Reevaluation of properties of acetyl-CoA carboxylase from rat liver

Rat liver acetyl-CoA carboxylase can be rapidly isolated by a new procedure which uses avidin-Sepharose affinity chromatography. The isolated enzyme has Mr = 260,000; none or very little of the proteolytic products of the carboxylase which are formed in conventional purification procedures are found in our preparations. It is apparent that the previously reported subunit of the carboxylase, with Mr = 230,000, is itself the product of proteolysis. The properties of the enzyme produced by our new method are quite different from those of the conventionally prepared enzyme. Our enzyme contains 6 mol of alkali-labile phosphate/mol of subunit, rather than 2 mol; the Km for acetyl-CoA is about 8-fold higher and the specific activity is only about one-fifth of that previously reported. The large amount of phosphate does not appear to cause the low specific activity of the new enzyme preparation, because alkaline phosphatase treatment reduces the number of phosphates/subunit from 6 to 3 mol but does not change the specific activity.     

The reversibility of cytosolic dehydrogenase reactions in hepatocytes from starved and fed rats. Effect of fructose.

The metabolism of [2-3H]lactate was studied in isolated hepatocytes from fed and starved rats metabolizing ethanol and lactate in the absence and presence of fructose. The yields of 3H in ethanol, water, glucose and glycerol were determined. The rate of ethanol oxidation (3 mumol/min per g wet wt.) was the same for fed and starved rats with and without fructose. From the detritiation of labelled lactate and the labelling pattern of ethanol and glucose, we calculated the rate of reoxidation of NADH catalysed by lactate dehydrogenase, alcohol dehydrogenase and triosephosphate dehydrogenase. The calculated flux of reducing equivalents from NADH to pyruvate was of the same order of magnitude as previously found with [3H]ethanol or [3H]xylitol as the labelled substrate [Vind & Grunnet (1982) Biochim. Biophys. Acta 720, 295-302]. The results suggest that the cytoplasm can be regarded as a single compartment with respect to NAD(H). The rate of reduction of acetaldehyde and pyruvate was correlated with the concentration of these metabolites and NADH, and was highest in fed rats and during fructose metabolism. The rate of reoxidation of NADH catalysed by lactate dehydrogenase was only a few per cent of the maximal activity of the enzymes, but the rate of reoxidation of NADH catalysed by alcohol dehydrogenase was equal to or higher than the maximal activity as measured in vitro, suggesting that the dissociation of enzyme-bound NAD+ as well as NADH may be rate-limiting steps in the alcohol dehydrogenase reaction.     

Quantitation of mitochondrial DNA,RNA, and protein in starved and starved-refed rat liver

The purpose of this study was to investigate the problem of mitochondrial biogenesis in rat liver. The approach consisted of isolating mitochondria from control, 6 day starved and 6 day starved-5 day refed rats and comparing their DNA, RNA and protein content. This was performed by isolating the mitochondria by reorienting rate zonal centrifugation in sucrose gradients. It was found that six days of starvation resulted in a loss of 30% of the body weight, 55% of the liver weight, 40% of the mitochondrial protein, 60% of the mitochondrial RNA, but only 20% of the mitrochondrial DNA. It was also shown that refeeding of the rats for five days resulted in a restoration to normal or near normal levels in all the parameters measured. Further experiments employing the incorporation of ³H-TTP into isolated mitochondria indicated that the maintenance of mitochondrial DNA was not the result of continuous DNA synthesis.     

Phosphoinositide inhibition of acetyl-CoA carboxylase from rat liver

When purified acetyl-CoA carboxylase was incubated with various phospholipids, the effects on carboxylase activity were quite diverse. Phosphatidic acid, phosphatidylcholine, and phosphatidylinositol were slightly stimulatory, whereas carboxylase was inhibited by polyphosphoinositides in a time- and concentration-dependent manner. Phosphatidylinositol 4,5-bisphosphate (TPI) was the most effective inhibitor; carboxylase activity was inhibited 50% after incubation with 1.5 μm TPI for 30 min. Incubation of carboxylase with citrate reduced the susceptibility to inhibition by TPI. The inhibition was reversed by removal of TPI from the inhibited enzyme. Incubation of TPI with divalent metal cations removed its ability to inhibit carboxylase. Sedimentation studies showed that TPI treatment shifts carboxylase to a less-polymerized form. The K_m for ATP, 24 μm, was not affected by the inhibitor. However, the apparent K_m for acetyl-CoA was decreased from 44 to 11 μm following incubation with TPI. The possibility that polyphosphoinositides may play a role in acetyl-CoA carboxylase regulation is discussed.     

Quantification of liver and kidney phosphofructokinase by radioimmunoassay in fed, starved and alloxan-diabetic rats.

A newly developed specific radioimmunoassay was used to quantify phosphofructokinase protein directly and independently of assayable activity in liver and kidney cytosol of normal fed, starved and alloxan-diabetic rats. In the fed state, liver phosphofructokinase concentration was 0.096 microM and the kidney enzyme was 0.086 microM (mumol/kg of tissue). In the starved state (24h), liver and kidney phosphofructokinase concentrations decreased by 30%. Prolonged starvation up to 72h did not further decrease enzyme concentration. In liver, total enzyme content during starvation declined by more than 50%, secondary also to a decrease in liver weight. In the alloxan-diabetic rats, there was a 22% decrease in enzyme protein concentration in liver and kidney. Total enzyme content per liver actually decreased much more (46%), because diabetes also resulted in a decrease in liver size. In conjunction with assayable activity measurements, the results of the radioimmunoassay allowed us to calculate the apparent specific activity of the enzyme. The specific activity of the kidney enzyme was 2-3 times that of the liver. Little or no change in specific activity of the liver or kidney enzyme occurred as a result of starvation or chemically induced diabetes. Tissue enzyme concentrations of phosphofructokinase unequivocally reconcile the ultimate results of changing rates of synthesis and degradation and are useful data in the design of spectrophotometric, kinetic, aggregation-disaggregation and other studies.     

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.     

Influence of ethanol on the liver metabolism of fed and starved rats

1. The influence of ethanol on the metabolism of livers from fed and starved rats has been studied in liver-perfusion experiments. Results have been obtained on oxygen consumption and carbon dioxide production, on glucose release and uptake by the liver and on changes in the concentrations of lactate and pyruvate and of β-hydroxybutyrate and acetoacetate in the perfusion medium. 2. Oxygen consumption and carbon dioxide production were lower in livers from starved rats than in livers from fed rats. Ethanol had no effect on the oxygen consumption of either type of liver. After the addition of ethanol to the perfusion medium carbon dioxide production ceased almost completely, the change being faster in livers from starved rats. 3. With livers from fed rats glucose was released from the liver into the perfusion medium. This release was slightly greater when ethanol was present. With livers from starved rats no release of glucose was observed, and when ethanol was added a marked uptake of glucose from the medium was found. A simultaneous release of glycolytic end products, lactate and pyruvate, into the medium occurred. 4. Acetate was the main metabolite accumulating in the perfusion medium when ethanol was oxidized. With livers from starved rats a slightly increased formation of ketone bodies was found when ethanol was present. 5. The lactate/pyruvate concentration ratio in the perfusion medium increased from 10 to 87 with livers from fed rats and from 20 to 171 with livers from starved rats when the livers were perfused with ethanol in the medium. The β-hydroxybutyrate/acetoacetate concentration ratio increased from 0·8 to 7·6 with livers from fed rats and from 1·0 to 9·5 with livers from starved rats when ethanol was added to the medium. 6. The effects of ethanol are discussed and related to changes in the redox state of the liver that produce new conditions for some metabolic pathways.     

The content of pentose-cycle intermediates in liver in starved, fed ad libitum and meal-fed rats.

Liver content of pentose-cycle intermediates and the activity of the three major cytoplasmic NADPH-producing enzymes and pentose-cycle enzymes were measured in three dietary states: 48 h-starved rats, rats fed on a standard diet ad libitum, and rats meal-fed with a low-fat high-carbohydrate diet. Measured tissue contents of pentose-cycle intermediates in starved liver were: 6-phosphogluconate, 4.7 +/- 0.5 nmol/g; ribulose 5-P, 3.7 +/- 0.5 nmol/g; xylulose 5-P, 4.3 +/- 0.4 nmol/g; sedoheptulose 7-P, 25.5 +/- 1.3 nmol/g; and combined sedoheptulose 7-P and ribose 5-P, 30.6 +/- 0.7 nmol/g. These values were in good agreement with values calculated from fructose 6-P and free glyceraldehyde 3-P, assuming the major transketolase, transaldolase, ribulose-5-P 3-epimerase and ribose-5-P isomerase reactions were all in near-equilibrium. Similar results were found in animals fed ad libitum. These relationships were not valid in animals fed on a low-fat high-carbohydrate diet, with tissue contents of metabolites in some cases being more than an order of magnitude higher than the calculated values. Measured tissue contents of pentose-cycle intermediates in these animals were: 6-phosphogluconate, 124.2 +/- 13.9 nmol/g; ribulose 5-P, 44.8 +/- 7.1 nmol/g; xylulose 5-P, 77.2 +/- 9.4 nmol/g; sedoheptulose 7-P, 129.9 +/- 10.1 nmol/g; and combined sedoheptulose 7-P and ribose 5-P, 157.0 +/- 11.3 nmol/g. In all animals, regardless of dietary state, tissue content of erythrose 4-P was less than 2 nmol/ml. Liver activities of glucose-6-P dehydrogenase and 6-phosphogluconate dehydrogenase were increased from 3.5 +/- 0.9 mumol/g and 7.3 +/- 0.5 mumol/min per g in starved animals to 13.2 +/- 1.1 and 10.5 +/- 0.7 mumol/min per g in low-fat high-carbohydrate-fed animals. Despite these changes, the activities of transaldolase (3.4 +/- 0.3 mumol/min per g), transketolase (7.8 +/- 0.2 mumol/min per g) and ribulose-5-P 3-epimerase (7.5 +/- 0.4 mumol/min per g) were not increased in meal-fed animals above those observed in starved animals (3.4 +/- 0.2, 7.1 +/- 0.3 and 8.6 +/- 0.4 mumol/min per g respectively). The increase in the activity of oxidative pentose-cycle enzymes in the absence of any change in the non-oxidative pentose cycle appeared to contribute to the observed disequilibrium in the pentose cycle in animals meal fed on a low-fat high-carbohydrate diet.     

Regulation of fatty acid synthesis in lactating rat mammary gland in the fed to starved transition: asynchronous control of pyruvate dehydrogenase, phosphofructokinase and acetyl-CoA carboxylase.

1. Withdrawal of food from lactating rats produced a rapid and dramatic decrease in the uptake of glucose by the mammary gland and an inhibition of the rate of fatty acid synthesis that could not be explained alone by decreased substrate supply to the tissue. 2. Within the first 6 hr starvation, fatty acid synthesis and pyruvate dehydrogenase activity were inhibited by 87 and 80%, respectively, but acetyl-CoA carboxylase activity did not change significantly. 3. Between 6 and 24 hr starvation, total and expressed activities of acetyl-CoA carboxylase decreased by 62 and 55%, respectively. 4. The ratio of fructose-6-phosphate/fructose-1,6-bisphosphate concentration in mammary tissue increased 9-fold during the first 6 hr starvation, indicating an inhibition of 6-phosphofructo-1-kinase. However, the major inhibition of this enzyme occurred between 6 and 24 hr starvation when this metabolite ratio increased a further 160-fold in parallel with increased tissue citrate concentration. 5. The increase in citrate concentration between 6 and 24 hr starvation correlated with acetyl-CoA carboxylase inactivation and ketone body accumulation in the mammary gland. 6. This study confirms the asynchronous control of three important regulatory steps in the pathway of glucose utilization and fatty acid synthesis in the lactating rat mammary gland.     

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.     

Heat activation of rat liver acetyl-CoA carboxylase in vitro.

Acetyl-CoA carboxylase in rat liver homogenates was activated in vitro in a time- and temperature-dependent manner. The activity of acetyl-CoA carboxylase in rat liver preparations was determined in a 1-min assay to preclude the possibility of citrate activation of the enzyme during the assay period. Activation of the enzyme occurred more rapidly in liver preparations continuously maintained at ambient or greater temperatures than in homogenates of liver which had been chilled. High speed supernatant (105,000 X g, 60 min) did not heat-activate, and reconstitution of the heat-activatable 27,000 X g, 20-min, fraction by recombining the high speed pellet with the high speed supernatant only partially restored the heat activatability. Elution of the 105,000 X g supernatant from Sephadex G-25 resulted in an enzyme preparation which was heat-activatable. Addition of boiled 105,000 X g supernatant to the Sephadex G-25-treated enzyme again prevented heat activation. Dilution of the enzyme 5-fold did not prevent heat activation.     

Mitochondrial acetyl-CoA carboxylase. Time course of mobilization/activation in liver of refed rats.

Fasted (48 h) rats were killed at 0, 2, 4, 6, 8, 12, 16, 20 and 24 h after they were refed on a high-carbohydrate diet. An increase in the maximal activity and quantity of cystolic acetyl-CoA carboxylase was found in liver of refed rats after a lag time of about 8 h. The increased quantity of cytosolic enzyme was attributable primarily to mobilization of mitochondrial storage forms and not to substantial increase in the rate of synthesis of acetyl-CoA carboxylase.     

Chromosome mapping and phylogenetic analysis of the cytosolic acetyl-CoA carboxylase loci in wheat

The cytosolic isoform of plant acetyl-CoA carboxylase is a multidomain enzyme involved in the synthesis of very-long-chain fatty acids and in secondary metabolism. Chromosome mapping of wheat identified one locus containing cytosolic acetyl-CoA carboxylase genes (Acc-2) and a related partially processed pseudogene (Psi-Acc-2) in the distal region of the long arm of wheat homoeologous group 3 chromosomes. Multiple copies of the Acc-2 genes, whose presence was suggested by sequence analysis, are likely to be arranged in tandem repeats. At least three out of five genes cloned from hexaploid wheat map to this locus. Another locus containing Acc-2--related sequences is present in the distal region of the long arm of chromosome 5D. The identity of the hybridizing DNA present at this locus remains unknown. A system based on PCR-cloning and DNA sequence analysis of acetyl-CoA carboxylase genes was developed to address various phylogenetic and systematics questions in grasses. It was applied to reconstruct the phylogeny of the Acc-2 genes from D- and S-genome Aegilops and A-genome Triticum diploid species, AABB- and AAGG-genome tetraploid wheat, and AABBDD-genome hexaploid wheat, as well as from rye and barley. The combined cytogenetic and molecular evolution approach allowed assignment of gene sequences included in phylogenetic analysis to specific loci on homoeologous chromosomes. Recurring gene duplication followed by chromosome translocation and/or possible loss of some gene copies, as well as loss of introns, occurred in the gene family in different plant lineages. Two major Acc-2 clades appeared before the divergence of barley and rye. Nucleotide substitution rates in different parts of the Acc-2 gene were assessed. This analysis of the Acc-2 loci provides detailed information regarding evolutionary events at a low--copy-number locus containing important functional genes. These events are likely to be common and to play a significant role in shaping grass genomes.     

Changes in the proportion of acetyl-CoA carboxylase in the active form in rat liver. Effect of starvation, lactation and weaning

1. The activity of acetyl-CoA carboxylase (EC 6.4.1.2) in extracts of freeze-clamped liver samples from fed or 24 h-starved virgin, pregnant, lactating and weaned rats was measured (i) immediately after preparation of extracts (;I activity'), (ii) after incubation of extracts with partially purified preparations of either rabbit muscle protein phosphatase 1 [Antoniw, Nimmo, Yeaman & Cohen (1977) Biochem. J.162, 423-433] or rabbit liver phosphatase [Brandt, Capulong & Lee (1975) J. Biol. Chem.250, 8038-8044] (;A activity') and (iii) after incubation with 20mm-potassium citrate before or after incubation with phosphatases (;C activity'). 2. Incubation of liver extracts at 30 degrees C without any additions resulted in activation of acetyl-CoA carboxylase that was shown to be due to dephosphorylation of the enzyme by endogenous protein phosphatase activity. This latter activity was not stimulated by Ca(2+) and/or Mg(2+) but was stimulated by 1 mm-Mn(2+). Incubation of extracts with either of the partially purified phosphatases (0.2-0.5 unit) resulted in faster dephosphorylation and activation. The activity achieved after incubation with either of the exogenously added phosphatases was similar. 3. The A and C activities increased during late pregnancy, were lower than in the virgin rat liver during early lactation and increased by 2-fold in liver of mid-lactating rats. Weaning of mid-lactating rats for 24 h resulted in no change in A and C activities but after 48 h weaning they were significantly lower than those in livers from suckled mothers. 4. The I activity followed a similar pattern of changes as the A and C activities during pregnancy and lactation such that, although the I/A and I/C activity ratios tended to be lower during late pregnancy and early lactation, there were no significant changes in I/A and I/C ratios between lactating and virgin animals. However, these ratios were significantly higher in liver from fed 24 h-weaned animals. 5. Starvation (24 h) resulted in a marked decrease in I activity for all animals studied except early-lactating rats. This was due to a combination of a decrease in the concentration of acetyl-CoA carboxylase in liver of starved animals (A and C activities) and a decrease in the fraction of the enzyme in the active form (lower I/C and I/A ratios). The relative importance of the two forms of regulation in mediating the starvation-induced fall in I activity was about equal in livers of virgin, pregnant and lactating animals. However, the decrease in I/A and I/C ratios was of dominating importance in livers of weaned animals. The A/C activity ratios were the same for livers from all animals studied. 6. The maximal activity of fatty acid synthase was also measured in livers and was highly and positively correlated with the A and C activities of acetyl-CoA carboxylase, suggesting that the concentrations of the two enzymes in the liver were controlled coordinately. 7. It is suggested that the lack of correlation between plasma insulin levels and rates of lipogenesis in the transition from the virgin to the lactating state may be explained by different effects of insulin and prolactin on the concentration of acetyl-CoA carboxylase in the liver and on the fraction of the enzyme in the active form.     

Subunit size and paracrystal structure of avian liver acetyl-CoA carboxylase.

The subunit molecular weight of chicken liver acetyl-CoA carboxylase has been redetermined by immunoprecipitation and sodium dodecyl sulfate gel electrophoresis. In the presence of parotid trypsin inhibitor, the immunoprecipitate gave a single band corresponding to a molecular weight of 230,000, which was also found to contain bound biotin. From the biotin content of the protomer (1.0 prosthetic group per 480,000 daltons) it appears that it consists of two non identical subunits, both with molecular weights of approximately 230,000.Electron microscopy has been carried out on the active filamentous form of the enzyme and on paracrystals formed under high-salt conditions. These indicate that the filaments are readily distortable helical ribbons, with an approximate axial repeat of 1100 Å, containing eight protomers. The paracrystals are made up of a staggered lateral packing of filaments.     

Enzymatically inactive forms of acetyl-CoA carboxylase in rat liver mitochondria.

Biotinyl proteins were labelled by incubation of SDS-denatured preparations of subcellular fractions of rat liver with [14C]methylavidin before polyacrylamide-gel electrophoresis. Fluorographic analysis showed that mitochondria contained two forms of acetyl-CoA carboxylase [acetyl-CoA:carbon dioxide ligase (ADP-forming) EC 6.4.1.2], both of which were precipitated by antibody to the enzyme. When both forms were considered, almost three-quarters of the total liver acetyl-CoA carboxylase was found in the mitochondrial fraction of liver from fed rats while only 3.5% was associated with the microsomal fraction. The remainder was present in cytosol, either as the intact active enzyme or as a degradation product. The actual specific activity of the cytosolic enzyme was approx. 2 units/mg of acetyl-CoA carboxylase protein while that of the mitochondrial enzyme was about 20-fold lower, indicating that mitochondrial acetyl-CoA carboxylase was relatively inactive. Fractionation of mitochondria with digitonin showed that acetyl-CoA carboxylase was associated with the outer mitochondrial membrane. The available evidence suggests that mitochondrial acetyl-CoA carboxylase represents a reservoir of enzyme which can be released and activated under lipogenic conditions.     

The effect of phenobarbitone on protein synthesis by liver polyribosomes in fed and starved rats.

1. The effect of phenobarbitone on the rate of protein synthesis and on the sedimentation patterns of various liver subcellular fractions containing ribosomes was studied in rats. 2. Phenobarbitone treatment increased the incorporation of [114C]leucine into protein by all preparations, provided they had not been subjected to preliminary treatment with Sephadex G-25. The phenobarbitone-induced effect on incorporation was associated with a gain in liver weight and a higher degree of polyribosomal aggregation. 3. Preparations that were treated with Sephadex G-25 incorporated more radioactivity into protein, but did not show the response to phenobarbitone treatment. 4. When the influence of starvation and phenobarbitone was studied separately on membrane-bound and membrane-free polyribosomes, it was shown that whereas both classes of polyribosomes were affected by starvation, apparently only the former class was susceptible to phenobarbitone stimulation of protein synthesis. 5. The decreased capacity for protein synthesis of polyribosomes from starved rats was independent of their association with the membranes of the endoplasmic reticulum, but resulted from polyribosomal disaggregation, from an intrinsic defect of the polyribosomes themselves and from changes in composition of the cell cap. 6. The results are discussed in relation to the problem of the control of protein biosynthesis and of the functional separation of membrane-bound and membrane-free polyribosomes.