The Unique and Complementary Roles of Sarcolipin and Uncoupling Protein 1 In

The Unique and Complementary Roles of Sarcolipin and Uncoupling Protein 1 in

Adaptive Thermogenesis

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Leslie Anne Rowland

Graduate Program in Biomedical Sciences Graduate Program

The Ohio State University

2015

Dissertation Committee:

Muthu Periasamy, PhD, Advisor

Tsonwin Hai, PhD

Kamal Mehta, PhD

Douglas Pfeiffer, PhD

Leslie Anne Rowland

2015

Abstract

Thermogenic mechanisms are well known to influence body temperature maintenance as well as energy balance, including body mass and composition.

Uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) is a substantial component of cold-induced thermogenesis, and loss of UCP1 in mice results in severe diet-induced obesity. However, UCP1-knockout (UCP1-KO) mice can readily adapt to cold by gradual acclimatization, and under certain conditions, UCP1-KO mice do not develop diet- induced obesity. Furthermore, BAT function is either absent (birds and marsupials) or is minimally present as in adult mammals, including humans, yet these organisms maintain thermogenesis, suggesting other components are involved. Sarcolipin (SLN), a regulator of the sarcoplasmic reticulum Ca2+ transport ATPase (SERCA) in muscle, was recently identified as a significant contributor to cold-induced thermogenesis and diet-induced energy expenditure. When bound to SERCA, SLN has been shown to uncouple Ca2+ transport from ATP hydrolysis and increase heat production. However, the mechanistic basis for SLN in thermogenesis has not been fully elucidated. The studies here sought to further our understanding of how SLN contributes to both cold-induced thermogenesis and diet-induced thermogenesis. The major goals were: 1) to demonstrate that SLN-based heat production in muscle is a major mechanism for thermogenesis especially when BAT function is minimal and 2) to demonstrate that SLN is involved in diet-induced ii thermogenesis. Because BAT is a dominant contributor to thermogenesis in mice, we utilized the UCP1-KO mouse model and a double knockout (DKO) mouse for SLN and

UCP1 to uncover the role of SLN. Our data showed that the DKO mice are severely cold- sensitive, indicating both SLN and UCP1 are important for cold-induced thermogenesis.

Importantly, we also found that UCP1 and SLN compensate for the loss of one another during cold adaptation, suggesting muscle and BAT play complementary thermogenic roles. Surprisingly, the DKO mice were able to survive gradual cold exposure, though at an extremely high energetic cost, with significant weight loss and depletion of fat stores.

Together, these studies suggest that UCP1 and SLN are required to maintain optimal thermogenesis and loss of both systems compromises survival of mice under cold stress.

We next sought to determine how the DKO mice would respond to diet-induced obesity by feeding a high fat diet (HFD). After 12 weeks of HFD-feeding UCP1-KO and SLN-

KO mice became obese and had similar weight gains and metabolic efficiencies.

Surprisingly, the DKO mice gained weight equally, but no more than, the single knockout mice. These data suggest that while SLN and UCP1 play additive roles in cold-induced thermogenesis, they do not appear to compensate for one another in response to diet. That is, both SLN and UCP1 are required for diet-induced thermogenesis, while either SLN or

UCP1 is sufficient for body temperature maintenance. The studies performed here establish that SLN is a significant contributor to thermogenesis and has broader implications in our overall understanding of muscle metabolism, energy expenditure, and obesity in animals where BAT content is limited, including humans.

iii

Dedication

This document is dedicated to my parents.

Acknowledgments

First and foremost, I would like to acknowledge Dr. Periasamy for his enduring support, unabated criticisms, and guidance over the years. I am very thankful for the independence I was given in my research projects, which has allowed me to develop the confidence to become an independent researcher. Secondly, I am extremely grateful for

Dr. Naresh Bal for his experimental guidance, thoughtful discussions, and help in performing my experiments. Dr. Bal’s work also set the groundwork for the entirety of this dissertation and for that, I am indebted. I would also like to acknowledge Dr. Santosh

Maurya and Dr. Sanjaya Sahoo, whom have provided helpful criticisms and ideas to improve my research projects. Importantly, I would like to thank my fellow graduate students: Meghna Pant, Danesh Sopariwala, Sana Shaikh, and Joseph Ostler, for their support, friendship, countless scientific discussions, and much-needed empathy. In addition, though our experimental efforts did not result in the completion of a successful study, I need to acknowledge Dr. Adam Rauckhorst for his countless hours teaching and helping me perform experiments and answering my many questions. Finally, I would like to acknowledge my family and close friends for their support, patience, and continued belief in me.

Vita

2002 ...... East Palestine High School

2006 ...... B.S. Biochemistry, Duquesne University

2009 to present ...... Graduate Associate, Department of

Physiology and Cell Biology, The Ohio

State University

Publications

Huang W, Bansode RR, Xie Y, Rowland L, Mehta M, Davidson MO, Mehta KD.

Disruption of the murine protein kinase Cbeta gene promotes gallstone formation and alters biliary lipid and hepatic metabolism. J Biol Chem. 2011 Jul 1; 286(26):22795-805.

Bal NC, Maurya SK, Sopariwala DH, Sahoo SK, Gupta SC, Shaikh SA, Pant M,

Rowland LA, Bombardier E, Goonasekera SA, Tupling AR, Molkentin JR, Periasamy

M. Sarcolipin is a newly identified regulator of muscle-based thermogenesis in mammals.

Nat. Med. 2012 Oct;18(10):1575-9.

Rowland LA, Bal NC, Periasamy M. The role of skeletal muscle-based thermogenic mechanisms in vertebrate endothermy. Biol Rev Camb Philos Soc. 2014 Nov 25.

Maurya SK, Bal NC, Sopariwala DH, Pant M, Rowland LA, Shaikh SA, Periasamy, M.

Sarcolipin is a key determinant of basal metabolic rate and its overexpression enhances energy expenditure and resistance against diet induced obesity. J Biol Chem. 2015 Feb

24.

Rowland LA, Bal NC, Kozak LP, Periasamy M. Uncoupling Protein 1 and Sarcolipin

Are Required to Maintain Optimal Thermogenesis and Loss of Both Systems

Compromises Survival of Mice. J Biol Chem. 2015 March 30.

Fields of Study

Major Field: Biomedical Sciences Graduate Program

vii

Table of Contents

Abstract ...... ii

Acknowledgments ...... v

Vita ...... vi

Table of Contents ...... viii

List of Tables ...... xiii

List of Figures ...... xiv

Chapter 1: Adaptive Thermogenesis and Obesity...... 1

1.1. General Introduction ...... 1

1.2. Adaptive Thermogenesis ...... 2

1.3. Brown adipose tissue (BAT) as a site of adaptive thermogenesis...... 3

1.3.1. BAT and cold-induced thermogenesis...... 4

1.3.2. BAT and diet-induced thermogenesis ...... 6

1.3.3. Beige/Brite cells: Inducible brown adipocytes ...... 7

1.3.4. Evidence for BAT-independent adaptive thermogenesis ...... 7

1.4. Skeletal muscle as a site of adaptive thermogenesis ...... 8 viii

1.4.1. Evolutionary origins of skeletal muscle-based thermogenesis ...... 9

1.4.2. Evidence for muscle-based NST ...... 10

1.4.3 Modes of thermogenesis in small versus large mammals...... 13

1.4.4. Calcium cycling as a mechanism for NST...... 15

1.4.5. Diet-Induced Thermogenesis in Muscle ...... 16

1.5. Evidence for Sarcolipin-mediated adaptive thermogenesis ...... 17

1.3.1. Sarcolipin is an uncoupler of the SERCA pump ...... 18

1.5.2. Loss of Sarcolipin increases cold-sensitivity of mice ...... 19

1.5.3. Sarcolipin protects against diet-induced obesity ...... 20

1.6. Gaps in knowledge ...... 20

Tables and Figures for Chapter 1 ...... 22

Chapter 2. Sarcolipin and Uncoupling Protein 1 are required to maintain optimal thermogenesis and loss of both systems compromises survival of mice under cold stress.

...... 26

2.1. Research Design and Methods ...... 28

2.1.1. Animals...... 28

2.1.2. Acute cold exposures...... 29

2.1.3. Gradual cold adaptation...... 29

2.1.4. Metabolic monitoring...... 30

2.1.5. Histology of adipose tissues ...... 30

2.1.6. Urinary catecholamine output...... 30

2.1.7. Western Blotting...... 30

2.1.8. Statistical Analysis...... 31

2.2. Results ...... 31

2.2.1. Gradual cold adaptation of UCP1-KO and SLN-KO mice...... 31

2.2.2. Increased energy expenditure in SLN-KO mice...... 33

2.2.3. UCP1/SLN double knockout (DKO) mice show poor survival rate at 22°C. ..33

2.2.4. Acute cold challenge of mice housed at 22°C versus 28°C...... 34

2.2.5. DKO mice can survive gradual cold exposure ...... 35

2.2.6. DKO and UCP1-KO mice have elevated catecholamine levels during their

exposure to cold...... 36

2.2.7. Activity-dependent thermogenesis is not different among genotypes...... 37

2.2.8. Loss of SLN leads to decreased reliance on muscle-based thermogenesis...... 37

2.3. Discussion ...... 39

Figures and Tables for Chapter 2 ...... 44

Chapter 3. Sarcolipin and Uncoupling Protein 1 contribute to DIT independently and do not compensate for one another in response to diet overload...... 55

3.1. Research Design and Methods ...... 56

3.1.1. Animals...... 56

3.1.2. High Fat Diet Feeding...... 57

3.1.3. Metabolic Monitoring...... 57

3.1.4. Blood glucose and lipid levels...... 57

3.1.5. Measurement of fat pad and muscle weights...... 58

3.1.6. Western Blotting...... 58

3.1.8. Statistical Analysis...... 58

3.2. Results ...... 59

3.2.1. DKO mice do not gain more weight on HFD than SLN-KO and UCP1-KO

littermates...... 59

3.2.2. Metabolic Rate...... 60

3.2.3. SLN and UCP1 do not compensate for one another in response to HFD

feeding...... 61

3.3. Discussion ...... 62

Figures and Tables for Chapter 3 ...... 65

Chapter 4. Sarcolipin in Non-Rodent Species ...... 72

4.1. Research Design and Methods ...... 73

4.1.1. Sequence analysis...... 73

4.1.2. Western blotting...... 73

4.1.3. Cloning of avian SLN DNA sequences...... 74

4.2. Results ...... 74

4.2.1. Sarcolipin-like protein sequences can be found in species from fish to man...74

4.2.2. Sarcolipin in large mammals ...... 75

4.2.3. Sarcolipin expression in birds ...... 76

4.3. Discussion ...... 77

Figures and Tables for Chapter 4 ...... 80

Chapter 5. Discussion ...... 85

5.1. Summary ...... 85

5.2. Significance of this work ...... 89

5.3. Future Directions ...... 90

Figures and Tables for Chapter 5 ...... 94

References ...... 95

xii

List of Tables

Table 1.1. Characteristics and compositions of tissues contributing to thermogenesis in extant endotherms...... 22

Table 2.2. Observed and predicted frequencies of offspring from UCP1+/-.SLN+/- intercrosses when reared at 22°C and 28°C...... 44

Table 3.1. Heart and muscle weights after 12-weeks of high-fat diet feeding...... 65

xiii

List of Figures

Figure 1.1. Components of total daily energy expenditure in mammals...... 23

Figure 1.2. Mechanism of UCP1...... 24

Figure 1.3. Mechanism of Sarcolipin...... 25

Figure 2.1. Changes in skeletal muscle after cold adaptation...... 45

Figure 2.2. Changes in adipose tissue after cold adaptation...... 46

Figure 2.3. Energetics of cold adaptation...... 47

Figure 2.4. White adipose tissue after cold adaptation...... 48

Figure 2.5. Acute cold exposure...... 49

Figure 2.6. Catecholamine levels throughout cold adaptation...... 50

Figure 2.7. Physical activity before and after cold adaptation...... 51

Figure 2.8. CPT1 and LDH expression in cold-adapted muscles...... 52

Figure 2.9. SERCA expression in cold-adapted muscles...... 53

Figure 2.10. Calsequestrin expression in cold-adapted muscles...... 54

Figure 3.1. Results of high fat diet feeding...... 66

Figure 3.2. Fat pad weights after HFD-feeding...... 67

xiv

Figure 3.3. Levels of blood metabolites...... 68

Figure 3.4. Metabolic rates of mice before and after HFD-feeding...... 69

Figure 3.5. UCP1 expression after HFD-feeding...... 70

Figure 3.6. SLN expression after HFD-feeding...... 71

Figure 4.1. Alignment of SLN protein sequences from various vertebrate species...... 80

Figure 4.2. Analyses of SLN and SERCA expression in rabbit skeletal muscle...... 81

Figure 4.3. Comparison of SLN expression in large mammals versus mice...... 82

Figure 4.4. Sarcolipin DNA cloning strategy and results...... 83

Figure 4.5. Western blot analysis SLN protein expression in house sparrow pectoralis, chicken pectoralis, rabbit quadriceps, and mouse atria...... 84

Figure 5.1. Proposed model showing relative contributions of UCP1 and SLN to thermogenesis in birds and mammals...... 94

Chapter 1: Adaptive Thermogenesis and Obesity

1.1. General Introduction

Obesity is rapidly becoming one of the most prevalent disease conditions; as of

2009, one-third of the United States population was clinically obese (1). Strikingly, a recent report by the World Health Organization found that, as of 2008, one of every nine humans worldwide are obese (2). Obesity is a disease arising from both genetic and environmental factors; although, it has been reported that 40-70% of the variation in body mass index (BMI), a measure of obesity, is a result of genetic factors (3). Unfortunately, these genetic and/or molecular mechanisms contributing to obesity are poorly understood.

In addition to the health burden of obesity itself, there are a host of comorbidities associated with the disease, further increasing obesity-associated healthcare costs.

Obesity and overweight are known risk factors for conditions including, but not limited to, type II diabetes, cardiovascular diseases, various cancers, asthma, gallbladder disease, osteoarthritis, and chronic back pain (4). Regrettably, there are few, if any, truly effective treatments for obesity. Most existing non-surgical treatment options come with undesirable or intolerable side effects, and surgical interventions often offer only temporary weight loss. For these reasons, it is becoming increasingly important to define

1 the genetic and/or molecular factors that contribute to the disease in order to develop more effective therapies.

Weight gain is a result of energy imbalance; that is, when energy (caloric) consumption exceeds energy expenditure. Methods to increase energy expenditure include physical activity and exercise. Another method is to increase energy expenditure at rest. To do this, the mechanisms contributing to metabolic rate must be identified (5).

The purpose of this dissertation is thus to better understand how skeletal muscle and brown adipose tissue uniquely and in concert contribute to energy expenditure to provide insight into developing weight loss therapies in the future.

1.2. Adaptive Thermogenesis

Whole-body energy expenditure has many contributing factors as detailed in

Figure 1.1. Basal metabolic rate is the energy utilized by all organs for basic functions in the resting state. Energy is also utilized for the digestion and absorption of food, physiological functions like reproduction and growth, and physical activity (5). Finally, energy can be utilized in response to changes in environmental temperature or food intake. These latter processes are collectively termed “adaptive thermogenesis.” Adaptive thermogenesis occurs in distinct tissues, namely brown adipose tissue and skeletal muscle, and is highly regulated, thus making it an attractive target to increase energy expenditure (5-6).

All endotherms must maintain constant body temperature. When the environmental temperature is low and basal metabolic rate becomes insufficient to

2 produce enough heat to maintain body temperature, adaptive thermogenesis is activated

(5,7). This so-called “cold-induced” adaptive thermogenesis has two components: shivering and nonshivering thermogenesis (NST). Shivering produces heat by repetitive muscle contractions and cannot occur for long periods of time. Thus, NST must be recruited to supplement and/or replace shivering. Classically, NST has been attributed to the function of brown adipose tissue, though mounting evidence suggests skeletal muscle plays a key role in NST (8-13).

The other type of adaptive thermogenesis is diet-induced thermogenesis (DIT).

DIT is defined as the increase in energy expenditure in response to high caloric intake

(5,14). Essentially, DIT is a calorie-wasting mechanism to combat the deleterious effects of an energy-dense diet and is a mechanism for the body to defend its “fat mass set point.” (14). As with cold-induced thermogenesis, the majority of studies have shown

DIT occurs in brown adipose tissue, though sufficient evidence exists for DIT occurring in skeletal muscle as well (8,15-17). The following sections will detail the existing knowledge of how brown adipose tissue and skeletal muscle contribute to both aspects of adaptive thermogenesis.

1.3. Brown adipose tissue (BAT) as a site of adaptive thermogenesis

The role of BAT as thermogenic organ has been recognized only rather recently, with its first description occurring in 1961 (8). BAT is a unique organ in that it is specialized for heat production and/or energy expenditure. To date, functional BAT has only been identified in mammalian species (18-20). In rodents, BAT is found in the

3 subcutaneous interscapular region (the largest depot) as well as in smaller depots dispersed throughout the body (perirenal, periaortic, axillary, etc.) (21-22). In most other mammals, BAT is also found in the neck region. BAT is different from white adipose tissue in that it is highly vascularized, has large numbers of mitochondria, contains multilocular lipid droplets, and expresses the protein Uncoupling Protein 1 (UCP1) (8).

Heat generation in BAT is dependent on the expression and activity of UCP1 (23-

24). UCP1 is located in the inner mitochondrial membrane and functions as a fatty acid/H+ symporter, transporting H+ ions generated by the electron transport chain from the intermembrane space to the mitochondrial matrix (23). Heat is produced due to the conversion of potential energy by UCP1 activity (Figure 1.2). Thus, UCP1 is so named because it uncouples fuel oxidation; i.e. electron transport chain activity, from ATP production.

1.3.1. BAT and cold-induced thermogenesis

The role of BAT in cold-induced adaptive thermogenesis has been well established over the past 50+ years. The majority of studies have been performed on rodents, so those studies will be the focus of this section. When rodents are exposed to temperatures below thermoneutrality; i.e. the temperature at which basal metabolic rate is sufficient to maintain body temperature, BAT is recruited. Recruitment of BAT is evident by a 10-fold increase in UCP1 protein expression, increased mitochondrial content and enzymes, increased substrate oxidation enzymes, increased sympathetic innervation and capillarization, and hyperplasia (8).

BAT activation and recruitment is mediated by the sympathetic nervous system.

Cold temperatures are sensed by peripheral thermoreceptors and reduced body temperature is sensed by the hypothalamus, leading to the release of norepinephrine (NE) by BAT neurons (25). NE activates the beta 3-adrenoreceptor, which is highly expressed in BAT. NE has multifold effects on BAT: 1) NE stimulates the differentiation of brown preadipocytes to brown adipocytes to mature brown adipocytes possessing the requirements for full thermogenic capacity, 2) NE directly regulates the expression of the

Ucp1 gene, 3) NE stimulates lipolysis and fatty acid oxidation to supply the energy for thermogenesis, and finally, 4) NE drives UCP1 activity through its lipolytic effects (fatty acids activate UCP1) (8,10,26).

Early studies on cold-adapted rats estimated BAT could account for ~60% of the cold-induced increase in oxygen consumption, suggesting BAT is the dominant site of

NST (27). The significance of BAT to cold-induced thermogenesis has more recently been substantiated by elegant studies performed using UCP1-knockout (UCP1-KO) mice developed by Dr. Leslie Kozak (24). Since UCP1 is required for BAT thermogenic function, UCP1-KO mice are considered a BAT-deficient model. These studies have shown that UCP1-KO mice are extremely sensitive to acute cold exposure, developing hypothermia within minutes to hours at 4°C (24,28). Though UCP1-KO mice are able to survive in the cold when gradually adapted, their survival comes at a much greater energetic cost, as indicated by greater oxygen consumption compared to wild-type controls (28-29). In addition, UCP1-KO mice have reduced longevity when housed in the cold (30).

1.3.2. BAT and diet-induced thermogenesis

Rothwell and Stock were the first to implicate BAT in diet-induced thermogenesis

(16). Their pioneering study showed that rats fed a cafeteria diet (a model of overfeeding) had a modest 27% increase in body weight, compared to control-fed rats, despite consuming 80% more calories. Moreover, the cafeteria rats had a 100% increase in total energy expenditure, even when body weight was taken into consideration. The authors astutely observed BAT mass increased in the cafeteria rats and further showed increased skin temperature over the interscapular BAT region of cafeteria-fed rats administered norepinephrine. These results are consistent with increased heat production or blood flow through the tissue, which are also seen cold-adapted rats (16).

Since this study, many others have reported on the recruitment of BAT in response to obesogenic diets, as well as defective BAT function in several models of obesity (31-32). An interesting study by Jan Nedergaard’s group showed that UCP1-KO mice became obese on a high-fat diet (HFD) when housed at thermoneutrality, which the authors concluded was a result of the lack of diet-induced thermogenesis (15). This study also showed that UCP1 protein was increased 4-fold in wild-type mice fed the HFD, compared to chow-fed controls. Interestingly, there was no difference in resting metabolic rate between the HFD-fed UCP1-KO and wild-type mice. However, HFD-fed wild-type mice had a strong augmentation in their thermogenic response to norepinephrine, compared to chow-fed mice - an effect that was abolished in the UCP1-

KO mice (15).

1.3.3. Beige/Brite cells: Inducible brown adipocytes

In more recent years it has come to light that certain white adipose tissue depots have the potential to acquire a thermogenic capacity, qualitatively similar to BAT. This phenomenon, termed “browning” or “beiging” of white adipose tissue occurs in response to cold exposure, as does classical BAT (33-34). Compensatory browning of white adipose tissue has been shown to occur in mouse models of defective classical BAT thermogenesis, indicating beige fat has a similar thermogenic function (35-36). In addition, studies have shown recruitment of beige fat confers similar metabolic benefits as classical BAT (37-38).

Beige fat can be induced by other physiological stimuli as well, including exercise, or pharmacologically, with chronic beta 3-adrenoreceptor agonism, peroxisome proliferator-activated receptor gamma (PPAR gamma) agonism, etc. (21,39).

Interestingly, classical brown adipose tissue and beige fat have different developmental origins: classical BAT arises from the skeletal muscle lineage, whereas beige fat has a smooth muscle origin (40-41). Recently, the regulation, origins, and functions of beige fat have become hot research topics, due to the finding that the molecular signature of human BAT more closely resembles that of rodent beige fat than classical BAT (42-43).

1.3.4. Evidence for BAT-independent adaptive thermogenesis

While BAT is unequivocally the dominant thermogenic organ, especially in rodents, there is evidence to support the existence of other sites of thermogenesis. Some of the most convincing evidence stems from studies on UCP1-KO mice that have

7 repeatedly shown though UCP1-KO mice are extremely sensitive to acute cold exposure, they can be gradually adapted to 4°C (28,44). Additionally, the cold sensitivity of UCP1-

KO mice is incompletely penetrant and highly dependent on genetic background: 32.1% of C57Bl/6 and 87.5% of 129SV/J are cold sensitive (28). Moreover, studies performed on UCP1-KO mice indicate the existence of a BAT-independent increase in metabolism in response to diet overload (8). Researchers have identified a few UCP1-independent thermogenic mechanisms, including Ca2+ cycling in skeletal muscle, thermogenesis from white adipose tissue (UCP1-independent), and substrate cycling in skeletal muscle, but none of these mechanisms were found to be dominant, or able to compensate fully for the lack of UCP1 in mice (28,45-47).

1.4. Skeletal muscle as a site of adaptive thermogenesis

It is well known that skeletal muscle produces heat. Muscle contractions from physical activity generate considerable amounts of heat, and contraction-based heat production is exploited by shivering during cold exposure (48-59). Indeed, when maximally recruited, as during exercise or an intense bout of shivering, muscle can account for up to 90% of whole-body oxygen uptake, an indirect measure of heat production (60). During muscle contraction, heat is generated by the hydrolysis of ATP from three different ATPases: myosin ATPase (61-63), which performs the contractile work, and SERCA (11,13,63-71) and Na+/K+ ATPase (72-77), which reset resting ion gradients and membrane potential. To sustain these processes, ATP generation must be

8 increased to match demand. These obligatory metabolic processes also generate heat, adding to total heat production.

However, shivering alone is insufficient for the maintenance of body temperature, and nonshivering mechanisms must be activated to sustain sufficient heat production. As discussed above, BAT is an important site of NST, but it is less well understood whether and/or how muscle contributes to NST, though there is much evidence throughout the animal kingdom to support a role for muscle in NST, as will be discussed below.

1.4.1. Evolutionary origins of skeletal muscle-based thermogenesis

Striated muscle can be considered the most primitive facultative thermogenic organ. Fish were among the earliest vertebrate species to evolve, and evidence for muscle-based heat production can be observed in certain fish species that show characteristics of endothermy. While the vast majority of fish are ectotherms, species from the families Lamnidae (sharks) and Scombridae (tunas) can achieve some level of whole-body homeothermy through a modified myotomal muscle (78-79). In these species, this slow-twitch, oxidative muscle is located in a medial location, in contrast to the myotomal muscle of ectothermic fish, permitting heat conservation (79-80). The muscle is also perfused by counter-current heat exchangers, which minimize heat loss

(79). A known characteristic of these species is their continuous swimming behaviour; these sustained contractions combined with the heat-conservation mechanisms described above result in net heat production and represent the source of endothermy in these fishes

(78-79,81-83).

Another fish species known to exhibit endothermy is the opah (Lampris guttatus).

Rather than achieving whole-body homeothermy, opahs have developed regional endothermy, which utilizes localized heat production specifically to warm the cranial region (78). Cranial endothermy is an important ecological adaptation that provides a selective advantage as it protects the central nervous system from rapid changes in ambient temperature and enhances vision and detection of prey. To achieve cranial endothermy, opahs employ contractions of the extraocular muscles in order to elevate cranial temperatures when water temperatures are low (78). The evidence thus shows that heat can be generated by muscle, and that muscle can be recruited, in non-homeothermic species; perhaps this represents the most primitive form of regulated heat production.

1.4.2. Evidence for muscle-based NST

The above sections emphasized that muscle can be recruited for thermogenesis, even in non-homeothermic species, but in these cases, the heat-producing capability of muscle is dependent upon contraction. However, muscle can be modified to produce heat in a nonshivering manner without being coupled to contraction. Evidence for muscle- based NST is found in certain fishes with the capacity for regional endothermy. These species, including billfishes, tuna, and mackerel, utilize modified muscle tissue as a means of achieving endothermy Block, 1986, 1994; Carey, 1982; Dickson and Graham,

2004). This specialized muscle tissue, termed the “heater organ”, is derived from eye muscle and is located in the cranial region. The heater organ is composed of cells that lack the typical myofibrillar structure and have very low expression of actin and myosin

(84). Heater organ cells are densely packed with mitochondria and sarcoplasmic reticulum (SR) networks (13,81). The extensive SR of heater organs is organized into tightly packed stacks, which optimizes surface area for SERCA. Studies have indicated that, in addition to SERCA, the heater organ expresses a Ca2+ release channel (CRC) and calsequestrin (CASQ), which are distributed rather homogeneously throughout the SR

(85-86). This is in contrast to skeletal muscle, where the CRC and CASQ are commonly located at specific sites in the SR, called triad junctions. Although the precise mechanism of heat production in heater organ cells has not been proven, it has been linked to Ca2+ transport across the SR membrane. The mechanism proposed is as follows: neuronal stimulation depolarizes the heater cell, which leads to Ca2+ release from the SR. The resulting increase in cytoplasmic Ca2+ stimulates SERCA to actively remove the Ca2+ by utilizing energy derived from ATP hydrolysis. Simultaneously, the free cytosolic Ca2+ can also enter through the mitochondrial uniporter and stimulate mitochondrial respiration (and heat generation) directly (13). Thus, in the heater organ, heat is produced by SERCA-catalysed ATP hydrolysis and the stimulation of mitochondrial metabolism required to replenish ATP. Hence, by coupling SR Ca2+-transport with ATP production by mitochondria, and with the loss of myofilaments, a new cell type evolved to produce heat. Interestingly, the heater organ originated independently in two lineages of fish

[billfishes and butterfly mackerel (Gasterochisma melampus)], a fact that highlights the relative ease with which muscle can be adapted to fulfill a thermogenic role (13).

Perhaps the most convincing evidence for NST in skeletal muscle comes from avian species (64,87-99). Birds occupy some of the most diverse climates yet maintain

11 the highest body temperatures (38–42°C) among homeotherms (100). Some birds including pigeons, geese, and starlings are able to maintain T c at ~44°C during flight even at ambient temperatures below 0°C (101-104). A common feature of birds is that their skeletal muscle is more massive than in comparably sized reptiles and mammals

(105-110): the Japanese quail (Coturnix coturnix japonica) has a skeletal muscle mass that constitutes more than 70% of its body mass (111). Therefore it is speculated that the expansion of avian skeletal muscle, particularly the breast and thigh muscles, provided a unique survival advantage that permitted more efficient heat generation. These muscles are highly specialized for aerobic respiration with high levels of myoglobin and mitochondria, making them well suited for flight (112). Cold-adaptation experiments on birds have shown that, while shivering is the first line of defense used to maintain body temperature as in mammals, birds also use NST mechanisms (95,97,113-115). Hence, the same characteristics that enable flight very likely provide the proper physiological milieu for NST in muscle.

Although others had previously recognized cold-induced NST in birds, C.

Duchamp and H. Barre were the first to identify skeletal muscle as the major site of NST

(95,114,116). Using blood-flow measurements from thermoneutral (TN, 25°C) and cold- acclimated (CA, 4°C for 5 weeks) ducklings (Cairina moschata) exposed to 8°C, they were able to show that total muscle blood flow increased equally in the TN and CA ducklings, although the CA ducklings did not shiver. Both groups were also able to maintain Tc in the optimal range (114). Thus, skeletal muscles from CA ducklings were able to produce the same amount of heat as muscles from shivering TN ducklings,

12 demonstrating the existence of NST in skeletal muscle. Similar results have also been found for sparrows (Passer domesticus), king penguin chicks (Aptenodytes patagonicus), and chickens (Gallus domesticus) (95,115-117). Although this work defined muscle as the site of NST, it did not provide a detailed mechanism for the source of heat production.

In a follow-up study, Dumonteil et al. (1995) performed timed cold exposures to determine how changes in gene expression patterns coincided with the activation of NST.

In their experiment, the onset of NST correlated with the timing of increases in SERCA and CRC expression, whereas CASQ levels were unaffected (64). Thus, these studies indicated that Ca2+ cycling could be responsible for muscle-based NST. Earlier studies substantiated these findings by showing increases in both SERCA and the CRC after 6 weeks of cold acclimation in ducklings (87). Further support for Ca2+ cycling in NST is the finding that long-chain fatty acyl-carnitines and related metabolites induce Ca2+ release from the avian CRC. During cold acclimation, long-chain fatty acids and their metabolites have been shown to be increased in muscle (88). Therefore, fatty acyl-

CoA/carnitine-induced Ca2+ release could be a mechanism by which Ca2+-cycling is activated in response to cold. Further research is required to define the exact mechanism of NST in avian skeletal muscle.

1.4.3 Modes of thermogenesis in small versus large mammals.

Comparisons of large and small mammals and birds yield striking differences in apparent means of thermogenesis (Table 1.1). Studies in large mammals, including adult rabbits, dogs, and marsupials, where BAT is less prevalent, underscore the importance of

13 skeletal muscle-based NST in facultative thermogenesis (18,118-119); whereas, studies on small mammals, namely rodents, focus on BAT as the principal contributor to NST.

These species-specific recruitment strategies are discussed in more detail below.

Rodents rely on the heat-generating capacity of BAT to maintain body temperature in response to temperatures below thermoneutrality (8,24). Unlike rodents, studies on thermogenesis in large mammals point to a much higher reliance on skeletal muscle. In large mammals such as rabbits, ruminants, and humans, BAT activity is downregulated after the neonatal period and is minimally present or not detectable in adulthood, suggesting that skeletal muscle is the primary site of heat production (18,120-

124). Interestingly, some species even lack a functional Ucp1 gene; pigs lost a functional

UCP1 protein ~20 million years ago due to a nonsense mutation (125). Pigs maintain Tc at ~37°C, and interestingly, are very susceptible to a deadly condition called malignant hyperthermia, where sustained Ca2+ release by ryanodine receptor 1 in skeletal muscle results in excessive heat production (126-127). This may suggest that pigs employ muscle-based thermogenesis and that its dysregulation can lead to hyperthermia.

Moreover, cold-exposure studies performed on large mammals provide evidence for skeletal-muscle-based NST. Cold-acclimation experiments performed on dogs in which the hindlimbs were denervated to prevent shivering showed that cold-acclimated dogs were able to significantly increase oxygen consumption of the hindlimbs, similar to levels before denervation. Thus, the authors concluded that the denervated skeletal muscle contributed to NST (118). Similar results were obtained for cold exposures on the

Tasmanian bettong Bettongia gaimardi, a marsupial in which thermogenic BAT has not

14 been identified (18). In addition, in bettongs adapted to cold, heat production and responsiveness to catecholamines are increased. While direct muscle respiration was not measured in this study, earlier experiments had shown that catecholamines stimulated muscle oxygen consumption (128-129); therefore, it was concluded that muscle contributes to NST in bettongs (18).

1.4.4. Calcium cycling as a mechanism for NST.

For muscle to be an effective site of NST, one or more of the processes present in muscle must be recruitable and adaptable to heat generation in the absence of a muscle contraction. However, it would be disadvantageous to muscle function and energetics to evolve an additional machinery devoted entirely to NST. There are published accounts that indicate a role for all three muscle ATPases as well as metabolic/mitochondrial heat in muscle-based NST (61,75,91,130-136). However, the evidence for SERCA in NST is much more convincing than that for the myosin ATPase, Na+/K+ ATPase, or mitochondria alone. In fact, SERCA has three unique properties that would permit NST without affecting muscle function: (i) SERCA is abundant in muscle (137-143), therefore, a portion of the SERCA population can be recruited without affecting muscle function, (ii) the energy liberated as heat from SERCA can be increased or decreased depending on the cellular conditions (67-68,71,119,144-147), and (iii) the Ca2+-uptake function of SERCA can be uncoupled from ATP hydrolysis by its regulatory partner, sarcolipin (SLN) (11,69-70,148-154).

A considerable amount of data has been published concerning the molecular and biochemical basis of NST in rabbits. Most work hinges on the unifying theme that Ca2+ cycling in muscle is the major thermogenic mechanism in rabbits, and by extrapolation, other large mammals where BAT is not a major thermogenic organ. In particular, ATP hydrolysis by SERCA is considered to be the dominant heat producer (67-68,71). When activated by high cytosolic Ca2+ levels, as during excitation–contraction coupling,

SERCA pumps Ca2+ into the SR, using the energy derived from ATP hydrolysis to restore the SR load. In the optimal “coupled” state, two Ca2+ ions are transported per

ATP hydrolyzed (142,155-158). However, SERCA has a unique ability to become

“uncoupled,” a state in which fewer (than two) Ca2+ ions are transported per ATP and the remaining energy from ATP hydrolysis is transformed into heat (144,159). Interestingly, when rabbits are acclimatized to cold, SERCA1 expression is increased in red muscle

(SERCA2 levels are unaffected) but not in white muscle (119). Further- more, in vitro preparations of these cold-acclimatized muscles show that cold exposure increased the heat released during ATP hydrolysis twofold in red muscle, where oxidative

(mitochondrial) capacity is at least twofold greater than white muscle. Thus, cold exposure increased the heat-generating capacity of rabbit red muscle (119). These results are intriguingly in agreement with those obtained on cold-exposed birds.

1.4.5. Diet-Induced Thermogenesis in Muscle

In addition to cold-induced thermogenesis, skeletal muscle has been implicated as a mediator of diet-induced thermogenesis (5-6,160). Like BAT, the same mechanisms

16 underlying muscle-based cold-induced thermogenesis are postulated to play a role in DIT

(5). Similarly, DIT is also regulated by the sympathetic nervous system (SNS). Evidence for SNS regulation of DIT came from studies on mice lacking all three beta-adrenergic receptors (beta-AR), the main effectors of SNS signaling (161). These mice had a complete failure of DIT, evidenced by a lack of an increase in oxygen consumption after high-fat diet feeding. Moreover, the beta-AR knockout mice had lower oxygen consumption and became obese on a standard chow diet, in addition to the high-fat diet

(161). Other studies have shown heat production in response to overfeeding is correlated with catecholamine levels (Wijers 2007), and in humans, catecholamine levels increased after an oral glucose load (162-163).

Studies have shown that skeletal muscle may contribute to DIT. In humans, forearm muscle oxygen consumption increases during epinephrine infusion (164), and beta-blockade inhibits forearm oxygen consumption after a meal (163). Further evidence comes from high-fat-diet-fed rats, which exhibited an increased oxidative capacity of skeletal muscle (165). These studies indicate that there is an adrenergically stimulated elevation in EE in response to feeding that is mediated by beta-receptor signaling in skeletal muscle. However, unlike BAT, the mechanistic basis for muscle-based DIT is not well understood.

1.5. Evidence for Sarcolipin-mediated adaptive thermogenesis

A pioneering study performed by our laboratory was the first to provide evidence that the protein Sarcolipin (SLN) could mediate cold- and diet-induced adaptive

17 thermogenesis in skeletal muscle (11). SLN functions as a regulator of the sarco/endoplasmic reticulum calcium ATPase (SERCA), thus playing a role in calcium cycling in muscle. As discussed above, calcium cycling has been suggested to be a mechanism for muscle-based thermogenesis, though the precise details are lacking. Our characterization of SLN function has revealed SLN as a missing link in muscle-based thermogenesis.

1.3.1. Sarcolipin is an uncoupler of the SERCA pump

SLN is a 31 amino acid single transmembrane protein that co-localizes with the

SERCA pump in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscles (137).

SERCA functions to maintain low cytosolic calcium levels and high luminal calcium levels in the SR by using the energy derived from ATP hydrolysis to transport calcium from the cytosol to lumen (70). When bound to SERCA, SLN can uncouple SERCA from

Ca2+ transport, i.e. SLN allows ATP hydrolysis to occur but interferes with calcium transport, resulting in the release of calcium back into the cytosol (Figure 1.3). As a result, SLN promotes futile cycling of SERCA, increasing ATP hydrolysis and heat production (148-149). In addition to heat production, due to its inhibitory effects on the

SERCA pump, SLN increases cytosolic calcium levels, which activate Ca2+-dependent signaling pathways that regulate muscle metabolism and mitochondrial activity (70,166).

1.5.2. Loss of Sarcolipin increases cold-sensitivity of mice

In vitro studies had shown that SLN increased heat production by SERCA; therefore, it was hypothesized that SLN could contribute to thermogenesis in vivo. Mice with a targeted deletion of SLN (SLN-knockout, SLN-KO) were thus tested for their ability to maintain body temperature when exposed to cold (11). Since BAT is a major contributor to thermogenesis in mice and can mask the contributions from other sites, the interscapular BAT (iBAT) depot was surgically removed prior to the test. At 22°C, both iBAT-ablated wild-type (WT) and iBAT-ablated SLN-KO mice were able to maintain normal body temperature. However, when challenged to an acute cold exposure at 4°C, the iBAT-ablated SLN-KO mice could not maintain normal body temperature, with the body temperature dropping from 37°C to 26.9°C after 6 hours of exposure. In all, 85% of the iBAT-ablated SLN-KO mice developed hypothermia after 10 hours of cold exposure, compared to iBAT-ablated WT mice, which were able to maintain a body temperature of

35.8°C and did not develop hypothermia. Interestingly, the SLN-KO mice with intact iBAT, though did not become hypothermic, exhibited an increased cold sensitivity, indicated by a reduced body temperature (33.8°C) during the cold exposure; whereas WT mice maintained a normal temperature of 36.3°C. These data show that even when compensated by BAT, the absence of SLN reduces thermogenesis sufficiently to increase cold sensitivity (11).

1.5.3. Sarcolipin protects against diet-induced obesity

Because SLN uncouples SERCA and increases heat generation, it was hypothesized that the presence of SLN would promote metabolic inefficiency of muscle.

Therefore, SLN-KO mice were challenged with high-fat diet (HFD) feeding (11). After

12 weeks of feeding, the SLN-KO mice gained significantly more weight than WT controls, despite a lower caloric intake. Magnetic resonance imaging (MRI) and histological analysis showed that the increased body weight was primarily due to increased fat deposition. In addition, the SLN-KO mice had elevated serum cholesterol, triglyceride, and glucose concentrations along with increased glucose intolerance.

Interestingly, WT mice upregulated SLN protein expression, suggesting SLN is recruited to increase energy expenditure in response to the HFD and could play a role in DIT (11).

1.6. Gaps in knowledge

While the mechanism of adaptive thermogenesis in BAT has been well studied and is now well understood, less is known about the mechanism(s) of muscle-based thermogenesis. Our laboratory’s previous studies identified a putative role for SLN in both cold- and diet-induced thermogenesis, though the exact details are still lacking.

Therefore, the goal of this dissertation was to develop a better understanding of muscle- based adaptive thermogenesis by addressing the following gaps in knowledge:

1) How does muscle contribute to cold-induced thermogenesis in animals with minimal contributions from BAT? What is the role of SLN in these animals? BAT, while

20 abundant in rodents and newborns, is much less prominent in adult large mammals, including humans. This suggests that these species cannot rely entirely on BAT thermogenesis to maintain body temperature. In fact, studies on large mammals, including rabbits, dogs, ruminants, marsupials, etc., suggest muscle is the major site of heat production. However, an ongoing challenge for researchers studying skeletal muscle thermogenesis has been the delineation of heat production originating from shivering versus nonshivering mechanisms. In addition, the study of large animal models does not permit the study of specific genes, due to the difficulty/unavailability of generating genetically modified animals. For these reasons, the role of muscle in adaptive thermogenesis has not been well elucidated.

2) What is the contribution of muscle and SLN, specifically, to DIT? In humans, attempts to increase energy expenditure through BAT have not been very successful, likely due to the lack of abundance of BAT in humans. However, it is becoming increasingly clear that skeletal muscle is a major consumer of metabolites and ATP, and therefore could be recruited to increase metabolism. A greater understanding of how muscle contributes to energy expenditure will provide novel therapeutic targets for DIT research and development of future anti-obesity therapeutics.

Tables and Figures for Chapter 1

Eutherian Mammals Characteristic Birds Small Large High (~16% Low (scant to None BAT (volume) weight/body mass) * <1%)* Low, restricted to High, present in SLN expression Unknown slow-twitch muscles most/all muscles Muscles are Partitioned fiber mostly mixed with types; Restricted high muscles with Muscle mitochondrial composition abundant number; (Fiber type & mitochondria; Most muscles rich Considerable mitochondrial Preponderance of in mitochondria reliance on Type IIB fibers; abundance) postural muscles; Lesser reliance on Type IIB fibers postural muscles minimal or absent Body Temperature ~37°C ~37-38°C 38-42°C Surface area to High Low High Volume ratio

High range of Long range of Usually bursts of activity (occupy activity (occupy Physical Activity activity (occupy small large ecological large ecological ecological space) space) space)

Table 1.1. Characteristics and compositions of tissues contributing to thermogenesis in extant endotherms. *Adapted from (22,167)

Figure 1.1. Components of total daily energy expenditure in mammals.

Figure 1.2. Mechanism of UCP1.

Complexes I (CI), III (CIII), and IV (CIV) of the electron transport chain in mitochondria translocate protons (H+) from the mitochondrial matrix to the intermembrane space, generating an electrochemical gradient. In the absence of UCP1 activity, the energy from this gradient is used to drive ATP synthesis by Complex V (CV), also known as ATP Synthase. However, when UCP1 is present and activated, it dissipates the proton gradient via symport of a fatty acid and H+ from the intermembrane space to the matrix. Heat is generated from the conversion of potential energy stored in the electrochemical (proton) gradient.

Figure 1.3. Mechanism of Sarcolipin.

When Ca2+ binds to SERCA, ATP is hydrolyzed. In the presence of SLN, the transition of SERCA from the E1 state to the E2 state is inhibited, resulting in premature release of Ca2+ back into the cytosol. Thus, in the presence of SLN, ATP is hydrolyzed without Ca2+ transport into the SR lumen. This research was originally published in the Journal of Biological Chemistry. Sahoo SK, Shaikh SA, Sopariwala DH, Bal NC, Periasamy, M. Sarcolipin protein interaction with sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) is distinct from phospholamban protein, and only sarcolipin can promote uncoupling of the SERCA pump. J Biol Chem. 2013; 288:6881-9. © the American Society for Biochemistry and Molecular Biology.

Chapter 2. Sarcolipin and Uncoupling Protein 1 are required to maintain optimal thermogenesis and loss of both systems compromises survival of mice under cold

stress.

Maintaining constant body temperature is an important homeostatic mechanism essential for normal physiological functions and survival of endotherms. This ability to regulate internal temperature has enabled mammals and birds to colonize vast areas of the world. To maintain body temperature when external temperatures fall below thermoneutrality (7,58), endotherms rely on facultative thermogenic mechanisms. Among these thermogenic mechanisms, shivering is a first line of defense. However, prolonged shivering can be detrimental to muscle health; therefore, the organism recruits nonshivering thermogenesis (NST) to replace and/or supplement shivering thermogenesis. The activation of NST from brown adipose tissue (BAT) is a well-studied and well-established phenomenon in mammals, in particular rodents (8,168). The thermogenic capacity of BAT is dependent on the activity of uncoupling protein 1

(UCP1), which generates heat by dissipating the mitochondrial proton gradient (10,23-

24). However, in many adult large mammals BAT activity is limited to neonatal stages by becoming downregulated in the adult and is even absent in some endothermic species.

Therefore, these animals, including humans, must rely on alternative thermogenic mechanisms to survive when exposed to cold environments (18-19,119-120,122,124).

Because many studies use rodents, which heavily rely on BAT, as experimental models to understand NST mechanisms, the importance of alternate thermogenic mechanisms has been overlooked. However, there is increasing evidence for the existence of BAT-independent sites of thermogenesis in mammals. This has become especially obvious from studies on large mammals that have limited BAT, as well as from UCP1-knockout (UCP1-KO) mice. Although UCP1-KO mice are extremely sensitive to acute cold exposure, they can be gradually adapted to 4°C (28,44).

Additionally, the cold sensitivity of UCP1-KO mice is incompletely penetrant and highly dependent on genetic background: 32.1% of C57Bl/6 and 87.5% of 129SV/J are cold sensitive (28). While BAT is unequivocally a dominant thermogenic mechanism in rodents, studies on cold-adapted UCP1-KO mice studies have suggested other mechanisms of heat production, including Ca2+ cycling in skeletal muscle (45-46).

Skeletal muscle has long been viewed as a significant contributor to thermogenesis. Studies on large mammals, including rabbits, dogs, ruminants, marsupials, etc., suggest muscle is the major site of heat production (13,18-19,118-

119,169). These species have reduced BAT content in adulthood, especially when compared to rodents; thus, they cannot rely entirely on BAT thermogenesis to maintain body temperature. Similarly, it is widely accepted that birds, which completely lack a

Ucp1 gene, predominately utilize muscle-based thermogenesis (64,87-88,92,113-115).

Thus, a major research interest of our laboratory has been to investigate the role of skeletal muscle as a site of thermogenesis, independent of shivering.

Recently, we showed that skeletal muscle is an important site for thermogenesis even in rodents, especially when BAT function is compromised (11). Our studies highlighted that sarcoplasmic reticulum Ca2+ transport serves as an important mechanism for heat production in muscle. We described that uncoupling of sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) Ca2+ transport from ATP hydrolysis by Sarcolipin

(SLN), a regulator of SERCA, can increase ATP hydrolysis and heat production via futile cycling of the pump (11). This novel discovery led us to the question: is SLN-mediated heat production the “missing link” underlying UCP1-independent thermogenesis? Thus, we wanted to investigate whether SLN-based thermogenesis could compensate and enable the UCP1-KO mice to adapt to the cold. Furthermore, we hypothesized that a loss of both UCP1 and SLN would render mice unable to survive in cold. To test this, we generated a UCP1; SLN double knockout (DKO) mouse model and challenged the single and DKO mice to acute and long-term cold exposures. Our studies highlight the compensatory and unique roles of these two thermogenic systems in a mouse model.

2.1. Research Design and Methods

2.1.1. Animals. The generation of UCP1-/- and SLN-/- mice have been described previously (24,166). UCP1-/-.SLN-/- mice on a C57Bl/6J background were generated by crossing UCP1-/-.SLN+/+ and UCP1+/+.SLN-/- to obtain the first generation of UCP1+/-

.SLN+/-. The double heterozygotes were intercrossed to obtain the DKO and littermate

28 controls. The mice were maintained in a temperature controlled room either at 22C or

28C and fed a chow diet (Harlan Labs, rodent diet 17% kcal/fat). Male and female mice were used for the acute cold exposure studies. Only male mice were used for the gradual cold adaptation experiments.

2.1.2. Acute cold exposures. All cold exposures were carried out in a temperature- controlled unit. Mice maintained at either 22C or 28C were transferred to the pre- cooled 4C unit and housed in individual cages. Body temperature was monitored every

20 minutes during the first hour, then every 30-60 minutes thereafter using implanted thermal transponders (11). Body weight was measured immediately before the cold exposure. Mice from 22C: WT (n=3), SLN-KO (n=4), UCP1-KO (n=10), DKO (n=12).

Mice from 28C: WT (n=11), SLN-KO (n=9), UCP1-KO (n=7), DKO (n=7).

2.1.3. Gradual cold adaptation. Mice previously maintained at 28C were individually housed in the temperature controlled unit where the temperature was decreased to 22C and maintained for 2 days, 18C for 2 days, then decreased by 2C/day until the temperature reached 4C. The mice were housed at 4C for a further 9-10 days until the termination of the experiment. Using implanted thermal transponders, body temperature was monitored at the same time everyday. Body weight was measured twice weekly. WT

(n=6), SLN-KO (n=6), UCP1-KO (n=5), DKO (n=6).

2.1.4. Metabolic monitoring. During the cold exposures, oxygen consumption, carbon dioxide production, and physical activity were continuously measured by the Oxymax

Comprehensive Lab Animal Monitoring System (CLAMS, Columbus Instruments,

Columbus, OH). Food intake was measured by physical weighing of the food provided.

2.1.5. Histology of adipose tissues. Brown adipose tissue, epidydymal, and inguinal fat pads were fixed in 10% formalin. Paraffin-embedded sections were stained with hematoxylin and eosin. Images were collected with Richter Optica U2D digital microscope with a digital camera (5MP Toupcam) attached.

2.1.6. Urinary catecholamine output. Urine was obtained by a one-time collection at the same time of day at four temperatures during the gradual cold adaptation: 28C, 18C,

10C, and 4C. Hydrochloric acid was added to a final concentration of 0.1M to maintain the integrity of the catecholamine levels until analysis. The collected urine was stored at -

80C until analysis. Norepinephrine and epinephrine levels were determined by ELISA

(Rocky Mountain Diagnostics). Catecholamine levels were normalized to creatinine content, determined by a creatinine (urinary) colorimetric assay (Cayman Chemical).

2.1.7. Western Blotting. Protein expression was analyzed as described previously (11).

Briefly, muscle homogenates were resolved by SDS- PAGE (16% Tris-tricine for SLN, and 10% Tris- glycine for others). Proteins were transferred to a nitrocellulose membrane

(0.2μm for SLN, 0.45μm for UCP1). For the UCP1 blots, immediately after transfer, the

30 membranes were stained with 0.5% Ponceau S. The membranes were blocked in 5%

BSA or milk, and probed with primary antibodies to SLN (Millipore, rabbit polyclonal),

UCP1 (rabbit polyclonal, a kind gift from Dr. Barbara Cannon), GAPDH

(ThermoScientific, mouse monoclonal), CPT1 (Alpha Diagnostics, rabbit polyclonal),

PFK-1 (Santa Cruz, rabbit polyclonal), LDH (Santa Cruz, rabbit polyclonal), SERCA1a

(custom-made), SERCA2a (custom-made), CASQ (custom-made). Secondary horseradish peroxidase antibodies were applied for 1 hour at a 1:25,000- 1:50,000 dilution. Expression was detected with chemiluminescent substrate.

2.1.8. Statistical Analysis. Data are expressed as means ± S.E.M. Differences among groups were determined by one-way ANOVA. Statistically significant differences were accepted at p<0.05.

2.2. Results

2.2.1. Gradual cold adaptation of UCP1-KO and SLN-KO mice.

It has previously been shown that UCP1-KO mice can readily adapt to cold

(12,28-29,44), but the mechanism(s) underlying their adaptation has remained incompletely understood. In this study, we reinvestigated the mechanism underlying cold adaptation in UCP1-KO mice. We tested the hypothesis that increased SLN expression could potentially compensate for loss of BAT function. We adapted WT, SLN-KO and

UCP1-KO mice to 4°C by gradually reducing the temperature from 28°C and then maintaining the mice at 4°C for ~10 days. After cold adaptation, we observed an

31 increased redness of the muscles of UCP1-KO mice, compared to WT (Figure 2.1A), accompanied by an increased myoglobin content (Figure 2.1B). Interestingly, we observed a strong induction of SLN in the red quadriceps (Figure 2.1C), and to a lesser extent in the whole gastrocnemius, infraspinatus, and soleus muscles (Figure 2.1C and data not shown). However, SLN expression was not altered in the diaphragm, which already expresses high levels in WT and UCP1-KO mice (Figure 2.1C).

As expected, the SLN-KO mice were able to adapt to gradual cold exposure.

Hematoxylin and eosin (H&E) staining of brown adipose tissue depots of cold-adapted

WT and SLN-KO mice showed extreme lipid depletion, which was slightly greater in the

SLN-KO (Figure 2.2A). UCP1 protein was ~2 fold greater in the cold- adapted SLN-KO compared to WT (Figure 2.2A). We next examined if there is additional recruitment of subcutaneous white adipose tissue (WAT) to increase thermogenesis by what is known as

“browning.” Interestingly, H&E staining of subcutaneous WAT in SLN-KO mice showed morphological characteristics consistent with browning: large areas of fat depletion and multilocular adipocytes (Figure 2.2B). Moreover, UCP1 expression was increased ~3 fold in the SLN-KO compared to WT and mitochondrial protein content was higher in the

SLN-KO (Figure 2.2B and data not shown). Therefore, in order to overcome the loss of

SLN-based thermogenesis, SLN-KO mice must increase UCP1-based thermogenesis by increasing BAT activity and browning of WAT.

2.2.2. Increased energy expenditure in SLN-KO mice.

We also investigated the energetic cost of cold adaptation in these mice.

Throughout the cold adaptation period, oxygen consumption, food intake, and body weight were monitored. We found that SLN-KO mice exhibited higher oxygen consumption at 4°C than WT mice (Figure 2.3A-D) and also trended toward greater food intake (Figure 2.3E). Interestingly, measurement of fat pad weights from thermoneutral controls and cold-adapted mice trended toward a greater depletion of fat stores from

SLN-KO than WT (Figure 2.4A). Similarly, UCP1-KO mice, as previously reported, exhibited higher oxygen consumption (Figure 2.3A-D) and significantly greater fat loss than WT (Figure 2.4A), though food intake was not significantly different (Figure 2.3E).

Together, these data suggest a loss of either SLN or UCP1 places a greater energetic cost to maintain thermogenesis and would be detrimental to the survival of mice during long- term cold exposure.

2.2.3. UCP1/SLN double knockout (DKO) mice show poor survival rate at 22°C.

We hypothesized that a loss of both UCP1 and SLN would render mice unable to survive in cold. To test this, we generated a UCP1; SLN double knockout (DKO) mouse model by breeding double heterozygotes (UCP1+/-.SLN+/-). When bred at standard vivarium temperature 22°C, very few DKO mice were obtained and were found to be less than the expected Mendelian frequencies, whereas single knockout mice were not affected (Table 2.1). Although 22°C is a mild cold stress, it is sufficient to cause death in newborns, since neonates are much more vulnerable to cold than adult mice (170-171).

Therefore, we bred the double heterozygotes at thermoneutrality (28°C) and found that the DKO mice were born at normal Mendelian frequencies. These findings suggest that a loss of both SLN and UCP1 reduces the thermogenic capacity significantly and compromises the survival of newborn mice.

2.2.4. Acute cold challenge of mice housed at 22°C versus 28°C.

It is known that UCP1-KO mice are sensitive to an acute cold challenge; however, their sensitivity is highly variable and is influenced by factors including housing temperature (28-29). Therefore, we maintained WT, SLN-KO, UCP1-KO, and DKO mice at two different temperatures: 22°C and 28°C and then challenged them to a 4°C exposure. When reared at 22°C, the DKO mice were severely cold-sensitive, as indicated by reduced body temperature and a greater number (75%) reaching early removal criteria

(ERC, defined by a body temperature reaching 30°C) within 8 hours of cold challenge

(Figure 2.5A and C). In agreement with published data, UCP1-KO mice were also cold- sensitive, though to a lesser degree than the DKO. As expected, SLN-KO mice were found to be cold-sensitive as indicated by a lower body temperature than WT mice, but did not develop hypothermia.

On the other hand, when maintained at thermoneutrality (28°C), NST mechanisms (BAT-dependent and -independent) are downregulated since there is no requirement for facultative thermogenesis to maintain body temperature (7). Therefore, when these mice were challenged acutely to 4°C, all mice showed increased cold- sensitivity as indicated by a greater drop in body temperature (Figure 2.5B), due to an

34 inability to fully recruit NST mechanisms. Interestingly, the DKO mice reared at 28°C were highly cold-sensitive: 43% had reached ERC within 2 hours of cold challenge, whereas only 14% of UCP1-KO mice reached ERC (Figure 2.5D). When the cold challenge was continued beyond 2 hours, 100% of the DKO and UCP1-KO mice reached

ERC (data not shown). Another interesting observation was when reared at 28°C, the effect of loss of SLN on thermogenic capacity became much more apparent. Here, we observed that ~33% of SLN-KO mice are cold-sensitive (reaching ERC within 4 hours of cold challenge, data not shown), compared to ~11% of WT. In contrast, none of the SLN-

KO mice reached ERC when reared at 22°C (Figure 2.5C).

2.2.5. DKO mice can survive gradual cold exposure.

Paradoxically, the majority of DKO mice were able to adapt to the cold by a gradual reduction in temperature; though one out of six developed hypothermia and lost

~26% of body weight within 20 days of cold adaptation. Overall, the DKO mice had similar oxygen consumption rates as WT when expressed per mouse (Figure 2.3A-D) throughout the adaptation period. However, the DKO mice lose the most weight (11% of starting weight, compared to 3.8% for WT) (Figure 2.3F). Considering this, the DKOs had the highest oxygen consumption per body weight (Figure 2.3B and D). Interestingly, they also consumed 39% more food than WT mice at 4°C (Figure 2.3E). Despite higher caloric intake, the white fat stores of the DKO mice were almost completely depleted by the end of the exposure (Figure 2.4A). Histological analysis of visceral fat depots further substantiate these data by showing much smaller adipocyte size in the DKO after cold

35 adaptation, compared to other genotypes (Figure 2.4B). These histological analyses along with fat mass quantification suggest that DKO mice have to completely mobilize their fat stores, despite increased food intake, to maintain thermogenesis at all costs. These data point out that mice lacking both SLN- and UCP1-based adaptive thermogenesis can survive in the cold but at an extremely high energetic cost.

2.2.6. DKO and UCP1-KO mice have elevated catecholamine levels during their exposure to cold.

The sympathoadrenal system is an important regulator of whole body temperature homeostasis, and this is achieved by activating heat generating as well as heat conservation mechanisms (172-173). We hypothesized that the ability of DKO mice to adapt to cold was due, in part, to higher sympathoadrenal tone/levels. Therefore, we measured sympathoadrenal output by quantifying epinephrine (E) and norepinephrine

(NE) content in the urine from four time points during the cold adaptation (28°C, 18°C,

10°C, and 4°C). We found that when housed at 28°C there was no difference in NE or E among the genotypes, whereas their levels were elevated upon cold stress (Figure 2.6).

During cold adaptation, beginning at 18°C, both UCP1-KO and DKO had elevated E and

NE levels over WT and SLN-KO. Both UCP1-KO and DKO reached maximal E and NE levels by 10°C; however, there was no difference between the two at any time point.

These findings suggest that in the absence of UCP1, elevated adrenergic signaling is required to recruit BAT-independent thermogenic mechanisms.

2.2.7. Activity-dependent thermogenesis is not different among genotypes.

Physical activity generates heat due to the thermic effect of muscle contractions.

Therefore, increased activity can increase total thermogenic output. Alternatively, reduced physical activity minimizes heat loss to the environment, when the animal takes on a compact, huddled posture. Thus, we hypothesized that differences in physical activity during cold exposure would provide clues as to how the mice were tolerating the cold. We found that while all mice decreased activity upon exposure to 4°C, there was no significant difference in activity among genotypes at thermoneutrality or in the cold

(Figure 2.7A and B).

2.2.8. Loss of SLN leads to decreased reliance on muscle-based thermogenesis.

Previous studies have shown that cold adaptation induces an endurance training- like effect on skeletal muscle, due to an increased reliance on muscle-based thermogenesis. These changes include increased respiratory capacity, due to increased mitochondrial content, activity and content of enzymes involved in oxidative metabolism, and oxygen delivery (12,46,119,174). Often, there are also transitions in the contractile machinery towards a “slower” muscle phenotype, though this is highly dependent on species and muscle type (29,44,90,119). Therefore, we analyzed the muscle phenotype in cold-adapted WT, SLN-KO, UCP1-KO, and DKO mice to determine how SLN is involved in these training-like effects.

Muscles from UCP1-KO mice become visibly redder after cold adaptation, compared to WT, which is indicative of a greater oxidative capacity and reliance on

37 muscle-based thermogenesis. We thus chose to focus on these muscles with obvious adaptations for biochemical analysis. A previous study by Nedergaard et al. showed that cold-adaptation of UCP1-KO mice induced an increase in the expression the mitochondrial fatty acid transporter CPT1 in skeletal muscle (12). Interestingly, we found that a loss of SLN blunted this effect, with reduced CPT1 in DKO muscle, though not significant, compared to UCP1-KO muscle (Figure 2.8A). We next examined enzymes of glycolytic metabolism, as a shift towards an oxidative phenotype would reduce the content of glycolytic enzymes. Here we found that cold-adapted SLN-KO muscle trended toward an ~1.5 fold greater lactate dehydrogenase levels compared to WT, suggesting that the SLN-KO muscle did not undergo the switch toward an oxidative phenotype

(Figure 2.8B).

Comparison of SERCA1a expression among genotypes showed no differences

(Figure 2.9B). Interestingly, a comparison of SERCA2a expression revealed significantly decreased levels in the SLN-KO and DKO muscle compared to WT and UCP1-KO, respectively (Figure 2.9A). In addition, SLN-KO muscle had approximately 2-fold greater levels of calsequestrin (CASQ) than WT (Figure 2.10). These data suggest that the loss of SLN affects the ability of the muscle to adapt to cold (to convert into a slow, oxidative phenotype) i.e. the muscle is not being recruited to the same extent as in the

WT and especially UCP1-KO. Thus, these data further demonstrate that in the absence of

SLN, mice cannot efficiently recruit muscle but must rely on other thermogenic mechanisms.

2.3. Discussion

Temperature homeostasis is a major homeostatic mechanism that is required for proper physiological functions. Despite its central importance to human physiology, the mechanisms that contribute to temperature homeostasis, including thermogenesis, are still poorly understood. However, during the past decade, there has been renewed excitement toward defining the mechanisms that contribute to thermogenesis. This is largely because thermogenic processes are unique in that they expend vast amounts of energy (releasing heat as a byproduct) when activated and can affect whole body metabolism and fat stores

(5-6). Thus, they are also ideal targets for increasing energy expenditure to combat weight gain and obesity.

BAT is a well-studied component of mammalian thermogenesis. Multiple lines of evidence demonstrate the existence of BAT-independent thermogenesis, though the mechanisms have remained less well understood. Studies have shown that UCP1-KO mice, generated by Kozak and colleagues (24), can be gradually adapted to survive and maintain body temperature during prolonged exposure to 4°C. It has been argued that constant shivering could be the major source of heat that replaces UCP1-mediated thermogenesis (12,175). However, prolonged shivering can compromise muscle function and is detrimental to muscle health (175). Furthermore, UCP1-KO mice are severely sensitive to acute cold exposure, despite shivering. This suggests that shivering alone is not sufficient and additional thermogenic mechanisms must be recruited to fully compensate for the absence of UCP1 thermogenesis.

Previous studies from our laboratory and others indicated that SLN is an important component of muscle thermogenesis (11,148-149,176). We found that SLN-

KO mice were sensitive to acute cold exposure and developed hypothermia when interscapular BAT (iBAT) was ablated, suggesting that both SLN and BAT are major contributors to rodent thermogenesis (11). However, it has been shown that neither SLN nor BAT is absolutely necessary for survival during cold exposure. In this study, we therefore studied whether the presence of one can compensate for a lack of the other.

Indeed, our studies suggest that SLN and UCP1 could compensate for each other’s deficiency, indicating a functional interplay between the two systems. We found that

SLN protein was upregulated in the skeletal muscles of UCP1-KO mice after cold adaptation, accompanied by a deep reddening of the muscle and increased myoglobin content, which is indicative of a greater oxidative capacity. This data is consistent with previous studies that have shown increased mitochondrial content and fatty acid oxidative capacity in muscles of cold-adapted UCP1-KO mice (12). In addition, we found compensatory browning occurring in the subcutaneous and visceral (data not shown) white adipose tissue of SLN-KO mice after cold adaptation, which suggests that classical

BAT alone is insufficient. Compensatory browning has been shown to occur in mouse models of defective thermogenesis (35-36), which further substantiates the imperative contribution of SLN to thermogenesis.

Our oxygen consumption data provided interesting insight into the process of gradual cold adaptation. SLN-KO mice can readily adapt to the cold and maintain body temperature close to that of WT mice, like UCP1-KO mice. Both UCP1-KO and SLN-

KO mice showed higher oxygen consumption than the WT mice, while both knockout groups had similar oxygen consumption rates. Higher oxygen consumption is suggestive of an increased reliance on less efficient heat-producing mechanisms. Therefore, the above data indicate that when either of these mechanisms is not present, heat production becomes energetically costly, in spite of the compensatory recruitment of the other mechanism. Based on this observation, we propose that while BAT is the major heat producer in rodents, SLN is also a considerable contributor to thermogenesis, especially when considering BAT constitutes less than 1% of mouse weight compared to muscle, which constitutes about 40% of mouse weight. This argument is further supported by the fact that both the knockouts lost similar amounts of their fat stores. Further, with muscle being dispersed throughout the body, it can provide heat locally, rather than needing to be circulated by the blood from a localized BAT. Hence, during the evolution of endothermy in individual species, a combination of BAT and muscle-based thermogenesis has been selected that provides the best survival outcome (19).

An important question that has not been addressed is whether animals can survive cold in the absence of both SLN and UCP1. Thus, we compared cold tolerance in single and double knockout (DKO) mice using littermates in the same genetic background. Our data showed that the DKO mice were extremely sensitive to an acute 4C cold challenge and developed hypothermia rapidly. Paradoxically, the DKO mice were able to maintain body temperature and survive during gradual cold exposure. Despite consuming the most food, the DKOs lost the most weight and had the highest oxygen consumption per body weight. In fact, at the end of the cold exposure, the DKO mice had almost completely

41 depleted their adipose tissue stores, suggesting that these mice have to mobilize their white fat, in addition to increasing food intake, to fuel thermogenesis. The finding that the DKO mice had significantly elevated catecholamine levels suggest that the mice are highly cold stressed and recruiting every other mechanism to maintain body temperature.

Thus, the ability to maintain body temperature without UCP1 and SLN came at a huge energetic cost. These data indicate that in the absence of UCP1 and SLN, the DKO mice must rely on extremely inefficient thermogenic mechanisms (possibly due to sustained elevation of metabolic heat) to maintain body temperature. It appears that the DKO mice would not be able to survive for longer periods of time in the cold, once fat stores are depleted. Hence, metabolic heat production will not be sufficient to meet the thermogenic demand during prolonged cold exposure, which is why mammals have evolved more energetically efficient thermogenic mechanisms.

Taken together, the present study shows that the presence of both UCP1 and SLN provides an effective combination to meet the thermogenic demand and loss of either one increases the energetic cost of cold adaptation. Loss of both mechanisms makes the system almost unsustainable, and after ~3 weeks of survival in the cold most of the WAT stores are depleted in the DKO mice. The unexpected survival of the DKO mice in this study highlights the resiliency of mice and how species have evolved with a multitude of mechanisms to accomplish vital physiological functions. A species will not rely on only one or 2 mechanisms but will have the ability to recruit additional methods to ensure survival at all costs. Finally, these studies further substantiate the critical role of SLN and

42 muscle in thermogenesis and provide exciting insight into mechanisms of energy expenditure and thermogenesis in animals with minimal BAT, such as humans.

Figures and Tables for Chapter 2

22°C 28°C

Observed Observed Genotype Predicted Predicted (N=136) (N=113)

UCP1+/+ SLN+/+ 15 8.5 9 7.0625

UCP1+/+ SLN-/- 11 8.5 11 7.0625

UCP1-/- SLN+/+ 11 8.5 9 7.0625

UCP1-/- SLN-/- 4 8.5 9 7.0625

Table 2.2. Observed and predicted frequencies of offspring from UCP1+/-.SLN+/- intercrosses when reared at 22°C and 28°C.

Figure 2.1. Changes in skeletal muscle after cold adaptation.

A. Representative images of cold adapted quadriceps and soleus muscles are presented to show the greater reddening of the muscle in UCP1-KO. B. Myoglobin protein expression in Quadriceps muscles of cold-adapted WT and UCP1-KO mice. C. SLN protein expression in the red quadriceps, soleus, and diaphragm muscles of WT and UCP1-KO mice after cold adaptation. GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Figure 2.2. Changes in adipose tissue after cold adaptation.

Hematoxylin and eosin staining of brown adipose tissue (BAT) (A) and inguinal (subcutaneous) white adipose tissue (WAT) from thermoneutral (28°C) and cold-adapted (4°C) WT and SLN-KO mice. UCP1 protein expression in thermoneutral (28°C) and cold-adapted (4°C) WT and SLN-KO mice from BAT (A) and inguinal WAT (B) is presented below. Scale bars are equal to 100μm.

Figure 2.3. Energetics of cold adaptation. A and B. Average oxygen consumption over 24 hour periods at each temperature indicated during the cold adaptation. Not normalized (A) and normalized to body weight (B). C and D. Oxygen consumption over a 24 hour period at 4°C. Not normalized (C) and normalized to body weight (D). * = p<0.05, ** = p<0.01, *** = p<0.001. E. Daily caloric intake per mouse at 4°C. *** = WT vs. DKO, p<0.001; SLN-KO vs. DKO, p<0.05; UCP1-KO vs. DKO, p<0.01. F. Change in body weight (in grams) from the start of the cold adaptation period to the end. * = WT vs. DKO, SLN-KO vs. DKO, UCP1-KO vs. DKO, p<0.05. 47

Figure 2.4. White adipose tissue after cold adaptation. A. Epidydymal (visceral white fat) fat pad weights from thermoneutral (Tn) control mice and cold-acclimated (Cold) mice at the end of the cold adaptation protocol. Fat pad weights are expressed as a percentage of body weight. * = UCP1-KO Tn vs. UCP1-KO Cold, DKO Tn vs. DKO Cold, p<0.05. B. Hematoxylin and eosin staining of epidydymal white adipose tissue from thermoneutral (28°C) and cold-adapted mice (4°C). Adipocyte size is mildly reduced in WT, SLN-KO, and UCP1-KO mice after cold adaptation; however, cold adaptation of DKO mice results in a severe reduction in adipocyte size. Scale bars are equal to 100μm.

Figure 2.5. Acute cold exposure. A and B. Average core body temperature during an acute cold exposure to 4°C in WT, SLN-KO, UCP1-KO, and DKO mice reared at 22°C (A) and 28°C (B). C and D. Percentage of mice reaching Early Removal Criteria (ERC), defined by a body temperature ≤ 30°C, during the acute cold challenge in mice reared at 22°C (C) and 28°C (D).

Figure 2.6. Catecholamine levels throughout cold adaptation. Urine was collected at each temperature shown during the cold adaptation. Norepinephrine and epinephrine levels were determined by ELISA and normalized to creatinine levels. * = significant difference compared to WT at the same temperature, # = significant difference compared to SLN-KO at the same temperature.

Figure 2.7. Physical activity before and after cold adaptation.

Average physical activity over 24 hours from mice before the cold adaptation (28°C) and after the cold adaptation at 4°C. Activity is the sum of the average X counts and Z counts.

Figure 2.8. CPT1 and LDH expression in cold-adapted muscles. A. Carnitine palmitoyl-CoA transferase 1 (CPT1) and B. lactate dehydrogenase (LDH) protein expression in red quadriceps muscles of cold-adapted mice with densiometric analysis. A.U.: arbitrary units. *** = p<0.001.

Figure 2.9. SERCA expression in cold-adapted muscles.

A. SERCA2A and B. SERCA1a protein expression in red quadriceps muscles of cold- adapted mice with densiometric analysis. A.U.: arbitrary units. * = p<0.05.

Figure 2.10. Calsequestrin expression in cold-adapted muscles.

Calsequestrin 1 (CASQ) protein expression in red quadriceps muscles of cold-adapted WT and SLN-KO mice with densiometric analysis. A.U.: arbitrary units. ** = p<0.01.

Chapter 3. Sarcolipin and Uncoupling Protein 1 contribute to DIT independently

and do not compensate for one another in response to diet overload.

Obesity is the result of an imbalance of energy expenditure and energy intake.

Therefore, obesity can be caused by increased food intake and/or defects in energy expenditure. Many studies have confirmed that rodents and humans have the ability to increase energy expenditure in response to overfeeding to prevent excessive weight gain, a phenomenon termed adaptive diet-induced thermogenesis (DIT) (5-6,16,161,177).

Thus, defects in DIT could lead to weight gain and obesity.

The first player in DIT to be identified was BAT in rodents. Numerous investigations have confirmed BAT/UCP1 (at least partially) mediate DIT in rodents, though their contribution to energy expenditure in humans is still under investigation

(8,15-16), due to the limited amount of BAT found in adult humans. Other components that contribute to DIT are largely unknown; though similar to cold-induced thermogenesis, skeletal muscle is suggested to play a role. Skeletal muscle comprises approximately 40% of human body mass and accounts for 20-30% of resting energy expenditure (during activity this number can increase to 90%) (60). As skeletal muscle has such a considerable contribution to body mass and metabolic rate, it has a significant potential to contribute to DIT. In fact, studies on humans have shown muscle oxygen

55 consumption increases after a meal, though the underlying mechanisms have not been identified (163-164).

Recent data from our laboratory, however, suggest that SLN plays a role in skeletal muscle-based DIT. By uncoupling SERCA, SLN increases ATP utilization, thereby increasing the energy demand of muscle. Our studies showed that SLN-KO mice, when fed a high fat diet (HFD), became more obese than WT controls in spite of consuming fewer calories (11). Interestingly, SLN protein levels were increased in the muscles of HFD-fed mice, indicating a recruitment of SLN in response to the HFD (11).

These studies led us to hypothesize that SLN is the missing link in UCP1-independent

DIT and more relevantly, in human DIT. Since our cold exposure studies had shown that

SLN and UCP1 played compensatory roles, we also hypothesized that SLN and UCP1 would compensate for one another during DIT. Therefore, we wanted to investigate whether a loss of both SLN and UCP1 would lead to severe diet-induced obesity, due to an absence of DIT. In addition, we sought to determine whether SLN-mediated DIT could compensate for a lack of UCP1 and vice versa. For this study, we utilized the

SLN,UCP1 double knockout (DKO) mouse model, and the results are discussed below.

3.1. Research Design and Methods

3.1.1. Animals. The generation of UCP1-/- and SLN-/- mice have been described previously (24,166). UCP1-/-.SLN-/- mice on a C57Bl/6J background were generated by crossing UCP1-/-.SLN+/+ and UCP1+/+.SLN-/- to obtain the first generation of UCP1+/-

.SLN+/-. The double heterozygotes were intercrossed to obtain the DKO and littermate

56 controls. The mice were maintained in a temperature-controlled room at 28C-30C and fed a chow diet (Harlan Labs, rodent diet 17% kcal/fat). To increase the number of mice for experimentation, heterozygotes were used; i.e., WT=UCP1+/-.SLN+/-, SLN-

KO=UCP1+/-.SLN-/-, UCP1-KO=UCP1-/-; SLN+/-, DKO=UCP1-/-.SLN-/-.Only male mice were used for experimentation.

3.1.2. High Fat Diet Feeding. 10-week old WT, SLN-KO, UCP1-KO, and DKO littermates were fed a high fat diet (45% kcal/fat, Research Diets) for 12 weeks. Body weight and food intake were measured weekly.

3.1.3. Metabolic Monitoring. Before and after HFD-feeding, oxygen consumption and carbon dioxide production were measured over a three-day period by the Oxymax

Comprehensive Lab Animal Monitoring System (CLAMS, Columbus Instruments,

Columbus, OH). The mice were housed at 29C for the duration of the measurements.

Only the middle 24-hour period was taken for analysis.

3.1.4. Blood glucose and lipid levels. After 12-weeks of HFD-feeding, blood glucose was measured in 16-hour fasted mice. The blood sample was obtained by a tail nick, and glucose was measured with the TrueTrack® blood glucose monitoring kit. For lipid analysis, mice were fasted for 4 hours, and blood was analyzed using the CardioChek® analyzer and test strips.

3.1.5. Measurement of fat pad and muscle weights. After HFD-feeding, the mice were sacrificed by carbon dioxide inhalation. White fat pads (epidydymal and inguinal) and the interscapular brown fat pad were removed and weighed. Skeletal muscles, including soleus, gastrocnemius, tibialis anterior (TA), extensor digitorum longus (EDL) were also removed and weighed. The ventricles were also removed and weighed.

3.1.6. Western Blotting. Protein expression was analyzed as described previously (29).

Briefly, muscle homogenates were resolved by SDS- PAGE (16% Tris-tricine for SLN, and 10% Tris- glycine for UCP1). Proteins were transferred to a nitrocellulose membrane

(0.2μm for SLN, 0.45μm for UCP1). For the UCP1 blots, immediately after transfer, the membranes were stained with 0.5% Ponceau S. The membranes were blocked in 5%

BSA or milk, and probed with primary antibodies to SLN (Millipore, rabbit polyclonal),

UCP1 (rabbit polyclonal, a kind gift from Dr. Barbara Cannon), and GAPDH

(ThermoScientific, mouse monoclonal). Secondary horseradish peroxidase antibodies were applied for 1 hour at a 1:25,000- 1:50,000 dilution. Expression was detected with chemiluminescent substrate.

3.1.8. Statistical Analysis. Data are expressed as means ± S.E.M. Differences among groups were determined by one-way ANOVA. Statistically significant differences were accepted at p<0.05.

3.2. Results

3.2.1. DKO mice do not gain more weight on HFD than SLN-KO and UCP1-KO littermates.

Previous studies have shown both SLN-KO and UCP1-KO mice become obese when fed a HFD (11,15). Therefore, we hypothesized that SLN;UCP1 DKO mice would be even more sensitive to HFD-induced obesity, resulting in more rapid weight gain and a greater cumulative weight gain. For this study, we placed WT, SLN-KO, UCP1-KO, and DKO littermates on a HFD (45% kcal from fat) for 12 weeks. When housed at 22-

25C (standard conditions), mice are under a constant thermal stress, which leads to elevated metabolic rates (7,178) due to activation of adaptive thermogenesis. Therefore, to eliminate this thermal stress and its effects on energy expenditure, the mice were housed at 28-30C for the duration of the experiment. This allowed us to visualize the full effect of DIT, or lack thereof, in these animals.

After 12 weeks of HFD-feeding, as expected, we found the SLN-KO and UCP1-

KO gained more weight than WT (Figure 3.1A). Interestingly, the SLN-KO and UCP1-

KO gained weight at the same rate, leading to similar obesity phenotype. However, unexpectedly, the DKO mice did not gain more weight than the single knockout controls

(Figure 3.1A). Food intake was not significantly different among genotypes (Figure

3.1B), thus the differences in weight gain could not be attributed to differences in caloric intake. Metabolic efficiency is an important measure of metabolic control-it is the ratio of body weight gained to total caloric intake, and represents the amount of ingested energy stored, rather than consumed and converted to heat. We found the SLN-KO and UCP1-

KO mice had significantly higher metabolic efficiencies than WT, and the DKO had a metabolic efficiency between the single knockouts and WT (Figure 3.1C).

Measurement of fat pad weights after HFD-feeding showed a large portion of the weight gained could be attributed to increased adipose tissue deposition. SLN-KO and

UCP1-KO mice had greater white adipose and brown adipose tissue weights, than WT and DKO mice, in agreement with the total weight gained (Figure 3.2A –D).

Interestingly, the greatest differences in white adipose tissue weights occurred in the subcutaneous fat depot, but not in the visceral (epidydymal) depot (Figure 3.2C and D).

Weights of skeletal muscles and ventricles of the heart were not different among genotypes (Table 3.1).

In addition, we measured serum cholesterol and triglycerides and fasting blood glucose levels at the end of the feeding protocol, though there were no significant differences among genotypes (Figure 3.3).

3.2.2. Metabolic Rate.

In order to determine if differences in basal metabolic rate could explain the differences in weight gain, we measured oxygen consumption both before and after 12 weeks of HFD-feeding. The data are presented in two ways: 1)normalized to body weight and 2) not normalized to body weight. Because there are such large variations in body weight by the end of the HFD, normalizing oxygen consumption to body weight can be misleading. There were no differences in metabolic rate among the genotypes before

HFD feeding (Figure 3.4A and B). As shown in Figure 3.4A, all genotypes displayed

60 increased oxygen consumption after HFD-feeding. The increase was significantly greater in the SLN-KO and UCP1-KO. This indicates the resulting obesity was not due exclusively to an inability to increase oxygen consumption in response to diet overload.

However, when oxygen consumption is normalized to body weight (Figure 3.4B), it appears the metabolic rate is decreased in response to HFD-feeding, though this an artifact of the large size difference of the animals Pre- and Post-HFD. All genotypes displayed a decreased respiratory exchange ratio (RER) after HFD-feeding (Figure 3.4C), as expected, indicating a shift in substrate utilization from carbohydrates to fats.

3.2.3. SLN and UCP1 do not compensate for one another in response to HFD feeding.

Cannon and Nedergaard previously observed a UCP1-independent increase in metabolism in response to diet overload, suggesting the presence of other mediators of

DIT in UCP1-KO mice (8). It has also been shown that both SLN and UCP1 are upregulated in response to HFD (11,15). Since we observed SLN and UCP1 could compensate for the loss of the other in cold-induced adaptive thermogenesis, we hypothesized that SLN and UCP1 could compensate for one another in response to HFD- feeding to blunt some of the obesogenic effects. We found that UCP1 protein was increased in response to the HFD (Figure 3.5), as previously reported. Initial analysis of the data indicated UCP1 expression was reduced in the HFD-fed SLN-KO mice; however, when the UCP1 protein levels were normalized to BAT weight, we found the total UCP1 content was equal between the WT and SLN-KO (Figure 3.5 and Figure

3.2A).

Surprisingly, our analysis of SLN levels following HFD-feeding showed SLN was not upregulated in WT mice (Figure 3.6A), in contrast to previous reports. However, the previous studies showing SLN upregulation had been performed with mice reared at

22°C (11), where the mice here were housed at 28-30°C. Moreover, SLN levels were not different between WT and UCP1-KO mice (Figure 3.6).

3.3. Discussion

UCP1 is a major contributor to diet-induced thermogenesis in rodents (15). Our previous studies suggested SLN is also a significant contributor to diet-induced thermogenesis (11). However, it was not known whether these two components; i.e.,

UCP1 and SLN, work together to mediate DIT. Therefore, the objective of this study was twofold: 1) to determine if SLN or UCP1 are required for DIT and 2) to determine if a loss of both SLN and UCP1 results in severe obesity, due to a lack of DIT.

Our cold exposure studies showed that the thermogenic contributions of SLN and

UCP1 are additive; that is, DKO mice are more cold-sensitive than SLN-KO and UCP1-

KO. Thus, we hypothesized that SLN and UCP1 would also contribute additively to DIT, and DKO mice would be severely sensitive to diet-induced obesity. Surprisingly, our data showed that the DKO mice were not more obese than single knockout controls and even showed a slight (not significant) reduction in the severity of obesity. Interestingly, a study by the Kozak group on another UCP1-deficient double knockout (Ucp1-/-; Gdm-/-) mouse model, showed that the double knockouts were resistant to diet-induced obesity, in contrast to the single knockout controls (179). Kozak and colleagues concluded that the

62 resistance to obesity was a result of the activation of alternative metabolically inefficient mechanisms. Therefore, the unexpected similar phenotype of the UCP1;SLN DKO mice to the single knockouts may be the result of an adaptive activation of alternative inefficient pathways to minimize the effects of diet overload. Intriguingly, we observed the DKO mice were also able to activate alternative thermogenic pathways to survive long-term cold exposure, which suggests there may be a common mechanism activated in the cold- and diet-challenged DKO mice.

Another interesting observation from this study was that both SLN-KO and

UCP1-KO developed similar weight gain suggesting that both contribute to diet-induced thermogenesis. Surprisingly, their metabolic efficiencies were almost identical. This indicates both SLN and UCP1 contribute to DIT to a similar degree. However, the presence of UCP1 or SLN is not sufficient to compensate for the lack of the other.

Accordingly, analysis of UCP1 expression in HFD-fed SLN-KO mice and SLN in HFD- fed UCP1-KO mice showed that there was no compensation; i.e. neither UCP1 nor SLN was upregulated in the absence of the other. The paradoxical observation that the DKOs were not more obese than the single knockouts is very interesting and may suggest that when adaptive thermogenic mechanisms cannot be effectively recruited, less efficient mechanisms involving other organs might be activated to dispose excess calories. At this time we could not perform an in-depth study and this area requires further investigation.

. Our analysis of the basal metabolic rates showed no significant difference among genotypes when fed a chow diet (before HFD-feeding). This indicates that neither SLN nor UCP1 significantly affect basal metabolic rate (BMR) at thermoneutrality, and a loss

63 of both SLN and UCP1 does not affect BMR. However, when fed a HFD, one can see the effects of a loss SLN and UCP1 on metabolic rate. This can be visualized by comparisons of the ratio of the increase in oxygen consumption (not normalized to body weight) to the increase in weight. WT mice thus have a ratio of 0.802. SLN-KO mice have a ratio of

0.704, the UCP1-KO of 0.738, and DKO of 0.721. Assuming the WT mice have a

“normal” or “ideal” ratio, one can see the increase in oxygen consumption is less than required to meet the ratio of the WT. Therefore, this suggests that the SLN-KO, UCP-

KO, and DKO are unable to fully activate DIT in response to HFD-feeding.

Figures and Tables for Chapter 3

WT SLN-KO UCP1-KO DKO Ventricle 137.7 ± 4.56 163.3 ± 13.9 156.7 ± 11.1 145.3 ± 14.1 Soleus 9.633 ± 0.558 10.48 ± 0.353 10.86 ± 0.838 10.59 ± 1.348 Tibialis Anterior 51.04 ± 1.238 52.65 ± 1.862 51.42 ± 2.791 50.43 ± 3.457 Extensor Digitorum Longus 11.28 ± 0.411 11.63 ± 0.434 11.69 ± 0.909 11.39 ± 0.588 Gastrocnemius 162.8 ± 1.826 169.2 ± 2.177 165.3 ± 8.57 165.8 ± 8.479

Table 3.1. Heart and muscle weights after 12-weeks of high-fat diet feeding.

Weights are in mg ± S.E.M.

Figure 3.1. Results of high fat diet feeding.

A. Weight gained at each week during high-fat diet feeding at 28°C. B. Total food intake in MJ during the 12-week high-fat diet feeding protocol. C. Metabolic efficiency (total weight gain/total food intake) following the high-fat diet. ** = p<0.01, *** = p<0.001.

Figure 3.2. Fat pad weights after HFD-feeding.

Wet tissue weights of A. brown adipose tissue (BAT), B. white adipose tissue (WAT) (sum of epidydymal and inguinal WAT), C. epidydymal WAT, and D. inguinal WAT after 12-weeks HFD feeding. Statistically significant differences are compared to WT. * = p<0.05, ** = p<0.01.

Figure 3.3. Levels of blood metabolites.

Serum cholesterol (A.) and triglycerides (B.) after a 4-hour fast after 12-weeks HFD feeding. Fasted (16-hour fast) blood glucose levels after HFD-feeding.

Figure 3.4. Metabolic rates of mice before and after HFD-feeding.

Average oxygen consumption over a 24-hour period before (Pre-HFD) and after (Post- HFD) 12-weeks of HFD-feeding. A. Oxygen consumption (ml/hr). B. Oxygen consumption expressed per body weight (ml/kg/hr). C. Respiratory exchange ratio before (Pre-HFD) and after (Post-HFD) HFD feeding. Statistically significant differences are between the same genotype Pre-HFD and Post-HFD. * = p<0.05, **** = p<0.0001.

Figure 3.5. UCP1 expression after HFD-feeding.

Uncoupling protein 1 (UCP1) protein expression in brown adipose tissue of chow-fed WT, HFD-fed WT, and HFD-fed SLN-KO mice. Ponceau S blot provided to show equal protein loading.

Figure 3.6. SLN expression after HFD-feeding.

Sarcolipin (SLN) protein expression in soleus muscles from A. Chow-fed WT and HFD- fed WT and B. HFD-fed WT and HFD-fed UCP1-KO mice. GAPDH: glyceraldehyde 3- phosphate dehydrogenase.

Chapter 4. Sarcolipin in Non-Rodent Species

Endothermy and the requirement for the maintenance of constant body temperature are not exclusive to mammals with BAT (19). Birds, for one, do not have functional BAT, due to the absence of a Ucp1 gene, yet maintain some of the highest body temperatures of endothermic species (20,100). In addition, pigs have a mutated, dysfunctional Ucp1 gene but are able to maintain normal body temperature, and to date, functional BAT has not been identified in marsupials (18,125). Adult large mammals, including humans, have much reduced BAT content, compared to rodents (180-181).

Moreover, even some non-endothermic species, have the capacity for regional endothermy, in the absence of BAT (13,19). In all these species, it has been suggested that skeletal muscle-based thermogenesis is the major contributor to body temperature maintenance (13,19). We therefore hypothesized that SLN could play a major thermogenic role in species with minimal or no BAT. However, little is known about

SLN expression in non-rodent species. Thus, to begin to address this question, the goals of the experiments outlined in this chapter are as follows:

1) to identify SLN protein sequences in non-rodent vertebrate species by analyzing published genomic sequences,

2) to thoroughly analyze SLN protein expression in large mammals, and

3) to identify SLN genomic sequences in two bird species: the house sparrow and chicken.

4.1. Research Design and Methods

4.1.1. Sequence analysis. All nucleotide and protein sequences were obtained from NCBI databases. Conserved Sarcolipin coding sequences (CDS) and protein sequences were identified by a BLAST® search of the mouse Sarcolipin CDS or protein sequence, respectively.

4.1.2. Western blotting. Protein expression was analyzed as described previously (11).

Briefly, muscle homogenates were resolved by SDS- PAGE (16% Tris-tricine for SLN, and 10% Tris-glycine for others). Proteins were transferred to a nitrocellulose membrane

(0.2μm for SLN, 0.45μm for others). The membranes were blocked in 5% BSA or milk, and probed with primary antibodies to SLN (Millipore, rabbit polyclonal), SERCA1a,

SERCA2a, CASQ, and PLB (rabbit polyclonal, custom-made), and GAPDH

(ThermoScientific, mouse monoclonal). Secondary horseradish peroxidase antibodies were applied for 1 hour at a 1:25,000- 1:50,000 dilution. Expression was detected with chemiluminescent substrate.

4.1.3. Cloning of avian SLN DNA sequences. Chicken and house sparrow DNA was prepared by Proteinase K (Sigma-Aldrich) digestion of pectoralis muscle tissue. Primers were designed using SLN-like coding sequences identified in published chicken (Gallus gallus) and zebra finch (Taeniopygia guttata) DNA sequences. The zebra finch sequence was used as a substitute for the house sparrow (Passer domesticus) sequence, since the house sparrow DNA had not been published at the time of experimentation. The primers

(~20-mer) were designed to flank the entirety of the SLN CDS, as shown in Figure 4.4A.

To account for mismatched sequences, the PCR annealing temperature was set at a gradient between 40°C and 50°C for the first 8 cycles, then increased to 56°C for 20 cycles. The PCR products were run on an agarose gel, excised, and purified. The purified

PCR products were then cloned using TOPO® TA cloning. Sequences of the PCR products were obtained by direct sequencing.

4.2. Results

4.2.1. Sarcolipin-like protein sequences can be found in species from fish to man

Given the putative role of SLN in thermogenesis, we searched for conserved SLN protein sequences throughout vertebrate species. High sequence conservation of a protein is indicative of high functional importance, as any mutations to that sequence may be fatal or lead to elimination via natural selection. We hypothesized that if SLN is an important component of muscle-based thermogenesis, it could contribute to heat production in all endothermic species, and possibly in heterotherms that possess regional endothermy. Indeed, alignment of predicted protein sequences indeed showed that SLN is

74 highly conserved from fish to mammals. (Figure 4.1). Importantly, the transmembrane and C-terminal domains, responsible for inhibition of SERCA, are remarkably conserved from amphibians to humans. The N-terminal region has unique conservation among different vertebrate groups; however, the physiological relevance of these unique residues has yet to be studied.

4.2.2. Sarcolipin in large mammals

SLN is expressed exclusively in skeletal and cardiac muscles. However, a previous study showed that the level of SLN expression as well as muscle-specific expression is highly species-dependent (137). This study showed that in rodents, SLN is expressed most highly in slow-twitch muscles, including the soleus, diaphragm, and red portions of the gastrocnemius and quadriceps, as well as the atria of the heart. However, in large mammals, including rabbit and dog, SLN is expressed at a much higher level and is not limited to slow-twitch muscles (137). Here, to more extensively characterize SLN expression, a large panel of rabbit muscles and non-muscle tissues were analyzed for

SLN protein levels. We observed SLN was in fact only expressed in muscle and was ubiquitously expressed at high levels (Figure 4.2).

Protein levels of SLN in human muscle has not been previously determined.

Analysis of human quadriceps tissue showed that SLN is expressed highly, at similar levels to the rabbit (Figure 4.3). A further comparison of human, rabbit, and mouse muscle tissue shows that SLN is much more highly expressed in humans and rabbits.

SLN protein is detectable in 0.5 and 1.0 μg of rabbit and human quadriceps muscles,

75 respectively, but is not detectable in 20 μg mouse quadriceps. In mouse muscles where

SLN protein is detectable (soleus and diaphragm), total SLN content is still significantly lower than in rabbit and human muscle.

4.2.3. Sarcolipin expression in birds

Skeletal muscle-based thermogenesis is considered to be the main contributor to body temperature maintenance in avian species (19,64,99,113,115). As the Ucp1 gene was lost during the evolution of birds, there is no thermogenic contribution from BAT

(20). Therefore, we hypothesized that SLN could significantly contribute to thermogenesis in birds. However, annotated genomic sequences are rare in bird species; thus, the expression of SLN and/or the presence of a functional protein sequence has not yet been determined. The only evidence suggesting SLN is expressed in bird muscle is a

SLN-like mRNA sequence in a chicken expressed sequence tag (EST) (BX935884.1).

Therefore, we sought to clone the SLN DNA sequence in two bird species: the house sparrow (Passer domesticus) and chicken (Gallus gallus) to show that an intact SLN coding sequence is present.

To clone the putative SLN DNA coding sequence from the house sparrow and chicken, primers were designed using existing published sequences from GenBank. As shown in Figure 4.4A, SLN has a short coding sequence (CDS) that is contained within one exon in all known annotated sequences. This made DNA cloning straightforward, as intronic sequences would not interrupt the CDS. Primers flanking the 5’ and 3’ ends of

CDS were used to amplify the DNA, and the PCR product was subsequently sequenced,

76 and the results are shown in Figure 4.4B. The house sparrow nucleotide and protein sequence was similar to other published bird sequences, and the chicken sequences were in exact agreement with published data. These data confirm that SLN-like protein sequences are present in avian species.

To determine if SLN protein is expressed in bird species, we attempted to perform western blotting using our custom-made antibody to SLN. However, our antibody is directed against the last 5 residues of the C-terminus of mouse SLN (RSYQY). As shown in Figure 4.1 and Figure 4.4B, avian SLN has some sequence variation in this region, compared to mouse. Thus, a western blot of house sparrow and chicken muscle did not show expression of SLN (Figure 4.5), though this is likely due to the inability of the antibody to recognize the avian C-terminal sequence. In contrast, the N-terminal region is well-conserved between mouse and birds; therefore, a future study using an antibody generated against this region could provide more conclusive results.

4.3. Discussion

Endothermy is not unique to mammals and maintaining constant body temperature is essential for other animals, including birds. It is well known that rodents primarily use BAT for thermogenesis, while non-rodent species, including large mammals (humans), birds, marsupials, and some endothermic fish species, predominately utilize muscle-based thermogenesis (19). For this reason, we hypothesized that SLN may be a critical contributor to heat production in these species. However, the expression of

SLN in non-rodent species has not previously been well documented.

Our studies show that SLN can be found in vertebrate species spanning from fish to humans. The sequence is remarkably conserved, suggesting SLN has played a critical role throughout evolution. Importantly, the transmembrane and C-terminal regions of

SLN, which mediate the interaction with SERCA, show very little variation among species. As SLN does not function unless it is bound to SERCA, major variations in this region would have resulted in a non-functional protein, which would likely have resulted in a loss of the species. It should also be noted that SERCA is very highly conserved among species, including the region where SLN interacts. On the other hand, the N- terminal region, while highly conserved among fish, birds, and some mammals, has some variation specifically in carnivorous mammals, including humans, cats, walrus, etc. The precise role of the N-terminal region is still under investigation, though it is suggested this region may mediate the uncoupling function of SLN. Future studies should be directed at determining how each of these residue variations affect SLN function, especially considering human SLN has different N-terminal residues than mouse SLN, on which most functional studies have been performed.

We next sought to more extensively characterize SLN expression in a large animal model. For this, rabbit tissue was examined. Rabbits, like humans, downregulate

UCP1 expression, and thus BAT activity soon after birth (120); therefore, we proposed that the SLN expression pattern in rabbits would more closely resemble that of humans than rodents. We found SLN was expressed in all striated muscle tissues collected but was not expressed in non-striated muscle tissue, including smooth muscle. A direct comparison of mouse skeletal muscle with rabbit muscle showed that SLN was expressed

78 at a much higher level in rabbits than mice. We also found that human muscle expressed

SLN at levels comparable to rabbit muscle. These data strongly suggest that SLN plays an important role in large animals, where BAT is lacking.

Some of the most conclusive data implicating skeletal muscle-based thermogenesis has been generated from studies on birds. However, whether SLN plays a role has not been determined. The studies performed here show that a well-conserved

SLN protein sequence is in fact present in avian species. Though our attempts here at determining whether SLN is expressed in bird muscle were unsuccessful, the identification and successful cloning of the DNA CDS holds promise for an important functional role of SLN. Future experiments to clone the entire SLN mRNA sequence in bird species will permit a more detailed study of SLN expression, by allowing for gene expression analyses. In addition, the generation of an antibody directed against the avian

SLN sequence would allow for the determination of SLN protein expression in bird tissues. Future studies could then be performed to determine if SLN expression correlates with exposure to cold temperatures.

Figures and Tables for Chapter 4

Figure 4.1. Alignment of SLN protein sequences from various vertebrate species. Red indicates complete conservation of a residue. Blue indicates conservative substitutions of a residue. Purple/dark blue indicate conservation of a residue across multiple similar species.

Figure 4.2. Analyses of SLN and SERCA expression in rabbit skeletal muscle.

PLB, phospholamban; CASQ, calsequestrin; EDL, extensor digitorum longus; TA, tibialis anterior.

Figure 4.3. Comparison of SLN expression in large mammals versus mice.

SLN protein is detectable in 0.5 and 1.0 μg of rabbit and human quadriceps muscles, respectively, but is not detectable in 20 μg mouse quadriceps. In mouse muscles where SLN protein is detectable (soleus and diaphragm), total SLN content is still significantly lower than in rabbit and human muscle. Quad, quadriceps; Diaph, diaphragm.

Figure 4.4. Sarcolipin DNA cloning strategy and results.

A. Sarcolipin gene schematic. Arrows indicate location of primers for DNA cloning. SLN coding sequences in expansion: M. mus (Mus musculus, mouse) obtained from NBCI; G. gallus (Gallus gallus, chicken) sequence confirmed by DNA cloning; T. gutt (Taeniopygia guttata, zebra finch) sequence from BLAST search; P. dom (Passer domesticus, house sparrow) sequence obtained by DNA cloning. Red nucleotides indicate mismatch with mouse sequence. B. Protein products of SLN CDS obtained in (A). Blue residues are conservatively substituted compared with mouse SLN, and red indicates non- conserved residues.

Figure 4.5. Western blot analysis SLN protein expression in house sparrow pectoralis, chicken pectoralis, rabbit quadriceps, and mouse atria.

Rabbit liver is provided as a negative control and bacterially expressed purified SLN is provided as a positive control. Rabbit quad: rabbit quadriceps; bac. SLN: bacterial SLN.

Chapter 5. Discussion

5.1. Summary

Adaptive thermogenesis, when activated, comprises a significant portion of whole body energy expenditure, thus making the mechanisms mediating adaptive thermogenesis attractive anti-obesity targets (5-6). Brown adipose tissue is well-known for its contribution to adaptive thermogenesis and recently has become the topic of intense research efforts to increase energy expenditure in humans. Unfortunately, to date, these efforts have not yielded major breakthroughs in the fight against human obesity. Part of the reason for this may be due to the relative paucity of brown adipose tissue in adult humans. Though humans possess brown adipose tissue, its abundance and thermogenic contribution are low, especially when compared to rodent brown adipose tissue (19,22).

Another confounding factor may be due to the species-specific differences in brown adipose tissue biology. A recent study showed that human brown adipose tissue is molecularly more similar to rodent beige fat than classical brown adipose tissue (42-43).

Therefore, the assumptions and conclusions made from studies on rodent classical brown adipose tissue may not directly translate to human brown fat. Thus, the identification and targeting of alternative adaptive thermogenic mechanisms is becoming increasingly important.

Studies in humans and large mammals suggest that skeletal muscle plays an important role in adaptive thermogenesis, though the mechanisms have not been identified (19). Muscle is often viewed only as a locomotory organ, though emerging data from our laboratory’s work, among others, suggest muscle is also a powerful regulator of metabolism as well as a significant component of adaptive thermogenesis (182). Thus, a major goal of the work in this dissertation was to better define the role(s) of skeletal muscle in adaptive thermogenesis. Since mammals, and importantly, humans, have both brown adipose tissue and skeletal muscle, another important goal of this dissertation was to understand whether and how these two systems work together in adaptive thermogenesis.

An earlier study by our laboratory suggested Sarcolipin was a major contributor to muscle-based adaptive thermogenesis; therefore, for these studies, we utilized the SLN- knockout (SLN-KO) mouse model (182). Studies on muscle-based thermogenesis in rodents are made difficult due to the dominant thermogenic contribution of brown adipose tissue. Therefore, we also took advantage of the UCP1-KO mouse model, which lacks brown adipose tissue-based thermogenesis (24). The absence of BAT thermogenesis thus allowed us to uncover alternative thermogenic mechanisms. We additionally made a UCP1;SLN double knockout mouse (DKO) to not only understand the unique contributions of UCP1; i.e. BAT, and SLN, but also to understand whether these two systems work in concert in adaptive thermogenesis. The major findings from this study include:

1) UCP1;SLN DKO mice are severely cold-sensitive when challenged to acute cold.

DKO mice are more cold-sensitive than UCP1-KO or SLN-KO controls. This shows that

UCP1 and SLN work additively to mount a full thermogenic response and are dominant thermogenic mechanisms.

2) UCP1 and SLN compensate for the loss of one another during cold adaptation. In the absence of UCP1, SLN levels are upregulated in muscle, and vice versa, UCP1 is upregulated in SLN-KO mice adapted to cold. These data show that UCP1 and SLN play complementary roles and indicate muscle and BAT may interact during cold-adaptation.

3) The absence of SLN increases the energetic cost of survival in the cold. SLN-KO mice exhibit elevated oxygen consumption (metabolic rate) compared to wild-type mice when adapted to 4°C. For optimal survival in cold, animals will recruit the most energetically efficient thermogenic mechanisms. The fact that the SLN-KO mice have increased oxygen consumption indicates these mice are relying on less-efficient thermogenic mechanisms that are activated secondarily to SLN.

4) DKO mice are able to survive gradual cold exposure but at an extremely high energetic cost. Though sensitive to acute cold exposure, DKO mice were able to survive in the cold as long as the temperature was gradually reduced to 4°C. However, as indicated by elevated oxygen consumption (above single knockout and wild-type controls), increased food intake, reduced body weight, and exhaustion of fat stores, it is

87 evident survival in the cold is extremely taxing on the DKO mice. The extreme energetic cost is due to the activation of inefficient thermogenic mechanisms to compensate for the loss of two major systems. It is likely survival in the cold for longer periods than performed in this study would not be feasible for the DKO mice.

5) DKO mice are not more sensitive to diet-induced obesity than UCP1-KO or SLN-KO controls. Despite a lack of both UCP1 and SLN-mediated thermogenesis, the DKO mice are equally sensitive to diet-induced obesity. This indicates both SLN and UCP1 are required for diet-induced thermogenesis.

6) SLN-KO and UCP1-KO mice develop obesity at similar rates. Our unique mouse model (DKO) allowed us to directly compare diet-induced thermogenesis in UCP1-KO and SLN-KO littermates. We found that these mice gained weight at the same rate and had almost identical metabolic efficiencies, indicating SLN and UCP1 contribute to DIT to a similar degree.

7) SLN and UCP1 are not compensatory in response to high-fat-diet-feeding. Unlike cold adaptation, neither SLN nor UCP1 was upregulated in the absence of the other during high-fat-diet-feeding. These data are in agreement with the equal sensitivities of the DKO and single knockouts to diet-induced obesity and indicate neither SLN nor UCP1 is sufficient but both are necessary for DIT.

8) SLN is expressed at high levels in large mammals. This indicates the contribution of

SLN to adaptive thermogenesis may be greater in large mammals, including humans, compared to rodents.

5.2. Significance of this work

This work was the first to show that Sarcolipin is an essential contributor to adaptive thermogenesis, especially in animals lacking brown adipose tissue function.

Because humans possess relatively small amounts of BAT, these studies have exciting implications. Our studies also found that SLN expression is much greater in adult large mammals (humans) where BAT is minimal. Together, this suggests that SLN may play a greater role in adaptive thermogenesis in humans. Based on these data, we propose a new model for the major thermogenic components of endotherms and their relative contributions (Figure 5.1). In adult non-hibernating mammals, including humans, where

UCP1 and BAT are limited, SLN is the dominant source of thermogenesis. By contrast, in species where UCP1 and BAT are abundant, the contribution of SLN to thermogenesis is secondary to that of BAT.

Another exciting observation with significant implications for human health is that SLN-KO and UCP1-KO mice develop comparable levels of diet-induced obesity, which appears to be a result of similar levels of metabolic dysfunction. Since BAT and

UCP1 have become attractive targets to increase energy expenditure in humans, these data suggest SLN is also an attractive target. Considering SLN expression levels in humans as well as skeletal muscle comprising 40% of body mass, activation of SLN-

89 mediated thermogenesis could have profound effects on energy expenditure and metabolism.

In addition to SLN, these studies also establish skeletal muscle as a target to increase energy expenditure, independent of exercise or activity. The work performed here shows that muscle is much more than a contractile organ, and we anticipate this will set a groundwork for future studies on the involvement of skeletal muscle in metabolism.

We hope our studies will highlight the importance of skeletal muscle to human health and the significance of maintaining healthy muscle mass.

5.3. Future Directions

The studies performed here established that SLN and UCP1 are important components of adaptive thermogenesis and are exciting targets for increasing energy expenditure. However, there still remain important gaps in our knowledge that require future investigation, as discussed below.

1) One of the foremost-unanswered questions from this study was the paradoxical survival of DKO mice during gradual cold exposure. Our analysis showed there was no difference in catecholamines between the UCP1-KO and DKO mice, suggesting increased sympathetic drive/output was not a mechanism mediated the DKO survival.

However, other neurohormonal factors not studied here have been shown to regulate thermogenesis (183). For one, thyroid hormone is a well-known regulator of both brown adipose tissue thermogenesis as well as skeletal muscle-based thermogenesis, thus the

90 role of thyroid hormone in the cold-exposed DKO mice requires investigation (119,184-

185). In our interpretation of the data, we concluded that the DKO survival was due to elevated metabolic heat production that was not due to a specific activation of an alternative thermogenic mechanism. However, it is possible there are other thermogenic mechanisms tertiary to SLN and UCP1. The DKO mouse is thus an invaluable tool to identify these mechanisms.

2) While these studies have shown the critical role of SLN in adaptive thermogenesis, the precise mechanism of SLN action has not yet been fully elucidated. In particular, these studies have not differentiated between the contribution of SLN to shivering versus nonshivering thermogenesis, due to the difficulty in separating the two mechanisms.

Regardless, we postulate that SLN contributes to both aspects of heat production by muscle. By uncoupling Ca2+ uptake from ATP hydrolysis, SLN can have a dual effect: it can both increase ATP hydrolysis and heat production (67,71,148-149) and intracellular

Ca2+ levels (166,186). Therefore, in addition to augmenting heat production by SERCA- mediated ATP hydrolysis, SLN could affect muscle heat production via Ca2+-dependent processes. For instance, the myosin ATPases present in muscle are significant energy consumers (61,187), and the SLN-mediated increase in intracellular Ca2+ could further enhance this heat production by activating more cross-bridge cycling, both at rest

(resulting in an increase in muscle tension) and during contraction. It is also known that

Ca2+ is a powerful stimulator of metabolism through allosteric regulation of metabolic enzymes and Ca2+-dependent signaling pathways (46,188-194). Thus, the SLN-mediated

91 increase in Ca2+ could serve as a regulator of ATP supply to the muscle during thermogenic demand. Future studies will be aimed at understanding the detailed mechanism of how SLN regulates muscle heat production at the molecular and cellular level.

3) The regulation of SLN expression has not been determined. In particular, how SLN is upregulated in the UCP1-KO mice is not known. Moreover, we have not identified the factors mediating browning in the SLN-KO mice. The mechanisms by which muscle and

BAT can stimulate each other’s activity require further investigation, in addition to whether this is mediated by factors/hormones originating from these tissues.

4) There remain important gaps in our knowledge with regard to the role of BAT and muscle-based thermogenesis in different mammalian species. Why certain species evolved a preferential utilization of BAT versus muscle or vice versa, why BAT evolved in some species but not others (birds, marsupials, etc.) remain unknown. An interesting question arises from hibernating animals. While these animals are known to use BAT- dependent thermogenesis, their muscles are remarkably protected from atrophy during hibernation, which may suggest the recruitment of muscle-based thermogenesis as well, though the differential or seasonal variations in recruitment of these two mechanisms have not been studied.

Moreover, our sequence comparison revealed that SLN is highly conserved from fish to man, suggesting that SLN could potentially be involved in thermogenesis in non-

92 mammalian endotherms. However, it remains to be determined if SLN has the same function in all vertebrates. In addition, relatively little information is available concerning thermogenic mechanisms in avian species, especially regarding the role of SLN in avian skeletal muscle. Although putative sequences have been identified in many bird and fish species, expression of SLN mRNA or protein has not been investigated. The most promising evidence, so far, has been the identification of SLN-like mRNA sequences in a chicken expressed sequence tag (EST) (BX935884.1) and catfish EST (AF227818.1), both from muscle, although experimental confirmation is lacking.

Figures and Tables for Chapter 5

Figure 5.1. Proposed model showing relative contributions of UCP1 and SLN to thermogenesis in birds and mammals.

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