Muscle Disorder: Inflammation-Induced

*Muscle Cells - Recent Advances and Future Perspectives*

[137] Yu P, Wang H, Xie Y, Zhou J, Yao J, Che L. Deregulated cardiac specific microRNAs in postnatal heart growth. BioMed Research International. 2016;**2016**:6241763. DOI: 10.1155/2016/6241763

[130] Boettger T, Beetz N, Kostin S, Schneider J, Krüger M, Hein L, et al. Acquisition of the contractile phenotype by murine arterial smooth muscle cells depends on the Mir143/145 gene cluster. Journal of Clinical Investigation.

[131] Elia L, Quintavalle M, Zhang J. The knockout of miR-143 and -145 alters smooth muscle cell maintenance and vascular homeostasis in mice: Correlates with human disease. Cell Death and Differentiation. 2009;**16**(12):1590-1598

[132] Wang K-C, Garmire LX, Young A, Nguyen P, Trinh A, Subramaniam S, et al. Role of microRNA-23b in flowregulation of Rb phosphorylation and endothelial cell growth. Proceedings of the National Academy of Sciences.

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[133] Ewan LC, Jopling HM, Jia H, Mittar S, Bagherzadeh A, Howell GJ, et al. Intrinsic tyrosine kinase activity is required for vascular endothelial growth factor receptor 2 ubiquitination, sorting and degradation in endothelial cells.

Traffic. 2006;**7**(9):1270-1282

[134] Takata H, Kato M, Denda K, Kitamura NA. Hrs binding protein having a Src homology 3 domain is involved in intracellular degradation of growth factors and their receptors. Genes to Cells. 2000;**5**(1):57-69

[135] Chen Y, Gorski D. Regulation of angiogenesis through a microRNA (miR-130a) that down-regulates antiangiogenic homeobox genes GAX and HOXA5. Blood.

[136] das Neves VJ, Fernandes T, Roque FR, Soci UPR, Melo SFS, de Oliveira EM. Exercise training in hypertension: Role of microRNAs. World Journal of Cardiology. 2014;**6**(8713):27. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/25228951 [Cited: September 10,

2008;**111**(3):1217-1226

**84**

2018]

**87**

**Chapter 5**

**Abstract**

inflammation

**1. Introduction**

Inflammatory Muscle Diseases

Inflammatory myopathies, also called idiopathic inflammatory myopathy or myositis, are rare conditions characterized by the involvement of various organs in addition to muscle tissue. These changes can lead to severe impairments and adversely impact the quality of life of affected individuals. The diagnosis and treatment of inflammatory myopathies involve the participation of an interdisciplinary team, due to the complexity of the disease and the high variety of possible signs and symptoms. In this chapter we will discuss the epidemiology and characteristics of the main subtypes of inflammatory myopathies, such as polymyositis, dermatomyositis, necrotizing myopathy, overlap myositis, and myositis of inclusion bodies. Next, we will discuss the existence of crosstalk between inflammatory processes in the oral cavity and their consequences on skeletal muscle. As oral inflammation can increase infiltration of macrophages in muscle tissue and this increase is related to the production of proinflammatory cytokines in this tissue, these cytokines can cause muscle weakness. It is important to consider the prevention of chronic inflammatory processes in order to maintain muscle integrity or even prevent the worsening of the clinical condition of patients with inflammatory muscle diseases.

**Keywords:** myositis, dermatomyositis, polymyositis, inclusion body myositis,

Inflammatory myopathies, also called idiopathic inflammatory myopathy or myositis, are rare conditions characterized by the involvement of various organs in addition to muscle tissue. These changes can lead to severe impairments and

The diagnosis and treatment of inflammatory myopathies involve the participation of an interdisciplinary team, due to the complexity of the disease and the high variety of possible signs and symptoms. The integration of subspecialties, such as rheumatologist, neurologist, dermatologist, pulmonologist, cardiologist, and physiotherapist, among others, is necessary to achieve the ideal treatment plan. Diagnosis of inflammatory myopathies involves several steps and often requires autoantibody testing and histological evaluation of a muscle tissue biopsy in addition to several other tests, including muscle magnetic resonance imaging and electromyography. Typical symptoms of inflammatory myopathies include muscle weakness in the arms and legs, which may manifest in a few days or even several weeks. Muscular weakness is reflected in difficulties in performing daily activities such as walking, climbing stairs, or lifting an object above the head. In addition to muscle weakness, it is observed that pain is also a frequent detectable symptom in a

adversely impact the quality of life of affected individuals [1, 2].

*Doris Hissako Sumida, Fernando Yamamoto Chiba* 

*and Maria Sara de Lima Coutinho Mattera*

## **Chapter 5** Inflammatory Muscle Diseases

*Doris Hissako Sumida, Fernando Yamamoto Chiba and Maria Sara de Lima Coutinho Mattera*

### **Abstract**

Inflammatory myopathies, also called idiopathic inflammatory myopathy or myositis, are rare conditions characterized by the involvement of various organs in addition to muscle tissue. These changes can lead to severe impairments and adversely impact the quality of life of affected individuals. The diagnosis and treatment of inflammatory myopathies involve the participation of an interdisciplinary team, due to the complexity of the disease and the high variety of possible signs and symptoms. In this chapter we will discuss the epidemiology and characteristics of the main subtypes of inflammatory myopathies, such as polymyositis, dermatomyositis, necrotizing myopathy, overlap myositis, and myositis of inclusion bodies. Next, we will discuss the existence of crosstalk between inflammatory processes in the oral cavity and their consequences on skeletal muscle. As oral inflammation can increase infiltration of macrophages in muscle tissue and this increase is related to the production of proinflammatory cytokines in this tissue, these cytokines can cause muscle weakness. It is important to consider the prevention of chronic inflammatory processes in order to maintain muscle integrity or even prevent the worsening of the clinical condition of patients with inflammatory muscle diseases.

**Keywords:** myositis, dermatomyositis, polymyositis, inclusion body myositis, inflammation

### **1. Introduction**

Inflammatory myopathies, also called idiopathic inflammatory myopathy or myositis, are rare conditions characterized by the involvement of various organs in addition to muscle tissue. These changes can lead to severe impairments and adversely impact the quality of life of affected individuals [1, 2].

The diagnosis and treatment of inflammatory myopathies involve the participation of an interdisciplinary team, due to the complexity of the disease and the high variety of possible signs and symptoms. The integration of subspecialties, such as rheumatologist, neurologist, dermatologist, pulmonologist, cardiologist, and physiotherapist, among others, is necessary to achieve the ideal treatment plan. Diagnosis of inflammatory myopathies involves several steps and often requires autoantibody testing and histological evaluation of a muscle tissue biopsy in addition to several other tests, including muscle magnetic resonance imaging and electromyography. Typical symptoms of inflammatory myopathies include muscle weakness in the arms and legs, which may manifest in a few days or even several weeks. Muscular weakness is reflected in difficulties in performing daily activities such as walking, climbing stairs, or lifting an object above the head. In addition to muscle weakness, it is observed that pain is also a frequent detectable symptom in a patient with inflammatory myopathies. Laboratory tests usually show a significant increase of creatine kinase and elevation in the concentration of liver enzymes that suggest the occurrence of damage to muscle cells [1, 3].

The adverse impact on quality of life highlights the importance of performing an accurate and reliable diagnosis from the combination of clinical and laboratory findings to establish the appropriate treatment for each individual [1, 2].

In this chapter we will discuss the epidemiology and subtypes of inflammatory myopathies. Next, we will discuss the existence of crosstalk between inflammatory processes in the oral cavity and their consequences on skeletal muscle.

### **2. Inflammatory myopathies**

### **2.1 Epidemiology of inflammatory muscle diseases**

All myositis subtypes can be considered rare diseases due to their relatively low prevalence. Studies indicate that overlap myositis represents the subtype of the disease that affects the largest number of people, comprising about half of the cases registered. Dermatomyositis accounts for more than a third of the cases of the disease and presents a prevalence of approximately 1–6 patients per 100,000 people in the United States [4–6].

It is important to emphasize that obtaining accurate epidemiological data is extremely difficult due to the different diagnostic criteria adopted in each study. Therefore, the information provided by the publications should be examined and evaluated with caution and attention [7].

A large study conducted from the analysis of 3067 patients from Belgium, China, Czech Republic, Hungary, Italy, Mexico, Norway, Sweden, Switzerland, the United Kingdom (UK), and Vietnam who were registered in the Euromyositis Registry demonstrated that the dermatomyositis was the most common disorder with 31% of the cases [7].

Data on the prevalence of necrotizing myopathy suggest that this subtype of the disease accounts for approximately one-fifth of the reported cases of inflammatory muscle diseases [4–6].

The information regarding the epidemiology of polymyositis varies and depends on the methodology and location of the study ranging from the largest fraction with prevalence of approximately 10 cases per 100,000 people in the United States [1–3], 27% in the Euromyositis Registry [7], to the rarest subtype that should be diagnosed only by exclusion [4–6].

Currently there is some consensus that overlap myositis, necrotizing myopathy, and dermatomyositis represent about 90% of the cases of inflammatory muscle diseases [4–6]. It is estimated that the inclusion body myositis occurs with a prevalence of up to 14 per million people [8].

### **2.2 Dermatomyositis**

Dermatomyositis is typically characterized by the development of proximal muscle weakness and cutaneous manifestations that may arise over a period of weeks to months. However, there are cases in which muscular impairment is not significant without signs and symptoms of muscle weakness, elevated muscle enzymes or changes in electromyography, magnetic resonance imaging (MRI), and muscle biopsy [9].

Skin signs frequently seen in dermatomyositis include an exacerbated periorbital rash with edematous features and erythematous lesions involving the extensor

**89**

*Inflammatory Muscle Diseases*

*DOI: http://dx.doi.org/10.5772/intechopen.86053*

surfaces of the joints. In some cases, myalgia and pruritus may also be observed as important symptoms of the disease. Muscle enzyme concentrations tend to be elevated, and electromyography commonly shows a myopathic pattern [10]. Intramuscular T2 hyperintensities resulting from inflammation or muscle necrosis can be observed on MRI. Dermatomyositis may present a characteristic less frequently observed in other types of inflammatory myopathies, which involves the presence of T2 hyperintensities around individual muscles due to fascial involvement [11].

may be characteristically prominent in perifascicular regions [14].

Studies have shown that dermatomyositis autoantibody can be found in a considerable proportion of patients with dermatomyositis [15]. Typical features of dermatomyositis, including proximal muscle weakness and prominent cutaneous manifestations have been associated with the presence of autoantibodies recognizing the nuclear antigen Mi2 [16]. Patients with dermatomyositis and autoantibodies that recognize nuclear matrix protein (NXP) 2 are more predisposed to be affected by proximal and distal muscular weakness, subcutaneous edema, and dysphagia [17]. Patients with dermatomyositis who are positive for anti-NXP2 or antitranscription intermediary factor (TIF)-1 autoantibodies are at increased risk for malignancy development; thus making comprehensive cancer screening 13–15 or positron emission tomography–computed tomography (PET-CT) scans is extremely

important in these cases [18]. In cases of dermatomyositis patients who have autoantibodies recognizing the small ubiquitin-like modifier activating enzyme or melanoma differentiation-associated gene 5 (MDA5), it is observed that cutaneous tissue impairment is more prominent than in muscle. In addition to most commonly present cutaneous manifestations, these patients may develop ulcerous lesions on

Most patients with anti-MDA5 autoantibodies are hypomyopathic or amyopathic. In addition, it should be noted that unlike patients with other autoantibodies of dermatomyositis, those who are anti-MDA5 positive often develop an aggressive form of interstitial lung disease, reinforcing the importance of assessment through periodic lung function tests and high-resolution computed tomography [20–22]. Although the etiology of dermatomyositis is not fully elucidated, it is suggested that a combination of genetic risk factors and exposure to environmental factors may trigger the disease. In this sense, several immunogenetic risk factors, including certain class-2 human leukocyte antigen (HLA) alleles, have been implicated in dermatomyositis pathogenesis [23]. Studies suggest that exposure to ultraviolet light may also be considered an important risk factor for the development of dermatomyositis [24]. Regardless of the origin of dermatomyositis, it is not known which mechanisms are involved in the development of muscle damage and weakness. Studies suggest

the flexor surface of the fingers and palm [19, 20].

Muscular biopsies in patients with dermatomyositis have perifascicular atrophy as a feature of high specificity [12]. Evidences show that the expression of perifascicular human myxovirus resistance protein 1 and retinoic acid-inducible gene 1 have higher diagnostic sensitivity than perifascicular atrophy with equivalent specificity [13]. Muscular biopsies of dermatomyositis patients usually present cellular infiltrates composed of plasmacytoid dendritic cells, B cells, CD4 T cells, and macrophages. These cells usually involve medium-sized blood vessels and invade the perimysium [14]. However, it is possible that dermatomyositis biopsy does not present this cellular infiltrate. Predominantly, necrotic pathologically indistinguishable from immune-mediated necrotizing myopathy may be observed. Some early features of dermatomyositis involve deposition of membrane attack complex and presence of microtubular inclusions on intramuscular capillaries [11]. In addition, like other inflammatory myopathies, class-1 major histocompatibility complex (MHC) is generally upregulated in the sarcolemma of muscle fibers. In patients with dermatomyositis, class-1 MHC upregulation and other pathological findings

### *Inflammatory Muscle Diseases DOI: http://dx.doi.org/10.5772/intechopen.86053*

*Muscle Cells - Recent Advances and Future Perspectives*

**2. Inflammatory myopathies**

in the United States [4–6].

with 31% of the cases [7].

muscle diseases [4–6].

only by exclusion [4–6].

**2.2 Dermatomyositis**

muscle biopsy [9].

lence of up to 14 per million people [8].

evaluated with caution and attention [7].

suggest the occurrence of damage to muscle cells [1, 3].

**2.1 Epidemiology of inflammatory muscle diseases**

patient with inflammatory myopathies. Laboratory tests usually show a significant increase of creatine kinase and elevation in the concentration of liver enzymes that

The adverse impact on quality of life highlights the importance of performing an accurate and reliable diagnosis from the combination of clinical and laboratory

In this chapter we will discuss the epidemiology and subtypes of inflammatory myopathies. Next, we will discuss the existence of crosstalk between inflammatory

All myositis subtypes can be considered rare diseases due to their relatively low prevalence. Studies indicate that overlap myositis represents the subtype of the disease that affects the largest number of people, comprising about half of the cases registered. Dermatomyositis accounts for more than a third of the cases of the disease and presents a prevalence of approximately 1–6 patients per 100,000 people

It is important to emphasize that obtaining accurate epidemiological data is extremely difficult due to the different diagnostic criteria adopted in each study. Therefore, the information provided by the publications should be examined and

A large study conducted from the analysis of 3067 patients from Belgium, China, Czech Republic, Hungary, Italy, Mexico, Norway, Sweden, Switzerland, the United Kingdom (UK), and Vietnam who were registered in the Euromyositis Registry demonstrated that the dermatomyositis was the most common disorder

Data on the prevalence of necrotizing myopathy suggest that this subtype of the disease accounts for approximately one-fifth of the reported cases of inflammatory

The information regarding the epidemiology of polymyositis varies and depends on the methodology and location of the study ranging from the largest fraction with prevalence of approximately 10 cases per 100,000 people in the United States [1–3], 27% in the Euromyositis Registry [7], to the rarest subtype that should be diagnosed

Currently there is some consensus that overlap myositis, necrotizing myopathy, and dermatomyositis represent about 90% of the cases of inflammatory muscle diseases [4–6]. It is estimated that the inclusion body myositis occurs with a preva-

Dermatomyositis is typically characterized by the development of proximal muscle weakness and cutaneous manifestations that may arise over a period of weeks to months. However, there are cases in which muscular impairment is not significant without signs and symptoms of muscle weakness, elevated muscle enzymes or changes in electromyography, magnetic resonance imaging (MRI), and

Skin signs frequently seen in dermatomyositis include an exacerbated periorbital

rash with edematous features and erythematous lesions involving the extensor

findings to establish the appropriate treatment for each individual [1, 2].

processes in the oral cavity and their consequences on skeletal muscle.

**88**

surfaces of the joints. In some cases, myalgia and pruritus may also be observed as important symptoms of the disease. Muscle enzyme concentrations tend to be elevated, and electromyography commonly shows a myopathic pattern [10]. Intramuscular T2 hyperintensities resulting from inflammation or muscle necrosis can be observed on MRI. Dermatomyositis may present a characteristic less frequently observed in other types of inflammatory myopathies, which involves the presence of T2 hyperintensities around individual muscles due to fascial involvement [11].

Muscular biopsies in patients with dermatomyositis have perifascicular atrophy as a feature of high specificity [12]. Evidences show that the expression of perifascicular human myxovirus resistance protein 1 and retinoic acid-inducible gene 1 have higher diagnostic sensitivity than perifascicular atrophy with equivalent specificity [13]. Muscular biopsies of dermatomyositis patients usually present cellular infiltrates composed of plasmacytoid dendritic cells, B cells, CD4 T cells, and macrophages. These cells usually involve medium-sized blood vessels and invade the perimysium [14]. However, it is possible that dermatomyositis biopsy does not present this cellular infiltrate. Predominantly, necrotic pathologically indistinguishable from immune-mediated necrotizing myopathy may be observed. Some early features of dermatomyositis involve deposition of membrane attack complex and presence of microtubular inclusions on intramuscular capillaries [11]. In addition, like other inflammatory myopathies, class-1 major histocompatibility complex (MHC) is generally upregulated in the sarcolemma of muscle fibers. In patients with dermatomyositis, class-1 MHC upregulation and other pathological findings may be characteristically prominent in perifascicular regions [14].

Studies have shown that dermatomyositis autoantibody can be found in a considerable proportion of patients with dermatomyositis [15]. Typical features of dermatomyositis, including proximal muscle weakness and prominent cutaneous manifestations have been associated with the presence of autoantibodies recognizing the nuclear antigen Mi2 [16]. Patients with dermatomyositis and autoantibodies that recognize nuclear matrix protein (NXP) 2 are more predisposed to be affected by proximal and distal muscular weakness, subcutaneous edema, and dysphagia [17].

Patients with dermatomyositis who are positive for anti-NXP2 or antitranscription intermediary factor (TIF)-1 autoantibodies are at increased risk for malignancy development; thus making comprehensive cancer screening 13–15 or positron emission tomography–computed tomography (PET-CT) scans is extremely important in these cases [18]. In cases of dermatomyositis patients who have autoantibodies recognizing the small ubiquitin-like modifier activating enzyme or melanoma differentiation-associated gene 5 (MDA5), it is observed that cutaneous tissue impairment is more prominent than in muscle. In addition to most commonly present cutaneous manifestations, these patients may develop ulcerous lesions on the flexor surface of the fingers and palm [19, 20].

Most patients with anti-MDA5 autoantibodies are hypomyopathic or amyopathic. In addition, it should be noted that unlike patients with other autoantibodies of dermatomyositis, those who are anti-MDA5 positive often develop an aggressive form of interstitial lung disease, reinforcing the importance of assessment through periodic lung function tests and high-resolution computed tomography [20–22].

Although the etiology of dermatomyositis is not fully elucidated, it is suggested that a combination of genetic risk factors and exposure to environmental factors may trigger the disease. In this sense, several immunogenetic risk factors, including certain class-2 human leukocyte antigen (HLA) alleles, have been implicated in dermatomyositis pathogenesis [23]. Studies suggest that exposure to ultraviolet light may also be considered an important risk factor for the development of dermatomyositis [24].

Regardless of the origin of dermatomyositis, it is not known which mechanisms are involved in the development of muscle damage and weakness. Studies suggest

that muscle damage may result from hypoperfusion due to endothelial destruction [14]. In addition, the presence of plasmacytoid dendritic cells, along with the increase in expression of type-1 interferon-inducible proteins in the perifascicular area, suggests that interferon may mediate perifascicular atrophy [12, 25].

Overlap myositis is being recognized as an individual form of myositis. This myositis manifests itself without a rash typical of dermatomyositis, with prominent pathologic changes in the perifascicular, interfascicular, and perimysial regions, and is frequently associated with anti-synthetase antibodies [2].

Laboratory evaluation shows a significant elevation of muscle enzymes including creatine kinase (CK), which is generally present [3]. Approximately 30% of patients with myositis were positive for Jo-1O antibody (most common of the eight anti-synthetase antibodies) [26].

### **2.3 Polymyositis**

Polymyositis is a rare disease, which belongs to the various idiopathic inflammatory myopathies. It is estimated that the incidence of polymyositis is 5% of all cases of myositis [2, 5, 27]. Polymyositis consists of muscle weakness, elevated creatine phosphokinase concentrations, and myopathic electromyography features [2]. However, rash or other signs of skin inflammation do not occur in polymyositis. Therefore, its diagnosis is by exclusion [3].

Histopathological hallmarks of polymyositis include invasion of endomysial cytotoxic CD8 T cells and widespread upregulation of class I MHC in muscle fibers [2, 24]. Polymyositis is a chronic, degenerative disease that has no cure. The treatment consists in the relief of the symptoms with the use of corticosteroids, such as prednisone, intravenous glucocorticoids (when weakness at onset is severe or rapidly worsening), azathioprine, methotrexate, mycophenolate, cyclosporine, and intravenous immune globulin [3].

### **2.4 Inclusion body myositis**

Inclusion body myositis is a very common disease among inflammatory myopathies affecting mainly men from the age of 50. The disease begins insidiously and develops over a period of years, sometimes asymmetrically; it may begin with unilateral affection of a leg or arm, progress steadily, and lead to deep muscular atrophy [2]. Laboratory evaluation shows that an elevated CK is much blander. Skin changes are not present [3].

There is a higher mortality rate in patients with inclusion body myositis, since muscle weakness (long flexors of the fingers, quadriceps, anterior tibial, and, to a lesser extent, all other muscles of the arms and legs) usually leads to harmful falls and dysphagia can cause aspiration pneumonia [3].

The antibody, identified a few years ago, that is present in inclusion body myositis is cN1A (5NT1A/5NTC1A) [3]. The frequency of this antibody is about 30%; other forms of myositis such as dermatomyositis and other conditions such as Sjögren's syndrome and systemic lupus erythematosus (SLE) were also positive even in the absence of any muscle symptoms [3, 28, 29]. Study suggested that the presence of cN1A is associated with a more severe course of disease, dysphagia [3, 30], and increased mortality [3, 31]. However, in another study in German patients, the presence of cN1A did not correlate with the severity of dysphagia or muscle impairment [3, 32].

In the histopathological hallmarks, the distribution and the immunophenotypic profile of the inflammatory cells are similar to those seen in polymyositis macrophages and CD8+ T cells which invade nonnecrotic muscle fibers that express MHC

**91**

*Inflammatory Muscle Diseases*

line inclusions [3, 34, 35].

clean the cell debris [3].

2 (M2) alternatively activated [45].

*DOI: http://dx.doi.org/10.5772/intechopen.86053*

**2.5 Immune-mediated necrotizing myopathy**

detection in certain cohorts was 60% [39].

**2.6 Crosstalk between oral cavity and skeletal muscle**

intolerance shown in animals with oral inflammation [43].

class I antigen on the sarcolemma [33], signs of protein accumulation by detection of amyloid (Congo red, thioflavin S, immunohistochemistry for p62 or TDP-43), detection of tubulofilaments on EM, vacuoles and signs of mitochondrial damage as evidenced by histochemical proof of COX-deficient muscle fibers, and paracrystal-

Immune-mediated necrotizing myopathy is an acute or subacute proximal weakness of the arms and legs, most prominent in the lower limbs [3]. It often affects adults, but it can also occur in children [3]. The progression of the disease is constantly more rapid and severe compared to other myopathies (dermatomyositis and polymyositis) [3]. Laboratory evaluation shows very high muscle enzymes, with an elevated CK of

Approximately 10–20% of patients with immune-mediated necrotizing myopathy have anti-signal recognition particle (SRP); however its detection varies from 0 to 54% [36]. This antibody may be associated with cardiomyopathy and a severe disease with muscle atrophy, interstitial lung disease, and dysphagia [37, 38]. Another antibody that has been identified is reductase (HMGCR) antibody; its

Histopathological hallmarks in necrotizing myopathy show dispersed necrotic myofibers of varying degrees; moderate and predominantly MHC class I focal regulation, particularly in areas with necrotic fibers; and complement binding to the sarcolemma [2, 3, 40–42]. Some inflammatory T cells and other immune cells may be present around these focal points, but there are no primary inflammatory lesions. Necrotic fibers typically exhibit a secondary invasion by macrophages to

In addition to these inflammatory muscular diseases mentioned above, a localized inflammation at a distance from the skeletal muscle may promote change in this tissue. Recent study proposed the existence of crosstalk between oral cavity and skeletal muscle [43]. The researchers induced oral inflammation in rats and observed that the skeletal muscle was affected by increased infiltration of macrophages, which was suggested by the authors as an explanation for the glucose

Research conducted over the last 15 years has investigated possible mechanisms that cause changes in macrophages polarization and the effects of these changes on insulin signaling in metabolic organs [44]. These cells exhibit a high degree of functional plasticity, so that the nature of an inflammatory trigger, as well as the cytokines present, can determine their polarization and their functional status [44]. In analogy to the nomenclature T-helper cells (Th), Th1 Th2, macrophages can be classified into two distinct phenotypes: type 1 (M1) classically activated and type

In vitro, these subsets can be induced by stimulation with interferon gamma (IFN-γ) and lipopolysaccharides (LPS) for M1 or interleukin-4 (IL-4) for M2 [46]. The M1/M2 dichotomy is often used to classify macrophages into pro-inflammatory (M1) or anti-inflammatory (M2) [44]. Among the functions performed by the M1 macrophages, tumor necrosis factor-alpha (TNF-α) production is outstanding [47]. Saghizadeh [48] and collaborators observed that diabetic or insulin-resistant patients have increased expression of TNF-α in skeletal muscle when compared to normoglycemic individuals, suggesting that cytokine plays an important role in the

20–50 times [3]. Neck muscle weakness and dysphagia are common [3].

### *Inflammatory Muscle Diseases DOI: http://dx.doi.org/10.5772/intechopen.86053*

*Muscle Cells - Recent Advances and Future Perspectives*

anti-synthetase antibodies) [26].

Therefore, its diagnosis is by exclusion [3].

intravenous immune globulin [3].

**2.4 Inclusion body myositis**

changes are not present [3].

and dysphagia can cause aspiration pneumonia [3].

**2.3 Polymyositis**

that muscle damage may result from hypoperfusion due to endothelial destruction [14]. In addition, the presence of plasmacytoid dendritic cells, along with the increase in expression of type-1 interferon-inducible proteins in the perifascicular

Overlap myositis is being recognized as an individual form of myositis. This myositis manifests itself without a rash typical of dermatomyositis, with prominent pathologic changes in the perifascicular, interfascicular, and perimysial regions, and

Laboratory evaluation shows a significant elevation of muscle enzymes including creatine kinase (CK), which is generally present [3]. Approximately 30% of patients with myositis were positive for Jo-1O antibody (most common of the eight

Polymyositis is a rare disease, which belongs to the various idiopathic inflammatory myopathies. It is estimated that the incidence of polymyositis is 5% of all cases of myositis [2, 5, 27]. Polymyositis consists of muscle weakness, elevated creatine phosphokinase concentrations, and myopathic electromyography features [2]. However, rash or other signs of skin inflammation do not occur in polymyositis.

Histopathological hallmarks of polymyositis include invasion of endomysial cytotoxic CD8 T cells and widespread upregulation of class I MHC in muscle fibers [2, 24]. Polymyositis is a chronic, degenerative disease that has no cure. The treatment consists in the relief of the symptoms with the use of corticosteroids, such as prednisone, intravenous glucocorticoids (when weakness at onset is severe or rapidly worsening), azathioprine, methotrexate, mycophenolate, cyclosporine, and

Inclusion body myositis is a very common disease among inflammatory myopathies affecting mainly men from the age of 50. The disease begins insidiously and develops over a period of years, sometimes asymmetrically; it may begin with unilateral affection of a leg or arm, progress steadily, and lead to deep muscular atrophy [2]. Laboratory evaluation shows that an elevated CK is much blander. Skin

There is a higher mortality rate in patients with inclusion body myositis, since muscle weakness (long flexors of the fingers, quadriceps, anterior tibial, and, to a lesser extent, all other muscles of the arms and legs) usually leads to harmful falls

In the histopathological hallmarks, the distribution and the immunophenotypic profile of the inflammatory cells are similar to those seen in polymyositis macrophages and CD8+ T cells which invade nonnecrotic muscle fibers that express MHC

The antibody, identified a few years ago, that is present in inclusion body myositis is cN1A (5NT1A/5NTC1A) [3]. The frequency of this antibody is about 30%; other forms of myositis such as dermatomyositis and other conditions such as Sjögren's syndrome and systemic lupus erythematosus (SLE) were also positive even in the absence of any muscle symptoms [3, 28, 29]. Study suggested that the presence of cN1A is associated with a more severe course of disease, dysphagia [3, 30], and increased mortality [3, 31]. However, in another study in German patients, the presence of cN1A did not correlate with the severity of dysphagia or muscle impair-

area, suggests that interferon may mediate perifascicular atrophy [12, 25].

is frequently associated with anti-synthetase antibodies [2].

**90**

ment [3, 32].

class I antigen on the sarcolemma [33], signs of protein accumulation by detection of amyloid (Congo red, thioflavin S, immunohistochemistry for p62 or TDP-43), detection of tubulofilaments on EM, vacuoles and signs of mitochondrial damage as evidenced by histochemical proof of COX-deficient muscle fibers, and paracrystalline inclusions [3, 34, 35].

### **2.5 Immune-mediated necrotizing myopathy**

Immune-mediated necrotizing myopathy is an acute or subacute proximal weakness of the arms and legs, most prominent in the lower limbs [3]. It often affects adults, but it can also occur in children [3]. The progression of the disease is constantly more rapid and severe compared to other myopathies (dermatomyositis and polymyositis) [3]. Laboratory evaluation shows very high muscle enzymes, with an elevated CK of 20–50 times [3]. Neck muscle weakness and dysphagia are common [3].

Approximately 10–20% of patients with immune-mediated necrotizing myopathy have anti-signal recognition particle (SRP); however its detection varies from 0 to 54% [36]. This antibody may be associated with cardiomyopathy and a severe disease with muscle atrophy, interstitial lung disease, and dysphagia [37, 38]. Another antibody that has been identified is reductase (HMGCR) antibody; its detection in certain cohorts was 60% [39].

Histopathological hallmarks in necrotizing myopathy show dispersed necrotic myofibers of varying degrees; moderate and predominantly MHC class I focal regulation, particularly in areas with necrotic fibers; and complement binding to the sarcolemma [2, 3, 40–42]. Some inflammatory T cells and other immune cells may be present around these focal points, but there are no primary inflammatory lesions. Necrotic fibers typically exhibit a secondary invasion by macrophages to clean the cell debris [3].

### **2.6 Crosstalk between oral cavity and skeletal muscle**

In addition to these inflammatory muscular diseases mentioned above, a localized inflammation at a distance from the skeletal muscle may promote change in this tissue. Recent study proposed the existence of crosstalk between oral cavity and skeletal muscle [43]. The researchers induced oral inflammation in rats and observed that the skeletal muscle was affected by increased infiltration of macrophages, which was suggested by the authors as an explanation for the glucose intolerance shown in animals with oral inflammation [43].

Research conducted over the last 15 years has investigated possible mechanisms that cause changes in macrophages polarization and the effects of these changes on insulin signaling in metabolic organs [44]. These cells exhibit a high degree of functional plasticity, so that the nature of an inflammatory trigger, as well as the cytokines present, can determine their polarization and their functional status [44]. In analogy to the nomenclature T-helper cells (Th), Th1 Th2, macrophages can be classified into two distinct phenotypes: type 1 (M1) classically activated and type 2 (M2) alternatively activated [45].

In vitro, these subsets can be induced by stimulation with interferon gamma (IFN-γ) and lipopolysaccharides (LPS) for M1 or interleukin-4 (IL-4) for M2 [46]. The M1/M2 dichotomy is often used to classify macrophages into pro-inflammatory (M1) or anti-inflammatory (M2) [44]. Among the functions performed by the M1 macrophages, tumor necrosis factor-alpha (TNF-α) production is outstanding [47]. Saghizadeh [48] and collaborators observed that diabetic or insulin-resistant patients have increased expression of TNF-α in skeletal muscle when compared to normoglycemic individuals, suggesting that cytokine plays an important role in the

pathogenesis of insulin resistance. TNF-α impairs the insulin signal by decreasing the phosphorylation of insulin receptor substrate 1 (IRS-1) in tyrosine residues [49]. In addition, TNF-α can stimulate some serine kinases including IκB kinase (IKK) and c-Jun amino-terminal kinase (JNK), which promote IRS-1 phosphorylation in serine residues, resulting in insulin signal attenuation [50]. On the other hand, M2 macrophages are associated with tissue repair, angiogenesis, reduction of inflammation, and the improvement of insulin signaling in adipose tissue [45, 51]. In addition to the studies that relate obesity to insulin resistance, there are studies in the literature that demonstrate a correlation between this hormonal resistance and inflammatory processes, such as rheumatoid arthritis and oral inflammations [52–54]. In this context, the apical periodontitis (AP), an oral inflammation, stands out. AP occurs as a consequence of various aggressions to the dental pulp, including physical, iatrogenic, infectious, and endodontic traumas. This inflammatory picture can cause a wide variety of immunological responses, in order to protect the dental pulp and periapical regions. The regulation of periapical inflammation is extremely complex, as it involves host mediators, including immunological components such as antibodies, cytokines, arachidonic acid metabolites, and neuropeptides [55]. The characteristic inflammatory process of AP presents different types of gram-negative anaerobic bacteria [56] with LPS in the cell wall [57]. Studies have reported that bacteria which are present in the oral cavity can release LPS into the systemic circulation [58]. This substance has the ability to activate toll-like receptors (TLRs), a cell surface receptor that activates innate immunity and induces inflammatory responses. LPS is a specific ligand for TLR2 and TLR4 but has a higher specificity for TLR4 [59, 60]. When released by gram-negative bacteria, LPS binds to a soluble plasma protein called LPS binding protein. LPS or LPS binding protein [61, 62] binds to the CD14 co-receptor via lipopolysaccharide binding protein (LPB), forming the LPS-CD14 complex. This complex, in turn, is recognized by the TLR4-MD-2 complex, present on the cell surface, which is capable of promoting intracellular recruitment of adapter molecules with N-terminal TIR domain, such as myeloid differentiation primary response 88 (MYD88). This molecule can activate the serine kinases JNK and IKKα/β, which promote activation of the activating proteins-1 (AP-1) and factor nuclear kappa B (NF-κB) transcription factors, respectively [63, 64]. NF-κB regulates the expression of several genes involved in different cellular processes such as inflammatory and immune responses and cell growth and development. In the absence of an NF-κB-activating stimulus, this protein is present in the cytoplasm inactive with an inhibitory protein, IκB [65]. Activation of NF-κB can occur not only by exposure of the cells to LPS but also by the action of inflammatory cytokines (TNF-α and IL-1), activation of T and B lymphocytes, UV radiation, and expression of products [66]. After stimulation, the IKK is phosphorylated and activated. The IKK complex consists of two catalytic subunits, IKK-α and IKK-β, in addition to the NF-kappa-B essential modulator (NEMO) or IKK-γ [67]. After activation, IKK recruits and phosphorylates the IκB that is recognized by the ubiquitin ligase machinery, which leads to its polyubiquitination and consequent degradation. In this way, the NF-κB dimers translocate to the nucleus, binding at specific sites of the deoxyribonucleic acid (DNA) and promoting the transcription of a large number of genes [65, 67].

In addition to activating the IKKα/β/NF-κB pathway, TLRs are capable of activating the JNK pathway [68]. The serine/threonine kinase group called JNK (JNK-1, JNK-2, and JNK-3) belongs to the family of mitogen-activated protein kinase (MAPKs), responsible for the regulation of various cellular functions. This regulation occurs largely because of its ability to control the transcription of specific genes by AP-1 [69]. AP-1 is a transcription factor that, when activated, promotes the expression of genes related to innate immunity [70]. In addition to LPS, the signaling pathway of TLRs can be activated by heat shock proteins [71]. Heat shock proteins

**93**

*Inflammatory Muscle Diseases*

*DOI: http://dx.doi.org/10.5772/intechopen.86053*

important anabolic hormone for protein metabolism [90].

homeostasis in patients with diabetes and insulin resistance [44].

The study performed by Boon et al. [91] with healthy lean individuals observed that only 5 days of hyperlipidic diet promoted increased messenger ribonucleic acid (mRNA) expression of macrophage markers in skeletal muscle and reduced expression of the glucose transporter type 4 (GLUT-4) glucose transporter protein in this tissue. Similarly, Patsouris et al. [92] demonstrated increased macrophage content in skeletal muscle in diabetic patients independently of body mass index (BMI). An increased macrophage content (assessed by F4/80 protein detection) was observed in muscle tissue of rats with AP in the absence of obesity, highlighting the key role of these cells in the etiology of insulin resistance. It should be noted that only F4/80 detection is not able to provide details on M1-type and M2-type macrophage polarization although evidence demonstrates that under obesity conditions, macrophages infiltrated into muscle tissue exhibit phenotype characteristic of M1 polarization [92–95]. The reprogramming of the M1 polarization toward the M2 polarization may represent a promising strategy for the treatment of glycemic

As previously reported, inflammation causes insulin resistance. According to Pereira et al., rats with AP had increased IKKα/β and JNK phosphorylation status

(HSP) are proteins characterized as chaperones because they have an important function in adaptation to stress and cellular protection, acting mainly in the synthesis and protein degradation, besides regulating fundamental cellular processes [72]. The family of HSPs is divided into subfamilies, classified according to the molecular mass, being small HSPs (8–27 kDa) and large HSPs (100–110 kDa), among which stand out HSP90, HSP70, and HSP60 [73]. In addition to its essential functions as a chaperone [74], HSP70 has an anti-inflammatory effect by inhibiting the activation of NF-KB when present in the intracellular environment [75]. However, stimuli such as cell necrosis and bacterial products such as LPS can cause the passage of HSP70 through the membrane into the extracellular environment [76, 77]. Studies have suggested that elevated serum levels of HSP70 may be correlated with cardiovascular disorder, pulmonary fibrosis, renal damage, oxidative stress, and inflammation [78]. The development of these conditions may occur due to the ability of HSP70 to bind to TLR2 and TLR4, promoting the activation of the NF-κB pathway which, as mentioned above, induces the expression of inflammatory mediators related to insulin resistance [79]. Studies suggest that insulin sensitivity may undergo regulatory action by the adaptive immune system [80, 81]. This system is composed of different types of cells, among which the B and T lymphocytes [82] stand out. T lymphocytes are classified into two main classes: helper T lymphocytes, also known as T helper (Th), and cytotoxic T lymphocytes. The "naïve" Th1 lymphocytes, when interacting with antigen presenting cells, undergo activation and can differentiate into different subtypes [83]. The Th1 subtype expresses proinflammatory cytokines, such as TNF-α and IFN-γ; Th2 expresses mainly anti-inflammatory cytokines, such as interleukin-4 (IL-4) and interleukin-13 (IL-13), and regulatory T cells secrete predominantly anti-inflammatory cytokine and transforming growth factor-β (TGF-β) [84]. Th1 cells play a central role in the recruitment of macrophages and induction of insulin resistance in obesity-induced diabetes models. These effects are counterbalanced by the function of Th2 and Treg cells that maintain an anti-inflammatory state and increase insulin sensitivity [85]. Appropriate regulation of Th cells is of extreme importance for the control and prevention of various diseases [86]. An increase or decrease in the Th1 or Th2 subtypes, as well as the cytokines produced by these cells, indicates an imbalance that may be one of the factors responsible for the development of insulin resistance [87]. It is known that insulin resistance is one of the main characteristics of diabetes mellitus [88]. This disease is also closely related to muscle weakness due to altered insulin action [89], standing out that insulin is an

### *Inflammatory Muscle Diseases DOI: http://dx.doi.org/10.5772/intechopen.86053*

*Muscle Cells - Recent Advances and Future Perspectives*

pathogenesis of insulin resistance. TNF-α impairs the insulin signal by decreasing the phosphorylation of insulin receptor substrate 1 (IRS-1) in tyrosine residues [49]. In addition, TNF-α can stimulate some serine kinases including IκB kinase (IKK) and c-Jun amino-terminal kinase (JNK), which promote IRS-1 phosphorylation in serine residues, resulting in insulin signal attenuation [50]. On the other hand, M2 macrophages are associated with tissue repair, angiogenesis, reduction of inflammation, and the improvement of insulin signaling in adipose tissue [45, 51]. In addition to the studies that relate obesity to insulin resistance, there are studies in the literature that demonstrate a correlation between this hormonal resistance and inflammatory processes, such as rheumatoid arthritis and oral inflammations [52–54]. In this context, the apical periodontitis (AP), an oral inflammation, stands out. AP occurs as a consequence of various aggressions to the dental pulp, including physical, iatrogenic, infectious, and endodontic traumas. This inflammatory picture can cause a wide variety of immunological responses, in order to protect the dental pulp and periapical regions. The regulation of periapical inflammation is extremely complex, as it involves host mediators, including immunological components such as antibodies, cytokines, arachidonic acid metabolites, and neuropeptides [55]. The characteristic inflammatory process of AP presents different types of gram-negative anaerobic bacteria [56] with LPS in the cell wall [57]. Studies have reported that bacteria which are present in the oral cavity can release LPS into the systemic circulation [58]. This substance has the ability to activate toll-like receptors (TLRs), a cell surface receptor that activates innate immunity and induces inflammatory responses. LPS is a specific ligand for TLR2 and TLR4 but has a higher specificity for TLR4 [59, 60]. When released by gram-negative bacteria, LPS binds to a soluble plasma protein called LPS binding protein. LPS or LPS binding protein [61, 62] binds to the CD14 co-receptor via lipopolysaccharide binding protein (LPB), forming the LPS-CD14 complex. This complex, in turn, is recognized by the TLR4-MD-2 complex, present on the cell surface, which is capable of promoting intracellular recruitment of adapter molecules with N-terminal TIR domain, such as myeloid differentiation primary response 88 (MYD88). This molecule can activate the serine kinases JNK and IKKα/β, which promote activation of the activating proteins-1 (AP-1) and factor nuclear kappa B (NF-κB) transcription factors, respectively [63, 64]. NF-κB regulates the expression of several genes involved in different cellular processes such as inflammatory and immune responses and cell growth and development. In the absence of an NF-κB-activating stimulus, this protein is present in the cytoplasm inactive with an inhibitory protein, IκB [65]. Activation of NF-κB can occur not only by exposure of the cells to LPS but also by the action of inflammatory cytokines (TNF-α and IL-1), activation of T and B lymphocytes, UV radiation, and expression of products [66]. After stimulation, the IKK is phosphorylated and activated. The IKK complex consists of two catalytic subunits, IKK-α and IKK-β, in addition to the NF-kappa-B essential modulator (NEMO) or IKK-γ [67]. After activation, IKK recruits and phosphorylates the IκB that is recognized by the ubiquitin ligase machinery, which leads to its polyubiquitination and consequent degradation. In this way, the NF-κB dimers translocate to the nucleus, binding at specific sites of the deoxyribonucleic acid (DNA) and promoting the transcription of a large number of genes [65, 67]. In addition to activating the IKKα/β/NF-κB pathway, TLRs are capable of activating the JNK pathway [68]. The serine/threonine kinase group called JNK (JNK-1, JNK-2, and JNK-3) belongs to the family of mitogen-activated protein kinase (MAPKs), responsible for the regulation of various cellular functions. This regulation occurs largely because of its ability to control the transcription of specific genes by AP-1 [69]. AP-1 is a transcription factor that, when activated, promotes the expression of genes related to innate immunity [70]. In addition to LPS, the signaling pathway of TLRs can be activated by heat shock proteins [71]. Heat shock proteins

**92**

(HSP) are proteins characterized as chaperones because they have an important function in adaptation to stress and cellular protection, acting mainly in the synthesis and protein degradation, besides regulating fundamental cellular processes [72]. The family of HSPs is divided into subfamilies, classified according to the molecular mass, being small HSPs (8–27 kDa) and large HSPs (100–110 kDa), among which stand out HSP90, HSP70, and HSP60 [73]. In addition to its essential functions as a chaperone [74], HSP70 has an anti-inflammatory effect by inhibiting the activation of NF-KB when present in the intracellular environment [75]. However, stimuli such as cell necrosis and bacterial products such as LPS can cause the passage of HSP70 through the membrane into the extracellular environment [76, 77]. Studies have suggested that elevated serum levels of HSP70 may be correlated with cardiovascular disorder, pulmonary fibrosis, renal damage, oxidative stress, and inflammation [78]. The development of these conditions may occur due to the ability of HSP70 to bind to TLR2 and TLR4, promoting the activation of the NF-κB pathway which, as mentioned above, induces the expression of inflammatory mediators related to insulin resistance [79]. Studies suggest that insulin sensitivity may undergo regulatory action by the adaptive immune system [80, 81]. This system is composed of different types of cells, among which the B and T lymphocytes [82] stand out. T lymphocytes are classified into two main classes: helper T lymphocytes, also known as T helper (Th), and cytotoxic T lymphocytes. The "naïve" Th1 lymphocytes, when interacting with antigen presenting cells, undergo activation and can differentiate into different subtypes [83]. The Th1 subtype expresses proinflammatory cytokines, such as TNF-α and IFN-γ; Th2 expresses mainly anti-inflammatory cytokines, such as interleukin-4 (IL-4) and interleukin-13 (IL-13), and regulatory T cells secrete predominantly anti-inflammatory cytokine and transforming growth factor-β (TGF-β) [84]. Th1 cells play a central role in the recruitment of macrophages and induction of insulin resistance in obesity-induced diabetes models. These effects are counterbalanced by the function of Th2 and Treg cells that maintain an anti-inflammatory state and increase insulin sensitivity [85]. Appropriate regulation of Th cells is of extreme importance for the control and prevention of various diseases [86]. An increase or decrease in the Th1 or Th2 subtypes, as well as the cytokines produced by these cells, indicates an imbalance that may be one of the factors responsible for the development of insulin resistance [87]. It is known that insulin resistance is one of the main characteristics of diabetes mellitus [88]. This disease is also closely related to muscle weakness due to altered insulin action [89], standing out that insulin is an important anabolic hormone for protein metabolism [90].

The study performed by Boon et al. [91] with healthy lean individuals observed that only 5 days of hyperlipidic diet promoted increased messenger ribonucleic acid (mRNA) expression of macrophage markers in skeletal muscle and reduced expression of the glucose transporter type 4 (GLUT-4) glucose transporter protein in this tissue. Similarly, Patsouris et al. [92] demonstrated increased macrophage content in skeletal muscle in diabetic patients independently of body mass index (BMI). An increased macrophage content (assessed by F4/80 protein detection) was observed in muscle tissue of rats with AP in the absence of obesity, highlighting the key role of these cells in the etiology of insulin resistance. It should be noted that only F4/80 detection is not able to provide details on M1-type and M2-type macrophage polarization although evidence demonstrates that under obesity conditions, macrophages infiltrated into muscle tissue exhibit phenotype characteristic of M1 polarization [92–95]. The reprogramming of the M1 polarization toward the M2 polarization may represent a promising strategy for the treatment of glycemic homeostasis in patients with diabetes and insulin resistance [44].

As previously reported, inflammation causes insulin resistance. According to Pereira et al., rats with AP had increased IKKα/β and JNK phosphorylation status in gastrocnemius muscle. These results are in agreement with the study of Yaspelkis et al. [96], who observed a higher IKKα/β phosphorylation status in the skeletal muscle of rats treated with a hyperlipidic diet for 12 weeks, and also the study by Todd et al. [97] that identified an increase in JNK activity in the skeletal muscle of rats subjected to 3 weeks of hyperlipidic diet. Kaneto et al. [98] reported that treatment of diabetic rats with JNK inhibitors improved the insulin sensitivity of the animals. Similarly, studies by Yuan et al. [99] and Hundal et al. [100] have reported that inhibition of IKK-β by the administration of salicylates improves insulin action in obese and diabetic human and rats. Furthermore, it has been demonstrated that genetically modified mice, which do not express IKK-β or JNK, are protected from obesity-induced insulin resistance [99, 101–103].

In addition to stimulating inhibitory effects on insulin signal transduction, TNF-α may interact with tumor necrosis factor receptor 1 (TNFR1) in skeletal muscle [104] and thereby stimulate the NF-κB and/or MAPK pathway [105, 106], which are related to the phosphorylation of IKK and JNK and, in their turn, may impair insulin action. Pereira et al. [43] evaluated the plasma concentrations of LPS and HSP70 in AP models. Rats with AP showed a significant increase in both LPS and HSP70 when compared to the control group. Research on diabetes suggests that chronic elevation of LPS levels may play a key role in the development of insulin resistance [107, 108].

Among the possible mechanisms involved in this alteration, we highlight the ability of LPS to bind to the TLR4 receptor, which may trigger the activation of inflammatory signaling pathways related to inhibition of the insulin signal [108]. Another mediator that plays an active role in the modulation of inflammation is the heat shock proteins. The study by Goodman et al. [109] reported higher expression of 44 HSP genes in periapical granulomas compared to healthy periodontal tissues. Elevation of HSP70 plasma concentrations observed in rats with AP may indicate that increased local HSP expression is associated with higher concentrations of this protein in serum [43]. Interestingly, studies have shown that serum concentrations of HSP70 are higher in diabetic patients [110, 111]. Asea et al. [79] reported that HSP70 can bind to the TLR4 receptor, suggesting a possible involvement of this protein in the development of insulin resistance. With regard to the adaptive immunity markers, animals from the AP groups showed an increase in the Th1 response represented by increased T-bet expression in the spleen and elevated plasma concentrations of INF-γ [43]. A study carried out with knockout animals for the T-bet gene treated with hypercaloric diet showed that even with weight gain and increased adiposity, the animals were protected from insulin resistance [112]. The authors attributed the lack of insulin resistance to reduced production of INF-γ. These results are consistent with studies that reported that IFN-γ deficiency may improve glycemic homeostasis under obesity conditions [113–115]. In addition, treatment of adipocytes (3 T3-L1) with interferon gamma (INF-γ) reduces insulin signal and glucose uptake [116]. The functions of Th1 cells are antagonized by the Th2 subpopulation presenting the transcription factor GATA3 and IL-4 as specific markers. The AP in rats promotes a reduction of IL-4 [43]. Chang et al. [117] reported that IL-4 treatment promotes improved insulin sensitivity and glucose tolerance and simultaneously reduces body weight in obese rats. These findings suggest that IL-4 plays beneficial effects on glycemic homeostasis. The role of Th2 cells in insulin sensitivity was demonstrated in the study by Gonzales et al. [118]. In this study, a model of inactivation of Th2 response was developed through the inhibition of the activator of transcription 6 (STAT6) protein, in which it was observed that animals with Th2 response deficiency were more prone to insulin resistance. Thus, the reduction of the Th2 response observed in rats with AP may contribute to the understanding of the mechanisms involved in insulin resistance observed in animals with AP [43, 54].

**95**

*Inflammatory Muscle Diseases*

**3. Conclusions**

**Conflict of interest**

**Funding**

*DOI: http://dx.doi.org/10.5772/intechopen.86053*

Studies suggest that TNF-α contributes to age-related muscle loss and that resistance exercise may attenuate this process by suppressing TNF-α expression in skeletal muscle [119]. Other findings demonstrate that decreased muscle strength in diabetic individuals is associated with elevated plasma concentrations of TNF-α and interleukin-6 (IL-6) [120]. Therefore, considering that oral inflammation, such as AP, may increase infiltration of macrophages in muscle tissue and this increase is related to the production of proinflammatory cytokines, it is possible to suggest that prevention of chronic inflammatory oral diseases contributes to the maintenance of muscle integrity.

The main subtypes of inflammatory muscular diseases are polymyositis, dermatomyositis, necrotizing myopathy, overlap myositis, and myositis of inclusion bodies. The origin of these diseases is idiopathic, making it difficult to prevent them. As oral inflammation can increase infiltration of macrophages in muscle tissue and this increase is related to the production of proinflammatory cytokines in this tissue, these cytokines can cause muscle weakness. It is important to consider the prevention of chronic inflammatory processes in order to maintain muscle integrity or even prevent the worsening of the clinical condition of patients with inflammatory muscle diseases.

This study was supported by the São Paulo Research Foundation (FAPESP)

The authors declare that there are no conflicts of interest.

[grant #2016/24829-2] São Paulo, SP, Brazil.

CD14 cluster of differentiation 14 CD4 cluster of differentiation 4 CD8 cluster of differentiation 8

HMGCR 3-hydroxy-3-methylglutaryl-CoA reductase

**Appendices and nomenclature**

AP apical periodontitis AP-1 activating proteins-1 BMI body mass index

CK creatine kinase COX cyclooxygenase DNA deoxyribonucleic acid GLUT4 glucose transporter type 4 HLA human leukocyte antigen

HSP heat shock proteins IFN-γ interferon gamma

IKK IkB kinase IL-13 interleukin-13 IL-4 interleukin-4 IL-6 interleukin-6

### *Inflammatory Muscle Diseases DOI: http://dx.doi.org/10.5772/intechopen.86053*

Studies suggest that TNF-α contributes to age-related muscle loss and that resistance exercise may attenuate this process by suppressing TNF-α expression in skeletal muscle [119]. Other findings demonstrate that decreased muscle strength in diabetic individuals is associated with elevated plasma concentrations of TNF-α and interleukin-6 (IL-6) [120]. Therefore, considering that oral inflammation, such as AP, may increase infiltration of macrophages in muscle tissue and this increase is related to the production of proinflammatory cytokines, it is possible to suggest that prevention of chronic inflammatory oral diseases contributes to the maintenance of muscle integrity.

### **3. Conclusions**

*Muscle Cells - Recent Advances and Future Perspectives*

obesity-induced insulin resistance [99, 101–103].

resistance [107, 108].

in gastrocnemius muscle. These results are in agreement with the study of Yaspelkis et al. [96], who observed a higher IKKα/β phosphorylation status in the skeletal muscle of rats treated with a hyperlipidic diet for 12 weeks, and also the study by Todd et al. [97] that identified an increase in JNK activity in the skeletal muscle of rats subjected to 3 weeks of hyperlipidic diet. Kaneto et al. [98] reported that treatment of diabetic rats with JNK inhibitors improved the insulin sensitivity of the animals. Similarly, studies by Yuan et al. [99] and Hundal et al. [100] have reported that inhibition of IKK-β by the administration of salicylates improves insulin action in obese and diabetic human and rats. Furthermore, it has been demonstrated that genetically modified mice, which do not express IKK-β or JNK, are protected from

In addition to stimulating inhibitory effects on insulin signal transduction, TNF-α may interact with tumor necrosis factor receptor 1 (TNFR1) in skeletal muscle [104] and thereby stimulate the NF-κB and/or MAPK pathway [105, 106], which are related to the phosphorylation of IKK and JNK and, in their turn, may impair insulin action. Pereira et al. [43] evaluated the plasma concentrations of LPS and HSP70 in AP models. Rats with AP showed a significant increase in both LPS and HSP70 when compared to the control group. Research on diabetes suggests that chronic elevation of LPS levels may play a key role in the development of insulin

Among the possible mechanisms involved in this alteration, we highlight the ability of LPS to bind to the TLR4 receptor, which may trigger the activation of inflammatory signaling pathways related to inhibition of the insulin signal [108]. Another mediator that plays an active role in the modulation of inflammation is the heat shock proteins. The study by Goodman et al. [109] reported higher expression of 44 HSP genes in periapical granulomas compared to healthy periodontal tissues. Elevation of HSP70 plasma concentrations observed in rats with AP may indicate that increased local HSP expression is associated with higher concentrations of this protein in serum [43]. Interestingly, studies have shown that serum concentrations of HSP70 are higher in diabetic patients [110, 111]. Asea et al. [79] reported that HSP70 can bind to the TLR4 receptor, suggesting a possible involvement of this protein in the development of insulin resistance. With regard to the adaptive immunity markers, animals from the AP groups showed an increase in the Th1 response represented by increased T-bet expression in the spleen and elevated plasma concentrations of INF-γ [43]. A study carried out with knockout animals for the T-bet gene treated with hypercaloric diet showed that even with weight gain and increased adiposity, the animals were protected from insulin resistance [112]. The authors attributed the lack of insulin resistance to reduced production of INF-γ. These results are consistent with studies that reported that IFN-γ deficiency may improve glycemic homeostasis under obesity conditions [113–115]. In addition, treatment of adipocytes (3 T3-L1) with interferon gamma (INF-γ) reduces insulin signal and glucose uptake [116]. The functions of Th1 cells are antagonized by the Th2 subpopulation presenting the transcription factor GATA3 and IL-4 as specific markers. The AP in rats promotes a reduction of IL-4 [43]. Chang et al. [117] reported that IL-4 treatment promotes improved insulin sensitivity and glucose tolerance and simultaneously reduces body weight in obese rats. These findings suggest that IL-4 plays beneficial effects on glycemic homeostasis. The role of Th2 cells in insulin sensitivity was demonstrated in the study by Gonzales et al. [118]. In this study, a model of inactivation of Th2 response was developed through the inhibition of the activator of transcription 6 (STAT6) protein, in which it was observed that animals with Th2 response deficiency were more prone to insulin resistance. Thus, the reduction of the Th2 response observed in rats with AP may contribute to the understanding of the mechanisms involved in insulin resistance observed in animals with AP [43, 54].

**94**

The main subtypes of inflammatory muscular diseases are polymyositis, dermatomyositis, necrotizing myopathy, overlap myositis, and myositis of inclusion bodies. The origin of these diseases is idiopathic, making it difficult to prevent them. As oral inflammation can increase infiltration of macrophages in muscle tissue and this increase is related to the production of proinflammatory cytokines in this tissue, these cytokines can cause muscle weakness. It is important to consider the prevention of chronic inflammatory processes in order to maintain muscle integrity or even prevent the worsening of the clinical condition of patients with inflammatory muscle diseases.

### **Conflict of interest**

The authors declare that there are no conflicts of interest.

### **Funding**

This study was supported by the São Paulo Research Foundation (FAPESP) [grant #2016/24829-2] São Paulo, SP, Brazil.

### **Appendices and nomenclature**



### **Author details**

Doris Hissako Sumida1 \*, FernandoYamamoto Chiba2 and Maria Sara de Lima Coutinho Mattera1

1 Multicenter Post-Graduate Program in Physiological Sciences (SBFis), Department of Basic Sciences, School of Dentistry, São Paulo State University (UNESP), Araçatuba, Brazil

2 Department of Child and Social Dentistry, School of Dentistry, São Paulo State University (UNESP), Araçatuba, Brazil

\*Address all correspondence to: doris.hissako@unesp.br

© 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.

**97**

*Inflammatory Muscle Diseases*

cei.12194

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10.1002/mus.23302

10.1002/art.40063

WNL.61.3.316

*DOI: http://dx.doi.org/10.5772/intechopen.86053*

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[4] Furst DE, Amato AA, Iorga Ş, Gajria K, Fernandes AW. Epidemiology of adult idiopathic inflammatory myopathies in a U.S. managed care plan. Muscle & Nerve. 2012;**45**:676-683. DOI:

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### **References**

*Muscle Cells - Recent Advances and Future Perspectives*

IRS-1 insulin receptor substrate 1 JNK c-jun amino-terminal kinase LPB lipopolysaccharide binding protein

M1 M1-type macrophage polarization M2 M2-type macrophage polarization MAPKs mitogen-activated protein kinase

MHC major histocompatibility complex MRI magnetic resonance imaging mRNA messenger ribonucleic acid

NEMO NF-kappa-B essential modulator

SLE systemic lupus erythematosus SRP signal recognition particle STAT6 activator of transcription 6

TDP-43 transactive DNA-binding protein 43 TGF-β transforming growth factor-β

TIF transcription intermediary factor

TNFR1 tumor necrosis factor receptor 1 TNF-α tumor necrosis factor-alpha

NF-κB factor nuclear kappa B NXP nuclear matrix protein

Th T-helper

**Author details**

Doris Hissako Sumida1

(UNESP), Araçatuba, Brazil

TLR toll-like receptors

UK United Kingdom

USA United States of America

and Maria Sara de Lima Coutinho Mattera1

University (UNESP), Araçatuba, Brazil

provided the original work is properly cited.

\*Address all correspondence to: doris.hissako@unesp.br

MDA5 melanoma differentiation-associated gene 5

MyD88 myeloid differentiation primary response 88

PET-CT positron emission tomography–computed tomography

\*, FernandoYamamoto Chiba2

1 Multicenter Post-Graduate Program in Physiological Sciences (SBFis), Department of Basic Sciences, School of Dentistry, São Paulo State University

2 Department of Child and Social Dentistry, School of Dentistry, São Paulo State

© 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,

IR insulin resistance

LPS lipopolysaccharides

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[102] Hirosumi J, Tuncman G, Chang L, Görgün CZ, Uysal KT, Maeda K, et al. A central role for JNK in obesity and insulin resistance. Nature. 2002;**420**:333-336. DOI: 10.1038/

[103] Singh R, Wang Y, Xiang Y, Tanaka KE, Gaarde WA, Czaja MJ. Differential effects of JNK1 and JNK2 inhibition on murine steatohepatitis and insulin resistance. Hepatology. 2009;**49**:87-96.

[104] Hardin BJ, Campbell KS, Smith JD,

[105] Tracey D, Klareskog L, Sasso EH, Salfeld JG, Tak PP. Tumor necrosis factor antagonist mechanisms of action: A comprehensive review. Pharmacology & Therapeutics. 2008;**117**:244-279. DOI: 10.1016/j.pharmthera.2007.10.001

[106] Aggarwal BB. Signalling pathways of the TNF superfamily: A double-edged sword. Nature Reviews. Immunology. 2003;**3**:745-775. DOI: 10.1038/nri1184

[107] Watanabe K, Iizuka T, Adeleke A,

experimental periodontitis. Journal of Periodontology. 2011;**46**:21-30. DOI: 10.1111/j.1600-0765.2010.01304.x

[108] Liang H, Hussey SE, Sanchez-Avila A, Tantiwong P, Musi N. Effect of lipopolysaccharide on inflammation and insulin action in human muscle. PLoS One. 2013;**8**:e63983. DOI: 10.1371/

[109] Goodman SC, Letra A, Dorn S, Araujo-Pires AC, Vieira AE, Chaves de

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[118] Ricardo-Gonzalez RR, Red Eagle A, Odegaard JI, Jouihan H, Morel CR, Heredia JE, et al. IL-4/ STAT6 immune axis regulates peripheral nutrient metabolism and insulin sensitivity. Proceedings of the National Academy of Sciences of the United States of America. 2010;**107**:22617- 22622. DOI: 10.1073/pnas.1009152108

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Section 3

Muscle Wasting:

Cancer-Induced

107

Section 3

## Muscle Wasting: Cancer-Induced

Chapter 6

Abstract

experimental data.

1. Introduction

109

Leucine and Its Importance for

Sarah Christine Pereira de Oliveira, Lais Rosa Viana,

Luiz Alberto Ferreira Ramos, Rafael Rossi Valentim

and Maria Cristina Cintra Gomes-Marcondes

Andre Gustavo Oliveira, Bread Cruz,

Natalia Angelo Da Silva Miyaguti,

Cell Signalling Pathways in Cancer

Cachexia-Induced Muscle Wasting

The anabolic effects of a supplemented diet with branched-chain amino acids, especially leucine, on skeletal muscle wasting and as a co-adjuvant in cancer treatment have been well-studied. Leucine is a precursor of protein synthesis and acts as a nutritional signal, affecting multiple metabolic processes (e.g., satiety, thermogenesis, energy efficiency, and body composition). Previous studies related to nutritional therapy have mainly focused on myopenia, which is the loss of skeletal muscle mass in some pathologies, including cancer. Leucine plays a role in the maintenance and even increase of lean body mass in healthy individuals as well as the prevention of disease states that culminate in myopenia. Herein, we review the available data addressing the mechanisms by which leucine acts as a cellular signal, thereby stimulating muscle protein synthesis, leading to the inhibition of muscle catabolism, especially in an experimental model of cancer cachexia. We also show differences found in the metabolomic and proteomic analyses, including the use of leucine in maternal diets as a preventative for muscle wasting as supported by our

Keywords: leucine, cell signalling, protein metabolism, protein synthesis, protein degradation, muscle wasting, experimental cachexia models

loss of muscle function in cancer patients. In every type of cachexia, the

Cancer remains an enigmatic pathology for some patient types and can also cause deleterious effects, e.g., in some cases ending in a cachexia state. Cancer cachexia is a complex syndrome that results from anorexia associated with glucose intolerance, depletion of body fat, and severe wasting of lean mass, which corresponds to the more significant proportion of metabolically active tissue—the muscle tissue. In particular, the loss of skeletal mass, which is referred to as myopenia in the pathological process, is clinically relevant as this process is directly related to the

### Chapter 6

## Leucine and Its Importance for Cell Signalling Pathways in Cancer Cachexia-Induced Muscle Wasting

Andre Gustavo Oliveira, Bread Cruz, Sarah Christine Pereira de Oliveira, Lais Rosa Viana, Natalia Angelo Da Silva Miyaguti, Luiz Alberto Ferreira Ramos, Rafael Rossi Valentim and Maria Cristina Cintra Gomes-Marcondes

### Abstract

The anabolic effects of a supplemented diet with branched-chain amino acids, especially leucine, on skeletal muscle wasting and as a co-adjuvant in cancer treatment have been well-studied. Leucine is a precursor of protein synthesis and acts as a nutritional signal, affecting multiple metabolic processes (e.g., satiety, thermogenesis, energy efficiency, and body composition). Previous studies related to nutritional therapy have mainly focused on myopenia, which is the loss of skeletal muscle mass in some pathologies, including cancer. Leucine plays a role in the maintenance and even increase of lean body mass in healthy individuals as well as the prevention of disease states that culminate in myopenia. Herein, we review the available data addressing the mechanisms by which leucine acts as a cellular signal, thereby stimulating muscle protein synthesis, leading to the inhibition of muscle catabolism, especially in an experimental model of cancer cachexia. We also show differences found in the metabolomic and proteomic analyses, including the use of leucine in maternal diets as a preventative for muscle wasting as supported by our experimental data.

Keywords: leucine, cell signalling, protein metabolism, protein synthesis, protein degradation, muscle wasting, experimental cachexia models

### 1. Introduction

Cancer remains an enigmatic pathology for some patient types and can also cause deleterious effects, e.g., in some cases ending in a cachexia state. Cancer cachexia is a complex syndrome that results from anorexia associated with glucose intolerance, depletion of body fat, and severe wasting of lean mass, which corresponds to the more significant proportion of metabolically active tissue—the muscle tissue. In particular, the loss of skeletal mass, which is referred to as myopenia in the pathological process, is clinically relevant as this process is directly related to the loss of muscle function in cancer patients. In every type of cachexia, the

pathogenesis of muscle loss is complex and multifactorial. Due to the high energy expenditure produced by neoplastic cells, the patient presents inefficiency of energy production, as well as so-called futile energy processes, which is a major cause of muscle wasting. These energy expenses include high glucose production via gluconeogenesis from lactate or gluconeogenic precursors where there is excessive consumption associated with reduced the production of ATP. Then, the cancer patient loses weight involuntarily with severe loss of muscle mass due to increased protein degradation, which produces gluconeogenic amino acids. Moreover, this process also includes lipolysis, resulting in glycerol as a glucose precursor, inducing a spoliation cycle. All these points lead the cancer patient into fatigue and asthenia, thereby leading to a worse prognosis. Recently, the number of studies on new cancer treatment therapies has increased; most of these studies focus on the patient's responses to conventional treatments and improvement of survival and quality of life.

with BCAA, especially leucine, has been shown to improve skeletal muscle mass because leucine plays an important role in skeletal muscle metabolism, regulates protein synthesis by stimulating the mTOR pathway, and inhibits the ubiquitin-

Leucine and Its Importance for Cell Signalling Pathways in Cancer Cachexia-Induced Muscle…

Muscle mass is a controlled balance of protein turnover by the cellular processes of protein synthesis and breakdown. In some pathological conditions, such as cancer cachexia, protein synthesis could also be compromised, which results in skeletal muscle atrophy and weakness [10, 11]. The regulation of protein turnover in skeletal muscle is a complex process, usually involving interactions between gene transcription, translation, and protein degradation. Stimulation and signalling processes are initiated by principal agents of these activities, such as anabolic hormones (e.g., insulin), growth factors, glucose, and amino acids. One such signalling pathway is triggered by insulin, which initiates protein synthesis after binding to its receptor, thereby activating several downstream components. The activated insulin receptor triggers the tyrosine phosphorylation of the insulin receptor substrate (IRS) 1 and 2, followed by activation of the phosphoinositol 3 kinase (PI3-kinase). Then, PI3 kinase activates phosphoinositide kinases-dependent 1 and 2 (PDK1/2) to phosphorylate the protein kinase Akt/PKB. The activated PKB phosphorylates tuberous sclerosis 2 (TSC2) inactivates the tuberous sclerosis complex 1 and 2 (TSC1/TSC2), which are no longer able to perform GTPase activity at Rheb (brain-enriched rashomologue), allowing Rheb to release and activate the mechanistic target of rapamycin (mTOR), the key component of this machinery [12]. Also, the Akt substrate, PRAS40, when phosphorylated by PKB, loses its inhibitory effect over mTOR. In fact, mTOR acts as a sensor and integrator of diverse inputs, such as nutrients, growth factors, and energy status. mTOR, which consists of the mTORC1 and mTORC2 complexes, is a master regulator of protein synthesis and is essential for the maintenance of muscle mass and function [13]. Upon encountering anabolic factors, such as amino acids, mTORC1 is activated and signals to ribosomal protein S6 protein kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4EBP1)—these are the best-known downstream effectors of mTOR signalling and control the protein synthesis pathway. Activated p70S6K subsequently leads to phosphorylation of the downstream target S6K1, which results in the translation of messenger RNA (mRNA) encoding for ribosomes and transcription factors [14]. In parallel, mTOR phosphorylates the dissociation of the 4E-BP1/eIF4E complex, releasing the eukaryotic initiation factor 4E (eIF4E), which subsequently binds to eIF4G, thus forming the eIF4F translation initiation complex, and allows the recruitment of the 40S ribosomal subunit to initiate protein translation [15].

Although the impairment of muscle protein synthesis in cachexia is not an obligate feature, many studies are working in strategies to improve the muscle mass and also the patient's muscle function which could imply in better prognosis and quality of life in those patients. Thereby, leucine together with valine and isoleucine, or even alone, can stimulate protein synthesis and act as cell signalling molecules in skeletal muscle by activating the mTOR pathway [16, 17]. Multiple studies have shown that leucine alone stimulates protein synthesis, mediating the translational control of protein synthesis in skeletal muscle independently of other BCAAs [18, 19]. For example, some studies have shown that the oral administration or infusion of leucine in adult humans or animals elevates muscular protein synthesis [20]. Moreover, leucine appears to have a much more potent anabolic effect (i.e., stimulating the mTOR pathway) than anabolic hormones, such as insulin. The administration of

proteasome pathway [8, 9].

111

3. Muscle protein synthesis and leucine

DOI: http://dx.doi.org/10.5772/intechopen.78990

A novel therapeutic approach to cancer involves preserving, restoring, or even an epigenetic influence to maintain an adequate nutritional status for cancer patients, thereby slowing the onset of muscle mass wasting. In this context, nutritional supplementation has been identified as a potentially useful intervention. During protein synthesis, branched-chain amino acids (BCAAs), mainly leucine, act as precursors of the carbon skeleton and nitrogen. Also, leucine can primarily be oxidised in the muscle for energy supply and contribute nitrogen for the synthesis of other amino acids. Leucine also plays an essential role in cell signalling, stimulates protein synthesis, and modulates catabolism, mainly in skeletal muscle. In an experimental cachexia model that includes a leucine-rich diet (data in print), we report an improvement in functional muscle tests (verified by CatWalk test) and the influence of maternal leucine supplementation on the offspring's adulthood responses in the improving of the muscle tissue response. Since recent works have indicated that the most important goal during cancer progression is the maintenance of lean body mass and considering the key role of leucine in modulating skeletal muscle protein synthesis and degradation, our research group has been evaluating the effects of a leucine-rich diet in an experimental model of cancer cachexia. Herein, we summarise the findings of our group as well as others that show that a leucine-rich diet can ameliorate the prognosis, reduce the risk of death, and help to maintain the quality of life in cancer patients.

### 2. Cancer cachexia

Cachexia is a condition characterised by reduced food intake; involuntary and progressive weight loss; and intense catabolism of carbohydrates, lipids, and proteins [1], thereby resulting in intense deterioration of host tissues, severe weight loss, and adipose tissue and muscle mass wasting [2]. Weight loss and malnutrition are the most common characteristics observed in advanced cancer patients [3]. Cachexia is responsible for almost 30% of all cancer-related deaths and associated with significantly decreased physical activity and psychological burden [3]. Cachexia is also related to other pathophysiological changes, such as systemic inflammation, insulin resistance, and oxidative stress [4]. Several pro-inflammatory cytokines (e.g., IL-6 and TNF-α) and pro-cachectic factors (e.g., factor inducing proteolysis [PIF] and lipid mobilisation factor [LMF]), which are considered mediators of muscle wasting, act during the cachectic process [5, 6]. Considering the high prevalence (50–80%) of cachexia in advanced cancer, the investigation of the molecular process of cancer cachexia is important when considering the most efficient targets of treatment. The use of nutritional interventions to minimise the side effects of cancer is a novel and promising approach [7]. As such, supplementation

with BCAA, especially leucine, has been shown to improve skeletal muscle mass because leucine plays an important role in skeletal muscle metabolism, regulates protein synthesis by stimulating the mTOR pathway, and inhibits the ubiquitinproteasome pathway [8, 9].

### 3. Muscle protein synthesis and leucine

pathogenesis of muscle loss is complex and multifactorial. Due to the high energy expenditure produced by neoplastic cells, the patient presents inefficiency of energy production, as well as so-called futile energy processes, which is a major cause of muscle wasting. These energy expenses include high glucose production via gluconeogenesis from lactate or gluconeogenic precursors where there is excessive consumption associated with reduced the production of ATP. Then, the cancer patient loses weight involuntarily with severe loss of muscle mass due to increased protein degradation, which produces gluconeogenic amino acids. Moreover, this process also includes lipolysis, resulting in glycerol as a glucose precursor, inducing a spoliation cycle. All these points lead the cancer patient into fatigue and asthenia, thereby leading to a worse prognosis. Recently, the number of studies on new cancer treatment therapies has increased; most of these studies focus on the patient's responses to

Muscle Cells - Recent Advances and Future Perspectives

conventional treatments and improvement of survival and quality of life.

and help to maintain the quality of life in cancer patients.

2. Cancer cachexia

110

an epigenetic influence to maintain an adequate nutritional status for cancer patients, thereby slowing the onset of muscle mass wasting. In this context, nutritional supplementation has been identified as a potentially useful intervention. During protein synthesis, branched-chain amino acids (BCAAs), mainly leucine, act as precursors of the carbon skeleton and nitrogen. Also, leucine can primarily be oxidised in the muscle for energy supply and contribute nitrogen for the synthesis of other amino acids. Leucine also plays an essential role in cell signalling, stimulates protein synthesis, and modulates catabolism, mainly in skeletal muscle. In an experimental cachexia model that includes a leucine-rich diet (data in print), we report an improvement in functional muscle tests (verified by CatWalk test) and the influence of maternal leucine supplementation on the offspring's adulthood responses in the improving of the muscle tissue response. Since recent works have indicated that the most important goal during cancer progression is the maintenance of lean body mass and considering the key role of leucine in modulating skeletal muscle protein synthesis and degradation, our research group has been evaluating the effects of a leucine-rich diet in an experimental model of cancer cachexia. Herein, we summarise the findings of our group as well as others that show that a leucine-rich diet can ameliorate the prognosis, reduce the risk of death,

A novel therapeutic approach to cancer involves preserving, restoring, or even

Cachexia is a condition characterised by reduced food intake; involuntary and progressive weight loss; and intense catabolism of carbohydrates, lipids, and proteins [1], thereby resulting in intense deterioration of host tissues, severe weight loss, and adipose tissue and muscle mass wasting [2]. Weight loss and malnutrition are the most common characteristics observed in advanced cancer patients [3]. Cachexia is responsible for almost 30% of all cancer-related deaths and associated with significantly decreased physical activity and psychological burden [3]. Cachexia is also related to other pathophysiological changes, such as systemic inflammation, insulin resistance, and oxidative stress [4]. Several pro-inflammatory cytokines (e.g., IL-6 and TNF-α) and pro-cachectic factors (e.g., factor inducing proteolysis [PIF] and lipid mobilisation factor [LMF]), which are considered mediators of muscle wasting, act during the cachectic process [5, 6]. Considering the high prevalence (50–80%) of cachexia in advanced cancer, the investigation of the molecular process of cancer cachexia is important when considering the most efficient targets of treatment. The use of nutritional interventions to minimise the side effects of cancer is a novel and promising approach [7]. As such, supplementation

Muscle mass is a controlled balance of protein turnover by the cellular processes of protein synthesis and breakdown. In some pathological conditions, such as cancer cachexia, protein synthesis could also be compromised, which results in skeletal muscle atrophy and weakness [10, 11]. The regulation of protein turnover in skeletal muscle is a complex process, usually involving interactions between gene transcription, translation, and protein degradation. Stimulation and signalling processes are initiated by principal agents of these activities, such as anabolic hormones (e.g., insulin), growth factors, glucose, and amino acids. One such signalling pathway is triggered by insulin, which initiates protein synthesis after binding to its receptor, thereby activating several downstream components. The activated insulin receptor triggers the tyrosine phosphorylation of the insulin receptor substrate (IRS) 1 and 2, followed by activation of the phosphoinositol 3 kinase (PI3-kinase). Then, PI3 kinase activates phosphoinositide kinases-dependent 1 and 2 (PDK1/2) to phosphorylate the protein kinase Akt/PKB. The activated PKB phosphorylates tuberous sclerosis 2 (TSC2) inactivates the tuberous sclerosis complex 1 and 2 (TSC1/TSC2), which are no longer able to perform GTPase activity at Rheb (brain-enriched rashomologue), allowing Rheb to release and activate the mechanistic target of rapamycin (mTOR), the key component of this machinery [12]. Also, the Akt substrate, PRAS40, when phosphorylated by PKB, loses its inhibitory effect over mTOR. In fact, mTOR acts as a sensor and integrator of diverse inputs, such as nutrients, growth factors, and energy status. mTOR, which consists of the mTORC1 and mTORC2 complexes, is a master regulator of protein synthesis and is essential for the maintenance of muscle mass and function [13]. Upon encountering anabolic factors, such as amino acids, mTORC1 is activated and signals to ribosomal protein S6 protein kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4EBP1)—these are the best-known downstream effectors of mTOR signalling and control the protein synthesis pathway. Activated p70S6K subsequently leads to phosphorylation of the downstream target S6K1, which results in the translation of messenger RNA (mRNA) encoding for ribosomes and transcription factors [14]. In parallel, mTOR phosphorylates the dissociation of the 4E-BP1/eIF4E complex, releasing the eukaryotic initiation factor 4E (eIF4E), which subsequently binds to eIF4G, thus forming the eIF4F translation initiation complex, and allows the recruitment of the 40S ribosomal subunit to initiate protein translation [15].

Although the impairment of muscle protein synthesis in cachexia is not an obligate feature, many studies are working in strategies to improve the muscle mass and also the patient's muscle function which could imply in better prognosis and quality of life in those patients. Thereby, leucine together with valine and isoleucine, or even alone, can stimulate protein synthesis and act as cell signalling molecules in skeletal muscle by activating the mTOR pathway [16, 17]. Multiple studies have shown that leucine alone stimulates protein synthesis, mediating the translational control of protein synthesis in skeletal muscle independently of other BCAAs [18, 19]. For example, some studies have shown that the oral administration or infusion of leucine in adult humans or animals elevates muscular protein synthesis [20]. Moreover, leucine appears to have a much more potent anabolic effect (i.e., stimulating the mTOR pathway) than anabolic hormones, such as insulin. The administration of

leucine after fasting or amino acid starvation stimulates protein synthesis and promotes the phosphorylation and activation of S6K1 via the rapamycin-sensitive mTOR in skeletal muscle [21, 22]. On the other hand, several studies have emphasised the specific contribution of cell membrane transport through the coupling of the amino acid transporter system 1 (LAT1 or SLC7A5), which carries leucine in exchange for glutamine [23]. The availability of amino acids (especially leucine and glutamine) is determined by its uptake by the cell, which appears to play an essential role in the entrance of Leu into the cell and the maintenance of a high intracellular concentration of glutamine [24]. Some evidence suggests that leucine uses the insulin signalling pathway, but the exact mechanism of triggering the mTOR complex remains under debate [25]. Nutritional supplementation with leucine stimulated the incorporation of phenylalanine in muscle in an experimental cachexia model, confirming an increased protein synthesis and also an increasing muscle mass [26, 27]. Subunits of the mTORC1 complex (i.e., Raptor and GβL) and substrates belonging to the downstream pathway (i.e., 4E-BPs, eIF4A, eIF4B, eIF4E, including S6K1), which represent the key points within the metabolism of proteins through mTOR, are highly increased in the muscle of Walker-256 tumour-bearing animals subjected to leucine nutritional supplementation [27–29]. In vitro cell culture studies have generated evidence relevant to the mechanism through which leucine affects mTOR [30, 31]. As such, leucine supplementation can stimulate protein synthesis and, consequently, might lead to a positive protein net balance, even within a high rate of protein degradation. In addition to increasing the protein synthesis in skeletal muscle, a leucine-rich diet has a protective effect in other tissues. In our previous works, we also observed improvement of protein synthesis in placenta tissue since leucine acted by improving the cell-signalling activity, thereby increasing placental protein synthesis and also reducing the placental proteolytic process [32, 33].

leucine tumour-bearing group showed lower muscle ATP5a1 content, likely indicating that the production of ATP must be derived from other metabolic processes. Therefore, the presence of tumour factors interferes in the cellular processes involved with obtaining energy. Since cell proliferation depends on the constant use of ATP for the duplication of all cellular machinery, the interference of tumour factors in the muscle mitochondria and the electron transport chain leads to less availability to energy for muscle cell activity. With leucine supplementation, a stimulating pathway occurs to obtain energy, thus contributing to the maintenance of adequate ATP supply and the ability maintain muscle activities. Since leucine is a ketogenic amino acid, its entire carbon skeleton is converted to keto acid or acetyl-CoA, which can be directed to participate in the Krebs cycle and beta-oxidation processes, both of which produce ATP as the final product. According to our results, the metabolic pathways in cachectic-tumour-bearing animals are related to ammonia recycling and the urea cycle, likely associated with protein degradation and directly associated with the futile cycle of energy production. In parallel, the metabolic activity of the leucine-tumour-bearing group was affected, which was related to ketone body and butyrate metabolism [35]. These points confirm the relationship to the increase in the muscle tissue's energy needs in tumour-bearing animals, which are minimised/modulated when the animal's diet is supplemented with leucine. Our proteomic results show that, in muscle tissue, mitochondria dysfunction occurs in the tumour-bearing host; however, under leucine supplementation, there

Leucine and Its Importance for Cell Signalling Pathways in Cancer Cachexia-Induced Muscle…

DOI: http://dx.doi.org/10.5772/intechopen.78990

are muscle mitochondrial biogenesis and activities improvements (data not published). Thus, we know that both insulin and leucine can independently affect the activation of mRNA translation and, consequently, the protein synthesis process [17, 37]. However, the real effect of amino acid signalling, especially leucine, on protein synthesis via the mTOR pathway remains complex and less understood, and there is a need for further studies, especially in vivo models. Moreover, as mentioned previously, our data for the CatWalk analysis showed an improvement in muscular functional activity; i.e., when rats with tumours were fed a leucine-rich diet, muscle function improved (data in print). Moreover, a leucine-supplemented maternal diet can influence and ameliorate the adult host response in tumourbearing rats. In this way, an improved understanding of muscle protein synthesis and how leucine influences it is essential when developing new targets and strate-

Muscle homeostasis is important because muscle makes up a large part of the whole organism and performs many functions and activities. Moreover, as one of the main structures of the body, it is the most significant source of the protein turnover process. As noted above, muscle maintenance occurs by the intense activity of both protein synthesis and degradation [38]. Accordingly, proteolytic systems also play a key role in the regulation of cellular homeostasis and cell recycling, differentiation, cell cycle, abnormal protein degradation, and amino acid supply for gluconeogenesis [39]. Myofibrillar protein degradation is performed by the following four different pathways: ubiquitin proteasome system (UPS), autophagy, Ca2+ dependent proteolysis, and caspase pathway [40]. The increase of protein catabolism in skeletal muscle contributes to a worse prognosis in cancer patients, especially those in a cachexia state, which is one of the most important causes of morbidity and mortality in these patients [41]. In cancer patients, the loss of either

gies to restore muscle in muscle wasting diseases.

4. Muscle protein degradation and leucine

4.1 Skeletal muscle wasting in cachexia

113

Interestingly, our previous works and other experimental studies have shown that leucine supplementation can work as an excellent nutritional strategy to treat or prevent muscle wasting in cancer cachexia. In this way, leucine also emerged as a potent stimulator of metabolism, leading to improvements in both oxidative metabolism and mitochondrial biogenesis [34]. Recently, our research group used metabolomic and proteomic analyses in an experimental cachexia model to better understand the benefits of leucine supplementation. Compared to a nonsupplemented group, tumour-bearing rats under leucine supplementation showed metabolic pathways diverted to ketone bodies and butyrate metabolism [35]. Since an excess of leucine might provide ketone precursors being utilised by muscle tissue as energy sources, this likely diverted the metabolism to improve muscle protein synthesis [35]. The ketone bodies could provide additional energy to skeletal muscle and host tissues; this energy source is not available to the non-leucinesupplemented group. Besides acting as a fuel source to supply energy for the cellular activity of several tissues, ketone bodies, especially acetoacetate, can also promote muscle cell proliferation [36], probably accounting for the benefits of leucine nutritional supplementation [35]. We also made important findings as part of our proteomic analysis of the muscle tissue of tumour-bearing rats fed a leucine-rich diet (data in print). These results show a significant action of leucine on modulation of the mitochondrial membrane proteins involving the production of ATP, such as the ATP synthase complex family. Proteins associated with ATP synthase (e.g., F1F0 or Complex V) participate in the synthesis of ATP from ADP in the presence of a proton gradient across the mitochondrial membrane. One protein from this family that stood out in our studies is the ATP5a1 synthase subunit alpha. The tumour-bearing group showed a higher concentration of ATP5a1, which indicates a higher mitochondrial activity for the production of ATP, which is associated with a greater availability of glucose from the gluconeogenesis process. In contrast, the

Leucine and Its Importance for Cell Signalling Pathways in Cancer Cachexia-Induced Muscle… DOI: http://dx.doi.org/10.5772/intechopen.78990

leucine tumour-bearing group showed lower muscle ATP5a1 content, likely indicating that the production of ATP must be derived from other metabolic processes. Therefore, the presence of tumour factors interferes in the cellular processes involved with obtaining energy. Since cell proliferation depends on the constant use of ATP for the duplication of all cellular machinery, the interference of tumour factors in the muscle mitochondria and the electron transport chain leads to less availability to energy for muscle cell activity. With leucine supplementation, a stimulating pathway occurs to obtain energy, thus contributing to the maintenance of adequate ATP supply and the ability maintain muscle activities. Since leucine is a ketogenic amino acid, its entire carbon skeleton is converted to keto acid or acetyl-CoA, which can be directed to participate in the Krebs cycle and beta-oxidation processes, both of which produce ATP as the final product. According to our results, the metabolic pathways in cachectic-tumour-bearing animals are related to ammonia recycling and the urea cycle, likely associated with protein degradation and directly associated with the futile cycle of energy production. In parallel, the metabolic activity of the leucine-tumour-bearing group was affected, which was related to ketone body and butyrate metabolism [35]. These points confirm the relationship to the increase in the muscle tissue's energy needs in tumour-bearing animals, which are minimised/modulated when the animal's diet is supplemented with leucine. Our proteomic results show that, in muscle tissue, mitochondria dysfunction occurs in the tumour-bearing host; however, under leucine supplementation, there are muscle mitochondrial biogenesis and activities improvements (data not published). Thus, we know that both insulin and leucine can independently affect the activation of mRNA translation and, consequently, the protein synthesis process [17, 37]. However, the real effect of amino acid signalling, especially leucine, on protein synthesis via the mTOR pathway remains complex and less understood, and there is a need for further studies, especially in vivo models. Moreover, as mentioned previously, our data for the CatWalk analysis showed an improvement in muscular functional activity; i.e., when rats with tumours were fed a leucine-rich diet, muscle function improved (data in print). Moreover, a leucine-supplemented maternal diet can influence and ameliorate the adult host response in tumourbearing rats. In this way, an improved understanding of muscle protein synthesis and how leucine influences it is essential when developing new targets and strategies to restore muscle in muscle wasting diseases.

### 4. Muscle protein degradation and leucine

### 4.1 Skeletal muscle wasting in cachexia

Muscle homeostasis is important because muscle makes up a large part of the whole organism and performs many functions and activities. Moreover, as one of the main structures of the body, it is the most significant source of the protein turnover process. As noted above, muscle maintenance occurs by the intense activity of both protein synthesis and degradation [38]. Accordingly, proteolytic systems also play a key role in the regulation of cellular homeostasis and cell recycling, differentiation, cell cycle, abnormal protein degradation, and amino acid supply for gluconeogenesis [39]. Myofibrillar protein degradation is performed by the following four different pathways: ubiquitin proteasome system (UPS), autophagy, Ca2+ dependent proteolysis, and caspase pathway [40]. The increase of protein catabolism in skeletal muscle contributes to a worse prognosis in cancer patients, especially those in a cachexia state, which is one of the most important causes of morbidity and mortality in these patients [41]. In cancer patients, the loss of either

leucine after fasting or amino acid starvation stimulates protein synthesis and promotes the phosphorylation and activation of S6K1 via the rapamycin-sensitive mTOR in skeletal muscle [21, 22]. On the other hand, several studies have emphasised the specific contribution of cell membrane transport through the coupling of the amino acid transporter system 1 (LAT1 or SLC7A5), which carries leucine in exchange for glutamine [23]. The availability of amino acids (especially leucine and glutamine) is determined by its uptake by the cell, which appears to play an essential role in the entrance of Leu into the cell and the maintenance of a high intracellular concentration of glutamine [24]. Some evidence suggests that leucine uses the insulin signalling pathway, but the exact mechanism of triggering the mTOR complex remains under debate [25]. Nutritional supplementation with leucine stimulated the incorporation of phenylalanine in muscle in an experimental cachexia model, confirming an increased

protein synthesis and also an increasing muscle mass [26, 27]. Subunits of the mTORC1 complex (i.e., Raptor and GβL) and substrates belonging to the downstream pathway (i.e., 4E-BPs, eIF4A, eIF4B, eIF4E, including S6K1), which represent the key points within the metabolism of proteins through mTOR, are highly increased in the muscle of Walker-256 tumour-bearing animals subjected to leucine nutritional supplementation [27–29]. In vitro cell culture studies have generated evidence relevant to the mechanism through which leucine affects mTOR [30, 31]. As such, leucine supplementation can stimulate protein synthesis and, consequently, might lead to a positive protein net balance, even within a high rate of protein degradation. In addition to increasing the protein synthesis in skeletal muscle, a leucine-rich diet has a protective effect in other tissues. In our previous works, we also observed improvement of protein synthesis in placenta tissue since leucine acted by improving the cell-signalling activity, thereby increasing placental protein synthesis and also

Interestingly, our previous works and other experimental studies have shown that leucine supplementation can work as an excellent nutritional strategy to treat or prevent muscle wasting in cancer cachexia. In this way, leucine also emerged as a potent stimulator of metabolism, leading to improvements in both oxidative metabolism and mitochondrial biogenesis [34]. Recently, our research group used metabolomic and proteomic analyses in an experimental cachexia model to better

supplemented group, tumour-bearing rats under leucine supplementation showed metabolic pathways diverted to ketone bodies and butyrate metabolism [35]. Since an excess of leucine might provide ketone precursors being utilised by muscle tissue as energy sources, this likely diverted the metabolism to improve muscle protein synthesis [35]. The ketone bodies could provide additional energy to skeletal muscle

supplemented group. Besides acting as a fuel source to supply energy for the cellular activity of several tissues, ketone bodies, especially acetoacetate, can also promote muscle cell proliferation [36], probably accounting for the benefits of leucine nutritional supplementation [35]. We also made important findings as part of our proteomic analysis of the muscle tissue of tumour-bearing rats fed a leucine-rich diet (data in print). These results show a significant action of leucine on modulation of the mitochondrial membrane proteins involving the production of ATP, such as the ATP synthase complex family. Proteins associated with ATP synthase (e.g., F1F0 or Complex V) participate in the synthesis of ATP from ADP in the presence of a proton gradient across the mitochondrial membrane. One protein from this family that stood out in our studies is the ATP5a1 synthase subunit alpha. The tumour-bearing group showed a higher concentration of ATP5a1, which indicates a higher mitochondrial activity for the production of ATP, which is associated with a greater availability of glucose from the gluconeogenesis process. In contrast, the

understand the benefits of leucine supplementation. Compared to a non-

and host tissues; this energy source is not available to the non-leucine-

reducing the placental proteolytic process [32, 33].

Muscle Cells - Recent Advances and Future Perspectives

112

skeletal or cardiac muscle mass might lead to cardiac and respiratory failure, in addition to the fact that it decreases the host's response after conventional treatments, such as chemotherapy or radiotherapy [42]. Moreover, it is well-established that the ubiquitin-proteasome system is a very important pathway in skeletal muscle degradation during cancer cachexia [43]. Furthermore, multiple studies have identified released factors that contribute to an increase in muscle protein degradation during cancer. The main factors that lead to protein degradation during cancer cachexia syndrome produced by the host are tumour necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-1 (IL-1), interferon gamma (IFN-γ) [44]. Meanwhile, the main factors produced by cancer cells are proteolysis-inducing factor (PIF), lipid-mobilising factor (LMF), and anaemia-inducing factor (AIS) [44].

Atrogin have been reported to be specifically expressed and increased in skeletal muscle under many catabolic conditions [60, 61]. Interestingly, acute or chronical leucine supplementation prevented the upregulation of proteasomal proteolysis in fasted aged rats as compared to younger adult controls [62]. We have recently verified that ageing causes additional proteasomal activity in an experimental model of cancer-induced cachexia (unpublished data). Thus, leucine supplementation might be a valuable tool to counteract higher susceptibility to cachexia in senescence. Also, MuRF1, atrogin, and other E3 ligases, such as MUSA1 and SMART1, have been associated with enhanced proteolysis during muscle wasting [63]. Moreover, the degradation of ubiquitin-tagged proteins occurs in proteasome 26S, formed by regulatory (19S) and catalytic (20S) subunits. Interaction of the 19S subunit with ubiquitin drives the target protein to the core of the proteasome, a cylindrical protein complex formed by two external alpha rings (alpha 1–7) and two central beta rings (beta 1–7), in which beta 1, 2, and 5 present caspase-like, trypsin, and chymotrypsin protease activity, respectively [64]. Proteasome degradation results in 7–9 amino acid peptides, which are subsequently degraded by cytosolic proteases. During muscle wasting, the activity of chymotrypsin is increased, as is the expression of 19S, 11S, and 20S, all of which are modulated by the nutritional

Leucine and Its Importance for Cell Signalling Pathways in Cancer Cachexia-Induced Muscle…

In addition to UPS in muscle wasting, autophagy is a degradation process led by lysosomes, and it manages the catabolism of long-life proteins, defective organelles, and protein aggregates. Three different autophagy pathways have been described, i.e., microautophagy, chaperone driven autophagy, and macroautophagy (herein referred as autophagy)—extensively reported as a key regulator of muscle mass. Autophagy involves complex protein machinery, including ATGs (autophagyrelated genes); ultimately, autophagy leads to the formation of phagophores, i.e., the formation of autophagosomes by the elongation of the lipid membrane, which is

followed by a fusion of the autophagosome to the lysosome, generating the autolysosome with many hydrolases and proteases (i.e., cathepsins). The first step of autophagy is the activation of ULK1, which, in turn, phosphorylates Beclin1, promoting its interaction with VPS34, VPS15, and ATG14. This complex activates VPS34, assembling the phagophore rich in PI3K class III enzyme to form PI3P, a signal to recruit other ATGs. The ubiquitin-like ATG5 brings together the final complex, i.e., ATG5/ATG12/ATG16, initiating and expanding the membrane extension of the phagophore. In parallel, the conjugated form of ATG8, also a homologue to LC3 in muscle, is tightly bound to the autophagosome membrane and later cleaved by ATG4, thereby converting LC3I to LC3II, which is necessary for the fusion of the autophagosome with the endocytic compartments, thus forming the lysosome. Inside the autolysosome, cargoes are degraded with cathepsins L and B being especially important for the degradation of myofibrils proteins [67]. Interestingly, the treatment of C2C12 myotubes with PIF-like increased the cathepsin B and chymotrypsin-like activity. The previous exposition of leucine PIF-like-treated myotubes prevented not only cathepsins and chymotrypsin enzymes activity but also proteasome activity [31], thereby highlighting another role for leucine in cancer-cachexia reversal. Indeed, the inhibition of cathepsin activity has been

suggested as a useful approach to treat cancer cachexia [68].

115

Calcium-dependent proteolysis is composed by cysteine-proteases, which are dominated by calpains and the endogenous inhibitor calpastatin [69]. Among the 14 calpains described, striated muscle contains considerable amounts of μ-calpain, mcalpain and calpain 3, which are activated by the intracellular concentration of calcium [70]. Above a certain threshold, Ca2+ intracellular levels interact with the Cterminal domain of the calpain large subunit, thereby promoting N-terminal autocleavage and leading to maximal protease activity. Therefore, there is a correlation

supplementation of leucine [27, 65, 66].

DOI: http://dx.doi.org/10.5772/intechopen.78990

Among the factors produced by a tumour, the proteolysis-inducing factor (PIF) has central importance. This protein, first described by Todorov et al. [6], is a 24 kDa glycoprotein isolated from the adenocarcinoma MAC16 tissue, an experimental model of cancer cachexia. Similarly, some studies found a PIF like those that were also verified in other cachexia models, such as in Walker-256 carcinosarcoma [31], in patients with gastrointestinal [45], pancreatic [46], and other types of cancer [47]. The injection of PIF in mice induces an intense loss of lean body mass, similar to that associated with MAC16 tumour growth [6]. After being synthesised and released by tumour cells, PIF reaches the bloodstream and binds to its cell membrane receptor in muscle cells [48], leading to activation of the ubiquitinproteasome pathway and a decrease in protein synthesis by stimulating the doublestranded RNA-dependent protein kinase (PKR) [49]. The activated PKR leads to phosphorylation of eukaryotic translation initiation factor (eIF2α) and, consequently, inhibition of protein synthesis [50]. Many studies have shown that, unlike starvation, a decrease in food intake is not sufficient to cause muscle mass wasting in cancer patients, such as that which occurs during cancer cachexia [51].

As mentioned above, despite the fact that leucine stimulates protein synthesis, leucine and its metabolite β-hydroxy-β-methylbutyrate (HMB) can also decrease the rate of protein degradation apparently by reducing the expression of proteins from the ubiquitin-proteasome system [52, 53] and the other proteolytic pathways, i.e., mainly autophagy [54]. This characteristic makes leucine a great tool in cancerinduced cachexia therapy. In fact, leucine or HMB, i.e., alone [55] or in combination with other nutrients [56], can prevent the decrease of lean mass in cancer patients; this has been verified by our group in an experimental cachexia model [32, 35].

### 4.1.1 Proteolytic pathways and leucine

As noted above, the UPS is responsible for degrading proteins and might be responsible for up to 80% of proteolysis during skeletal muscle wasting [57, 58]. Since the UPS depends on linking the target protein to a ubiquitin tag and subsequent recognition and degradation by the proteasome core, leucine cell signalling can affect multiple steps. Ubiquitin conjugation to target proteins involves the action of a ubiquitin-activating enzyme (E1), which uses ATP to form thioester ubiquitin; conjugating to the ubiquitin-conjugating protein family of enzymes (E2), which in concert with ubiquitin protein ligase (E3), mediates the binding of the ubiquitin C terminal end to the targeted protein. The specificity of the substrate recognition is mainly dependent on E3 interaction with the targeted protein, giving relevance to this class of enzymes in studies of muscle atrophy affected by tumour evolution [59]. Our previous studies have shown that leucine supplementation can minimise the E2 activity in the muscle of Walker-256 tumour-bearing rats, suggesting a beneficial effect of this cell signal (data in print). Although approximately 1000 members of the E3 ligase family have been described, MuRF1 and

### Leucine and Its Importance for Cell Signalling Pathways in Cancer Cachexia-Induced Muscle… DOI: http://dx.doi.org/10.5772/intechopen.78990

Atrogin have been reported to be specifically expressed and increased in skeletal muscle under many catabolic conditions [60, 61]. Interestingly, acute or chronical leucine supplementation prevented the upregulation of proteasomal proteolysis in fasted aged rats as compared to younger adult controls [62]. We have recently verified that ageing causes additional proteasomal activity in an experimental model of cancer-induced cachexia (unpublished data). Thus, leucine supplementation might be a valuable tool to counteract higher susceptibility to cachexia in senescence. Also, MuRF1, atrogin, and other E3 ligases, such as MUSA1 and SMART1, have been associated with enhanced proteolysis during muscle wasting [63]. Moreover, the degradation of ubiquitin-tagged proteins occurs in proteasome 26S, formed by regulatory (19S) and catalytic (20S) subunits. Interaction of the 19S subunit with ubiquitin drives the target protein to the core of the proteasome, a cylindrical protein complex formed by two external alpha rings (alpha 1–7) and two central beta rings (beta 1–7), in which beta 1, 2, and 5 present caspase-like, trypsin, and chymotrypsin protease activity, respectively [64]. Proteasome degradation results in 7–9 amino acid peptides, which are subsequently degraded by cytosolic proteases. During muscle wasting, the activity of chymotrypsin is increased, as is the expression of 19S, 11S, and 20S, all of which are modulated by the nutritional supplementation of leucine [27, 65, 66].

In addition to UPS in muscle wasting, autophagy is a degradation process led by lysosomes, and it manages the catabolism of long-life proteins, defective organelles, and protein aggregates. Three different autophagy pathways have been described, i.e., microautophagy, chaperone driven autophagy, and macroautophagy (herein referred as autophagy)—extensively reported as a key regulator of muscle mass. Autophagy involves complex protein machinery, including ATGs (autophagyrelated genes); ultimately, autophagy leads to the formation of phagophores, i.e., the formation of autophagosomes by the elongation of the lipid membrane, which is followed by a fusion of the autophagosome to the lysosome, generating the autolysosome with many hydrolases and proteases (i.e., cathepsins). The first step of autophagy is the activation of ULK1, which, in turn, phosphorylates Beclin1, promoting its interaction with VPS34, VPS15, and ATG14. This complex activates VPS34, assembling the phagophore rich in PI3K class III enzyme to form PI3P, a signal to recruit other ATGs. The ubiquitin-like ATG5 brings together the final complex, i.e., ATG5/ATG12/ATG16, initiating and expanding the membrane extension of the phagophore. In parallel, the conjugated form of ATG8, also a homologue to LC3 in muscle, is tightly bound to the autophagosome membrane and later cleaved by ATG4, thereby converting LC3I to LC3II, which is necessary for the fusion of the autophagosome with the endocytic compartments, thus forming the lysosome. Inside the autolysosome, cargoes are degraded with cathepsins L and B being especially important for the degradation of myofibrils proteins [67]. Interestingly, the treatment of C2C12 myotubes with PIF-like increased the cathepsin B and chymotrypsin-like activity. The previous exposition of leucine PIF-like-treated myotubes prevented not only cathepsins and chymotrypsin enzymes activity but also proteasome activity [31], thereby highlighting another role for leucine in cancer-cachexia reversal. Indeed, the inhibition of cathepsin activity has been suggested as a useful approach to treat cancer cachexia [68].

Calcium-dependent proteolysis is composed by cysteine-proteases, which are dominated by calpains and the endogenous inhibitor calpastatin [69]. Among the 14 calpains described, striated muscle contains considerable amounts of μ-calpain, mcalpain and calpain 3, which are activated by the intracellular concentration of calcium [70]. Above a certain threshold, Ca2+ intracellular levels interact with the Cterminal domain of the calpain large subunit, thereby promoting N-terminal autocleavage and leading to maximal protease activity. Therefore, there is a correlation

skeletal or cardiac muscle mass might lead to cardiac and respiratory failure, in addition to the fact that it decreases the host's response after conventional treatments, such as chemotherapy or radiotherapy [42]. Moreover, it is well-established that the ubiquitin-proteasome system is a very important pathway in skeletal muscle degradation during cancer cachexia [43]. Furthermore, multiple studies have identified released factors that contribute to an increase in muscle protein degradation during cancer. The main factors that lead to protein degradation during cancer cachexia syndrome produced by the host are tumour necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-1 (IL-1), interferon gamma (IFN-γ) [44]. Meanwhile, the main factors produced by cancer cells are proteolysis-inducing factor (PIF), lipid-mobilising factor (LMF), and anaemia-inducing factor (AIS) [44].

Muscle Cells - Recent Advances and Future Perspectives

Among the factors produced by a tumour, the proteolysis-inducing factor (PIF)

has central importance. This protein, first described by Todorov et al. [6], is a 24 kDa glycoprotein isolated from the adenocarcinoma MAC16 tissue, an experimental model of cancer cachexia. Similarly, some studies found a PIF like those that were also verified in other cachexia models, such as in Walker-256 carcinosarcoma [31], in patients with gastrointestinal [45], pancreatic [46], and other types of cancer [47]. The injection of PIF in mice induces an intense loss of lean body mass, similar to that associated with MAC16 tumour growth [6]. After being synthesised and released by tumour cells, PIF reaches the bloodstream and binds to its cell membrane receptor in muscle cells [48], leading to activation of the ubiquitinproteasome pathway and a decrease in protein synthesis by stimulating the doublestranded RNA-dependent protein kinase (PKR) [49]. The activated PKR leads to phosphorylation of eukaryotic translation initiation factor (eIF2α) and, consequently, inhibition of protein synthesis [50]. Many studies have shown that, unlike starvation, a decrease in food intake is not sufficient to cause muscle mass wasting

in cancer patients, such as that which occurs during cancer cachexia [51].

4.1.1 Proteolytic pathways and leucine

114

As mentioned above, despite the fact that leucine stimulates protein synthesis, leucine and its metabolite β-hydroxy-β-methylbutyrate (HMB) can also decrease the rate of protein degradation apparently by reducing the expression of proteins from the ubiquitin-proteasome system [52, 53] and the other proteolytic pathways, i.e., mainly autophagy [54]. This characteristic makes leucine a great tool in cancerinduced cachexia therapy. In fact, leucine or HMB, i.e., alone [55] or in combination with other nutrients [56], can prevent the decrease of lean mass in cancer patients; this has been verified by our group in an experimental cachexia model [32, 35].

As noted above, the UPS is responsible for degrading proteins and might be responsible for up to 80% of proteolysis during skeletal muscle wasting [57, 58]. Since the UPS depends on linking the target protein to a ubiquitin tag and subsequent recognition and degradation by the proteasome core, leucine cell signalling can affect multiple steps. Ubiquitin conjugation to target proteins involves the action of a ubiquitin-activating enzyme (E1), which uses ATP to form thioester ubiquitin; conjugating to the ubiquitin-conjugating protein family of enzymes (E2), which in concert with ubiquitin protein ligase (E3), mediates the binding of the ubiquitin C terminal end to the targeted protein. The specificity of the substrate recognition is mainly dependent on E3 interaction with the targeted protein, giving relevance to this class of enzymes in studies of muscle atrophy affected by tumour evolution [59]. Our previous studies have shown that leucine supplementation can

minimise the E2 activity in the muscle of Walker-256 tumour-bearing rats, suggesting a beneficial effect of this cell signal (data in print). Although approximately 1000 members of the E3 ligase family have been described, MuRF1 and

between calpain activity and protein turnover in muscle, thereby suggesting an important role in muscle mass maintenance and partially accounting for muscle wasting in some pathologies [71]. Cancer patients and tumour-bearing rats present higher calcium-dependent proteolysis, which is linked with increased calpain and decreased calpastatin protein content [72, 73]. Moreover, there is evidence that calpains, mainly μ-calpain, is localised to the Z-disk in the sarcomere, which is anchored by myofibrils such as p35, nebulin, troponin-T, α-activin, and desmine. Thus, Ca2+ dependent proteolysis seems essential to the initial disaggregation of the sarcomere structure, thereby releasing contractile proteins for further degradation by other proteolytic systems. Similarly, leucine supplementation improves muscle mass, thereby minimising the muscle wasting by inhibiting the calpain activity.

catabolic state and resulting in widespread and progressive atrophy, including myocardial tissue. Studies have shown that cardiac atrophy can be a result of cancer evolution and its treatments [89–91]. These damages result in symptoms that can include breathlessness, lethargy, reduced exercise tolerance, congestive cardiac failure, and mortality [92]. Because the alterations in cardiac muscle structure and metabolism induced by cancer cachexia are poorly understood, cardiologists and oncologists are working together to explore models of care to improve outcomes. Some findings show that pancreatic, lung, and colorectal cancer patients have a reduced heart mass with a reduced left ventricular (LV) and wall thickness and are associated with smaller heart cell size and numbers and increased extracellular stroma surrounding the myocytes [92]. Indeed, this cardiac atrophy is part of a complex systemic metabolic syndrome caused by cancer damage, resulting in severe muscle wasting, including of the myocardium. Rodent models of cancer cachexia also show characteristics of cardiac atrophy, including decreased heart weight and LV mass; the thinning of septal, interventricular, and posterior walls; and chamber dilation as demonstrated by echocardiography [91, 93]. Cardiac atrophy in cancer cachexia is likely driven by cellular atrophy, including the activation of UPS [93] and the imbalance of protein turnover [91]. Cancer cachectic mice presented decreased cardiac contractile function and heart rate with concomitantly increased heart tissue fibrosis, which was associated with higher pro-inflammatory cytokine content and enhanced oxidative stress [94, 95]. Therefore, the use of an experimental cancer cachexia model allows us to evaluate how leucine supplementation counteracts cachexia damage in the heart. Recently, we reported relevant data related to the benefits of leucine supplementation for reverting/maintaining cardiac mass for both tumour-bearing rats fed a leucine-rich diet [91] and adult offspring whose mothers had been fed a leucine-rich diet (data not published). More interestingly, we observed improvement in enzyme activities related to the heart function via electrocardiography as a positive effect of leucine in tumourbearing rats [91]. We know that leucine stimulates protein synthesis through activation of the mTOR pathway, thereby stimulating the intracellular signalling pathways that modulate cellular metabolism and apoptosis; this supports our data since the activation of mTOR is also essential for mediating physiologic cardiac hypertrophy and preventing cardiac dysfunction in the face of pressure overload [91, 96], thus supporting the cardioprotective effects of leucine over the cancer-cachexia-

DOI: http://dx.doi.org/10.5772/intechopen.78990

Leucine and Its Importance for Cell Signalling Pathways in Cancer Cachexia-Induced Muscle…

induced cardiac damages [91].

widely studied [99].

117

6. Leucine maternal diet influence over muscle wasting

Since the number of new cases of cancer is increasing every year, and most of these are attributed to environmental factors and lifestyle, prevention is a major target of cancer studies [97, 98]. In addition to maintaining a balanced diet throughout life, the influence of maternal diet on offspring's adulthood has been

Among environmental factors, nutritional composition is the main factor in the modulation of gene expression, especially those related to metabolic pathways. The periods of gestation and lactation are considered crucial because the maternal diet exerts influence on the development and the plasticity of organs and tissues of the foetus/newborn [100]. The energy composition of foods, fatty acid composition, proteins, and micronutrients can modify several aspects of metabolism. Poor or imbalanced maternal diet, e.g., undernutrition, might contribute to a change in the metabolic programming of the offspring [101, 102], thereby increasing the risk

of metabolic diseases (e.g., insulin resistance, obesity, type II diabetes),

Other points of the proteolysis processes that should be mentioned include those related to other intracellular pathways that govern cancer cachexia. For example, AKT phosphorylation causes FoxOs inactivation and translocation from the nucleus to the cytosol [74]. FoxOs (Foxo1, 3 and 4) are transcription factors that regulate energy metabolism, the cell cycle, antioxidant defence, cell death, and longevity [75]. Repression of FoxOs by AKT is a key step in the anticatabolic action of insulin/IGF1 signalling [76]. Therefore, genetic ablation of FoxOs specifically in skeletal muscle reverses muscle atrophy caused by starvation and denervation, indicating that FoxOs are necessary to the expression of several atrophic genes, such as atrogin1, MuRF1, proteasome subunits, and lysosomal enzymes [75]. FoxO directly upregulated many proteasome subunits and E3 ligases in cancer-induced muscle wasting. Interestingly, FoxOs also increased autophagy in tumour-bearing mice [77], inducing the expression of such genes as Cathepsin-L and other genes related to the lysosomal/autophagy pathway (e.g., Gabarapl1 and Bnip3). Thus, FoxOs seem to mediate crosstalk between proteasomal and autophagy-dependent proteolysis in cancer-induced cachexia since the inhibition of FoxO3 or FoxO1 by RNA interference entirely prevents muscle loss [78]. Also, pro-inflammatory cytokines contribute to mass muscle decline under several conditions [79]. TNFα increases muscle protein degradation by activating the transcription factor NFκB [38]. The blockade of NFκB signalling in tumour-bearing mice partially attenuated cancer-induced muscle loss, thereby enhancing longevity [80]. Activation of NFκB enhanced atrophy by the transcription of MuRF1, ubiquitin, UbcH2 (E2), proteasome subunits, and autophagy-related genes [81]. Likewise, higher levels of myostatin and activin-A (i.e., members of the TGF-B family, share the receptor ACTRIIβ, and are known to regulate muscle mass) are related to muscle atrophy [82, 83]. Moreover, the inhibition of the bioactivity of activin-A and myostatin by inhibin and follistatin prevents muscle loss independent of tumour growth [84]. Additionally, several human tumour cell lines secrete considerable amounts of myostatin and activin-A [85], which are correlated with muscle strength loss [86]. Moreover, myostatin acting due to ACTRIIβ downstream effectors Smad2/3 activity also enhances skeletal muscle loss by phosphorylating the Smad2/3 and transcription of MuRF1, atrogin1, and autophagy induction [87], which corroborates our data, thereby highlighting the modulatory effect of leucine supplementation in C2C12 cells treated with PIF-like. Interestingly, myostatin effects might depend on the suppression of PI3K/Akt signalling [88], where we also find some beneficial effects of leucine supplementation, such as restoring the inhibitory effect of Akt and minimising proteolysis in PIF-like-treated C2C12 cells.

### 5. Myocardial muscle in cachexia and leucine

Recently, the number of studies addressing cancer and cardiac failure has increased. This is because cancer has significant effects on skeletal muscle, causing a

### Leucine and Its Importance for Cell Signalling Pathways in Cancer Cachexia-Induced Muscle… DOI: http://dx.doi.org/10.5772/intechopen.78990

catabolic state and resulting in widespread and progressive atrophy, including myocardial tissue. Studies have shown that cardiac atrophy can be a result of cancer evolution and its treatments [89–91]. These damages result in symptoms that can include breathlessness, lethargy, reduced exercise tolerance, congestive cardiac failure, and mortality [92]. Because the alterations in cardiac muscle structure and metabolism induced by cancer cachexia are poorly understood, cardiologists and oncologists are working together to explore models of care to improve outcomes. Some findings show that pancreatic, lung, and colorectal cancer patients have a reduced heart mass with a reduced left ventricular (LV) and wall thickness and are associated with smaller heart cell size and numbers and increased extracellular stroma surrounding the myocytes [92]. Indeed, this cardiac atrophy is part of a complex systemic metabolic syndrome caused by cancer damage, resulting in severe muscle wasting, including of the myocardium. Rodent models of cancer cachexia also show characteristics of cardiac atrophy, including decreased heart weight and LV mass; the thinning of septal, interventricular, and posterior walls; and chamber dilation as demonstrated by echocardiography [91, 93]. Cardiac atrophy in cancer cachexia is likely driven by cellular atrophy, including the activation of UPS [93] and the imbalance of protein turnover [91]. Cancer cachectic mice presented decreased cardiac contractile function and heart rate with concomitantly increased heart tissue fibrosis, which was associated with higher pro-inflammatory cytokine content and enhanced oxidative stress [94, 95]. Therefore, the use of an experimental cancer cachexia model allows us to evaluate how leucine supplementation counteracts cachexia damage in the heart. Recently, we reported relevant data related to the benefits of leucine supplementation for reverting/maintaining cardiac mass for both tumour-bearing rats fed a leucine-rich diet [91] and adult offspring whose mothers had been fed a leucine-rich diet (data not published). More interestingly, we observed improvement in enzyme activities related to the heart function via electrocardiography as a positive effect of leucine in tumourbearing rats [91]. We know that leucine stimulates protein synthesis through activation of the mTOR pathway, thereby stimulating the intracellular signalling pathways that modulate cellular metabolism and apoptosis; this supports our data since the activation of mTOR is also essential for mediating physiologic cardiac hypertrophy and preventing cardiac dysfunction in the face of pressure overload [91, 96], thus supporting the cardioprotective effects of leucine over the cancer-cachexiainduced cardiac damages [91].

### 6. Leucine maternal diet influence over muscle wasting

Since the number of new cases of cancer is increasing every year, and most of these are attributed to environmental factors and lifestyle, prevention is a major target of cancer studies [97, 98]. In addition to maintaining a balanced diet throughout life, the influence of maternal diet on offspring's adulthood has been widely studied [99].

Among environmental factors, nutritional composition is the main factor in the modulation of gene expression, especially those related to metabolic pathways. The periods of gestation and lactation are considered crucial because the maternal diet exerts influence on the development and the plasticity of organs and tissues of the foetus/newborn [100]. The energy composition of foods, fatty acid composition, proteins, and micronutrients can modify several aspects of metabolism. Poor or imbalanced maternal diet, e.g., undernutrition, might contribute to a change in the metabolic programming of the offspring [101, 102], thereby increasing the risk of metabolic diseases (e.g., insulin resistance, obesity, type II diabetes),

between calpain activity and protein turnover in muscle, thereby suggesting an important role in muscle mass maintenance and partially accounting for muscle wasting in some pathologies [71]. Cancer patients and tumour-bearing rats present higher calcium-dependent proteolysis, which is linked with increased calpain and decreased calpastatin protein content [72, 73]. Moreover, there is evidence that calpains, mainly μ-calpain, is localised to the Z-disk in the sarcomere, which is anchored by myofibrils such as p35, nebulin, troponin-T, α-activin, and desmine. Thus, Ca2+ dependent proteolysis seems essential to the initial disaggregation of the sarcomere structure, thereby releasing contractile proteins for further degradation by other proteolytic systems. Similarly, leucine supplementation improves muscle mass,

Muscle Cells - Recent Advances and Future Perspectives

thereby minimising the muscle wasting by inhibiting the calpain activity.

of Akt and minimising proteolysis in PIF-like-treated C2C12 cells.

Recently, the number of studies addressing cancer and cardiac failure has increased. This is because cancer has significant effects on skeletal muscle, causing a

5. Myocardial muscle in cachexia and leucine

116

Other points of the proteolysis processes that should be mentioned include those related to other intracellular pathways that govern cancer cachexia. For example, AKT phosphorylation causes FoxOs inactivation and translocation from the nucleus to the cytosol [74]. FoxOs (Foxo1, 3 and 4) are transcription factors that regulate energy metabolism, the cell cycle, antioxidant defence, cell death, and longevity [75]. Repression of FoxOs by AKT is a key step in the anticatabolic action of insulin/IGF1 signalling [76]. Therefore, genetic ablation of FoxOs specifically in skeletal muscle reverses muscle atrophy caused by starvation and denervation, indicating that FoxOs are necessary to the expression of several atrophic genes, such as atrogin1, MuRF1, proteasome subunits, and lysosomal enzymes [75]. FoxO directly upregulated many proteasome subunits and E3 ligases in cancer-induced muscle wasting. Interestingly, FoxOs also increased autophagy in tumour-bearing mice [77], inducing the expression of such genes as Cathepsin-L and other genes related to the lysosomal/autophagy pathway (e.g., Gabarapl1 and Bnip3). Thus, FoxOs seem to mediate crosstalk between proteasomal and autophagy-dependent proteolysis in cancer-induced cachexia since the inhibition of FoxO3 or FoxO1 by RNA interference entirely prevents muscle loss [78]. Also, pro-inflammatory cytokines contribute to mass muscle decline under several conditions [79]. TNFα increases muscle protein degradation by activating the transcription factor NFκB [38]. The blockade of NFκB signalling in tumour-bearing mice partially attenuated cancer-induced muscle loss, thereby enhancing longevity [80]. Activation of NFκB enhanced atrophy by the transcription of MuRF1, ubiquitin, UbcH2 (E2), proteasome subunits, and autophagy-related genes [81]. Likewise, higher levels of myostatin and activin-A (i.e., members of the TGF-B family, share the receptor ACTRIIβ, and are known to regulate muscle mass) are related to muscle atrophy [82, 83]. Moreover, the inhibition of the bioactivity of activin-A and myostatin by inhibin and follistatin prevents muscle loss independent of tumour growth [84]. Additionally, several human tumour cell lines secrete considerable amounts of myostatin and activin-A [85], which are correlated with muscle strength loss [86]. Moreover, myostatin acting due to ACTRIIβ downstream effectors Smad2/3 activity also enhances skeletal muscle loss by phosphorylating the Smad2/3 and transcription of MuRF1, atrogin1, and autophagy induction [87], which corroborates our data, thereby highlighting the modulatory effect of leucine supplementation in C2C12 cells treated with PIF-like. Interestingly, myostatin effects might depend on the suppression of PI3K/Akt signalling [88], where we also find some beneficial effects of leucine supplementation, such as restoring the inhibitory effect

cardiovascular disorders (e.g., hypertension and atherosclerosis), hormonal imbalance, and even cancer incidence in the offspring [103]. Thus, some amount of prevention may be achieved through a balanced maternal diet, considering not only the proper nutrition but the nutritional scheme; this can be viewed as a longterm investment that benefits both the current generation and its descendants, i.e., one that can minimise the risk of diseases (e.g., cancer) in the mother and her adult children [104, 105]. Thereby, due to foetal and lifetime nutrition there are epigenetic modifications [101], which are stable heritable patterns of gene expression in the DNA and histone proteins [106], and may result in DNA methylation, histone modifications, and RNA interference. Global hypomethylation, global miRNA downregulation, specific promoter hypermethylation, histone deacetylation, and upregulation of epigenetic machinery have been reported in cancer [107, 108], which are related to epigenetic silencing of detoxifying enzymes, suppressor tumour genes, cellular cycle regulators, apoptosis inducers, and DNA repair genes [109].

Knowing the benefits of leucine for attenuating the cachectic state and preventive interventions [110–112], previous studies using animal models of cachexia indicate that maternal nutrition affects the development of cancer cachexia and its effects in offspring adulthood [113, 114]. In our previous work, a maternal diet supplemented with leucine had a positive impact on the adult offspring's ability to respond to a Walker-256 tumour, diminishing the cachexia index, modulating markers of hepatic damage functions, and increasing the antioxidant response of the liver [112]. In this same experimental procedure concerning muscle wasting, our unpublished data show that maternal leucine supplementation can minimise the cachectic index by preserving the skeletal muscle mass in adult offspring. These results are confirmed by the stimulatory effect on the expression of mTOR pathway proteins. We observed a significant activation of mTOR and p70S6K, which indicates the preservation of protein synthesis and a decrease in proteolysis (i.e., we also verified less tyrosine release in the perfusion procedure) in the gastrocnemius muscle of these adulthood Walker-256 tumour-bearing rats subjected to a leucine enriched maternal diet (data in print). In fact, these findings indicate that leucine supplementation can modulate the mTOR pathway, resulting in the preservation of protein synthesis (data in print) and protection against the damaging effects of the Walker-256 tumour. Thus, maternal leucine supplementation shows promise in terms of improving the response to cachexia, i.e., preventing muscle loss, and further studies are needed to better understand the epigenetic mechanisms involved in this modulation and how the parental influence can counteract the damages caused by cachexia.

Acknowledgements

Figure 1.

061-990-754-255).

119

Conflict of interest

The authors are thankful for the financial support of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq #302524/2016-9), and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP #2014/13334-7; #2015/ 06766-0; #2015/21890-0; #2017/02739-4; #2017/10809-2; #2017/23065-1). The authors gratefully thank Biol. R.W. Santos for technical support and A.C.G. Marcondes for the graphic art of tissue and organs. The main text has been edited by native speakers from American Manuscript Editors (Editing Certificate # 241-

Summary of the effects of leucine (and maternal diet) on protein synthesis, degradation, and cardiac cachexia. The arrows indicate the principal impact of leucine over the process, and the bar-headed lines show the inhibitory effect of leucine. All cited processes mentioned in the main text are summarised in these four blocks.

Leucine and Its Importance for Cell Signalling Pathways in Cancer Cachexia-Induced Muscle…

DOI: http://dx.doi.org/10.5772/intechopen.78990

The authors declare that there are no conflicts of interest.

### 7. Conclusion

In summary, in Figure 1, we present evidence demonstrating the key role of leucine in improving skeletal muscle protein synthesis and minimising muscle degradation; we also report some metabolomic and proteomic findings, which are ameliorated by a diet supplemented with leucine. Also, these data show the benefits of leucine supplementation in cases of cardiac cachexia and the potential that a leucine supplemented maternal diet has for improvement of the host response to cancer-cachexia-induced muscle damage. As found in our studies and reported by other research groups, leucine is a suitable co-adjuvant treatment in an experimental model of cancer cachexia. However, more translation human studies are needed to determine whether leucine supplementation is capable of modulating muscle mass in cancer cachexia patients.

Leucine and Its Importance for Cell Signalling Pathways in Cancer Cachexia-Induced Muscle… DOI: http://dx.doi.org/10.5772/intechopen.78990

### Figure 1.

cardiovascular disorders (e.g., hypertension and atherosclerosis), hormonal imbalance, and even cancer incidence in the offspring [103]. Thus, some amount of prevention may be achieved through a balanced maternal diet, considering not only the proper nutrition but the nutritional scheme; this can be viewed as a longterm investment that benefits both the current generation and its descendants, i.e., one that can minimise the risk of diseases (e.g., cancer) in the mother and her adult children [104, 105]. Thereby, due to foetal and lifetime nutrition there are epigenetic modifications [101], which are stable heritable patterns of gene expression in the DNA and histone proteins [106], and may result in DNA methylation, histone modifications, and RNA interference. Global hypomethylation, global

Muscle Cells - Recent Advances and Future Perspectives

miRNA downregulation, specific promoter hypermethylation, histone

and DNA repair genes [109].

caused by cachexia.

mass in cancer cachexia patients.

7. Conclusion

118

deacetylation, and upregulation of epigenetic machinery have been reported in cancer [107, 108], which are related to epigenetic silencing of detoxifying enzymes, suppressor tumour genes, cellular cycle regulators, apoptosis inducers,

Knowing the benefits of leucine for attenuating the cachectic state and preventive interventions [110–112], previous studies using animal models of cachexia indicate that maternal nutrition affects the development of cancer cachexia and its effects in offspring adulthood [113, 114]. In our previous work, a maternal diet supplemented with leucine had a positive impact on the adult offspring's ability to respond to a Walker-256 tumour, diminishing the cachexia index, modulating markers of hepatic damage functions, and increasing the antioxidant response of the liver [112]. In this same experimental procedure concerning muscle wasting, our unpublished data show that maternal leucine supplementation can minimise the cachectic index by preserving the skeletal muscle mass in adult offspring. These results are confirmed by the stimulatory effect on the expression of mTOR pathway proteins. We observed a significant activation of mTOR and p70S6K, which indicates the preservation of protein synthesis and a decrease in proteolysis (i.e., we also verified less tyrosine release in the perfusion procedure) in the gastrocnemius muscle of these adulthood Walker-256 tumour-bearing rats subjected to a leucine enriched maternal diet (data in print). In fact, these findings indicate that leucine supplementation can modulate the mTOR pathway, resulting in the preservation of protein synthesis (data in print) and protection against the damaging effects of the Walker-256 tumour. Thus, maternal leucine supplementation shows promise in terms of improving the response to cachexia, i.e., preventing muscle loss, and further studies are needed to better understand the epigenetic mechanisms involved in this modulation and how the parental influence can counteract the damages

In summary, in Figure 1, we present evidence demonstrating the key role of leucine in improving skeletal muscle protein synthesis and minimising muscle degradation; we also report some metabolomic and proteomic findings, which are ameliorated by a diet supplemented with leucine. Also, these data show the benefits of leucine supplementation in cases of cardiac cachexia and the potential that a leucine supplemented maternal diet has for improvement of the host response to cancer-cachexia-induced muscle damage. As found in our studies and reported by other research groups, leucine is a suitable co-adjuvant treatment in an experimental model of cancer cachexia. However, more translation human studies are needed to determine whether leucine supplementation is capable of modulating muscle

Summary of the effects of leucine (and maternal diet) on protein synthesis, degradation, and cardiac cachexia. The arrows indicate the principal impact of leucine over the process, and the bar-headed lines show the inhibitory effect of leucine. All cited processes mentioned in the main text are summarised in these four blocks.

### Acknowledgements

The authors are thankful for the financial support of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq #302524/2016-9), and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP #2014/13334-7; #2015/ 06766-0; #2015/21890-0; #2017/02739-4; #2017/10809-2; #2017/23065-1). The authors gratefully thank Biol. R.W. Santos for technical support and A.C.G. Marcondes for the graphic art of tissue and organs. The main text has been edited by native speakers from American Manuscript Editors (Editing Certificate # 241- 061-990-754-255).

### Conflict of interest

The authors declare that there are no conflicts of interest.

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Leucine and Its Importance for Cell Signalling Pathways in Cancer Cachexia-Induced Muscle…

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10.1016/j.biocel.2013.05.032

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[4] Argilés JM, López-Soriano FJ, Busquets S, Argiles JM, Lopez-Soriano FJ, Busquets S. Mechanisms and treatment of cancer cachexia. Nutrition, Metabolism, and

### Author details

Andre Gustavo Oliveira†, Bread Cruz†, Sarah Christine Pereira de Oliveira†, Lais Rosa Viana†, Natalia Angelo Da Silva Miyaguti†, Luiz Alberto Ferreira Ramos†, Rafael Rossi Valentim† and Maria Cristina Cintra Gomes-Marcondes\* Department of Structural and Functional Biology, Laboratory of Nutrition and Cancer, Institute of Biology, University of Campinas (UNICAMP), Campinas, Sao Paulo, Brazil

\*Address all correspondence to: cintgoma@unicamp.br

† All authors are considered the first author, as all contributed equally to write, research, and develop data from experimental procedures for this work.

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

Leucine and Its Importance for Cell Signalling Pathways in Cancer Cachexia-Induced Muscle… DOI: http://dx.doi.org/10.5772/intechopen.78990

### References

[1] Tisdale MJ. Are tumoral factors responsible for host tissue wasting in cancer cachexia? Future Oncology. 2010;6:503-513. DOI: 10.2217/fon.10.20

[2] Johns N, Stephens NA, Fearon KC. Muscle wasting in cancer. The International Journal of Biochemistry & Cell Biology. 2013;45:2215-2229. DOI: 10.1016/j.biocel.2013.05.032

[3] Skipworth RJE, Stewart GD, Dejong CHC, Preston T, Fearon KCH. Pathophysiology of cancer cachexia: Much more than host-tumour interaction? Clinical Nutrition. 2007;26: 667-676. DOI: 10.1016/j.clnu.2007. 03.011

[4] Argilés JM, López-Soriano FJ, Busquets S, Argiles JM, Lopez-Soriano FJ, Busquets S. Mechanisms and treatment of cancer cachexia. Nutrition, Metabolism, and Cardiovascular Diseases. 2013;23(Suppl 1):S19-S24. DOI: 10.1016/j.numecd. 2012.04.011

[5] Deans DAC, Wigmore SJ, Gilmour H, Paterson-Brown S, Ross JA, Fearon KCH. Elevated tumour interleukin-1beta is associated with systemic inflammation: A marker of reduced survival in gastro-oesophageal cancer. British Journal of Cancer. 2006; 95:1568-1575. DOI: 10.1038/sj.bjc. 6603446

[6] Todorov P, Cariuk P, McDevitt T, Coles B, Fearon K, Tisdale M. Characterization of a cancer cachectic factor. Nature. 1996;379:739-742. DOI: 10.1038/379739a0

[7] Maschke J, Kruk U, Kastrati K, Kleeberg J, Buchholz D, Erickson N, et al. Nutritional care of cancer patients: A survey on patients' needs and medical care in reality. International Journal of Clinical Oncology. 2017;22:200-206. DOI: 10.1007/s10147-016-1025-6

[8] Columbus DA, Fiorotto ML, Davis TA. Leucine is a major regulator of muscle protein synthesis in neonates. Amino Acids. 2015;47:259-270. DOI: 10.1007/s00726-014-1866-0

[9] Moro T, Ebert SM, Adams CM, Rasmussen BB. Amino acid sensing in skeletal muscle. Trends in Endocrinology and Metabolism. 2016; 27:796-806. DOI: 10.1016/j.tem.2016. 06.010

[10] Cohen S, Nathan JA, Goldberg AL. Muscle wasting in disease: Molecular mechanisms and promising therapies. Nature Reviews. Drug Discovery. 2015; 14:58-74. DOI: 10.1038/nrd4467

[11] Argilés JM, Busquets S, Stemmler B, López-Soriano FJ. Cancer cachexia: Understanding the molecular basis. Nature Reviews. Cancer. 2014;14: 754-762. DOI: 10.1038/nrc3829

[12] Wang XJ, Yang X, Wang RX, Jiao HC, Zhao JP, Song ZG, et al. Leucine alleviates dexamethasoneinduced suppression of muscle protein synthesis via synergy involvement of mTOR and AMPK pathways. Bioscience Reports. 2016;36(3):1-11. DOI: 10.1042/ BSR20160096

[13] Mahoney SJ, Narayan S, Molz L, Berstler LA, Kang SA, Vlasuk GP, et al. A small molecule inhibitor of Rheb selectively targets mTORC1 signaling. Nature Communications. 2018;9(1): 548-559. DOI: 10.1038/s41467-018- 03035-z

[14] Mieulet V, Roceri M, Espeillac C, Sotiropoulos A, Ohanna M, Oorschot V, et al. S6 kinase inactivation impairs growth and translational target phosphorylation in muscle cells maintaining proper regulation of protein turnover. American Journal of Physiology. 2007;293:C712-C722. DOI: 10.1152/ajpcell.00499.2006

Author details

Sao Paulo, Brazil

120

Andre Gustavo Oliveira†, Bread Cruz†, Sarah Christine Pereira de Oliveira†,

Department of Structural and Functional Biology, Laboratory of Nutrition and Cancer, Institute of Biology, University of Campinas (UNICAMP), Campinas,

† All authors are considered the first author, as all contributed equally to write, research, and develop data from experimental procedures for this work.

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

Rafael Rossi Valentim† and Maria Cristina Cintra Gomes-Marcondes\*

\*Address all correspondence to: cintgoma@unicamp.br

Muscle Cells - Recent Advances and Future Perspectives

provided the original work is properly cited.

Lais Rosa Viana†, Natalia Angelo Da Silva Miyaguti†, Luiz Alberto Ferreira Ramos†,

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[26] Ventrucci G, Mello MAR, Gomes-Marcondes MCC. Proteasome activity is altered in skeletal muscle tissue of tumour-bearing rats fed a leucine-rich diet. Endocrine-Related Cancer. 2004; 11(4):887-895. DOI: 10.1677/erc.1.00828

[27] Cruz B, Oliveira A, Gomes-Marcondes MCC. L-leucine dietary supplementation modulates muscle protein degradation and increases pro-inflammatory cytokines in tumour-bearing rats. Cytokine. 2017; 96:253-260. DOI: 10.1016/j.cyto.2017. 04.019

Leucine and Its Importance for Cell Signalling Pathways in Cancer Cachexia-Induced Muscle… DOI: http://dx.doi.org/10.5772/intechopen.78990

[28] Soliman GA. The mammalian target of rapamycin signaling network and gene regulation. Current Opinion in Lipidology. 2005;16:317-323

[15] Gran P, Cameron-Smith D. The actions of exogenous leucine on mTOR signalling and amino acid transporters in human myotubes. BMC Physiology. 2011;11:10-19. DOI: 10.1186/1472-6793-

Muscle Cells - Recent Advances and Future Perspectives

[21] Kimball SR, Shantz LM, Horetsky RL, Jefferson LS. Leucine regulates translation of specific mRNAs in L6 myoblasts through mTORmediated changes in availability of eIF4E and phosphorylation of ribosomal protein S6. The Journal of Biological Chemistry. 1999. DOI: 10.1074/jbc.

[22] Anthony JC, Yoshizawa F, Anthony TG, Vary TC, Jefferson LS, Kimball SR. Leucine stimulates

translation initiation in skeletal muscle of postabsorptive rats via a rapamycinsensitive pathway. The Journal of Nutrition. 2000;130:2413-2419

[23] Nicklin P, Bergman P, Zhang B, Triantafellow E, Wang H, Nyfeler B, et al. Bidirectional transport of amino acids regulates mTOR and autophagy. Cell. 2009;136:521-534. DOI: 10.1016/j.

[24] Dodd KM, Tee AR. Leucine and mTORC1: A complex relationship. The American Journal of Clinical Nutrition.

[25] Shimobayashi M, Hall MN. Making new contacts: The mTOR network in metabolism and signalling crosstalk. Nature Reviews. Molecular Cell

Biology. 2014;15:155-162. DOI: 10.1038/

[26] Ventrucci G, Mello MAR, Gomes-Marcondes MCC. Proteasome activity is altered in skeletal muscle tissue of tumour-bearing rats fed a leucine-rich diet. Endocrine-Related Cancer. 2004; 11(4):887-895. DOI: 10.1677/erc.1.00828

[27] Cruz B, Oliveira A, Gomes-Marcondes MCC. L-leucine dietary supplementation modulates muscle protein degradation and increases pro-inflammatory cytokines in tumour-bearing rats. Cytokine. 2017; 96:253-260. DOI: 10.1016/j.cyto.2017.

2004;79:418-423. DOI: 10.1152/

274.17.11647

cell.2008.11.044

ajpendo.00525.2011

nrm3757

04.019

[16] Hang YZ, Obayashi HK, Awatari KM, Ato JS, Ajotto GB, Itaura YK, et al. Effects of branchedchain amino acid supplementation on plasma concentrations of free amino acids, insulin, and energy substrates in young men. Journal of Nutritional Science and Vitaminology. 2011;57:

[17] Kimball SR, Jefferson LS. New functions for amino acids: Effects on gene transcription and translation. The American Journal of Clinical Nutrition.

Columbus DA, Manjarín R, Nguyen HV, Suryawan A, Orellana RA, et al. Leucine supplementation stimulates protein synthesis and reduces degradation signal activation in muscle of newborn pigs during acute endotoxemia. American Journal of Physiology. Endocrinology and Metabolism. 2016;

[19] Kimball SR, Jefferson LS. Branched-

11-10

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[97] Bail J, Meneses K, Demark-Wahnefried W. Nutritional status and diet in cancer prevention. Seminars in Oncology Nursing. 2016;32:206-214. DOI: 10.1016/j.soncn.2016.05.004

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[99] Remely M, Stefanska B, Lovrecic L, Magnet U, Haslberger AG. Nutriepigenomics. Current Opinion in Clinical Nutrition and Metabolic Care. 2015;18:328-333. DOI: 10.1097/ MCO.0000000000000180

[100] Eberle C, Ament C. Diabetic metabolic programming: Mechanisms altering the intrauterine milieu. ISRN Pediatrics. 2012;2012:1-11. DOI: 10.5402/2012/975685

[101] Vanden Berghe W. Epigenetic impact of dietary polyphenols in cancer chemoprevention: Lifelong remodeling of our epigenomes. Pharmacological Research. 2012;65:565-576. DOI: 10.1016/j.phrs.2012.03.007

[102] Mathias PCF, Elmhiri G, De Oliveira JC, Delayre-Orthez C, Barella LF, Tófolo LP, et al. Maternal diet, bioactive molecules, and exercising as reprogramming tools of metabolic programming. European Journal of Nutrition. 2014;53:711-722. DOI: 10.1007/s00394-014-0654-7

[103] Wu G, Bazer FW, Cudd TA, Meininger CJ, Spencer TE.

Maternal nutrition and fetal development. The Journal of Nutrition. 2004:2169-2172

synthesis. Cancer. 1986:147-157. DOI:

DOI: http://dx.doi.org/10.5772/intechopen.78990

Leucine and Its Importance for Cell Signalling Pathways in Cancer Cachexia-Induced Muscle…

[112] Miyaguti NA da S, de Oliveira SCP, Gomes-Marcondes MCC. Maternal nutritional supplementation with fish oil and/or leucine improves hepatic function and antioxidant defenses, and minimizes cachexia indexes in Walker-256 tumor-bearing rats offspring. Nutrition Research. 2018;51:29-39. DOI:

10.1002/1097-0142

10.1016/j.nutres.2017.12.003

rats supplemented with N-3 polyunsaturated fatty acids for one generation. Nutrition and Cancer. 2003; 46:52-58. DOI: 10.1207/S15327914NC4

[114] Folador A, Hirabara SM,

Curi R, et al. Effect of fish oil

Bonatto SJR, Aikawa J, Yamazaki RK,

supplementation for 2 generations on changes in macrophage function induced by Walker 256 cancer cachexia in rats. International Journal of Cancer. 2007;120:344-350. DOI: 10.1002/ijc.

601\_07

22333

129

[113] Togni V, Ota CCC, Folador A, Júnior OT, Aikawa J, Yamazaki RK, et al. Cancer cachexia and tumor growth reduction in Walker 256 tumor-bearing

[104] Victora CG, Adair L, Fall C, Hallal PC, Martorell R, Richter L, et al. Maternal and child undernutrition: Consequences for adult health and human capital. Lancet. 2008;371: 340-357. DOI: 10.1016/S0140-6736(07) 61692-4

[105] Perera F, Herbstman J. Prenatal environmental exposures, epigenetics, and disease. Reproductive Toxicology. 2011;31:363-373. DOI: 10.1016/j. reprotox.2010.12.055

[106] Tammen SA, Friso S, Choi SW. Epigenetics: The link between nature and nurture. Molecular Aspects of Medicine. 2013;34:753-764. DOI: 10.1016/j.mam.2012.07.018

[107] Taby R, Issa JJ. Cancer epigenetics. CA: A Cancer Journal for Clinicians. 2010;60:376-392. DOI: 10.3322/ caac.20085

[108] Esteller M. Cancer epigenomics: DNA methylomes and histonemodification maps. Nature Reviews. Genetics. 2007;8:286-298

[109] Gerhauser C. Cancer chemoprevention and nutri-epigenetics: State of the art and future challenges. In: Pezzuto MJ, Suh N, editors. Natural Products in Cancer Prevention and Therapy. Berlin, Heidelberg: Springer Berlin Heidelberg; 2012. pp. 73-132. DOI: 10.1007/128\_2012\_360

[110] Hunter DC, Weintraub M, Blackburn GL, Bistrian BR. Branched chain amino acids as the protein component of parenteral nutrition in cancer cachexia. The British Journal of Surgery. 1989;76:149-153

[111] Tayek JA, Bistrian BR, Hehir DJ, Martin R, Moldawer LL, Blackburn GL. Improved protein kinetics and albumin

Leucine and Its Importance for Cell Signalling Pathways in Cancer Cachexia-Induced Muscle… DOI: http://dx.doi.org/10.5772/intechopen.78990

synthesis. Cancer. 1986:147-157. DOI: 10.1002/1097-0142

35:932-941. DOI: 10.1093/eurheartj/

[97] Bail J, Meneses K, Demark-Wahnefried W. Nutritional status and diet in cancer prevention. Seminars in Oncology Nursing. 2016;32:206-214. DOI: 10.1016/j.soncn.2016.05.004

[98] World Health Organization. Global Status Report on Noncommunicable Diseases 2010. World Health

Organization; 2011. p. 176. DOI: ISBN:

[99] Remely M, Stefanska B, Lovrecic L,

Nutriepigenomics. Current Opinion in Clinical Nutrition and Metabolic Care. 2015;18:328-333. DOI: 10.1097/ MCO.0000000000000180

[100] Eberle C, Ament C. Diabetic metabolic programming: Mechanisms altering the intrauterine milieu. ISRN Pediatrics. 2012;2012:1-11. DOI:

[101] Vanden Berghe W. Epigenetic impact of dietary polyphenols in cancer chemoprevention: Lifelong remodeling of our epigenomes. Pharmacological Research. 2012;65:565-576. DOI: 10.1016/j.phrs.2012.03.007

[102] Mathias PCF, Elmhiri G, De Oliveira JC, Delayre-Orthez C, Barella LF, Tófolo LP, et al. Maternal diet, bioactive molecules, and exercising as reprogramming tools of metabolic programming. European Journal of Nutrition. 2014;53:711-722. DOI: 10.1007/s00394-014-0654-7

[103] Wu G, Bazer FW, Cudd TA, Meininger CJ, Spencer TE.

128

978 92 4 156422 9

Magnet U, Haslberger AG.

10.5402/2012/975685

[96] Huang Y, Zhou M, Sun H, Wang Y. Branched-chain amino acid metabolism in heart disease: An epiphenomenon or a real culprit? Cardiovascular Research. 2011;90:220-223. DOI: 10.1093/cvr/

Muscle Cells - Recent Advances and Future Perspectives

Maternal nutrition and fetal

2004:2169-2172

61692-4

caac.20085

reprotox.2010.12.055

development. The Journal of Nutrition.

[104] Victora CG, Adair L, Fall C, Hallal PC, Martorell R, Richter L, et al. Maternal and child undernutrition: Consequences for adult health and human capital. Lancet. 2008;371: 340-357. DOI: 10.1016/S0140-6736(07)

[105] Perera F, Herbstman J. Prenatal environmental exposures, epigenetics, and disease. Reproductive Toxicology. 2011;31:363-373. DOI: 10.1016/j.

[106] Tammen SA, Friso S, Choi SW. Epigenetics: The link between nature and nurture. Molecular Aspects of Medicine. 2013;34:753-764. DOI: 10.1016/j.mam.2012.07.018

[107] Taby R, Issa JJ. Cancer epigenetics. CA: A Cancer Journal for Clinicians. 2010;60:376-392. DOI: 10.3322/

[108] Esteller M. Cancer epigenomics: DNA methylomes and histonemodification maps. Nature Reviews.

chemoprevention and nutri-epigenetics: State of the art and future challenges. In: Pezzuto MJ, Suh N, editors. Natural Products in Cancer Prevention and Therapy. Berlin, Heidelberg: Springer Berlin Heidelberg; 2012. pp. 73-132. DOI: 10.1007/128\_2012\_360

Genetics. 2007;8:286-298

[109] Gerhauser C. Cancer

[110] Hunter DC, Weintraub M, Blackburn GL, Bistrian BR. Branched chain amino acids as the protein component of parenteral nutrition in cancer cachexia. The British Journal of

[111] Tayek JA, Bistrian BR, Hehir DJ, Martin R, Moldawer LL, Blackburn GL. Improved protein kinetics and albumin

Surgery. 1989;76:149-153

eht302

cvr070

[112] Miyaguti NA da S, de Oliveira SCP, Gomes-Marcondes MCC. Maternal nutritional supplementation with fish oil and/or leucine improves hepatic function and antioxidant defenses, and minimizes cachexia indexes in Walker-256 tumor-bearing rats offspring. Nutrition Research. 2018;51:29-39. DOI: 10.1016/j.nutres.2017.12.003

[113] Togni V, Ota CCC, Folador A, Júnior OT, Aikawa J, Yamazaki RK, et al. Cancer cachexia and tumor growth reduction in Walker 256 tumor-bearing rats supplemented with N-3 polyunsaturated fatty acids for one generation. Nutrition and Cancer. 2003; 46:52-58. DOI: 10.1207/S15327914NC4 601\_07

[114] Folador A, Hirabara SM, Bonatto SJR, Aikawa J, Yamazaki RK, Curi R, et al. Effect of fish oil supplementation for 2 generations on changes in macrophage function induced by Walker 256 cancer cachexia in rats. International Journal of Cancer. 2007;120:344-350. DOI: 10.1002/ijc. 22333

Chapter 7

Abstract

development of CC.

ECM, browning

1. Introduction

131

Cancer Cachexia

Adipose Tissue Remodeling during

Cancer-induced cachexia (CC), characterized by systemic inflammation, body weight loss, adipose tissue (AT) remodeling, and muscle wasting, is a malignant metabolic syndrome with an undefined etiology. There is a consensus that multiple factors contribute to cancer-induced AT remodeling, and longitudinal studies show that patients lose AT before they start losing muscle mass. In CC, AT remodeling occurs predominantly through adipocyte atrophy, impairment of fatty acid turnover, inflammation, rearrangement of extracellular matrix (ECM), and browning of AT. More recently, some studies have shown that AT is affected early in the course of cachexia. Additionally, studies using experimental models have consistently indicated that the alterations in adipocyte metabolism begin quite early, followed by the downregulation of adipogenic and thermogenic genes. These sets of changes, in addition to metabolites derived from this process, maybe the initial (sterile) trigger of the sequence of events that result in the remodeling and dysfunction of AT in cachexia. Therefore, the present chapter aims to describe state of the art related to the subject of interest by analyzing the primary studies that have addressed the possible interface between inflammation and morphofunctional alterations of AT, in addition to the possible repercussions of this process during the

Keywords: adipose tissue, cachexia, inflammation, metabolism, adipogenesis,

Cachexia is a common occurrence in the advanced stages of cancer and contrib-

utes to reduce the quality of life and life expectancy of patients [1, 2]. Cancerrelated cachexia (CC) is a significant cause of morbidity and mortality, affecting more than 80% of individuals with advanced cancer and accounting for more than 20% of deaths [3–5]. Notably, the severity of cachexia does not correlate with tumor size [6]. Although there are descriptions of cachectic individuals dating back to more than 2000 years ago, increased attention has been focused on this syndrome in the recent decades. Nevertheless, its etiology remains unknown, and there is no

Cachexia is understood as a complex metabolic syndrome associated with underlying diseases and is characterized by a reduction of muscle mass and

1.1 Etiology of cachexia syndrome and adipose tissue remodeling

treatment that is able to revert this condition [7, 8].

Miguel Luiz Batista Júnior and Felipe Henriques

### Chapter 7

## Adipose Tissue Remodeling during Cancer Cachexia

Miguel Luiz Batista Júnior and Felipe Henriques

### Abstract

Cancer-induced cachexia (CC), characterized by systemic inflammation, body weight loss, adipose tissue (AT) remodeling, and muscle wasting, is a malignant metabolic syndrome with an undefined etiology. There is a consensus that multiple factors contribute to cancer-induced AT remodeling, and longitudinal studies show that patients lose AT before they start losing muscle mass. In CC, AT remodeling occurs predominantly through adipocyte atrophy, impairment of fatty acid turnover, inflammation, rearrangement of extracellular matrix (ECM), and browning of AT. More recently, some studies have shown that AT is affected early in the course of cachexia. Additionally, studies using experimental models have consistently indicated that the alterations in adipocyte metabolism begin quite early, followed by the downregulation of adipogenic and thermogenic genes. These sets of changes, in addition to metabolites derived from this process, maybe the initial (sterile) trigger of the sequence of events that result in the remodeling and dysfunction of AT in cachexia. Therefore, the present chapter aims to describe state of the art related to the subject of interest by analyzing the primary studies that have addressed the possible interface between inflammation and morphofunctional alterations of AT, in addition to the possible repercussions of this process during the development of CC.

Keywords: adipose tissue, cachexia, inflammation, metabolism, adipogenesis, ECM, browning

### 1. Introduction

### 1.1 Etiology of cachexia syndrome and adipose tissue remodeling

Cachexia is a common occurrence in the advanced stages of cancer and contributes to reduce the quality of life and life expectancy of patients [1, 2]. Cancerrelated cachexia (CC) is a significant cause of morbidity and mortality, affecting more than 80% of individuals with advanced cancer and accounting for more than 20% of deaths [3–5]. Notably, the severity of cachexia does not correlate with tumor size [6]. Although there are descriptions of cachectic individuals dating back to more than 2000 years ago, increased attention has been focused on this syndrome in the recent decades. Nevertheless, its etiology remains unknown, and there is no treatment that is able to revert this condition [7, 8].

Cachexia is understood as a complex metabolic syndrome associated with underlying diseases and is characterized by a reduction of muscle mass and

a depletion of fat stores [9–11]. Thus, the main clinical symptoms of CC are body weight loss in adults (corrected for water retention) and impaired (substandard) growth in children (after exclusion of endocrine disorders) [12]. Anorexia, inflammation, insulin resistance, and increased degradation of muscle proteins are frequently associated with cachexia [1, 2]. Although anorexia exhibits different characteristics when compared with starvation, muscle mass loss due to aging (sarcopenia), primary depression, malnutrition, and hyperthyroidism is correlated with increased morbidity associated with asthenia and metabolic disorders [3, 13].

An accurate understanding of the fundamental mechanisms that underlie CC is essential for the development of new pharmacological and nutritional therapies. In this way, several studies [19–23] have suggested that AT is the target of local and systemic factors derived from the host and by the tumor, including pro-cachectic factors [tumor necrosis factor α (TNF-α); interleukin (IL) 6; IL-1ß; IL-8; interferon-γ (INF-γ); ciliary neurotrophic factor; and leukemia inhibitory factor], anticachectic factors [soluble TNF-α receptor (sTNFR); soluble IL-6 receptor (sIL-6R); IL-1 receptor antagonist (IL-1RA); IL-4; IL-10; and IL-15], and tumor products [proteolysis-inducing factor (PIF); lipid-mobilizing factor (LMF); zinc-α 2-glycoprotein (ZAG); toxohormone-L; and anemia-inducing substance (AIS)]. Such factors are involved in the etiology and progression of cachexia [8, 14, 24–29]. Upon positing the inflammatory model as the hypothesis to be tested, some studies have recently demonstrated the relevance of subcutaneous adipose tissue (scAT) as both an important source of inflammatory mediators (particularly IL-6 and adiponectin) and an important source of biomarkers for cachexia (clinical progression), as adiponectin expression exhibits a correlation with the magnitude of the total body mass reduction [20]. More recently, a consistent modification consisting of inflammatory cell presence and fibrosis in scAT induced by cachexia was demonstrated in gastrointestinal cancer patients [30, 31]. The fibrosis was characterized by the presence of "crown-like structures" composed of CD68 positive AT macrophages (ATMϕs) surrounding adipocytes, and increased CD3 Ly, both of which were more evident in the fibrotic areas. In addition, some of these changes were already present in the cancer group, suggesting that AT inflammation may occur at an early stage of cachexia, even before the detection of pre-cachexic clinical features. Thus, alterations in AT inflammation seem to play a crucial role in the changes resulting in fat mass reduction, in addition to other morphofunctional alterations related to this tissue [19, 21]. Moreover, these changes appear to start quite early, long before any

Adipose Tissue Remodeling during Cancer Cachexia DOI: http://dx.doi.org/10.5772/intechopen.79979

local tissue and/or systemic (circulatory system) changes can be detected. Nevertheless, most studies on this topic have been restricted to assessing inflammation from the systemic point of view, only investigating plasma parameters, while neglecting tissue inflammation and, particularly, the events that precede the appearance of these alterations, although they may significantly contribute to disorders that result in AT remodeling, such as impairment of lipid metabolism, tissue cells turnover and inflammation, fibrosis, and subsequent systemic inflammation [2, 28]. Additionally, considering the important relationship among infiltrating cells (inflammatory mediators), the regulation of adipocyte metabolism, and the consequent remodeling of the AT, few or no studies have investigated this relationship and its role in the various stages of cachexia to our knowledge.

In general, AT remodeling in CC comprises a set of morphostructural modifications characterized by adipocyte atrophy, a result of an unbalance of lipids turnover, main due to increased lipolysis [10, 32]; impairment of adipocyte cellular turnover (adipogenesis) [18, 19]; enhanced inflammatory signaling [21, 30, 32]; modification of extracellular matrix (ECM) component generally resulting in fibrosis [30, 31]; and

The basic structure of the various types of AT includes mature adipocytes, stromal vascular cells (mesenchymal precursor cells, preadipocytes, fibroblasts, and immune system cells), blood vessels, lymph nodes, and nerves [36]. Fat cells

browning phenotype related to increase thermogenic effect [16, 33–35].

1.2.1 Adipose tissue atrophy is involved in the etiology of cachexia

133

1.2 General features of adipose tissue remodeling

Weight loss is the foremost independent predictor of mortality in cancer patients [8, 12, 14], beginning with the loss of both a fat mass (adipose tissue—AT) and the lean body mass (skeletal muscle tissue). Over the last few years, the former has often been proposed to proceed more rapidly in the patient than the latter [11, 15, 16]. By the late 1980s and early 1990s, cachexia was being approached from a different perspective, leading to a new conception according to which it is considered a chronic inflammatory syndrome. It is currently believed that factors produced by both the tumor and the host cause anorexia and the metabolic abnormalities that result in cachexia [3, 17, 18]. Based on the knowledge obtained regarding cachexia and its complexity, the latest international consensus defined standard diagnostic criteria for the disease [1]. According to this consensus, the condition is categorized as pre-cachexia (early stage), cachexia, or refractory cachexia (late stage) based on the severity of the following parameters: (1) the reduction of total body mass; (2) the presence of metabolic disorders; (3) anorexia; and (4) systemic inflammation, as illustrated in Figure 1A.

### Figure 1.

Model of cachexia development from a translational point of view. (A) Morphofunctional changes in adipose tissue described in cancer cachexia patients. These alterations are associated with the stages of syndrome development, according to [1]. (B) Compilation of main metabolic and inflammatory changes described in the experimental model of cachexia induced by Walker 256 tumor.

### Adipose Tissue Remodeling during Cancer Cachexia DOI: http://dx.doi.org/10.5772/intechopen.79979

a depletion of fat stores [9–11]. Thus, the main clinical symptoms of CC are body weight loss in adults (corrected for water retention) and impaired (substandard) growth in children (after exclusion of endocrine disorders) [12]. Anorexia, inflammation, insulin resistance, and increased degradation of muscle proteins are frequently associated with cachexia [1, 2]. Although anorexia exhibits different characteristics when compared with starvation, muscle mass loss due to aging (sarcopenia), primary depression, malnutrition, and hyperthyroidism is correlated with increased morbidity associated with asthenia and metabolic

Muscle Cells - Recent Advances and Future Perspectives

Weight loss is the foremost independent predictor of mortality in cancer patients [8, 12, 14], beginning with the loss of both a fat mass (adipose tissue—AT) and the lean body mass (skeletal muscle tissue). Over the last few years, the former has often been proposed to proceed more rapidly in the patient than the latter [11, 15, 16]. By the late 1980s and early 1990s, cachexia was being approached from a different perspective, leading to a new conception according to which it is considered a chronic inflammatory syndrome. It is currently believed that factors produced by both the tumor and the host cause anorexia and the metabolic abnormalities that result in cachexia [3, 17, 18]. Based on the knowledge obtained regarding cachexia and its complexity, the latest international consensus defined standard diagnostic criteria for the disease [1]. According to this consensus, the condition is categorized as pre-cachexia (early stage), cachexia, or refractory cachexia (late stage) based on the severity of the following parameters: (1) the reduction of total body mass; (2) the presence of metabolic disorders; (3) anorexia;

Model of cachexia development from a translational point of view. (A) Morphofunctional changes in adipose tissue described in cancer cachexia patients. These alterations are associated with the stages of syndrome development, according to [1]. (B) Compilation of main metabolic and inflammatory changes described in the

experimental model of cachexia induced by Walker 256 tumor.

and (4) systemic inflammation, as illustrated in Figure 1A.

disorders [3, 13].

Figure 1.

132

An accurate understanding of the fundamental mechanisms that underlie CC is essential for the development of new pharmacological and nutritional therapies. In this way, several studies [19–23] have suggested that AT is the target of local and systemic factors derived from the host and by the tumor, including pro-cachectic factors [tumor necrosis factor α (TNF-α); interleukin (IL) 6; IL-1ß; IL-8; interferon-γ (INF-γ); ciliary neurotrophic factor; and leukemia inhibitory factor], anticachectic factors [soluble TNF-α receptor (sTNFR); soluble IL-6 receptor (sIL-6R); IL-1 receptor antagonist (IL-1RA); IL-4; IL-10; and IL-15], and tumor products [proteolysis-inducing factor (PIF); lipid-mobilizing factor (LMF); zinc-α 2-glycoprotein (ZAG); toxohormone-L; and anemia-inducing substance (AIS)]. Such factors are involved in the etiology and progression of cachexia [8, 14, 24–29]. Upon positing the inflammatory model as the hypothesis to be tested, some studies have recently demonstrated the relevance of subcutaneous adipose tissue (scAT) as both an important source of inflammatory mediators (particularly IL-6 and adiponectin) and an important source of biomarkers for cachexia (clinical progression), as adiponectin expression exhibits a correlation with the magnitude of the total body mass reduction [20]. More recently, a consistent modification consisting of inflammatory cell presence and fibrosis in scAT induced by cachexia was demonstrated in gastrointestinal cancer patients [30, 31]. The fibrosis was characterized by the presence of "crown-like structures" composed of CD68 positive AT macrophages (ATMϕs) surrounding adipocytes, and increased CD3 Ly, both of which were more evident in the fibrotic areas. In addition, some of these changes were already present in the cancer group, suggesting that AT inflammation may occur at an early stage of cachexia, even before the detection of pre-cachexic clinical features. Thus, alterations in AT inflammation seem to play a crucial role in the changes resulting in fat mass reduction, in addition to other morphofunctional alterations related to this tissue [19, 21]. Moreover, these changes appear to start quite early, long before any local tissue and/or systemic (circulatory system) changes can be detected.

Nevertheless, most studies on this topic have been restricted to assessing inflammation from the systemic point of view, only investigating plasma parameters, while neglecting tissue inflammation and, particularly, the events that precede the appearance of these alterations, although they may significantly contribute to disorders that result in AT remodeling, such as impairment of lipid metabolism, tissue cells turnover and inflammation, fibrosis, and subsequent systemic inflammation [2, 28]. Additionally, considering the important relationship among infiltrating cells (inflammatory mediators), the regulation of adipocyte metabolism, and the consequent remodeling of the AT, few or no studies have investigated this relationship and its role in the various stages of cachexia to our knowledge.

### 1.2 General features of adipose tissue remodeling

In general, AT remodeling in CC comprises a set of morphostructural modifications characterized by adipocyte atrophy, a result of an unbalance of lipids turnover, main due to increased lipolysis [10, 32]; impairment of adipocyte cellular turnover (adipogenesis) [18, 19]; enhanced inflammatory signaling [21, 30, 32]; modification of extracellular matrix (ECM) component generally resulting in fibrosis [30, 31]; and browning phenotype related to increase thermogenic effect [16, 33–35].

### 1.2.1 Adipose tissue atrophy is involved in the etiology of cachexia

The basic structure of the various types of AT includes mature adipocytes, stromal vascular cells (mesenchymal precursor cells, preadipocytes, fibroblasts, and immune system cells), blood vessels, lymph nodes, and nerves [36]. Fat cells

(adipocytes) are the main cell type in AT, while the presence of other cell types varies as a function of the tissue location [mesenteric (meAT), epididymal (epiAT), retroperitoneal (rpAT), or scAT], animal species, and disease (e.g., obesity and cachexia) [37]. Additionally, the importance of AT in the control of adiposity is well established, as is the role of the adipokines (leptin, adiponectin, and resistin, among others) released by the adipose tissue [38]. While alterations in the development and metabolism of adipocytes have been implicated in the pathogenesis of human immunodeficiency virus (HIV)-related lipodystrophy [39], very little is known about the molecular mechanisms involved in the occurrence of lipodystrophy associated with other conditions, such as cachexia.

when compared with visceral AT (epiAT and meAT), at least considering tissue

of classic signs of cachexia. In this sense, the main morphological alteration observed as a result of AT atrophy is the alterations in the area and perimeter of adipocytes in both animal models [18, 42] and CC patients [20, 30]. Still in this context, depot-specific changes in adipocyte ultrastructure [42] were also

1.2.2 Metabolic features of adipocytes in the course of cachexia

shorter survival time during cancer progression [49].

minate in NEFA and glycerol release [10].

135

In fact, as described above, AT atrophy is a well-characterized clinical variable in cachexia syndrome. In addition, this tissue is affected early before the appearance

The body weight loss in CC has been thought to be a result of profound changes in metabolic pathways of tissues and organs, which cannot be solely explained by enhanced energy expenditure or malnutrition [45]. In this regard, the role of early AT dysfunction seems to have gained importance in the onset and progression of many alterations induced by the syndrome. Different mechanistic possibilities have been proposed to explain the changes in AT in cachexia, such as increased lipolytic activity, decreased the activity of LPL, reduced de novo lipogenesis, and, consequently, decreased lipid triacylglycerol (TG) deposition [32, 45–47]. Adipocyte lipid turnover [i.e., the balance between incorporation and removal of TG into adipocytes, in which lipolysis (hydrolysis of intracellular TGs)] and is the most important factor for lipid removal [48]. In CC, human and animal models [11] have shown an increased rate of lipid mobilization, and longitudinal studies have shown that patients with CC lose AT mass before wasting of the muscle mass can be detected [15]. In addition, an accelerated rate of AT loss is believed to be associated with

Most of the volume (>90%) of a white adipocyte is represented by a single fat droplet, which is mainly composed of TG. During periods of stress and/or nutrient deprivation, such as in metabolic disorders, the adipocytes activate mechanisms that lead to lipolysis, with a consequent release of non-esterified fatty acids

(NEFAs) and glycerol originating in the TG stored in these cells. NEFAs are immediately released into the bloodstream and subsequently serve as a substrate for energy production of muscle tissues, or alternatively, they are taken up into the liver, where they are oxidized, esterified, or transformed into ketone bodies [50]. The reactions that result in NEFA release are mainly catalyzed by two enzymes: adipocyte triglyceride lipase (ATGL), which catalyzes the first step of the pathway, resulting in the formation of diacylglycerol, and hormone-sensitive lipase (HSL), which is responsible for additional hydrolysis and catalyzes the reactions that cul-

Among the mechanisms that may be involved in the metabolic disorders that cause fat mass reduction in cachexia, increased lipolysis appears to be the most evident and is being described as an increasing frequency [9, 11]. Das and colleagues found that in ATGL and HSL knockout mice, there was a greater resistance to the development of tumor-induced cachexia, which was more evident in ATGLdeficient animals. These authors also observed a positive correlation between ATGL activity and the severity of cachexia. Even more interestingly, they only detected a

reduction of lean body mass in animals that exhibited severe cachexia. This phenomenon followed the events that led to a reduction of fat body mass, most noticeably involving an increase of TG hydrolysis in AT. The results of this study corroborate findings previously reported in individuals with cancer-related cachexia [15], thus increasing the consistency of the evidence demonstrating the role of AT as a target tissue in cachexia. Nevertheless, neither the exact time when

inflammation parameters [44].

Adipose Tissue Remodeling during Cancer Cachexia DOI: http://dx.doi.org/10.5772/intechopen.79979

described.

As mentioned above, in the course of cachexia, the observed body weight loss predominantly results from a reduction in the fat (AT) and lean (skeletal muscle) mass [10, 40]. More recently, AT loss was found to precede the reduction of the lean body mass [11, 15], and thus, a more accurate understanding of this process began to emerge. Several factors have been suggested as the cause of the changes that lead to a reduction of AT mass, including (1)increased lipid mobilization due to increased lipolysis in adipocytes [9, 10]; (2) reduced lipogenesis, resulting from decreased lipoprotein lipase (LPL) activity [17]; and (3) impaired adipocyte turnover, most likely as a function of an adipogenesis-apoptosis imbalance in AT [41].

Taking the factors that are most likely to be involved in the observed fat mass loss and the relevance of AT in cachexia into consideration, the recent studies have utilized an experimental animal model that allows temporal assessment of the main variables that are potentially involved in cachexia-induced AT disorders, with an emphasis on the parameters related to adipogenesis, metabolism, and inflammation [18, 23, 27, 32], as shown in Figure 1A. These data demonstrate that these alterations start early, at 4 days after cachexia induction by inoculation of Walker 256 tumor cells in Wistar rats [19, 32]. It is worth noting that this is the period that precedes the appearance of the classic symptoms of cachexia, such as dyslipidemia, and a reduction of the total body weight, as well as inflammatory alterations in the AT in these animals, which begin to predominate starting on Day 7. In Lewis lung carcinoma (LLC) tumor-bearing mice, upregulation of genes related to lipid turnover and adipose browning is evident even before the detection of the body weight loss of the animals [16, 33]. Similarly, using several experimental models of cachexia (syngeneic and genetic), K5-SOS mice showed reduction in fat mass and spleen enlargement in the pre-cachexic period, that is, before the detection of body weight loss and the development of cachexia [33]. Taking the aforementioned findings into consideration, the temporal characterization of some of the alterations that occur in different AT depots in the course of cachexia has effectively pointed to actual pathways and mediators that might be involved in the earliest changes, in addition to serving as biomarkers for the clinical progression of cachexia.

As mentioned above, accentuated reduction of fat mass is a significant clinical sign of CC. Although it has not yet been well established, these alterations depend on the location of the adipose tissue involved (e.g., visceral or subcutaneous). Taking this into account, it was recently shown that, considering results found in the experimental animal model of cachexia (Walker 256 tumors cells-induced), visceral fat depots were affected in different ways. Thus, epiAT and meAT [25] exhibited higher reductions in relative weight (percentage of total body mass), respectively, while rpAT did not show any change [42]. Furthermore, in cachectic patients, assessment of the adipose tissue area by means of computed tomography in humans showed that visceral AT was reduced in cachectic individuals with gastrointestinal carcinosarcoma when compared with controls [43]. On the other hand, in individuals with gastrointestinal cancer, scAT seems to be more affected

(adipocytes) are the main cell type in AT, while the presence of other cell types varies as a function of the tissue location [mesenteric (meAT), epididymal (epiAT), retroperitoneal (rpAT), or scAT], animal species, and disease (e.g., obesity and cachexia) [37]. Additionally, the importance of AT in the control of adiposity is well established, as is the role of the adipokines (leptin, adiponectin, and resistin, among others) released by the adipose tissue [38]. While alterations in the development and metabolism of adipocytes have been implicated in the pathogenesis of human immunodeficiency virus (HIV)-related lipodystrophy [39], very little is known about the molecular mechanisms involved in the occurrence of lipodystrophy asso-

As mentioned above, in the course of cachexia, the observed body weight loss predominantly results from a reduction in the fat (AT) and lean (skeletal muscle) mass [10, 40]. More recently, AT loss was found to precede the reduction of the lean body mass [11, 15], and thus, a more accurate understanding of this process began to emerge. Several factors have been suggested as the cause of the changes that lead to a reduction of AT mass, including (1)increased lipid mobilization due to increased lipolysis in adipocytes [9, 10]; (2) reduced lipogenesis, resulting from decreased lipoprotein lipase (LPL) activity [17]; and (3) impaired adipocyte turnover, most likely as a function of an adipogenesis-apoptosis imbalance

Taking the factors that are most likely to be involved in the observed fat mass loss and the relevance of AT in cachexia into consideration, the recent studies have utilized an experimental animal model that allows temporal assessment of the main variables that are potentially involved in cachexia-induced AT disorders, with an emphasis on the parameters related to adipogenesis, metabolism, and inflammation [18, 23, 27, 32], as shown in Figure 1A. These data demonstrate that these alterations start early, at 4 days after cachexia induction by inoculation of Walker 256 tumor cells in Wistar rats [19, 32]. It is worth noting that this is the period that precedes the appearance of the classic symptoms of cachexia, such as dyslipidemia, and a reduction of the total body weight, as well as inflammatory alterations in the AT in these animals, which begin to predominate starting on Day 7. In Lewis lung carcinoma (LLC) tumor-bearing mice, upregulation of genes related to lipid turnover and adipose browning is evident even before the detection of the body weight

loss of the animals [16, 33]. Similarly, using several experimental models of cachexia (syngeneic and genetic), K5-SOS mice showed reduction in fat mass and spleen enlargement in the pre-cachexic period, that is, before the detection of body weight loss and the development of cachexia [33]. Taking the aforementioned findings into consideration, the temporal characterization of some of the alterations that occur in different AT depots in the course of cachexia has effectively pointed to actual pathways and mediators that might be involved in the earliest changes, in addition to serving as biomarkers for the clinical progression of cachexia.

As mentioned above, accentuated reduction of fat mass is a significant clinical sign of CC. Although it has not yet been well established, these alterations depend on the location of the adipose tissue involved (e.g., visceral or subcutaneous). Taking this into account, it was recently shown that, considering results found in the experimental animal model of cachexia (Walker 256 tumors cells-induced), visceral fat depots were affected in different ways. Thus, epiAT and meAT [25] exhibited higher reductions in relative weight (percentage of total body mass), respectively, while rpAT did not show any change [42]. Furthermore, in cachectic patients, assessment of the adipose tissue area by means of computed tomography in humans showed that visceral AT was reduced in cachectic individuals with gastrointestinal carcinosarcoma when compared with controls [43]. On the other hand, in individuals with gastrointestinal cancer, scAT seems to be more affected

ciated with other conditions, such as cachexia.

Muscle Cells - Recent Advances and Future Perspectives

in AT [41].

134

when compared with visceral AT (epiAT and meAT), at least considering tissue inflammation parameters [44].

In fact, as described above, AT atrophy is a well-characterized clinical variable in cachexia syndrome. In addition, this tissue is affected early before the appearance of classic signs of cachexia. In this sense, the main morphological alteration observed as a result of AT atrophy is the alterations in the area and perimeter of adipocytes in both animal models [18, 42] and CC patients [20, 30]. Still in this context, depot-specific changes in adipocyte ultrastructure [42] were also described.

### 1.2.2 Metabolic features of adipocytes in the course of cachexia

The body weight loss in CC has been thought to be a result of profound changes in metabolic pathways of tissues and organs, which cannot be solely explained by enhanced energy expenditure or malnutrition [45]. In this regard, the role of early AT dysfunction seems to have gained importance in the onset and progression of many alterations induced by the syndrome. Different mechanistic possibilities have been proposed to explain the changes in AT in cachexia, such as increased lipolytic activity, decreased the activity of LPL, reduced de novo lipogenesis, and, consequently, decreased lipid triacylglycerol (TG) deposition [32, 45–47]. Adipocyte lipid turnover [i.e., the balance between incorporation and removal of TG into adipocytes, in which lipolysis (hydrolysis of intracellular TGs)] and is the most important factor for lipid removal [48]. In CC, human and animal models [11] have shown an increased rate of lipid mobilization, and longitudinal studies have shown that patients with CC lose AT mass before wasting of the muscle mass can be detected [15]. In addition, an accelerated rate of AT loss is believed to be associated with shorter survival time during cancer progression [49].

Most of the volume (>90%) of a white adipocyte is represented by a single fat droplet, which is mainly composed of TG. During periods of stress and/or nutrient deprivation, such as in metabolic disorders, the adipocytes activate mechanisms that lead to lipolysis, with a consequent release of non-esterified fatty acids (NEFAs) and glycerol originating in the TG stored in these cells. NEFAs are immediately released into the bloodstream and subsequently serve as a substrate for energy production of muscle tissues, or alternatively, they are taken up into the liver, where they are oxidized, esterified, or transformed into ketone bodies [50]. The reactions that result in NEFA release are mainly catalyzed by two enzymes: adipocyte triglyceride lipase (ATGL), which catalyzes the first step of the pathway, resulting in the formation of diacylglycerol, and hormone-sensitive lipase (HSL), which is responsible for additional hydrolysis and catalyzes the reactions that culminate in NEFA and glycerol release [10].

Among the mechanisms that may be involved in the metabolic disorders that cause fat mass reduction in cachexia, increased lipolysis appears to be the most evident and is being described as an increasing frequency [9, 11]. Das and colleagues found that in ATGL and HSL knockout mice, there was a greater resistance to the development of tumor-induced cachexia, which was more evident in ATGLdeficient animals. These authors also observed a positive correlation between ATGL activity and the severity of cachexia. Even more interestingly, they only detected a reduction of lean body mass in animals that exhibited severe cachexia. This phenomenon followed the events that led to a reduction of fat body mass, most noticeably involving an increase of TG hydrolysis in AT. The results of this study corroborate findings previously reported in individuals with cancer-related cachexia [15], thus increasing the consistency of the evidence demonstrating the role of AT as a target tissue in cachexia. Nevertheless, neither the exact time when

these changes occur in the course of cachexia nor the various affected depots have yet been adequately described.

fatty acid-binding protein (aP2), LPL, fatty acid transport proteins (FATPs), and fatty acid-binding proteins (FABPs), among others. Activation of genes associated with glucose transport, such as Glut4, and thermogenesis, such as the mitochondrial

In AT, a balance between the growth/differentiation (adipogenesis) and death of its cells (generally by apoptosis) regulates the cellular turnover [41, 59]. In this aspect, the impairment of adipogenesis in the course of cachexia has been recently addressed. Some studies have elucidated the adipogenic marker profile during the development of cachexia syndrome [19, 25]. However, few studies have addressed the apoptotic processes and/or AT turnover during cachexia [43]. It has been known that adipogenic genes are downregulated in CC in epiAT [18] and rpAT [19]. On the other hand, scAT apoptosis did not change in cancer patients [43]. Therefore, considering that AT depots respond heterogeneously to CC and several metabolic and inflammatory pathways are involved in AT remodeling, there is no consensus if such effects induced by cachexia would be a result of secreted products directly by the tumor and tumor-host relationship. Thus, a recent study has analyzed in vitro adipogenesis in a co-culture system to mimic the effects of CC on adipocytes [60]. Co-culture of LLC cells promoted a decreased volume of the lipid droplets in 3 T3-L1 cells, compromising its maturation process

(adipogenesis) in vitro. This result was followed by the downregulation of adipogenic and lipolytic gene expression, increased in apoptosis markers and proinflammatory cytokines secretion by both tumor cells and adipocytes. In this sense, these data suggest that the presence of the tumor cells was able to inhibit the adipocytes' maturation, which was associated with the increased levels of

strated to be adequate for investigating cachexia-induced alterations of

the adipose tissue of individuals with cachexia.

1.2.4 Adipose tissue inflammatory profile in the course of cachexia

as the pathways and regulatory factors involved. Besides, modifications in adipogenesis appear to precede the appearance of the classic signs of cachexia as well as the signs of tissue inflammation in AT, such as increases in infiltrated macrophages and the production of inflammatory cytokines [21]. Thus, the factors that "silence" the genes involved in the differentiation of preadipocytes, and, consequently, in the maintenance of adipose cells turnover in AT might play a central role in the genesis of the damaging changes (metabolism and function) that occur in

In this way, the findings generated in the experimental model have demon-

adipogenesis and point to the need to widen the scope of the assessed genes as well

According to the abovementioned findings from animal models and cancer cachexia patients, a metabolic and morphological dysfunction that results in the AT remodeling occurs during the development of CC [2]. More recently, another relevant aspect of cachexia-induced AT remodeling is the establishment of AT inflammation, which is characterized by increased recruitment of ATMϕs, including activated M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophages [32]. In this way, an inflammatory profile predominates the end-stage of cachexia, particularly in visceral AT, as most of the characteristic changes of this syndrome are already established at this time [19, 27]. More recently, the immune cell infiltration profile was analyzed in greater detail, which was found to be characterized by an increase in M1-polarized macrophages [20]. In that study, the profile of chemokines specific to polymorphonuclear cells was also investigated, in addition to the presence of neutrophils in the various AT depots. The results revealed an increase in the chemokines CCL3 and CXCL2 at 7 days after tumor cell inoculation. The presence

inflammatory cytokines.

137

uncoupling proteins (Ucp2 and Ucp3), has also been described [18].

Adipose Tissue Remodeling during Cancer Cachexia DOI: http://dx.doi.org/10.5772/intechopen.79979

To elucidate these aspects, a study was conducted in which the lipolytic capacity of isolated adipocytes was assessed at 4 and 14 days after the inoculation of tumor cells. Two particular visceral fat stores were selected (meAT and rpAT) based on the results of previous studies that demonstrated their relevance for the development of cachexia. Day 4 was selected to perform the analysis, also based on previous results [19], which demonstrated downregulation of the genes involved in adipocyte metabolism, while no changes were found in the assessed morphological and inflammatory parameters. Interestingly, the tumor-bearing animals exhibited a reduction in basal lipolysis 4 days after inoculation, while on Day 14, the cachectic animals exhibited a considerable increase in the basal lipolysis rate (Figure 1B). These findings corroborate with the results of other studies showing increased lipolysis in the subcutaneous AT of cachectic patients [9, 51].

In this regard, another important aspect was a deregulation of lipolysis (in vitro) revealed a distinct profile, depending on the degree of disease progression. In the first, the basal rate of lipolysis was reduced and was accompanied by increased p-HSL (Ser565) expression, which is regulated by AMPK activation and, consequently, inhibits HSL activity. Patients with CC show reduced spontaneous basal lipolysis with elevated ex vivo responses to catecholamine and natriuretic peptides [40, 52]. In this aspect, an elegant study showed that the lack of AMPK activity is a common feature of adipose tissue dysfunction in cachectic mice and is triggered, at least partially, through the aberrant induction of Cidea and the subsequent degradation of AMPK in this tissue [53]. The authors suggest that treatment of cachectic animals with a peptide specifically targeting the white adipose tissue AMPK-CIDEA interaction prevents AT loss under CC conditions.

### 1.2.3 Downregulation of adipogenic genes in cancer cachexia

Adipogenesis may be defined as the process of the differentiation of precursor cells (preadipocytes) into new adipose cells (adipocytes) that are able to store TG and synthesize and secrete various proteins called adipokines [54]. In fact, impaired adipogenesis might contribute to the development of obesity-related metabolic disorders, such as peripheral insulin resistance, hyperlipidemia, and type 2 diabetes [55, 56]. The process of adipocyte differentiation involves the activation of a cascade of transcription factors that coordinate the expression of genes that are responsible for adipocyte function [55, 57]. The initial events include transient increases in CCAAT/enhancer-binding proteins beta and delta (C/EBPβ and δ), which allow preadipocytes to be distinguished from non-adipogenic precursor cells and subsequently activate the expression of the peroxisome proliferator-activated receptor gamma-2 (Pparγ-2), which in turn stimulates the expression of C/ebpα, which exerts a synergic effect with PPARγ-2 on the control of terminal differentiation [54, 57]. Local and endocrine factors might regulate adipogenesis through the modulation of these transcriptional events [58].

Adipocyte maturation is accompanied by intracellular lipid accumulation, which is mainly mediated by sterol regulatory element-binding protein-1C (SREBP-1C). In addition to activating the expression of Pparγ-2, SREBP-1C also activates the lipogenic pathway by stimulating the expression of the genes that encode the main enzymes of that pathway, such as ATP-citrate lyase (Acly), acetyl-CoA carboxylase (Acaca), fatty acid synthase (Fasn), and stearoyl-CoA desaturase-1 (Scd-1) [18, 57]. In addition to its direct participation in adipocyte differentiation, PPARγ-2 also plays an important role in the transcriptional regulation of genes associated with the lipogenic pathway by inducing the transcription of genes that encode adipocyte

these changes occur in the course of cachexia nor the various affected depots have

lipolysis in the subcutaneous AT of cachectic patients [9, 51].

interaction prevents AT loss under CC conditions.

modulation of these transcriptional events [58].

136

1.2.3 Downregulation of adipogenic genes in cancer cachexia

To elucidate these aspects, a study was conducted in which the lipolytic capacity of isolated adipocytes was assessed at 4 and 14 days after the inoculation of tumor cells. Two particular visceral fat stores were selected (meAT and rpAT) based on the results of previous studies that demonstrated their relevance for the development of cachexia. Day 4 was selected to perform the analysis, also based on previous results [19], which demonstrated downregulation of the genes involved in adipocyte metabolism, while no changes were found in the assessed morphological and inflammatory parameters. Interestingly, the tumor-bearing animals exhibited a reduction in basal lipolysis 4 days after inoculation, while on Day 14, the cachectic animals exhibited a considerable increase in the basal lipolysis rate (Figure 1B). These findings corroborate with the results of other studies showing increased

In this regard, another important aspect was a deregulation of lipolysis (in vitro) revealed a distinct profile, depending on the degree of disease progression. In the first, the basal rate of lipolysis was reduced and was accompanied by increased p-HSL (Ser565) expression, which is regulated by AMPK activation and, consequently, inhibits HSL activity. Patients with CC show reduced spontaneous basal lipolysis with elevated ex vivo responses to catecholamine and natriuretic peptides [40, 52]. In this aspect, an elegant study showed that the lack of AMPK activity is a common feature of adipose tissue dysfunction in cachectic mice and is triggered, at least partially, through the aberrant induction of Cidea and the subsequent degradation of AMPK in this tissue [53]. The authors suggest that treatment of cachectic animals with a peptide specifically targeting the white adipose tissue AMPK-CIDEA

Adipogenesis may be defined as the process of the differentiation of precursor cells (preadipocytes) into new adipose cells (adipocytes) that are able to store TG and synthesize and secrete various proteins called adipokines [54]. In fact, impaired adipogenesis might contribute to the development of obesity-related metabolic disorders, such as peripheral insulin resistance, hyperlipidemia, and type 2 diabetes [55, 56]. The process of adipocyte differentiation involves the activation of a cascade of transcription factors that coordinate the expression of genes that are responsible for adipocyte function [55, 57]. The initial events include transient increases in CCAAT/enhancer-binding proteins beta and delta (C/EBPβ and δ), which allow preadipocytes to be distinguished from non-adipogenic precursor cells and subsequently activate the expression of the peroxisome proliferator-activated receptor gamma-2 (Pparγ-2), which in turn stimulates the expression of C/ebpα, which exerts a synergic effect with PPARγ-2 on the control of terminal differentiation [54, 57]. Local and endocrine factors might regulate adipogenesis through the

Adipocyte maturation is accompanied by intracellular lipid accumulation, which is mainly mediated by sterol regulatory element-binding protein-1C (SREBP-1C). In addition to activating the expression of Pparγ-2, SREBP-1C also activates the lipogenic pathway by stimulating the expression of the genes that encode the main enzymes of that pathway, such as ATP-citrate lyase (Acly), acetyl-CoA carboxylase (Acaca), fatty acid synthase (Fasn), and stearoyl-CoA desaturase-1 (Scd-1) [18, 57]. In addition to its direct participation in adipocyte differentiation, PPARγ-2 also plays an important role in the transcriptional regulation of genes associated with the lipogenic pathway by inducing the transcription of genes that encode adipocyte

yet been adequately described.

Muscle Cells - Recent Advances and Future Perspectives

fatty acid-binding protein (aP2), LPL, fatty acid transport proteins (FATPs), and fatty acid-binding proteins (FABPs), among others. Activation of genes associated with glucose transport, such as Glut4, and thermogenesis, such as the mitochondrial uncoupling proteins (Ucp2 and Ucp3), has also been described [18].

In AT, a balance between the growth/differentiation (adipogenesis) and death of its cells (generally by apoptosis) regulates the cellular turnover [41, 59]. In this aspect, the impairment of adipogenesis in the course of cachexia has been recently addressed. Some studies have elucidated the adipogenic marker profile during the development of cachexia syndrome [19, 25]. However, few studies have addressed the apoptotic processes and/or AT turnover during cachexia [43]. It has been known that adipogenic genes are downregulated in CC in epiAT [18] and rpAT [19]. On the other hand, scAT apoptosis did not change in cancer patients [43]. Therefore, considering that AT depots respond heterogeneously to CC and several metabolic and inflammatory pathways are involved in AT remodeling, there is no consensus if such effects induced by cachexia would be a result of secreted products directly by the tumor and tumor-host relationship. Thus, a recent study has analyzed in vitro adipogenesis in a co-culture system to mimic the effects of CC on adipocytes [60]. Co-culture of LLC cells promoted a decreased volume of the lipid droplets in 3 T3-L1 cells, compromising its maturation process (adipogenesis) in vitro. This result was followed by the downregulation of adipogenic and lipolytic gene expression, increased in apoptosis markers and proinflammatory cytokines secretion by both tumor cells and adipocytes. In this sense, these data suggest that the presence of the tumor cells was able to inhibit the adipocytes' maturation, which was associated with the increased levels of inflammatory cytokines.

In this way, the findings generated in the experimental model have demonstrated to be adequate for investigating cachexia-induced alterations of adipogenesis and point to the need to widen the scope of the assessed genes as well as the pathways and regulatory factors involved. Besides, modifications in adipogenesis appear to precede the appearance of the classic signs of cachexia as well as the signs of tissue inflammation in AT, such as increases in infiltrated macrophages and the production of inflammatory cytokines [21]. Thus, the factors that "silence" the genes involved in the differentiation of preadipocytes, and, consequently, in the maintenance of adipose cells turnover in AT might play a central role in the genesis of the damaging changes (metabolism and function) that occur in the adipose tissue of individuals with cachexia.

### 1.2.4 Adipose tissue inflammatory profile in the course of cachexia

According to the abovementioned findings from animal models and cancer cachexia patients, a metabolic and morphological dysfunction that results in the AT remodeling occurs during the development of CC [2]. More recently, another relevant aspect of cachexia-induced AT remodeling is the establishment of AT inflammation, which is characterized by increased recruitment of ATMϕs, including activated M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophages [32]. In this way, an inflammatory profile predominates the end-stage of cachexia, particularly in visceral AT, as most of the characteristic changes of this syndrome are already established at this time [19, 27]. More recently, the immune cell infiltration profile was analyzed in greater detail, which was found to be characterized by an increase in M1-polarized macrophages [20]. In that study, the profile of chemokines specific to polymorphonuclear cells was also investigated, in addition to the presence of neutrophils in the various AT depots. The results revealed an increase in the chemokines CCL3 and CXCL2 at 7 days after tumor cell inoculation. The presence

of CD11b-positive cells, which were tested to detect the presence of neutrophils, was observed in the same period. Taking the temporal changes identified in the cachexia model into consideration, as a function of the assessed parameters, the results indicate that inflammation starts on Day 7 and is established by Day 14, a period during which a series of disorders characteristic of cachexia become evident (e.g., a reduction of the total body and fat mass, dyslipidemia, and hypoglycemia). In this regard, even more recently, these findings were presented in greater detail in cachexia induced by LLC cells, showing ATMϕs polarization tends to be directed to M1 phenotype [61, 62].

membrane type-1 matrix metalloproteinase (Mt1-Mmp) gene in the course of the mouse development results in the formation of a rigid collagen fibril chain and changes in vivo adipogenesis [68]. This condition is an example of the relationship

ECM remodeling plays a central role in the differentiation of adipocytes. Although the similar molecular mechanisms are only partially understood, ECM remodeling occurs concomitantly with the activation and/or repression of a transcriptional "network" involved in adipogenesis, which may be activated or repressed due to extracellular stimuli [69]. While some such alterations, such as changes in collagen content deposition, an increase in the number of infiltrated cells and insulin resistance, are also present in cancer-related cachexia, very little is known regarding the possible relationship between these cell types (e.g. fibroblasts, pre-adipocytes, immune cells, and others) and the processes that lead to ECM

In MAC16 tumor-bearing mice, a cancer cachexia animal model, shrunken adi-

pocytes and increased collagen-fibril content in AT were reported [18]. In CC patients, our group recently showed that the total type I collagen content of the scAT is rearranged in cachectic individuals with gastrointestinal cancer, which is associated with an increase in macrophage and lymphocyte contents. Interestingly, the total collagen content exhibited discrete changes in cancer patients without cachexia, but the expression of the Ccl2 gene was found to be increased [20]. Another exciting finding demonstrates that ECM remodeling of AT in cancer cachexia results in augmented collagen fiber content. Excessive synthesis of mature elastic fibers accompanies such morphological scenery, besides strong labeling for collagen type I (COL1) and III (COL3) in the AT from cachectic patients [30, 31]. Besides, the presence of fibrosis was also associated with an increased number of myofibroblasts and an activated TGFβ/SMAD pathway in the subcutaneous AT of

These findings indicate that the morphological changes that lead to AT remodeling in CC patients are evident, albeit discretely, before the onset of the earliest characteristic symptoms of cachexia (Figure 1). However, to the best of our knowledge, no study has yet investigated the causes and repercussions of fat

has recently been shown that cachexia induces browning of AT in addition to changes in immune-modulatory activity. In this scenario, chronic inflammation and β-adrenergic activation of thermogenesis functionally cooperate in the pathogenesis of cachexia [16, 33, 62, 71]. In general, browning of AT has been described as responsible for the increase in total caloric expenditure [72], and the induction of browning has therapeutic potential in promoting the reduction of body fat [16, 33, 34, 73]. However, this fact refers to conditions of diseases characterized by the presence of metabolic disorders, usually associated with high caloric intake, over-

Within the set of morphofunctional changes that result in the AT remodeling, it

In this sense, in CC, the presence of the browning phenotype has shown to be detected very early [16] in different experimental models [33, 34, 35]. Also, it has demonstrated a significant role in altering the metabolism of this tissue because this process is related to the increase in energy expenditure and mobilization of fatty acids [17] by adipocytes. Another interesting new fact was that, in this same study,

AT from cachectic mice showed upregulation of particular genes for brown

gastrointestinal cancer cachectic patients [31, 70].

remodeling in the course of cachexia in full detail.

weight, and/or obesity [74].

139

1.2.6 Browning of adipose tissue induced by cancer cachexia

between structural changes in ECM and adipocyte differentiation.

Adipose Tissue Remodeling during Cancer Cachexia DOI: http://dx.doi.org/10.5772/intechopen.79979

remodeling.

Despite the increasing perception of the importance of the relationship between inflammation and CC, and systemic inflammation in particular, there is still no consensus regarding its source and also the role of inflammation of TA in the establishment and development of cachexia, among cancer patients in particular [9, 43]. Limitations in experimental designs, the selection of control groups, and the techniques used to analyze markers of inflammation have most likely been responsible for preventing a more precise investigation of the presence of inflammation in AT. Addressing this question, a study has recently demonstrated increased gene expression of phenotypic markers of ATMϕs and inflammatory cytokines, such as IL-6 and TNF-α, in cachectic patients with gastrointestinal cancer [20]. Interestingly, increased gene levels of IL-6 were positively correlated to increase plasmatic levels of this cytokine, indicating that in cachectic patients, scAT may be an important source of inflammatory mediators. Even more recently, the same group revealed an increase in ATMϕs forming crown-like structures in the same AT depot from cachectic patients [44], which is a characteristic finding in fat tissue in experimental animal models of obesity and in obese patients [63]. In addition, an increase in chemoattractant for ATMϕs gene expression in scAT, such as Ccl2, was detected only in cancer patients without cachexia, showing no changes in cachectic ones. However, despite the relevance of local inflammation, in AT in particular, the mechanisms responsible for both cachexia and inflammation remain to be elucidated. The characterization and understanding of the process of inflammation in cachexia are also relevant to establish whether it is secondary to or the "trigger" for the development of cachexia syndrome.

### 1.2.5 Alterations in the extracellular matrix of adipose tissue

Extracellular matrix (ECM) remodeling is the result of the processes of matrix synthesis and degradation during which specific proteins are deposited, such as tenascin and fibrin, and occurs under both physiological (e.g., tissue repair) and pathological conditions (e.g., inflammation) [64]. The ECM consists of a complex network of multifunctional and structural molecules, including various collagen isoforms, adhesive glycoproteins, and proteoglycans. This network provides support to cells and to the signaling pathways that control their migration, proliferation, and differentiation. Also, the ECM might serve as a reservoir of cytokines and other growth factors, which are released into the system in variable amounts depending on the pathological condition.

ECM remodeling plays a central role in the differentiation of adipocytes. Although the corresponding molecular mechanisms are only partially understood, ECM remodeling occurs concomitantly with the activation and/or repression of a transcriptional "network" involved in adipogenesis, which may be activated or repressed due to extracellular stimuli [65]. In the 3 T3-L1 mouse cell line, the differentiation of adipocytes is associated with a reduction in the fibronectin-rich matrix and basal lamina formation [66, 67]. Silencing of the pericellular collagen

### Adipose Tissue Remodeling during Cancer Cachexia DOI: http://dx.doi.org/10.5772/intechopen.79979

of CD11b-positive cells, which were tested to detect the presence of neutrophils, was observed in the same period. Taking the temporal changes identified in the cachexia model into consideration, as a function of the assessed parameters, the results indicate that inflammation starts on Day 7 and is established by Day 14, a period during which a series of disorders characteristic of cachexia become evident (e.g., a reduction of the total body and fat mass, dyslipidemia, and hypoglycemia). In this regard, even more recently, these findings were presented in greater detail in cachexia induced by LLC cells, showing ATMϕs polarization tends to be directed to

Despite the increasing perception of the importance of the relationship between

inflammation and CC, and systemic inflammation in particular, there is still no consensus regarding its source and also the role of inflammation of TA in the establishment and development of cachexia, among cancer patients in particular [9, 43]. Limitations in experimental designs, the selection of control groups, and the techniques used to analyze markers of inflammation have most likely been responsible for preventing a more precise investigation of the presence of inflammation in AT. Addressing this question, a study has recently demonstrated increased gene expression of phenotypic markers of ATMϕs and inflammatory cytokines, such as IL-6 and TNF-α, in cachectic patients with gastrointestinal cancer [20]. Interestingly, increased gene levels of IL-6 were positively correlated to increase plasmatic levels of this cytokine, indicating that in cachectic patients, scAT may be an important source of inflammatory mediators. Even more recently, the same group revealed an increase in ATMϕs forming crown-like structures in the same AT depot from cachectic patients [44], which is a characteristic finding in fat tissue in experimental animal models of obesity and in obese patients [63]. In addition, an increase in chemoattractant for ATMϕs gene expression in scAT, such as Ccl2, was detected only in cancer patients without cachexia, showing no changes in cachectic ones. However, despite the relevance of local inflammation, in AT in particular, the mechanisms responsible for both cachexia and inflammation remain to be elucidated. The characterization and understanding of the process of inflammation in cachexia are also relevant to establish whether it is secondary to or the "trigger" for

M1 phenotype [61, 62].

Muscle Cells - Recent Advances and Future Perspectives

the development of cachexia syndrome.

depending on the pathological condition.

138

1.2.5 Alterations in the extracellular matrix of adipose tissue

Extracellular matrix (ECM) remodeling is the result of the processes of matrix synthesis and degradation during which specific proteins are deposited, such as tenascin and fibrin, and occurs under both physiological (e.g., tissue repair) and pathological conditions (e.g., inflammation) [64]. The ECM consists of a complex network of multifunctional and structural molecules, including various collagen isoforms, adhesive glycoproteins, and proteoglycans. This network provides support to cells and to the signaling pathways that control their migration, proliferation, and differentiation. Also, the ECM might serve as a reservoir of cytokines and other growth factors, which are released into the system in variable amounts

ECM remodeling plays a central role in the differentiation of adipocytes. Although the corresponding molecular mechanisms are only partially understood, ECM remodeling occurs concomitantly with the activation and/or repression of a transcriptional "network" involved in adipogenesis, which may be activated or repressed due to extracellular stimuli [65]. In the 3 T3-L1 mouse cell line, the differentiation of adipocytes is associated with a reduction in the fibronectin-rich matrix and basal lamina formation [66, 67]. Silencing of the pericellular collagen

membrane type-1 matrix metalloproteinase (Mt1-Mmp) gene in the course of the mouse development results in the formation of a rigid collagen fibril chain and changes in vivo adipogenesis [68]. This condition is an example of the relationship between structural changes in ECM and adipocyte differentiation.

ECM remodeling plays a central role in the differentiation of adipocytes. Although the similar molecular mechanisms are only partially understood, ECM remodeling occurs concomitantly with the activation and/or repression of a transcriptional "network" involved in adipogenesis, which may be activated or repressed due to extracellular stimuli [69]. While some such alterations, such as changes in collagen content deposition, an increase in the number of infiltrated cells and insulin resistance, are also present in cancer-related cachexia, very little is known regarding the possible relationship between these cell types (e.g. fibroblasts, pre-adipocytes, immune cells, and others) and the processes that lead to ECM remodeling.

In MAC16 tumor-bearing mice, a cancer cachexia animal model, shrunken adipocytes and increased collagen-fibril content in AT were reported [18]. In CC patients, our group recently showed that the total type I collagen content of the scAT is rearranged in cachectic individuals with gastrointestinal cancer, which is associated with an increase in macrophage and lymphocyte contents. Interestingly, the total collagen content exhibited discrete changes in cancer patients without cachexia, but the expression of the Ccl2 gene was found to be increased [20]. Another exciting finding demonstrates that ECM remodeling of AT in cancer cachexia results in augmented collagen fiber content. Excessive synthesis of mature elastic fibers accompanies such morphological scenery, besides strong labeling for collagen type I (COL1) and III (COL3) in the AT from cachectic patients [30, 31]. Besides, the presence of fibrosis was also associated with an increased number of myofibroblasts and an activated TGFβ/SMAD pathway in the subcutaneous AT of gastrointestinal cancer cachectic patients [31, 70].

These findings indicate that the morphological changes that lead to AT remodeling in CC patients are evident, albeit discretely, before the onset of the earliest characteristic symptoms of cachexia (Figure 1). However, to the best of our knowledge, no study has yet investigated the causes and repercussions of fat remodeling in the course of cachexia in full detail.

### 1.2.6 Browning of adipose tissue induced by cancer cachexia

Within the set of morphofunctional changes that result in the AT remodeling, it has recently been shown that cachexia induces browning of AT in addition to changes in immune-modulatory activity. In this scenario, chronic inflammation and β-adrenergic activation of thermogenesis functionally cooperate in the pathogenesis of cachexia [16, 33, 62, 71]. In general, browning of AT has been described as responsible for the increase in total caloric expenditure [72], and the induction of browning has therapeutic potential in promoting the reduction of body fat [16, 33, 34, 73]. However, this fact refers to conditions of diseases characterized by the presence of metabolic disorders, usually associated with high caloric intake, overweight, and/or obesity [74].

In this sense, in CC, the presence of the browning phenotype has shown to be detected very early [16] in different experimental models [33, 34, 35]. Also, it has demonstrated a significant role in altering the metabolism of this tissue because this process is related to the increase in energy expenditure and mobilization of fatty acids [17] by adipocytes. Another interesting new fact was that, in this same study, AT from cachectic mice showed upregulation of particular genes for brown

adipocytes when compared to samples of brown adipose tissue. This fact is not usual because the one would expect an increase in genes specific to beige adipocytes. Also, regarding thermogenesis, the rectal temperature was reduced when the main clinical signs of the diseases were already established (refractory cachexia). Interestingly, this hypothermic phenotype has previously been described in the Walker 256 tumor-induced cachexia model, in the final stage of cachexia [75]. On the other hand, additional studies are needed to clarify the immuno-metabolic changes resulting in thermogenic adjustments induced by the syndrome, as well as the particular clinical consequences.

Conflict of interest

The authors declare no conflicts of interest.

Adipose Tissue Remodeling during Cancer Cachexia DOI: http://dx.doi.org/10.5772/intechopen.79979

sTNFR soluble tumor necrosis factor-α receptor

Appendices and nomenclature

AT adipose tissue

IL interleukin

Ly lymphocytes

LPL lipoprotein lipase LLC Lewis lung carcinoma

TG triacylglycerol

ECM extracellular matrix ATGL adipose triglycerides lipase HSL hormone-sensitive lipase CC cancer-related cachexia

sIL-6R soluble IL-6 receptor IL-1RA IL-1 receptor antagonist

PIF proteolysis-inducing factor LMF lipid-mobilizing factor ZAG zinc-α2-glycoprotein AIS anemia-inducing substance scAT subcutaneous adipose tissue CD cluster of differentiation ATMϕs adipose tissue macrophage CD3 cluster of differentiation 3

HIV human immunodeficiency virus

NEFAs non-esterified fatty acids AMPK

GPAT glycerol-3-phosphate acetyltransferase aP2 adipocyte fatty acid-binding protein

UCP mitochondrial uncoupling proteins

ACC acetyl-CoA carboxylase FAS fatty acid synthase SCD-1 stearoyl-CoA desaturase-1

FATPs fatty acid transport proteins FABPs fatty acid-binding proteins

GLUT-4 glucose transport 4

141

M1 polarized macrophages 1 M2 polarized macrophages 2 CCL chemokine (C-C motif) ligand CXCL chemokine (C-X-C motif) ligand

K5-SOS-F keratinocyte-specific expression of an HA tagged dominant

3 T3-L1 embryo fibroblast cells with a continuous substrain (L1) of 3T3 (Swiss albino) developed through clonal isolation

form of the human SOS1

AMPK 5<sup>0</sup> adenosine monophosphate-activated protein CIDEA cell death-inducing DFFA-like effector a C/EBP CCAAT/enhancer-binding proteins

PPARγ peroxisome proliferator-activated receptor gamma SREBP-1C sterol regulatory element-binding protein-1C

In this same study [33], the browning phenotype has also been described in samples of visceral adipose tissue from cancer cachexia patients. However, there is a need for analysis in a larger cohort and additional characterizations about the possible physiological repercussions for these patients. Also, there is still a need to characterize the real contribution of AT browning to overall energy expenditure during cancer cachexia. In this sense, an elegant study has evaluated, in several experimental models, that although the studies above detected mild induction of Ucp1 mRNA levels in tumor-exposed AT, such changes appear to be discrete in thermogenic terms [53]. In this scenario, the overall effect of AT UCP1-dependent thermogenesis on systemic energy homeostasis may not be the principal actor during cancer cachexia.

### 2. Concluding remarks

In summary, several studies have shown that AT is significantly affected during the development of cachexia. The main alterations related to metabolic disorders, particularly those involving early adipocyte lipid turnover dysfunction of AT, increases in immune cell infiltration followed by increased local production of inflammatory mediators and remodeling of ECM components. More recently, some studies have shown that cachexia-induced browning of AT is a characteristic phenotype that arises from alterations that result in the AT remodeling, although its function is still not well characterized. Nevertheless, studies using those experimental models have consistently indicated that the modifications in the adipocyte metabolism begin quite early, and the metabolites derived from this process may be the initial (sterile) trigger of the sequence of events that result in the remodeling and consequent dysfunction of AT in cachexia. Finally, a deeper understanding of the initial stimulus that triggers AT dysfunction, in particular, inflammation and remodeling, needs to be further studied because evidence indicates that AT dysfunction plays a significant role in cachexia and may be a potential modulator of the process that could be explored therapeutically.

### Acknowledgements

We would like to thank all the members of Laboratory of Adipose Tissue Biology for helpful discussions and critical reading of the chapter. We would also like to thank Alexander H. Bedard for the revision of the chapter as a native of the English language. This study was supported by São Paulo Research Foundation (FAPESP) Grants: 2010/51078-1, 2015/19259-0, and CNPq 311966/2015-2 to MLB Jr. The contents of this chapter are solely the responsibility of the authors and do not necessarily represent the official views of FAPESP and CNPq.

Adipose Tissue Remodeling during Cancer Cachexia DOI: http://dx.doi.org/10.5772/intechopen.79979

### Conflict of interest

adipocytes when compared to samples of brown adipose tissue. This fact is not usual because the one would expect an increase in genes specific to beige adipocytes. Also, regarding thermogenesis, the rectal temperature was reduced when the main clinical signs of the diseases were already established (refractory cachexia). Interestingly, this hypothermic phenotype has previously been described in the Walker 256 tumor-induced cachexia model, in the final stage of cachexia [75]. On the other hand, additional studies are needed to clarify the immuno-metabolic changes resulting in thermogenic adjustments induced by the syndrome, as well as the

In this same study [33], the browning phenotype has also been described in samples of visceral adipose tissue from cancer cachexia patients. However, there is a need for analysis in a larger cohort and additional characterizations about the possible physiological repercussions for these patients. Also, there is still a need to characterize the real contribution of AT browning to overall energy expenditure during cancer cachexia. In this sense, an elegant study has evaluated, in several experimental models, that although the studies above detected mild induction of Ucp1 mRNA levels in tumor-exposed AT, such changes appear to be discrete in thermogenic terms [53]. In this scenario, the overall effect of AT UCP1-dependent thermogenesis on systemic energy homeostasis may not be the principal actor

In summary, several studies have shown that AT is significantly affected during the development of cachexia. The main alterations related to metabolic disorders, particularly those involving early adipocyte lipid turnover dysfunction of AT, increases in immune cell infiltration followed by increased local production of inflammatory mediators and remodeling of ECM components. More recently, some studies have shown that cachexia-induced browning of AT is a characteristic phenotype that arises from alterations that result in the AT remodeling, although its function is still not well characterized. Nevertheless, studies using those experimental models have consistently indicated that the modifications in the adipocyte metabolism begin quite early, and the metabolites derived from this process may be the initial (sterile) trigger of the sequence of events that result in the remodeling and consequent dysfunction of AT in cachexia. Finally, a deeper understanding of the initial stimulus that triggers AT dysfunction, in particular, inflammation and remodeling, needs to be further studied because evidence indicates that AT dysfunction plays a significant role in cachexia and may be a potential modulator of the

We would like to thank all the members of Laboratory of Adipose Tissue Biology for helpful discussions and critical reading of the chapter. We would also like to thank Alexander H. Bedard for the revision of the chapter as a native of the English language. This study was supported by São Paulo Research Foundation (FAPESP) Grants: 2010/51078-1, 2015/19259-0, and CNPq 311966/2015-2 to MLB Jr. The contents of this chapter are solely the responsibility of the authors and do not

particular clinical consequences.

Muscle Cells - Recent Advances and Future Perspectives

during cancer cachexia.

2. Concluding remarks

Acknowledgements

140

process that could be explored therapeutically.

necessarily represent the official views of FAPESP and CNPq.

The authors declare no conflicts of interest.

### Appendices and nomenclature


### Muscle Cells - Recent Advances and Future Perspectives


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Society. 2009;68:385-392

### Author details

Miguel Luiz Batista Júnior<sup>1</sup> \* and Felipe Henriques<sup>2</sup>

1 Laboratory of Adipose Tissue Biology, Integrated Group of Biotechnology, University of Mogi das Cruzes, Mogi das Cruzes, Brazil

2 Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA

\*Address all correspondence to: migueljr4@me.com

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

Adipose Tissue Remodeling during Cancer Cachexia DOI: http://dx.doi.org/10.5772/intechopen.79979

### References

MT1-MMP collagen membrane type-1 matrix metalloproteinase

SMAD small worm phenotype mothers against decapentaplegic

\* and Felipe Henriques<sup>2</sup>

1 Laboratory of Adipose Tissue Biology, Integrated Group of Biotechnology,

2 Program in Molecular Medicine, University of Massachusetts Medical School,

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

University of Mogi das Cruzes, Mogi das Cruzes, Brazil

\*Address all correspondence to: migueljr4@me.com

provided the original work is properly cited.

MAC16 murine adenocarcinoma 16 CCR2 (MCP-1) C-C chemokine receptor type 2

Muscle Cells - Recent Advances and Future Perspectives

TGFβ transforming growth factor beta

COL1 collagen type I COL3 collagen type III

Author details

Miguel Luiz Batista Júnior<sup>1</sup>

Worcester, MA, USA

142

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Section 4

Muscle Markers:

Immuno-Analysis

Section 4
