A new mechanism of regulation of rat liver acetyl-CoA carboxylase activity

A factor has been found in rat liver supernatant solution which inhibits acetyl-CoA carboxylase activity regardless of the presence or absence of Mg2+ and ATP. Inactivation of the enzyme has been demonstrated via radiochemical and spectrophotometric assay procedures. The inactivation of acetyl-CoA carboxylase is not attributable to either malonyl-CoA decarboxylase activity, to phosphorylation of the enzyme, or to action on substrates or cofactors of the reaction. The activity of the inhibitor is destroyed by heating to 70-80 degrees C for 5 min or by treatment with trypsin. Dialyzing the inhibitor for 24 h at 4 degrees C does not alter its activity in inhibiting acetyl-CoA carboxylase. Hence, it appears that the inhibitor is a regulatory protein that acts directly on acetyl-CoA carboxylase.     

Hepatic zonation of acetyl-CoA carboxylase activity.

The activities of several hepatic enzymes are preferentially zonated to the periportal or perivenous cells of the liver acinus. Employing dual-digitonin-pulse perfusion of rat liver in the study of acetyl-CoA carboxylase (ACC), we have identified a heretofore unrecognized feature of hepatic zonation, namely an intrahepatic gradient in enzyme specific activity. ACC activity shows a relative periportal localization in normally feeding rats, even when corrected for ACC protein mass. In contrast with results previously reported by us [Evans, Quistorff & Witters (1989) Biochem. J. 259, 821-829], the total mass of both hepatic ACC isoenzymes was not found to differ between the two hepatic zones in the present study. In perfusion eluates from fed animals, periportal ACC displays enhanced citrate reactivity and two kinetic components of acetyl-CoA reactivity; the largest periportal/perivenous gradient (5-fold) is accounted for by a species with a lower Km for acetyl-CoA. The zonal gradient in ACC maximal velocity, measured in eluates from fed rats, does not persist after ACC purification, although the isolated periportal enzyme, like dephosphorylated ACC, has a lower activation constant for citrate. Total ACC protein phosphatase activity is higher in periportal eluates, but no differences in the activities of either a 5'-AMP-activated ACC kinase or the cyclic-AMP-dependent protein kinase are noted between the hepatic zones. The induction of total hepatic ACC mass and specific activity, on fasting/refeeding with a high-carbohydrate diet, abolishes the periportal/perivenous activity gradient, largely owing to a selective activation of perivenous enzyme. Nutritional induction is also accompanied by a marked alteration in ACC acetyl-CoA kinetics and abolition of the gradient in total ACC phosphatase. These studies indicate that hepatic enzyme zonation, which is often attributed to differential expression of enzyme protein, may result from zonal variations in enzyme specific activity, owing to differences in allosteric regulation and/or covalent modification.     

Measurement of acetyl-CoA carboxylase activity in isolated hepatocytes

An assay is described for acetyl-CoA carboxylase activity in isolated hepatocytes. The assay is based on two principles: The hepatocytes are made permeable by digitonin. 64 micrograms of digitonin per mg of cellular protein were most effective in exposing enzyme activity without a significant effect on mitochondrial permeability. Enzyme activity is measured by coupling the carboxylase reaction to the fatty acid synthase reaction. The advantages offered by this procedure over existing assays are: rapidity, no need to prepare cell extracts, absence of product inhibition, no interference by mitochondrial enzymes, useful in systems with bicarbonate buffers, and simple separation of radioactive substrate from labelled products. Using this coupled enzyme assay a good correlation was observed between changes in the activity of acetyl-CoA carboxylase and changes in the rate of fatty acid synthesis in hepatocytes as effected by short-term modulators.     

A biotin analog inhibits acetyl-CoA carboxylase activity and adipogenesis

Acetyl-CoA carboxylase catalyzes the first committed step in the synthesis of long chain fatty acids. In this study, we observed that treatment of 3T3-L1 cells with biotin chloroacetylated at the 1' nitrogen reduced the enzymatic activity of cytosolic acetyl-CoA carboxylase and concomitantly inhibited the differentiation of 3T3-L1 cells in a dose-dependent manner. Treatment with chloroacetylated biotin blocked the induction of PPARgamma, STAT1, and STAT5A expression that normally occurs with adipogenesis. Moreover, addition of chloroacetylated biotin inhibited lipid accumulation, as judged by Oil Red O staining. Our results support recent studies that indicate that acetyl-CoA carboxylase may be a suitable target for an anti-obesity therapeutic.     

The effects of clofibrate on acetyl-CoA carboxylase activity in hepatocytes

Summary Cytochemical localization techniques and electron microscopy were used to evaluate the effects of clofibrate on acetyl-CoA carboxylase activity. It was demonstrated that the drug inhibited the activity of acetyl-CoA carboxylase in rat hepatocytes. Although the results in one of these experiments were somewhat variable, it is suggested that the inhibition of acetyl-CoA carboxylase may be the mechanism by which clofibrate exerts its hypolipidemic effects.Research supported by USPHS Grants HE 12751, NS 05665 and 00690.Recipient of Career Research Development Award 1K3 GM 28064. The authors would like to express their appreciation to Richard L. Hogg of the Ayerst Laboratories for supplying the drug.     

Sethoxydim affects lipid synthesis and acetyl-CoA carboxylase activity in soybean

With rare exceptions, dicot plastids have been reported to contain only a multisubunit (prokaryotic) form of acetyl-coA carboxylase (ACCase), the first committed step of lipid biosynthesis. The sensitivity of most monocots to cyclohexanediones (CHDs) such as sethoxydim, has been shown to be associated with the presence in their plastids of a multifunctional (eukaryotic) form of ACCase. Little is known about the effects of sethoxydim on lipid metabolism and ACCase activity in dicots. Here it is shown that foliar lipid biosynthesis is differentially affected by the herbicide treatment in two dicot species, Nicotiana sylvestris (wild tobacco) and Glycine max (soybean). In N. sylvestris, the total lipid content of neoformed leaves harvested 2 weeks after the sethoxydim treatment was unaffected by doses of up to 10(-3) M sethoxydim. In soybean, lipid content decreased by 45% when 10(-5) M sethoxydim was used, and this was associated with a 30% reduction in fatty acid synthesis activity. ACCase activity of soybean plastidial preparations was 60% reduced in the presence of sethoxydim, whereas that of N. sylvestris was unaffected. Finally, the presence of a biotinylated 220 kDa polypeptide, corresponding in size to multifunctional ACCase, was observed in soybean plastids. Possible relationships between sensitivity of plastidial soybean ACCase towards sethoxydim, plastidial protein content, and altered de novo lipid biosynthesis in herbicide-treated plants are discussed.     

The polymerization of acetyl-CoA carboxylase

Citrate, an allosteric activator of acetyl-CoA carboxylase, induces polymerization of an inactive protomeric form of the enzyme into an active filamentous form composed of 10-20 protomers. The light-scattering properties of the carboxylase were used to study the kinetics of its polymerization and depolymerization. From stopped flow kinetic studies, we have established that polymerization is a second order process, with a second order rate constant of 597,000 M-1 s-1. There appear to be two steps which limit polymerization of the inactive carboxylase protomer: 1) a rapid citrate-induced conformational change which is independent of enzyme concentration and leads to an active protomeric form of the enzyme (Beaty, N. B., and Lane, M. D. (1983) J. Biol. Chem. 258, 13043-13050, preceding paper) and 2) the dimerization of the active protomer, which constitutes the first step of polymerization and is enzyme concentration-dependent. Dimerization is the rate-limiting step of acetyl-CoA carboxylase polymerization. Depolymerization of fully polymerized acetyl-CoA carboxylase is caused by malonyl-CoA, ATP X Mg, and Mg2+. Both malonyl-CoA and ATP X Mg (and HCO-3) compete with citrate in the maintenance of a given state of the protomer-polymer equilibrium apparently by carboxylating the enzyme to form enzyme-biotin-CO-2 which destablizes the polymeric form. Free citrate is the species responsible for polymerizing the enzyme and Mg2+ causes depolymerization of the enzyme by lowering the concentration of free citrate.     

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.     

Changes in the activity of acetyl-CoA carboxylase during rape-seed formation.

During the formation of rape-seeds, lipid accumulated in the cotyledons from 16 days after pollination, rising to a plateau after 28 days. The accumulation of lipid was preceded by a marked rise in acetyl-CoA carboxylase activity, which declined rapidly, correlating with the decline in rate of lipid formation. Incubation of rape-seed extracts with avidin-agarose resulted in a decrease in acetyl-CoA carboxylase activity in the extract. Polyacrylamide-gel electrophoresis of polypeptides bound to avidin-agarose showed the presence of a polypeptide of Mr 225 000. The intensity of this band increased during the period of increase of acetyl-CoA carboxylase activity in the seeds.     

Stimulation by a tumor-promoting phorbol ester of acetyl-CoA carboxylase activity in isolated rat hepatocytes

Acetyl-CoA carboxylase (EC 6.4.1.2) in hepatocytes from meal-fed rats was activated by phorbol myristate acetate (PMA) in a time- and concentration-dependent fashion. This activation can account for the PMA-induced stimulation of de novo fatty acid synthesis. Purified rat-liver acetyl-CoA carboxylase was found to be phosphorylated and activated by protein kinase C, thus providing a possible mechanism for the metabolic action of PMA in intact hepatocytes.     

Hormonal regulation of acetyl-CoA carboxylase activity in the liver cell.

Chick liver cell monolayers synthesize fatty acids at in vivo rates and are responsive to insulin and glucagon. High rates of fatty acid synthesis are maintained with insulin present and lost slowly without insulin. Glucagon or 3',5'-cyclic AMP cause immediate cessation of fatty acid synthesis. The site of inhibition appears to be cytoplasmic acetyl-CoA carboxylase which catalyzes the first committed step of fatty acid synthesis. Liver carboxylase exists either as catalytically inactive protomers or active filamentous polymers. Citrate, an allosteric activator of the enzyme, is required for both catalysis and polymerization. Glucagon and cAMP cause an immediate decrease in the cytoplasmic citrate concentration of chick liver cells apparently by inhibiting the conversion of glucose to citrate at the phosphofructokinase reaction. Since fatty acid synthesis and citrate level are closely correlated, citrate appears to be a feed-forward activator of the carboxylase in vivo. Compelling evidence indicates that carboxylase filaments are present in the intact cell when citrate levels are high and depolymerize when citrate levels fall. Hence, carboxylase activity and fatty acid synthetic rate appear to be determined by cytoplasmic citrate level.     

Regulation of acetyl-CoA carboxylase by ADP-ribosylation

Because of certain similarities between acetyl-CoA carboxylase (ACC) and tubulin, and the recent demonstration of the ADP-ribosylation of tubulin by cholera toxin, we have investigated a potential role for ADP-ribosylation in the regulation of ACC activity. Incubation of purified rat liver ACC with cholera toxin in the presence of millimolar concentrations of [adenylate-32P]NAD results in a time-dependent incorporation of ADP-ribose into ACC of greater than 2 mol/mol of enzyme subunit, accompanied by a marked inactivation of enzyme activity. This effect is not mimicked by pertussis toxin, ADP-ribose, or ribose 5-phosphate. Incubation of labeled ACC with snake venom phosphodiesterase and alkaline hydrolysis release 32P-products tentatively identified by high-performance liquid chromatography as 5'-[32P]AMP and [32P]ADP-ribose, respectively. These data are consistent with a mono-ADP-ribosylation of ACC catalyzed by cholera toxin. Phosphodiesterase treatment of inactivated ACC partially restores enzyme activity. The effects of ADP-ribosylation of ACC are expressed both as a decrease in the enzyme Vmax and as an increase in the apparent Ka for citrate. These results suggest that ACC might be a substrate for endogenous ADP-ribosyltransferases and that this covalent modification could be an important regulatory mechanism for the modulation of fatty acid synthesis in vivo.     

An autocrine factor from Reuber hepatoma cells that stimulates DNA synthesis and acetyl-CoA carboxylase. Characterization of biologic activity and evidence for a glycan structure

Conditioned medium from Reuber H-35 or Fao hepatoma cells contains autocrine factors that both stimulate DNA synthesis and activate acetyl-coenzyme A (CoA) carboxylase in serum-deprived Fao cells. The factor(s), which appears within 4 h of serum-free culture, also increases the cell number and the mitotic index. The effects of the conditioned medium are insulinomimetic, both with respect to stimulation of DNA synthesis and acetyl-CoA carboxylase activity. However, no induction of tyrosine aminotransferase activity or stimulation of aminoisobutyric acid uptake is seen in response to the conditioned medium. Insulin over a 4-h period does not increase the concentration of DNA synthesis stimulating activity that is observed in the medium. This activity is dialyzable and is resistant to acid treatment or to heating to 60-100 degrees C and to trypsin digestion; it is not extracted with chloroform/methanol nor adsorbed by charcoal or by a C18 reverse-phase column. Fractionation of the conditioned medium derived from Reuber H-35 hepatoma cells by gel filtration chromatography reveals two low molecular weight (less than 1000) compounds that both stimulate DNA synthesis in Fao hepatoma cells. The larger compound (peak I) also stimulates the activity of acetyl-CoA carboxylase. The stimulatory effects of the peak I compound are destroyed by nitrous acid deamination, periodate oxidation, and methanolysis. Biosynthetic labeling studies indicate the probable presence of glucosamine, galactose, and perhaps phosphate in the peak I-activating material. No significant incorporation of either myoinositol or mannose into the active material has been observed. These data, taken together, are consistent with a glycan structure for this autocrine factor, which bears strong resemblance to similar insulinomimetic factors generated in BC3H1 myocytes and H-35 hepatoma cells in response to insulin and on digestion of membranes with a phosphatidylinositol-specific phospholipase C. Further characterization of this factor may provide insight into different pathways of insulin action and could provide a strategy to check autocrine-stimulated tumor growth.     

Bimodal activation of acetyl-CoA carboxylase by glutamate

Acetyl-CoA carboxylase (ACC) catalyzes the formation of malonyl-CoA, an essential substrate for fatty acid biosynthesis and a potent inhibitor of fatty acid oxidation. Here, we provide evidence that glutamate may be a physiologically relevant activator of ACC. Glutamate induced the activation of both major isoforms of ACC, prepared from rat liver, heart, or white adipose tissue. In agreement with previous studies, a type 2A protein phosphatase contributed to the effects of glutamate on ACC. However, the protein phosphatase inhibitor microcystin LR did not abolish the effects of glutamate on ACC activity. Moreover, glutamate directly activated purified preparations of ACC when protein phosphatase activity was excluded. Phosphatase-independent ACC activation by glutamate was also reflected by polymerization of the enzyme as judged by size-exclusion chromatography. The sensitivity of ACC to direct activation by glutamate was diminished by treatment in vitro with AMP-activated protein kinase or cAMP-dependent protein kinase or by beta-adrenergic stimulation of intact adipose tissue. We conclude that glutamate, an abundant intracellular amino acid, induces ACC activation through complementary actions as a phosphatase activator and as a direct allosteric ligand for dephosphorylated ACC. This study supports the general hypothesis that amino acids fulfill important roles as signal molecules as well as intermediates in carbon and nitrogen metabolism.