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• W2530145254 abstract "AMP-activated protein kinase (AMPK) is an energy sensor and master regulator of metabolism. AMPK functions as a fuel gauge monitoring systemic and cellular energy status. Activation of AMPK occurs when the intracellular AMP/ATP ratio increases and leads to a metabolic switch from anabolism to catabolism. AMPK phosphorylates and inhibits acetyl-CoA carboxylase (ACC), which catalyzes carboxylation of acetyl-CoA to malonyl-CoA, the first and rate-limiting reaction in de novo synthesis of fatty acids. AMPK thus regulates homeostasis of acetyl-CoA, a key metabolite at the crossroads of metabolism, signaling, chromatin structure, and transcription. Nucleocytosolic concentration of acetyl-CoA affects histone acetylation and links metabolism and chromatin structure. Here we show that activation of AMPK with the widely used antidiabetic drug metformin or with the AMP mimetic 5-aminoimidazole-4-carboxamide ribonucleotide increases the inhibitory phosphorylation of ACC and decreases the conversion of acetyl-CoA to malonyl-CoA, leading to increased protein acetylation and altered gene expression in prostate and ovarian cancer cells. Direct inhibition of ACC with allosteric inhibitor 5-(tetradecyloxy)-2-furoic acid also increases acetylation of histones and non-histone proteins. Because AMPK activation requires liver kinase B1, metformin does not induce protein acetylation in liver kinase B1-deficient cells. Together, our data indicate that AMPK regulates the availability of nucleocytosolic acetyl-CoA for protein acetylation and that AMPK activators, such as metformin, have the capacity to increase protein acetylation and alter patterns of gene expression, further expanding the plethora of metformin's physiological effects. AMP-activated protein kinase (AMPK) is an energy sensor and master regulator of metabolism. AMPK functions as a fuel gauge monitoring systemic and cellular energy status. Activation of AMPK occurs when the intracellular AMP/ATP ratio increases and leads to a metabolic switch from anabolism to catabolism. AMPK phosphorylates and inhibits acetyl-CoA carboxylase (ACC), which catalyzes carboxylation of acetyl-CoA to malonyl-CoA, the first and rate-limiting reaction in de novo synthesis of fatty acids. AMPK thus regulates homeostasis of acetyl-CoA, a key metabolite at the crossroads of metabolism, signaling, chromatin structure, and transcription. Nucleocytosolic concentration of acetyl-CoA affects histone acetylation and links metabolism and chromatin structure. Here we show that activation of AMPK with the widely used antidiabetic drug metformin or with the AMP mimetic 5-aminoimidazole-4-carboxamide ribonucleotide increases the inhibitory phosphorylation of ACC and decreases the conversion of acetyl-CoA to malonyl-CoA, leading to increased protein acetylation and altered gene expression in prostate and ovarian cancer cells. Direct inhibition of ACC with allosteric inhibitor 5-(tetradecyloxy)-2-furoic acid also increases acetylation of histones and non-histone proteins. Because AMPK activation requires liver kinase B1, metformin does not induce protein acetylation in liver kinase B1-deficient cells. Together, our data indicate that AMPK regulates the availability of nucleocytosolic acetyl-CoA for protein acetylation and that AMPK activators, such as metformin, have the capacity to increase protein acetylation and alter patterns of gene expression, further expanding the plethora of metformin's physiological effects. Acetylation is one of the epigenetic post-translational modifications of histones; it affects chromatin structure and regulates diverse cellular functions, such as gene expression, DNA replication and repair, and cellular proliferation (1.Strahl B.D. Allis C.D. The language of covalent histone modifications.Nature. 2000; 403: 41-45Crossref PubMed Scopus (6605) Google Scholar, 2.Kouzarides T. Chromatin modifications and their function.Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (8060) Google Scholar). Acetylation and deacetylation of chromatin histones, mediated by histone acetyltransferases (HATs) 3The abbreviations used are: HAT, histone acetyltransferase; HDAC, histone deacetylase; AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase; ACCA, acetyl-CoA carboxylase-α; LKB1, liver kinase B1; AICAR, 5-amino-1-β-d-ribofuranosyl-1H-imidazole-4-carboxamide; TOFA, 5-(tetradecyloxy)-2-furoic acid; mTOR, mechanistic target of rapamycin; CBP, cAMP-response element-binding protein (CREB)-binding protein; βOHB, β-hydroxybutyrate; acH3, histone H3 acetylated at Lys14; acH4, hyperacetylated histone H4; acp65, p65 NFκB acetylated at Lys310; acTubulin, tubulin acetylated at Lys40; pAMPK, AMPK phosphorylated at Thr172; pACCA, ACCA phosphorylated at Ser79. and histone deacetylases (HDACs), respectively, represent the major mechanisms for epigenetic gene regulation. The dynamic balance between histone acetylation and deacetylation, mediated by the activities of HATs and HDACs, is stringently regulated in healthy cells but is often dysregulated in cancer (3.Farria A. Li W. Dent S.Y. KATs in cancer: functions and therapies.Oncogene. 2015; 34: 4901-4913Crossref PubMed Scopus (85) Google Scholar, 4.Hsu Y.C. Hsieh Y.H. Liao C.C. Chong L.W. Lee C.Y. Yu Y.L. Chou R.H. Targeting post-translational modifications of histones in cancer therapy.Cell. Mol. Biol. 2015; 61: 69-84PubMed Google Scholar). Histone acetylation depends on intermediary metabolism for supplying acetyl-CoA in the nucleocytosolic compartment (5.Takahashi H. McCaffery J.M. Irizarry R.A. Boeke J.D. Nucleocytosolic acetyl-coenzyme A synthetase is required for histone acetylation and global transcription.Mol. Cell. 2006; 23: 207-217Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). In mammalian cells, the nucleocytosolic enzyme ATP-citrate lyase is the major source of acetyl-CoA for histone acetylation (6.Wellen K.E. Hatzivassiliou G. Sachdeva U.M. Bui T.V. Cross J.R. Thompson C.B. ATP-citrate lyase links cellular metabolism to histone acetylation.Science. 2009; 324: 1076-1080Crossref PubMed Scopus (1469) Google Scholar). Another mechanism for generation of acetyl-CoA in the nucleus involves translocation of pyruvate dehydrogenase from mitochondria to the nucleus (7.Sutendra G. Kinnaird A. Dromparis P. Paulin R. Stenson T.H. Haromy A. Hashimoto K. Zhang N. Flaim E. Michelakis E.D. A nuclear pyruvate dehydrogenase complex is important for the generation of acetyl-CoA and histone acetylation.Cell. 2014; 158: 84-97Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). In yeast, global histone acetylation depends on nucleocytosolic acetyl-CoA produced by acetyl-CoA synthetase (5.Takahashi H. McCaffery J.M. Irizarry R.A. Boeke J.D. Nucleocytosolic acetyl-coenzyme A synthetase is required for histone acetylation and global transcription.Mol. Cell. 2006; 23: 207-217Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). In both yeast and mammalian cells, the nucleocytosolic acetyl-CoA is the link among cellular energy, carbon metabolism, histone acetylation, and chromatin regulation (8.Pietrocola F. Galluzzi L. Bravo-San Pedro J.M. Madeo F. Kroemer G. Acetyl coenzyme A: a central metabolite and second messenger.Cell Metab. 2015; 21: 805-821Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar9.Janke R. Dodson A.E. Rine J. Metabolism and epigenetics.Annu. Rev. Cell Dev. Biol. 2015; 31: 473-496Crossref PubMed Scopus (122) Google Scholar, 10.Cai L. Sutter B.M. Li B. Tu B.P. Acetyl-CoA induces cell growth and proliferation by promoting the acetylation of histones at growth genes.Mol. Cell. 2011; 42: 426-437Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar11.Wellen K.E. Thompson C.B. A two-way street. Reciprocal regulation of metabolism and signaling.Nat. Rev. Mol. Cell Biol. 2012; 13: 270-276Crossref PubMed Scopus (378) Google Scholar). The nucleocytosolic acetyl-CoA is a critical precursor of several anabolic processes, including de novo synthesis of fatty acids. Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the first and rate-limiting reaction in the de novo synthesis of fatty acids (12.Kim K.H. Regulation of mammalian acetyl-coenzyme A carboxylase.Annu. Rev. Nutr. 1997; 17: 77-99Crossref PubMed Scopus (313) Google Scholar). The ACC activity affects the concentration of nucleocytosolic acetyl-CoA. We have previously shown that attenuated expression of yeast ACC increases global acetylation of chromatin histones and alters transcriptional regulation (13.Galdieri L. Vancura A. Acetyl-CoA carboxylase regulates global histone acetylation.J. Biol. Chem. 2012; 287: 23865-23876Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Moreover, chronic inhibition of ACC in mouse hepatocytes increases protein acetylation (14.Chow J.D. Lawrence R.T. Healy M.E. Dominy J.E. Liao J.A. Breen D.S. Byrne F.L. Kenwood B.M. Lackner C. Okutsu S. Mas V.R. Caldwell S.H. Tomsig J.L. Cooney G.J. Puigserver P.B. et al.Genetic inhibition of hepatic acetyl-CoA carboxylase activity increases liver fat and alters global protein acetylation.Mol. Metab. 2014; 3: 419-431Crossref PubMed Scopus (72) Google Scholar). The human genome encodes two tissue-specific ACC isoforms, ACCα (ACCA) and ACCβ (ACCB) (15.Travers M.T. Barber M.C. Tissue-specific control of the acetyl-CoA carboxylase gene.Biochem. Soc. Trans. 1997; 25: 1215-1219Crossref PubMed Scopus (6) Google Scholar). ACCA activity is controlled by AMP-activated protein kinase (AMPK), a conserved cellular energy sensor and master regulator of metabolism. A hallmark of AMPK activation is phosphorylation of ACCA at Ser79, which results in reduced activity of ACCA and inhibition of fatty acid synthesis (16.Carling D. Zammit V.A. Hardie D.G. A common bicyclic protein kinase cascade inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis.FEBS Lett. 1987; 223: 217-222Crossref PubMed Scopus (439) Google Scholar, 17.Hardie D.G. AMP-activated protein kinase: an energy sensor that regulates all aspects of cell function.Genes Dev. 2011; 25: 1895-1908Crossref PubMed Scopus (1174) Google Scholar). In yeast, inactivation of SNF1, the budding yeast ortholog of mammalian AMPK, results in increased ACC activity, a reduced pool of cellular acetyl-CoA, and globally decreased histone acetylation (18.Zhang M. Galdieri L. Vancura A. The yeast AMPK homolog SNF1 regulates acetyl coenzyme A homeostasis and histone acetylation.Mol. Cell. Biol. 2013; 33: 4701-4717Crossref PubMed Scopus (64) Google Scholar). The main objective of this study was to test the hypothesis that inhibition of ACC activity in human cells increases the nucleocytosolic pool of acetyl-CoA and histone acetylation. We show that suppression of ACC activity either by direct inhibition or by metformin-mediated AMPK activation increases acetylation of histones and non-histones proteins and induces transcriptional changes in prostate and ovarian cancer cells. Metformin, widely used for diabetes type 2 treatment, decreases ATP production by inhibiting mitochondrial respiratory chain complex I, leading to AMPK activation (19.El-Mir M.Y. Nogueira V. Fontaine E. Avéret N. Rigoulet M. Leverve X. Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I.J. Biol. Chem. 2000; 275: 223-228Abstract Full Text Full Text PDF PubMed Scopus (1069) Google Scholar20.Owen M.R. Doran E. Halestrap A.P. Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain.Biochem. J. 2000; 348: 607-614Crossref PubMed Scopus (1628) Google Scholar, 21.Hardie D.G. AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy.Nat. Rev. Mol. Cell Biol. 2007; 8: 774-785Crossref PubMed Scopus (1762) Google Scholar, 22.Foretz M. Hébrard S. Leclerc J. Zarrinpashneh E. Soty M. Mithieux G. Sakamoto K. Andreelli F. Viollet B. Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state.J. Clin. Investig. 2010; 120: 2355-2369Crossref PubMed Scopus (910) Google Scholar23.Viollet B. Guigas B. Sanz Garcia N. Leclerc J. Foretz M. Andreelli F. Cellular and molecular mechanisms of metformin: an overview.Clin. Sci. 2012; 122: 253-270Crossref PubMed Scopus (1221) Google Scholar). The metformin therapy is associated with a reduced risk of cancer in diabetes type 2 patients; however, the mechanisms are not completely understood (24.Evans J.M. Donnelly L.A. Emslie-Smith A.M. Alessi D.R. Morris A.D. Metformin and reduced risk of cancer in diabetic patients.BMJ. 2005; 330: 1304-1305Crossref PubMed Scopus (1865) Google Scholar). Our results indicate that some of the physiological effects of metformin may involve increased acetylation of histone and non-histone proteins and altered patterns of transcriptional regulation. Histone acetylation depends on intermediary metabolism for supplying acetyl-CoA as a substrate for HATs in the nucleocytosolic compartment (5.Takahashi H. McCaffery J.M. Irizarry R.A. Boeke J.D. Nucleocytosolic acetyl-coenzyme A synthetase is required for histone acetylation and global transcription.Mol. Cell. 2006; 23: 207-217Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 6.Wellen K.E. Hatzivassiliou G. Sachdeva U.M. Bui T.V. Cross J.R. Thompson C.B. ATP-citrate lyase links cellular metabolism to histone acetylation.Science. 2009; 324: 1076-1080Crossref PubMed Scopus (1469) Google Scholar). Cytosolic acetyl-CoA is also used by acetyl-CoA carboxylase to yield malonyl-CoA, a precursor for de novo synthesis of fatty acids (25.Tehlivets O. Scheuringer K. Kohlwein S.D. Fatty acid synthesis and elongation in yeast.Biochim. Biophys. Acta. 2007; 1771: 255-270Crossref PubMed Scopus (338) Google Scholar, 26.Beld J. Lee D.J. Burkart M.D. Fatty acid biosynthesis revisited: structure elucidation and metabolic engineering.Mol. Biosyst. 2015; 11: 38-59Crossref PubMed Google Scholar). We have previously shown that acetyl-CoA carboxylase Acc1p regulates homeostasis of nucleocytosolic acetyl-CoA and acetylation of histones and nonhistone proteins in yeast (13.Galdieri L. Vancura A. Acetyl-CoA carboxylase regulates global histone acetylation.J. Biol. Chem. 2012; 287: 23865-23876Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). To investigate whether ACCA regulates histone acetylation also in mammalian cells, we analyzed histone acetylation in prostate cancer PC3 and ovarian cancer OVCAR3 cells treated with 5-(tetradecyloxy)-2-furoic acid (TOFA), an allosteric ACCA inhibitor that decreases conversion of acetyl-CoA to malonyl-CoA and induces apoptosis in lung and colon cancer cells (27.Wang C. Xu C. Sun M. Luo D. Liao D.F. Cao D. Acetyl-CoA carboxylase-α inhibitor TOFA induces human cancer cell apoptosis.Biochem. Biophys. Res. Commun. 2009; 385: 302-306Crossref PubMed Scopus (86) Google Scholar). Our results show that inhibition of ACCA significantly increases acetylation levels of histones H3 and H4 in PC3 cells and to a lesser extent in OVCAR3 cells (Fig. 1A). In addition to histones, many other proteins are acetylated (3.Farria A. Li W. Dent S.Y. KATs in cancer: functions and therapies.Oncogene. 2015; 34: 4901-4913Crossref PubMed Scopus (85) Google Scholar, 8.Pietrocola F. Galluzzi L. Bravo-San Pedro J.M. Madeo F. Kroemer G. Acetyl coenzyme A: a central metabolite and second messenger.Cell Metab. 2015; 21: 805-821Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). To determine whether ACCA inhibition selectively affects only histone acetylation or has a similar effect on acetylation of other proteins, we assayed acetylation of α-tubulin and p65 NFκB. α-Tubulin is acetylated at Lys40 by a conserved α-tubulin acetyltransferase, increasing stability of microtubules (28.Al-Bassam J. Corbett K.D. α-Tubulin acetylation from the inside out.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 19515-19516Crossref PubMed Scopus (24) Google Scholar). The transcription factor NFκB regulates expression of genes involved in inflammation, growth, development, and apoptosis (29.Verma I.M. Stevenson J.K. Schwarz E.M. Van Antwerp D. Miyamoto S. Rel/NF-κB/IκB family: intimate tales of association and dissociation.Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1660) Google Scholar, 30.Ghosh S. May M.J. Kopp E.B. NF-κB and Rel proteins: evolutionarily conserved mediators of immune responses.Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4601) Google Scholar). Acetylation of p65 at Lys310 is required for the full transcriptional activity of NFκB (31.Chen L.F. Mu Y. Greene W.C. Acetylation of RelA at discrete sites regulates distinct nuclear functions of NF-κB.EMBO J. 2002; 21: 6539-6548Crossref PubMed Scopus (635) Google Scholar). Our results show that the acetylation levels of α-tubulin and p65 are increased after TOFA treatment in PC3 and OVCAR3 cells without affecting the total cellular levels of these proteins (Fig. 1A). ACCA activity is inhibited by AMPK phosphorylation (32.Davies S.P. Carling D. Munday M.R. Hardie D.G. Diurnal rhythm of phosphorylation of rat liver acetyl-CoA carboxylase by the AMP activated protein kinase, demonstrated using free-clamping. Effects of high fat diets.Eur. J. Biochem. 1992; 203: 615-623Crossref PubMed Scopus (143) Google Scholar, 33.Woods A. Munday M.R. Scott J. Yang X. Carlson M. Carling D. Yeast SNF1 is functionally related to mammalian AMP-activated protein kinase and regulates acetyl-CoA carboxylase in vivo.J. Biol. Chem. 1994; 269: 19509-19515Abstract Full Text PDF PubMed Google Scholar34.Hardie D.G. Scott J.W. Pan D.A. Hudson E.R. Management of cellular energy by the AMP-activated protein kinase system.FEBS Lett. 2003; 546: 113-120Crossref PubMed Scopus (713) Google Scholar). A hallmark of AMPK activation is ACCA phosphorylation at Ser79, resulting in ACCA inactivation and inhibition of fatty acid biosynthesis (35.Ha J. Daniel S. Broyles S.S. Kim K.H. Critical phosphorylation sites for acetyl-CoA carboxylase activity.J. Biol. Chem. 1994; 269: 22162-22168Abstract Full Text PDF PubMed Google Scholar). We have shown that inactivation of the yeast AMPK homolog SNF1 results in a decreased level of nucleocytosolic acetyl-CoA, leading to hypoacetylation of chromatin histones and non-histone proteins (18.Zhang M. Galdieri L. Vancura A. The yeast AMPK homolog SNF1 regulates acetyl coenzyme A homeostasis and histone acetylation.Mol. Cell. Biol. 2013; 33: 4701-4717Crossref PubMed Scopus (64) Google Scholar). Because SNF1 modulates acetyl-CoA homeostasis in yeast cells, we speculated that activation of AMPK in mammalian cells might decrease ACCA activity, leading to increased acetylation of histones. As expected, stimulation of AMPK in PC3 and OVCAR3 cells with the AMP homolog 5-amino-1-β-d-ribofuranosyl-1H-imidazole-4-carboxamide (AICAR) increased AMPK phosphorylation at Thr172, a hallmark of AMPK activation by liver kinase B1 (LKB1), the primary upstream kinase that activates the AMPK pathway (34.Hardie D.G. Scott J.W. Pan D.A. Hudson E.R. Management of cellular energy by the AMP-activated protein kinase system.FEBS Lett. 2003; 546: 113-120Crossref PubMed Scopus (713) Google Scholar). Activation of AMPK resulted also in phosphorylation of ACCA at Ser79 (Fig. 1B), known to decrease ACCA enzymatic activity (35.Ha J. Daniel S. Broyles S.S. Kim K.H. Critical phosphorylation sites for acetyl-CoA carboxylase activity.J. Biol. Chem. 1994; 269: 22162-22168Abstract Full Text PDF PubMed Google Scholar). Importantly, AMPK activation by AICAR increased acetylation of histones H3 and H4, α-tubulin, and p65 in both PC3 and OVCAR3 cells (Fig. 1B). AMPK can also be activated by drugs that inhibit the mitochondrial electron transport pathway and oxidative phosphorylation and reduce the cellular ATP level. One of these drugs is metformin, a widely used antidiabetic drug that inhibits mitochondrial complex I, reducing ATP production and increasing AMP levels (20.Owen M.R. Doran E. Halestrap A.P. Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain.Biochem. J. 2000; 348: 607-614Crossref PubMed Scopus (1628) Google Scholar, 23.Viollet B. Guigas B. Sanz Garcia N. Leclerc J. Foretz M. Andreelli F. Cellular and molecular mechanisms of metformin: an overview.Clin. Sci. 2012; 122: 253-270Crossref PubMed Scopus (1221) Google Scholar). Upon AMP binding, AMPK becomes a better substrate for its activator kinase LKB1 (34.Hardie D.G. Scott J.W. Pan D.A. Hudson E.R. Management of cellular energy by the AMP-activated protein kinase system.FEBS Lett. 2003; 546: 113-120Crossref PubMed Scopus (713) Google Scholar). In PC3 and OVCAR3 cells, metformin activated AMPK as shown by increased AMPK phosphorylation at Thr172 and ACCA phosphorylation at Ser79 (Fig. 2A). In accordance with ACCA inactivation, 0.03 and 0.3 mm metformin decreased cellular malonyl-CoA levels to 80 and 20% compared with untreated cells, respectively (Fig. 2B). Similarly to TOFA and AICAR, treatment of PC3 and OVCAR3 cells with metformin increased acetylation of histones H3 and H4, α-tubulin, and p65 (Fig. 2A). The lowest concentration of metformin effective in increasing protein acetylation was about 30 μm (Fig. 2), which corresponds to the metformin concentration in human plasma following a therapeutic dose of around 30 mg/kg (36.Fogarty S. Hardie D.G. Development of protein kinase activators: AMPK as a target in metabolic disorders and cancer.Biochim. Biophys. Acta. 2010; 1804: 581-591Crossref PubMed Scopus (310) Google Scholar). Together, our results indicate that, by regulating ACCA activity, AMPK controls acetyl-CoA homeostasis and protein acetylation. To investigate whether activation of AMPK, rather than a modulation of other cellular activities, accounts for the AICAR-induced increase in protein acetylation in PC3 cells, we analyzed global acetylation of histones and non-histone proteins after small interfering RNA (siRNA)-mediated silencing of both AMPKα1 and AMPKα2. As shown in Fig. 3A, AMPK siRNA silencing suppressed the cellular AMPK level by about 50% in untreated cells and by about 70% in AICAR-treated PC3 cells. Cells with suppressed AMPK expression exhibited significantly reduced acetylation of histones H3 and H4, tubulin, and p65 after AICAR treatment (Fig. 3A). These results indicate that activation of AMPK is responsible for the increased protein acetylation. Treatment of PC3 and OVCAR3 cells with TOFA results in increased protein acetylation (Fig. 1A). To confirm that the mechanism responsible involves inhibition of the ACCA activity, we analyzed protein acetylation in PC3 cells transfected with ACCA siRNA as well as with control non-silencing siRNA. Cell transfection with ACCA siRNA suppressed the ACCA protein levels by about 60%. The ACCA suppression significantly increased acetylation of histones H3 and H4 as well as increased acetylation of tubulin and p65 (Fig. 3B). These results are consistent with the effect of TOFA on protein acetylation (Fig. 1A); we interpret these results to mean that decreased activity of ACCA results in increased protein acetylation. These results are also consistent with increased protein acetylation upon repression of yeast ACC (13.Galdieri L. Vancura A. Acetyl-CoA carboxylase regulates global histone acetylation.J. Biol. Chem. 2012; 287: 23865-23876Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The tumor suppressor serine/threonine LKB1 activates AMPK by phosphorylation at Thr172. LKB1 is a low energy sensor that regulates tumorigenesis and apoptosis by regulating AMPK and mTOR pathways (37.Shaw R.J. Kosmatka M. Bardeesy N. Hurley R.L. Witters L.A. DePinho R.A. Cantley L.C. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 3329-3335Crossref PubMed Scopus (1441) Google Scholar). LKB1-deficient cells have increased mTOR signaling due to the lack of tuberous sclerosis 2 protein phosphorylation by AMPK, which results in increased growth and tumorigenic potential. To investigate whether metformin-induced protein acetylation requires LKB1-dependent activation of AMPK, we analyzed global protein acetylation of histones and non-histone proteins in PC3 cells transfected with LKB1 siRNA as well as with control non-silencing siRNA. As shown in Fig. 4A, LKB1 siRNA silencing suppressed the cellular LKB1 level by about 80% in untreated cells and by about 95% in metformin-treated PC3 cells. The LKB1 suppression abolished the increase in protein acetylation after metformin treatment (Fig. 4A), suggesting that LKB1 activity is required for metformin-induced protein acetylation. To further investigate the role of LKB1 in metformin-induced protein acetylation, we used HeLa S3 cells that lack LKB1 expression (38.Tiainen M. Ylikorkala A. Mäkelä T.P. Growth suppression by LKB1 is mediated by a G1 cell cycle arrest.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 9248-9251Crossref PubMed Scopus (263) Google Scholar). Metformin did not induce AMPK phosphorylation at Thr172 or ACCA phosphorylation at Ser79 and did not increase acetylation of histones H3 and H4, α-tubulin, and p65 in HeLa S3 cells (Fig. 4B). However, inhibition of ACCA with TOFA in HeLa S3 cells increased acetylation of histones H3 and H4, α-tubulin, and p65 (Fig. 4C). Taken together, our results suggest that the metformin-induced protein acetylation in PC3 and OVCAR3 cells is due to the LKB1-dependent activation of AMPK and AMPK-dependent inactivation of ACCA. To test whether metformin regulates histone acetylation globally or only at specific loci, we used chromatin immunoprecipitation (ChIP) to evaluate the occupancy of histone H3 acetylated at Lys14 (acH3) as well as hyperacetylated histone H4 (acH4; acetylated at Lys5,8,12,16) in the promoter regions of β-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclin-dependent kinase inhibitor p21, apoptosis regulator Bcl-2, proinflammatory genes IL6 and IL8, the transcription factor Bcl-3, the transcriptionally inactive euchromatin gene MYOD1 encoding myogenic differentiation 1 protein, and the transcriptionally inactive heterochromatin gene SAT2 encoding spermidine/spermine N1-acetyltransferase. We used anti-H3 antibody that recognizes the C-terminal region of histone H3, which is not post-translationally modified. The ChIP signal obtained with this antibody thus represents the total H3 occupancy and can be used to calculate the histone acetylation levels per nucleosome content (18.Zhang M. Galdieri L. Vancura A. The yeast AMPK homolog SNF1 regulates acetyl coenzyme A homeostasis and histone acetylation.Mol. Cell. Biol. 2013; 33: 4701-4717Crossref PubMed Scopus (64) Google Scholar). To account for differences in nucleosome density at different genomic loci, we corrected the acH3 and acH4 occupancies for histone H3 content and generated values that represent acetylation per nucleosome. In metformin-treated PC3 cells, acetylation of histone H3 was increased 4- and 10-fold in the promoters of IL8 and IL6, respectively. The acetylation status of histone H3 in the other promoters was not altered. Acetylation of histone H4 was increased 1.3–7.5 times in all examined promoters (Fig. 5). Upon treatment of PC3 cells with AICAR, acetylation of histone H3 was increased 2–3.4 times in the promoters of p21, IL8, ACTB, and Bcl2, whereas acetylation of histone H4 was increased 1.2–5.2 times in all examined promoters (Fig. 6). The fact that the increased acetylation of histone H4 was not always accompanied by increased acetylation of histone H3 is in an agreement with the notion that different acetylation levels of histones H3 and H4 are due to different affinity of individual HATs for acetyl-CoA (8.Pietrocola F. Galluzzi L. Bravo-San Pedro J.M. Madeo F. Kroemer G. Acetyl coenzyme A: a central metabolite and second messenger.Cell Metab. 2015; 21: 805-821Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar, 39.Tanner K.G. Langer M.R. Kim Y. Denu J.M. Kinetic mechanisms of the histone acetyltransferase GCN5 from yeast.J. Biol. Chem. 2000; 275: 22048-22055Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar40.Henry R.A. Kuo Y.M. Andrews A.J. Differences in specificity and selectivity between CBP and p300 acetylation of histone H3 and H3/H4.Biochemistry. 2013; 52: 5746-5759Crossref PubMed Scopus (114) Google Scholar, 41.Lee J.V. Carrer A. Shah S. Snyder N.W. Wei S. Venneti S. Worth A.J. Yuan Z.-F. Lim H.-W. Liu S. Jackson E. Aiello N.M. Haas N.B. Rebbeck T.R. Judkins A. et al.Akt-dependent metabolic reprogramming regulates tumor cell histone acetylation.Cell Metab. 2014; 20: 306-319Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar42.Galdieri L. Zhang T. Rogerson D. Lleshi R. Vancura A. Protein acetylation and acetyl-CoA metabolism in budding yeast.Eukaryot. Cell. 2014; 13: 1472-1483Crossref PubMed Scopus (76) Google Scholar). Individual genes differed in the acetylation levels, and as expected the transcriptionally inactive heterochromatin gene SAT2 displayed the lowest acetylation. This result is consistent with the general correlation between acetylation of promoter histones and transcriptional activity (43.Struhl K. Histone acetylation and transcriptional regulatory mechanisms.Genes Dev. 1998; 12: 599-606Crossref PubMed Scopus (1550) Google Scholar).FIGURE 6.AICAR-treated cells display increased untargeted acetylation of chromatin histones. PC3 cells were treated with 0 and 3 mm AICAR for 24 h. ChIP experiments were performed with antibodies against total histone H3, acH3, and acH4. Occupancies of H3, acH3, and acH4 were determined in the promoter regions of p21, IL6, IL8, ACTB, G" @default.
• W2530145254 created "2016-10-21" @default.
• W2530145254 creator A5019782659 @default.
• W2530145254 creator A5042117627 @default.
• W2530145254 creator A5054346929 @default.
• W2530145254 creator A5071871609 @default.
• W2530145254 date "2016-11-01" @default.
• W2530145254 modified "2023-10-10" @default.
• W2530145254 title "Activation of AMP-activated Protein Kinase by Metformin Induces Protein Acetylation in Prostate and Ovarian Cancer Cells" @default.
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• W2530145254 doi "https://doi.org/10.1074/jbc.m116.742247" @default.
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• W2530145254 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/27733682" @default.
• W2530145254 hasPublicationYear "2016" @default.
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