat liver acetyl CoA carboxylase [7-91 . Furthermore the inactivation can be reversed by incubation with a protein phosphatase preparation partially purified from hen oviduct [9] . If isolated fat cells are incubated with 32P, a protein with a subunit molecular weight corresponding to acetyl CoA carboxylase becomes labelled, and this protein can be precipitated using a monospecific antibody raised to acetyl CoA carboxylase from rat mammary gland [lo] . This experiment shows that the enzyme is phosphorylated in intact cells. We describe here an extremely simple method for the purification of large amounts of undergraded acetyl CoA carboxylase and demonstrate that this preparation can be phosphorylated by two distinct protein kinase activities that are present as trace endogenous contaminants in the preparation.

Preincubation stimulates fatty acid synthesis by liver slices and isolated hepatocytes from fasted and diabetic rats.

We have observed that preincubation of 48 hour-fasted or alloxan diabetic rat liver slices, with no exogenous energy supply, for 3 hours resulted in an increased rate of incorporation of [1- 14C] acetate into fatty acids and cholesterol during the following 2 hours. This preincubation effect was enhanced by the presence of glucose (25mM) in or prevented by the addition of dibutyryl cyclic adenosine 3′,5′ monophosphate (10 −4M) to the preincubation medium. Preincubation of normal rat liver slices did not change their rate of incorporation of [1- 14C] acetate into fatty acids or cholesterol. The rate of 14CO 2 synthesized by normal, fasted or diabetic liver slices was little affected by preincubation. The preincubation effect, i.e. enhanced fatty acid synthesis was also observed in suspensions of hepatocytes from fasted and diabetic rats, preincubated for 2 hours, followed by a 1 hour incubation with either [1- 14C] acetate or [ 3H] H 2O as precursor. We conclude from these data that there is concurrent and coordinated short- and long-term regulation of fatty acid biosynthesis in fasted and diabetic rat livers. Further, we suggest that the release of inhibition by preincubation of these tissues provides a useful tool for studying the coordinated control

Comparative studies on lipogenesis and cholesterogenesis in lipemic BHE rats and normal wistar rats

The BHE strain of rat is characterized by early hyperinsulinemia and maturity onset hyperlipemia and hyperglycemia. Since we have previously shown that insulin is required for the coordinate regulation of a number of lipogenic enzymes in rat liver, a comparative study of the hepatic activities of the rate-limiting enzymes of lipid synthesis and the in vivo rates of fatty acid and cholesterol synthesis in the liver and the adipose tissue has been conducted in BHE and Wistar rats. In the liver, BHE rats had 25–28% higher acetyl-CoA carboxylase and fatty acid synthetase activities as measured in vitro but a 100% greater rate of fatty acid synthesis in vivo as compared to Wistar animals. These results strongly suggest that factors other than the amount of acetyl-CoA carboxylase, such as allosteric effectors, must be operating in vivo, thereby facilitating the carboxylase to function at its maximal capacity in BHE rats. Such a regulation of fatty acid biosynthesis by allosteric modifiers of acetyl-CoA carboxylase is already known, although the mechanism of this regulation is not fully understood. BHE rats also exhibited a twofold greater rate of fatty acid synthesis in the adipose tissue compared to the Wistar rats. Thus, increased lipogenic capacity and increased lipogenesis in BHE rats are consistent with early hyperinsulinemia in this strain. Furthermore, BHE rats had 71% more 3-hydroxy-3-methylglutaryl CoA reductase activity with a 97% greater rate of cholesterol synthesis as compared to Wistar rats. In contrast, cholesterol 7α-hydroxylase activity was only 20% greater in BHE rats compared to Wistar rats, suggesting that the BHE rat does not have the capacity to degrade cholesterol to bile acids at a rate commensurate with the increased rate of cholesterol synthesis. This difference in synthesis versus degradation might account for the hypercholesterolemia which occurs in BHE rats, but not in Wistar rats.

Regulation of fatty acid biosynthesis in Ehrlich cells by ascites tumor plasma lipoproteins

Fatty acid biosynthesis in Ehrlich cells in vitro was reduced when very low density lipoproteins (VLDL) isolated from the ascites tumor plasma were added to the incubation medium. The degree of inhibition was dependent on the VLDL concentration. At the VLDL concentrations usually present in the ascites plasma, there was a 30% decrease in biosynthesis as measured by (3)H(2)O incorporation into fatty acids. Analysis of the labeled fatty acids by gas liquid chromatography indicated that this decrease was due to a reduction in fatty acid de novo biosynthesis and that chain elongation actually was increased when VLDL were present. Although ascites plasma low- and high density lipoproteins also produced a concentration-dependent inhibition of fatty acid biosynthesis, their effects were much smaller than those of the VLDL. Studies employing VLDL and radioactive free fatty acids indicated that the cells took up utlilzed fatty acids derived from these lipoproteins. When VLDL were present, labeled free fatty acid incorporation into cell phospholipids, cholesteryl esters, and CO(2) decreased, whereas its incorporation into the cell free fatty acid pool increased. By contrast, the cells incorporated only very small amounts of fatty acid from either low- or high density lipoproteins. This suggests that the VLDL exert their inhibitory effect on fatty acid synthesis by supplying exogenous fatty acids to the cells.

Acyl carrier protein metabolism and regulation of fatty acid biosynthesis by Lactobacillus plantarum.

Endogenous fatty acid biosynthesis in the bacterium Lactobacillus plantarum is greatly decreased upon addition of exogenous fatty acids (Henderson, T.A., and McNeil, J.J. (1966) Biochem. Biophys. Res. Commun. 25, 662-669). We have demonstrated the presence of five pantothenate-containing compounds in L. plantarum which have been identified by co-chromatography with authentic samples: pantothenate, 4'-phosphopantetheine, 3'-dephosphocoenzyme A, coenzyme A, and acyl carrier protein (ACP). The concentrations of the above pantothenate-containing compounds were found to be: 0.009, 0.13, 0.067, 0.69, and 0.22 nmol/mg of protein, respectively. L. plantarum ACP was shown to have a molecular weight near that of Escherichia coli ACP but to have a lower isoelectric point (pI = 3.75). Oleate in the presence of Triton X-100 was found to reduce the concentration of ACP by 80% with little effect on the concentrations of the other pantothenate-containing compounds. The synthesis of ACP apparently ceased soon after the addition of oleate and the rate of decrease in concentration of ACP was quanitatively consistent with the previously observed rate of decrease in the initial rate of fatty acid biosynthesis in this organism (Weeks, G., and Wakil, S.J. (1970) J. Biol. Chem. 245, 1913-1921). Thus, the change in rate of fatty acid biosynthesis in L. plantarum upon addition of oleate to the medium can be quantitatively related to the concentration of ACP (and probably to the concentration of co-repressible enzymes of fatty acid biosynthesis).

Lipid metabolism during bacterial growth, sporulation, and germination: an obligate nutritional requirement in Bacillus thuringiensis for compounds that stimulate fatty acid synthesis.

The regulation of fatty acid biosynthesis by compounds that are required for growth of Bacillus thuringiensis was investigated using an vivo assay developed to measure fatty acid synthesis in germinating spores. A minimal glucose-ammonium-salts medium does not support growth even though previous radiorespirometric studies have shown B. thuringiensis to possess intact tricarboxylic acid and Embden-Meyerhof-Parnas pathways. Abundant growth does occur, however, when this medium is supplemented with either glutamate, aspartate, citrate, thiosulfate, cystine, or ethylenediaminetetraacetic acid. Cells held under nongrowing conditions incorporate acetate into fatty acids; fatty acid synthesis is stimulated by the compounds that permit growth. These alternate nutritional requirements are not manifestations of a vitamin or trace metal deficiency and do not reflect a chelation phenomenon. These results indicate a direct correlation between the capacity of these compounds to promote growth and to stimulate formation of fatty acids.

Regulation of fatty acid biosynthesis in the hydrocarbon oxidizing microorganism, Acinetobacter sp.

1. The regulation of fatty acid biosynthesis was studied inAcinetobacter strainHOl-N, a microorganism which grows on long chain paraffinic hydrocarbons. Dual-label experiments with acetate-3H and palmitate-1-14C were designed to assess the differences in fatty acid metabolism between hexadecane-and acetategrown cells: 2. Experiments involving non-growing conditions indicated a preferential labeling of the cellular lipids with acetate-3H in acetate-grown cells and palmitate-1-14C in hexadecane grown cells. In addition to this relationship, non-growing cells assimilated exogenous lipid precursors (acetate) or preformed lipid (palmitate) almost exclusively into the cellular neutral lipids. 3. Under growing conditions acetate-grown cells exhibited a transfer of lipid radioactivity from the neutral lipid fraction to the phospholipid fraction. 4. Hexadecane-grown cells preferentially labeled with palmitate-1-14C in the cellular lipid exhibited a 98–100% loss of neutral lipid radioactivity. 5. Palmitate derived directly from hexadecane oxidation diluted the radioactive free fatty acid pool 4 times faster in hexadecane-grown cells than in acetate-grown cells. 6. These results indicate that cells grown at the expense of hexadecane are characterized by mechanisms more efficient in the transport and assimilation of exogenous fatty acid and thatde novo fatty acid biosynthesis is significantly reduced in hexadecane-grown cells.

The Regulation of Fatty Acid Synthesis in Human Skin Fibroblasts: INHIBITION OF FATTY ACID SYNTHESIS BY FREE FATTY ACIDS

Abstract The short term regulation of fatty acid biosynthesis has been studied using primary cultures of human skin fibroblasts. These cells show rapid changes in rates of acetate incorporation into fatty acids depending upon the lipid content of the medium. When cells are incubated in medium containing lipid-deficient fetal calf serum overnight and then are switched to Eagle's minimum essential medium, the rate of acetate incorporation into fatty acids is unaffected. Addition of 5 µm albumin-bound palmitic, stearic, oleic, linoleic, or arachadonic acids to the minimum essential medium results in a 26 to 67% inhibition in the rate of acetate incorporation depending upon the fatty acid used. These results are obtained when cells are incubated with [1-14C]acetate from 0.5 to 10.5 min after addition of fatty acid to the medium. When cells are labeled from 5 to 15 min after addition of fatty acid, there was a greater inhibition of acetate incorporation to 70 to 85%. Several lines of evidence suggest that the site of action of fatty acids is intracellular. The effect of added fatty acids on the intracellular concentration of citrate, long chain acyl-CoA compounds, and free fatty acids were measured in these cells. Long chain acyl-CoA levels and citrate levels of cells incubated in Eagle's minimum essential medium for 10.5 min are 73.3 ± 2.5 pmoles per mg of total cellular protein and 2.1 ± 0.3 nmoles per mg of total cellular protein, respectively. When albumin-bound palmitic acid at a concentration of 2.1 µm is included in the incubation medium the levels of long chain acyl-CoA compounds (69.0 ± 3.0 pmoles per mg of total cellular protein) and of citrate (1.9 ± 0.1 nmoles per mg of total cellular protein) remain unchanged despite inhibition of acetate incorporation into fatty acid by this time. Furthermore, incubation of cells in medium containing albumin-bound palmitic acid results in only slight increases in the intracellular free fatty acid levels compared to cells incubated in fatty acid-free medium. These results suggest that total cellular levels of these metabolites are not directly responsible for regulation of the rate of acetate incorporation into fatty acids.

The Regulation of Fatty Acid Biosynthesis in Human Skin Fibroblasts

Fatty acid biosynthesis has been studied using primary cultures of human skin fibroblasts. Acetyl coenzyme A carboxylase activity of cells incubated in medium containing 15% fetal calf serum was 0.17 ± 0.01 nmole of malonyl coenzyme A formed per mg of protein per min whereas in cells incubated for 2 to 3 days in a medium with 15% lipid-deficient fetal calf serum acetyl-CoA carboxylase activity increased to a level of 0.29 ± 0.02 nmole of malonyl-CoA formed per mg of protein per min. Equivalence point determinations using an antibody against acetyl-CoA carboxylase indicate that the increase in enzyme activity results from increased enzyme content in the cells rather than from activation of preformed enzyme. The effect of lipid-deficient medium on acetyl-CoA carboxylase activity was reversed by the addition of normal fetal calf serum or by the addition of any of a number of different albumin-bound free fatty acids to the lipid-deficient medium. When cells originally incubated in medium containing normal fetal calf serum were switched to medium containing lipid-deficient fetal calf serum, an 8-fold increase in the rate of acetate incorporation into lipid was observed in the first 7 hours, whereas cells switched from lipid-deficient to normal serum-containing medium or medium supplemented with free fatty acids showed an almost immediate decrease in the rate of acetate incorporation. The changes in acetate incorporation occurred before any change in enzyme activity was seen, indicating that two types of control are exerted on fatty acid biosynthesis in these cells.

The Regulation of Fatty Acid Biosynthesis in Rat Hepatomas

Abstract The dietary regulation of fatty acid synthesis de novo in two transplantable hepatomas, 7777 and 9618A, has been studied by comparison of the acetyl coenzyme A carboxylase and fatty acid synthetase activities of tumor and host liver, both in animals fasted 48 hours and in those subsequently refed a fat-free diet for 48 hours. Neither acetyl-CoA carboxylase nor fatty acid synthetase of these hepatomas was subject to the changes in enzyme concentration normally observed in host liver following dietary alteration. Thus, in livers of refed animals acetyl-CoA carboxylase activity was 12- to 29-fold greater than in the livers of fasted animals. Neither tumor studied showed any change in enzyme levels upon refeeding 48-hour fasted animals; 1.4 mµmoles of malonyl-CoA were formed per mg per min for hepatoma 7777 and 3.3 mµmoles of malonyl-CoA were formed per mg per min for hepatoma 9618A. These activities of enzyme are 5- to 10-fold greater than fasting levels in host liver. Fatty acid synthetase levels in host livers increased 12- to 20-fold after refeeding. Again, neither tumor showed significant changes in enzyme activity upon refeeding fasted animals. Both acetyl-CoA carboxylase and fatty acid synthetase from hepatoma were inhibited by palmityl-CoA despite their lack of response to dietary alteration. Acetyl-CoA carboxylase was purified 164-fold from rat liver and 67-fold from hepatoma 7777 to define further this lack of regulation. Studies of the purified enzymes disclosed that the tumor enzyme was essentially identical with that derived from liver in terms of heat inactivation, affinities for acetyl-CoA and ATP, activation and aggregation by citrate, product inhibition by malonyl-CoA, pH optima, and intrinsic specific activity as determined from biotin content. These experiments suggest that the acetyl-CoA carboxylase from the tumors is the same enzyme as that from host liver, and that the increased activity in the tumors is due to an increased amount of enzyme rather than a structurally altered enzyme.

Feedback inhibition of key glycolytic enzymes in liver: action of free fatty acids.

Increasing concentrations of sodium octanoate were progressively inhibitory to the activities of glucokinase, hexokinase, phosphofructokinase, and pyruvate kinase. Glucose-6-phosphate and 6-phosphogluconate dehydrogenases were also markedly inhibited. Other enzymes of carbohydrate metabolism such as lactate dehydrogenase, phosphohexose isomerase, and fructose-1,6-diphosphatase were not decreased. Among the key glycolytic enzymes, the inhibition of pyruvate kinase by the fatty acid was most marked. The biological significance of the inhibition of the key glycolytic enzymes is interpreted as a feedback inhibitory mechanism in regulation of fatty acid biosynthesis. The mechanism may function for rapid adaptation by which the organism can use the fatty acid level as a metabolic directional switch in decreasing glycolysis and turning on gluconeogenesis.