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beyond reason
Critical Review
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The sirtuin class of histone deacetylases: Regulation and roles in lipid metabolism
First published: 03 February 2014
Abstract
After the completion of the human genome sequence and that from many other organisms, last decade has witnessed a spectacular gain of knowledge on gene functions. These studies provided new insights on the roles of genes in physiology and disease. Nonetheless, the availability of genetically modified models and of “omics” technologies such as next generation sequencing unveiled clear evidences on epigenetic regulation of many cellular functions. At this regard, sirtuins, belonging to class III histone deacetylase family, have emerged as regulators of metabolism as well as other cellular processes and seem ideally suited as targets of future therapeutical interventions. This review deals on general aspects of the biology of sirtuins and focuses on their relevance in lipid metabolism in different tissues, pointing to their exploitation as potential pharmacological targets of compounds that could be used as new therapeutic alternatives in several disorders ranging from type 2 diabetes and obesity to age‐related cardiovascular and neurodegenerative diseases.
© 2014 IUBMB Life, 66(2):89–99, 2014
The Histone Deacetylase Superfamily
In eukaryotic cells, chromatin is a well‐organized and highly dynamic structure susceptible to epigenetic processes that modify its architecture, regulating gene activity without changing DNA sequence. The basic unit of chromatin is the nucleosome, a histone octamer core wrapped twice by DNA. Covalent modification of DNA, histones or the association of nonhistone proteins regulate the transition between heterochromatin, a highly condensed and transcriptionally inhibited state, and euchromatin, being transcriptionally active since more accessible to transcription factors 1. Post‐translational modifications, including acetylation, ADP‐ribosylation, methylation, phosphorylation, and ubiquitination occurring at amino acids in the N‐terminal of histone tails strongly regulate chromatin packaging and consequently gene expression.
One of the most studied post‐translational modifications is acetylation of lysine residues of histone tails resulting from the balance between HAT (histone acetyltransferase) and HDAC (histone deacetylase) activity 2; these two classes of enzymes reduce and increase respectively chromatin packaging, thus regulating the accessibility of transcription factors and RNA polymerase II to local chromatin regions and ultimately gene transcription 3. Eukaryotic HDACs belong to an ancient family of proteins comprising two subfamilies with different HDAC activity: the classical HDAC family and the sirtuins family 4. The eleven members of the classical HDAC family are Zn2+‐dependent enzymes harboring a Zn2+ ion in their catalytic pocket and they are divided in three classes: class I (HDAC1, 2, 3, and 8) closely related to the yeast transcriptional regulator Rpd3, class II (HDAC4, 5, 6, 7, 9, and 10) similar to the yeast Hda1 and class IV (HDAC11) whose role is still not clearly understood 5. Sirtuins belong to class III HDACs.
Sirtuins
Sirtuins are related to the yeast orthologous silent information regulator 2 (Sir2) and are characterized by a NAD+‐dependent deacetylase activity 6; in fact, despite other classes of HDACs, which use the Zn2+ active site and bind a water molecule on acetylated lysines, sirtuins transfer the acetyl group from the lysine side chain of a substrate to NAD+ cofactor, generating nicotinamide, 2′‐O‐acetyl‐ADP‐ribose and a deacetylated substrate (Fig. 1). Mammalian sirtuins also catalyze reactions for a number of different protein substrates and certain mammalian sirtuins possess ADP‐ribosyltransferase activity 7. Since the discovery of Sir2 involvement in increasing yeast longevity 8 and that calorie restriction activates Sir2 leading to lifespan extension 9, a growing number of studies on sirtuins have been published, revealing their important role in calorie restriction, aging‐related diseases and metabolic homeostasis 10-14.
Figure 1
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Sirtuin‐mediated regulation of hepatic lipid metabolism. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Classification and Localization
Sir2 homolog family in mammals comprises seven proteins with different localizations and functions that can be classified in four classes based on phylogenetic analysis: SIRT1, 2, and 3 belong to class I, SIRT4 to class II, SIRT5 to class III, and SIRT6 and 7 to class IV 15.
SIRT1 is expressed in different organs, such as liver, brain, pancreas, heart, muscle, and adipose tissue. The subcellular localization of SIRT1 is influenced by different factors. In fact, although it is mainly localized in the nucleus, under specific conditions SIRT1 can shuttle to the cytoplasm 16 controlling not only nuclear but also cytoplasmic target proteins.
SIRT2, highly expressed in heart, brain, and skeletal muscle, is primarily cytosolic, where deacetylates microtubules α‐tubulin, but it shuttles to the nucleus during G2/M phase transition and deacetylases histone H4 thus controlling chromatin condensation in metaphase 17.
SIRT3, SIRT4, and SIRT5 are localized in mitochondria, the key organelles in energy metabolism and aging. SIRT3 is highly expressed in kidney, brain, heart, liver and brown adipose tissue and it is the first sirtuin whose localization was found in the mitochondrial matrix. SIRT4 was found in vascular smooth muscle and striated muscle, heart, liver and pancreatic β‐cells. SIRT5 is mainly expressed in brain, heart, muscle, liver and kidney. SIRT3 is considered the major mitochondrial deacetylase, in fact while Sirt4 and Sirt5 knock out mice do not show any changes in mitochondrial protein acetylation level, a dramatic increase of mitochondria protein acetylation is shown in mice lacking Sirt3 18.
SIRT6 is particularly abundant in brain and liver, even though the highest amount of protein is found in muscle, suggesting either higher protein stability or increased rate of translation of its mRNA as compared to other tissues. Localization studies demonstrated that SIRT6 is predominantly nuclear and that it is excluded from nucleolus 7.
SIRT7 is a nucleolar protein mainly found in metabolically active tissues such as liver and spleen, while its expression is low in nonproliferating tissues such as muscle, heart, and brain 19.
Structure and Enzymatic Activity
Mammalian sirtuins contain an evolutionarily conserved core domain homologous to Sir2 including their catalytic structure; notably, the seven‐polypeptide chains share several regions of sequence homology within this core. Sirtuins appear disordered in the N‐ and C‐ terminal regions, interfering with crystallization of full‐length proteins 20. Crystal structures of the core region of human sirtuins show a large well‐organized and conserved Rossmann fold domain, characteristic of NAD+/NADH binding proteins, and a more variable smaller zinc‐binding domain. Between these domains, several loops form a pocket that can bind NAD+ and acetylated peptides 21, 22. The great variability among the zinc domains of different sirtuins is the reason of the different activities revealed in some of them such as SIRT5 that, despite its weak deacetylase activity, is a more efficient lysine desuccinylase and demalonylase 23 due to Arg‐105 and Tyr‐102 residues in the small domain that binds succinyllysine‐containing peptides.
Furthermore, N‐ and C‐terminal flanking regions differ in length and sequence among sirtuins, which explains some differences among sirtuin activities with protein‐specific regulatory roles. In particular, SIRT1 N‐ and C‐terminal regions contain approximately 240–500 residues that, by interacting with the core domain and forming SIRT1 holoenzyme, potentiate its catalytic activity 24.
Human SIRT6 displays, instead, a peculiar structure containing a splayed zinc‐binding domain not connected to the Rossmann fold domain because of the lack of a helix group, consequently generating a more open conformation that could explain the mild deacetylase efficiency (Table 1) 25. The deletion of the helix has also been observed in SIRT7, another class IV sirtuin‐like SIRT6, not yet structurally and functionally characterized.
Table 1. Structural features of sirtuins: SIRT 1, 2, 3, and 5 versus SIRT6
SIRT 1, 2, 3, 5SIRT6
Disordered N‐ and C‐ terminal regions | Disordered N‐ and C‐ terminal regions |
Well organized Rossman fold domain | Well organized Rossman fold domain |
Small zinc‐binding domain connected to Rossman fold domain | Splayed zinc‐binding domain not connected to Rossman fold domain |
Pocket for NAD+ and acetylated peptides binding formed by loops between Rossman fold domain and zinc‐binding domain | Stable single helix that reduces flexibility of the substrate binding pocket and allows interaction with ADP‐ribose |
NAD+ binding in presence of acetylated substrates | NAD+ binding in absence of acetylated substrates |
Moreover, contrary to SIRT1, SIRT2, and SIRT3, which need a substrate binding to form an ordered NAD+‐binding pocket before NAD+ binding, SIRT6 binds NAD+ in the absence of acetylated substrates. In fact, instead of the highly conserved flexible NAD+‐binding loop that facilitates NAD+‐binding, SIRT6 possesses a stable and ordered single helix conferring less flexibility to the substrate binding pocket and interacting with ADP‐ribose (Table 1) 25.
To catalyze substrate‐deacetylation reaction that removes an acetyl group from the lysine of the substrate, sirtuins need NAD+ as cosubstrate that is cleaved to nicotinamide and 2′‐O‐acetyl‐ADP‐ribose. NAD+ binding to the catalytic site is the first step of NAD+ dependent deacetylation reaction. Sirtuins not only deacetylate histones H3 and H4, but also other proteins with different subcellular localizations and moreover, as described above, their structural differences confer to each subtype a specific enzymatic activity. In fact, although all sirtuins, apart from SIRT4, possess deacetylase activity, only SIRT1, 2, and 3 are true deacetylating enzymes; in contrast SIRT6 acts as an auto‐ADP ribosyltransferase with weak deacetylation activity 7. SIRT5 was thought to deacetylate carbamoyl phosphate synthetase (CPS1), but it was shown to act mainly as demalonylase and desuccinylase on CPS1 and other protein substrates 23. The activity of SIRT7 has been debated until its role in histone H3K18 deacetylation was demonstrated 26. The mitochondrial SIRT4 is, instead, an ADP‐ribosyltransferase that transfers an ADP‐ribosyl group to substrates 27.
Regulation
Sirtuin activity is regulated at different levels; at this regard, SIRT1 is the sirtuin with the best‐characterized regulation occurring at multiple levels (Fig. 2).
Figure 2
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Sirtuin modulation: positive and negative regulators of sirtuin activity.
Transcriptional Regulation
Several mechanisms (Fig. 2) have been shown to contribute to changes of SIRT1 expression occurring during energy state modifications. Under calorie restriction SIRT1 levels are induced by forkhead box O1 (FOXO1), peroxisome proliferator‐activated receptors (PPAR) α and β/δ and cAMP response element‐binding protein (CREB), whereas it is reduced by PPARγ and carbohydrate response element binding protein (ChREBP) 28, 29. Moreover, SIRT1 expression is reduced by poly (ADP‐ribose) polymerase 2 (PARP2), which is involved in DNA repair and apoptosis. Another level of transcriptional modulation is played by microRNAs (miRNAs), in particular miR‐34a and miR‐199a, which repress SIRT1 mRNA levels 30, 31.
In mice, Sirt3 expression is induced by estrogen‐related receptor‐α (Errα) that interacts with PPARγ coactivator 1α (PGC‐1α) and controls the expression of genes for brown adipose tissue development and function 32.
Post‐Transcriptional Modifications
Sirtuin activity is also regulated by post‐translational modifications that are able to direct sirtuins to their own specific targets and to increase their activity (Fig. 2). Cyclin B–cyclin‐dependent kinase 1 (CDK1) complex phosphorylates SIRT1 affecting cell cycle 33. During oxidative stress SIRT1 phosphorylation is also mediated by c‐Jun N‐terminal kinase (JNK) that, by phosphorylating three residues of SIRT1, leads to deacetylation of histone H3, but not of p53 34; in contrast, SIRT1 phosphorylation at threonine 522 by the dual specificity tyrosine phosphorylation‐regulated kinase (DYRK) 1 and 2 causes p53 deacetylation 35, highlighting how post‐translational modifications direct SIRT1 to specific targets.
In addition, SIRT1 activity is increased by sumoylation, while desumoylation by sentrin‐specific protease (SENP) represses the activity and leads to cell death 36.
Complex Formation
The formation of complexes between sirtuins and other protein partners constitutes another layer of modulation of sirtuin activity (Fig. 2). Usually, the formation of these complexes negatively regulates sirtuins activity: deleted in breast cancer 1 (DBC1) binds the SIRT1 in the catalytic domain blocking its ability to deacetylate p53, thus working as a negative regulator 37. The only example of direct positive regulation of sirtuin activity by complex formation is the active regulator of SIRT1 (AROS) that stimulates SIRT1 activity and inhibits p53 38. Moreover, the complexes with other proteins can influence sirtuins activity, for example, the complex containing SIRT1, PPARγ and the nuclear receptor corepressor 1 (NCoR1) or the silencing mediator of retinoid and thyroid hormone receptors (SMRT, also known as NCoR2) represses PPARγ‐mediated adipogenesis 39.
NAD+ Influence
An important player in the regulation of sirtuin activity is NAD+ availability. During the fasted state NAD+ levels in liver, muscle and white adipose tissue increase and activate sirtuins 40, 41, whereas NAD+ levels decrease on high fat diet 42. Moreover, when DNA damage occurs, Parps catalyze ADP‐ribose unit transfer from NAD+ to the substrates, reducing intracellular NAD+ levels and inhibiting sirtuin activity. On one side, Parp1 deletion induces Sirt1 since it elevates NAD+ cellular content, on the other side the deletion of Parp2, which is a Sirt1 expression repressor, increases Sirt1 transcription 43.
Sirtuins in LIPID Metabolism
In recent years, several experimental evidences suggested that sirtuins regulate energy metabolism and insulin sensitivity, through their ability to act as energy sensors and regulators in several metabolic tissues. As described before, high NAD+ levels, a condition reflecting low cellular energy status, can activate sirtuins. Their activity is therefore linked with metabolism, and different sirtuins act to maintain cellular energy homeostasis. Concerning lipid metabolism, SIRT1 and SIRT3 action is the most studied and well characterized among members of the sirtuin family. Nowadays, little is known about SIRT2, SIRT4 and SIRT6, while SIRT5 and SIRT7 most likely play a minor role in metabolic homeostasis.
Generally, SIRT1 is identified as a key regulatory sensor of nutrient availability 44 that counteracts obesity 45 and it is regulated by fasting and feeding. Due to its pivotal role in energy metabolism, different groups pinpointed SIRT1 as a crucial player in both glucose and lipid metabolism 46, 47, suggesting that SIRT1 protects from inflammation and obesity under normal feeding conditions and from the progression to metabolic dysfunction under dietary stress and aging 44. Insights in the effect of up‐ and downregulation of SIRT1 provide strong evidences of its importance in metabolic homeostasis. Sirt1 heterozygous null mice on 40% fat diet become obese and insulin resistant, display increased serum cytokine levels and develop hepatomegaly; these effects are due to elevated hepatic gluconeogenesis and oxidative stress summed with altered expression of genes related to steroid and glycerolipid metabolism 48. On the other hand, Banks et al. 49 demonstrated that transgenic mice overexpressing Sirt1 (SirBACO) in all tissues are protected from developing liver steatosis and insulin resistance after high‐fat feeding. Controversially, SirBACO mice under atherogenic diet maintain better glucose homeostasis but develop worse lipid profiles and larger atherosclerotic lesions than controls. This effect results from Sirt1‐dependent deacetylation of CREB that leads to reduced expression of gluconeogenic genes and hepatic lipid accumulation and secretion 50.
SIRT3 is generally identified as a positive modulator of β‐oxidation as it regulates hydroxyacyl‐CoA dehydrogenase (Schad), short/branched chain acyl‐CoA dehydrogenase (Acadsb), very long chain acyl‐CoA dehydrogenase (Acadvl) and long chain acyl‐CoA dehydrogenase (Acadl) 51, 52; it also deacetylates and activates acyl‐CoA synthetase short‐chain (AceCS2) 53.
Concerning the findings about sirtuins and their link with energy metabolism, important roles of different members of the sirtuin family can be identified specifically in the liver, in adipose tissue, in brain‐adipose tissue axis and in skeletal muscle.
Sirtuins in Hepatic Lipid Metabolism
SIRT1 and SIRT3 play a prominent role in hepatic lipid metabolism; however, some evidences demonstrate that also SIRT4 and SIRT6 could be important players in the liver. In this section, we will focus the discussion on the role of SIRT1 and SIRT3 in the liver.
Role of SIRT1 in Hepatic Lipid Metabolism and Dysmetabolism
Liver is involved in the regulation of different aspects of lipid metabolism including fatty acid β‐oxidation, lipogenesis and lipoprotein uptake and secretion, in response to nutritional and hormonal signals. Dysregulation of lipid metabolic pathways results in the development of hepatic steatosis and contributes to the development of chronic hepatic inflammation, insulin resistance and liver damage 54, 55.
In recent years, the central role of SIRT1 in lipid metabolism regulation has become clear, as confirmed by the high number of publications on this topic. SIRT1 plays a prominent role in the regulation of hepatic fatty acid metabolism and inhibits hepatic lipogenesis by different mechanisms (Fig. 3). It activates the AMPK/LKB1 signaling pathway leading to increased fatty acid oxidation and preventing lipogenic pathways 56. Another study demonstrated that SIRT1 interacts, especially during fasting, with sterol regulatory element binding transcription factor 1 (SREBP‐1c), a key lipogenic transcription factor. The result of this interaction is decreased SREBP‐1c acetylation, with a consequent reduction in SREBP‐1c occupancy at the lipogenic genes in mouse liver 57. Furthermore, Xu et al. 58 demonstrated that the partial lack of Sirt1 (Sirt1+/− mice) leads to liver steatosis and hepatomegaly when mice were fed a medium‐fat diet (11% fat wt/wt), suggesting that alteration of Sirt1 activity promotes hepatic lipid deposition.
Figure 3
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Sirtuin‐mediated regulation of hepatic lipid metabolism. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Li et al. 59 demonstrated that SIRT1 deaceylates liver X receptors (LXRs) at lysine K432 in a loop region adjacent to the ligand activation domain, acting as a positive regulator of LXRs. For this reason the authors hypothesized that SIRT1 could play a role in cholesterol homeostasis. As a consequence, SIRT1 activates the transcription of the LXR target gene encoding the ATP‐binding cassette transporter (Abca1), which is important for HDL synthesis and reverse cholesterol transport. The same authors demonstrated that SIRT1 exerts some of its effects by modulating LXRs. In fact, Sirt1‐deficient mice (Sirt1−/−) had significantly lower total plasma cholesterol levels, due primarily to 40% reduction in HDL‐cholesterol, the main cholesterol‐rich lipoprotein (80–90% of total) in murine plasma, whereas cholesterol levels increased in the liver.
In this scenario, the phenotypical characterization of mice lacking SIRT1 in liver (Sirt1 LKO mice) by Chen et al. 41 showed that the lack of SIRT1 prevents deacetylation and activation of LXR and in fact the authors detected reduced hepatic expression of known LXR target genes such as Abca1, Srebp1c and of the lipogenic enzyme fatty acid synthase (Fas), regulated by SREBP‐1c. Consistent with the role of SIRT1 in LXR activation demonstrated by Li et al. 59, Sirt1 LKO mice displayed alterations in cholesterol metabolism, showing hepatic cholesterol accumulation when fed a western diet. They also accumulated cholesterol in liver when fed a high‐fat diet without cholesterol, suggesting an increase in de novo cholesterol synthesis. Purushotham et al. 60 further investigated the metabolic profile of this mouse model demonstrating that hepatic SIRT1 positively regulates PPARα, the nuclear receptor that mediates the adaptive response to fasting and starvation. Hepatocyte specific deletion of SIRT1 impaired PPARα signaling and decreased fatty acid β‐oxidation. When fed high‐fat diet, Sirt1 LKO mice developed hepatic steatosis, hepatic inflammation and endoplasmic reticulum stress. Overall, the SIRT1‐dependent induced activity of PPARα and the concomitant reduced activity of deacetylated SREBP‐1c may counterbalance the lipogenic effect of LXR activation.
SIRT1 participates also in the regulation of bile acid metabolism. Kemper et al. demonstrated in fact that farnesoid X receptor (FXR), a nuclear receptor that regulates bile acid homeostasis, is an important target of SIRT1, which deacetylates FXR on lysine 217. FXR deacetylation inhibits its dimerization with RXRα and its binding to target sites. Consequently, downregulation of hepatic SIRT1 increased FXR acetylation with deleterious metabolic outcomes. In mouse models of metabolic disease (ob/ob mouse), FXR interaction with SIRT1 is altered, leading to FXR hyperacetylation. Overexpression of SIRT1 or resveratrol treatment reduces acetylated FXR levels 61. Moreover, Purushotham et al. demonstrated that hepatic deletion of SIRT1 reduced the expression of FXR decreasing the binding of the hepatocyte nuclear factor 1α (Hnf1α) to the FXR promoter. Furthermore, when Sirt1 LKO mice were challenged with lithogenic diet they developed cholesterol gallstones, suggesting that hepatocyte‐specific deletion of SIRT1 leads to derangements of bile acid homeostasis 62.
All these findings demonstrate that SIRT1 plays a central role in the regulation of hepatic lipid pathways, highlighting that a lack of SIRT1 may compromise profoundly liver functionality.
Interestingly, a number of publications underlined that SIRT1 is an important target of ethanol action in liver. A chronic ethanol exposure decreases SIRT1 expression levels and inhibits its deacetylase activity in liver 63. Recently, Yin et al. 64 performed an in depth study on hepatic signaling molecules affected by ethanol. The authors identified microRNA‐217 as a regulator of the effects of ethanol in liver, both in a cellular model (AML‐12 hepatocytes) and in the livers of chronically ethanol‐fed mice. Ethanol exposure increased miR‐217 levels, inducing a miR‐217‐mediated loss of function of SIRT1 and SIRT1‐regulated genes encoding lipogenic or fatty acid oxidation enzymes. The overall outcome was increased fat accumulation in hepatocytes. These data underscore how SIRT1 protects against liver damage induced by fat accumulation. Accordingly, Bordone et al. 65 generated transgenic mice by knocking‐in SIRT1 cDNA in the β‐actin locus. These mice displayed some phenotypes similar to mice on a calorie‐restricted diet: they are leaner than littermate controls, more metabolically active and more glucose tolerant. Moreover, Pfluger et al. 66 demonstrated that overexpression of SIRT1 protected liver from high‐fat diet induced damage, suggesting that activation of SIRT1 signaling could be a useful approach for the treatment of nonalcoholic fatty liver disease (NAFLD), and in general a moderate systemic SIRT1 expression under its own promoter protects mice from HFD‐induced glucose intolerance.
Role of SIRT3 in Hepatic Lipid Metabolism and Dysmetabolism
SIRT3 expression in the liver is activated by fasting and induces fatty acid oxidation through the deacetylation of Acadl, as mice lacking Sirt3 show lower fatty acid oxidation rate 52. Another study confirmed the importance of Sirt3 in hepatic lipid metabolism as its overexpression in hepatocytes, by phosphorylating AMPK and acetyl‐CoA carboxylase (Acc), decreases lipid accumulation 67. In addition, upon fasting SIRT3 deacetylates the ketogenic enzyme hydroxymethylglutaryl CoA synthase 2 (HMGCS2) and increases the production of ketone bodies in wild‐type mice but not in Sirt3 KO mice 68.
The role of SIRT‐mediated regulation of acetylation in hepatic pathological conditions caused by nutrient excess is still poorly understood. At this regard, by analyzing acetylated proteins from total liver proteome, Kendrick 69 observed that proteins involved in gluconeogenesis, mitochondrial oxidative metabolism, liver injury, and the endoplasmic reticulum stress response were preferentially acetylated in mice fed high‐fat diet (HFD) compared with controls. Livers of mice fed HFD showed decreased levels of NAD+ with a concomitant reduction of SIRT3 activity. Interestingly these authors did not observe any alteration in Sirt1 or histone acetyltransferase activity, suggesting that Sirt3 could be the major responsible for the observed phenotype. These experimental evidences pinpoint the role of SIRT3 in the control of liver functionality and lipotoxicity in response to metabolic fuels (Fig. 3).
Roles of Other Sirtuins in Hepatic Lipid Metabolism and Dysmetabolism
Experiments with knock down of SIRT4 in mouse primary hepatocytes increased expression of mitochondrial and fatty acid metabolism enzymes, suggesting that SIRT4 may play a role in hepatic lipid metabolism. These effects might be explained by the increase in SIRT1 mRNA and protein levels in cells depleted of SIRT4, leading to higher oxidative metabolism 70.
In 2010, Kim et al. demonstrated that SIRT1‐FOXO3A and NRF1 participate in the formation of a complex on Sirt6 promoter, activating its expression (Fig. 3). Sirt6 ablation in liver induced accumulation of triglycerides, typically associated with fatty liver disease. The authors showed that SIRT6 deficiency increases expression of genes involved in hepatic long‐chain fatty acid uptake with a concomitant decrease in β‐oxidation genes. Moreover, they highlighted that Sirt6 ablation induces the hyperacetylation of histone H3 on lysine 9 in the promoters and consequently to increased expression of the genes encoding glycerol kinase (Gk), liver pyruvate kinase (Lpk), fatty acid synthase (Fas), acetyl‐CoA carboxylase (Acc1), fatty acid elongase 6 (Elovl6) and stearoyl‐CoA desaturase (Scd1), and prevented the fasting‐mediated deacetylation of H3K9. Changes of the expression of these enzymes lead to increased glycolysis, triglyceride synthesis, reduced β‐oxidation, and ultimately fatty liver 71.
Sirtuins in Adipose Tissue Lipid Metabolism
Adipose tissue is no longer regarded simply as a site for lipid storage. It is now clear that fat depots are linked and communicate with other tissues involved in the energy metabolism. SIRT1 (Fig. 4) and SIRT3 act in adipose tissue to influence lipid metabolism.
Figure 4
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Sirt1‐mediated regulation of adipose tissue lipid metabolism. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
High‐fat diet induces the cleavage of SIRT1 protein in adipose tissue by the inflammation‐activated caspase‐1, suggesting a link between dietary stress and predisposition to metabolic dysfunctions 44. In the epididymal adipose tissue of the SHRSP/IDmcr‐fa rats, an animal model of metabolic syndrome, under food restriction, SIRT1 and adiponectin expression are enhanced 72. In white adipocytes SIRT1 inhibits differentiation by interacting with NCoR1 and SMRT and by repressing the transcriptional activity of PPARγ, ultimately favoring lipolysis and fatty acid mobilization in response to fasting 39, 73. Moreover in differentiated adipocytes, overexpression of SIRT1 triggers lipolysis and loss of fat 39. To confirm the lipolytic role of SIRT1, Picard et al. 39 showed that whole‐body deletion of Sirt1 gene impairs mobilization of free fatty acids from white adipose tissue in response to fasting. Another evidence of the role of SIRT1 in lipid homeostasis in adipose tissue comes from the observation that it deacetylates and activates FOXO1, which binds to and regulates the expression of adipose triglyceride lipase gene (Atgl), encoding the rate‐limiting lipolytic enzyme 74. SIRT1 is also involved in adipose tissue inflammatory processes. It has been reported that in vivo reduction of SIRT1 expression elicits macrophage recruitment to adipose tissue, whereas overexpression of SIRT1 prevents adipose tissue macrophage accumulation caused by high‐fat feeding 75. Furthermore, in human subcutaneous fat SIRT1 expression is inversely related to adipose tissue macrophage infiltration 75. A recent work demonstrated that SIRT1 deacetylates PPARγ and promotes the “browning” of subcutaneous white adipose tissue (WAT). Deacetylation of PPARγ Lys268 and Lys293 allows the recruitment of the BAT program coactivator PR domain containing 16 (PRDM16) to PPARγ, resulting in induction of brown adipose tissue (BAT) specific genes and repression of visceral WAT marker genes, typically associated with the onset of insulin resistance 76.
There are evidences indicating that other sirtuins regulate adipose tissue functions. SIRT3 shows important role in brown adipose tissue (BAT), where it is activated by caloric restriction or cold exposure and increases the expression of PGC‐1α and uncoupling protein 1 (UCP1), leading to elevated thermogenesis and oxygen consumption 77. Finally, little is known about SIRT2 activity in adipose tissue in vivo, although it has been demonstrated that SIRT2 inhibits adipogenesis and accumulation of lipids in 3T3‐L1 adipocytes 78 by deacetylating FOXO1 79.
Sirtuins in the Brain‐Adipose Tissue Axis
During fast‐feed cycles, central nervous system and adipose tissue communicate and mutually affect their functions. It has been shown that SIRT1 regulates food intake and influences body weight through the central melanocortin system 80, and brain‐specific Sirt1 KO mice are more sensitive to diet‐induced obesity 81. The deletion of Sirt1 in POMC neurons abolished the capacity of leptin to activate phosphoinositol 3‐kinase (PI3K) signaling altering the metabolism of BAT and perigonadal WAT 81. On the other hand, overexpression of SIRT1 in striatum and hippocampus driven by the CaMKIIα promoter leads to increased fat accumulation and upregulation of adipogenic genes in WAT 82. Interestingly, the phenotype of these mice transgenic was not limited to adipose tissue as they displayed reduction of GLUT4 expression, of energy expenditure and altered motor activity, due to modifications of mitochondrial gene expression also in skeletal muscle.
Sirtuins in Skeletal Muscle Lipid Metabolism
Lipid metabolism is an important source of energy also in skeletal muscles. When skeletal muscle is nutrient‐deprived, glucose oxidation shifts to fatty acid oxidation, and sirtuins are crucial players of this metabolic balance, confirming their action as metabolic sensors also in this tissue.
In fact, in a cellular model of skeletal muscle SIRT1 upregulates mitochondrial and fatty acid oxidation genes by deacetylating and activating PGC‐1α 83, also during prolonged fasting. In this scenario, the finding that SIRT1 reduces transcription of uncoupling protein 3 (UCP3) 84, a protein postulated to protect mitochondria against an overload of fatty acids, seemed paradoxical. However, the authors speculate that this can be a physiological mechanism, independent of PGC‐1α, to control metabolic homeostasis: when NAD+/NADH ratio increased as a consequence of enhanced muscle catabolism, SIRT1 is activated. This, in turn, inhibits Ucp3 transcription leading to reduced NAD+/NADH ratio, thus preserving energy homeostasis of the tissue.
On the other hand, sirtuin action as metabolic sensors is confirmed when, depending on the muscle nutritional state, SIRT1 activates AceCS1 53 and regulates SREBP‐1c expression through the deacetylation of LXR 85, inducing fatty acid and triglycerides synthesis. Concerning other sirtuins, the lack of SIRT3 inactivates AMPK and phospo‐cAMP responsive element binding protein (pCREB) leading to inhibition of PGC‐1α activity in muscles 86.
Sirtuins as Therapeutic TargetsGenetic Polymorphisms of SIRT1 Gene: A Link with Obesity
Given the importance of SIRT1 in the regulation of energy metabolism, Peeters et al. 87 investigated whether genetic variations in SIRT1 could be correlated with obesity. In this study, the authors included 1,068 obese patients (BMI ≥ 30 kg/m2) and 313 lean controls (BMI between 18.5 and 25 kg/m2) and they found that only in male subjects the single nucleotide polymorphism (SNP) rs7069102 correlates with increased visceral adiposity. Moreover a recent study by Figarska et al. 88 investigated the associations between variation in single nucleotide polymorphisms (SNPs) in the Sirt1 gene and human survival. The purpose of these studies was the investigation of SIRT1 role in lifespan in humans, but since it is known that SIRT1 controls glucose metabolism, the authors investigated associations between SIRT1 and glucose tolerance. They found that carriers of the minor allele of rs12778366 had a significantly reduced risk of mortality compared to carriers of wild type allele (both in male and female subjects), and the effect was significantly protective in overweight/obese subjects. This result might be explained by the greater glucose tolerance found in this subpopulation as compared to wild type subjects. A larger study by Zillikens et al. 89 demonstrated that there are two Sirt1 variants associated with obesity. They analyzed 6,251 elderly subjects from the population‐based Rotterdam Study and they found that minor alleles rs7895833 and rs1467568 were associated with lower BMI and decreased risk of obesity.
Pharmacological Modulation of Sirtuins
Since several evidences have shown the crucial role of sirtuins in metabolic regulation, pharmacological modulation of sirtuins has emerged as a useful approach for the treatment of metabolic disorders. Several groups have focused their attention on the polyphenol resveratrol, proposed as a potent activator of SIRT1 90. Even though it was demonstrated that administration of resveratrol in vivo ameliorated glucose intolerance, insulin resistance and protected against severe metabolic derangement induced by high fat feeding 91, it was not clear whether the effects of resveratrol administration were due to a direct activation of SIRT1. Recently, it has been shown that resveratrol could indirectly activate several proteins such as AMPK, and its activation by resveratrol could mediate SIRT1 activation. According to this hypothesis, Um et al. 92 showed that resveratrol failed to improve insulin sensitivity, glucose tolerance and mitochondrial biogenesis in Ampkα1 and Ampkα2 KO mice.
In recent years, sirtuin synthetic activators have been described mostly for SIRT1 and proposed as therapeutic strategy. These compounds may help in the treatment and prevention of metabolic diseases like T2D, obesity as well as age‐related impaired heart function and neurological disorders 93. Yamazaki et al. 94 demonstrated that treatment of mice affected by nonalcoholic fatty liver disease (NAFLD) with the activator SRT1720 reduced expression of lipogenic enzymes and genes for oxidative stress and contributed to reduce hepatic lipid accumulation. Moreover, it was shown that SRT1720 administration enhanced oxidative metabolism in skeletal muscle, liver and brown adipose tissue, thus protecting mice from high fat diet‐induced obesity and insulin resistance 95. On the other hand, synthetic molecules with inhibiting activity towards SIRT1, SIRT2, SIRT3, and SIRT5 (splitomicin, sirtinol, AGK2, cambinol, suramin, tenovin, salermide) have been reported 93. These compounds could be potentially employed in several diseases such as cancer and neurodegenerative disorders. For instance, upregulation of SIRT1 has been noticed in several cancer cell lines 96 suggesting that inhibition of SIRT1 activity could be used in certain types of tumors as suppressor of cell proliferation. At this regard, Audrito et al. 96 demonstrated that the SIRT1 inhibitor nicotinamide blocks proliferation and induces apoptosis of leukemic cells via activation of the tumor suppressor p53 pathway. Mellini et al. showed that pseudopetidic inhibitors of SIRT1–3 inhibit lung and breast carcinoma growth by arresting cell cycle in G1 phase 97.
Conclusions
In the past decade, epigenetic modifications have emerged as fundamental modulators of metabolic functions. Sirtuins, belonging to class III histone deacetylases, regulate metabolic pathways at different points. This review pointed out the recent evidences on the important roles of sirtuins in lipid metabolism in different districts of the organism. Sirtuins lay at essential intersections of different metabolic pathways, which can be promoted or inhibited by sirtuins depending on the energy status. Concerning lipid metabolism, the pivotal role of SIRT1 and SIRT3 as key regulators has clearly emerged. These two sirtuins are well characterized, and in particular SIRT1 appears as an attractive target for the treatment of metabolic disorders. However, it will be extremely important to investigate further the actions of the other members of the sirtuin family, in order to provide new insights on the modulation of the metabolic pathways mediated by these enzymes and possibly treat their alterations.
Acknowledgements
We apologize for the many important papers that we did not cite in this review because of space constraint. This work was supported by grants from the European Union (FP6 LSHM‐CT2006–037498, FP7 602757‐HUMAN, and FP7 606806‐NR‐NET to Maurizio Crestani), Fondazione Cariplo (2008.2511 and 2009.2727 to Maurizio Crestani), the Giovanni Armenise‐Harvard Foundation (to Nico Mitro), the Italian Ministry of University (PRIN 2008 ZTN724 to Emma De Fabiani, PRIN2009 K7R7NA to Maurizio Crestani). We wish to thank Miss Elda Desiderio Pinto (Università degli Studi di Milano) for invaluable help in administrative management.
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