**Introduction**

**Chapter 1**

**Provisional chapter**

**Introductory Chapter: Current Status of Research Field**

**Introductory Chapter: Current Status of Research Field** 

Skeletal muscle tissue accounts for almost half of the human body mass. Muscle contractions of the skeletal muscle enable to move body and maintain homeostasis. Human health is markedly affected by any deterioration in the material, metabolic, and contractile properties of skeletal muscle. Skeletal muscle is a highly plastic organ that is modulated by various pathways controlling cell and protein turnover. Nowadays, the autophagy-dependent system and ubiquitin-proteasome signaling are well known as a major intracellular degradation system, and its appropriate function is crucial to health and muscle homeostasis. Indeed, muscle wasting and weakness such as cachexia, atrophy, and sarcopenia are characterized by marked decreases in the protein content, myonuclear number, muscle fiber size, and muscle strength [1]. Muscle wasting elicits a poor functional status and reduces the quality of life. Thirty-five percent of all cancer patients directly die because of cachexia and not from cancer. Different types of molecular triggers/catabolic factors such as pro-inflammatory cytokines and myostatin also seem to involve muscle wasting [2]. In contrast, mTOR- or serum response factor (SRF)-dependent signaling are positive regulators to promote protein synthesis and skeletal muscle-specific mRNA transcription. Interestingly, a functional defect in autophagy-dependent signaling in sarcopenic mice and humans are recently suggested [3, 4]. Such a condition accumulates the denaturing protein and nonfunctional mitochondria eventually result in the atrophy of sarcopenic muscle fibers because of the deterioration of

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

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

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

DOI: 10.5772/intechopen.79771

**in Muscle Tissue**

**in Muscle Tissue**

Additional information is available at the end of the chapter

Kunihiro SakumaAdditional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.79771

Kunihiro Sakuma

**1. Introduction**

homeostasis.

#### **Introductory Chapter: Current Status of Research Field in Muscle Tissue Introductory Chapter: Current Status of Research Field in Muscle Tissue**

DOI: 10.5772/intechopen.79771

### Kunihiro Sakuma

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

http://dx.doi.org/10.5772/intechopen.79771

### **1. Introduction**

Skeletal muscle tissue accounts for almost half of the human body mass. Muscle contractions of the skeletal muscle enable to move body and maintain homeostasis. Human health is markedly affected by any deterioration in the material, metabolic, and contractile properties of skeletal muscle. Skeletal muscle is a highly plastic organ that is modulated by various pathways controlling cell and protein turnover. Nowadays, the autophagy-dependent system and ubiquitin-proteasome signaling are well known as a major intracellular degradation system, and its appropriate function is crucial to health and muscle homeostasis. Indeed, muscle wasting and weakness such as cachexia, atrophy, and sarcopenia are characterized by marked decreases in the protein content, myonuclear number, muscle fiber size, and muscle strength [1]. Muscle wasting elicits a poor functional status and reduces the quality of life. Thirty-five percent of all cancer patients directly die because of cachexia and not from cancer. Different types of molecular triggers/catabolic factors such as pro-inflammatory cytokines and myostatin also seem to involve muscle wasting [2]. In contrast, mTOR- or serum response factor (SRF)-dependent signaling are positive regulators to promote protein synthesis and skeletal muscle-specific mRNA transcription. Interestingly, a functional defect in autophagy-dependent signaling in sarcopenic mice and humans are recently suggested [3, 4]. Such a condition accumulates the denaturing protein and nonfunctional mitochondria eventually result in the atrophy of sarcopenic muscle fibers because of the deterioration of homeostasis.

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

### **2. Various therapeutic approaches for muscle wasting**

To attenuate various forms of muscle wasting, many researchers have investigated exercisebased, supplemental, and pharmacological approaches. For example, the combination of resistance training and amino acid-containing supplements is thought to effectively prevent sarcopenia. In addition, myostatin inhibition for sarcopenic patients was successful in phase II trials, but the effect on muscular dystrophy is unclear. The administrations of ghrelin and megestrol acetate have shown good results against cancer cachexia [5]. Furthermore, recent studies [6, 7] indicated the possible application of novel supplements such as soy isoflavone and ursolic acid to prevent muscle atrophy in rodents. More recently, pharmacological treatment with fibroblast growth factor 19 markedly ameliorated two different type of muscle atrophy after aging and glucocorticoid treatment, probably via an obligate co-receptor for fibroblast growth factor 15/19, β-Klotho.

This book deals with current progress and perspectives in a variety topic of skeletal and smooth muscle, stem cells, growth, regeneration, disease, biomaterials, or therapeutics. Novel applications for cell and tissue engineering including cell therapy, tissue models, and disease pathology modeling are welcomed. The molecular mechanism of hypertrophy and atrophy in muscle cell would be also discussed by linking with the signal pathway of protein synthesis

Introductory Chapter: Current Status of Research Field in Muscle Tissue

http://dx.doi.org/10.5772/intechopen.79771

5

Institute for Liberal Arts, Environment and Society, Tokyo Institute of Technology, Tokyo,

[1] Zamboni M, Rossi AP, Corzato F, Bambace C, Mazzali G, Fantin F. Sarcopenia, cachexia and congestive heart failure in the elderly. Endocrine, Metabolic & Immune Disorders

[2] Sakuma K, Aoi W, Yamaguchi A. Molecular mechanism of sarcopenia and cachexia:

[3] Carnio S, LoVerso F, Baraibar MA, Longa E, Khan MM, Maffei M, Reischl M, Canepari M, Loefler S, Kern H, Blaauw B, Friguet B, Bottinelli R, Rudolf R, Sandri M. Autophagy impairment in muscle induces neuromuscular junction degeneration and precocious

[4] Sakuma K, Kinoshita M, Ito Y, Aizawa M, Aoi W, Yamaguchi A. p62/SQSTM1 but not LC3 is accumulated in sarcopenic muscle of mice. Journal of Cachexia, Sarcopenia and

[5] Temel JS, Abernethy AP, Currow DC, Friend J, Duus EM, Yan Y, Fearon KC. Anamorelin in patients with non-small-cell lung cancer and cachexia (ROMANA 1 and ROMANA 2): Results from two randomized, double-blind, phase 3 trials. The Lancet Oncology. 2016;

[6] Tabata S, Aizawa M, Kinoshita M, Ito Y, Kawamura Y, Takebe M, Pan W, Sakuma K. The influence of isoflavone for denervation-induced muscle atrophy. European Journal of

[7] Yu R, Chen JA, Xu J, Cao J, Wang Y, Thomas SS, Hu Z. Suppression of muscle wasting by the plant-derived compound ursolic acid in a model of chronic kidney disease. Journal

Recent research advances. Pflügers Archiv. 2017;**469**(5-6):573-591

Nutrition. in press. 2018. DOI: 10.1007/s00394-017-1593-x

of Cachexia, Sarcopenia and Muscle. 2017;**8**(2):327-341

and degradation.

**Author details**

Kunihiro Sakuma

Japan

**References**

Address all correspondence to: sakuma@ila.titech.ac.jp

Drug Targets. 2013;**13**(1):58-67

Muscle. 2016;**7**(2):204-212

**17**(4):519-531

aging. Cell Reports. 2014;**8**(5):1509-1521

### **3. The function of smooth muscle cells**

Our circulatory system is modulated of the heart, lungs, and vasculature. These components serve crucial roles in controlling blood and lymph flow and in the delivery of gases, hormone, and essential nutrients (i.e., glucose, fat, or amino acids). Vascular smooth muscle cells (VSMCs) are the most numerous cell types in blood vessels. They are located in the medial layer of the vascular wall, i.e., in the tunica media. The media also contains sparse fibroblasts and macrophages along with an interstitial matrix consisting collagens; chondroitin sulfate proteoglycans including versican; glycoproteins such as tenascin, vitronectin, and fibronectin; and elastic laminae. VSMCs serve critical regulatory roles of blood vessels, particularly for vasoconstriction, vasodilatation, and synthesis of vascular extracellular matrix. Adult blood vessels are normally contractile, static, and quiescent. However, under cardiovascular disease including atherosclerosis, hypertension, and diabetic angiopathy, VSMCs undergo phenotypic alterations and revert to a growth-promoting, synthetic nature. Indeed, after biochemical or mechanical damage to blood vessels, VSMCs undergo phenotypic modulation, characterized by increased proteosynthesis and by activation of the migration and growth of VSMCs [8, 9]. These changes often lead to severe damage to blood vessels, including stenosis and occlusion. Ischemia of the tissues supplied by the damaged vessels is then manifested by serious disorders, e.g., heart failure, brain stroke, or necrosis of leg tissues, which can result in amputation of the leg.

Vascular remodeling is an adaptive alternating process of vascular wall architecture and is caused by various stimuli such as vascular injury, oxidative stress, and hemodynamic stress [10]. VSMCs and endothelial cells compose the arteries and have essential roles in vascular remodeling in conjunction with inflammatory cells (macrophages, monocytes, leucocytes, and lymphocytes) [11]. During vascular remodeling, the infiltration of macrophages and monocytes, synthetic or contractile phenotypic changes of VSMCs, and the EC dysfunction promote vascular diseases such as atherosclerosis. Therefore, modulation of VSMC phenotype, maintenance of ECs, and regulation of inflammation in the vessel wall are important in arterial function and homeostasis.

This book deals with current progress and perspectives in a variety topic of skeletal and smooth muscle, stem cells, growth, regeneration, disease, biomaterials, or therapeutics. Novel applications for cell and tissue engineering including cell therapy, tissue models, and disease pathology modeling are welcomed. The molecular mechanism of hypertrophy and atrophy in muscle cell would be also discussed by linking with the signal pathway of protein synthesis and degradation.

## **Author details**

**2. Various therapeutic approaches for muscle wasting**

fibroblast growth factor 15/19, β-Klotho.

4 Muscle Cell and Tissue - Current Status of Research Field

arterial function and homeostasis.

**3. The function of smooth muscle cells**

To attenuate various forms of muscle wasting, many researchers have investigated exercisebased, supplemental, and pharmacological approaches. For example, the combination of resistance training and amino acid-containing supplements is thought to effectively prevent sarcopenia. In addition, myostatin inhibition for sarcopenic patients was successful in phase II trials, but the effect on muscular dystrophy is unclear. The administrations of ghrelin and megestrol acetate have shown good results against cancer cachexia [5]. Furthermore, recent studies [6, 7] indicated the possible application of novel supplements such as soy isoflavone and ursolic acid to prevent muscle atrophy in rodents. More recently, pharmacological treatment with fibroblast growth factor 19 markedly ameliorated two different type of muscle atrophy after aging and glucocorticoid treatment, probably via an obligate co-receptor for

Our circulatory system is modulated of the heart, lungs, and vasculature. These components serve crucial roles in controlling blood and lymph flow and in the delivery of gases, hormone, and essential nutrients (i.e., glucose, fat, or amino acids). Vascular smooth muscle cells (VSMCs) are the most numerous cell types in blood vessels. They are located in the medial layer of the vascular wall, i.e., in the tunica media. The media also contains sparse fibroblasts and macrophages along with an interstitial matrix consisting collagens; chondroitin sulfate proteoglycans including versican; glycoproteins such as tenascin, vitronectin, and fibronectin; and elastic laminae. VSMCs serve critical regulatory roles of blood vessels, particularly for vasoconstriction, vasodilatation, and synthesis of vascular extracellular matrix. Adult blood vessels are normally contractile, static, and quiescent. However, under cardiovascular disease including atherosclerosis, hypertension, and diabetic angiopathy, VSMCs undergo phenotypic alterations and revert to a growth-promoting, synthetic nature. Indeed, after biochemical or mechanical damage to blood vessels, VSMCs undergo phenotypic modulation, characterized by increased proteosynthesis and by activation of the migration and growth of VSMCs [8, 9]. These changes often lead to severe damage to blood vessels, including stenosis and occlusion. Ischemia of the tissues supplied by the damaged vessels is then manifested by serious disorders, e.g., heart failure, brain stroke, or necrosis of leg tissues, which can result in amputation of the leg.

Vascular remodeling is an adaptive alternating process of vascular wall architecture and is caused by various stimuli such as vascular injury, oxidative stress, and hemodynamic stress [10]. VSMCs and endothelial cells compose the arteries and have essential roles in vascular remodeling in conjunction with inflammatory cells (macrophages, monocytes, leucocytes, and lymphocytes) [11]. During vascular remodeling, the infiltration of macrophages and monocytes, synthetic or contractile phenotypic changes of VSMCs, and the EC dysfunction promote vascular diseases such as atherosclerosis. Therefore, modulation of VSMC phenotype, maintenance of ECs, and regulation of inflammation in the vessel wall are important in Kunihiro Sakuma

Address all correspondence to: sakuma@ila.titech.ac.jp

Institute for Liberal Arts, Environment and Society, Tokyo Institute of Technology, Tokyo, Japan

### **References**


[8] Schwartz SM, Campbell GR, Campbell JH. Replication of smooth muscle cells in vascular disease. Circulation Research. 1986;**58**(4):427-444

**Section 2**

**Plasticity of the Skeletal Muscle**


**Plasticity of the Skeletal Muscle**

[8] Schwartz SM, Campbell GR, Campbell JH. Replication of smooth muscle cells in vascu-

[9] Campbell JH, Campbell GR. Smooth muscle phenotypic modulation—A personal experience. Arteriosclerosis, Thrombosis, and Vascular Biology. 2012;**32**(8):1784-1789

[10] Salabei JK, Hill BG.Autophagic regulation of smooth muscle cell biology. Redox Biology.

[11] Nazari-Jahantigh M, Wei Y, Schober A. The role of microRNAs in arterial remodeling.

lar disease. Circulation Research. 1986;**58**(4):427-444

6 Muscle Cell and Tissue - Current Status of Research Field

Thrombosis and Haemostasis. 2012;**107**(4):611-618

2015;**4**:97-103

**Chapter 2**

Provisional chapter

**The Role of Glucose and Fatty Acid Metabolism in the**

DOI: 10.5772/intechopen.75904

The Role of Glucose and Fatty Acid Metabolism in the

**Development of Insulin Resistance in Skeletal Muscle**

The rapid rise in the prevalence of obesity and diabetes has significantly contributed to the increasing global burden of noncommunicable diseases. Insulin resistance is a major underpinning etiology of both obesity and type 2 diabetes. Insulin resistance is characterized by a reduced response of skeletal, liver, and fat tissues to the actions of insulin hormone. Although detailed mechanisms implicated in the development of insulin resistance remain plausible, skeletal muscles have been identified to play an integral role in the improvement of insulin sensitivity in the diseased state. The effective modulation of glucose and fatty acid metabolism in the skeletal muscle through exercise or by certain therapeutics has been associated with reversal of insulin resistance and amelioration of diabetes associated complications such as inflammation and oxidative stress. This chapter will briefly discuss the role of glucose and fatty acid metabolism in the development of

Keywords: skeletal muscle, insulin resistance, glucose and fatty acid metabolism

According to the World Health Organization, type 2 diabetes mellitus (T2D) contributes to approximately 90% of all diabetes mellitus cases and is amongst the top 10 leading causes of death worldwide [1]. Symptoms such as enhanced thirst, polyuria, fatigue, and impaired wound healing are identified in those with T2D. The recent International Diabetes Federation (IDF) report projects an astonishing increase in cases of diabetes [2]. An estimated 425 million

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

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

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

Development of Insulin Resistance in Skeletal Muscle

Sithandiwe Eunice Mazibuko-Mbeje,

Sithandiwe Eunice Mazibuko-Mbeje,

Nnini Obonye and Johan Louw

Nnini Obonye and Johan Louw

http://dx.doi.org/10.5772/intechopen.75904

Abstract

1. Introduction

Phiwayinkosi V. Dludla, Bongani B. Nkambule,

Phiwayinkosi V. Dludla, Bongani B. Nkambule,

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

insulin resistance in the skeletal muscle.

**Chapter 2** Provisional chapter

#### **The Role of Glucose and Fatty Acid Metabolism in the Development of Insulin Resistance in Skeletal Muscle** The Role of Glucose and Fatty Acid Metabolism in the Development of Insulin Resistance in Skeletal Muscle

DOI: 10.5772/intechopen.75904

Sithandiwe Eunice Mazibuko-Mbeje, Phiwayinkosi V. Dludla, Bongani B. Nkambule, Nnini Obonye and Johan Louw Sithandiwe Eunice Mazibuko-Mbeje, Phiwayinkosi V. Dludla, Bongani B. Nkambule, Nnini Obonye and Johan Louw

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

http://dx.doi.org/10.5772/intechopen.75904

#### Abstract

The rapid rise in the prevalence of obesity and diabetes has significantly contributed to the increasing global burden of noncommunicable diseases. Insulin resistance is a major underpinning etiology of both obesity and type 2 diabetes. Insulin resistance is characterized by a reduced response of skeletal, liver, and fat tissues to the actions of insulin hormone. Although detailed mechanisms implicated in the development of insulin resistance remain plausible, skeletal muscles have been identified to play an integral role in the improvement of insulin sensitivity in the diseased state. The effective modulation of glucose and fatty acid metabolism in the skeletal muscle through exercise or by certain therapeutics has been associated with reversal of insulin resistance and amelioration of diabetes associated complications such as inflammation and oxidative stress. This chapter will briefly discuss the role of glucose and fatty acid metabolism in the development of insulin resistance in the skeletal muscle.

Keywords: skeletal muscle, insulin resistance, glucose and fatty acid metabolism

### 1. Introduction

According to the World Health Organization, type 2 diabetes mellitus (T2D) contributes to approximately 90% of all diabetes mellitus cases and is amongst the top 10 leading causes of death worldwide [1]. Symptoms such as enhanced thirst, polyuria, fatigue, and impaired wound healing are identified in those with T2D. The recent International Diabetes Federation (IDF) report projects an astonishing increase in cases of diabetes [2]. An estimated 425 million

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

people are currently living with diabetes, with a projected 1.5 fold increase in the prevalence of diabetes, while a total of 629 million adults are expected to be diabetic by the year 2045 [2]. In Africa, the prevalence of diabetes is estimated at 16 million and is expected to rise to 41 million by 2045 [2]. Diabetes is characterized by hyperglycemia resulting from an inadequate production of insulin and insulin utilization in a T2D state. An unhealthy lifestyle such as lack of physical activity and a diet containing excessive fat content, including refined carbohydrates has been associated with an increased risk for developing insulin resistance (IR) [2, 3]. Carbohydrate-rich diets with a high glycemic index may contribute to obesity, impaired glucose tolerance, and hyperinsulinemia. This consequence can impair glucose and lipid metabolism, and accelerate the progression of IR. During a state of IR, insulin levels are elevated due to the rising glucose levels, but over time, a state of relative inadequate production of insulin can develop [2]. IR is regarded as one of the early phenotypes associated with development of obesity, and it is normally present in high-risk individual's years before development of T2D. Although maintenance of healthy lifestyle, as well as the use of hypolipidemic and hypoglycemic drugs, remains effective at attenuating insulin-resistant complications, the escalating incidence of the metabolic syndrome (MetS) warrants further exploration into pathological mechanisms implicated in the development of IR. Accumulative evidence suggests that effective modulation of energy substrates such as glucose and free fatty acids (FFAs) remains crucial in the amelioration of lifestyle diseases, including T2D [4–6]. This chapter will discuss the role of glucose and lipid metabolism in the development of IR.

32 molecules of ATP during the processes of glycolysis and oxidative phosphorylation. Glycolysis is the metabolic whereby glucose is metabolized into pyruvate or to lactate; this process yields higher capacity for ATP generation [12]. During glycolysis, glucose 6-phosphate is converted to fructose-6-phosphate by phosphohexose, and then to fructose-1,6-biphosphate by phosphofructokinase. This reaction is irreversible and is a major point of regulation during glycolysis. Energy utilization by adult skeletal muscle is tightly controlled, with muscle fibers having the ability to switch between different substrates for ATP production. This is highly dependent on the availability of energy substrates and the energy requirements [13, 14]. Skeletal muscles are able to utilize both glucose and FFAs as a source of ATP production. However, utilization of glucose and FFAs as a primary source of ATP production depends on the metabolic state of an individual, i.e., whether the individual is at a fed or fasting state [15]. During the fasting state, glucose uptake in skeletal muscle is reduced while plasma FFA levels are increased due to lipolysis in adipose tissue. This subsequently leads to the utilization of FFAs as the predominant source of ATP production [16]; whereas, during a fed state, plasma glucose levels are elevated, which stimulates insulin secretion and enhances glucose uptake by skeletal muscle. This also leads to reduced lipolysis in adipose tissue and a reduction in plasma FFAs. The ability of switching between substrates in the fasted and fed state is crucial in promoting skeletal muscle glucose oxidation. Consequently, it has been reported that muscle of insulin-resistant or diabetic subjects fails to switch between the substrates, showing metabolic inflexibility [8]. This metabolic inflexibility can result in impaired glucose and fatty acid

The Role of Glucose and Fatty Acid Metabolism in the Development of Insulin Resistance in Skeletal Muscle

http://dx.doi.org/10.5772/intechopen.75904

11

Several mechanisms, which include glucose transportation, are implicated in the regulation of skeletal muscle glucose metabolism and have been a therapeutic target for the reversal IR and improvement of skeletal muscle function. Briefly, postprandial glucose is transported actively across the plasma membrane by specific carrier proteins, which belong to the glucose transporter (GLUT) family. There are several types of glucose transporters located in the plasma membrane of myocytes. Each glucose transporter isoform plays a specific role in glucose transportation that is determined by its tissue distribution, substrate specificity, and transport kinetics [17]. Glucose transporter isoform 1 (GLUT1) is present in all cells and is largely responsible for regulating basal glucose and ensuring a steady influx of glucose into cells. The glucose transporter isoform 2 (GLUT2) is a high-Km glucose transporter expressed in hepatocytes, pancreatic beta cells, and the basolateral membranes of intestinal and renal epithelial cells. In contrast to other transporters, GLUT2 facilitates bidirectional glucose transport into and out of the cell [17]. GLUT3 is a low-capacity glucose transporter that is responsible for glucose uptake in neurons, while glucose transporter protein isoform 4 (GLUT4) is expressed exclusively in muscle and fat cells, and is responsible for increased glucose uptake into these

In skeletal muscle, insulin stimulation induces translocation of GLUT4 from intracellular vesicles within the cytoplasm to the plasma membrane and thereby increases glucose uptake [18, 19]. When insulin levels decrease in the blood and insulin receptors are no longer occupied, the glucose transporters are recycled back into the cytoplasm. Failure of GLUT4 to translocate to the plasma membrane results in IR [20]. The crucial step for effective modulation of GLUT4 translocation has been the binding of insulin or insulin-like growth factor 1 (IGF1) to its receptor or IGF1 receptor, leading to the activation of phosphatidylinositol-4,5-bisphosphate

metabolism, leading to the development of IR.

tissues postprandially, thereby maintaining normoglycemia [17].

### 2. Skeletal muscle and its role in modulating insulin resistance

Skeletal muscles are comprised of an intricate tissue, with diverse network of fibers, which have different mechanical and metabolic functions. Skeletal muscles contribute to approximately 40% of the total body weight and contain 50–75% of all body proteins [7]. Skeletal muscles account for more than 80% of insulin-stimulated glucose uptake [8], and using combined oral and intravenous glucose tolerance testing, Himsworth and Kerr were able to demonstrate that tissue-specific insulin sensitivity was lower in T2D individuals [9]. Therefore, IR in skeletal muscle has a major impact on whole-body metabolic homeostasis and it is the main element for the development of T2D. However, the underlying molecular mechanisms remain elusive. Several mechanisms that play a role in the development of IR in skeletal muscle have been proposed, and these include accumulation of intracellular lipid derivatives (diacylglycerol and ceramides) as a result of elevated plasma FFAs, oxidative stress, pro-inflammatory signals, and impaired gene transcription [8, 10]. Moreover, mitochondrial dysfunction has associated with IR [11]. The following section will focus on glucose regulation and fatty acid metabolism, in relation to the development of IR in skeletal muscle.

#### 3. Glucose metabolism in skeletal muscle

Glucose is a monosaccharide used as a biological fuel during aerobic and anaerobic respiration or fermentation. Aerobic respiration is the most efficient means of glucose utilization, yielding 32 molecules of ATP during the processes of glycolysis and oxidative phosphorylation. Glycolysis is the metabolic whereby glucose is metabolized into pyruvate or to lactate; this process yields higher capacity for ATP generation [12]. During glycolysis, glucose 6-phosphate is converted to fructose-6-phosphate by phosphohexose, and then to fructose-1,6-biphosphate by phosphofructokinase. This reaction is irreversible and is a major point of regulation during glycolysis. Energy utilization by adult skeletal muscle is tightly controlled, with muscle fibers having the ability to switch between different substrates for ATP production. This is highly dependent on the availability of energy substrates and the energy requirements [13, 14]. Skeletal muscles are able to utilize both glucose and FFAs as a source of ATP production. However, utilization of glucose and FFAs as a primary source of ATP production depends on the metabolic state of an individual, i.e., whether the individual is at a fed or fasting state [15]. During the fasting state, glucose uptake in skeletal muscle is reduced while plasma FFA levels are increased due to lipolysis in adipose tissue. This subsequently leads to the utilization of FFAs as the predominant source of ATP production [16]; whereas, during a fed state, plasma glucose levels are elevated, which stimulates insulin secretion and enhances glucose uptake by skeletal muscle. This also leads to reduced lipolysis in adipose tissue and a reduction in plasma FFAs. The ability of switching between substrates in the fasted and fed state is crucial in promoting skeletal muscle glucose oxidation. Consequently, it has been reported that muscle of insulin-resistant or diabetic subjects fails to switch between the substrates, showing metabolic inflexibility [8]. This metabolic inflexibility can result in impaired glucose and fatty acid metabolism, leading to the development of IR.

people are currently living with diabetes, with a projected 1.5 fold increase in the prevalence of diabetes, while a total of 629 million adults are expected to be diabetic by the year 2045 [2]. In Africa, the prevalence of diabetes is estimated at 16 million and is expected to rise to 41 million by 2045 [2]. Diabetes is characterized by hyperglycemia resulting from an inadequate production of insulin and insulin utilization in a T2D state. An unhealthy lifestyle such as lack of physical activity and a diet containing excessive fat content, including refined carbohydrates has been associated with an increased risk for developing insulin resistance (IR) [2, 3]. Carbohydrate-rich diets with a high glycemic index may contribute to obesity, impaired glucose tolerance, and hyperinsulinemia. This consequence can impair glucose and lipid metabolism, and accelerate the progression of IR. During a state of IR, insulin levels are elevated due to the rising glucose levels, but over time, a state of relative inadequate production of insulin can develop [2]. IR is regarded as one of the early phenotypes associated with development of obesity, and it is normally present in high-risk individual's years before development of T2D. Although maintenance of healthy lifestyle, as well as the use of hypolipidemic and hypoglycemic drugs, remains effective at attenuating insulin-resistant complications, the escalating incidence of the metabolic syndrome (MetS) warrants further exploration into pathological mechanisms implicated in the development of IR. Accumulative evidence suggests that effective modulation of energy substrates such as glucose and free fatty acids (FFAs) remains crucial in the amelioration of lifestyle diseases, including T2D [4–6]. This chapter will discuss the role of glucose and lipid metabolism in the development of IR.

10 Muscle Cell and Tissue - Current Status of Research Field

2. Skeletal muscle and its role in modulating insulin resistance

relation to the development of IR in skeletal muscle.

3. Glucose metabolism in skeletal muscle

Skeletal muscles are comprised of an intricate tissue, with diverse network of fibers, which have different mechanical and metabolic functions. Skeletal muscles contribute to approximately 40% of the total body weight and contain 50–75% of all body proteins [7]. Skeletal muscles account for more than 80% of insulin-stimulated glucose uptake [8], and using combined oral and intravenous glucose tolerance testing, Himsworth and Kerr were able to demonstrate that tissue-specific insulin sensitivity was lower in T2D individuals [9]. Therefore, IR in skeletal muscle has a major impact on whole-body metabolic homeostasis and it is the main element for the development of T2D. However, the underlying molecular mechanisms remain elusive. Several mechanisms that play a role in the development of IR in skeletal muscle have been proposed, and these include accumulation of intracellular lipid derivatives (diacylglycerol and ceramides) as a result of elevated plasma FFAs, oxidative stress, pro-inflammatory signals, and impaired gene transcription [8, 10]. Moreover, mitochondrial dysfunction has associated with IR [11]. The following section will focus on glucose regulation and fatty acid metabolism, in

Glucose is a monosaccharide used as a biological fuel during aerobic and anaerobic respiration or fermentation. Aerobic respiration is the most efficient means of glucose utilization, yielding Several mechanisms, which include glucose transportation, are implicated in the regulation of skeletal muscle glucose metabolism and have been a therapeutic target for the reversal IR and improvement of skeletal muscle function. Briefly, postprandial glucose is transported actively across the plasma membrane by specific carrier proteins, which belong to the glucose transporter (GLUT) family. There are several types of glucose transporters located in the plasma membrane of myocytes. Each glucose transporter isoform plays a specific role in glucose transportation that is determined by its tissue distribution, substrate specificity, and transport kinetics [17]. Glucose transporter isoform 1 (GLUT1) is present in all cells and is largely responsible for regulating basal glucose and ensuring a steady influx of glucose into cells. The glucose transporter isoform 2 (GLUT2) is a high-Km glucose transporter expressed in hepatocytes, pancreatic beta cells, and the basolateral membranes of intestinal and renal epithelial cells. In contrast to other transporters, GLUT2 facilitates bidirectional glucose transport into and out of the cell [17]. GLUT3 is a low-capacity glucose transporter that is responsible for glucose uptake in neurons, while glucose transporter protein isoform 4 (GLUT4) is expressed exclusively in muscle and fat cells, and is responsible for increased glucose uptake into these tissues postprandially, thereby maintaining normoglycemia [17].

In skeletal muscle, insulin stimulation induces translocation of GLUT4 from intracellular vesicles within the cytoplasm to the plasma membrane and thereby increases glucose uptake [18, 19]. When insulin levels decrease in the blood and insulin receptors are no longer occupied, the glucose transporters are recycled back into the cytoplasm. Failure of GLUT4 to translocate to the plasma membrane results in IR [20]. The crucial step for effective modulation of GLUT4 translocation has been the binding of insulin or insulin-like growth factor 1 (IGF1) to its receptor or IGF1 receptor, leading to the activation of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT) pathway. Activation of this pathway has been subject to ongoing research for its role in skeletal muscle tissue growth, and most importantly, in the regulation of insulin signaling [21]. This has been verified on various models showing that knockout of insulin receptor, PI3K and AKT genes, especially in skeletal muscle, is associated with growth retardation as well as with impairment of insulin action [22, 23]. Therefore, effective modulation of glucose transportation and activation of PI3K/AKT pathway remains important to improve glucose tolerance and also skeletal muscle function.

### 4. Fatty acid metabolism in skeletal muscle

FFAs are elongated hydrocarbon chains with a terminal carboxylate group. Apart from being one of the major sources of fuel in the body, FFAs can perform a number of other functions, including serving as building blocks for phospholipids and also acting as hormones as well as intracellular messengers [24]. FFAs can exist in unsaturated or saturated form, depending on the number of bonds the hydrocarbon chain contains. While unsaturated fats such as oleic acid, mostly available in vegetable oils, are considered beneficial to the body [25], saturated fats, namely palmitic acid, are associated with the development of IR [26]. For the latter, it is widely used in experimental models to induce IR [19, 27]. Exposure of cultured skeletal muscle cells to high palmitate concentrations has been linked with the activation of protein kinase C (PKC), one of the main enzymes involved in impaired insulin signaling [26, 28]. Briefly, by phosphorylating insulin receptor substrate 1, PKC can alter the whole downstream effect of insulin response, ultimately leading to impaired GLUT4 translocation and reduced glucose uptake in skeletal muscle. Evidence shows that PKC activation by 12-deoxyphorbol 13 phenylacetate 20-acetate is associated with a reduction in insulin-stimulated glucose uptake, whereas PKC inhibition with GF 109203X results in enhanced insulin action in cultured human skeletal muscle [29]. An abnormal reduction of glucose uptake in skeletal muscle, mainly due to an impaired switch in substrate preference, as explained by Randle [6], remains an important contributing factor to the development of IR and subsequent metabolic complications. Thus, it is a viable option to target glucose uptake improvement, concomitant to reducing glycogen stores to reverse IR in skeletal muscle.

Some polyphenols, including those from grape extracts, can influence muscle lipid metabolism by reducing CD36 and regulating CPT1 expression in high fat diet (HFD) fed rats, leading to upregulated GLUT4 protein expression and improved insulin signaling [33]. Similarly, metformin, a commonly used antidiabetic drug, has demonstrated increased capacity to reverse IR and improve skeletal muscle function through the modulation of CPT1 and 5<sup>0</sup> AMP-activated protein kinase (AMPK) [34, 35]. Like PI3K/AKT, AMPK is also a target of ongoing research for its role in preventing metabolic disease through modulation of substrate metabolism in various tissues [36, 37]. Intracellular energy fluctuations, represented by changing AMP/ATP ratio, such as those identified in an IR state, remain monumental for the activation or deactivation of AMPK activity [38]. A number of natural products [39–41], including metformin, are known to activate AMPK, leading to the phosphorylation of acetyl-CoA carboxylase and to effective modulation of beta oxidation. However, the activity of AMPK is tissue specific and is tightly controlled in a T2D state, with its activation demonstrated to be important in reversing IR and

isoform 4, GO: glucose oxidation, IR: insulin receptor, and PKC: protein kinase C.

Figure 1. Glucose and free fatty acids (FFAs) are the predominant substrates that are oxidized to generate acetyl-CoA, which is then utilized by the electron transport chain to generate adenosine triphosphate (ATP). An insulin-resistant state is characterized by an impaired substrate utilization, a process termed metabolic inflexibility. Modulation of phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/AKT) and 5<sup>0</sup> AMP-activated protein kinase (AMPK) signaling mechanisms are increasingly targeted by various pharmacological compounds to reverse insulin resistance and improve skeletal muscle function. Abbreviations: CD36: cluster of differentiation 36, CPT1: carnitine palmitoyltransferase 1, DAG: diacyl glycerol, FAO: fatty acid oxidation, FATP: fatty acid transport protein 1, GLUT4: glucose transporter

The Role of Glucose and Fatty Acid Metabolism in the Development of Insulin Resistance in Skeletal Muscle

http://dx.doi.org/10.5772/intechopen.75904

13

Skeletal muscle forms the largest insulin-sensitive tissue in the body and remains the key site for insulin-stimulated glucose uptake. Glucose and FFAs are the prominent substrates

improving signaling in skeletal muscle [19, 36].

5. Conclusions

Beta oxidation, the main catabolic process by which fats are broken down in the body, is another system crucial for the control of substrate switch within many cells, including skeletal muscle (Figure 1). Generally, during periods of fasting, a substrate switch occurs where FFAs become a predominant source for ATP production via beta oxidation [30]. Although FFAs are hydrophobic in nature and can passively diffuse across the lipophilic cell membrane, transporters such as plasma membrane fatty acid-binding protein (FABP), fatty acid transport protein 1 (FATP1), and cluster of differentiation 36 (CD36) are widely expressed in rodent and human skeletal muscle [31, 32]. By controlling entry of long chain fatty acids across the barrier of the inner mitochondrial membrane for subsequent beta oxidation, the carnitine shuttle system can influence skeletal muscle substrate switch. Some of the well-investigated components of the system include the malonyl-CoA sensitive carnitine palmitoyltransferase 1 (CPT1) that resides on the mitochondrial outer membrane (Figure 1).

The Role of Glucose and Fatty Acid Metabolism in the Development of Insulin Resistance in Skeletal Muscle http://dx.doi.org/10.5772/intechopen.75904 13

Figure 1. Glucose and free fatty acids (FFAs) are the predominant substrates that are oxidized to generate acetyl-CoA, which is then utilized by the electron transport chain to generate adenosine triphosphate (ATP). An insulin-resistant state is characterized by an impaired substrate utilization, a process termed metabolic inflexibility. Modulation of phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/AKT) and 5<sup>0</sup> AMP-activated protein kinase (AMPK) signaling mechanisms are increasingly targeted by various pharmacological compounds to reverse insulin resistance and improve skeletal muscle function. Abbreviations: CD36: cluster of differentiation 36, CPT1: carnitine palmitoyltransferase 1, DAG: diacyl glycerol, FAO: fatty acid oxidation, FATP: fatty acid transport protein 1, GLUT4: glucose transporter isoform 4, GO: glucose oxidation, IR: insulin receptor, and PKC: protein kinase C.

Some polyphenols, including those from grape extracts, can influence muscle lipid metabolism by reducing CD36 and regulating CPT1 expression in high fat diet (HFD) fed rats, leading to upregulated GLUT4 protein expression and improved insulin signaling [33]. Similarly, metformin, a commonly used antidiabetic drug, has demonstrated increased capacity to reverse IR and improve skeletal muscle function through the modulation of CPT1 and 5<sup>0</sup> AMP-activated protein kinase (AMPK) [34, 35]. Like PI3K/AKT, AMPK is also a target of ongoing research for its role in preventing metabolic disease through modulation of substrate metabolism in various tissues [36, 37]. Intracellular energy fluctuations, represented by changing AMP/ATP ratio, such as those identified in an IR state, remain monumental for the activation or deactivation of AMPK activity [38]. A number of natural products [39–41], including metformin, are known to activate AMPK, leading to the phosphorylation of acetyl-CoA carboxylase and to effective modulation of beta oxidation. However, the activity of AMPK is tissue specific and is tightly controlled in a T2D state, with its activation demonstrated to be important in reversing IR and improving signaling in skeletal muscle [19, 36].

### 5. Conclusions

3-kinase (PI3K)/protein kinase B (AKT) pathway. Activation of this pathway has been subject to ongoing research for its role in skeletal muscle tissue growth, and most importantly, in the regulation of insulin signaling [21]. This has been verified on various models showing that knockout of insulin receptor, PI3K and AKT genes, especially in skeletal muscle, is associated with growth retardation as well as with impairment of insulin action [22, 23]. Therefore, effective modulation of glucose transportation and activation of PI3K/AKT pathway remains

FFAs are elongated hydrocarbon chains with a terminal carboxylate group. Apart from being one of the major sources of fuel in the body, FFAs can perform a number of other functions, including serving as building blocks for phospholipids and also acting as hormones as well as intracellular messengers [24]. FFAs can exist in unsaturated or saturated form, depending on the number of bonds the hydrocarbon chain contains. While unsaturated fats such as oleic acid, mostly available in vegetable oils, are considered beneficial to the body [25], saturated fats, namely palmitic acid, are associated with the development of IR [26]. For the latter, it is widely used in experimental models to induce IR [19, 27]. Exposure of cultured skeletal muscle cells to high palmitate concentrations has been linked with the activation of protein kinase C (PKC), one of the main enzymes involved in impaired insulin signaling [26, 28]. Briefly, by phosphorylating insulin receptor substrate 1, PKC can alter the whole downstream effect of insulin response, ultimately leading to impaired GLUT4 translocation and reduced glucose uptake in skeletal muscle. Evidence shows that PKC activation by 12-deoxyphorbol 13 phenylacetate 20-acetate is associated with a reduction in insulin-stimulated glucose uptake, whereas PKC inhibition with GF 109203X results in enhanced insulin action in cultured human skeletal muscle [29]. An abnormal reduction of glucose uptake in skeletal muscle, mainly due to an impaired switch in substrate preference, as explained by Randle [6], remains an important contributing factor to the development of IR and subsequent metabolic complications. Thus, it is a viable option to target glucose uptake improvement, concomitant to reducing

Beta oxidation, the main catabolic process by which fats are broken down in the body, is another system crucial for the control of substrate switch within many cells, including skeletal muscle (Figure 1). Generally, during periods of fasting, a substrate switch occurs where FFAs become a predominant source for ATP production via beta oxidation [30]. Although FFAs are hydrophobic in nature and can passively diffuse across the lipophilic cell membrane, transporters such as plasma membrane fatty acid-binding protein (FABP), fatty acid transport protein 1 (FATP1), and cluster of differentiation 36 (CD36) are widely expressed in rodent and human skeletal muscle [31, 32]. By controlling entry of long chain fatty acids across the barrier of the inner mitochondrial membrane for subsequent beta oxidation, the carnitine shuttle system can influence skeletal muscle substrate switch. Some of the well-investigated components of the system include the malonyl-CoA sensitive carnitine palmitoyltransferase 1

important to improve glucose tolerance and also skeletal muscle function.

4. Fatty acid metabolism in skeletal muscle

12 Muscle Cell and Tissue - Current Status of Research Field

glycogen stores to reverse IR in skeletal muscle.

(CPT1) that resides on the mitochondrial outer membrane (Figure 1).

Skeletal muscle forms the largest insulin-sensitive tissue in the body and remains the key site for insulin-stimulated glucose uptake. Glucose and FFAs are the prominent substrates responsible for ATP production in the skeletal muscle. However, in an insulin-resistant state, the utilization of both glucose and FFAs is impaired, leading to abnormally enhanced intramuscular substrate storage. Modulation of the PI3K/AKT and AMPK signaling appears to be the driving mechanism responsible for the regulation of substrate metabolism, as well as associated downstream effects such as generation of oxidative stress. Interestingly, some pharmacological compounds such as metformin are known to exert their therapeutic effects through the modulation of these pathways, leading to improved control of energy substrates. Therefore, it is imperative that more research be directed at exploring signaling mechanisms implicated in the control of energy substrates, especially in the skeletal muscle, since it is known to be the major "hub" for energy metabolism.

GLUT glucose transporter

IR insulin resistance

PKC protein kinase C

MetS metabolic syndrome

T2D type 2 diabetes mellitus

UCP uncoupling protein

Nnini Obonye1 and Johan Louw1,4

Author details

Tygerberg, South Africa

Tygerberg, South Africa

South Africa

References

IDF International Diabetes Federation

PI3K phosphatidylinositol-4,5-bisphosphate 3-kinase

Sithandiwe Eunice Mazibuko-Mbeje1,2\*, Phiwayinkosi V. Dludla1

\*Address all correspondence to: sithandiwe.mazibuko@mrc.ac.za

University of KwaZulu-Natal, Durban, South Africa

ua=1 [Accessed: 30 January 2018]

1 Biomedical Research and Innovation Platform, South African Medical Research Council,

The Role of Glucose and Fatty Acid Metabolism in the Development of Insulin Resistance in Skeletal Muscle

3 School of Laboratory Medicine and Medical Sciences (SLMMS), College of Health Sciences,

4 Department of Biochemistry and Microbiology, University of Zululand, KwaDlangezwa,

[1] World Health Organizatio. The Top 10 Causes of Death. 2017. Available from: http://

[2] International Diabetes Federation. IDF Diabetes Atlas Eighth Edition. Available from: http://www.diabetesatlas.org/resources/2017-atlas.html [Accessed: 30 January 2018]

[3] World Health Organization. Global Status Report on Noncommunicable Diseases 2014. Available from: http://apps.who.int/iris/bitstream/10665/148114/1/9789241564854\_eng.pdf?

www.who.int/mediacentre/factsheets/fs310/en/ [Accessed: 31 January 2018]

2 Division of Medical Physiology, Faculty of Health Sciences, Stellenbosch University,

, Bongani B. Nkambule<sup>3</sup>

http://dx.doi.org/10.5772/intechopen.75904

,

15

IGF-1 insulin-like growth factor 1

HFD high fat diet

### Acknowledgements

This work was supported by the Biomedical Research and Innovation Platform of the South African Medical Research Council (SAMRC) baseline funding and the South African National Research Foundation (NRF: grant number 87836 to SE Mazibuko-Mbeje). The grant holders acknowledge that opinions, findings, and conclusions or recommendations expressed in any publication generated by the SAMRC or NRF supported research are those of the authors, and that the SAMRC and NRF accept no liability whatsoever in this regard. PVD was partially supported as a postdoctoral fellow by funding from the SAMRC.

### Conflict of interest

The authors report no conflict of interest. All authors are responsible for the content and writing of the paper.

### Abbreviations



### Author details

responsible for ATP production in the skeletal muscle. However, in an insulin-resistant state, the utilization of both glucose and FFAs is impaired, leading to abnormally enhanced intramuscular substrate storage. Modulation of the PI3K/AKT and AMPK signaling appears to be the driving mechanism responsible for the regulation of substrate metabolism, as well as associated downstream effects such as generation of oxidative stress. Interestingly, some pharmacological compounds such as metformin are known to exert their therapeutic effects through the modulation of these pathways, leading to improved control of energy substrates. Therefore, it is imperative that more research be directed at exploring signaling mechanisms implicated in the control of energy substrates, especially in the skeletal muscle, since it is known to be the major

This work was supported by the Biomedical Research and Innovation Platform of the South African Medical Research Council (SAMRC) baseline funding and the South African National Research Foundation (NRF: grant number 87836 to SE Mazibuko-Mbeje). The grant holders acknowledge that opinions, findings, and conclusions or recommendations expressed in any publication generated by the SAMRC or NRF supported research are those of the authors, and that the SAMRC and NRF accept no liability whatsoever in this regard. PVD was partially

The authors report no conflict of interest. All authors are responsible for the content and

supported as a postdoctoral fellow by funding from the SAMRC.

"hub" for energy metabolism.

14 Muscle Cell and Tissue - Current Status of Research Field

Acknowledgements

Conflict of interest

writing of the paper.

Abbreviations

AKT protein kinase B

FFAs free fatty acids

AMPK 5<sup>0</sup> AMP-activated protein kinase

CPT1 carnitine palmitoyltransferase 1

ATP adenosine triphosphate

CD36 cluster of differentiation 36

FABP fatty acid binding protein

FATP1 fatty acid transport protein 1

Sithandiwe Eunice Mazibuko-Mbeje1,2\*, Phiwayinkosi V. Dludla1 , Bongani B. Nkambule<sup>3</sup> , Nnini Obonye1 and Johan Louw1,4

\*Address all correspondence to: sithandiwe.mazibuko@mrc.ac.za

1 Biomedical Research and Innovation Platform, South African Medical Research Council, Tygerberg, South Africa

2 Division of Medical Physiology, Faculty of Health Sciences, Stellenbosch University, Tygerberg, South Africa

3 School of Laboratory Medicine and Medical Sciences (SLMMS), College of Health Sciences, University of KwaZulu-Natal, Durban, South Africa

4 Department of Biochemistry and Microbiology, University of Zululand, KwaDlangezwa, South Africa

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**Chapter 3**

**Provisional chapter**

**Molecular and Cellular Markers in Skeletal Muscle**

**Molecular and Cellular Markers in Skeletal Muscle** 

DOI: 10.5772/intechopen.76384

**Damage after Acute Voluntary Exercise Containing**

**Damage after Acute Voluntary Exercise Containing** 

In eccentric muscle contraction, the muscle is lengthening while contracting. For example, in downhill walking, the thigh muscles are contracting eccentrically. It is well known that unaccustomed eccentric exercise causes pain and may lead to inflammation reactions on muscles few days after the exercise. The theme of the present chapter is molecular and cellular markers in skeletal muscle damage after voluntary exercise containing eccentric muscle contractions. The chapter contains three topics: In the first topic, the damaging process followed by regeneration is demonstrated with antibody stainings of connective tissue, plasma membrane, and cytoskeletal proteins. The second topic is infiltration of inflammatory cells in damaged skeletal muscle. Neutrophils are usually the first inflammatory cells mostly present in the injured tissues; however, neutrophils are not present in exercise-induced skeletal muscle damage. Finally, the relationship between skeletal muscle damage and systematic markers, serum creatine kinase and voluntary maximal

**Keywords:** skeletal muscle damage, eccentric exercise, infiltration of inflammatory cells,

In movements such as walking, running, and jumping, both eccentric (the muscle is actively lengthened/stretched) and concentric (the muscle is actively shortened) muscle contractions are present as the muscles undergo repeated stretching-shortening cycles. [1] Typical physical activities, which contain a great deal of eccentric contractions, are going down stairs, walking

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

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

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

**Eccentric Muscle Contractions**

**Eccentric Muscle Contractions**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Satu O.A. Koskinen and Maarit Lehti

Satu O.A. Koskinen and Maarit Lehti

http://dx.doi.org/10.5772/intechopen.76384

force production, is described.

neutrophils, monocytes/macrophages, extracellular matrix

**Abstract**

**1. Introduction**


#### **Molecular and Cellular Markers in Skeletal Muscle Damage after Acute Voluntary Exercise Containing Eccentric Muscle Contractions Molecular and Cellular Markers in Skeletal Muscle Damage after Acute Voluntary Exercise Containing Eccentric Muscle Contractions**

DOI: 10.5772/intechopen.76384

Satu O.A. Koskinen and Maarit Lehti Satu O.A. Koskinen and Maarit Lehti

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

http://dx.doi.org/10.5772/intechopen.76384

#### **Abstract**

[35] Collier CA et al. Metformin counters the insulin-induced suppression of fatty acid oxidation and stimulation of triacylglycerol storage in rodent skeletal muscle. American Journal

[36] Mihaylova MM, Shaw RJ. The AMPK signalling pathway coordinates cell growth,

[37] Heidrich F et al. AMPK—Activated protein kinase and its role in energy metabolism of the

[38] Zungu M et al. Regulation of AMPK by the ubiquitin proteasome system. The American

[39] Ahn J et al. The anti-obesity effect of quercetin is mediated by the AMPK and MAPK signaling pathways. Biochemical and Biophysical Research Communications. 2008;373(4):

[40] Dugdale HF, et al. The role of resveratrol on skeletal muscle cell differentiation and myo-

[41] Johnson R et al. Aspalathin, a dihydrochalcone C-glucoside, protects H9c2 cardiomyocytes against high glucose induced shifts in substrate preference and apoptosis. Molecular

of Physiology. Endocrinology and Metabolism. 2006;291(1):E182-E189

autophagy and metabolism. Nature Cell Biology. 2011;13(9):1016-1023

tube hypertrophy during glucose restriction. 2017;444(1-2):109-123

heart. Current Cardiology Reviews. 2010;6(4):337-342

Journal of Pathology. 2011;178(1):4-11

18 Muscle Cell and Tissue - Current Status of Research Field

Nutrition & Food Research. 2016;60(4):922-934

545-549

In eccentric muscle contraction, the muscle is lengthening while contracting. For example, in downhill walking, the thigh muscles are contracting eccentrically. It is well known that unaccustomed eccentric exercise causes pain and may lead to inflammation reactions on muscles few days after the exercise. The theme of the present chapter is molecular and cellular markers in skeletal muscle damage after voluntary exercise containing eccentric muscle contractions. The chapter contains three topics: In the first topic, the damaging process followed by regeneration is demonstrated with antibody stainings of connective tissue, plasma membrane, and cytoskeletal proteins. The second topic is infiltration of inflammatory cells in damaged skeletal muscle. Neutrophils are usually the first inflammatory cells mostly present in the injured tissues; however, neutrophils are not present in exercise-induced skeletal muscle damage. Finally, the relationship between skeletal muscle damage and systematic markers, serum creatine kinase and voluntary maximal force production, is described.

**Keywords:** skeletal muscle damage, eccentric exercise, infiltration of inflammatory cells, neutrophils, monocytes/macrophages, extracellular matrix

### **1. Introduction**

In movements such as walking, running, and jumping, both eccentric (the muscle is actively lengthened/stretched) and concentric (the muscle is actively shortened) muscle contractions are present as the muscles undergo repeated stretching-shortening cycles. [1] Typical physical activities, which contain a great deal of eccentric contractions, are going down stairs, walking

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

and running downhill, lowering weights, and the downward motion of squats and push-ups. It is fair to say that muscles contracting eccentrically produce "more for less." High mechanical muscle tension produced by eccentric muscle contraction is generated at lower metabolic cost and at a greatly reduced oxygen requirement compared to concentric muscle contraction. It has been reported that the oxygen requirement of submaximal eccentric cycling is only 1/6– 1/7 of that for concentric cycling at the same workload [2]. Furthermore, eccentric training can increase the size and strength of muscles with very little demand on the cardiovascular system [3]. Therefore, eccentric training is potential training method for elderly and patients suffering from diseases that limit either the uptake or delivery of oxygen, e.g., chronic obstructive pulmonary disease or chronic heart failure. Any exercise that requires a significant increase in respiration and in cardiac output may not be only uncomfortable but also impossible for fragile individuals. However, it is not only fragile individuals who benefit from eccentric training: It is advantageous for anyone since less time is needed and training feels less strenuous compared to concentric type of training. As a disadvantage, unaccustomed eccentric exercise causes muscle pain and may lead to inflammation reaction on muscles for few days postexercise. Likely, one bout of eccentric exercise induces protective effect against muscle pain and against skeletal muscle fiber injury for several weeks or even months [4].

muscle damage and systematic markers, serum creatine kinase and voluntary maximal force

Molecular and Cellular Markers in Skeletal Muscle Damage after Acute Voluntary Exercise…

http://dx.doi.org/10.5772/intechopen.76384

21

The first changes in the sarcoplasm of skeletal muscle fibers occur within few hours after the damaging exercise such as myofibrillar loss (**Figure 1D**). Furthermore, accumulation of mitochondria, local loss of myofilaments, Z line streaming (**Figure 1E**), swelling of mitochondria, disconnection of plasma membrane, swelling of sarcoplasmic reticulum, infiltration of inflammatory cells, complete loss of plasma membrane (**Figure 1F**), and disrupted muscle fibers may appear depending on how severe the damage is. In the next subsections, antibody stainings for HSP27, dystrophin, and type IV collagen are given as an example for visualizing changes after eccentric exercise in sarcoplasm, plasma membrane, and extracellular matrix,

In general, HSPs are considered to be the cellular protein quality control machinery. They can stabilize proteins during cellular damage, contribute to protein folding during increased protein synthesis, and protect proteins from aggregation. HSP27 can interact with actin and with many actin-binding proteins such as tropomyosin and troponin T [10]. HSP27 immunostainings in longitudinal sections of unexercised skeletal muscle fibers appear as fine lines (**Figure 2A**) indicating that HSP27 is localized to the Z-disks and/or I-band [11]. Immediately and 3 h after the exercise, in this case continuous drop jumping unilaterally on a sledge apparatus with a submaximal height until complete exhaustion, HSP27 immunostainings showed intensively stained and variable-sized clusters in both cross-sectional and longitudinal sections of skeletal muscle fiber HSP27 (**Figure 2C–F**) [12]. These stained clusters were probably formed due to translocation and accumulation of HSP27 on cytoskeletal/myofibril-

Dystrophin is part of dystrophin protein complex, which transmits force laterally from contractile filaments to extracellular matrix through sarcolemma. Dystrophin is located beneath the sarcolemma, and it strengthens muscle fibers and protects them from injury. Immediately after forced lengthening contractions, the immunostaining of antibody against C-terminus of dystrophin fades out or disappeared partially before immunostaining of antibody against rod domain from few fibers in muscle sections from rat tibialis anterior muscle (**Figure 3**). This sequence of structural disturbance after eccentric exercise is interesting in relation to recent finding of new membrane-binding domain in dystrophin C-terminus [13]. In addition, proteinase-resistant regions in the rod domain of dystrophin make it more resistant against

**2. Molecular markers for exercise-induced skeletal muscle damage**

production, is described.

respectively.

lar structures [11].

degradation.

**2.1. Sarcoplasm of the skeletal muscle**

**2.2. Plasma membrane of the skeletal muscle**

Maximal eccentric exercise with the knee extensors or elbow flexors on an isokinetic dynamometer has frequently been used to induce and to study skeletal muscle damage in humans. A typical response to such high-force, single-joint eccentric exercise protocols is on average a 50% reduction of the force-generating capacity immediately post exercise, followed by gradual recovery over the next days or weeks [5]. There are clearly individual differences both in force-generating capacity immediately post exercise and in the length of the force recovery period [6, 7]. It is not known why the same amount of eccentric exercise induces skeletal muscle fiber damage, loss in force-generating capacity, and prolonged force recovery period for some individuals, whereas only short-term decrease in muscle force-generating capacity was observed in other subjects [6]. Prolonged recovery of muscle force is thought to be related to distortion of the myofibrillar structure and disturbed calcium homoeostasis and/or prolonged inflammatory response [5]. Consequently, it has been suggested that reduction in muscle force-generating capacity may be a valuable indicator for monitoring muscle damage following exercise [8]. Physiological adaptation takes place after a single bout of unaccustomed eccentric exercise by making muscles more resistant against structural changes from the second bout of eccentric exercise [9].

In the next sections, few examples are presented for visualizing skeletal muscle fiber injury in muscle biopsies after eccentric exercise. Heat shock protein 27 (HSP27) antibody staining shows abnormal sarcoplasmic staining pattern already during the first hour after the exercise. Plasma membrane protein (dystrophin)-negative skeletal muscle fiber indicates quite severe muscle damage, while basement membrane proteins laminin and type IV collagen are intact and are keeping the muscle fiber together. Counting the number of infiltrated inflammatory cells in a damaged skeletal muscle is often reported after different exercise protocols. However, antibodies used for counting neutrophils and monocytes/macrophages do not always exclusively recognize the studied leukocyte. Finally, the relationship between skeletal muscle damage and systematic markers, serum creatine kinase and voluntary maximal force production, is described.

### **2. Molecular markers for exercise-induced skeletal muscle damage**

The first changes in the sarcoplasm of skeletal muscle fibers occur within few hours after the damaging exercise such as myofibrillar loss (**Figure 1D**). Furthermore, accumulation of mitochondria, local loss of myofilaments, Z line streaming (**Figure 1E**), swelling of mitochondria, disconnection of plasma membrane, swelling of sarcoplasmic reticulum, infiltration of inflammatory cells, complete loss of plasma membrane (**Figure 1F**), and disrupted muscle fibers may appear depending on how severe the damage is. In the next subsections, antibody stainings for HSP27, dystrophin, and type IV collagen are given as an example for visualizing changes after eccentric exercise in sarcoplasm, plasma membrane, and extracellular matrix, respectively.

#### **2.1. Sarcoplasm of the skeletal muscle**

and running downhill, lowering weights, and the downward motion of squats and push-ups. It is fair to say that muscles contracting eccentrically produce "more for less." High mechanical muscle tension produced by eccentric muscle contraction is generated at lower metabolic cost and at a greatly reduced oxygen requirement compared to concentric muscle contraction. It has been reported that the oxygen requirement of submaximal eccentric cycling is only 1/6– 1/7 of that for concentric cycling at the same workload [2]. Furthermore, eccentric training can increase the size and strength of muscles with very little demand on the cardiovascular system [3]. Therefore, eccentric training is potential training method for elderly and patients suffering from diseases that limit either the uptake or delivery of oxygen, e.g., chronic obstructive pulmonary disease or chronic heart failure. Any exercise that requires a significant increase in respiration and in cardiac output may not be only uncomfortable but also impossible for fragile individuals. However, it is not only fragile individuals who benefit from eccentric training: It is advantageous for anyone since less time is needed and training feels less strenuous compared to concentric type of training. As a disadvantage, unaccustomed eccentric exercise causes muscle pain and may lead to inflammation reaction on muscles for few days postexercise. Likely, one bout of eccentric exercise induces protective effect against muscle pain

and against skeletal muscle fiber injury for several weeks or even months [4].

the second bout of eccentric exercise [9].

20 Muscle Cell and Tissue - Current Status of Research Field

Maximal eccentric exercise with the knee extensors or elbow flexors on an isokinetic dynamometer has frequently been used to induce and to study skeletal muscle damage in humans. A typical response to such high-force, single-joint eccentric exercise protocols is on average a 50% reduction of the force-generating capacity immediately post exercise, followed by gradual recovery over the next days or weeks [5]. There are clearly individual differences both in force-generating capacity immediately post exercise and in the length of the force recovery period [6, 7]. It is not known why the same amount of eccentric exercise induces skeletal muscle fiber damage, loss in force-generating capacity, and prolonged force recovery period for some individuals, whereas only short-term decrease in muscle force-generating capacity was observed in other subjects [6]. Prolonged recovery of muscle force is thought to be related to distortion of the myofibrillar structure and disturbed calcium homoeostasis and/or prolonged inflammatory response [5]. Consequently, it has been suggested that reduction in muscle force-generating capacity may be a valuable indicator for monitoring muscle damage following exercise [8]. Physiological adaptation takes place after a single bout of unaccustomed eccentric exercise by making muscles more resistant against structural changes from

In the next sections, few examples are presented for visualizing skeletal muscle fiber injury in muscle biopsies after eccentric exercise. Heat shock protein 27 (HSP27) antibody staining shows abnormal sarcoplasmic staining pattern already during the first hour after the exercise. Plasma membrane protein (dystrophin)-negative skeletal muscle fiber indicates quite severe muscle damage, while basement membrane proteins laminin and type IV collagen are intact and are keeping the muscle fiber together. Counting the number of infiltrated inflammatory cells in a damaged skeletal muscle is often reported after different exercise protocols. However, antibodies used for counting neutrophils and monocytes/macrophages do not always exclusively recognize the studied leukocyte. Finally, the relationship between skeletal In general, HSPs are considered to be the cellular protein quality control machinery. They can stabilize proteins during cellular damage, contribute to protein folding during increased protein synthesis, and protect proteins from aggregation. HSP27 can interact with actin and with many actin-binding proteins such as tropomyosin and troponin T [10]. HSP27 immunostainings in longitudinal sections of unexercised skeletal muscle fibers appear as fine lines (**Figure 2A**) indicating that HSP27 is localized to the Z-disks and/or I-band [11]. Immediately and 3 h after the exercise, in this case continuous drop jumping unilaterally on a sledge apparatus with a submaximal height until complete exhaustion, HSP27 immunostainings showed intensively stained and variable-sized clusters in both cross-sectional and longitudinal sections of skeletal muscle fiber HSP27 (**Figure 2C–F**) [12]. These stained clusters were probably formed due to translocation and accumulation of HSP27 on cytoskeletal/myofibrillar structures [11].

#### **2.2. Plasma membrane of the skeletal muscle**

Dystrophin is part of dystrophin protein complex, which transmits force laterally from contractile filaments to extracellular matrix through sarcolemma. Dystrophin is located beneath the sarcolemma, and it strengthens muscle fibers and protects them from injury. Immediately after forced lengthening contractions, the immunostaining of antibody against C-terminus of dystrophin fades out or disappeared partially before immunostaining of antibody against rod domain from few fibers in muscle sections from rat tibialis anterior muscle (**Figure 3**). This sequence of structural disturbance after eccentric exercise is interesting in relation to recent finding of new membrane-binding domain in dystrophin C-terminus [13]. In addition, proteinase-resistant regions in the rod domain of dystrophin make it more resistant against degradation.

of skeletal muscle fibers (see **Figure 1C**, small gray dots between muscle fibers), whereas nonfibrillar type IV collagen is in basement membranes of skeletal muscle fibers (see **Figure 1C**, small black arrow) and capillaries. Type IV collagen is present in swollen, necrotic (**Figure 4**), and regenerated fibers, similarly as in undamaged skeletal muscle fibers. This suggests that basement membrane including type IV collagen holds on skeletal muscle fiber during the process of fiber damage, when the cytoskeleton is disrupted, contractile proteins are disorganized, and

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The first structural changes appear within few hours after eccentric exercise in sarcoplasm. This can be visualized using, for example, HSP27 immunostainings, which can be seen as intensively stained and variable-sized clusters (**Figure 2C–F**). During the next few days after the damaging exercise, antibody stainings for dystrophin fade out or disappear partially (**Figure 3**) and in severe damaged skeletal muscle fibers disappear totally (**Figure 4B**), whereas basement membrane including type IV collagen serves as a supportive structure

**Figure 2.** HSP27 (red) immunostaining as a marker for muscle damage. In biopsies obtained from human vastus lateralis before the exercise, no HSP27-stained clusters were observed (A and B). HSP27 was localized to the Z-disks of skeletal muscle fibers. After the exercise, intensively stained and variably sized clusters of HSP27 were observed immediately (C–E) and 3 hours post exercise (F). Dystrophin (green) immunostaining was used to visualize the borders of muscle

fibers and DAPI (blue) stained nuclei. Published in Koskinen et al. [12].

**2.4. What to remember about molecular markers for exercise-induced skeletal** 

during skeletal muscle fiber injury (**Figure 4A**) and regeneration.

inflammatory cells are infiltrated [14].

**muscle damage**

**Figure 1.** Electron micrograph of longitudinal sections of epoxy-embedded human skeletal muscle biopsy from vastus lateralis. (A–C) Non-exercised muscles. Skeletal muscle fiber (long two-headed arrow), endomysium (space between the two small arrows facing each other), myonuclei/satellite cell (m), capillary (c), plasma membrane is the fine dark line (white small arrow), basement membrane is the thick gray line (black small arrow). (D–F) Eccentric exercised muscle. (D) Five hours after the exercise, some myofibrils are lost in skeletal muscle fiber (lower left corner). (E) One day after the exercise, Z line smearing. (F) Eight days after the exercise, only basement membrane can be seen in damaged skeletal muscle fiber (upper small black arrow). Cell between two muscle fibers is probably fibroblast containing rough endoplasmic reticulum.

#### **2.3. Basement membrane of the skeletal muscle**

The extracellular matrix provides mechanical support for skeletal muscle fibers and plays an important role in force transmission. Fibrillar type I and III collagen are present in endomysium of skeletal muscle fibers (see **Figure 1C**, small gray dots between muscle fibers), whereas nonfibrillar type IV collagen is in basement membranes of skeletal muscle fibers (see **Figure 1C**, small black arrow) and capillaries. Type IV collagen is present in swollen, necrotic (**Figure 4**), and regenerated fibers, similarly as in undamaged skeletal muscle fibers. This suggests that basement membrane including type IV collagen holds on skeletal muscle fiber during the process of fiber damage, when the cytoskeleton is disrupted, contractile proteins are disorganized, and inflammatory cells are infiltrated [14].

### **2.4. What to remember about molecular markers for exercise-induced skeletal muscle damage**

The first structural changes appear within few hours after eccentric exercise in sarcoplasm. This can be visualized using, for example, HSP27 immunostainings, which can be seen as intensively stained and variable-sized clusters (**Figure 2C–F**). During the next few days after the damaging exercise, antibody stainings for dystrophin fade out or disappear partially (**Figure 3**) and in severe damaged skeletal muscle fibers disappear totally (**Figure 4B**), whereas basement membrane including type IV collagen serves as a supportive structure during skeletal muscle fiber injury (**Figure 4A**) and regeneration.

**Figure 2.** HSP27 (red) immunostaining as a marker for muscle damage. In biopsies obtained from human vastus lateralis before the exercise, no HSP27-stained clusters were observed (A and B). HSP27 was localized to the Z-disks of skeletal muscle fibers. After the exercise, intensively stained and variably sized clusters of HSP27 were observed immediately (C–E) and 3 hours post exercise (F). Dystrophin (green) immunostaining was used to visualize the borders of muscle fibers and DAPI (blue) stained nuclei. Published in Koskinen et al. [12].

**2.3. Basement membrane of the skeletal muscle**

22 Muscle Cell and Tissue - Current Status of Research Field

The extracellular matrix provides mechanical support for skeletal muscle fibers and plays an important role in force transmission. Fibrillar type I and III collagen are present in endomysium

**Figure 1.** Electron micrograph of longitudinal sections of epoxy-embedded human skeletal muscle biopsy from vastus lateralis. (A–C) Non-exercised muscles. Skeletal muscle fiber (long two-headed arrow), endomysium (space between the two small arrows facing each other), myonuclei/satellite cell (m), capillary (c), plasma membrane is the fine dark line (white small arrow), basement membrane is the thick gray line (black small arrow). (D–F) Eccentric exercised muscle. (D) Five hours after the exercise, some myofibrils are lost in skeletal muscle fiber (lower left corner). (E) One day after the exercise, Z line smearing. (F) Eight days after the exercise, only basement membrane can be seen in damaged skeletal muscle fiber (upper small black arrow). Cell between two muscle fibers is probably fibroblast containing rough endoplasmic reticulum.

after damaging exercise (e.g., see [15–20]). It is often concluded that the inflammatory cell reaction in skeletal muscle fiber injury caused by unaccustomed eccentric exercise is initiated by infiltration of neutrophils. However, the antibodies for CD11b, CD16, neutrophil elastase, and myeloperoxidase recognize also other leukocytes than neutrophils in leukocyte blood smears, whereas antibody for CD66b recognized only neutrophils (**Figure 5**) [6]. Therefore, the CD66b antibody is more suitable for detecting neutrophils in skeletal muscle sections than antibody for CD11b, CD16, myeloperoxidase, and neutrophil elastase. The CD68 antibody, the marker for monocytes/macrophages, recognized monocytes/macrophages and a portion of cells with bilobed nuclei (basophils or eosinophils) on leukocyte blood smears. In skeletal muscle biopsies after eccentric exercise, the CD68 antibody recognized more cell types than

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**Figure 5.** Immunostaining of CD11b, CD16, CD66b, CD68, myeloperoxidase, and neutrophil elastase antibodies on circulating leukocytes extracted from whole blood. Secondary antibody for CD66b Alexa Fluor 488 anti-mouse; for CD11b, CD16, CD68, and neutrophil elastase Alexa Fluor 594 anti-mouse; and for myeloperoxidase Alexa Fluor 594 anti-

rabbit. Scale bar of 20 μm. Published in Paulsen et al. [6].

monocytes/macrophages [6].

**Figure 3.** Immunostaining of antibody against C-terminus dystrophin (green) faded out or disappeared partially from individual muscle fiber (rat tibialis anterior muscle immediately after forced lengthening contractions), before than the staining for antibody against rod domain of dystrophin (red) that was still partially as bright as in non-exercised muscles.

**Figure 4.** Injured skeletal muscle fiber in rat tibialis anterior muscle 2 days after forced lengthening contractions. Type IV collagen immunohistochemical staining is visible in the borders of basement membranes of skeletal muscle fibers (A), while dystrophin staining is negative (B). Infiltration of inflammatory cells inside the damaged fiber (D, hematoxylin– eosin staining). Bar = 25 μm. Published in Koskinen et al. [14].

### **3. Inflammatory cell markers in exercise-induced skeletal muscle damage**

Immunostainings of leukocyte markers for neutrophils (CD11b, CD16, CD66b, neutrophil elastase, and myeloperoxidase) and for monocytes/macrophages (CD68) have been applied for localizing and counting the number of these inflammatory cells in human skeletal muscles after damaging exercise (e.g., see [15–20]). It is often concluded that the inflammatory cell reaction in skeletal muscle fiber injury caused by unaccustomed eccentric exercise is initiated by infiltration of neutrophils. However, the antibodies for CD11b, CD16, neutrophil elastase, and myeloperoxidase recognize also other leukocytes than neutrophils in leukocyte blood smears, whereas antibody for CD66b recognized only neutrophils (**Figure 5**) [6]. Therefore, the CD66b antibody is more suitable for detecting neutrophils in skeletal muscle sections than antibody for CD11b, CD16, myeloperoxidase, and neutrophil elastase. The CD68 antibody, the marker for monocytes/macrophages, recognized monocytes/macrophages and a portion of cells with bilobed nuclei (basophils or eosinophils) on leukocyte blood smears. In skeletal muscle biopsies after eccentric exercise, the CD68 antibody recognized more cell types than monocytes/macrophages [6].

**Figure 5.** Immunostaining of CD11b, CD16, CD66b, CD68, myeloperoxidase, and neutrophil elastase antibodies on circulating leukocytes extracted from whole blood. Secondary antibody for CD66b Alexa Fluor 488 anti-mouse; for CD11b, CD16, CD68, and neutrophil elastase Alexa Fluor 594 anti-mouse; and for myeloperoxidase Alexa Fluor 594 antirabbit. Scale bar of 20 μm. Published in Paulsen et al. [6].

**3. Inflammatory cell markers in exercise-induced skeletal muscle** 

eosin staining). Bar = 25 μm. Published in Koskinen et al. [14].

24 Muscle Cell and Tissue - Current Status of Research Field

Immunostainings of leukocyte markers for neutrophils (CD11b, CD16, CD66b, neutrophil elastase, and myeloperoxidase) and for monocytes/macrophages (CD68) have been applied for localizing and counting the number of these inflammatory cells in human skeletal muscles

**Figure 4.** Injured skeletal muscle fiber in rat tibialis anterior muscle 2 days after forced lengthening contractions. Type IV collagen immunohistochemical staining is visible in the borders of basement membranes of skeletal muscle fibers (A), while dystrophin staining is negative (B). Infiltration of inflammatory cells inside the damaged fiber (D, hematoxylin–

**Figure 3.** Immunostaining of antibody against C-terminus dystrophin (green) faded out or disappeared partially from individual muscle fiber (rat tibialis anterior muscle immediately after forced lengthening contractions), before than the staining for antibody against rod domain of dystrophin (red) that was still partially as bright as in non-exercised muscles.

**damage**

#### **3.1. Neutrophils in damaged skeletal muscle**

The number of CD66b-positive cells is very low in both exercised and non-exercised skeletal muscles [6]. As an example, only 34 of 122 biopsies from eccentrically exercised and non-exercised human biceps brachii muscles contained CD66b-positive cells. A closer examination showed that in 23 of these biopsies, the CD66b stained cells (varied from 1 to 4 CD66bpositive cells per 100 muscle fibers) were located inside capillaries or vessels (**Figure 6B**), attached to the wall of the vessels, or were detected in blood clots (**Figure 6A**). In the remaining 11 biopsies, single CD66b-positive cells (varied from 1 to 30 CD66b-positive cells per 100 muscle fibers) were observed in the endomysium of affected muscle fibers or in the sarcoplasm of damaged fibers. There was no consistent pattern regarding how CD66b-positive cells were distributed between exercised and non-exercised samples or between different time points. These results indicated that neutrophils were not involved in exercise-induced skeletal muscle fiber injury. Unusual high numbers of CD66b stained cells located in the endomysium or inside fibers may indicate trauma from the previous biopsy.

showed that the sarcoplasmic CD68 immunostaining observed in single cells aligned next to the laminin (**Figure 7B** arrow) was most likely satellite cells (in electron microscopy pictures, these cells were located between the plasma membrane and the basement membrane of skeletal muscle fibers). Furthermore, CD68-positive cells with long extensions located in endomysium (**Figure 7B** arrowhead) were probably fibroblasts or myofibroblasts. According to the observation from electron microcopy pictures, these types of cells contained prominent rough endoplasmic reticulum indicating cells with high capacity for protein synthesis such as fibroblasts/myofibroblasts. Both satellite cells and fibroblasts were most likely involved in the skeletal muscle adaptation to increased mechanical loading. The number of CD68-positive cells is not optimal to be applied as

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Neutrophils are not involved in exercise-induced skeletal muscle fiber injury, although in other injured tissues neutrophils are usually the first inflammatory cells present. CD66b antibody seems to exclusively recognize neutrophils both in leukocyte blood smears and skeletal muscle biopsies. Numerous monocytes/macrophages are present in damaged skeletal muscle fibers, typically totally occupying the entire sarcoplasm of damaged fibers. CD68 antibody is not optimal to be applied as a quantitative value for monocytes/macrophages, because it

**Figure 7.** CD68 (red), laminin (basement membrane, green), and DAPI (nuclei, blue) staining on eccentric exercised human skeletal muscle. (A) Damaged skeletal muscle fiber infiltrated by CD68 stained cells (asterisk). In the endomysium, CD68 stained cells seemed to form a chain around necrotic fiber (arrow heads). (B) CD68 stained cells with long extensions in sarcoplasm (arrow) and in the endomysium (arrow head). (C) CD68 stained cell (arrow heads) inside capillary. (D)

Vessel surrounded by CD68 stained cells. Scale bar of 20 μm. Published in Paulsen et al. [6].

a quantitative value for monocytes/macrophages in human skeletal muscles.

recognizes also satellite cells and fibroblasts in skeletal muscle biopsies.

**3.3. What to remember about inflammatory cells in damaged skeletal muscle**

#### **3.2. Monocytes/macrophages in damaged skeletal muscle**

CD68-positive cell counts are widely used for indication of monocyte/macrophage infiltration in skeletal muscle biopsies after single bout of eccentric exercise (e.g., see [19]). However, it has been shown that the CD68 antibody recognized more cell types than monocytes/macrophages in human skeletal muscle biopsies after eccentric exercise [6]. The highest individual CD68 positive cell counts were related to skeletal muscle fiber injury, which was observed in exercised biopsies at 4 and 7 days after acute eccentric exercise. In these biopsies, CD68-positive cells typically occupied the entire sarcoplasm of damaged skeletal muscle fibers (**Figure 7A**). Therefore, monocytes/macrophages were probably the most prominent CD68-positive cell type in these biopsies. In addition, CD68-positive cells inside capillaries (**Figure 7C**) and vessels (**Figure 7D**) were most likely monocytes/macrophages. In the exercised biopsies without damaged skeletal muscle fibers, the determination of the monocytes/macrophages proportion of CD68-positive cells is not straightforward. Comparison of cells with similar appearance and location between light microscopy pictures of CD68-positive cells and transmission electron microscopy pictures

**Figure 6.** The nuclei of neutrophils are divided in few parts, which makes them easily recognizable. (A) Semi-thin toluidine blue stained section contains a cluster of red blood cells and single neutrophil (non-exercised skeletal muscle biopsy from human biceps brachii muscle). (B) Leukocytes inside a blood vessel (electron micrograph of epoxy-embedded exercised skeletal muscle biopsy from biceps brachii muscle). Endothelial cell (E), neutrophil (Nf), red blood cell (R).

showed that the sarcoplasmic CD68 immunostaining observed in single cells aligned next to the laminin (**Figure 7B** arrow) was most likely satellite cells (in electron microscopy pictures, these cells were located between the plasma membrane and the basement membrane of skeletal muscle fibers). Furthermore, CD68-positive cells with long extensions located in endomysium (**Figure 7B** arrowhead) were probably fibroblasts or myofibroblasts. According to the observation from electron microcopy pictures, these types of cells contained prominent rough endoplasmic reticulum indicating cells with high capacity for protein synthesis such as fibroblasts/myofibroblasts. Both satellite cells and fibroblasts were most likely involved in the skeletal muscle adaptation to increased mechanical loading. The number of CD68-positive cells is not optimal to be applied as a quantitative value for monocytes/macrophages in human skeletal muscles.

#### **3.3. What to remember about inflammatory cells in damaged skeletal muscle**

Neutrophils are not involved in exercise-induced skeletal muscle fiber injury, although in other injured tissues neutrophils are usually the first inflammatory cells present. CD66b antibody seems to exclusively recognize neutrophils both in leukocyte blood smears and skeletal muscle biopsies. Numerous monocytes/macrophages are present in damaged skeletal muscle fibers, typically totally occupying the entire sarcoplasm of damaged fibers. CD68 antibody is not optimal to be applied as a quantitative value for monocytes/macrophages, because it recognizes also satellite cells and fibroblasts in skeletal muscle biopsies.

**Figure 7.** CD68 (red), laminin (basement membrane, green), and DAPI (nuclei, blue) staining on eccentric exercised human skeletal muscle. (A) Damaged skeletal muscle fiber infiltrated by CD68 stained cells (asterisk). In the endomysium, CD68 stained cells seemed to form a chain around necrotic fiber (arrow heads). (B) CD68 stained cells with long extensions in sarcoplasm (arrow) and in the endomysium (arrow head). (C) CD68 stained cell (arrow heads) inside capillary. (D) Vessel surrounded by CD68 stained cells. Scale bar of 20 μm. Published in Paulsen et al. [6].

**Figure 6.** The nuclei of neutrophils are divided in few parts, which makes them easily recognizable. (A) Semi-thin toluidine blue stained section contains a cluster of red blood cells and single neutrophil (non-exercised skeletal muscle biopsy from human biceps brachii muscle). (B) Leukocytes inside a blood vessel (electron micrograph of epoxy-embedded exercised

skeletal muscle biopsy from biceps brachii muscle). Endothelial cell (E), neutrophil (Nf), red blood cell (R).

**3.1. Neutrophils in damaged skeletal muscle**

26 Muscle Cell and Tissue - Current Status of Research Field

The number of CD66b-positive cells is very low in both exercised and non-exercised skeletal muscles [6]. As an example, only 34 of 122 biopsies from eccentrically exercised and non-exercised human biceps brachii muscles contained CD66b-positive cells. A closer examination showed that in 23 of these biopsies, the CD66b stained cells (varied from 1 to 4 CD66bpositive cells per 100 muscle fibers) were located inside capillaries or vessels (**Figure 6B**), attached to the wall of the vessels, or were detected in blood clots (**Figure 6A**). In the remaining 11 biopsies, single CD66b-positive cells (varied from 1 to 30 CD66b-positive cells per 100 muscle fibers) were observed in the endomysium of affected muscle fibers or in the sarcoplasm of damaged fibers. There was no consistent pattern regarding how CD66b-positive cells were distributed between exercised and non-exercised samples or between different time points. These results indicated that neutrophils were not involved in exercise-induced skeletal muscle fiber injury. Unusual high numbers of CD66b stained cells located in the

CD68-positive cell counts are widely used for indication of monocyte/macrophage infiltration in skeletal muscle biopsies after single bout of eccentric exercise (e.g., see [19]). However, it has been shown that the CD68 antibody recognized more cell types than monocytes/macrophages in human skeletal muscle biopsies after eccentric exercise [6]. The highest individual CD68 positive cell counts were related to skeletal muscle fiber injury, which was observed in exercised biopsies at 4 and 7 days after acute eccentric exercise. In these biopsies, CD68-positive cells typically occupied the entire sarcoplasm of damaged skeletal muscle fibers (**Figure 7A**). Therefore, monocytes/macrophages were probably the most prominent CD68-positive cell type in these biopsies. In addition, CD68-positive cells inside capillaries (**Figure 7C**) and vessels (**Figure 7D**) were most likely monocytes/macrophages. In the exercised biopsies without damaged skeletal muscle fibers, the determination of the monocytes/macrophages proportion of CD68-positive cells is not straightforward. Comparison of cells with similar appearance and location between light microscopy pictures of CD68-positive cells and transmission electron microscopy pictures

endomysium or inside fibers may indicate trauma from the previous biopsy.

**3.2. Monocytes/macrophages in damaged skeletal muscle**

### **4. Serum creatine kinase and voluntary maximal force production as indirect indicator for severity of skeletal muscle damage**

Decrease in muscle force-generating capacity after eccentric exercise is a valuable tool as an indirect indicator for severity of skeletal muscle damage together with serum creatine kinase. As an example, 23 subjects were divided into 3 categories, mild (n = 6), moderate (n = 10), and severe (n = 7) effect of eccentric exercise, depending on the muscle force loss immediately after performing 70 maximal eccentric actions with elbow flexors on an isokinetic dynamometer and how fast muscle force was recovered during the following 7 days [6]. Muscle force loss immediately after the exercise was 64, 53, and 50% of pre-exercise in mild, moderate, and severe categories, respectively (**Figure 8**). After 7 days post exercise, muscle force was totally recovered in the category mild effect of eccentric exercise, whereas in the categories moderate and severe effect of eccentric exercise, force was still clearly below the baseline level (**Figure 8**).

The average values of serum creatine kinase (207, 228, and 140 U/l; mild, moderate, and severe, respectively) before eccentric exercise were similar in all three categories (**Figure 9**). The average values of serum creatine kinase changed the most in categories moderate and severe effects of eccentric exercise. The highest average creatine kinase values, 2929 and 10,266 U/l, in these categories were observed 4 days after eccentric exercise, whereas in the mild effects of eccentric exercise category, the highest average value, 538 U/l, was observed 7 days after the exercise.

> Eight of total 23 subjects showed skeletal muscle fiber injury detected at light microscopy level as dystrophin-negative fibers and infiltration of CD68-positive cells at 4 or 7 days after the exercise. These subjects belonged to the categories moderate (five subjects of ten showed dystrophinnegative fibers) and severe (three subjects of seven showed dystrophin-negative fibers) effects of eccentric exercise. If the muscle force-generating capacity and serum creatine kinase have not totally recovered to the baseline level, it is most likely that the muscle is undergoing regeneration.

> **Figure 9.** Serum creatine kinase. Subjects were divided into three categories, mild (n = 6), moderate (n = 10), and severe (n = 7) effect of eccentric exercise, based on the loss and the recovery of muscle force. Vertical dashed line indicates the time of eccentric exercise. Y-axis is logarithmic. Error bars are standard deviation. Published in Paulsen et al. [6].

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Skeletal muscle fiber injury and prolonged regeneration process are probably the reasons for impaired peripheral muscle function after high-force eccentric exercise. Muscle force-generating capacity after single bout of eccentric exercise is a good indirect indicator of muscle damage in humans together with serum creatine kinase analysis. In the future studies, more attention should be paid for making sure that subjects' muscle force has recovered to the baseline level

The authors thank LIKES Research Centre for Physical Activity and Health, Jyväskylä, Finland for the support to the current publication and the skeletal muscle damage project. Furthermore, special thanks to Jyrki Komulainen, Timo Takala and Veikko Vihko for setting

**4.1. What to remember about indirect indicators for severity of skeletal muscle** 

**damage**

after the experiment is over.

**Acknowledgements**

**Figure 8.** Post-exercise muscle force-generating capacity of the exercised arm. Subjects were divided into three categories, mild (n = 6), moderate (n = 10), and severe (n = 7) effect of eccentric exercise, based on the loss and the recovery of muscle force. Vertical dashed line indicates the time of eccentric exercise bout was performed and horizontal dashed line is the baseline for muscle force. Error bars are standard deviation. Published in Paulsen et al. [6].

**Figure 9.** Serum creatine kinase. Subjects were divided into three categories, mild (n = 6), moderate (n = 10), and severe (n = 7) effect of eccentric exercise, based on the loss and the recovery of muscle force. Vertical dashed line indicates the time of eccentric exercise. Y-axis is logarithmic. Error bars are standard deviation. Published in Paulsen et al. [6].

Eight of total 23 subjects showed skeletal muscle fiber injury detected at light microscopy level as dystrophin-negative fibers and infiltration of CD68-positive cells at 4 or 7 days after the exercise. These subjects belonged to the categories moderate (five subjects of ten showed dystrophinnegative fibers) and severe (three subjects of seven showed dystrophin-negative fibers) effects of eccentric exercise. If the muscle force-generating capacity and serum creatine kinase have not totally recovered to the baseline level, it is most likely that the muscle is undergoing regeneration.

### **4.1. What to remember about indirect indicators for severity of skeletal muscle damage**

Skeletal muscle fiber injury and prolonged regeneration process are probably the reasons for impaired peripheral muscle function after high-force eccentric exercise. Muscle force-generating capacity after single bout of eccentric exercise is a good indirect indicator of muscle damage in humans together with serum creatine kinase analysis. In the future studies, more attention should be paid for making sure that subjects' muscle force has recovered to the baseline level after the experiment is over.

### **Acknowledgements**

**4. Serum creatine kinase and voluntary maximal force production as** 

Decrease in muscle force-generating capacity after eccentric exercise is a valuable tool as an indirect indicator for severity of skeletal muscle damage together with serum creatine kinase. As an example, 23 subjects were divided into 3 categories, mild (n = 6), moderate (n = 10), and severe (n = 7) effect of eccentric exercise, depending on the muscle force loss immediately after performing 70 maximal eccentric actions with elbow flexors on an isokinetic dynamometer and how fast muscle force was recovered during the following 7 days [6]. Muscle force loss immediately after the exercise was 64, 53, and 50% of pre-exercise in mild, moderate, and severe categories, respectively (**Figure 8**). After 7 days post exercise, muscle force was totally recovered in the category mild effect of eccentric exercise, whereas in the categories moderate and severe effect of eccentric exercise, force was still clearly below

The average values of serum creatine kinase (207, 228, and 140 U/l; mild, moderate, and severe, respectively) before eccentric exercise were similar in all three categories (**Figure 9**). The average values of serum creatine kinase changed the most in categories moderate and severe effects of eccentric exercise. The highest average creatine kinase values, 2929 and 10,266 U/l, in these categories were observed 4 days after eccentric exercise, whereas in the mild effects of eccentric exercise category, the highest average value, 538 U/l, was observed 7 days after the exercise.

**Figure 8.** Post-exercise muscle force-generating capacity of the exercised arm. Subjects were divided into three categories, mild (n = 6), moderate (n = 10), and severe (n = 7) effect of eccentric exercise, based on the loss and the recovery of muscle force. Vertical dashed line indicates the time of eccentric exercise bout was performed and horizontal dashed line is the

baseline for muscle force. Error bars are standard deviation. Published in Paulsen et al. [6].

**indirect indicator for severity of skeletal muscle damage**

the baseline level (**Figure 8**).

28 Muscle Cell and Tissue - Current Status of Research Field

The authors thank LIKES Research Centre for Physical Activity and Health, Jyväskylä, Finland for the support to the current publication and the skeletal muscle damage project. Furthermore, special thanks to Jyrki Komulainen, Timo Takala and Veikko Vihko for setting up the research environment for muscle damage projects and opportunity to conduct dissertation research on this topic.

elastase in eccentric exercised human skeletal muscles. Histochemistry and Cell Biology.

Molecular and Cellular Markers in Skeletal Muscle Damage after Acute Voluntary Exercise…

http://dx.doi.org/10.5772/intechopen.76384

31

[7] Sayers SP, Clarkson PM. Force recovery after eccentric exercise in males and females.

[8] Warren GL, Ingalls CP, Shah SJ, Armstrong RB. Uncoupling of in vivo torque production from EMG in mouse muscles injured by eccentric contractions. The Journal of

[9] Clarkson PM, Byrnes WC, Gillisson E, Harper E. Adaptation to exercise-induced muscle

[10] Mymrikov EV, Seit-Nebi AS, Gusev NB. Large potentials of small heat shock proteins.

[11] Paulsen G, Lauritzen F, Bayer ML, Kalhovde JM, Ugelstad I, Owe SG, Hallén J, Bergersen LH, Raastad T. Subcellular movement and expression of HSP27, alphaB-crystallin, and HSP70 after two bouts of eccentric exercise in humans. The Journal of Applied

[12] Koskinen SOA, Kyröläinen H, Flink R, Selänne HP, Gagnon SS, Ahtiainen JP, Nindl BC, Lehti M. Human skeletal muscle type 1 fibre distribution and response of stress-sensing proteins along the titin molecule after submaximal exhaustive exercise. Histochemistry

[13] Zhao J, Kodippili K, Yue Y, Hakim CH, Wasala L, Pan X, Zhang K, Yang NN, Duan D, Lai Y. Dystrophin contains multiple independent membrane-binding domains. Human

[14] Koskinen SO, Ahtikoski AM, Komulainen J, Hesselink MK, Drost MR, Takala TE. Shortterm effects of forced eccentric contractions on collagen synthesis and degradation in rat

[15] Malm C, Nyberg P, Engstrom M, Sjodin B, Lenkei R, Ekblom B, Lundberg I.Immunological changes in human skeletal muscle and blood after eccentric exercise and multiple biop-

[16] Malm C, Sjödin TL, Sjöberg B, Lenkei R, Renström P, Lundberg IE, Ekblom B. Leukocytes, cytokines, growth factors and hormones in human skeletal muscle and blood after uphill

[17] Paulsen G, Crameri R, Benestad HB, Fjeld JG, Mørkrid L, Hallén J, Raastad T. Time course of leukocyte accumulation in human muscle after eccentric exercise. Medicine and Science in Sports and Exercise. 2010;**42**:75-85. DOI: 10.1249/MSS.0b013e3181ac7adb

[18] Paulsen G, Egner IM, Drange M, Langberg H, Benestad HB, Fjeld JG, Hallén J, Raastad T.A COX-2 inhibitor reduces muscle soreness, but does not influence recovery and adaptation after eccentric exercise. Scandinavian Journal of Medicine & Science in Sports.

Physiological Reviews. 2011;**1**:123-1159. DOI: 10.1152/physrev.00023.2010

Physiology (1985). 2009;**107**:570-582. DOI: 10.1152/japplphysiol.00209.2009

and Cell Biology. 2017;**148**:545-555. DOI: 10.1007/s00418-017-1595-z

Molecular Genetics. 2016;**25**:3647-3655. DOI: 10.1093/hmg/ddw210

or downhill running. The Journal of Physiology. 2004;**556**:983-1000

skeletal muscle. Pflügers Archiv. 2002;**444**:59-72

sies. The Journal of Physiology. 2000;**529**:243-262

2010;**20**:195-207. DOI: 10.1111/j.1600-0838.2009.00947.x

2013;**139**:691-715. DOI: 10.1111/j.1600-0838.2009.00947.x

European Journal of Applied Physiology. 2001;**84**:122-126

damage. Clinical Science (London, England). 1987;**73**:383-386

Physiology. 1999;**515**:609-619

### **Conflict of interest**

The authors declare they have no conflict of interest.

## **Abbreviations**

HSP Heat shock protein

### **Author details**

Satu O.A. Koskinen\* and Maarit Lehti

\*Address all correspondence to: satuosmianneli@hotmail.com

LIKES Research Centre for Physical Activity and Health, Jyväskylä, Finland

### **References**


elastase in eccentric exercised human skeletal muscles. Histochemistry and Cell Biology. 2013;**139**:691-715. DOI: 10.1111/j.1600-0838.2009.00947.x

[7] Sayers SP, Clarkson PM. Force recovery after eccentric exercise in males and females. European Journal of Applied Physiology. 2001;**84**:122-126

up the research environment for muscle damage projects and opportunity to conduct disser-

tation research on this topic.

30 Muscle Cell and Tissue - Current Status of Research Field

The authors declare they have no conflict of interest.

\*Address all correspondence to: satuosmianneli@hotmail.com

in Physiology. 2017;**8**:447. DOI: 10.3389/fphys.2017.00447

LIKES Research Centre for Physical Activity and Health, Jyväskylä, Finland

[1] Franchi MV, Reeves ND, Narici MV. Skeletal muscle remodeling in response to eccentric vs. concentric loading: Morphological, molecular, and metabolic adaptations. Frontiers

[2] Bigland-Ritchie B, Woods JJ. Integrated electromyogram and oxygen uptake during

[3] LaStayo PC, Pierotti DJ, Pifer J, Hoppeler H, Lindstedt SL. Eccentric ergometry: Increases in locomotor muscle size and strength at low training intensities. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 2000;**278**:

[4] McHugh HP. Recent advances in the understanding of the repeated bout effect: The protective effect against muscle damage from a single bout of eccentric exercise.

[5] Clarkson PM, Hubal MJ. Exercise-induced muscle damage in humans. American Journal

[6] Paulsen G, Egner I, Raastad T, Reinholt F, Owe S, Lauritzen F, Brorson SH, Koskinen S. Inflammatory markers CD11b, CD16, CD66b, CD68, myeloperoxidase and neutrophil

positive and negative work. The Journal of Physiology. 1976;**260**:267-277

Scandinavian Journal of Medicine & Science in Sports. 2003;**13**:88-97

of Physical Medicine & Rehabilitation. 2002;**8**:S52-S69

HSP Heat shock protein

Satu O.A. Koskinen\* and Maarit Lehti

**Conflict of interest**

**Abbreviations**

**Author details**

**References**

R1282-R1288


[19] Crameri RM, Langberg H, Teisner B, Magnusson P, Schrøder HD, Olesen JL, Jensen CH, Koskinen S, Suetta C, Kjaer M. Enhanced procollagen processing in skeletal muscle after a single bout of eccentric loading in humans. Matrix Biology. 2004;**23**:259-264

**Chapter 4**

**Provisional chapter**

H, 13C, and 31P) *in vivo* mag-

**Multinuclear Magnetic Resonance Spectroscopy of**

**Multinuclear Magnetic Resonance Spectroscopy of** 

DOI: 10.5772/intechopen.77107

**Human Skeletal Muscle Metabolism in Training and**

**Human Skeletal Muscle Metabolism in Training and** 

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

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

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

**Disease**

**Disease**

Martin Krššák

Martin Krššák

**Abstract**

metabolic flexibility.

**1. Introduction**

Ladislav Valkovič, Radka Klepochová and

Ladislav Valkovič, Radka Klepochová and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

In this chapter, techniques and application of multinuclear (1

netic resonance spectroscopy (MRS) for the assessment of skeletal muscle metabolism in health and disease are described. Studies focusing on glucose transport and utilization, lipid storage and consumption, handling of energy rich phosphates, and measurements of newly emerging noninvasive biomarkers, i.e., acetylcarnitine and carnosine are summarized. Muscle metabolism connections to exercise physiology and the development as well as possible treatment of metabolic diseases, such as obesity and diabetes are also discussed. Taken together, multinuclear *in vivo* MRS on humans helped to uncover defects in skeletal muscle metabolic pathways in insulin-resistant conditions; and to discover links between defects in mitochondrial activity/capacity and lipid metabolism, as well as defects in whole-body and/or muscle glucose metabolism. There is also to mention that several of the MR-derived readouts are affected by both training status and metabolic disease in a specific way, and thus could serve as potential markers of training status and

**Keywords:** magnetic resonance spectroscopy, skeletal muscle, energy metabolism, training status, pathophysiology, glucose, lipids, diabetes mellitus, obesity, exercise

Skeletal muscle is the key human tissue responsible for the body weight bearing and movement and plays a central role in whole-body energy metabolism. Even in the resting conditions

http://dx.doi.org/10.5772/intechopen.77107

[20] Mikkelsen UR, Langberg H, Helmark IC, Skovgaard D, Andersen LL, Kjaer M, Mackey AL, Local NSAID. Infusion inhibits satellite cell proliferation in human skeletal muscle after eccentric exercise. The Journal of Applied Physiology (1985). 2009;**107**:1600-1611. DOI: 10.1152/japplphysiol.00707.2009

#### **Multinuclear Magnetic Resonance Spectroscopy of Human Skeletal Muscle Metabolism in Training and Disease Multinuclear Magnetic Resonance Spectroscopy of Human Skeletal Muscle Metabolism in Training and Disease**

DOI: 10.5772/intechopen.77107

Ladislav Valkovič, Radka Klepochová and Martin Krššák Ladislav Valkovič, Radka Klepochová and Martin Krššák

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

http://dx.doi.org/10.5772/intechopen.77107

#### **Abstract**

[19] Crameri RM, Langberg H, Teisner B, Magnusson P, Schrøder HD, Olesen JL, Jensen CH, Koskinen S, Suetta C, Kjaer M. Enhanced procollagen processing in skeletal muscle after

[20] Mikkelsen UR, Langberg H, Helmark IC, Skovgaard D, Andersen LL, Kjaer M, Mackey AL, Local NSAID. Infusion inhibits satellite cell proliferation in human skeletal muscle after eccentric exercise. The Journal of Applied Physiology (1985). 2009;**107**:1600-1611.

a single bout of eccentric loading in humans. Matrix Biology. 2004;**23**:259-264

DOI: 10.1152/japplphysiol.00707.2009

32 Muscle Cell and Tissue - Current Status of Research Field

In this chapter, techniques and application of multinuclear (1 H, 13C, and 31P) *in vivo* magnetic resonance spectroscopy (MRS) for the assessment of skeletal muscle metabolism in health and disease are described. Studies focusing on glucose transport and utilization, lipid storage and consumption, handling of energy rich phosphates, and measurements of newly emerging noninvasive biomarkers, i.e., acetylcarnitine and carnosine are summarized. Muscle metabolism connections to exercise physiology and the development as well as possible treatment of metabolic diseases, such as obesity and diabetes are also discussed. Taken together, multinuclear *in vivo* MRS on humans helped to uncover defects in skeletal muscle metabolic pathways in insulin-resistant conditions; and to discover links between defects in mitochondrial activity/capacity and lipid metabolism, as well as defects in whole-body and/or muscle glucose metabolism. There is also to mention that several of the MR-derived readouts are affected by both training status and metabolic disease in a specific way, and thus could serve as potential markers of training status and metabolic flexibility.

**Keywords:** magnetic resonance spectroscopy, skeletal muscle, energy metabolism, training status, pathophysiology, glucose, lipids, diabetes mellitus, obesity, exercise

### **1. Introduction**

Skeletal muscle is the key human tissue responsible for the body weight bearing and movement and plays a central role in whole-body energy metabolism. Even in the resting conditions

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

skeletal muscle accounts for ~30% of metabolic rate of human body [1]. In particular, as the main target of insulin activity, skeletal muscle effectively regulates the glucose uptake, while serving also as a glycogen storage [2]. The extent to which skeletal muscle fulfills these roles is affected by many physiological and pathophysiological factors, which can change over time. Several diseases largely manifesting in skeletal muscle pathology have a rapidly increasing socioeconomic impact, as they start to affect not only the elderly, e.g., sarcopenia, but also young productive, population, e.g., insulin resistance and type 2 diabetes mellitus (T2DM). Insulin resistance and T2DM are rapidly reaching epidemic proportions worldwide and the associated treatment costs of T2DM also continue to grow. The cost of diabetes (with over 85% attributable to T2DM) was in 2012 over £1.5 milion an hour or 10% of the entire National Health Service budget for England and Wales [3]. In order to improve the understanding and clinical management of such disorders, it is vital to be able to assess muscle function and metabolism *in vivo* noninvasively, to support their diagnosis, monitor changes in tissue status during disease progression and interventions, and above all, to establish robust markers that can be used in disease prevention [4].

**2. Methods of magnetic resonance spectroscopy**

lular lipids (IMCL) [16–18]. Among other typical uses of 1

*H MRS*

intramyocellular (IMCL) and extramyocellular (EMCL) lipids [0.9 and 1.1 ppm (CH3

Next to water, lipid accounts for the strongest signals in a <sup>1</sup>

spectrum of skeletal muscle is given in **Figure 1**.

H MRS that is used in exercise and nutrition research, just as often as

Multinuclear Magnetic Resonance Spectroscopy of Human Skeletal Muscle Metabolism…

H MRS. An example of high resolved *in vivo* acquired 1

H-MRS spectrum from an athlete acquired from the vastus lateralis muscle at 7 T showing

groups)], AcC at 2.13 ppm, Cr at 3.03 and 3.9 ppm, trimethyl ammonium (TMA) groups of carnitine, AcC, and choline at 3.20 ppm, residual water peak at 4.7 ppm, removed in postprocessing, and carnosine spectral lines at 7 and 8 ppm.

H MRS belong: (a) detection of lac-

http://dx.doi.org/10.5772/intechopen.77107

H spectrum of skeletal muscle at rest.

groups) 1.5 and 1.3 ppm (CH2

H MR

35

in studying the etiology of insulin resistance and T2DM, is the quantification of intramyocel-

tate (Lac) formation during exercise [8, 19–21]; (b) measurement of total creatine (Cr) content [22–24]; (c) assessment of muscle fiber orientation using dipolar coupling [25]; (d) measurement of intramyocellular metabolite diffusion [26]; and (e) the dynamic measurement of tissue (de)oxygenation using the signal of deoxymyoglobin (DMb) [27–29]. Furthermore, detection of resting muscle carnosine [30, 31] and acetylcarnitine (AcC) [32, 33] has been recently pro-

However, even with optimal tissue selection, not all lipid signals in the spectrum are intramyocellular (IMCL). Fortunately, it is possible to differentiate between IMCL and extramyocellular

**2.1. 1**

**H MRS**

The main application of 1

moted as a promising use of 1

*2.1.1. Static examinations by 1*

**Figure 1.** A representative 1

Magnetic resonance spectroscopy (MRS) represents an advanced noninvasive technology that allows for assessment of tissue metabolism in the healthy as well as diseased conditions [5]. In particular, MRS techniques are able to noninvasively monitor intramyocellular storage and turnover of important energy storage pools, namely lipids and glycogen. In addition, MRS is uniquely suited to quantitatively assess adenosine-triphosphate (ATP) production reactions in the muscle, i.e., mitochondrial oxidative phosphorylation, glycolysis, and creatine kinase activity. Among other things, proton (1 H) MRS is best suited to quantify intramyocellular lipid (IMCL) storage, carbon (13C) MRS is optimal for glycogen reserves measurements and phosphorus (31P) is ideal for investigations of ATP metabolism. This chapter briefly describes the basic principles and availability of these measurements, and further focuses on applications of MRS techniques for studying functional properties of skeletal muscle in health and disease. Obesity, type 2 diabetes mellitus, and skeletal muscle insulin resistance serve as good model for pathologic conditions, while the summary of MRS observable adaption to training is brought as positive control or contrast to aforementioned circumstances.

While most of the described methods and measurements have been introduced at lower field strengths 20–30 years ago [6–8], recent development in MR technology, namely the transition towards ultra-high field MR systems (B<sup>0</sup> ≥ 7 T), meant significant improvements to *in vivo* MRS [9–13]. Next to the linear gain in signal-to-noise ratio (SNR), which can be translated to significantly shorten data acquisition time [12] or improved signal localization [14], higher field strength also provides improved spectral resolution, reducing metabolite overlapping, and thus, improving quantification accuracy. The increase in SNR is of particular importance to nonproton MRS, which is limited mainly by SNR in its applicability [15]. 31P MRS benefits from additional increase in SNR per unit of time due to shortening of the T<sup>1</sup> relaxation of 31P metabolites at 7 T [11]. MR systems (B<sup>0</sup> ≥ 7 T) equipped with multinuclear broadband capabilities hold great potential for investigations of the not yet well-understood mechanisms of tissue metabolism.

### **2. Methods of magnetic resonance spectroscopy**

#### **2.1. 1 H MRS**

skeletal muscle accounts for ~30% of metabolic rate of human body [1]. In particular, as the main target of insulin activity, skeletal muscle effectively regulates the glucose uptake, while serving also as a glycogen storage [2]. The extent to which skeletal muscle fulfills these roles is affected by many physiological and pathophysiological factors, which can change over time. Several diseases largely manifesting in skeletal muscle pathology have a rapidly increasing socioeconomic impact, as they start to affect not only the elderly, e.g., sarcopenia, but also young productive, population, e.g., insulin resistance and type 2 diabetes mellitus (T2DM). Insulin resistance and T2DM are rapidly reaching epidemic proportions worldwide and the associated treatment costs of T2DM also continue to grow. The cost of diabetes (with over 85% attributable to T2DM) was in 2012 over £1.5 milion an hour or 10% of the entire National Health Service budget for England and Wales [3]. In order to improve the understanding and clinical management of such disorders, it is vital to be able to assess muscle function and metabolism *in vivo* noninvasively, to support their diagnosis, monitor changes in tissue status during disease progression and interventions, and above all, to establish robust markers that

Magnetic resonance spectroscopy (MRS) represents an advanced noninvasive technology that allows for assessment of tissue metabolism in the healthy as well as diseased conditions [5]. In particular, MRS techniques are able to noninvasively monitor intramyocellular storage and turnover of important energy storage pools, namely lipids and glycogen. In addition, MRS is uniquely suited to quantitatively assess adenosine-triphosphate (ATP) production reactions in the muscle, i.e., mitochondrial oxidative phosphorylation, glycoly-

quantify intramyocellular lipid (IMCL) storage, carbon (13C) MRS is optimal for glycogen reserves measurements and phosphorus (31P) is ideal for investigations of ATP metabolism. This chapter briefly describes the basic principles and availability of these measurements, and further focuses on applications of MRS techniques for studying functional properties of skeletal muscle in health and disease. Obesity, type 2 diabetes mellitus, and skeletal muscle insulin resistance serve as good model for pathologic conditions, while the summary of MRS observable adaption to training is brought as positive control or contrast to

While most of the described methods and measurements have been introduced at lower field strengths 20–30 years ago [6–8], recent development in MR technology, namely the transition towards ultra-high field MR systems (B<sup>0</sup> ≥ 7 T), meant significant improvements to *in vivo* MRS [9–13]. Next to the linear gain in signal-to-noise ratio (SNR), which can be translated to significantly shorten data acquisition time [12] or improved signal localization [14], higher field strength also provides improved spectral resolution, reducing metabolite overlapping, and thus, improving quantification accuracy. The increase in SNR is of particular importance to nonproton MRS, which is limited mainly by SNR in its applicability [15]. 31P MRS benefits

metabolites at 7 T [11]. MR systems (B<sup>0</sup> ≥ 7 T) equipped with multinuclear broadband capabilities hold great potential for investigations of the not yet well-understood mechanisms of

from additional increase in SNR per unit of time due to shortening of the T<sup>1</sup>

H) MRS is best suited to

relaxation of 31P

sis, and creatine kinase activity. Among other things, proton (1

can be used in disease prevention [4].

34 Muscle Cell and Tissue - Current Status of Research Field

aforementioned circumstances.

tissue metabolism.

The main application of 1 H MRS that is used in exercise and nutrition research, just as often as in studying the etiology of insulin resistance and T2DM, is the quantification of intramyocellular lipids (IMCL) [16–18]. Among other typical uses of 1 H MRS belong: (a) detection of lactate (Lac) formation during exercise [8, 19–21]; (b) measurement of total creatine (Cr) content [22–24]; (c) assessment of muscle fiber orientation using dipolar coupling [25]; (d) measurement of intramyocellular metabolite diffusion [26]; and (e) the dynamic measurement of tissue (de)oxygenation using the signal of deoxymyoglobin (DMb) [27–29]. Furthermore, detection of resting muscle carnosine [30, 31] and acetylcarnitine (AcC) [32, 33] has been recently promoted as a promising use of 1 H MRS. An example of high resolved *in vivo* acquired 1 H MR spectrum of skeletal muscle is given in **Figure 1**.

#### *2.1.1. Static examinations by 1 H MRS*

Next to water, lipid accounts for the strongest signals in a <sup>1</sup> H spectrum of skeletal muscle at rest. However, even with optimal tissue selection, not all lipid signals in the spectrum are intramyocellular (IMCL). Fortunately, it is possible to differentiate between IMCL and extramyocellular

**Figure 1.** A representative 1 H-MRS spectrum from an athlete acquired from the vastus lateralis muscle at 7 T showing intramyocellular (IMCL) and extramyocellular (EMCL) lipids [0.9 and 1.1 ppm (CH3 groups) 1.5 and 1.3 ppm (CH2 groups)], AcC at 2.13 ppm, Cr at 3.03 and 3.9 ppm, trimethyl ammonium (TMA) groups of carnitine, AcC, and choline at 3.20 ppm, residual water peak at 4.7 ppm, removed in postprocessing, and carnosine spectral lines at 7 and 8 ppm.

lipids (EMCL). Inside myocytes, lipids form small droplets in the cytoplasm, whereas EMCLs are found layered between myocytes along the main muscle orientation, and are tubular in shape. This difference in spherical versus cylindrical geometry influences the bulk magnetic susceptibility of these lipid compartments making the differentiation possible [34, 35]. The IMCL/EMCL peak separation depends on the angular orientation of EMCL to the external magnetic field as a result of anisotropy effects [36] which results into maximum of 0.2 ppm frequency shift in case of fully parallel orientation [4], as is the case in tibialis anterior [25, 37].

resonance lines. Of particular interest has been the formation of lactate (Lac) during exercise challenge or ischaemia [8, 19, 20, 53, 54], because lactate is the end-product of anaerobic

in good agreement with tissue extracts analysis [53], due to overlapping lipid signals, dipolar coupling and relaxation effects, quantification of Lac levels in skeletal muscle *in vivo* is

It can often be unclear whether the measured results reflect real change in skeletal muscle metabolism or just manifest inadequate oxygenation state of the muscle. This query can be

state of human skeletal muscle under stress through the measurement of deoxymyoglobin (DMb) [29]. Very low concentration of DMb is not an obstacle, as DMb resonates substantially downfield away from the typical spectral range, securing no overlap with other metabolites

Formation of AcC during strenuous exercise and its slow decay after exercise has also been

nance line that gets resolved after strenuous exercise [56], 7T allows direct observation of split in resonance lines of AcC and carnitine in the TMA region, providing the option to quantify

On the longer time scale of few 10 minutes during prolonged submaximal exercise and fol-

The presence of carbon nuclei in almost every organic structure, the nonzero spin of carbon-13 (13C) nuclei, and a very wide chemical shift range of up to 200 ppm have made 13C MRS well-suited for studies of molecular structure and biochemistry in cellular and animal models since the early days of biochemical MRS. The dynamic assessment of biochemical pathways

Due to the different magnetic properties of 13C compared to protons, the resonance frequency

the natural abundance of carbon nuclei is very high in living tissues, i.e., almost matching the abundance of protons, the ratio of MR visible 13C to MR invisible 12C is extremely low (approx. 1:99). Lower gyromagnetic ratio and consecutively lower intrinsic sensitivity of 13C MRS, together with lower natural abundance of 13C nuclei leads to inherently low SNR, and thus, hampers the spatial and temporal resolution of 13C MRS experiments. Techniques to increase low SNR of 13C MRS include: (a) increased volume of interests and/or averaging of the MRS signal using a high number of repetitions, (b) elimination of the spin-spin coupling

polarization transfer techniques; (d) the use of a higher field-strength MR apparatus; and (e) increasing the abundance of the 13C isotope by systemic infusion of 13C-enriched metabolic

lowing recovery decrease and replenishment of IMCL pool can be observed [58, 59].

in particular, forms the basis for the current application of 13C MRS in humans.

of 13C at a given magnetic field is approximately one-quarter that of <sup>1</sup>

interaction between 13C-nuclei and its coupled protons by the 1

period of 13C signal acquisition; (c) the utilization of the 1

H MRS, which can serve to noninvasively monitor the (de)oxygenation

Multinuclear Magnetic Resonance Spectroscopy of Human Skeletal Muscle Metabolism…

H MRS [56, 57]. While at lower fields, it is only the 2.13 ppm reso-

H signal of AcC at this resonance is twice as strong, improving sensi-

, and thus, it plays an important role in skeletal muscle

H MRS measurements of Lac were shown to be

http://dx.doi.org/10.5772/intechopen.77107

37

H MRS. Although

H decoupling pulses in the

H-13C magnetic interaction with

metabolism and a source of free H+

also answered by 1

and has extremely short T1

under investigation using 1

their ratio. Besides, the <sup>1</sup>

**2.2. 13C MRS**

substrates.

tivity of the measurement [57].

metabolism and pH regulation. Although 1

extremely challenging, and thus, prone to inaccurate estimation [4].

[55].

In general, to maximize the acquired signal, MRS sequences with short echo time (TE) are often used for IMCL quantification [38–40]. This requires suppression of water signal and can also lead to broad resonances of various shapes and strong IMCL/EMCL overlap, which can cause inaccurate quantification of IMCLs [41, 42]. Contamination from subcutaneous adipose tissue or bone marrow can make this even more challenging. Moreover, if the water signal is to be used as an internal concentration reference, additional acquisition of water signal is necessary. Better separation of EMCLs and IMCLs and improved fitting of lipid resonances was suggested and observed when using an MRS acquisition with longer TEs [10, 42, 43]. This improved separation is a result of the different T<sup>2</sup> relaxation times of IMCL and EMCL resonances and the line width narrowing effect [10]. Thus, the long-TE acquisition has a major advantage in increased spectral resolution [10, 34] and provides the possibility to omit water suppression, reducing energy deposition in tissues. On the other hand, absolute quantification from the long-TE MR spectra requires precise T2 relaxation correction, which can be inaccurate especially for signals with short T2 , i.e., water signal [10, 44]. Thus, an ideal acquisition combines a short TE measurement of water signal with long-TE detection of lipids [14].

Another muscle metabolite that greatly benefits from long-TE acquisition is acetylcarnitine (AcC). This relatively low concentrated metabolite fulfills a major role in translocation of longchain fatty acids from cytosol to the mitochondrial matrix [45] and in maintaining pyruvate dehydrogenation activity [46], and is, therefore, of high interest in skeletal muscle research. The straight forward detection and quantification of AcC is challenging, due to the strong overlap of the 2.13 ppm line with lipid resonances, and the fact that the line at 3.20 ppm represents a combination of the trimethylammonium (TMA) groups of carnitine, AcC, and choline. Fortunately, the differences in T<sup>2</sup> relaxation times of AcC and lipids allow the detection of the 2.13 ppm line at rest, using long-TE 1 H MRS [32, 33].

The downfield region of the <sup>1</sup> H spectrum, i.e., left to the water signal, gets often overlooked as the detectable signals belong to low concentrated metabolites, e.g., carnosine, and can be easily mistaken for noise. This is very unfortunate, as carnosine is a pH-buffering metabolite that can be manipulated externally [47, 48]. The concentration of carnosine is mainly determined by muscle fiber type composition, with fast-twitch glycolytic fibers containing up to twice as much carnosine as slow-twitch oxidative fibers [49, 50]. In addition, chemical shift of carnosine is sensitive to pH, and thus, carnosine signal can be also used to assess intramyocellular pH [51, 52]. While it is possible to detect carnosine using clinical systems [30, 51], the increased SNR of ultra-high fields, provides high repeatability [31].

#### *2.1.2. Dynamic examinations by 1 H MRS*

While most of the metabolite signals can be observed in basal resting conditions, metabolic adaption to stress induces by exercise and/or ischemia may alleviate the visibility of specific resonance lines. Of particular interest has been the formation of lactate (Lac) during exercise challenge or ischaemia [8, 19, 20, 53, 54], because lactate is the end-product of anaerobic metabolism and a source of free H+ , and thus, it plays an important role in skeletal muscle metabolism and pH regulation. Although 1 H MRS measurements of Lac were shown to be in good agreement with tissue extracts analysis [53], due to overlapping lipid signals, dipolar coupling and relaxation effects, quantification of Lac levels in skeletal muscle *in vivo* is extremely challenging, and thus, prone to inaccurate estimation [4].

It can often be unclear whether the measured results reflect real change in skeletal muscle metabolism or just manifest inadequate oxygenation state of the muscle. This query can be also answered by 1 H MRS, which can serve to noninvasively monitor the (de)oxygenation state of human skeletal muscle under stress through the measurement of deoxymyoglobin (DMb) [29]. Very low concentration of DMb is not an obstacle, as DMb resonates substantially downfield away from the typical spectral range, securing no overlap with other metabolites and has extremely short T1 [55].

Formation of AcC during strenuous exercise and its slow decay after exercise has also been under investigation using 1 H MRS [56, 57]. While at lower fields, it is only the 2.13 ppm resonance line that gets resolved after strenuous exercise [56], 7T allows direct observation of split in resonance lines of AcC and carnitine in the TMA region, providing the option to quantify their ratio. Besides, the <sup>1</sup> H signal of AcC at this resonance is twice as strong, improving sensitivity of the measurement [57].

On the longer time scale of few 10 minutes during prolonged submaximal exercise and following recovery decrease and replenishment of IMCL pool can be observed [58, 59].

### **2.2. 13C MRS**

lipids (EMCL). Inside myocytes, lipids form small droplets in the cytoplasm, whereas EMCLs are found layered between myocytes along the main muscle orientation, and are tubular in shape. This difference in spherical versus cylindrical geometry influences the bulk magnetic susceptibility of these lipid compartments making the differentiation possible [34, 35]. The IMCL/EMCL peak separation depends on the angular orientation of EMCL to the external magnetic field as a result of anisotropy effects [36] which results into maximum of 0.2 ppm frequency shift in case of fully parallel orientation [4], as is the case in tibialis anterior [25, 37]. In general, to maximize the acquired signal, MRS sequences with short echo time (TE) are often used for IMCL quantification [38–40]. This requires suppression of water signal and can also lead to broad resonances of various shapes and strong IMCL/EMCL overlap, which can cause inaccurate quantification of IMCLs [41, 42]. Contamination from subcutaneous adipose tissue or bone marrow can make this even more challenging. Moreover, if the water signal is to be used as an internal concentration reference, additional acquisition of water signal is necessary. Better separation of EMCLs and IMCLs and improved fitting of lipid resonances was suggested and observed when using an MRS acquisition with longer TEs [10, 42, 43].

resonances and the line width narrowing effect [10]. Thus, the long-TE acquisition has a major advantage in increased spectral resolution [10, 34] and provides the possibility to omit water suppression, reducing energy deposition in tissues. On the other hand, absolute quantifica-

Another muscle metabolite that greatly benefits from long-TE acquisition is acetylcarnitine (AcC). This relatively low concentrated metabolite fulfills a major role in translocation of longchain fatty acids from cytosol to the mitochondrial matrix [45] and in maintaining pyruvate dehydrogenation activity [46], and is, therefore, of high interest in skeletal muscle research. The straight forward detection and quantification of AcC is challenging, due to the strong overlap of the 2.13 ppm line with lipid resonances, and the fact that the line at 3.20 ppm represents a combination of the trimethylammonium (TMA) groups of carnitine, AcC, and choline.

combines a short TE measurement of water signal with long-TE detection of lipids [14].

H MRS [32, 33].

increased SNR of ultra-high fields, provides high repeatability [31].

*H MRS*

as the detectable signals belong to low concentrated metabolites, e.g., carnosine, and can be easily mistaken for noise. This is very unfortunate, as carnosine is a pH-buffering metabolite that can be manipulated externally [47, 48]. The concentration of carnosine is mainly determined by muscle fiber type composition, with fast-twitch glycolytic fibers containing up to twice as much carnosine as slow-twitch oxidative fibers [49, 50]. In addition, chemical shift of carnosine is sensitive to pH, and thus, carnosine signal can be also used to assess intramyocellular pH [51, 52]. While it is possible to detect carnosine using clinical systems [30, 51], the

While most of the metabolite signals can be observed in basal resting conditions, metabolic adaption to stress induces by exercise and/or ischemia may alleviate the visibility of specific

relaxation times of IMCL and EMCL

relaxation correction, which can be inac-

, i.e., water signal [10, 44]. Thus, an ideal acquisition

relaxation times of AcC and lipids allow the detection of the

H spectrum, i.e., left to the water signal, gets often overlooked

This improved separation is a result of the different T<sup>2</sup>

tion from the long-TE MR spectra requires precise T2

curate especially for signals with short T2

36 Muscle Cell and Tissue - Current Status of Research Field

Fortunately, the differences in T<sup>2</sup>

The downfield region of the <sup>1</sup>

*2.1.2. Dynamic examinations by 1*

2.13 ppm line at rest, using long-TE 1

The presence of carbon nuclei in almost every organic structure, the nonzero spin of carbon-13 (13C) nuclei, and a very wide chemical shift range of up to 200 ppm have made 13C MRS well-suited for studies of molecular structure and biochemistry in cellular and animal models since the early days of biochemical MRS. The dynamic assessment of biochemical pathways in particular, forms the basis for the current application of 13C MRS in humans.

Due to the different magnetic properties of 13C compared to protons, the resonance frequency of 13C at a given magnetic field is approximately one-quarter that of <sup>1</sup> H MRS. Although the natural abundance of carbon nuclei is very high in living tissues, i.e., almost matching the abundance of protons, the ratio of MR visible 13C to MR invisible 12C is extremely low (approx. 1:99). Lower gyromagnetic ratio and consecutively lower intrinsic sensitivity of 13C MRS, together with lower natural abundance of 13C nuclei leads to inherently low SNR, and thus, hampers the spatial and temporal resolution of 13C MRS experiments. Techniques to increase low SNR of 13C MRS include: (a) increased volume of interests and/or averaging of the MRS signal using a high number of repetitions, (b) elimination of the spin-spin coupling interaction between 13C-nuclei and its coupled protons by the 1 H decoupling pulses in the period of 13C signal acquisition; (c) the utilization of the 1 H-13C magnetic interaction with polarization transfer techniques; (d) the use of a higher field-strength MR apparatus; and (e) increasing the abundance of the 13C isotope by systemic infusion of 13C-enriched metabolic substrates.

### *2.2.1. 13C MRS natural abundance studies*

The use of 13C MRS for *in vivo* studies of skeletal muscle without artificial isotope enrichment is essentially limited to measurements of metabolites present at high concentrations, in particular glycogen and triglycerides [4]. Despite its high molecular weight, the glycogen C-1 resonance line is 100% MR visible [60, 61] due to the high intramolecular mobility of its glucose residues. Skeletal muscle glycogen is present at approximately 80–120 mM concentrations, depending on the muscle and physiological conditions [62–64]. Good reproducibility of natural abundance muscle glycogen measurements by 13C MRS [65] favors the use of dynamic experimental protocols to assess the depletion of glycogen during exercise (**Figure 2**) and its resynthesis over the course of several hours during post-exercise recovery [58, 59, 66, 67].

#### *2.2.2. 13C MRC labeling studies*

To overcome the low SNR due to low natural abundance of 13C nuclei and increase the measurement sensitivity, it is common to use an isotope enriched infusion in 13C MRS studies [4]. After an infusion of 13C-labeled glucose under steady-state conditions, glycogen synthesis in skeletal gastrocnemius muscle has been quantified and correlated with whole-body carbohydrate consumption [7, 69, 70].

Another exciting use of 13C MRS *in vivo* is the quantification of the flux through the tricarboxylic acid (TCA) cycle, which serves as a surrogate for the rate of mitochondrial oxygen consumption by the cellular respiration that is vital for skeletal muscle function. The labeling of substrates in the TCA by infusing [2-13C]-acetate and observing the enrichment of the C4 position of glutamate, has been performed in muscle. These measurements can easily be combined with experiments in which undirectional flux through the skeletal muscle ATPsynthase is measured by means of 31P saturation transfer [4].

Alternative approach for further improvement of signal-to-noise and localization is the application of so called indirect 13C measurements, where high sensitivity and low chemical shift displacement of 1 H MRS is used for signal excitation and detection and chemical specificity is introduced exploiting magnetic interaction with coupling 13C atoms. Proof of the principle for this approach has been demonstrated the measurements of fatty acid composition of human subcutaneous tissue [71], while application of similar methodology with the sensitivity enhancement by concomitant 13C label infusion has been demonstrated in the study focused on postprandial lipid partitioning in liver and skeletal muscle in prediabetic and diabetic rats [72].

#### **2.3. 31P MRS**

Skeletal muscle was the first human tissue studied by 31P MRS *in vivo*, mainly because of its high metabolic activity, physiological importance, and relatively simple access [6, 73, 74]. 31P MR spectra of skeletal muscle typically depict five major resonances from inorganic phosphate (Pi), phosphocreatine (PCr), and adenosine-triphosphate (ATP).

glycerolphosphocholine (GPC) and glycerol-phosphoethanolamine (GPE) [11] (**Figure 3**). Besides, using the chemical shift between PCr and Pi, intramyocellular pH can be calculated

**Figure 2.** Transversal MRI of calf muscle (a) and natural abundance 13C MR spectra acquired at 7T depicting glycogen signals from soleus and gastrocnemius muscle (b) (pulse-acquire block pulse MRS, acquisition time approx. 4 min). The glycogen signal is decreased after 90 s of toe raising exercise by approx. 30%. Adapted from Goluch et al. [68].

Multinuclear Magnetic Resonance Spectroscopy of Human Skeletal Muscle Metabolism…

http://dx.doi.org/10.5772/intechopen.77107

39

Next to the analysis of resting 31P MR spectra, for metabolite concentration determination, it is very frequent to obtain the 31P MR spectra during exercise and consecutive recovery [6, 77]. Such dynamic 31P MR experiments provide a measure of skeletal muscle oxidative metabo-

noninvasively [75].

lism, through quantification of mitochondrial capacity.

Other detectable 31P metabolites include cell membrane precursors, i.e., phosphomonoesters (PMEs) combined from—phosphocholine (PC) and phosphoethanolamine (PE) and cell membrane degradation products, i.e., phosphodiesters (PDEs) in particular Multinuclear Magnetic Resonance Spectroscopy of Human Skeletal Muscle Metabolism… http://dx.doi.org/10.5772/intechopen.77107 39

*2.2.1. 13C MRS natural abundance studies*

38 Muscle Cell and Tissue - Current Status of Research Field

*2.2.2. 13C MRC labeling studies*

drate consumption [7, 69, 70].

displacement of 1

**2.3. 31P MRS**

The use of 13C MRS for *in vivo* studies of skeletal muscle without artificial isotope enrichment is essentially limited to measurements of metabolites present at high concentrations, in particular glycogen and triglycerides [4]. Despite its high molecular weight, the glycogen C-1 resonance line is 100% MR visible [60, 61] due to the high intramolecular mobility of its glucose residues. Skeletal muscle glycogen is present at approximately 80–120 mM concentrations, depending on the muscle and physiological conditions [62–64]. Good reproducibility of natural abundance muscle glycogen measurements by 13C MRS [65] favors the use of dynamic experimental protocols to assess the depletion of glycogen during exercise (**Figure 2**) and its resynthesis over the course of several hours during post-exercise recovery [58, 59, 66, 67].

To overcome the low SNR due to low natural abundance of 13C nuclei and increase the measurement sensitivity, it is common to use an isotope enriched infusion in 13C MRS studies [4]. After an infusion of 13C-labeled glucose under steady-state conditions, glycogen synthesis in skeletal gastrocnemius muscle has been quantified and correlated with whole-body carbohy-

Another exciting use of 13C MRS *in vivo* is the quantification of the flux through the tricarboxylic acid (TCA) cycle, which serves as a surrogate for the rate of mitochondrial oxygen consumption by the cellular respiration that is vital for skeletal muscle function. The labeling of substrates in the TCA by infusing [2-13C]-acetate and observing the enrichment of the C4 position of glutamate, has been performed in muscle. These measurements can easily be combined with experiments in which undirectional flux through the skeletal muscle ATP-

Alternative approach for further improvement of signal-to-noise and localization is the application of so called indirect 13C measurements, where high sensitivity and low chemical shift

is introduced exploiting magnetic interaction with coupling 13C atoms. Proof of the principle for this approach has been demonstrated the measurements of fatty acid composition of human subcutaneous tissue [71], while application of similar methodology with the sensitivity enhancement by concomitant 13C label infusion has been demonstrated in the study focused on postprandial lipid partitioning in liver and skeletal muscle in prediabetic and diabetic rats [72].

Skeletal muscle was the first human tissue studied by 31P MRS *in vivo*, mainly because of its high metabolic activity, physiological importance, and relatively simple access [6, 73, 74]. 31P MR spectra of skeletal muscle typically depict five major resonances from inorganic phos-

Other detectable 31P metabolites include cell membrane precursors, i.e., phosphomonoesters (PMEs) combined from—phosphocholine (PC) and phosphoethanolamine (PE) and cell membrane degradation products, i.e., phosphodiesters (PDEs) in particular

phate (Pi), phosphocreatine (PCr), and adenosine-triphosphate (ATP).

H MRS is used for signal excitation and detection and chemical specificity

synthase is measured by means of 31P saturation transfer [4].

**Figure 2.** Transversal MRI of calf muscle (a) and natural abundance 13C MR spectra acquired at 7T depicting glycogen signals from soleus and gastrocnemius muscle (b) (pulse-acquire block pulse MRS, acquisition time approx. 4 min). The glycogen signal is decreased after 90 s of toe raising exercise by approx. 30%. Adapted from Goluch et al. [68].

glycerolphosphocholine (GPC) and glycerol-phosphoethanolamine (GPE) [11] (**Figure 3**). Besides, using the chemical shift between PCr and Pi, intramyocellular pH can be calculated noninvasively [75].

Next to the analysis of resting 31P MR spectra, for metabolite concentration determination, it is very frequent to obtain the 31P MR spectra during exercise and consecutive recovery [6, 77]. Such dynamic 31P MR experiments provide a measure of skeletal muscle oxidative metabolism, through quantification of mitochondrial capacity.

Alike 13C MRS, 31P MRS also has a lower gyromagnetic ratio in comparison to protons, and thus, suffers from lower intrinsic sensitivity. Therefore, SNR enhancing approaches, e.g., <sup>1</sup>

Multinuclear Magnetic Resonance Spectroscopy of Human Skeletal Muscle Metabolism…

decoupling, at lower fields, or benefit from the SNR boost of higher magnetic fields are uti-

The quantification of static 31P-MR spectra was repeatedly exploited in the past to gather information about skeletal muscle fiber composition using the PCr/Pi ratio, however, the observed scattering in metabolite content is large and the final conclusions vary [79–83], thus severely

On the other hand, 31P MRS of skeletal muscle can provide valuable information about wholebody training status, metabolic health, and/or muscle integrity. In particular, the concentration of phospholipids-phosphodiesters seems to provide a valuable surrogate of metabolism

pling, the signal of main PDE in human skeletal muscle—GPC—can be separated and used directly rather than the total PDE signal [86]. Another very recent approach for the determination of skeletal muscle oxidative metabolism from resting 31P MR spectra that profits from the increased spectral resolution of the ultra-high field systems (i.e., 7 T), is the assessment

, and small contribution of extracellular space to skeletal muscle signal, the mitochondrial matrix has been recognized as the likely origin of this pool [91]. As such, it should be able to provide direct information about changes in mitochondrial density in response to training or

in the quadriceps of the trained subjects [92] and decreased in sedentary subjects [86] in com-

31P MRS can also assess the reaction kinetics of energy metabolism through a technique called saturation transfer (ST). ST exploits the transfer of magnetization between nuclei that are in direct chemical exchange, thus estimating the unidirectional exchange rates and fluxes under steady-state conditions [4]. Unfortunately, ST experiment in skeletal muscle does not yield a net oxidative flux, as the measured flux contains a major glycolytic component and both turnover reactions operate close to equilibrium, i.e., the net rates of both glycolytic and oxidative ATP synthesis are low at rest [93]. On the other hand, the resting fluxes were correlated with parameters of oxidative metabolism [94, 95], and follow changes of oxidative metabolism

31P MRS measured during muscle contraction and recovery, i.e., dynamic 31P-MRS, can be used to observe the kinetics of intramyocellular pH and of the cytosolic concentrations of PCr, Pi, and ADP during perturbations of metabolic equilibrium. These measurements offer understanding of pH homeostasis, as well as insight into the oxidative ATP synthesis regulation driven by ATP demand. In short, to preserve stable ATP concentration, hydrolyzed ATP is resynthesized from PCr, causing PCr levels to decrease and Pi levels to increase during exercise. After the

) [91]. Based on its chemical shift (~5.1 ppm), relatively short

/Pi ratio was showed to be increased

http://dx.doi.org/10.5772/intechopen.77107

or systemic muscle damage [82, 84–90]. At ultra-high fields (i.e., 7 T), or by using <sup>1</sup>

lized [11, 12, 78].

*2.3.1. 31P MRS of resting muscle*

of alkaline pool of Pi signal (Pi2

observed in disease [96].

T1

limiting the reliability of these measurements [15].

defects of mitochondrial metabolism [15]. Thus far, Pi2

parison to normals, thus, supporting this hypothesis.

*2.3.2. Dynamic 31P MRS during exercise-recovery challenge*

H

41

H decou-

**Figure 3.** (A) A representative highly spectrally resolved static 31P-MRS spectra acquired at 7T. (B) Time course of a 31P MR spectra during a knee extension exercise with depicted depletion of the PCr signal and its subsequent resynthesis during the recovery period. (C) Saturation transfer spectra showing the effect of γ-ATP saturation, at approximately −2.48 ppm (solid line) on the Pi signal compared to the control experiment with saturation at approximately 12.52 ppm (dashed line). Adapted and reproduced from Klepochová et al. [76].

Alike 13C MRS, 31P MRS also has a lower gyromagnetic ratio in comparison to protons, and thus, suffers from lower intrinsic sensitivity. Therefore, SNR enhancing approaches, e.g., <sup>1</sup> H decoupling, at lower fields, or benefit from the SNR boost of higher magnetic fields are utilized [11, 12, 78].

### *2.3.1. 31P MRS of resting muscle*

The quantification of static 31P-MR spectra was repeatedly exploited in the past to gather information about skeletal muscle fiber composition using the PCr/Pi ratio, however, the observed scattering in metabolite content is large and the final conclusions vary [79–83], thus severely limiting the reliability of these measurements [15].

On the other hand, 31P MRS of skeletal muscle can provide valuable information about wholebody training status, metabolic health, and/or muscle integrity. In particular, the concentration of phospholipids-phosphodiesters seems to provide a valuable surrogate of metabolism or systemic muscle damage [82, 84–90]. At ultra-high fields (i.e., 7 T), or by using <sup>1</sup> H decoupling, the signal of main PDE in human skeletal muscle—GPC—can be separated and used directly rather than the total PDE signal [86]. Another very recent approach for the determination of skeletal muscle oxidative metabolism from resting 31P MR spectra that profits from the increased spectral resolution of the ultra-high field systems (i.e., 7 T), is the assessment of alkaline pool of Pi signal (Pi2 ) [91]. Based on its chemical shift (~5.1 ppm), relatively short T1 , and small contribution of extracellular space to skeletal muscle signal, the mitochondrial matrix has been recognized as the likely origin of this pool [91]. As such, it should be able to provide direct information about changes in mitochondrial density in response to training or defects of mitochondrial metabolism [15]. Thus far, Pi2 /Pi ratio was showed to be increased in the quadriceps of the trained subjects [92] and decreased in sedentary subjects [86] in comparison to normals, thus, supporting this hypothesis.

31P MRS can also assess the reaction kinetics of energy metabolism through a technique called saturation transfer (ST). ST exploits the transfer of magnetization between nuclei that are in direct chemical exchange, thus estimating the unidirectional exchange rates and fluxes under steady-state conditions [4]. Unfortunately, ST experiment in skeletal muscle does not yield a net oxidative flux, as the measured flux contains a major glycolytic component and both turnover reactions operate close to equilibrium, i.e., the net rates of both glycolytic and oxidative ATP synthesis are low at rest [93]. On the other hand, the resting fluxes were correlated with parameters of oxidative metabolism [94, 95], and follow changes of oxidative metabolism observed in disease [96].

### *2.3.2. Dynamic 31P MRS during exercise-recovery challenge*

**Figure 3.** (A) A representative highly spectrally resolved static 31P-MRS spectra acquired at 7T. (B) Time course of a 31P MR spectra during a knee extension exercise with depicted depletion of the PCr signal and its subsequent resynthesis during the recovery period. (C) Saturation transfer spectra showing the effect of γ-ATP saturation, at approximately −2.48 ppm (solid line) on the Pi signal compared to the control experiment with saturation at approximately 12.52 ppm

(dashed line). Adapted and reproduced from Klepochová et al. [76].

40 Muscle Cell and Tissue - Current Status of Research Field

31P MRS measured during muscle contraction and recovery, i.e., dynamic 31P-MRS, can be used to observe the kinetics of intramyocellular pH and of the cytosolic concentrations of PCr, Pi, and ADP during perturbations of metabolic equilibrium. These measurements offer understanding of pH homeostasis, as well as insight into the oxidative ATP synthesis regulation driven by ATP demand. In short, to preserve stable ATP concentration, hydrolyzed ATP is resynthesized from PCr, causing PCr levels to decrease and Pi levels to increase during exercise. After the challenge, the PCr buffer is restored primarily through oxidative phosphorylation allowing assessment of mitochondrial function [97]. The fitted PCr time recovery rate constant provides a good estimate on its own, however, it is pH dependent [98]. Using the calculated intracellular pH and consecutively the free ADP concentration [99], maximal oxidative capacity can be estimated providing a more robust parameter of mitochondrial capacity [15].

Unlike in static investigations, it is common to use only single spectral transient in dynamic examinations due to the high temporal resolution required (on the order of seconds) to sufficiently sample the PCr recovery time course. To boost the SNR for these experiments, highly sensitive surface receive coils are deployed and 31P signal is "localized" by their restricted sensitivity volume. However, this type of localization does not allow to differentiate signals that originate from different anatomic and/or morphologic compartments, nor between muscle groups that are recruited differently in the performed exercise (e.g., soleus and gastrocnemius during plantar flexion [100–103]). Quantification of combined signal from differently active muscles significantly skews the measurement of mitochondrial capacity [103–105]. Over the last few years, many different localization techniques have been developed for dynamic 31P MRS [103, 105–107], but as localization decreases available tissue volume and consecutively SNR, they are mainly used at ultra-high fields, i.e., 7T.

Examinations of skeletal muscle metabolism provide not only important information about muscle physiology, but can also be used to observe the effects of aging [108, 109] and/or to help define the training status [86, 110]. In addition, dynamic 31P MR examinations can identify mitochondrial defects in muscular diseases and can uncover decreased oxidative metabolism of skeletal muscle.

### **3. Muscle MRS and training**

Skeletal muscle demonstrates remarkable plasticity in functional adaptation and remodeling in response to contractile activity, i.e., exercise. Training-induced adaptations are reflected by changes in metabolic regulation, intracellular signaling, transcriptional responses and contractile protein and function [111]. Muscle mitochondrial density increases along with concomitant changes in organelle composition in just after 6 weeks of exercise training. Overall, the major metabolic consequences of the adaptations of muscle to endurance exercise are a slower utilization of muscle glycogen and blood glucose, a greater reliance on fat oxidation, and less lactate production during exercise of a given submaximal intensity [112]. Many of the named changes in skeletal muscles caused by exercise may be explored, identified, and potentially quantified by MRS (**Figure 4**). The effect of exercise can be studied from three angles: (i) direct comparison of differently trained subjects; (ii) exploration of acute exercise challenge effects; and (iii) longitudinal studies involving exercise intervention. The effect of dietary interventions on muscle metabolism and the role of MRS will also be discussed.

utilized as an energy source, similarly to glycogen [113, 114]. The use of these substrates depends heavily on the exercise intensity, and both are replenished in the recovery phase post-exercise. Similarly to IMCL, glycogen levels are also elevated in endurance-trained subjects, which promote their fatigue resistance [115, 116]. The phenomenon of increased IMCL was also termed athlete's paradox, because increased IMCL observed in obese, sedentary subjects are indicative of insulin resistance [17]; however, insulin sensitivity is not impaired in endurance-trained people [18]. IMCL content differs between individual muscle groups, depending on muscle fiber composition. In particular, lower IMCL content has been found in predominantly glycolytic, fiber type II-tibialis anterior, gastrocnemius, and vastus lateralis compared to the predominantly oxidative, fiber type I-soleus and vastus intermedius muscles [117–119]. As the concentration of carnosine is also fiber composition dependent [48, 49], it is no surprise that explosive athletes have 30% higher carnosine levels in gastrocnemius muscle compared to a reference population, whereas it is 20% lower than normal in typical endurance athletes [120]. No significant difference has been reported in acetylcarnitine (AcC) concentration between endurance-trained and

A; G-6-P, glucose 6 phosphate.

**Figure 4.** Summary of skeletal muscle metabolic processes exploitable by MRS. Linked in-figure legends denote observable effects, correlations with whole-body metabolic readouts, suggested mechanism in healthy trained, systemic metabolic disease or skeletal muscle myophaties/dystrophies and respective nucleus for MRS. Please note that several of the readouts are affected by both training status and metabolic disease and thus could serve as potential markers of training status and metabolic flexibility. Metabolites are abbreviated as follows: LCFA-CoA, long-chain fatty acid coenzyme A; IMCL, intramyocellular lipids; Cr, creatine; PCr, Phosphocreatine; ATP, adenosine triphosphate; ADP, adenosine diphosphosphate; Pi, inorganic phosphate; GPC, glycerophosphocholine; PDE, phosphodiester; Glu, glutamate; Gln, glutamine; TCA cycle, tricarboxyacid cycle; AcCarnitine, acetylcarnitine; Acetyl CoA, acetyl coenzyme

Multinuclear Magnetic Resonance Spectroscopy of Human Skeletal Muscle Metabolism…

http://dx.doi.org/10.5772/intechopen.77107

43

### **3.1. Metabolic differences in training status**

Increased IMCL content has been reported in endurance-trained muscle indicating the switch to higher utilization and efficiency of fat oxidation, as during long-lasting exercise, IMCL stores are Multinuclear Magnetic Resonance Spectroscopy of Human Skeletal Muscle Metabolism… http://dx.doi.org/10.5772/intechopen.77107 43

challenge, the PCr buffer is restored primarily through oxidative phosphorylation allowing assessment of mitochondrial function [97]. The fitted PCr time recovery rate constant provides a good estimate on its own, however, it is pH dependent [98]. Using the calculated intracellular pH and consecutively the free ADP concentration [99], maximal oxidative capacity can be esti-

Unlike in static investigations, it is common to use only single spectral transient in dynamic examinations due to the high temporal resolution required (on the order of seconds) to sufficiently sample the PCr recovery time course. To boost the SNR for these experiments, highly sensitive surface receive coils are deployed and 31P signal is "localized" by their restricted sensitivity volume. However, this type of localization does not allow to differentiate signals that originate from different anatomic and/or morphologic compartments, nor between muscle groups that are recruited differently in the performed exercise (e.g., soleus and gastrocnemius during plantar flexion [100–103]). Quantification of combined signal from differently active muscles significantly skews the measurement of mitochondrial capacity [103–105]. Over the last few years, many different localization techniques have been developed for dynamic 31P MRS [103, 105–107], but as localization decreases available tissue volume and consecutively

Examinations of skeletal muscle metabolism provide not only important information about muscle physiology, but can also be used to observe the effects of aging [108, 109] and/or to help define the training status [86, 110]. In addition, dynamic 31P MR examinations can identify mitochondrial defects in muscular diseases and can uncover decreased oxidative metabo-

Skeletal muscle demonstrates remarkable plasticity in functional adaptation and remodeling in response to contractile activity, i.e., exercise. Training-induced adaptations are reflected by changes in metabolic regulation, intracellular signaling, transcriptional responses and contractile protein and function [111]. Muscle mitochondrial density increases along with concomitant changes in organelle composition in just after 6 weeks of exercise training. Overall, the major metabolic consequences of the adaptations of muscle to endurance exercise are a slower utilization of muscle glycogen and blood glucose, a greater reliance on fat oxidation, and less lactate production during exercise of a given submaximal intensity [112]. Many of the named changes in skeletal muscles caused by exercise may be explored, identified, and potentially quantified by MRS (**Figure 4**). The effect of exercise can be studied from three angles: (i) direct comparison of differently trained subjects; (ii) exploration of acute exercise challenge effects; and (iii) longitudinal studies involving exercise intervention. The effect of dietary interventions on muscle metabolism and the role of MRS will also be discussed.

Increased IMCL content has been reported in endurance-trained muscle indicating the switch to higher utilization and efficiency of fat oxidation, as during long-lasting exercise, IMCL stores are

mated providing a more robust parameter of mitochondrial capacity [15].

SNR, they are mainly used at ultra-high fields, i.e., 7T.

42 Muscle Cell and Tissue - Current Status of Research Field

lism of skeletal muscle.

**3. Muscle MRS and training**

**3.1. Metabolic differences in training status**

**Figure 4.** Summary of skeletal muscle metabolic processes exploitable by MRS. Linked in-figure legends denote observable effects, correlations with whole-body metabolic readouts, suggested mechanism in healthy trained, systemic metabolic disease or skeletal muscle myophaties/dystrophies and respective nucleus for MRS. Please note that several of the readouts are affected by both training status and metabolic disease and thus could serve as potential markers of training status and metabolic flexibility. Metabolites are abbreviated as follows: LCFA-CoA, long-chain fatty acid coenzyme A; IMCL, intramyocellular lipids; Cr, creatine; PCr, Phosphocreatine; ATP, adenosine triphosphate; ADP, adenosine diphosphosphate; Pi, inorganic phosphate; GPC, glycerophosphocholine; PDE, phosphodiester; Glu, glutamate; Gln, glutamine; TCA cycle, tricarboxyacid cycle; AcCarnitine, acetylcarnitine; Acetyl CoA, acetyl coenzyme A; G-6-P, glucose 6 phosphate.

utilized as an energy source, similarly to glycogen [113, 114]. The use of these substrates depends heavily on the exercise intensity, and both are replenished in the recovery phase post-exercise. Similarly to IMCL, glycogen levels are also elevated in endurance-trained subjects, which promote their fatigue resistance [115, 116]. The phenomenon of increased IMCL was also termed athlete's paradox, because increased IMCL observed in obese, sedentary subjects are indicative of insulin resistance [17]; however, insulin sensitivity is not impaired in endurance-trained people [18]. IMCL content differs between individual muscle groups, depending on muscle fiber composition. In particular, lower IMCL content has been found in predominantly glycolytic, fiber type II-tibialis anterior, gastrocnemius, and vastus lateralis compared to the predominantly oxidative, fiber type I-soleus and vastus intermedius muscles [117–119]. As the concentration of carnosine is also fiber composition dependent [48, 49], it is no surprise that explosive athletes have 30% higher carnosine levels in gastrocnemius muscle compared to a reference population, whereas it is 20% lower than normal in typical endurance athletes [120]. No significant difference has been reported in acetylcarnitine (AcC) concentration between endurance-trained and untrained lean sedentary or obese sedentary volunteers [32, 121]. A recent study performed in trained and normally active subjects showed significant differences between AcC concentrations measured after overnight fast or after lunch [33]. This makes the comparison difficult and emphasizes the need for strict standardization of measurement time, dietary conditions, and physical activity (explained below) for the measurement of AcC/carnitine.

ergometer exercise. While, during 40-min recovery period, the AcC signal decayed rapidly in the trained group, it continued to rise in the untrained group [121]. Exercise that results in muscle glycogen depletion are followed by adenine nucleotide loss and muscle fatigue [116, 128]. Later on, depending on the diet and exercise regimen during the recovery, glycogen super-compensation can be seen. Comparing trained cyclists with untrained subjects, it has been shown that endurance-trained subjects resynthesize glycogen faster and are able to accu-

Multinuclear Magnetic Resonance Spectroscopy of Human Skeletal Muscle Metabolism…

or cycling [38, 58, 129, 130], but not during the sprints or repetitive bouts of strenuous exercise [129, 131], supporting the notion that increased IMCL stores serve as important energy reserves for endurance athletes. Following the exercise, repletion of IMCL stores was shown

Interventional studies focused on endurance training show an increase in IMCL after the intervention period of 4–6 weeks [133, 134]. On the other hand, 12 weeks of high-intensity training does not seem to have a similar effect [135]. This is potentially due to relative increase of type I oxidative muscle fibers during endurance training and the fact that IMCL concentration is fiber dependent, as discussed earlier. A recent overview of effects of a varying periods and different training types on the carnosine content in the vastus lateralis muscle showed that in most of them carnosine levels did not change after training. Only 8 weeks of power-training led to an increase of muscle carnosine levels [136]. Examining muscle glycogen resynthesis rate and levels after a glycogen-depleting exercise before and after 10 weeks of endurance training exposed higher glycogen concentration as well as an accumulation rate in trained than in untrained state [128], what is in good agreement with studies directly comparing trained and untrained subjects [116]. Eight weeks of endurance training also leads to lower PCr depletion and increased pH levels after exercise [137]. Similarly, the PCr resynthesis rate

max) running

45

http://dx.doi.org/10.5772/intechopen.77107

mulate more muscle glycogen during the super-compensation period [116].

IMCL depletion can be observed during prolonged submaximal (60–70% of VO<sup>2</sup>

to be dependent on the diet composition in recovery period [16, 58, 130, 132].

and muscle mitochondrial capacity can be improved by regular exercise [138].

and thus can potentially be considered an affective ergogenic aid [141].

Alternative approach to alter muscle metabolism without changing the physical activity pattern of an individual is a dietary intervention. This includes calorie restricting diets, carbohydrate loading, as well as substrate supplementation studies. Even very short, but rigorous calorie restriction in obese sedentary subjects leads to decrease in IMCL stores [139]. Although one could expect an improvement in muscular oxidative metabolism to accompany the IMCL reduction, it has been demonstrated using biopsies that mitochondrial capacity is unaltered by diet alone and can be improved only if combined with exercise intervention [140]. Creatine supplementation is often advertised as a tool to increase body mass in body building and physical sports [141]. An increase in total creatine and PCr levels in the muscle can be demonstrated [22], however no improvement in PCr resynthesis has been found after creatine supplementation [22, 142], off-putting the effect on muscle oxidative metabolism. Still, creatine supplementation leads to an increase in glycogen super-compensation [143],

**3.3. Training interventions**

**3.4. Dietary interventions**

Endurance-trained athletes also have a higher volume of mitochondrial density, and, therefore, faster oxidative metabolism which is mirrored by faster PCr resynthesis following submaximal exercise [122]. Faster PCr resynthesis has been demonstrated in comparison to untrained [123–125], and even sprint-trained athletes reflecting superior oxidative metabolism function of endurance-trained subjects [122, 126]. Gradually decreasing training status is also mirrored in decreasing 31P MRS derived measures of mitochondrial capacity and Pi2 /Pi ratio when comparing endurance-trained, lean sedentary and overweight-to-obese sedentary volunteers [86, 92, 110]. Sedentary lifestyle, if accompanied by overweight, type 2 diabetes mellitus or in connection to different muscle specific disease, gives also rise to higher PDE levels in skeletal muscle [84, 86]. Increased PDE levels, although to a much lesser extent, have been also reported in professional cyclists in comparison to normally trained men [85] and in long-distance runners compared to sprinters [82]. These increased PDE levels in highly trained or pathology hampered subjects can potentially indicate persistently damaged (and actively remodeling) muscles as the result of their training or disease. As yet, the connection of PDE to oxidative metabolism and/or muscle integrity is not completely understood.

#### **3.2. Acute exercise challenge**

From the metabolic point of view acute exercise challenge relates to changes of concentrations in energy storage pools, e.g., glycogen, lipids, or phosphocreatine, boost in the aerobic and anaerobic metabolism, lactate formation, following pH changes and effects on cell osmotic equilibrium.

From the MRS point of view: although carnosine concentration in gastrocnemius nor in soleus muscles could be influenced by a 1-h-long submaximal street run, the carnosine peak was shown to change in shape, demonstrating an exercise-induced change in pH [31]. The appearance of the second line of carnosine peak can potentially mirror the existence of two skeletal muscle compartments with different pH, possibly as a result of oxidative (slow-twitch) and glycolytic (fast-twitch) fiber composition. Acute exercise has been also shown to alter carnitine metabolism. Low-intensity exercise (below the individual's lactate threshold) does not cause significant changes in the MR detectable muscle carnitine pool, however, after only 10 min of high-intensity exercise, majority of muscle carnitine pool is redistributed to short-chain acylcarnitine. This redistribution is highlighted over a further 20 min of exercise and has long recovery period (over a 60-min) [45, 127]. Likewise, no changes in creatine (Cr) concentration were detected during exhaustive exercise, but a specific change in its methylene (Cr2) resonance line advocate for detection of compartmentation of Cr pool to bound and free sections [23].

Recently, high-intensity exercise challenge to the vastus lateralis muscle by performing squats continuously for 10 min also showed an increase in the AcC level and approximately 15 min after the cessation of exercise, AcC depletion or washout was observed [33]. Similar effect of increasing AcC levels was observed in trained and untrained subject after 30 min of cycle ergometer exercise. While, during 40-min recovery period, the AcC signal decayed rapidly in the trained group, it continued to rise in the untrained group [121]. Exercise that results in muscle glycogen depletion are followed by adenine nucleotide loss and muscle fatigue [116, 128]. Later on, depending on the diet and exercise regimen during the recovery, glycogen super-compensation can be seen. Comparing trained cyclists with untrained subjects, it has been shown that endurance-trained subjects resynthesize glycogen faster and are able to accumulate more muscle glycogen during the super-compensation period [116].

IMCL depletion can be observed during prolonged submaximal (60–70% of VO<sup>2</sup> max) running or cycling [38, 58, 129, 130], but not during the sprints or repetitive bouts of strenuous exercise [129, 131], supporting the notion that increased IMCL stores serve as important energy reserves for endurance athletes. Following the exercise, repletion of IMCL stores was shown to be dependent on the diet composition in recovery period [16, 58, 130, 132].

### **3.3. Training interventions**

/Pi

untrained lean sedentary or obese sedentary volunteers [32, 121]. A recent study performed in trained and normally active subjects showed significant differences between AcC concentrations measured after overnight fast or after lunch [33]. This makes the comparison difficult and emphasizes the need for strict standardization of measurement time, dietary conditions, and

Endurance-trained athletes also have a higher volume of mitochondrial density, and, therefore, faster oxidative metabolism which is mirrored by faster PCr resynthesis following submaximal exercise [122]. Faster PCr resynthesis has been demonstrated in comparison to untrained [123–125], and even sprint-trained athletes reflecting superior oxidative metabolism function of endurance-trained subjects [122, 126]. Gradually decreasing training status is also mirrored in decreasing 31P MRS derived measures of mitochondrial capacity and Pi2

ratio when comparing endurance-trained, lean sedentary and overweight-to-obese sedentary volunteers [86, 92, 110]. Sedentary lifestyle, if accompanied by overweight, type 2 diabetes mellitus or in connection to different muscle specific disease, gives also rise to higher PDE levels in skeletal muscle [84, 86]. Increased PDE levels, although to a much lesser extent, have been also reported in professional cyclists in comparison to normally trained men [85] and in long-distance runners compared to sprinters [82]. These increased PDE levels in highly trained or pathology hampered subjects can potentially indicate persistently damaged (and actively remodeling) muscles as the result of their training or disease. As yet, the connection of PDE to oxidative metabolism and/or muscle integrity is not completely understood.

From the metabolic point of view acute exercise challenge relates to changes of concentrations in energy storage pools, e.g., glycogen, lipids, or phosphocreatine, boost in the aerobic and anaerobic metabolism, lactate formation, following pH changes and effects on cell osmotic

From the MRS point of view: although carnosine concentration in gastrocnemius nor in soleus muscles could be influenced by a 1-h-long submaximal street run, the carnosine peak was shown to change in shape, demonstrating an exercise-induced change in pH [31]. The appearance of the second line of carnosine peak can potentially mirror the existence of two skeletal muscle compartments with different pH, possibly as a result of oxidative (slow-twitch) and glycolytic (fast-twitch) fiber composition. Acute exercise has been also shown to alter carnitine metabolism. Low-intensity exercise (below the individual's lactate threshold) does not cause significant changes in the MR detectable muscle carnitine pool, however, after only 10 min of high-intensity exercise, majority of muscle carnitine pool is redistributed to short-chain acylcarnitine. This redistribution is highlighted over a further 20 min of exercise and has long recovery period (over a 60-min) [45, 127]. Likewise, no changes in creatine (Cr) concentration were detected during exhaustive exercise, but a specific change in its methylene (Cr2) resonance line

advocate for detection of compartmentation of Cr pool to bound and free sections [23].

Recently, high-intensity exercise challenge to the vastus lateralis muscle by performing squats continuously for 10 min also showed an increase in the AcC level and approximately 15 min after the cessation of exercise, AcC depletion or washout was observed [33]. Similar effect of increasing AcC levels was observed in trained and untrained subject after 30 min of cycle

physical activity (explained below) for the measurement of AcC/carnitine.

44 Muscle Cell and Tissue - Current Status of Research Field

**3.2. Acute exercise challenge**

equilibrium.

Interventional studies focused on endurance training show an increase in IMCL after the intervention period of 4–6 weeks [133, 134]. On the other hand, 12 weeks of high-intensity training does not seem to have a similar effect [135]. This is potentially due to relative increase of type I oxidative muscle fibers during endurance training and the fact that IMCL concentration is fiber dependent, as discussed earlier. A recent overview of effects of a varying periods and different training types on the carnosine content in the vastus lateralis muscle showed that in most of them carnosine levels did not change after training. Only 8 weeks of power-training led to an increase of muscle carnosine levels [136]. Examining muscle glycogen resynthesis rate and levels after a glycogen-depleting exercise before and after 10 weeks of endurance training exposed higher glycogen concentration as well as an accumulation rate in trained than in untrained state [128], what is in good agreement with studies directly comparing trained and untrained subjects [116]. Eight weeks of endurance training also leads to lower PCr depletion and increased pH levels after exercise [137]. Similarly, the PCr resynthesis rate and muscle mitochondrial capacity can be improved by regular exercise [138].

### **3.4. Dietary interventions**

Alternative approach to alter muscle metabolism without changing the physical activity pattern of an individual is a dietary intervention. This includes calorie restricting diets, carbohydrate loading, as well as substrate supplementation studies. Even very short, but rigorous calorie restriction in obese sedentary subjects leads to decrease in IMCL stores [139]. Although one could expect an improvement in muscular oxidative metabolism to accompany the IMCL reduction, it has been demonstrated using biopsies that mitochondrial capacity is unaltered by diet alone and can be improved only if combined with exercise intervention [140]. Creatine supplementation is often advertised as a tool to increase body mass in body building and physical sports [141]. An increase in total creatine and PCr levels in the muscle can be demonstrated [22], however no improvement in PCr resynthesis has been found after creatine supplementation [22, 142], off-putting the effect on muscle oxidative metabolism. Still, creatine supplementation leads to an increase in glycogen super-compensation [143], and thus can potentially be considered an affective ergogenic aid [141].

Increase in skeletal muscle glycogen super-compensation by carbohydrate loading due to the preceding depletion exercise was also detected in longitudinal study applying 13C MRS [144]. Similar study setup where carbohydrate loading yielded glycogen super-compensation and insulin-stimulated glycogen synthesis as well as glucose-6-phosphate (G-6-P) accumulation was measured by 13C/31P MRS during hyperinsulinemic-euglycemic clamp confirmed the hypothesis that glycogen limits its own synthesis through feedback inhibition of glycogen synthase activity, as reflected by an accumulation of intramuscular G-6-P, which is then shunted into aerobic and anaerobic glycolysis [145]. Sequential 13C MRS measurement could also show that caffeine ingestion 90 min before prolonged exercise did not exert a muscle glycogen-sparing effect in athletes with high muscle glycogen content [63].

bout of aerobic exercise normalized insulin-stimulated glucose fluxes along with the normalization of whole-body insulin sensitivity in insulin-resistant offspring of T2DM patients [152], while troglitazone treatment improved the skeletal muscle glucose transport and the

Multinuclear Magnetic Resonance Spectroscopy of Human Skeletal Muscle Metabolism…

http://dx.doi.org/10.5772/intechopen.77107

Unlike in endurance-trained volunteers, where IMCLs act as an important energy source for prolonged exercise [113], accumulation of ectopic lipids inside muscle cells in untrained subjects is detrimental. Starting with obesity, through the insulin resistance toward T2DM, IMCL have an increasing tendency, showing a clear correlation between IMCL and insulin sensitivity in sedentary subjects [17], making IMCL a very good indicator of metabolic defect. However, due to the fact that endurance training also leads to increased IMCLs, i.e., due to the athletesparadox, high IMCL levels cannot be used as a marker of metabolic disorder on their alone. Muscle acetylcarnitine (AcC) levels measured at rest could be potentially used to tip the scales in the right direction, as it has been shown that while T2DM subjects have low muscle AcC concentration, endurance-trained subjects have high stores of muscle AcC [32]. Unfortunately, as the AcC levels are dependent on dietary status and physical activity [33], more studies accounting for these dependencies are required to support these initial findings. Multinuclear

MRS studies have also revealed a link between IMCL accumulation measured by 1

also been studied in different states of insulin resistance and physical fitness [155].

skeletal muscle glucose metabolism [17, 118, 154] assessed by 13C and/or 31P MRS, which has

The role of free fatty acids (FFA) and amino acids (AA) serum over-abundance on skeletal muscle glucose metabolism has been investigated in studies simulating the metabolic conditions of T2DM in young healthy men. An experimentally induced increase in plasma FA concentrations showed that substrate over-abundance decreased glucose transport and phosphorylation [156–158], and impaired skeletal muscle glycogen synthesis [156], which precedes the decrease in whole-body glucose uptake in a dose-dependent manner [157]. The observed effect of over-abundance also holds true in various conditions of insulinemia [156–158], as well as with depleted skeletal muscle glycogen [159]. Measuring skeletal glucose transport/ phosphorylation and glycogen synthesis in the skeletal muscle of young healthy men during an experimental AA challenge showed a direct effect of AA on glucose transport or phosphorylation [160] and reduced skeletal muscle glycogen synthesis. Substrate over-abundance and defects in lipid oxidation can lead to increased lipid accumulation inside the skeletal muscle. Exchange kinetics between Pi and ATP, measured by 31P MRS ST, are also decreased in T2DM in basal and glucose/insulin challenged conditions [161] as well as in the presence of increased serum FFA in healthy volunteers and hyperinsulinemic-euglycemia [162]. Slower PCr recovery rate after exercise and lower mitochondrial capacity also accompanies obesity [86] and insulin resistance [163, 164]. Similarly, increased muscle PDE levels were found in T2DM and shown to correlate with insulin resistance [84]. However, the PDE dependence on age [86, 165] has to be taken into account when using PDE to compare different metabolic groups.

Skeletal muscle pathologies are often characterized by muscle pain, weakness, and defects in skeletal muscle energetic metabolism. From the MRS point of view, changes in relative 31P

H MRS and

47

glycogen metabolism of patients with T2DM [70, 153].

**4.2. Skeletal muscle myopathies**

### **4. Muscle MRS in metabolic and skeletal muscular disease**

Variations in skeletal muscle metabolism are not only connected to training, but are also indicative of many health conditions. Whole-body metabolic disorders, e.g., insulin resistance, T2DM and metabolic syndrome are accompanied by impaired skeletal muscle metabolism [17, 84]. Similarly, skeletal muscle myopathies effect the metabolic health of skeletal muscles [146, 147]. The usability of MRS to monitor these two major groups of diseases influencing muscle metabolism will be discussed now.

#### **4.1. Insulin resistance, T2DM and substrate over-abundance**

Insulin-resistant states are characterized by hampered reactions of skeletal muscle to increased peripheral serum insulin concentrations. Insulin signaling, glucose transport and/or phosphorylation, glycogen synthesis, and glycolysis rates are reduced. Many 13C MRS studies have characterized the defects in skeletal muscle metabolism in insulin-resistant states, including experimental manipulations. These studies revealed a ~60% decrease of insulin-stimulated glycogen synthesis in overt T2DM patients [7], as well as a comparable impairment in their lean insulin-resistant offspring [62, 148] and in obese nondiabetic insulin-resistant volunteers [149]. Similar 13C MRS approaches have shown decreased postprandial skeletal muscle glycogen synthesis under normal physiologic conditions after a standard carbohydrate rich mixed meal regimen in T2DM patients [64]. In combination with 31P MRS measurement focused on glucose phosphorylation, i.e., the formation of intramuscular glucose-6-phosphate [148], 13C MRS measurements of intra- and extracellular glucose demonstrated that the lowered glucose transport is one of the main defects effecting whole skeletal muscle glucose metabolism in T2DM [150]. Excellent time resolution of labeled 13C MRS measurements of skeletal muscle resynthesis following a depleting exercise could reveal early insulin independent and subsequent insulin dependent phases of this process [151], from which the latter, insulin dependent, is impeded in insulin-resistant offspring of individuals with T2DM [62].

Combined 13C and 31P MRS has also been used to monitor the effect of lifestyle changes and pharmacological insulin-sensitizing therapy on skeletal muscle glucose metabolism. One bout of aerobic exercise normalized insulin-stimulated glucose fluxes along with the normalization of whole-body insulin sensitivity in insulin-resistant offspring of T2DM patients [152], while troglitazone treatment improved the skeletal muscle glucose transport and the glycogen metabolism of patients with T2DM [70, 153].

Unlike in endurance-trained volunteers, where IMCLs act as an important energy source for prolonged exercise [113], accumulation of ectopic lipids inside muscle cells in untrained subjects is detrimental. Starting with obesity, through the insulin resistance toward T2DM, IMCL have an increasing tendency, showing a clear correlation between IMCL and insulin sensitivity in sedentary subjects [17], making IMCL a very good indicator of metabolic defect. However, due to the fact that endurance training also leads to increased IMCLs, i.e., due to the athletesparadox, high IMCL levels cannot be used as a marker of metabolic disorder on their alone. Muscle acetylcarnitine (AcC) levels measured at rest could be potentially used to tip the scales in the right direction, as it has been shown that while T2DM subjects have low muscle AcC concentration, endurance-trained subjects have high stores of muscle AcC [32]. Unfortunately, as the AcC levels are dependent on dietary status and physical activity [33], more studies accounting for these dependencies are required to support these initial findings. Multinuclear MRS studies have also revealed a link between IMCL accumulation measured by 1 H MRS and skeletal muscle glucose metabolism [17, 118, 154] assessed by 13C and/or 31P MRS, which has also been studied in different states of insulin resistance and physical fitness [155].

The role of free fatty acids (FFA) and amino acids (AA) serum over-abundance on skeletal muscle glucose metabolism has been investigated in studies simulating the metabolic conditions of T2DM in young healthy men. An experimentally induced increase in plasma FA concentrations showed that substrate over-abundance decreased glucose transport and phosphorylation [156–158], and impaired skeletal muscle glycogen synthesis [156], which precedes the decrease in whole-body glucose uptake in a dose-dependent manner [157]. The observed effect of over-abundance also holds true in various conditions of insulinemia [156–158], as well as with depleted skeletal muscle glycogen [159]. Measuring skeletal glucose transport/ phosphorylation and glycogen synthesis in the skeletal muscle of young healthy men during an experimental AA challenge showed a direct effect of AA on glucose transport or phosphorylation [160] and reduced skeletal muscle glycogen synthesis. Substrate over-abundance and defects in lipid oxidation can lead to increased lipid accumulation inside the skeletal muscle. Exchange kinetics between Pi and ATP, measured by 31P MRS ST, are also decreased in T2DM in basal and glucose/insulin challenged conditions [161] as well as in the presence of increased serum FFA in healthy volunteers and hyperinsulinemic-euglycemia [162]. Slower PCr recovery rate after exercise and lower mitochondrial capacity also accompanies obesity [86] and insulin resistance [163, 164]. Similarly, increased muscle PDE levels were found in T2DM and shown to correlate with insulin resistance [84]. However, the PDE dependence on age [86, 165] has to be taken into account when using PDE to compare different metabolic groups.

### **4.2. Skeletal muscle myopathies**

Increase in skeletal muscle glycogen super-compensation by carbohydrate loading due to the preceding depletion exercise was also detected in longitudinal study applying 13C MRS [144]. Similar study setup where carbohydrate loading yielded glycogen super-compensation and insulin-stimulated glycogen synthesis as well as glucose-6-phosphate (G-6-P) accumulation was measured by 13C/31P MRS during hyperinsulinemic-euglycemic clamp confirmed the hypothesis that glycogen limits its own synthesis through feedback inhibition of glycogen synthase activity, as reflected by an accumulation of intramuscular G-6-P, which is then shunted into aerobic and anaerobic glycolysis [145]. Sequential 13C MRS measurement could also show that caffeine ingestion 90 min before prolonged exercise did not exert a muscle

Variations in skeletal muscle metabolism are not only connected to training, but are also indicative of many health conditions. Whole-body metabolic disorders, e.g., insulin resistance, T2DM and metabolic syndrome are accompanied by impaired skeletal muscle metabolism [17, 84]. Similarly, skeletal muscle myopathies effect the metabolic health of skeletal muscles [146, 147]. The usability of MRS to monitor these two major groups of diseases influencing

Insulin-resistant states are characterized by hampered reactions of skeletal muscle to increased peripheral serum insulin concentrations. Insulin signaling, glucose transport and/or phosphorylation, glycogen synthesis, and glycolysis rates are reduced. Many 13C MRS studies have characterized the defects in skeletal muscle metabolism in insulin-resistant states, including experimental manipulations. These studies revealed a ~60% decrease of insulin-stimulated glycogen synthesis in overt T2DM patients [7], as well as a comparable impairment in their lean insulin-resistant offspring [62, 148] and in obese nondiabetic insulin-resistant volunteers [149]. Similar 13C MRS approaches have shown decreased postprandial skeletal muscle glycogen synthesis under normal physiologic conditions after a standard carbohydrate rich mixed meal regimen in T2DM patients [64]. In combination with 31P MRS measurement focused on glucose phosphorylation, i.e., the formation of intramuscular glucose-6-phosphate [148], 13C MRS measurements of intra- and extracellular glucose demonstrated that the lowered glucose transport is one of the main defects effecting whole skeletal muscle glucose metabolism in T2DM [150]. Excellent time resolution of labeled 13C MRS measurements of skeletal muscle resynthesis following a depleting exercise could reveal early insulin independent and subsequent insulin dependent phases of this process [151], from which the latter, insulin dependent, is impeded in insulin-resis-

Combined 13C and 31P MRS has also been used to monitor the effect of lifestyle changes and pharmacological insulin-sensitizing therapy on skeletal muscle glucose metabolism. One

glycogen-sparing effect in athletes with high muscle glycogen content [63].

**4. Muscle MRS in metabolic and skeletal muscular disease**

muscle metabolism will be discussed now.

46 Muscle Cell and Tissue - Current Status of Research Field

tant offspring of individuals with T2DM [62].

**4.1. Insulin resistance, T2DM and substrate over-abundance**

Skeletal muscle pathologies are often characterized by muscle pain, weakness, and defects in skeletal muscle energetic metabolism. From the MRS point of view, changes in relative 31P metabolite concentrations, i.e., drop in PCr and increase in Pi, were observed in patients with mitochondrial myopathy [97] and Duchenne dystrophy [166]. Increased levels of PDE measured at rest can be indicative of congenital lipodystrophy [87], fibromyalgia [90, 167], or various muscular dystrophies [166, 168, 169]. Slower PCr recovery and decreased mitochondrial capacity was found in patients with chronic fatigue syndrome [170], as well as in patients with lipodystrophy [87]. Pathologic defects in muscle trimethylamine compounds-to-creatine ratio were found in facioscapulohumeral muscular dystrophy already prior to macroscopic muscle fat infiltration [171]. Furthermore, analytic *in vitro* MRS could detect alteration of lipid metabolism in patients with muscular dystrophy in early phase of the disease [172].

**Author details**

Oxford, United Kingdom

Austria

**References**

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Ladislav Valkovič1,2, Radka Klepochová3,4 and Martin Krššák3,4,5\* \*Address all correspondence to: martin.krssak@meduniwien.ac.at

1 Oxford Centre for Clinical Magnetic Resonance Research (OCMR), University of Oxford,

Multinuclear Magnetic Resonance Spectroscopy of Human Skeletal Muscle Metabolism…

http://dx.doi.org/10.5772/intechopen.77107

49

2 Department of Imaging Methods, Institute of Measurement Science, Slovak Academy of

3 High-Field MR Centre, Department of Biomedical Imaging and Image-guided Therapy,

4 Christian Doppler Laboratory for Clinical Molecular MR Imaging, MOLIMA, Vienna,

5 Division of Endocrinology and Metabolism, Department of Internal Medicine III, Medical

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#### **5. Summary**

Summarizing the knowledge gained from skeletal muscle magnetic resonance spectroscopic studies, we can say that the combination of 1 H, 13C, and 31P MRS: (i) can measure intramyocellular lipids deposition, which can be either utilized as a useful energy source in endurance-trained athletes, or is an indication of metabolic disorder (athletes-paradox); (ii) enables quantification of acetylcarnitine that may help to resolve the athletes-paradox; (iii) can improve the knowledge about buffering capacities of skeletal muscle by observing changes in lactate and carnosine metabolism; (iv) can measure glycogen metabolism and glycogenic substrate flux in the skeletal muscle under various conditions; (v) can assess oxidative and nonoxidative energy fluxes in basal and exercise challenged conditions. Taken together, it has helped to uncover defects in skeletal muscle metabolic pathways in insulin-resistant conditions; and to discover links between defects in mitochondrial activity/capacity and lipid metabolism, as well as defects in whole-body and/or muscle glucose metabolism. There is also to mention that several of the MR-derived readouts are affected by both training status and metabolic disease, and thus could serve as potential markers of training status and metabolic flexibility.

#### **Acknowledgements**

The financial support for the research of the authors at their home institutions by the Christian Doppler Society—Clinical Molecular MR Imaging (MOLIMA), by the OeNB Jubilaeumsfond (grant #15363 and #15455), by the Slovak Grant Agencies VEGA (grant #2/0001/17) and APVV (grant #15-0029), and by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society (grant #098436/Z/12/B), is gratefully acknowledged. The support of Dr. Martin Meyerspeer with adaption of **Figure 2** is also gratefully acknowledged.

#### **Conflict of interest**

None of the authors or authors' institutions have any conflicts of interest to disclose.
