Insulin and the regulation of adipose-tissue acetyl-coenzyme A carboxylase

Rat epididymal fat-pads were incubated for 30min with glucose (2mg/ml) in the presence or absence of insulin. A twofold or greater increase in acetyl-CoA carboxylase activity was observed in extracts from insulin-treated tissue provided that assays were performed rapidly after extraction. This effect of insulin was evident whether or not extracts were prepared with albumin, and was not noticeably diminished by the presence of citrate or albumin or both in the assay. Incubation of extracts before assay led to activation of acetyl-CoA carboxylase and a marked diminution in the insulin effect. The enzyme in extracts was very sensitive to reversible inhibition by palmitoyl-CoA even in the presence of albumin (10mg/ml); inhibition persisted on dilution of enzyme and inhibitor. It is suggested that the observed activation of acetyl-CoA carboxylase by insulin may reflect changes in enzyme activity in the fat-cell resulting from the reduction of long-chain fatty-acyl-CoA that occurs in the presence of insulin. Activation of the enzyme with loss of the insulin effect on incubation of the extracts may be due to the slow dissociation of long-chain fatty acyl-CoA from the enzyme.     

The synthesis and hydrolysis of long-chain fatty acyl-coenzyme A thioesters by soluble and microsomal fractions from the brain of the developing rat.

1. The specific activities of long-chain fatty acid-CoA ligase (EC6.2.1.3) and of long-chain fatty acyl-CoA hydrolase (EC3.1.2.2) were measured in soluble and microsomal fractions from rat brain. 2. In the presence of either palmitic acid or stearic acid, the specific activity of the ligase increased during development; the specific activity of this enzyme with arachidic acid or behenic acid was considerably lower. 3. The specific activities of palmitoyl-CoA hydrolase and of stearoyl-CoA hydrolase in the microsomal fraction decreased markedly (75%) between 6 and 20 days after birth; by contrast, the corresponding specific activities in the soluble fraction showed no decline. 4. Stearoyl-CoA hydrolase in the microsomal fraction is inhibited (99%) by bovine serum albumin; this is in contrast with the microsomal fatty acid-chain-elongation system, which is stimulated 3.9-fold by albumin. Inhibition of stearoyl-CoA hydrolase does not stimulate stearoyl-CoA chain elongation. Therefore it does not appear likely that the decline in the specific activity of hydrolase during myelogenesis is responsible for the increased rate of fatty acid chain elongation. 5. It is suggested that the decline in specific activity of the microsomal hydrolase and to a lesser extent the increase in the specific activity of the ligase is directly related to the increased demand for long-chain acyl-CoA esters during myelogenesis as substrates in the biosynthesis of myelin lipids.     

Steady-state concentrations of coenzyme A, acetyl-coenzyme A and long-chain fatty acyl-coenzyme A in rat-liver mitochondria oxidizing palmitate

1. Fluorimetric assays are described for CoASH, acetyl-CoA and long-chain fatty acyl-CoA, and are sensitive to at least 50mumumoles of each. 2. Application of these assays to rat-liver mitochondria oxidizing palmitate in the absence and presence of carnitine indicated two pools of intramitochondrial CoA. One pool could be acylated by palmitate and ATP, and the other pool acylated by palmitate with ATP and carnitine, or by palmitoylcarnitine alone. 3. The intramitochondrial content of acetyl-CoA is increased by the oxidation of palmitate both in the absence and presence of l-malate. 4. The conversion of palmitoyl-CoA into acetyl-CoA by beta-oxidation takes place without detectable accumulation of acyl-CoA intermediates.     

Vasoactive intestinal peptide stimulates long-chain fatty acid oxidation and inhibits acetyl-coenzyme A carboxylase activity in isolated rat enterocytes

The effects of vasoactive intestinal peptide (VIP) on fatty acid oxidation in isolated rat enterocytes were investigated. VIP (10(-7) M) increased more than 2-fold the production of 14CO2 from [U-14C]palmitate. This effect was dose-dependent (K0.5 = 5.10(-11) M) and appeared to be related to the stimulation of cAMP production since it was mimicked by forskolin (10(-4) M). VIP also stimulated oxygen consumption of the cells, an effect accounted for by the stimulation of the oxidation of both exogenous added palmitate (0.12 mM) and endogenous fatty acids produced by lipolysis. VIP appeared to specifically enhance the oxidation of long-chain fatty acids since its effects were counteracted by 5.10(-5) M sodium 2-[6-(chlorophenoxy)hexyl]oxirane-2-carboxylate, a potent inhibitor of carnitine palmitoyltransferase 1, and since VIP did not affect cell respiration in the presence of octanoate. These results suggested that VIP stimulated long-chain fatty acid oxidation by increasing their translocation into the mitochondria. Therefore, we examined the effect of VIP on the activity of acetyl-coenzyme A carboxylase, the enzyme responsible for the biosynthesis of malonyl-CoA, a physiological inhibitor of carnitine acyltransferase 1. VIP produced an acute, dose-dependent (Ki = 3.10(-11) M), 90% inhibition of acetyl-coenzyme A carboxylase activity. These results allow us to elucidate the mechanism of the recently reported inhibitory effect of VIP on glucose oxidation (Vidal, H., Comte, B., Beylot, M., and Riou, J. P. (1988) J. Biol. Chem. 263, 9206-9211) and demonstrate for the first time that balance between fatty acids and glucose as energetic fuels is under neurohormonal control in isolated rat enterocytes.     

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.     

Hormonal and stress induction of the gene encoding common bean acetyl-coenzyme A carboxylase

Regulation of the cytosolic acetyl-coenzyme A carboxylase (ACCase) gene promoter from common bean (Phaseolus vulgaris) was studied in transgenic Arabidopsis (Arabidopsis thaliana) plants using a beta-glucuronidase (GUS) reporter gene fusion (PvACCase::GUS). Under normal growth conditions, GUS was expressed in hydathodes, stipules, trichome bases, flowers, pollen, and embryos. In roots, expression was observed in the tip, elongation zone, hypocotyl-root transition zone, and lateral root primordia. The PvACCase promoter was induced by wounding, Pseudomonas syringae infection, hydrogen peroxide, jasmonic acid (JA), ethylene, or auxin treatment. Analysis of PvACCase::GUS expression in JA and ethylene mutants (coronatine insensitive1-1 [coi1-1], ethylene resistant1-1 [etr1-1], coi1-1/etr1-1) suggests that neither JA nor ethylene perception participates in the activation of this gene in response to wounding, although each of these independent signaling pathways is sufficient for pathogen or hydrogen peroxide-induced PvACCase gene expression. We propose a model involving different pathways of PvACCase gene activation in response to stress.     

Beta-oxidation of long-chain fatty acids by human fibroblasts: evidence for a novel long-chain acyl-coenzyme A dehydrogenase.

Fibroblasts from patients with long-chain acyl-CoA dehydrogenase deficiency were found to oxidize [1-14C]linoleate at an average rate of 60% of normal but [9,10(n)-3H]myristate at an average rate of only 37% of normal, a relationship reverse from that predicted by the chain-length specificities of the three known straight-chain mitochondrial acyl-CoA dehydrogenases. The residual long-chain beta-oxidative activity was found to be mitochondrial and associated with the accumulation of tetradecadienoate (C14:2w6) when the mutant fibroblasts were incubated with 100 mumol/L linoleate (C18:2w6) or eicosadienoate (C20:2w6). The results suggest the presence in human fibroblasts of a novel acyl-CoA dehydrogenase with activity toward 15 to 20 carbon-length fatty acids.     

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.     

Regulation of acetyl-coenzyme A carboxylase. I. Purification and properties of two forms of acetyl-coenzyme A carboxylase from rat liver

Acetyl-CoA carboxylase of animal tissues is known to be dependent on citrate for its activity. The observation that dephosphorylation abolishes its citrate dependence (Thampy, K. G., and Wakil, S. J. (1985) J. Biol. Chem. 260, 6318-6323) suggested that the citrate-independent form might exist in vivo. We have purified such a form from rapidly freeze-clamped livers of rats. Sodium dodecyl sulfate gel electrophoresis of the enzyme gave one protein band (Mr 250,000). The preparation has high specific activity (3.5 units/mg in the absence of citrate) and low phosphate content (5.0 mol of Pi/mol of subunit). The enzyme isolated from unfrozen liver or liver kept in ice-cold sucrose solution for 10 min and then freeze-clamped has low activity (0.3 unit/mg) and high phosphate content (7-8 mol of Pi/mol of subunit). Citrate activated such preparations with half-maximal activation at greater than 1.6 mM, well above physiological range. The low activity may be due to its high phosphate content because dephosphorylation by [acetyl-CoA carboxylase]-phosphatase 2 activates the enzyme and reduces its dependence on citrate. Since freeze-clamping the liver yields enzyme with lower phosphate content and higher activity, it is suggested that the carboxylase undergoes rapid phosphorylation and consequent inactivation after the excision of the liver. The carboxylase is made up of two polymeric forms of Mr greater than or equal to 10 million and 2 million based on gel filtration on Superose 6. The former, which predominates in preparations from freeze-clamped liver, has higher activity and lower phosphate content (5.3 units/mg and 4.0 mol of Pi/mol of subunit, respectively) than the latter (2.0 units/mg and 6.0 mol of Pi/mol of subunit, respectively). The latter, which predominates in preparations from unfrozen liver, is converted to the active polymer (Mr greater than or equal to 10 million) by dephosphorylation. Thus, the two polymeric forms are interconvertible by phosphorylation/dephosphorylation and may be important in the physiological regulation of acetyl-CoA carboxylase.     

Short-term regulation of acetyl CoA carboxylase: is the key enzyme in long-chain fatty acid synthesis regulated by an existing physiological mechanism?

Acetyl CoA carboxylase, the rate-limiting enzyme in regulating fatty acid synthesis, is thought to be controlled by allosteric effectors, its state of aggregation, covalent modulation and protein inhibitors. It is still obscure whether citrate, a positive allosteric effector, and long-chain fatty acyl CoA esters, negative allosteric effectors, function physiologically to regulate acetyl CoA carboxylase activity. New evidence from several laboratories reveals that the covalent phosphorylation may not involve regulation of acetyl CoA carboxylase activity. Protein inhibitors from liver cytosol and a peptide from fat cells were found to regulate acetyl CoA carboxylase both in vivo and in vitro. Coenzyme A, guanosine 5-monophosphate and phosphatidylinositol 4,5-bisphosphate may have an indirect effect, but certainly no direct involvement, on carboxylase activity.     

The role of acyl-coenzyme A carboxylase complex in lipstatin biosynthesis of Streptomyces toxytricini

Streptomyces toxytricini produces lipstatin, a specific inhibitor of pancreatic lipase, which is derived from two fatty acid moieties with eight and 14 carbon atoms. The pccB gene locus in 10.6 kb fragment of S. toxytricini chromosomal DNA contains three genes for acyl-coenzyme A carboxylase (ACCase) complex accA3, pccB, and pccE that are presumed to be involved in secondary metabolism. The pccB gene encoding a β subunit of ACCase [carboxyltransferase (CT)] was identified upstream of pccE gene for a small protein of ε subunit. The accA3 encoding the α subunit of ACCase [biotin carboxylase (BC)] was also identified downstream of pccB gene. When the pccB and pccE genes were inactivated by homologous recombination, the lipstatin production was reduced as much as 80%. In contrast, the accumulation of another compound, tetradeca-5.8-dienoic acid (the major lipstatin precursor), was 4.5-fold increased in disruptant compared with wild-type. It implies that PccB of S. toxytricini is involved in the activation of octanoic acid to hexylmalonic acid for lipstatin biosynthesis.     

Stimulation of hepatic lipogenesis and acetyl-coenzyme A carboxylase by vasopressin.

The effect of vasopressin on the short-term regulation of fatty acid synthesis was studied in isolated hepatocytes from rats fed ad libitum. Vasopressin stimulates fatty acid synthesis by 30-110%. This increase is comparable with that obtained with insulin. Angiotensin also stimulates fatty acid synthesis, whereas phenylephrine does not. The dose-response curve for vasopressin-stimulated lipogenesis is similar to the dose-response curve for glycogenolysis and release of lactate plus pyruvate. Vasopression also stimulates acetyl-CoA carboxylase activity in a dose-dependent manner. Vasopressin does not relieve glucagon-inhibited lipogenesis, whereas insulin does. The action of vasopressin on hepatic lipogenesis is decreased, but not suppressed, in Ca2+-depleted hepatocytes. The results suggest that vasopressin acts on lipogenesis by increasing availability of lipogenic substrate (lactate + pyruvate) and by activating acetyl-CoA carboxylase.     

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

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

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.     

The sensitivity of acetyl-coenzyme A carboxylase to citrate stimulation in a homogenate of rat liver containing subcellular particles

1. Although citrate is known to activate purified preparations of acetyl-CoA carboxylase, it had no stimulatory effect on the incorporation of [¹⁴C]acetate into long-chain fatty acids in a whole homogenate of rat liver (S_0.7) under conditions in which the activity of acetyl-CoA carboxylase was rate-limiting for fatty acid synthesis. 2. The rate of incorporation of acetyl carbon into fatty acids was estimated in S_0.7 preparations incubated with [¹⁴C]acetate, by measuring the specific radioactivity of the acetyl carbon of acetyl-CoA and the incorporation of ¹⁴C into fatty acids. These estimates were compared with estimates of acetyl-CoA carboxylase activity in the S_0.7 preparation obtained by direct assay in conditions in which the enzyme was in the fully activated state. 3. In the absence of citrate, incorporation of acetyl carbon into fatty acids was about 75% of the value expected if the acetyl-CoA carboxylase in the S_0.7 preparation were in the fully activated state. 4. Incorporation of acetyl carbon into fatty acids in the S_0.7 preparation was stimulated by citrate, but the effect was many times less than the stimulation of [¹⁴C]acetate incorporation by citrate in particle-free preparations. 5. When the mitochondria and microsomes were removed from the S_0.7 preparation, [¹⁴C]acetate incorporation into fatty acids fell to a negligible value and the preparation became highly sensitive to stimulation by citrate. 6. It is suggested that in the presence of mitochondria and microsomes, and in the intact liver cell, the degree of activation of acetyl-CoA carboxylase is such that citrate activation may not be of physiological significance.