Fine-tuning acetyl-CoA carboxylase 1 activity through localization: functional genomics reveals a role for the lysine acetyltransferase NuA4 and sphingolipid metabolism in regulating Acc1 activity and localization

Acetyl-CoA Carboxylase 1 catalyzes the conversion of acetyl-CoA to malonyl-CoA, the committed step of de novo fatty acid synthesis. As a master regulator of lipid synthesis, acetyl-CoA carboxylase 1 has been proposed to be a therapeutic target for numerous metabolic diseases. We have shown that acetyl-CoA carboxylase 1 activity is reduced in the absence of the lysine acetyltransferase NuA4 in Saccharomyces cerevisiae. This change in acetyl-CoA carboxylase 1 activity is correlated with a change in localization. In wild-type cells, acetyl-CoA carboxylase 1 is localized throughout the cytoplasm in small punctate and rod-like structures. However, in NuA4 mutants, acetyl-CoA carboxylase 1 localization becomes diffuse. To uncover mechanisms regulating acetyl-CoA carboxylase 1 localization, we performed a microscopy screen to identify other deletion mutants that impact acetyl-CoA carboxylase 1 localization and then measured acetyl-CoA carboxylase 1 activity in these mutants through chemical genetics and biochemical assays. Three phenotypes were identified. Mutants with hyper-active acetyl-CoA carboxylase 1 form 1 or 2 rod-like structures centrally within the cytoplasm, mutants with mid-low acetyl-CoA carboxylase 1 activity displayed diffuse acetyl-CoA carboxylase 1, while the mutants with the lowest acetyl-CoA carboxylase 1 activity (hypomorphs) formed thick rod-like acetyl-CoA carboxylase 1 structures at the periphery of the cell. All the acetyl-CoA carboxylase 1 hypomorphic mutants were implicated in sphingolipid metabolism or very long-chain fatty acid elongation and in common, their deletion causes an accumulation of palmitoyl-CoA. Through exogenous lipid treatments, enzyme inhibitors, and genetics, we determined that increasing palmitoyl-CoA levels inhibits acetyl-CoA carboxylase 1 activity and remodels acetyl-CoA carboxylase 1 localization. Together this study suggests yeast cells have developed a dynamic feed-back mechanism in which downstream products of acetyl-CoA carboxylase 1 can fine-tune the rate of fatty acid synthesis.     

Effect of tumor necrosis factor on acetyl-coenzyme A carboxylase gene expression and preadipocyte differentiation

Tumor necrosis factor (TNF) is secreted by macrophages in response to various stimuli and blocks lipid accumulation during the conversion of preadipocytes to adipocytes in culture. In the present report, we investigate the effect of recombinant TNF on the expression of acetyl-coenzyme-A (CoA) carboxylase, the rate-limiting enzyme for long-chain fatty acid biosynthesis. We used a preadipocyte cell line, 30A-5, derived from 10T1/2 mouse fibroblasts after treatment with 5-azacytidine. Treatment of the preadipocyte cell line with dexamethasone and insulin triggers the conversion of these cells to mature adipocytes as evidenced by the accumulation of lipid. The mRNA and enzyme levels of acetyl-CoA carboxylase as well as the enzyme activity increase markedly during the conversion process. TNF prevents the conversion of preadipocytes to adipocytes with a concomitant inhibition in the accumulation of acetyl-CoA carboxylase mRNA and decrease in enzyme activity. This observed reduction in acetyl-CoA carboxylase mRNA levels is reversible upon removal of TNF. Acetyl-CoA carboxylase mRNA levels and enzyme activity also decrease when fully differentiated adipocytes are exposed to TNF but to a much lesser extent. These results suggest that TNF affects de novo lipid synthesis in part by altering the mRNA levels of acetyl-CoA carboxylase.     

The source of malonyl-CoA in rat heart. The calcium paradox releases acetyl-CoA carboxylase and not propionyl-CoA carboxylase

The formation of malonyl-CoA in rat heart is catalyzed by cytosolic acetyl-CoA carboxylase. The existence of this enzyme in heart is difficult to prove by the abundant occurrence of mitochondrial propionyl-CoA carboxylase, which is also able to catalyze the carboxylation of acetyl-CoA. We used the calcium paradox as a tool to separate cytosolic components from the remaining heart, and found that acetyl-CoA carboxylase activity was preferentially released, like lactate dehydrogenase and carnitine, while propionyl-CoA carboxylase was almost fully retained. Acetyl-CoA carboxylase activity was determined after activation by citrate ion and Mg2+. The activity decreased to 64% by 48 h of fasting.     

Initiation of lipogenic enzyme activities in rat mammary glands.

The activities of acetyl-CoA carboxylase, ATP citrate-lyase and fatty acid synthetase remained low until parturition at 22 days of gestation and increased significantly within 1 day post partum. Administration of progesterone on days 20 and 21 and at parturition abolished the increases for at least 48 h after parturition. Removal of the pups of normal rats prevented the increases in activities of acetyl-CoA carboxylase and ATP citrate-lyase, but not of fatty acid synthetase, and administration of prolactin corticosterone or insulin did not stimulate activity. Tissue from suckled glands in which the ducts had been ligated at parturition showed no increase in the activities of acetyl-CoA carboxylase and ATP citrate-lyase within 24 h, whereas fatty acid synthetase activity was similar to that in the sham-operated contralateral glands. Foetoplacentectomy on day 18 increased the activity of fatty acid synthetase but not of acetyl-CoA carboxylase and ATP citrate-lyase; suckling of these dams by foster pups increased both acetyl-CoA carboxylase and ATP citrate-lyase.     

Physiological regulation of acetyl-CoA carboxylase gene expression: effects of diet, diabetes, and lactation on acetyl-CoA carboxylase mRNA

We measured acetyl-CoA carboxylase mRNA levels in various tissues of the rat under different nutritional and hormonal states using a cDNA probe. We surveyed physiological conditions which are known to alter carboxylase activity, and thus fatty acid synthesis, to determine whether changes in the levels of carboxylase mRNA are involved. The present studies include the effects of fasting and refeeding, diabetes and insulin, and lactation on carboxylase mRNA levels. Northern blot analysis of liver RNA revealed that fasting followed by refeeding animals a fat-free (high carbohydrate) diet dramatically increased the amount of carboxylase mRNA compared to the fasted condition. These changes in the level of mRNA correspond to changes in the activity and amount of acetyl-CoA carboxylase. Acetyl-CoA carboxylase mRNA levels in epididymal fat tissue decreased upon fasting and increased to virtually normal levels after 72 h of refeeding, closely resembling the liver response. The amount of acetyl-CoA carboxylase mRNA decreased markedly in epididymal fat tissue of diabetic rats as compared to nondiabetic animals. However, 6 h after injection of insulin the mRNA level returned to that of the nondiabetic animals. Gestation and lactation also affected the levels of carboxylase mRNA in both liver and mammary gland. Maximum induction in both tissues occurred 5 days postpartum. These studies suggest that these diverse physiological conditions affect fatty acid synthesis in part by altering acetyl-CoA carboxylase gene expression.     

葡萄糖和维生素C对普安银鲫卵黄囊仔鱼ACC、FAS及CPTⅠ活性的影响

本试验旨在探究普安银鲫(Carassius auratus )卵黄囊仔鱼发育过程中ACC、FAS及CPT I活性变化及葡萄糖和维生素C溶液分别浸泡对它们的影响。采用酶学方法研究了普安银鲫卵黄囊仔鱼过程中ACC、FAS及CPT I活性变化的变化特点。结果显示：在卵黄囊仔鱼发育过程中,对照组与维生素C组中ACC和FAS活性呈上升趋势,CPT I活性呈“下降-上升”变化趋势,而葡萄糖组ACC、FAS及CPT I活性均呈上升趋势,且3种酶的活性均显著高于对照组(P<0.05)。维生素C组ACC活性在内源营养期显著高于对照组,FAS活性在混合营养期和外源营养期显著高于对照组,CPT I活性在内源营养期和外源营养期显著高于对照组(P<0.05)。研究表明：ACC、FAS及CPT I在维持普安银鲫卵黄囊仔鱼发育中脂质代谢的动态平衡起着重要作用,15g/L的葡萄糖溶液可通过调节仔鱼体内脂质代谢酶的活性而形成新的脂质代谢水平,以满足仔鱼生长发育需要;而30mg/L的维生素C对维持仔鱼发育中体内正常的脂质代谢具有重要作用。     

Calmodulin mediated activation of acetyl-CoA carboxylase during aflatoxin production by Aspergillus parasiticus

The relevance of Ca2+-calmodulin-mediated processes in channelling acetate for aflatoxin formation was investigated by studying the influence of trifluoperazine (an anticalmodulin agent) on [14C]-acetate incorporation and activity of acetyl-CoA carboxylase in Aspergillus parasiticus NRRL 2999. Culturing the organism in presence of 0.14 mmol l-1 trifluoperazine resulted in 55% decrease of [14C]-acetate incorporation into aflatoxin B1, along with an 80% decrease in acetyl-CoA carboxylase activity at periods corresponding to maximal aflatoxin production. Concomitant decrement (35%) in the activity of glucose-6-phosphate dehydrogenase indicated decreased availability of reduction potential (NADPH) required for aflatoxin biosynthesis. The ability of calmodulin to activate and trifluoperazine to inhibit acetyl-CoA carboxylase activity in a dose-dependent manner was also noted under in vitro conditions. The combined results suggest calmodulin-mediated activation of acetyl-CoA carboxylase as an important event for aflatoxin production.     

Possible role of acetyl-CoA-carboxylase in biosynthesis of mevalonic acid and sterols in rat liver]

Effect of citrate on acetyl-CoA incorporation into mevalonic acid, sterols and fatty acids after preliminary incubation of rat liver extracts under conditions optimal for acetyl-CoA carboxylase activation, was studied. 30 min preincubation with the citrate at 37 degrees C results in a 2--3-fold stimulation of the mevalonic acid biosynthesis from acetyl-CoA in the microsomal and soluble (140 000 g) fraction, and in that of sterols precipitated by digitonin or isolated by TLC in the mitochondria--free fraction. 2-14C-malonyl-CoA incorporation into the mevalonic acid and sterols and biosynthesis of sterols from 2-14C-mevalonic acid were not stimulated under those conditions. A correlation was shown to exist between the activity of acetyl-CoA carboxylase and the rate of acetyl-CoA incorporation into mevalonate and sterols; the activity of beta-hydroxy-beta-methylglutaryl-CoA reductase, limiting the rate of the sterol biosynthesis, was not changed. The stimulating effect of citrate was found to depend on the concentration of acetyl-CoA and NADPH in the medium. The data obtained suggest that the mevalonic acid biosynthesis in rat liver may occur in the presence of acetyl-CoA carboxylase through the formation of malonyl-CoA.     

A multienzyme complex for CO2 fixation.

Acetyl-coenzyme A carboxylase from Euglena gracilis strain Z was isolated as a component of a multienzyme complex which includes phosphoenolpyruvate carboxylase and malate dehydrogenase. The multienzyme complex was shown to exist in crude extracts and was purified to a homogeneous protein with a molecular weight of 360,000 by gel filtration. The ratio of the activities of the constituent enzymes was acetyl-CoA carboxylase:phosphoenolpyruvate carboxylase:malate dehydrogenase, 1:25:500. The complex is proposed to operate in conjunction with malic enzyme, which is present in Euglena, to facilitate the formation of substrates, malonyl-CoA, and NADPH, for fatty acid biosynthesis. The interaction of the enzymes may represent a means of control of acetyl-CoA carboxylase activity in organisms which do not possess an enzyme subject to allosteric regulation. The acetyl-CoA carboxylase activity from Euglena is unaffected by citrate and isocitrate.     

Hepatocyte heterogeneity in the metabolism of fatty acids: discrepancies on zonation of acetyl-CoA carboxylase.

Lipid metabolism appears to be less zonated than carbohydrate and protein metabolism. Studies on the zonation of lipid metabolism have been centered in particular on fatty acid synthesis which, according to the concept of metabolic zonation, should be a predominantly perivenous process while fatty acid oxidation should be periportal. There are, however, conflicting data on the activity gradients of lipogenic enzymes as well as measurements of actual synthesis of fatty acid and very low density lipoprotein. Data obtained by microdissection show a 1.5- to 2-fold higher activity of acetyl-CoA carboxylase and citrate lyase in the perivenous zone in agreement with measurements of the actual rate of fatty acid synthesis in preparations of hepatocyte, enriched in periportal or perivenous cells. On the other hand, results obtained with the dual-digitonin-pulse perfusion technique demonstrate the opposite gradient in the form of a 2- to 3-fold higher specific activity of acetyl-CoA carboxylase in the periportal zone based on measurements of the acetyl-CoA carboxylase protein proper. This specific activity gradient, which applies to male and not female rats, disappears almost completely in the fasted-refed animal, were lipogenesis is strongly induced. In this review we attempt to rationalize these discrepancies in the results as methodological differences which in particular apply to the following parameters: (1) expression of results (reference substance); (2) selectivity of zonal sampling, and (3) differences in methodology of acetyl-CoA carboxylase measurements. It is concluded that these factors could account for the discrepancies, but further studies, in particular on the zonation acetyl-CoA carboxylase mRNA, are required in order to further understand the zonation of lipid metabolism and its possible role in the metabolic regulation of the liver.     

Citrate carrier and lipogenic enzyme activities in lead nitrate-induced proliferative and apoptotic phase in rat liver.

After in vivo administration of lead nitrate, functional changes of the mitochondrial tricarboxylate carrier and of the cytosolic lipogenic enzymes acetyl-CoA carboxylase and fatty acid synthetase have been detected in rat liver. The rate of citrate transport was greatly reduced in rats during both the proliferative phase (3 days after the lead nitrate administration) and the involutive phase (5 days after the metal injection), which follows hepatic hyperplasia and corresponds to the peak of hepatocyte apoptosis. In both phases, a decrease of the lipogenic enzyme activities has been detected. In treated animals, an alteration of mitochondrial lipid composition has also been found. The modified lipid microenvironment could be responsible for the decreased carrier activity which, in turn, may account for the reduced activities of the lipogenic enzymes.     

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.     

Acetyl-CoA carboxylase exerts strong flux control over lipid synthesis in plants

The importance of acetyl-CoA carboxylase in regulation of lipid synthesis for barley and maize leaves has been quantitatively assessed using, as specific inhibitors, the herbicides fluazifop and sethoxydim. Apparent flux control coefficients of about 0.58 and 0.52 were determined for acetyl-CoA carboxylase in barley and maize leaves, respectively. These results show that acetyl-CoA carboxylase is the major flux controlling enzyme for light-stimulated lipid synthesis in these tissues.     

Insulin-like effects of fatty acid synthesis in liver of hamsters infected with plerocercoids of the tapeworm, Spirometra mansonoides

Elevated serum lipids are associated with infections of laboratory rodents with plerocercoids of Spirometra mansonoides. The effect of infection with these larval tapeworms on triglyceride degradation and hepatic de novo fatty acid synthesis was investigated in Syrian hamsters. Serum lipoprotein electrophoresis revealed a consistent elevation in very low density lipoproteins in the infected animals. Lipoprotein lipase activity was enhanced in the infected animals. After seven days of plerocercoid infection the activity of acetyl-CoA carboxylase (E.C. 6.4.1.2) was significantly elevated after 6, 12 and 18 hours of fasting. Fatty acid synthetase was significantly increased after 0, 6, 12, 18 and 24 hours of fasting. Therefore, a chronic insulin-like activity on lipid metabolism of hamsters is associated with plerocercoid infection.     

Lipid biosynthesis in liver slices of the foetal guinea pig.

Lipid synthesis as measured by the incorporation of acetate or 3H2O into slices of foetal liver, is much higher than in slices of adult liver and shows a peak at about two-thirds of gestation. At this time the synthesis from glucose was low and reached a peak 10 days later. The changes in the activity of ATP citrate lyase, which mirrored acetate incorporation, and the effect of glucose and pyruvate on acetate corporation into lipid suggests that some of the lipid synthesis occurs via intramitochondrial acetyl-CoA production from acetate. Despite this, lipid synthesis was not inhibited by (-)-hydroxycitrate. The low rate of synthesis from glucose at two-thirds of gestation is ascribed to the low activity of pyruvate carboxylase at this time and a role for a phosphoenolpyruvate carboxykinase in providing oxaloacetate for lipogenesis is proposed. The activity of fatty acid synthetase broadly agreed with the changes in lipid synthesis, whereas the activity of acetyl-CoA carboxylase was barely sufficient to account for the rates of lipid synthesis in vivo. Acetate and short-chain fatty acids are likely to be the major precursors for lipid synthesis in vivo.     

Changes in the activities of various lipid and carbohydrate biosynthetic enzymes in the diatom Cyclotella cryptica in response to silicon deficiency

The effects of silicon deficiency on the activities of several enzymes involved in lipid and storage carbohydrate synthesis in the diatom Cyclotella cryptica were determined. The activity of UDPglucose pyrophosphorylase was not affected after 4 h of silicon-deficient growth, but the activity of UDPglucose: beta-(1----3)-glucan-beta-3-glucosyltransferase (chrysolaminarin synthase) was reduced by 31% during this period. Acetyl-CoA synthetase, acetyl-CoA hydrolase, and citrate synthase activities were present in cell-free extracts of C. cryptica, but did not change in response to 4 h of silicon deficiency. However, the activity of acetyl-CoA carboxylase increased approximately two- and fourfold after 4 and 15 h of silicon-deficient growth, respectively. This induction could be blocked by cycloheximide (20 micrograms/ml) and actinomycin D (10 micrograms/ml), suggesting that silicon deficiency may induce an increase in the rate of acetyl-CoA carboxylase synthesis. These changes in enzymatic activity may be partially responsible for the accumulation of lipids that has been observed in C. cryptica and other diatoms in response to silicon deficiency.     

Phosphorylation of pea chloroplast acetyl-CoA carboxylase

We have examined whether chloroplast acetyl-CoA carboxylase is a phosphoprotein. Pea ( Pisum sativum ) chloroplasts were incubated in the presence of [γ- ³³ P]-ATP and radiolabeled proteins were examined after immunoprecipitation with antibodies against all four known subunits of heteromeric chloroplast acetyl-CoA carboxylase. The β-subunit of the carboxyltransferase was found to be labeled by ³³ P. Phosphoamino acid analysis of the immunoprecipitated β-subunit of the carboxyltransferase indicates that it is phosphorylated on serine residues. Incorporation of ³³ P into carboxyltransferase β-subunit decreased in chloroplasts transferred to dark conditions after labeling in the light. Dephosphorylation of pea chloroplast extracts by an alkaline phosphatase-agarose conjugate reduced in vitro acetyl-CoA carboxylase activity by 67%. Furthermore, while acetyl-CoA carboxylase activity and its carboxyltransferase half-reaction were reduced in dephosphorylated extracts, the biotin carboxylase half-reaction was not inhibited. The evidence presented here points to the carboxyltransferase β-subunit of chloroplast acetyl-CoA carboxylase as a candidate for regulation by protein phosphorylation/dephosphorylation.     

The role of pyruvate dehydrogenase and acetyl-coenzyme A synthetase in fatty acid synthesis in developing Arabidopsis seeds

Acetyl-coenzyme A (acetyl-CoA) formed within the plastid is the precursor for the biosynthesis of fatty acids and, through them, a range of important biomolecules. The source of acetyl-CoA in the plastid is not known, but two enzymes are thought to be involved: acetyl-CoA synthetase and plastidic pyruvate dehydrogenase. To determine the importance of these two enzymes in synthesizing acetyl-CoA during lipid accumulation in developing Arabidopsis seeds, we isolated cDNA clones for acetyl-CoA synthetase and for the ptE1alpha- and ptE1beta-subunits of plastidic pyruvate dehydrogenase. To our knowledge, this is the first reported acetyl-CoA synthetase sequence from a plant source. The Arabidopsis acetyl-CoA synthetase preprotein has a calculated mass of 76,678 D, an apparent plastid targeting sequence, and the mature protein is a monomer of 70 to 72 kD. During silique development, the spatial and temporal patterns of the ptE1beta mRNA level are very similar to those of the mRNAs for the plastidic heteromeric acetyl-CoA carboxylase subunits. The pattern of ptE1beta mRNA accumulation strongly correlates with the formation of lipid within the developing embryo. In contrast, the level of mRNA for acetyl-CoA synthetase does not correlate in time and space with lipid accumulation. The highest level of accumulation of the mRNA for acetyl-CoA synthetase during silique development is within the funiculus. These mRNA data suggest a predominant role for plastidic pyruvate dehydrogenase in acetyl-CoA formation during lipid synthesis in seeds.     

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.     

Protein-serine kinase from rat epididymal adipose tissue which phosphorylates and activates acetyl-CoA carboxylase. Possible role in insulin action.

1. Most of the cyclic-nucleotide-independent acetyl-CoA carboxylase kinase activity in an extract of rat epididymal adipose tissue was evaluated from a Mono Q column by 0.175 M-NaCl at pH 7.4. The activity of the kinase in this fraction (fraction 1) was increased after exposure of intact tissue to insulin. 2. Incubation of purified adipose-tissue acetyl-CoA carboxylase with [gamma-32P]ATP and samples of fraction 1 led to the incorporation of up to 0.4 mol of 32P/mol of enzyme subunit. Most of the phosphorylation was on serine residues within a single tryptic peptide. This peptide, on the basis of two-dimensional t.l.c. analysis, h.p.l.c. and Superose 12 chromatography, appeared to be the same as the acetyl-CoA carboxylase peptide ('I'-peptide) which exhibits increased phosphorylation in insulin-treated tissue. 3. Phosphorylation of purified acetyl-CoA carboxylase by the kinase in fraction 1 was found to be associated with a parallel 4-fold increase in activity. However, increases in both phosphorylation and activity were much diminished if fraction 1 was treated by Centricon centrifugation to remove low-Mr components. Among these components was a potent inhibitor of acetyl-CoA carboxylase activity which appeared to be necessary for the kinase in fraction 1 to be fully active. 4. The inhibitor remains to be identified, but inhibition requires MgATP, although the inhibitor itself does not cause any phosphorylation of the carboxylase. No effects of insulin were observed on the activity of the inhibitor. 5. It is concluded that the kinase probably plays an important role in the mechanism whereby insulin brings about the well-established increases in phosphorylation and activation of acetyl-CoA carboxylase in adipose tissue.