β-adrenergic regulation of glucose transporters199952/FULLTEXT01.pdf · coupled receptor (GPCR)...

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Doctoral thesis from the Department of Physiology, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden β-adrenergic regulation of glucose transporters Olof Dallner Stockholm 2008

Transcript of β-adrenergic regulation of glucose transporters199952/FULLTEXT01.pdf · coupled receptor (GPCR)...

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Doctoral thesis from the Department of Physiology, The Wenner-Gren Institute,

Stockholm University, Stockholm, Sweden

β-adrenergic regulation of

glucose transporters

Olof Dallner

Stockholm 2008

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©Olof Dallner, Stockholm 2008

Cover by Olof Dallner

ISBN 978-91-7155-766-7

Printed in Sweden by Universitetsservice AB, Stockholm 2008

Distributor: Stockholm University Library

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“The cloning of humans is on most of the lists of

things to worry about from Science, along with

behaviour control, genetic engineering,

transplanted heads, computer poetry and the

unrestrained growth of plastic flowers.”

Lewis Thomas

“All men dream, but not equally. Those who dream

by night, in the dusty recesses of their minds, awake

in the day to find that it was vanity. But the

dreamers of the day are dangerous men, for they

may act their dreams with open eyes to make it

reality.”

T.E. Lawrence

To my family

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ABSTRACT

The transport of glucose across the plasma membrane is a fundamental mechanism to provide cells

with its basic requirements for energy yielding processes. It is also vital for clearing glucose from blood

into tissues, a process normally stimulated by the hormone insulin in mammals. In disease states such as

diabetes mellitus, the insulin mechanism is not functional, resulting in severe complications. This has

been a driving force for understanding how insulin regulates glucose transport, and to find insulin-

independent glucose transport mechanisms. The sympathetic nervous system, normally activated during

stress, also regulates glucose transport. In contrast to insulin, less is known about the sympathetic

regulation of glucose uptake. The sympathetic neurotransmitter noradrenaline, act on the G protein-

coupled receptor (GPCR) family of adrenergic receptors (ARs). An important subtype of the AR family is

the β-AR which is further subdivided into the β1, β2, and β3-AR. The β3-AR is the predominant subtype in

adipocytes, and the β2-AR is predominant in skeletal muscle.

Glucose is a polar molecule that has to be transported across the hydrophobic plasma membrane by the

13 member family of facilitative glucose transporters (GLUTs). GLUT1 is ubiquitously expressed and

provides cells with basal glucose uptake for maintaining energy levels. GLUT4 is the major GLUT

isoform that is rapidly regulated by insulin signaling. In this thesis, I investigated the β-AR regulation of

GLUT1 and 4, and glucose uptake. I studied these events using skeletal muscle cells and brown

adipocytes in culture, model systems which correspond to metabolically active, sympathetically

innervated and insulin-sensitive tissues.

Stimulation with β-adrenergic agonists increased glucose uptake in skeletal muscle cells and brown

adipocytes. In brown adipocytes this can be divided into two separate mechanisms. Activation of the β3-

ARs and production of cyclic AMP (cAMP) induced the expression of GLUT1, resulting in an increase of

GLUT1 protein in the plasma membrane, and a large increase of glucose uptake after 5 hours of

stimulation. However, there was a faster mechanism of glucose uptake that surprisingly was not due to an

increase of GLUT1 or GLUT4 protein at the plasma membrane, indicating that β3-adrenergic stimulation

may regulate other GLUTs or the intrinsic activity of GLUTs already present at the plasma membrane. In

skeletal myotubes, we postulate there is a possible mechanism where β2-ARs can regulate the intrinsic

activity of GLUT1.

Investigating the insulin and β-adrenergic signaling to glucose uptake revealed that they are two

separate pathways, in both skeletal muscle and brown adipocytes. We found that insulin signaling, but not

β-adrenergic signaling, mediated glucose uptake through class I phosphatidylinositol 3-kinase (PI3K).

The β-adrenergic signaling to glucose uptake appeared to involve a PI3K related kinase (PIKK), in

skeletal muscle and brown adipocytes. Furthermore, the increase of glucose uptake by β3-ARs in brown

adipocytes is partially mediated by AMP-activated protein kinase (AMPK), which is not activated by

insulin.

In an artificially constructed system, consisting of Chinese hamster ovary (CHO) cells stably

expressing GLUT4 and transiently expressing β2-ARs, both insulin and a β-adrenergic activation

regulated GLUT4 and increased glucose uptake. These results suggest that β-adrenergic stimulation can

translocate GLUT4 in certain systems, perhaps through atypical signaling from the C-terminal of the β2-

AR.

These results show that β-adrenergic signaling increase glucose uptake by regulating glucose

transporters. This occurs through distinct signaling pathways, and separately from insulin signaling to

glucose uptake.

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This thesis is based on the following papers, which are referred to in the text by their

respective Roman numerals.

I. Dallner, O.S.., Chernogubova, E., Brolinson, K.A., and Bengtsson, T. (2006)

β3-adrenergic receptors stimulate glucose uptake in brown adipocytes by two mechanisms

independently of GLUT4 translocation

Endocrinology 147(12): 5730-9.

II. Dallner, O.S., Chernogubova, E., Yamamoto, D.L., Tore Bengtsson (2008)

β1- and β3-adrenergic receptor stimulated glucose uptake is dependent on a PI3K related

kinase in brown adipocytes.

Manuscript

III. Hutchinson, D., Chernogubova E, Dallner, O.S., Cannon, B. and Bengtsson, T. (2005):

β-adrenoceptors, but not α-adrenoceptors, stimulate AMP-activated protein kinase in

brown adipocytes independently of uncoupling protein-1.

Diabetologia 48(11): 2386-95

IV. Dallner, O.S., Hutchinson, D.S., and Bengtsson, T. (2008)

Involvement of a PI3K related kinase in β2-adrenergic receptor stimulated glucose uptake

in L6 myotubes.

Manuscript

V. Nevzorova, J., Dallner, O.S., Hutchinson, D.S., Evans B.A., Summers, R.J., and

Bengtsson, T. (2008)

Stimulation of β2-adrenergic receptors leads to translocation of GLUT4 and glucose uptake

in CHO-GLUT4myc cells.

Manuscript

VI. Dallner, O.S., Mäe, M., Eiríksdóttir, E., Langel, U., and Bengtsson, T. (2008)

β-adrenergic modulation of glucose uptake through GLUT1

Manuscript

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β-ADRENERGIC REGULATION OF GLUCOSE TRANSPORTERS

1. INTRODUCTION 11

2. GLUCOSE TRANSPORTERS (GLUTs) 12

2.1. Introduction to GLUTs 12

2.2. Structure of GLUTs 14

2.3. Regulation of GLUT gene expression 15

2.3.1. GLUT1 gene expression 15

2.3.2. GLUT4 gene expression 17

2.3.3. Expression of other GLUTs in skeletal muscle 19

and/or adipose tissues

2.3.4. Relevance of GLUT gene expression 21

2.4. Regulation of GLUT translocation 22

2.4.1. Conventional insulin signaling 22

2.4.2. TC10 pathway 25

2.4.3. Tethering of vesicles: SNARES 26

2.4.4. Translocation of other GLUTs 27

2.5. Regulation of GLUT intrinsic activity 28

2.5.1. GLUT1 28

2.5.1.1. ATP/nucleotides 28

2.5.1.2. Structural interaction of GLUT1 29

2.5.1.3. Other pathways regulating GLUT1 intrinsic activity 29

2.5.2. GLUT4 29

3. ADRENERGIC SIGNALING 30

3.1. Introduction to the sympathetic nervous system 30

3.2. Adrenergic receptors and signaling 30

3.2.1. Adrenergic receptors 30

3.2.1.1. The α1-adrenergic receptors 30

3.2.1.2. The α2-adrenergic receptors 31

3.2.1.3. The β-adrenergic receptors 32

3.2.2. Desensitization and atypical signaling 32

3.2.3. Phosphoinositide 3-kinases (PI3Ks) 34

and PI3K related kinases (PIKKs)

4. β-ADRENERGIC REGULATION OF GLUCOSE TRANSPORT 36

4.1. Introduction 36

4.2. In brown adipose tissue 37

4.2.1. AMP activated kinase (AMPK) 40

4.3. In white adipose tissue 41

4.4. In skeletal muscle 42

5. SUMMARY AND CONCLUSIONS 43

6. ACKNOWLEDGEMENTS 46

7. REFERENCES 48

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ABBREVIATIONS

AC Adenylyl cyclase

AMPK AMP-activated protein kinase

ANS Autonomic nervous system

AR Adrenergic receptor

ASE Adipocyte tissue specific element

AS160 AKT substrate 160

BAT Brown adipose tissue

cAMP Cyclic adenosine monophosphate

CAP Cbl associated protein

C/EBP CCAAT/enhancer binding protein

CRE cAMP responsive element

CREB cAMP-response element binding protein

DNA-PK DNA protein kinase

DAG Diacylglycerol

Epac Exchange proteins directly activated by cAMP

GEF GLUT4 enhancer factor

GLUT Glucose transporter

GPCR G-protein coupled receptor

GRK2 G-protein-coupled receptor kinase 2

HFRE High fat responsive element

HMIT H+-coupled myo-inositol symporter

IP3 Inositol 1, 4, 5-triphosphate

IR Insulin receptor

IRAP Insulin-regulated aminopeptidase

IRE Insulin response element

IRS-1 Insulin receptor substrate 1

MAPK Mitogen-activated protein kinase

MEF2 Myocyte enhancer factor 2

MG1E Muscle specific GLUT1 element

mTOR Mammalian target of rapamycin

NF1 Nuclear factor 1

O/E Olf-1/Early B cell factor

PDE Phosphodiesterase

PDGF Platelet derived growth factor

PDK1 3’-phosphoinositide-dependant kinase 1

PI3K Phosphatidylinositol 3-kinase

PIKK PI3K related kinase

PIP3 Phosphatidylinositol-3,4,5-phosphate

PKA Protein kinase A

PKB Protein kinase B (or Akt)

PKC Protein kinase C

PLA2 Phospholipase A2

PLCβ Phospholipase Cβ

PLD Phospholipase D

PPAR Peroxisome proliferator-activated receptor

PTEN Phosphatase and tensin homolog deleted on chromosome 10

RICTOR Rapamycin insensitive companion of mTOR

SHIP Src homology 2 domain containing inositol 5’-phosphatase 2

SM Skeletal muscle

SNARES Soluble NSF attachement protein receptors

SNS Sympathetic nervous system

SRE Serum response element

WAT White adipose tissue

TPA 12-O-tetradecanoylphorbol-13-acetate

TRE TPA responsive element/Thyroid hormone responsive element

UCP1 Uncoupling protein 1

VAMP Vesicle-associated membrane protein 2

VMH Ventromedial hypothalamic nucleus

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1. INTRODUCTION

The sympathetic nervous system (SNS) is a part of the autonomic nervous system (ANS)

and regulates important processes in mammals, including humans. Activation stimulates the

release of primarily adrenaline and to some extent noradrenaline from the adrenal medulla,

and focal release of noradrenaline from sympathetic nerve endings. Adrenaline circulating in

the blood mediates the acute effects of stress during isolated events to prepare the body for a

reaction. However, noradrenaline released from sympathetic nerves regulate processes on a

daily basis to uphold homestasis. Target tissues express adrenergic receptors (ARs)

responsible for mediating the effects of adrenaline and noradrenaline, accelerating heart rate,

releasing triglycerides from fat and glucose from liver, increasing blood flow to skeletal

muscle, dilating pupils and airways, and reducing gut movements. Adrenergic activation of

white adipose tissue (WAT) promotes β-oxidation and release of triglycerides to mobilize

energy for other organs. On the contrary, activation of adrenergic receptors on skeletal muscle

cells results in an activation of processes to mobilize energy for the tissue itself, one such

process being glucose uptake as discussed in this thesis. Heavily sympathetically innervated

brown adipocyte tissue (BAT) is present in rodents, hibernating animals, and human infants

and is characterized by its ability to uncouple the electron transport chain in mitochondria and

produce heat, stimulated by sympathetic activation in response to cold. Adrenergic activation

of BAT not only activates heat production and hypertrophy of the tissue, but also increases

glucose uptake.

Increasing or decreasing glucose uptake in target cells is achieved by regulating the

glucose transporters (GLUTs) responsible for facilitating the transport of the polar glucose

molecule across the hydrophobic plasma membrane. Signals can alter the expression of the

transporter, the translocation and levels of the transporter at the plasma membrane, or the

intrinsic activity of the transporter.

The insulin signaling pathway to translocation of GLUTs and increase in glucose uptake

into target tissues has been in focus in glucose transporter biology and has given us

knowledge in the regulation of GLUTs. The increase in the prevalence of diabetes type II

encourages the research in the field to further understand the complex mechanisms involved.

Furthermore, this incentive has led to the search for alternative ways to stimulate glucose

uptake, and the identification of other signaling pathways regulating GLUTs. The sympathetic

regulation of blood glucose homeostasis encourages further investigation into signaling and

mechanisms involved. This is of particular interest in skeletal muscle and brown adipose

tissue. Skeletal muscle is the largest tissue in the human body, and hence accounts for the

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most part of insulin induced reduction of blood glucose levels after feeding. Recently, studies

indicate the presence of active brown adipose tissue not only in infants, but also in adult

humans (Nedergaard, et al. 2007). Furthermore, there is a recent interesting development in

the research into BAT/WAT/skeletal muscle cell lineage, suggesting there is a common

progenitor of skeletal muscle and brown adipocytes. There might be possibilities in the future

to promote the brown adipocyte lineage as an energy wasting therapy for obesity, and perhaps

as a blood glucose clearing tissue. There are clearly many reasons that emphasize the

importance of understanding skeletal muscle and brown adipocyte physiology.

In this thesis, I focus on defining parts of the β-adrenergic receptor signaling and

mechanisms regulating glucose transporters and ultimately glucose transport, in skeletal

muscle and brown adipocyte cells.

2. GLUCOSE TRANSPORTERS (GLUTS)

2.1. Introduction to GLUTs

Transport of monosaccharides over the plasma membrane is a fundamental mechanism in

providing the eukaryotic cell with its basic requirements for energy yielding processes. In

mammals, glucose is the primary monosaccharide, although fructose and other sugars can also

be transported into cells. Transport of glucose into cells is vital in reducing glucose levels and

maintaining the homeostasis in the blood plasma after feeding. Glucose cannot penetrate the

hydrophobic plasma membrane and has to be transported by facilitative glucose transporters

(GLUTs) by diffusion. The GLUT family is comprised of 13 known proteins, GLUT1-12 and

HMIT (gene symbols SLC2A1-13), and it is now generally considered that all members of the

family are identified (Joost and Thorens 2001; Uldry and Thorens 2004) (Table 1).

Since the first isolation of a glucose transporter (GLUT1, (Mueckler, et al. 1985) ) other

members has been included, and subsequently a consensus for their terminology has evolved.

The GLUTs all share a structure prediction of 12 hydrophobic -helical membrane-spanning

regions with intracellular amino- and carboxyl-termini. The family is divided into three

classes based on multiple sequence alignments and common sequence motifs, class I

(GLUT1-4), class II (GLUT5, GLUT7, GLUT9, and GLUT11), and class III (GLUT6,

GLUT8, GLUT10, GLUT12, and HMIT) (Table 1). They display different substrate

specificities and tissue distribution.

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A multitude of stimuli can modulate glucose transport in different tissues, in response to

different physiological conditions. Three distinct mechanisms have been identified by which

this can be achieved (further discussed in this chapter).

Table 1. The family of facilitative glucose transporters (GLUT). WAT, white adipose tissue; SM, skeletal

muscle; BAT, brown adipose tissue (adapted from (Joost and Thorens 2001)).

First, the gene expression of the GLUT can be altered resulting in an increase/decrease in

the number of transporters in the cell and at the plasma membrane.

Secondly, the glucose transporters can be shuttled between intracellular compartments and

the plasma membrane in vesicles, a process referred to as translocation.

The third possible mechanism is a change in the intrinsic activity of the glucose

transporter located in the plasma membrane.

Specific signals can trigger responses changing glucose uptake into tissues by one or

several of these mechanisms.

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2.2. Structure of GLUTs

Determining the structure of GLUTs has proven difficult using methods like x-ray

crystallography or NMR spectroscopy. However, there are studies on specific members of the

GLUT family based on alternative approaches such as homology comparison, mutagenesis

studies, and 3D modeling. These studies have contributed with information about their

structure that ultimately is important for investigating their physiological context.

Figure 1. The structure of glucose transporters (GLUTs). Each transmembrane segment is numbered 1-12. N-

glycosylation sites are indicated by CHO. Black line indicates structure of class I-II GLUTs, grey line

indicates structures that are different in class III GLUTs, compared to class I-II.

Data that confirms the tertiary structure of GLUT1 has been derived from both extensive

cysteine-scanning mutagenesis studies (Heinze, et al. 2004; Mueckler and Makepeace 2005),

and structure prediction based on similarities with glycerol phosphate transporter and glucose-

6-phosphate translocase structure (Salas-Burgos, et al. 2004). These studies predict GLUT1 is

a water-filled channel that selectively transports glucose over the plasma membrane. GLUT1

has a N-glycosylation site at residue N-45 in a large extracellular loop, between

transmembrane segments 1 and 2, a motif shared by all class I-II GLUTs (Figure 1).

Glycosylation of this site has been shown to be important for GLUT1 intracellular targeting,

protein stability, and transport efficiency (Asano, et al. 1991; Asano, et al. 1993). Class III

GLUTs have a shorter loop between transmembrane segments 1 and 2, and a longer loop

between transmembrane segments 9 and 10, with the characteristic N-glycosylation site.

Current theory states that glucose binds to specific sites on the extracellular side of GLUT1,

inducing a conformal change that promotes transport over the plasma membrane.

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A theory on the structure of GLUT3 made by homology modeling further supports the

similarities within the GLUT family. Comparison between GLUT3 and a mechanosensitive

ion channel (MscL protein) together with general information from aquaporin structure

describes GLUT3 as a 12 transmembrane helix protein, with a central hydrophilic pore where

glucose can cross the plasma membrane (Dwyer 2001).

GLUT4 has been extensively studied for expression, translocation and transport

characteristics, but not much is known about the structure, except for homology comparisons.

To date, no direct studies on structure of other GLUTs expressed in skeletal muscle- and

adipose-tissue are known to the author.

2.3. Regulation of GLUT gene expression

Regulation of transcriptional activity, post-transcriptional events and translation can alter

the number of glucose transporters in cells. This may also correspond to changes in the

glucose transport, and have a physiological relevance.

2.3.1. GLUT1 gene expression

GLUT1 was the first glucose transporter to be cloned (Mueckler, et al. 1985), it is

ubiquitously expressed, with high expression patterns in erythrocytes and the endothelial cells

in the blood-brain barrier (Maher, et al. 1994). GLUT1 is generally expressed at high levels in

fetal or proliferative cells, and lower levels in the differentiated cells, of adipose and muscle

tissues (Santalucia, et al. 1992; Castello, et al. 1993; Postic, et al. 1994) (Paper II)

The GLUT1 promoter contains a number of recognition sequences for transcription

factors and transcription is induced by SP1, cAMP, serum and thyroid hormone in both

adipocyte and skeletal muscle cells (Romero, et al. 2000; Sanchez-Feutrie, et al. 2004; Vinals,

et al. 1997b)(Vinals, et al. 1997a) (Figure 2). SP1-SP4 is a family of zinc finger (DNA-

binding domain) transcription factors, both SP1 and SP3 has been shown to be involved in

regulation of GLUT1 mRNA expression in skeletal muscle cells (Sanchez-Feutrie, et al. 2004;

Vinals, et al. 1997a; Vinals, et al. 1997a; Fandos, et al. 1999; Fandos, et al. 1999).

Hyperosmotic stimulation has been shown to increase GLUT1 gene expression, by

activation and binding of SP1 to the GC rich region located –95/-83 of the GLUT1 proximal

promoter, in Clone9 cells (Hwang and Ismail-Beigi 2006). Another site designated MG1E

(muscle specific GLUT1 element) has been identified in L6E9 myoblasts, which leads to

induction of the GLUT1 gene by SP1, cAMP or serum (Sanchez-Feutrie, et al. 2004).

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SP3 is proposed to be a negative regulator of GLUT1 transcription, when associated with

the GLUT1 proximal promoter. SP3 is induced during differentiation when GLUT1 gene

expression is down-regulated, and binds to a GC box located –91/-86 in the GLUT1 proximal

promoter (Fandos, et al. 1999), presumably acting as a repressor of transcription. It is

noticeable that these recognition sequences overlap and further studies is required to elucidate

the relationship between these two transcription factors.

Serum, platelet derived growth factor (PDGF), and insulin also has been shown to be able

to activate GLUT1 transcription in 3T3/NIH adipocytes. Two enhancer elements are

identified for this effect, and enhancer 1 seems to be most important (Murakami, et al. 1992;

Todaka, et al. 1994). Enhancer 1 contains several homologues sequences that can be regulated

by different stimuli. A cAMP responsive element (CRE) is present, and cAMP has been

shown to increase GLUT1 transcription in L6E9 skeletal myocytes, 3T3-NIH adipocytes and

brown adipocytes (Vinals, et al. 1997b; Cornelius, et al. 1991) (Paper I). A serum response

element is also located in enhancer 1, responding to a variety of growth factors. There is also

a TPA responsive element (TRE), sensitive to stimulation with 12-O-tetradecanoylphorbol-

13-acetate (TPA). TPA activates conventional and novel protein kinase C (PKC) isoforms,

and the TRE site in enhancer 1 can be activated by platelet-derived growth factor. Enhancer 2

is located between exon 1 and 2 of the GLUT1 gene, and also contains putative TRE and CRE

sites.

A transcriptional activation and a de novo synthesis of GLUT1 protein is subsequently

followed by transport of the protein to the plasma membrane, this is not considered to be a

translocation mechanism. The c-terminus of GLUT1 can interact with the GLUT1 transporter

binding protein GLUT1CBP (GIPC1), coupling the GLUT1 vesicles to myosin VI movement

on actin filaments (Reed, et al. 2005).

Figure 2. The 5’ flanking sequence of the GLUT1 gene. TRE; TPA responsive element, SRE; serum

response element, CRE; cAMP responsive element, MG1E; muscle specific GLUT1 element.

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2.3.2. GLUT4 gene expression

The GLUT4 gene was cloned and characterized in rat, mouse and human in 1989 by a

number of groups (Birnbaum 1989; Charron, et al. 1989; Fukumoto, et al. 1989; James, et al.

1989b; Kaestner, et al. 1989). It is expressed in insulin sensitive tissues such as heart, skeletal

muscle and adipose tissue, where insulin stimulation translocates GLUT4 to the plasma

membrane to increase glucose transport into the cell. Expression of the transporter is

increased in differentiating tissues and coincides with the time when the cells acquire insulin

sensitivity (Santalucia, et al. 1992; Castello, et al. 1993; Postic, et al. 1994) (Paper I).

GLUT4 is also regulated on a transcriptional level by a vast number of growth factors and

hormones. Many factors are involved in binding to specific regulatory elements in the GLUT4

promoter and altering the transcription rate.

Figure 3. The 5’ flanking sequence of the GLUT4 gene. C/EBP; C/EBP binding site, TRE; thyroid hormone

responsive element, MEF2; MEF2 binding site, ASE; adipocyte tissue-specific element, HFRE; high-fat

responsive element, NF1; nuclear factor 1 binding site, O/E; Olf-1/Early B Cell Factor binding site, IRE; insulin

responsive element.

CCAAT/enhancer binding proteins (C/EBPs) is involved in regulating the differentiation

of adipocytes (Christy, et al. 1991), and GLUT4 is a target gene of C/EBP. C/EBP bind to

a recognition sequence located –258/-250 in the GLUT4 promoter and enhances transcription

in 3T3-NIH/L1 adipocytes (Gross, et al. 2004; Kaestner, et al. 1990) (Figure 3).

In C2C12 skeletal muscle cells, it has been shown that myocyte enhancer factor 2 (MEF2)

binds specifically to a site located –437/-428, which confers tissue specific expression (Liu, et

al. 1994). The same reporter gene study also indicated that the MEF2 site alone, without the

two downstream thyroid hormone responsive elements located –419/-414, and –409/-404

(Ezaki 1997) reduced expression to 60 %. Thyroid hormone has been shown to induce

GLUT4 expression by increased transcriptional activity in C2C12 myocytes, 3T3-L1

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adipocytes, brown adipocytes, and in vivo rat skeletal muscle (Romero, et al. 2000;

Richardson and Pessin 1993; Shimizu and Shimazu 2002; Weinstein, et al. 1994).

An adipocyte tissue-specific element (ASE), essential for expression of GLUT4 in white

adipocytes, has been located at -551/-506 by examining mutated GLUT4 minigene expression

in transgenic mice (Miura, et al. 2003). The study also found evidence for a high-fat

responsive element (HFRE) at -701/-551. The existence of such an element correlates with in

vivo experiments when mice fed with a high fat diet for three months caused decreased

GLUT4 mRNA expression in WAT by 50-70% (Ikemoto, et al. 1995). A GLUT4 minigene

with the nuclear factor 1 (NF1) site at -700/-688 mutated results in no expression of the

GLUT4 minigene mRNA exclusively in WAT, suggesting that both the ASE and the NF1

sites are necessary for white adipocyte tissue GLUT4 expression (Miura, et al. 2004).

Interfering with the NF1 binding does however not affect the repression of GLUT4 mRNA

expression by high-fat feeding, and needs to be investigated further.

Prolonged exposure to cAMP or insulin down-regulate expression of the GLUT4 gene in

3T3-L1 adipocytes. This has been shown to be partly mediated by the NF1-binding site in the

GLUT4 promoter (Cooke and Lane 1999a; Cooke and Lane 1999b), and stimulation with

cAMP also repress GLUT4 transcription in skeletal myocytes and brown adipocytes (Vinals,

et al. 1997b; Kaestner, et al. 1991) (Paper I). Further studies of the repression of GLUT4

transcription by insulin and cAMP has also implicated the transcription factor Olf-1/Early B

Cell Factor (O/E) in 3T3-L1 adipocytes (Dowell and Cooke 2002), but the relationship

between O/E and NF1 is not yet understood. A study indicate, that in the human GLUT

promoter there is a domain located -724/-712 where a novel transcription factor designated

GLUT4 enhancer factor (GEF) binds, and is required for GLUT4 expression in cooperation

with MEF2 (Oshel, et al. 2000). However, there are conflicting data as this domain

corresponds to the NF1 binding site in the mouse GLUT4 promoter, and disruption of the

GEF binding site also disrupts the NF1 site of human GLUT4.

AMP-activated protein kinase (AMPK) is thought to be a key sensor of the energy status

in cells (for a recent review see (Kahn, et al. 2005). It is activated by changes in the

AMP/ATP ratio and possibly by exercise, and regulates several important cellular processes

in skeletal muscle (see further discussion in chapter 4). Physical training has in several studies

induced GLUT4 expression, in skeletal muscle (Host, et al. 1998; Kawanaka, et al. 1997; Ren,

et al. 1994). It is a possibility that AMPK activation in response to exercise can induce the

observed increase in GLUT4, and also GLUT1 protein (Fryer, et al. 2002). AMPK activation

can activate GEF and myogenic factor MEF2, and increase their binding to the GLUT4

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promoter (Holmes, et al. 2005). However, there are also indications that AMPK is not

required for exercise-induced increases in GLUT4 expression (Holmes, et al. 2004).

2.3.3. Expression of other GLUTs in skeletal muscle and/or adipose tissues

Although nearly all research on GLUTs in skeletal muscle and adipose tissues investigates

GLUT1 and/or GLUT4, several of the other members of the GLUT family are also expressed

in these tissues.

GLUT3 gene expression

In humans, GLUT3 mRNA is abundant in brain where it is specifically expressed in

neurons, and at lower levels in adipose tissue (for a recent review see, (Simpson, et al. 2008)).

GLUT3 was originally cloned by low stringency hybridization with a GLUT1 probe from a

human fetal muscle cDNA library (Kayano, et al. 1988). However, there are conflicting

studies concerning the expression in skeletal muscle, with results showing no expression or

moderate expression and a possible regulation by insulin growth factor-1 (IGF-1) (Haber, et

al. 1993; Shepherd, et al. 1992; Stuart, et al. 2000; Copland, et al. 2007). Even though the

expression pattern of GLUT3 suggests it is primarily a “neuronal glucose transporter” results

from heterozygous GLUT3+/-

mice indicate it has a possible role in metabolism (Ganguly and

Devaskar 2008). GLUT3 is vital in fetal development and the heterozygous mice are exposed

to limited fetal glucose supply resulting in sexually dimorphic (only male mice are affected)

adult onset adiposity and insulin resistance. Further studies into GLUT3 physiology would be

of interest not only to elucidate the importance in neurons, but also in metabolism.

GLUT5 gene expression

The GLUT5 gene was first isolated from human small intestine (Kayano, et al. 1990), and

then from rat (Rand, et al. 1993), rabbit (Miyamoto, et al. 1994), and mouse (Corpe, et al.

2002), cDNA libraries. GLUT5 has a high specificity for fructose transport and is present in

small intestine, kidney, and brain, but also in skeletal muscle and adipose tissues (for a recent

review see, (Douard and Ferraris 2008)). Long-term stimulations with insulin increase

expression of GLUT5 and fructose transport by transcriptional activation in L6 skeletal

myocytes, and the 5’ flanking sequence of the gene has several putative recognition sites for

transcription factors such as, IRES, C/EBP, CREB, and PPAR (Hajduch, et al. 2003). There is

an ongoing discussion of the relevance of the transition from glucose to fructose in food and

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beverage products and the possible relation to the worldwide increase in obesity and type 2

diabetes (Douard and Ferraris 2008; Stanhope and Havel 2008). This may also implicate the

regulation and physiology of GLUT5 in the role as a very specific fructose transporter in

skeletal muscle and adipose tissues.

GLUT8 gene expression

The GLUT8 gene was cloned from mouse with an approach of database mining (Doege, et

al. 2000). It has a high affinity for glucose, and is expressed at high levels in the testis, and at

low levels in cerebellum, adrenal gland, liver, spleen, adipose and skeletal muscle tissue.

Little evidence exists regarding regulation of expression, but gonadotropins seem to increase

GLUT8 expression, correlating with the high expression in testis (Doege, et al. 2000).

GLUT10 gene expression

The GLUT10 gene was cloned and characterized as a GLUT that primarily transports

glucose, and is expressed in human heart, lung, brain, liver, skeletal muscle, pancreas,

placenta, and kidney (Dawson, et al. 2001). Promoter analysis of the 5’-flanking sequence of

the gene reveals putative regulation of several transcription factors as SP1, SP3, AP2, and a

possible upstream transcription silencer (Segade, et al. 2005). There are very few publications

on GLUT10, but studies on polymorphisms in humans are correlated with changes in

angiogenesis and the emergence of arterial tortuosity (Coucke, et al. 2006).

GLUT11 gene expression

The GLUT11 gene is expressed primarily in human heart and skeletal muscle (cloned from

human heart), has closest homology to GLUT5 (42 % identical amino acids), and presumably

transports both glucose and fructose (Doege, et al. 2001). Interestingly, there is evidence of

alternative splicing, resulting in three isoforms expressed in different tissues (Sasaki, et al.

2001; Wu, et al. 2002). Very few studies are made on this novel GLUT, and further

investigation is necessary to define its physiological significance.

GLUT12 gene expression

The GLUT12 gene was characterized recently from MCF-7 cells (human breast cancer cell

line), and is primarily expressed in human skeletal muscle, heart and prostate, but also adipose

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tissues (Rogers, et al. 2002). GLUT12 is together with GLUT4 and GLUT5 the major isoform

expressed in human skeletal muscle. Functional data suggests that GLUT12 transports

glucose (Rogers, et al. 2003a), and its expression patterns in tissues with high energy

demands is interesting for future studies.

HMIT gene expression

The H+-coupled myo-inositol symporter (HMIT) was cloned from from cDNA libraries of

spleen and frontal cortex, and is predominantly expressed in brain, but also kidney and

adipose tissues (Uldry, et al. 2001). It selectively transports myo-inositol, which is an

important precursor for different types of phosphatidylinositol molecules, important in a vast

number of signaling steps, including insulin signaling.

2.3.4. Relevance of GLUT gene expression

The physiological importance of the expression levels of GLUTs are stressed by a number

of studies, indicating the relevance of changes in GLUT expression for glucose transport. The

expression of GLUTs may change in response to changes in the energy demand of the tissue,

or during disease and abnormal physiological states, an effect observed in obesity and cancer.

Decreased levels of GLUT4 expression in adipose tissue is observed in obesity (Olefsky, et

al. 1988; Garvey, et al. 1991; Sinha, et al. 1991), and may be related to the insulin resistance

caused by this state. On the contrary, high expression levels of GLUT4 in skeletal muscle or

adipose tissues of transgenic mice increase these animals capacity for glucose disposal, and

renders them highly insulin sensitive (Shepherd, et al. 1993; Tsao, et al. 1996; Tsao, et al.

2001). Exercise increase the expression of GLUT4 in skeletal muscle, presumably to meet the

increased energy demand of the tissue (Ren, et al. 1994; Kraniou, et al. 2004). The nerve

system is also implicated in regulating expression of GLUT expression in metabolically active

tissues. Denervating muscle in rat changes expression levels of both GLUT1 and GLUT4,

resulting in a change of glucose transport (Block, et al. 1991; Coderre, et al. 1992).

A known marker for oncogenic transformation is a rapid acceleration of glucose transport

to meet the increased energy demand for the acute growth. The major mechanism behind this

phenomenon is an increase in GLUT gene expression (for reviews see (Macheda, et al. 2005;

Airley and Mobasheri 2007), and several oncogenes are involved and are able to induce this

response (Birnbaum, et al. 1987; Flier, et al. 1987; Hiraki, et al. 1989). Oncogenes ras and src

can both activate GLUT1 expression by interaction with enhancer 1 in the GLUT1 promoter

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(Murakami, et al. 1992). There are also examples of GLUTs that are expressed in malignant,

but not in the normal cells. GLUT3 has been identified by immunohistochemical analysis in

lung, ovarian, and gastric cancers, but are not normally found in these tissues (Younes, et al.

1997). GLUT12 has also been suggested as a GLUT involved in tumor growth, and has been

detected in breast cancer cells (Rogers, et al. 2003b). The tumor suppressor p53, known to be

mutated in many types of cancers, repress transcriptional activity of the GLUT1 and GLUT4

genes (Schwartzenberg-Bar-Yoseph, et al. 2004). Glucose uptake might be rate-limiting in the

development and growth of cancers and could serve as a major target for therapeutic

intervention (Airley and Mobasheri 2007).

The changes in expression of glucose transporters and correlating glucose transport

observed in rodents could be of importance for human physiology. Therapeutic strategies

targeting the expression of GLUTs to counteract diseases are not yet explored, but could

provide great possibilities for treatment.

2.4. Regulation of GLUT translocation

Translocation is the mechanism where stimuli shift the equilibrium of glucose transporters,

located in vesicles, from intracellular compartments to the plasma membrane. The stimuli

often activate a receptor coupling to an intracellular pathway that subsequently interacts with

proteins in the GLUT vesicle. When the stimulation is abolished, the glucose transporters are

withdrawn in vesicles from the plasma membrane to the intracellular compartment again. This

mechanism has been extensively studied for insulin stimulation causing GLUT4 translocation

in skeletal muscle and adipose tissues.

2.4.1. Conventional insulin signaling

The involvement of the pancreas in glucose metabolism has been postulated since the 19th

century and 1921 Banting and Best discovered insulin, a success that subsequently earned

them the Nobel Prize 1923 (Banting, et al. 1991; Banting and Best 1990; Rosenfeld 2002).

The first theories describing translocation of an insulin-sensitive glucose transporter were

published in 1980 (Cushman and Wardzala 1980; Suzuki and Kono 1980). A vast number of

studies on GLUT4 biology is published, mainly as a result of the aim to prevent the emerging

problem with diabetes mellitus, and has yielded profound knowledge in insulin signaling and

the mechanism of glucose transporter translocation. Diabetes mellitus is a syndrome where

defects in the production of or response to insulin results in dysfunctional regulation of blood

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glucose homeostasis. Type I diabetes has a strong genetic association and is often caused by a

T-cell mediated autoimmune attack on insulin producing β-cells in the islets of Langerhans in

the pancreas. Type II diabetes is connected to lifestyle, and insulin resistance (loss of response

from target tissues) is the key abnormality. The insulin signaling pathway and the mechanism

of GLUT4 vesicle translocation has been described in several excellent reviews (Bryant, et al.

2002; Watson, et al. 2004), for a recent review see (Zaid, et al. 2008).

The insulin receptor (IR) is a receptor tyrosine kinase, and consists of two extra cellular -

and two transmembrane -subunits (Figure 4). When insulin binds to the extracellular -

subunits of the receptor, a conformational change is induced, which cause an

autophosphorylation of tyrosine residues in the -subunits. The activated state of the IR

recruits various scaffolding proteins, such as insulin receptor substrate 1 (IRS1) (White 2002).

As IRS-1 is associated with the activated IR it is phosphorylated, which then provides a

binding site for the p85 subunit of class 1A phosphatidylinositol 3-kinase (PI3K). PI3K

signals and regulates a number of cellular responses, making it a central molecule in many of

these processes (Cantley 2002). In turn, the p110 subunit of PI3K catalyzes the formation of

Figure 4. The insulin signaling pathway. Activation of the insulin receptor and downstream signaling shifts the

equilibrium of GLUT4 vesicles from an intracellular location to the plasma membrane (translocation), causing an

increase in glucose uptake.

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phosphatidylinositol-3,4,5-phosphate (PIP3) which recruits Akt (also referred to as protein

kinase B; PKB) to the plasma membrane. PI3K isoforms are further discussed in chapter III,

but recent studies provide strong evidence that PI3K-p110α is the main p110 isoform for

insulin signaling (Knight, et al. 2006). PIP3 also recruits 3’-phosphoinositide-dependant

kinase 1(PDK1) which phosphorylates threonine 308 (T308), a key phosphorylation site in

the activation loop of Akt (Storz and Toker 2002). Akt has a second phosphorylation site,

serine 473 (S473), which is also required for full activation (Welsh, et al. 2005). A study also

suggests that mammalian target of rapamycin and rapamycin insensitive companion of mTOR

(mTOR/RICTOR) can be the second PDK that phosphorylate S473 of Akt in response to

insulin (Hresko and Mueckler 2005). Of the three isoforms of Akt (1, 2 or 3), Akt2 emerges

as the most important isoform in insulin signaling. Depletion of Akt2 by RNAi in 3T3L1

adipocytes inhibits insulin signaling, and knockout of Akt2 results in mice with impaired

glucose uptake in skeletal muscle (Cho, et al. 2001; Jiang, et al. 2003).

Another target that seems to be downstream of PI3K is protein kinase C (PKC). The

family of PKC consists of at least 12 members of serine/threonine kinases that are involved in

many signaling events (Way, et al. 2000). The members are divided into three groups,

conventional PKC isoforms , I, II, (Ca+-dependent, activated by phosphatidylserine and

diacylglycerol), novel PKC isoforms , , , (Ca+-independent, activated by

phosphatidylserine and diacylglycerol), and atypical PKC isoforms and / (Ca+-

independent, regulated by phosphatidylserine but not diacylglycerol). PKC signaling has

emerged as important steps in insulin signaling and GLUT4 translocation, in particular

atypical PKCs (aPKCs)(Farese, et al. 2005), and novel PKC (Braiman, et al. 1999). Insulin

stimulation induces a rapid remodeling of the actin filaments in L6 skeletal muscle cells, and

inhibiting cortical actin almost abolish insulin induced increases of GLUT4 at the plasma

membrane (Khayat, et al. 2000; Tong, et al. 2001). PKC may be involved in this process in

skeletal muscle cells (Liu, et al. 2006), but the effect is not as apparent in adipose cells.

Although the involvement of AKT in insulin-stimulated GLUT4-vesicle translocation is

supported by a number of studies in both adipocytes and skeletal myocytes, the precise

mechanism is unclear. A link in the interaction between PKB and GLUT4 vesicles are the

Rab GTPase-activating proteins AKT substrate 160 (AS160 or TBC1D4) and TBC1D1, both

phosphorylated by AKT in response to insulin stimulation, and proposed to be negative

regulators of GLUT4 vesicle translocation (Eguez, et al. 2005; Sakamoto and Holman 2008).

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Another putative downstream target of AKT, involved in GLUT4 translocation, is the

phosphoinositide 5-kinase PIKfyve. Insulin stimulation phosphorylates PIKfyve at serine 318,

an effect that is abolished by wortmannin (PI3K antagonist) (Berwick, et al. 2004). Kinase-

dead mutants of PIKfyve inhibit GLUT4 translocation, which although its precise

involvement is not clear, makes it an interesting target in the insulin signaling.

Two factors that may negatively influence insulin signaling is the tumor suppressor

phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and src homology 2

domain containing inositol 5’-phosphatase 2 (SHIP2). These phosphatases can

dephosphorylate PIP3 to PIP2 and suppress insulin signaling, thus inhibition of PTEN and

SHIP2 may be a strategy for treating insulin resistance (Sasaoka, et al. 2006).

The insulin-regulated aminopeptidase (IRAP) has also been implicated in insulin signaling

and GLUT4 translocation (Keller 2004). It is localized in intracellular compartments under

basal conditions, and is rapidly translocated to the plasma membrane in response to insulin.

The role of IRAP in GLUT4 translocation is not clear, but it is hypothezised that the

aminopeptidase activity is functional at the plasma membrane, where it initiates degradation

of non-insulin peptide hormones.

2.4.2. TC10 pathway

There is evidence of a second insulin signaling pathway leading to GLUT4 translocation

(primarily in adipocytes), possibly associated with caveolae and lipid raft microdomains.

When insulin receptors in these microdomains are activated and undergo autophosphorylation

of tyrosine residues, adaptor protein containing a PH and SH2 domain (APS) is recruited and

phosphorylated (Liu, et al. 2002) (Figure 5). This in turn recruits Cbl and CAP (Cbl

associated protein) to form a complex, and through CAP this complex interacts with flotillin

and lipid rafts (Baumann, et al. 2000; Liu, et al. 2005). Cbl associates with another adaptor

protein, CrkII, a protein that is constitutively associated to the nucleotide exchange factor

C3G (Ribon, et al. 1996). As C3G also localizes to the lipid rafts, it activates the small G-

protein TC10 (Chiang, et al. 2001), which in turn can interact with the exocyst complex. The

exocyst complex is comprised of several proteins, Exo70, Sec6, Sec8, and SAP97, and is

thought to be involved in the tethering of GLUT4 vesicles to the plasma membrane (Inoue, et

al. 2003; Inoue, et al. 2006). Furthermore, activation of the TC10 pathway seems to be able to

activate and recruit aPKCs to the plasma membrane in a PI3K independent way (Kanzaki, et

al. 2004), presumably to participate in GLUT4 translocation events.

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Figure 5. The TC10 pathway. Insulin receptor activation recruits APS-CAP-Cbl-CrkII/C3G activating TC10.

This recruits the exocyst complex involved in stimulating vesicle tethering, and atypical PKCs (aPKCs) involved

in actin remodeling.

However, there are some opposing results. There is evidence that ablation of APS results in

inhibition of insulin stimulated glucose uptake (Ahn, et al. 2004), while in another study no

effect could be seen when siRNA knockdown of Cbl or CAP was achieved (Zhou, et al.

2004). In spite of this, the TC10 pathway appears to be an important part of insulin signaling.

2.4.3. Tethering of vesicles: SNARES

Fusion of vesicles to the plasma membrane is not a random event, but requires a specific

interaction between molecules in the vesicles and the plasma membrane (Figure 6). Two types

of SNARES (Soluble NSF attachement protein receptors), t-SNARES (target membrane

SNARES) and v-SNARES (vesicle membrane SNARES), are the core of the mechanism to

bring the vesicles in close proximity to the plasma membrane to allow lipid bilayer mixing.

Two potential GLUT4 vesicles v-SNARES are expressed in adipocytes, synaptobrevin 2

(VAMP2; vesicle-associated membrane protein 2) and cellubrevin (VAMP3), but VAMP2

has been found to be the primary v-SNARE involved in GLUT4 vesicle translocation (Martin,

et al. 1998). VAMP2 bind to a complex of t-SNARES Syntaxin 4 and SNAP23 in adipocytes.

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A number of SNARE associated proteins, Munc18c, Synip, and Tomosyn, (Thurmond and

Pessin 2000; Widberg, et al. 2003; Yamada, et al. 2005) have also been implicated in the

process of vesicle/plasma membrane fusion. Furthermore, it is suggested that cortical actin

also is involved in the tethering of the vesicles close to the plasma membrane (reviewed in

(Zaid, et al. 2008) ).

Figure 6. The three steps of GLUT4 vesicle exocytosis. Insulin stimulation promotes the tethering, docking,

and fusion of GLUT4 vesicles to the plasma membrane.

2.4.4. Translocation of other GLUTs

A very interesting finding is that the GLUT4 deficient mice (GLUT4-/-

) are growth

retarded and has decreased longevity, but does not develop diabetes (Katz, et al. 1995;

Stenbit, et al. 1996). Although these mice possibly show insulin resistance, GLUT4-/-

mice

clear oral glucose tolerance tests as efficiently as wild type controls. This suggests that there

are one or several other insulin-responsive GLUTs, able to compensate for the lack of

GLUT4. Candidates are GLUT3, 8, 10-12, but there is up to date no definitive theory for the

phenotype of the GLUT4 null mice. Expression patterns of GLUT10-12 in insulin-sensitive

tissues indicate they may be involved, but there is no evidence of translocation in response to

insulin. GLUT8 has been shown to be insulin-responsive, but only in the blastocyst

(Carayannopoulos, et al. 2000), and GLUT3 is suggested to translocate and be insulin

responsive in neurons (Heather West Greenlee, et al. 2003; Uemura and Greenlee 2006) .

However, hetezygous GLUT4+/-

or conditional depletion of GLUT4 in skeletal muscle or

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adipose tissue of mice show the expected insulin resistance and tendency for diabetes

(Stenbit, et al. 1997; Li, et al. 2000; Zisman, et al. 2000; Abel, et al. 2001), and

overexpression of GLUT4 in GLUT4+/-

mice normalizes insulin sensitivity and glucose

tolerance (Tsao, et al. 1999). It is evident that GLUT4 has a central role in insulin stimulated

glucose uptake during normal physiologic conditions although the observed compensation for

ablation of GLUT4 in GLUT4-/-

mice is not yet explained.

2.5. Regulation of GLUT intrinsic activity

The least investigated area of glucose transporter biology is the regulation of putative

changes in GLUT intrinsic activity, and little direct evidence of such a mechanism exists.

However, there are a number of observations that cannot readily be explained by altering

expression or translocation of GLUTs.

2.5.1. GLUT1

Changes in the intrinsic activity of GLUT1 have been proposed in a number of studies,

and may account for observed increases in glucose transport. In general, this increase in

glucose transport cannot be explained by GLUT1 expression or translocation. A majority of

the studies has been carried out in erythrocytes, and only little is known about intrinsic

activity changes in adipocyte and skeletal muscle tissue. Possible changes in intrinsic activity

of GLUT1 protein in 3T3-L1 adipocytes has been observed after treatment with protein

synthesis blockers, indicating this mechanism also could be present in this tissue (Harrison, et

al. 1992).

2.5.1.1. ATP/nucleotides

GLUT1 has three predicted ATP-binding sites (domain I-III) at residues 111-118, 225-

229, and 332-338 (Liu, et al. 2001), which are homologues to Walker ATP binding domains

(Walker, et al. 1982). There are evidence indicating that domain I and III are true ATP

binding sites, and that ATP binding there is important for GLUT1 mediated glucose transport

(Liu, et al. 2001; Levine, et al. 1998). It is unclear if ATP exerts an effect directly on GLUT1

or in cooperation with extrinsic proteins.

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2.5.1.2. Structural interaction of GLUT1

It has been observed that GLUT1 protein can form oligomeric structures, tetramers, and

that disrupting this association inhibits glucose transport activity in erythrocytes (Zottola, et

al. 1995). It is however not concluded whether this mechanism exists in adipocytes and

skeletal muscle tissue.

Association of GLUT1 to lipid rafts has been observed, and there is evidence of an

increase in localization to these plasma membrane domains during glucose deprivation, in

3T3-L1 adipocytes (Kumar, et al. 2004). This distribution of GLUT1 seems to be regulated by

the N-terminal part of the protein, as a chimeric GLUT1 with the GLUT3 N-terminus is not

associated to lipid rafts (Sakyo, et al. 2007). A redistribution of GLUT1 transporters from

lipid raft domains in plasma membranes correlates with an increase in glucose transport

without changing the number of transporters in the plasma membrane, suggesting a change in

the intrinsic activity (Rubin and Ismail-Beigi 2003). This theory is also supported by the

negative effect on GLUT1 mediated glucose transport by stomatin, a protein that is enriched

in lipid raft domains (Zhang, et al. 1999; Zhang, et al. 2001). There is no direct evidence that

this causes an alteration in GLUT1 transport activity, but it raises an interesting theory that

these domains may be favorable for activation.

2.5.1.3 Other pathways regulating GLUT1 intrinsic activity

In brown adipocytes, there have been studies proposing a mechanism where

norepinephrine acting on adrenergic receptors can increase glucose uptake by altering GLUT1

intrinsic activity (Shimizu, et al. 1996; Shimizu, et al. 1998) (Paper II). It is possible β-

adrenergic receptors also regulate intrinsic activity of GLUT1 in skeletal muscle (Paper VI).

Furthermore, it has been proposed that activation of the serum- and glucocorticoid-

inducible kinase SGK1 may be involved in altering GLUT1 intrinsic activity in insulin-

sensitive tissues (Palmada, et al. 2006).

2.5.2. GLUT4

Although GLUT4 is translocated and regulates glucose uptake by an increase in of the

number of transporters at the plasma membrane, studies suggest GLUT4 intrinsic activity can

be a part of the increase of glucose uptake. Insulin-stimulated docking of GLUT4 vesicles to

the plasma membrane precedes the increase in glucose uptake in L6 skeletal myocytes,

suggesting there is a signal regulating GLUT4 intrinsic activity (Somwar, et al. 2001b). The

mitogen-activated protein kinase (MAPK) p38 has been implicated in this effect, and using

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inhibitors on p38 inhibits insulin-stimulated glucose uptake without affecting the translocation

of GLUT4 (Hausdorff, et al. 1999; Somwar, et al. 2001a). Another possible event that could

alter the intrinsic activity of GLUT4 is the direct nucleotide binding to the transporter (James,

et al. 1989a; Lawrence, et al. 1990; Piper, et al. 1993)

3. ADRENERGIC SIGNALING

3.1 Introduction to the sympathetic nervous system

The sympathetic nerves are a part of the autonomic nervous system (ANS) and innervate

many organs in the human body. Postganglionic neurons release the primary sympathetic

neurotransmitter noradrenaline to accelerate heart beat, relax airways, stimulate

glycogenolysis and glucose output from the liver, and stimulates secretion of the hormone

adrenaline, but to some extent also noradrenaline, from the medulla of the adrenal gland. In

mammals, sympathetic activation in response to cold activates brown adipose tissue (BAT) to

produce heat and regulate body temperature. It is generally accepted that blood vessels in

skeletal muscle are sympathetically innervated, but it has also been discovered that

sympathetic nerves directly innervate skeletal muscle in mammals (Barker and Saito 1981).

3.2. Adrenergic receptors and signaling

3.2.1. Adrenergic receptors

The endogenous natural receptors for the catecholamines adrenaline and noradrenaline are

adrenergic receptors that belong to the superfamily of seven-transmembrane G-protein

coupled receptors (GPCRs). Adrenergic receptors mediate responses both centrally and

peripherally, and are expressed many tissues in the human body. The first subdivision of

adrenergic receptors was into α-adrenergic and β-adrenergic receptors based on

pharmacological characteristics (Ahlquist 1948).Subsequently, further studies led to the

identification of three pharmacologically distinct types (α1, α2, and β) (Figure 7), and

currently there is 9 members of the family (Bylund, et al. 1994).

3.2.1.1. The α1-adrenergic receptors

Three subtypes of α1-adrenergic receptors are cloned and genetically defined, α1A, α1B, and

α1D (Hieble, et al. 1995). They are expressed in many different tissues in humans, α1A is

predominantly expressed in heart, brain, and prostate, α1B in spleen and kidney, and α1D in

aorta (Garcia-Sainz, et al. 1999). The three α1-adrenergic receptor subtypes primarily couples

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to Gq/11 proteins to activate phospholipase Cβ (PLCβ) (Wu, et al. 1992; Hubbard and Hepler

2006). PLCβ catalyses the formation of inositol 1, 4, 5-triphosphate (IP3) and diacylglycerol

(DAG) that mediates many responses as second messengers. Increases in the levels of IP3

trigger release of Ca2+

from the endoplasmatic reticulum, and DAG activates protein kinase C

(PKC). However, α1-adrenergic receptors can also couple to pertussis toxin sensitive Gi and

Go proteins, and Gs and G12/13 proteins, and activate phospholipase A2 (PLA2) and

phospholipase D (PLD) (Hein and Michel 2007). The α1-adrenergic receptor subtypes display

selective signaling which has been observed as differences in the activation of PLC (Zhong, et

al. 2001; Richardson, et al. 2003; Taguchi, et al. 1998).

3.2.1.2. The α2-adrenergic receptors

Three subtypes of α2-adrenergic receptors have been cloned and defined, α2A, α2B, and α2C.

Noradrenaline and adrenaline target these receptors which couples to Gi/Go proteins and

signaling pathways that inhibit adenylyl cyclase (AC) and cAMP production, activate

receptor-operated K+-channels, and inhibit voltage-gated Ca

2+-channels (Limbird 1988). The

negative coupling of α2-adrenergic receptors seems to be important in a short negative

feedback to inhibit adrenaline release from the chromaffin cells of the adrenal medulla and

noradrenaline from sympathetic nerves (Brede, et al. 2003).

Figure 7. Conventional adrenergic receptor signaling

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3.2.1.3. The β-adrenergic receptors

Three subtypes of β-adrenergic receptors are identified, β1, β2 and β3. They are

differentially expressed in many tissues, where β1-adrenergic receptors are highly expressed

in heart and brain, β2-adrenergic receptors are expressed in uterus, skeletal muscle, and lungs,

and β3-adrenergic receptors are expressed at high levels in adipose tissues and gall bladder

(Dixon, et al. 1986; Frielle, et al. 1987; Emorine, et al. 1989; Strosberg 1997). It has been

known since the 80’s that β-adrenergic receptors primarily couple to stimulatory Gα proteins

(Gαs), causing an activation of adenylyl cyclase and production of cAMP (Gilman 1987).

In contrast to α1, and α2, β-adrenergic receptors display a difference in the pharmacological

response to their natural agonists. In several model systems, adrenaline has a greater effect on

β2-adrenergic receptors, and is less potent on β1 and β3, while noradrenaline is most effective

on β3-adrenergic receptors, and has less effect on β1 and β2 (Bylund, et al. 1994; Strosberg

1997).

The existence of cAMP and its function as a classical intracellular “second messenger”

(neurotransmitters and hormones being “first transmitters”) to catecholamines was pioneered

by E. W. Sutherland during the late 50’s and 60’s, and for these discoveries he was awarded

the Nobel Prize in 1971 {{189 RALL,T.W. 1958;190 Sutherland,E.W. 1966;}}. cAMP

triggers a plethora of effects downstream of the GPCR/G-protein/AC pathway, and discussing

all of them is beyond the scope of this thesis. Rising levels of cAMP in response to AC

activation initiates signaling by ion channels, cAMP-dependant protein kinase A (PKA), and

exchange proteins directly activated by cAMP (Epac) (Beavo and Brunton 2002). PKA

phophorylates cAMP-response element binding protein (CREB) that binds to CRE sites in

gene promoters, initiating transcription of many target genes (Sands and Palmer 2008).

Recently, the complexity of cAMP signaling has become increasingly evident as cAMP

concentration is varying throughout the cell, and occurs compartmentalized. This can be

correlated to the localization of proteins involved in cAMP signaling and regulation, as AC,

phosphodiesterases (hydrolyzes cAMP to AMP), and PKA (Zaccolo, et al. 2006; Lynch, et al.

2007).

3.2.2. Desensitization and atypical signaling

During the 1980s it was made clear that the β2-adrenergic receptor was phosphorylated

following stimulus, and this appeared to be related to the strictly controlled mechanism of

receptor desensitization (Lefkowitz 2004)(Figure 8). The proteins that mediate the

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33

phosphorylation of the β2-adrenergic receptor were identified as PKA and β-adrenoceptor

kinase (βARK), later renamed G-protein-coupled receptor kinase 2 (GRK2) (Benovic, et al.

1985; Benovic, et al. 1986). β-arrestin1 and β-arrestin2 was also identified as cofactors in the

mechanism of desensitization (Lohse, et al. 1992; Attramadal, et al. 1992). Studies on visual

arrestins (arrestins that are recruited to the GPCR rhodopsin in rod and cone photoreceptors)

show that phosphorylation and the ligand induced conformational change of the receptor

recruits β-arrestins, changing the β-arrestin to an active state (Gurevich and Benovic 1992;

Hirsch, et al. 1999). Only phosphorylation on the c-terminal tail of the β2-adrenergic receptor

by GRK2, and not PKA, recruits β-arrestin (Lohse, et al. 1992), but both GRK2 and PKA

mediated phosphorylation cause desensitization (Roth, et al. 1991). Desensitization of the

agonist induced response is in part due to β-arrestins ability to block or uncouple the G-

protein dependent response from the β-adrenergic receptor (Krupnick and Benovic 1998), but

also involve internalization. It was observed before the discovery of GRKs and arrestins that

β2-adrenergic receptors were rapidly internalized into the cytosol of frog erythrocytes when

exposed to the agonist isoproterenol (Chuang and Costa 1979). Internalized β2-adrenergic

receptors are detected after only 2 minutes of agonist exposure and co-localize with the the

transferrin receptor (TrR), which is an endosomal marker (von Zastrow and Kobilka 1992).

Activated β-arrestins also recruits phosphodiesterases (PDEs), enzymes with an activity for

hydrolyzing cAMP (Perry, et al. 2002), and this also seems to cause the β2-adrenergic

Figure 8. The desensitization mechanism of β-adrenergic receptors

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34

receptor to switch from coupling to Gs to Gi (Baillie, et al. 2003). The increased degradation

of cAMP and G-protein subtype swithching also diminishes the agonist response, displaying

the several different actions of β-arrestins in β-adrenergic receptor desensitization.

The similarity of β1 and β2-adrenergic receptor c-terminal tails correlates with the

mechanism of desensitization as GRK2 and PKA phosphorylation, and β-arrestin recruitment,

also occurs at the β1-adrenergic receptor (Freedman, et al. 1995). The β3-adrenergic receptor

on the other hand, has fewer sites for phosphorylation by GRK or PKA (Strosberg 1997;

Liggett, et al. 1993).

Recently, β-arrestins was found not only a central protein in receptor desensitization but

also gained a new role as signal transducer. The atypical signaling of seven transmembrane g-

protein coupled receptors through β-arrestins implicates a growing number of proteins

(reviewed in (Lefkowitz and Shenoy 2005)).

3.2.3. Phosphoinositide 3-kinases (PI3Ks) and PI3K related kinases (PIKKs)

An important participant in a growing number of signaling pathways, and possibly atypical

GPCR signaling, are the family of phosphoinositide 3-kinases (PI3Ks) that phosphorylate

inositol phospholipids at the 3’-OH position of the inositol ring. They are tightly bound

heterodimers of a regulatory and a catalytic subunit or monomers and are divided into three

classes based on homology, where the major focus has been on class I PI3Ks, and less is

known about class II-III PI3Ks (Vanhaesebroeck, et al. 1997; Hawkins, et al. 2006)(Table 2).

Class Regulatory subunit Catalytic subunit

Class IA PI3K

p85α

p55α

p50α

p110α

p100β

p110

Class IB PI3K p101

p84 P110

Class II PI3K

C2α

C2β

C2

Class III PI3K p150 hVPS34

Table 2. The regulatory and catalytic subunits of the three classes of PI3Ks

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Classification of PI3Ks is generally based on the catalytic subunit present in the

heterodimer (e.g. p85α-p110α heterodimer is referred to as PI3Kα), since there is limited data

of the specific function of the different regulatory subunits. There are four members of Class I

PI3K catalytic subunits and they are divided into class 1A (p110α, p110β, p110 and class 1B

(p110 with class IA being primarily activated by tyrosine kinases, and class IB primarily

activated by GPCRs. Subunits p110α and β are ubiquitously expressed, while subunits p110

and expression are primarily restricted to cells of myeloid origin in the hematopoietic

system. This is supported as transgenic mice lacking p110α or p110β are embryonic lethal,

while mice lacking p110 and p110 subunits survive, but has defects in leukocyte,

lymphocyte, and mast cell function (Vanhaesebroeck, et al. 2005). Class IA PI3Ks has an

important role in insulin signaling, and inhibitors of the PI3K-p110 as the potent inhibitor

wortmannin and the reversible p110 subunit ATP pocket binding inhibitor LY294002 has

extensively been used to probe this (Srivastava 1998). However, these inhibitors are pan-

specific and inhibit other kinases in the PI3K family, members of the PI3K related kinase

(PIKK) family, and some unrelated kinases. Recently, more specific small cell permeable

inhibitors were developed from molecular modeling of the interaction of LY294002 with

PI3K-p110 isofoms and high throughput screening, and such inhibitors has high potential for

drug therapy. Several such inhibitors were used in paper II and paper IV and in vitro IC50

values (inhibitory concentration 50%) from lipid kinase assays are summarized in table 3.

Such approaches have yielded evidence for p110α as the primary isoform involved in insulin

signaling (Knight, et al. 2006). As a GPCR is activated and the classical Gα signaling occurs,

the β subunit of the G-protein is also able to mediate signaling (atypical signaling).

Observations suggest Gβ can activate PI3Kβ (Kurosu, et al. 1997). However, the primary

Class I PI3K isoform implicated in GPCR signaling through the G-protein β subunit is the

PI3K the only class 1b member (Stoyanov, et al. 1995; Stephens, et al. 1997). Recently, it

was suggested that PI3Kβ indeed signals downstream of GPCRs, being functionally

redundant with the GPCR activated PI3K (Guillermet-Guibert, et al. 2008). The PIKKs are a

family of six proteins, mammalian target of rapamycin (mTOR), ataxia-telangiectasia mutated

(ATM), ataxia-telangiectasia and Rad3 related (ATR), suppressor of morphogenesis in

genitalia (SMG-1), DNA protein kinase catalytic subunit (DNA-PKcs), and

transformation/transcription domain associated protein (TRRAP). All PIKKs but TRRAP are

identified as serine/threonine protein kinases, sharing sequence similarities with PI3Ks, and

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have established themselves as stress response proteins primarily related to DNA and RNA

damage, but also growth and proliferation (Bakkenist and Kastan 2004; Abraham 2004).

PI3Ks LY294002 C15e TGX221 AS605240 PI-103

p110α 0.63-9.3(3) 0.0020 (0.58) 5 0.06 0.002-0.008 (~0.5)

p110β 0.31-2.9 0.016 0.005 (0.02) 0.27 0.003-0.088

p110 1.33-6.0 0.66 0.1 0.3 0.003-0.048

P110 7.26-38 >10 0.008 (0.09) 0.015-0.15

PI3KC2α 15-100 >10 ~1

PI3KC2β 2.1-30 0.22 0.026-0.043

PI3KC2 40

hsVPS34 2.3-3.9

PIKKs

ATR 100 0.85

ATM 100 0.92

DNA-PK 0.66 0.002-0.014

mTORC1 8.9 0.02 (0.1)

mTORC2 0.083

The family of PI3Ks seems to be a central participant in many pathways, as tyrosine kinase

receptor and GPCR signaling, even though the roles of the different isoforms are constantly

changing. The evident involvement in GPCR signaling suggests PI3Ks may be a participant in

the β-adrenergic pathway to glucose uptake, perhaps through atypical signaling.

4. β-ADRENERGIC REGULATION OF GLUCOSE TRANSPORT

4.1. Introduction

Focally released norepinephrine from sympathetic nerves and epinephrine released from

the adrenal medulla affect glucose transport and metabolism in liver, brown adipose tissue,

white adipose tissue, and skeletal muscle (Nonogaki 2000; Philipson 2002). Administering β-

agonists to humans are likely to cause hyperglycemia due to the acute effect on liver, resulting

Table 3. IC50 values of inhibitors for PI3K and PIKK family members as determined in in vitro assays. Values

are compiled from several studies and show a range when different values were reported (Knight, et al. 2006;

Raynaud, et al. 2007; Jackson, et al. 2005; Camps, et al. 2005; Hayakawa, et al. 2006; Knight, et al. 2004).

Values in parenthesis are reported IC50 in cellular assays and grey color indicates the primary target of inhibitor.

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in a net output of glucose from the liver to blood (Philipson 2002). However, there are several

studies concerning effects of β-agonists or sympathetic activation on fat and muscle that are

primary tissues for clearing glucose from the blood, and main target for insulin stimulated

glucose uptake. Activation of the ventromedial hypothalamic nucleus (VMH) cause an

increased peripheral sympathetic nerve activity and increase glucose uptake in an insulin

independent way in brown adipocytes, heart, and skeletal muscle, but not in white adipose

tissue or brain (Sudo, et al. 1991).

4.2. In brown adipose tissue

Brown adipose tissue is the organ responsible for non-shivering thermogenesis by

uncoupling mitochondrial respiration, through a mechanism mediated by uncoupling protein 1

(UCP1) (Cannon and Nedergaard 2004). It is metabolically active, highly vascularized, rich in

mitochondria, and under direct hypothalamic control via sympathetic nerves. In response to

cold exposure, norepinephrine from sympathetic nerve endings activates BAT to increase

metabolism and produce heat. BAT is primarily an organ in rodents, hibernating animals, and

neonatal humans, but there are novel theories of it being present in adult humans (Nedergaard,

et al. 2007).

Glucose uptake in BAT is markedly increased by both norepinephrine and insulin (Marette

and Bukowiecki 1989; Liu, et al. 1994). Extensive studies of insulin signaling in skeletal

muscle and white adipocytes show that insulin translocates GLUT4 vesicles to the plasma

membrane to increase the influx of glucose. This mechanism is also displayed by brown

adipocytes (Slot, et al. 1991; Omatsu-Kanbe, et al. 1996). Furthermore, crucial components of

insulin signaling in skeletal muscle and white adipocytes are important for insulin stimulated

uptake in brown adipocytes. Insulin stimulated glucose uptake is impaired in immortalized

brown adipocytes from IRS2-KO mice, and retroviral re-expression of IRS2 partially restores

the response (Fasshauer, et al. 2000). Similar approaches with a Cre-LoxP system abolishing

PDK1 or using brown adipocytes from AKT2-KO mice implies importance of PDK1 and

AKT2 (Sakaue, et al. 2003; Bae, et al. 2003). Insulin signaling in brown adipocytes seems to

be mediated by the PI3K p110α catalytic subunit causing phosphorylation on threonine 308

and serine 473 of PKB/Akt (Paper II), as seen in skeletal myotubes and white adipocytes

(Knight, et al. 2006). In many ways, brown adipose tissue display all the characteristics and

signaling steps of an insulin sensitive tissue, such as white adipose and skeletal muscle tissue.

Exposing rats to cold stimulates a dramatic increase of glucose uptake into brown adipose

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38

tissue (Vallerand, et al. 1990; Vallerand, et al. 1990; Greco-Perotto, et al. 1987). This tissue

has an exceptionally high uptake of glucose per tissue gram in response to cold exposure or

norepinephrine in rats, which reinforces significance in regulation of blood glucose

homeostasis (Liu, et al. 1994; Shibata, et al. 1989). Brown adipocytes express α1, α2, β1 and

β3-adrenergic receptors (Lafontan and Berlan 1993). The effect of norepinephrine on this

tissue is mainly mediated by the β3-adrenergic receptor, although α1-adrenergic receptors may

be of some importance (Zhao, et al. 1997). Norepinephrine-stimulated glucose uptake is

mediated by the β3-adrenergic receptor subtype in brown adipocytes, but α1- and β1-

adrenergic receptors can compensate for β3-adrenergic receptor deficiency in β3-KO models

(Chernogubova, et al. 2004; Chernogubova, et al. 2005). The response is mimicked by

stimulation with 8-Br cAMP (cAMP analog) and diminished by 4-cyano-3-

methylisoquinoline (4CM, PKA inhibitor), which indicates the involvement of classical β-

adrenergic signaling through Gsα, cAMP and PKA. The observed sensitivity of β-

adrenergically and 8-Br-cAMP stimulated glucose uptake to PI3K inhibitor LY294002

suggests the involvement of PI3Ks or related kinases. An interpretation of this observation

could be that β3-adrenergic and insulin signaling pathways may converge at the level of PI3K,

with GLUT4 translocation possibly being responsible for both increases in glucose uptake. In

white adipocytes, β3-adrenergic agonists inhibit insulin stimulated glucose uptake, but this is

not observed in brown adipocytes (Carpene, et al. 1993). To further elucidate the mechanism

Figure 9. Glucose uptake in response to the adrenergic natural ligand norepinephrine and

insulin during 0-8 hours of stimulation, in brown adipocyte primary cultures.

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of norepinephrine stimulated glucose uptake in brown adipocytes and address the question of

convergence between insulin and β3-adrenergic signaling, I studied the effects of

norepinephrine on GLUT1 and GLUT4 in brown adipocytes (Paper I). Initially we observed a

dramatic effect of norepinephrine on GLUT1 and GLUT4 mRNA transcription, stimulating a

potent 8-fold increase of GLUT1 and a surprising 2-fold decrease of GLUT4 mRNA levels.

The effect of norepinephrine GLUT expression is mediated by the classical β3-adrenergic

signaling pathway with the second messenger cAMP. Fractionation and analysis of protein

levels of primary brown adipocytes after norepinephrine stimulation for 0, 2 and 5 h show an

increase of GLUT1 protein in the plasma membrane fraction and low density microsomes,

which is consistent with a de novo synthesis of GLUT1. The dramatic increase of GLUT1

mRNA and protein corresponds to a very high glucose uptake at 5 hours that exceeds insulin

stimulated uptake in primary brown adipocytes (Figure 9). Interestingly, when we

investigated the transcriptional influence of GLUT1 on norepinephrine stimulated glucose

uptake, actinomycin D did not completely block the response. Our results indicate the

existence of two separate mechanisms of norepinephrine stimulated glucose uptake, a faster

actinomycin D insensitive mechanism and a slower actinomycin sensitive mechansism. The

second mechanism is actinomycin sensitive due to the de novo synthesis of GLUT1, but the

first mechanism is still elusive. The first mechanism may be due to a translocation of another

GLUT or a change in intrinsic activity of a GLUT. We find no evidence that GLUT4

translocation occurs, but changes in GLUT1 intrinsic activity in response to norepinephrine

stimulation has been proposed earlier (Shimizu, et al. 1996; Shimizu, et al. 1998). To keep

brown adipose tissue primaries viable 4 nM insulin is added to the culture media and this is

sufficient to promote some GLUT4 translocation. Norepinephrine stimulation seems to

antagonize insulin signaling and cause retention of GLUT4 protein from the plasma

membrane to an intracellular location (Figure 10). This observation further supports the

Figure 10. Subcellular distribution of GLUT4

and GLUT1 with norepinephrine-stimulation.

Isolated brown adipocytes stimulated with 0.1

M norepinephrine for 0, 2, or 5 hours. 10 g

of protein from plasma membrane (PM) or

low-density microsome (LDM) fractions was

subjected to SDS-PAGE and immunoblotting

with GLUT4, GLUT1, 3-AR, or ERK 1/2

antibodies.

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40

apparent differences between insulin and β-adrenergic signaling to glucose uptake.

Insulin and β-adrenergic signaling do not converge to regulate glucose transporters in a

similar way. However, enigmatically not only insulin, but also β-adrenergically stimulated

glucose uptake is inhibited by PI3K inhibitors LY294002 and wortmannin. The possible

involvement of PI3K family members indicated by the ability of these PI3K inhibitors to

inhibit β-adrenergic glucose uptake could be attributed to an unspecific effect. This notion

seems unlikely as the very potent and specific inhibitor PI-103, completely inhibits both

insulin and β-adrenergic signaling in brown adipocyte primary cultures (Paper II). PI-103

selectively targets PI3K-P110 PI3KC2β, PI3K related kinases (PIKKs) rapamycin-sensitive

mTORC1 and rapamycin-insensitive mTORC2 mTOR complexes, and DNA protein kinase

(DNA-PK) (Knight, et al. 2006). Its high selectivity is indicated by the absence of activity

toward a panel of 70 protein kinases (Raynaud, et al. 2007). Furthermore, the specific

mTORC1 inhibitor rapamycin does not affect insulin or β-adrenergically stimulated glucose

uptake. Class I PI3K activation seems to universally cause phosphorylation of Akt t308 and

s473. Intriguingly, 2 hours stimulation with isoproterenol induced a significant

phosphorylation of Akt residue s473, but not t308. Several studies suggest both DNA-PK and

mTORC2 specifically phosphorylates Akt on this residue (Hresko and Mueckler 2005;

Sarbassov, et al. 2005; Bozulic, et al. 2008; Feng, et al. 2004).

In conclusion, β-adrenergic stimulation causes a potent increase of glucose uptake in

brown adipocytes consisting of two mechanisms. Mechanism 1 is not due to transcriptional

activation or translocation of GLUT1 or GLUT4, and mechanism 2 is through a de novo

synthesis of GLUT1. It also seems unlikely that β-adrenergic and insulin signaling converge,

but seem to be completely separate signaling pathways. Our data suggest the involvement of a

PI3K related kinase.

4.2.1. AMP-activated protein kinase (AMPK)

AMP-activated protein kinase (AMPK) is considered to be an energy sensor, responding to

changes in cellular energy levels. Although ubiquitously found, its high expression levels in

metabolically important tissues as skeletal muscle, adipose tissue, liver, heart, pancreas, and

brain, indicates its importance in energy homeostasis. Low levels of energy activate AMPK to

increase glucose uptake, feeding, fatty acid oxidation, and inhibit lipogenesis, all processes

increasing available energy and restoring homeostasis. AMPK consists of a catalytic α

subunit, and regulatory β and subunits, and is allosterically activated by AMP by

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phosphorylation on threonine 172, when energy levels are decreasing in the cell (Kahn, et al.

2005). Recently, it has become appreciated that AMPK can be activated by GPCRs, including

β-adrenergic receptors (Hutchinson, et al. 2008). The β-adrenergic agonist isoproterenol

stimulates AMPK activity in adipocytes (Moule and Denton 1998; Yin, et al. 2003), and it has

been proposed that in brown adipocytes, serial activation of UCP1 and AMPK is necessary

for glucose utilization (Inokuma, et al. 2005). However, we show that noradrenaline, acting

via β-adrenergic receptors, increases AMPK phosphorylation independently of UCP1

expression and function (Paper III). The activation of AMPK is partially responsible for the β-

adrenergically stimulated glucose uptake in brown adipocytes.

4.3. In white adipose tissue

White adipose tissue (WAT) is the main organ for fuel storage when energy intake exceeds

energy expenditure in mammals. Fatty acids can be synthesized or taken up, converted to

triacylglycerol, and stored in lipid droplets. It is also a target for postprandial increases in

glucose uptake in response to insulin stimulation. BAT is more innervated by sympathetic

nerves then WAT, though SNS is presumed to have a significant role in WAT as well (Slavin

and Ballard 1978; Fliers, et al. 2003; Garofalo, et al. 1996). The SNS acts directly via

norepinephrine on β-adrenergic receptors in WAT (for a recent review, see (Collins, et al.

2004). Fasting or sustained physical activity can trigger sympathetic activation in WAT,

promoting catabolic processes and fuel mobilization through increased lipolysis and

triglyceride release, but lipolysis during fasting is not exclusively regulated through β-

adrenergic receptors (Jimenez, et al. 2002). There is a clear role for the SNS as a regulator of

lipolysis, and a more elusive role in the regulation of glucose transport in WAT. Infusion of

norepinephrine for 10-12 days increase glucose uptake independently of insulin, and chronic

administration of the selective β3-agonist CL-316243 seems to increase both basal and

insulin-stimulated glucose uptake in WAT (Liu, et al. 1994; de Souza, et al. 1997; Liu, et al.

1998). However, most evidence indicates an antagonistic effect of acute β3-adrenergic

receptor activation on insulin signaling. β-adrenergic agonists inhibit insulin stimulated

glucose uptake and GLUT4 translocation in rat adipocytes and 3T3-L1 adipocytes via the β3-

adrenergic receptor (Carpene, et al. 1993; Ohsaka, et al. 1997; Mulder, et al. 2005), an

observation we also have seen in mouse mature white adipocytes (Nevzorova, unpublished

observations). The inhibition of insulin signaling has also been shown in human primary

cultures of mammary adipocytes, although insulin stimulated glucose uptake was not altered

(Jost, et al. 2005). In conclusion, the effects of the sympathetic nervous system on glucose

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transport in WAT are primarily mediated by the β3-adrenergic receptor subtype. Longer

exposures to norepinephrine or β-adrenergic agonists have positive effects on glucose uptake,

and shorter exposures does not increase glucose uptake. In fact, it is possible that acute effects

of the sympathetic nervous system negatively influences insulin signaling and glucose uptake.

4.4. In skeletal muscle

It seems evident the sympathetic nervous system can regulate glucose transport in skeletal

muscle in an insulin-independent way. Cold acclimation can increase glucose uptake into

skeletal muscle without shivering thermogenesis or contractile activity, suggesting

involvement of sympathetic nerves (Vallerand, et al. 1990). Central neurochemical

stimulation or electrical stimulation of the VMH in rats increase glucose uptake in skeletal

muscle without elevating plasma insulin concentrations (Sudo, et al. 1991; Minokoshi, et al.

1994; Lang, et al. 1995). This effect is abolished by the norepinephrine-release inhibitor

guanethidine, but not by adrenal demedullation, thus indicating that the response is mediated

by norepinephrine and not circulating epinephrine (Minokoshi, et al. 1994). Furthermore,

administering the β2-AR agonist clenbuterol improves glucose tolerance due to increased

glucose uptake in skeletal muscle (Castle, et al. 2001; Pan, et al. 2001). This may partially be

explained as clenbuterol has a hypertrophic effect on skeletal muscle and an increase in the

actual mass will correspond to the increased glucose uptake (Yang and McElligott 1989).

Most studies on glucose transport effects with β-agonists in rodents are long term, and do not

investigate possible acute short term effects.

Skeletal muscle express high levels of the β2-adrenergic receptor subtype and low levels of

the β1-adrenergic receptor subtype, and the effects of endogenous catecholamines as well as

adrenergic agonists are primarily mediated by the β2-adrenergic receptor (Liggett, et al. 1988;

Roberts and Summers 1998; Nevzorova, et al. 2002). A significant body of evidence now

suggests the existence of muscle glucose uptake in response to sympathetic nerve activation

and focal noradrenaline release, and this is mediated by the β2-adrenergic receptor. The β2-

adrenergic receptors in skeletal muscle mediates the conventional β-adrenergic signaling,

activation of adenylyl cyclase, production of cAMP and activation of PKA, resulting in many

metabolic effects. Selective β-adrenergic agonists and 8-Br-cAMP increase glucose uptake in

L6 myotubes, but only the former is inhibited by wortmannin and LY294002 (Nevzorova, et

al. 2002; Nevzorova, et al. 2006). These compounds are inhibitors of the PI3K family,

suggesting there is an atypical activation of PI3K in the β-adrenergic response, not mediated

by cAMP. Investigating this possibility with more specific PI3K-P110 catalytic subunit

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43

inhibitors, we believe class I PI3Ks are not involved in β-adrenergically stimulated glucose

uptake (paper IV). Although there seems to be distinct differences between insulin and β2-

adrenergically stimulated glucose uptake in L6 myotubes, we also investigated the regulation

of glucose transport by the β2-adrenergic receptor in CHO cells stably expressing

GLUT4myc, and transiently transfected with β2-adrenergic receptors (Paper V). In this

artificial system, β2-adrenergic receptor activation seems to cause GLUT4 translocation and

increase of glucose uptake, possibly by atypical signaling through the desensitization

mechanism.

Interestingly, the dual mTOR/PI3K inhibitor PI-103 abolishes not only glucose uptake in

response to insulin, but also in response to the β-adrenergic agonist isoproterenol in L6

myotubes. The inhibitory profile of PI-103 suggest the involvement of mTOR1C, mTOR2C,

PI3KC2β, or DNA-PK. Pretreatment with the mTOR1C inhibitor rapamycin fails to inhibit

glucose uptake in response to isoproterenol indicating mTOR1C is not a factor in the β-

adrenergic signaling. Class II PI3KC2β is not a plausible candidate to explain the LY294002

inhibition of β-adrenergically stimulated glucose uptake, as it is relatively insensitive to this

inhibitor, with reported IC50 values of up to 30 µM (Falasca and Maffucci 2007). These

results suggest that a distinct member of the PIKK family is crucial to β2-adrenergically

stimulated glucose uptake.

5. SUMMARY AND CONCLUSIONS

β-adrenergic receptors are among the most ubiquitously expressed receptors, responsible

for mediating the effects of the sympathetic nervous system in many tissues, focally as well as

to circulating catecholamines. They are important in regulating vital functions in the body,

heartbeat and blood flow, dilation of airways, and mobilizing energy from liver and adipose

tissues. Although their existence was shown more than 50 years ago, there are constantly new

discoveries changing the way we consider β-adrenergic receptor regulation and signaling. It

has also been known for quite some time, that β-adrenergic stimulation can regulate glucose

uptake in target tissues, but there is not much known about the signaling and regulation in this

respect.

In this thesis, I have reviewed and compiled an overlook of the field of glucose transporters

and β-adrenergic receptors, and how they are connected. I (and my co-authors) show that β-

adrenergic receptor stimulation activates distinct signaling pathways, regulating glucose

transporters mechanisms, ultimately altering glucose transport into cells. We are also able to

show that this occurs in insulin sensitive tissues, but separately from insulin signaling.

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44

Paper I describes the mechanism behind norepinephrines potent effect on glucose uptake in

brown adipocytes. We show that norepinephrine acting on β3-adrenergic receptors induces a

markedly increased glucose uptake in brown adipocyte primary cultures, which can be

separated into two distinct mechanisms. First, an acute mechanism that is resistant to

actinomycin D (transcriptional blocker), and is not due to GLUT4 or GLUT1 translocation.

Secondly, there is a slower actinomycin D sensitive mechanism caused by transcriptional

activation and protein synthesis of GLUT1. Furthermore, we also conclude that β-

adrenergically and insulin stimulated glucose uptake are produced by completely different

mechanisms, and that β-adrenergic stimulation in fact antagonizes insulin dependent GLUT4

translocation.

Paper II concerns the previously observed sensitivity of β-adrenergically stimulated

glucose uptake to PI3K inhibitors, and if this is correlated with the different signaling

properties of the two β-adrenergic receptor subtypes (β1- and β3-adrenergic receptors)

expressed in brown adipocytes. We show that although β3- and β3-adrenergic receptors differ

in the production and desensitization of cAMP, glucose uptake stimulated by both receptor

subtypes is sensitive to the pan-specific (does not discriminate between isoforms) PI3K

family inhibitor LY294002. As the mechanism of desensitization is involved in mediating

atypical signaling, and the β1- but probably not the β3-adrenergic receptor recruits this

mechanism, we conclude that this type of atypical signaling from β-adrenergic receptors is not

involved in mediating glucose uptake. By investigating the sensitivity of β-adrenergically

stimulated glucose uptake to LY294002 with several PI3K isoform selective inhibitors, we

show that insulin activated class I PI3Ks are not involved in β-adrenergically stimulated

glucose uptake, but is most likely dependent on a PIKK.

Paper III show that noradrenaline acts on β3-adrenergic receptors to activate AMPK,

independently of UCP1 expression and function in brown adipocytes. This activation partially

contributes to β-adrenergically stimulated glucose uptake, and is likely mediated by cAMP.

Paper IV reveals the differences between insulin and β-adrenergic signaling to glucose

uptake in L6 myotubes, and identifies possible candidates in the family of PI3K related

kinases (PIKKs) crucial to β-adrenergically stimulated glucose uptake. By using PI3K

isoform selective inhibitors, we conclude that class I PI3Ks does not participate in β-

adrenergically stimulated glucose uptake, and does not explain the high sensitivity of β-

adrenergically stimulated glucose uptake to PI3K inhibitor LY294002. However, we show

that a novel inhibitor with a highly selective profile for class I PI3Ks, and members of the

PIKK family, inhibits both insulin and β-adrenergically stimulated glucose uptake. We

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conclude that the increase of glucose uptake in response to β2-adrenergic receptor stimulation

is dependent on a member of the PIKK family, in L6 myotubes.

Paper V deal with the mechanism of β-adrenergically stimulated glucose uptake in an

artificially constructed system, and investigate the importance of the C-terminal tail of β-

adrenergic receptors in this response C-terminal. In this constructed system, consisting of

Chinese hamster ovary (CHO) cells stably expressing GLUT4myc, and transiently transfected

with the β2-receptor, not only insulin, but also isoproterenol stimulation is able to induce

GLUT4 translocation resulting in an increase in glucose uptake. Transfecting these cells with

β2-receptors with a truncated C-terminal tail abolishes the β-adrenergically stimulated glucose

uptake, without affecting production of cAMP. This indicates that the C-terminal part of the

receptor is important for the glucose uptake observed in this system, possibly due to atypical

signaling.

Paper VI investigates the possibility that β-adrenergic receptors can modulate the intrinsic

activity of GLUT1 in L6 myotubes. Adopting a new approach of introducing cell penetrating

peptides that are homolugues to parts of the intracellular sequences of GLUT1 to compete for

proteins that interact with these sequences, reveal the possibility that a β-adrenergic signaling

pathway may be able not only to increase GLUT1 intrinsic activity as suggested in brown

adipocytes, but also decrease it. We show that introducing a cell penetrating peptide

homologues to a specific sequence in the large intracellular loop of GLUT1 augments β-

adrenergically stimulate glucose uptake, suggesting such a mechanism exists in L6 myotubes.

In conclusion of these studies, it is evident that β-adrenergic receptors are able to regulate

glucose transporters in insulin sensitive tissues, such as skeletal muscle and adipose tissues.

This is achieved in distinct mechanisms and separate from insulin signaling. Understanding

the complex events in this physiological response could further our knowledge of β-

adrenergic signaling and the regulation of glucose transporters, which may provide valuable

information in trying to understand diseases such as diabetes mellitus.

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6. ACKNOWLEDGEMENTS

So many people has contributed to and supported the realization of this thesis, and I would like to thank all of

you. I hope I remember to mention you all.

First of all I would like to thank my supervisor Tore Bengtsson. What a journey! Through ups and downs

you have been there with “Nu kör vi!!”, and a never ending enthusiasm for our own small world of science. If we

would only remember some of the weird ideas and strange theories we came up with during those afternoons we

were half awake. Thank you for backing me up so many times and letting me freely explore my own ideas. You

have always been, and will continue to be, a source of inspiration for me.

My research group (the GLUT group), past and present members and collaborators

Dana Hutchinson, the aussie girl that taught me so many things in, as well as outside (drinking-lessons) the

lab. We had a great time when you where post-doc at “zoofys”. My visit to Australia was super, I learnt so much

during those three months, and I hope I will return some time. Thank you everyone else at Monash University,

Molecular Pharmacology too. Professor Roger J. Summers and Sybil, thank you for always taking time to give

some advice, for great hospitality, and for introducing me to the mighty “Buxton burger”. Masato (Masa) for

great times in the lab, lots of laughter, and a few good beers. Mitch, Emma, Ree, and everyone else in the lab,

thank you! And to Rob, I hope the tigers will do better. I´m sorry, is still like the magpies.

Ekaterina “Katja” Chernogubova, my superb brown fat glucose uptake teacher. I wish you the best of luck

in work and life. You always helped me whenever there were questions, and never hesitated to give me a hand.

May the strange canned fish you always bring, and the vodka, be plenty.

Julia Nevzorova, we had a lot of fun! You don’t really hold your liquor well, compared to other Russians.

Maybe that’s what you would expect when you have a group-meeting on a boat to Finland? I wish you the best

of luck!

Daniel Yamamoto, my lab-bench neighbor with only black clothes, and party friend. We had lots of fun (and

consumed a lot of drinks!), and I have the scars to show for it. I will miss the pointless practical pranks and dirty

jokes. Good luck with your research in Italy!

Robert Csikasz, the true connoisseur that does not really settle for second best. You will shoulder the

responsibility of being the most experienced PhD student in our group. I’m sure you will cope with this task

well, with some help from extremely rare and expensive beers and “tryffelolja”. When will we ever attempt the

“lion-club”(24 beers in 6 hours)?

Anette “Nettan” Östberg, the “new kid on the block”. I trust you will shoulder the important GLUT

research with grace, and most importantly, continue the “kiss-labben” saga. I know you will do well, but please,

don’t forget to breathe when you are laughing.

Claudia Böhme, our visiting student from the alps! No worries, your glucose uptake experiments will soon

be publishing quality (or I might help you).

All the people at physiology

Professors Barbara Cannon and Jan Nedergaard, thank you for your guidance, support and brilliant

Christmas dinners! I will truly miss the Christmas pudding.

Professor Anders Jacobsson, thank you for making scientific discussions very philosophical and dreamy. It

sort of added an extra dimension to whatever was being discussed. And for your brilliant crayfish parties!

Damir Zadravec, the PUFA wizard. Will never ever forget the great Irish-coffe nights we had at gärdet, a

tradition that must go on! Will never forget the tupplapukki-evening at “zoofys” (unfortunately there is evidently

picture evidence), that was truly a party. You have been a great friend at “zoofys”, and talking to you made all

the difference when things were not going my way. And don´t forget, you can always get “snus” from me!

Charlotte Mattson, thank you for always being a good friend since we started at the University and during

our time at NF, for always caring, and always baking cakes. One of those cakes saved this thesis!

My original roommates, Therese Holmström, Annelie Brolinson, and Johanna Jörgensen. We had a lot of

serious discussions, and we talked about pretty much everything. Will never forget the conversations about

babies and various aspects of male/female physiology I was forced to have. Perhaps I was forced to recognize

my feminine sides, and if not, I recognized yours (candy-cravings).

Katarina Lennström, missed you when you left! You are a great friend to talk to when it is really needed,

and a good friend.

Emma Backlund, you are a good friend and without competition the funniest person when drunk. There are

many quotes that could be listed here, but they are simply not suited for a doctoral thesis.

Andreas Jacobsson, we had some really great moments. I think it was a brilliant idea to see if we could fold

Annelie together and put her in a waste basket, it actually worked!

Ida Östlund, I think you will be married, soon. You planned it well (for at least a year during our coffee

breaks), now you just have execute the plan! Furthermore, I think you look good in the dirndl!

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Tomas Waldén, the man with the funniest excuse ever, “jag är ju från Fittja”. Good luck with the

“kiss/kidney physiology” lectures, you will do well.

Birgitta Leksell, extra mom and caring lab assistant. You have always been supportive and I am very

thankful. When will I get to play the bagpipe in your garden?

Helene Allbrink, you knew how to keep me in order, I missed you when you left. And you are yet another

person that is very funny after a couple of drinks. I think twice before I get into a discussion with you then.

And everyone else, Helena Feldmann, Natasa Petrovic, Yanling Wang, Victor Sabanov, Irina Sabanova,

Tatiana Kramarova, Ludmilla Kramarova, Irina Mona Wilcke, Irina Shabalina, Anna-Lena Järva, Natalia

Gibanova, Veronika Hruschka.

I also want to express my gratitude to some persons outside the department.

Kerstin Brismar, you gave me the chance to work during one summer at your lab, and that meant a lot. You

have been so very supportive and encouraging since then. Thank you for all your help!

Karl “Kalle” Lindberg, friend since way back during the “KM”-days at the University. Mats Bergström, a

very good friend since growing up in Bollnäs. The friends I have shared many nights with working as

security/ordningsvakt. Fredrik Lindberg and Tomas Wiik, friends since way back as subway security. The

cheerful people I worked with at the Dubliner, Janne, Bolme, Herr Rodhe, Glavare, Lelle, Denis, Emelie.

The Thistle Pipe Band and all its members, the traditional military style Pipes and Drums band where I

learnt to play bagpipe and still do. Pergite!

My climbing gang, Magnus Carlebjörk, Roland Revsäter och Michael Wåhlin. We have climed ice and

rock in Sweden, and all over the world, for nearly 10 years now. I hope we still have a lot of impressive routes,

breathtaking peaks, clear water fall ice, solid rock, and really, really bad jokes ahead of us.

The friends from molecular biology h98, NF, and spexet. Åse och Johan, Benita, Anders, Slavena, André

och Lina (André and me used to read Conan comics during general chemistry lectures), Dan and Susanne,

David Nord and Linda, and everyone else.

Last, but definitely not least, I want to thank the closest of my family and loved ones. This was truly not

possible without their love and support. My father Gustav Dallner, who helped me in every possible way in my

work. Some scientific advice, dinners with a lot of food, lots of red wine (for medicinal purposes), laughter, and

occasionally really breathtaking and “exciting” rides with an old red Volvo through the streets of Stockholm. My

mother Britt Herlin Dallner, who always worries about me, making sure I have all that I need, supported me

when life was hard, and loved me despite all the crazy things I’ve come up with. My big brother Björn “Nalle”

Dallner (and Jonna), who always supported me, and listened when it was needed. I know I can rely on you when

things are tough and help is needed. My girlfriend Linda, who supported me immensely during the last

gruesome part of writing this thesis. Showing nothing but love and caring when it was really needed. Puss min

älskling!

Thank you all!

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