ATP Citrate Lyase – Biology and Implication in Human Pathology

Revista Român ă de Medicin ă de Laborator Vol. 12, Nr. 3, Septembrie 2008 17

ATP citrate lyase – biology and implication in human pathology

ATP citrat liaza – biologie i implicare în patologia uman ă Liviu S. Enache * Institut Fédératif de Recherche 128 ‘BioSciences Gerland – Lyon Sud’, INSERM, U851, Lyon, France Emergency Clinical Hospital, Medical Analysis Laboratory, Tîrgu Mure , Romania

Abstract ATP citrate lyase (ACLY) is a cytoplasmic enzyme that catalyzes the cleavage of citrate into oxaloacetate and acetyl-CoA. This reaction provides most of the acetyl-CoA required for the biosynthesis of fatty acids and cholesterol. Since its discovery half a century ago, many of the mechanisms involved in the regulation of ACLY activity have remained unclear. Various attempts to biochemically characterize the enzyme gave highly heterogeneous results. The development of expression systems for the recombinant human protein offered a pow- erful tool for research on this enzyme. Although the reaction catalyzed by ACLY is not rate limiting in either lipogenesis or cholesterologenesis, its „strategic” metabolic position makes ACLY an appealing target for hypolipidemic intervention. ACLY inhibition experiments showing hypolipidemic and hypocholoesterolemic effects in animal models, together with data suggesting the role of the enzyme in cancer biology, cell growth and differentiation, brought it in the attention of scientific community. This paper makes a review of ACLY biology and its potential implication in human pathology. Keywords: ATP citrate lyase, lipogenesis, enzyme inhibitors, hipolipidemic agents

Rezumat ATP citrat liaza (ACLY) este o enzim ă situat ă în citoplasm ă, care catalizeaz ă scindarea citratului în oxaloacetat i acetil-coenzima A. Aceast ă reac Ńie ofer ă majoritatea necesarului de acetil-coenzim ă A pentru bio- sinteza acizilor gra i i a colesterolului. Începând cu descoperirea sa în urm ă cu o jum ătate de secol, mai multe mecanisme implicate în reglarea activit ăŃ ii ACLY r ămân inc ă neclare. Încerc ările de a caracteriza enzima din punct de vedere biochimic au dat rezultate foarte variabile. Dezvoltarea sistemelor de expresie a proteinei uma- ne recombinante au oferit un instrument puternic pentru studierea acestei enzime. De i reac Ńia catalizat ă de ACLY nu este limitant ă de vitez ă nici pentru lipogenez ă, nici pentru colesterologenez ă, pozi Ńia ei metabolic ă strategic ă face din ACLY o Ńint ă atr ăgătoare pentru interven Ńia hipolipemiant ă. Experimente de inhibare a ACLY care au demonstrat efecte hipolipemiante i hipocolesterolemiante în modelele animale i date sugerând rolul enzimei în biologia cancerului, în cre terea i diferen Ńierea celular ă, au adus-o în aten Ńia comunit ăŃ ii tiin Ńifice. Aceast ă lucrare constituie o prezentare general ă asupra biologiei ACLY i a implic ării sale în patologia uman ă. Cuvinte-cheie: ATP citrat liaza, lipogenez ă, inhibitori enzimatici, agen Ńi hipolipemian Ńi.

*Address for correspondence: Emergency Clinical Hospital, Str. Gh. Marinescu, Nr. 50, Tîrgu Mure , Romania Tel. 0040724882779, E-mail: [email protected] 18 Revista Român ă de Medicin ă de Laborator Vol. 12, Nr. 3, Septembrie 2008

Fatty acids synthesis

Glucose Cholesterologenesis Malonyl-CoA Gluconeogenesis Glucose-6-P ACETYL-CoA CARBOXYLASE

Fructose-6-P Oxaloacetate Acetyl-CoA Acetate NADH+H + phosphorylation Pyruvate MDH CoA Insulin ATP CITRATE NAD + ATP Glucagon LYASE + Allosteric Malate Citrate activation Phosphorylated sugars P T +

PYRUVATE Malate DEHYDROGENASE Pyruvate Acetyl-CoA

Citrate Oxaloacetate

Citric acid cycle Malate

MITOCHONDRION

Figure 1. Involvement of ATP citrate lyase in several metabolic pathways. MDH = malate dehydrogenase, P = pyruvate transporter, T = tricarboxylate transporter ATP citrate lyase (ACLY) i is a cyto- catabolism of the carbon skeletons of amino plasmic enzyme that catalyses the cleavage of acids. The mitochondrial inner membrane is im- citrate into oxaloacetate and acetyl-CoA, the permeable to acetyl-CoA, so an indirect shuttle latter being the major building block for both transfers acetyl group equivalents across the in- cholesterologenesis and lipogenesis ( Figure 1 ). ner membrane. Intramitochondrial acetyl-CoA Acetyl-CoA provided by ACLY is also used in first reacts with oxaloacetate to form citrate in the biosynthesis of acetylcholine. Oxaloacetate the citric acid cycle, reaction catalyzed by cit- enters the gluconeogenic pathway through rate synthase. Citrate then passes through the phosphoenolpyruvate carboxykinase. inner membrane on the citrate transporter. In In nonruminants, nearly all the acetyl- the cytosol, citrate cleavage by ACLY regener- CoA used in fatty acid synthesis is formed in ates acetyl-CoA in an ATP-dependent reac- mitochondria from pyruvate oxidation and from tion 24 . Although the reaction catalyzed by i EC 2.3.3.8 (created in 1965 as EC 4.1.3.8, modified ACLY is not rate limiting in either lipo- or in 1986, transferred in 2002 to EC 2.3.3.8). System- cholesterologenesis, its „strategic” metabolic atic name: acetyl-CoA:oxaloacetate C-acetyltrans- position upstream of acetyl-CoA carboxylase ferase [(pro-S)-carboxymethyl-forming, ADP-phos- and hydroxymethyl glutaryl coenzyme A reduc- phorylating]. Synonyms: ATP-citrate synthase, tase attracted researchers to select this enzyme ATP-citrate (pro-S-)-lyase, Citrate cleavage enzyme Revista Român ă de Medicin ă de Laborator Vol. 12, Nr. 3, Septembrie 2008 19

as a target for hypolipidemic intervention. Re- CoA synthetase is not reported to involve the sults of ACLY inhibition experiments showing production of a covalent succinyl-enzyme as hypolipidemic and hypocholoesterolemic ef- part of the mechanism 36 . The structural and fects in animal models, together with data sug- functional similarity between the two enzymes gesting the role of the enzyme in cancer biolo- led to further investigations which showed that gy, cell growth and differentiation, brought it the mechanism of the reaction for ACLY pro- again in the attention of scientific community. ceeds as far as the production of an enzyme - This paper makes a review of ACLY citryl-phosphate complex. However, the next biology and its potential implication in human stage of the reaction is therefore most likely to pathology. be the direct attack of CoA on the citrate phosphate. Therefore, only one covalent enzyme- Reaction mechanism bound intermediate occurs in the ATP-citrate- lyase-catalysed reaction 36 . The novel proposed mechanism was also confirmed by the failure in The reaction catalysed by ACLY is the attempts to inhibit enzyme activity using shown in Equation 1 . compounds that interacted with the active site A five-step reaction mechanism was thiol nucleophile 10 . initially proposed, where enzyme was first phosphorylated by ATP and the phosphate was transferred to citrate to form a non-covalent cit- Structure ryl phosphate within the active site. The citrate was then transferred to a thiol nucleophile en- Since the early work of Srere and his zyme active site to form a covalent citryl-en- collaborators 30 who purified ACLY from chick- zyme complex which was then attacked by CoA en liver, the methods of production and purifi- to generate citryl-CoA within the active site. Fi- cation of the active enzyme have evolved, per- nally, the enzyme catalysed the retro-Claisen mitting detailed analysis of its structure and cleavage of citryl-CoA to produce acetyl-CoA function. Cloning and expression of human and oxaloacetate 36 . ACLY have been achieved 8 and large quantities The primary sequence of rat liver of active enzyme can now be obtained in bac- ACLY has been obtained from a cDNA clone ulovirus expression system 19 . and its amino acid sequence was showed to The enzyme is a tetramer (molecular have a high similarity with the alpha chain of mass of 440 KDa as determined by sedimenta- succinyl-CoA synthetase of Escherichia coli 7. tion equilibrium) of four apparently identical Both enzymes catalyse similar reactions. The subunits 29 . Each subunit has a sequence of 1101 first reaction step for both of them is autophos- amino acids and a calculated mass of 120,8 phorylation of the enzyme by ATP, followed by KDa 34 . Human ACLY amino acid sequence is phosphorylation of the substrate (citrate and 96,3% identical to rat ACLY and the inclusion succinate, respectively) by the phosphoenzyme of conservative replacements increases the de- formed. CoA reacts with citrate/succinate phos- gree of similarity to 98% 8. phate to form citryl-/succinyl- CoA. ACLY has Studies on this enzyme showed that the one additional step in that citryl-CoA is cleaved isolated protein was a phosphoprotein contain- into acetyl-CoA and oxaloacetate before re- ing usually two phosphate groups per lease. However, the mechanism of succinyl- tetramer 18 .

++ Citrate + ATP + CoA ←Mg →acetyl − CoA + oxaloaceta te + ADP + Pi Equation 1. Reaction catalyzed by ATP citrate lyase 20 Revista Român ă de Medicin ă de Laborator Vol. 12, Nr. 3, Septembrie 2008

Table 1. Features of ATP citrate lyase chain 34 Feature Key Position Length Description Regions Nucleotide binding 701-721 21 ATP (by similarity) Nucleotide binding 752 - 778 27 ATP (by similarity) Region 779 - 789 11 CoA-binding (potential) Sites Active site 760 1 Tele-phosphohystidine intermediate (by similarity) Metal binding 718 1 Magnesium (by similarity) Amino acid modifications Modified residue 131 1 Phosphotyrosine Modified residue 455 1 Phosphoserine Modified residue 481 1 Phosphoserine Modified residue 682 1 Phosphotyrosine Natural variations Natural variant 175 1 Glu →Asp

A summary of sequence annotations in ACLY mRNA. In young individuals, ACLY (features) on the chain of an ACLY subunit as mRNA is increased in brain, as lipogenesis is described in UniProtKB (Protein Knowledge- also an important pathway in the developing base) 34 are presented in Table 1 . brain, particularly with respect to myelination 7. A diurnal variation in ACLY mRNA in rat liver Enzyme distribution in tissues and was demonstrated. However, no diurnal variation in activity of the enzyme was observed 9. cellular localization The activity of ACLY is lower in human tissues compared to rat tissues. However, ACLY distribution in animal and vege- rat metabolic rates are in general higher than in tal tissues has been extensively studied. Ex- human. Early studies reported that human adi- tracts of rat, rabbit, chicken, and beef tissues pose tissue, unlike that of the rat, contains little were first compared by Srere 30 . The greatest en- or no ACLY activity. The mean activity of zyme activities were observed in extracts of ACLY in subcutaneous adipose tissue in hu- chicken liver and rat liver. Extracts of spinach mans was found approximately 50 times lower and of mushroom had no enzyme activity. versus the rat maintained on a normal laborato- Chicken liver frozen for as long as 3 months re- ry diet. It is known that diet influences li- tained most of its activity. In general, there is pogenic enzymes activity, which is lowered by little ACLY in ruminants, probably because in a high fat intake or by fasting and increased by these species acetate (derived from the rumen carbohydrates. The normal diet of laboratory and activated to acetyl-CoA extramitochondri- rats contains a high concentration of carbohy- ally) is the main source of acetyl-CoA 23 . drate and a low concentration of fat. The human The liver has been reported to be the diet contains a much higher level of fat and organ with the highest ACLY enzyme activity, most of the studies have been performed on hu- followed by brain and kidney 30 . The level of man adipose tissue taken after an overnight fast, ACLY mRNA in adult male rats has been which preceded the surgical removal of the tis- found to be the highest in liver. The adrenals, sue. Thus, one can suppose that if rats ate a nor- lung, brain and large intestine are relatively rich Revista Român ă de Medicin ă de Laborator Vol. 12, Nr. 3, Septembrie 2008 21

Table 2. Reaction conditions used in various experiments for the assessment of ATP citrate lyase activity Article Srere, 1959 30 Linn, 1979 18 Gribble, 1996 9 Ki, 2000 14 Potassium citrate 21 mM 20 mM 0.1 mM 5 mM ATP 5 mM 5 mM 0.250 mM 5 mM CoA 0.3 mM 0.33 mM 0.2 mM 0.33 mM Malate dehydrogenase 500 U 0.5 U Present 2 U NADH 0.1-0.2 mM 0.14 mM 35 mM 0.14 mM

MgCl 2 10 mM 10 mM 10 mM 10 mM KCl - - 10 mM - Tris HCl 100 mM, pH=7.3 100 mM, pH=8.7 100 mM, pH=8.0 100 mM, pH=8.5 Dithiothreitol 10 mM thioethanol 10 mM 10 mM 10 mM Total volume 1 ml 1 ml 100 µl mal human diet, their lipogenic enzyme activi- larger range of enzyme activities can be assayed ties would be significantly reduced. Indeed, when oxaloacetate is reduced with malate de- when rats were fed a high-fat diet and fasted hydrogenase (MDH) and NADH while monit- overnight, their ACLY activity in the adipose oring the decreasing NADH level at 340 nm us- tissue was only 8 times higher than in humans 32 . ing a spectrophotometer 30 . In human liver, ACLY activity per The two linked reactions used in the gram of tissue is approximately 10 times higher latter model of assay are presented in Figure 2 . than in adipose tissue, due to a higher total pro- The reaction medium contains TrisHCl tein concentration. Corrected for the total pro- as a buffer, dithiothreitol, potassium citrate, tein concentration, liver and adipose tissue coenzyme A, magnesium chloride, ATP, ACLY activities are very similar 32 . NADH and MDH. The reaction temperature At cellular level, ACLY is a cytoplas- used by most of the authors is 25ºC. A pH of 8 mic enzyme bound to the endoplasmic reticu- or above is used to favorize the dissociation of lum 17 . citrate salt 26 . The reducing agent dithiothreitol is needed for the protection of sulfhydryl 6 Assessment of ACLY activity groups of the enzyme . Optical density decreases linearly with time when ATP is used to start the reaction (the last component added). When ACLY enzymatic activity can be as- citrate or the enzyme preparation is the last ad- sayed by trapping acetyl-CoA as acetylhydroxa- dition, a lag phase occurres. The rate of NADH mate and measuring the latter by the color pro- oxidation is proportional to enzyme concentra- duced with FeCl , but this assay is useful only 3 tion 30 . Table 2 shows the reaction conditions for a small range of enzyme concentration. A ACLY Citrate + CoA Acetyl-CoA + Oxaloacetate

ATP ADP+Pi NADH+H + MDH

NAD +

Malate Figure 2. Principle of the ATP citrate lyase activity assay. ACLY = ATP citrate lyase, MDH = malate dehydrogenase, CoA = coenzyme A 22 Revista Român ă de Medicin ă de Laborator Vol. 12, Nr. 3, Septembrie 2008

used in various experiments. Ser454 residues to be specifically phosphorylat- Attempts to use the MDH-coupled ed in vitro 19 . These sites are different from the method in the assessment of ACLY activity in catalytic autophosphorylation site His760, but renal cortex lysate were unsuccessful because their physiological role is not completely under- of high native renal NADH dehydrogenase ac- stood. Ser454 is phosphorylated by cAMP-de- tivity. In this case, hydroxamate assay was per- pendent protein kinase (PKA) and an insulin- formed 20 . stimulated kinase. Thr446 and Ser450 are phos- The presence of the chloride ion in the phorylated by glycogen synthase kinase-3 reaction mixture is necessary and its concentra- (GSK-3) 19, 27 . Thr446 and Ser450 phosphorylation influences the kinetic behavior of ACLY 26 . tion is decreased by insulin 4, 27 . The enzyme partially loses its activity Results reported on kinetic properties by oxydation upon storage and it is reactivated of the enzyme and a possible regulatory role of by dithiothreitol 6. Some authors incubated the its phosphorylation have been variable. A possi- enzyme with 10 mM 2-mercaptoethanol before ble reason for this is the fact that ACLY was the assay, because under normal conditions it isolated from mammalian tissues or eukaryote may take up to 15 minutes to fully reactivate 36 . systems that were able to phosphorylate the en- Concentrated lyase solutions tend to polymerize zyme prior to extraction and purification. Thus, after a few days, probably due to sulfhydryl variable phosphorylation and/or degradation group oxidation. This phenomenon is more status may have conducted to highly hetero- likely to occur at pH values above 7,5 and is ac- genic results. In this case, the use of an expres- companied by considerable loss of activity even sion system that is unable to posttranslationally when the enzyme is redissolved in the presence modify the enzyme perfectly makes sense. of additional dithiothreitol 18 . Potapova et al. 27 obtained an unphosphorylated human recombinant ACLY using a bacterial recombinant system which relied on the lack of Regulation of enzyme activity posttranslational processing of eukaryotic proteins expressed in protease-deficient bacteria. It Enzyme levels in liver and adipose tis- has been shown that unphosphorylated ACLY sue vary over a wide range under the control of cannot be the substrate of GSK-3 alone. Phos- nutrients and hormones 13 , declining sharply in phorylation of ACLY by PKA introduces a starved animals and rising many fold when the phosphate group on one of the 4 subunits of the animals are fed a high carbohydrate/ low fat di- enzyme. This first step allows further phospho- et 14 . These changes in ACLY activity were at- rylation of the enzyme by GSK-3, which further tributed mainly to alterations in the rate of incorporates another 2 phosphate groups in the ACLY biosynthesis. In fasted rats refed a car- same subunit as PKA. Phosphorylation of one bohydrate/ protein diet, the transcriptional rate subunit of ACLY inhibits the phosphorylation was reported to increase 4,5-fold above the fast- of the other 3 subunits 27 . ed level within 2h and mRNA concentration Phosphorylation was found to increase reached a maximum level of approximately 30- 6-fold the activity of ACLY, without affecting fold increased in 16h 9. its Michaelis-Menten constants for citrate, ATP ACLY has been purified from a variety or coenzyme A. The unphosphorylated ACLY of tissues and expression systems and the phos- tetramer shows a negative cooperativity with phorylation of the enzyme has been shown to respect to citrate. This means that binding of the be enhanced by glucagon, insulin, vasopressin first molecule of citrate to one of the four po- and transforming growth factor β18. Further in- tential catalytic sites decreases the affinity of vestigation has identified Thr446, Ser450, and Revista Român ă de Medicin ă de Laborator Vol. 12, Nr. 3, Septembrie 2008 23

the other 3 catalytic sites for citrate. This phe- phosphorylation, and Akt expression stimulated nomenon allows a constant ACLY activity over this phosphorylation event in those cells 3. a wide range of cytoplasmic citrate concentra- Human ACLY activity was also found tions. Phosphorylation of the enzyme eliminates to be regulated allosterically in vitro by phos- the negative cooperation and transforms ACLY phorylated sugars. Fructose 6-phosphate was into a noncooperative form which is active even found to be a potent activator of the unphospho- at low concentrations of citrate 27 . rylated recombinant enzyme at concentrations It seemed paradoxal that insulin and close to physiological intracellular levels 27 . glucagon both promote phosphorylation of Experiments performed on rats showed ACLY at the same serine residue 31 (Ser454). that chronic metabolic acidosis increases corti- During fasting state, glucagon activates ACLY cal renal ACLY activity and protein abundance by phosphorylation of Ser454 via PKA, thus in- as determined by immunoblotting, but does not creasing oxaloacetate availability for gluconeo- affect its mRNA abundance. Chronic K + defi- genesys. Glucagon also inhibits acetyl-CoA ciency also increases the enzyme activity. carboxylase via its phosphorylation, preventing These studies suggest that intracellular, rather the use of acetyl-CoA in the synthesis of fatty than extracellular, acidosis is responsible for the acids 24 . The rise of acetyl-CoA levels, a potent increase in ACLY activity in the kidney. activator of pyruvate carboxylase, activates glu- Chronic metabolic acidosis did not influenece coneogenesys from oxaloacetate. On the other the enzyme activity in liver and did not affect hand, during the fed state, insulin activates ACLY mRNA abundance in either organ 20 . ACLY via phosphorylation of Ser454 by a reported „insulin-stimulated protein kinase”, Regulation of gene expression which has been shown to be Akt (PKB, cAMP- independent protein kinase) 5. The identity be- De novo transcription and translation tween the „insulin-stimulated protein kinase” appears to be one major mode of regulation of and Akt had been presumed previously by ACLY activity, as well as for other lipogenic Potapova et al 27 . The mechanism proposed by enzymes 7, 9 . Diet manipulation of rats demon- Berwick et al. is that insulin receptor activation strates that ACLY mRNA can be highly in- leads to the phosphorylation of insulin receptor duced, the liver mRNA concentration in substrates, recruitment of phosphatidylinositol fasted/refed rats being much higher than in fast- 3’-kinase (PI3K), production of plasma mem- ed 9. brane phosphoinositide (3,4,5)-trisphosphate, The ACLY gene is expressed in most activation of Akt, and finally phosphorylation tissues 7. In fact, this gene has a character of a of ACLY Ser454 5. Insulin also promotes the housekeeping gene; that is, the TATA box is phosphorylation and subsequent activation of absent in the ACLY promoter and it would be a acetyl-CoA carboxylase 5, leading the acetyl- reason that transcription of ACLY is initiated CoA generated by the activated ACLY to the over several bases 15 . pathway of fatty acids synthesis. The ability of Akt to phosphorilate and Nutrients and hormones activate ACLY makes the latter potentially re- sponsive to cytokine signalling. In a study per- It was suggested that ACLY gene ex- formed on cultured IL-3-dependent cells, IL-3 pression is strongly carbohydrate-dependent signaling led to a dose and time-dependent in- and glycolytic metabolites primarily stimulate crease in ACLY Ser454 phosphorylation. A transcription 9. PI3K inhibitor reduced IL-3-dependent ACLY ACLY appears to be induced by feed- ing with a carbohydrate-only diet, primarily at 24 Revista Român ă de Medicin ă de Laborator Vol. 12, Nr. 3, Septembrie 2008

the transcriptional level 9. In cultured rat hepato- cholesterol or fatty acid metabolism is regulated cytes in was found that high concentrations (25 by two sterol regulatory element - binding pro- and 50 mM) of glucose without insulin could teins (SREBPs), designated SREBP-1 and -2. increase the ACLY protein level. ACLY SREBP-1 mainly regulates lipogenic gene ex- mRNA level was increased from 6h after the pression and SREBP-2 regulates cholestero- addition of glucose in the medium and then its genic gene expression. Two isoforms of levels were maintained until 48h 15 . Activation SREBP-1 (SREBP-1a and SREBP-1c) are pro- of the ACLY promoter by glucose may be an duced by alternate promoters. SREBP-1a has a effect in the changes in the expression of Sp1 longer and therefore more potent transactivation family proteins 21 . domain than SREBP-1c, but is less abundant in The fructose diet in diabetic rats, even mouse and human tissues 28 . without insulin treatment, increased the tran- The amounts of SREBP-1c protein and scriptional rate more greatly and the mRNA mRNA, but not those of SREBP-2, dramatically concentration, comparably to levels reached by increase when mice are placed on a high carbo- insulin treatment, but it did not increase enzyme hydrate diet or after refeeding fasted mice, in a activity 9. similar manner to changes in mRNAs of li- Fat present in the diet of the rats pogenic enzymes 2. markedly decreases the transcriptional rate, In experiments with transgenic mice, mRNA concentration and enzyme activity of ACLY gene was profoundly activated in the ACLY, suggesting a primary decrease at the presence of SREBP-1a overexpression and, to a transcriptional level 9. lesser extent, in the case of SREBP-1c and It has been reported that insulin acti- SREBP-2 2. Further investigations, conducted on vates expression of mRNA for ACLY as well Hep G2 cell cultures, showed that expression of as other lipogenic enzymes. Insulin treatment to ACLY mRNA is regulated by sterols: under the the diabetic rats increased the ACLY mRNA sterol-depleted conditions, expression of ACLY fourfold in 4 h and induced enzyme activity mRNA was up-regulated 2–3-fold 28 . eightfold within 48 h 9. Insulin added to culture Studies with SREBP-1 knockout mice medium of rat hepatocytes had no effect alone, demonstrated that SREBP-1 plays a crucial role but in the presence of glucose, it increased the in nutritional induction of lipogenic enzymes ACLY protein level further than glucose including ACL. In the absence of SREBP-1, the alone 15 . Several investigations have demonstrat- ACLY mRNA level was profoundly sup- ed that insulin transcriptionally stimulates pressed. However, SREBP-2 has some signifi- SREBP-1c expression and that the insulin-de- cant contribution to ACLY gene transcription in pendent hepatic expression of lipogenesis-relat- a nonfasted state. As a cytosolic enzyme that ed genes including fatty acid synthase, acetyl- provides acetyl-CoA for both cholesterol and CoA carboxylase, and L-pyruvate kinase is me- fatty acid de novo synthesis, ACLY is transcrip- diated by SREBP-1c. It is not clear, however, if tionally maintained in a sterol-regulatory fash- hepatic ACLY gene expression is under the ion under the control of SREBP-2, even though control of SREBP-1c, a predominant form of the main regulation of ACLY is mediated by SREBP in the liver and mediator of insulin ac- SREBP-1 in a lipogenic regulatory manner 28 . tion 28 . The analysis of the promoter of ACLY gene showed that its sequence is highly similar Transcription factors in human and rat, suggesting that the regulation at transcriptional level takes place in the same The transcription of a number of genes 21 encoding enzymes and proteins involved in manners in these two species . In the region Revista Român ă de Medicin ă de Laborator Vol. 12, Nr. 3, Septembrie 2008 25

from transcription start site to -419 bp region, for development and that other enzymes poten- six Sp1 binding sites and a CCAAT box exist. tially capable of supplying cytosolic acetyl- NF-Y (a ubiquitous transcription factor) binds CoA, such as acetyl-CoA synthetase, were un- to CCAAT box of ACLY 22 . It is likely that tran- able to functionally complement ACLY 3. Het- scription of ACLY gene is regulated by a com- erozygous mice had half-normal amounts of bination of SREBPs and NF-Y across species. ACLY mRNA and appeared healthy. ACLY Several genes, including SREBP-2, farnesyl py- was expressed at high levels throughout embry- rophosphate synthase, squalene synthase, and onic development and β -galactosidase staining HMG-CoA synthase have been shown to be revealed that ACLY was expressed at high lev- regulated by the same combination of transcrip- els in the neural tube. In the adult brain, ACLY tion factors 28 . It has been also suggested that expression was in general much lower, consis- ACLY transcriptional activity during ACLY in- tent with reduced levels of lipogenesis; howev- duction by refeeding low fat/high carbohydrate er, it remained very high in cholinergic neurons, diet may be regulated by Sp1 and/or the ratio of likely reflecting the importance of ACLY in Sp1/Sp3 22 . generating acetyl-CoA for acetylcholine synthesis. Viral infections The surprise was that adult heterozy- ACLY gene expression seems to be al- gous (ACLY+/-) mice had the same body tered in liver cells presenting an active replica- weight and the same levels of plasma choles- tion of various types of hepatitis viruses. ACLY terol, triglycerides and fatty acids as mice nor- gene (along with other genes involved in lipid mally expressing ACLY (ACLY +/+). Diet ma- biosynthesis: fatty acid synthase, SREBP-2, and nipulations meant to stimulate the activity of retinol binding protein 1) has been found to be ACLY did not result in any differences between upregulated in the liver of hepatitis B virus ACLY+/- mice and ACLY +/+ mice. That (HBV) transgenic mice that replicate HBV 12 . means that either ACLY is synthesized in abun- ACLY mRNA was induced 3-fold in Huh-7 dant quantities and half-normal levels are not cells containing the full-length genotype 1b limiting for the downstream reactions, or the hepatitis C virus (HCV) replicon, as compared activation of the enzyme produced in low quantities rises its activity enough for the metabolic with IFN-cured replicon cells, which were neg- 3 ative for HCV RNA 14 . needs . Obesity and hyperlipidemia Implication of ACLY in human pathology Since ACLY is the primary enzyme re- and its role as a potential therapeutic tar- sponsible for the production of cytosolic acetyl- get CoA, a precursor required for de novo biosyn- theses of cholesterol and fatty acids, inhibition ACLY role in development of ACLY has the potential to reduce cholesterol In order to further elucidate the in vivo and triglyceride levels and possibly exert an im- role of ACLY and its expression patterns in tis- pact on obesity via reduction of lipogenic fac- sues, ACLY-deficient mice have been created, tors. Several theoretical and experimental as- in which a β-galactosidase marker was ex- pects sustain this theory. pressed under the control of ACLY regulatoroy The importance of the cholesterol syn- sequences 4. Homozygous ACLY knockout mice thesis pathway in controlling plasma lipid lev- died early in development. It was concluded els has been clearly demonstrated with 3-hy- that ACLY expression in embryos was required droxy-3-methylglutaryl-CoA (HMG-CoA) re- 26 Revista Român ă de Medicin ă de Laborator Vol. 12, Nr. 3, Septembrie 2008

ductase inhibitors, statins. Their hypocholes- levels in vivo . The best of these is able to in- terolaemic activity results from an increase in duce clear hypocholesterolemic and hy- hepatic LDL receptors as a consequence of the potriglyceridaemic responses when adminis- inhibition of cholesterol synthesis. tered orally to rat and dog 11 . ACLY inhibition would theoretically A novel series of 2-hydroxy-N-arylben- decrease the amount of substrate for acetyl- zenesulfonamides have been identified as CoA carboxylase and lower malonyl-CoA lev- ACLY inhibitors. When administered to mice els, the product of this enzyme. Malonyl-CoA fed on a high-fat diet, a compound from this negatively regulates the entry of acyl moieties class produced an approximate 20%-30% low- into the mitochondrial β-oxidation spiral (at the ering in plasma cholesterol and triglycerides. level of carnitine-palmitoyl transferase I) and a Chronic administration of this compound decrease in malonyl-CoA levels can thus lower showed a gradual inhibition of weight gain the amount of liver free fatty acids available for along with a reduction in adiposity without ap- VLDL synthesis. As VLDL are the precursor parent changes in food intake 16 . for LDL, this is another mechanism of lowering circulating LDL levels, supplimentary to the in- Cancer crease in LDL receptor numbers. Experimental Growing cells face a high biosynthetic evidence sustains this mechanism: inhibitors of challenge; at each division they must double fatty acid synthesis like RO-22-0654 have been their macromolecular mass, including phospho- shown to decrease hepatic VLDL output and lipids, sphingolipids, cholesterol, and lipid moi- plasma triglyceride levels. eties for protein modification that are essential Studies on (-)-hydroxycitrate, a natural for membrane integrity, permeability, and sig- citrate analogue and potent ACLY inhibitor, nal transduction 3. Transformed cells au- have proved its ability to inhibit cholesterol tonomously activate growth factor signaling synthesis in Hep G2 cells, increase in HMG- pathways involved in this process. The phos- CoA reductase activity and up-regulate the phatidylinositol 3’-kinase/protein kinase B LDL receptor activity in these cells. It is also (PI3K/Akt) pathway is autonomously upregu- able to stimulate the oxidation of palmitate and lated in many cancer cells and stimulates glu- decrease its esterification in pancreatic islets. cose metabolism in excess of bioenergetic de- However, (-)-hydroxycitrate is not the best mand. This signaling pathway stimulates li- model of ACLY inhibitor as it poorly penetrates pogenic gene transcription through activation of the cellular membrane due to its polarity and is the lipogenic transcription factor SREBP-1 and also an allosteric activator of acetyl-CoA car- 35 directly activates lipogenic enzymes such as boxylase . ACLY 33 . In cells that have undergone a gly- SB-204990, the γ-lactone prodrug of an colytic conversion, i.e., have shifted from an ACLY inhibitor caused a dose-dependent de- oxidative to a glycolytic mode of metabolism crease in the rates of cholesterol and fatty acid under aerobic conditions, much of the bioener- 25 synthesis in Hep G2 cells . getic supply is provided through glycolytic In another study, a series of (3R*,5S*)- ATP production, and glycolytically-derived ω-substituted-3-carboxy-3,5-dihydroxyalkanoic pyruvate enters a truncated tricarboxylic acids acids have been synthesized and evaluated as cycle where citrate is preferentially exported to inhibitors of the recombinant human form of the cytosol via the tricarboxylate transporter 13 . ACLY. A number of these compounds are able Once in the cytoplasm, citrate is cleaved by the to inhibit cholesterol and fatty acid synthesis in stimulated ACLY, providing a high amount of HepG2 cells and reduce plasma triglyceride acetyl-CoA for de novo lipid synthesis. This Revista Român ă de Medicin ă de Laborator Vol. 12, Nr. 3, Septembrie 2008 27

phenomenon links the upregulation of lipogene- Renal lithyasis sis in cancer cells to the well known tumor-associated increase in glycolysis 33 . It has already It has been found that ACLY mediates 20 been shown that glucose-dependent lipogenesis hypocitraturia in rats . Urinary citrate plays an is an important component of cell growth. Ex- important role in preventing formation of calci- periments with IL-3-dependent hematopoietic um-containing kidney stones and low urinary cells showed that knocking-down the ACLY citrate occurs in approximately half of patients led to a significant impairment of glucose-de- with renal stone disease. Citrate is freely fil- pendent lipogenesis 3. tered by the glomerulus and reabsorbed in the Due to its key position in several renal proximal tubule, the rate of the reabsorp- metabolic pathways, ACLY is an appealing tar- tion being the major determinant of urinary cit- get for the treatment of malignancy. Stable rate excretion. Once reabsorbed across the api- knockdown of endogenous ACLY levels by cal membrane, citrate is either transported into shRNA has been found to suppress tumor mitochondria and metabolized in Krebs cycle or growth in vivo and to induce glandular differen- is cleaved by ACLY to oxaloacetate and acetyl- tiation in A549 lung adenocarcinoma cells. It CoA. As kidney tissue homogenates have been also induced differentiation in the K562 chronic found to possess the third highest activity of myelogenous leukemia cell line in vitro . Phar- ACLY after liver and brain, it has been thought macologic inhibition of ACLY activity in sev- that the modulation of ACLY might influence eral cancer cell lines resulted in a dose-depen- citrate renal excretion. Indeed, specific inhibi- dent inhibition of cell proliferation in vitro and tion of ACLY with 4S-hydroxycitrate in a rat tumor growth in vivo 13 . model attenuated hypocitraturia of chronic + 20 In another study, hematopoietic cells metabolic acidosis and K depletion , two con- stably expressing an activated form of Akt ditions known to be associated with hypocitra- known to predispose to tumorigenesis, have turia. been injected to athymic mice and produced leukemia 3. The same type of cells, but in which Albeit not a rate-limiting enzyme, ACLY gene had been knocked-down by ACLY is an interesting metabolic target as it is shRNA, either failed to or needed a longer time known that small changes in the rates of en- to induce leukemia than control cells expressing zymes at the beginning of a biochemical path- ACLY. way may produce significant changes in the rate The ability of ACLY inhibition to sup- of production of the end product of a pathway 27 press tumor growth correlates with the glycolyt- composed of many enzymes . ic phenotype of the tumor. Cancer cells displaying high rates of glucose metabolism are more Abbreviations severely affected, whereas those displaying a low rate of aerobic glycolysis are largely unaf- ACLY = ATP citrate lyase fected. An explanation may be the fact that Akt = cAMP-independent protein kinase, protein ki- there are alternative salvage lipogenic pathways nase B that tumor cells can use to produce acetyl-CoA CoA = coenzyme A independently of ACLY, and cytosolic acetyl- GSK-3 = glycogen synthase kinase-3 CoA production from acetate via acetyl-CoA HBV = hepatitis B virus HCV = hepatitis C virus synthetase is one of them 13 . HMG-CoA = 3-hydroxy-3-methylglutaryl-CoA IL-3 = interleukin 3 LDL = low density lipoproteins 28 Revista Român ă de Medicin ă de Laborator Vol. 12, Nr. 3, Septembrie 2008

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