Physiology of Adipose Tissue

Chapter 2

Abstract

Alina Kurylowicz

related complications.

1. Introduction

therapies [1].

13

Role of Sirtuins in Adipose Tissue

Sirtuins (silent information regulators, sirts) via modification of histones, as well

genes, particularly those involved in the organism response to stress. Detection of sirtuin expression in adipocytes initiated interest in their role in adipose tissue development and metabolism. This chapter presents how sirtuins control the critical steps of preadipocytes' differentiation and proliferation, as well as the process of adipose tissue browning. Moreover, it shows in vitro and in vivo data proving that sirtuins are involved in the regulation of lipogenesis, lipolysis, and secretory activity of adipose tissue. Due to all these reasons, sirtuins may constitute potential targets in the treatment of obesity and

Keywords: sirtuins, adipocytes, adipogenesis, lipid metabolism, adipokines

Recent research widened our understanding of the role of adipose tissue from the simple energy storage to the metabolically and hormonally active organ that in response to environmental stimuli is able not only to activate lipolysis/ lipogenesis but also to secrete several factors to communicate with and regulate the function of other organs. These findings allowed to understand the link between excess adiposity and the development of obesity-related complications and renewed interest in adipose tissue as a possible target for obesity-orientated

However, despite the constant progress in understanding its pathogenesis, the therapeutic potential to prevent and combat obesity is limited. Behavioral interventions, calorie restriction (CR) combined with the increased physical activity, do not assure persistent, long-term effects, while available pharmacological treatments allow for loss of 5–10% of initial weight. Therefore, there is a need for novel

Studies on the influence of CR on the whole body function allow to identify sirtuins (silent information regulators, sirts)—essential players in different cellular metabolic pathways that seem to be crucial for the proper function of adipose tissue and in this way may constitute attractive therapeutic targets in the treatment of

methods of treatment of obesity and its complications.

obesity and related complications.

Development and Metabolism

as transcription factors and co-regulators, control expression of other

#### Chapter 2

## Role of Sirtuins in Adipose Tissue Development and Metabolism

Alina Kurylowicz

#### Abstract

Sirtuins (silent information regulators, sirts) via modification of histones, as well as transcription factors and co-regulators, control expression of other genes, particularly those involved in the organism response to stress. Detection of sirtuin expression in adipocytes initiated interest in their role in adipose tissue development and metabolism. This chapter presents how sirtuins control the critical steps of preadipocytes' differentiation and proliferation, as well as the process of adipose tissue browning. Moreover, it shows in vitro and in vivo data proving that sirtuins are involved in the regulation of lipogenesis, lipolysis, and secretory activity of adipose tissue. Due to all these reasons, sirtuins may constitute potential targets in the treatment of obesity and related complications.

Keywords: sirtuins, adipocytes, adipogenesis, lipid metabolism, adipokines

#### 1. Introduction

Recent research widened our understanding of the role of adipose tissue from the simple energy storage to the metabolically and hormonally active organ that in response to environmental stimuli is able not only to activate lipolysis/ lipogenesis but also to secrete several factors to communicate with and regulate the function of other organs. These findings allowed to understand the link between excess adiposity and the development of obesity-related complications and renewed interest in adipose tissue as a possible target for obesity-orientated therapies [1].

However, despite the constant progress in understanding its pathogenesis, the therapeutic potential to prevent and combat obesity is limited. Behavioral interventions, calorie restriction (CR) combined with the increased physical activity, do not assure persistent, long-term effects, while available pharmacological treatments allow for loss of 5–10% of initial weight. Therefore, there is a need for novel methods of treatment of obesity and its complications.

Studies on the influence of CR on the whole body function allow to identify sirtuins (silent information regulators, sirts)—essential players in different cellular metabolic pathways that seem to be crucial for the proper function of adipose tissue and in this way may constitute attractive therapeutic targets in the treatment of obesity and related complications.

#### 2. A short review of the sirt system

The sirts are highly conserved regulatory proteins present almost in all species. Initially, they have been identified as class III histone deacetylases, nicotinamide adenine dinucleotide (NAD)-dependent enzymes responsible for the removal of acetyl groups from lysine residues in proteins, while some members of this family act also as mono-ADP-ribosyltransferases. Since acetylation and deacetylation are essential mechanisms of posttranslational modifications of proteins determining their activity, sirts were found to be involved in the regulation of distinct cellular pathways including, among others, those related to cell survival, apoptosis, inflammatory and stress responses, as well as lipid and glucose homeostases [2].

3.1 Types of adipocytes

during the process of β-oxidation.

DOI: http://dx.doi.org/10.5772/intechopen.88467

Role of Sirtuins in Adipose Tissue Development and Metabolism

of obesity and related metabolic disorders [13].

perspective in the treatment of excess adiposity [18].

ever, such compounds have not been developed yet.

adipocyte dysfunction [20].

15

3.2 sirts and preadipocyte differentiation

In mammals, there are two main types of adipose tissue that differ in their structure, physiology, and function. White adipose tissue (WAT) acts mainly as energy storage that releases FA for the production of adenosine triphosphate (ATP)

Small mammals and human newborns, apart from white adipocytes, possess large deposits of brown adipose tissue (BAT) responsible for the non-shivering (adaptive) thermogenesis which is for them the most important regulatory mechanism for maintaining body temperature. The energy produced due to the oxidation of lipolysis-derived FA in the BAT mitochondria is released as heat, mostly thanks to uncoupling proteins (UCP). Age progression in humans was believed to be associated with complete atrophy of BAT; however, novel methods of imaging led to the identification of BAT stores in several areas of the adult human body, as well as of cells reminding brown adipocytes dispersed within WAT also known as beige/brite adipocytes (BeAT). These cells share common morphological features of white and brown adipocytes, and their number may increase upon different stimuli (e.g., cold, exercise, thyroid hormones, resveratrol). There are two theories regarding BeAT origin: they (i) differentiate from the progenitor cells resident in WAT or (ii) arise due to the transdifferentiation of white adipocytes. Given the role of adaptive thermogenesis in the whole body energy expenditure, stimulation of white adipocytes browning seems to be an attractive therapeutic pathway in the treatment

Peroxisome proliferator-activated receptor γ (PPARγ) is considered to be the

Another sirtuin family member—sirt2—has also shown an inhibitory effect on adipocyte differentiation [14]. In this process, sirt2 deacetylates forkhead box O1 (FOXO1) transcription factor and subsequently represses PPARγ transcriptional activity [19]. Therefore, sirt2 overexpression inhibits adipogenesis, while its silencing has an opposite effect in 3 T3-L1 preadipocytes. Moreover, this inhibitory influence of sirt2 on adipocyte differentiation discloses under CR that indicates the role of this sirtuin in the maintenance of energy homeostasis and suggests that sirt2 activators could provide novel therapeutics of obesity and its complications; how-

sirt3 is essential for the activation of bioenergetic function of mitochondria at the early stage of adipocyte differentiation. Silencing of sirt3 decreases the protein

downregulates the expression of several antioxidant enzymes and increases oxidative stress in MSCs after adipogenic induction. In this way, sirt3 depletion diminishes the ability of MSCs to undergo adipogenic differentiation and leads to

level of forkhead box O3a (FoxO3a) transcription factor and subsequently

main transcription factor responsible for promoting adipogenesis. sirt1, by interacting with two PPARγ corepressors, nuclear receptor corepressor (N-CoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT), can attenuate adipogenesis [14]. Consistently, overexpression of ectopic sirt1 blocks adipogenesis in 3T3-L1 cells, a culture of mouse adipocytes used as a model of adipocyte differentiation [15, 16]. Additionally, via activation of the Wnt signaling pathway, sirt1 determinates mesenchymal stem cells (MSC) differentiation toward myogenic cells, while its inhibition in MSC promotes adipogenesis [17]. MicroRNA 146b (miR-146b) acts as a negative regulator of sirt1 during adipocyte differentiation, giving a hope that interference with this miRNA may constitute a therapeutic

In human, seven sirt genes (sirts) have been identified that encode seven sirt enzymes of different structure, cellular localization, and tissue expression. All of them share a common conserved catalytic core region consisting of approximately 275 amino acids, forming a Rossmann fold domain (characteristic of NAD+ /NADH-binding proteins) and a zinc-binding domain connected by several loops [2]. Outside the catalytic core, sirt enzymes possess variable N- and C-terminal regions that decide about their enzymatic activities, binding partners and substrates, as well as subcellular localization [3]. sirt1, sirt6, and sirt7 localize predominantly in the nucleus where via modifications of transcription factors, cofactors, and histones they participate in the regulation of energy metabolism, stress and inflammatory responses, DNA repair (sirt1 and sirt6), and rDNA transcription (sirt7) [4]. sirt2 is a cytoplasmic sirtuin and plays a role in cell cycle control [5]. sirt3 can be found in mitochondria where it takes part in the regulation of enzymes involved, e.g., in glycolysis, fatty acid (FA) oxidation, ketone body synthesis, and the catabolism of amino acids as well as of apoptosis and oxidative stress pathways. This sirtuin also has as a nuclear full-length form (FL-sirt3) that is processed to the short mitochondrial form. Therefore, sirt3 may regulate cellular metabolism both at the transcriptional and posttranscriptional levels. sirt4 is also localized in mitochondria and acts as ADP-ribosylase. Another mitochondrial sirtuin—sirt5 —has a potent demalonylation and desuccinylation enzymatic activity and is involved in the regulation of amino acid catabolism [6]. Importantly, the subcellular localization of sirts may vary in different cell types and may depend on their molecular interactions as it was shown in the case of sirt1, sirt2, and sirt3 that can be found both in the nucleus and in the cytoplasm [4].

Expression of sirts was detected in various human tissues, including those crucial for the regulation of metabolism, e.g., hypothalamus, liver, pancreatic islets, skeletal muscles, and adipocytes [7–10]. In these tissues, sirts control the expression of other genes, particularly those involved in the organism response to stress. It was shown that sirt expression and activity of sirt enzymes are highly sensitive to several environmental factors, CR, exercise, and cold exposure that represents an adaptive mechanism in response to environmental stress [3]. Fluctuations in intracellular NAD+ levels in response to nutrient availability are believed to mediate in this phenomenon. When nutrients are plentiful, cellular metabolism relies on glycolysis to produce energy, leading to the generation of ATP and conversion of NAD+ to NADH. Low levels of NAD+ and high levels of NADH result in inactivation of the enzymatic activity of sirts. In turn CR leads to the elevation of NAD+ levels in most metabolically active tissues resulting in the increased sirt activity [11]. In humans, obesity leads to downregulation of sirt1 level in adipose tissue that can be restored by the weight loss [12].

#### 3. sirts and adipogenesis

sirts are considered as potential targets for the treatment of obesity that results from their involvement in the regulation of adipogenesis and adipocyte browning.

#### 3.1 Types of adipocytes

2. A short review of the sirt system

Adipose Tissue - An Update

and in the cytoplasm [4].

3. sirts and adipogenesis

14

The sirts are highly conserved regulatory proteins present almost in all species. Initially, they have been identified as class III histone deacetylases, nicotinamide adenine dinucleotide (NAD)-dependent enzymes responsible for the removal of acetyl groups from lysine residues in proteins, while some members of this family act also as mono-ADP-ribosyltransferases. Since acetylation and deacetylation are essential mechanisms of posttranslational modifications of proteins determining their activity, sirts were found to be involved in the regulation of distinct cellular pathways including, among others, those related to cell survival, apoptosis, inflam-

/NADH-binding

matory and stress responses, as well as lipid and glucose homeostases [2]. In human, seven sirt genes (sirts) have been identified that encode seven sirt enzymes of different structure, cellular localization, and tissue expression. All of them share a common conserved catalytic core region consisting of approximately 275

proteins) and a zinc-binding domain connected by several loops [2]. Outside the catalytic core, sirt enzymes possess variable N- and C-terminal regions that decide about their enzymatic activities, binding partners and substrates, as well as subcellular localization [3]. sirt1, sirt6, and sirt7 localize predominantly in the nucleus where via modifications of transcription factors, cofactors, and histones they participate in the regulation of energy metabolism, stress and inflammatory responses, DNA repair (sirt1 and sirt6), and rDNA transcription (sirt7) [4]. sirt2 is a cytoplasmic sirtuin and plays a role in cell cycle control [5]. sirt3 can be found in mitochondria where it takes part in the regulation of enzymes involved, e.g., in glycolysis, fatty acid (FA) oxidation, ketone body synthesis, and the catabolism of amino acids as well as of apoptosis and oxidative stress pathways. This sirtuin also has as a nuclear full-length form (FL-sirt3) that is processed to the short mitochondrial form. Therefore, sirt3 may regulate cellular metabolism both at the transcriptional and posttranscriptional levels. sirt4 is also localized in mitochondria and acts as ADP-ribosylase. Another mitochondrial sirtuin—sirt5 —has a potent demalonylation and desuccinylation enzymatic activity and is involved in the regulation of amino acid catabolism [6]. Importantly, the subcellular localization of sirts may vary in different cell types and may depend on their molecular interactions as it was shown in the case of sirt1, sirt2, and sirt3 that can be found both in the nucleus

Expression of sirts was detected in various human tissues, including those crucial for the regulation of metabolism, e.g., hypothalamus, liver, pancreatic islets, skeletal muscles, and adipocytes [7–10]. In these tissues, sirts control the expression of other genes, particularly those involved in the organism response to stress. It was shown that sirt expression and activity of sirt enzymes are highly sensitive to several environmental factors, CR, exercise, and cold exposure that represents an adaptive mechanism in response to environmental stress [3]. Fluctuations in intracellular NAD+ levels in response to nutrient availability are believed to mediate in this phenomenon. When nutrients are plentiful, cellular metabolism relies on glycolysis to produce energy, leading to the generation of ATP and conversion of NAD+ to NADH. Low levels of NAD+ and high levels of NADH result in inactivation of the enzymatic activity of sirts. In turn CR leads to the elevation of NAD+ levels in most metabolically active tissues resulting in the increased sirt activity [11]. In humans, obesity leads to downregulation

of sirt1 level in adipose tissue that can be restored by the weight loss [12].

sirts are considered as potential targets for the treatment of obesity that results from their involvement in the regulation of adipogenesis and adipocyte browning.

amino acids, forming a Rossmann fold domain (characteristic of NAD+

In mammals, there are two main types of adipose tissue that differ in their structure, physiology, and function. White adipose tissue (WAT) acts mainly as energy storage that releases FA for the production of adenosine triphosphate (ATP) during the process of β-oxidation.

Small mammals and human newborns, apart from white adipocytes, possess large deposits of brown adipose tissue (BAT) responsible for the non-shivering (adaptive) thermogenesis which is for them the most important regulatory mechanism for maintaining body temperature. The energy produced due to the oxidation of lipolysis-derived FA in the BAT mitochondria is released as heat, mostly thanks to uncoupling proteins (UCP). Age progression in humans was believed to be associated with complete atrophy of BAT; however, novel methods of imaging led to the identification of BAT stores in several areas of the adult human body, as well as of cells reminding brown adipocytes dispersed within WAT also known as beige/brite adipocytes (BeAT). These cells share common morphological features of white and brown adipocytes, and their number may increase upon different stimuli (e.g., cold, exercise, thyroid hormones, resveratrol). There are two theories regarding BeAT origin: they (i) differentiate from the progenitor cells resident in WAT or (ii) arise due to the transdifferentiation of white adipocytes. Given the role of adaptive thermogenesis in the whole body energy expenditure, stimulation of white adipocytes browning seems to be an attractive therapeutic pathway in the treatment of obesity and related metabolic disorders [13].

#### 3.2 sirts and preadipocyte differentiation

Peroxisome proliferator-activated receptor γ (PPARγ) is considered to be the main transcription factor responsible for promoting adipogenesis. sirt1, by interacting with two PPARγ corepressors, nuclear receptor corepressor (N-CoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT), can attenuate adipogenesis [14]. Consistently, overexpression of ectopic sirt1 blocks adipogenesis in 3T3-L1 cells, a culture of mouse adipocytes used as a model of adipocyte differentiation [15, 16]. Additionally, via activation of the Wnt signaling pathway, sirt1 determinates mesenchymal stem cells (MSC) differentiation toward myogenic cells, while its inhibition in MSC promotes adipogenesis [17]. MicroRNA 146b (miR-146b) acts as a negative regulator of sirt1 during adipocyte differentiation, giving a hope that interference with this miRNA may constitute a therapeutic perspective in the treatment of excess adiposity [18].

Another sirtuin family member—sirt2—has also shown an inhibitory effect on adipocyte differentiation [14]. In this process, sirt2 deacetylates forkhead box O1 (FOXO1) transcription factor and subsequently represses PPARγ transcriptional activity [19]. Therefore, sirt2 overexpression inhibits adipogenesis, while its silencing has an opposite effect in 3 T3-L1 preadipocytes. Moreover, this inhibitory influence of sirt2 on adipocyte differentiation discloses under CR that indicates the role of this sirtuin in the maintenance of energy homeostasis and suggests that sirt2 activators could provide novel therapeutics of obesity and its complications; however, such compounds have not been developed yet.

sirt3 is essential for the activation of bioenergetic function of mitochondria at the early stage of adipocyte differentiation. Silencing of sirt3 decreases the protein level of forkhead box O3a (FoxO3a) transcription factor and subsequently downregulates the expression of several antioxidant enzymes and increases oxidative stress in MSCs after adipogenic induction. In this way, sirt3 depletion diminishes the ability of MSCs to undergo adipogenic differentiation and leads to adipocyte dysfunction [20].

Knockout of sirt4 (encoding sirt4) leads to the decreased expression of adipogenic differentiation marker genes during differentiation of bovine adipocytes, suggesting that this sirtuin is crucial for the proper adipogenesis too [21].

sirt6 and sirt7 were also found to be necessary for adipocyte differentiation, and their deficiency inhibits the development of preadipocytes toward white adipocytes. sirt6 inhibits the expression of kinesin family member 5C (KIF5C) and enhances casein kinase 2 (CK2) and in this way promotes mitotic clonal expansion of adipocytes [22]. Deletion of sirt7 or inhibition of sirt7 diminishes the ability of mouse embryo fibroblasts and 3T3L1 cells to undergo adipogenesis. However, its overexpression did not rescue the preadipocyte differentiation, suggesting that sirt7 is required but not sufficient to perform a full program of adipogenesis. Interestingly, sirt7 is a metabolic target for miR-93, a negative regulator of adipogenesis, which expression is decreased in genetically obese ob/ob mice [23].

Experimental data suggest a direct interaction between sirt1 and sirt7 proteins at the molecular level as it was shown in immunoprecipitation assays and in vivo, where sirt7 knockout (KO) mice have increased sirt1 protein levels and enzymatic activity in WAT. Loss of sirt7 leads to increased sirt1 activity and recruitment to the PPARγ promoter, causing downregulation of its expression, that can explain the lipodystrophic phenotype in sirt7 KO mice [24].

in the induction of genes typical for BAT and repression of WAT genes associated with insulin resistance [25]. Therefore, silencing of sirt1 in 3T3-L1 preadipocytes leads to their hyperplasia and increased expression of WAT and inflammatory markers with a parallel decrease in BAT markers, whereas its activation results in

Role of sirtuins in adipocyte browning. PGC-1α, PPARγ coactivator 1α; PPARγ, peroxisome proliferatoractivated receptor γ; Prdm16, PR domain containing 16; sirt, sirtuin; UCP, uncoupling protein 1; ↑,

Role of Sirtuins in Adipose Tissue Development and Metabolism

DOI: http://dx.doi.org/10.5772/intechopen.88467

Cooperation among different sirtuins is crucial for the proper differentiation of brown adipocytes. For example, nutritional and thermal stress induces sirt1, which, by its deacetylation, activates PPARγ coactivator 1α (PGC-1α) which upregulates transcription of sirt3. In cultures of brown adipocyte precursors (HIB1B cells), overexpression of sirt3 resulted in the increased phosphorylation of the cAMP response element-binding protein (CREB) which then directly activates PGC-1α promoter, resulting in the increased expression of UCP1 and in promotion of mitochondrial respiration [27]. However, subsequent experiments showed that the protein produced based on the cDNA used in this experiment lacked proper deacetylase activity, so this finding should be treated with caution [28]. Moreover,

sirt3 KO mice, despite mitochondrial protein hyperacetylation, showed no

sirt5 was found to be essential for activation of brown adipogenic genes, and adipocyte differentiation in vitro and its knockout leads to the decrease in intracellular α-ketoglutarate concentration, which results in elevated histone methylation and transcriptional repression of pparγ and Prdm16. Therefore sirt5 KO mice present diminished browning capacity of WAT with subsequent cold intoler-

Finally, depletion of sirt6 in primary brown adipocytes reduces binding of the activating transcription factor 2 (ATF2) to the PGC-1α promoter and in this way decreases basal mitochondrial respiration and maximal mitochondrial capacity [31]. The role of sirts in adipocyte browning is schematically shown in Figure 2.

Both in vitro and in vivo studies have implicated sirts in the regulation of adipose tissue metabolism. These studies let us understand the complexity of sirt

significant disturbances of the adaptive thermogenesis [29].

4. sirts in control of adipose tissue function

increased adipocyte browning [26].

ance [30].

17

Figure 2.

upregulation and stimulation.

The role of sirts in preadipocyte differentiation is schematically shown in Figure 1.

#### 3.3 sirts and adipocyte browning

One of the approaches to the treatment of obesity is based on the activation in preadipocyte genes specific to BAT, which is characterized by high metabolic activity. Browning (brightening or beiging) of white adipocytes is an adaptive and reversible process that occurs in response to various stimuli.

Since sirt1, by direct deacetylation of PPARγ, recruits the BAT program coactivator Prdm16 (PR domain containing 16) to PPARγ, it also plays a crucial role

#### Figure 1.

Role of sirtuins in adipocyte differentiation. CK2, casein kinase 2; FOXO1, forkhead box O1; FOXO3a, forkhead box O3a; KI5FC, kinesin family member 5C; PPARγ, peroxisome proliferator-activated receptor γ; ROS, reactive oxygen species; sirt, sirtuin; Wnt, signaling pathway; ↑, upregulation and stimulation; ↓, downregulation and inhibition.

Role of Sirtuins in Adipose Tissue Development and Metabolism DOI: http://dx.doi.org/10.5772/intechopen.88467

#### Figure 2.

Knockout of sirt4 (encoding sirt4) leads to the decreased expression of adipogenic differentiation marker genes during differentiation of bovine adipocytes, suggesting that this sirtuin is crucial for the proper adipogenesis too [21]. sirt6 and sirt7 were also found to be necessary for adipocyte differentiation, and

Experimental data suggest a direct interaction between sirt1 and sirt7 proteins at

the molecular level as it was shown in immunoprecipitation assays and in vivo, where sirt7 knockout (KO) mice have increased sirt1 protein levels and enzymatic activity in WAT. Loss of sirt7 leads to increased sirt1 activity and recruitment to the PPARγ promoter, causing downregulation of its expression, that can explain the

The role of sirts in preadipocyte differentiation is schematically shown in

One of the approaches to the treatment of obesity is based on the activation in

preadipocyte genes specific to BAT, which is characterized by high metabolic activity. Browning (brightening or beiging) of white adipocytes is an adaptive and

Since sirt1, by direct deacetylation of PPARγ, recruits the BAT program coactivator Prdm16 (PR domain containing 16) to PPARγ, it also plays a crucial role

Role of sirtuins in adipocyte differentiation. CK2, casein kinase 2; FOXO1, forkhead box O1; FOXO3a, forkhead box O3a; KI5FC, kinesin family member 5C; PPARγ, peroxisome proliferator-activated receptor γ; ROS, reactive oxygen species; sirt, sirtuin; Wnt, signaling pathway; ↑, upregulation and stimulation; ↓,

reversible process that occurs in response to various stimuli.

lipodystrophic phenotype in sirt7 KO mice [24].

3.3 sirts and adipocyte browning

their deficiency inhibits the development of preadipocytes toward white adipocytes. sirt6 inhibits the expression of kinesin family member 5C (KIF5C) and enhances casein kinase 2 (CK2) and in this way promotes mitotic clonal expansion of adipocytes [22]. Deletion of sirt7 or inhibition of sirt7 diminishes the ability of mouse embryo fibroblasts and 3T3L1 cells to undergo adipogenesis. However, its overexpression did not rescue the preadipocyte differentiation, suggesting that sirt7 is required but not sufficient to perform a full program of adipogenesis. Interestingly, sirt7 is a metabolic target for miR-93, a negative regulator of adipogenesis, which expression is decreased in genetically obese

ob/ob mice [23].

Adipose Tissue - An Update

Figure 1.

Figure 1.

16

downregulation and inhibition.

Role of sirtuins in adipocyte browning. PGC-1α, PPARγ coactivator 1α; PPARγ, peroxisome proliferatoractivated receptor γ; Prdm16, PR domain containing 16; sirt, sirtuin; UCP, uncoupling protein 1; ↑, upregulation and stimulation.

in the induction of genes typical for BAT and repression of WAT genes associated with insulin resistance [25]. Therefore, silencing of sirt1 in 3T3-L1 preadipocytes leads to their hyperplasia and increased expression of WAT and inflammatory markers with a parallel decrease in BAT markers, whereas its activation results in increased adipocyte browning [26].

Cooperation among different sirtuins is crucial for the proper differentiation of brown adipocytes. For example, nutritional and thermal stress induces sirt1, which, by its deacetylation, activates PPARγ coactivator 1α (PGC-1α) which upregulates transcription of sirt3. In cultures of brown adipocyte precursors (HIB1B cells), overexpression of sirt3 resulted in the increased phosphorylation of the cAMP response element-binding protein (CREB) which then directly activates PGC-1α promoter, resulting in the increased expression of UCP1 and in promotion of mitochondrial respiration [27]. However, subsequent experiments showed that the protein produced based on the cDNA used in this experiment lacked proper deacetylase activity, so this finding should be treated with caution [28]. Moreover, sirt3 KO mice, despite mitochondrial protein hyperacetylation, showed no significant disturbances of the adaptive thermogenesis [29].

sirt5 was found to be essential for activation of brown adipogenic genes, and adipocyte differentiation in vitro and its knockout leads to the decrease in intracellular α-ketoglutarate concentration, which results in elevated histone methylation and transcriptional repression of pparγ and Prdm16. Therefore sirt5 KO mice present diminished browning capacity of WAT with subsequent cold intolerance [30].

Finally, depletion of sirt6 in primary brown adipocytes reduces binding of the activating transcription factor 2 (ATF2) to the PGC-1α promoter and in this way decreases basal mitochondrial respiration and maximal mitochondrial capacity [31].

The role of sirts in adipocyte browning is schematically shown in Figure 2.

#### 4. sirts in control of adipose tissue function

Both in vitro and in vivo studies have implicated sirts in the regulation of adipose tissue metabolism. These studies let us understand the complexity of sirt actions and gave hope that the modulation of their activity may constitute a new therapeutic strategy for the treatment of obesity and its metabolic complications including hyperlipidemia and chronic inflammation.

proper mitochondrial FA oxidation [41, 42], while downregulation of sirt4 level results in the increased expression of genes involved in FA oxidation [43]. In experimental animals, sirt6 deficiency leads to impaired lipolytic activity and subsequent adipocyte hypertrophy [44]. On the molecular level, sirt6 deficiency increases the acetylation and phosphorylation of FOXO1, leading to its nuclear exclusion and decrease in its transcriptional activity that downregulates the expression of the gene encoding ATGL [44]. In turn, sirt6 overexpression in adipose tissue counteracts lipotoxicity caused by the high-fat diet by decreasing PPARγ signaling and diacylglycerol acyltransferase 1 (DGAT1) activity [45]. The role of sirt7 in lipid metabolism is yet to be determined. In some studies sirt7 KO mice, due to the impaired management of the endoplasmic reticulum stress, have increased lipogenesis in the liver that results in liver steatosis and dyslipidemia [23], while sirt7 upregulation restores hepatic homeostasis in diet-induced obesity [46]. On the contrary, other researchers showed that sirt7 via inhibition of testicular receptor 4 (TR4) degradation promotes FA uptake, triglyceride biosynthesis, and storage [47]. These results constituted the basis for studies on the use of sirtuin-activating

Role of Sirtuins in Adipose Tissue Development and Metabolism

DOI: http://dx.doi.org/10.5772/intechopen.88467

compounds in order to increase lipolysis and to prevent excess adiposity.

complications.

19

encoding inhibitor α of κB (IκBα) [50].

4.2 sirts in control of adipose tissue inflammation and secretory activity

Recent years widened our understanding of the role of WAT which is now considered not only an energy storage but also an important endocrine organ that via secreted mediators (e.g., cytokines and adipokines) may influence the function of the whole organism and be responsible for the development of obesity-related

sirt1, by interference with the nuclear factor κB (NF-κB) signaling pathway, represses inflammatory gene expression in adipocytes and in macrophages infiltrating adipose tissue, which results in the improvement of insulin signaling and in the reduction of hyperinsulinemia accompanied by an increase in insulin sensitivity in vivo [48, 49]. sirt1 can inhibit NF-κB signaling both directly and indirectly. Acting directly sirt1 deacetylates the RelA/p65 subunit of the NF-κB, leading to its subsequent ubiquitination and degradation. Indirect inhibition of NF-κB by sirt1 takes place by increasing activity of repressive transcriptional complexes, e.g., PPARα, which can bind and inactivate RelA/p65 or increase expression of the gene

Similarly, overexpression of sirt6 suppresses activation of the NF-κB signaling in cell lines, firstly, by the direct interaction with NF-κB subunit and, secondly, by deacetylation of histone H3 lysine 9 at target gene promoters leading to inhibition of the transcription of the proinflammatory genes [51]. Moreover, sirt6 by binding to the c-Jun downregulates expression of its target genes including interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and monocyte chemoattractant protein 1 (MCP-1) [52]. Subsequently, in model animals, sirt1 and sirt6 deficiency increases macrophage infiltration in adipose tissue and subsequent inflammation [44]. Moreover, sirt1 deficiency in adipocytes (probably due to the decreased expression

This last adipokine is a protein hormone with many desirable metabolic properties (including anti-inflammatory and anti-oxidative effects) almost exclusively produced in adipocytes. sirt1 tightly regulates the expression and secretion of adiponectin by adipocytes: enhancing formation of the complex between FOXO1

of IL-4) led to the shift between the profiles of macrophages from the antiinflammatory (M2) to the proinflammatory (M1) [53]. Therefore, sirt1- and sirt6 deficient adipocytes are more potent in promoting macrophages migration than wild-type cells that can be reversed by addition of MCP1 or adiponectin.

#### 4.1 sirts in lipid metabolism

sirts are expressed in tissues and organs involved in lipid metabolism including the liver, skeletal muscle, and white and brown adipose tissues, where they control lipid synthesis, storage, and utilization both directly and indirectly (via control of insulin secretion).

During fasting sirt1, by deacetylation of PPARγ corepressors (FOXO1 and PGC-1α), stimulates in the adipose tissue transcription of the gene encoding adipose triglyceride lipase (ATGL) and subsequent lipolysis. This process is impaired in sirt1 KO mice [15]. However, the results of animal studies regarding sirt1 overexpression on body weight and composition are inconsistent [32, 33]. It is suggested that these discrepancies may be attributed to the different levels of sirt1 expression between the transgenic animals as well as to the differences between strains and species used in the experiments.

Apart from the regulation of PPARα-related pathways, sirt1 may influence FA metabolism via downregulation of sterol regulatory element-binding proteins 1 and 2 (SREBP-1 and SREBP-2) transcription factors. sirt1 overexpression or its activation by, e.g., resveratrol (RSV), prevents cleavage-induced activation of SERBs and their translocation to the nucleus where they promote transcription of genes crucial for sterol biosynthesis [34]. sirt1 KO mice have lower SREBP-1 mRNA levels in the liver that correlates with decreased serum triglyceride concentrations [35]. Activation of sirt1 also induces phosphorylation of AMP-activated protein kinase (AMPK) that protects against FA synthase induction and lipid accumulation caused by high glucose [36].

sirt1 also promotes deacetylation of liver X receptor (LXR) proteins and transcription factors that act as cholesterol sensors and regulate whole body cholesterol and lipid homeostasis [37]. LXR deacetylation is necessary both for their activation and induction of LXR target genes and for their subsequent ubiquitination. sirt1 KO animals have reduced mRNA levels of LXR target genes that result in impaired reverse cholesterol transport—a process by which excess cholesterol is removed from the peripheral cells and transported to the liver where it can be converted to bile and excreted [38].

Fasting and cold exposure were found to increase the expression of sirt2 in WAT. That results in the deacetylation of FOXO1 and subsequent repression of PPARγ activity, lipolysis, and release of FA. Similar effect can be obtained by administration of isoproterenol that confirms the role of adrenergic signaling in the regulation of sirt2 expression in WAT [19]. sirt2 may also inhibit lipogenesis by deacetylation of ATP-citrate lyase (ACLY), an enzyme crucial for FA synthesis. A deacetylated form of ACLY is then ubiquitinated and degraded, while lipogenesis is reduced [39].

Livers from sirt3 KO mice showed higher levels of FA oxidation intermediate products and triglycerides during fasting that was associated with decreased levels of FA oxidation when compared to wild-type animals. These findings are consistent with the fact that deacetylation of the long-chain acyl-coenzyme A dehydrogenase by sirt3 was found to determine proper mitochondrial FA oxidation [40].

There are experimental data that other sirts are also involved in lipid metabolism: in adipose tissue, e.g., deacetylation of malonyl-CoA-decarboxylase by sirt4 and desuccinylation of the hydroxyl-coenzyme A dehydrogenase by sirt5 determine

#### Role of Sirtuins in Adipose Tissue Development and Metabolism DOI: http://dx.doi.org/10.5772/intechopen.88467

actions and gave hope that the modulation of their activity may constitute a new therapeutic strategy for the treatment of obesity and its metabolic complications

sirts are expressed in tissues and organs involved in lipid metabolism including the liver, skeletal muscle, and white and brown adipose tissues, where they control lipid synthesis, storage, and utilization both directly and indirectly (via control of

During fasting sirt1, by deacetylation of PPARγ corepressors (FOXO1 and PGC-

Apart from the regulation of PPARα-related pathways, sirt1 may influence FA metabolism via downregulation of sterol regulatory element-binding proteins 1 and 2 (SREBP-1 and SREBP-2) transcription factors. sirt1 overexpression or its activation by, e.g., resveratrol (RSV), prevents cleavage-induced activation of SERBs and their translocation to the nucleus where they promote transcription of genes crucial for sterol biosynthesis [34]. sirt1 KO mice have lower SREBP-1 mRNA levels in the liver that correlates with decreased serum triglyceride concentrations [35]. Activation of sirt1 also induces phosphorylation of AMP-activated protein kinase (AMPK) that protects against FA synthase induction and lipid accumulation caused by high

sirt1 also promotes deacetylation of liver X receptor (LXR) proteins and transcription factors that act as cholesterol sensors and regulate whole body cholesterol and lipid homeostasis [37]. LXR deacetylation is necessary both for their activation and induction of LXR target genes and for their subsequent ubiquitination. sirt1 KO animals have reduced mRNA levels of LXR target genes that result in impaired reverse cholesterol transport—a process by which excess cholesterol is removed from the peripheral cells and transported to the liver where it can be converted to

Fasting and cold exposure were found to increase the expression of sirt2 in WAT. That results in the deacetylation of FOXO1 and subsequent repression of PPARγ activity, lipolysis, and release of FA. Similar effect can be obtained by administration of isoproterenol that confirms the role of adrenergic signaling in the regulation of sirt2 expression in WAT [19]. sirt2 may also inhibit lipogenesis by deacetylation of ATP-citrate lyase (ACLY), an enzyme crucial for FA synthesis. A deacetylated form of ACLY is then ubiquitinated and degraded, while lipogenesis is

Livers from sirt3 KO mice showed higher levels of FA oxidation intermediate products and triglycerides during fasting that was associated with decreased levels of FA oxidation when compared to wild-type animals. These findings are consistent with the fact that deacetylation of the long-chain acyl-coenzyme A dehydrogenase

There are experimental data that other sirts are also involved in lipid metabolism: in adipose tissue, e.g., deacetylation of malonyl-CoA-decarboxylase by sirt4 and desuccinylation of the hydroxyl-coenzyme A dehydrogenase by sirt5 determine

by sirt3 was found to determine proper mitochondrial FA oxidation [40].

1α), stimulates in the adipose tissue transcription of the gene encoding adipose triglyceride lipase (ATGL) and subsequent lipolysis. This process is impaired in sirt1 KO mice [15]. However, the results of animal studies regarding sirt1 overexpression on body weight and composition are inconsistent [32, 33]. It is suggested that these discrepancies may be attributed to the different levels of sirt1 expression between the transgenic animals as well as to the differences between strains and species used

including hyperlipidemia and chronic inflammation.

4.1 sirts in lipid metabolism

Adipose Tissue - An Update

insulin secretion).

in the experiments.

glucose [36].

bile and excreted [38].

reduced [39].

18

proper mitochondrial FA oxidation [41, 42], while downregulation of sirt4 level results in the increased expression of genes involved in FA oxidation [43]. In experimental animals, sirt6 deficiency leads to impaired lipolytic activity and subsequent adipocyte hypertrophy [44]. On the molecular level, sirt6 deficiency increases the acetylation and phosphorylation of FOXO1, leading to its nuclear exclusion and decrease in its transcriptional activity that downregulates the expression of the gene encoding ATGL [44]. In turn, sirt6 overexpression in adipose tissue counteracts lipotoxicity caused by the high-fat diet by decreasing PPARγ signaling and diacylglycerol acyltransferase 1 (DGAT1) activity [45]. The role of sirt7 in lipid metabolism is yet to be determined. In some studies sirt7 KO mice, due to the impaired management of the endoplasmic reticulum stress, have increased lipogenesis in the liver that results in liver steatosis and dyslipidemia [23], while sirt7 upregulation restores hepatic homeostasis in diet-induced obesity [46]. On the contrary, other researchers showed that sirt7 via inhibition of testicular receptor 4 (TR4) degradation promotes FA uptake, triglyceride biosynthesis, and storage [47].

These results constituted the basis for studies on the use of sirtuin-activating compounds in order to increase lipolysis and to prevent excess adiposity.

#### 4.2 sirts in control of adipose tissue inflammation and secretory activity

Recent years widened our understanding of the role of WAT which is now considered not only an energy storage but also an important endocrine organ that via secreted mediators (e.g., cytokines and adipokines) may influence the function of the whole organism and be responsible for the development of obesity-related complications.

sirt1, by interference with the nuclear factor κB (NF-κB) signaling pathway, represses inflammatory gene expression in adipocytes and in macrophages infiltrating adipose tissue, which results in the improvement of insulin signaling and in the reduction of hyperinsulinemia accompanied by an increase in insulin sensitivity in vivo [48, 49]. sirt1 can inhibit NF-κB signaling both directly and indirectly. Acting directly sirt1 deacetylates the RelA/p65 subunit of the NF-κB, leading to its subsequent ubiquitination and degradation. Indirect inhibition of NF-κB by sirt1 takes place by increasing activity of repressive transcriptional complexes, e.g., PPARα, which can bind and inactivate RelA/p65 or increase expression of the gene encoding inhibitor α of κB (IκBα) [50].

Similarly, overexpression of sirt6 suppresses activation of the NF-κB signaling in cell lines, firstly, by the direct interaction with NF-κB subunit and, secondly, by deacetylation of histone H3 lysine 9 at target gene promoters leading to inhibition of the transcription of the proinflammatory genes [51]. Moreover, sirt6 by binding to the c-Jun downregulates expression of its target genes including interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and monocyte chemoattractant protein 1 (MCP-1) [52]. Subsequently, in model animals, sirt1 and sirt6 deficiency increases macrophage infiltration in adipose tissue and subsequent inflammation [44]. Moreover, sirt1 deficiency in adipocytes (probably due to the decreased expression of IL-4) led to the shift between the profiles of macrophages from the antiinflammatory (M2) to the proinflammatory (M1) [53]. Therefore, sirt1- and sirt6 deficient adipocytes are more potent in promoting macrophages migration than wild-type cells that can be reversed by addition of MCP1 or adiponectin.

This last adipokine is a protein hormone with many desirable metabolic properties (including anti-inflammatory and anti-oxidative effects) almost exclusively produced in adipocytes. sirt1 tightly regulates the expression and secretion of adiponectin by adipocytes: enhancing formation of the complex between FOXO1

and C/EBPα (CCAAT/enhancer binding protein α) increases expression of the ADIPOQ gene, while inhibition of endoplasmic reticulum oxidoreductase Ero-Lα decreases secretion of the high-molecular-weight (HMW) adiponectin [54]. Omentin-1 (intelectin-1) is another adipokine secreted, but not only by adipose tissue with anti-inflammatory properties that via activation of sirt1 exert its molecular effects on target genes [55].

insulin resistance in animal models of type 2 diabetes [63, 65, 66]. Apart from the favorable influence on glucose metabolism, SRT1720, by decreasing expression of lipogenic genes, occurred to be effective in the treatment of animal models of liver steatosis [67]. However, some studies question the beneficial effect of SRT1720 on metabolic parameters in animals fed a high-fat diet [68]. Moreover, RSV and other sirt1 activators (SRT1720, SRT2183, SRT1460) were found not to activate sirt1 directly but by the activation of AMPK that increases intracellular NAD+ levels and in this way induces deacetylation of sirt1 targets [69]. However, studies on sirt1 mutations that influence the protein structure suggest that there is also a direct interaction of RSV derivates with the sirt1 enzyme molecule [64]. In humans, administration of SRT2104 (another RSV analogue) caused a decrease in serum total cholesterol and triglycerides levels as well as a significant reduction of the

Despite their beneficial effects on adipose tissue metabolism, the critical issue that may arise during the use of sirt1 activators in everyday practice is their limited target specificity that might result in unexpected adverse effects [71]. That is why sirt modulators are still under consideration before they can be approved for the

muscle. This concept is based on animal studies where sirt1 KO mice display higher

Recently there has been a rapidly growing interest in the role of miRNAs in fat cell development and obesity, and there is also evidence that miRNA plays a role in the regulation of sirt activity [18, 23, 46, 72]. Therefore, one can assume that strategies based on modifying the action of sirts by specific miRNAs may also be useful in treating obesity. However, these studies are still at a preliminary stage.

If the remarkable effects of sirts on adipose tissue development and metabolism coming from animal studies hold up in humans, their activators and inhibitors may revolutionize the treatment of obesity and associated complications. However, one should remember that sirt activities are not limited to the regulation of metabolism and include, also, e.g., control of longevity, oncogenesis as well as the function of neural and cardiovascular systems. Therefore, compounds targeting sirts'system in order to combat excess adiposity have to be adipose tissue-specific to avoid potentially harmful and counterproductive side effects of global sirt activation/inactivation. Till now such compounds have not been accepted for the clinical practice; however, many of them are under evaluation, and it is very likely that shortly new therapeutic strategies aimed at selective and tissue-specific modulation of sirt activity will be registered for the treatment of obesity and its complications.

Till now, the only aspects in which sirt inhibitors can be used to treat obesity-associated metabolic disorders are to induce favorable changes in body

composition. sirt1-inhibiting compounds such as splitomycin, suramin, salermide, EX-527, or sirtinol can be used to increase the amount of skeletal

muscle growth than wild-type animals and mice with muscle-specific sirt1 overexpression [64]. However, sirt1 inhibitors were not tested for that purpose

inflammatory response to lipopolysaccharide stimulation [70].

routine treatment of obesity and metabolic disorders.

Role of Sirtuins in Adipose Tissue Development and Metabolism

DOI: http://dx.doi.org/10.5772/intechopen.88467

6. Final remarks and conclusions

in humans.

21

In contrary, resistin is a hormone with biological characteristics opposite to adiponectin and omentin. It is secreted, apart from other sites, by adipose tissue; however, resistin expression in isolated human adipocytes is low, and its content in adipose tissue is proportional to the intensity of macrophages infiltration, which are the primary source of this adipokine [56]. Stimulation of sirt1 by RSV reduces resistin mRNA level and protein expression in macrophages, whereas sirt1 KO results in the opposite effect. On the molecular level, sirt1 interacts directly with the resistin promoter region at an activator protein 1 (AP-1) transcription factor response element as well as inhibits transactivation of the resistin gene by c-Jun pathway [57]. In animal model RSV, via activation of sirt1 was also found to decrease expression of visfatin—another adipokine secreted by macrophages infiltrating adipose tissue [58].

#### 5. Sirtuins as targets for obesity treatment

Given their role in the regulation of lipid metabolism, adipogenesis and secretory activity of adipose tissue sirts constitute promising targets for novel therapies, targeting excess adiposity and associated metabolic disorders. However, the discovery of a compound that would be able to activate some sirt isoforms and to inhibit others is still a challenge. Another obstacle is to obtain tissue specificity of action for these compounds, since sirt activity may depend on the cell type and environmental factors.

Several sirt isoforms bear the potential for being used as therapeutic targets, but to date, only modulators of sirt1 have entered into the clinic. The most effective sirtuin-activating compound able to increase sirt1 activity in vitro by >10 fold is RSV [59]. RSV, naturally present in grapes and red wine, successfully inhibited maturation of preadipocytes and induced adipocyte apoptosis in cell cultures [60]. When administered to mice on the high-calorie diet, RSV was able to improve their metabolic and inflammatory profiles [61]. A reformulated version of RSV (resVida) with improved bioavailability was effective in decreasing glucose and triglyceride levels, reducing the intensity of inflammation and liver steatosis in obese men [62]. Another micronized formulation of RSV, SRT501, via activation of the similar set of genes as in the case of CR, was able to counteract negative consequences of a high-calorie diet in mice [63]. A composition containing RSV, leucine, β-hydroxymethyl butyrate (HMB), and ketoisocaproic acid synergistically activating sirt1 and sirt3 can induce FA oxidation and mitochondrial biogenesis. This combination, when tested on 3LT3-L1 preadipocytes, was more effective in activation of sirt1 than RSV alone but also able to activate sirt3. In c57/BL6 mice, treatment with a combination of low doses of RSV with either HMB or leucine resulted in a reduction of body weight and improvement of body composition accompanied by increased insulin sensitivity [64].

A variety of synthetic RSV derivatives with lower toxicity and higher potency to activate sirt1 have been invented. The example of them is SRT1720, able to increase deacetylation of sirt1 substrates in vitro and successfully applied in vivo to treat

Role of Sirtuins in Adipose Tissue Development and Metabolism DOI: http://dx.doi.org/10.5772/intechopen.88467

and C/EBPα (CCAAT/enhancer binding protein α) increases expression of the ADIPOQ gene, while inhibition of endoplasmic reticulum oxidoreductase Ero-Lα decreases secretion of the high-molecular-weight (HMW) adiponectin [54]. Omentin-1 (intelectin-1) is another adipokine secreted, but not only by adipose tissue with anti-inflammatory properties that via activation of sirt1 exert its molec-

In contrary, resistin is a hormone with biological characteristics opposite to adiponectin and omentin. It is secreted, apart from other sites, by adipose tissue; however, resistin expression in isolated human adipocytes is low, and its content in adipose tissue is proportional to the intensity of macrophages infiltration, which are the primary source of this adipokine [56]. Stimulation of sirt1 by RSV reduces resistin mRNA level and protein expression in macrophages, whereas sirt1 KO results in the opposite effect. On the molecular level, sirt1 interacts directly with the resistin promoter region at an activator protein 1 (AP-1) transcription factor response element as well as inhibits transactivation of the resistin gene by c-Jun pathway [57]. In animal model RSV, via activation of sirt1 was also found to decrease expression of visfatin—another adipokine secreted by macrophages

Given their role in the regulation of lipid metabolism, adipogenesis and secretory activity of adipose tissue sirts constitute promising targets for novel therapies, targeting excess adiposity and associated metabolic disorders. However, the discovery of a compound that would be able to activate some sirt isoforms and to inhibit others is still a challenge. Another obstacle is to obtain tissue specificity of action for these compounds, since sirt activity may depend on the cell type and

Several sirt isoforms bear the potential for being used as therapeutic targets,

but to date, only modulators of sirt1 have entered into the clinic. The most effective sirtuin-activating compound able to increase sirt1 activity in vitro by >10 fold is RSV [59]. RSV, naturally present in grapes and red wine, successfully inhibited maturation of preadipocytes and induced adipocyte apoptosis in cell cultures [60]. When administered to mice on the high-calorie diet, RSV was able to improve their metabolic and inflammatory profiles [61]. A reformulated version of RSV (resVida) with improved bioavailability was effective in decreasing glucose and triglyceride levels, reducing the intensity of inflammation and liver steatosis in obese men [62]. Another micronized formulation of RSV, SRT501, via activation of the similar set of genes as in the case of CR, was able to counteract negative consequences of a high-calorie diet in mice [63]. A composition containing RSV, leucine, β-hydroxymethyl butyrate (HMB), and ketoisocaproic acid synergistically activating sirt1 and sirt3 can induce FA oxidation and mitochondrial biogenesis. This combination, when tested on 3LT3-L1 preadipocytes, was more effective in activation of sirt1 than RSV alone but also able to activate sirt3. In c57/BL6 mice, treatment with a combination of low doses of RSV with either HMB or leucine resulted in a reduction of body weight and improvement of

body composition accompanied by increased insulin sensitivity [64].

A variety of synthetic RSV derivatives with lower toxicity and higher potency to activate sirt1 have been invented. The example of them is SRT1720, able to increase deacetylation of sirt1 substrates in vitro and successfully applied in vivo to treat

ular effects on target genes [55].

Adipose Tissue - An Update

infiltrating adipose tissue [58].

environmental factors.

20

5. Sirtuins as targets for obesity treatment

insulin resistance in animal models of type 2 diabetes [63, 65, 66]. Apart from the favorable influence on glucose metabolism, SRT1720, by decreasing expression of lipogenic genes, occurred to be effective in the treatment of animal models of liver steatosis [67]. However, some studies question the beneficial effect of SRT1720 on metabolic parameters in animals fed a high-fat diet [68]. Moreover, RSV and other sirt1 activators (SRT1720, SRT2183, SRT1460) were found not to activate sirt1 directly but by the activation of AMPK that increases intracellular NAD+ levels and in this way induces deacetylation of sirt1 targets [69]. However, studies on sirt1 mutations that influence the protein structure suggest that there is also a direct interaction of RSV derivates with the sirt1 enzyme molecule [64]. In humans, administration of SRT2104 (another RSV analogue) caused a decrease in serum total cholesterol and triglycerides levels as well as a significant reduction of the inflammatory response to lipopolysaccharide stimulation [70].

Despite their beneficial effects on adipose tissue metabolism, the critical issue that may arise during the use of sirt1 activators in everyday practice is their limited target specificity that might result in unexpected adverse effects [71]. That is why sirt modulators are still under consideration before they can be approved for the routine treatment of obesity and metabolic disorders.

Till now, the only aspects in which sirt inhibitors can be used to treat obesity-associated metabolic disorders are to induce favorable changes in body composition. sirt1-inhibiting compounds such as splitomycin, suramin, salermide, EX-527, or sirtinol can be used to increase the amount of skeletal muscle. This concept is based on animal studies where sirt1 KO mice display higher muscle growth than wild-type animals and mice with muscle-specific sirt1 overexpression [64]. However, sirt1 inhibitors were not tested for that purpose in humans.

Recently there has been a rapidly growing interest in the role of miRNAs in fat cell development and obesity, and there is also evidence that miRNA plays a role in the regulation of sirt activity [18, 23, 46, 72]. Therefore, one can assume that strategies based on modifying the action of sirts by specific miRNAs may also be useful in treating obesity. However, these studies are still at a preliminary stage.

#### 6. Final remarks and conclusions

If the remarkable effects of sirts on adipose tissue development and metabolism coming from animal studies hold up in humans, their activators and inhibitors may revolutionize the treatment of obesity and associated complications. However, one should remember that sirt activities are not limited to the regulation of metabolism and include, also, e.g., control of longevity, oncogenesis as well as the function of neural and cardiovascular systems. Therefore, compounds targeting sirts'system in order to combat excess adiposity have to be adipose tissue-specific to avoid potentially harmful and counterproductive side effects of global sirt activation/inactivation. Till now such compounds have not been accepted for the clinical practice; however, many of them are under evaluation, and it is very likely that shortly new therapeutic strategies aimed at selective and tissue-specific modulation of sirt activity will be registered for the treatment of obesity and its complications.

Adipose Tissue - An Update

### Author details

Alina Kurylowicz Department of Human Epigenetics, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland

References

mce.2009.08.018

[2] Sanders BD, Jackson B,

10.1101/gad.1467506

pathol.4.110807.092250

12.004

23

to oxidative stress and caloric

[1] Galic S, Oakhill JS, Steinberg GR. Adipose tissue as an endocrine organ. Molecular and Cellular Endocrinology. 2010;316:129-139. DOI: 10.1016/j.

DOI: http://dx.doi.org/10.5772/intechopen.88467

Role of Sirtuins in Adipose Tissue Development and Metabolism

Hepatology. 2013;59:1315-1322. DOI:

10.1016/j.jhep.2013.07.027

[9] Caton PW, Richardson SJ, Kieswich J, Bugliani M, Holland ML, Marchetti P, et al. Sirtuin 3 regulates mouse pancreatic beta cell function and is suppressed in pancreatic islets isolated from human type 2 diabetic patients. Diabetologia. 2013;56:1068-1077. DOI:

10.1007/s00125-013-2851-y

[10] Acs Z, Bori Z, Takeda M, Osvath P, Berkes I, Taylor AW, et al. High altitude exposure alters gene expression levels of DNA repair enzymes, and modulates fatty acid metabolism by sirt4 induction in human skeletal muscle. Respiratory Physiology & Neurobiology. 2014;196: 33-37. DOI: 10.1016/j.resp.2014.02.006

[11] Chalkiadaki A, Guarente L. Sirtuins

mediate mammalian metabolic responses to nutrient availability. Nature Reviews Endocrinology. 2012;8: 287-296. DOI: 10.1038/nrendo.2011.225

[12] Kurylowicz A, Owczarz M, Polosak J, Jonas MI, Lisik W, Jonas M, et al. sirt1 and sirt7 expression in adipose tissues of obese and normalweight individuals is regulated by microRNAs but not by methylation status. International Journal of Obesity. 2016;40:1635-1642. DOI: 10.1038/

[13] Zwick RK, Guerrero-Juarez CF, Horsley V, Plikus MV. Anatomical, physiological, and functional diversity of adipose tissue. Cell Metabolism. 2018; 27:68-83. DOI: 10.1016/j.cmet.2017.

[14] Jing E, Gesta S, Kahn CR. sirt2 regulates adipocyte differentiation through FoxO1 acetylation/

deacetylation. Cell Metabolism. 2007;6: 105-114. DOI: 10.1016/j.cmet.2007.

ijo.2016.131

12.002

07.003

Marmorstein R. Structural basis for sirtuin function: What we know and what we don't. Biochimica et Biophysica Acta. 2010;1804:1604-1616. DOI: 10.1016/j.bbapap.2009.09.009

[3] Haigis MC, Guarente LP. Mammalian sirtuins—Emerging roles in physiology, aging, and calorie restriction. Genes & Development. 2006;20:2913-2921. DOI:

[4] Haigis MC, Sinclair DA. Mammalian sirtuins: Biological insights and disease relevance. Annual Review of Pathology. 2010;5:253-295. DOI: 10.1146/annurev.

[5] Wang F, Nguyen M, Qin FX, Tong Q. sirt2 deacetylates FOXO3a in response

restriction. Aging Cell. 2007;6:505-514. DOI: 10.1111/j.1474-9726.2007.00304.x

[6] Parihar P, Solanki I, Mansuri ML, Parihar MS. Mitochondrial sirtuins: Emerging roles in metabolic regulations, energy homeostasis and diseases. Experimental Gerontology. 2015;61: 130-141. DOI: 10.1016/j.exger.2014.

[7] Zakhary SM, Ayubcha D, Dileo JN, Jose R, Leheste JR, Horowitz JM, et al. Distribution analysis of deacetylase sirt1 in rodent and human nervous systems. The Anatomical Record. 2010;293: 1024-1032. DOI: 10.1002/ar.21116

[8] Moschen AR, Wieser V, Gerner RR, Bichler A, Enrich B, Moser P, et al. Adipose tissue and liver expression of sirt1, 3, and 6 increase after extensive weight loss in morbid obesity. Journal of

\*Address all correspondence to: akurylowicz@imdik.pan.pl

© 2019 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.

Role of Sirtuins in Adipose Tissue Development and Metabolism DOI: http://dx.doi.org/10.5772/intechopen.88467

#### References

[1] Galic S, Oakhill JS, Steinberg GR. Adipose tissue as an endocrine organ. Molecular and Cellular Endocrinology. 2010;316:129-139. DOI: 10.1016/j. mce.2009.08.018

[2] Sanders BD, Jackson B, Marmorstein R. Structural basis for sirtuin function: What we know and what we don't. Biochimica et Biophysica Acta. 2010;1804:1604-1616. DOI: 10.1016/j.bbapap.2009.09.009

[3] Haigis MC, Guarente LP. Mammalian sirtuins—Emerging roles in physiology, aging, and calorie restriction. Genes & Development. 2006;20:2913-2921. DOI: 10.1101/gad.1467506

[4] Haigis MC, Sinclair DA. Mammalian sirtuins: Biological insights and disease relevance. Annual Review of Pathology. 2010;5:253-295. DOI: 10.1146/annurev. pathol.4.110807.092250

[5] Wang F, Nguyen M, Qin FX, Tong Q. sirt2 deacetylates FOXO3a in response to oxidative stress and caloric restriction. Aging Cell. 2007;6:505-514. DOI: 10.1111/j.1474-9726.2007.00304.x

[6] Parihar P, Solanki I, Mansuri ML, Parihar MS. Mitochondrial sirtuins: Emerging roles in metabolic regulations, energy homeostasis and diseases. Experimental Gerontology. 2015;61: 130-141. DOI: 10.1016/j.exger.2014. 12.004

[7] Zakhary SM, Ayubcha D, Dileo JN, Jose R, Leheste JR, Horowitz JM, et al. Distribution analysis of deacetylase sirt1 in rodent and human nervous systems. The Anatomical Record. 2010;293: 1024-1032. DOI: 10.1002/ar.21116

[8] Moschen AR, Wieser V, Gerner RR, Bichler A, Enrich B, Moser P, et al. Adipose tissue and liver expression of sirt1, 3, and 6 increase after extensive weight loss in morbid obesity. Journal of Hepatology. 2013;59:1315-1322. DOI: 10.1016/j.jhep.2013.07.027

[9] Caton PW, Richardson SJ, Kieswich J, Bugliani M, Holland ML, Marchetti P, et al. Sirtuin 3 regulates mouse pancreatic beta cell function and is suppressed in pancreatic islets isolated from human type 2 diabetic patients. Diabetologia. 2013;56:1068-1077. DOI: 10.1007/s00125-013-2851-y

[10] Acs Z, Bori Z, Takeda M, Osvath P, Berkes I, Taylor AW, et al. High altitude exposure alters gene expression levels of DNA repair enzymes, and modulates fatty acid metabolism by sirt4 induction in human skeletal muscle. Respiratory Physiology & Neurobiology. 2014;196: 33-37. DOI: 10.1016/j.resp.2014.02.006

[11] Chalkiadaki A, Guarente L. Sirtuins mediate mammalian metabolic responses to nutrient availability. Nature Reviews Endocrinology. 2012;8: 287-296. DOI: 10.1038/nrendo.2011.225

[12] Kurylowicz A, Owczarz M, Polosak J, Jonas MI, Lisik W, Jonas M, et al. sirt1 and sirt7 expression in adipose tissues of obese and normalweight individuals is regulated by microRNAs but not by methylation status. International Journal of Obesity. 2016;40:1635-1642. DOI: 10.1038/ ijo.2016.131

[13] Zwick RK, Guerrero-Juarez CF, Horsley V, Plikus MV. Anatomical, physiological, and functional diversity of adipose tissue. Cell Metabolism. 2018; 27:68-83. DOI: 10.1016/j.cmet.2017. 12.002

[14] Jing E, Gesta S, Kahn CR. sirt2 regulates adipocyte differentiation through FoxO1 acetylation/ deacetylation. Cell Metabolism. 2007;6: 105-114. DOI: 10.1016/j.cmet.2007. 07.003

Author details

Adipose Tissue - An Update

Alina Kurylowicz

22

Polish Academy of Sciences, Warsaw, Poland

provided the original work is properly cited.

\*Address all correspondence to: akurylowicz@imdik.pan.pl

Department of Human Epigenetics, Mossakowski Medical Research Centre,

© 2019 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,

[15] Picard F, Kurtev M, Chung N, Topark-Ngarm A, Senawong T, Machado De Oliveira R, et al. Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-gamma. Nature. 2004;429:771-776. DOI: 10.1038/nature02583

[16] Puri N, Sodhi K, Haarstad M, Kim DH, Bohinc S, Foglio E, et al. Heme induced oxidative stress attenuates sirtuin1 and enhances adipogenesis in mesenchymal stem cells and mouse preadipocytes. Journal of Cellular Biochemistry. 2012;113:1926-1935. DOI: 10.1002/jcb.24061

[17] Zhou Y, Zhou Z, Zhang W, Hu X, Wei H, Peng J, et al. sirt1 inhibits adipogenesis and promotes myogenic differentiation in C3H10T1/2 pluripotent cells by regulating Wnt signaling. Cell & Bioscience. 2015;5:61. DOI: 10.1186/s13578-015-0055-5

[18] Ahn J, Lee H, Jung CH, Jeon TI, Ha TY. MicroRNA-146b promotes adipogenesis by suppressing the sirt1- FOXO1 cascade. EMBO Molecular Medicine. 2013;5:1602-1612. DOI: 10.1002/emmm.201302647

[19] Wang F, Tong Q. sirt2 suppresses adipocyte differentiation by deacetylating FOXO1 and enhancing FOXO1's repressive interaction with PPARgamma. Molecular Biology of the Cell. 2009;20:801-808. DOI: 10.1091/ mbc.E08-06-0647

[20] Wu YT, Chi KT, Lan YW, Chan JC, Ma YS, Wei YH. Depletion of Sirt3 leads to the impairment of adipogenic differentiation and insulin resistance via interfering mitochondrial function of adipose-derived human mesenchymal stem cells. Free Radical Research. 2018; 52:1398-1415. DOI: 10.1080/ 10715762.2018.1489130

[21] Hong J, Li S, Wang X, Mei C, Zan L. Study of expression analysis of sirt4 and the coordinate regulation of bovine adipocyte differentiation by sirt4 and its transcription factors. Bioscience Reports. 2018;38(6):pii: BSR20181705. DOI: 10.1042/BSR20181705

localization, and tissue distribution of the longer form of mouse sirt3. Protein Science. 2009;18:514-525. DOI: 10.1002/

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Role of Sirtuins in Adipose Tissue Development and Metabolism

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Antagonistic crosstalk between NF-κB

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[43] Nasrin N, Wu X, Fortier E, Feng Y, Bare' OC, Chen S, et al. sirt4 regulates fatty acid oxidation and mitochondrial gene expression in liver and muscle cells. The Journal of Biological

Chemistry. 2010;285:31995-32002. DOI:

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[46] Cioffi M, Vallespinos-Serrano M, Trabulo SM, Fernandez-Marcos PJ, Firment AN, Vazquez BN, et al. MiR-93 controls adiposity via inhibition of Sirt7

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molcel.2013.06.001

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2009.00544.x

celrep.2015.08.006

712-721. DOI: 10.1016/j. cmet.2014.03.006

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26

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[62] Timmers S, Konings E, Bilet L, Houtkooper RH, van de Weijer T, Goossens GH, et al. Calorie restrictionlike effects of 30 days of resveratrol supplementation on energy metabolism and metabolic profile in obese humans. Cell Metabolism. 2011;14:612-622. DOI: 10.1016/j.cmet.2011.10.002

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[66] Feige JN, Lagouge M, Canto C, Strehle A, Houten SM, Milne JC, et al. Specific sirt1 activation mimics low energy levels and protects against dietinduced metabolic disorders by enhancing fat oxidation. Cell Metabolism. 2008;8:347-538. DOI: 10.1016/j.cmet.2008.08.017

[67] Yamazaki Y, Usui I, Kanatani Y, Matsuya Y, Tsuneyama K, Fujisaka S, et al. Treatment with SRT1720, a sirt1 activator, ameliorates fatty liver with reduced expression of lipogenic enzymes in MSG mice. American Journal of Physiology. Endocrinology and Metabolism. 2009;297:E1179-E1186. DOI: 10.1152/ajpendo.90997.2008

[68] Pacholec M, Bleasdale JE, Chrunyk B, Cunningham D, Flynn D, Garofalo RS, et al. SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of sirt1. The Journal of Biological Chemistry. 2010;285: 8340-8351. DOI: 10.1074/jbc. M109.088682

[69] Cantó C, Gerhart-Hines Z, Feige JN, Lagouge M, Noriega L, Milne JC, et al. AMPK regulates energy expenditure by modulating NAD+ metabolism and sirt1

#### Adipose Tissue - An Update

activity. Nature. 2009;458:1056-1060. DOI: 10.1038/nature07813

[70] Dai H, Sinclair DA, Ellis JL, Steegborn C. Sirtuin activators and inhibitors: Promises, achievements, and challenges. Pharmacology & Therapeutics. 2018;188:140-154. DOI: 10.1016/j.pharmthera.2018.03.004

[71] Villalba JM, Alcaín FJ. Sirtuin activators and inhibitors. BioFactors. 2012;38:349-359. DOI: 10.1002/ biof.1032

[72] Kuryłowicz A, Wicik Z, Owczarz M, Jonas MI, Kotlarek M, Świerniak M, et al. NGS reveals molecular pathways affected by obesity and weight lossrelated changes in miRNA levels in adipose tissue. International Journal of Molecular Sciences. 2017;19:E66. DOI: 10.3390/ijms19010066

**29**

**1. Introduction**

**Chapter 3**

Novel Aspects of Follistatin/

Transforming Growth Factor-β

(TGF-β) Signaling in Adipose

*Shehla Pervin, Wilson Nyah, Srinivasa T. Reddy* 

Metabolic Health

obesity and related metabolic syndromes.

*and Rajan Singh*

**Abstract**

Tissue Metabolism: Implications in

Obesity is a major risk factor for several metabolic disorders including insulin resistance, diabetes, and cardiovascular diseases. Chronic imbalance of calorie intake and expenditure results in storage of excess unused energy resulting in obesity and related metabolic dysfunctions. While most obesity therapies are focused on reducing the calorie intake and exercise, recent studies suggest that targeting cellular energy expenditure could be a fascinating alternative approach. Brown adipose tissue (BAT) not only has a remarkable calorie burning capacity, but it could also promote triglyceride clearance and glucose disposal. Induction of brown adipose mass and activity in relevant tissues are linked to relieve symptoms of various metabolic disorders such as diabetes, insulin resistance, and cardiovascular diseases. Follistatin (Fst), an extracellular protein that binds and antagonizes several members of the transforming growth factor beta (TGF-β)/myostatin (Mst) superfamily, promotes brown adipose characteristics in both white and brown adipose tissues by targeting distinct molecular pathways. Inhibition of Mst, on the other hand, leads to significant upregulation of adipose browning in white adipose tissues. This chapter will summarize most recent developments in targeting adipose tissue and their functional characteristics to explore therapeutic potential of Fst and TGF-β/Mst signaling to modulate adipose tissue metabolic functions to combat

**Keywords:** adipocyte, follistatin, myostatin, transforming growth factor beta, adipose browning, uncoupling protein 1, thermogenesis, insulin sensitivity

Obesity is a global health problem that results from chronic imbalance between energy intake and its expenditure. Obesity is a major risk factor for several metabolic diseases including diabetes, dyslipidemia, insulin resistance, cardiovascular diseases, nonalcoholic fatty liver, and even some form of cancer. According to most recent global estimates, by year 2030 roughly 2.16 billion individuals will be obese

#### **Chapter 3**

activity. Nature. 2009;458:1056-1060.

Therapeutics. 2018;188:140-154. DOI: 10.1016/j.pharmthera.2018.03.004

[72] Kuryłowicz A, Wicik Z, Owczarz M, Jonas MI, Kotlarek M, Świerniak M, et al. NGS reveals molecular pathways affected by obesity and weight lossrelated changes in miRNA levels in adipose tissue. International Journal of Molecular Sciences. 2017;19:E66. DOI:

[71] Villalba JM, Alcaín FJ. Sirtuin activators and inhibitors. BioFactors. 2012;38:349-359. DOI: 10.1002/

DOI: 10.1038/nature07813

Adipose Tissue - An Update

[70] Dai H, Sinclair DA, Ellis JL, Steegborn C. Sirtuin activators and inhibitors: Promises, achievements, and

challenges. Pharmacology &

10.3390/ijms19010066

biof.1032

28

## Novel Aspects of Follistatin/ Transforming Growth Factor-β (TGF-β) Signaling in Adipose Tissue Metabolism: Implications in Metabolic Health

*Shehla Pervin, Wilson Nyah, Srinivasa T. Reddy and Rajan Singh*

#### **Abstract**

Obesity is a major risk factor for several metabolic disorders including insulin resistance, diabetes, and cardiovascular diseases. Chronic imbalance of calorie intake and expenditure results in storage of excess unused energy resulting in obesity and related metabolic dysfunctions. While most obesity therapies are focused on reducing the calorie intake and exercise, recent studies suggest that targeting cellular energy expenditure could be a fascinating alternative approach. Brown adipose tissue (BAT) not only has a remarkable calorie burning capacity, but it could also promote triglyceride clearance and glucose disposal. Induction of brown adipose mass and activity in relevant tissues are linked to relieve symptoms of various metabolic disorders such as diabetes, insulin resistance, and cardiovascular diseases. Follistatin (Fst), an extracellular protein that binds and antagonizes several members of the transforming growth factor beta (TGF-β)/myostatin (Mst) superfamily, promotes brown adipose characteristics in both white and brown adipose tissues by targeting distinct molecular pathways. Inhibition of Mst, on the other hand, leads to significant upregulation of adipose browning in white adipose tissues. This chapter will summarize most recent developments in targeting adipose tissue and their functional characteristics to explore therapeutic potential of Fst and TGF-β/Mst signaling to modulate adipose tissue metabolic functions to combat obesity and related metabolic syndromes.

**Keywords:** adipocyte, follistatin, myostatin, transforming growth factor beta, adipose browning, uncoupling protein 1, thermogenesis, insulin sensitivity

#### **1. Introduction**

Obesity is a global health problem that results from chronic imbalance between energy intake and its expenditure. Obesity is a major risk factor for several metabolic diseases including diabetes, dyslipidemia, insulin resistance, cardiovascular diseases, nonalcoholic fatty liver, and even some form of cancer. According to most recent global estimates, by year 2030 roughly 2.16 billion individuals will be obese

as defined by body mass index (BMI) of 30 or higher [1]. The economic impact of obesity and related metabolic complications has been estimated between 4 and 8% of gross domestic product which is comparable to 2018 defense budget (\$643 billion) and Medicare (\$588 billion) in the United States [2]. Thus the toll of obesity imposes massive and rapidly growing economic cost beyond human suffering. This economic burden of obesity, therefore, significantly impacts low-income and otherwise disadvantaged population. Staying physically active and maintaining a healthy diet are well accepted and proven strategies to prevent weight gain; however, an alarming increase of global obesity urgently requires the development of novel and highly effective anti-obesity therapies. According to the laws of thermodynamics, any treatment for obesity must require reduced energy intake, increased energy expenditure, or both. Recent data suggest that targeting cellular bioenergetics may provide attractive therapeutic avenues for the treatment and prevention of obesity. White adipose tissue (WAT) and brown adipose tissue (BAT) are two distinct adipose tissue types present in mammals. While WAT with larger unilocular lipid droplets store excess energy in the form of triglycerides, BAT consisting of multilocular smaller lipid droplets enriched with mitochondria that express uncoupling protein 1 (UCP1) has specialized capacity to dissipate excess energy via activating non-shivering thermogenesis. Pockets of UCP1-positive adipocytes have also been found within WAT depots which are called beige or brite (brown within white) adipocytes. These beige adipocytes show some morphological and functional similarities to classical brown adipocytes present with the BAT. Several molecular signaling pathways are reported to play significant roles in the development and differentiation of these white, beige, and brown adipose cells. Transforming growth factor beta (TGF-β) controls the development, growth, and cellular functions of diverse cell types by transmitting signals via dual serine/threonine kinase receptors and transcription factors called Smads, especially Smad3. TGF-β expression levels are significantly elevated in adipose tissues from obese mice [3], and blocking of TGF-β/Smad3 signaling results in protection from obesity and diabetes. These metabolic benefits are associated with increased appearance of brown-like adipocytes within the WAT [4]. Inactivation of myostatin (Mst) also called growth and differentiation factor 8 (GDF8), a key member of the TGF-β superfamily in both differentiating mouse embryonic fibroblast (MEF) primary cultures from wild type (WT) and Mst knockout (Mst KO) embryos, as well as in white adipose tissues of Mst KO mouse models, displays beige adipocyte phenotype and upregulation of key beige markers compared to the wild type [5]. Blockade of activin receptor IIB (ActRIIB) that integrates the actions of Mst and TGF-β-related ligands has been demonstrated to activate functional brown adipogenesis and thermogenesis [6]. Inhibition of Smad3 signaling, which has been identified as canonical pathway for Mst, induced WAT browning [7]. It therefore suggests that antagonizing TGF-β/Smad3/Mst signaling pathway would lead to significant favorable metabolic alterations by promoting adipose browning. Since follistatin (Fst) is a well-known inhibitor of TGF-β signaling pathway in a variety of cell lines [8–10], and a key antagonist of Mst, Braga et al. [11] hypothesized that Fst may promote browning of white adipocytes, and using differentiating MEF primary cultures from WT and Fst KO embryos provided the first evidence that Fst is a novel inducer of brown adipose characteristics. Subsequent studies using Fst-transgenic (Fst-Tg) mice overexpressing Fst under the control of skeletal muscle-specific myosin light chain promoter, the authors demonstrated that Fst targets distinct pathways to promote brown adipose characteristics in both BAT and WAT [11]. Combined together, these findings support the idea that targeting TGF-β/Smad3/Mst signaling either via direct genetic or pharmacological inhibition of this pathways or via directly upregulating Fst could be attractive therapeutic options for the treatment of obesity and related metabolic diseases.

**31**

distinct cellular origins.

*Novel Aspects of Follistatin/Transforming Growth Factor-β (TGF-β) Signaling in Adipose…*

**2. Developmental origin, transcriptional regulators, and molecular** 

Although white and brown adipocytes share many common features such as PPAR-γ-driven transcriptional control of adipogenesis, their gene expression profiles are distinct, and they do not share a direct common progenitor. Recent genetic studies using fat-mapping experiments have shown that while brown fat in the interscapular region and skeletal muscle share some common features and are derived from Myf5 expressing (Myf5+) cells (previously assumed to be exclusively present in committed skeletal muscle precursors), these Myf5+ precursor cells were absent in white and beige cells [12, 13]. Studies from several other laboratories using global gene expression as well as mitochondrial proteomics signature confirmed that BAT is highly related to the skeletal muscle and not WAT [14, 15]. The divergence of brown adipocyte precursor and skeletal muscle was investigated using Pax7, another myogenic marker in pulse-chase experiments, and it was reported that this divergence occurred between embryonic day 9.5 and 11.5 in mice [16]. UCP1-expressing beige adipocytes present in epididymal WAT (Epi WAT) are thought to be derived through the proliferation and differentiation of platelet-derived growth factor receptor α (PDGRF α), CD44, and SC1 precursor cells [17]. Beige or brown-like cells in the inguinal WAT, on the other hand, are suggested to be derived from Myf5-negative (Myf5-) precursor cells [12]. However, this view has recently been challenged by various groups on the basis of linage analysis studies that suggest that subsets of white adipocytes are derived from both Myf5+ and Myf5- precursors and respond to beta-3 adrenergic receptor (β3-AR) signaling suggesting that these beige adipocytes may have multiple origins [18–20]. A subset of UCP1-positive beige adipocytes is also recently reported to arise from Myh11, selectively expressed in smooth muscle cells [21]. It is also possible that beige cells can either originate from mesodermal stem cells or trans-differentiation of mature white adipocytes [22]. Beige cells may also originate from Ebf2+ precursors located in the subcutaneous adipose tissue (SAT) population characterized by specific markers Cd137 and Tmeme26 [23]. Furthermore, it is also possible that thermogenic adipocytes may arise from endothelial cells and capillaries where retinoic acid (RA) could induce adipose browning by activating VEGF signaling pathways [24]. RA is known to trigger angiogenesis and facilitate de novo generation of *Pdgrfα* expressing adipocyte precursors mediated via VEGFA/ VEGFR2 signaling [25]. These findings, therefore, collectively suggest that beige adipocytes which are composed of heterogeneous cell populations may have

The acquisition of morphological and molecular features of brown and beige fat is under the control of PPARγ-coactivator 1α (PGC-1α) [26]. PGC-1α is induced early in brown fat differentiation and is preferentially expressed in mature brown adipocytes. PGC1-1α ectopic expression is sufficient to promote various aspects of differentiation toward the brown fat lineage. PGC-1α is also rapidly and highly induced by cold exposure and turns on several key components of the adaptive thermogenic program including fatty acid oxidation, mitochondrial biogenesis, and increased oxygen consumption [27]. The expression levels of 140kD zinc figure containing transcription factor called PR domain containing 16 (PRDM16) are very high in BAT compared to the visceral WAT and appear to play a major role in brown adipose/skeletal muscle fate determination [28]. Ectopic expression of PRDM16 in cultured mesenchymal cells including white preadipocytes induced a complete brown fat differentiation program and activation of key thermogenic (*Ucp1*, *Pgc-1α*, *cidea*, and *elov3*) genes and coactivates the transcriptional activity

*DOI: http://dx.doi.org/10.5772/intechopen.88294*

**signature of beige and brown adipocytes**

*Novel Aspects of Follistatin/Transforming Growth Factor-β (TGF-β) Signaling in Adipose… DOI: http://dx.doi.org/10.5772/intechopen.88294*

#### **2. Developmental origin, transcriptional regulators, and molecular signature of beige and brown adipocytes**

Although white and brown adipocytes share many common features such as PPAR-γ-driven transcriptional control of adipogenesis, their gene expression profiles are distinct, and they do not share a direct common progenitor. Recent genetic studies using fat-mapping experiments have shown that while brown fat in the interscapular region and skeletal muscle share some common features and are derived from Myf5 expressing (Myf5+) cells (previously assumed to be exclusively present in committed skeletal muscle precursors), these Myf5+ precursor cells were absent in white and beige cells [12, 13]. Studies from several other laboratories using global gene expression as well as mitochondrial proteomics signature confirmed that BAT is highly related to the skeletal muscle and not WAT [14, 15]. The divergence of brown adipocyte precursor and skeletal muscle was investigated using Pax7, another myogenic marker in pulse-chase experiments, and it was reported that this divergence occurred between embryonic day 9.5 and 11.5 in mice [16]. UCP1-expressing beige adipocytes present in epididymal WAT (Epi WAT) are thought to be derived through the proliferation and differentiation of platelet-derived growth factor receptor α (PDGRF α), CD44, and SC1 precursor cells [17]. Beige or brown-like cells in the inguinal WAT, on the other hand, are suggested to be derived from Myf5-negative (Myf5-) precursor cells [12]. However, this view has recently been challenged by various groups on the basis of linage analysis studies that suggest that subsets of white adipocytes are derived from both Myf5+ and Myf5- precursors and respond to beta-3 adrenergic receptor (β3-AR) signaling suggesting that these beige adipocytes may have multiple origins [18–20]. A subset of UCP1-positive beige adipocytes is also recently reported to arise from Myh11, selectively expressed in smooth muscle cells [21]. It is also possible that beige cells can either originate from mesodermal stem cells or trans-differentiation of mature white adipocytes [22]. Beige cells may also originate from Ebf2+ precursors located in the subcutaneous adipose tissue (SAT) population characterized by specific markers Cd137 and Tmeme26 [23]. Furthermore, it is also possible that thermogenic adipocytes may arise from endothelial cells and capillaries where retinoic acid (RA) could induce adipose browning by activating VEGF signaling pathways [24]. RA is known to trigger angiogenesis and facilitate de novo generation of *Pdgrfα* expressing adipocyte precursors mediated via VEGFA/ VEGFR2 signaling [25]. These findings, therefore, collectively suggest that beige adipocytes which are composed of heterogeneous cell populations may have distinct cellular origins.

The acquisition of morphological and molecular features of brown and beige fat is under the control of PPARγ-coactivator 1α (PGC-1α) [26]. PGC-1α is induced early in brown fat differentiation and is preferentially expressed in mature brown adipocytes. PGC1-1α ectopic expression is sufficient to promote various aspects of differentiation toward the brown fat lineage. PGC-1α is also rapidly and highly induced by cold exposure and turns on several key components of the adaptive thermogenic program including fatty acid oxidation, mitochondrial biogenesis, and increased oxygen consumption [27]. The expression levels of 140kD zinc figure containing transcription factor called PR domain containing 16 (PRDM16) are very high in BAT compared to the visceral WAT and appear to play a major role in brown adipose/skeletal muscle fate determination [28]. Ectopic expression of PRDM16 in cultured mesenchymal cells including white preadipocytes induced a complete brown fat differentiation program and activation of key thermogenic (*Ucp1*, *Pgc-1α*, *cidea*, and *elov3*) genes and coactivates the transcriptional activity

*Adipose Tissue - An Update*

as defined by body mass index (BMI) of 30 or higher [1]. The economic impact of obesity and related metabolic complications has been estimated between 4 and 8% of gross domestic product which is comparable to 2018 defense budget (\$643 billion) and Medicare (\$588 billion) in the United States [2]. Thus the toll of obesity imposes massive and rapidly growing economic cost beyond human suffering. This economic burden of obesity, therefore, significantly impacts low-income and otherwise disadvantaged population. Staying physically active and maintaining a healthy diet are well accepted and proven strategies to prevent weight gain; however, an alarming increase of global obesity urgently requires the development of novel and highly effective anti-obesity therapies. According to the laws of thermodynamics, any treatment for obesity must require reduced energy intake, increased energy expenditure, or both. Recent data suggest that targeting cellular bioenergetics may provide attractive therapeutic avenues for the treatment and prevention of obesity. White adipose tissue (WAT) and brown adipose tissue (BAT) are two distinct adipose tissue types present in mammals. While WAT with larger unilocular lipid droplets store excess energy in the form of triglycerides, BAT consisting of multilocular smaller lipid droplets enriched with mitochondria that express uncoupling protein 1 (UCP1) has specialized capacity to dissipate excess energy via activating non-shivering thermogenesis. Pockets of UCP1-positive adipocytes have also been found within WAT depots which are called beige or brite (brown within white) adipocytes. These beige adipocytes show some morphological and functional similarities to classical brown adipocytes present with the BAT. Several molecular signaling pathways are reported to play significant roles in the development and differentiation of these white, beige, and brown adipose cells. Transforming growth factor beta (TGF-β) controls the development, growth, and cellular functions of diverse cell types by transmitting signals via dual serine/threonine kinase receptors and transcription factors called Smads, especially Smad3. TGF-β expression levels are significantly elevated in adipose tissues from obese mice [3], and blocking of TGF-β/Smad3 signaling results in protection from obesity and diabetes. These metabolic benefits are associated with increased appearance of brown-like adipocytes within the WAT [4]. Inactivation of myostatin (Mst) also called growth and differentiation factor 8 (GDF8), a key member of the TGF-β superfamily in both differentiating mouse embryonic fibroblast (MEF) primary cultures from wild type (WT) and Mst knockout (Mst KO) embryos, as well as in white adipose tissues of Mst KO mouse models, displays beige adipocyte phenotype and upregulation of key beige markers compared to the wild type [5]. Blockade of activin receptor IIB (ActRIIB) that integrates the actions of Mst and TGF-β-related ligands has been demonstrated to activate functional brown adipogenesis and thermogenesis [6]. Inhibition of Smad3 signaling, which has been identified as canonical pathway for Mst, induced WAT browning [7]. It therefore suggests that antagonizing TGF-β/Smad3/Mst signaling pathway would lead to significant favorable metabolic alterations by promoting adipose browning. Since follistatin (Fst) is a well-known inhibitor of TGF-β signaling pathway in a variety of cell lines [8–10], and a key antagonist of Mst, Braga et al. [11] hypothesized that Fst may promote browning of white adipocytes, and using differentiating MEF primary cultures from WT and Fst KO embryos provided the first evidence that Fst is a novel inducer of brown adipose characteristics. Subsequent studies using Fst-transgenic (Fst-Tg) mice overexpressing Fst under the control of skeletal muscle-specific myosin light chain promoter, the authors demonstrated that Fst targets distinct pathways to promote brown adipose characteristics in both BAT and WAT [11]. Combined together, these findings support the idea that targeting TGF-β/Smad3/Mst signaling either via direct genetic or pharmacological inhibition of this pathways or via directly upregulating Fst could be attractive therapeutic options for the treatment of

**30**

obesity and related metabolic diseases.

of PGC-1α/PGC-1β, as well as PPARα and PPARγ [12, 28]. Coincident with these changes, PRDM16 expression also led to suppression of several white fat and muscle-selective markers [29]. On the other hand, genetic ablation of PRDM16 in brown fat leads to significant increase in white adipose and muscle-specific genes [28]. These findings, therefore, suggest that PRDM16 acts as a critical cell fate regulator of brown fat, and careful analysis of its embryonic expression pattern will be extremely valuable to dissect out the putative brown fat-skeletal muscle precursors. Signaling molecules that control the timing and specificity of PRDM16 expression during development are unknown. Certain growth factors like bone morphogenetic proteins (BMPs), members of the TGF-β superfamily of secreted factors, are reported to influence both brown and white adipocyte differentiation [30–32]. While BMP2 and BMP4 are reported to promote white adipose cell differentiation, BMP7 is reported to selectively induce brown adipogenesis in committed precursor cells [32, 33]. BMP7 exposure to fibroblast cultures results in induction of full brown fat differentiation program, including induction of PRDM16 and UCP1 expression [30]. Importantly, significantly reduced amounts of BAT mass were observed in BMP7-deficient mice. However, it is not clear whether BMP7 plays any role in the regulation of PRDM16.

Under basal conditions both beige and brown adipocytes share some of the same key markers including UCP1 and PRDM16; however, data from clonal cell lines suggest that beige and brown adipose cells express related but distinctly different gene expression profiles [23]. Beige cells are highly enriched in Tmem26, Tbx1, and CD137 expression [23]. Comprehensive gene expression analysis of adipose tissues isolated from interscapular BAT and inguinal fat revealed several other beige-selective genes including *Ear2*, *Sp100*, *Klh113*, and *Slc27a* [23]. Molecular profiling and histological analysis of human BAT identified additional beige-selective markers HoxC8, HoxC9, Cited1, and Shox2 [34, 35]. On the other hand, classical brown adipocytes selectively express epithelial V-like antigen (Eva 1), Zic1, Lhx1, and Epsti [23, 36–38]. Using adipose tissues isolated from white and interscapular BAT from 129SVE mice, Wu et al. identified additional genes including *Hspb7*, *Ebf3*, *Pdk4*, *Fbxo31*, and *Oplah* that were enriched in BAT [23]. Using a combination of in silico, in vitro, as well as in vivo approaches, Ussar et al. reported the identification of three new cell surface markers of adipose tissues [39]. In this study, amino acid transporter Asc1 was identified as a white adipocyte-specific cell surface protein with very low to undetectable levels in brown adipocytes, whereas amino acid transporter PAT2 and the purinergic receptor P2RX5 are cell surface markers expressed in classical brown and beige adipocytes.

Studies from microRNA (miRNA) signature analysis between beige and brown adipogenesis have provided significant differences in their molecular signature. MiRNA-193b-365 cluster is expressed in brown fat tissues and initially thought to be involved in the regulation of brown fat differentiation by inhibiting Runx1t1, which inhibits BAT differentiation [40]. However, subsequent in vivo studies show a normal BAT function in the absence of miRNA-193b-365 [41]. Inhibition of miRNA-182 and miRNA-203 in brown adipocytes led to downregulation of several genes involved in oxidative phosphorylation and electron transport [42]. Inhibition of miRNA-106b-93 led to induced expression of several adipogenic markers [43]. Similarly, positive (miRNA-196b) and negative (miRNA-26) regulation of beige adipogenesis have been identified [44, 45]. miRNAs that positively (miRNA-30 family) and negatively (miRNA-27 and miRNA-34a) regulate both brown and beige adipocytes are also identified [46–48]. Thus, there appear to be clear differences between BAT and beige miRNA gene signature in mouse and human tissues and cells.

**33**

*Novel Aspects of Follistatin/Transforming Growth Factor-β (TGF-β) Signaling in Adipose…*

**3. Role of transforming growth factor-β (TGF-β) superfamily in adipose** 

The TGF-β superfamily consists of more than 33 members including TGFβ1, TGFβ2, and TGFβ3, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), and activins that play important roles in growth, development, and function of diverse cell types including adipocytes [49, 50]. These evolutionary highly conserved superfamily members transmit their signals via dual serine/threonine kinase receptors and transcription factors called Smads. TGF-β superfamily members control various aspects of adipocyte biology. Adipose tissues from obese mice were reported to express elevated levels of TGF-β [3]. The binding of TGF-β family to their membrane receptors could be somewhat promiscuous and may allow 7 type I and 5 type II receptors to transduce signaling from these TGF-β superfamily members. Multiple cell types including adipose progenitors, preadipocytes, and adipocytes along with various immune cells are known to express protein belonging to TGF-β superfamily and their antagonists [51]. The role of TGF-β/Smad3 signaling in regulating beige adipocyte phenotype and metabolic characteristics were elegantly demonstrated by Yadav et al. [4]. They observed significant positive correlation between TGF-β1 levels and adiposity in both rodents and human subjects. Using Smad3<sup>−</sup>/<sup>−</sup> mice, they provided interesting link between Smad3 loss and protection against diet-induced obesity and related metabolic syndromes. These changes in metabolic parameters were associated with induction of white to brown phenotype and significantly increased mitochondrial biogenesis. In the same study, examination of a total of 184 nondiabetic human subjects from diverse ethnic groups, the authors identified direct relationship between circulating TGF-β1 levels and BMI, fat mass, and VO2 consumption. Furthermore, anti-TGF-β antibody in Lepob/ob and diet-induced obesity mouse models resulted in significantly reduced body weight, improved glucose and insulin tolerance, as well as significantly reduced fasting glucose and insulin levels. These metabolic improvements were associated with elevated expression of BAT/ mitochondria-specific proteins in white adipose tissues. Such links between TGF-β signaling and mitochondrial energy metabolism pathway have also been reported by several other laboratories [52, 53]. Extracellular matrix protein microfibril-associated glycoprotein (MAGP) was found to be significantly altered in obese humans, and inactivation of MAGP1 gene (Mfap2<sup>−</sup>/<sup>−</sup>) resulted in adipocyte hypertrophy and predisposition to metabolic diseases. Mfap2<sup>−</sup>/<sup>−</sup> mice had significantly lower expression of UCP1 expression in BAT and display reduced subcutaneous adipose browning and defective adaptation to cold exposure [53]. Treatment of these Mfap2<sup>−</sup>/<sup>−</sup> mice with neutralizing concentrations of anti-TGF-β antibody led to decreased adiposity and improved body temperature. Administration of a novel activin receptor type II B (ActRIIB) decoy receptor containing the extracellular domain of ActRIIB fused to human Fc (ActRIIB-Fc) resulted in suppression of diet-induced obesity and associated metabolic functions in mice [54]. In the same study, significantly increased adipose browning in epididymal white fat displaying robustly increased expression of UCP1 and PGC 1-α was observed following ActRIIB-Fc treatment. Furthermore, protection from diet-induced obesity in ActRIIB-Fc-treated mice was demonstrated to result from increased energy expenditure and not decreased caloric intake. Combined together, these interesting findings suggest novel insights into the role of TGF-β signaling in suppressing adipose browning program within white fat tissues in both mouse models and human subjects suggesting that efficient blockade of TGF-β activity could serve as an effective treatment strategy for obesity and diabetes.

Myostatin (Mst) is a key member of the TGF-β superfamily which is known to play a major role in the regulation of skeletal muscle growth. However, recent studies have clearly indicated that the effect of Mst extends beyond its role in skeletal

*DOI: http://dx.doi.org/10.5772/intechopen.88294*

**browning and metabolic health**

*Novel Aspects of Follistatin/Transforming Growth Factor-β (TGF-β) Signaling in Adipose… DOI: http://dx.doi.org/10.5772/intechopen.88294*

#### **3. Role of transforming growth factor-β (TGF-β) superfamily in adipose browning and metabolic health**

The TGF-β superfamily consists of more than 33 members including TGFβ1, TGFβ2, and TGFβ3, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), and activins that play important roles in growth, development, and function of diverse cell types including adipocytes [49, 50]. These evolutionary highly conserved superfamily members transmit their signals via dual serine/threonine kinase receptors and transcription factors called Smads. TGF-β superfamily members control various aspects of adipocyte biology. Adipose tissues from obese mice were reported to express elevated levels of TGF-β [3]. The binding of TGF-β family to their membrane receptors could be somewhat promiscuous and may allow 7 type I and 5 type II receptors to transduce signaling from these TGF-β superfamily members. Multiple cell types including adipose progenitors, preadipocytes, and adipocytes along with various immune cells are known to express protein belonging to TGF-β superfamily and their antagonists [51]. The role of TGF-β/Smad3 signaling in regulating beige adipocyte phenotype and metabolic characteristics were elegantly demonstrated by Yadav et al. [4]. They observed significant positive correlation between TGF-β1 levels and adiposity in both rodents and human subjects. Using Smad3<sup>−</sup>/<sup>−</sup> mice, they provided interesting link between Smad3 loss and protection against diet-induced obesity and related metabolic syndromes. These changes in metabolic parameters were associated with induction of white to brown phenotype and significantly increased mitochondrial biogenesis. In the same study, examination of a total of 184 nondiabetic human subjects from diverse ethnic groups, the authors identified direct relationship between circulating TGF-β1 levels and BMI, fat mass, and VO2 consumption. Furthermore, anti-TGF-β antibody in Lepob/ob and diet-induced obesity mouse models resulted in significantly reduced body weight, improved glucose and insulin tolerance, as well as significantly reduced fasting glucose and insulin levels. These metabolic improvements were associated with elevated expression of BAT/ mitochondria-specific proteins in white adipose tissues. Such links between TGF-β signaling and mitochondrial energy metabolism pathway have also been reported by several other laboratories [52, 53]. Extracellular matrix protein microfibril-associated glycoprotein (MAGP) was found to be significantly altered in obese humans, and inactivation of MAGP1 gene (Mfap2<sup>−</sup>/<sup>−</sup>) resulted in adipocyte hypertrophy and predisposition to metabolic diseases. Mfap2<sup>−</sup>/<sup>−</sup> mice had significantly lower expression of UCP1 expression in BAT and display reduced subcutaneous adipose browning and defective adaptation to cold exposure [53]. Treatment of these Mfap2<sup>−</sup>/<sup>−</sup> mice with neutralizing concentrations of anti-TGF-β antibody led to decreased adiposity and improved body temperature. Administration of a novel activin receptor type II B (ActRIIB) decoy receptor containing the extracellular domain of ActRIIB fused to human Fc (ActRIIB-Fc) resulted in suppression of diet-induced obesity and associated metabolic functions in mice [54]. In the same study, significantly increased adipose browning in epididymal white fat displaying robustly increased expression of UCP1 and PGC 1-α was observed following ActRIIB-Fc treatment. Furthermore, protection from diet-induced obesity in ActRIIB-Fc-treated mice was demonstrated to result from increased energy expenditure and not decreased caloric intake. Combined together, these interesting findings suggest novel insights into the role of TGF-β signaling in suppressing adipose browning program within white fat tissues in both mouse models and human subjects suggesting that efficient blockade of TGF-β activity could serve as an effective treatment strategy for obesity and diabetes.

Myostatin (Mst) is a key member of the TGF-β superfamily which is known to play a major role in the regulation of skeletal muscle growth. However, recent studies have clearly indicated that the effect of Mst extends beyond its role in skeletal

*Adipose Tissue - An Update*

role in the regulation of PRDM16.

classical brown and beige adipocytes.

of PGC-1α/PGC-1β, as well as PPARα and PPARγ [12, 28]. Coincident with these changes, PRDM16 expression also led to suppression of several white fat and muscle-selective markers [29]. On the other hand, genetic ablation of PRDM16 in brown fat leads to significant increase in white adipose and muscle-specific genes [28]. These findings, therefore, suggest that PRDM16 acts as a critical cell fate regulator of brown fat, and careful analysis of its embryonic expression pattern will be extremely valuable to dissect out the putative brown fat-skeletal muscle precursors. Signaling molecules that control the timing and specificity of PRDM16 expression during development are unknown. Certain growth factors like bone morphogenetic proteins (BMPs), members of the TGF-β superfamily of secreted factors, are reported to influence both brown and white adipocyte differentiation [30–32]. While BMP2 and BMP4 are reported to promote white adipose cell differentiation, BMP7 is reported to selectively induce brown adipogenesis in committed precursor cells [32, 33]. BMP7 exposure to fibroblast cultures results in induction of full brown fat differentiation program, including induction of PRDM16 and UCP1 expression [30]. Importantly, significantly reduced amounts of BAT mass were observed in BMP7-deficient mice. However, it is not clear whether BMP7 plays any

Under basal conditions both beige and brown adipocytes share some of the same key markers including UCP1 and PRDM16; however, data from clonal cell lines suggest that beige and brown adipose cells express related but distinctly different gene expression profiles [23]. Beige cells are highly enriched in Tmem26, Tbx1, and CD137 expression [23]. Comprehensive gene expression analysis of adipose tissues isolated from interscapular BAT and inguinal fat revealed several other beige-selective genes including *Ear2*, *Sp100*, *Klh113*, and *Slc27a* [23]. Molecular profiling and histological analysis of human BAT identified additional beige-selective markers HoxC8, HoxC9, Cited1, and Shox2 [34, 35]. On the other hand, classical brown adipocytes selectively express epithelial V-like antigen (Eva 1), Zic1, Lhx1, and Epsti [23, 36–38]. Using adipose tissues isolated from white and interscapular BAT from 129SVE mice, Wu et al. identified additional genes including *Hspb7*, *Ebf3*, *Pdk4*, *Fbxo31*, and *Oplah* that were enriched in BAT [23]. Using a combination of in silico, in vitro, as well as in vivo approaches, Ussar et al. reported the identification of three new cell surface markers of adipose tissues [39]. In this study, amino acid transporter Asc1 was identified as a white adipocyte-specific cell surface protein with very low to undetectable levels in brown adipocytes, whereas amino acid transporter PAT2 and the purinergic receptor P2RX5 are cell surface markers expressed in

Studies from microRNA (miRNA) signature analysis between beige and brown adipogenesis have provided significant differences in their molecular signature. MiRNA-193b-365 cluster is expressed in brown fat tissues and initially thought to be involved in the regulation of brown fat differentiation by inhibiting Runx1t1, which inhibits BAT differentiation [40]. However, subsequent in vivo studies show a normal BAT function in the absence of miRNA-193b-365 [41]. Inhibition of miRNA-182 and miRNA-203 in brown adipocytes led to downregulation of several genes involved in oxidative phosphorylation and electron transport [42]. Inhibition of miRNA-106b-93 led to induced expression of several adipogenic markers [43]. Similarly, positive (miRNA-196b) and negative (miRNA-26) regulation of beige adipogenesis have been identified [44, 45]. miRNAs that positively (miRNA-30 family) and negatively (miRNA-27 and miRNA-34a) regulate both brown and beige adipocytes are also identified [46–48]. Thus, there appear to be clear differences between BAT and beige miRNA gene signature in mouse and human tissues

**32**

and cells.

muscle. Genetic deletion of Mst displays favorable changes in several metabolic parameters including decreased fat deposition, enhanced fatty acid oxidation, improved insulin sensitivity, and increased resistance to diet-induced obesity besides increased skeletal muscle mass [55, 56]. Since Mst is expressed at very low levels in adipose tissues [57], it remains unclear how depletion of Mst can suppress fat accumulation. Earlier studies by Kim et al. show that treatment of mouse primary brown preadipocytes with recombinant Mst led to significant inhibition of brown adipogenic differentiation and reduced expression of markers *Ucp1*, *Prdm16*, and *Pgc-1α* [58]. A comparison of key thermogenic markers obtained from epididymal (Epi) and subcutaneous (SC) white adipose tissues shows significantly increased expression of UCP1 and PRDM16 in Mst KO mice compared to the WT littermates [54]. Using differentiating primary cultures isolated from WT and Mst KO mouse embryonic fibroblasts (MEFs) in the same study, Braga et al. further confirmed upregulation of key thermogenic markers in Mst KO mice compared to the WT mice [5]. Furthermore, recombinant Mst protein treatment of the differentiating MEFs significantly downregulated several key thermogenic markers including UCP1, PRDM16, PGC-1α/PGC-1β, and BMP7. Also, protein expression of adiponectin and phosphorylated AMP-activated protein kinase (pAMPK), which control the expression of genes involved in energy metabolism in coordination with NAD+-dependent sirtuin 1 (SirT1), were upregulated in Mst KO MEFs compared to the WT group [59, 60]. In another study, Chio et al. reported significantly increased energy expenditure and leptin sensitivity in Mst-deficient mice that could explain low fat mass in these mice compared to the WT group [61]. Shan et al. demonstrated that inhibition of Mst signaling in WAT SVF cells failed to induce browning of white adipocytes in vitro, suggesting that loss or inhibition of Mst signaling in preadipocytes does not account for adipose browning in white adipocyte tissues in Mst KO mice [62]. In order to test the possible non-cell autonomous effects of Mst, the authors thoroughly analyzed various muscle-derived circulating factors that could account for the browning phenotype. They reported that skeletal muscle-derived Fndc5 (irisin) plays a central role in mediating white adipose browning in Mst KO mice via activation of AMPK-PGC1α-Fndc5 pathway, suggesting the involvement of muscle-adipose cross talk during the process [63, 64]. Fndc5/irisin was initially identified as a PGC-1α-dependent myokine that is responsible for adipose browning both in vitro and in vivo and protects diet-induced obesity in obesity [65]. Possible involvement of Fndc5 in mediating adipose browning in Mst loss-of-function models has emerged from other laboratories. Dong et al. also confirmed possible intermediate role for Fndc5/irisin-mediated adipose browning in Mst KO mice. In another study, Mst signaling was shown to regulate Fndc5 expression and adipose browning via upregulation of miRNA-34a. Several laboratories have demonstrated that the absence of Mst in both in vitro and in vivo models improves insulin sensitivity [58, 66]. Several other laboratories provided additional evidence to support the view that Mst loss-of-function results in significant metabolic improvements resulting from adipose browning. Increased insulin sensitivity and WAT browning were reported in Meishan pigs with Mst functional deletion [67]. Several browningrelated genes including UCP1, PGC-1α, PRDM16, Cidea, CD137, and Tmem26 were significantly upregulated in these Mst-deficient pigs. Protein expression levels of insulin receptor (IR) and insulin receptor substrate (IRS) were significantly induced in the skeletal muscle of these Mst-deficient pigs. Interestingly, serum irisin levels and skeletal muscle protein expression of irisin precursor protein FNDC5 were significantly higher in Mst-null pigs than wild-type pigs. These authors also demonstrated that inhibition of irisin expression was unable to block the activation of insulin signaling pathway, thus, implying that irisin may not be required for activation of insulin signaling in Mst-deficient skeletal muscle [67]. Genetic

**35**

*Novel Aspects of Follistatin/Transforming Growth Factor-β (TGF-β) Signaling in Adipose…*

disruption of Mst (Mst<sup>−</sup>/<sup>−</sup>) in LDLR<sup>−</sup>/<sup>−</sup> (Mst<sup>−</sup>/<sup>−</sup>/LDLR<sup>−</sup>/<sup>−</sup>) mice was shown to reduce the development of proatherogenic dyslipidemia, improve insulin-mediated glucose disposal, and protect against hepatic steatosis [68]. Furthermore, Guo et al. demonstrated that administration of adeno-associated virus 9 (AAV9)-mediated Mst-pro-peptide in adult LDLR<sup>−</sup>/<sup>−</sup> mice reduced diet-induced hepatosteatosis and progression of atherosclerosis [69]. In both these reports, the beneficial metabolic effects were claimed to result from enlarged muscle mass following inactivation of functional Mst in LDLR<sup>−</sup>/<sup>−</sup> mice. Several recent reports have provided strong evidence suggesting that brown fat activation could reduce hypercholesterolemia and protect from atherosclerosis development [70–72]. Therefore, it is possible that observed beneficial effects of Mst inactivation in LDLR<sup>−</sup>/<sup>−</sup> background could be

More recently, Mst expression has been linked to mediate BAT-muscle cross talk [73]. Induction of Mst following loss of interferon regulatory factor 4 (IRF4) in BAT leads to significantly reduced exercise capacity, ribosomal protein synthesis, and mitochondrial function [73]. On the other hand, reduced serum levels of Mst resulting from IRF4 overexpression significantly increased exercise capacity in muscle. IRF4 expression was found to be induced in brown adipocytes following cold exposure and β3-adrenergic receptor (AR) agonist [74]. IRF4 expression was reported to be sufficient to induce thermogenic program in BAT, and loss of IRF4 in brown fat leads to significantly reduced energy expenditure. Also, IRF4 was shown to physically and functionally interact with PGC-1α to upregulate transcription of *Ucp1* gene and drive mitochondrial biogenesis and thermogenic program in BAT. In light of these exciting reports establishing IRF4 as a novel inhibitor of Mst, it is not surprising that IRF4 could antagonize the bioactivity of secreted Mst present in the

**4. Follistatin regulation of white and brown adipose characteristics**

Follistatin (Fst) is a soluble secreted glycoprotein that is known to bind and neutralize the activity of several members of the TGF-β superfamily including activins and Mst in a variety of cell lines [9–10, 77]. Several genetic studies have convincingly demonstrated an essential role of Fst in the regulation of muscle mass. Elegant initial studies led by Lee and McPherron demonstrated that inhibition of Mst either by genetic manipulation or overexpressing Fst resulted in significantly increased muscle mass in mice [75]. The direct role for Fst in the regulation of muscle mass was also verified by several laboratories [8, 9, 76]. Fst was identified as a downstream target of testosterone during its pro-myogenic action in both in vitro and in vivo studies [8, 9]. Testosterone treatment of mouse mesenchymal multipotent C3H 10T1/C3H 10T2 cells led to upregulation of Fst and altered the expression of several key members of TGF-β superfamily [8]. Testosteroneinduced upregulation of key myogenic markers MyoD and myosin heavy chain II proteins in C3H 10T1/C3H 10 T2 cells was abolished in cells simultaneously treated with anti-Fst antibody, suggesting an essential role of Fst during testosterone regulation of myogenic differentiation. The essential role of Fst was also established in in vivo studies using castrated male mice, where Fst gene expression level significantly reduced in the levator ani (LA) muscle compared to the sham-operated male mice, but testosterone supplementation in castrated mice upregulated Fst mRNA expression in LA muscle to the baseline levels [8]. Subsequent studies by Braga et al. reported that primary culture of muscle satellite cells express Fst and respond to testosterone treatment. Fst blocked TGF-β-induced inhibition of MHC II expression and induction of Smad2/Smad3 phosphorylation in satellite cells [9]. These

*DOI: http://dx.doi.org/10.5772/intechopen.88294*

mediated at least in part via adipose browning.

blood to promote overall thermogenic program.

#### *Novel Aspects of Follistatin/Transforming Growth Factor-β (TGF-β) Signaling in Adipose… DOI: http://dx.doi.org/10.5772/intechopen.88294*

disruption of Mst (Mst<sup>−</sup>/<sup>−</sup>) in LDLR<sup>−</sup>/<sup>−</sup> (Mst<sup>−</sup>/<sup>−</sup>/LDLR<sup>−</sup>/<sup>−</sup>) mice was shown to reduce the development of proatherogenic dyslipidemia, improve insulin-mediated glucose disposal, and protect against hepatic steatosis [68]. Furthermore, Guo et al. demonstrated that administration of adeno-associated virus 9 (AAV9)-mediated Mst-pro-peptide in adult LDLR<sup>−</sup>/<sup>−</sup> mice reduced diet-induced hepatosteatosis and progression of atherosclerosis [69]. In both these reports, the beneficial metabolic effects were claimed to result from enlarged muscle mass following inactivation of functional Mst in LDLR<sup>−</sup>/<sup>−</sup> mice. Several recent reports have provided strong evidence suggesting that brown fat activation could reduce hypercholesterolemia and protect from atherosclerosis development [70–72]. Therefore, it is possible that observed beneficial effects of Mst inactivation in LDLR<sup>−</sup>/<sup>−</sup> background could be mediated at least in part via adipose browning.

More recently, Mst expression has been linked to mediate BAT-muscle cross talk [73]. Induction of Mst following loss of interferon regulatory factor 4 (IRF4) in BAT leads to significantly reduced exercise capacity, ribosomal protein synthesis, and mitochondrial function [73]. On the other hand, reduced serum levels of Mst resulting from IRF4 overexpression significantly increased exercise capacity in muscle. IRF4 expression was found to be induced in brown adipocytes following cold exposure and β3-adrenergic receptor (AR) agonist [74]. IRF4 expression was reported to be sufficient to induce thermogenic program in BAT, and loss of IRF4 in brown fat leads to significantly reduced energy expenditure. Also, IRF4 was shown to physically and functionally interact with PGC-1α to upregulate transcription of *Ucp1* gene and drive mitochondrial biogenesis and thermogenic program in BAT. In light of these exciting reports establishing IRF4 as a novel inhibitor of Mst, it is not surprising that IRF4 could antagonize the bioactivity of secreted Mst present in the blood to promote overall thermogenic program.

#### **4. Follistatin regulation of white and brown adipose characteristics**

Follistatin (Fst) is a soluble secreted glycoprotein that is known to bind and neutralize the activity of several members of the TGF-β superfamily including activins and Mst in a variety of cell lines [9–10, 77]. Several genetic studies have convincingly demonstrated an essential role of Fst in the regulation of muscle mass. Elegant initial studies led by Lee and McPherron demonstrated that inhibition of Mst either by genetic manipulation or overexpressing Fst resulted in significantly increased muscle mass in mice [75]. The direct role for Fst in the regulation of muscle mass was also verified by several laboratories [8, 9, 76]. Fst was identified as a downstream target of testosterone during its pro-myogenic action in both in vitro and in vivo studies [8, 9]. Testosterone treatment of mouse mesenchymal multipotent C3H 10T1/C3H 10T2 cells led to upregulation of Fst and altered the expression of several key members of TGF-β superfamily [8]. Testosteroneinduced upregulation of key myogenic markers MyoD and myosin heavy chain II proteins in C3H 10T1/C3H 10 T2 cells was abolished in cells simultaneously treated with anti-Fst antibody, suggesting an essential role of Fst during testosterone regulation of myogenic differentiation. The essential role of Fst was also established in in vivo studies using castrated male mice, where Fst gene expression level significantly reduced in the levator ani (LA) muscle compared to the sham-operated male mice, but testosterone supplementation in castrated mice upregulated Fst mRNA expression in LA muscle to the baseline levels [8]. Subsequent studies by Braga et al. reported that primary culture of muscle satellite cells express Fst and respond to testosterone treatment. Fst blocked TGF-β-induced inhibition of MHC II expression and induction of Smad2/Smad3 phosphorylation in satellite cells [9]. These

*Adipose Tissue - An Update*

muscle. Genetic deletion of Mst displays favorable changes in several metabolic parameters including decreased fat deposition, enhanced fatty acid oxidation, improved insulin sensitivity, and increased resistance to diet-induced obesity besides increased skeletal muscle mass [55, 56]. Since Mst is expressed at very low levels in adipose tissues [57], it remains unclear how depletion of Mst can suppress fat accumulation. Earlier studies by Kim et al. show that treatment of mouse primary brown preadipocytes with recombinant Mst led to significant inhibition of brown adipogenic differentiation and reduced expression of markers *Ucp1*, *Prdm16*, and *Pgc-1α* [58]. A comparison of key thermogenic markers obtained from epididymal (Epi) and subcutaneous (SC) white adipose tissues shows significantly increased expression of UCP1 and PRDM16 in Mst KO mice compared to the WT littermates [54]. Using differentiating primary cultures isolated from WT and Mst KO mouse embryonic fibroblasts (MEFs) in the same study, Braga et al. further confirmed upregulation of key thermogenic markers in Mst KO mice compared to the WT mice [5]. Furthermore, recombinant Mst protein treatment of the differentiating MEFs significantly downregulated several key thermogenic markers including UCP1, PRDM16, PGC-1α/PGC-1β, and BMP7. Also, protein expression of adiponectin and phosphorylated AMP-activated protein kinase (pAMPK), which control the expression of genes involved in energy metabolism in coordination with NAD+-dependent sirtuin 1 (SirT1), were upregulated in Mst KO MEFs compared to the WT group [59, 60]. In another study, Chio et al. reported significantly increased energy expenditure and leptin sensitivity in Mst-deficient mice that could explain low fat mass in these mice compared to the WT group [61]. Shan et al. demonstrated that inhibition of Mst signaling in WAT SVF cells failed to induce browning of white adipocytes in vitro, suggesting that loss or inhibition of Mst signaling in preadipocytes does not account for adipose browning in white adipocyte tissues in Mst KO mice [62]. In order to test the possible non-cell autonomous effects of Mst, the authors thoroughly analyzed various muscle-derived circulating factors that could account for the browning phenotype. They reported that skeletal muscle-derived Fndc5 (irisin) plays a central role in mediating white adipose browning in Mst KO mice via activation of AMPK-PGC1α-Fndc5 pathway, suggesting the involvement of muscle-adipose cross talk during the process [63, 64]. Fndc5/irisin was initially identified as a PGC-1α-dependent myokine that is responsible for adipose browning both in vitro and in vivo and protects diet-induced obesity in obesity [65]. Possible involvement of Fndc5 in mediating adipose browning in Mst loss-of-function models has emerged from other laboratories. Dong et al. also confirmed possible intermediate role for Fndc5/irisin-mediated adipose browning in Mst KO mice. In another study, Mst signaling was shown to regulate Fndc5 expression and adipose browning via upregulation of miRNA-34a. Several laboratories have demonstrated that the absence of Mst in both in vitro and in vivo models improves insulin sensitivity [58, 66]. Several other laboratories provided additional evidence to support the view that Mst loss-of-function results in significant metabolic improvements resulting from adipose browning. Increased insulin sensitivity and WAT browning were reported in Meishan pigs with Mst functional deletion [67]. Several browningrelated genes including UCP1, PGC-1α, PRDM16, Cidea, CD137, and Tmem26 were significantly upregulated in these Mst-deficient pigs. Protein expression levels of insulin receptor (IR) and insulin receptor substrate (IRS) were significantly induced in the skeletal muscle of these Mst-deficient pigs. Interestingly, serum irisin levels and skeletal muscle protein expression of irisin precursor protein FNDC5 were significantly higher in Mst-null pigs than wild-type pigs. These authors also demonstrated that inhibition of irisin expression was unable to block the activation of insulin signaling pathway, thus, implying that irisin may not be required for activation of insulin signaling in Mst-deficient skeletal muscle [67]. Genetic

**34**

reports provide conclusive evidence that Fst plays an important role in promoting myogenic differentiation and increasing muscle mass. In spite of several reports demonstrating as essential role of Fst in regulating muscle mass and its function, its role in lipid metabolism and energy balance was largely unknown. Fst-deficient mice die within hours after birth and have several defects including reduced size of diaphragm muscle [76]. These severe musculoskeletal defects were suggested to account for the neonatal death of these Fst-deficient pups. Since maintenance of body temperature through thermogenesis during early hours of neonatal life is extremely important, and both skeletal muscle and thermogenic brown fat share Myf5+ precursor cells, it is logical to test whether Fst could play a role in regulating the thermogenic program along with its established role in muscle development. Based on this logic and several published reports that Fst can bind and antagonize the biological actions of TGF-β/Mst signaling [8, 9], which are known inhibitors of thermogenic program, Braga et al. hypothesized that Fst may promote adipose browning and favorably alter energy metabolism [11]. Initial quantitative analysis of Fst gene expression in a mouse tissue panel consisting of several metabolic tissues demonstrated that Fst expression was highest in BAT along with skeletal muscle and was also expressed at a substantial level in inguinal WAT and the liver [11]. The expression levels in other tissues including the heart, intestine, and testis were significantly lower. This finding for the first time suggested a possible novel role of Fst in BAT and WAT and led to a series of subsequent in vitro and in vivo experimental approaches to delineate the precise role of Fst in adipose tissues of both origins. Using immortalized mouse brown preadipocytes, the authors clearly demonstrated that Fst protein expression was significantly induced in differentiated BAT cells displaying characteristic multilocular lipid droplets compared to the undifferentiated cells [11]. As expected, levels of key brown adipose markers such as UCP1 and PRDM16 were also significantly induced after differentiation of BAT cells. Furthermore, Fst gene expression in BAT was dramatically induced following cold exposure of the mice, suggesting that Fst is a novel cold-inducible gene and could play important role in regulating key metabolic functions. Since Fst KO mice are not viable, Braga et al. utilized primary cultures of differentiating mouse embryonic fibroblast (MEF) cultures isolated from Fst KO and WT embryos to test whether Fst loss-of-function results in defective thermogenic program [11]. Significant impairment in adipogenic differentiation and upregulation of BAT-specific markers were noted in Fst-deficient MEF cultures compared to the WT. Exogenous recombinant Fst protein treatment was able to rescue the thermogenic genes and proteins in Fst-deficient MEF cultures and further induced the expression of several BAT-specific genes in differentiated mouse BAT cells. Affymetrix global gene expression profiling clearly demonstrated lipid metabolism as the most significantly altered pathway and identified several genes involved in lipid metabolism and energy production such as *Adn*, *Thrsp*, *Hp*, *Acsl1*, *Fabp4*, *Pparg,* and *Cd36* were significantly downregulated in Fst KO MEFs compared to the WT. Significantly lower basal mitochondrial respiration in Fst KO MEFs compared to the WT cultures was rescued by exogenous recombinant Fst, suggesting that Fst increases cellular respiration [11].

In subsequent experiments, Singh et al. explored the in vivo actions of Fst overexpression on both white and brown adipose tissues using Fst-transgenic (Fst-Tg) mice to determine whether Fst promotes adipose browning and brown adipose mass and function in these mice and identify possible molecular targets of Fst in these adipose tissues. Fst-Tg mice express Fst under a muscle-specific promoter [75] in which the circulating Fst levels are 1.5-fold higher along with ~70% increased interscapular BAT mass compared to the WT mice [77]. BAT signature genes and several key proteins involved in mitochondrial biogenesis, fatty acid oxidation (FAO),

**37**

*Novel Aspects of Follistatin/Transforming Growth Factor-β (TGF-β) Signaling in Adipose…*

were significantly upregulated in iBAT as well as in Epi and SC adipose tissues of Fst-Tg mice compared to the WT mice [77]. The BAT marker UCP1 and beigespecific markers CD137 were significantly higher in both WAT depots of Fst-Tg mice compared to WT, with relatively larger differences observed in SC adipose depots. Several other markers involved in mitochondrial biogenesis and FAO were also found to be induced in both adipose depots from Fst-Tg mice compared to the WT mice. These observed differences in adipose browning capacity between the two

The actions of Fst in regulating WAT and BAT adipose characteristics were shown to be mediated via two distinct mechanisms. Fst increased phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signalregulated kinase (ERK1/ERK2) in both WAT depots, while it increased Myf5 expression in iBAT of Fst-Tg mice [77]. The authors utilized in vitro studies to further confirm the obligatory and mechanistic basis for these distinctly different Fst targets. In differentiating 3T3-L1 cells, recombinant Fst treatment led to significant induction of UCP1 and beige-specific marker CD137. Pharmacological inhibition of p38 MAPK and ERK1/ERK2 phosphorylation by SB023580 (10 μM) and PD98059 (10 μM), respectively, either alone or in combination, led to significant blockade of Fst-induced (i) phosphorylation of both these proteins as expected and (ii) upregulation of UCP1 protein [77]. On the other hand, in BAT and differentiated mouse BAT cells, Fst increased Myf5 protein expression. Knockdown of Myf5 expression led to significant inhibition of recombinant Fst-mediated increase in UCP1 protein expression in differentiated mouse BAT cells. Additionally, Fst treatment was able to rescue Myf5 gene and protein expression in Fst KO MEFs, reinforcing that Myf5 is a critical mediator of Fst action in BAT [77]. Since BAT and skeletal muscle share Myf5-expressing progenitor cells [78, 79], the authors proposed that Fst promotes BAT activation and skeletal muscle growth by upregulating Myf5. Based on these novel findings, the authors proposed that Fst induces Myf5 expression in BAT and Myf5-positive progenitor cells to increase classical BAT activation, whereas it promotes phosphorylation of p38MAPK and ERK1/ERK2 in WAT to promote adipose browning. It is also possible that Fst could efficiently enhance the production of one or more of several myokines which are shown to induce white adipose browning including irisin (encoded by *Fndc5* gene), IL6, or FGF21 [80–82]. Both Fst and FGF21 were shown to be induced and secreted following exercise [81]. Also, secretion of irisin by skeletal muscle in response to exercise was reported to induce phosphorylation of p38 MAPK and ERK1/ERK2 leading to white adipose browning [80]. Upregulation of *Fndc5* gene expression was reported in skeletal muscle after treatment with both recombinant Fst protein and anti-Mst antibody [62], suggesting that Fst could target irisin/Mst-mediated pathway in muscle tissue to promote adipose browning mediated via muscle-adipose cross talk. Using similar MEF-based primary cultures obtained from WT and Fst KO and Mst KO, Braga et al. showed reciprocal regulation of BMP7 [5, 11], a key driver of brown adipogenesis and energy metabolism by Fst and Mst. These findings were confirmed by other laboratories in support of Fst-induced upregulation of BMP7 [83] and its downregulation by Mst [84]. Gene expression analysis of MEF primary cultures from WT and Fst KO versus WT and Mst KO shows several genes that were reciprocally regulated by Fst and Mst as identified by Affymetrix gene expression analysis and further validated by quantitative real-time PCR analysis (**Figure 1**). Analysis of basal oxygen consumption rate (OCR) in differentiated MEF cultures from these WT and Fst/ Mst KO groups further suggests reciprocal effects of Fst and Mst on mitochondrial respiration (**Figure 2**). Combined together, these findings support the view that Fst may exert its pro-browning effects at least in part by inhibiting Mst signaling. Follistatin-like-3 (FSTL3) has been reported as another Mst binding protein that

WAT depots were found to be consistent with previous reports [78].

*DOI: http://dx.doi.org/10.5772/intechopen.88294*

#### *Novel Aspects of Follistatin/Transforming Growth Factor-β (TGF-β) Signaling in Adipose… DOI: http://dx.doi.org/10.5772/intechopen.88294*

were significantly upregulated in iBAT as well as in Epi and SC adipose tissues of Fst-Tg mice compared to the WT mice [77]. The BAT marker UCP1 and beigespecific markers CD137 were significantly higher in both WAT depots of Fst-Tg mice compared to WT, with relatively larger differences observed in SC adipose depots. Several other markers involved in mitochondrial biogenesis and FAO were also found to be induced in both adipose depots from Fst-Tg mice compared to the WT mice. These observed differences in adipose browning capacity between the two WAT depots were found to be consistent with previous reports [78].

The actions of Fst in regulating WAT and BAT adipose characteristics were shown to be mediated via two distinct mechanisms. Fst increased phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signalregulated kinase (ERK1/ERK2) in both WAT depots, while it increased Myf5 expression in iBAT of Fst-Tg mice [77]. The authors utilized in vitro studies to further confirm the obligatory and mechanistic basis for these distinctly different Fst targets. In differentiating 3T3-L1 cells, recombinant Fst treatment led to significant induction of UCP1 and beige-specific marker CD137. Pharmacological inhibition of p38 MAPK and ERK1/ERK2 phosphorylation by SB023580 (10 μM) and PD98059 (10 μM), respectively, either alone or in combination, led to significant blockade of Fst-induced (i) phosphorylation of both these proteins as expected and (ii) upregulation of UCP1 protein [77]. On the other hand, in BAT and differentiated mouse BAT cells, Fst increased Myf5 protein expression. Knockdown of Myf5 expression led to significant inhibition of recombinant Fst-mediated increase in UCP1 protein expression in differentiated mouse BAT cells. Additionally, Fst treatment was able to rescue Myf5 gene and protein expression in Fst KO MEFs, reinforcing that Myf5 is a critical mediator of Fst action in BAT [77]. Since BAT and skeletal muscle share Myf5-expressing progenitor cells [78, 79], the authors proposed that Fst promotes BAT activation and skeletal muscle growth by upregulating Myf5. Based on these novel findings, the authors proposed that Fst induces Myf5 expression in BAT and Myf5-positive progenitor cells to increase classical BAT activation, whereas it promotes phosphorylation of p38MAPK and ERK1/ERK2 in WAT to promote adipose browning. It is also possible that Fst could efficiently enhance the production of one or more of several myokines which are shown to induce white adipose browning including irisin (encoded by *Fndc5* gene), IL6, or FGF21 [80–82]. Both Fst and FGF21 were shown to be induced and secreted following exercise [81]. Also, secretion of irisin by skeletal muscle in response to exercise was reported to induce phosphorylation of p38 MAPK and ERK1/ERK2 leading to white adipose browning [80]. Upregulation of *Fndc5* gene expression was reported in skeletal muscle after treatment with both recombinant Fst protein and anti-Mst antibody [62], suggesting that Fst could target irisin/Mst-mediated pathway in muscle tissue to promote adipose browning mediated via muscle-adipose cross talk. Using similar MEF-based primary cultures obtained from WT and Fst KO and Mst KO, Braga et al. showed reciprocal regulation of BMP7 [5, 11], a key driver of brown adipogenesis and energy metabolism by Fst and Mst. These findings were confirmed by other laboratories in support of Fst-induced upregulation of BMP7 [83] and its downregulation by Mst [84]. Gene expression analysis of MEF primary cultures from WT and Fst KO versus WT and Mst KO shows several genes that were reciprocally regulated by Fst and Mst as identified by Affymetrix gene expression analysis and further validated by quantitative real-time PCR analysis (**Figure 1**). Analysis of basal oxygen consumption rate (OCR) in differentiated MEF cultures from these WT and Fst/ Mst KO groups further suggests reciprocal effects of Fst and Mst on mitochondrial respiration (**Figure 2**). Combined together, these findings support the view that Fst may exert its pro-browning effects at least in part by inhibiting Mst signaling. Follistatin-like-3 (FSTL3) has been reported as another Mst binding protein that

*Adipose Tissue - An Update*

reports provide conclusive evidence that Fst plays an important role in promoting myogenic differentiation and increasing muscle mass. In spite of several reports demonstrating as essential role of Fst in regulating muscle mass and its function, its role in lipid metabolism and energy balance was largely unknown. Fst-deficient mice die within hours after birth and have several defects including reduced size of diaphragm muscle [76]. These severe musculoskeletal defects were suggested to account for the neonatal death of these Fst-deficient pups. Since maintenance of body temperature through thermogenesis during early hours of neonatal life is extremely important, and both skeletal muscle and thermogenic brown fat share Myf5+ precursor cells, it is logical to test whether Fst could play a role in regulating the thermogenic program along with its established role in muscle development. Based on this logic and several published reports that Fst can bind and antagonize the biological actions of TGF-β/Mst signaling [8, 9], which are known inhibitors of thermogenic program, Braga et al. hypothesized that Fst may promote adipose browning and favorably alter energy metabolism [11]. Initial quantitative analysis of Fst gene expression in a mouse tissue panel consisting of several metabolic tissues demonstrated that Fst expression was highest in BAT along with skeletal muscle and was also expressed at a substantial level in inguinal WAT and the liver [11]. The expression levels in other tissues including the heart, intestine, and testis were significantly lower. This finding for the first time suggested a possible novel role of Fst in BAT and WAT and led to a series of subsequent in vitro and in vivo experimental approaches to delineate the precise role of Fst in adipose tissues of both origins. Using immortalized mouse brown preadipocytes, the authors clearly demonstrated that Fst protein expression was significantly induced in differentiated BAT cells displaying characteristic multilocular lipid droplets compared to the undifferentiated cells [11]. As expected, levels of key brown adipose markers such as UCP1 and PRDM16 were also significantly induced after differentiation of BAT cells. Furthermore, Fst gene expression in BAT was dramatically induced following cold exposure of the mice, suggesting that Fst is a novel cold-inducible gene and could play important role in regulating key metabolic functions. Since Fst KO mice are not viable, Braga et al. utilized primary cultures of differentiating mouse embryonic fibroblast (MEF) cultures isolated from Fst KO and WT embryos to test whether Fst loss-of-function results in defective thermogenic program [11]. Significant impairment in adipogenic differentiation and upregulation of BAT-specific markers were noted in Fst-deficient MEF cultures compared to the WT. Exogenous recombinant Fst protein treatment was able to rescue the thermogenic genes and proteins in Fst-deficient MEF cultures and further induced the expression of several BAT-specific genes in differentiated mouse BAT cells. Affymetrix global gene expression profiling clearly demonstrated lipid metabolism as the most significantly altered pathway and identified several genes involved in lipid metabolism and energy production such as *Adn*, *Thrsp*, *Hp*, *Acsl1*, *Fabp4*, *Pparg,* and *Cd36* were significantly downregulated in Fst KO MEFs compared to the WT. Significantly lower basal mitochondrial respiration in Fst KO MEFs compared to the WT cultures was rescued by exogenous recombinant Fst, suggest-

**36**

ing that Fst increases cellular respiration [11].

In subsequent experiments, Singh et al. explored the in vivo actions of Fst overexpression on both white and brown adipose tissues using Fst-transgenic (Fst-Tg) mice to determine whether Fst promotes adipose browning and brown adipose mass and function in these mice and identify possible molecular targets of Fst in these adipose tissues. Fst-Tg mice express Fst under a muscle-specific promoter [75] in which the circulating Fst levels are 1.5-fold higher along with ~70% increased interscapular BAT mass compared to the WT mice [77]. BAT signature genes and several key proteins involved in mitochondrial biogenesis, fatty acid oxidation (FAO),

#### **Figure 1.**

*Reciprocal effects of Fst and Mst on several genes involved in lipid and energy metabolism. Primary cultures obtained from differentiating WT and Fst/Mst KO cells were analyzed by Affymetrix global gene profiling. (A) Van diagram showing 27 common genes that were reciprocally regulated by Fst and Mst. (B) Heat map showing differential expression of those 27 genes. (C) List of reciprocally regulated common genes. (D, E) Validation of Affymetrix data real-time PCR analysis. n = 3; \*p* ≤ *0.05; \*\*p* ≤ *0.01.*

could play an important role in regulating fat mass and glucose homeostasis [85]. FSTL3 knockout mice display distinct phenotype including decreased fat mass and improved insulin sensitivity. However, it is not known whether FSTL3 regulates BAT mass and thermogenic activity, suggesting that the role of Fst may be more complicated [86].

Activation of p38MAPK pathway that promotes adipose browning by β3-adrenergic receptor (β3-AR) has been well documented [87, 88]. Since intraperitoneal injection of β3-AR agonist CL316,243 in Fst-Tg mice resulted in additive response to UCP1 levels in WAT and BAT compared to the WT mice, it is possible that β3-AR signaling could play an important role upstream of p38 MAPK pathway during Fst-induced adipose browning. More recently, elegant studies by Liu et al. demonstrated that inhibition of follicle-stimulating hormone (FSH) through a polyclonal antibody induced adipose browning and activated BAT and thermogenesis [89]. Since Fst was initially isolated from follicular fluid and found to inhibit secretion of FSH from anterior pituitary, this recent report further validates the identification of Fst as a novel inducer of adipose browning.

**39**

**diseases**

**Figure 2.**

*Novel Aspects of Follistatin/Transforming Growth Factor-β (TGF-β) Signaling in Adipose…*

**5. Metabolic profiling of Fst overexpression and relevance to metabolic** 

*Reciprocal effects of Fst (A, B) and Mst (C, D) on basal oxygen consumption rate (OCR) as analyzed by the seahorse bioscience XF24 extracellular flux analyzer. Data are expressed as mean +/− SEM. \*p* ≤ *0.05; \*\*p* ≤ *0.01.*

In order to get better understanding of observed adipose browning and its metabolic consequences in Fst overexpressing in mice (Fst-Tg) as well as in differentiated 3T3-L1 (3T3-L1 Fst) preadipocyte, Singh et al. initially performed quantitative analysis of abdominal fat volume by CT scan, glucose clearance, and serum lipid profiles [90]. Fst-Tg mice displayed significant decrease in abdominal fat mass, increased glucose clearance, and significantly lower triglyceride (TG) and free fatty acid (FFA) levels compared to the WT control mice. A comparison of the overall lipidomic profiles using gas chromatography time-of-flight technology (GC TOF) shows a general reduction in diglycerides (DG), triglycerides (TG), ceramide (D42:0), fatty acids (FA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and lysophosphatidylethanolamines (LPE: 16.0) in Fst overexpressing 3T3-L1 (3T3-L1 Fst) cells compared to the basal 3T3-L1 cells after adipogenic differentiation (ref). On the other hand, a significant increase in several lysophosphatidylcholines (LPC) including LPC 16:0, LPC 18:0, and LPC 18:1 was observed in 3T3-L1 Fst cells in comparison to 3T3-L1 cells. The decreased levels of several of these lipid metabolites observed in 3T3-L1 Fst cells are known contributors toward the development of obesity and related metabolic diseases [91, 92]. Increased levels of several of these LPCs following Fst overexpression also suggest a beneficial role for Fst as these LPCs are reported to be significantly reduced in obesity and type 2 diabetes [93, 94]. Comprehensive analysis of metabolites obtained from epi and SC adipose tissue between WT and Fst-Tg mice provided significant differences between metabolites involved in energy and

*DOI: http://dx.doi.org/10.5772/intechopen.88294*

*Novel Aspects of Follistatin/Transforming Growth Factor-β (TGF-β) Signaling in Adipose… DOI: http://dx.doi.org/10.5772/intechopen.88294*

**Figure 2.**

*Adipose Tissue - An Update*

**38**

complicated [86].

**Figure 1.**

adipose browning.

could play an important role in regulating fat mass and glucose homeostasis [85]. FSTL3 knockout mice display distinct phenotype including decreased fat mass and improved insulin sensitivity. However, it is not known whether FSTL3 regulates BAT mass and thermogenic activity, suggesting that the role of Fst may be more

*Affymetrix data real-time PCR analysis. n = 3; \*p* ≤ *0.05; \*\*p* ≤ *0.01.*

*Reciprocal effects of Fst and Mst on several genes involved in lipid and energy metabolism. Primary cultures obtained from differentiating WT and Fst/Mst KO cells were analyzed by Affymetrix global gene profiling. (A) Van diagram showing 27 common genes that were reciprocally regulated by Fst and Mst. (B) Heat map showing differential expression of those 27 genes. (C) List of reciprocally regulated common genes. (D, E) Validation of* 

Activation of p38MAPK pathway that promotes adipose browning by β3-adrenergic receptor (β3-AR) has been well documented [87, 88]. Since intraperitoneal injection of β3-AR agonist CL316,243 in Fst-Tg mice resulted in additive response to UCP1 levels in WAT and BAT compared to the WT mice, it is possible that β3-AR signaling could play an important role upstream of p38 MAPK pathway during Fst-induced adipose browning. More recently, elegant studies by Liu et al. demonstrated that inhibition of follicle-stimulating hormone (FSH) through a polyclonal antibody induced adipose browning and activated BAT and thermogenesis [89]. Since Fst was initially isolated from follicular fluid and found to inhibit secretion of FSH from anterior pituitary, this recent report further validates the identification of Fst as a novel inducer of

*Reciprocal effects of Fst (A, B) and Mst (C, D) on basal oxygen consumption rate (OCR) as analyzed by the seahorse bioscience XF24 extracellular flux analyzer. Data are expressed as mean +/− SEM. \*p* ≤ *0.05; \*\*p* ≤ *0.01.*

#### **5. Metabolic profiling of Fst overexpression and relevance to metabolic diseases**

In order to get better understanding of observed adipose browning and its metabolic consequences in Fst overexpressing in mice (Fst-Tg) as well as in differentiated 3T3-L1 (3T3-L1 Fst) preadipocyte, Singh et al. initially performed quantitative analysis of abdominal fat volume by CT scan, glucose clearance, and serum lipid profiles [90]. Fst-Tg mice displayed significant decrease in abdominal fat mass, increased glucose clearance, and significantly lower triglyceride (TG) and free fatty acid (FFA) levels compared to the WT control mice. A comparison of the overall lipidomic profiles using gas chromatography time-of-flight technology (GC TOF) shows a general reduction in diglycerides (DG), triglycerides (TG), ceramide (D42:0), fatty acids (FA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and lysophosphatidylethanolamines (LPE: 16.0) in Fst overexpressing 3T3-L1 (3T3-L1 Fst) cells compared to the basal 3T3-L1 cells after adipogenic differentiation (ref). On the other hand, a significant increase in several lysophosphatidylcholines (LPC) including LPC 16:0, LPC 18:0, and LPC 18:1 was observed in 3T3-L1 Fst cells in comparison to 3T3-L1 cells. The decreased levels of several of these lipid metabolites observed in 3T3-L1 Fst cells are known contributors toward the development of obesity and related metabolic diseases [91, 92]. Increased levels of several of these LPCs following Fst overexpression also suggest a beneficial role for Fst as these LPCs are reported to be significantly reduced in obesity and type 2 diabetes [93, 94]. Comprehensive analysis of metabolites obtained from epi and SC adipose tissue between WT and Fst-Tg mice provided significant differences between metabolites involved in energy and lipid metabolism of the groups. Several components of the Krebs cycle including citrate, succinylcarnitine, and fumarate were significantly downregulated in Fst-Tg Epi tissues compared to the WT tissues, whereas only reduced levels of succinate were found in SC adipose tissues form Fst-Tg mice compared to the WT mice. Also, several long-chain FAs, components of the carnitine metabolism, glycerolipids, ketone bodies, and lysolipids were selectively found to be lower in Fst-Tg Epi tissues than the WT group. Interestingly, levels of Epi cholesterol levels were also selectively reduced in Epi tissues only. Several key amino acids including tyrosine, components of tryptophan, branched-chain amino acid (BCAA), and urea cycle metabolism were also found to be dramatically reduced in Epi adipose tissues in Fst-Tg mice compared to the WT mice. A comparison of omega-3 polyunsaturated fatty acid (ω-3 PUFAs) levels between the groups shows highly significant increase selectively in SC adipose tissues of Fst-Tg mice. Interestingly, ω-3 PUFAs are reported to improve not only obesity-associated metabolic disorders including insulin resistance and dyslipidemia but also several aspects of energy and lipid metabolism and inflammation [95–97]. Significantly decreased levels of beta-hydroxybutyric acid (BHBA), the end product of FA beta-oxidation and key contributor to metabolic syndrome, were also observed in the Epi adipose tissues of Fst-Tg mice compared to the WT. Combined together, these comprehensive metabolomic profilings of Fst in vitro and in vivo overexpression show a clear pattern of favorable changes in several metabolites implicated in metabolic complications and provide future impetus to thoroughly investigate the novel therapeutic role of Fst.

#### **6. Conclusions**

Several lines of evidence support the view that brown and beige adipocytes play important roles in regulating various aspects of lipid and glucose metabolism. The browning process in WAT that entails a shift in WAT primary function from storing excess energy to the dissipation of energy has been linked to the prevention of progression and development of obesity and related metabolic abnormalities including insulin resistance, hyperlipidemia, and type 2 diabetes. Data generated from several laboratories collectively suggest that blocking of TGF-β/Smad3/Mst signaling efficiently increases brown adipose phenotypic and metabolic characteristics and possible downstream mediators during the process as summarized in **Figure 3**.

Accordingly, new strategies to identify and develop novel TGF-β/Mst inhibitors to increase BAT and beige adipose mass and activities are currently being explored with the hope that blockade of this signaling pathway could lead to the development of therapeutic avenues. Since Fst has been demonstrated to efficiently antagonize Mst and inhibit overall TGF-β signaling in several in vitro and in vivo models, the novel therapeutic potential of Fst for the treatment of obesity and related metabolic diseases needs to be thoroughly explored in preclinical studies. It is, therefore, necessary to identify the key molecular and cellular targets of Fst responsible for its regulation of overall thermogenic program. Although phosphorylation and activation of the p38MAPK/ERK1/ERK2 signaling by Fst in WAT have recently been linked to Fst-induced browning, the possible intermediate role of irisin during the process could not be ruled out. Similar to Fst, secretion of irisin by skeletal muscle is induced following exercise which could induce p38MAPK/ERK1/ERK2 phosphorylation and lead to browning of WAT [80]. Fibroblast growth factor 21 (FGF21), another exercise-induced secretory protein linked to adipose browning and which promotes brown adipose characteristics, has also been shown to be influenced by recombinant Fst (rFst) treatment in 3T3-L1 as well as in WAT of

**41**

metabolic diseases.

**Figure 3.**

**Acknowledgements**

R. Drew University of Medicine and Science.

*and brown adipose tissue and their metabolic implications.*

*Novel Aspects of Follistatin/Transforming Growth Factor-β (TGF-β) Signaling in Adipose…*

Fst-transgenic (Fst-Tg) mice. Robust upregulation of FGF21/adiponectin/AMPK signaling pathway observed under both conditions suggests a possible mechanistic link between Fst and FGF21. Preclinical studies using an alternatively spliced cDNA of follistatin (FS344) delivered by adeno-associated virus (AAV) to muscle in both human patients with certain degenerative muscle disorders [98, 99] and nonhuman primates [100] show no apparent structural or functional aberrations in a variety of organs, suggesting the potential of Fst use in clinical trials, although these studies did not assess adipose tissue metabolic parameters. Therefore, novel antagonists of TGF-β/Mst signaling pathways, including Fst [101], hold a great promise for the treatment of not only muscle loss and dysfunction but also for obesity and related

*Proposed hypothetical model for Fst and Mst/TGF-β regulation of adipose browning characteristics of white* 

This work was supported by the National Institute of Health Grants SC1AG049682 (RS), SC1CA1658650 (SP), UHI grant S21MD000103 and

Accelerating Excellence in Translational Science (AXIS) U54MD007598 to Charles

*DOI: http://dx.doi.org/10.5772/intechopen.88294*

*Novel Aspects of Follistatin/Transforming Growth Factor-β (TGF-β) Signaling in Adipose… DOI: http://dx.doi.org/10.5772/intechopen.88294*

#### **Figure 3.**

*Adipose Tissue - An Update*

therapeutic role of Fst.

**6. Conclusions**

lipid metabolism of the groups. Several components of the Krebs cycle including citrate, succinylcarnitine, and fumarate were significantly downregulated in Fst-Tg Epi tissues compared to the WT tissues, whereas only reduced levels of succinate were found in SC adipose tissues form Fst-Tg mice compared to the WT mice. Also, several long-chain FAs, components of the carnitine metabolism, glycerolipids, ketone bodies, and lysolipids were selectively found to be lower in Fst-Tg Epi tissues than the WT group. Interestingly, levels of Epi cholesterol levels were also selectively reduced in Epi tissues only. Several key amino acids including tyrosine, components of tryptophan, branched-chain amino acid (BCAA), and urea cycle metabolism were also found to be dramatically reduced in Epi adipose tissues in Fst-Tg mice compared to the WT mice. A comparison of omega-3 polyunsaturated fatty acid (ω-3 PUFAs) levels between the groups shows highly significant increase selectively in SC adipose tissues of Fst-Tg mice. Interestingly, ω-3 PUFAs are reported to improve not only obesity-associated metabolic disorders including insulin resistance and dyslipidemia but also several aspects of energy and lipid metabolism and inflammation [95–97]. Significantly decreased levels of beta-hydroxybutyric acid (BHBA), the end product of FA beta-oxidation and key contributor to metabolic syndrome, were also observed in the Epi adipose tissues of Fst-Tg mice compared to the WT. Combined together, these comprehensive metabolomic profilings of Fst in vitro and in vivo overexpression show a clear pattern of favorable changes in several metabolites implicated in metabolic complications and provide future impetus to thoroughly investigate the novel

Several lines of evidence support the view that brown and beige adipocytes play important roles in regulating various aspects of lipid and glucose metabolism. The browning process in WAT that entails a shift in WAT primary function from storing excess energy to the dissipation of energy has been linked to the prevention of progression and development of obesity and related metabolic abnormalities including insulin resistance, hyperlipidemia, and type 2 diabetes. Data generated from several laboratories collectively suggest that blocking of TGF-β/Smad3/Mst signaling efficiently increases brown adipose phenotypic and metabolic characteristics and possible downstream mediators during the process as summarized in **Figure 3**.

Accordingly, new strategies to identify and develop novel TGF-β/Mst inhibitors to increase BAT and beige adipose mass and activities are currently being explored with the hope that blockade of this signaling pathway could lead to the development of therapeutic avenues. Since Fst has been demonstrated to efficiently antagonize Mst and inhibit overall TGF-β signaling in several in vitro and in vivo models, the novel therapeutic potential of Fst for the treatment of obesity and related metabolic diseases needs to be thoroughly explored in preclinical studies. It is, therefore, necessary to identify the key molecular and cellular targets of Fst responsible for its regulation of overall thermogenic program. Although phosphorylation and activation of the p38MAPK/ERK1/ERK2 signaling by Fst in WAT have recently been linked to Fst-induced browning, the possible intermediate role of irisin during the process could not be ruled out. Similar to Fst, secretion of irisin by skeletal muscle is induced following exercise which could induce p38MAPK/ERK1/ERK2 phosphorylation and lead to browning of WAT [80]. Fibroblast growth factor 21 (FGF21), another exercise-induced secretory protein linked to adipose browning and which promotes brown adipose characteristics, has also been shown to be influenced by recombinant Fst (rFst) treatment in 3T3-L1 as well as in WAT of

**40**

*Proposed hypothetical model for Fst and Mst/TGF-β regulation of adipose browning characteristics of white and brown adipose tissue and their metabolic implications.*

Fst-transgenic (Fst-Tg) mice. Robust upregulation of FGF21/adiponectin/AMPK signaling pathway observed under both conditions suggests a possible mechanistic link between Fst and FGF21. Preclinical studies using an alternatively spliced cDNA of follistatin (FS344) delivered by adeno-associated virus (AAV) to muscle in both human patients with certain degenerative muscle disorders [98, 99] and nonhuman primates [100] show no apparent structural or functional aberrations in a variety of organs, suggesting the potential of Fst use in clinical trials, although these studies did not assess adipose tissue metabolic parameters. Therefore, novel antagonists of TGF-β/Mst signaling pathways, including Fst [101], hold a great promise for the treatment of not only muscle loss and dysfunction but also for obesity and related metabolic diseases.

#### **Acknowledgements**

This work was supported by the National Institute of Health Grants SC1AG049682 (RS), SC1CA1658650 (SP), UHI grant S21MD000103 and Accelerating Excellence in Translational Science (AXIS) U54MD007598 to Charles R. Drew University of Medicine and Science.

*Adipose Tissue - An Update*

#### **Author details**

Shehla Pervin1,2, Wilson Nyah3 , Srinivasa T. Reddy1,4 and Rajan Singh1,2\*

1 Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

2 Division of Endocrinology and Metabolism, Charles R. Drew University of Medicine and Science, Los Angeles, CA, USA

3 College of Science and Health, Charles R. Drew University of Medicine and Science, Los Angeles, CA, USA

4 Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

\*Address all correspondence to: rajansingh@mednet.ucla.edu

© 2019 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.

**43**

*Novel Aspects of Follistatin/Transforming Growth Factor-β (TGF-β) Signaling in Adipose…*

androgen receptor/beta-catenin and follistatin/transforming growth factorbeta signaling pathways. Endocrinology.

[9] Braga M, Bhasin S, Jasuja R, Pervin S,

transforming growth factor-β signaling during myogenic differentiation and proliferation of mouse satellite cells: Potential role of follistatin in mediating testosterone action. Molecular and Cellular Endocrinology. 2012;**350**:39-52

[10] Pervin S, Singh V, Tucker A, Collazo J, Singh R. Modulation of transforming growth factor-β/follistatin signaling and white adipose browning: Therapeutic implications for obesity related

disorders. Hormone Molecular Biology and Clinical Investigation. 2017;**31**(2)

[11] Braga M, Reddy ST, Vergnes L, Pervin S, Grijalva V, Stout D, et al. Follistatin promotes adipocyte

2014;**55**:375-384

2010;**11**:257-262

differentiation, browning, and energy metabolism. Journal of Lipid Research.

[12] Seale P, Bjork B, Yang W, Kajimura S, Chin S, Kuang S, et al. PRDM16 controls a brown fat/skeletal muscle switch. Nature. 2008;**454**:961-967

[13] Kajimura S, Seale P, Spiegelman BM. Transcriptional control of brown fat development. Cell Metabolism.

[14] Timmons JA, Wennmalm K, Larsson

O, Walden TB, Lassmann T, Petrovic N, et al. Myogenic gene expression signature establishes that brown and white adipocytes originate from distinct cell lineages. Proceedings of the National Academy of Sciences of the United States of America.

2007;**104**:4401-4406

[15] Forner F, Kumar C, Luber CA, Fromme T, Klingenspor M, Mann M.

Singh R. Testosterone inhibits

2009;**150**:1259-1268

*DOI: http://dx.doi.org/10.5772/intechopen.88294*

[1] Tam CS, Lecoultre V, Ravussin E. Brown adipose tissue: Mechanisms and potential therapeutic targets. Circulation. 2012;**125**:2782-2791

[2] The Toll of America's Obesity. The New York Times. Available from: https://www.nytimes.com/2018/08/09/ opinion/cost-diabetes-obesity-budget.

[3] Samad F, Yamamoto K, Pandey M, Loskutoff DJ. Elevated expression of transforming growth factor-beta in adipose tissue from obese mice. Molecular Medicine. 1997;**3**:37-48

[4] Yadav H, Quijano C, Kamaraju AK, Gavrilova O, Malek R, Chen W, et al. Protection from obesity and diabetes by blockade of TGF-β/Smad3 signaling.

[5] Braga M, Pervin S, Norris K, Bhasin S, Singh R. Inhibition of in vitro and in vivo brown fat differentiation program by myostatin. Obesity.

[6] Fournier B, Murray B, Gutzwiller S, Marcaletti S, Marcellin D, Bergling S, et al. Blockade of the activin receptor IIb activates functional brown adipogenesis

and thermogenesis by inducing mitochondrial oxidative metabolism. Molecular and Cellular Biology.

[7] Tiano JP, Springer DA, Rane SG. SMAD3 negatively regulates serum irisin and skeletal muscle FNDC5 and peroxisome proliferatoractivated receptor γ coactivator 1-α (PGC-1α) during exercise. The Journal of Biological Chemistry.

Cell Metabolism. 2011;**14**:67-79

2013;**21**:1180-1188

2012;**32**:2871-2879

2015;**290**:7671-7684

[8] Singh R, Bhasin S, Braga M, Artaza JN, Pervin S, Taylor WE, et al. Regulation of myogenic differentiation by androgens: Cross talk between

**References**

html

*Novel Aspects of Follistatin/Transforming Growth Factor-β (TGF-β) Signaling in Adipose… DOI: http://dx.doi.org/10.5772/intechopen.88294*

#### **References**

*Adipose Tissue - An Update*

**Author details**

Shehla Pervin1,2, Wilson Nyah3

UCLA, Los Angeles, CA, USA

Science, Los Angeles, CA, USA

Medicine and Science, Los Angeles, CA, USA

Medicine at UCLA, Los Angeles, CA, USA

provided the original work is properly cited.

\*Address all correspondence to: rajansingh@mednet.ucla.edu

, Srinivasa T. Reddy1,4 and Rajan Singh1,2\*

1 Department of Obstetrics and Gynecology, David Geffen School of Medicine at

2 Division of Endocrinology and Metabolism, Charles R. Drew University of

3 College of Science and Health, Charles R. Drew University of Medicine and

4 Department of Molecular and Medical Pharmacology, David Geffen School of

© 2019 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,

**42**

[1] Tam CS, Lecoultre V, Ravussin E. Brown adipose tissue: Mechanisms and potential therapeutic targets. Circulation. 2012;**125**:2782-2791

[2] The Toll of America's Obesity. The New York Times. Available from: https://www.nytimes.com/2018/08/09/ opinion/cost-diabetes-obesity-budget. html

[3] Samad F, Yamamoto K, Pandey M, Loskutoff DJ. Elevated expression of transforming growth factor-beta in adipose tissue from obese mice. Molecular Medicine. 1997;**3**:37-48

[4] Yadav H, Quijano C, Kamaraju AK, Gavrilova O, Malek R, Chen W, et al. Protection from obesity and diabetes by blockade of TGF-β/Smad3 signaling. Cell Metabolism. 2011;**14**:67-79

[5] Braga M, Pervin S, Norris K, Bhasin S, Singh R. Inhibition of in vitro and in vivo brown fat differentiation program by myostatin. Obesity. 2013;**21**:1180-1188

[6] Fournier B, Murray B, Gutzwiller S, Marcaletti S, Marcellin D, Bergling S, et al. Blockade of the activin receptor IIb activates functional brown adipogenesis and thermogenesis by inducing mitochondrial oxidative metabolism. Molecular and Cellular Biology. 2012;**32**:2871-2879

[7] Tiano JP, Springer DA, Rane SG. SMAD3 negatively regulates serum irisin and skeletal muscle FNDC5 and peroxisome proliferatoractivated receptor γ coactivator 1-α (PGC-1α) during exercise. The Journal of Biological Chemistry. 2015;**290**:7671-7684

[8] Singh R, Bhasin S, Braga M, Artaza JN, Pervin S, Taylor WE, et al. Regulation of myogenic differentiation by androgens: Cross talk between

androgen receptor/beta-catenin and follistatin/transforming growth factorbeta signaling pathways. Endocrinology. 2009;**150**:1259-1268

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[73] Kong X, Yao T, Zhou P, Kazak L, Tenen D, Lyubetskaya A, et al. Brown adipose tissue controls skeletal muscle function via the secretion of myostatin. Cell Metabolism. 2018;**28**:631-643

[74] Kong X, Banks A, Liu T, Kazak L, Rao RR, Cohen P, et al. IRF4 is a key thermogenic transcriptional partner of PGC-1α. Cell. 2014;**158**:69-83

[75] Lee SJ, McPherron AC. Regulation of myostatin activity and muscle growth. Proceedings of the National Academy of Sciences of the United States of America. 2001;**98**:9306-9311

[76] Matzuk MM, Lu N, Vogel H, Sellheyer K, Roop DR, Bradley A. Multiple defects and perinatal death in mice deficient in follistatin. Nature. 1995;**374**:360-363

[77] Singh R, Braga M, Reddy ST, Lee SJ, Parveen M, Grijalva V, et al. Follistatin targets distinct pathways to promote brown adipocyte characteristics in brown and white adipose tissues. Endocrinology. 2017;**158**:1217-1230

[78] Lo KA, Sun L. Turning WAT into BAT: A review on regulators controlling the browning of white adipocytes. Bioscience Reports. 2013;**33**(5):pii: e00065

[79] Sanchez-Gurmaches J, Guertin DA. Adipocyte lineages: Tracing back the origins of fat. Biochimica et Biophysica Acta. 1842;**2014**:340-351

[80] Zhang Y, Li R, Meng Y, Li S, Donelan W, Zhao Y, et al. Irisin

stimulates browning of white adipocytes through mitogen-activated protein kinase p38 MAP kinase and ERK MAP kinase signaling. Diabetes. 2014;**63**:514-525

[81] Hansen JS, Pedersen BK, Xu G, Lehmann R, Weigert C, Plomgaard P. Exercise-induced secretion of FGF21 and follistatin are blocked by pancreatic clamp and impaired in type 2 diabetes. The Journal of Clinical Endocrinology and Metabolism. 2016;**101**:2816-2825

[82] Reza MM, Subramaniyam N, Sim CM, Ge X, Sathiakumar D, McFarlane C, et al. Irisin is a promyogenic factor that induces skeletal muscle hypertrophy and rescues denervation-induced atrophy. Nature Communications. 2017;**8**:1104

[83] Amthor H, Christ B, Rashid-Doubell F, Kemp CF, Lang E, Patel K. Follistatin regulates bone morphogenetic protein-7 (BMP-7) activity to stimulate embryonic muscle growth. Developmental Biology. 2002;**243**:115-127

[84] Rebbapragada A, Benchabane H, Wrana JL, Celeste AJ, Attisano L. Myostatin signals through a transforming growth factor beta-like signaling pathway to block adipogenesis. Molecular and Cellular Biology. 2003;**23**:7230-7242

[85] Brown ML, Bonomi L, Ungerleider N, Zina J, Kimura F, Mukherjee A, et al. Follistatin and follistatin like-3 differentially regulate adiposity and glucose homeostasis. Obesity. 2011;**19**:1940-1949

[86] Mukherjee A, Sidis Y, Mahan A, Raher MJ, Xia Y, Rosen ED, et al. FSTL3 deletion reveals roles for TGFbeta family ligands in glucose and fat homeostasis in adults. Proceedings of the National Academy of Sciences of the United States of America. 2007;**104**(4):1348-1353

[87] Collins S, Surwit RS. The betaadrenergic receptors and the control of adipose tissue metabolism and thermogenesis. Recent Progress in Hormone Research. 2001;**56**:309-328

[88] Cao W, Medvedev AV, Daniel KW, Collins S. Beta-adrenergic activation of p38 MAP kinase in adipocytes: cAMP induction of the uncoupling protein 1 (UCP1) gene requires p38 MAP kinase. The Journal of Biological Chemistry. 2001;**276**:27077-27082

[89] Liu P, Ji Y, Yuen T, Rendina-Ruedy E, DeMambro VE, Dhawan S, et al. Blocking FSH induces thermogenic adipose tissue and reduces body fat. Nature. 2017;**546**:107-112

[90] Singh R, Pervin S, Lee SJ, Kuo A, Grijalva V, David J, et al. Metabolic profiling of follistatin overexpression: A novel therapeutic strategy for metabolic diseases. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy. 2018;**11**:65-84

[91] Xia JY, Holland WL, Kusminski CM, Sun K, Sharma AX, Pearson MJ, et al. Targeted induction of ceramide degradation leads to improved systemic metabolism and reduced hepatic steatosis. Cell Metabolism. 2015;**22**:266-278

[92] Chimin P, Andrade ML, Belchior T, Paschoal VA, Magdalon J, Yamashita AS, et al. Adipocyte mTORC1 deficiency promotes adipose tissue inflammation and NLRP3 inflammasome activation via oxidative stress and de novo ceramide synthesis. Journal of Lipid Research. 2017;**58**:1797-1807

[93] Barber MN, Risis S, Yang C, Meikle PJ, Staples M, Febbraio MA, et al. Plasma lysophosphatidylcholine levels are reduced in obesity and type 2 diabetes. PLoS One. 2012;**7**:e41456

[94] Kim JY, Park JY, Kim OY, Ham BM, Kim HJ, Kwon DY, et al. Metabolic

**49**

*Novel Aspects of Follistatin/Transforming Growth Factor-β (TGF-β) Signaling in Adipose…*

strength in nonhuman primates. Science Translational Medicine. 2009;**1**:6ra15

[101] Singh R. Composition and methods for treating or preventing metabolic syndrome disorders. US 9,682,093 B2 patent; 2017. Available from: https:// patentimages.storage.googleapis. com/67/27/b3/e5ee5a9cd485d9/

US9682093.pdf

*DOI: http://dx.doi.org/10.5772/intechopen.88294*

profiling of plasma in overweight/obese and lean men using ultra performance liquid chromatography and Q-TOF mass spectrometry (UPLC-Q-TOF MS). Journal of Proteome Research.

[95] Martínez-Fernández L, Laiglesia LM, Huerta AE, Martínez JA, Moreno-Aliaga MJ. Omega-3 fatty acids and adipose tissue function in obesity and metabolic syndrome. Prostaglandins & Other Lipid Mediators. 2015;**121**

[96] Kunesová M, Braunerová R, Hlavatý P, Tvrzická E, Stanková B, Skrha J, et al. The influence of n-3 polyunsaturated fatty acids and very low calorie diet during a short-term weight reducing regimen on weight loss and serum fatty acid composition in severely obese women. Physiological Research.

[97] Krebs JD, Browning LM, McLean NK, Rothwell JL, Mishra GD, Moore CS, et al. Additive benefits of long-chain n-3 polyunsaturated fatty acids and weight-loss in the management of cardiovascular disease risk in overweight hyperinsulinaemic women. International Journal of Obesity.

[98] Mendell JR, Sahenk Z, Al-Zaidy S, Rodino-Klapac LR, Lowes LP, Alfano LN, et al. Follistatin gene therapy for sporadic inclusion body myositis improves functional outcomes. Molecular Therapy. 2017;**25**:870-879

[99] Mendell JR, Sahenk Z, Malik V, Gomez AM, Flanigan KM, Lowes LP, et al. A phase 1/2a follistatin gene therapy trial for Becker muscular dystrophy. Molecular Therapy.

[100] Kota J, Handy CR, Haidet AM, Montgomery CL, Eagle A, Rodino-Klapac LR, et al. Follistatin gene delivery enhances muscle growth and

2010;**9**:4368-4375

(Pt A):24-41

2006;**55**:63-72

2006;**30**:1535-1544

2015;**23**:192-201

*Novel Aspects of Follistatin/Transforming Growth Factor-β (TGF-β) Signaling in Adipose… DOI: http://dx.doi.org/10.5772/intechopen.88294*

profiling of plasma in overweight/obese and lean men using ultra performance liquid chromatography and Q-TOF mass spectrometry (UPLC-Q-TOF MS). Journal of Proteome Research. 2010;**9**:4368-4375

*Adipose Tissue - An Update*

2014;**63**:514-525

stimulates browning of white

adipocytes through mitogen-activated protein kinase p38 MAP kinase and ERK MAP kinase signaling. Diabetes.

[87] Collins S, Surwit RS. The betaadrenergic receptors and the control of adipose tissue metabolism and thermogenesis. Recent Progress in Hormone Research. 2001;**56**:309-328

[88] Cao W, Medvedev AV, Daniel KW, Collins S. Beta-adrenergic activation of p38 MAP kinase in adipocytes: cAMP induction of the uncoupling protein 1 (UCP1) gene requires p38 MAP kinase. The Journal of Biological Chemistry.

[89] Liu P, Ji Y, Yuen T, Rendina-Ruedy E,

DeMambro VE, Dhawan S, et al. Blocking FSH induces thermogenic adipose tissue and reduces body fat.

[90] Singh R, Pervin S, Lee SJ, Kuo A, Grijalva V, David J, et al. Metabolic profiling of follistatin overexpression: A novel therapeutic strategy for metabolic diseases. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy.

[91] Xia JY, Holland WL, Kusminski CM,

[92] Chimin P, Andrade ML, Belchior T, Paschoal VA, Magdalon J, Yamashita AS, et al. Adipocyte mTORC1 deficiency promotes adipose tissue inflammation and NLRP3 inflammasome activation via oxidative stress and de novo ceramide synthesis. Journal of Lipid

Research. 2017;**58**:1797-1807

[93] Barber MN, Risis S, Yang C, Meikle PJ, Staples M, Febbraio MA, et al. Plasma lysophosphatidylcholine levels are reduced in obesity and type 2 diabetes. PLoS One. 2012;**7**:e41456

[94] Kim JY, Park JY, Kim OY, Ham BM, Kim HJ, Kwon DY, et al. Metabolic

Sun K, Sharma AX, Pearson MJ, et al. Targeted induction of ceramide degradation leads to improved systemic metabolism and reduced hepatic steatosis. Cell Metabolism.

2001;**276**:27077-27082

Nature. 2017;**546**:107-112

2018;**11**:65-84

2015;**22**:266-278

[81] Hansen JS, Pedersen BK, Xu G, Lehmann R, Weigert C, Plomgaard P. Exercise-induced secretion of FGF21 and follistatin are blocked by pancreatic clamp and impaired in type 2 diabetes. The Journal of Clinical Endocrinology and Metabolism. 2016;**101**:2816-2825

[82] Reza MM, Subramaniyam N, Sim CM, Ge X, Sathiakumar D, McFarlane C, et al. Irisin is a promyogenic factor that induces skeletal muscle hypertrophy and rescues denervation-induced atrophy. Nature

Communications. 2017;**8**:1104

2002;**243**:115-127

2003;**23**:7230-7242

2011;**19**:1940-1949

2007;**104**(4):1348-1353

[83] Amthor H, Christ B, Rashid-Doubell F, Kemp CF, Lang E, Patel K. Follistatin regulates bone morphogenetic protein-7 (BMP-7) activity to stimulate embryonic muscle growth. Developmental Biology.

[84] Rebbapragada A, Benchabane H, Wrana JL, Celeste AJ, Attisano L. Myostatin signals through a

transforming growth factor beta-like signaling pathway to block adipogenesis.

[85] Brown ML, Bonomi L, Ungerleider N, Zina J, Kimura F, Mukherjee A, et al. Follistatin and follistatin like-3 differentially regulate adiposity and glucose homeostasis. Obesity.

[86] Mukherjee A, Sidis Y, Mahan A, Raher MJ, Xia Y, Rosen ED, et al. FSTL3 deletion reveals roles for TGFbeta family ligands in glucose and fat homeostasis in adults. Proceedings of the National Academy of Sciences of the United States of America.

Molecular and Cellular Biology.

**48**

[95] Martínez-Fernández L, Laiglesia LM, Huerta AE, Martínez JA, Moreno-Aliaga MJ. Omega-3 fatty acids and adipose tissue function in obesity and metabolic syndrome. Prostaglandins & Other Lipid Mediators. 2015;**121** (Pt A):24-41

[96] Kunesová M, Braunerová R, Hlavatý P, Tvrzická E, Stanková B, Skrha J, et al. The influence of n-3 polyunsaturated fatty acids and very low calorie diet during a short-term weight reducing regimen on weight loss and serum fatty acid composition in severely obese women. Physiological Research. 2006;**55**:63-72

[97] Krebs JD, Browning LM, McLean NK, Rothwell JL, Mishra GD, Moore CS, et al. Additive benefits of long-chain n-3 polyunsaturated fatty acids and weight-loss in the management of cardiovascular disease risk in overweight hyperinsulinaemic women. International Journal of Obesity. 2006;**30**:1535-1544

[98] Mendell JR, Sahenk Z, Al-Zaidy S, Rodino-Klapac LR, Lowes LP, Alfano LN, et al. Follistatin gene therapy for sporadic inclusion body myositis improves functional outcomes. Molecular Therapy. 2017;**25**:870-879

[99] Mendell JR, Sahenk Z, Malik V, Gomez AM, Flanigan KM, Lowes LP, et al. A phase 1/2a follistatin gene therapy trial for Becker muscular dystrophy. Molecular Therapy. 2015;**23**:192-201

[100] Kota J, Handy CR, Haidet AM, Montgomery CL, Eagle A, Rodino-Klapac LR, et al. Follistatin gene delivery enhances muscle growth and strength in nonhuman primates. Science Translational Medicine. 2009;**1**:6ra15

[101] Singh R. Composition and methods for treating or preventing metabolic syndrome disorders. US 9,682,093 B2 patent; 2017. Available from: https:// patentimages.storage.googleapis. com/67/27/b3/e5ee5a9cd485d9/ US9682093.pdf

**51**

**Chapter 4**

**Abstract**

Effect of Alcohol on Gut-Liver

Adipose tissue comprises of large volumes of biologically functioning fat globule, which employs substantial systemic effect. Adipocytes and adipokines play an active role in autocrine, paracrine, or endocrine metabolic functions. Recent studies demonstrated that the hormonal role of adipocyte and adipose tissue dysfunction contributes to the pathogenesis of alcoholic liver disease (ALD) by the activation of CYP2E1. The gut microbiome and adipose tissue response play a pivotal role in the pathogenesis of ALD. Enteric dysbiosis increases plasma levels of metabolites that activate Kupffer cells. Recent literature suggested that chronic alcohol consumption is also correlated with oxidative stress in adipose tissue, inflammation, and adipocyte cell death, decrease in adiponectin, increase level of leptin and resistin, adipose tissue mass, and insulin resistance that acts on the muscle and liver. Dysbiosis combined with non-nutritional diet has an effect on the luminal metabolism causing immunological changes in the gut that might also contribute to pathogenesis of nonalcoholic fatty liver disease (NAFLD). Understanding the interaction between the altered gut microbiota, diet, environmental factors, and their effects on the gutliver axis can provide an insight toward the pathogenesis of liver-associated disease.

**Keywords:** alcoholic liver disease, adipose tissue, adipokines, gut microbiota,

Alcohol is considered the fifth leading risk factor for premature death and various disorders universally. It is psychoactive substance that leads to overuse of alcohol or alcoholism abuse. Alcohol related liver diseases are the primary cause of every third person undergoing liver transplants worldwide. Worldwide, alcohol liver disease (ALD) causes 14.5 million disability-reduced life years and approximately 500,000 deaths in 2010 [1]. Depending on behavior, genetics, and comorbidities, individuals who consume alcohol develop hepatic steatosis, an early stage of alcoholic hepatitis [2]. Although ALD is a disease that requires an intention for consumption of alcohol, there are various other factors, including genetic host system characteristics involved in the development and progression. The amount of

Adipose tissue comprises of large volumes of biologically functioning fat globule, which employs considered to be submissive [3]. Researchers have established a remarkable understanding of adipocytes being an acute component of metabolic pathways and functioning of endocrine organs. Recent studies have given an insight on the hormonal role of adipocytes. Adipose tissue is identified to secrete proteins that are termed as

pure alcohol consumption and duration is directly linked to cirrhosis.

Axis and Adipose Tissue

*Dhara Patel and Palash Mandal*

nonalcoholic fatty liver disease

**1. Introduction**

#### **Chapter 4**

## Effect of Alcohol on Gut-Liver Axis and Adipose Tissue

*Dhara Patel and Palash Mandal*

#### **Abstract**

Adipose tissue comprises of large volumes of biologically functioning fat globule, which employs substantial systemic effect. Adipocytes and adipokines play an active role in autocrine, paracrine, or endocrine metabolic functions. Recent studies demonstrated that the hormonal role of adipocyte and adipose tissue dysfunction contributes to the pathogenesis of alcoholic liver disease (ALD) by the activation of CYP2E1. The gut microbiome and adipose tissue response play a pivotal role in the pathogenesis of ALD. Enteric dysbiosis increases plasma levels of metabolites that activate Kupffer cells. Recent literature suggested that chronic alcohol consumption is also correlated with oxidative stress in adipose tissue, inflammation, and adipocyte cell death, decrease in adiponectin, increase level of leptin and resistin, adipose tissue mass, and insulin resistance that acts on the muscle and liver. Dysbiosis combined with non-nutritional diet has an effect on the luminal metabolism causing immunological changes in the gut that might also contribute to pathogenesis of nonalcoholic fatty liver disease (NAFLD). Understanding the interaction between the altered gut microbiota, diet, environmental factors, and their effects on the gutliver axis can provide an insight toward the pathogenesis of liver-associated disease.

**Keywords:** alcoholic liver disease, adipose tissue, adipokines, gut microbiota, nonalcoholic fatty liver disease

#### **1. Introduction**

Alcohol is considered the fifth leading risk factor for premature death and various disorders universally. It is psychoactive substance that leads to overuse of alcohol or alcoholism abuse. Alcohol related liver diseases are the primary cause of every third person undergoing liver transplants worldwide. Worldwide, alcohol liver disease (ALD) causes 14.5 million disability-reduced life years and approximately 500,000 deaths in 2010 [1]. Depending on behavior, genetics, and comorbidities, individuals who consume alcohol develop hepatic steatosis, an early stage of alcoholic hepatitis [2]. Although ALD is a disease that requires an intention for consumption of alcohol, there are various other factors, including genetic host system characteristics involved in the development and progression. The amount of pure alcohol consumption and duration is directly linked to cirrhosis.

Adipose tissue comprises of large volumes of biologically functioning fat globule, which employs considered to be submissive [3]. Researchers have established a remarkable understanding of adipocytes being an acute component of metabolic pathways and functioning of endocrine organs. Recent studies have given an insight on the hormonal role of adipocytes. Adipose tissue is identified to secrete proteins that are termed as

adipokines, which play an active role in autocrine, paracrine, or endocrine metabolic functions. Adiponectin, leptin, and resistin are the most affected functional adipokines.

The body as a whole is affected on the consumption of alcohol. It has been demonstrated that mainly enteric dysbiosis plays a significant role in the development of ALD. Due to an increased intestinal gut permeability of microbes like *Clostridiales*, *Ruminococcaceae*, and *Bifidobacterium* spp., this leads to an elevated plasma levels of metabolites like lipopolysaccharide (LPS), Toll like receptors (TLR-4, TLR-2), cell surface receptor and differentiation marker 14 (CD-14), NADPH oxidase homolog 4 (Nox-4), glucose transporter-4 (GLUT4), and short-chain fatty acid (SCFA) which activates Kupffer cells along with the consequent effects of inflammation, necrosis, and oxidative stress. The activation of cytochrome P450 2E1 (CYP2E1) mediated by ethanol breakdown leads to adipokine dysfunction. Adiponectin acts as an antiinflammatory cytokine while leptin and resistin act as pro-inflammatory cytokines that trigger adenosine monophosphate-activated protein kinase (AMPK) pathway which activates fatty acid oxidation and decreased hepatic lipid influx and de novo lipogenesis. Studies have reported that chronic alcohol consumption leads to reduce levels of adiponectin and an increase in leptin secretion and macrophage migration inhibitory factor (MIF) leading to reduction in adipose tissue mass and increase in fatty acid uptake by hepatocytes [3, 4]. Compounds like rosiglitazone, a PPAR-ϒ agonist that targets the adipocytes exogenously, have shown to attenuate alcohol-induced fatty liver [5, 6]. Inflammation due to bacterial translocation is the main contributor to the development of alcoholic liver disease. Cytokines like tumor necrosis factor alpha (TNF-α), interleukins (IL-1β, IL6, IL8), induced nitric oxide synthase (iNOS), reactive oxygen species (ROS), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and heme oxygenase (HO-1) increase adipocyte lipolysis and systemic insulin resistance by stimulating the release of free fatty acids from adipose tissue into the blood stream, which acts on the muscle and liver [4].

The gut microbiome and adipose tissue responses play an essential role in the pathogenesis of alcohol liver disease. The mechanism between adipose tissue and alcohol consumption is yet to be answered.

#### **2. Adipose tissue**

An obese person can have up to 80 L volume of adipose tissue, which contains about 24 L volume of biologically active adipose tissue [7, 8]. The factors that affect the distribution and volume of adipose tissue mainly vary by gender and location. For example, body fat is found more in women than in men. Similarly people from southeast Asia have less body fat compared to white people of identical body mass index (BMI) [9].

Depending upon the anatomy, adipose tissue is classified as follows:


Visceral adipose tissue corresponds with insulin resistance and diabetes mellitus [10].

#### **2.1 What does adipose tissue composed of ?**

Adipocytes are considered to be the building blocks of adipose tissue. Adipocyte stores energy from non-esterified fatty acids (NEFA) and esterification of triglyceride [11]. Lipotoxicity refers to as uptake of circulating lipids, which prevents accumulation

**53**

*Effect of Alcohol on Gut-Liver Axis and Adipose Tissue DOI: http://dx.doi.org/10.5772/intechopen.89340*

**2.2 Which cytokines are released by adipocytes?**

**2.3 Different kind of adipokines:**

glucose and lipid homeostasis [3].

and patient with clinical complications [22].

*2.3.1 Leptin*

*2.3.2 Adiponectin*

*2.3.3 Resistin*

*2.3.4 Omentin*

of NEFA in the organs [12]. There are various processes that take place in adipose tissue which are controlled by hormonal pathways and are useful for metabolic demand [13]:

• Hydrolysis of triglycerides (lipolysis) takes place during fasting or exercise.

Adipose tissue secretes adipokines that play a central role in metabolism of energy. The secretion of adipokines can be altered due to obesity and insulin resistance. Out of several adipokines, leptin, adiponectin, and resistin are the primary

Leptin receptor is located in numerous tissues, which controls expenditure of energy, food consumption, lipolysis, fatty acid oxidation, lipogenesis, and insulin sensitivity signifying as a paracrine and autocrine hormonal function [15]. It also

Adiponectin has a significant role in insulin sensitizing by altering the signaling pathway of AMPK and metabolism of glucose and fatty acid oxidation in tissues. It is also responsible for adiopogenesis, prevention of ectopic fat storage and decline in Kupffer cell activation [14, 16]. The onset of chronic exposure to ethanol can lead to the disruption of adiponectin which is proven to contribute toward the imbalance of pro-inflammatory pathways [3]. A preventive study was performed on the mice along with the treatment of adiponectin and chronic ethanol exposure and signifies the prevention of the liver injury indicating the decrease in both steatosis and TNF-α expression in the liver [17]. Though the mechanism of the therapeutic adiponectin is not well understood, the hypothesis suggests the vital role of a adiponectin in decreasing steatosis which is related to

Resistin can acquire insulin resistance and regulates food intake, thus acting as an antagonist to adiponectin [18]. Resistin, a 12.5-kDa polypeptide, is secreted by white adipose tissue in female [19]. In human, resistin gene is mainly found in the bone marrow and lung with untraceable levels in adipose tissue [20]. Resistin gene expression was provoked during adipocyte differentiation [21]. Thus, serum resistin can act as a powerful diagnostic marker to access the severity of liver disease

Omentin secretion increases insulin sensitivity in adipocytes [16]. It is mainly secreted from the stromal vascular fraction of adipose tissue which enhances glucose uptake mainly activated by insulin [23]. The concentration of omentin was

increased in portal vein which is a consistent marker for ALD [24].

• Synthesis of triglyceride (lipogenesis) takes place during fed state.

ones that are responsible for insulin resistance as well as in ALD [14].

helps in enhancing the release of a TNF-α by Kupffer cells [16].

of NEFA in the organs [12]. There are various processes that take place in adipose tissue which are controlled by hormonal pathways and are useful for metabolic demand [13]:


#### **2.2 Which cytokines are released by adipocytes?**

Adipose tissue secretes adipokines that play a central role in metabolism of energy. The secretion of adipokines can be altered due to obesity and insulin resistance. Out of several adipokines, leptin, adiponectin, and resistin are the primary ones that are responsible for insulin resistance as well as in ALD [14].

#### **2.3 Different kind of adipokines:**

#### *2.3.1 Leptin*

*Adipose Tissue - An Update*

adipokines, which play an active role in autocrine, paracrine, or endocrine metabolic functions. Adiponectin, leptin, and resistin are the most affected functional adipokines. The body as a whole is affected on the consumption of alcohol. It has been demonstrated that mainly enteric dysbiosis plays a significant role in the development of ALD. Due to an increased intestinal gut permeability of microbes like *Clostridiales*, *Ruminococcaceae*, and *Bifidobacterium* spp., this leads to an elevated plasma levels of metabolites like lipopolysaccharide (LPS), Toll like receptors (TLR-4, TLR-2), cell surface receptor and differentiation marker 14 (CD-14), NADPH oxidase homolog 4 (Nox-4), glucose transporter-4 (GLUT4), and short-chain fatty acid (SCFA) which activates Kupffer cells along with the consequent effects of inflammation, necrosis, and oxidative stress. The activation of cytochrome P450 2E1 (CYP2E1) mediated by ethanol breakdown leads to adipokine dysfunction. Adiponectin acts as an antiinflammatory cytokine while leptin and resistin act as pro-inflammatory cytokines that trigger adenosine monophosphate-activated protein kinase (AMPK) pathway which activates fatty acid oxidation and decreased hepatic lipid influx and de novo lipogenesis. Studies have reported that chronic alcohol consumption leads to reduce levels of adiponectin and an increase in leptin secretion and macrophage migration inhibitory factor (MIF) leading to reduction in adipose tissue mass and increase in fatty acid uptake by hepatocytes [3, 4]. Compounds like rosiglitazone, a PPAR-ϒ agonist that targets the adipocytes exogenously, have shown to attenuate alcohol-induced fatty liver [5, 6]. Inflammation due to bacterial translocation is the main contributor to the development of alcoholic liver disease. Cytokines like tumor necrosis factor alpha (TNF-α), interleukins (IL-1β, IL6, IL8), induced nitric oxide synthase (iNOS), reactive oxygen species (ROS), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and heme oxygenase (HO-1) increase adipocyte lipolysis and systemic insulin resistance by stimulating the release of free fatty acids from adipose

tissue into the blood stream, which acts on the muscle and liver [4].

alcohol consumption is yet to be answered.

• Visceral adipose tissue (VAT)

• Subcutaneous adipose tissue (SAT)

**2.1 What does adipose tissue composed of ?**

**2. Adipose tissue**

index (BMI) [9].

litus [10].

The gut microbiome and adipose tissue responses play an essential role in the pathogenesis of alcohol liver disease. The mechanism between adipose tissue and

An obese person can have up to 80 L volume of adipose tissue, which contains about 24 L volume of biologically active adipose tissue [7, 8]. The factors that affect the distribution and volume of adipose tissue mainly vary by gender and location. For example, body fat is found more in women than in men. Similarly people from southeast Asia have less body fat compared to white people of identical body mass

Visceral adipose tissue corresponds with insulin resistance and diabetes mel-

Adipocytes are considered to be the building blocks of adipose tissue. Adipocyte stores energy from non-esterified fatty acids (NEFA) and esterification of triglyceride [11]. Lipotoxicity refers to as uptake of circulating lipids, which prevents accumulation

Depending upon the anatomy, adipose tissue is classified as follows:

**52**

Leptin receptor is located in numerous tissues, which controls expenditure of energy, food consumption, lipolysis, fatty acid oxidation, lipogenesis, and insulin sensitivity signifying as a paracrine and autocrine hormonal function [15]. It also helps in enhancing the release of a TNF-α by Kupffer cells [16].

#### *2.3.2 Adiponectin*

Adiponectin has a significant role in insulin sensitizing by altering the signaling pathway of AMPK and metabolism of glucose and fatty acid oxidation in tissues. It is also responsible for adiopogenesis, prevention of ectopic fat storage and decline in Kupffer cell activation [14, 16]. The onset of chronic exposure to ethanol can lead to the disruption of adiponectin which is proven to contribute toward the imbalance of pro-inflammatory pathways [3]. A preventive study was performed on the mice along with the treatment of adiponectin and chronic ethanol exposure and signifies the prevention of the liver injury indicating the decrease in both steatosis and TNF-α expression in the liver [17]. Though the mechanism of the therapeutic adiponectin is not well understood, the hypothesis suggests the vital role of a adiponectin in decreasing steatosis which is related to glucose and lipid homeostasis [3].

#### *2.3.3 Resistin*

Resistin can acquire insulin resistance and regulates food intake, thus acting as an antagonist to adiponectin [18]. Resistin, a 12.5-kDa polypeptide, is secreted by white adipose tissue in female [19]. In human, resistin gene is mainly found in the bone marrow and lung with untraceable levels in adipose tissue [20]. Resistin gene expression was provoked during adipocyte differentiation [21]. Thus, serum resistin can act as a powerful diagnostic marker to access the severity of liver disease and patient with clinical complications [22].

#### *2.3.4 Omentin*

Omentin secretion increases insulin sensitivity in adipocytes [16]. It is mainly secreted from the stromal vascular fraction of adipose tissue which enhances glucose uptake mainly activated by insulin [23]. The concentration of omentin was increased in portal vein which is a consistent marker for ALD [24].

#### *2.3.5 Chemerin*

Chemerin helps pre-adipocyte differentiation and contributes to immune cell trafficking. It is also proven to increase the sensitivity of insulin as well as provides anti-inflammatory effects on endothelium immune cell [25].

#### **2.4 The role of non-adipocytes**

Non-adipocyte cells are present in a considerable amount of overall cellularity of the adipose tissue. They include cells from perivascular, endothelium, immune, and stem cells. These clusters of cells are known as stromal vascular fraction (SVF) [14].

#### *2.4.1 Macrophages as inflammatory mediators*

Macrophages make up the majority of the resident immune cells in the adipose tissue [26]. The systemic insulin resistance and inflammation are linked with increased macrophage infiltration into the adipose tissue indicating M1 pro-inflammatory state [27]. "Crown-like structure" is formed where macrophages present in adipose tissue encircle dying adipocytes [28]. Adiponectin suppresses macrophage activity using several ways; one of them is to prevent proliferation of myelomonocytic progenitor cells. This reduces the upregulation of endothelial adhesion molecules in response to cytokine production by macrophages [3].

There are other immune cells like B cells, T cells, and dendritic cells which contribute to the obesity-related inflammation. Dendritic cell in particular promotes CD4+ T helper cells to activate macrophage recruitment [29]. Neutrophils are seen in lean and obese individuals, which are primary defense cells in a high-fructose diet mice model [30]. Thus, cytokine expression in adipose tissue is predominately from SVF, while in case of obese individuals, there is an increased expression of cytokines [31, 32]. Studies suggested the important link between the adiponectin and IL-10, the two main critical anti-inflammatory mediators. For instance, adiponectin stimulates IL-10 mRNA and protein expression in RAW264.7 macrophages. In the same cells, gAcrp-mediated desensitization to LPS is prevented due to the immunoneutralization of IL-10 [3]. HO-1 shows antiproliferative, anti-inflammatory, and anti-apoptotic properties. HO-1 is considered as a vital downstream mediator of the anti-inflammatory effects of IL-10 in macrophages [33].

#### **3. What is gut microbiota comprised of ?**

The human gut contains more than 400 different species, comprising of four major bacterial families that play important roles like defining the physiology of the host [34]. The majority of mammalian gut microbiota belongs to the two bacterial phyla, the gram-negative *Bacteroidetes* and the gram-positive *Firmicutes*, which play a major role in the maintenance of normal health condition, metabolism, and disease. Mainly four major families play an important role, an dthey are comprised of:

**55**

molecular patterns (PAMPs) [43].

*Effect of Alcohol on Gut-Liver Axis and Adipose Tissue DOI: http://dx.doi.org/10.5772/intechopen.89340*

in host adiposity and nutrient uptake [37].

gluconeogenesis and lipogenesis [39].

–104

**3.1 Gut microflora in well-being, metabolism, and disease**

*3.1.1 How does gut microflora gets affected in nonalcoholic fatty liver disease?*

Nonalcoholic fatty liver disease is the liver disorder whose pathogenesis is not well understood due to the portal system interaction with the intestinal lumen and liver. Therefore, it is considered that gut microbiome plays an important role in the pathogenesis of NAFLD. Also, diet has a potential to modify the gut microbiome and several metabolic pathways. Thus, the combination of diet, gut, and liver associates directly with the progression of NAFLD or T2D. Most of the diabetic patients are diagnosed with high blood glucose levels in context with insulin resistance and

Westernized diet and pattern of eating are the main driving forces for the increased prevalence of insulin resistance and increased obesity. Studies have suggested that the diet rich in saturated fats are directly proportional to weight gain, insulin resistance, and hyperlipidemia in humans and animal models [40]. In addition, diet specifically high in sugars like fructose and sucrose has contributed to the metabolic alterations in animal models resulting in weight gain hyperlipidemia and hypertension [41]. An overconsumption of fructose hampers glycolysis and glucose uptake pathways in the liver. This leads to an enhanced rate of de novo lipogenesis and triacylglycerol synthesis leading to insulin resistance through fructose catabolism. Increased activity of the inflammatory pathways is a very important mechanism for insulin resistance. An increase in the activity of the nuclear factor kB (NF-kB) pathway and the maintenance of a subacute inflammatory state are associated with obesity. These cytokines and chemokine activate intracellular pathways which promote the development of T2D [42]. Pattern recognition receptors (PRRs) play an important role for identification of commensals versus pathogenic microbes, which reside in the gastrointestinal tract. TLR recognize extracellular patterns, whereas NOD-like receptors (NLRs) recognize intracellular (cytosolic) pathogen Associated

diverse microflora and higher bacterial strains from 107

microbes in the range of 100

(pH 5.0–7.0) [35].

insulin deficiency.

Proximal two thirds of the small intestine and stomach contain less number of

of species of obligate anaerobe reside in the colon due to a low oxidation-reduction potential of the colon. Thus, a subsequent increase in microbes from the stomach to colon has been observed, as the human gastrointestinal tract pH has shown an increase from the stomach (pH 2.0) to duodenum, jejunum, ileum, and colon

*Bacteroidetes* contain variety of enzymes like hydrolase, dehydrogenase, and dehydroxylase that play a major role in the biotransformation of bile acids. *Firmicutes* play an important role in the energy extraction from undigested carbohydrates in the form of production of short-chain fatty acids [36]. Far from being a static ecosystem, the content of this phylum radially shifts in the response to change

Changes in the intake of diet clearly affect composition of an individual's gut microbiota and its body physiology [38]. Complex carbohydrates are metabolized by the colonic microorganism. *Bifidobacteria* convert complex carbohydrates into oligosaccharides and monosaccharide, further fermenting into the short-chain fatty acid end products like acetate, propionate, and butyrate. Colon absorbs SCFA, where butyrate provides energy for colonic epithelial cells; acetate and propionate migrate to the liver and other peripheral organs, where they act as substrates for

cfu/ml due to acidic pH. The ileum contains more

to 108

cfu/ml. Most amounts


*Effect of Alcohol on Gut-Liver Axis and Adipose Tissue DOI: http://dx.doi.org/10.5772/intechopen.89340*

*Adipose Tissue - An Update*

**2.4 The role of non-adipocytes**

*2.4.1 Macrophages as inflammatory mediators*

Chemerin helps pre-adipocyte differentiation and contributes to immune cell trafficking. It is also proven to increase the sensitivity of insulin as well as provides

Non-adipocyte cells are present in a considerable amount of overall cellularity of the adipose tissue. They include cells from perivascular, endothelium, immune, and stem cells. These clusters of cells are known as stromal vascular fraction (SVF) [14].

Macrophages make up the majority of the resident immune cells in the adipose

There are other immune cells like B cells, T cells, and dendritic cells which contribute to the obesity-related inflammation. Dendritic cell in particular promotes

 T helper cells to activate macrophage recruitment [29]. Neutrophils are seen in lean and obese individuals, which are primary defense cells in a high-fructose diet mice model [30]. Thus, cytokine expression in adipose tissue is predominately from SVF, while in case of obese individuals, there is an increased expression of cytokines [31, 32]. Studies suggested the important link between the adiponectin and IL-10, the two main critical anti-inflammatory mediators. For instance, adiponectin stimulates IL-10 mRNA and protein expression in RAW264.7 macrophages. In the same cells, gAcrp-mediated desensitization to LPS is prevented due to the immunoneutralization of IL-10 [3]. HO-1 shows antiproliferative, anti-inflammatory, and anti-apoptotic properties. HO-1 is considered as a vital downstream mediator of the

The human gut contains more than 400 different species, comprising of four major bacterial families that play important roles like defining the physiology of the host [34]. The majority of mammalian gut microbiota belongs to the two bacterial phyla, the gram-negative *Bacteroidetes* and the gram-positive *Firmicutes*, which play a major role in the maintenance of normal health condition, metabolism, and disease. Mainly four major families play an important role, an dthey are comprised of:

tissue [26]. The systemic insulin resistance and inflammation are linked with increased macrophage infiltration into the adipose tissue indicating M1 pro-inflammatory state [27]. "Crown-like structure" is formed where macrophages present in adipose tissue encircle dying adipocytes [28]. Adiponectin suppresses macrophage activity using several ways; one of them is to prevent proliferation of myelomonocytic progenitor cells. This reduces the upregulation of endothelial adhesion

molecules in response to cytokine production by macrophages [3].

anti-inflammatory effects of IL-10 in macrophages [33].

**3. What is gut microbiota comprised of ?**

anti-inflammatory effects on endothelium immune cell [25].

*2.3.5 Chemerin*

CD4+

**54**

• *Bacteroidetes*

• *Actinobacteria*

• *Proteobacteria*

• *Firmicutes*

Proximal two thirds of the small intestine and stomach contain less number of microbes in the range of 100 –104 cfu/ml due to acidic pH. The ileum contains more diverse microflora and higher bacterial strains from 107 to 108 cfu/ml. Most amounts of species of obligate anaerobe reside in the colon due to a low oxidation-reduction potential of the colon. Thus, a subsequent increase in microbes from the stomach to colon has been observed, as the human gastrointestinal tract pH has shown an increase from the stomach (pH 2.0) to duodenum, jejunum, ileum, and colon (pH 5.0–7.0) [35].

*Bacteroidetes* contain variety of enzymes like hydrolase, dehydrogenase, and dehydroxylase that play a major role in the biotransformation of bile acids. *Firmicutes* play an important role in the energy extraction from undigested carbohydrates in the form of production of short-chain fatty acids [36]. Far from being a static ecosystem, the content of this phylum radially shifts in the response to change in host adiposity and nutrient uptake [37].

Changes in the intake of diet clearly affect composition of an individual's gut microbiota and its body physiology [38]. Complex carbohydrates are metabolized by the colonic microorganism. *Bifidobacteria* convert complex carbohydrates into oligosaccharides and monosaccharide, further fermenting into the short-chain fatty acid end products like acetate, propionate, and butyrate. Colon absorbs SCFA, where butyrate provides energy for colonic epithelial cells; acetate and propionate migrate to the liver and other peripheral organs, where they act as substrates for gluconeogenesis and lipogenesis [39].

#### **3.1 Gut microflora in well-being, metabolism, and disease**

#### *3.1.1 How does gut microflora gets affected in nonalcoholic fatty liver disease?*

Nonalcoholic fatty liver disease is the liver disorder whose pathogenesis is not well understood due to the portal system interaction with the intestinal lumen and liver. Therefore, it is considered that gut microbiome plays an important role in the pathogenesis of NAFLD. Also, diet has a potential to modify the gut microbiome and several metabolic pathways. Thus, the combination of diet, gut, and liver associates directly with the progression of NAFLD or T2D. Most of the diabetic patients are diagnosed with high blood glucose levels in context with insulin resistance and insulin deficiency.

Westernized diet and pattern of eating are the main driving forces for the increased prevalence of insulin resistance and increased obesity. Studies have suggested that the diet rich in saturated fats are directly proportional to weight gain, insulin resistance, and hyperlipidemia in humans and animal models [40]. In addition, diet specifically high in sugars like fructose and sucrose has contributed to the metabolic alterations in animal models resulting in weight gain hyperlipidemia and hypertension [41]. An overconsumption of fructose hampers glycolysis and glucose uptake pathways in the liver. This leads to an enhanced rate of de novo lipogenesis and triacylglycerol synthesis leading to insulin resistance through fructose catabolism.

Increased activity of the inflammatory pathways is a very important mechanism for insulin resistance. An increase in the activity of the nuclear factor kB (NF-kB) pathway and the maintenance of a subacute inflammatory state are associated with obesity. These cytokines and chemokine activate intracellular pathways which promote the development of T2D [42]. Pattern recognition receptors (PRRs) play an important role for identification of commensals versus pathogenic microbes, which reside in the gastrointestinal tract. TLR recognize extracellular patterns, whereas NOD-like receptors (NLRs) recognize intracellular (cytosolic) pathogen Associated molecular patterns (PAMPs) [43].

TLRs, extracellular (innate) pattern recognition receptors, are expressed nearly on all the cell types. In total, 13 different TLRs are present in human genome, which remain specific for unique class of PAMPs. Among the TLRs, TLR2 and TLR4 are considered to be vital for the pathogenesis of insulin resistance and diabetes in both clinical and experimental conditions. TLR2 specifically binds to peptidoglycan (gram-positive bacteria), and TLR4 binds to lipopolysaccharide (gram-negative bacteria) [43]. High-fat or high carbohydrate food intake increases the concentration of plasma LPS levels and LPS binding protein, which increases the expression of TLR2 and TLR4 at mRNA and protein level [44]. The study has also shown that the absence of TLR4 protects against the detrimental effects of obesity and lipids on the insulin resistance [44]. A study on TLR4 null mice demonstrated a reduced adiposity and hepatic steatosis compared with the wild-type control when fed on high fat diet (HFD) [45].

NLRs are intracellular or cytoplasmic pattern recognition receptors, which exhibit specificity toward one or more PAMPs. In gastrointestinal epithelial cells, nucleotidebinding oligomerization domain (NOD) is mainly characterized by NLRs. Caspase activation and recruitment domain (CARD) is unique for each NOD protein.


NOD1 and NOD2 are essential since they were the first NLRs reported as potential sensors of bacterial components. It has been reported that NOD1 and NOD2 are also involved in high fat diet induced-inflammation and insulin intolerance [46]. NOD1 agonist causes inflammation and insulin resistance in a primary hepatocytes of the wild-type mice, but this effect was absent in NOD1 knockout mice [47].

The downstream pathway that follows after the engagement of NLRs and TLRs with their respective ligands leads to the activation of NF-kB-mediated inflammatory pathways through adaptor protein MyD88 and secretes the major pro-inflammatory cytokines like TNF-α and IL-6. Pro-inflammatory cytokines phosphorylate the serine/ threonine residue of insulin and downregulate the insulin signaling pathway, which finally leads to the insulin resistance and occurrence of T2D [48]. In vivo studies in mice have shown that the gut tight junction between the cells loosens up when the population of *Bifidobacteria* is decreased. These loose junctions increase the gut permeability and allow lipopolysaccharide present in microbes to pass through the gut epithelial resulting into metabolic endotoxemia causing a low-grade inflammation which is responsible to induce a metabolic disorder including the insulin resistance [49].

Increased body weight with other metabolic phenotypes was observed in the germ-free mice who were fed with either low-fat mouse chow or with different levels of saturated fat and fruits along with vegetables in different groups [50]. In another study, group of mice developed hyperglycemia and high plasma concentration of pro-inflammatory cytokines when HFD was induced. Hyperglycemia resulted in hepatic macro-vesicular steatosis, elevated hepatic triglycerides, and de novo lipogenesis [51].

When an adult germ-free C57BL/6 mouse was orally fed with normal microbiota harvested from the distal intestine of any normal animals, they developed 60% increase in body fat content with insulin resistance. Fasting-induced adipocyte factor (FIAF), a circulating protein of angiopoietin, is essential for the microbiotainduced deposition of triglycerides in adipocytes [52]. Deficiency of choline is usually liked with NAFLD and nonalcoholic steatohepatitis (NASH) [53].

**57**

*Effect of Alcohol on Gut-Liver Axis and Adipose Tissue DOI: http://dx.doi.org/10.5772/intechopen.89340*

ways as follows:

• By the use of CYP2E1

the alcoholic liver disease [55].

documented [57].

ALD [61, 62].

dependence

dependence

• By the use of mitochondrial catalase

genetic factors in correlation with the liver diseases.

*3.1.2 How does a whole body responds to alcoholic liver disease?*

• By the use of an enzyme alcohol dehydrogenase (ADH)

The above-mentioned examples show the significance of the environment and

The liver is a vital organ of the body, as it metabolizes alcohol in three different

ADH and CYP2E1 are two significant ways through which alcohol gets converted into acetaldehyde; ADH is used when the consumption of alcohol is limited, while on the consumption of an excess alcohol, CYP2E1 metabolism plays a role [54]. ADH is not only present in the liver but, it also is expressed in the gastric mucosa. It is an assumption that people with lower gastric ADH are more prone to

Alcoholic liver disease includes various stages like alcoholic hepatitis, steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Alcohol consumption, diet, nutrition, and genetics determine the severity and prognosis of ALD. Morbidity and mortality remain higher in the liver cirrhosis than in benign liver disease (i.e., liver steatosis) [54]. Twenty percent of the patients with simple steatosis with continuous abuse can develop fibrosis within a period of 10 years [56]. In an absence of alcohol for a few weeks, simple steatosis is reversible, while a fibrogenic process of steatohepatitis can induce cirrhosis. Human trials on reversing the steatohepatitis for the treatment of chronic hepatitis C and NASH are well

Oxidative stress mainly occurs due to CYP2E1 accompanied along with the shortage of antioxidants in the hepatocytes and an altered inflammatory cytokines [58]. It has been known that changes in the lipid metabolism and adipose tissue will also enhance the process of liver injury [59]. Genetics of an individual is also another factor that is taken into account for the susceptibility of alcoholism. Lately correlations between the genetic polymorphism of alcoholic metabolizing enzymes

and ALD have shown a significant association [60]. Family studies in Asian population have shown association of the following two genes in particular with

• Alcohol dehydrogenase ADH1B\*1 allele: responsible for increase in alcohol

• Alcohol dehydrogenase ADH2B\*2 allele: responsible for decrease in alcohol

Diet is also one of the significant factors affecting the structure and functionality of gut microbiota. Alcohol and its degradation products can contribute toward the gut dysbiosis [63]. Patients with ALD have shown decrease in commensal groups like *Roseburia*, *Faecalibacterium*, *Blautia*, and *Bacteroides*, while increase in *Proteobacteria* and *Bacilli* resulting in an increased gut permeability, tight junction barrier dysfunctioning, and inflammation [64, 65]. One of the proposed mechanisms is the direct interaction of gut and endotoxins from the liver via

The above-mentioned examples show the significance of the environment and genetic factors in correlation with the liver diseases.

#### *3.1.2 How does a whole body responds to alcoholic liver disease?*

The liver is a vital organ of the body, as it metabolizes alcohol in three different ways as follows:


*Adipose Tissue - An Update*

high fat diet (HFD) [45].

TLRs, extracellular (innate) pattern recognition receptors, are expressed nearly on all the cell types. In total, 13 different TLRs are present in human genome, which remain specific for unique class of PAMPs. Among the TLRs, TLR2 and TLR4 are considered to be vital for the pathogenesis of insulin resistance and diabetes in both clinical and experimental conditions. TLR2 specifically binds to peptidoglycan (gram-positive bacteria), and TLR4 binds to lipopolysaccharide (gram-negative bacteria) [43]. High-fat or high carbohydrate food intake increases the concentration of plasma LPS levels and LPS binding protein, which increases the expression of TLR2 and TLR4 at mRNA and protein level [44]. The study has also shown that the absence of TLR4 protects against the detrimental effects of obesity and lipids on the insulin resistance [44]. A study on TLR4 null mice demonstrated a reduced adiposity and hepatic steatosis compared with the wild-type control when fed on

NLRs are intracellular or cytoplasmic pattern recognition receptors, which exhibit specificity toward one or more PAMPs. In gastrointestinal epithelial cells, nucleotidebinding oligomerization domain (NOD) is mainly characterized by NLRs. Caspase activation and recruitment domain (CARD) is unique for each NOD protein.

• NOD1 (CARD 4): senses peptidoglycan contents in gram-negative bacteria

• NOD2 (CARD 15): senses muramyl dipeptide, the common molecular motif

NOD1 and NOD2 are essential since they were the first NLRs reported as potential sensors of bacterial components. It has been reported that NOD1 and NOD2 are also involved in high fat diet induced-inflammation and insulin intolerance [46]. NOD1 agonist causes inflammation and insulin resistance in a primary hepatocytes of the wild-type mice, but this effect was absent in NOD1 knockout mice [47]. The downstream pathway that follows after the engagement of NLRs and TLRs with their respective ligands leads to the activation of NF-kB-mediated inflammatory pathways through adaptor protein MyD88 and secretes the major pro-inflammatory cytokines like TNF-α and IL-6. Pro-inflammatory cytokines phosphorylate the serine/ threonine residue of insulin and downregulate the insulin signaling pathway, which finally leads to the insulin resistance and occurrence of T2D [48]. In vivo studies in mice have shown that the gut tight junction between the cells loosens up when the population of *Bifidobacteria* is decreased. These loose junctions increase the gut permeability and allow lipopolysaccharide present in microbes to pass through the gut epithelial resulting into metabolic endotoxemia causing a low-grade inflammation which is responsible to

Increased body weight with other metabolic phenotypes was observed in the germ-free mice who were fed with either low-fat mouse chow or with different levels of saturated fat and fruits along with vegetables in different groups [50]. In another study, group of mice developed hyperglycemia and high plasma concentration of pro-inflammatory cytokines when HFD was induced. Hyperglycemia resulted in hepatic macro-vesicular steatosis, elevated hepatic triglycerides, and de

When an adult germ-free C57BL/6 mouse was orally fed with normal microbiota

harvested from the distal intestine of any normal animals, they developed 60% increase in body fat content with insulin resistance. Fasting-induced adipocyte factor (FIAF), a circulating protein of angiopoietin, is essential for the microbiotainduced deposition of triglycerides in adipocytes [52]. Deficiency of choline is usually liked with NAFLD and nonalcoholic steatohepatitis (NASH) [53].

specifically meso-diaminopimelic acid (meso-DAP)

both in gram-positive and gram-negative bacteria.

induce a metabolic disorder including the insulin resistance [49].

**56**

novo lipogenesis [51].

• By the use of mitochondrial catalase

ADH and CYP2E1 are two significant ways through which alcohol gets converted into acetaldehyde; ADH is used when the consumption of alcohol is limited, while on the consumption of an excess alcohol, CYP2E1 metabolism plays a role [54]. ADH is not only present in the liver but, it also is expressed in the gastric mucosa. It is an assumption that people with lower gastric ADH are more prone to the alcoholic liver disease [55].

Alcoholic liver disease includes various stages like alcoholic hepatitis, steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Alcohol consumption, diet, nutrition, and genetics determine the severity and prognosis of ALD. Morbidity and mortality remain higher in the liver cirrhosis than in benign liver disease (i.e., liver steatosis) [54]. Twenty percent of the patients with simple steatosis with continuous abuse can develop fibrosis within a period of 10 years [56]. In an absence of alcohol for a few weeks, simple steatosis is reversible, while a fibrogenic process of steatohepatitis can induce cirrhosis. Human trials on reversing the steatohepatitis for the treatment of chronic hepatitis C and NASH are well documented [57].

Oxidative stress mainly occurs due to CYP2E1 accompanied along with the shortage of antioxidants in the hepatocytes and an altered inflammatory cytokines [58]. It has been known that changes in the lipid metabolism and adipose tissue will also enhance the process of liver injury [59]. Genetics of an individual is also another factor that is taken into account for the susceptibility of alcoholism. Lately correlations between the genetic polymorphism of alcoholic metabolizing enzymes and ALD have shown a significant association [60]. Family studies in Asian population have shown association of the following two genes in particular with ALD [61, 62].


Diet is also one of the significant factors affecting the structure and functionality of gut microbiota. Alcohol and its degradation products can contribute toward the gut dysbiosis [63]. Patients with ALD have shown decrease in commensal groups like *Roseburia*, *Faecalibacterium*, *Blautia*, and *Bacteroides*, while increase in *Proteobacteria* and *Bacilli* resulting in an increased gut permeability, tight junction barrier dysfunctioning, and inflammation [64, 65]. One of the proposed mechanisms is the direct interaction of gut and endotoxins from the liver via

hepatic artery, as well as mechanism of bile acids that contributes toward ALD [66], although the mechanism of the latter interaction is yet to be elucidated.

Due to an exposure of alcohol, intestinal microbiome is getting affected by causing bacterial (gram negative) overgrowth in animal models and humans. Particularly the genus *Lactobacillus* is on a lower side due to the onset of chronic alcohol consumption [67]. It has been recently demonstrated that supplementing saturated long-chain fatty acid with commensal *Lactobacilli* stabilizes the intestinal gut barrier and tight junction barrier in ethanol-induced liver disease in mice [68]. NOD2 is mainly responsible for increasing bacterial peritonitis and bacterascites in cirrhosis, which primarily affects the survival [69]. PAMPs or damage-associated molecular patterns (DAMPs) are recognized by inflammasomes and activate the pro-inflammatory cytokines such as pro-interleukin (IL-1, IL-18) [70]. Chemokine (C-C motif) ligand causes inflammation in colon due to intestinal dysbiosis [71]. TNF-receptor-1 (TNFR-1) present on the intestinal epithelial cells are crucial mediator for ALD and also cause an intestinal barrier dysbiosis [72]. Thus, inflammation can lead to intestinal permeability, which is associated with the translocation of microbial products to TLRs in the liver, which is related to aggravated hepatic steatosis.

#### **4. Role of immune system in intestinal membrane**

Maintaining the balance and symbiotic relation between the immune system and host intestinal microbiome is a very important aspect. This is because they maintain a balance of an immune system by restricting the overgrowth of pathogenic microbiota, as well as the bacteria that reaches the intestinal barriers, chemical barriers, and physical barriers [73]. Innate signaling by MyD88 in T cells directs IgA-mediated microbiota to promote the healthy gut. In IgA-deficient mice, it has been observed that TLR-5 and host protein programmed cell death 1 (PD1) regulate the modulation of IgA homeostasis by differentiating B cells into IgA producing antibodies [74, 75]. The importance of IgA in the microbiota composition in chronic liver disease is yet to be studied.

In liver cirrhosis patient, buccal origin microbes were found in intestine, taxonomically signifying the translocation or invasion from mouth to intestine. Simultaneously these patients also observed to have compromised innate immune system, reduced bile flow and impaired AMP production [76, 77]. The production of AMP is mainly regulated by the gut microbiota that includes defensins, C-type lectins (Reg3b and Reg3g), ribonucleases, and S100 proteins, which rapidly inactivate microbes [78]. In MYD88-deficient mice, NOD2 altered AMP production, which was closely marked [79, 80]. Chronic alcohol administration results in decreased expression of the intestinal C-type lectins in mice, and similar results were observed in the duodenum [81, 82].

Mucin is secreted by the goblet cells which are glycosylated and accountable for the construction of the inner and outer layer of mucus. In mice and humans, mainly mucin-2 is responsible for the mucus layer formation. Upon interaction with the lectin, they are responsible for bacterial composition of the host that promotes formation of glycosidase and metabolic enzymes which are used as a source of energy [83]. Innate immune system is activated to maintain the intestinal homeostasis in the absence of mucin-2. An experiment in the mucin-2-deficient mice demonstrated higher expression of antimicrobial proteins and protected the intestinal barrier from bacterial overgrowth and dysbiosis. This interconnection between the different intestinal defense layers and microbiota helps in decreasing ALD [81].

**59**

fibrosis [85].

**Figure 1.**

*Effect of microbiome dysbiosis on liver disease.*

*Effect of Alcohol on Gut-Liver Axis and Adipose Tissue DOI: http://dx.doi.org/10.5772/intechopen.89340*

Interestingly, there is no single assigned bacterial species that marks the begin-

Ethanol consumptions lead to the elevation of lipopolysaccharide and endotoxin in the portal blood circulation that sensitizes Kupffer cells to activate the inflammatory mediators like TNF-α, IL6, and ROS. Another factor that facilitates the liver disease is the loss of anti-inflammatory mediators. A study has shown IL-10 deficient mice to be more sensitive to ethanol liver injury [3]. The alterations in the intestinal microbiota composition are significant for the pathogenesis of chronic

The energy value of alcohol is equal to those of other nutrients, so when the alcohol consumption is increased, the overall calorie intake excessed the expenditure of energy, which leads to adiposity [86]. It has been established that chronic cirrhosis patient shifts toward the lipid oxidation instead of carbohydrate as fuel to meet the energy requirements which in turn reduces the overall fat mass in an individual [87]. Thus, malnutrition with low adipose skeletal muscle mass is a symptom for an advancement of the liver disease [88]. Alterations in the body mass may also depend on the type of drinking pattern; for instance, a person drinking beer or spirits gains more body mass than wine consumption [89]. Thus, excess alcohol

ning or development of the liver disease. It is always marked by an increased percentage of gram-negative bacteria especially *Proteobacteria* which is known for accelerating cholestatic liver fibrosis [84]. To study the importance of the intestinal microbiota for chronic liver disease, liver fibrosis was induced into the germ-free mice model via the administration of thioacetamide in the drinking water. As a result in comparison to conventional mice, germ-free mice showed elevated liver

liver disease which is demonstrated and briefed in **Figure 1**.

**5. Effect of alcohol on liver and adipose tissue**

*Effect of Alcohol on Gut-Liver Axis and Adipose Tissue DOI: http://dx.doi.org/10.5772/intechopen.89340*

*Adipose Tissue - An Update*

hepatic steatosis.

liver disease is yet to be studied.

were observed in the duodenum [81, 82].

hepatic artery, as well as mechanism of bile acids that contributes toward ALD [66],

Maintaining the balance and symbiotic relation between the immune system and host intestinal microbiome is a very important aspect. This is because they maintain a balance of an immune system by restricting the overgrowth of pathogenic microbiota, as well as the bacteria that reaches the intestinal barriers, chemical barriers, and physical barriers [73]. Innate signaling by MyD88 in T cells directs IgA-mediated microbiota to promote the healthy gut. In IgA-deficient mice, it has been observed that TLR-5 and host protein programmed cell death 1 (PD1) regulate the modulation of IgA homeostasis by differentiating B cells into IgA producing antibodies [74, 75]. The importance of IgA in the microbiota composition in chronic

In liver cirrhosis patient, buccal origin microbes were found in intestine, taxonomically signifying the translocation or invasion from mouth to intestine. Simultaneously these patients also observed to have compromised innate immune system, reduced bile flow and impaired AMP production [76, 77]. The production of AMP is mainly regulated by the gut microbiota that includes defensins, C-type lectins (Reg3b and Reg3g), ribonucleases, and S100 proteins, which rapidly inactivate microbes [78]. In MYD88-deficient mice, NOD2 altered AMP production, which was closely marked [79, 80]. Chronic alcohol administration results in decreased expression of the intestinal C-type lectins in mice, and similar results

Mucin is secreted by the goblet cells which are glycosylated and accountable for the construction of the inner and outer layer of mucus. In mice and humans, mainly mucin-2 is responsible for the mucus layer formation. Upon interaction with the lectin, they are responsible for bacterial composition of the host that promotes formation of glycosidase and metabolic enzymes which are used as a source of energy [83]. Innate immune system is activated to maintain the intestinal homeostasis in the absence of mucin-2. An experiment in the mucin-2-deficient mice demonstrated higher expression of antimicrobial proteins and protected the intestinal barrier from bacterial overgrowth and dysbiosis. This interconnection between the different intestinal defense layers and microbiota helps in decreasing

Due to an exposure of alcohol, intestinal microbiome is getting affected by causing bacterial (gram negative) overgrowth in animal models and humans. Particularly the genus *Lactobacillus* is on a lower side due to the onset of chronic alcohol consumption [67]. It has been recently demonstrated that supplementing saturated long-chain fatty acid with commensal *Lactobacilli* stabilizes the intestinal gut barrier and tight junction barrier in ethanol-induced liver disease in mice [68]. NOD2 is mainly responsible for increasing bacterial peritonitis and bacterascites in cirrhosis, which primarily affects the survival [69]. PAMPs or damage-associated molecular patterns (DAMPs) are recognized by inflammasomes and activate the pro-inflammatory cytokines such as pro-interleukin (IL-1, IL-18) [70]. Chemokine (C-C motif) ligand causes inflammation in colon due to intestinal dysbiosis [71]. TNF-receptor-1 (TNFR-1) present on the intestinal epithelial cells are crucial mediator for ALD and also cause an intestinal barrier dysbiosis [72]. Thus, inflammation can lead to intestinal permeability, which is associated with the translocation of microbial products to TLRs in the liver, which is related to aggravated

although the mechanism of the latter interaction is yet to be elucidated.

**4. Role of immune system in intestinal membrane**

**58**

ALD [81].

**Figure 1.** *Effect of microbiome dysbiosis on liver disease.*

Interestingly, there is no single assigned bacterial species that marks the beginning or development of the liver disease. It is always marked by an increased percentage of gram-negative bacteria especially *Proteobacteria* which is known for accelerating cholestatic liver fibrosis [84]. To study the importance of the intestinal microbiota for chronic liver disease, liver fibrosis was induced into the germ-free mice model via the administration of thioacetamide in the drinking water. As a result in comparison to conventional mice, germ-free mice showed elevated liver fibrosis [85].

Ethanol consumptions lead to the elevation of lipopolysaccharide and endotoxin in the portal blood circulation that sensitizes Kupffer cells to activate the inflammatory mediators like TNF-α, IL6, and ROS. Another factor that facilitates the liver disease is the loss of anti-inflammatory mediators. A study has shown IL-10 deficient mice to be more sensitive to ethanol liver injury [3]. The alterations in the intestinal microbiota composition are significant for the pathogenesis of chronic liver disease which is demonstrated and briefed in **Figure 1**.

#### **5. Effect of alcohol on liver and adipose tissue**

The energy value of alcohol is equal to those of other nutrients, so when the alcohol consumption is increased, the overall calorie intake excessed the expenditure of energy, which leads to adiposity [86]. It has been established that chronic cirrhosis patient shifts toward the lipid oxidation instead of carbohydrate as fuel to meet the energy requirements which in turn reduces the overall fat mass in an individual [87]. Thus, malnutrition with low adipose skeletal muscle mass is a symptom for an advancement of the liver disease [88]. Alterations in the body mass may also depend on the type of drinking pattern; for instance, a person drinking beer or spirits gains more body mass than wine consumption [89]. Thus, excess alcohol

#### **Figure 2.**

*Association of adipose tissue in alcoholism due to metabolic, endocrine, and immune dysbiosis.*

intake increases the amount of visceral adipose tissue as compared to the changes observed in case of obesity [90].

In vivo mice experimental alcoholic model has shown the significant increase in the number of adipocyte death in white adipose tissue. The mechanism for death of adipocytes involves interaction between CYP2E1, BH3-an interaction domain agonist for death (BID) and C1Q complement pathway. As sequence, these interaction lead to adipose tissue inflammation, insulin resistance, lipolysis, NEFA and release of proinflammatory cytokines [91]. Hence, increase in uptake of fatty acids in the adipose tissue will lead to an increase in hypertrophy, hypoxia, and inflammation ultimately leading to the cell death [92]. However, alcohol uptake in the moderation has been associated with insulin sensitivity [93]. Thus, acute or chronic alcohol consumption is associated with metabolic, endocrine, and immune dysbiosis as shown in **Figure 2**.

#### **5.1 Influence of alcohol on metabolic dysbiosis**

Metabolically, an increase of NEFA is seen in ALD patients [94]. Increase in NEFA depends on the increase in expression of adipose triglyceride lipase (ATGL) but is independent of lipase [95]. Lipolysis is particularly marked by acute alcoholic hepatitis (AAH), but it may decrease during the advanced cirrhosis. The molecular mechanism that leads to lipolysis with excess ethanol consumption is not clearly understood; the possible primary factor may be the ethanol-mediated insulin resistance. Contradicting effect is seen with the use of catecholamine, which may reduce lipolysis or remain unchanged [96]. Consequently, higher level of circulating NEFA shows reduced capacity of the adipose tissue to esterify alcohol and store up free fatty acid [97]. Further these unsaturated fatty acids are delivered to the liver which contributes to hepatic steatosis as they get converted to triglyceride [98]. c-Jun N-terminal kinase (JNK) pathway triggers the hepatocyte apoptosis by increase in number of saturated fatty acids with enhanced hepatotoxic effect [99]. Hepatic de novo lipogenesis increases due to the transcription of sterol regulatory

**61**

*Effect of Alcohol on Gut-Liver Axis and Adipose Tissue DOI: http://dx.doi.org/10.5772/intechopen.89340*

> **(mouse and human)**

**Acute alcoholic model**

Leptin In both models ↓ ↓ [102]

Visfatin In both models ↑ ↑ [105] Omentin In human model ↑ ↑ [24]

*Adiponectin data are in contrast with the observation in individual with obesity and metabolic syndrome due to* 

Chemerin In human model ↑ ↑ Chronic alcoholic

**Chronic alcoholic** 

**Reference**

[106]

**model**

patient

↓ Cirrhosis patient [107]

In human model ↑ ↑ [103] In mouse model ↑ ↑ [104]

**Adipokine In vivo model** 

(high fat diet

Adiponectin\*

and alcohol)

*\**

**Table 1.**

myeloid cells stimulating cytokine release [101].

*changes in liver function while affecting the bile obstruction.*

Kupffer cell sensitivity toward LPS [67].

phages, and expression of CD4+

**5.3 Immune dysbiosis due to alcohol intake**

**5.2 Endocrine imbalance due to consumption of alcohol**

*The changes in adipokines in mouse and human models with severity of alcohol abuse.*

element-binding protein 1 (*Srebf1*) [100]. The mechanism that follows in hepatocytes on increase in NEFA activates the hepatic stellate cells (HSCs) which lead to the deposition of collagen and fibrosis, which in turn exerts the inflammatory pathway through stimulation of NF-ĸB and the activation of Kupffer cells and

Acute or chronic alcohol intake has an important difference in both animal and

Oxidative stress due to the consumption of alcohol leads to adipose tissue hypoxia which in turn increases the expression of TNF-α, CCL2, IL-6, infiltration of macro-

The secretion of pro-inflammatory cytokines alters the hepatic immunology via hepatic inflammation affecting the role of parenchymal and non-parenchymal liver cells. TNF-α activation triggers ALD pathogenesis, which induces apoptosis through the activation of JNK and NFκB pathways [111]. A protective mechanism of the hepatocytes is exerted by IL-6 through promoting the hepatic survival, proliferation, and improved hepatic steatosis [112]. Nevertheless, an excessive exposure of IL-6 can lead to the liver carcinogenesis [113]. In CCL2 knockout mice, there is a reduced level of hepatic inflammation, proving CCL2 to not play any protective role in hepatic inflammation [114]. The role of CCL2 is much more clear as an inflammatory factor through

an insulin signaling in NASH, but its role in ALD is yet to be determined [115].

T cells and dendritic cells in the adipose tissue [110].

Due to the change in an endocrine function, the liver fibrosis takes place by promoting HSC activation [108]. Leptin contributes to the activation of TNF-alpha and the Kupffer cells, thereby causing hepatic inflammation by stimulating CCL2 release from HSCs [109]. The administration of adiponectin and recombinant adiponectin in ethanol-fed mice reduced the circulating NEFA level as well as decreased weight loss, steatosis, and hepatic inflammation due to the inhibition of

human models with respect to the endocrine aspect as shown in **Table 1**.

#### *Effect of Alcohol on Gut-Liver Axis and Adipose Tissue DOI: http://dx.doi.org/10.5772/intechopen.89340*


*\* Adiponectin data are in contrast with the observation in individual with obesity and metabolic syndrome due to changes in liver function while affecting the bile obstruction.*

#### **Table 1.**

*Adipose Tissue - An Update*

observed in case of obesity [90].

**5.1 Influence of alcohol on metabolic dysbiosis**

shown in **Figure 2**.

**Figure 2.**

intake increases the amount of visceral adipose tissue as compared to the changes

*Association of adipose tissue in alcoholism due to metabolic, endocrine, and immune dysbiosis.*

Metabolically, an increase of NEFA is seen in ALD patients [94]. Increase in NEFA depends on the increase in expression of adipose triglyceride lipase (ATGL) but is independent of lipase [95]. Lipolysis is particularly marked by acute alcoholic hepatitis (AAH), but it may decrease during the advanced cirrhosis. The molecular mechanism that leads to lipolysis with excess ethanol consumption is not clearly understood; the possible primary factor may be the ethanol-mediated insulin resistance. Contradicting effect is seen with the use of catecholamine, which may reduce lipolysis or remain unchanged [96]. Consequently, higher level of circulating NEFA shows reduced capacity of the adipose tissue to esterify alcohol and store up free fatty acid [97]. Further these unsaturated fatty acids are delivered to the liver which contributes to hepatic steatosis as they get converted to triglyceride [98]. c-Jun N-terminal kinase (JNK) pathway triggers the hepatocyte apoptosis by increase in number of saturated fatty acids with enhanced hepatotoxic effect [99]. Hepatic de novo lipogenesis increases due to the transcription of sterol regulatory

In vivo mice experimental alcoholic model has shown the significant increase in the number of adipocyte death in white adipose tissue. The mechanism for death of adipocytes involves interaction between CYP2E1, BH3-an interaction domain agonist for death (BID) and C1Q complement pathway. As sequence, these interaction lead to adipose tissue inflammation, insulin resistance, lipolysis, NEFA and release of proinflammatory cytokines [91]. Hence, increase in uptake of fatty acids in the adipose tissue will lead to an increase in hypertrophy, hypoxia, and inflammation ultimately leading to the cell death [92]. However, alcohol uptake in the moderation has been associated with insulin sensitivity [93]. Thus, acute or chronic alcohol consumption is associated with metabolic, endocrine, and immune dysbiosis as

**60**

*The changes in adipokines in mouse and human models with severity of alcohol abuse.*

element-binding protein 1 (*Srebf1*) [100]. The mechanism that follows in hepatocytes on increase in NEFA activates the hepatic stellate cells (HSCs) which lead to the deposition of collagen and fibrosis, which in turn exerts the inflammatory pathway through stimulation of NF-ĸB and the activation of Kupffer cells and myeloid cells stimulating cytokine release [101].

#### **5.2 Endocrine imbalance due to consumption of alcohol**

Acute or chronic alcohol intake has an important difference in both animal and human models with respect to the endocrine aspect as shown in **Table 1**.

Due to the change in an endocrine function, the liver fibrosis takes place by promoting HSC activation [108]. Leptin contributes to the activation of TNF-alpha and the Kupffer cells, thereby causing hepatic inflammation by stimulating CCL2 release from HSCs [109]. The administration of adiponectin and recombinant adiponectin in ethanol-fed mice reduced the circulating NEFA level as well as decreased weight loss, steatosis, and hepatic inflammation due to the inhibition of Kupffer cell sensitivity toward LPS [67].

#### **5.3 Immune dysbiosis due to alcohol intake**

Oxidative stress due to the consumption of alcohol leads to adipose tissue hypoxia which in turn increases the expression of TNF-α, CCL2, IL-6, infiltration of macrophages, and expression of CD4+ T cells and dendritic cells in the adipose tissue [110]. The secretion of pro-inflammatory cytokines alters the hepatic immunology via hepatic inflammation affecting the role of parenchymal and non-parenchymal liver cells. TNF-α activation triggers ALD pathogenesis, which induces apoptosis through the activation of JNK and NFκB pathways [111]. A protective mechanism of the hepatocytes is exerted by IL-6 through promoting the hepatic survival, proliferation, and improved hepatic steatosis [112]. Nevertheless, an excessive exposure of IL-6 can lead to the liver carcinogenesis [113]. In CCL2 knockout mice, there is a reduced level of hepatic inflammation, proving CCL2 to not play any protective role in hepatic inflammation [114]. The role of CCL2 is much more clear as an inflammatory factor through an insulin signaling in NASH, but its role in ALD is yet to be determined [115].

#### **5.4 Role of microRNA**

Exosomes that contain small biologically active but noncoding RNA, i.e., microRNA (miRNA), are released by adipocytes which regulate various intracellular processes. These miRNAs are able to temper the distant tissues and organs representing the alteration between adipose tissue and liver function as well as immune responses [116]. In an animal model, miRNA-122 and miRNA-192 expressions are elevated in the ALD, while miRNA-155 expression is increased in the adipose tissues in particular, which contributes to the hepatic steatosis and fibrosis [117].

#### **6. Conclusion**

Chronic alcohol consumption not only disturbs the metabolism of whole body but also has a prominent effect on the function of gut microbiota and adipose tissue. These alterations have direct as well as indirect effects on the liver functions, which contribute to the advancement of ALD. Cessation of alcohol intake can quickly reverse inflammatory reaction in the adipose tissue and halt the progression of ALD. In addition to that, pharmacological treatment can also help to improve ALD. There is a significant overlapping in an alteration of the adipose tissue between obesity, NAFLD, and ALD mechanism. The physicians who are dealing with patients of ALD should keep an eye on the adipose tissue dysfunction and its effect on the liver and consider the therapeutic treatment accordingly. Understanding the fundamental mechanism of the alcohol and metabolic syndrome in the pathogenesis of liver disease will help in pursuing an effective treatment for liver diseases.

#### **Author details**

Dhara Patel and Palash Mandal\* Department of Biological Sciences, P.D. Patel Institute of Applied Sciences, Charotar University of Science and Technology, Anand, Gujarat, India

\*Address all correspondence to: palashmandal.bio@charusat.ac.in

© 2019 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.

**63**

*Effect of Alcohol on Gut-Liver Axis and Adipose Tissue DOI: http://dx.doi.org/10.5772/intechopen.89340*

> [8] Molina DK, DiMaio VJM. Normal organ weights in men. The American Journal of Forensic Medicine and Pathology. 2012;**33**:368-372. DOI: 10.1097/PAF.0b013e31823d29ad

[9] Nazare J-A, Smith JD, Borel A-L, Haffner SM, Balkau B, Ross R, et al. Ethnic influences on the relations between abdominal subcutaneous and visceral adiposity, liver fat, and cardiometabolic risk profile: The international study of prediction of intra-abdominal adiposity and its relationship with cardiometabolic risk/intra-abdominal adiposity. The American Journal of Clinical Nutrition.

2012;**96**:714-726. DOI: 10.3945/

[10] Raji A, Seely EW, Arky RA, Simonson DC. Body fat distribution and insulin resistance in healthy Asian Indians and Caucasians. The Journal of Clinical Endocrinology and Metabolism. 2001;**86**:5366-5371. DOI: 10.1210/

[11] Rutkowski JM, Stern JH, Scherer PE. The cell biology of fat expansion. The Journal of Cell Biology. 2015;**208**:501-512. DOI: 10.1083/

[12] Cusi K. Role of obesity and lipotoxicity in the development of nonalcoholic steatohepatitis: Pathophysiology and clinical implications. Gastroenterology. 2012;**142**:711-725. DOI: 10.1053/j.

[14] Patel D, Patel F, Mandal P. Potential molecular mechanism of

[13] Nielsen TS, Jessen N, Jorgensen JOL, Moller N, Lund S. Dissecting adipose tissue lipolysis: Molecular regulation and implications for metabolic disease. Journal of Molecular Endocrinology. 2014;**52**:R199-R222. DOI: 10.1530/

ajcn.112.035758

jcem.86.11.7992

jcb.201409063

gastro.2012.02.003

JME-13-0277

[1] Rehm J, Samokhvalov AV, Shield KD. Global burden of alcoholic liver diseases. Journal of Hepatology. 2013;**59**:160-168.

DOI: 10.1016/j.jhep.2013.03.007

McCullough AJ, Practice Guideline Committee of the American Association for the Study of Liver Diseases, Practice Parameters Committee of the American College of Gastroenterology. Alcoholic liver disease. Hepatology. 2010;**51**: 307-328. DOI: 10.1002/hep.23258

[3] Mandal P, Park P-H, McMullen MR,

inflammatory effects of adiponectin are mediated via a heme oxygenase-1 dependent pathway in rat Kupffer cells. Hepatology. 2010;**51**:1420-1429. DOI:

[4] Steiner J, Lang C. Alcohol, adipose tissue and lipid dysregulation.

Biomolecules. 2017;**7**:16. DOI: 10.3390/

[6] Zhang W, Zhong W, Sun X, Sun Q, Tan X, Li Q, et al. Visceral white adipose tissue is susceptible to alcoholinduced lipodystrophy in rats: Role of acetaldehyde. Alcoholism, Clinical and Experimental Research. 2015;**39**: 416-423. DOI: 10.1111/acer.12646

[7] Molina DK, DiMaio VJM. Normal organ weights in women. The American Journal of Forensic Medicine and Pathology. 2015;**36**:176-181. DOI: 10.1097/PAF.0000000000000174

[5] Sun X, Tang Y, Tan X, Li Q, Zhong W, Sun X, et al. Activation of peroxisome proliferator-activated receptor-γ by rosiglitazone improves lipid homeostasis at the adipose tissue-liver axis in ethanol-fed mice. American Journal of Physiology. Gastrointestinal and Liver Physiology. 2012;**302**:G548-G557. DOI: 10.1152/

[2] O'Shea RS, Dasarathy S,

Pratt BT, Nagy LE. The anti-

10.1002/hep.23427

biom7010016

ajpgi.00342.2011

**References**

*Effect of Alcohol on Gut-Liver Axis and Adipose Tissue DOI: http://dx.doi.org/10.5772/intechopen.89340*

### **References**

*Adipose Tissue - An Update*

**5.4 Role of microRNA**

**6. Conclusion**

liver diseases.

**Author details**

Dhara Patel and Palash Mandal\*

provided the original work is properly cited.

Exosomes that contain small biologically active but noncoding RNA, i.e., microRNA (miRNA), are released by adipocytes which regulate various intracellular processes. These miRNAs are able to temper the distant tissues and organs representing the alteration between adipose tissue and liver function as well as immune responses [116]. In an animal model, miRNA-122 and miRNA-192 expressions are elevated in the ALD, while miRNA-155 expression is increased in the adipose tissues

in particular, which contributes to the hepatic steatosis and fibrosis [117].

Chronic alcohol consumption not only disturbs the metabolism of whole body

but also has a prominent effect on the function of gut microbiota and adipose tissue. These alterations have direct as well as indirect effects on the liver functions, which contribute to the advancement of ALD. Cessation of alcohol intake can quickly reverse inflammatory reaction in the adipose tissue and halt the progression of ALD. In addition to that, pharmacological treatment can also help to improve ALD. There is a significant overlapping in an alteration of the adipose tissue between obesity, NAFLD, and ALD mechanism. The physicians who are dealing with patients of ALD should keep an eye on the adipose tissue dysfunction and its effect on the liver and consider the therapeutic treatment accordingly. Understanding the fundamental mechanism of the alcohol and metabolic syndrome in the pathogenesis of liver disease will help in pursuing an effective treatment for

Department of Biological Sciences, P.D. Patel Institute of Applied Sciences, Charotar University of Science and Technology, Anand, Gujarat, India

© 2019 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,

\*Address all correspondence to: palashmandal.bio@charusat.ac.in

**62**

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DOI: 10.1038/35053000

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2006;**101**:1244-1252. DOI: 10.1111/j.1572-0241.2006.00543.x

ajpendo.00572.2004

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10.1016/j.tem.2010.08.001

DOI: 10.1172/JCI19246

DOI: 10.1172/JCI29881

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Saltiel AR. Obesity induces a phenotypic switch in adipose tissue macrophage polarization. The Journal of Clinical Investigation. 2007;**117**:175-184.

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[17] Xu A, Wang Y, Keshaw H, Xu LY, Lam KSL, Cooper GJS. The fat-derived hormone adiponectin alleviates alcoholic and nonalcoholic fatty liver diseases in mice. The Journal of Clinical Investigation. 2003;**112**:91-100. DOI:

[18] Vázquez MJ, González CR, Varela L, Lage R, Tovar S,

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10.1016/j.lfs.2005.04.004

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2005;**54**:3458-3465

2001;**25**(1):83-88

**70**

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**73**

Section 3

Diseases Due to

Disturbances in Adipose

Tissue

Section 3
