Reversible Acetylation of PGC-1: Connecting Energy Sensors and Effectors to Guarantee Metabolic ﬂexibility

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Oncogene (2010) 29, 4617–4624 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc REVIEW Reversible acetylation of PGC-1: connecting energy sensors and effectors to guarantee metabolic ﬂexibility

EH Jeninga, K Schoonjans and J Auwerx

Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), Laboratory of Integrative and Systems Physiology, Lausanne, Switzerland

Organisms adapt their metabolism to meet ever changing of diseases, such as metabolic disorders and cancer. environmental conditions. This metabolic adaptation Metabolic homeostasis is to a large extent controlled by involves at a cellular level the ﬁne tuning of mitochondrial transcriptional regulatory mechanisms, which involve function, which is mainly under the control of the the coordinated action of transcription factors, cofac- transcriptional co-activator proliferator-activated receptors and the transcription initiation machinery regulat- tor c co-activator (PGC)-1a. Changes in PGC-1a activity ing the expression of genes involved in mitochondrial coordinate a transcriptional response, which boosts biogenesis and function (Kelly and Scarpulla, 2004). mitochondrial activity in times of energy needs and The transcriptional co-activator proliferator-activated attenuates it when energy demands are low. Reversible receptor g co-activator 1a (PGC-1a) is the master acetylation has emerged as a key way to alter PGC-1a regulator of mitochondrial biogenesis and function activity. Although it is well established that PGC-1a is (Puigserver et al., 1998; Wu et al., 1999). PGC-1a deacetylated and activated by Sirt1 and acetylated and expression and activity is regulated at multiple levels and inhibited by GCN5, less is known regarding how these by diverse signaling events. Within the whole array of enzymes themselves are regulated. Recently, it became regulatory events that converge on PGC-1a, reversible clear that the energy sensor, AMP-activated kinase acetylation only recently emerged as the key modiﬁer (AMPK) translates the effects of energy stress into of PGC-1a activity. The acetylation state of PGC-1a altered Sirt1 activity by regulating the intracellular level is regulated by the deacetylase Sirt1 and the acetyl- of its co-substrate nicotinamide adenine dinucleotide transferase GCN5. Interestingly, the activity of both (NAD) þ . Conversely, the enzyme ATP citrate lyase the Sirt1 and GCN5 proteins are modulated by the (ACL), relates energy balance to GCN5, through energy status of the cell. This review will focus on the the control of the nuclear production of acetyl-CoA, the direct regulators of the PGC1-a acetylation state, Sirt1 substrate for GCN5’s acetyltransferase activity. We and GCN5, as well as on the pathways that provide the review here how these metabolic signaling pathways, substrates for the deacetylation and acetylation reaction, affecting GCN5 and Sirt1 activity, allow the reversible respectively. acetylation–deacetylation of PGC-1a and the adaptation of mitochondrial energy homeostasis to energy levels. Oncogene (2010) 29, 4617–4624; doi:10.1038/onc.2010.206; published online 7 June 2010 PGC-1a, an important regulator of energy homeostasis

Keywords: AMPK; ATP citrate lyase; GCN5; mito- As exemplified by its name, peroxisome proliferator- chondria; PGC-1a; SIRT1 activated receptor g co-activator 1a (PGC-1a) was originally identified as a transcriptional co-activator of the nuclear receptor PPARg (Puigserver et al., 1998). It is now established that PGC-1a interacts with several Introduction other transcription factors, including PPARa, glucocor- ticoid receptor, hepatic nuclear factor 4a, estrogen When energy levels are limiting, such as during fasting receptor related a and FOXO1 (Vega et al., 2000; Yoon and exercise, peripheral tissues switch to use fatty acids et al., 2001; Puigserver et al., 2003). By binding and as fuel for mitochondrial oxidation to preserve blood modulating the activity of these different transcription glucose for cells that strictly rely on glucose as their factors, PGC-1a fine tunes the expression of a number main energy source, such as neurons and red blood cells. of genes involved in diverse metabolic pathways, such as This metabolic flexibility is critically important, as fatty acid oxidation, gluconeogenesis, glycolysis and inappropriate adaptation is often the basis for the onset fatty acid synthesis. In brown adipose tissue, PGC-1a controls adaptive thermogenesis by increasing mito- Correspondence: Dr J Auwerx, Ecole Polytechnique Federale de chondrial fatty acid oxidation and heat production Lausanne, EPFL-SV-IBI1—LISP—NCEM1, Baˆtiment AI (AI 1.145), through the expression of uncoupling protein 1 after Station 15, CH-1015 Lausanne, Switzerland. E-mail: admin.auwerx@epfl.ch cold exposure (Puigserver et al., 1998). In liver, fasting Received 8 March 2010; revised 22 April 2010; accepted 25 April 2010; increases PGC-1a expression thereby promoting gluco- published online 7 June 2010 neogenesis and repressing glycolysis, which results in an Cofactors as energy sensors EH Jeninga et al 4618 increased hepatic glucose output (Yoon et al., 2001). Canto et al., 2009). These findings indicate that PGC-1a is Fasting and exercise induces PGC-1a-mediated mito- activated by Sirt1-mediated deacetylation in times of chondrial biogenesis and fatty acid oxidation in the energy demand, such as fasting and physical activity. muscle (Wu et al., 1999; Gerhart-Hines et al., 2007; As Sirt1 requires the coenzyme nicotinamide adenine Canto et al., 2009). The expression and activity of PGC- dinucleotide (NAD þ ) as a co-substrate for its function 1a is regulated by both cell-autonomous factors, such as (Imai et al., 2000), this suggests that NAD þ , NADH or AMP-activated kinase (AMPK) and mTOR (Herzig the NAD þ /NADH ratio modulates its activity, thereby et al., 2001; Yoon et al., 2001; Canto et al., 2009), as well linking Sirt1 activity to the energy status of the cell. as by systemic factors of hormonal origin, such as Intracellular NAD þ levels are regulated by the balance insulin, glucagon and glucocorticoids and centrally between NAD þ synthesis, either de novo or by recycling regulated neural outputs (Puigserver et al., 2003). In through the NAD þ salvage pathway, and NAD þ addition to the regulation of its expression level, PGC- consumption by NAD þ -dependent enzymes (reviewed 1a activity is controlled by a variety of posttranslational in Houtkooper et al. (2010)). In addition, AMPK can modifications, including phosphorylation (Puigserver modulate this balance through altering metabolic et al., 2003; Jager et al., 2007; Li et al., 2007), ubiquiti- pathways, as recently shown (Fulco et al., 2008; Canto nation (Olson et al., 2008) and acetylation (Rodgers et al., 2009, 2010). et al., 2005; Lerin et al., 2006). Of all these posttranslational events reversible acetylation has emerged as a key modifier of PGC-1a activity. Tandem mass spectro- NAD þ synthesis and NAD þ consumption metry analysis of PGC-1a identified 13 acetylation sites (Rodgers et al., 2005) and so far only two proteins have Although NAD þ can be synthesized de novo from the been unequivocally shown to be involved in the amino acid tryptophan derived from the diet, it is reversible acetylation of PGC-1a, the deacetylase Sirt1 assumed that the main source of NAD þ is produced (Nemoto et al., 2005; Rodgers et al., 2005) and the through the so-called NAD þ salvage pathways. This acetyltransferase GCN5 (Lerin et al., 2006). requires the dietary uptake of NAD þ precursors, such as the niacin-derived nicotinic acid (NA), nicotinamide (NAM) and nicotinamide riboside (NR), which in Deacetylation and activation of PGC-1a by Sirt1 mammals are converted into NAD þ through the salvage pathway (see Figure 1). In this pathway, NAM is Up to date, the only protein identified to be able to thought to be the most important contributor to NAD þ deacetylate PGC-1a is Sirt1 (Nemoto et al., 2005; synthesis. The conversion of NAM to NAD þ is different Gerhart-Hines et al., 2007; Rodgers and Puigserver, between yeast and mammals. In yeast, NAM, the end 2007). Sirt1 belongs to a family of class III histone/ product of reactions catalyzed by NAD þ -consuming protein deacetylase proteins, compromised of seven enzymes, is converted to NA by the enzyme pyrazina- members in mammals (that is, Sirt1–Sirt7), with midase/nicotinamidase 1 (Pnc1), followed by the different cellular localization. Sirt1 is the mammalian conversion to NAM mononucleotide. In contrast, in homolog of the Silent information regulator 2 (Sir2), mammals NAM is directly converted to NAM mono- which was initially identified as a trans-acting factor nucleotide by one of the NAM phosphoribosyltransfer- involved in repression of the silent mating-type loci in ase (Nampt) enzymes. In both yeast and mammals, yeast (Shore et al., 1984). In addition to silencing, Sir2 NAM mononucleotide is subsequently converted to activity is linked to lifespan extension in yeast (Kae- NAD þ . Interestingly, mutating either Sir2 or Pnc1 berlein et al., 1999), worms (Tissenbaum and Guarente, abolishes lifespan expansion after caloric restriction in 2001) and flies (Rogina and Helfand, 2004), indicating yeast (Lin et al., 2000). In addition, in skeletal muscle that Sir2 has conserved its function throughout evolu- the level of Nampt increases upon exercise (Costford tion. In contrast to mammals, in yeast only five et al., 2009; Canto et al., 2010) as well as in muscle of homologs exits; ySir2 and Hst1–4 (homologous to mice on fasting (Canto et al., 2010), all conditions in Sir2). Sirt1 is the most studied mammalian orthologue which Sirt1 is active. This might suggest that an increase and, as mentioned earlier, responsible for deacetylation in the Nampt-dependent NAD þ salvage pathway of PGC-1a. Unlike histone deacetylation, which con- contributes to the increased Sirt1 activity under these denses chromatin structure and attenuates general conditions. This could occur, either by an increase in transcriptional activity, PGC-1a deacetylation enhances NAD þ levels, which activates Sirt1, or by a decrease in its activity to co-activate transcription factors that in the levels of NAM, which act as a potent inhibitor of turn will induce transcription of its target genes. Sirt1 (Bitterman et al., 2002; Anderson et al., 2003). In Consistent with this fact, overexpression of Sirt1 in addition to NAM, NA and NR can also function as livers of mice significantly increases the expression of the precursors in the salvage pathway. For NA, the pathway gluconeogenic genes that are under the control of PGC- starts with the conversion of NA to NA mononucleotide 1a, whereas Sirt1 knockdown attenuates this effect and then converges with the pathway of de novo NAD þ (Rodgers and Puigserver, 2007). Also in skeletal muscle synthesis from tryptophan. When NR is used, NAM Sirt1 has been shown to be required for the PGC-1a- mononucleotide formation from NR is first required, mediated induction of the fatty acid oxidation genes in before entering the NAM-dependent salvage pathway response to fasting and exercise (Gerhart-Hines et al., 2007; (Figure 1).

Oncogene Cofactors as energy sensors EH Jeninga et al 4619 NAD+ synthesis NAD+ consumption

Tryptophan NA Mononucleotide NAD+ Nicotinic Acid (NA) AMPK Nicotinamide Riboside NAM Mononucleotide Sirtuins NADH PARP1-2 CD38/ CD157

Nampt Nicotinamide (NAM) Nampt Pnc1 Nicotinic Acid Figure 1 Regulation of intracellular NAD þ levels. Intracellular NAD þ levels are regulated by the balance between NAD þ synthesis and consumption. NAD þ synthesis can occur either de novo from tryptophan, or through the ‘salvage pathways’ that use nicotinic acid (NA), nicotinamide riboside, or nicotinamide (NAM). NAD þ is consumed by enzymes, such as the sirtuins, CD38, CD157, PARP1 and PARP2, which use it as a substrate for their catalytic reaction and convert it into NAM. Furthermore the ratio of NAD þ to NADH can be modulated by metabolic pathways, such as those activated by AMPK.

NAD þ is not only synthesized, it is also consumed by activation leads to an increase in intracellular NAD þ several enzymes. In addition to the sirtuins, poly(ADP- levels. An increase in NAD þ might be achieved by an ribose) polymerases (PARPs) and two cADP-ribose increase in the NAM-dependent NAD þ salvage path- synthetases, CD38 and CD157, use NAD þ as a way. In line with this, Nampt expression increases after substrate. The PARPs, of which PARP1 and PARP2 AICAR-mediated AMPK activation (Fulco et al., 2008) are most widely studied, catalyze a reaction in which the as well as after exercise in muscle of mice after a 20-h ADP-ribose moiety of NAD þ is transferred to a fast (Canto et al., 2010). Consistent with the importance substrate protein. In addition, the multifunctional of Nampt in generating NAD þ , shRNA-mediated enzymes, CD38 and CD157 use NAD þ as a substrate knockdown of Nampt blocked the increase in NAD þ / to generate second messengers, such as cADP-ribose, NADH ratio in 24-h glucose restricted cells in which which contributes to calcium mobilization. The func- AMPK is activated (Fulco et al., 2008). In apparent tions of these enzymes, which also generate NAM as a contradiction to this last observation, acute pharmaco- by-product, will not be discussed here as they have been logical inhibition of Nampt activity did not seem to reviewed elsewhere (Ortolan et al., 2002; Schreiber et al., affect AICARs capacity to modulate PGC-1a activity or 2006; Malavasi et al., 2008). By using NAD þ as a NAD þ /NADH ratio (Canto et al., 2009). Rather substrate, these enzymes seem under certain conditions inhibition of mitochondrial fatty acid uptake blocked to be able to modulate intracellular NAD þ and NAM the acute AICAR-mediated increase in NAD þ /NADH levels, as recently reviewed (Houtkooper et al., 2010). ratio (Canto et al., 2009), suggesting that under these conditions an increase in mitochondrial oxidative phosphorylation is required for AMPK to increase the þ AMPK modulates Sirt1 and NAD þ levels NAD /NADH ratio. Combining the above evidence, it is likely that the AMPK-mediated increase in NAD þ Very recently, activation of AMPK has been shown to levels is boosted through a ‘faster’ metabolic adaptation increase intracellular NAD þ levels in C2C12 myotubes (Canto et al., 2009), and sustained by the ‘slower’ and mouse skeletal muscle (Fulco et al., 2008; Canto induction of Nampt transcription, which maintains et al., 2009, 2010). In eukaryotic cells, AMPK has an NAD þ levels for a longer period (Fulco et al., 2008; essential role in the maintenance of cellular energy Canto et al., 2010). stores. On activation, AMPK switches on catabolic pathways that produce ATP, such as oxidative phosphorylation, while switching off anabolic pathways that Acetylation and inhibition of PGC-1a by GCN5 consume ATP (Hardie, 2007). In line with these actions, AMPK activation increases PGC-1a expression (Suwa p300, Steroid receptor co-activator (SRC)-1, GCN5 and et al., 2003), and activates PGC-1a by direct phosphor- SRC-3 are all acetyltransferases that have been shown to ylation (Jager et al., 2007). In addition, AMPK interact with PGC-1a (Puigserver et al., 1999; Lerin activation also induces Sirt1-mediated PGC-1a deace- et al., 2006; Coste et al., 2008), but only GCN5 was tylation (Canto et al., 2009). In keeping with the shown to acetylate and inhibit PGC-1a (Lerin et al., dependence of Sirt1 on cellular NAD þ , the NAD þ 2006). GCN5 was discovered as a histone acetyltrans- levels in C2C12 myotubes and skeletal muscle were ferase (HAT) in Tetrahymena and shown to be the increased on AMPK activation (Canto et al., 2009, homolog of yeast Gcn5, thereby being the ﬁrst enzyme 2010). Interestingly, the AMPK-mediated phosphoryla- linking transcriptional activation to histone acetylation tion of PGC-1a serves as a priming signal for the (Brownell et al., 1996). Histone acetylation results in subsequent PGC-1a deacetylation that AMPK induces unwinding the chromatin making it accessible for through boosting intracellular NAD þ levels (Canto the transcriptional machinery to initiate transcription et al., 2009). The question remains how AMPK (Kouzarides, 2000). Besides histone acetylation, GCN5

Oncogene Cofactors as energy sensors EH Jeninga et al 4620 has been shown to acetylate a large array of transcrip- Acs2p and Acs1p, respectively. AceCS2 is mainly tion factors (reviewed in Nagy and Tora (2007)) expressed in brown adipose tissue, skeletal muscle and including the transcriptional cofactors, PGC-1a (Lerin heart and localizes to the mitochondria, in which it et al., 2006) and PGC-1b (Kelly et al., 2009). GCN5- generates acetyl-CoA for oxidation in the tricarboxylic mediated PGC-1a acetylation suppresses its transcrip- acid (TCA) cycle to generate ATP and CO2 (Fujino tional activity in vitro and in vivo resulting in reduced et al., 2001). In contrast, AceCS1 is highly expressed in gluconeogenesis and hepatic glucose output (Lerin et al., the liver (Luong et al., 2000) and present in the 2006). Although, it is clear that GCN5 acetylates PGC- cytoplasm, in which it produces acetyl-CoA for fatty 1a, it cannot be excluded that other acetyltransferases acid synthesis. Although, a homolog of yeast Acs2p is can acetylate PGC-1a as well under certain conditions. thus present in higher eukaryotes, the cellular concen- In addition to the expression of GCN5, which is trations of acetate are relatively low in mammals, regulated by nutritional/metabolic factors (Coste et al., suggesting that the contribution of AceCS1 to the 2008), another link between GCN5 and metabolism generation of the nuclear acetyl-CoA pool is relatively could come from the fact that, like all acetyltransferases, low. Interestingly, both AceCS2 and AceCS1 are it requires acetyl-CoA as a substrate for the acetylation acetylated proteins themselves and are deacetylated by reaction. Remarkably, the regulation of the acetylation SIRT3 and SIRT1, respectively (Hallows et al., 2006). reaction and its dependence on acetyl-CoA as a Unlike in yeast, mammals can produce acetyl-CoA by substrate is still not well understood. the enzyme ACL, which generates acetyl-CoA from TCA-derived citrate in the cytoplasm. Interestingly, ACL and AceCS1 were recently also reported to be present in the nucleus (Wellen et al., 2009). As both Acetyl-CoA synthetase regulates nucleocytosolic acetate and citrate are small molecules able to diffuse acetyl-CoA levels in yeast freely through the nucleopore complex (Paine et al., 1975), this suggests that acetyl-CoA production may In yeast acetyl-CoA can be produced by at least three occur in both the cytoplasm and nucleus (see Figure 2). major pathways. Using yeast strains in which the critical Given that siRNA-mediated knockdown of ACL enzymes of either of these pathways were deleted, acetyl- decreased histone acetylation, it seems that ACL is CoA synthetase 2 (Acs2p) was shown to be essential for critical for nuclear acetyl-CoA production and that histone acetylation by HATs (Takahashi et al., 2006). AceCS1 cannot compensate under basal conditions Two different acetyl-CoA synthetases, which catalyze (Wellen et al., 2009). ACL knockdown in 3T3-L1 cells the ligation of acetate and CoA to acetyl-CoA, exist in moreover blocks adipocyte differentation (Wellen et al., yeast, that is, Acs1p and Acs2p. Consistent with the 2009), a process in which histone acetylation normally presence of a putative mitochondrial localization signal, Acs1p localizes to mitochondria (Kumar et al., 2002), whereas Acs2p is present in the nucleus (Kals et al., 2005; Takahashi et al., 2006). Although glucose represses the mitochondrial Acs1p, Acs2p is active in Glycolysis the presence of glucose (van den Berg et al., 1996), suggesting that high nutrient availability drives acetyl- Pyruvate Acetyl-CoA CoA production and concomitant histone acetylation. TCA Indeed, glucose induces overall histone acetylation mainly by the stimulation of the two HATs, Gcn5 Citrate (GCN5) and Esa1 (TIP60), acting in two different complexes (Friis et al., 2009). Interestingly, this required glycolysis, indicating that glucose-induced overall histone acetylation is due to direct metabolic induction of Citrate Citrate HATs. In line with this, PKA and TORC signal ACL ACL transduction pathways had little effect on the glucose- induced overall histone acetylation, whereas the deletion Acetyl-CoA Acetyl-CoA Lipid of either Acs2p or Gcn5 in yeast abolished the glucose- synthesis HAT induced H3 acetylation (Friis et al., 2009). All together this indicates that H3 acetylation depends on the Ac Ac Ac Ac enzyme that require acetyl-CoA (Gcn5) and the enzyme that generate the nuclear acetyl-CoA (Acs2p). NUCLEUS

Figure 2 ACL generates a nuclear/cytosolic pool of acetyl-CoA in In mammals ATP citrate lyase (ACL) regulates nuclear mammals. In times when glucose is around, TCA-derived citrate acetyl-CoA levels can be converted into aceteyl-CoA by ACL, both in the nucleus and cytosol, thereby providing a nuclear/cytosolic pool of acetyl- CoA. This pool of acetyl-CoA, which seems to be independent of In mammals also two acetyl-CoA synthetases exist, the mitochondrial acetyl-CoA pool, is used for histone acetylation AceCS1 and AceCS2, which are the homologs of yeast by histone acetyltransferases, such as GCN5.

Oncogene Cofactors as energy sensors EH Jeninga et al 4621 increases (Yoo et al., 2006), whereas it induces activity, the expression of both SRC-3 and GCN5 was differentiation of C2C12, which is normally associated shown to increase concomitant with the induction of with reduced histone acetylation (Bracha et al., 2010). PGC-1a acetylation levels in muscle of mice on a high- Furthermore, under glucose-deprived conditions histone fat diet. The converse, a reduction in the expression of acetylation decreases in MEF cells (Wellen et al., 2009). GCN5 and SRC-3, associated with PGC-1a deacetyla- Under these conditions cells can use fatty acid oxidation tion and activation, was observed in muscle of fasted as a source of energy. However, fatty acid oxidation animals (Coste et al., 2008). These observations indicate results in the production of mitochondrial, but not that SRC-3 and GCN5 expression could in fact nucleocytosolic acetyl-CoA and consistent with this contribute to modulate PGC-1a acetylation state and notion, supplementation of fatty acids to glucose- mitochondrial activity pending on energy levels, in a starved cells could not rescue histone acetylation (Well- synergistic manner with Sirt1, ultimately resulting in the en et al., 2009). This suggests that, like in yeast, a inhibition of mitochondrial function in situations with mitochondrial and a nuclear/cytosolic pool of acetyl- high energy supplies favouring fat accumulation (Coste CoA exist regulating acetylation reactions in different et al., 2008). cellular compartments. Interestingly, ACL seems to Although, SRC-1 has been shown to interact with provide acetyl-CoA only for certain acetyl transferases, PGC-1a (Puigserver et al., 1999), and SRC-2À/À mice as acetylation of p53, which is regulated by the p300, have increased energy expenditure, a phenotype that was not affected by ACL knockdown. As the combined was reminiscent of Sirt1 activation (Picard et al., 2002), knockdown of ACL and GCN5 did not result in an it remains to be analyzed, whether these SRC proteins additive inhibition of histone acetylation, GCN5 and also affect on GCN5 activity in a manner analogous to ACL function in the same pathway (Wellen et al., 2009). SRC-3. As mentioned earlier, GCN5 can also acetylate non- histone proteins, such as PGC-1a. Conspicuously, ACL mediates histone acetylation when glucose (energy) is present, conditions in which PGC-1a is also acetylated. A role for PGC-1a and its partners in cancer This suggests that ACL may not only provide acetyl- CoA for GCN5-mediated histone acetylation, but also A common feature of human cancer is a metabolic shift for the GCN5-mediated acetylation of non-histone from the more efﬁcient oxidative metabolism to the proteins such as PGC-1a and that this occurs in a faster glycolytic metabolism to meet the high energy nutrient-dependent manner. demand of tumor cells. As the transcriptional coactivator PGC-1a triggers a shift from glycolytic to oxidative metabolism through the increase in mitochondrial biogenesis and activity, this might suggest that Other regulators of GCN5 activity PGC-1a expression levels are downregulated in cancer. This hypothesis is supported by the reduced PGC-1a SRC1–3 are transcriptional co-activators that bind to expression levels in a variety of cancers, including nuclear receptors and other transcription factors thereby breast (Jiang et al., 2003; Watkins et al., 2004), colon enhancing their transcriptional activity (Spiegelman (Feilchenfeldt et al., 2004), ovary (Zhang et al., 2007) and Heinrich, 2004; Xu et al., 2009). Although SRCs and liver cancer (Yin et al., 2004). In addition, PGC-1a have been reported to possess HAT activity (Chen et al., overexpression suppressed cancer cell growth (Zhang 1997; Spencer et al., 1997), this activity is much weaker et al., 2007) further supporting a role for PGC-1a in than that of other HATs. Consistent with this, SRCs do cancer. As described earlier, the activity of PGC-1a is not seem to contain a prototypical acetyl-CoA binding modulated by reversible acetylation–deacetylation. The site as other HATs, such as GCN5 (Trievel et al., 1999). deacetylase Sirt1 activates PGC-1a in times of energy These SRC proteins therefore rather seem to act as demand thereby switching on oxidative phosphoryla- platform proteins that bind to transcription factors and tion, whereas the acetyltransferase GCN5 represses recruit other proteins with HAT activity, thereby PGC-1a in excess of energy. In turn, the activity of enhancing transcription. Interestingly, SRC-3 has been Sirt1 and GCN5 seem to depend on the activity of reported to positively inﬂuence GCN5 activity in AMPK and ACL, the metabolic enzymes that provide mammalian cells (Coste et al., 2008). GCN5 expression the substrates for the deacetylation and acetylation was decreased in muscle and brown adipose tissue of reaction, respectively. This implies that modulation of SRC-3À/À mice. In line with the reduction in GCN5, the activity of Sirt1, GCN5, AMPK and/or ACL might mitochondrial function and energy expenditure was potentially prevent and/or suppress tumor formation or increased in SRC-3À/À mice, which nicely correlated with growth. In line with this, overexpression of Sirt1 a decrease in PGC-1a acetylation levels (Coste et al., suppresses intestinal tumor formation and growth 2008). Whereas overexpression of SRC-3 by itself did (Firestein et al., 2008) and the AMPK activator not increase PGC-1a acetylation in myotubes, it Metformin inhibits the growth of human breast cancer enhanced the GCN5-mediated PGC-1a acetylation. cells (Zakikhani et al., 2006). In addition, the use of Together these data indicate that SRC-3 acts as a Metformin in the treatment of type II diabetes and modulator of GCN5 activity. Supportive of a physiolo- physical activity, which are both known to activate gical role of SRC-3/GCN5 in the control of PGC-1a AMPK, have been associated with a reduced risk in the

Oncogene Cofactors as energy sensors EH Jeninga et al 4622 development of cancer (Evans et al., 2005; Brown et al., Calorie restriction Calorie excess 2007; Libby et al., 2009). Interestingly, the tumor Ac suppressor LKB-1 that is mutated in the human AMPK NAD+ Acetyl-CoA ACL Peutz–Jeghers cancer syndrome (Hawley et al., 2003), PGC1α has been identified as the upstream activating kinase of SIRT1 GCN5 AMPK, supporting the idea that modulation of AMPK deacetylases acetyltransferases might be anticancerous. Next to activation of LKB-1, SRC3 AMPK and/or Sirt1, inhibition of ACL and/or GCN5 PGC1α will result in PGC-1a activation and potentially prevent ERR NRF1 Mitochondrial biogenesis carcinogenesis. At present, it is unknown of GCN5 and activity inhibition can prevent tumorigenesis as specific GCN5 inhibitors are lacking. However, pharmacological inhibition of ACL has been shown to suppress tumor cell growth (Hatzivassiliou et al., 2005). Moreover, ACL Figure 3 Energy sensing mediated by cofactors. In times of high expression is increased in breast and bladder carcinomas energy demand, an AMPK-mediated increase in NAD þ activates (Szutowicz et al., 1979; Turyn et al., 2003). In view of all Sirt1 resulting in the deacetylation and activation of PGC-1a leading ultimately to mitochondrial biogenesis and improved these findings that suggest that modulation of the mitochondrial function. Conversely, GCN5-mediated acetylation activity of AMPK, Sirt1, GCN5 and ACL all affect on inactivates PGC-1a when plenty of energy is around. The acetyl- carcinogenesis, it is of interest to analyze if these effects CoA necessary for this acetylation reaction is provided by ACL, are accomplished by changes in the PGC-1a acetylation which acts as rate limiting for this reaction. status and PGC-1a-mediated oxidative phosphorylation, thereby ultimately shifting cancer cells away from pro-tumorigenic glycolytic pathways. If this is the case, acetyl-CoA for GCN5-catalyzed histone acetylation in strategies to induce PGC-1a expression and/or activity the nucleus is ACL (Wellen et al., 2009). The ACL- could become attractive to treat cancer and should incite mediated histone acetylation depends on glucose avail- the exploration of the therapeutic potential of com- ability as this was decreased in glucose-deprived pounds that interfere with PGC-1a activity, such as conditions (Wellen et al., 2009). Whether the pool of ACL inhibitors, GCN5 inhibitors, Sirt1 activators and ACL-generated acetyl-CoA is also used by GCN5 for AMPK agonists. the acetylation of PGC-1a needs to be further examined. If this is the case, these observations support the idea that the co-substrates of Sirt1 and GCN5 are regulated in a yin-yang manner by nutrients, with high levels of Conclusions and future perspectives NAD þ and low levels of acetyl-CoA when glucose is limited, whereas this is the opposite when glucose is The maintenance of metabolic homeostasis is controlled around. by transcriptional events that enable the cell to adapt its Teleologically, such a regulation of PGC-1a by gene expression to its energetic status. The transcrip- nutrients makes sense as it inhibits mitochondrial tional cofactor PGC-1a, which acts as a master function in situations with high-energy supplies, favor- regulator of mitochondrial function, acts in coherence ing fat accumulation, whereas it solicits fat release from with the energy status of the cell, being activated in adipose stores and their use in oxidative metabolism in times of energy demand, such as during fasting, physical situations of energy need (Coste et al., 2008). The activity and cold exposure, and being inhibited when relative level of activity of Sirt1 and GCN5, which seems energy is around. We have summarized here the wide to be determined by changes in the supply of their co- body of evidence in support of the regulation of PGC-1a substrates as a function of cellular metabolic activity, activity by changes in its acetylation status. In fact, hence ultimately will inform PGC-1a regarding the PGC-1a activity is conversely regulated by the Sirt1 energetic status of the cell. PGC-1a then will adapt deacetylase, which activates PGC-1a, and the GCN5 cellular energy production through its role in mitochon- acetyltransferase, which represses PGC-1a activity. drial biogenesis and function, so that energy require- GCN5 and Sirt1 seem therefore to act as the yin-yang ments are exactly met. Although our review mainly that controls the activity of PGC-1a (see Figure 3). As focussed on the regulation of PGC-1a as a prototypical the activity of PGC-1a is reflected by the metabolic example of metabolic regulation of protein activity by status of the cell, it is of interest to analyze if also the reversible acetylation–deacetylation, it needs to be activity of GCN5 and Sirt1 is regulated in an opposite underscored that such regulation may be more wide- manner by nutrients. In the case of Sirt1, cellular levels spread and affect other proteins that control metabo- of its co-substrate NAD þ are shown to determine its lism, such as the FOXO family of transcription factors, activity. This seems to depend on nutrient availability, which already have been shown to be controlled by as NAD þ levels are increased in C2C12 myotubes on Sirt1-mediated acetylation (Brunet et al., 2004). low glucose, whereas this is reversed after addition of In conclusion, unraveling the mechanisms by which glucose (Canto et al., 2010). GCN5 requires acetyl-CoA PGC-1a is regulated by the energy status of the cell will as a co-substrate for its acetylation reaction. In contribute to our understanding of how cellular meta- mammals, the main metabolic enzyme providing bolic processes are regulated and as such might help to

Oncogene Cofactors as energy sensors EH Jeninga et al 4623 design novel therapeutics strategies to combat metabolic Acknowledgements diseases and cancer. This work was supported by grants from the Ecole Poly- Conﬂict of interest technique Fe´de´rale de Lausanne, Swiss National Science Foundation, NIH (DK59820) and the European Research The authors declare no conﬂict of interest. Council Ideas programme (Sirtuins; ERC-2008-AdG23118).

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