Regulation of acetyl-coenzyme A carboxylase. II. Effect of fasting and refeeding on the activity, phosphate content, and aggregation state of the enzyme

Acetyl-CoA carboxylase isolated from freeze-clamped livers of fed rats has relatively low phosphate content (5.0 mol of Pi/mol of subunit) and high specific activity (3.5 units/mg in the absence of citrate). The enzyme from rats fasted for 12, 18, 24, and 48 h exhibited decreasing specific activities of 2.75, 1.85, 1.7, and 0.9 units/mg, respectively. Citrate activated all preparations of carboxylase, with most activation observed with the least active preparation. There was no significant change in the sensitivity of the enzyme to citrate since half-maximal activation was observed at 0.2 mM for carboxylase from fed as well as fasted rats. With the decrease in activity as a function of fasting, there was a concomitant increase in the phosphate content of carboxylase, with values of 5.3, 5.6, 6.7, and 7.6 mol of Pi/mol of subunit obtained for preparations from rats fasted for 12, 18, 24, and 48 h, respectively. Refeeding the fasted rats resulted in increased specific activity of carboxylase (3.4 units/mg) and decreased phosphate content (5.1 mol of Pi/mol of subunit). Moreover, dephosphorylation by [acetyl-CoA carboxylase]-phosphatase 2 activated the carboxylase from 48-h fasted rats to a value of 2.9 units/mg, assayed in the absence of citrate, indicating that the low activity of carboxylase from fasted rats was due to its increased phosphate content. Superose 6 chromatography showed that the enzyme exists in two polymeric forms, a highly active polymer of greater than or equal to 40 subunits and less active octamer. The former predominates in livers of fed rats, whereas the latter predominates in livers of fasted rats. The octamer could be converted to the highly active polymer by dephosphorylation. These observations indicate that fasting/refeeding results in phosphorylation/dephosphorylation of acetyl-CoA carboxylase with concomitant depolymerization/polymerization of the protein and ultimately decreasing or increasing its specific activity.     

Formation of malonyl coenzyme A in rat heart. Identification and purification of an isozyme of A carboxylase from rat heart

Acetyl-CoA carboxylase is thought to be absent in the heart since the latter is highly catabolic and nonlipogenic. It has been suggested that the high level of malonyl-CoA that is found in the heart is derived from mitochondrial propionyl-CoA carboxylase, which also uses acetyl-CoA. In the present study, acetyl-CoA carboxylase was identified and purified from homogenates of rat heart. The isolated enzyme had little activity in the absence of citrate (specific activity, less than 0.1 units/mg); however, citrate stimulated its activity (specific activity, 1.8 units/mg in the presence of 10 mM citrate). Avidin inhibited greater than 95% of activity, and addition of biotin reversed this inhibition. Further, malonyl-CoA (1 mM) and palmitoyl-CoA (100 microM) inhibited greater than 90% of carboxylase activity. Similar to acetyl-CoA carboxylase of lipogenic tissues, the heart enzyme could be activated greater than 6-fold by preincubation with liver (acetyl-CoA carboxylase)-phosphatase 2. The activation was accompanied by a decrease in the K0.5 for citrate to 0.68 mM. These observations suggest that the activity in preparations from heart is due to authentic acetyl-CoA carboxylase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the preparation from heart showed the presence of one major protein band (Mr 280,000) and a minor band (Mr 265,000) while that from liver gave a major protein band (Mr 265,000). A Western blot probed with avidin-peroxidase suggested that both the 280- and 265-kDa species contained biotin. Antibodies to liver acetyl-CoA carboxylase, which inhibited greater than 95% of liver carboxylase activity, inhibited only 35% of heart enzyme activity. In an immunoblot (using antibodies to liver enzyme) the 265-kDa species, and not the major 280-kDa species, in the heart preparation was specifically stained. These observations suggest the presence of two isoenzymes of acetyl-CoA carboxylase that are immunologically distinct, the 265-kDa species being predominant in the liver and the 280-kDa species being predominant in the heart.     

Activation of acetyl-CoA carboxylase. Purification and properties of a Mn2+-dependent phosphatase

Acetyl-CoA carboxylase was isolated from rat liver by polyethylene glycol precipitation and avidin affinity chromatography. Sodium dodecyl sulfate electrophoresis of the enzyme gives one protein band (Mr 250,000). Phosphate analysis of the carboxylase showed the presence of 8.3 mol of phosphate/mol of subunit (Mr 250,000). The purified carboxylase has low activity in the absence of citrate (specific activity = 0.3 units/mg). However, addition of 10 mM citrate activates the carboxylase 10-fold, with half-maximal activation observed at 2 mM citrate, well above the physiological citrate level. Using this carboxylase as a substrate, we have isolated from rat liver a protein that activates the enzyme about 10-fold. This protein has been purified to near homogeneity (Mr 90,000). Incubation of this protein with 32P-labeled acetyl-CoA carboxylase results in a time-dependent activation of carboxylase with concomitant release of 32Pi, indicating that this protein is a phosphoprotein phosphatase. Both activation and dephosphorylation are dependent on Mn2+, but not citrate. This phosphatase does not hydrolyze p-nitrophenyl phosphate but does show high affinity for acetyl-CoA carboxylase (Km = 0.2 microM) as compared to its action on phosphorylase a (Km = 5.5 microM) and phosphohistone (Km = 20 microM). Activated acetyl-CoA carboxylase was isolated after dephosphorylation by the phosphatase. Such preparations contain about 5 mol of phosphate/mol of subunit and have specific activities of 2.6-3.0 units/mg in the absence of citrate. These activities are comparable to those of the phosphorylated carboxylase in the presence of 10 mM citrate. Thus, dephosphorylation by the Mn2+-dependent phosphatase renders the carboxylase citrate-independent, as compared to the phosphorylated form, which is citrate-dependent. To our knowledge this is the first report of a preparation of animal acetyl-CoA carboxylase that has substantial catalytic activity independent of citrate.     

Estrogen induces hyperlipidemia in fasted chicks

To determine whether the estrogen-induced hyperlipidemia is affected by fasting, male growing chicks were administered subcutaneously a single dose of 17 beta-estradiol (25 mg/kg body wt), and the hormone treatment lasted for 2 days with or without feed (Experiment 1). In the second experiment, chicks were initially fasted for 1 or 3 days, and then treated with the same dosage of 17 beta-estradiol as in Experiment 1 for 2 days without feed. Plasma and liver lipids, and the activities of hepatic malic enzyme, glucose-6-phosphate dehydrogenase, and hormone-sensitive lipase in the adipose tissue were determined. Compared with fed control chicks, estrogen treatment in fed birds resulted in a marked elevation of plasma lipids, especially triglyceride during the 2-day period (137 vs 2263 mg/dl). In fasted chicks, the present finding that estrogen also induced a marked hyperlipidemia is noteworthy. Upon estrogen treatment (Experiment 1), the level of plasma triglyceride in fasted birds increased about 16 times over that of the fasted control group (133 vs 2093 mg/dl). Even in chicks fasted for 5 days (Experiment 2), estrogen treatment resulted in a persistent hypertriglyceridemia (75 vs 1369 mg/dl). In fed chicks, estrogen treatment also induced a fatty liver with massive accumulation of triglyceride, but the liver of estrogen-treated/fasted chicks appeared to be normal. In both fed and fasted chicks, malic enzyme was found to be the major NADPH producing enzyme in the liver. Upon fasting, both malic enzyme and glucose-6-phosphate dehydrogenase activities decreased significantly (P less than 0.05). In fed chicks, the total activities of both enzymes increased with estrogen treatment, whereas the effect of hormone on these enzymes was less obvious in fasted chicks. The hormone-sensitive lipase activity in the adipose tissue was much lower in fed chicks compared with that of fasted birds (0.15 vs 0.33 nmol of oleic acid released/min/mg protein). Estrogen treatment in fed chicks had no effect on the hormone-sensitive lipase activity, but its activity was enhanced by the hormone treatment in fasted chicks. The present finding that hyperlipidemia persisted in estrogenized chicks during the fasting seems to indicate the complex nature of this hormonal influence on lipid metabolism.     

Acute hormonal control of acetyl-CoA carboxylase. The roles of insulin, glucagon, and epinephrine

Acetyl-CoA carboxylase, purified from rapidly freeze-clamped livers of rats maintained on a normal laboratory diet and given 0-5 units of insulin shortly before death, gives a major protein band (Mr 265,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carboxylase from untreated rats has relatively low activity (0.8 unit/mg protein when assayed in the absence of citrate) and high phosphate content (8.5 mol of Pi/mol of subunit), while the enzyme from livers of rats that received 5 units of insulin has higher activity (2.0 units/mg protein) and lower phosphate content (7.0 mol of Pi/mol of subunit). Addition of citrate activates both preparations with half-maximal activation (K0.5) at 1.0 and 0.6 mM citrate, respectively. The enzyme from rats that did not receive insulin is mainly in the octameric state (Mr approximately 2 x 10(6)), while that from rats that received insulin is mainly in the polymeric state (Mr approximately 10 x 10(6)). Thus, short-term administration of insulin results in activation of acetyl-CoA carboxylase, lowering of its citrate requirement, and dephosphorylation and polymerization of the protein. The insulin-induced changes in the carboxylase are probably due to dephosphorylation of the protein since similar changes are observed when the enzyme from rats that did not receive insulin is dephosphorylated by the Mn2(+)-dependent [acetyl-CoA carboxylase]-phosphatase 2. The effect of glucagon or epinephrine administration on acetyl-CoA carboxylase was also investigated. The carboxylase from fasted/refed rats has a relatively high specific activity (3.4 units/mg protein in the absence of citrate), lower phosphate content (4.9 mol of Pi/mol of subunit), and is present mainly in the polymeric state (Mr approximately 10 x 10(6)). Addition of citrate activates the enzyme with K0.5 = 0.2 mM citrate. Glucagon or epinephrine injection of fasted/refed rats yielded carboxylase with lower specific activity (1.4 or 1.9 units/mg, respectively, in the absence of citrate), higher phosphate content (6.4 or 6.7 mol of Pi/mol of subunit, respectively), and mainly in the octameric state (Mr approximately 2 x 10(6)). Treatment of these preparations with [acetyl-CoA carboxylase]-phosphatase 2 reactivated the enzyme (specific activity approximately 8 units/mg protein in the absence of citrate) and polymerized the protein (Mr approximately 10 x 10(6]. These observations indicate that insulin and glucagon, by altering the phosphorylation state of the acetyl-CoA carboxylase, play antagonistic roles in the acetyl-control of its activity and therefore in the regulation of fatty acid synthesis.     

Purification, characterization, and ontogeny of acetyl-CoA carboxylase isozyme of chick embryo brain.

Acetyl-CoA carboxylase catalyzes the first committed step in the synthesis of fatty acids. Because fatty acids are required during myelination in the developing brain, it was proposed that the level of acetyl-CoA carboxylase may be highest in embryonic brain. The presence of acetyl-CoA carboxylase activity was detected in chick embryo brain. Its activity varied with age, showing a peak in the 17-18-day-old embryo and decreasing thereafter. The enzyme, affinity-purified from 18-day-old chick embryo brain, appeared as a major protein band on polyacrylamide electrophoresis gels in the presence of sodium dodecyl sulfate (Mr 265,000), indistinguishable from the 265 kDa isozyme of liver acetyl-CoA carboxylase. It had significant activity (Sp act = 1.1 mumol/min per mg protein) in the absence of citrate. There was a maximum stimulation of only 25% in the presence of citrate. Dephosphorylation using [acetyl-CoA carboxylase] phosphatase 2 did not result in activation of the enzyme. Palmitoyl-CoA (0.1 mM) and malonyl-CoA (1 mM) inhibited the activity to 95% and 71%, respectively. Palmitoylcarnitine, however, did not show significant inhibition. The enzyme was inhibited (greater than 95%) by avidin; however, avidin did not show significant inhibition in the presence of excess biotin. The enzyme was also inhibited (greater than 90%) by antibodies against liver acetyl-CoA carboxylase. An immunoblot or avidin-blot detected only one protein band (Mr 265,000) in preparations from chick embryo brain or adult liver. These observations suggest that acetyl-CoA carboxylase is present in embryonic brain and that the enzyme appears to be similar to the 265 kDa isozyme of liver.     

Effect of pyruvate, octanoate and glucose on leucine degradation in skeletal muscle from fed and fasted chicks

1. Pyruvate at 5 mM decreased the rate of leucine oxidative decarboxylation and increased the rate of 2-oxoisocaproate production in extensor digitorum communis (EDC) muscles from both fed and 24-hr fasted chicks. Pyruvate at 5 mM increased the net rate of leucine transamination in EDC muscle from fed chicks and had no effect in EDC muscles from 24-hr fasted chicks. 2. Octanoate at 0.2 and 1 mM markedly increased the rates of net leucine transamination, leucine oxidative decarboxylation and oxidation of decarboxylated leucine carbons 2-6 in EDC muscles from fed chicks, but had no effect on these parameters of leucine degradation in muscles from 24-hr fasted chicks. 3. Glucose at 5 and 12 mM decreased the rates of leucine oxidative decarboxylation and oxidation of decarboxylated leucine carbons 2-6, and increased the net rate of 2-oxoisocaproate production as compared to control (no glucose) group in muscles from fed chicks. Glucose had no effect on these parameters of leucine degradation in muscles from 24-hr fasted chicks.     

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.     

Quantitation by immunoblotting of the in vivo induction and subcellular distribution of hepatic acetyl-CoA carboxylase

The in vivo induction of rat liver acetyl-CoA carboxylase (ACC) the rate-limiting enzyme of fatty acid biosynthesis, has been examined by immunoblotting, avidin blotting, and enzyme isolation. Three high-molecular-weight immunoreactive bands (Mr 220,000-260,000) were recognized in liver extracts by an anti-carboxylase polyclonal antiserum. Two bands, A and B, comigrated on sodium dodecyl sulfate polyacrylamide gels with purified acetyl-CoA carboxylase, were avidin binding, and were dramatically induced following high carbohydrate refeeding. Only band A was recognized on immunoblots using a monoclonal antibody directed against acetyl-CoA carboxylase, suggesting that band B is a proteolytic fragment in which the epitope recognized by the monoclonal antibody is absent. Following refeeding, approximately 57% of acetyl-CoA carboxylase mass (band A + band B) was present in the high-speed supernatant fraction, while 34 and 9% were in the high-speed (microsomal) and low-speed pellet fractions, respectively. Refeeding caused a large increase in total acetyl-CoA carboxylase mass, the magnitude of which differed in the various fractions. In the low-speed supernatant, a 20-fold increase in ACC mass was observed, while a 12-fold increase was seen in the high-speed supernatant. The fold increase in the high-speed pellet was even greater (greater than 27-fold). Acetyl-CoA carboxylase purified by avidin-Sepharose chromatography from fasted/refed rats had an approximate 4-fold higher Vmax and a significantly lower Ka for citrate than enzyme purified from fasted animals. The results of this study indicate that the induction of hepatic ACC that occurs during high carbohydrate refeeding of the fasted rat predominantly involves increases in enzyme content in both cytosol and microsomes, but is also accompanied by an increase in enzyme specific activity.     

Ketone bodies inhibit leucine degradation in chick skeletal muscle

1. DL-beta-hydroxybutyrate (4 mM) increased the net rate of leucine transamination and the net rate of 2-oxoisocaproate (KIC) production in extensor digitorum communis muscles from fed chicks. 2. DL-beta-hydroxybutyrate at 1 and 4 mM inhibited leucine oxidative decarboxylation in muscles from fed chicks. 3. Acetoacetate at 1 and 4 mM inhibited leucine oxidative decarboxylation and total leucine oxidation, but increased net KIC production in muscles from fed chicks. 4. Both DL-beta-hydroxybutyrate and acetoacetate at 1 and 4 mM inhibited the net rate of leucine transamination and the rates of leucine oxidative decarboxylation and total leucine oxidation in muscles from 24-hr fasted chicks.     

Phosphorylation state of acetyl-coenzyme A carboxylase. II. Variation with nutritional condition

Acetyl-CoA carboxylase from liver exhibits a linear inverse relationship between the ratio of enzymic activities at 0 and 2 mM citrate and the extent of phosphorylation by its kinase, and this citrate activity ratio method was used to examine the effect of nutritional conditions on the phosphorylation state of the enzyme. This method showed that the calculated phosphorylation state, being the extent of phosphorylation at sites accessible to carboxylase kinase, was highest in the livers of starved rats, lower in those fed normally, and lower still in starved rats which had been refed for 48 h on a fat-free diet. The actual values were 0.44, 0.26, and 0 mol of P/subunit, respectively, provided that liver samples were frozen rapidly to liquid nitrogen temperatures and extracted with stopping buffers at temperatures well below freezing. Normal homogenization with stopping buffers (containing inhibitors for protein kinases and phosphatases) resulted in much higher calculated phosphorylation states. The effect of nutritional conditions on the phosphorylation state as estimated reported above was confirmed by purifying the carboxylase from livers of rats, measuring the amount of phosphate which could be incorporated by carboxylase kinase, and comparing this with the phosphorylation state calculated from the citrate activity ratio method or the specific activity. Furthermore, treatment with protein phosphatase of carboxylase from starved rats resulted in the largest increase in specific activity, that from the starved/refed rats in the least. Finally, the effects of hyperglycemia on carboxylase and phosphorylase characteristics in the livers of intact rats were ascertained by taking liver samples and preparing crude extracts by the rapid freezing method described above. Hyperglycemia caused a rapid increase in the activity of the carboxylase and a rapid decrease in its putative phosphorylation state as measured by the citrate activity ratio method. Phosphorylase was also dephosphorylated, as indicated by a decrease in phosphorylase a activity. We conclude that the citrate activity ratio method is a valid test for the phosphorylation state of acetyl-CoA carboxylase in crude extracts of tissue.     

The effect of ketone bodies on protein turnover in isolated skeletal muscle from the fed and fasted chick

1. The addition of 4 mM acetoacetate or DL-beta-hydroxybutyrate to the incubation medium decreased the rate of protein synthesis without influencing the rate of protein degradation in extensor digitorum communis (EDC) muscles from fed chicks and decreased the rates of protein synthesis and degradation in muscles from fasted chicks. 2. Ketone bodies markedly decreased intracellular concentrations of glutamine in EDC muscles from fed chicks by increasing glutamine oxidation. 3. The addition of 0.5 mM glutamine to incubation media containing 1.0 mM glutamine reversed the ketone body-induced decrease in intracellular glutamine concentration to the control value and blocked the inhibiting effect of ketone bodies on protein synthesis in skeletal muscles from fed chicks. 4. The addition of 5 mM pyruvate blocked the ability of ketone bodies to increase glutamine oxidation and prevented the associated decrease in intracellular glutamine concentration and the rate of protein synthesis in EDC muscles from fed chicks. 5. These results suggest that ketone bodies can act directly on skeletal muscle to inhibit the rate of protein synthesis in muscles from fed chicks by decreasing intracellular glutamine concentration by increasing its oxidation.     

Organ and intracellular localization of short-chain acyl-CoA synthetases in rat and guinea-pig.

1. Homogenates of rat epididymal fat pad, heart, kidney, lactating mammary gland, liver, skeletal muscle and small intestinal mucosa have been partitioned into a particulate and supernatant fraction. With reliable marker enzymes for the mitochondrial matrix and the cytosol: propionyl-CoA carboxylase and pyruvate kinase, the distributions of the acyl-CoA synthetase activities measured at 1 and 10 mM C2, C3 and C4 over mitochondria and cytosol have been calculated. From these values an estimate was made of the K0.5 of the fatty acids. 2. A distinct fatty acid-activating enzyme was assumed to be present in one of the compartments when that fatty acid was activated with a K0.5 less than or equal to 1.5 mM in an amount of greater than 13% of the total cellular activity. Adipose tissue, gut, liver and mammary gland, all organs of a high lipogenetic capacity, contained a cytosolic acetyl-CoA synthetase. At 1 mM acetate 60, 31, 77 and 83% of the total cellular activities in these organs were cytosolic in nature, with activities of 0.021, 0.32, 0.37 and 1.16 mumol C2 activated per min per g wet weight, respectively. 3. Mitochondrial acetyl-CoA and butyryl-CoA synthetases were found in adipose tissue, gut, heart, kidney, mammary gland and muscle. They were absent in liver. Adipose tissue and liver contained a mitochondrial propionyl-CoA synthetase with activities at 1 mM C3 of 0.014 and 1.50 mumol C3 activated per min per g wet weight, respectively. 4. At 1 mM, C2 was activated with decreasing rates by kidney, heart, mammary gland and gut (7.6-1.0 mumol C2 activated per min per g wet weight). C3 (1 mM) activation was about equal (1.6-1.9 mumol C3 activated per min per g wet weight) in liver, kidney and heart. C4 (1 mM) was activated with decreasing rates by heart, liver, kidney and gut (4.0-0.5 mumol C4 activated per min per g wet weight) in the order given. 5. The influence of the isolation method and the diet on fatty acid activation in small intestinal mucosal scrapings have been studied. To demonstrate the existence of cytosolic acetyl-CoA synthetase in fed animals a pre-treatment of everted intestine by low amplitude vibration has been found essential. Also C16 activation was highly (95%) decreased in a non-pre-vibrated preparation. 24 h starvation lowered cytosolic C2 and total C16 activation by 90 and 80%, respectively. Refeeding of starved rats with a balanced fat-free diet, and not with sucrose only, gave the same cytosolic C2 and total C16 activation as normally fed rats. 6. In guienea-pig heart, kidney, liver and muscle about the same partitions have been found as in the respective rat organs. The acetate activation in liver was factor 6 lower. Acetate and butyrate activation in guinea-pig muscle was much higher (6 and 37 times, respectively).     

Leucine: Effector of phosphate activated glutaminase in rat cerebral cortex

Phosphate activated glutaminase (PAG) was assayed in whole homogenate and synaptosomes of cerebral cortex from normal or fasted for 120 h rats. The specific activity (s.a.) of PAG was found diminished by 25% in the whole homogenate from the fasted animals compared to the normal. On the contrary, fasting did not affect PAG s.a. of the synaptosomal fraction. Reconstitution experiments showed that when the deproteinized supernatant of the 12,500g centrifugation from the fasted rats was added to the synaptosomes from either fed or fasted animals the PAG activity was diminished but there was no change when the corresponding supernatant from the fed animals was added to the synaptosomes from both conditions. When leucine at 5mM was added to the homogenate or to synaptosomes from fed or fasted animals the s.a. of PAG was significantly decreased. Even in the presence of aminooxyacetate the effect of leucine was observed. Branched chain amino acids i.e. leucine, isoleucine and valine at 0.5 mM each added to synaptosomes again decreased PAG activity. The addition of ketone bodies had no effect. It is suggested that leucine, because PAG has been implicated in the supply of transmitter glutamate, might be an important regulator of the pool of this neurotransmitter.     

Phosphorylation state of acetyl-coenzyme A carboxylase. I. Linear inverse relationship to activity ratios at different citrate concentrations

Acetyl-CoA carboxylase and its associated kinase have been purified to homogeneity from rat liver and, together with the catalytic subunit of liver protein phosphatase, used to study the effect of phosphorylation on the carboxylase activity. Phosphatase increases the carboxylase activity, whereas the kinase decreases it. A linear inverse relationship (correlation coefficient = 0.98) exists between phosphate incorporated by the kinase and the specific activity. The kinetics of activation by citrate show an increased Ka and a decreased Vmax for carboxylase preparations with increasing levels of phosphate. On this basis an enzymic test was devised for phosphate incorporated by the kinase. Thus the ratio of activities at 0 and 2 mM citrate is inversely proportional to the phosphate incorporated (correlation coefficient = -0.95), with 0.8 mol of P incorporated per mol of subunit decreasing the activity ratio from 0.5 to 0. This activity ratio method has an inherent internal control which makes it suitable for determining the level of protein-bound phosphate affecting the carboxylase activity in crude tissue extracts, and hence it should be useful for physiological studies. Tryptic maps of carboxylase labeled with radioactive phosphate by the carboxylase kinase indicate that the slightly less than 1 mol of P/mol of subunit is distributed equally between two peptides, whereas cAMP-dependent protein kinase phosphorylates these two sites and a third which may not affect activity.     

Heterogeneous distribution of ATP citrate lyase in rat-liver parenchyma. Microradiochemical determination in microdissected periportal and perivenous liver tissue

1. A radiochemical microtest was established for the determination of ATP citrate lyase in tissue samples of 0.2-1.0 micrograms dry weight. The specificity of this test system was guaranteed by its coenzyme A dependence as well as by inhibition of the activity measured in presence of a specific antibody. 2. Using this test system ATP citrate lyase activity was determined in microdissected periportal and perivenous liver tissue of fed, fasted and refed animals. The perivenous activity was 1.8-fold and 2.4-fold higher than the periportal one in fed male and female rats respectively. 3. The perivenous to periportal gradient was decreased during starvation-dependent reduction of the ATP citrate lyase activity. On the other hand it was not only restored but enhanced up to 2.8 after refeeding-dependent enhancement of the enzyme activity. 4. The predominance of the ATP citrate lyase activity in the perivenous, mainly glycolytic zone supports the hypothesis of the coordinate zonation of the carbohydrate and the lipid metabolism in the liver parenchyma.     

Mechanism of glucagon inhibition of liver acetyl-CoA carboxylase. Interrelationship of the effects of phosphorylation, polymer-protomer transition, and citrate on enzyme activity

The short-term regulation of rat liver acetyl-CoA carboxylase by glucagon has been studied in hepatocytes from rats that had been fasted and refed a fat-free diet. Glucagon inhibition of the activity of this enzyme can be accounted for by a direct correlation between phosphorylation, polymer-protomer ratio, and activity. Glucagon rapidly inactivates acetyl-CoA carboxylase with an accompanying 4-fold increase in the phosphorylation of the enzyme and 3-fold increase in the protomer-polymer ratio of enzyme protein. Citrate, an allosteric activator of acetyl-CoA carboxylase required for enzyme activity, has no effect on these phenomena, indicating a mechanism that is independent of citrate concentration within the cell. The observation of these effects of glucagon on acetyl-CoA carboxylase activity is absolutely dependent upon the minimization of proteolytic degradation of the enzyme after cell lysis. Therefore, for the first time, an interrelationship has been demonstrated between phosphorylation, protomer-polymer ratio, and citrate for the inactivation of acetyl-CoA carboxylase by glucagon.     

Accumulation of neutral lipids in Saccharomyces carlsbergensis by myo-inositol deficiency and its mechanism. Reciprocal regulation of yeast acetyl-CoA carboxylase by fructose bisphosphate and citrate.

The abnormal accumulation of lipids due to myo-inositol deficiency in Saccharomyces carlsbergensis, and the mechanism involved was investigated. The deficient cells contained much more neutral lipids with a greater ratio of unsaturated fatty acids compared to the supplemented cells, whereas there was no significant change in their phospholipid contents. The biosynthesis of fatty acids and sterols from acetate, and of triacylglycerols and sterol esters from palmitate was markedly augmented in the deficient cells. Acetyl-CoA carboxylase activity of the deficient supernatant was 2- to 5-fold higher than that of the supplemented. However, the activity from both sources was not significantly different after Sephadex G-25 gel filtration of the supernatant, suggesting the presence of low molecular effector(s) in the deficient supernatant. There was a great increase in acid-soluble glycogen, trehalose, and fructose-1,6-P2, as well as a drastic decrease in citrate in the deficient cells. Their intracellular levels were calculated so that their effects on acetyl-CoA carboxylase was examined over the range of physiological concentration. Citrate strongly inhibited the enzyme activity of the supernatant, but it had no effect on the preparation after gel filtration. On the other hand, fructose-1,6-P2 stimulated the enzyme activity both before and after gel filtration. The acetyl-CoA carboxylase activity in the gel filtrate was measured as a function of citrate concentration at several fixed concentrations of fructose-1,6-P2. Citrate counteracted the activation by fructose-1,6-P2 in a dose-dependent manner. Citrate lacked the inhibitory effect in the absence of fructose-1,6-P2. It was concluded from these results that neutral lipid accumulation in the deficient cells reflected an increase in the synthesis of fatty acids, at least partly based on an enhancement of acetyl-CoA carboxylase activity, and that the operation of a reciprocal regulation of the enzyme by fructose-1,6-P2 and citrate caused a marked elevation of the enzyme activity in the deficient cells with a high fructose-1,6-P2 level and a low citrate level.     

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.     

Glutathione oxidation and activation of pentose phosphate cycle during hydroperoxide metabolism. A comparison of livers from fed and fasted rats

Perfusion of livers from fed and fasted rats with 0.07--0.1 mM t-butyl hydroperoxide for 15 min decreased the levels of reduced glutathione (GSH) by 1.5 mumol/g liver in both nutritional states. Glutathione disulfide (GSSG) was increased by 70 and 140 nmol/g liver and glutathione mixed disulfides enhanced by 45 and 150 nmol/g liver in livers from fed and fasted animals, respectively. The ratio of GSH/GSSG was decreased from 243 to 58 in fed animals, and from 122 to 8 in fasted animals. The increase of GSSG and the mixed disulfides was nearly parallel until an apparently critical low GSH content of 1.5 mumol/g was reached. Only in livers from fasted rats 14CO2-production from [1-14C]glucose was stimulated upon t-butyl hydroperoxide infusion at the employed rates. Flux of glucose through pentose phosphate cycle rose from 8 to 12% of glucose utilization via glycolysis, whereas in livers from fed animals this portion remained unchanged at 8% Dithio-erythritol reversed pentose phosphate cycle activity as well as GSSG and protein-bound glutathione contents to the original levels. In livers from fasted rats the activity of glucose-6-phosphate dehydrogenase was increased by 34% by t-butyl hydroperoxide infusion.