Adipose tissue is a functionally diverse organ with remarkable plasticity to adapt to changes in energy balance and contribute to systemic regulation of metabolism. It is capable of expanding in response to over-nutrition preventing ectopic fat deposition in non-adipose tissues, as well as mobilizing stored lipids during starvation or energy demand. Apart from its storage function, its ability to produce multiple adipokines as an endocrine organ, influences functions of other metabolic tissues. The ability of adipose tissue to respond to changes in energy balance is finely regulated by molecular mechanisms linked to nutrient response and

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the redox status of the tissue. Sirtuins are a group of NAD+ -dependent protein-deacetylating enzymes that play a key role in metabolic homeostasis. They influence genome stability, transcription and activity of several enzymes contributing to epigenetic regulation and act as key nutrient sensors.

silencing and cellular metabolism and led to establishing sirtuins as important epigenetic regulators. This was followed by identification of *Sir2* orthologues in mammalian systems; these

On the basis of sequence similarity, mammalian sirtuins are classified into four classes, class I– IV. Mammalian SIRT1–3 belong to class I, SIRT4 to class II, SIRT5 to class III and SIRT6 and 7 to class IV (**Table 1**) [9, 16]. All these enzymes share a conserved catalytic core structure, but they differ in their enzymic activity. While class I sirtuins (SIRT1–3) show robust deacetylase activity in vitro, SIRT4–7 show weak deacetylase activity; SIRT4 shows mono ADP–ribosyl transferase activity and SIRT2, 3 and 6 exhibit both deacetylase and ribosyl transferase activity. SIRT5 removes succinyl, malonyl and glutaryl groups from acylated protein–lysine residues. SIRT6 is more efficient in removing long chain fatty acyl groups such as myristoyl and palmitoyl groups. The enzymic reaction proceeds through the formation of a ternary complex involving acetyl protein substrate and NAD at the active site, which decomposes to form deacylated protein, nicotinamide and 2-*O*-acetyl-ADP ribose. They also differ in tissue distribution and subcellular localization (**Table 1**) [6, 9]. SIRT1 is expressed in metabolic tissues such as liver, muscle, adipose tissue and other organs such as heart, brain, pancreas; it is localized mainly in the nucleus, but shuttles from nucleus to cytosol. SIRT2, primarily a cytosolic protein, is highly expressed in heart, brain and skeletal muscle. SIRT3–5 are expressed in mitochondria. SIRT3 and 5 are mainly expressed in kidney, brain, liver and heart. In contrast to white adipose tissue, brown adipose tissue (BAT) expresses SIRT3. SIRT4 is expressed in heart, liver, pancreas and vascular smooth muscle. SIRT6 and 7 are localized predominantly in the nucleus. While SIRT6 is expressed in brain, liver and muscle, SIRT7 is mainly found in liver and spleen.

Mammalian sirtuins influence various cellular processes such as chromatin silencing, cell cycle regulation, differentiation and survival, mitochondrial biogenesis, metabolism, inflammation and stress response. While most studies in model organisms indicate a role for sirtuins in mediating the increased longevity affected by calorie restriction [17], a similar role for mammalian sirtuins is debated [18]. Sirtuins deacetylate transcription factors and regulate their activities either by influencing their cytoplasmic-nuclear distribution, their binding to

SIRT1 is by far the best characterized among all mammalian sirtuins. It has been linked to hypothalamic control of energy balance [19], has a role in adipogenesis and fat mobilization, as well as regulation of carbohydrate and lipid metabolism [6, 7]. SIRT1 promotes vasodilation and regenerative function in endothelial and smooth muscle cells of vascular wall by targeting eNOS for deacetylation [20]. In cardiomyocytes, SIRT1, 3 and 7 play a critical role in

Important targets of SIRT1 include p53, forkhead box type O transcription factors (FOXO), PPARγ co-activator-1α (PGC1α), NFkB, androgen receptor and their co-regulatory molecules (**Table 1**). Apart from its effect on PGC1α, a master regulator of mitochondrial biogenesis,

Sirtuins in Adipose Tissue Metabolism http://dx.doi.org/10.5772/intechopen.75704 31

were demonstrated to be NAD+

*2.1.2. Biological effects of sirtuins*

DNA or changing their interaction with regulatory proteins.

promoting cardiomyocyte resistance to stress and toxicity [21].

conserved across bacteria to humans.

Acetylation and deacetylation of histones and other DNA associated proteins are key epigenetic processes that can influence chromatin structure regulating the transition between highly condensed and transcriptionally less active heterochromatin state to loose and transcriptionally active euchromatin structure [1]. The extent of acetylation of histones depends, on one hand, on the balance between the activity of acetyl transferases such as histone acetyl transferase and deacetylases such as histone deacetylases (HDACs) [2, 3] and on the other hand on acetyl CoA which is an important metabolite at the junction of several metabolic pathways. Phylogenetically, eukaryotic HDACs are ancient proteins comprising of two subfamilies of proteins with different structure and function [4, 5]. While the enzymes belonging to the classical HDAC family are generally Zn-dependent enzymes which remove acetyl group from lysine residues on acetylated protein substrates with the addition of a water molecule, those belonging to the sirtuin family remove acetyl moieties bound to protein substrates to another substrate viz. NAD+ cleaving it to nicotinamide and *O*-acetyl ADP–ribose. So far 11 members of the classical HDAC enzymes, subdivided into three classes (HDAC I, III and IV), and 7 members of sirtuins belonging to HDAC class III family have been identified.

Sirtuins, particularly SIRT1, play a key role in adipogenesis of white adipose tissue (WAT) and browning of WAT, metabolism of glucose and fat, insulin sensitivity, control of inflammation and energy homeostasis. Dysregulation of these physiological processes have major implications in the development of obesity and related metabolic diseases. The role of sirtuins in the regulation of adipose tissue biology and its implications in the development of obesity form the subject matter of this chapter. A number of reviews on the structure and functions of various sirtuins and their implications in aging and several pathological conditions are available [6–11].

#### **2. Sirtuins in adipose tissue biology**

#### **2.1. Biochemistry and molecular biology of sirtuins**

#### *2.1.1. Classification and tissue distribution*

Interest in understanding the role of sirtuins in mammalian system was driven by findings in calorie-restricted conditions in model organisms. The discovery of a protein in yeast, referred to as yeast-silencing information regulator-2 (Sir2) [12] followed by the demonstration of its localization in the nucleolus and telomeres [13] and low histone acetylation level in the genetic loci highlighted the importance of this group of proteins in regulating chromatin structure in specific loci. These findings were followed by demonstration of the importance of the protein in regulating yeast lifespan [14] and identification of Sir2 as an NAD-dependent histone deacetylase [15]. This revealed the connection between a molecule involved in gene silencing and cellular metabolism and led to establishing sirtuins as important epigenetic regulators. This was followed by identification of *Sir2* orthologues in mammalian systems; these were demonstrated to be NAD+ -dependent protein deacetylating enzymes that are highly conserved across bacteria to humans.

On the basis of sequence similarity, mammalian sirtuins are classified into four classes, class I– IV. Mammalian SIRT1–3 belong to class I, SIRT4 to class II, SIRT5 to class III and SIRT6 and 7 to class IV (**Table 1**) [9, 16]. All these enzymes share a conserved catalytic core structure, but they differ in their enzymic activity. While class I sirtuins (SIRT1–3) show robust deacetylase activity in vitro, SIRT4–7 show weak deacetylase activity; SIRT4 shows mono ADP–ribosyl transferase activity and SIRT2, 3 and 6 exhibit both deacetylase and ribosyl transferase activity. SIRT5 removes succinyl, malonyl and glutaryl groups from acylated protein–lysine residues. SIRT6 is more efficient in removing long chain fatty acyl groups such as myristoyl and palmitoyl groups. The enzymic reaction proceeds through the formation of a ternary complex involving acetyl protein substrate and NAD at the active site, which decomposes to form deacylated protein, nicotinamide and 2-*O*-acetyl-ADP ribose. They also differ in tissue distribution and subcellular localization (**Table 1**) [6, 9]. SIRT1 is expressed in metabolic tissues such as liver, muscle, adipose tissue and other organs such as heart, brain, pancreas; it is localized mainly in the nucleus, but shuttles from nucleus to cytosol. SIRT2, primarily a cytosolic protein, is highly expressed in heart, brain and skeletal muscle. SIRT3–5 are expressed in mitochondria. SIRT3 and 5 are mainly expressed in kidney, brain, liver and heart. In contrast to white adipose tissue, brown adipose tissue (BAT) expresses SIRT3. SIRT4 is expressed in heart, liver, pancreas and vascular smooth muscle. SIRT6 and 7 are localized predominantly in the nucleus. While SIRT6 is expressed in brain, liver and muscle, SIRT7 is mainly found in liver and spleen.

#### *2.1.2. Biological effects of sirtuins*

the redox status of the tissue. Sirtuins are a group of NAD+

nutrient sensors.

30 Adipose Tissue

substrate viz. NAD+

**2. Sirtuins in adipose tissue biology**

*2.1.1. Classification and tissue distribution*

**2.1. Biochemistry and molecular biology of sirtuins**

enzymes that play a key role in metabolic homeostasis. They influence genome stability, transcription and activity of several enzymes contributing to epigenetic regulation and act as key

Acetylation and deacetylation of histones and other DNA associated proteins are key epigenetic processes that can influence chromatin structure regulating the transition between highly condensed and transcriptionally less active heterochromatin state to loose and transcriptionally active euchromatin structure [1]. The extent of acetylation of histones depends, on one hand, on the balance between the activity of acetyl transferases such as histone acetyl transferase and deacetylases such as histone deacetylases (HDACs) [2, 3] and on the other hand on acetyl CoA which is an important metabolite at the junction of several metabolic pathways. Phylogenetically, eukaryotic HDACs are ancient proteins comprising of two subfamilies of proteins with different structure and function [4, 5]. While the enzymes belonging to the classical HDAC family are generally Zn-dependent enzymes which remove acetyl group from lysine residues on acetylated protein substrates with the addition of a water molecule, those belonging to the sirtuin family remove acetyl moieties bound to protein substrates to another

of the classical HDAC enzymes, subdivided into three classes (HDAC I, III and IV), and 7

Sirtuins, particularly SIRT1, play a key role in adipogenesis of white adipose tissue (WAT) and browning of WAT, metabolism of glucose and fat, insulin sensitivity, control of inflammation and energy homeostasis. Dysregulation of these physiological processes have major implications in the development of obesity and related metabolic diseases. The role of sirtuins in the regulation of adipose tissue biology and its implications in the development of obesity form the subject matter of this chapter. A number of reviews on the structure and functions of various sirtuins and their implications in aging and several pathological conditions are available [6–11].

Interest in understanding the role of sirtuins in mammalian system was driven by findings in calorie-restricted conditions in model organisms. The discovery of a protein in yeast, referred to as yeast-silencing information regulator-2 (Sir2) [12] followed by the demonstration of its localization in the nucleolus and telomeres [13] and low histone acetylation level in the genetic loci highlighted the importance of this group of proteins in regulating chromatin structure in specific loci. These findings were followed by demonstration of the importance of the protein in regulating yeast lifespan [14] and identification of Sir2 as an NAD-dependent histone deacetylase [15]. This revealed the connection between a molecule involved in gene

members of sirtuins belonging to HDAC class III family have been identified.

cleaving it to nicotinamide and *O*-acetyl ADP–ribose. So far 11 members

Mammalian sirtuins influence various cellular processes such as chromatin silencing, cell cycle regulation, differentiation and survival, mitochondrial biogenesis, metabolism, inflammation and stress response. While most studies in model organisms indicate a role for sirtuins in mediating the increased longevity affected by calorie restriction [17], a similar role for mammalian sirtuins is debated [18]. Sirtuins deacetylate transcription factors and regulate their activities either by influencing their cytoplasmic-nuclear distribution, their binding to DNA or changing their interaction with regulatory proteins.

SIRT1 is by far the best characterized among all mammalian sirtuins. It has been linked to hypothalamic control of energy balance [19], has a role in adipogenesis and fat mobilization, as well as regulation of carbohydrate and lipid metabolism [6, 7]. SIRT1 promotes vasodilation and regenerative function in endothelial and smooth muscle cells of vascular wall by targeting eNOS for deacetylation [20]. In cardiomyocytes, SIRT1, 3 and 7 play a critical role in promoting cardiomyocyte resistance to stress and toxicity [21].

Important targets of SIRT1 include p53, forkhead box type O transcription factors (FOXO), PPARγ co-activator-1α (PGC1α), NFkB, androgen receptor and their co-regulatory molecules (**Table 1**). Apart from its effect on PGC1α, a master regulator of mitochondrial biogenesis,

**Sirt 1**

**Biological**

**effects (BAT)**

Brown

adipogenesis

Browing Whitening β-oxidation

Oxphos

Stimulates mitochondrial

biogenesis

Thermogenesis

Glucose

Improves glucose

metabolism

**Effects on**

**sirtuins**

Obesity

CR Gastric banding

Increased

surgery

References

**Table 1.**

Sirtuins – targets, enzymic activity and biological effects.

[6, 7, 11, 31, 52, 53, 77, 78,

[6, 7, 11, 26, 31]

[6, 7, 11, 27, 31]

[6, 7, 11, 31, 65, 66]

[6, 7, 11, 31]

[6, 7, 11, 31]

[6, 7, 11, 31, 108]

Sirtuins in Adipose Tissue Metabolism http://dx.doi.org/10.5772/intechopen.75704 33

100, 102]

Increased

Decreased

Decreased

Increased

Decreased (?)

Increased

Increased

Decreased?

Decreased

Increased

Increased

Increased/

decreased

tolerance

Stimulates

Stimulates

Stimulates

Stimulates

Stimulates Increases on cold

Increases

Decreases glucose

glucose uptake

tolerance

exposure

Inhibits

Inhibits

Stimulates

**Sirt 2**

**Sirt 3** Stimulates

Stimulates

Inhibits (expressed

more in BAT than

WAT)

(expressed more in

BAT than WAT)

(expressed more

in BAT than WAT)

**Sirt 4**

**Sirt 5**

**Sirt 6**

**Sirt 7**

**Sirt 1**

> Class

Subcellular

Nucleus, cytoplasm

Cytoplasm,

Mitochondria

Mitochondria

Mitochondria

Nucleus

Nucleolus

nucleus

location

Tissue

Liver, heart, brain,

Heart, brain,

Brown adipose

Vascular smooth

Brain, heart,

Brain, liver,

Liver, spleen

muscle

muscle, liver,

muscle, skeletal

muscle, heart, liver,

kidney

pancreas

tissue, kidney

brain, heart, liver

skeletal muscle

pancreas, muscle,

adipose tissue

Activity

Target

PGC1-α, PPARγ, PPARα,

Tubulin,

LCAD, HMGCS-2,

GDH, Malonyl CoA

CPS1

H3KK9, H3K56

SIRT 1

decarboxylase

SOD-2, GDH,

PEPCK,

FOXO-1,PAR-3

IDH2

FOXO1, FOXO3, p53,

notch, NF-kB, HIF-1α,

LXR, FXR, SREBP1c

**Biological**

**effects (WAT)**

Adipogenesis

Lipogenesis

Lipolysis β-oxidation

Oxphos

Glucose

Improves insulin

Decreases insulin

sensitivity

Decreases

Increases

Increases

Decreases leptin

adiponectin

sensitivity

metabolism

Inflammation

Adipokines

Increases adiponectin

Increases

adiponectin

Decreases leptin

Decreases

Stimulates (FOXO1,

Stimulates

ATGL)

Inhibition (decreases

Stimulates malonyl

decarboxylase

AC1)

Inhibition

Inhibition

Stimulates

Deacetylation

Deacetylation

Deacetylation

ADP-ribosylation

Deacetylation,

Deacetylation,

Deacetylation

ADPribosylation

demalonylation

desuccinylation

I

**Sirt 2**

I

I

II

**Sirt 3**

**Sirt 4**

**Sirt 5**

III

IV

IV

**Sirt 6**

**Sirt 7**

32 Adipose Tissue

**Table 1.** Sirtuins – targets, enzymic activity and biological effects. 33

SIRT1 acts on several transcription factors like estrogen like receptors, the nuclear respiratory factors 1 and 2 to induce mitochondrial gene expression [22].

through interaction with different protein complexes such as DBC1 (nuclear protein deleted in breast cancer-1), AROS (active regulator of SIRT1) and NCoR1 [35, 36]. SIRT1 can also be

families, PARP and cADP ribose synthase [37]. SIRT1 can enhance its own activity by autodeacetylation. This is inhibited by SIRT7 suggesting coordinated action of different sirtuins [38].

*vitro* studies using malignant cell lines, have reported the role of different miRNAs in the post-transcriptional regulation of SIRT1 [31]. Mir34a is reported to regulate SIRT1 at different levels; it inhibits translation of SIRT1 mRNA; it indirectly suppresses transcription and enzyme activity of SIRT1 by modulating the levels of NAD through regulation of the biosyn-

Unlike SIRT1, information on the regulation of other sirtuins is limited. Cyclin(E-Cdk2 and A-Cdk2) mediated phosphorylation at S331 inhibits SIRT2 activity whereas Erk1/2 enhanced its activity [31]. Cyclic AMP–PKA pathway, which is activated in response to various stimuli, causes upregulation of SIRT3 expression through PGC1α. cAMP may also activate SIRT3 by direct binding [31]. Activated AMPK also positively regulates SIRT3 by increasing NAD. Similar to SIRT3, expression of SIRT5 is also upregulated by PGC1α, while AMPK appears to negatively regulate its expression [31]. Several transcription factors and miRNAs appear to regulate expression of SIRT6. While Nuclear Respiratory factor 1 (NRF-1) and coactivators induce SIRT6 expression during nutrient deprivation, cAMP signaling reduces

SIRT6 expression. A relation between SIRT6 and mir122 has also been suggested [31].

Adipose tissue is classified into white adipose tissue (WAT) which serves as the principal energy storage organ and brown adipose tissue (BAT) whose principal function is maintaining temperature by non-shivering thermogenesis. A third category includes beige or brite (brown in white) adipocytes within WAT which can potentially differentiate into cells of

The cellular and molecular mechanisms that govern the adipocyte life cycle have been extensively studied [42–45]. Both white and brown adipocytes develop by a highly regulated process of differentiation of mesenchymal stem cells (MSC). During the early phase of adipogenesis the pluripotent MSCs are committed to unipotent pre-adipocytes which in the latter phase undergo terminal differentiation acquiring the characteristic phenotype and functions of mature adipocytes. The complex process of adipocyte differentiation is coordinated by myriad factors. Isoforms of bone morphogenetic protein, BMP2 and BMP4 are the key positive regulators of commitment to white pre-adipocytes [46, 47]. This early phase of differentiation is also subject to negative regulation by several transcription factors including members of GATA and Forkhead family, Wnt and Notch signaling, Kruppel-like factors 2 and 7 (KLF2,

synthesis from NAD+

precursors like nicotinic

Sirtuins in Adipose Tissue Metabolism http://dx.doi.org/10.5772/intechopen.75704 35

consumption by two alternate enzyme

[39] and by nicotinamide [40]. *In*

activated indirectly by either increasing NAD+

acid, NAM, nicotinamide riboside or decreasing NAD+

SIRT1 is inhibited by NADH which competes with NAD<sup>+</sup>

thetic enzyme nicotinamide phosphoribosyl transferase [41].

**2.2. Sirtuins regulate WAT development and metabolism**

brown like phenotype.

*2.2.1. Sirtuins and WAT development*

KLF7) and CHOP proteins [48].

SIRT1 appears to directly block lipid anabolism by interfering with PPARγ and LXR signaling. The repressive effect of SIRT1 on PPARγ activity requires the formation of a co-represssor complex that involves NCoR1 [23]. SIRT1 also has a role in reverse cholesterol transport; it stimulates cholesterol efflux from macrophages and the hepatic conversion of cholesterol to bile acids through LXR [24] . It is also present in the cytosol of many cell types and regulates major cytoplasmic enzymes such as acetyl CoA synthase and eNOS by deacetylation [20].

Compared to SIRT1, not much is known about the physiological effect of other mammalian sirtuins. SIRT2 shows similarity to SIRT1 in several of its biological effects. It appears to increase hepatic glucose which is beneficial in starving conditions where SIRT2 activity increases. While it suppresses glycolysis by deacetylating and destabilizing glucokinase, it activates gluconeogenesis by enhancing the action of a key gluconeogenic enzyme PEPCK by its deacetylation [25]. It also appears to have a role in the control of microtubule stability and cell cycle oscillations by deacetylating α-tubulin [26].

SIRT3 is a major regulator of mitochondrial function; it deacetylates several mitochondrial proteins which are critical in mitochondrial oxidative metabolism. Calorie restriction increases activity of SIRT3 which alters the mitochondrial acetylome [27, 28]. SIRT4 is a mitochondrial matrix protein with remarkable ADP-ribosyltransferase activity [29]. It is reported to regulate insulin secretion [30], the activity of glutamate dehydrogenase, and serves as a metabolic regulator by inhibiting the activity of several metabolic enzymes such as pyruvate dehydrogenase opposing the effect of SIRT3 [31]. Although recent studies have identified several SIRT5 target proteins, not much is known about its biological function. SIRT5 appears to play a role in energy homeostasis and free radical metabolism [32]. SIRT6 deficient mice aged prematurely and its overexpression increased life span of male mice apparently by altering IGF signaling [31].

#### *2.1.3. Regulation of sirtuins*

Sirtuins are regulated in response to nutritional and metabolic challenges, oxidative stress, and inflammation in a cell and tissue specific manner. They are subject to transcriptional control, post-transcriptional regulation by miRNA and post-translational modulation [31]. These regulatory events can either alter the levels of each sirtuin or their enzyme activity or both. Their activity can be modulated either directly by post-translational modifications(PTMs) such as phosphorylation and acetylation, protein interactions and by compounds that activate them, or indirectly by modulating NAD+ levels. The substrates themselves appear to regulate sirtuin expression indicating the possible formation of feedback regulatory loops.

Some of the key factors involved in metabolic homeostasis that cause upregulation of SIRT1 include CREB, FOXO1, FOX3a, C/EBPα, PPARα and PPARβ/δ while the negative regulators include ChREBP, C/EBP. NFkB, EGR1, APE1 positively regulate transcription of SIRT1 during stress conditions. SIRT1 in turn can modulate the activity of several of these transcription factors. The enzymatic activity of SIRT1 is enhanced by post-translational modification by phosphorylation [33] and SUMOylation (Lys734) [34]. The activity of SIRT1 can also be controlled through interaction with different protein complexes such as DBC1 (nuclear protein deleted in breast cancer-1), AROS (active regulator of SIRT1) and NCoR1 [35, 36]. SIRT1 can also be activated indirectly by either increasing NAD+ synthesis from NAD+ precursors like nicotinic acid, NAM, nicotinamide riboside or decreasing NAD+ consumption by two alternate enzyme families, PARP and cADP ribose synthase [37]. SIRT1 can enhance its own activity by autodeacetylation. This is inhibited by SIRT7 suggesting coordinated action of different sirtuins [38]. SIRT1 is inhibited by NADH which competes with NAD<sup>+</sup> [39] and by nicotinamide [40]. *In vitro* studies using malignant cell lines, have reported the role of different miRNAs in the post-transcriptional regulation of SIRT1 [31]. Mir34a is reported to regulate SIRT1 at different levels; it inhibits translation of SIRT1 mRNA; it indirectly suppresses transcription and enzyme activity of SIRT1 by modulating the levels of NAD through regulation of the biosynthetic enzyme nicotinamide phosphoribosyl transferase [41].

Unlike SIRT1, information on the regulation of other sirtuins is limited. Cyclin(E-Cdk2 and A-Cdk2) mediated phosphorylation at S331 inhibits SIRT2 activity whereas Erk1/2 enhanced its activity [31]. Cyclic AMP–PKA pathway, which is activated in response to various stimuli, causes upregulation of SIRT3 expression through PGC1α. cAMP may also activate SIRT3 by direct binding [31]. Activated AMPK also positively regulates SIRT3 by increasing NAD. Similar to SIRT3, expression of SIRT5 is also upregulated by PGC1α, while AMPK appears to negatively regulate its expression [31]. Several transcription factors and miRNAs appear to regulate expression of SIRT6. While Nuclear Respiratory factor 1 (NRF-1) and coactivators induce SIRT6 expression during nutrient deprivation, cAMP signaling reduces SIRT6 expression. A relation between SIRT6 and mir122 has also been suggested [31].

#### **2.2. Sirtuins regulate WAT development and metabolism**

Adipose tissue is classified into white adipose tissue (WAT) which serves as the principal energy storage organ and brown adipose tissue (BAT) whose principal function is maintaining temperature by non-shivering thermogenesis. A third category includes beige or brite (brown in white) adipocytes within WAT which can potentially differentiate into cells of brown like phenotype.

#### *2.2.1. Sirtuins and WAT development*

SIRT1 acts on several transcription factors like estrogen like receptors, the nuclear respiratory

SIRT1 appears to directly block lipid anabolism by interfering with PPARγ and LXR signaling. The repressive effect of SIRT1 on PPARγ activity requires the formation of a co-represssor complex that involves NCoR1 [23]. SIRT1 also has a role in reverse cholesterol transport; it stimulates cholesterol efflux from macrophages and the hepatic conversion of cholesterol to bile acids through LXR [24] . It is also present in the cytosol of many cell types and regulates major cytoplasmic enzymes such as acetyl CoA synthase and eNOS by deacetylation [20].

Compared to SIRT1, not much is known about the physiological effect of other mammalian sirtuins. SIRT2 shows similarity to SIRT1 in several of its biological effects. It appears to increase hepatic glucose which is beneficial in starving conditions where SIRT2 activity increases. While it suppresses glycolysis by deacetylating and destabilizing glucokinase, it activates gluconeogenesis by enhancing the action of a key gluconeogenic enzyme PEPCK by its deacetylation [25]. It also appears to have a role in the control of microtubule stability and

SIRT3 is a major regulator of mitochondrial function; it deacetylates several mitochondrial proteins which are critical in mitochondrial oxidative metabolism. Calorie restriction increases activity of SIRT3 which alters the mitochondrial acetylome [27, 28]. SIRT4 is a mitochondrial matrix protein with remarkable ADP-ribosyltransferase activity [29]. It is reported to regulate insulin secretion [30], the activity of glutamate dehydrogenase, and serves as a metabolic regulator by inhibiting the activity of several metabolic enzymes such as pyruvate dehydrogenase opposing the effect of SIRT3 [31]. Although recent studies have identified several SIRT5 target proteins, not much is known about its biological function. SIRT5 appears to play a role in energy homeostasis and free radical metabolism [32]. SIRT6 deficient mice aged prematurely and its overexpression increased life span of male mice apparently by altering IGF signaling [31].

Sirtuins are regulated in response to nutritional and metabolic challenges, oxidative stress, and inflammation in a cell and tissue specific manner. They are subject to transcriptional control, post-transcriptional regulation by miRNA and post-translational modulation [31]. These regulatory events can either alter the levels of each sirtuin or their enzyme activity or both. Their activity can be modulated either directly by post-translational modifications(PTMs) such as phosphorylation and acetylation, protein interactions and by compounds that activate

Some of the key factors involved in metabolic homeostasis that cause upregulation of SIRT1 include CREB, FOXO1, FOX3a, C/EBPα, PPARα and PPARβ/δ while the negative regulators include ChREBP, C/EBP. NFkB, EGR1, APE1 positively regulate transcription of SIRT1 during stress conditions. SIRT1 in turn can modulate the activity of several of these transcription factors. The enzymatic activity of SIRT1 is enhanced by post-translational modification by phosphorylation [33] and SUMOylation (Lys734) [34]. The activity of SIRT1 can also be controlled

sirtuin expression indicating the possible formation of feedback regulatory loops.

levels. The substrates themselves appear to regulate

factors 1 and 2 to induce mitochondrial gene expression [22].

cell cycle oscillations by deacetylating α-tubulin [26].

*2.1.3. Regulation of sirtuins*

34 Adipose Tissue

them, or indirectly by modulating NAD+

The cellular and molecular mechanisms that govern the adipocyte life cycle have been extensively studied [42–45]. Both white and brown adipocytes develop by a highly regulated process of differentiation of mesenchymal stem cells (MSC). During the early phase of adipogenesis the pluripotent MSCs are committed to unipotent pre-adipocytes which in the latter phase undergo terminal differentiation acquiring the characteristic phenotype and functions of mature adipocytes. The complex process of adipocyte differentiation is coordinated by myriad factors. Isoforms of bone morphogenetic protein, BMP2 and BMP4 are the key positive regulators of commitment to white pre-adipocytes [46, 47]. This early phase of differentiation is also subject to negative regulation by several transcription factors including members of GATA and Forkhead family, Wnt and Notch signaling, Kruppel-like factors 2 and 7 (KLF2, KLF7) and CHOP proteins [48].

The differentiation of white pre-adipocytes to mature adipocytes is mediated by multiple transcription factors. The key transcription factors involved are CCAAT enhancer binding protein (C/EBP), PPARγ and sterol-regulatory element binding protein 1 (SREBP1) [43]. C-EBP, which exists in six different isoforms, is activated and translocated to the nucleus in response to cAMP mediated signaling [43]. Hormones induce transient expression of C/EBPβ and C/EBPδ which upregulate expression of PPARγ and C/EBPα [43] PPARγ, (particularly the isoform PPARγ1) that belongs to the nuclear receptor superfamily, heterodimerises with retinoid receptor α (RXRα), another nuclear receptor, and binds to DNA to promote expression of adipocyte specific genes such as leptin, adiponectin, FABP4, and perilipin. Activation of C/ EBPα leads to transcriptional activation of several genes encoding proteins such as GLUT4, SCD1, FABP4 which are critical in establishing adipocyte phenotype [43].

*2.2.2. Sirtuins and WAT metabolism*

and other metabolic organs respond to energy needs [8].

thus affect nutrient flux into the adipose tissue.

During times of positive energy balance, storage of lipids as triglycerides in lipid droplets in adipocytes leads to expansion of adipose tissue. It leads to changes in the levels of adipokines such as leptin and adiponectin that affect metabolic functions of other organs particularly liver, muscle and brain, which in turn can affect the adipose tissue function [43, 45]. The stored lipid is mobilized in response to hormones and systemic energy needs during periods of negative energy balance. Metabolism of adipose tissue is thus linked with that of other tissues. SIRT1 appears to serve as an important metabolic switch through which adipose tissue

Sirtuins in Adipose Tissue Metabolism http://dx.doi.org/10.5772/intechopen.75704 37

When energy stores are high, excess nutrients particularly glucose and amino acids are used to synthesize fatty acids de novo primarily in liver and are exported to WAT where they are stored as TG [6] SIRT1 deacetylates sterol-responsive element binding protein 1c (SREBP1c) causing reduction of its transcriptional ability and suppression of fatty acid synthesis [6]. Further, by increasing PGC1α-mediated mitochondrial biogenesis, SIRT1 facilitates fatty acid oxidation. SIRT1 promotes gluconeogenesis in the liver through deacetylation and activation of PGC1α and FOXO1, and increasing expression of gluconeogenic enzymes [22] and inhibits glycolysis [6] This increases hepatic production of glucose during fasting. Apart from SIRT1, SIRT6 also has been shown to repress glycolysis in liver [31]. Further, SIRT1, by deacetylating STAT3, reduces its repressive effect on gluconeogenesis [60].The hepatic effects of sirtuins can

Sirtuins are not only critical in WAT adipogenesis, they also play a key role in maintaining the functions of differentiated adipocytes by regulating the expression of several PPARγresponsive genes involved in metabolism. Activation of SIRT1 reduces expression of acetyl coA carboxylase and other lipogenic genes involved in the de novo synthesis of FAs in pre-adipocytes. Multiple studies support the role of PPARγ in regulating adipose tissue metabolism [61]. Over-expression of a dominant negative form of PPARγ downregulates the expression of key genes involved in lipid metabolism, insulin signaling and decreases lipid content in 3T3L1 differentiated adipocytes [62]. Further, selective ablation of PPARγ in mature white and brown adipocytes results in adipocyte death without any effect on pre-adipocyte differentiation indicating the requirement of PPARγ for maintenance of differentiated functions of adipocytes [63]. Insulin dependent glucose uptake in the adipocyte takes place through the GLUT4 transporter. Expression of GLUT4 gene is regulated by PPARγ. SIRT1 appears to regulate glucose–induced secretion of insulin by transcriptional repression of UCP2 which uncouples mitochondrial ATP production [64]. SIRT1 by deacetylating SREBP1, destabilizes and reduces its occupancy on the lipogenic gene promoters suppressing fatty acid synthesis [6]. Although SIRT1 inhibits lipogenesis in WAT, SIRT4 appears to have an opposite effect. Deletion of mitochondrial SIRT4 decreased lipogenesis apparently by regulating malonyl CoA decarboxylase, thereby altering the level of malonyl CoA that represses fatty acid oxidation [65, 66]. While an increase in SIRT4 in fed state results in deacetylation and inactivation of malonyl CoA decarboxylase resulting in enhanced FA synthesis, during fasting, reduction of SIRT4 expression

All seven sirtuin genes are expressed in human and rodent WAT. A difference in their level of expression, particularly a reduction in SIRT1, has been observed, in experimental obese models as well as obese human subjects [11]. Studies in experimental model systems suggest that sirtuins, particularly SIRT1, are negative modulators of WAT adipogenesis.  Resveratrol, an activator of SIRT1 reduces osteoblastic differentiation of MSC to adipocytes [49]. SIRT1 is upregulated in WAT in calorie-restricted mice model in which there was significant reduction in fat mass [50, 51]. Further, transgenic mice overexpressing SIRT1showed lower body weight and reduction of fat mass [52], while ablation of SIRT1 in WAT resulted in gain in body weight, increase in fat mass and an increase in the size of individual adipocytes [53]. Studies using bone marrow-derived MSC with SIRT1 deletion showed impaired self- renewal and differentiation to osteoblasts without significantly affecting their differentiation to adipocytes [54]. In the light of these observations it has been suggested that although SIRT1 inhibits adipocyte differentiation, its expression is critical for maintenance of MSC pool [11].

PPARγ is an important substrate for SIRT1 [55]. SIRT1 dependent deacetylation of lysine residues (268 and 293 K) on PPARγ is critical in the regulation of its transcriptional activity by its co-repressors NCoR and SMRT. Sirt1 can thus inhibit white adipogenesis by suppressing the transcriptional activity of PPARγ by promoting the binding of its co-repressors NCoR and SMRT [23]. Further, C/EBPα, a PPARγ dependent factor, regulates the expression of SIRT1 during adipogenesis [56]. miRNAs can also regulate effects of SIRT1 on adipogenesis. For example, mir 146b can promote adipogenesis by suppressing SIRT1-FOXO1 cascade [57]. SIRT2, the predominant sirtuin in adipose tissue, has also been shown to inhibit adipocyte differentiation by deacetylating FOXO1 and enhancing its repressive interaction with PPARγ [58, 59]. Unlike SIRT1 and SIRT2, SIRT7 knockdown in human pre-adipocytes reduced lipid content and the number of FABP4<sup>+</sup> differentiated adipocytes indicating a contrasting effect of SIRT7. Further, SIRT7 knockout mice had significantly reduced WAT and increased SIRT1 activity. This suggests that SIRT7 influences adipogenesis in mice by inhibiting autocatalytic activation of SIRT1, further highlighting the importance of cross talk among sirtuins in the control of adipose tissue maintenance [38].

#### *2.2.2. Sirtuins and WAT metabolism*

The differentiation of white pre-adipocytes to mature adipocytes is mediated by multiple transcription factors. The key transcription factors involved are CCAAT enhancer binding protein (C/EBP), PPARγ and sterol-regulatory element binding protein 1 (SREBP1) [43]. C-EBP, which exists in six different isoforms, is activated and translocated to the nucleus in response to cAMP mediated signaling [43]. Hormones induce transient expression of C/EBPβ and C/EBPδ which upregulate expression of PPARγ and C/EBPα [43] PPARγ, (particularly the isoform PPARγ1) that belongs to the nuclear receptor superfamily, heterodimerises with retinoid receptor α (RXRα), another nuclear receptor, and binds to DNA to promote expression of adipocyte specific genes such as leptin, adiponectin, FABP4, and perilipin. Activation of C/ EBPα leads to transcriptional activation of several genes encoding proteins such as GLUT4,

All seven sirtuin genes are expressed in human and rodent WAT. A difference in their level of expression, particularly a reduction in SIRT1, has been observed, in experimental obese models as well as obese human subjects [11]. Studies in experimental model systems suggest that sirtuins, particularly SIRT1, are negative modulators of WAT adipogenesis.  Resveratrol, an activator of SIRT1 reduces osteoblastic differentiation of MSC to adipocytes [49]. SIRT1 is upregulated in WAT in calorie-restricted mice model in which there was significant reduction in fat mass [50, 51]. Further, transgenic mice overexpressing SIRT1showed lower body weight and reduction of fat mass [52], while ablation of SIRT1 in WAT resulted in gain in body weight, increase in fat mass and an increase in the size of individual adipocytes [53]. Studies using bone marrow-derived MSC with SIRT1 deletion showed impaired self- renewal and differentiation to osteoblasts without significantly affecting their differentiation to adipocytes [54]. In the light of these observations it has been suggested that although SIRT1 inhibits adipocyte differentiation, its expression is critical for maintenance

PPARγ is an important substrate for SIRT1 [55]. SIRT1 dependent deacetylation of lysine residues (268 and 293 K) on PPARγ is critical in the regulation of its transcriptional activity by its co-repressors NCoR and SMRT. Sirt1 can thus inhibit white adipogenesis by suppressing the transcriptional activity of PPARγ by promoting the binding of its co-repressors NCoR and SMRT [23]. Further, C/EBPα, a PPARγ dependent factor, regulates the expression of SIRT1 during adipogenesis [56]. miRNAs can also regulate effects of SIRT1 on adipogenesis. For example, mir 146b can promote adipogenesis by suppressing SIRT1-FOXO1 cascade [57]. SIRT2, the predominant sirtuin in adipose tissue, has also been shown to inhibit adipocyte differentiation by deacetylating FOXO1 and enhancing its repressive interaction with PPARγ [58, 59]. Unlike SIRT1 and SIRT2, SIRT7 knockdown in human pre-adipocytes reduced lipid

of SIRT7. Further, SIRT7 knockout mice had significantly reduced WAT and increased SIRT1 activity. This suggests that SIRT7 influences adipogenesis in mice by inhibiting autocatalytic activation of SIRT1, further highlighting the importance of cross talk among sirtuins in the

differentiated adipocytes indicating a contrasting effect

SCD1, FABP4 which are critical in establishing adipocyte phenotype [43].

of MSC pool [11].

36 Adipose Tissue

content and the number of FABP4<sup>+</sup>

control of adipose tissue maintenance [38].

During times of positive energy balance, storage of lipids as triglycerides in lipid droplets in adipocytes leads to expansion of adipose tissue. It leads to changes in the levels of adipokines such as leptin and adiponectin that affect metabolic functions of other organs particularly liver, muscle and brain, which in turn can affect the adipose tissue function [43, 45]. The stored lipid is mobilized in response to hormones and systemic energy needs during periods of negative energy balance. Metabolism of adipose tissue is thus linked with that of other tissues. SIRT1 appears to serve as an important metabolic switch through which adipose tissue and other metabolic organs respond to energy needs [8].

When energy stores are high, excess nutrients particularly glucose and amino acids are used to synthesize fatty acids de novo primarily in liver and are exported to WAT where they are stored as TG [6] SIRT1 deacetylates sterol-responsive element binding protein 1c (SREBP1c) causing reduction of its transcriptional ability and suppression of fatty acid synthesis [6]. Further, by increasing PGC1α-mediated mitochondrial biogenesis, SIRT1 facilitates fatty acid oxidation. SIRT1 promotes gluconeogenesis in the liver through deacetylation and activation of PGC1α and FOXO1, and increasing expression of gluconeogenic enzymes [22] and inhibits glycolysis [6] This increases hepatic production of glucose during fasting. Apart from SIRT1, SIRT6 also has been shown to repress glycolysis in liver [31]. Further, SIRT1, by deacetylating STAT3, reduces its repressive effect on gluconeogenesis [60].The hepatic effects of sirtuins can thus affect nutrient flux into the adipose tissue.

Sirtuins are not only critical in WAT adipogenesis, they also play a key role in maintaining the functions of differentiated adipocytes by regulating the expression of several PPARγresponsive genes involved in metabolism. Activation of SIRT1 reduces expression of acetyl coA carboxylase and other lipogenic genes involved in the de novo synthesis of FAs in pre-adipocytes. Multiple studies support the role of PPARγ in regulating adipose tissue metabolism [61]. Over-expression of a dominant negative form of PPARγ downregulates the expression of key genes involved in lipid metabolism, insulin signaling and decreases lipid content in 3T3L1 differentiated adipocytes [62]. Further, selective ablation of PPARγ in mature white and brown adipocytes results in adipocyte death without any effect on pre-adipocyte differentiation indicating the requirement of PPARγ for maintenance of differentiated functions of adipocytes [63]. Insulin dependent glucose uptake in the adipocyte takes place through the GLUT4 transporter. Expression of GLUT4 gene is regulated by PPARγ. SIRT1 appears to regulate glucose–induced secretion of insulin by transcriptional repression of UCP2 which uncouples mitochondrial ATP production [64]. SIRT1 by deacetylating SREBP1, destabilizes and reduces its occupancy on the lipogenic gene promoters suppressing fatty acid synthesis [6]. Although SIRT1 inhibits lipogenesis in WAT, SIRT4 appears to have an opposite effect. Deletion of mitochondrial SIRT4 decreased lipogenesis apparently by regulating malonyl CoA decarboxylase, thereby altering the level of malonyl CoA that represses fatty acid oxidation [65, 66]. While an increase in SIRT4 in fed state results in deacetylation and inactivation of malonyl CoA decarboxylase resulting in enhanced FA synthesis, during fasting, reduction of SIRT4 expression leads to activation of malonyl CoA decarboxylase resulting in enhanced lipid oxidation. It therefore appears that SIRT4 exhibits a dual effect regulating anabolism and catabolism of fatty acids [65]. SIRT6 also appears to regulate lipogenesis. In SIRT6 overexpressing mice, expression of diacylglycerol acyl transferase a key enzyme involved in TG synthesis was down regulated along with certain PPARγ responsive genes involved in lipogenesis [31, 67].

The ability of human white adipocytes to acquire brown fat-like phenotype, termed browning, in response to β-adrenergic stimulation, cold exposure [43] and by several molecules such as muscle derived irisin [71, 72] liver derived FGF21 [73] and small molecules such as β-aminoisobutyric acid [74] has been observed. These type of cells called beige or brite adiopcytes express genes involved in thermogenesis such as UCP1, deiodinase type II and PGC1α

Sirtuins in Adipose Tissue Metabolism http://dx.doi.org/10.5772/intechopen.75704 39

Sirtuins also appear to play a role in the differentiation and function of BAT. Like WAT, brown adipose tissue also has been shown to express all the members of the mammalian SIRT family [11]. While the relative level of expression of SIRT3 and 5 are higher, that of SIRT1 and 7 are lower in BAT than those in WAT. Calorie restriction (CR) and cold exposure upregulated the expression of SIRT3 present in the mitochondria in BAT [75]; SIRT1 and SIRT2 also showed an upregulation under such conditions. Conversely, SIRT3 is down regulated in BAT in high fat diet induced obese mice. SIRT1 also appears to have a role in differentiation of pre-adipocyte to brown adipocytes. It appears that SIRT1 influences BAT differentiation through repression of the MyoD-mediated myogenic gene expression signature and stimulation of PGC-1α mediated mitochondrial gene expression [76]. SIRT1 appeared to improve glucose homeostasis in SIRT1 transgenic mice and brown adipocytes derived from them due to an enhanced response of brown adipocytes to β3-adrenergic stimuli rather than differences in differentiation status [77]. Of the different sirtuins, SIRT3 appeared to be critical in the differentiation of brown adipocytes. SIRT3 has been shown to activate PGC1α mediated thermogenic response

Brown remodeling of white fat in response to cold exposure is shown to be regulated by SIRT1-dependent deacetylation of PPARγ. SIRT1-dependent deacetylation of Lys 268 and Lys 293 of PPARγ is required to recruit the co-activator PRDM16 to PPARγ, leading to upregulation of BAT-specific genes and repression of WAT genes [78].In response to different environmental stimuli, SIRT1 can differentially modulate PPARγ in WAT. SIRT1 inhibits PPARγ through local modulation of acetylation status of histones and recruitment of co-repressor NCoR in response to caloric restriction; but on cold exposure, it directly enhances PPARγ signaling through deacetylation of PPARγ itself [79]. SIRT1 deficiency in mice results in accumulation of lipid droplets and reduction of mitochondrial content in BAT indicating a role for SIRT1 in the white remodeling of BAT which appears to occur in obese conditions [80].

Grossly elevated fat stores in adipose tissue with hypertrophic or hyperplastic adipocytes and concomitant development of blood vessels result in obesity. The relevance of sirtuins in adipose tissue development and metabolism and their effects on metabolism of glucose and lipids primarily in the liver, and insulin function suggest a possible link between sirtuins and

The level of expression and activity of SIRT1 decrease in adipose tissue in different obesity models. Expression of SIRT1 in adipose tissue of db/db leptin resistant obese mice and in mice fed on HFD was significantly low [53, 81]. Overexpression of SIRT1 in HFD-induced obese animals caused less inflammation and better glucose tolerance. SIRT1 expression in obese pigs

in response to stimulation of β3–adrenergic receptors [43].

in differentiating brown adipocytes.

**2.4. Sirtuins and obesity**

obesity.

#### *2.2.3. Mobilization of depot fat*

During periods of negative energy balance, the triglycerides stored in the lipid droplets, are hydrolysed to FFA which are released into circulation. In the basal state or during TG synthesis, perilipin, the structural protein on lipid droplet, is bound to protein CGI-58 (comparative gene identification-58) which is a co-activator of the adipose triglyceride lipase (ATGL). In response to cAMP dependent PKA-mediated phosphorylation of perilipin, CGI-58 is released leading to activation of ATGL [68]. Activated ATGL moves to the lipid droplet membrane surface to hydrolyse TG to diacyl glycerol. Further activation of hormone sensitive lipase (HSL) by PKA mediated phosphorylation causes binding of HSL to perilipin and continues lipolysis forming monoacyl glycerol which is acted upon by another specific monoacyl glycerol lipase (MGL) forming glycerol and FFA which are released to plasma for systemic utilization.

Unlike its suppressive effect on adipogenesis in WAT, SIRT1 appears to promote mobilization and utilization of depot fat. Overexpression of SIRT1 in differentiated 3T3L1 cells resulted in decreased triglyceride levels and increased release of FFAs [23], while knockdown of SIRT1 decreased basal and stimulated lipolysis in adipocytes in culture.  Further, over expression of SIRT1 inhibited diet induced accumulation of fat [6, 31, 52]. SIRT1 regulates the expression of ATGL gene and thereby lipolysis in adipocytes through modulation of the acetylation and transcriptional activity of FOXO1 [70]. SIRT2 also appears to show similar effect on fat mobilization [58, 59].Although sirtuins influence mitochondrial oxidative metabolism in other metabolic tissues such as liver, their role in WAT mitochondrial metabolism is poorly understood.

#### **2.3. Role of sirtuins in BAT development and browning of WAT**

Unlike white adipogenesis, BAT biogenesis involves BMP7-stimulated commitment of Myf 5+ cells to brown pre-adipocytes that mature into mitochondria–rich brown adipocytes.BMP7 stimulation of progenitor cells leads to down regulation of early adipogenic inhibitors such as Pref1, Nectin, Wnt signaling molecules [43]. This is followed by upregulation of transcription factors such as PPARγ and C/EBPα which cause upregulation of expression of PRDM16, a key factor involved in adipocyte–myocyte switch through activation of expression of BAT specific genes (PGC1α, UCP-1, ZIC1) and downregulation of myogenic genes such as Myf5 and MyoD or myogenin [43]. This leads to increased biogenesis of mitochondria. PRDM16 is a 140 kDa protein that binds and activates the transcriptional function of PPARγ and PGC1α. PGC1α co-activates PPARγ-RXRα heterodimer stimulating the expression of BAT-specific genes UCP1and UCP3.

The ability of human white adipocytes to acquire brown fat-like phenotype, termed browning, in response to β-adrenergic stimulation, cold exposure [43] and by several molecules such as muscle derived irisin [71, 72] liver derived FGF21 [73] and small molecules such as β-aminoisobutyric acid [74] has been observed. These type of cells called beige or brite adiopcytes express genes involved in thermogenesis such as UCP1, deiodinase type II and PGC1α in response to stimulation of β3–adrenergic receptors [43].

Sirtuins also appear to play a role in the differentiation and function of BAT. Like WAT, brown adipose tissue also has been shown to express all the members of the mammalian SIRT family [11]. While the relative level of expression of SIRT3 and 5 are higher, that of SIRT1 and 7 are lower in BAT than those in WAT. Calorie restriction (CR) and cold exposure upregulated the expression of SIRT3 present in the mitochondria in BAT [75]; SIRT1 and SIRT2 also showed an upregulation under such conditions. Conversely, SIRT3 is down regulated in BAT in high fat diet induced obese mice. SIRT1 also appears to have a role in differentiation of pre-adipocyte to brown adipocytes. It appears that SIRT1 influences BAT differentiation through repression of the MyoD-mediated myogenic gene expression signature and stimulation of PGC-1α mediated mitochondrial gene expression [76]. SIRT1 appeared to improve glucose homeostasis in SIRT1 transgenic mice and brown adipocytes derived from them due to an enhanced response of brown adipocytes to β3-adrenergic stimuli rather than differences in differentiation status [77]. Of the different sirtuins, SIRT3 appeared to be critical in the differentiation of brown adipocytes. SIRT3 has been shown to activate PGC1α mediated thermogenic response in differentiating brown adipocytes.

Brown remodeling of white fat in response to cold exposure is shown to be regulated by SIRT1-dependent deacetylation of PPARγ. SIRT1-dependent deacetylation of Lys 268 and Lys 293 of PPARγ is required to recruit the co-activator PRDM16 to PPARγ, leading to upregulation of BAT-specific genes and repression of WAT genes [78].In response to different environmental stimuli, SIRT1 can differentially modulate PPARγ in WAT. SIRT1 inhibits PPARγ through local modulation of acetylation status of histones and recruitment of co-repressor NCoR in response to caloric restriction; but on cold exposure, it directly enhances PPARγ signaling through deacetylation of PPARγ itself [79]. SIRT1 deficiency in mice results in accumulation of lipid droplets and reduction of mitochondrial content in BAT indicating a role for SIRT1 in the white remodeling of BAT which appears to occur in obese conditions [80].

#### **2.4. Sirtuins and obesity**

leads to activation of malonyl CoA decarboxylase resulting in enhanced lipid oxidation. It therefore appears that SIRT4 exhibits a dual effect regulating anabolism and catabolism of fatty acids [65]. SIRT6 also appears to regulate lipogenesis. In SIRT6 overexpressing mice, expression of diacylglycerol acyl transferase a key enzyme involved in TG synthesis was down

During periods of negative energy balance, the triglycerides stored in the lipid droplets, are hydrolysed to FFA which are released into circulation. In the basal state or during TG synthesis, perilipin, the structural protein on lipid droplet, is bound to protein CGI-58 (comparative gene identification-58) which is a co-activator of the adipose triglyceride lipase (ATGL). In response to cAMP dependent PKA-mediated phosphorylation of perilipin, CGI-58 is released leading to activation of ATGL [68]. Activated ATGL moves to the lipid droplet membrane surface to hydrolyse TG to diacyl glycerol. Further activation of hormone sensitive lipase (HSL) by PKA mediated phosphorylation causes binding of HSL to perilipin and continues lipolysis forming monoacyl glycerol which is acted upon by another specific monoacyl glycerol lipase (MGL) forming glycerol and FFA which are released to plasma for

Unlike its suppressive effect on adipogenesis in WAT, SIRT1 appears to promote mobilization and utilization of depot fat. Overexpression of SIRT1 in differentiated 3T3L1 cells resulted in decreased triglyceride levels and increased release of FFAs [23], while knockdown of SIRT1 decreased basal and stimulated lipolysis in adipocytes in culture.  Further, over expression of SIRT1 inhibited diet induced accumulation of fat [6, 31, 52]. SIRT1 regulates the expression of ATGL gene and thereby lipolysis in adipocytes through modulation of the acetylation and transcriptional activity of FOXO1 [70]. SIRT2 also appears to show similar effect on fat mobilization [58, 59].Although sirtuins influence mitochondrial oxidative metabolism in other metabolic tissues such as liver, their role in WAT mitochondrial metabo-

Unlike white adipogenesis, BAT biogenesis involves BMP7-stimulated commitment of Myf

 cells to brown pre-adipocytes that mature into mitochondria–rich brown adipocytes.BMP7 stimulation of progenitor cells leads to down regulation of early adipogenic inhibitors such as Pref1, Nectin, Wnt signaling molecules [43]. This is followed by upregulation of transcription factors such as PPARγ and C/EBPα which cause upregulation of expression of PRDM16, a key factor involved in adipocyte–myocyte switch through activation of expression of BAT specific genes (PGC1α, UCP-1, ZIC1) and downregulation of myogenic genes such as Myf5 and MyoD or myogenin [43]. This leads to increased biogenesis of mitochondria. PRDM16 is a 140 kDa protein that binds and activates the transcriptional function of PPARγ and PGC1α. PGC1α co-activates PPARγ-RXRα heterodimer stimulating the expression of BAT-specific

regulated along with certain PPARγ responsive genes involved in lipogenesis [31, 67].

*2.2.3. Mobilization of depot fat*

38 Adipose Tissue

systemic utilization.

lism is poorly understood.

genes UCP1and UCP3.

5+

**2.3. Role of sirtuins in BAT development and browning of WAT**

Grossly elevated fat stores in adipose tissue with hypertrophic or hyperplastic adipocytes and concomitant development of blood vessels result in obesity. The relevance of sirtuins in adipose tissue development and metabolism and their effects on metabolism of glucose and lipids primarily in the liver, and insulin function suggest a possible link between sirtuins and obesity.

The level of expression and activity of SIRT1 decrease in adipose tissue in different obesity models. Expression of SIRT1 in adipose tissue of db/db leptin resistant obese mice and in mice fed on HFD was significantly low [53, 81]. Overexpression of SIRT1 in HFD-induced obese animals caused less inflammation and better glucose tolerance. SIRT1 expression in obese pigs is reported to be lesser than that in lean pigs [82]. Apart from decrease in SIRT1 levels, its function is also affected by changes in its post translational modification in obesity. One of the important post-translational modifications of SIRT1 which has been shown to be affected in obesity leading to inhibition of its nuclear localization is casein kinase mediated phosphorylation of ser-164 which is enhanced in obese and not in lean animals [83]. Unlike SIRT1 which is decreased in WAT in obesity, there is no consensus on the changes in other sirtuins in obese WAT; while some reports show decrease in SIRT2–6, other reports do not show any significant differences between obese and respective controls. But in obese BAT, SIRT1 and SIRT3 are down regulated and SIRT7 is upregulated. It has been shown that mir34a, which regulates the expression and activity of Sirt1, is elevated in obesity [41]. A possible association of sirtuins with obesity and obesity-associated pathological conditions in humans has also been indicated mostly from observational studies [84–88]. There is significant reduction in sirtuins in adipose tissue and other metabolic tissues in obese subjects and that weight loss or long term fasting can result in increase in their expression.

*2.4.2. Sirtuins and inflammation in adipose tissue*

for SIRT1 and 7 [107].

**3. Conclusions**

insulin resistance have been highlighted.

*2.4.3. Sirtuin activators for therapy*

Inflammation of adipose tissue in obesity is a major contributor to insulin resistance and pathogenesis of the metabolic syndrome [98]. SIRT1 could act as a transcriptional regulator of inflammation in multiple tissues, particularly adipose tissue as well as macrophages and endothelial cells [90, 99]. The levels of SIRT1 is inversely related to inflammation in adipose tissue. SIRT1 expression in human subcutaneous adipose tissue was less in cases where macrophage infiltration was high [100–102]. The decrease in SIRT1 in obese conditions in adipose tissue is suggested to be due to its proteosomal degradation. Activation of C-jun N terminal kinase (JNK1), which is a key component in inflammation associated signaling pathway, leads to phosphorylation of SIRT1, followed by its degradation in proteasomes [53, 103]. The molecular basis of the beneficial effect of SIRT1 on inflammation is related to suppression of NFkB activation [104]. SIRT1 inhibits transcriptional activity of NFkB directly by deacetylating the RelA/p65 subunit of NFkB at Lys 310 [105]. Moreover mir34a dependent decrease in SIRT1 activity can increase NFkB activity [41]. This suggests that SIRT1 and inflammatory signals interact at various levels and that SIRT1 is an important molecular link between nutrients, inflammation and metabolic dysfunction of the tissue. Though not much data on the role of other sirtuins in inflammation in human subjects is available, SIRT5 expression levels also correlate inversely with markers of inflammation [106] Deletion of SIRT7 in mice reduced WAT inflammatory gene expression in HFD induced obesity, suggesting opposing functions

Sirtuins in Adipose Tissue Metabolism http://dx.doi.org/10.5772/intechopen.75704 41

Since sirtuins play an important role in regulation of adipogenesis, and adipose tissue metabolism, pharmacological activation of sirtuins could be a useful approach for the treatment of obesity and related metabolic disorders. Sirt1 is an allosteric enzyme which is regulated by ligand binding. Resveratrol, which is a naturally occurring polyphenol with anti-oxidant property, increased the enzyme activity of SIRT1 by binding to its allosteric site [108]. High throughput screening has identified several small molecular activators of SIRT1 [109]. The most potent of these is SRT1720 which, protects against diet induced obesity [6]. The administration of SRT1720 reduced expression of lipogenic enzymes and reduced hepatic lipid accumulation, it also enhanced oxidative metabolism in skeletal muscle, liver and BAT in mice, protecting from HFD induced obesity and insulin resistance [110]. However, increasing sirtuin activity could result in indiscriminate deacetylation of histones and several other key proteins in different tissues. Sirtuin activating therapies would therefore have to be target specific.

Epigenetic modifications have emerged as fundamental modulators of metabolic functions, and sirtuins, a group of class III histone deacetylases, play a key role in this context. In this chapter the regulatory effects of sirtuins on adipose tissue metabolism in both WAT and BAT, and implication of alterations in their expression and activity in obesity, inflammation, and

#### *2.4.1. Sirtuins, insulin response and energy homeostasis*

Insulin resistance is a hallmark of obesity and a major factor contributing to obesity associated pathological conditions. *In vitro* and *in vivo* studies suggest that SIRT1 regulates insulin response. In insulin resistant cells where SIRT1 is down regulated, induction of SIRT1 expression increased insulin sensitivity [89]. SIRT1 regulated insulin-dependent glucose uptake in adipocytes. Increase in SIRT1 activity improved insulin sensitivity [90]. Adipose tissue-specific SIRT1 knockout mice were reported to be more prone to developing insulin resistance. In experimentally induced diabetic animals, overexpression of SIRT1 increased insulin sensitivity. Mechanistically, SIRT1 effect appears to involve transcriptional repression of protein tyrosine phosphatase 1B gene which is critical in insulin signaling [91] Along with SIRT1in WAT, SIRT3 and SIRT5 contribute to systemic glucose homeostasis. As indicated before, SIRT1 also regulates insulin secretion by β-cells of pancreas by repressing UCP-2 [64]. Inhibition of SIRT1 expression reduced insulin secretion in β-cell lines; conversely overexpression of SIRT1 increased it. *In vivo*, transgenic mice over expressing SIRT1 in pancreatic β-cells showed increase in glucose-stimulated insulin secretion [92]. Further, SIRT1 deficiency impaired insulin secretion apparently by disrupting glucose sensing and impairing response to fluctuations in glucose levels [93].

In addition to its effect on peripheral tissue metabolism, SIRT1 in hypothalamus appears to act as a key regulator of central control of energy homeostasis. Evidence in support of this include (a) increase in the expression and activity of SIRT1 in hypothalamus in both calorie restriction and fasting [19, 94] (b) inhibition of hypothalamic SIRT1 expression, specifically in anorexigenic POMC neurons, resulted in loss of response to leptin and reduced energy expenditure indicating requirement of SIRT1 in POMC neurons for homeostatic defense against diet- induced obesity [95] (c) deletion of SIRT1 expression specifically in orexigenic Agouti-related peptide (AgRP)-expressing neurons, which promotes feeding in response to fasting, decreased AgRP neuronal activity resulting in decreased food intake and body weight [96] (d) Central inhibition of SIRT1 in rodents on a high fat diet caused decreased body weight and increased energy expenditure. This is mediated through increased acetylated-FoxO1-mediated increased production of POMC and its active product αMSH which in turn augmented TRH and T3 levels suggesting a hypothalamic–pituitary-thyroid axis which stimulates energy expenditure [97].

#### *2.4.2. Sirtuins and inflammation in adipose tissue*

is reported to be lesser than that in lean pigs [82]. Apart from decrease in SIRT1 levels, its function is also affected by changes in its post translational modification in obesity. One of the important post-translational modifications of SIRT1 which has been shown to be affected in obesity leading to inhibition of its nuclear localization is casein kinase mediated phosphorylation of ser-164 which is enhanced in obese and not in lean animals [83]. Unlike SIRT1 which is decreased in WAT in obesity, there is no consensus on the changes in other sirtuins in obese WAT; while some reports show decrease in SIRT2–6, other reports do not show any significant differences between obese and respective controls. But in obese BAT, SIRT1 and SIRT3 are down regulated and SIRT7 is upregulated. It has been shown that mir34a, which regulates the expression and activity of Sirt1, is elevated in obesity [41]. A possible association of sirtuins with obesity and obesity-associated pathological conditions in humans has also been indicated mostly from observational studies [84–88]. There is significant reduction in sirtuins in adipose tissue and other metabolic tissues in obese subjects and that weight loss or long term fasting

Insulin resistance is a hallmark of obesity and a major factor contributing to obesity associated pathological conditions. *In vitro* and *in vivo* studies suggest that SIRT1 regulates insulin response. In insulin resistant cells where SIRT1 is down regulated, induction of SIRT1 expression increased insulin sensitivity [89]. SIRT1 regulated insulin-dependent glucose uptake in adipocytes. Increase in SIRT1 activity improved insulin sensitivity [90]. Adipose tissue-specific SIRT1 knockout mice were reported to be more prone to developing insulin resistance. In experimentally induced diabetic animals, overexpression of SIRT1 increased insulin sensitivity. Mechanistically, SIRT1 effect appears to involve transcriptional repression of protein tyrosine phosphatase 1B gene which is critical in insulin signaling [91] Along with SIRT1in WAT, SIRT3 and SIRT5 contribute to systemic glucose homeostasis. As indicated before, SIRT1 also regulates insulin secretion by β-cells of pancreas by repressing UCP-2 [64]. Inhibition of SIRT1 expression reduced insulin secretion in β-cell lines; conversely overexpression of SIRT1 increased it. *In vivo*, transgenic mice over expressing SIRT1 in pancreatic β-cells showed increase in glucose-stimulated insulin secretion [92]. Further, SIRT1 deficiency impaired insulin secretion apparently by disrupting glucose sensing and impairing response to fluctuations in glucose levels [93].

In addition to its effect on peripheral tissue metabolism, SIRT1 in hypothalamus appears to act as a key regulator of central control of energy homeostasis. Evidence in support of this include (a) increase in the expression and activity of SIRT1 in hypothalamus in both calorie restriction and fasting [19, 94] (b) inhibition of hypothalamic SIRT1 expression, specifically in anorexigenic POMC neurons, resulted in loss of response to leptin and reduced energy expenditure indicating requirement of SIRT1 in POMC neurons for homeostatic defense against diet- induced obesity [95] (c) deletion of SIRT1 expression specifically in orexigenic Agouti-related peptide (AgRP)-expressing neurons, which promotes feeding in response to fasting, decreased AgRP neuronal activity resulting in decreased food intake and body weight [96] (d) Central inhibition of SIRT1 in rodents on a high fat diet caused decreased body weight and increased energy expenditure. This is mediated through increased acetylated-FoxO1-mediated increased production of POMC and its active product αMSH which in turn augmented TRH and T3 levels suggesting a hypothalamic–pituitary-thyroid axis which stimulates energy expenditure [97].

can result in increase in their expression.

40 Adipose Tissue

*2.4.1. Sirtuins, insulin response and energy homeostasis*

Inflammation of adipose tissue in obesity is a major contributor to insulin resistance and pathogenesis of the metabolic syndrome [98]. SIRT1 could act as a transcriptional regulator of inflammation in multiple tissues, particularly adipose tissue as well as macrophages and endothelial cells [90, 99]. The levels of SIRT1 is inversely related to inflammation in adipose tissue. SIRT1 expression in human subcutaneous adipose tissue was less in cases where macrophage infiltration was high [100–102]. The decrease in SIRT1 in obese conditions in adipose tissue is suggested to be due to its proteosomal degradation. Activation of C-jun N terminal kinase (JNK1), which is a key component in inflammation associated signaling pathway, leads to phosphorylation of SIRT1, followed by its degradation in proteasomes [53, 103]. The molecular basis of the beneficial effect of SIRT1 on inflammation is related to suppression of NFkB activation [104]. SIRT1 inhibits transcriptional activity of NFkB directly by deacetylating the RelA/p65 subunit of NFkB at Lys 310 [105]. Moreover mir34a dependent decrease in SIRT1 activity can increase NFkB activity [41]. This suggests that SIRT1 and inflammatory signals interact at various levels and that SIRT1 is an important molecular link between nutrients, inflammation and metabolic dysfunction of the tissue. Though not much data on the role of other sirtuins in inflammation in human subjects is available, SIRT5 expression levels also correlate inversely with markers of inflammation [106] Deletion of SIRT7 in mice reduced WAT inflammatory gene expression in HFD induced obesity, suggesting opposing functions for SIRT1 and 7 [107].

#### *2.4.3. Sirtuin activators for therapy*

Since sirtuins play an important role in regulation of adipogenesis, and adipose tissue metabolism, pharmacological activation of sirtuins could be a useful approach for the treatment of obesity and related metabolic disorders. Sirt1 is an allosteric enzyme which is regulated by ligand binding. Resveratrol, which is a naturally occurring polyphenol with anti-oxidant property, increased the enzyme activity of SIRT1 by binding to its allosteric site [108]. High throughput screening has identified several small molecular activators of SIRT1 [109]. The most potent of these is SRT1720 which, protects against diet induced obesity [6]. The administration of SRT1720 reduced expression of lipogenic enzymes and reduced hepatic lipid accumulation, it also enhanced oxidative metabolism in skeletal muscle, liver and BAT in mice, protecting from HFD induced obesity and insulin resistance [110]. However, increasing sirtuin activity could result in indiscriminate deacetylation of histones and several other key proteins in different tissues. Sirtuin activating therapies would therefore have to be target specific.

#### **3. Conclusions**

Epigenetic modifications have emerged as fundamental modulators of metabolic functions, and sirtuins, a group of class III histone deacetylases, play a key role in this context. In this chapter the regulatory effects of sirtuins on adipose tissue metabolism in both WAT and BAT, and implication of alterations in their expression and activity in obesity, inflammation, and insulin resistance have been highlighted.

In spite of considerable advances in molecular biology of mammalian sirtuins, many questions remain unanswered. Among the different sirtuins, the role of SIRT1 in development and metabolism of white adipose tissue is reasonably well known. Although all the other sirtuins are expressed in WAT, their role in WAT function is not clear. Similarly, the role of SIRT1 and 3 in brown remodeling of white fat has been elucidated, but the regulatory effects of the other sirtuins are still unknown.

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Different sirtuins control similar cellular processes in adipose tissue. Unraveling the potential crosstalk and coordination between them will require further study. The significance of this in the possible gene regulatory network and coordinated action among sirtuins in metabolic regulation is evident from the antagonistic interaction between SIRT1 and 7 in adipose tissue metabolism.

Apart from functional differences between WAT and BAT, variations in the status of different depots of WAT are also related to a risk for obesity-associated diseases. It is not clear whether there is any depot dependent variation in sirtuin action.

As the expansion of a vascular tissue like adipose tissue is associated with neovascularization, adipogenesis and angiogenesis are interrelated. Understanding the role of sirtuins in adipose tissue angiogenesis is of paramount importance especially in brown tissue, where both the mitochondrial activity and oxygen demand are high.

Sirtuins appear to be an attractive target for the treatment of obesity and related metabolic disorders. Increasing sirt activity in adipose tissue by identifying natural compounds, or engineering small molecular activators is an area which needs intensive research; increasing intracellular levels of NAD+ , a substrate for sirtuins, is an alternate approach.

#### **Acknowledgements**

Fellowship received from ICMR, New Delhi to MS under TSS Fellowship scheme is gratefully acknowledged.

### **Conflict of interest**

The authors declare that there is no conflict of interest.

#### **Author details**

Manu Sudhakar<sup>1</sup> \*, Santhi Silambanan<sup>1</sup> and Ramya Ramakrishnan2

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Biochemistry, Sri Ramachandra Medical College and Research Institute, Chennai, India

2 Department of Surgery, Sri Ramachandra Medical College and Research Institute, Chennai, India

#### **References**

In spite of considerable advances in molecular biology of mammalian sirtuins, many questions remain unanswered. Among the different sirtuins, the role of SIRT1 in development and metabolism of white adipose tissue is reasonably well known. Although all the other sirtuins are expressed in WAT, their role in WAT function is not clear. Similarly, the role of SIRT1 and 3 in brown remodeling of white fat has been elucidated, but the regulatory effects of the other sirtuins are still unknown. Different sirtuins control similar cellular processes in adipose tissue. Unraveling the potential crosstalk and coordination between them will require further study. The significance of this in the possible gene regulatory network and coordinated action among sirtuins in metabolic regulation is evident from the antagonistic interaction between SIRT1 and 7 in adipose tissue metabolism. Apart from functional differences between WAT and BAT, variations in the status of different depots of WAT are also related to a risk for obesity-associated diseases. It is not clear whether

As the expansion of a vascular tissue like adipose tissue is associated with neovascularization, adipogenesis and angiogenesis are interrelated. Understanding the role of sirtuins in adipose tissue angiogenesis is of paramount importance especially in brown tissue, where both the

Sirtuins appear to be an attractive target for the treatment of obesity and related metabolic disorders. Increasing sirt activity in adipose tissue by identifying natural compounds, or engineering small molecular activators is an area which needs intensive research; increasing intra-

, a substrate for sirtuins, is an alternate approach.

Fellowship received from ICMR, New Delhi to MS under TSS Fellowship scheme is gratefully

and Ramya Ramakrishnan2

1 Department of Biochemistry, Sri Ramachandra Medical College and Research Institute,

2 Department of Surgery, Sri Ramachandra Medical College and Research Institute,

there is any depot dependent variation in sirtuin action.

mitochondrial activity and oxygen demand are high.

The authors declare that there is no conflict of interest.

\*, Santhi Silambanan<sup>1</sup>

\*Address all correspondence to<EMAIL_ADDRESS>
cellular levels of NAD+

**Acknowledgements**

**Conflict of interest**

**Author details**

Manu Sudhakar<sup>1</sup>

Chennai, India

Chennai, India

acknowledged.

42 Adipose Tissue

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1363-1374

**Chapter 4**

**Provisional chapter**

**Browning of Adipose Tissue and Sirtuin1 Involvement**

Obesity is an important risk factor for many diseases, including cardiovascular diseases, metabolic syndrome and cancers. Excessive dietary intake of caloric food results in its accumulation in white adipose tissue (WAT), whereas energy expenditure by fat utilization and oxidation predominately occurs in brown adipose tissue (BAT). Reducing obesity has become an important prevention strategy of research interest, focusing in the recent years, mainly on browning of WAT, the process during which the enhance of the mitochondria biogenesis occurs and then white adipocytes are converted to metabolically active beige adipocytes. Sirtuin1 (SIRT1), the most known isoform of sirtuin deacetylases, is implied in the browning of WAT process. In fact, it is a sensitive sensor of cell energy metabolism and, together with other sirtuin isoforms, contributes to this differentiation process. This chapter provides an overview about SIRT1 involvement in

Lipids are stored in the body by two types of adipose tissue such as white adipose tissue (WAT) and brown adipose tissue (BAT) [1–3]; the main parenchymal cells of the adipose tissue are adipocytes, and so far two fat cell types, white and brown cells, have been reported, respectively [4–6]. The white and brown adipocytes arise from separate progenitor cell lines, present distinct structure, morphology, localization and functions [1, 5, 7] and these differences contribute to the maintenance and modulation of energy and of metabolic health [4, 8, 9].

browning of WAT as a target molecule that can thereby contrast obesity.

**Keywords:** adipose tissue, browning, obesity, resveratrol, sirtuin1

**Browning of Adipose Tissue and Sirtuin1 Involvement**

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.74760

Gaia Favero, Kristína Krajčíková,

Gaia Favero, Kristína Krajčíková,

http://dx.doi.org/10.5772/intechopen.74760

**Abstract**

**1. Introduction**

Francesca Bonomini, Luigi Fabrizio Rodella, Vladimíra Tomečková and Rita Rezzani

Francesca Bonomini, Luigi Fabrizio Rodella, Vladimíra Tomečková and Rita Rezzani

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

#### **Browning of Adipose Tissue and Sirtuin1 Involvement Browning of Adipose Tissue and Sirtuin1 Involvement**

DOI: 10.5772/intechopen.74760

Gaia Favero, Kristína Krajčíková, Francesca Bonomini, Luigi Fabrizio Rodella, Vladimíra Tomečková and Rita Rezzani Gaia Favero, Kristína Krajčíková, Francesca Bonomini, Luigi Fabrizio Rodella, Vladimíra Tomečková and Rita Rezzani

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74760

#### **Abstract**

Obesity is an important risk factor for many diseases, including cardiovascular diseases, metabolic syndrome and cancers. Excessive dietary intake of caloric food results in its accumulation in white adipose tissue (WAT), whereas energy expenditure by fat utilization and oxidation predominately occurs in brown adipose tissue (BAT). Reducing obesity has become an important prevention strategy of research interest, focusing in the recent years, mainly on browning of WAT, the process during which the enhance of the mitochondria biogenesis occurs and then white adipocytes are converted to metabolically active beige adipocytes. Sirtuin1 (SIRT1), the most known isoform of sirtuin deacetylases, is implied in the browning of WAT process. In fact, it is a sensitive sensor of cell energy metabolism and, together with other sirtuin isoforms, contributes to this differentiation process. This chapter provides an overview about SIRT1 involvement in browning of WAT as a target molecule that can thereby contrast obesity.

**Keywords:** adipose tissue, browning, obesity, resveratrol, sirtuin1

#### **1. Introduction**

Lipids are stored in the body by two types of adipose tissue such as white adipose tissue (WAT) and brown adipose tissue (BAT) [1–3]; the main parenchymal cells of the adipose tissue are adipocytes, and so far two fat cell types, white and brown cells, have been reported, respectively [4–6]. The white and brown adipocytes arise from separate progenitor cell lines, present distinct structure, morphology, localization and functions [1, 5, 7] and these differences contribute to the maintenance and modulation of energy and of metabolic health [4, 8, 9].

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Despite these differences, both types of adipocytes share the activity of accumulation and release of fatty acids and both express the rather specific adrenergic receptor β3 [10, 11].

**3. Brown adipose tissue**

Since the 1970s, BAT has been increasingly recognized as the main site of nonshivering thermogenesis in mammals [35, 38] and it is probably the outcome of a single evolutionary development, in fact, unlike WAT, BAT is only found in mammals [35]. BAT adipocytes are smaller (15–60 μm) than white adipocytes [39] and present a characteristic brown color due to its high content of mitochondria and a lobulated surface that is innervated and very well vascularized [6, 8, 30]. BAT has the physiological role of metabolizing fatty acids in order to produce heat [4, 26], that is why it is often referred to as "good" fat, since it helps burn, not store, calories. This specific role of BAT is supported by the high content in its mitochondria of uncoupling protein1 (UCP1), uniquely expressed in these cells [4, 8, 26, 40]. The activation of UCP1 and transcriptional induction of the genes encoding UCP1 induce uptake of lipids and glucose

Browning of Adipose Tissue and Sirtuin1 Involvement http://dx.doi.org/10.5772/intechopen.74760 53

Functional BAT is more common in women than men and its mass and activity are reduced in overweight, obese and aging people [4, 41, 42].  Browning of WAT is an adaptive and reversible response to numerous stimuli, including noradrenaline stimulation by cold exposure, exercise, natriuretic peptides, thyroid hormones, bile acids and nutritional compounds (resveratrol, menthol, capsaicin, etc.) [46, 47]. Other stimuli are due to pharmacological molecules, such as β3-adrenergic agonists, peroxisome proliferator-activated receptor γ (PPARγ) agonists thiazolidinediones and cannabinoid antagonist (rimonabant) [30, 48]. As a result of these stimuli, the transcriptional machinery of the browning program activates the expression of characteristic thermogenic genes (such as

Two theories exist about the origin of beige adipocytes: (1) *de novo* differentiation from resident progenitor cells and (2) transdifferentiation. In detail, beige adipocytes can originate from progenitors resident within WAT that are differentiated in response to browning stimuli [39, 49–51] or, alternatively, they can arise via transdifferentiation, a process that involves the direct conversion of existing white adipocytes into brown-like fat cells [50]. This capacity of transdifferentiation is highly dependent on environment stimuli and also on the physiopa-

Beige adipocytes have a predominant lipid vacuole in the cytoplasm and numerous mitochondria, so exhibiting several intermediate features between white and brown adipocytes [33, 36, 47] (**Figure 1**), but these cells expressed characteristic and distinct gene markers that distinguished them from both white and brown fat cells [30, 39, 53, 54]. These genes encode proteins with very distinct cellular functions, including transcription factors (Zic1, Tbx15, etc.), metabolism-related proteins (Slc27a1, etc.) and proteins associated with inflammatory pathways

from the circulation to sustain oxidation and thermogenesis [35, 41].

to be a good tool to counteract obesity and its related diseases.

UCP1), leading to a beige adipocyte phenotype [30, 45, 47].

thology aging [30, 52].

**4. Browning of white adipose tissue**

The adipose tissue is highly dynamic in the sense that changes in mass of an order of magnitude can occur, this happen during physiological (like pregnancy/lactation or aging) and pathological states (like metabolic syndrome, obesity, etc.) [12, 13]. In physiological condition, functional WAT and BAT adipose tissues control and modulate the energy balance with important effects on metabolic health and longevity [6, 14]. Aging is typically associated with a body redistribution of adipose tissue (increased central, visceral and ectopic adiposity) [4, 6, 15, 16] and chronic low-grade inflammation [17, 18]. Aging is also related to an increase of lipotoxicity due to the reduced capacity of adipose depots to store free fatty acids [16, 19]. These conditions contribute to increased risk for metabolic perturbations such as insulin resistance, impaired glucose tolerance and diabetes [18, 20]. Of importance, key processes of adipose tissue physiology affect molecular pathways that regulate lifespan [21], such as sirtuins (SIRTs) levels decline with age in several tissues, including adipose tissue, and this reduction induces adipocyte dysfunctions leading to obesity [6, 22].

To date, in both advanced and developing countries, obesity has become a major health problem [7, 12, 23], mainly because it carries an increased risk of death for the associated disorders [24, 25]. In fact, excess WAT throughout the body is associated with an increased risk of cardiovascular diseases, breast, colon, oesophageal, gall bladder and pancreatic cancers, sleep apnea and physical disabilities, such as knee arthritis [26–28].

In the following sections, we present firstly the main differences between WAT and BAT and the WAT browning process and then we discuss the potential SIRT1 involvement in this process as a target molecule that can thereby contrast obesity, introducing also the effects of resveratrol as a SIRT1 exogenous inducer.

#### **2. White adipose tissue**

WAT is now known to be highly dynamic, synthesizing and secreting multiple lipids, proteins and autocrine, endocrine, paracrine and neuroendocrine factors which are involved in the regulation of a wide range of physiological and metabolic processes [4, 29]. In particular, white adipocytes, characterized by a large unilocular lipid droplet, have a low density of mitochondria and variable size (25–200 μm) [6, 7, 12, 29, 30] and they might secrete adipokines that are pro- and anti-inflammatory cytokines fundamental in the regulation of metabolic functions and also in the communication from adipose tissue to other tissues [7, 12, 31]. Other lipid molecules are also secreted by WAT, including prostanoids, cholesterol and retinol, which are stored in order to be subsequently released [4, 32].

The development of subcutaneous WAT begins in uterus, but primarily occurs after birth, when specialized fat storage cells are needed to provide fuel during fasting periods. Hyperplastic and hypertrophic white adipocyte processes occur during WAT development, throughout the organism's lifespan and in conditions of positive energy balance [33–35]. If the caloric excess is not reconciled by increased energy utilization, cellular hypertrophy and hyperplasia occur and then lead to adipocytes dysfunctions and so obesity [36, 37].

#### **3. Brown adipose tissue**

Despite these differences, both types of adipocytes share the activity of accumulation and release of fatty acids and both express the rather specific adrenergic receptor β3 [10, 11].

The adipose tissue is highly dynamic in the sense that changes in mass of an order of magnitude can occur, this happen during physiological (like pregnancy/lactation or aging) and pathological states (like metabolic syndrome, obesity, etc.) [12, 13]. In physiological condition, functional WAT and BAT adipose tissues control and modulate the energy balance with important effects on metabolic health and longevity [6, 14]. Aging is typically associated with a body redistribution of adipose tissue (increased central, visceral and ectopic adiposity) [4, 6, 15, 16] and chronic low-grade inflammation [17, 18]. Aging is also related to an increase of lipotoxicity due to the reduced capacity of adipose depots to store free fatty acids [16, 19]. These conditions contribute to increased risk for metabolic perturbations such as insulin resistance, impaired glucose tolerance and diabetes [18, 20]. Of importance, key processes of adipose tissue physiology affect molecular pathways that regulate lifespan [21], such as sirtuins (SIRTs) levels decline with age in several tissues, including adipose tissue, and this reduction

To date, in both advanced and developing countries, obesity has become a major health problem [7, 12, 23], mainly because it carries an increased risk of death for the associated disorders [24, 25]. In fact, excess WAT throughout the body is associated with an increased risk of cardiovascular diseases, breast, colon, oesophageal, gall bladder and pancreatic cancers, sleep

In the following sections, we present firstly the main differences between WAT and BAT and the WAT browning process and then we discuss the potential SIRT1 involvement in this process as a target molecule that can thereby contrast obesity, introducing also the effects of

WAT is now known to be highly dynamic, synthesizing and secreting multiple lipids, proteins and autocrine, endocrine, paracrine and neuroendocrine factors which are involved in the regulation of a wide range of physiological and metabolic processes [4, 29]. In particular, white adipocytes, characterized by a large unilocular lipid droplet, have a low density of mitochondria and variable size (25–200 μm) [6, 7, 12, 29, 30] and they might secrete adipokines that are pro- and anti-inflammatory cytokines fundamental in the regulation of metabolic functions and also in the communication from adipose tissue to other tissues [7, 12, 31]. Other lipid molecules are also secreted by WAT, including prostanoids, cholesterol and

The development of subcutaneous WAT begins in uterus, but primarily occurs after birth, when specialized fat storage cells are needed to provide fuel during fasting periods. Hyperplastic and hypertrophic white adipocyte processes occur during WAT development, throughout the organism's lifespan and in conditions of positive energy balance [33–35]. If the caloric excess is not reconciled by increased energy utilization, cellular hypertrophy and hyperplasia occur

induces adipocyte dysfunctions leading to obesity [6, 22].

apnea and physical disabilities, such as knee arthritis [26–28].

retinol, which are stored in order to be subsequently released [4, 32].

and then lead to adipocytes dysfunctions and so obesity [36, 37].

resveratrol as a SIRT1 exogenous inducer.

**2. White adipose tissue**

52 Adipose Tissue

Since the 1970s, BAT has been increasingly recognized as the main site of nonshivering thermogenesis in mammals [35, 38] and it is probably the outcome of a single evolutionary development, in fact, unlike WAT, BAT is only found in mammals [35]. BAT adipocytes are smaller (15–60 μm) than white adipocytes [39] and present a characteristic brown color due to its high content of mitochondria and a lobulated surface that is innervated and very well vascularized [6, 8, 30]. BAT has the physiological role of metabolizing fatty acids in order to produce heat [4, 26], that is why it is often referred to as "good" fat, since it helps burn, not store, calories. This specific role of BAT is supported by the high content in its mitochondria of uncoupling protein1 (UCP1), uniquely expressed in these cells [4, 8, 26, 40]. The activation of UCP1 and transcriptional induction of the genes encoding UCP1 induce uptake of lipids and glucose from the circulation to sustain oxidation and thermogenesis [35, 41].

Functional BAT is more common in women than men and its mass and activity are reduced in overweight, obese and aging people [4, 41, 42]. Thus recruitment and activation of BAT seem to be a good tool to counteract obesity and its related diseases.

#### **4.  Browning of WAT is an adaptive and reversible response to numerous stimuli, including noradrenaline stimulation by cold exposure, exercise, natriuretic peptides, thyroid hormones, bile acids and nutritional compounds (resveratrol, menthol, capsaicin, etc.) [46, 47]. Other stimuli are due to pharmacological molecules, such as β3-adrenergic agonists, peroxisome proliferator-activated receptor γ (PPARγ) agonists thiazolidinediones and cannabinoid antagonist (rimonabant) [30, 48]. As a result of these stimuli, the transcriptional machinery of the browning program activates the expression of characteristic thermogenic genes (such as UCP1), leading to a beige adipocyte phenotype [30, 45, 47].

Two theories exist about the origin of beige adipocytes: (1) *de novo* differentiation from resident progenitor cells and (2) transdifferentiation. In detail, beige adipocytes can originate from progenitors resident within WAT that are differentiated in response to browning stimuli [39, 49–51] or, alternatively, they can arise via transdifferentiation, a process that involves the direct conversion of existing white adipocytes into brown-like fat cells [50]. This capacity of transdifferentiation is highly dependent on environment stimuli and also on the physiopathology aging [30, 52].

Beige adipocytes have a predominant lipid vacuole in the cytoplasm and numerous mitochondria, so exhibiting several intermediate features between white and brown adipocytes [33, 36, 47] (**Figure 1**), but these cells expressed characteristic and distinct gene markers that distinguished them from both white and brown fat cells [30, 39, 53, 54]. These genes encode proteins with very distinct cellular functions, including transcription factors (Zic1, Tbx15, etc.), metabolism-related proteins (Slc27a1, etc.) and proteins associated with inflammatory pathways

localization (mitochondrion, cytoplasm or nucleus) and tissue distribution [81, 83–88] (**Table 1**).

deacetylase activities [89, 90]; however, some particular isoforms, like SIRT4 or SIRT6, also hold ADP-ribosyl transfer activity [91, 92]. SIRT isoforms share a conserved core catalytic

differences are manifested at N- and C-terminal regions, which are variable among homologs

So far, more than 30 SIRTs substrates were identified and, in general, are divided into two groups: histones and non-histone substrates and deacetylation of both is a quick response to stress stimuli or activators [95, 96]. SIRTs also serve as the regulators of transcription of genes

Among the SIRT isoforms, the most attention has been focused on SIRT1, the ortholog of yeast Sir2 [64, 98]. During aging, its levels decrease [99] and this reduction occurs also during age-associated diseases (like neurodegenerative pathologies, cardiovascular diseases, metabolic syndrome, etc.) [100–102] making SIRT1 a possible treatment target. In fact, the beneficial effects of SIRT1 activation have been shown on numerous animal models [103–106] as well as humans [107–109]. Additionally, the positive effects of SIRT1 activation include also browning of WAT [110–112].

All seven SIRT isoforms are expressed in adipose tissue [113–118], where they hydrolyse acetyl- and/or other acyl-group from the lysine residue of target substrate [119–121]. After particular stimuli, as summarized in **Figure 2**, the activation of SIRT1 at WAT level occurs and leads to the modulation (deacetylation) of PPARγ [110, 122], that with PRDM16 and PGC-1α,

Furthermore, PPARγ induces binding of C/EBPα and carboxy-terminal binding proteins 1 and 2 (CtBP 1 and 2) and represses transcription of genes which are specific of WAT [61, 124]. Thus, through PPARγ, SIRT1 is involved not only in enhancement of transcription of BAT genes, but also in repression of WAT genes. Furthermore, SIRT1 promotes mito-

SIRT4 Mitochondria, cytoplasm ADP-ribosyltransferase, lipoamidase, deacetylase

deacylase

Deacetylase, ADP-ribozyltransferase, long-chain fatty acids

SIRT5 Mitochondria, cytoplasm, nucleus Malonyl-, sukcinyl-, glutaryl-deacylase

55

Browning of Adipose Tissue and Sirtuin1 Involvement http://dx.doi.org/10.5772/intechopen.74760

Biochemically, SIRTs are a class of proteins that posses mainly NAD+

and enable them to possess more than one type of catalytic activity [94].

**5.1. Sirtuin1 involvement in browning of white adipose tissue**

promote transcription of genes specific of BAT [61, 123].

**Enzyme Localization Activity** SIRT1 Nucleus, cytoplasm Deacetylase SIRT2 Nucleus, cytoplasm Deacetylase SIRT3 Mitochondria, nucleus, Cytoplasm Deacetylase

SIRT7 Nucleus (nucleolus), cytoplasm Deacetylase

**Table 1.** Localization and catalytic activity of mammalian sirtuin isoforms.

SIRT6 Nucleus, endoplasmatic reticulum,

cytoplasm

in complex with other transcription factors [97].

domain, a NAD+

**Figure 1.** Main morphological characteristics of white, beige and brown adipocytes. UCP1: uncoupling protein 1.

(CD40, CD137, etc.) [35, 53–55]. It has been proved that more than 50 transcriptional molecules have been identified and their action mechanisms have been defined necessary in the browning transcriptional process [35, 56]. Among these, it is important to mention: PPARγ [50, 57], several members of the bone morphogenic protein family (BMP) [35, 58, 59], peroxisome proliferatoractivated receptor gamma coactivator-1 alpha (PGC-1α), which is involved in mitochondrial biogenesis [30, 33, 35] and also some transcription factors, such as C/EBPα and PRDM16 [58, 60, 61]. Even though the current terminology stresses the differences in cell lineage and localization, the evidence suggests that the beige adipocytes function as true thermogenic brown adipocytes [35, 47]. However, there is not a precise bioenergetic analysis of beige fat cells.

To date, the metabolic benefits of browning of WAT in humans remain to be fully established and the safety is a concern that must first be addressed regarding any method used to induce WAT browning. Cold exposure is a classic and efficient way to induce browning [62, 63], but its obvious discomfort, together with risks of hypothermia, makes it almost impractical for clinical use [50]. Therefore, browning agents, either endogenous or exogenous, provide an attractive alternative for limiting metabolic diseases.

Following paragraphs describe the potential SIRT1 involvement as a target molecule that can thereby contrast obesity, introducing briefly the effects of resveratrol as a SIRT1 inducer.

#### **5. Sirtuins**

SIRTs are NAD+ -dependent deacetylases present in all prokaryotic and eukaryotic cells (with the exception of several red *algae* and *archaea*) [64, 65]. Mammals possess seven isoforms, from 1 to 7, mainly known as anti-aging molecules [66–69]; however they are involved also in the regulation of numerous other cellular processes, such as integrity of chromatin [70, 71], cell cycle [72, 73], apoptosis [74, 75], energy metabolism [76–78], inflammation [79, 80] or detoxification [81, 82]. Their expression occurs throughout whole body differing in their cellular localization (mitochondrion, cytoplasm or nucleus) and tissue distribution [81, 83–88] (**Table 1**). Biochemically, SIRTs are a class of proteins that posses mainly NAD+ -dependent lysine deacetylase activities [89, 90]; however, some particular isoforms, like SIRT4 or SIRT6, also hold ADP-ribosyl transfer activity [91, 92]. SIRT isoforms share a conserved core catalytic domain, a NAD+ -binding place, consists of two subdomains [93]. SIRT isoforms' structural differences are manifested at N- and C-terminal regions, which are variable among homologs and enable them to possess more than one type of catalytic activity [94].

So far, more than 30 SIRTs substrates were identified and, in general, are divided into two groups: histones and non-histone substrates and deacetylation of both is a quick response to stress stimuli or activators [95, 96]. SIRTs also serve as the regulators of transcription of genes in complex with other transcription factors [97].

Among the SIRT isoforms, the most attention has been focused on SIRT1, the ortholog of yeast Sir2 [64, 98]. During aging, its levels decrease [99] and this reduction occurs also during age-associated diseases (like neurodegenerative pathologies, cardiovascular diseases, metabolic syndrome, etc.) [100–102] making SIRT1 a possible treatment target. In fact, the beneficial effects of SIRT1 activation have been shown on numerous animal models [103–106] as well as humans [107–109]. Additionally, the positive effects of SIRT1 activation include also browning of WAT [110–112].

#### **5.1. Sirtuin1 involvement in browning of white adipose tissue**

(CD40, CD137, etc.) [35, 53–55]. It has been proved that more than 50 transcriptional molecules have been identified and their action mechanisms have been defined necessary in the browning transcriptional process [35, 56]. Among these, it is important to mention: PPARγ [50, 57], several members of the bone morphogenic protein family (BMP) [35, 58, 59], peroxisome proliferatoractivated receptor gamma coactivator-1 alpha (PGC-1α), which is involved in mitochondrial biogenesis [30, 33, 35] and also some transcription factors, such as C/EBPα and PRDM16 [58, 60, 61]. Even though the current terminology stresses the differences in cell lineage and localization, the evidence suggests that the beige adipocytes function as true thermogenic brown adipocytes [35, 47]. However, there is not a precise bioenergetic analysis of beige fat cells.

**Figure 1.** Main morphological characteristics of white, beige and brown adipocytes. UCP1: uncoupling protein 1.

To date, the metabolic benefits of browning of WAT in humans remain to be fully established and the safety is a concern that must first be addressed regarding any method used to induce WAT browning. Cold exposure is a classic and efficient way to induce browning [62, 63], but its obvious discomfort, together with risks of hypothermia, makes it almost impractical for clinical use [50]. Therefore, browning agents, either endogenous or exogenous, provide an

Following paragraphs describe the potential SIRT1 involvement as a target molecule that can thereby contrast obesity, introducing briefly the effects of resveratrol as a SIRT1 inducer.

the exception of several red *algae* and *archaea*) [64, 65]. Mammals possess seven isoforms, from 1 to 7, mainly known as anti-aging molecules [66–69]; however they are involved also in the regulation of numerous other cellular processes, such as integrity of chromatin [70, 71], cell cycle [72, 73], apoptosis [74, 75], energy metabolism [76–78], inflammation [79, 80] or detoxification [81, 82]. Their expression occurs throughout whole body differing in their cellular

attractive alternative for limiting metabolic diseases.

**5. Sirtuins**

54 Adipose Tissue

SIRTs are NAD+