Time course of hormonal effects on acetyl-CoA carboxylase as measured in digitonin-permeabilized rat hepatocytes

Acetyl-CoA carboxylase activity was measured in digitonin-permeabilized rat hepatocytes by coupling the carboxylase reaction to the fatty acid synthase reaction. Using this assay the activity of acetyl-CoA carboxylase was covariant with the rate of fatty acid synthesis. Insulin and the tumor promotor phorbol myristate acetate were found to stimulate, and glucagon and noradrenaline to inhibit both cellular parameters. The stimulation of acetyl-CoA carboxylase by insulin developed slowly (15 to 30 min) whereas the phorbol myristate acetate effect developed faster (within 15 min). The inhibition of the enzyme caused by glucagon was already apparent within 1 min after hormone addition. Inhibition by noradrenaline, in the presence of propranolol, was also quite rapid and occurred within 2 min after addition of the agonist.     

Studies on the assay, activity and sedimentation behaviour of acetyl-CoA carboxylase from isolated hepatocytes incubated with insulin or glucagon.

The activity of acetyl-CoA carboxylase, measured in various ways, was studied in 15000g extracts of rat liver hepatocytes and compared with the rate of fatty acid synthesis in intact hepatocytes incubated with insulin or glucagon. Hepatocyte extracts were prepared by disruption of cells with a Dounce homogenizer or by solubilization with 1.5% (v/v) Triton X-100. Sucrose-density-gradient centrifugation demonstrated that the sedimentation coefficient of acetyl-CoA carboxylase from cell extracts was 30-35S, regardless of the conditions of incubation or disruption of hepatocytes. Solubilization of cells with 1.5% Triton X-100 yielded twice as much enzyme activity (measured by [14C]bicarbonate fixation) in the sucrose-gradient fractions as did cell disruption by the Dounce homogenizer. Analysis by high-performance liquid chromatography of acetyl-CoA carboxylase reaction mixtures showed that [14C]malonyl-CoA accounted for 10-60% of the total acid-stable radioactivity, depending on the method for disrupting hepatocytes and on the preincubation of the 15000g extract, with or without citrate, before assay. Under conditions in which incubation of cells with insulin or glucagon caused an activation or inhibition, respectively, of acetyl-CoA carboxylase, only 25% of the acid-stable radioactivity was [14C]malonyl-CoA and enzyme activity was only 13% (control), 16% (insulin), and 57% (glucagon) of the rate of fatty acid synthesis. Under conditions when up to 60% of the acid-stable radioactivity was [14C]malonyl-CoA and acetyl-CoA carboxylase activity was comparable with the rate of fatty acid synthesis, there was no effect of insulin or glucagon on enzyme activity.     

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.     

Fatty acid inhibition of hormonal induction of acetyl-coenzyme A carboxylase in hepatocyte monolayers

Primary cultures of adult rat hepatocytes were utilized to ascertain the impact of free fatty acids on the insulin plus dexamethasone induction of acetyl-CoA carboxylase. Lipogenesis was induced threefold by the combination of insulin and dexamethasone. The rise in fatty acid synthesis was accompanied by a comparable increase in the rate-determining enzyme acetyl-CoA carboxylase. Dexamethasone was required for the insulin induction of acetyl-CoA carboxylase. Under the permissive action of glucocorticoid, 10(-7) M insulin maximally increased enzyme activity. Half-maximum stimulation occurred with 5 X 10(-9) M insulin. Media containing 0.2 mM palmitate, oleate, linoleate, arachidonate, or docosahexaenoate significantly suppressed the hormonal induction of acetyl-CoA carboxylase. The extent of suppression was only 30-35% and did not vary with chain length or degree of unsaturation. Carboxylase activity was not suppressed further by raising the concentration of linoleate to 0.5 mM; however, 0.5 mM palmitate depleted the cells of ATP and abolished acetyl-CoA carboxylase activity. Therefore, based upon the inhibitory characteristics of the various fatty acids and the lack of a concentration dependency of the fatty acid inhibition, it would appear that fatty acid inhibition of the induction of acetyl-CoA carboxylase activity may not be a direct, physiological regulatory mechanism.     

Periportal zonation of the cytosolic acetyl-CoA synthetase of male rat liver.

Several important metabolic functions of the mammalian liver have been shown to be located in zones with respect to the complex microcirculation of the organ. The zonal distribution of the cytosolic component of the acetyl-CoA synthetase activity has been investigated using the dual-digitonin-pulse-perfusion technique, which allows highly zone-selective sampling of cytosol from the periportal and perivenous zone of rat liver. Approximately 80% of the cytosolic enzymes are eluted from the hepatocytes in the periportal and perivenous sub-zones affected by digitonin, while less than 1% of the glutamate dehydrogenase activity (a marker enzyme of the mitochondrial compartment) is eluted. A twofold higher activity of the cytosolic form of acetyl-CoA synthetase is found in the periportal zone compared to the perivenous zone in fed male rats. Following a fasting/refeeding transition, this activity gradient is abolished in a manner similar to that observed for the enzyme acetyl-CoA carboxylase. Since the latter enzyme is utilizing the product of acetyl-CoA synthetase, acetyl-CoA, the similarity in the observed regulation suggests a functional coupling between cytosolic acetate activation and fatty-acid synthesis.     

Regulation of Plant Acetyl-CoA Carboxylase by Adenylate Nucleotides

The assay of acetyl-CoA carboxylase (EC 6.4.1.2) does not follow ideal zero-order kinetics when assayed in a crude extract from wheat (Triticum aestivum L.) germ. Our results show that the lack of ideality is the consequence of contamination by ATPase and adenylate kinase. These enzyme activities generate significant amounts of ADP and AMP in the assay mixture, thus limiting the availability of ATP for the carboxylase reaction. Moreover, ADP and AMP are competitive inhibitors, with respect to ATP, of acetyl-CoA carboxylase. Similar relationships between adenylate nucleotides and acetyl-CoA carboxylase are found in isolated chloroplasts. There is no evidence that acetyl-CoA carboxylase activity in the extracts of the plant systems examined is altered by covalent modification, such as a phosphorylation-dephosphorylation cycle. A scheme is presented that illustrates the dependency of acetyl-CoA carboxylase and fatty acid synthesis on the energy demands of the chloroplasts in vivo.     

Zonation of hepatic lipogenic enzymes identified by dual-digitonin-pulse perfusion.

The zonal distribution within rat liver of acetyl-CoA carboxylase, ATP citrate-lyase and fatty acid synthase, the principal enzymes of fatty acid synthesis, was investigated by using dual-digitonin-pulse perfusion. Analysis of enzyme mass by immunoblotting revealed that, in normally feeding male rats, the periportal/perivenous ratio of acetyl-CoA carboxylase mass was 1.9. The periportal/perivenous ratio of ATP citrate-lyase mass was 1.4, and fatty acid synthase exhibited the largest periportal/perivenous mass gradient, having a ratio of 3.1. This pattern of enzyme distribution was observed in male rats only; in females, the periportal/perivenous ratio of enzyme mass was nearly equal. The periportal/perivenous gradients for acetyl-CoA carboxylase, ATP citrate-lyase and fatty acid synthase observed in fed (and fasted) males were abolished when animals were fasted (48 h) and refed (30 h) with a high-carbohydrate/low-fat diet. As determined by enzyme assay of eluates obtained from the livers of normally feeding male rats, there is also periportal zonation of acetyl-CoA carboxylase activity, expressed either as units per mg of eluted protein or units per mg of acetyl-CoA carboxylase protein, suggesting the existence of gradients in both enzyme mass and specific activity. From these results, we conclude that the enzymes of fatty acid synthesis are zonated periportally in the liver of the normally feeding male rat.     

Simultaneous stimulation of fatty acid synthesis and oxidation in rat hepatocytes by vanadate

When added to the hepatocyte incubation medium, vanadate increased the rate of fatty acid synthesis de novo as well as the activity of acetyl-CoA carboxylase, whereas it had no effect on the activity of fatty acid synthase. On the other hand, and despite elevating the intracellular levels of malonyl-CoA, vanadate diverted exogenous fatty acids into the oxidation pathway at the expense of the esterification route. This was concomitant to an increase in carnitine palmitoyltransferase I activity. All these effects were not significantly different between periportal and perivenous hepatocytes and were also evident in cells incubated in Ca2(+)-free medium. Nevertheless, Ca2+ ions enhanced carnitine palmitoyltransferase I activity in isolated liver mitochondria. In addition, the effects of vanadate on acetyl-CoA carboxylase and carnitine palmitoyltransferase I were only evident in a permeabilized-cell assay, disappearing upon cell disruption and isolation of the corresponding cell subfraction for enzyme assay. Results show that vanadate exerts specific insulin-like and non-insulin-like effects on hepatic fatty acid metabolism, and suggest that the intracellular concentration of malonyl-CoA is not the only factor responsible for the regulation of the fatty-acid-oxidative process in the liver.     

Regulation of fatty acid synthesis.

Acetyl-CoA carboxylase and fatty acid synthetase are the two major enzymes involved in the synthesis of fatty acids in animals. The activities of both enzymes are affected by nutritional manipulations. Although acetyl-CoA carboxylase is considered generally to be the rate-limiting step in lipogenesis, there is evidence that suggests that fatty acid synthetase may become rate limiting under certain conditions. The principal support for the view that acetyl-CoA carboxylase is the rate-limiting enzyme for lipogenesis is that the activity of the enzyme is controlled by allosteric effectors that change the catalytic efficiency of the enzyme. Until recently, the only known control of fatty acid synthetase was through changes in rate of enzyme synthesis. Data are reviewed that show that fatty acid synthetase can exist in forms possessing different catalytic activities. Thus fatty acid synthetase appears to be subject to the type of control necessary for an enzyme to serve as a regulator of the rate of a biological process over a short term.     

Inactivation of hepatic acetyl-CoA carboxylase by catecholamine and its agonists through the alpha-adrenergic receptors

The effects of adrenergic agonists on acetyl-CoA carboxylase and fatty acid synthesis were studied in isolated rat hepatocytes from mature rats (300 to 350 g). Norepinephrine and phenylephrine inactivate acetyl-CoA carboxylase activity and inhibit fatty acid synthesis. The effects of both norepinephrine and phenylephrine were blocked by the alpha-adrenergic receptor blockers, phentolamine and phenoxybenzamine, and unaffected by the beta-receptor blocker propranolol. This inactivation was not mimicked by the beta-agonist isoproterenol. The measurable increase in cyclic AMP levels caused by norepinephrine and phenylephrine was abolished by the alpha-antagonist phentolamine and diminished by the beta-antagonist propranolol. Calcium depletion potentiated the increase in cyclic AMP levels by phenylephrine but abolished the phenylephrine inactivation of the carboxylase. The inactivation of acetyl-CoA carboxylase by phenylephrine was correlated with an increase in the incorporation of [32P]phosphate into the enzyme. Thus, catecholamines and their agonists promote phosphorylation and inactivation of acetyl-CoA carboxylase through the alpha-adrenergic receptor, and the inactivation requires calcium.     

The citrate cleavage pathway and lipogenesis in rat adipose tissue: replenishment of oxaloacetate

Fatty acid synthesis via the citrate cleavage pathway requires the continual replenishment of oxaloacetate within the mitochondria, probably by carboxylation of pyruvate. Malic enzyme, although present in adipose tissue, is completely localized in the cytoplasm and has insufficient activity to support lipogenesis. Pyruvate carboxylase was found to be active in both the mitochondria and cytoplasm of epididymal adipose tissue cells; it was dependent on both ATP and biotin. Alteractions in dietary conditions induced no significant changes in mitochondrial pyruvate carboxylase activity, but the soluble activity was depressed in fat-fed animals. The possible importance of the soluble activity in lipogenesis lies in its participation in a soluble malate transhydrogenation cycle with NAD malate dehydrogenase and malic enzyme, whereby a continual supply of NADPH is produced. Consequently, the pyruvate carboxylase in adipose tissue both generates mitochondrial oxaloacetate for the citrate cleavage pathway and supplies soluble NADPH for the conversion of acetyl-CoA to fatty acid.     

Estrogen regulation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and acetyl-CoA carboxylase in xenopus laevis

Administration of estradiol-17 beta to male Xenopus laevis evokes the proliferation of the endoplasmic reticulum and the Golgi apparatus and the synthesis and secretion by the liver of massive amounts of the egg yolk precursor phospholipoglycoprotein, vitellogenin. We have investigated the effects of estrogen on three key regulatory enzymes in lipid biosynthesis, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, the major regulatory enzyme in cholesterol and isoprenoid synthesis, and acetyl-CoA carboxylase and fatty acid synthetase, which regulate fatty acid biosynthesis. HMG-CoA reductase activity and cholesterol synthesis increase in parallel following estrogen administration. Reductase activity in estrogen stimulated Xenopus liver cells peaks at 40-100 times the activity observed in control liver cells. The increased rate of reduction of HMG-CoA to mevalonic acid is not due to activation of pre-existing HMG-CoA reductase by dephosphorylation, as the fold induction is unchanged when reductase from control and estrogen-stimulated animals is fully activated prior to assay. The estrogen-induced increase of fatty acid synthesis is paralleled by a 16- to 20-fold increase of acetyl-CoA carboxylase activity, indicating that estrogen regulates fatty acid synthesis at the level of acetyl-CoA carboxylase. Fatty acid synthetase activity was unchanged during the induction of fatty acid biosynthesis by estrogen. The induction of HMG-CoA reductase and of acetyl-CoA carboxylase by estradiol-17 beta provides a useful model for regulation of these enzymes by steroid hormones.     

Structural changes of isolated hepatocytes during treatment with digitonin

The structural changes accompanying digitonin-induced release of enzymes and metabolites from isolated hepatocytes have been studied by scanning and transmission electron microscopy. In the initial phase, characterized by total release of the cytosolic marker enzyme, lactate dehydrogenase, the plasma membrane was immediately damaged, rapidly followed by extensive damage to the endoplasmic reticulum. The shape of the cell, however, was maintained, and the mitochondria and nucleus remained tightly held together by the cytoskeleton. Mitochondria remained intact initially, whereas the cytosol became less electron dense and the nuclear chromatin was more dispersed. An intermediate phase was characterized by total release of adenylate kinase and most of the glucose-6-phosphatase, marker enzymes for the mitochondrial intermembrane space and the endoplasmic reticulum, respectively. The outer mitochondrial membrane was ruptured, but mitochondria maintained their normal matrix electron density. In the final phase, characterized by the beginning of citrate synthase release from the mitochondrial matrix space, the mitochondria became swollen, and only the nucleus, inner and outer mitochondrial membranes, and the cytoskeleton could be clearly distinguished. Although the plasma membrane could not be readily discerned in electron micrographs after the initial phase, the plasma membrane marker enzyme 5'-nucleotidase remained associated with digitonin-treated hepatocytes. Acetyl-CoA carboxylase was released much more slowly than lactate dehydrogenase, indicating some severe restriction on its release. The release of acetyl-CoA carboxylase closely paralleled the release of glucose-6-phosphatase. The controlled exposure of hepatocytes to digitonin, therefore, leads to the sequential release of soluble, compartmentalized cellular components and some membrane-bound components, but the mitochondrial membrane, cytoskeleton and the nucleoskeleton survive even long-term digitonin treatment.     

Structural changes of isolated hepatocytes during treatment with digitonin

The structural changes accompanying digitonin-induced release of enzymes and metabolites from isolated hepatocytes have been studied by scanning and transmission electron microscopy. In the initial phase, characterized by total release of the cytosolic marker enzyme, lactate dehydrogenase, the plasma membrane was immediately damaged, rapidly followed by extensive damage to the endoplasmic reticulum. The shape of the cell, however, was maintained, and the mitochondria and nucleus remained tightly held together by the cytoskeleton. Mitochondria remained intact initially, whereas the cytosol became less electron dense and the nuclear chromatin was more dispersed. An intermediate phase was characterized by total release of adenylate kinase and most of the glucose-6-phosphatase, marker enzymes for the mitochondrial intermembrane space and the endoplasmic reticulum, respectively. The outer mitochondrial membrane was ruptured, but mitochondria maintained their normal matrix electron density. In the final phase, characterized by the beginning of citrate synthase release from the mitochondrial matrix space, the mitochondria became swollen, and only the nucleus, inner and outer mitochondrial membranes, and the cytoskeleton could be clearly distinguished. Although the plasma membrane could not be readily discerned in electron micrographs after the initial phase, the plasma membrane marker enzyme 5′-nucleotidase remained associated with digitonin-treated hepatocytes. Acetyl-CoA carboxylase was released much more slowly than lactate dehydrogenase, indicating some severe restriction on its release. The release of acetyl-CoA carboxylase closely paralleled the release of glucose-6-phosphatase. The controlled exposure of hepatocytes to digitonin, therefore, leads to the sequential release of soluble, compartmentalized cellular components and some membrane-bound components, but the mitochondrial membrane, cytoskeleton and the nucleoskeleton survive even long-term digitonin treatment.     

Both insulin and epidermal growth factor stimulate fatty acid synthesis and increase phosphorylation of acetyl-CoA carboxylase and ATP-citrate lyase in isolated hepatocytes

Insulin and EGF cause identical stimulation (congruent to 40%) of fatty acid synthesis in hepatocytes isolated from rats which have been starved and then refed a low-fat diet. In both cases this stimulation is associated with increased phosphorylation of ATP-citrate lyase and of a specific site on acetyl-CoA carboxylase. However, the altered phosphorylation of acetyl-CoA carboxylase is not associated with a change in kinetic parameters which is detectable in the purified enzyme. Whatever the mechanism involved, stimulation of fatty acid synthesis by growth factors may have a role in providing new phospholipid for growth of membranes.     

Coordinate regulation of acetyl coenzyme A carboxylase and fatty acid synthetase in liver cells of the developing chick in vivo and in culture.

The activities of hepatic acetyl-CoA carboxylase and fatty acid synthetase undergo two distinct types of development in the perinatal chick. The first increase begins prior to hatching, continues after hatching in the starved chick, and is independent of feeding. The second increase is caused by feeding and is reversed by starvation (A. G. Goodridge (1973) J. Biol. Chem.248, 1932–1938). We have purified these enzymes to homogeneity and raised antibodies to them in rabbits. Using immunochemical techniques we have established that the activity changes in both types of development were a function of changes in the concentrations of enzyme proteins. All activity changes were accompanied by similar changes in the relative rates of synthesis of the two enzymes. Regulation of the activities of acetyl-CoA carboxylase and fatty acid synthetase was further characterized in liver cells from 19-day-old embryos maintained in culture in a chemically defined medium. After 3 days in culture in the absence of hormones, the activities of the enzymes increased significantly with respect to the activities of the freshly prepared cells. Addition of either insulin or triiodothyronine alone caused additional small increases. Insulin plus triiodothyronine caused 8- and 15-fold increases in acetyl-CoA carboxylase and fatty acid synthetase, respectively, relative to cells incubated without hormones. In the presence of insulin alone glucagon had no effect on the activity of either enzyme. In the presence of insulin plus triiodothyronine, glucagon inhibited the increase in enzyme activities by about 75%. The results of quantitative immunoprecipitin tests indicated that activity changes caused by the various hormones were functions of changes in the concentrations of the enzyme proteins. The effects of the hormones on enzyme activities were accompanied by comparable or larger changes in the relative rates of synthesis of the enzymes. Under a wide variety of experimental conditions, both in vivo and in culture, the relative rates of synthesis of acetyl-CoA carboxylase and fatty acid synthetase are regulated coordinately. Under some of these conditions, synthesis of malic enzyme also is regulated coordinately with the syntheses of acetyl-CoA carboxylase and fatty acid synthetase. The common intracellular mechanisms underlying the coordinate control remain to be elucidated.     

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.     

Short-term regulation of carnitine palmitoyltransferase activity in isolated rat hepatocytes

An assay procedure for carnitine palmitoyltransferase is described which allows rapid measurement of the overt activity of this enzyme in isolated rat hepatocytes. In a one-step procedure digitonin permeabilizes the plasma membrane and at the same time carnitine palmitoyltransferase activity is measured. The use of the present procedure shows that carnitine palmitoyltransferase activity is regulated on the short term by different types of agonists. Thus, insulin, epidermal growth factor, vasopressin and the phorbol ester PMA inhibit carnitine palmitoyltransferase activity, whereas glucagon treatment renders the enzyme more active. These changes in enzyme activity coincide with corresponding changes in the rate of fatty acid oxidation.     

Effects of dietary nutrients on substrate and effector levels of lipogenic enzymes, and lipogenesis from tritiated water in rat liver

When fasted rats were refed for 4 days with a carbohydrate and protein diet, a carbohydrate diet (without protein) or a protein diet (without carbohydrate), the effects of dietary nutrients on the fatty acid synthesis from injected tritiated water, the substrate and effector levels of lipogenic enzymes and the enzyme activities were compared in the livers. In the carbohydrate diet group, although acetyl-CoA carboxylase was much induced and citrate was much increased, the activity of acetyl-CoA carboxylase extracted with phosphatase inhibitor and activated with 0.5 mM citrate was low in comparison to the carbohydrate and protein diet group. The physiological activity of acetyl-CoA carboxylase seems to be low. In the protein diet group, the concentrations of glucose 6-phosphate, acetyl-CoA and malonyl-CoA were markedly higher than in the carbohydrate and protein group, whereas the concentrations of oxaloacetate and citrate were lower. The levels of hepatic cAMP and plasma glucagon were high. The activities of acetyl-CoA carboxylase and also fatty acid synthetase were low in the protein group. By feeding fat, the citrate level was not decreased as much as the lipogenic enzyme inductions. Comparing the substrate and effector levels with the Km and Ka values, the activities of acetyl-CoA carboxylase and fatty acid synthetase could be limited by the levels. The fatty acid synthesis from tritiated water corresponded more closely to the acetyl-CoA carboxylase activity (activated 0.5 mM citrate) than to other lipogenic enzyme activities. On the other hand, neither the activities of glucose-6-phosphate dehydrogenase and malic enzyme (even though markedly lowered by diet) nor the levels of their substrates appeared to limit fatty acid synthesis of any of the dietary groups. Thus, it is suggested that under the dietary nutrient manipulation, acetyl-CoA carboxylase activity would be the first candidate of the rate-limiting factor for fatty acid synthesis with the regulations of the enzyme quantity, the substrate and effector levels and the enzyme modification.