Hormonal regulation of fatty acid synthetase, acetyl-CoA carboxylase and fatty acid synthesis in mammalian adipose tissue and liver.

The major objectives of this study were to define the roles of adrenal glucocorticoids and glucagon in the long-term regulation of fatty acid synthetase and acetyl-CoA carboxylase of mammalian adipose tissue and liver. Particular emphasis was given to elucidation of the mechanisms whereby these hormones produce their regulatory effects on enzymatic activity. To dissociate mental manipulation, nutritional conditions were ridgidly controlled in the experiments described. Administration of glucocorticoids to adult rats led to a marked reductionin activities of fatty acid synthetase and carboxylase in adipose in adipose tissue but no change occurred in liver. Adrenalectomy produced an increase in activities of these lipogenic enzymes in adipose tissure, but, again, no change was noted in liver. The decrease in enzymatic activities in adipose tissue with glucocorticoid administration correlated well with a decrease in fatty acid synthesis, determined in vivo by the 3-H2O method. The mechanisms whereby glucocorticoids led to a decrease in fatty acid synthetase activity were elucidated by the use of immunochemical techniques. Thus, the decrease in fatty acid synthetase activity observed in adipose tissue was shown to reflect a decrease in content of enzyme, and not a change in catalytic efficiency. The mechanism underlying the decrease in enzyme content is a decrease in synthesis of the enzyme. The relation of the effects of glucocorticoids to the effects of certain other hormones involved in regulation of lipogenesis was investigated in hypophysectomized and in diabetic animals. Thus, the observation that the glucocorticoid effect on synthetase and carboxylase occurred in adipose tissue of hypophysectomized rats indicated that alterations in levels of other pituitary-regulated hormones were not necessary for the effect. That glucocorticoids play some role in regulation of synthetase and carboxylase in liver, at lease in the diabetic state, was shown by the observation that the low activities of these enzymes in diabetic animals could be restored to normal by adrenalectomy. An even more pronounced restorative effect was apparent in adipose tissue of adrenalectomized, diabetic animals. Administration of glucagon during the refeeding of starved rats resulted in a marked reduction in the induction of fatty acid synthetase, acetyl-CoA carboxylase and in the rate of incorporation of 3-H from 3-H2O into fatty acids in liver, but no change in these parameters occurred in adipose tissue. Administration of theophylline resulted in intermediate reduction in liver. The mechanisms whereby glucagon led tto a decrease in fatty acid synthetase activity were elucidated by the use of immunochemical techniques. Thus, the changes in fatty acid synthetase activity were shown to reflect reductions in content of enzyme. The mechanism underlying these reductions in content is reduced synthesis of enzyme.     

Long-term regulation by theophylline of fatty acid synthetase, acetyl-CoA carboxylase and lipid synthesis in cultured glial cells.

The long-term regulation of fatty acid synthetase and acetyl-CoA carboxylase and of fatty acid and sterol synthesis was studied in C-6 glial cells in culture. When theophylline (10(-3) M) was added to the culture medium of these cells, rates of lipid synthesis from acetate and activities of synthetase and carboxylase became distinctly lower than in cells that were untreated. This effect appeared after approximately 12 h, and after 48 h enzymatic activities were reduced approx. 2-fold and rates of lipid synthesis from acetate 3- to 4-fold. The likelihood that the decrease in fatty acid synthesis from acetate was caused by the decrease in activities of fatty acid synthetase and acetyl-CoA carboxylase was established by several observations. These indicated that the locus of the effect probably did not reside at the level of acetate uptake into the cell, alterations in acetate pool sizes or conversion of acetate to acetyl-CoA. Moreover, de novo fatty acid synthesis was found to be the predominant pathway in these glial cells, whether treated with theophylline or not. The mechanism of the effect of theophylline on fatty acid synthetase was shown by immunochemical techniques to involve an alteration in content of enzyme rather than in catalytic efficiency. The change in content of fatty acid synthetase was shown by isotopic-immunochemical experiments to involve a decrease in synthesis of the enzyme. The mechanism whereby theophylline leads to a decrease in lipogenesis and in the synthesis of fatty acid synthetase may not be mediated entirely by inhibition of phosphodiesterase and an increase in cyclic AMP levels, because dibutyryl cyclic AMP (10(-3) M) only partially reproduced the effect.     

Regulation of palmitic acid synthesis in cultured glial cells: effects of glucocorticoid on fatty acid synthetase, acetyl-CoA carboxylase, fatty acid and sterol synthesis.

Abstract— C-6 glial cells in culture were utilized to define the role of glucocorticoid in the regulation of palmitic acid synthesis and the important lipogenic enzymes, fatty acid synthetase and acetyl-CoA carboxylase. Particular emphasis was given to fatty acid synthetase which exhibited more than a 50% reduction in specific activity when cells were exposed to hydrocortisone (10 μg/ml) for 1 week. Coordinate changes in acetyl-CoA carboxylase activity and in palmitic acid (and sterol) synthesis from acetate accompanied the alterations in fatty acid synthetase. Immunochemical techniques were utilized to show that the decrease in synthetase activity involved an alteration in enzyme content, not in catalytic efficiency. The changes in content of fatty acid synthetase were caused by alterations in enzyme synthesis. Glucocorticoids may regulate fatty acid synthetase in C-6 glial cells by a mechanism similar to that suggested for adipose tissue. The inhibition of palmitic acid synthesis may be relevant to other effects of glucocorticoids on developing brain.     

Regulation of palmitic acid synthesis in cultured glial cells: effects of lipid on fatty acid synthetase, acetyl-CoA carboxylase, fatty acid and sterol synthesis.

Abstract— C6 glial cells in culture were utilized to study the regulation of the important lipogenic enzymes, fatty acid synthetase and acetyl-CoA carboxylase, and the synthesis of fatty acids and sterols. Regulation of these phenomena by lipid was demonstrated by the following observations. First, removal of serum from the culture medium was accompanied over the next five days by 2–3-fold increases in the lipogenic enzymatic activities and in 5–15-fold increases in rates of incorporation of acetate into fatty acids and sterols. Second, cells grown in delipidated serum exhibited approx 2-fold higher levels of activity of the lipogenic enzymes and 5–10-fold higher rates of synthesis of fatty acids and sterols than cells grown in normal calf serum. Third, cells grown in serum-free medium supplemented with concentrations of fatty acid comparable to those present in medium supplemented with serum exhibited activities of fatty acid synthetase comparable to those exhibited by cells grown in the serum-supplemented medium. The mechanism of these effects on fatty acid synthetase was shown by immunochemical techniques to involve alterations in content rather than in catalytic efficiency of the enzyme. The changes in content of the synthetase were caused by alterations in enzyme synthesis. In view of morphological and biochemical data suggesting that C6 cells are related to differentiating cells with properties of both astrocytes and oligodendroglia, the present data may indicate that regulation of palmitic acid synthesis by fatty acid or a product thereof occurs in brain during development.     

Studies on the synthesis of rat liver fatty acid synthetase.

The synthesis of the multienzyme complex rat liver fatty acid synthetase was investigated utilizing modifications of methods developed in the laboratory of Schimke (Schimke, R. T. (1964) J. Biol. Chem. 239, 3808-3817 and Arias, I. M., Doyle, D., and Schimke, R. T. (1969) J. Biol. Chem. 244, 3303-3315). The relative amounts of radioactivity from a pulse of labeled lysine appearing in polypeptides derived from purified synthetase complex can be measured compensating for the varying amounts of lysine per polypeptide chain. The results show that labeled amino acid is incorporated into polypeptides derived from the complex at heterogeneous rates. However, 10 to 15 hours after the administration of a pulse, the amount of label per lysine residue in these polypeptides is identical. The results support the previously proposed model of this multienzyme complex (Tweto, J., Dehlinger, P., and Larrabee, A. R. (1972) Biochem. Biophys. Res. Commun. 48, 1371-1377). The previous work and that reported here suggests the existence of a pool of synthetase subunits which is an obligatory intermediate in both synthesis and turnover of the complex. The results obtained in this work are consistent with this model if the exchange of subunits into the intact complex is a relatively slow process requiring several hours to reach equilibrium.     

Fatty acid-mediated disaggregation of acetyl-CoA carboxylase in isolated liver cells

In recent years the rapid regulation of acetyl-CoA (AcCoA) carboxylase (EC 6.4.1.2) has become of major interest because of the important role of malonyl-CoA in fatty acid synthesis, ketogenesis, and triglyceride production. AcCoA carboxylase is acutely regulated by two mechanisms: 1) phosphorylation-dephosphorylation and 2) polymer-protomer transition. Until recently polymer-protomer transition of AcCoA carboxylase in vivo has escaped detection. We developed a technique that estimates the intracellular proportion of polymer and protomer forms of AcCoA carboxylase based on the differential sensitivity of polymeric and protomeric AcCoA carboxylase to avidin inactivation. When the enzyme is in its highly aggregated conformation, the biotin prosthetic group of AcCoA carboxylase is protected from avidin binding. Thus the polymeric AcCoA carboxylase is more resistant than the protomeric conformation to avidin inactivation. Utilizing this technique with isolated liver cells we have been able to develop a model for the involvement of free fatty acids and glucagon in regulating polymer-protomer transition of AcCoA carboxylase, and the role of polymer as an intracellular determinant of AcCoA carboxylase activity. Our data suggest that the physiological regulation of AcCoA carboxylase involves the interaction of the phosphorylation mechanism with fatty acid-induced depolymerization. We propose that during periods of food deprivation the elevation in fatty acid-CoA esters promotes depolymerization of AcCoA carboxylase. In addition, glucagon induces phosphorylation of AcCoA carboxylase, which inhibits the enzyme's activity and facilitates acyl-CoA binding and depolymerization. The two separate mechanisms for regulating hepatic AcCoA carboxylase may work in concert to modulate the level of the regulatory metabolite malonyl-CoA.     

A novel acetyl-CoA carboxylase inhibitor reduces de novo fatty acid synthesis in HepG2 cells and rat primary hepatocytes

To identify the novel inhibitor of de novo lipogenesis in hepatocytes, we screened for inhibitory activity of triglyceride (TG) synthesis using [¹⁴C]acetate in the human hepatoma cell line, HepG2. Using this assay system we discovered the novel compound, benzofuranyl α-pyrone (TEI-B00422). TEI-B00422 also inhibited the incorporation of acetate into the triglyceride (TG) fraction in rat primary hepatocytes. In HepG2 cells, the incorporation of oleate into TG was unaffected. TEI-B00422 inhibited rat hepatic acetyl-CoA carboxylase (ACC), K_i = 3.3 μM, in a competitive manner with respect to acety-CoA but not fatty acid synthase and acyl-CoA transferase/diacylglycerol. Thus, these results suggest that the inhibition of TG synthesis by TEI-B00422 is based on the inhibitory action of ACC. The structure of TEI-B00422 is totally different from the known inhibitors of ACC and may be useful in the development of therapeutic agents to combat a number of metabolic disorders.     

Interaction of acetyl-CoA fragments with rat liver acetyl-CoA carboxylase

The interaction of acetyl-CoA fragments with rat liver acetyl-CoA carboxylase has been studied. Dephosphorylated acetyl-CoA did not actually differ from acetyl-CoA in its substrate properties. Non-nucleotide analogues of the substrate, S-acetylpantatheine and it's 4'-phosphate, also possess substrate properties (Vmax = 1.5% and 15% of the maximal rate value of acetyl-CoA carboxylation, respectively). The nucleotide fragment in the acetyl-CoA molecule produces a marked effect on the thermodynamics of the substrate-enzyme interaction, and is apparently involved in activation and appropriate orientation of the acetyl group in the active site. The better substrate properties of S-acetylpantetheine 4'-phosphate and the inhibitory properties of pantetheine 4'-phosphate, compared to the unphosphorylated analogues, evidence an important role of the 5'-beta-phosphate of 3'-phosphorylated ADP residue in acetyl-CoA binding to the enzyme.     

Purification and physicochemical properties of fatty acid synthetase and acetyl-CoA carboxylase from lactating rabbit mammary gland.

Fatty acid synthetase was purified 13-fold from lactating rabbit mammary glands by a procedure which involved chromatography on DEAE-cellulose, ammonium sulphate precipitation and gel filtration on Sepharose 4B. The preparation was completed within two days and over 100 mg of enzyme was isolated from 100--150 g of mammary tissue, which represented a yield of over 40%. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis and ultracentrifugal analysis. The sedimentation constant, S20,w was 13.3 S, the absorption coefficient, A280nm1%, measured refractometrically was 10.0 +/- 0.1, and the amino acid composition was determined. The subunit molecular weight determined by gel electrophoresis in the presence of sodium dodecyl sulphate was 252,000 +/- 6,000, and the molecular weight of the native enzyme measured by sedimentation equilibrium was 515,000. These experiments indicate that at the concentrations which exist in mammary tissue (2--4 mg/ml) fatty acid synthetase is a dimer. The purified enzyme did however show a tendency to dissociate to a monomeric 9-9S species on storage for several days or following exposure to a low ionic strength buffer at pH 8.3. There was only a small quantity of alkali labile phosphate (0.2 molecules per subunit) bound covalently to the purified enzyme. Acetyl-CoA carboxylase was purified 300-fold in a 50% yield within 24 h by ammonium sulphate and polyethylene glycol precipitations [Hardie, D.G. and Cohen, P. (1978) FEBS Lett. 91, 1--7]. The preparation was in a state approaching homogeneity as judged by polyacrylamide gel electrophoresis, gel filtration on Sepharose 4B and ultracentrifugal analysis. The sedimentation constant, S20,w, was 50.5 S, the absorption index, A280nm1%, was 14.5 +/- 0.7, and the amino acid composition was determined. The subunit molecular weight of acetyl-CoA carboxylase determined by gel electrophoresis in the presence of sodium dodecyl sulphate was identical to that of fatty acid synthetase (252,000) as shown by electrophoresis of a mixture of the two proteins. The preparations also contained two minor components of molecular weight 235,000 and 225,000, which appear to be derived from the major species of mol. wt 252,000. A large emount of phosphate (3.2 molecules per subunit) was found to be bound covalently to the purified enzyme. The properties of fatty acid synthetase and acetyl-CoA carboxylase are compared to those obtained by other workers.     

Role of reversible phosphorylation of acetyl-CoA carboxylase in long-chain fatty acid synthesis

Acetyl-CoA carboxylase, the rate-limiting enzyme in the biogenesis of long-chain fatty acids, is regulated by phosphorylation and dephosphorylation. The major phosphorylation sites that affect carboxylase activity and the specific protein kinases responsible for phosphorylation of different sites have been identified. A form of acetyl-CoA carboxylase that is independent of citrate for activity occurs in vivo. This active form of carboxylase becomes citrate-dependent upon phosphorylation under conditions of reduced lipogenesis. Therefore, phosphorylation-dephosphorylation of acetyl-CoA carboxylase is the enzyme's primary short-term regulatory mechanism; this control mechanism together with cellular metabolites such as CoA, citrate, and palmitoyl-CoA serves to fine-tune the synthesis of long-chain fatty acids under different physiological conditions.     

Cell-free synthesis of pigeon liver fatty acid synthetase.

Pigeon liver fatty acid synthetase proteins (apo- and holo-forms) have been synthesized in a cell-free system reconstituted from polysomes and a soluble enzyme fraction. Identification of the cell-free synthesized products as fatty acid synthetase was achieved by affinity chromatography, by immuno-precipitation and by the simultaneous conversion of both the authentic carrier protein and the $\text{in vitro}$ synthesized products from the holo- to the apo-form of the synthetase. The reverse conversion was also effected.