Adrenergic signaling in insulin-sensitive tissues
Daniel L. Yamamoto
The Wenner-Gren Institute The Arrhenius laboratories F3 Stockholm University SE-106 91 Stockholm Sweden
Stockholm 2007 Doctoral Dissertation 2007 Department of Physiology, The Wenner-Gren Institute The Arrhenius Laboratories Stockholm University SE-106 91 Stockholm
ABSTRACT
Glucose metabolism in insulin-sensitive tissues such as skeletal muscle and adipose tissue is tightly regulated by external stimuli. Metabolic changes in these tissues have direct effects on whole body metabolism. Such metabolic changes can be induced or influenced by adrenergic stimulation.
In L6 skeletal muscle cells, we have seen that the β2-adrenergic receptor increases glycogen synthesis to the same extent as insulin. The β2-adrenergically mediated effect is independent of cyclic AMP but dependent on PI3K. In brown adipocytes, our data suggest that signaling from the β-adrenergic receptors consists of an acute cyclic AMP effect that is rapidly desensitized and then a prolonged signal involving PI3K. In skeletal muscle cells in culture, we have shown that DPI (a NADPH oxidase inhibitor) increases glucose uptake through a signaling pathway independent of NADPH oxidase and insulin signaling. DPI instead inhibits complex 1 in the mitochondrial respiratory chain, which lowers ATP levels. This activates AMPK, an activator of glucose uptake. Furthermore, we have developed a model system for ordered fusion of skeletal muscle cells in culture. In this system, differentiating skeletal muscle cells can be studied separately. This system is optimal for microscopy techniques and easily adaptable for micromanipulations. We have seen that the myogenic factor MyoD can have different expression of the protein in different nuclei within the same myotube. This system could be used with advantage for intracellular signaling and metabolic studies.
© Daniel L. Yamamoto 2007 Cover by Daniel L. Yamamoto and Robert I. Csikasz ISBN 91-7155-395-9 Printed in Sweden by Universitetsservice US-AB, Stockholm 2007 Distributor: Stockholm University Library “A Jedi’s strength flows from the Force. But beware of the dark side. Anger, fear, aggression; the dark side of the Force are they. Easily they flow, quick to join you in a fight. If once you start down the dark path, forever will it dominate your destiny, consume you it will.” - Master Yoda, The Empire Strikes Back This thesis is based on the following papers, referred to in the text as papers I-IV
I. Daniel L. Yamamoto, Dana S. Hutchinson and Tore Bengtsson (2007)
β2-adrenergic activation increases glycogen synthesis in L6 skeletal muscle cells through a signalling pathway independent of cyclic AMP. Diabetologia 50 (1): 158-167
II. Daniel L. Yamamoto, Ekaterina Chernogubova, Dana S. Hutchinson, Julia Nevzorova and Tore Bengtsson β-adrenergic receptor cAMP and PI3K signaling in primary cultures of brown adipocytes. Manuscript
III. Dana S. Hutchinson, Robert I. Csikasz, Daniel L. Yamamoto, Irina G Shabalina, Per Wikström, Mona Wilcke and Tore Bengtsson Diphenylene iodonium stimulates glucose uptake in skeletal muscle cells through mitochondrial complex I inhibition and activation of AMP-activated protein kinase. Accepted for publication in Cellular Signalling
IV. Daniel L. Yamamoto, Robert I. Csikasz, Yu Li, Gunjana Sharma, Klas Hjort, Roger Karlsson and Tore Bengtsson Myotube Formation on UV-lithographically Micro-patterned Glass: A New Experimental System for Studies of Muscle differentiation in vitro. Submitted for publication Doctoral Thesis - Adrenergic signaling in insulin-sensitive tissues
CHAPTER I – Introduction 9 Adrenergic impact on insulin-sensitive tissues 9
CHAPTER II – Adipose and Muscle tissues 11 Adipose tissue – White and brown fat 11 White adipose tissue 11 Brown adipose tissue 11 Thermogenesis 12 Skeletal muscle 13 Skeletal muscle morphology 13 Factors involved in skeletal muscle growth 13 Differentiation and fusion of skeletal muscle cells – from myogenic precursor to myotube 14 - Fusion and myogenic cells 14 - Myogenic factors 15
CHAPTER III - Model systems 20 Skeletal muscle model systems 20 Primary culturing of skeletal muscle cells 20 Clonal cell lines 21 Spatially organized cell cultures 22 Primary cultures of brown adipocytes 24
CHAPTER IV – Insulin-sensitive tissues as a model system for intracellular signaling 26
CHAPTER V – Intracellular signaling 28 Signaling lessons from the skeletal muscle 28 Insulin signaling 28 - The classical pathway 28 - The TC10 pathway 28 Protein kinase C 29 Adrenergic receptors 30 Adrenergic receptors in insulin signaling tissues 31 Signaling kinetics 31 Molecular mechanisms of agonist binding to the GPCRs 31 - Activation of adrenergic receptors 32 - Desensitization of adrenergic receptors 33 Adrenergic signaling 35
α1-adrenoceptor signaling 35
α2-adrenoceptor signaling 35 Classical β-adrenergic signaling 36 Atypical β-adrenergic signaling 37
Compensation of β1- β3-adrenoceptor signaling 37 Biphasic adrenergic signaling 37
CHAPTER VI – Metabolism 39 Muscle and fat metabolism – Gathering, wasting, storing 39 Factors involved in insulin-sensitive tissue energy management 39 - Facilitative glucose transporters 39 - Glucose transport – Harvesting the energy 40 - Glucose metabolism – Using the energy 42 - Glycogen metabolism – Storing the energy 43 - AMPK – The energy sensor 43
CHAPTER VII – Adrenergic Effects 45 The involvement of adrenergic signaling in muscle growth 45 Protein degradation 45 Hyperplasia and hypertrophy 45 Adrenergic effects on myogenic factors 46 Adrenergic effects on brown adipocyte thermogenesis 46 Adrenergic regulation of metabolism in insulin-sensitive tissues 46 Adrenergic regulation of skeletal muscle energy management 46 Adrenergic regulation of brown adipocyte energy management 47 Glucose uptake 47 Glycolysis / Glycogenolysis 48 Glycogen synthesis 49
CHAPTER VIII – Summary and Conclusions 51
Acknowledgments 55
References 57 Abbreviations
BAT Brown adipose tissue WAT White adipose tissue UCP Uncoupling protein PI3K Phosphatidylinositol 3-kinase IGF-1 Insulin-like growth factor 1 SP Side population MRF Myogenic regulatory factor MEF2 Myocyte enhancer factor-2 PKA Protein kinase A PKB/Akt Protein kinase B PKC Protein kinase C PIP3 Phosphatidylinositol (3,4,5)-trisphosphate PDK PIP3-dependent kinase IR Insulin receptor c-Cbl Casitas b-lineage lymphoma CAP c-Cbl-associated protein GEF Guanyl nucleotide exchange factor AC Adenylyl cyclase PLC Phospholipase C GAP GTPase-activating protein TM Transmembrane GPCR G-protein coupled receptor β-ARK1/GRK2 β-adrenergic receptor kinase 1 AR Adrenergic receptor/Adrenoceptor KO Knock-out PDE Phosphodiesterase IP3 Inositol 1,4,5-trisphosphate DAG Diacylglycerol GLUT Glucose transporter AMPK AMP-activated protein kinase PP Protein phosphatase GP Glycogen phosphorylase GS Glycogen synthase GSK3 Glycogen synthase kinase 3 PK Phosphorylase kinase HSL Hormone sensitive lipase 2DMS 2-Dimensional muscle syncytium 9
CHAPTER I – Introduction
Insulin-sensitive tissues (including different types of skeletal muscle and adipose tissue) have a dynamic glucose metabolism that is regulated by insulin. The regulation of metabolism in these tissues can have an impact, not only on the state of the cells, but also of the whole body since it can lower or increase the blood concentration of glucose. Skeletal muscle is important due to it being a large part of the body, as well as having a high energy demand. Adipose tissue also has a great impact on whole body metabolism since it is the major tissue for energy storage. In certain metabolic disorders, such as type II diabetes, regulation of blood glucose is impaired. However, factors apart from insulin play important roles in regulating metabolism in these tissues. Increased cellular activity, such as contraction of skeletal muscle or thermogenesis of brown adipocytes (a thermogenic organ) also increases the energy demand of the cell. Adrenergic signals, coming either from the sympathetic nervous system or from the blood stream, also have a great impact on metabolism in muscular and adipose tissues. Historically, adrenergic signaling has been thought to oppose the actions of insulin. Insulin is known to be an anabolic signal and adrenergic signaling has been thought to be a catabolic signal either by itself (Challiss et al., 1986; Chasiotis and Hultman, 1985) or by negative effects on insulin signaling (Ahren, 1999). However, adrenergic signaling has now also been found to have anabolic effects. This will be discussed in this thesis.
Adrenergic impact on insulin-sensitive tissues Adrenergic signaling is induced by the release of epinephrine from the adrenal gland or release of norepinephrine from the sympathetic nervous system. There are several different effects of adrenergic stimulation on tissues such as an increased heart rate (Adams and Brown, 2001), hypertrophic growth of skeletal muscle cells (Hinkle et al., 2002), regulation of thermogenesis in brown adipocytes (Cannon and Nedergaard, 2004) and regulation of glucose metabolism in brown fat and skeletal muscle (Chernogubova et al., 2004; Nevzorova et al., 2002). The adrenergic system also plays a role in the regulation of myogenic factors controlling differentiation of skeletal muscle cells. 10
The main focus of this thesis is the adrenergic signaling pathways regulating different metabolic effects in insulin-sensitive tissues. 11
CHAPTER II – Adipose and Muscle tissues
Muscular and adipose tissues share the same origin. Mesenchymal stem cells can differentiate into adipocytes and myocytes, as well as other cell types (Alhadlaq and Mao, 2004; Pittenger et al., 1999; Saltiel, 2003). Both white and brown adipocytes differentiate from non-identical preadipocytes (Rosen and Spiegelman, 2000; Timmons et al., 2007) while skeletal muscle cells originate from myogenic precursors called satellite cells (Seale and Rudnicki, 2000). Both adipose tissue (Cinti, 2005) and skeletal muscle (Nonogaki, 2000) are innervated by the sympathetic nervous system.
Adipose tissues – White and brown fat There are two types of mammalian adipose tissue, white adipose tissue (WAT) and brown adipose tissue (BAT). The two tissues are both capable of storing triglycerides in lipid droplets. The main functions of WAT are energy storage, hormone production and insulation while the main function of BAT is thermogenesis. Of the adipose tissues, BAT has denser vascularisation, as well as sympathetic innervation (Cinti, 2005).
White adipose tissue WAT is responsible for the major triglyceride storage in the mammalian body. Energy is stored as triacylglycerols in intracellular fat droplets. The lipid droplets stored in WAT are not used for a specific task (such as thermogenesis in BAT) but instead as an energy reserve for the entire body (Trayhurn, 2005). Historically, WAT has been seen mainly as tissue for energy storage and insulation; however, it now appears that WAT has other important functions in the body, such as functioning as an endocrine organ since several important hormones are produced by WAT, among them the adipokines leptin (Ahima, 2006; Flier, 2004) and adiponectin (Maeda et al., 1996).
Brown adipose tissue BAT is responsible for non-shivering thermogenesis in mammals. It is found in several parts of the body, among them the interscapular, the periaortic and the perineal BAT depots. This subject has been extensively reviewed by Cannon and Nedergaard 12
(Cannon and Nedergaard, 2004). Brown adipocytes mature from preadipocytes in a similar manner to white adipocytes and start accumulating lipid droplets but in contrast to WAT these droplets are multilocular (Nechad et al., 1983). A unique feature of BAT is the expression of the mitochondrial uncoupling protein 1 (UCP1) (Cannon and Nedergaard, 2004). In comparison with WAT, BAT has abundant mitochondria (Cinti, 2005). In similarity to WAT, BAT can also produce leptin and adiponectin (Trayhurn, 2005). The regulation of the release of leptin is potentially important for the development of diabetes. In rodents, leptin is an important hormone for the regulation of whole body energy balance (Ahima, 2006; Trayhurn, 2005). Increased leptin levels inhibit insulin release (Emilsson et al., 1997) and glycogen synthesis in skeletal muscle (Liu et al., 1997).
Thermogenesis Thermogenesis produces heat in order to maintain the temperature of the body. The two tissues capable of producing heat are skeletal muscle (shivering thermogenesis) (Cannon and Nedergaard, 2004; Florez-Duquet and McDonald, 1998) and BAT (non-shivering thermogenesis) (Ahima, 2006; Cannon and Nedergaard, 2004). The UCP1 protein is required for the thermogenic function of the brown adipocyte and critical for non-shivering thermogenesis in the whole animal (Golozoubova et al., 2001; Matthias et al., 2000). Brown adipose thermogenesis is fueled by fatty acids supplied by lipolysis of the triglycerides stored in lipid droplets within the cell (Cannon and Nedergaard, 2004). UCP1 works as a mitochondrial uncoupler and allows for a higher proton flux through the inner mitochondrial membrane (Kopecky et al., 2001; Ricquier and Bouillaud, 2000). Uncoupling of the mitochondria is dependent on free fatty acids (Jezek, 1999) but the exact mechanism is not fully understood (Cannon and Nedergaard, 2004; Ricquier and Bouillaud, 2000). Several models have been proposed, either the fatty acids shuttle protons, act as allosteric regulators or as cofactors. Apart from maintaining body temperature, it has been discussed that brown adipose thermogenesis also has the function of expending excess energy. The subject of mitochondrial uncoupling has been extensively reviewed by Cannon and Nedergaard (Cannon and Nedergaard, 2004). 13
Skeletal muscle Skeletal muscle tissue is responsible for movement through contraction. A skeletal muscle is formed by several muscle fibers growing in parallel. Skeletal muscle cells are often referred to as striated muscle cells, the striation seen in the skeletal muscle cell coming from the organization of the contraction-producing sarcomeres. The functionality of the skeletal muscle tissue is critical for survival.
Skeletal muscle morphology Skeletal muscle consists of parallel muscle fibers capable of contraction. However, skeletal muscle also contains stem cell-like satellite cells. The satellite cells
A B C
Fig. 1 Differentiation of C2C12 skeletal muscle cells during different stages of differentiation A. Mononucleated myocytes. B. The fusing of myoblasts to form fibers. C. The multinucleated myotubes. are thought to form myotubes by proliferating and fusing together. In the mature muscle, satellite cells are located between the basement membrane and the sarcolemma (Goldring et al., 2002). In neonatal muscle, satellite cells make up around 30 % of the total cell number but in adult muscle this drops to near 5 % (Seale and Rudnicki, 2000). In need of repair or regeneration, satellite cells go into a proliferative stage and migrate to the site of damage (Goldring et al., 2002), where they differentiate into myoblasts, align and fuse with each other or with existing myotubes (Seale and Rudnicki, 2000). Factors involved in skeletal muscle growth In response to load-bearing exercise, skeletal muscle adapts by increasing its size (Glass, 2003). Two factors are involved in skeletal muscle growth, an increase in the number of cells it consists of (hyperplasia) and an increase in the size of each of its muscle fibers (hypertrophy). 14
Hypertrophy can be discussed at two different levels, the hypertrophy of a whole muscle or the hypertrophy of a single muscle fiber. To understand the increase in the mass of the whole muscle, one must consider a reduction of the muscle by cell death and protein degradation, as well as an increased protein synthesis or cell proliferation. The hypertrophic effects in adult muscle tissue are thought to be mainly due to increased protein synthesis, in view of the low amount of proliferating cells. The hypertrophy of the single muscle cell is dependent on the rate of protein synthesis and the rate of protein degradation. Skeletal muscle growth is regulated by exercise via the release of insulin-like growth factor 1 (IGF-1) (Glass, 2003) from skeletal muscle cells (Perrone et al., 1995). The release of IGF-1 is sufficient for inducing hypertrophic growth in skeletal muscle (Glass, 2003). Skeletal muscle hypertrophy can be induced by the Akt/mTOR signaling pathway (Bodine et al., 2001), which is induced by IGF-1 via phosphatidylinositol 3-kinase (PI3K) (Rommel et al., 2001). The increase in muscle mass induced by IGF-1 is dependent on two factors; protein synthesis of the mature myotubes and the hyperplastic and regenerative effects of the satellite cells (Barton- Davis et al., 1999). Hypertrophy is also regulated by the adrenergic system (Glass, 2003). One gene involved in hypertrophy of skeletal muscle is myostatin, which has negative effects on the rate of proliferation and protein synthesis of skeletal muscle cells. This effect can be seen in myoblasts, as well as in myotubes (Taylor et al., 2001). Myostatin works as a growth repressor and knock-out of the gene will cause abnormal hypertrophic and hyperplastic muscle growth (McPherron and Lee, 1997). Myostatin down-regulates the expression of MyoD and myogenin. The down- regulation of MyoD is followed by a decrease in myoblast proliferation (Langley et al., 2002).
Differentiation and fusion of skeletal muscle cells – from myogenic precursor to myotube
Fusion and myogenic cells Myogenic differentiation is dependent on the fusion of mononucleated cells (Figure 1). The fusion of myogenic cells has been extensively reviewed by Wakelam 15
(Wakelam, 1985). The myogenic fusion is cell-specific, and the cells cannot fuse with other cell types. The amount of cell nuclei increases within the myotubes during normal growth (Enesco and Puddy, 1964; Macconnachie et al., 1964). However, the cell nuclei within the myotubes are not capable of division; proliferative nuclei may instead be provided by satellite cells, reviewed by Campion (Campion, 1984). Historically, mammalian myogenic precursors are often simply termed satellite cells. Satellite cells are usually seen as dedicated to a myogenic fate (Asakura et al., 2001; Wada et al., 2002). Skeletal muscle tissue contains two distinct cell types capable of repair and myogenesis, satellite cells and side population (SP) cells (Asakura et al., 2001; Bachrach et al., 2004). Multipotential SP cells (Goodell et al., 1996) have been found in several types of tissue, including skeletal muscle (Asakura et al., 2001; Gussoni et al., 1999). Initially, SP cells do not express any myogenic markers (Asakura et al., 2001; Gussoni et al., 1999) but can differentiate into satellite cells (Asakura et al., 2001). SP cells are capable of acting systemically and may reach their target via the bloodstream (Gussoni et al., 1999), while satellite cells, situated within the skeletal muscle (Goldring et al., 2002), may be involved in local regeneration and myogenesis. Muscle repair is not limited to skeletal muscle SP cells; bone marrow SP cells are also capable of muscle repair (Gussoni et al., 1999). Historically, there have been different views of the fusion process. One theory is that cells fuse directly upon contact. It has also been proposed that fusion is the sum of a series of distinct recognition and adhesion events. In vivo, the myogenic cells are influenced by factors ensuring that the myotubes are orientated in a specific direction in order to form a functional muscle. In cell culture, such factors are not present and cultured myotubes are randomly arranged and are often branching. Both the composition and structure of the extracellular matrix (ECM) may be important for a proper spatial arrangement.
Myogenic factors The formation of skeletal muscle is regulated by myogenic regulatory factors (MRFs). Of special interest are the members of the MyoD family: MyoD (Pinney et al., 1988), myogenin (Wright et al., 1989), Myf5 (Braun et al., 1989) and MRF4/Myf6/Herculin (Braun et al., 1990; Miner and Wold, 1990). The MyoD family 16 belongs to a larger group of basic helix-loop-helix (bHlH) DNA binding proteins (Berkes and Tapscott, 2005). Most likely, the MRFs have different functions during myogenesis.
The expression of different MRFs can affect the expression of others. MRF4 induces MyoD expression and represses Myf5 expression (Block and Miller, 1992). Myf5 is important for the expression of myogenin (Braun et al., 1992). MyoD is also known to autoregulate its own expression. Due to the cross- and auto-regulatory loops between the MRFs (Thayer et al., 1989) matters are complicated and the exact function of the proteins is not fully understood. The different myogenic factors may also have overlapping functions (Dedieu et al., 2002), which further complicates the understanding of these factors. The myogenic factors may be put into two different groups, MyoD and Myf5 being primary factors and in general important for myogenic determination, while myogenin and MRF4 may be required downstream of MyoD and Myf5 to function as differentiation factors, reviewed by (Sabourin and Rudnicki, 2000). MyoD and Myf5 are thought to be regulated by the cell cycle and they are expressed in proliferating myoblasts (Lindon et al., 1998) and considered myogenic commitment factors (Berkes and Tapscott, 2005). While Myf5 is highly expressed in proliferating myoblasts, it is down-regulated during cell fusion (Lindon et al., 1998), but is important for myoblast fusion in the C2C12 skeletal muscle cell line (Dedieu et al., 2002), MyoD protein expression is down-regulated in multinucleated myotubes (Paper IV). L6 skeletal muscle cells lack MyoD and express only low levels of Myf5 but are to some extent capable of differentiation and fusion (Braun et al., 1989). Animals lacking MyoD have normal skeletal muscle development (Rudnicki et al., 1992) and the same is true for animals lacking Myf5 (Braun et al., 1992). This suggests that Myf5 and MyoD may compensate for each other. In contrast to MyoD and Myf5, myogenin is not expressed in undifferentiated myoblasts (Braun et al., 1989; Edmondson and Olson, 1989; Montarras et al., 1989; Wright et al., 1989). Myogenin is important for myotube formation (Wright et al., 1989) but the role of MRF4 is complicated (Berkes and Tapscott, 2005). In addition to the MRFs, there is another family of proteins important for myogenesis, the myocyte enhancer factor-2 (MEF2) family (Olson et al., 1995). 17
MEF2 proteins interact with MRFs and give different responses dependent on the combination of interacting proteins. Many skeletal muscle and cardiac muscle genes have a MEF2 binding site. Skeletal muscle cells in culture proliferate, migrate and align to fuse (Kalderon and Gilula, 1979). In C2C12 cells, the expression of MyoD appears to be more or less stable while Myf5 protein was only found during the fusion process and myogenin protein expression was seen at the start of fusion, increased until the cells were fully fused and then dropped over time (Dedieu et al., 2002).
Interestingly, in C2C12 myoblasts, the nuclear expression of MyoD is not equal between cells (Yoshida et al., 1998). Rather, the proliferating myoblasts are divided into subpopulations positive or negative for MyoD ((Yoshida et al., 1998); Paper IV; Figure 2).
Figure 2. MyoD protein expression in undifferentiated C2C12 myoblasts
The two subpopulations will be referred to as MyoD+ (expressing MyoD) and MyoD- (not expressing MyoD). It has been postulated that MyoD+ cells start to express myogenin and enter myogenic cell fusion, while MyoD- cells remain in an undifferentiated state to act as a reserve cell population which may later regain their myogenic potential (Yoshida et al., 1998). It is worth mentioning that quiescent satellite cells do not express any member of the MyoD family (Smith et al., 1994; 18
Yablonka-Reuveni and Rivera, 1994). MyoD- cells (reserve cells) share some of the properties of satellite cells. Differentiated C2C12 myotubes have lost their MyoD expression ((Yoshida et al., 1998); Paper IV). In contrast to the postulation made by Yoshida and coworkers that only MyoD+ cells may fuse with each other, we have seen that this may not be the case. Careful examination of C2C12 skeletal muscle cells in culture during early fusion suggests that skeletal muscle myotubes have different expression of MyoD protein in different nuclei, within the same cell (Figure 3; Paper IV).
Figure 3. Different protein expression of MyoD in cell nuclei within the same myotube. Green: filamentous actin, Blue: cell nuclei, Red: MyoD
This finding raises some interesting points of discussion. It is possible that myotubes are formed by the fusion of both MyoD+ and MyoD- myoblasts. One hypothesis is that MyoD- cells may take the role of a “receptive” cell while MyoD+ cells are “giver” cells fusing with MyoD- cells to form a myotube. It may be possible for a “template” myotube to act as a “receiver” in one part of the cell while other parts have other tasks. This model could explain the linear skeletal muscle formation seen in vivo by allowing for fusion in the cell ends. It is also possible that the myotube 19 defines a length by terminal fusion and then shifts the fusion “hotspots” to other parts of the cell to allow for skeletal muscle repair by satellite cells. A myotube is a large structure and there could be a vast separation of the nerve- muscular connections. It is unlikely that the signaling of a single neuron would regulate all cell nuclei in a muscle fiber; more likely the nervous system can affect different parts of the cell differently. External factors, such as the nervous system, may be able to regulate nuclei separately or at least locally. 20
CHAPTER III – Model systems
Skeletal muscle model systems The study of signaling in skeletal muscle cells can be investigated either in vivo or in vitro. There are advantages with both types of systems; in the in vivo system there is a risk of influences from surrounding cell types. The in vitro system may be less physiological but has the advantage of allowing studies on isolated cell types. In vivo systems include whole animal experiments or perfusion of a specific muscle. In vitro systems include organ-bath setups and tissue culture. Primary culturing of skeletal muscle cells has been performed since the mid 20th century but the protocols for isolating skeletal muscles are not yet optimized. Apart from primary cultures, there are a few skeletal muscle cell lines available, such as the mouse C2C12 and rat L6 skeletal muscle cell lines.
Primary culturing of skeletal muscle cells Primary cultures of skeletal muscle cells can be prepared in several different ways and from several types of muscle tissue. The growth of skeletal muscle cells is based on isolation of satellite cells. Several protocols for isolation of muscle cells from several different species have been reported, a subject extensively reviewed by Burton and coworkers (Burton et al., 2000). The isolation of satellite cells starts with the dissection of specific skeletal muscles. Different muscles contain satellite cells with different characteristics with respect to proliferation, differentiation and sensitivity to growth factors (Burton et al., 2000). When culturing skeletal muscle cells, it is important to remove contamination from surrounding tissues such as fat and fibroblasts. We have developed a primary skeletal muscle culture protocol yielding multinucleated contractile myotubes from both rat and mouse (Yamamoto et al., unpublished). Muscle tissue is cleaned from other tissue types during dissection (Paper III) and during a series of centrifugation and filtration steps (Paper III). The tissue is minced before being subjected to digestive enzymes that liberate the satellite cells (Paper III; Braiman et al., 1999). The cells are plated in culturing vessels that may or may not be coated with different attachment factors. 21
Substrates are sometimes used for increasing cell attachment. Some common substrata are Matrigel, fibronectin and denatured collagen. Muscle cultures can also be plated on substrates such as laminin and gelatin alone or gelatin supplemented with collagen. The primary cultured skeletal muscle cells obtained proliferate extensively in a myoblast stage and spontaneously form contractive myotubes (Yamamoto, unpublished).
Clonal cell lines The L6 skeletal muscle cell line was isolated from rat skeletal muscle in 1968 by David Yaffe (Yaffe, 1968). A few years later the skeletal muscle cell line C2 was isolated by Yaffe and Saxel (Yaffe and Saxel, 1977). A subclone of the C2 cells termed C2C12 (Blau et al., 1983) is now widely used as an in vitro skeletal muscle cell system. C2C12 skeletal muscle cells also have a spindle-shaped morphology in the proliferative state. C2C12 cells are capable of extensive proliferation and differentiate into multinucleated myotubes capable of spontaneous and electrically induced contraction (Yamamoto, unpublished). The L6 cells proliferate slower than the C2C12 cells and do not always form the characteristic multinucleated myotubes that the
C2C12 cells do (Yamamoto unpublished). The discussion of primary cultures versus cell lines can be seen from different angles. The most fitting model system is perhaps defined by the study that the researcher wishes to perform. Whether primary cultures or cell lines are most physiological systems can be debated. The immortalization of the clonal cell lines may induce changes in the cells physiology but on the other hand, the enzymatic liberation of the satellite cells may also affect the cells. Primary cultures may also contain a portion of non-myogenic cells (Yamamoto, unpublished). During the course of this thesis research, several attempts have been made to isolate a primary culture of skeletal muscle showing similar metabolic characteristics as one expects in vivo (Yamamoto unpublished). However, while the intact skeletal muscle is clearly insulin-sensitive, the isolated cell culture does not show any glucose uptake in response to insulin (Yamamoto unpublished). On the other hand, when obtaining clean cultures, there is high differentiation efficiency. These cells have clear myotube morphology and are multinucleated (Yamamoto, unpublished). 22
Between the cell lines L6 and C2C12, only L6 cells show any insulin-mediated glucose uptake in response to insulin. Still only the C2C12 cells are morphologically similar to skeletal muscle cells; the L6 cells are most often seen to possess a fibroblast shape, even after differentiation.
Spatially organized cell cultures One feature of skeletal muscle cells is the clear polarization; these cells cannot work properly if they do not have a proper spatial organization. Structural influence on orientation has been shown in cell types such as rat calvarial cells (Chesmel and Black, 1995), human gingival cells (Brunette et al., 1983) and astroglial cells (Recknor et al., 2004). Different types of skeletal muscle have also been used, such as immature myoblasts (Evans et al., 1999) and primary cultures of chicken muscle cells (Isaeva, 1980). Skeletal muscle cells grown on micropatterned laminin surfaces show some orientation during myogenesis (Clark et al., 1997). It has been seen in several cell types that surfaces with physical ridges and grooves can align cells along the ridges (Brunette et al., 1983; Chesmel and Black, 1995; Evans et al., 1999; Mulder et al., 1998). The degree of orientation may depend on several factors such as cell type, groove width, depth and material (Brunette et al., 1983; Chesmel and Black, 1995; Evans et al., 1999; Mulder et al., 1998).
In cell culture, one can study a population of cells; in some cases cell culture also allows for studies of mature cells, as well as undifferentiated cells. Skeletal muscle cell differentiation is dependent on cell-cell interactions and a mature skeletal muscle cell is defined by the fusion of multiple cells. Naturally, it is not possible to fully differentiate a skeletal muscle cell in a sparsely populated culture. No model system allows the study of differentiation of a single skeletal muscle cell isolated from all surrounding cells. However, limiting the cell-cell interactions to only the ends of the cell would resolve this problem. 23
Figure 4.
The effect of channels on myoblast and myotube organization in C2C12 skeletal muscle cells. Phase-contrast photography. (Paper IV) We have established a system for controlling cell arrangement of skeletal muscle cells in culture (Paper IV; Figure 4). This system is based on the introduction of physical ridges within a cell culture system; the ridges form channels in which cell attachment is permitted. When grown under these conditions, skeletal muscle cells can be followed during differentiation and are separated from each other. This enables the observer to study a single cell from the first stage of differentiation to the mature myotube. Cells grown with this technique have a linear morphology and the branching morphology seen in conventional culturing is abolished (Figure 5). Using this system, we have seen that the cell nuclei within the myotube are found both solitarily and in clusters. The finding that different cell nuclei can express different amounts of the MyoD protein within the same cell was also made in studies on spatially arranged C2C12 skeletal muscle cells. Having a linear myotube separated from the surrounding cells makes it easy to specify and define the microcompartment of interest. The system enables studies on the different functions of the different nuclei (or nuclei clusters) within the cell, as well as studies on microcompartments within the cell and especially regions of the cell that are normally difficult to study 24 due to interference by surrounding cells (such as filopodia or cell attachment regions).
Figure 5. Immunofluorescent picture of C2C12 myotubes grown in cultures with and without patterned surfaces. The random myogenic fusion in the non-patterned system induces a branching phenotype of the cells. This is abolished in the patterned system.
Studies on a single myotube in culture would be a good model system for several issues, such as cell differentiation, attachment, cell motility, and studies on a number of microcompartments within the cell.
Primary cultures of brown adipocytes Similar to white fat, it is possible to isolate brown adipocytes that proliferate and differentiate in culture (Nechad et al., 1983). Special care must be taken when isolating brown adipose tissue. For most studies such as cell signaling experiments, studies on glucose metabolism and different blotting techniques, it proves advantageous to use cell culture. The culturing of brown adipocytes is based on the dissection of the tissue and the isolation of precursor cells. The precursors isolated are referred to as preadipocytes. These cells are cleansed from surrounding tissues such as 25 skeletal muscle and mature adipocytes through a series of filtration and centrifugation steps. The cells are grown in medium containing factors such as newborn calf serum and insulin. The cells grow as a monolayer and proliferate until they reach confluence. At the time of confluence, the brown adipocytes accumulate a multitude of small triglyceride droplets. Isolation of primary cultures of brown adipocytes is extensively descrived by Néchad (Nechad et al., 1983). 26
CHAPTER IV - Insulin-sensitive tissues as a model system for intracellular signaling
Insulin-sensitive tissues are metabolically and cell biologically interesting since events such as metabolism and cell growth are tightly regulated by external signals. The tight regulation of glucose metabolism in skeletal muscle and brown adipocytes is a good example of a signaling cascade that is important but not yet fully understood.
Bloodstream
Hormonal stimulation
Various metabolic and cell biological effects Stimulation by neurotransmitters
Figure 6. External stimuli on a target tissue
From a medical point of view, research on insulin signaling is important for the understanding of diseases such as type II diabetes. It is known that factors such as IRS-1 (Backer et al., 1992), serotonin (Hajduch et al., 1999) and adrenergic signaling (Hutchinson et al., 2005; Nevzorova et al., 2002; Nevzorova et al., 2006) have effects on metabolism in insulin-sensitive tissues.
In vivo experiments give indications for how a factor such as insulin influences a tissue; however, this experimental setup may also show a response from surrounding tissues. While this may be physiologically relevant, it makes it difficult to study very specific intracellular events. Choosing a model system for studies on intracellular signaling is not a trivial decision, although, many scientists now favor cell culture. In cell culture, all cells have equal access to the drug or substrate and the risk of interference from surrounding tissues is reduced. This makes cell culture an ideal system for studying intracellular signaling. Using a clonal cell line theoretically gives a high amount of identical cells. This is equivalent to an amplification of the signal one would see if one were to study a single cell. Cell culture is ideal for studies on signal transduction, since metabolites, 27 proteins and mRNA can be easily measured. Cells in culture are also easily treated with inhibitors directed against signaling intermediates or can be treated with siRNA, both great advantages for studies of signal transduction. All physiological effects seen in an animal have explanations on a cellular level. To fully understand the response of a cell to an external signal, one must first understand the inner workings of the cell. Intracellular signaling regulates most of the metabolic effects presently known and understanding these events is crucial for development of treatment of many metabolic disorders. 28
CHAPTER V – Intracellular signaling
Signaling lessons from the skeletal muscle
Insulin signaling The insulin-signaling pathway is anabolic and has several metabolic effects such as regulating glucose transport and glycogen management. The insulin-signaling pathway is divided into two different pathways, one pathway is dependent on PI3K (Czech and Corvera, 1999), while the other signals via the small G-protein TC10, in parallel with the PI3K-dependent pathway (Chiang et al., 2001). The activation of both the classical and the TC10 signaling pathway is required for the full insulin effect on glucose uptake (Chiang et al., 2001).
The classical pathway The classical pathway starts with the binding of insulin to the insulin receptor (IR). When insulin binds the IR, it signals to insulin receptor substrate 1 (IRS1) and activates PI3K (Shepherd et al., 1998). PI3K signals through PIP3-dependent kinase (PDK) (Shepherd et al., 1998), which phosphorylates protein kinase B (PKB/Akt) (Glass, 2003). It has been suggested that the pathway diverges downstream of PI3K. At least two different pathways have been suggested, one dependent on Akt and one signaling via protein kinase C λ (PKCλ) (Kotani et al., 1998). The translocation of GLUT4 involves Akt (Whiteman et al., 2002). Different forms of PKC might be involved in glucose uptake; two of the candidates are PKCζ (Braiman et al., 2001) and PKCδ (Rosenzweig et al., 2004).
The TC10 pathway The binding of insulin to the insulin receptor phosphorylates the Casitas b-lineage lymphoma (c-Cbl) protein (Chiang et al., 2001) via the c-Cbl-associated protein (CAP) (Ribon et al., 1998). Through an interaction with CAP, Cbl is translocated to plasma membrane subdomains, where flotillin is colocalized with caveolin, (Baumann et al., 2000). Caveolin is mainly found in flask-like invaginations in the plasma membrane called caveolae. C3G is translocated to caveolin-enriched compartments in response to insulin; this translocation is dependent on CAP (Chiang et al., 2001). The 29
CrkII part of the CrkII-C3G complex is also recruited to these domains in response to insulin (Chiang et al., 2001). It has been seen that C3G can act as a guanyl nucleotide exchange factor (GEF) for the TC10 protein (Chiang et al., 2001). The GEF activity of C3G is necessary for activity of the TC10 protein.
Figure 7. The insulin-signaling pathway
Protein kinase C There are at least 12 isoforms of the serine/threonine kinase protein kinase C (PKC) (Way et al., 2000). Protein kinase C isoforms have been shown to modulate insulin signaling in skeletal muscle (Cortright et al., 2000). The isoforms are divided into 3 groups, conventional PKCs (PKC isoforms α, β1, β2, and γ), novel PKCs (PKC isoforms δ, ε, η and θ) and atypical PKCs (ζ and ι/λ) (Way et al., 2000). PKC isoforms are differentially distributed in different tissues (Way et al., 2000). The conventional and novel PKCs contain twin C1 domains, which bind DAG. The atypical PKCs have a variant of the C1 domain, which does not bind DAG. The conventional PKCs also contain a C2 domain, which is required for responsiveness to calcium (Marquez and Blumberg, 2003). 30
Adrenergic receptors The 7-transmembrane spanning adrenergic receptors (adrenoceptors) are members of the G-protein coupled receptor family. There are two natural ligands of the adrenergic receptors, epinephrine and norepinephrine. Epinephrine is released from the adrenal medulla and norepinephrine is released by the sympathetic nervous system. The adrenergic receptors were first divided into α- and β-adrenoceptors in the late 40’s (Ahlquist, 1948). Nowadays, the adrenergic receptors are divided into three main classes, α1-, α2- and β-adrenoceptors. The α-adrenoceptors can be further subdivided into the α1A-, α1B-, α1D-, α2A-, α2B- and α2C- adrenoceptors. The β-adrenoceptors have three subtypes, the β1-, β2- and β3-adrenoceptors (Morris and Malbon, 1999). The expression pattern of the adrenoceptors varies between different tissues. The adrenoceptors bind to different types of G-proteins, Gs-, Gi- and Gq-proteins.
α1-adrenoceptors are Gq-coupled and have effects mainly through phospholipase C
(PLC). α2-adrenoceptors are Gi-coupled and have inhibitory effects on adenylyl cyclase (AC), which inhibits the production of cyclic AMP. All forms of the β- adrenoceptors are Gs-coupled and thus induce a production of cyclic AMP via activation of AC (Johnson, 1998). The regulation of AC is illustrated in Figure 8. G-proteins consist of an α-subunit of about 45 kDa and a combined β/γ-subunit of 35 kDa for the β-subunit and 8-10 kDa for the γ-subunit. There are several isoforms of the α-subunit and at least 5 different β-subunits and 12 different γ subunits exist (Milligan and Kostenis, 2006). The β− and γ−subunits can form complexes; however, not all of the β-subunits can form complexes with all of the γ-subunits but this has not been fully investigated. The expression pattern in different tissues also remains to be investigated. The function of the different combinations is probably important for G- protein signaling. G-proteins signal via a GTP-dependent step to affect the second messengers. A GDP is exchanged for a GTP on the inactive G-protein to activate it. This exchange takes place by interaction of the G-protein with a GEF. The GTP-bound α-subunit is then detached from the β/γ-subunit and is able to affect the second messenger signals. The α-subunit is inactivated by hydrolysis of the GTP to GDP, often performed by GTPase-activating proteins (GAPs). The α-subunit is then attached to a β/γ-subunit 31 and ready to start the cycle. The β/γ-subunit, when released from the α-subunit, affect β/γ-effectors until reunited with an α-subunit (Milligan and Kostenis, 2006).
Stimulatory Inhibitory
αs AC αi
γ β γ β
GTP GTP GDP GDP
cAMP
Figure 8. Activation and inactivation of adenylyl cyclase by Gs- and Gi-coupled receptors
Adrenergic receptors in insulin-sensitive tissues
Skeletal muscle expresses α2-adrenoceptors (Eason and Liggett, 1993) and low levels of α1-adrenoceptors (Rokosh et al., 1994). Competition binding studies show that skeletal muscle exclusively expresses the β2-adrenergic receptor protein (Liggett et al., 1988). Skeletal muscle expresses negligible amounts of the β3-adrenoceptor (McNeel and Mersmann, 1999).
WAT expresses low levels of α1-adrenoceptors (in rat) and higher levels of α2-,
β1- and β 2-adrenoceptors (Lafontan and Berlan, 1993) also β3-adrenoceptors have been found in human adipose tissue (Chamberlain et al., 1999). Brown adipocytes express α1- and α2-adrenoceptors, as well as both β1- and β3-adrenoceptors (Lafontan and Berlan, 1993). β2-adrenoceptors, which are critical in the skeletal muscle system, are not expressed in brown adipocytes themselves but can be found in the blood vessels within BAT (Cannon and Nedergaard, 2004).
Signaling kinetics
Molecular mechanisms of agonist binding to the GPCRs G-protein coupled receptors (GPCRs) share some common features, an extracellular N-terminus, an intracellular C-terminus, seven hydrophobic transmembrane (TM) α-helices, three extracellular and three intracellular loops 32
(Baldwin et al., 1997). Studies on the β2-adrenoceptor have revealed that the transmembrane regions contain amino acid residues most likely important for ligand binding and activation of the receptor, especially Asp113 in the 3-TM region and Ser 204 and Ser 207 in the 5-TM region, which are thought to interact with the amino and aromatic regions of the catecholamine agonists (Strader et al., 1988; Strader et al., 1989).
Activation of adrenergic receptors Most of our understanding concerning adrenergic activation comes from studies of the β2-adrenoceptor. The β2-adrenoceptor continuously changes between different conformational stages in the absence of agonist (Johnson, 1998; McGraw and Liggett, 2005). The active and inactive forms form an equilibrium depending on the binding of a ligand to the receptor (Johnson, 1998; Onaran et al., 1993). In an unstimulated cell, the inactive form is predominant (Onaran et al., 1993). The presence of an agonist shifts the equilibrium to a more active state (Johnson, 1998; McGraw and Liggett, 2005) and this induces a downstream cascade.
Net shift
Inactive form Active form
Agonist stabilizing the active form
Antagonist stabilizing the inactive form
Figure 9. Regulation of the β-adrenergic receptor conformations. The receptor has an equilibrium with a higher abundance to the left in the unstimulated state. Addition of either an agonist or an antagonist stabilizes the receptor in a certain state and shifts the equilibrium.
There are several different possible conformations capable of producing downstream signaling via different signaling pathways (ligand-directed signaling) (McGraw and Liggett, 2005), which could explain the different effects of different synthetic agonists. The most well-known downstream signaling goes via the α- subunit of the G-protein, which couples to AC to produce cyclic AMP. The agonist/antagonist stabilization of the different states is summarized in Figure 9. 33
When the β-adrenoceptor is in an activated state, it is associated to the α-subunit of the Gs protein, and one molecule of GTP. The G-protein then exchanges the GTP for a GDP. Through this reaction, the AC becomes activated and catalyze the conversion of an ATP to cyclic AMP. At the same time the affinity of the β2- adrenoceptor for the α-subunit of the G-protein is reduced and the α-subunit dissociates.
One important detail in the activity of the β2-adrenoceptor is that it is not the binding of an agonist that induces a conformational change but instead, the binding of an agonist temporarily stabilizes the receptor in an already existing active state until the receptor returns to its inactive state. The introduction of an agonist into the system simply shifts the equilibrium. This raises an interesting question regarding antagonists. It is possible that antagonists and agonists are not competing for the same binding sites but instead antagonists stabilize the inactive receptors. This shifts the equilibrium to more inactive receptors and thus the agonists have fewer active receptors to stabilize, extensively reviewed by Johnson (Johnson, 1998). It is possible that many GPCRs are present in different conformations and that agonists act to stabilize the receptors in one or more possible active conformations, however this remains to be fully investigated.
Desensitization of adrenergic receptors Adrenergic receptors only signal for a limited time. The receptor then undergoes an agonist-induced desensitisation that limits the signaling. Most of the studies have been performed on the β2-adrenoceptor but it is possible that other adrenergic receptors have similar mechanisms. There are two types of β2-adrenoceptor desensitisation, functional uncoupling and sequestration (Lohse et al., 1990). Sequestration is the internalisation form of a receptor, which can occur within a few minutes. Two types of kinases have been found, β-adrenergic receptor kinase 1 (β-ARK1 or GRK2) and cyclic AMP-dependent protein kinase A (PKA), which both phosphorylate β-adrenoceptors (Rapacciuolo and Rockman, 2000). Desensitization of the β2-adrenoceptor is carried out by phosphorylation of PKA phosphorylation sites on the third intracellular loop and of β-ARK phosphorylation sites on the cytoplasmic tail (Hausdorff et al., 1989). Following phosphorylation, the receptors are removed 34 from the plasma membrane and transported to cytosolic compartments (internalization or sequestration). Sequestration can take place even in the absence of phosphorylation (Lohse et al., 1990). Receptors are internalized to vesicular compartments from which they can be recycled to the plasma membrane when dephosphorylated. An acute stimulation makes it more likely that the receptor ends up in transferrin-containing endosomes, while a longer stimulation induces a translocation to lysosomes (Kallal et al., 1998).
The functional uncoupling of the β2-adrenoceptor is in some cases even quicker than sequestration (Waldo et al., 1983). The uncoupling of the receptor is divided into homologous and heterologous desensitization. The homologous desensitization is a receptor-specific negative feedback while heterologous desensitization can affect multiple receptor types (Lohse et al., 1990). Uncoupling occurs following a phosphorylation of the receptor (Benovic et al., 1988). β-ARK1 plays a role in the homologous desensitization of β-adrenoceptors (Rapacciuolo and Rockman, 2000), since they are specifically desensitized by β- ARK1 ((Lefkowitz, 1998; Rapacciuolo and Rockman, 2000). PKA-mediated uncoupling is a heterologous desensitization since it is activated by numerous signals and it does not only phosphorylate β2-adrenoceptors (Clark et al., 1997;Rapacciuolo and Rockman, 2000). β-ARK1 phosphorylates the C-terminal tail and allows β- arrestin to bind to the receptor. Binding of β-arrestin prevents GSα from coupling to AC (Rapacciuolo and Rockman, 2000) and β-arrestin is thought to be required for homologous desensitization (Lohse et al., 1990). These factors contribute to a decrease in plasma membrane expression of active β-adrenoceptors. It is worth mentioning that β3-adrenoceptor lack many of the phosphorylation sites associated with the β1- and β2-adrenoceptors (Nahmias et al., 1991). Considering the fact that the
β3-adrenoceptor cyclic AMP response is thus only decreased by breakdown of cyclic
AMP, as compared to the β1- and β2-adrenoceptors are also subject to desensitisation, it seems logical that the β3-ARs have a slower decline of the cyclic AMP response (Paper II).
In some systems, both β1- and β 3-adrenoceptors can be found (Paper II). In undifferentiated cells, the β1-adrenoceptor is predominant but this changes during differentiation and the β3-adrenoceptor is upregulated (Bronnikov et al., 1999). It has been suggested that the β1-adrenoceptors in the latter system are not functionally 35
coupled to AC and that the β3-adrenoceptors take over the full control of the system
(Bronnikov et al., 1999). In Paper II, we have studied the β1-adrenergic effect on cyclic AMP production in a knockout mouse model lacking β3-adrenoceptors (β3-
KO), compared to the response in cells expressing both β1- and β3-adrenoceptors. In
β3-KO brown adipocytes, the β1-adrenergic signaling demonstrate an earlier peak in cyclic AMP production compared to the response in a system expressing both β1- and
β3-adrenoceptors (Paper II). It is possible that the β1-adrenoceptors are coupled only in the absence of β3-adrenoceptors. Studying the shape of the curves, we saw that the cyclic AMP levels are decreased more quickly in the β1-adrenergic system compared to the β1/β3-adrenoceptors system (Paper II). We saw that direct activation of AC gave similar decreases in cyclic AMP over time when comparing the β1-adrenoceptor system to the β1/β3-adrenoceptor system. This suggests that the PDE activity may not be affected by the knock-out of the β3- adrenoceptor. This indicates that the β1-adrenoceptor is quickly desensitized while the β3-adrenoceptor has a prolonged signaling, possibly as a result of the lesser effect of desensitization on this receptor (Paper II).
Adrenergic signaling
α1-Adrenoceptor signaling
α1-Adrenoceptors are Gq/11-coupled (Zhong and Minneman, 1999). Gq/11 activates phospholipase C (PLC) (Garcia-Sainz et al., 2000). This increases the levels of inositol 1, 4, 5-trisphosphate (IP3) and diacylglycerol (DAG). The increase in IP3 2+ releases Ca from the endoplasmatic reticulum (ER) (Neves et al., 2002). α1- Adrenoceptor signaling also has a branch that goes via diglyceride and activates PKC, which in turn activates CREB (Cannon and Nedergaard, 2004).
α2-Adrenoceptor signaling
α2-Adrenoceptors couple to Gi proteins and inhibit the production of cyclic AMP
(Cannon and Nedergaard, 2004). When studying cells possessing α 2- and β- adrenoceptors, it is seen that the cyclic AMP levels achieved by addition of norepinephrine being an agent acting on both receptor types, are lower than upon stimulation with β-AR specific agonists (Cannon and Nedergaard, 2004). 36
Figure 10.
Classical and atypical β2-adrenergic signaling
Classical β-adrenergic signaling The Gs-coupled β-adrenoceptors activate AC (Reisine et al., 1983). AC catalyses the formation of cyclic AMP from ATP in a Mg2+-dependent reaction (Sutherland et al., 1962). Changes in cyclic AMP levels activate cyclic AMP dependent kinases (PKAs) (Kuo and Greengard, 1969; Walsh et al., 1968). This is summarized in Figure 10. The cyclic AMP-mediated activation of PKA results in a number of responses in the cell, such as gene regulation (Roesler et al., 1988), phosphorylation of the β- adrenoceptor (Rapacciuolo and Rockman, 2000) and metabolism (Krebs and Beavo, 1979). The cyclic AMP signal in brown adipocytes peaks at around 10-15 minutes, rapidly decreases and reaches near basal values within 1.5-2 hours (Paper II). The cyclic AMP signal is not only capable of being mediated by PKA. The exchange protein directly activated by cyclic AMP (cAMP-GEF/Epac) protein family was recently discovered (de Rooij et al., 1998; de Rooij et al., 2000; Kawasaki et al., 1998). Epac acts as a guanine nucleotide exchange factor (GEF) for the Ras-like small GTP-binding proteins Rap1 and Rap2 (de Rooij et al., 1998; de Rooij et al., 2000; Kawasaki et al., 1998). Epac is thought to be involved in the mediation of many cellular effects (Brennesvik et al., 2005; Christensen et al., 2003; Cullen et al., 2004; Evans et al., 2001), however, not much is known of its full potential. In the case of brown adipocyte thermogenesis, it is thought that β3-adrenoceptor signaling goes via the AC-cAMP-PKA system (Cannon and Nedergaard, 2004). This is supported by the fact that forskolin, an AC activator, can induce thermogenesis (Connolly et al., 1986; 37
Scarpace and Matheny, 1996). Several possible AC subtypes may be responsible for the signal transduction. The activity of the AC subtypes may also change during differentiation (Cannon and Nedergaard, 2004).
Atypical β-adrenergic signaling Apart from the classical cyclic AMP signaling pathway, there is evidence that some subtypes of β-adrenoceptors can also couple to Gi and signal through β-arrestin to activate MAPK (Johnson, 2006). It is possible that the β2-adrenoceptor can couple to PI3K and crosstalk with the insulin signaling pathway (Wymann et al., 2003). It has also been shown that this connection can be made via the Gs protein, although the coupling mechanism remains unclear (Paper I). β2-adrenoceptors in skeletal muscle are possibly capable of coupling to both Gs and Gi-proteins.
Compensation of β1- β3-adrenoceptor signaling
Brown preadipocytes express β1-adrenoceptors, and they are also present to a certain degree in mature brown adipocytes. Studies on glucose uptake show that β1- adrenoceptors may not be significantly coupled in mature brown adipocytes (Cannon and Nedergaard, 2004). By the use of a β3-KO mouse we have found that in the absence of β3-adrenoceptors, the signaling is compensated by β1- and α 1- adrenoceptors (Chernogubova et al., 2005). It is still unclear whether the β 1- adrenoceptor is functionally coupled to cyclic AMP production in the wild-type mature brown adipocytes.
Biphasic β-adrenergic signaling β-adrenergic effects are not always mediated via cyclic AMP. In skeletal muscle, the β2-adrenoceptor can activate PI3K through a signaling pathway independent of cyclic AMP (Paper I). β-adrenergic effects on glucose uptake (Chernogubova et al., 2004) and glycogen synthesis (Paper I) are mediated via PI3K and many of the adrenergic effects such as glucose uptake and glycogen synthesis are seen long after the cyclic AMP levels have declined to near basal levels (Paper II; Paper I; Figure 11). 38
Figure 11. β-adrenergic signaling is divided into an acute phase and long-term effects It is possible that cyclic AMP induces a signaling pathway capable of signaling for a prolonged period of time. It is also possible that there is a prolonged signal independent of cyclic AMP from the β-adrenergic receptors. The effects attributed to the PI3K-mediated signal could be seen as long-term effects compared to the acute effects of cyclic AMP (Paper II). In skeletal muscle cells, the PI3K signal can be independent of the cyclic AMP signal (Paper I). 39
CHAPTER VI – Metabolism
Muscle and fat metabolism – Gathering, wasting, storing
Factors involved in energy management in insulin-sensitive tissues Skeletal muscle metabolism has a great impact on total blood glucose levels due to its large energy demand and relatively large mass. Skeletal muscle has two forms of energy storage, glycogen and triacylglycerol (Langfort et al., 2003). Glucose uptake is mainly regulated via two signaling pathways, contraction and the insulin-signaling pathway (Pereira and Lancha, 2004). Glucose uptake can also be regulated via the β-adrenergic pathway (Tanishita et al., 1997). Insulin and epinephrine reach the muscle via the bloodstream. The skeletal muscle is innervated by sympathetic nerves (Nonogaki, 2000), which release norepinephrine. Brown adipocytes have a high glucose uptake considering the small amount of tissue and may be important in a thermogenically active state (Cannon and Nedergaard, 2004). WAT can be a considerable part of the body and has effects on total blood glucose levels. Brown and white adipocytes are similar to skeletal muscle in respect of glucose management, although with a few important differences. The adipose tissues rely on both glucose and lipids as a source of energy but while brown adipocytes use lipids as fuel in thermogenesis, white adipocytes store lipids. Skeletal muscle tissues in contrast, differ in their fuel preference based on their fiber types (Quiroz-Rothe and Rivero, 2004).
Facilitative glucose transporters Glucose enters cells via two different types of glucose transporters, the facilitative glucose transporters (GLUTs) and the sodium-dependent glucose transporters (SGLTs) (Wood and Trayhurn, 2003). Most of the glucose uptake in skeletal muscle and fat is dependent on the facilitative glucose transporters. GLUTs are 12 membrane- spanning proteins that passively transport glucose (Mueckler, 1994). In skeletal muscle, several forms of GLUTs can be found, among them GLUT1, GLUT3, GLUT4 (Wood and Trayhurn, 2003) and GLUT8 (Doege et al., 2000). GLUT1 and GLUT4 are most probably the key players in insulin-mediated glucose uptake in skeletal muscle. 40
The ubiquitously expressed glucose transporter GLUT1 is responsible for basal glucose uptake. This transporter is mainly located in the plasma membrane. The insulin-sensitive glucose transporter GLUT4 is mainly expressed in skeletal muscle, and adipose tissues (Fukumoto et al., 1989) and insulin-mediated glucose uptake is due to GLUT4 translocation (Mueckler, 1994). The translocation of GLUT4 may not be enough to increase glucose uptake. Both GLUT4 activation and translocation are regulated by insulin (Funaki et al., 2004). The GLUT4 protein might be inactivated, either by cyclic AMP (shown by dibutyryl cyclic AMP) mediating a signal to the C- terminus (Piper et al., 1993) or by cyclic AMP-dependent protein kinase (Reusch et al., 1993). Adipose tissues have a GLUT expression pattern similar to skeletal muscle. As seen in skeletal muscle, GLUT1 is responsible for basal glucose uptake, and GLUT4 is the main transporter for induced glucose uptake (Santalucia et al., 1992). As with skeletal muscle, glucose uptake in brown adipocytes is regulated via insulin (Chernogubova et al., 2004;Valverde et al., 2005) and glucose uptake in BAT is stimulated by cold exposure (Greco-Perotto et al., 1987; Olichon-Berthe et al., 1992; Shibata et al., 1989; Shimizu et al., 1993; Vallerand et al., 1990). Interestingly, this effect is most likely not dependent on insulin, since it is still present in animals with very low levels of insulin (Shibata et al., 1989). This indicates that there is another signaling pathway involved in the regulation of glucose uptake. Sympathetic denervation of BAT abolishes the cold-induced glucose uptake and GLUT4 expression. The cold-induced effects on glucose uptake can clearly be regulated via the sympathetic nervous system (Shimizu et al., 1993). The expression of the glucose transporters GLUT1 and GLUT4 in skeletal muscle varies during differentiation (Paper IV; Figure 12).
Glucose transport – Harvesting the energy Insulin regulation of glucose uptake is brought about by translocation of the GLUT4 protein from intracellular compartments to the plasma membrane (Cushman and Wardzala, 1980; Suzuki and Kono, 1980), as seen in Figure 13. The localization of GLUT4 is complex since it may be found in several different cellular compartments. The trafficking of GLUT4 between intracellular compartments and the plasma membrane is even more complex and has been extensively reviewed by Bryant and co-workers (Bryant et al., 2002). Bryant proposes a model where GLUT4 41 is localized in the trans-Golgi network, recycling endosomes and in the plasma membrane. In the absence of insulin, the GLUT4 protein is mainly found in the intracellular compartments but upon insulin stimulation, the protein is increasingly located in the plasma membrane.
Figure 12. The expression of the glucose transporters GLUT1 and GLUT4 during myogenesis
The model proposed is not a static system; rather this is a dynamic process where GLUT4 continuously moves between the compartments. Bryant has divided this into two cycles. In one cycle, the GLUT4 protein is budded off from the trans-Golgi network to form GLUT4 storage vesicles. These vesicles are capable of being translocated directly to the plasma membrane in response to insulin. In addition to this transport, the vesicles can also fuse with the recycling endosomes. From the recycling endosomes, transport vesicles are budded off and return to the trans-Golgi network. In the other cycle, GLUT4 is continuously cycled from the recycling endosomes to the plasma membrane and back via endocytosis. GLUT4-containing vesicles are captured by the target membranes via the binding of v-SNAREs (vesicular) to t-SNAREs (found in the target membrane). Fusion then occurs. However, the precise mechanism is not yet clear (Bryant et al., 2002). 42
Plasma membrane Fusion
Recycling endosome GLUT4 storage vesicle Transport vesicle
Trans-Golgi network
Figure 13. Proposed model for the trafficking of GLUT4 from intracellular stores to the plasma membrane and back. Picture adapted from Bryant et al., 2002
Glucose metabolism – Using the energy Breakdown of glucose is induced by several factors, among them, insulin and contraction (Rider et al., 2004). Glycolysis is carried out by several steps, which can be divided into two phases, the preparatory phase and the payoff phase. In the preparatory phase, the breakdown of glucose to glyceraldehyde-3-phosphate (G-3-P) requires 2 ATP. In the payoff phase, the conversion of G-3-P to pyruvate yields 4 ATP and 4 NADH. Overall, the conversion of glucose to pyruvate yields a total amount of 2 ATP and 2 NADH. Glycolysis is stimulated by fructose-2,6-bisphosphate (F-2,6-P) which inactivates fructose-1,6-bisphosphatase, a key enzyme in the glyconeogenesis. F-2,6-P levels are increased by insulin and contraction. Both contraction by exercise and electrical stimulation increase the levels of F-2,6-P. It has been suggested that this rise is a secondary effect of increased levels of fructose-6- phosphate (F-6-P). 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK- 2/FBPase-2) is a bifunctional enzyme regulating the synthesis and degradation of F- 2,6-P from fructose-6-phosphate respectively (Rider et al., 2004). 43
Glycogen metabolism – Storing the energy The liver stores glycogen as an energy reservoir available for the whole body, while skeletal muscle glycogen is exclusively available for muscle. Glycogen synthesis is regulated by catecholamines and insulin (Yeaman et al., 2001). In skeletal muscle, glucose is phosphorylated to glucose-6-phosphate which has two alternative fates, either being stored as glycogen or going into glycolysis (Nielsen and Richter, 2003). The formation of glycogen is catalysed by glycogen synthase (GS), which is inactivated by phosphorylation and activated by dephosphorylation. The dephosphorylation and activation of glycogen synthase is carried out by the protein phosphatase 1 (PP1) (Nielsen and Richter, 2003). There are several factors that phosphorylate and thus inactivate GS, among them AMP-activated protein kinase (AMPK) (Wojtaszewski et al., 2002). AMPK is an energy sensor, activated by increased AMP levels in the cell (Hardie, 2003). Glycogen synthase can also be phosphorylated by glycogen synthase kinase 3 (GSK3) (Nielsen and Richter, 2003), calmodulin-dependent glycogen synthase kinase (Woodgett et al., 1982) and protein kinase A (PKA) (Fang et al., 2000). Insulin inactivates GSK3 via Akt-induced phosphorylation (Cross et al., 1995). IGF-1 also inhibits GSK3 by phosphorylation via the PI3K/Akt pathway (Rommel et al., 2001). Glycogenolysis breaks down glycogen to produce the substrate for glycolysis. The glucose residues of glycogen are converted to glucose-1-phosphate by glycogen phosphorylase (GP). Glycogen phosphorylase is a key enzyme in the breakdown of glycogen (Ferrer et al., 2003). The GP is phosphorylated and activated by phosphorylase kinase (PK) and dephosphorylated by phosphorylase phosphatase (PP). The regulation of glycogen metabolism is thus carried out by affecting two opposing enzymes, glycogen synthase and glycogen phosphorylase. Signals regulating the activity of these enzymes direct the metabolism into making glycogen or breaking down glycogen. Not much is known about glycogen metabolism in brown adipose tissue; however it has been shown that adrenergic stimulation has a negative effect on insulin- mediated glycogen synthesis (Jost et al., 2002).
AMPK – The energy sensor It is known that AMPK can regulate glucose uptake (Bergeron et al., 1999; Merrill et al., 1997). Interestingly this regulation is independent of insulin (Hayashi et al., 44
1998). AMPK is a heterotrimer consisting of a catalytic α- and regulatory β- and γ- subunits (Carling, 2004; Hardie et al., 1998; Kemp et al., 1999). Apart from the AMP/ATP ratio switch, AMPK can also be regulated by phosphorylation by enzymes such as LKB1 (Hawley et al., 2003; Hong et al., 2003; Shaw et al., 2004) and Ca2+/calmodulin kinase kinase CaMKK (Hawley et al., 2005; Hurley et al., 2005; Woods et al., 2005). It is also possible that contraction-induced glucose uptake is coupled to AMPK. Inhibition of the mitochondrial complex I by rotenone induces glucose uptake via AMPK (Hayashi et al., 2000), also demonstrating that lowering the energy levels in the cell activates AMPK. Skeletal muscle has an important role in metabolic diseases such as type II diabetes. Impaired glucose transport in the skeletal muscle cells results in impaired clearance of glucose from the blood. It has been shown that in rodents, diabetes is improved by the administration of NADPH oxidase inhibitors such as apocynin and DPI (diphenylene iodonium) (Furukawa et al., 2004; Hayes et al., 1985; Holland et al., 1973). We have seen that DPI increases glucose uptake in L6 cells, C2C12 cells and primary cultured skeletal muscle cells (Paper III). One must carefully consider many aspects before assuming a mechanism. It is generally regarded that DPI acts as an NADPH oxidase inhibitor (Cross and Jones, 1986; O'Donnell et al., 1994). However, it has various inhibitory effects on other enzymes, such as mitochondrial proteins responsible for ROS production (Li and Trush, 1998; Majander et al., 1994). It has long been thought that these effects could be attributed to the effects on NADPH oxidase. In our study, we have seen that L6 cells do not express NADPH oxidase and thus the effects must be indepedent of NADPH oxidase (Paper III). We have shown that the effect is not through the insulin-signaling pathway via PI3K-Akt (Paper III) but that DPI lowers ATP levels and induces glucose uptake via the energy regulator AMPK (Paper III). Interestingly, metformin, a drug used as treatment for type II diabetes, also induces glucose uptake through activation of AMPK (Fryer et al., 2002). We have seen that DPI decreases mitochondrial respiration (Paper III) through an inhibition of the mitochondrial complex I in the respiratory chain (Paper III). The DPI mediated signal is interesting since it does not only induce glucose uptake but also decreases glycogen synthesis (Paper III). 45
CHAPTER VII – Adrenergic effects
Adrenergic signaling has been shown to have great impact on both skeletal muscle growth and on metabolism in both skeletal muscle and brown adipocytes. The effect of adrenergic signaling is interesting since it has been debated whether it is anabolic, catabolic or maybe both, under different circumstances.
The involvement of adrenergic signaling in skeletal muscle growth and the force of contraction The adrenergic system has great impact on skeletal muscle growth regulation. The β-adrenergic agonist clenbuterol induces hypertrophy in skeletal muscle; this effect is mediated via the β2-adrenergic receptor (Choo et al., 1992). β 2-adrenergically mediated hypertrophy increases protein synthesis and decreases proteolysis (or protein degradation) (Navegantes et al., 1999).
Protein degradation Atrophic effects, i.e. a loss of muscle protein content, a reduction of muscle size and a decrease in the force of contraction are important for negative regulation of muscle function (Jackman and Kandarian, 2004). β2-adrenergic signaling inhibits proteolysis through cyclic AMP-dependent signaling (Navegantes et al., 1999). β- adrenergic signaling decreases proteolysis in general and Ca2+-dependent proteolysis in particular (Navegantes et al., 1999).
Hyperplasia and hypertrophy
Proliferation of mononucleated myocytes increases after β2-adrenergic signaling. Hyperplasia is also induced by cyclic AMP (Izevbigie and Bergen, 2000). Skeletal muscle hypertrophy is mediated via the β2-adrenergic signaling pathway (Hinkle et al., 2002). Stimulation of the β 2-adrenergic signaling pathway by clenbuterol increased the level of IGF-1 and the hypertrophic effect may be due to IGF-1 (Awede et al., 2002). 46
Adrenergic effect on myogenic factors
β2-adrenergic signaling can regulate factors that influence myogenesis and fusion, such as the myogenic factor MyoD (Bricout et al., 2004). In skeletal muscle, both the mRNA (Hughes et al., 1993;Mozdziak et al., 1998) and the protein expression of
MyoD increase in response to β2-adrenergic signaling. The β2-adrenergically induced increase of MyoD is associated with a slow-to-fast MHC transition (Bricout et al., 2004). This connects MyoD to postnatal skeletal muscle growth, as well as fiber to type switching.
Adrenergic effects on brown adipocyte thermogenesis The adrenergic system regulates brown adipocyte thermogenesis both negatively and positively. α2-Adrenoceptors inhibit thermogenesis while β3-adrenoceptors increase it. The slow desensitization of the β3-adrenoceptor may prove advantageous, since the stimulation of the thermogenesis may need to be prolonged. PKA phosphorylates the transcription factor CREB, which regulates the transcription of several genes, among them the uncoupling protein 1 (UCP1), which is important in thermogenesis. Apart from CREB, PKA also activates the Erk1/2- the P38- and the JNK-pathways. The subject of adrenergic regulation of thermogenesis has been extensively reviewed (Cannon and Nedergaard, 2004). Clearly adrenergic signaling may interact with various signaling pathways.
Adrenergic regulation of metabolism in insulin-sensitive tissues
Adrenergic regulation of skeletal muscle energy management
β2-Adrenoceptors are involved in the regulation of glucose uptake in skeletal muscle (Nevzorova et al., 2002). It has been shown that α1-adrenoceptors can also increase glucose uptake in skeletal muscle cells (Hutchinson and Bengtsson, 2005;Liu et al., 2001). It is possible that novel and conventional PKCs are involved in this signaling. The adrenergic system is also involved in lipolysis, glycolysis and in regulating HSL (Donsmark et al., 2004) and glycogen synthase (Fang et al., 2000). These factors are all involved in the energy regulation of energy balance in the skeletal muscle cell. 47
Adrenergic regulation of brown adipocyte energy management As in skeletal muscle, brown adipocytes also show metabolic responses to adrenergic stimulation. The adrenergic system has effects on both glucose and lipid metabolism. The sympathethic nervous system is important for the brown adipose tissue since the release of norepinephrine induces thermogenesis (Cannon and Nedergaard, 2004).
Glucose uptake
Glucose uptake in skeletal muscle can be regulated by both α1- and β 2- adrenoceptors (Hutchinson and Bengtsson, 2005; Nevzorova et al., 2002; Nevzorova et al., 2006). The α1-adrenoceptor stimulation of glucose uptake in skeletal muscle cells activates PI3K but the mechanism for this is not known. This activation of PI3K did not phosphorylate Akt (Hutchinson and Bengtsson, 2005). This is interesting since Akt is involved in most known signaling pathways downstream of PI3K. It has been suggested that the mechanism is dependent on PLC, which activates DAG and calcium, which activates DAG sensitive PKCs (Hutchinson and Bengtsson, 2005).
Interestingly, the α1-adrenoceptor induced glucose uptake in skeletal muscle is mediated by AMPK (Hutchinson and Bengtsson, 2006).
Glucose uptake is regulated via β2-adrenoceptors in L6 skeletal muscle cells
(Nevzorova et al., 2002; Nevzorova et al., 2006). The full mechanism of the β2- adrenergic signaling is unclear. The action of the Gs-protein in the signaling to glucose uptake has yet to be investigated. As the Gs-protein releases both an α- subunit and a β/γ-subunit there are two signaling pathways to consider. The β2- adrenergically mediated glucose uptake is not dependent on cyclic AMP but requires PI3K (Nevzorova et al., 2002; Nevzorova et al., 2006). These results are reflected by studies in brown adipocytes, which show that PI3K is involved in the stimulation of glucose uptake by α1-, β1- and β3-adrenergic receptors (Chernogubova et al., 2004; Chernogubova et al., 2005). It has also been shown that PKA, which acts downstream of cyclic AMP, is not directly involved in the β2-adrenergic regulation of glucose uptake (Nevzorova et al., 2006). Glucose uptake in BAT is regulated via the sympathetic nervous system, since the addition of norepinephrine and other adrenergic agonists in cell culture (Marette and Bukowiecki, 1989; Marette and Bukowiecki, 1991; Omatsu-Kanbe and Kitasato, 48
1992) and in vivo (Cooney et al., 1985; Liu et al., 1994; Ma and Foster, 1986) increases glucose uptake. As in skeletal muscle, glucose uptake is regulated via β- adrenoceptors (Cawthorne, 1989) and goes via cyclic AMP (Chernogubova et al., 2004; Marette and Bukowiecki, 1989). In contrast to the GLUT4 translocation response to insulin, adrenergic signaling appears to function through a different mechanism. Norepinephrine-induced glucose uptake in BAT independent of the translocation of GLUT4 to the plasma membrane (Cannon and Nedergaard, 2004). The GLUT4 location is not affected by adrenergic stimulation. It has been proposed that GLUT1, the transporter responsible for the basal uptake may be activated by NE treatment (Shimizu et al., 1996; Shimizu et al., 1998). However, recent evidence shows that it is not the translocation or activation but instead increased transcription of the GLUT1 gene itself that causes an increase in glucose uptake (Dallner et al., 2006).
Glycolysis/Glycogenolysis Adrenergic stimulation regulates glycogen breakdown via cyclic AMP. There are no α -adrenergic effects on glycogenolysis (Nonogaki, 2000). PKA activation following β2-adrenergic stimulation activates phosphorylase kinase, which activates GP (Purves et al., 1997). The dephosphorylation of glycogen phosphorylase is carried out by phosphorylase a phosphatase. Adrenergic signaling inactivates phosphorylase a phosphatase (Lehninger et al., 1993) indicating that β-adrenergic signaling has positive effects on the activation of GP and negative effects on the inactivation. Adrenergic stimulation induces glycolysis. In addition, adrenergic signaling increases F-2,6-P levels (Rider et al., 2004). PFK-2/FBPase-2 has different isoforms expressed in different tissues. It has been shown in liver, that the PFK-2/F-2,6-BPase has a N- terminus, which has a PKA-phosphorylation site. The muscle form is a splice variant of the liver isoform, the liver isoform does not have the PKA-phosphorylation site. The liver isoform of PFK-2/F-2,6-BPase is phosphorylated by PKA (Costa Rosa et al., 1995). However, both the liver variant and the skeletal muscle variant have been found in skeletal muscle cells (Rider et al., 2004). This indicates that PFK-2/F-2,6- BPase in skeletal muscle can be regulated in the same way as in liver. 49
Glycogen synthesis GS inactivation through phosphorylation is regulated via adrenergic receptors. It has been suggested that α1-adrenoceptor signaling phosphorylates GSK3, through a signal independent of PI3K and Akt (Ballou et al., 2001). The raised Ca2+ levels following activation of α1-adrenoceptors result in the activation of calmodulin- dependent protein kinase. It is well known that adrenergic signaling can have glycogenolytic effects in skeletal muscle (Challiss et al., 1986). However, recent data show that activation of the β2-adrenergic signaling pathway can increase glycogen synthesis and that this signal is not dependent on cyclic AMP but dependent upon PI3K (Paper I). Our results show that adrenergically mediated glycogen synthesis is not dependent on PKC, even though there are PKC isoforms capable of producing an increase in glycogen synthesis (Paper I). It is also worth noting that AMPK is not involved (Paper I). Insulin inactivates GSK3 in a PI3K/Akt dependent manner
(Cohen, 2002). It is also possible that the α1-adrenoceptor inhibition of glycogen synthase (GS) is mediated by suppression of the PI3K/Akt signaling pathway (Ballou et al., 2001). The opposing signaling pathways leading to decreased glycogen synthesis (acting through cyclic AMP) and to increased glycogen synthesis (acting through PI3K) most probably work under different conditions. Interestingly, the glycogen synthesis effect is still seen following 2 hours of stimulation (Paper I), while the cyclic AMP response declines rapidly (Paper II). Taken together, this indicates that the inhibitory effects acting through cyclic AMP might be an acute effect and the PI3K effect appear at a later stage. Similar to skeletal muscle regulation, brown adipocyte glycogen synthesis may also be induced by β-adrenergic signaling (Paper II). It is possible that the adrenergic signal goes through GSK3 in brown adipocytes, as well as in skeletal muscle (Yamamoto et al., unpublished data). We have suggested that the activation of PI3K does not necessarily lead to phosphorylation of Akt (Paper II). Insulin-mediated activation of PI3K increases Akt phosphorylation but interestingly, adrenergic stimulation does not. Even though the
β2-adrenergic system is capable of activating glucose uptake and glycogen synthesis through a signaling pathway dependent on PI3K, the signal is not dependent on Akt phosphorylation. Other proteins downstream of PI3K must be responsible for the signal transduction. These signaling proteins remain unidentified. In the case of 50 glycogen synthesis, a protein named CISK/SGLT/SGK3 may be involved, since it is reported to be downstream of PI3K (Zhou et al., 2004) and can also activate GSK3 (Wyatt et al., 2006). 51
CHAPTER VIII – Summary and Conclusions
Insulin-sensitive tissues are among the most metabolically active tissues known and are of great interest for studies on energy management. Metabolic diseases such as type II diabetes and obesity are increasing global problems. Much is known of the regulation of whole body glucose metabolism, however, much is still unknown about the regulation at the cellular level. The skeletal muscle cell is an interesting system since there are numerous signals capable of regulating glucose metabolism. Apart from the classical insulin-signaling pathway, the skeletal muscle cell is also innervated by the sympathetic nervous system signaling through adrenergic receptors. These two signaling systems interact with each other but the exact mechanisms are not known. In addition to these signals, skeletal muscle contraction has an inherent ability to regulate glucose metabolism. Together, these components form a very complex system with different effects on glucose metabolism.
In my thesis, I have reviewed the physiology of the insulin-sensitive tissues skeletal muscle and brown adipose tissue and have focused on intracellular signaling pathways involved in glucose metabolism. There are a few signaling molecules frequently mentioned in signaling pathways regulating metabolism. Among them we find PI3K, found in the insulin-signaling pathway, and PKC, found in several different isoforms regulated in different ways including Ca2+. Recent research has also revealed the importance of the energy sensor AMPK, which is regulated by a decreased ATP/AMP ratio. All of these signaling molecules are known to regulate glucose uptake in skeletal muscle cells. Skeletal muscle has a tight regulation of glucose metabolism and a dynamic demand for glucose. The glucose is obtained by passive transport through glucose transporter proteins and is either broken down to produce energy or stored as glycogen. The insulin-signaling system is involved in the regulation of both glucose transport and glycogen synthesis. It is now clear that these factors can also be regulated by the adrenergic system. Historically, insulin has been seen as an anabolic hormone mainly acquiring and storing energy while adrenergic signaling has been seen as a catabolic signal opposing the actions of insulin. Recently, it has been found that the adrenergic system, 52 particularly the β-adrenergic signaling system also has anabolic effects on glucose metabolism. We have seen that not only can the adrenergic system induce glucose uptake but it can also induce the incorporation of glucose into glycogen. Interestingly, the adrenergic system is not independent from the insulin signaling system in its regulation of either glucose uptake or glycogen synthesis. The β-adrenergic system may utilize parts of the insulin-signaling pathway. We have shown that the β2- adrenergic signaling system is dependent on activation of PI3K to induce glycogen synthesis. However, our results raise further questions: How does the β-adrenergic signaling pathway activate PI3K? We have shown that in the case of glycogen synthesis, the β-adrenergic signal does not signal via cyclic AMP. It is possible that the β/γ-subunit of the Gs-protein is responsible for this cross-talk. We have also shown that although some forms of PKC are capable of inducing glycogen synthesis, β-adrenergic signaling does not utilize PKC for its actions.
The energy regulator AMPK is involved in regulating glucose uptake in a signaling pathway that is independent of insulin. This is worth pointing out since this may be a potential target for treatment of type II diabetic patients with impaired insulin sensitivity. The NADPH oxidase inhibitor DPI is capable of inducing glucose uptake by itself and also to potentiate insulin sensitivity in skeletal muscle cells. We have seen that the action of DPI is not via inhibition of NADPH oxidase but rather through the inhibition of the mitochondrial respiratory chain (more specifically complex 1). This lowers the intracellular ATP levels and activates AMPK. AMPK functions as an energy switch capable of maximizing the amount of glucose in the cell. This glucose is taken both from the bloodstream and from the intracellular glycogen depots. When the cell has low levels of ATP, it activates AMPK. AMPK induces glucose uptake and reduces glycogen synthesis. This increases the inflow of glucose from the blood and stops glucose from being incorporated into glycogen. In order to study skeletal muscle cells in detail, we have developed a system for separating and arranging fused skeletal muscle cells in culture. This system is termed the 2-Dimensional Muscle Syncytium (2DMS) technique. The 2DMS can be used to separate and arrange multinucleated myotubes in parallel. This simplifies imaging 53 microscopy of the skeletal muscle cells and allows for micromanipulations of a specific nucleus in a region of the cells. We have used this system to study the protein expression of a myogenic factor, MyoD (which is under adrenergic control). Following the skeletal muscle cells throughout the differentiation process, we have found that the MyoD protein is found in the nucleus of some but not all of the myoblasts. In the mature myotubes, the protein was absent from all cell nuclei. Interestingly, in the fusing myotubes, the MyoD protein was found in individual nuclei and was absent in other nuclei within the same cell. This finding shows that different nuclei in a myotube can have different protein expression patterns, and it is also likely that different nuclei have different responsibilities depending on their location. The 2DMS technique is a powerful tool for studies on cellular location of different proteins. Combining this system with live cell imaging, one could study fusion processes and movement of specific proteins over a long time-period.
The β-adrenergic regulation in skeletal muscle cells is mediated by the β2- adrenoceptor. In BAT, there is expression of both β 1-adrenoceptors and β3- adrenoceptors and both receptors are capable of inducing an increase in cyclic AMP.
We have suggested that the β1-adrenoceptor may be rapidly desensitized while the β3- adrenoceptor has a prolonged response. This could be due to the fact that β3- adrenoceptors has fewer phosphorylation sites (as compared to β1- and β2- adrenoceptors) important for desensitization of the receptor. Interestingly, metabolic responses such as glucose uptake and glycogen synthesis are all seen at time points when even the β3-adrenergic cyclic AMP response has reached near-basal levels. We have seen that stimulation of adrenergic receptors in brown adipocytes lead to a PI3K- dependent production of PIP3. The PIP3 production reaches a maximum after a short time and stays up for a long time-period, which could explain the long-term adrenergic effects. A preliminary experiment suggests that PI3K activity is not dependent on cyclic AMP. This is supported by the fact that in skeletal muscle, glycogen synthesis is not dependent on cyclic AMP but goes through PI3K.
An interesting question is how the adrenergic system can both inhibit and induce glucose metabolism, as in the case of glycogen synthesis. We have seen that the β2- adrenergic signaling system is capable of inducing signaling both through cyclic AMP and PI3K. 54
Based on our results and the results of others, I propose a hypothesis concerning the different adrenergic effects on glucose metabolism. It is possible that the switch between anabolic and catabolic pathways may be time-dependent. This could be discussed in relation to the fight-or-flight system. Stress is an important signal for an animal facing a potentially dangerous situation such as a predator. Admittedly, skeletal muscle action may not be the instant response in acute stress. Instead other functions may be more important such as the liver releasing glucose into the blood and the brain making quick decisions. The stress- response may also depend on the species studied. However, after the initial moment, it is time for skeletal muscle action. In the case of cyclic AMP signaling, this is an acute signal responding rapidly (within 5 minutes) to sympathetic activity or stress. During a physical response to a threat, the muscle requires energy. All systems are induced to maximize the intracellular glucose concentration. Glucose uptake is induced to provide the cell with energy while glycogen breakdown is induced to provide glucose as a substrate in ATP production. After the acute stress signal, the immediate need of energy is over. However, after an energy-consuming burst of activity, skeletal muscle energy storage will be diminished. It seems logical that the long term PI3K dependent signal takes over to refill skeletal muscle cells with energy storage in preparation for the next threat. Glucose uptake increases to provide the substrate and also glycogen synthesis increases to store glucose as an energy buffer. 55
Acknowledgments So many people have made this work possible, a few of them will read these acknowledgments, fewer will read the thesis and a negligible lot will understand it. I hope I haven´t forgot anyone, no offense meant! Don´t get excited over the order of appearance, it does not reflect how much I like you. Dock måste jag först ta upp the GLUT4 superfamily! Tore “Dr. T” Bengtsson, jag vill tacka dig för en massa olika saker. Först ett stort tack för det vetenskapliga tänkandet som jag har lärt mig under de här fem (och lite till) åren. Ett stort tack även för alla insikter om forskning och livet i allmänhet som kommit fram under tiden som vi har suttit och jobbat med den ena svåra frågan efter den andra. Speciellt gillar jag de nya ordspråken som fötts “Det enda som spelar nån roll är hur mycket smärta man är beredd att ta” och “Referee nummer 1, han är både dum och elak”, mycket viktigt att ha i bakhuvudet i fortsatta livet. Även tack för att du alltid har haft tid för mina frågor och vilda teorier. Ett stort tack också för att du har ställt upp i vått och torrt och stöttat mig under hela doktorandtiden både jobbmässigt och personligt när saker har skitit sig. Jag tror aldrig att jag har träffat någon som har varit så lätt att arbeta med, socialt kompetent (fungerar med nästan alla), humoristisk, empatisk, klartänkt... listan kan göras låååååång men resten får tas över personal communication. Sammanfattningsvis är du en mästare inom en mängd områden och det har varit ett sant nöje att få arbeta med dig! Till en man med så många kvalitéer passar ett uttryck speciellt bra “The sky is the limit”. TACK!!!!!! Dana “Danamite” Hutchinson, G´day mate! I don´t know where I would be without your teachings and help, not to forget the gossiping. Now we have a few papers together and that’s great. Apart from work, I am grateful to acknowledge your humor, your taste for EtOH, the groupmeetings and the fact that I have picked up an annoying Australian accent that comes up now and then. Another thing you have provided is the master carpenter Rob. Julia “The Princess” Nevzorova. It’s difficult to place this half-Russian, Australian bred, Swedish import. Anyway, it has been a pleasure to work with you and I hope that everything will be fine once you go back to Australia. Olof “Olle” Dallner, your happiness and your outlook on life are intertwined. Nu drar jag härifrån för ett tag och lämnar alltså uppdraget åt dig att hålla stilen på gruppen. Som du skrev till mig i din Lic, nu är vi på gång! Och är vi inte det så är vi i alla fall världsbäst på att festa! Ekaterina “Katja” Chernogubova, during the time of my PhD studies, you have been a valuable support, teacher, colleague and friend. Never have I seen anyone make better brown fat cultures. I wish you good luck with your family, work and life. Robert “Hattori Hanzo” Csikasz, den nyaste rekryten till gruppen. Nu blir det dags för dig att axla labbrocken och dra Hattori Hanzo-klingan mot vetenskapsvärlden. Glöm inte att göra Chihuahua tecknet innan du labbar bara. Se till att folk lär sig uppskatta det goda i livet!
Zoofys (gillar det namnet bäst) har varit ett underbart ställe att arbeta på så tack alla som jobbar och har jobbat här! Barbara Cannon and Jan Nedergaard, I am grateful for our very interesting discussions and the teachings that I have (sometimes reluctantly) accepted. I have learned a lot from both of you, thank you! Damir Zadravec, uh uh uh uh sorry sorry, det har varit ett sant nöja att jobba, festa och umgås med den bästa kompis man kan önska sig! Glöm aldrig de saker som gör livet värt att leva som typ: Tuplapukki, White and Nerdy, JibJab etc. Jag är övertygad om att vi kommer att fortsätta slå världen med häpnad, vart än vi nu är på väg... hän. Tomas “Skalman” Walden, det blir aldrig trist när du är med. Glöm aldrig Tomas reflexbåge. Charlotte “Lotta” Mattson, Jahapp, några visa ord att tänka på tills vi ses igen (snart): I shall call him squishy and he shall be mine, and he shall be my squishy. Come on, squishy Come here, little squishy. Katarina “Kattis” Lennström, både jobbande och lunchande var mycket trivsammmare när du var med. Johanna Jörgensen, Det har varit kul under tiden på zoofys. Sorry att du fick ta hand om budgeten, jag är evigt tacksam att jag slapp. Annelie Brolinson, jag tror inte att vi någonsin haft samma åsikt om någonting förutom katter... Trots det har vi kommit bra överens och haft väldigt trevligt. Emma “Lilla Tigern” Backlund, det blev klart tristare sen du slutade och jag önskar dig lycka till i världen utanför. Therese Holmström, vad skulle man göra utan tjejerna på zoofys?! Lycka till i 56 fortsättningen. Ida Östlund, lycka till med grodorna och glöm inte ryggraden! Tatiana Kramarova. never forget to order frogs by the way. Helena “Francine” Feldmann, Glöm aldrig Futuramakvällen! I am the man with no name, Zapp Brannigan. Natasa Petrovic, you have a great sense of humor and this is something you need when you are a scientist. The entire Sabanov/Sabanova clan, Victor, your dissertation was great (could you show me that graph again). I have had the pleasure of talking to the rest of the family at work (thanks for the Kung-Fu talks Jana). I can just say: fina fisken! Elisabeth Bergner, tack för all hjälp även med att förstå maskineriet bakom vetenskapsvärlden. Anders Jacobsson, tack för den här tiden, det har varit ett sant nöje att jobba med dig. Ibland har jag till och med förstått vad du menar... Andreas Jakobsson, såg att du gått och blivit företagspamp, lycka till, hoppas att det går fortsatt bra för dig såhär Post-Zoofys. Helene Allbrink, ska hålla mig kortfattad som jag vet att en äkta norrländska uppskattar, tack! Birgitta Leksell, tack för att jag aldrig behövt titta på någon dokusåpa, dina kompisars liv är tillräckliga för att fylla den kvoten. Tack för allt stöd under de här fem och ett halvt åren. Rickard, Robert, Stig, tack för all teknisk hjälp. Bengt Håkansson, för pratstunderna. Britta Alsterman, för hjälp med det byråkratiska. Martina Jones, lycka till med jobbandet! Joris Hoeks, keep the mitochondria happy and the will help you while you’re biking. Thanks to all the others (Irina Shabalina, Tsusomu Kobayashi, Hideki Nikami, Satoru, Juha Halonen, Magnus Fredrikson, Mona Wilcke, Camilla Scheele) as well for everything.
Tack till alla trevliga studenter som utmärkt sig lite extra mycket: Martin “Martin-Per” Virhage dom snor min saft, Daniel Nilsson, Emelie Lingebrant, Frida Sahlén, Sharon Lind, Regina Rasmanis, Ylva Rosengarten och Sara Olsson: för inspiration och skön atmosfär. Och en del av er vet att ni tackas lite extra mycket för. Tack till en massa folk inom NF men speciellt: André, Linus, Malin, Kalle, Miche, Lisa, Lina, Ylva, Jenny, Miki, Jonas, HD, Åse, Marmar, Fredrik, Benita, Livia, Marika, Erik, Gustav, Simon: tack för att det finns ett liv utanför forskningen även om det inte är utanför universitetet, vem hade klarat sig utan KM? Som tur är så finns det en värld utanför universitetet också och jag vill speciellt tacka: Hela familjen Källberg (Inger, Krister, Källe, Malin, Mivke), Janne, Elin, Quensel, Marie, Alex, Hans-Emil, Monica, Rebbis, Svamp-omas, Görel Utan dessa människor hade inte det här varit möjligt och de förtjänar ett jättestort tack allihop. Andra som gjort livet trevligt under doktorandtiden: J.R.R. Tolkien, Matt Groening och George Lucas.
Tack till mina släktingar för att ni alltid har stöttat när det har behövts och speciellt stort tack till min mormor Ulla-Britta som alltid har varit ett stort stöd i allt jag gjort. Ett enormt stort tack till min familj som alltid funnits där och livat upp livet när det kännts hängigt och tagit emot! Tack till min mamma Ann-Sofie för allt som du sagt och gjort och för att du har försökt förstå mig även när det inte har varit så lätt. Tack till min bror Robin, utan dig hade det inte gått så bra som det har gjort. En bättre familj kan man inte önska sig! Tack! Tack även till min flickvän Kerstin I. Mehnert som alltid stöttat mig under den här tiden och även hjälpt till med korrekturläsning och många andra saker. Tack även för att du tar ner mig på jorden när jag blir för kaxig. 57
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