Effects of in vitro exercise and modulation of SERCA on energy metabolism in human skeletal muscle cells

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Effects of in vitro exercise and modulation of SERCA on energy metabolism in human skeletal muscle

cells

Abel M. Mengeste

Thesis for the degree of Philosophiae Doctor (Ph.D.)

Section for Pharmacology and Pharmaceutical Biosciences Department of Pharmacy

Faculty of Mathematics and Natural Sciences University of Oslo

2022

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© Abel M. Mengeste, 2022

Series of dissertations submitted to the

Faculty of Mathematics and Natural Sciences, University of Oslo No. 2534

ISSN 1501-7710

reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard.

Print production: Graphics Center, University of Oslo.

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Acknowledgements

This thesis is a summation of work carried out at Section for Pharmacology and Pharmaceutical Biosciences, Department of Pharmacy, University of Oslo in collaboration with Astra Zeneca during the period of 2018-2022. I am grateful for the opportunity to perform this Ph.D.

This journey would not have been possible without the support of my beloved family, friends, and colleagues. I take this opportunity to express my gratitude to my supervisors Arild C.

Rustan, Hege Thoresen, Xiao-Rong Peng, and Vigdis Aas for your support, valuable input, and guidance. I would especially like to show my greatest appreciation to my principal supervisor Arild for your inspiring attitude, enthusiasm, and responding to my questions even on weekends. This thesis would not have been materialized without your infinite dedication, encouragement, and guidance. I owe my sincere thanks also to my co-supervisor Hege, for carrying about the progression of my projects and for always offering your help whenever I needed it.

Further, I wish to thank all the co-authors for their instrumental contributions to this accomplishment, especially Eili T. Kase, Hege Bakke, Stefano Bartesaghi, Gavin O’ Mahony, Nataša Nikolić, Jenny Lund, Andrea Dalmao Fernandez, and Parmeshwar Katare for their collaboration and constructive comments. I also want to thank the rest of my colleagues, Solveig Krapf, Christine Skagen, Nimo Osoble, and Stanislava Stevanovic. I am grateful for the amazing people at the department working at Gydas vei. In particular, the PK group for your positive energy and for allowing me to be an unofficial member of your team.

Last but not least, my deepest and sincere gratitude to my family and friends for your support, listening, and for offering me help to get my mind off of research. I also hope you can forgive me for the times when I chose work over you. Afomia, Meron, and Sofonias for your thoughts and for showing interest in my work. My mother Rahel, for your unconditional love and always believing in me. Emily, I am forever grateful for your patience, understanding, and for putting up with me through this entire journey.

Oslo, March 2022 Abel M. Mengeste

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List of publications

Paper I

Abel M. Mengeste, Jenny Lund, Parmeshwar Katare, Roya Ghobadi, Hege G. Bakke, Per Kristian Lunde, Lars Eide, Gavin O’ Mahony, Sven Göpel, Xiao-Rong Peng, Eili Tranheim Kase, G. Hege Thoresen, Arild C. Rustan.

The small molecule SERCA activator CDN1163 increases energy metabolism in human skeletal muscle cells.

Current Research in Pharmacology and Drug Discovery, ISSN 2590-2571. DOI:

10.1016/j.crphar.2021.100060.

Paper II

Abel M. Mengeste, Parmeshwar Katare^†, Andrea Dalmao Fernandez^†, Jenny Lund, Hege G.

Bakke, David Baker, Stefano Bartesaghi, Xiao-Rong Peng, Arild C. Rustan, G. Hege Thoresen, Eili Tranheim Kase.

Knockdown of sarcolipin (SLN) impairs substrate utilization in human skeletal muscle cells.

Molecular Biology Reports, PMID: 35364719. DOI: 10.1007/s11033-022-07387-0

Paper III

Abel M. Mengeste, Nataša Nikolić, Andrea Dalmao Fernandez, Yuan Z. Feng, Tuula A.

Nyman, Sander Kersten, Fred Haugen, Eili Tranheim Kase, Vigdis Aas, Arild C. Rustan, G.

Hege Thoresen.

Insight into the metabolic adaptations of electrically pulse-stimulated human myotubes using global analysis of the transcriptome and proteome.

Manuscript

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III

Abbreviations

ACC Acyl-CoA carboxylase

ACS Acyl-CoA synthetase

AMPK AMP-activated protein kinase

ATGL Adipose triglyceride lipase

ATP Adenosine triphosphate

CaMK Ca²⁺/calmodulin-dependent kinases

CD36/FAT Cluster of differentiation 36/Fatty acid translocase

CE Cholesteryl ester

CPT1B Carnitine palmitoyltransferase 1B

CYC1 Cytochrome c1

DAG Diacylglycerol

DEGs Differentially expressed genes DGAT Diacylglycerol acyltransferase ECAR Extracellular acidification rate

EPS Electrical pulse stimulation

ETC Electron transport chain

FA Fatty acid

FABPc Cytoplasmic-associated fatty acid -binding protein

FAS Fatty acid synthase

FCCP Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone

FFA Free fatty acids

FOXO1 Forkhead box O1

GLUT4 Glucose transporter 4

GO Gene ontology

HSL Hormone-sensitive lipase

IMTG Intramyocellular triacylglycerol IRS-1 Insulin receptor substrate-1

LC-MS/MS Liquid chromatography-tandem mass spectrometry

LD Lipid droplet

LIF Leukemia inhibitory factor

MAG Monoacylglycerol

MHC Myosin heavy chain

NADH Nicotinamide adenine dinucleotide

NST Non-shivering thermogenesis

OA Oleic acid

OCR Oxygen consumption rate

OXPHOS Oxidative phosphorylation

PDC Pyruvate dehydrogenase complex

PDK4 Pyruvate dehydrogenase kinase 4

PGC-1α Peroxisome proliferator-activated receptor gamma coactivator 1-alpha

PI3K Phosphoinositide 3-kinase

PKB/Akt Protein kinase B

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PL Phospholipids

ROS Reactive oxygen species

SCD1 Stearoyl-CoA desaturase 1

SERCA Sarco-/endoplasmic reticulum Ca²⁺-ATPase

shRNA Small hairpin RNA

SLN Sarcolipin

SLN-KD Sarcolipin knockdown

SR Sarcoplasmic reticulum

TAG Triacylglycerol

TCA Tricarboxylic acid

TNFα Tumor necrosis factor α

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Abstract

The increasing prevalence of obesity in both developed and undeveloped countries poses a major health issue worldwide. Chronic obesity is often associated with many comorbidities, including type 2 diabetes (T2D), and a positive energy balance, for example, due to lack of exercise, is considered to be a significant factor predisposing for its development. In the obese state, ectopic lipid deposition may occur as a result of energy overload. This accumulation of lipids in skeletal muscle has been shown to disrupt cellular functions and is associated with insulin resistance. On the other hand, exercise has proven to offer an effective strategy to counter the rate of obesity and associated disorders by enhancing energy expenditure. Recently, a thermogenic mechanism involving futile cycling of sarco-/endoplasmic reticulum Ca²⁺- ATPase (SERCA) pump activity by sarcolipin (SLN) has also received more attention due to its potential to increase cellular energy expenditure without the need for muscle contraction.

Thus, in this thesis, we aimed to investigate regulation of energy metabolism in cultured human skeletal muscle cells in response to SERCA modulation and an in vitro model of exercise, electrical pulse stimulation (EPS).

Both acute (4 h) and chronic (5 days) treatment of human myotubes with the SERCA activating compound, CDN1163, increased cellular uptake and oxidation of glucose. With respect to fatty acid (FA) metabolism, oxidation of FA in the presence of the mitochondrial uncoupler, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), was enhanced following both acute and chronic CDN1163-treatment, indicating increased oxidative spare capacity. Similarly, enhanced oxidative spare capacity was observed in CDN1163-treated myotubes by respirometry. Moreover, cells chronically exposed to CDN1163 had improved cellular FA uptake, higher rate of FA β-oxidation, and lower intracellular lipid accumulation. In contrast, depletion of SLN, a key regulator of SERCA activity in skeletal muscle, markedly diminished glucose and FA oxidation in human myotubes. Compared to control myotubes, intracellular accumulation of FA was observed to be higher in SLN-depleted cells. Moreover, total lipid formation and incorporation of FA into complex lipids were increased in these cells, and they were also more prone to de novo lipogenesis, establishing the important role of SLN in the regulation of skeletal muscle energy metabolism. Furthermore, an increase in glucose and FA oxidation was observed when cultured myotubes were challenged with EPS, resembling some of the changes in oxidative metabolism mediated by exercise in vivo. Although the underlying molecular mechanisms are still not fully elucidated, exercise is known to promote a myriad of

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adaptive responses in skeletal muscle which can ameliorate metabolic dysfunctions. We identified a number of genes, cellular proteins, as well as secreted proteins to be regulated by in vitro exercise. These regulatory molecules may have a role in coordinating the beneficial metabolic outcome following exercise. Indeed, we observed several biological processes related to muscle contraction, positive regulation of FA oxidation, oxidative phosphorylation, oxidative stress, autophagy and glycolytic pathways to be enriched following EPS. It has also been apparent that skeletal muscle produces and secrete myokines in response to exercise to induce systemic adaptations. We found leukemia inhibitory factor (LIF) to be one of the myokines increased by EPS and also showed an enhanced glucose uptake in cultured myotubes, indicating an autocrine function of LIF.

In summary, the work presented in this thesis provided a valuable resource to uncover cellular processes and regulatory molecules that underpin the beneficial metabolic adaptations of skeletal muscle to exercise. We also provided evidence for the crucial role of SLN in maintaining metabolic homeostasis in human skeletal muscle, as depletion showed to have detrimental effects on overall fuel handling. Conversely, stimulation of SERCA by CDN1163 ameliorated substrate utilization, enhanced mitochondrial efficacy, and improved lipid profile in human myotubes. Thus, intervention based on SERCA modulation may hold therapeutic potential to overcome disorders related to metabolic dysfunctions such as obesity and T2D.

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1 Introduction

1.1 Obesity, insulin resistance, and type 2 diabetes

Worldwide, the prevalence of overweight and obesity has increased dramatically over the decades [1]. According to the estimates by WHO, more than 1.9 billion adults are overweight, and of those 650 million are considered to be obese [1-3]. Even more alarming than the rise in obesity among adults is the increased prevalence of overweight and obesity among children and adolescents, reaching epidemic levels [4, 5]. Obesity is characterized by abnormal or excessive accumulation of body fat that may impair health [6] and is in practice accessed by body mass index (BMI) [7]. While BMI between 18.5 kg/m² and 24.9 kg/m²is considered normal body weight, BMI between 25-30 kg/m² and BMI over 30 kg/m² are considered overweight and obese, respectively [2, 8]. However, limitation of BMI to assess health risks for individuals is also recognized as it can vary based on ethnicity, body composition, and age [2, 6]. Thus, it has been proposed that the waist circumference might be a better and simpler measure for abdominal (visceral) fat and obesity risk [9, 10]. Among populations of European descent, the threshold values for waist circumference (≥ 102 cm in men and ≥ 88 cm in woman) implicates obesity and predicts increased risk for comorbidities [2, 10, 11].

Obesity usually arises due to an imbalance between energy intake and energy expenditure, although several other contributing factors including genetics, gut microbiota, and side effects from medications are also known to be associated with the pathophysiology of obesity [12, 13].

In addition to reducing the quality of life [14] and life expectancy [15], obesity is considered to be a major risk factor for the development of several diseases, such as cardiovascular disease, hypertension, type 2 diabetes (T2D), and certain types of cancer [8]. Of these diseases, T2D is most strongly associated with obesity, and obesity-related diabetes is projected to double to 300 million by 2025 [16]. T2D is a complex endocrine and metabolic disorder characterized by chronic hyperglycemia resulting from defects in insulin secretion, insulin action, or both [17].

While insulin sensitivity is determined by the ability of insulin to promote glucose uptake and utilization, insulin-resistant conditions occur when there is a decrease in glucose clearance in response to insulin, leading to hyperglycemia [18, 19]. The pathologic hallmark of T2D often involves the vasculature, with hyperglycemia leading to both microvascular and macrovascular complications [20, 21]. These complications cause severe damaging effects and failure of various organs resulting in loss of vision [22], kidney dysfunction [23], heart disease [24],

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amputations [25], and several other long-term consequences that contribute to morbidity and premature death [20, 21, 25]. Given the dramatic increase in the prevalence of T2D is likely caused by the increased incidence of obesity, the harmful relationship between obesity and T2D is widely investigated. It has been reported that obesity, in particular excess visceral adiposity, increases the risk of T2D through the induction of insulin resistance [19, 26]. On the other hand, subcutaneous adipose tissue may have a protective role [27], suggesting the regional distribution of body fat itself is a critical determinant of insulin sensitivity. The differences in the characteristics of adipose tissue from these two depots might explain, in part, why the metabolic effects of visceral and subcutaneous adipose differ. Visceral adipose tissue is more prone to lipolysis than subcutaneous adipose tissue and is also less sensitive to the antilipolytic effect of insulin [28-30]. As a result, during conditions of energy overload, excess fatty acids (FAs) from visceral adipose tissue are released into the circulation, leading to elevated plasma free fatty acids (FFA) [26, 28, 30, 31]. The increased FFA flux can in turn inhibit insulin signaling in liver and skeletal muscle due to excessive lipotoxicity and/or ectopic accumulation of lipid in these tissues [32-34].

In addition, obesity is associated with a state of chronic, low-grade inflammation [35]. During the development of obesity, adipose tissue can expand by increasing recruitment of new adipocytes (hyperplasia) or by increasing the size of adipocytes (hypertrophy) as an adaptive response to excess amounts of nutrients [36]. Nonetheless, adipocytes do not have an unlimited capacity to expand. Thus, the overexpansion of adipocytes in obesity may lead to the failure of adipocytes to adequately sequester excess lipids, resulting in lipid deposition in the visceral depots as well as ectopic sites, such as liver, skeletal muscle, and pancreatic β-cell [31, 35, 36].

This adipocyte hypertrophy is also accompanied by increased secretion of pro-inflammatory cytokines, including tumor necrosis factor α (TNFα), and subsequent recruitment and infiltration of macrophages, which can lead to uncontrolled inflammatory response and insulin resistance [36, 37]. Since the majority of people with insulin resistance and T2D are overweight or obese, lifestyle interventions, such as restricted calorie intake and physical activity are considered key therapeutic approaches in the prevention and treatment of insulin resistance and T2D.

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3 1.2 Energy metabolism in skeletal muscle

Skeletal muscle comprises about 40-50% of human body weight and is the most abundant insulin-sensitive organ with a crucial role in glycemic control and metabolic homeostasis.

Approximately 30% of resting metabolic rate occurs in skeletal muscle [38]. It is also a major organ responsible for the conversion of glucose and lipids. Indeed, skeletal muscle accounts for more than 80% of insulin-stimulated glucose uptake in the postprandial state [38, 39]. In addition to being the main site of glucose disposal, skeletal muscle also functions as the largest glycogen storage organ, with 4-fold the capacity of the liver [38]. Furthermore, skeletal muscle is responsible for a large proportion of whole-body lipid oxidation [40]. Given the size of skeletal muscle and its metabolic function, any change in skeletal mass, or factors affecting substrate utilization and mitochondrial function would significantly affect the whole-body energy store and metabolic homeostasis.

The initial step of glucose utilization requires the transport of glucose across the plasma membrane, which is considered to be the rate-limiting step for glucose metabolism in the basal state [41, 42]. The family of glucose transporters (GLUT) proteins facilitates this step, specifically GLUT1 and GLUT4 [43]. In skeletal muscle, GLUT1 is expressed at a low level and strictly localized to the cell surface, whereas GLUT4 is at least 10-fold more abundant than GLUT1 and is sequestered intracellularly where it is translocated to the cell surface in response to insulin stimulation and contraction/exercise [42-44]. Insulin-mediated recruitment of GLUT4 to the cell surface is initiated when insulin binds to its receptor, activating receptor kinase activity, and autophosphorylation of specific tyrosine residues, which in turn leads to phosphorylation/activation of insulin receptor substrate-1 (IRS-1), phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB/Akt) [45, 46]. Of the three Akt isoforms (Akt1, Akt2, and Akt3), Akt 2 seems to be the most important kinase for insulin-stimulated glucose transport. It has been reported that Akt2 catalyzes the phosphorylation of many proteins, including a Rab GTPase-activating protein called Akt substrate of 160 kDa (AS160, also known as TBC1D4), which is perhaps one of the most important downstream effector molecules of Akt in insulin signaling and glucose uptake [47, 48].

Once inside the cell, the cellular fate of glucose depends on one of these three major pathways.

These include 1) oxidation to pyruvate, which may undergo further oxidation in the citric acid cycle (TCA cycle); 2) storage as glycogen during rest/inactivity, and can be available for rapid utilization at a later time; and 3) conversion to intermediates essential for triacylglycerol (TAG)

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and phospholipid synthesis [18]. The first step in degradation of glucose following cell entry is its phosphorylation to glucose 6-phosphate (G-6-P) by the enzyme hexokinase, followed by glycolysis to generate pyruvate, ATP, and NADH (Figure 1). In aerobic state, the produced pyruvate is transferred into the mitochondria, where it is converted to acetyl-CoA by the pyruvate dehydrogenase complex (PDC) [49]. The activity of PDC is a major determinant of the glucose oxidation rate, as it plays the role of a gatekeeper by regulating the entry as well as the transformation of pyruvate into acetyl-CoA [18, 49]. This activity is suppressed or enhanced by pyruvate dehydrogenase kinase (PDK) and phosphatase, respectively [50]. In skeletal muscle, negative regulation of PDC activity by PDK4 inhibits the entry of pyruvate into the TCA cycle and subsequently blunts glucose oxidation [51, 52]. Once in the mitochondria, acetyl-CoA can be used in the TCA cycle for complete oxidation of glucose. However, under conditions of excess glucose, acetyl-CoA is converted to citrate and directed away from the mitochondria and back to the cytosol for synthesis of FA and cholesterol [53].

In skeletal muscle, FA flux across the plasma membrane occurs through passive diffusion or facilitated by transport proteins (Figure 1). A number of FA transporters have been identified, including plasma membrane-associated fatty acid binding proteins (FABPpm), fatty acid translocase/cluster of differentiation 36 (FAT/CD36), and a family of fatty acid transport proteins (FATP1-6) [54, 55]. These proteins, however, exhibit different capacity for FA transport and has been suggested that FAT/CD36 and FATP4 are the most efficient in skeletal muscle [56]. After transport inside the muscle cell, FAs are reversibly bound to the abundantly expressed cytoplasmic fatty acid-binding protein (FABPc), which protects against lipotoxic accumulation of FAs and traffics the FAs throughout the cellular compartments [57]. The fate of FAs within the cell depends on the metabolic status of the cell, where they either can be oxidized to provide energy, esterified to monoacylglycerol (MAG) and diacylglycerol (DAG) or stored as TAG in lipid droplets (LDs) (discussed further under “Dynamics of skeletal muscle lipid pools”) [57, 58]. In order to be oxidized, intracellular FAs are first activated to fatty acyl-CoA (acyl-CoA) by the enzyme acyl-CoA synthase (ACS), before shuttled to the outer mitochondrial membrane by acyl-CoA-binding protein (ACBP) [57, 59]. As the mitochondrial membrane is impermeable to acyl-CoA, FAs must be conjugated to carnitine palmitoyltransferase 1 (CPT1) that resides on the mitochondrial outer membrane forming acylcarnitines, allowing FAs to be transported into the mitochondria [60]. In addition, FAT/CD36 has also been found on the mitochondrial membrane and has been implicated in delivering FAs to ACSs for subsequent import into the mitochondrial matrix by CPT1 [61, 62].

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5 Once inside the mitochondria, CPT2, located in the inner mitochondrial membrane, removes carnitine from acylcarnitines to re-generate acyl-CoA [60]. Furthermore, FA acyl-CoA is degraded via β-oxidation producing acetyl-CoA, acyl-CoA shortened by two carbon atoms, NADH, and flavin adenine dinucleotide (FADH2). While the produced acetyl-CoA enters the TCA cycle, NADH and FADH2 are delivered to the electron transport chain (ETC) to produce ATP [63].

Figure 1. Overview of energy metabolism in skeletal muscle. Both glucose and fatty acids (FAs) are used as a substrate for ATP production. The binding of insulin to its receptor activates a signaling cascade, which result in the translocation of glucose transporter 4 (GLUT4) to the plasma membrane, facilitating glucose uptake. In contrast, the entry of FA is facilitated by protein-mediated mechanism.

Once in the cell, glucose is converted into pyruvate, a process catalyzed by the enzyme hexokinase (HK) or stored as glycogen. Pyruvate is transferred into the mitochondria, where it is decarboxylated into acetyl-CoA by pyruvate dehydrogenase complex (PDC) before entering the tricarboxylic acid (TCA) cycle. Intracellular FA, on the other hand, are bound to cytosolic FA binding proteins (FABPc) after entering the cell. FAs can either be oxidized or undergo esterification to form triacyglycerol (TAG) stored as lipid droplets (LDs). To do so, FAs must be activated by acyl-CoA synthase (ACS) to FA-CoA first. The formation of TAG involves the incorporation of FA-CoA in to diacylglycerol (DAG), which is catalyzed by the enzyme monoacylglycerol acyltransferase (MGAT) and the conversion of DAG into TAG by diacylglycerol acyltransferase (DGAT). Upon energy demand, TAG, DAG, and monoacylglycerol (MAG) are hydrolyzed in a sequential process by adipose triglyceride lipase (AGTL),

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hormone-sensitive lipase (HSL), and monoacylglycerol lipase (MGL), respectively. FA-CoA derived from the plasma and intracellular TAG can be shuttled to the mitochondria by acyl-CoA-binding protein (ACBP) for oxidation after transported into the matrix by the action of carnitine palmitoyltransferase (CPT) 1 and 2. Once in the mitochondria, acyl-CoA undergo β-oxidation producing acetyl-CoA, which enters the TCA cycle. Nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) generated during TCA cycle and β-oxidation are oxidized by the complexes of electron transport chain (ETC) in the oxidative phosphorylation pathway, yielding ATP. In contrast, under conditions of excess energy, acetyl-CoA generated from both glucose and FA β-oxidation is converted to citrate and released from the mitochondria and back to the cytosol for the synthesis of malonyl-CoA.

This process is catalyzed by ATP citrate lyase (ACL) and acetyl-CoA carboxylase (ACC). While citrate exert a negative feedback on glycolysis by inhibiting formation of pyruvate, malonyl-CoA acts as an inhibitor of CPT1 to reduce FA oxidation. Elevated levels of FAs can also induce expression of pyruvate dehydrogenase kinase 4 (PDK4) to suppress PDC, thereby reducing glucose oxidation. Image was created with Biorender.com.

1.3 Dynamics of skeletal muscle lipid pools

FAs stored as LDs are important fuel source of energy substrate in working muscle. It is estimated that around 50-60% of the FAs taken up by skeletal muscle are stored in LDs as TAG, where the remainder is oxidized through β-oxidation in the mitochondria [64]. In addition to TAG, myocellular LDs are also composed of DAG, cholesteryl ester (CE), long-chain acyl CoA, and ceramides, enveloped by a monolayer phospholipid membrane and different members of LD-associating protein family (referred as PAT family) [58, 65, 66]. The most characterized members of the PAT family are perilipins (PLINs) and are important in LD synthesis [58]. Two major pathways have been suggested to be involved in TAG biogenesis; the glycerol-3- phosphate and the MAG-DAG pathway [67, 68]. Both pathways use fatty acyl-CoAs, synthesized by ACSs, where the final step in these pathways is esterification of DAG into TAG [69, 70]. This reaction is conducted by the enzymes diacylglycerol acyltransferase (DGAT) 1 and 2 [70]. While the production of TAG in most tissue occurs through glycerol-3-phosphate pathway, the MAG-DAG pathway plays the main role in TAG synthesis from the absorption of food-derived fat in the small intestine [71]. In muscle, TAG in LDs is predominantly synthesized by the MAG-DAG pathway, where it begins with the acylation of MAG with fatty acyl-CoA by the enzyme monoacylglycerol acyltransferase (MGAT) to form DAG, followed sequentially by further acylation by DGAT to yield TAG [65, 72].

Myocellular LDs are considered dynamic organelles that store and release FAs upon changes in energy demand and supply [73]. When energy demand from FAs is increased, e.g. during exercise, the release of FAs from LDs through lipolysis is also increased. Lipolysis, which is

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7 the hydrolysis of TAG, is regulated by the co-ordinate actions of lipases and their interactions with LD-associated proteins [74]. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are the major lipases involved in myocellular lipolysis [58, 65], where ATGL catalyzes the initial step of lipolysis, converting TAG to DAG and the second step is catalyzed by HSL, hydrolyzing DAG to MAG [74, 75]. HSL is also capable of cleaving TAG and was once considered to be the only rate-limiting enzyme for the catabolism of TAG. However, this enzyme is most active against DAG which is hydrolyzed ∼ 10-fold faster than TAG, suggesting that this enzyme is more important as a DAG-hydrolase than TAG-hydrolase [74-76]. In the final step of lipolysis, monoacylglycerol lipase (MGL) promotes the breakdown of MAG to glycerol and FA (Figure 1).

1.4 Metabolic flexibility of skeletal muscle

As described earlier, skeletal muscles are able to utilize both glucose and FAs as a source of energy. However, which substrate is used as a primary source of energy is determined by the metabolic state such as fed or fasting state. While glucose metabolism provides the principal source of energy and substrate storage in the fed state, FA oxidation is of importance during fasting and sustained exercise [18, 63]. Nevertheless, the rate of FA oxidation declines during high-intensity exercise, as muscle glycogen became the primary energy source utilized [77].

This ability to efficiently switch between substrates for fuel and adapt to conditional changes in metabolic demand is an important feature of healthy skeletal muscle and is referred to as metabolic flexibility [78, 79]. Conversely, the loss of this ability or inadequate responses to metabolic challenges is termed metabolic inflexibility and is often associated with many pathological conditions including obesity, insulin resistance, and T2D [80].

The glucose-FA or Randle cycle provides an important link between glucose and FA metabolism involving the competition of glucose and FA for their oxidation in muscle and adipose tissue, particularly the inhibition of glucose oxidation by FAs [81]. It was proposed that increased availability of FAs could stimulate FA oxidation and decrease glucose oxidation by suppressing PDC activity via upregulation of PDK4 [52] (Figure 1). Enhanced FA oxidation can further lead to accumulation of cytosolic citrate, which in turn inhibits the rate-limiting enzyme of glycolysis, 6-phosphofructo-1-kinase, followed by an increase in G-6-P, thereby decreasing the use of glucose as a substrate while increasing the incorporation of glucose into glycogen [18, 81, 82]. On contrary, the increased glucose availability on the suppression of FA

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oxidation is often referred to as the “reverse Randle cycle” [83]. This mechanism is reflected by the citrate escaping from glucose oxidation is shuttled back to the cytosol where it is converted to acetyl-CoA by ATP citrate lyase (ACL), and ultimately carboxylated to malonyl- CoA by acetyl-CoA carboxylase (ACC). Malonyl-CoA is both an intermediate in the de novo long-chain fatty acid (LCFA) synthesis and a potent inhibitor of CPT1, which governs FA entry into mitochondria and therefore mediate an increase in FA esterification and reduced FA oxidation [84, 85].

1.5 Skeletal muscle fiber type composition

Skeletal muscle is an intricate tissue composed of a heterogeneous collection of muscle fiber types with significant variability in the mechanical, biochemical and physiological properties [86, 87]. Each muscle fiber is composed of the myofibrillar proteins actin (thin filaments) and myosin (thick filaments), where the interaction of these two filaments allows the muscle to contract [87]. Based on their speed of shortening, muscle fibers can be broadly classified as slow-twitch (type I) or fast-twitch (type II) fibers [88, 89]. The most frequently used classification is however determined by the predominant myosin heavy chain (MHC) protein isoforms (MHC I, MHC IIa, and MHC IIx) expressed in human skeletal muscle [90], combined with the contractile and metabolic properties of the fiber. This classification includes three fiber types: type I fibers (slow, oxidative, fatigue-resistant), type IIa (fast, oxidative, intermediate), and type IIx (fastest, glycolytic, fatigable) [86, 87, 89, 90]. Both type I and IIa fibers contain high number of mitochondria and obtain ATP primarily from oxidative phosphorylation, whereas the type IIx fiber possess higher level of glycolytic enzymes and glycogen content as they rely upon glycolytic processes to generate ATP [90]. There is also a positive correlation between the proportions of type I fiber and insulin sensitivity as well as glucose transporter 4 (GLUT4) protein expression [91, 92], indicating that the slow oxidative fibers are more important than type II fibers for the regulation of glucose homeostasis in response to insulin.

Moreover, a decreased proportion of type I muscle fibers is associated with various insulin- resistant states such as obesity and T2D [93-95]. Similarly, aging and physical inactivity can influence MHC isoform expression toward fast-glycolytic fiber phenotype [96, 97].

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9 1.6 Skeletal muscle thermogenesis

Specialized heat production in response to diet alterations and cold exposure is referred to as facultative or adaptive thermogenesis [98]. In rodents, heat production primarily occurs in the brown adipose tissue (BAT) through the activity of uncoupling protein 1 (UCP-1) [99, 100].

UCP-1 allows a proton leak across the inner mitochondrial membrane, which leads to heat generation instead of ATP synthesis [98-101]. In contrast to rodents, the presence of BAT in larger mammals and adult human is minimal, and have to rely on skeletal muscle as their primary site for thermogenesis [99, 102]. Skeletal muscle can be recruited to produce heat through shivering, which is a repetitive muscle contraction-relaxation process, and non- shivering thermogenic (NST) mechanisms [102, 103]. It is known that exercise-mediated muscle contractions produce a substantial amount of heat, and this contraction-based thermogenesis has been exploited by shivering during cold exposure [104-106]. However, prolonged muscle activity cannot be sustained as it results in overheating and muscle fatigue, suggesting the importance of NST mechanisms, which are independent of muscle contractions, for thermoregulation. An important mechanism for muscle-based NST in mammals involves futile cycling of Ca²⁺ between the cytosol and sarcoplasmic reticulum (SR) mediated by uncoupling of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) [102, 105, 107-109]

(discussed further under “The role of SERCA and SLN in skeletal muscle energy metabolism”).

Furthermore, futile metabolic cycle of TAG/FA, which is the cycling of two opposing and simultaneous processes of TAG synthesis and lipolysis, has been proposed to be another possible adaptive thermogenic mechanism in skeletal muscle [110-112]. In this cycle, the energy lost by the esterification of TAG followed by hydrolysis leads to heat liberation. In addition, skeletal muscle also plays an important role in diet-induced thermogenesis, mechanisms that dissipate excess calories as heat, and protect against diet-induced obesity [102, 113]. In general, the production of heat is accompanied by a concomitant increase in lipolysis of TAG and oxidation of FAs [114], which results in enhanced energy expenditure. Thus, increasing energy expenditure in muscle through shivering and non-shivering thermogenic mechanisms can affect whole-body energy metabolism and could be a useful tool to counteract obesity.

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1.7 The role of SERCA and sarcolipin in skeletal muscle energy metabolism

Over 99% of total Ca²⁺ in a normal adult human body is contained within bones and teeth as calcium-phosphate complexes, providing strength and structure through mineralization [115].

In addition to its role in the skeleton, < 1% of total body Ca²⁺ found in soft tissues and body fluids, is considered to have a pivotal role in a wide range of cellular processes crucial to the survival of all living cells [115-117]. These include cell proliferation, motility, gene transcription, muscle contraction, secretion, and apoptosis [116, 118]. Cellular Ca²⁺

concentration is tightly regulated by complex interactions among pumps, channels, binding proteins, and transporters within a narrow physiological range, and disruption of cellular Ca²⁺

homeostasis can have an important implication for disease pathogenesis [119, 120].

The cytosolic free Ca²⁺ concentration of a resting cell is preserved at very low levels (~100 nM), which is about 10.000-fold lower compared to plasma [117]. In skeletal muscle, the SERCA pump plays an important role in maintaining this low concentration of intracellular Ca²⁺-ions [105, 121, 122]. SERCA is an ATP-driven pump that actively transports Ca²⁺-ions from the cell lumen into the SR [122]. To date, more than 10 isoforms of SERCA encoded by three separate genes; ATP2A1 (SERCA1), ATP2A2 (SERCA2), and ATP2A3 (SERCA3) have been reported [123, 124]. While SERCA1a and SERCA2a are the principal isoforms present in fast-twitch glycolytic (type II) fibers and slow-twitch oxidative (type I) muscle fibers, respectively, isoforms of SERCA3 are mainly expressed in non-muscle tissues, such as platelets and epithelial cells [123, 124]. When stimulated by elevated cytosolic Ca²⁺ levels, such as during excitation-contraction coupling, SERCA removes Ca²⁺ back into the SR by utilizing the energy derived from ATP hydrolysis [108, 121, 123, 125]. This allows the restoration of Ca²⁺

levels in the cytosol, as well as SR storage. The maintenance of Ca²⁺-ion gradient in skeletal muscle by SERCA requires considerable amounts of ATP even in resting skeletal muscles, contributing to more than 40 - 50% of the resting metabolic rate [126].

The hydrolysis of ATP is coupled to the transport of two Ca²⁺-ions across the SR membrane [122]. However, SERCA has a unique ability to become “uncoupled”, a state in which less than two Ca²⁺-ions are translocated per ATP hydrolyzed, transforming the remaining energy from ATP hydrolysis into heat [103, 125]. Uncontrolled SR Ca²⁺release and sequestration can induce excessive heat production and is associated with a pathological condition referred to as malignant hyperthermia [127]. Several lines of evidence suggest that this uncoupling of SERCA in skeletal muscle is predominantly modulated by the presence of a small peptide called

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11 sarcolipin (SLN) [128-133], proposing SLN-SERCA interaction as an important mechanism for the basis of muscle NST (Figure 2). SLN is a 31-amino-acid protein expressed solely in striated and cardiac muscle [134] and is highly expressed in all skeletal muscles of adult humans [135]. In comparison to rodents, SLN mRNA expression is several fold higher in muscles of larger mammals, including humans, where UCP1 and BAT are limited, suggesting that SLN is the dominant source of thermogenesis [103, 134, 135].

Figure 2. Proposed mechanism for sarcolipin (SLN)-based non-shivering thermogenesis to enhance energy metabolism in skeletal muscle. The release of Ca²⁺ from sarcoplasmic reticulum (SR) through ryanodine receptors (RyR) leads to elevated Ca²⁺levels in the cytosol, promoting muscle contraction. Following contraction, Ca²⁺-ions are rapidly sequestered back into the SR lumen by sarco- /endoplasmic reticulum Ca²⁺-ATPase (SERCA) to mediate relaxation and re-filling of the SR Ca²⁺ store.

SERCA utilizes energy from ATP hydrolysis for the transport of Ca²⁺-ions. However, the binding of SLN to SERCA uncouples ATP hydrolysis and promote a slippage of Ca²⁺ back to the cytosol. By this mechanism, SLN induce futile cycling of Ca²⁺ and heat generation. The rise in cytosolic Ca²⁺due to slippage can further activate Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) signaling, which increase the transcriptional regulators peroxisome proliferator-activated receptor γ coactivator 1α (PGC- 1α) and peroxisome proliferator-activated receptor δ (PPARδ), thereby amplifying oxidative metabolism and mitochondrial biogenesis. Adapted from Maurya et al. [136]. Image was created with Biorender.com.

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Although the thermogenic mechanism involving SLN interaction and regulation of SERCA is not fully elucidated, recent studies have shown that when SLN binds to the transmembrane groove of SERCA, it allows ATP hydrolysis to occur, but the Ca²⁺ transport across the SR is diminished due to “slippage” of Ca²⁺ back to the cytosol [126, 129-133, 137]. Thus, the presence of SLN mediates futile cycling of the SERCA pump consuming more ATP to transport the released Ca²⁺, which result in increased heat production in muscle. In addition to heat production, prolonged cytosolic Ca²⁺ transient mediated by the uncoupling of SERCA by SLN can induce muscle oxidative metabolism and mitochondrial biogenesis [136, 138, 139].

Specifically, elevated cytosolic Ca²⁺ can activate Ca²⁺-dependent signaling pathways including calcineurin and CaMKII, which in turn promote the upregulation of PGC-1α and peroxisome proliferator-activated receptor δ (PPARδ) expressions that are known key transcriptional activators of mitochondrial biogenesis and oxidative metabolism [140-143]. At the same time, free cytoplasmic Ca²⁺ ions can also enter through the mitochondria uniporter and act as a second messenger, electing a stimulatory effect of oxidative processes and substrate catabolism [144, 145] (Figure 2). These findings have led to the search for pharmacological agents aiming to increase skeletal muscle SERCA activity as a potential approach for the treatment of disorders related to metabolic dysfunction. High-throughput screening of a compound library identified CDN1163 as a potent, allosteric SERCA-activating compound that directly binds to SERCA and upturn the activity at saturating [Ca²⁺] (Vmax) in a concentration-dependent manner [146, 147].

1.8 Metabolic adaptations associated with exercise

Regular exercise exerts a wide variety of physiological responses on whole-body metabolism that provide undisputed beneficial health effects and is known to prevent and treat many diseases, including cardiovascular disease, obesity, and T2D [148]. These effects are considered to be mediated by the adaptation of skeletal muscle to exercise. Stimulation of skeletal muscle by exercise bouts is integrated by a multitude of complex signaling networks, where the functional consequences vary by the frequency, intensity, and duration of the exercise [149].

Generally, exercise can be categorized as aerobic/endurance or anaerobic/strength-based activities [150]. While endurance exercise is typically performed against a relatively low-load with high frequency (repetition) over a long duration, strength/resistance exercise imposes a high-load with low frequency for a short duration [38, 151]. Consequently, the functional adaptations to classical endurance exercise protocols are in some ways different from the

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13 adaptations to resistance exercise, representing two extremes of the energy system (glycolytic versus oxidative) used [150]. However, both types of exercise are potent stimuli for skeletal muscle adaptions and possess the ability to stimulate mitochondrial biogenesis, improved oxidative capacity, as well as enhanced glucose uptake [38, 42, 152].

Enhanced uptake of glucose during exercise has been suggested to be mediated by an insulin- independent translocation of GLUT4 to the cellular membrane [153]. Although the exact mechanism is unclear, there is a shred of strong evidence showing that muscle contraction induces the activation of AMP-activated protein kinase (AMPK), thereby stimulating glucose uptake [154, 155]. The mechanism involves phosphorylation of TBC1D1 and AS160 (TBC1D4), which can activate the Rab GTPase and subsequent translocation of GLUT4 vesicles to the membrane (Figure 3) [42]. However, it has also been shown that mice with blunted AMPK activity had normal contraction-induced glucose uptake [156, 157], suggesting that AMPK-independent mechanisms for the stimulation of glucose transport by contraction exist. In addition, the contraction of skeletal muscle fibers triggers the release of Ca²⁺ from sarcoplasmic reticulum (SR), which leads to an increase in myocellular Ca²⁺ concentrations [158]. The spike in cytosolic Ca²⁺ has been proposed to be a signal in the induction of contraction-mediated glucose uptake through activation of Ca²⁺/calmodulin-dependent kinases (CaMK), which are upstream regulators of AMPK and Akt [158-160]. In addition to increasing glucose uptake, exercise is also associated with improved insulin sensitivities. While acute improvements (2-72 h post-exercise) in insulin sensitivity can occur after a single bout of exercise, long-term chronic improvements to insulin sensitivity are evident following repeated exercise bouts [161].

Both FA uptake, lipid synthesis, and oxidation have been shown to be enhanced in exercising skeletal muscle (Figure 3) [162-165]. During exercise with high intensity, glycogen provides the main source of energy, freeing glucose molecules for the production of ATP needed through oxidation [162]. However, during prolonged exercise at lower intensities, FA utilization becomes more important as glycogen storage is depleted [162, 164]. This action might be beneficial, as muscle fatigue is associated with glycogen depletion [166]. The increase in FA utilization in skeletal muscle during exercise is facilitated by a coordinated increase in FFA delivery, surface membrane FA transport, and intracellular substrate flux through mitochondrial β-oxidation or storage as intracellular lipids [167]. Moreover, it has been shown that type I fibers possess 3-to 4-fold higher lipid content than type II muscle fibers [168], indicating that

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the enhanced fatty acid oxidative capacity in type I fibers is also accompanied by higher intramyocellular triacylglycerol (IMTG) storage. Even though the regulation of IMTG turnover and utilization upon muscle contraction and during exercise is not clear, FAT/CD36 (probably also other lipid binding proteins) act as dynamic regulators of FA transport by relocating from intracellular compartment to the membrane in response to muscle contraction [167, 169], thereby increasing intracellular FA availability. At rest, the FA taken up by muscle enter the IMTG pool before oxidation in the mitochondria [170]. However, during muscle contraction, both exogenous FA taken up into muscle and IMTG are directly used as a substrate for energy production [164, 170]. Lipolysis of IMTG for energy production requires the activity of lipases (HSL and ATGL, discussed under “Dynamics of skeletal muscle lipid pools”). HSL and ATGL collectively account for the majority of TAG hydrolysis and are shown to be activated in response to muscle contraction [74, 75, 164, 169]. FA oxidation during exercise is also determined by the availability of FA to the mitochondria, which is facilitated by the enzyme CPT1 [60]. Increased activity of CPT1, as well as decreased content of its potent inhibitor, malonyl Co-A, has been reported to be mediated by exercise [171, 172]. Thus, the lower content of malonyl Co-A means a reduced inhibitory effect on CPT1 and an increased entry of acylated FA into the mitochondria, thereby enhancing availability and capacity for FA oxidation (Figure 1).

Skeletal muscle is a dynamic tissue with remarkable plasticity, capable of pronounced metabolic and morphological adaptations to changes in response to external stimuli, such as exercise [173]. This adaptive malleability for instance involves differentiation of the muscle fibers towards a phenotype with high mitochondrial density after endurance exercise [173, 174].

Mitochondrial remodeling by exercise can lead to changes in the metabolic properties of skeletal muscles, which are likely to be accompanied by a number of signaling cascades and transcription factors, including the activation of CaMKII and upregulation of a master regulator of mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator 1- alpha (PGC-1α) [38, 149, 173, 175].

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15 Figure 3. Illustration of contraction-stimulated cellular events involved in skeletal muscle energy metabolism. At the onset of muscle contraction, the AMP/ATP ratio and intracellular Ca²⁺ levels increases, leading to the activation of Ca²⁺/calmodulin-dependent protein kinases (CaMK) and AMP- activated protein kinase (AMPK), respectively. Activated AMPK phosphorylates TBC1 domain family member 1 and 4 (TBC1D1 and TBC1D4) to promote glucose transporter 4 (GLUT4) translocation to the membrane through activation of the Rab GTPase, which in turn increase glucose uptake. Contraction also stimulate glycogen breakdown for energy production. Simultaneously, increased AMP/ATP ratio and cytosolic Ca²⁺ levels alters the transcriptional status of various nuclear genes, including peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and peroxisome proliferator-activated receptor δ (PPARδ), thereby increasing mitochondrial biogenesis and oxidative metabolism. Furthermore, upon contraction, the activity of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase increases to stimulate triacylglycerol (TAG) lipolysis. At the same time, contraction promotes relocation of cluster of differentiation 36/fatty acid translocase (CD36/FAT) and increase the expression of carnitine palmitoyltransferase 1 (CPT1), enhancing fatty acid (FA) uptake into the cytosol and mitochondria, respectively. Image was created with Biorender.com.

1.9 Skeletal muscle as an endocrine organ

It is now well recognized that skeletal muscle is an endocrine organ capable of producing and secreting hormones, cytokines, chemokines, and peptides, which are collectively termed myokines, as a response and exercise or muscle contraction [176-178]. These molecules can in turn exert their biological effects on distant organs (endocrine) or act locally (paracrine and/or autocrine) [176, 179]. Mounting evidence suggests that myokines released from skeletal muscle

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participate in mediating the systemic benefits of exercise through their action on various tissues, such as liver, pancreas, heart, brain, and adipose tissue [179-182], providing evidence for muscle-organ crosstalk. Among these interactions, the crosstalk with adipose tissue has received more attention due to the ability of adipose tissues in exerting endocrine effect through the secretion of adipokines [183]. Adipokines, which are mostly pro-inflammatory cytokines, are secreted during obesity and physical inactivity; promote pathological conditions such as insulin resistance [36, 37]. Conversely, myokines are released during exercise to promote beneficial effects of exercise [179], leading to the hypothesis in which myokines perhaps are important in counteracting the harmful effects of pro-inflammatory adipokines. In addition to myokines, skeletal muscle also secretes other factors including amino acids, metabolites, and microRNAs (miRNA), which also are involved in cell communication [184-186]. Although myokines are hypothesized to be implicated in exercise-mediated physiological responses and whole-body metabolism, the specific biological functions of myokines in humans are not well explored.

To date, more than 650 myokines have been identified [178, 187], including myostatin, various interleukins (IL-6, IL-8, and IL-15), irisin, leukemia inhibitory factor (LIF), brain-derived neurotrophic factor (BDNF), angiopoietin-like 4 (ANGPTL4), follistatin-like protein-1 (FSTL1), and fibroblast growth factor 21 (FGF21) [177, 188-190]. While myostatin was the first myokine to be discovered, interleukin 6 (IL-6) was the first one found to be secreted into the circulation as a response to muscle contraction and is also the most characterized [179].

Although IL-6 is generally classified as a pro-inflammatory cytokine, exercise-induced IL-6 has been reported to inhibit the production of pro-inflammatory cytokines, such as TNFα and IL-1β [178, 189, 191], thereby inducing anti-inflammatory effects. In addition to its role in inflammation, contraction-induced IL-6 improves glucose and lipid metabolism via AMPK activation [192] and stimulation of GLUT4 translocation [193]. Another exercise-induced myokine that has received more attention is irisin, which is a precursor protein transcribed and translated from the FNDC5 gene [177]. Irisin is a prominent PGC-1α-dependent myokine suggested to primarily drive the browning of white adipose tissue, but has also been shown to improve insulin sensitivity as well as increase total body energy expenditure [194]. Moreover, irisin stimulates glucose uptake through the induction of AMPK activation [195], recapitulating the metabolic role of exercise-induced myokines. Other myokines such as ANGPTL4 [196], IL-15 [197], and FGF21[198] have also been shown to have a role in regulating systemic metabolism.

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2 Aims

The overall purpose of this work was to explore and uncover molecular and cellular mechanisms underlying the metabolic effects of in vitro exercise using differentiated human skeletal muscle cells and to examine a potential target that can be modulated by therapeutic interventions to increase energy expenditure (thermogenesis) in skeletal muscle. Following are the specific aims of the papers included in the thesis:

Paper I: To assess the effects of stimulating sarco-/endoplasmic reticulum Ca²⁺-ATPase (SERCA) on energy metabolism in human myotubes using a small molecule SERCA activator, CDN1163.

Paper II: To investigate the role of sarcolipin (SLN) in glucose and lipid metabolism by generating human skeletal muscle cells with stable knockdown of SLN.

Paper III: To examine energy metabolism in human skeletal muscle cells following an in vitro models of exercise and identify differentially expressed genes and proteins associated with biological processes that could be involved in mediating the beneficial metabolic effects of exercise.

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3 Summary of papers

Paper I: The small molecule SERCA activator CDN1163 increases energy metabolism in human skeletal muscle cells

This study aimed to investigate the effects of SERCA-activating compound, CDN1163, on substrate metabolism in differentiated human myotubes. Findings from both acute (4 h) and chronic (5 days) treatment with CDN1163 showed increased cellular uptake and oxidation of glucose, as well as complete oleic acid (OA) oxidation in the presence of FCCP. This was associated with enhanced oxidative spare capacity. Cellular uptake and β-oxidation of OA were also observed to increase following chronic treatment of myotubes with CDN1163. Moreover, myotubes exposed chronically to CDN1163 treatment had lower incorporation of OA into diacylglycerol (DAG) and decreased de novo lipogenesis from acetate. The improved metabolic profile in CDN1163 treated myotubes was accompanied by upregulation of the mitochondria- related genes CPT1B and PDK4, as well as increased phosphorylation of AMPK.

In conclusion, our study showed that SERCA activation by CDN1163 enhanced energy metabolism in human skeletal muscle cells, suggesting SERCA as a potential therapeutic target to overcome disorders that are related to metabolic dysfunction such as obesity.

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Paper II: Knockdown of sarcolipin (SLN) impairs substrate utilization in human skeletal muscle cells

Uncoupling of SERCA by SLN has been suggested to increase ATP consumption and heat liberation, thereby affecting whole-body energy metabolism. This work was undertaken to explore the role of SLN on energy metabolism using sarcolipin knockdown (SLN-KD) human skeletal muscle cells. Following generation of myotubes with stable SLN-KD, functional studies were conducted to investigate their metabolic properties. Our results demonstrated that depletion of SLN diminished glucose and oleic acid (OA) oxidation. These results were supported by consistently lower basal oxygen consumption observed by respirometry, indicating that the mitochondrial efficacy in SLN-KD cells was reduced. Furthermore, both endogenous and exogenous lipid synthesis were increased in SLN-KD myotubes. Additionally, Oil red O staining of the cells demonstrated higher accumulation of lipid in SLN-KD myotubes compared to control (SCR) cells. The observed metabolic perturbation in SLN-KD cells was reflected by reduced mRNA expression levels of PGC-1α and FOXO1, elucidating, at least in part, the molecular mechanism behind hampered fuel handling in these cells.

In summary, we corroborated that SLN plays an important role in regulating energy metabolism in human skeletal muscle cells. Although more studies are required for understanding the implication of SLN-SERCA interaction, SLN-based modulation of energy expenditure may represent a novel therapeutic strategy to treat obesity and obesity-related disorders.

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21 Paper III: Insight into the metabolic adaptations of electrically pulse-stimulated human myotubes using global analysis of the transcriptome and proteome.

The aim of this work was to unravel regulatory proteins and genes involved in the complex cellular processes and mechanisms underlying the metabolic adaptation of skeletal muscle to exercise. In this study, a comprehensive characterization of the expression levels of human skeletal muscle proteome, secretome, and transcriptome following in vitro exercise (electrical pulse stimulation - EPS) was performed using multi-omics approaches.

Exposure of myotubes to EPS for 24 h enhanced oxidative metabolism, resembling certain metabolic features of exercise in vivo. The proteomic and high-throughput RNA sequencing analysis revealed 81 proteins and 952 genes to be differentially expressed in myotubes submitted to EPS for 24 h. Another EPS protocol with higher voltage and duration was employed to compare the transcriptional changes between these two protocols. We found some degree of overlap between the two EPS interventions, specifically 61 genes were observed to be commonly shared between the two protocols. Further, biological processes affected by EPS- regulated proteins and genes were identified using Gene Ontology (GO) functional analysis.

Among processes important for skeletal muscle energy metabolism, muscle contraction, autophagy/mitophagy, oxidative stress, and calcium homeostasis were enriched in this analysis.

Moreover, glycolytic pathways, positive regulation of fatty acid oxidation, and oxidative phosphorylation were affected by EPS-induced protein and gene alterations. Further analysis of the secretome by proteomics revealed 137 proteins were elevated in conditioned media obtained from EPS-treated myotubes. Of these secreted proteins, our investigation of leukemia inhibitory factor (LIF) on skeletal muscle glucose uptake showed that LIF could play a role in maintaining glucose homeostasis.

In summary, our data provided new insight into the comprehensive profile of genes and proteins expressed following EPS, as well as their contribution to biological processes associated with skeletal muscle energy metabolism. However, more work is needed to understand the complex pathways and molecular mechanisms mediating metabolic adaptations in exercising muscle.

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4 Methodological considerations

4.1 Cultured skeletal muscle as an in vitro model

In skeletal muscle tissue, satellite cells have been identified as the key source of new muscle nuclei [199], and cultures of human myotubes established from satellite cells represent an essential model system for intact human muscle [200]. These cell cultures are widely used as a model of choice to elucidate muscle biology, disease mechanisms, as well as to identify and validate pharmacological compounds. As opposed to rodent cell cultures, primary human myotubes possess the most relevant genetic background and are also not immortalized, which allows the donor cells to retain several characteristics of the donors [200]. These factors combined with the controlled extracellular environment permit investigation of in vivo biochemical signaling more closely and make the utility of this cellular system very advantageous.

The studies presented in this thesis were performed using human satellite cells isolated from biopsy samples of the musculus (m.) vastus lateralis (paper I-III), m. interspinales (paper I), and m. obliquus internus abdominis (paper III). Activation, proliferation, and differentiation of satellite cells into multinucleated myotubes was achieved based on the method described by Henry et al. [201] with modifications according to Gaster et al. [202, 203]. Upon differentiation of myoblast, the progeny of satellite cells, expressions of key proteins for both glucose [204]

and lipid metabolism [205] are observed to increase. As this expression pattern of myotubes displays a characteristic resemblance to mature skeletal muscle, experimental use of myotubes is thus preferred when aiming to study skeletal muscle energy metabolism [200, 206]. The ability to shift between glucose and fatty acid (FA) oxidation (metabolic flexibility) is another characteristic that has been shown to be conserved in cultured myotubes [207, 208], resembling in vivo phenotypes.

It has been reported that primary human myotubes are generally characterized by low mitochondrial oxidative capacity, possibly due to their preference for glycolytic metabolism rather than oxidative phosphorylation which could be attributed to the absence of appropriate environmental stimuli in vitro [200, 209]. Moreover, oxidative capacity of skeletal muscle in vivo depends on fiber type composition, where the slow type I fibers have higher oxidative capacity than fast type II fibers [90]. However, myotubes in culture co-express both fast and slow MHCs independent of the fiber type from which they originated [210]. In agreement, both

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slow and fast fiber types are observed to be present in cells from our laboratory [200, 211], indicating the suitability of cultured human myotubes to determine alterations in oxidative metabolism. Nonetheless, it is important to point out that mRNA expression levels of genes involved in glucose and lipid metabolism, such as PGC-1α, CPT1B, PDK4, and CD36, have been found to be lower in cultured myotubes compared to skeletal muscle biopsies [200]. The ratio of GLUT4:GLUT1 is also lower in human myotubes when compared with skeletal muscle, resulting in lower insulin-stimulated glucose uptake in vitro compared to in vivo [204, 212].

Despite the differences in insulin responsiveness, the molecular mechanism involved in regulating glucose uptake seems to be conserved in cultured myotubes [204]. Importantly, a number of physiological functions such as the capacity of myoblasts to differentiate into myotubes, glucose uptake and oxidation, as well as FA β-oxidation deteriorates with increased passage number [213]. The studies presented in this thesis were thus performed on cells with low passage numbers (two-four passages). Collectively, although some limitations, human myotubes are well suited as a model system that allows investigations that are not otherwise possible to perform in vivo.

Furthermore, in paper II, human satellite cells obtained from biopsies from m. vastus lateralis were used to elucidate the role of sarcolipin (SLN) in skeletal muscle energy metabolism. Toward this goal, we have generated myoblasts with stable knockdown of SLN through lentiviral-mediated introduction of small hairpin RNA (shRNA) targeting SLN.

Transduction of muscle cells with an empty vector backbone (scrambled shRNA) was also performed, serving as a negative control. Lentiviral vectors are extensively used for gene knockdown in mammalian cells. This method of transduction is convenient as lentiviruses are capable of integrating into the genomes, which allows for stable integration of shRNA and long- term knockdown of the targeted gene in cultured cells [214, 215]. As shown in paper II, knockdown of SLN was successfully achieved without affecting the viability of the cells, permitting further investigation of metabolic outcomes.

4.2 Donor characteristics

Muscle biopsies from adult donors of different ages (21-68 years) were taken to establish cultured myotubes used in this thesis. The subjects used in paper II were all male, whereas both genders were included in paper I and III. Selected characteristics of the donors across the papers are summarized in Table 1.

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25 Table 1. Selected characteristics of donors used in this thesis. Range of values for selected features of the donors. M, men; W, woman; n, the number of subjects; BMI, body mass index.

Paper n Gender Age (years) Weight (kg) Height (cm) BMI (kg/m²) I

6 M 21 – 28 70.8 – 97.3 179 – 202 19.7 – 26.9

2 W 42 – 48 50 – 78 164 – 165 18.4 – 28.7

II 6 M 21 – 28 70.8 – 97.3 179 – 202 19.7 – 26.9

III

13 M 21 – 63 65 – 106.4 172 – 202 19.6 – 32.1

12 W 37 – 68 52 – 133 160 – 175 20.2 – 48.8

In skeletal muscle, several metabolic processes are affected by aging. Increasing age has been reported to be accompanied by increased content of IMTG [216], decreased skeletal muscle mass [217], reduced mitochondrial content and function [216, 218] as well as insulin sensitivity [218]. Recent data from our laboratory also shed some light on the relationship between aging and energy metabolism in cultured human myotubes, where donor age was found to be negatively associated with substrate oxidation [219]. However, many of these detrimental effects could secondary be attributed to physical inactivity and obesity [220], as regular exercise negates the effect of age-associated decline in mitochondrial function and insulin sensitivity [221].

Another important factor that has a profound influence on metabolism in vivo is gender. It has been shown that whole-body resting energy expenditure and the mass of skeletal muscle were greater in men than women [222]. With respect to muscle morphology, women seem to have more of type I muscle fibers in the vastus lateralis muscle compared to men [223], whereas the proportion of type IIa and type IIx are greater in men [224]. The levels of IMTG [224] and insulin sensitivity [225] have also been found to be higher in women than men. However, cultured human myotubes do not appear to retain intrinsic gender differences, as glucose and lipid metabolism in cultured human myotubes have been shown to be the same for men and women unless treated with sex hormones [226, 227].

In addition to gender, the donors used in paper III also varied in BMI. A study from our laboratory has previously shown a positive correlation between insulin sensitivity with BMI, whereas lipid oxidation correlate negatively to BMI when myotubes were challenged with an in vitro model of exercise [228]. Moreover, skeletal muscle from individuals with obesity

Effects of in vitro exercise and modulation of SERCA on energy metabolism in human skeletal muscle cells