ENGAGEMENT OF STIMULATION OF GLUCOSE a-LIPOIC ACID. · ENGAGEMENT OF THE INSULIN-SENSITIVE PATHWAY IN THE STIMULATION OF GLUCOSE TRANSPORT BY a-LIPOIC ACID. A MeSc. thesis by Karen

ENGAGEMENT OF THE INSULIN-SENSITIVE PATHWAY IN THE

STIMULATION OF GLUCOSE TRANSPORT BY a-LIPOIC ACID.

Karen Lynne Yaworsky

A M. Sc. thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Biochemistry

University of Toronto

O Copyright by Karen Yaworsky 1999

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STIMULATION OF GLUCOSE TRANSPORT BY a-LIPOIC ACID.

A MeSc. thesis by Karen Lynne Yaworsky submitted in conformity with the

requirements for the degree of Master of Science, Graduate Department of

Biochemistry, University of Toronto, 1999.

ABSTRACT

A pnmary metabolic response to insulin is the acute stimulation of glucose transport in

muscle and adipose tissue. Activation of an intracellular signalling cascade by insulin results in

recruitment of glucose transporters to the plasma membrane; a process impaired in type 2 diabetes.

In search of other relevant insulin-mimetic agents our attention has focused on a-lipoic acid. a-

Lipoic acid, a cofactor of oxidative rnetabolism and a potent antioxidant, was shown to enhance

insulin-stimulated glucose rnetabolism and to stimulate glucose transport. Therefore, in an attempt

to undentand the mechanism underlying the stimulation of glucose transport by a-lipoic acid, the

effect of this compound on glucose transporter localization and intracellular signals involved in the

stimulation of glucose transport were examined. The results presented in this thesis suggest that

a-lipoic acid stimulates glucose transport in a unique manner, by directly targetting elements of the

insulin-sensitive signalling pathway.

The work presented in this M.Sc. thesis was perfonned from 1997- 1999 in the Programme

in Ce11 Biology, The Hospital for Sick Children, Toronto, ON, Canada, under the

supervision of Dr. Amira Klip. Financial support was provided by the Canadian Diabetes

Association and ASTA Medica, Germany.

The results of this Thesis have been presented in one publication:

K. Yaworsky, R. Somwar, T. Ramlal, H.J. Triischler, and A. Klip. 1999. Unique action of an

anti-diabetic agent: Engagement of the insulin-sensitive pathway in the stimulation of glucose

ansport by a-lipoic acid. Diabetologia -- Submitted. (Chapter one)

ACKNOWLEDGMENTS

A mere note of thanks cannot justifiably express my profound gratitude to everyone

who has helped me with this degree. To begin with, 1 would like to thank the members of

the Klip lab, past and present, for making the everyday so enjoyable. Special thanks to my

'big sister' Celia for continual support; Leonard, for your patience when explaining how a

computer actually works; Toolsie, for laughter and for always having a solution; and to

Rome1 for answering my endless questions. 1 would also like to thank the rnembers of my

advisory committee and especially rny supervisor, Amira Klip. Arnim's enthusiasm,

generosity and continual support are unparalleled. Lastly, to my greatest supporters,

Jonathan, Dodie, Kim and Alberta - words cannot even begin to express my sincere

appreciation. 1 really could not have achieved this without you.

TABLE OF CONTENTS

A B S T R A C T ................................................................................... I I

PREFACE ..................................................................................... III

A C K N O W L E D G M E N T S ................................................................. IV

................................................................. TABLE OF CONTENTS V

......................................................................... LIST OF TABLES IX

....................................................................... LIST OF FIGURES X

........................................................... LIST OF ABBREVIATIONS XII

BACKGROUND ............................................................................... 1

..................................................................... TYPE 2 DIABETES 1

............................................. BIOLOGICAL ACTIONS OF WSULIN 4

Glucose Transport And Transporters ......................................... 4

Structure. Function and Tissue-Specific Expression of Glucose

Transporter Isoforms .................................................. 6 GLUT 1 ......................................................... 6

GLUT4 ......................................................... 6 Acute Regulation of Glucose Transport by Insulin ................. 9

SIGNALLING MECHANISMS REGULATING INSULIN-STIMULATED GLUCOSE TRANSPORT .............................................................. 10

The Insulin Receptor ............................................................ 12

Insulin Receptor Subsuate Proteins ........................................... 15

Insulin Receptor Substrate- 1 (IRS- 1) ................................ 16 lnsulin Receptor Substrate-2 (ES-2) ................................ 20

................................ Insulin Receptor Substrate-3 (IRS-3) 21

Insulin Receptor Substrate-4 (IRS-4) ................................ 21 Role of lnsulin Receptor Substrate Proteins in Insulin-Stimulated Glucose Transport ...................................................... 22

Phosphatidylinositol 3-Kinase (PI 3-kinase) ................................. 24 Pharmacological Inhibitors of PI 3-Kinase .......................... 26

The Role of PI 3-kinase in Insulin-Regulated Glucose Transport . 27 Signals Downstream of PI 3-kinase: lnvolvement of Senne and Threonine

Kinases ........................................................................... 30 ...................................................................... Akt -30

Role of Akt in the Stimulation of Glucose Transport by

......................................................... Insulin -35

Atypical PKC's: Emerging Role in Insulin-Stimulated Glucose

................................................................ Transport 36

GLUCOSE TRANSPORTERS IN TYPE 2 DIABETES ........................... 39 ............................................................ GLUT4 Expression -39

.......................................................... GLUT4 Translocation 41

................................................................ Insulin Signalling 41

Factors That May Trigger Insulin Resistance ............................... $43

Anti-Diabetic Drugs: Potential nierapies for Insulin Resistance and Type 2

Diabetes ........................................................................... 46 .......................................................... a-Lipoic Acid -47

CHAPTER ONE ................................................................................ 52 RATION ALE AND HYPOTHESIS ................................................... 53

EXPERIMENTAL PROCEDURES ................................................... 55 .......................................................................... Materials 55

Methods ......................................................................... -56 .......................................... Cell Culture and Incubations 56

2 - ~ e o x ~ - 3 ~ - ~ - ~ l u c o s e Uptake ...................................... 56 SubcelluIar Fractionation of 3T3-L1 Adipocytes ................... 57 Immunoprecipitation and Assay of Phosphatidylinositol 3-Kinase

.................................................................. Activity 57

Immunoprecipitation and Assay of Aktl Protein Kinase Activity . 58 ....... Detection of Insulin Receptor Substrate- 1 Phosphorylation 59

Detection of Insulin Receptor Phosphorylation ..................... 60 Insulin Receptor Autophosphory lation .............................. 60

..................................................... Statistical Analysis 61

................................................................................ RESULTS -62 a-Lipoic Acid Stimulates Glucose Uptake in 3T3-L1 Adipocytes ....... -62 The Effeci of a-Lipoic acid on Insulin-Stimulated Glucose Uptake in 3T3- Ll Adipocytes ................................................................... 64

a-Lipoic Acid Stimulates the Translocation of GLUTl and GLUT4 to the

............................................................... Plasma Membrane 66 Wortmannin Prevents the Stimulation of Glucose Transport by cc-Lipoic

................................................... Acid in 3T3-L1 Adipocytes -69 Activation of PI 3-Kinase by a-Lipoic Acid in 3T3-L 1 Adipocytes ...... 71

.......... Effect of a-Lipoic Acid on Akt 1 Activity in 3T3-L1 Adipocytes 73 Effect of Erbstatin on a-Lipoic Acid-Stimulated Glucose Uptake in 3T3-LI

...................................................................... Adipocytes -75 Effect of a-Lipoic Acid on ES- 1 Phosphorylation in 3T3-L 1 Adipocytes.77

Induction of Tyrosine Phosphorylation of the Insulin Receptor by a-Lipoic

.............................................................. Acid in Intact Cells 79 Effect of a-Lipoic Acid on Phosphorylation of the Insulin Receptor In

............................................................................... Vitro 81

DISCUSSION ........................................................................... -83 a) Action of a-Lipoic Acid on Glucose Transporters and Glucose Transport

.................................................................................... -83

....... b) Effect of a-Lipoic Acid on Lipid and Sennemireonine Kinases 84

c) Effect of a-lipoic Acid on Tyrosine Kinases ............................. 85 d) Possible Effects of a-Lipoic Acid on Protein Tyrosine Phosphatases . 88

e) Does a-Lipoic Acid Function as an Antioxidant? ........................ 90

0 Does a-Lipoic Acid Affect Glucose Uptake Through a Metabolic Action

................................. on the Pyruvate Dehydrogenase Cornplex? -90

g) Concluding Remarks ....................................................... -94

FUTURE DIRECTIONS ................................................................ 96 ..................................................................................... APPENDIX -98

RATIONALE/HYPOTHESIS ......................................................... -99 ................................................................................ METHODS 104

...................................................................... Cell Culture lû4

Total Membrane Reparation and Immunoblotting .......................... 104

Recombinant Fusion Proteins .................................................. 105 . . ........................................................ In vitro Binding Assays 105

Lysate Preparation and Anti-Phosphotyrosine Immunoprecipitation ...... 106 .............................................................. RESULTS/DISCUSSION 107

Expression and Subcellular Distribution of Hw-2 in L6 Muscle Cells and

.............................................................. 3T3-L 1 Adipocytes 107 In Vitro Binding of Recombinant SNAP25 and SNAP23 to Hrs-2 ....... 110

Phosphorylation of Hrs-2 in L6 Myotubes in Response to Insulin ....... 1 12 ............................................................ Concluding Remarks 114

................................................................................. REFERENCES 115

LIST OF TABLES

BACKGROUND

Table B . 1 Mammalian Facilitative Glucose Transporters: Major Sites of

.................................... Expression and Physiological Functions 8

.......... Table 8.2 Classification of Phosphatidylinosiiol (PI) 3-Kinase Members 25

Table B.3 Anti-Diabetic Drugs: Mechanisms and Sites of Action ..................... 46

APPENDIX

Table A . 1 Functiond Characteristics of Rat Hrs.2. Mouse Hrs. and

..................................................................... Human Hrs 102

BACKGROUND . ............ Figure B.1 . Metabolic Actions of Insulin: Relationship to Type 2 Diabetes 3

Figure B.2. The Insulin Signalling Cascade ................................................ 11

Figure B.3. The Insulin Receptor ............................................................ 13

Figure BA . Structural Features of Insulin Receptor Substrate-1 (IRS-1) ............... 17

.......... Figure B.5. Activation of Akt/PKB by PI 3-Kinase-Dependent Mechanisms 34

Figure B.6. The Insulin Signalling Cascade: Proposed Signals Necessary for

........................................ Insulin-Stimulated Glucose Transport 38

Figure B.7. Schematic Representation of a-Lipoic Acid. Dihydrolipoic Acid

..................................................... and the Reduction Process 48

CHAPTER ONE . ......... Figure 1.1. a-Lipoic Acid S tirnulates Glucose Uptake in 3T3-L 1 Adipocytes 63

Figure 1.2. The Effect of a-Lipoic Acid on Insulin-Stimulated Glucose Uptake

in 3T3-L1 Adipocytes .......................................................... 65

Figure 1 3 . a-Lipoic Acid Stimulates the Translocation of GLUTl and GLUT4

to the Plasma Membrane ....................................................... 67

Figure 1.4. Wortmannin Prevents the Stimulation of Glucose Transport by

a-Lipoic Acid in 3T3-L1 Adipocytes ......................................... 70

Figure 1.5. Activation of PI 3-Kinase by a-Lipoic Acid in 3T3-Ll Adipocytes ....... 72

Figure 1.6. Effect of a-Lipoic Acid on Akt 1 Activity in 3T3-L 1 Adipocytes .......... 74

Figure 1.7. Effecr of Erbstatin on a-Lipoic Acid Stimulated Glucose Uptake

in 3T3-L1 Adipocytes .......................................................... 76

Figure 1.8. Effect of a-lipoic Acid on IRS-1 Phosphorylation in 3T3-L1

Adipocytes ...................................................................... -78

Fipre 1.9. Induction of Tyrosine Phosphorylation of the Insulin Receptor

by a-Lipoic acid in Intact Cells ............................................... 80

Figure 1.10. Effect of a-Lipoic Acid on Phosphorylation of the Insulin

Receptor In Vitro ................................................................ 82

Figure 1.11. The Signalling Pathway of a-Lipoic Acid ................................... 87

Fipre 1.12. Relationship of the Pyruvate Dehydrogenase Complex

Reaction and Glucose Metabolism ........................................... -92

Figure 1.13. a-Lipoic Acid: Potential Mechanisms of Action ............................ 95

APPENDIX

Figure A.1. Schematic Structures of Mouse Hrs. Hurnan Hrs.

and Rat Hrs-2 .................................................................... 101

Figure A.2. Schematic Mode1 of the Proposed Involvernent of

Hrs-2 in Insulin-Regulated Vesicle Traffic ................................... 103

Figure A.3. Expression and Subcellular Distribution of Hrs-2 in L6 Muscle Cells .... 108

Fipre A.4. Expression and Subcellular Distribution of Hrs-2 in

.............................................................. 3T3-L 1 Adipocytes 109

Figure A.5. In Vitro Binding of Recombinant SNAP25 and SNAP23 to Hrs-2 ........ 111

Figure A.6. Phosphorylation of Hrs-2 in L6 Myotubes in Response to Insulin ........ 113

LIST OF ABBREVIATIONS

2DG

a-MEM

ATP

CHO

cDNA

DMEM

EGF

FBS

FFA

G-protein

GAP

GLUT

Grb2

GTP

IC50

IGF-1

IRS

IRS- 1

IRS-2

IRS-3

IRS-4

kDa

LAR

LY294002

NSF

PPARy

2-Deoxy -D-glucose

Minimal essential medium-a

Adenosine triphosphate

Chinese hamster ovary

Complimentas, DNA

Dulbecco's modified Eagle's medium

Epidermd growth factor

Fetal bovine senim

Free fatty acids

Guanosine triphosphate-binding protein

GTPase-activating protein

Glucose transporter

Growth factor receptor-bound protein 2

Guanosine triphosphate

50% Inhibitory concentration

Insulin-like growth factor-1

Insulin receptor substrate

Insulin receptor substrate- 1

Insulin receptor substrate-2



Kilodd ton

Leukocyte comrnon antigen-related

2-(4-morpholiny1)-8-phenyl-4H- 1 -benzopyran-4-one

N-ethylmaleimide-sensitive factor

Peroxisome proliferator-activated receptor y

XII

PBS

PDC

PDGF

PDK- 1

PH

PI 3-kinase

PI

PIK

P m

PKC

PMSF

PTB

PTP

SDS-PAGE

SE

SH2

SH3

Shc

SNARE

SHP2

TNF-a

Phosphate-buffered saline

Pyruvate dehydrogenase complex

Platelet-derived growth factor

Phosphoinosi tide-dependent protein kinase- l

Pleckstrin homology

Phosphatidylinositol3-kinase

Phosphatidylinositol

PI-specific PI 3-kinase

Protein kinase A

Protein kinase C

Phen y lmethanesulfony lfluonde

Phosphotyrosine binding

Protein tyrosine phosphatase

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Standard error

Src homology 2

Src homology 3

Src homology-collagen-like

Soluble NEM sensitive factor-attachment protein receptor

SH2-containing protein tyrosine phosphatase 2

Tumour necrosis factor alpha

BACKGROUND

TYPE 2 DIABETES

Noninsulin-dependent diabetes mellitus (NIDDM, type 2 diabetes) is the most common

endocrine disorder, affecting over 5% of the population in western counûies. The incidence

increases as the population ages and becomes more sedentary and obese (345). This disease is

associated with devastating complications which severely influence the quality of life. In addition

to, or because of this, type 2 diabetes imposes an enormous burden on the health care system

worldwide (96). Despite intense research. the primary defects in the pathogenesis of type 2

diabetes remajns unknown. The genetic susceptibility of this disease is of a polygenic nature with

superimposed environmental influences (143) which contribute to the manifestation of this

progressive metabolic disorder.

Type 2 diabetes is characterized by: resistance to the stimulation of glucose uptake by

insulin in skelrtal muscle and adipose tissue; impaired insulin-dependent inhibition of hepatic

glucose production; dysregulated insulin secretion (143). A schematic diagram of insulin action

and relationship to type 2 diabetes is illustrated in Figure B. 1. It is largely acknowledged that

insulin resistance is a pnmary factor responsible for glucose intolerance in the pre-diabetic state.

Initially, to compensate for the insulin resistance, insulin secretion increases to maintain normal

glycemia. However, when the insulin secretory capacity fails to adequately compensate for the

impaired insulin action, hyperglycemia, a hallmark of type 2 diabetes, ensues. This, in tum,

further exacerbates the pnmary insulin resistance, through the effects of high glucose coupled to

elevated levels of circulating factors such as fatty acids. Hence, insulin resistance has both pnmary

and secondary ongins, and represents a cntical element, in the pathogenesis of type 2 diabetes.

To develop new strategies for the prevention and treatment of diabetes and its

complications, it is important to gain an understanding of the molecular buis of insulin resistance

in muscle and fat cells. This c m be accomplished, in part, by studying the normal mechanisrns of

insulin action at the cellular level. This thesis will examine some aspects of insulin action including

the stimulation of glucose transport in adipose cells in culture. In particular, it will focus on

understanding the mechanism of action of a physiologically relevant cofactor, a-lipoic acid, which

2

has been demonstrated to have beneficial effects on glucose utilization. highlighting the thenpeutic

potential of this agent in the treatment of type 2 diabetes.

3

Figure B.1. Metabolic Actions of Insulin: Relationship to Type 2 Diabetes.

fat synthesis - glucose uptake

lnsulin lnsulin

Inappropriate Secretory

Hepatic Glucose Output

Insulin

// lnsulin Reslstance \ //secmtory Defects --b

flycogen starage Inapproprlate Hepatlc /' Diabetes hepatic gluconeogenesis //GIUCOW output

In response to an increase in blood glucose concentrations, the fl cells of the pancreatic islets

release insulin into the bloodstrearn, through which it travels to its primary targets- adipose tissue, skeletal muscle and the liver. In these tissues, insulin promotes the influx of nuvients and blocks

the release of stored forms of energy. In skeletal muscle, insulin increases glucose uptake and

glycogen synthesis. In adipose tissue, insulin favours fat synthesis and increases glucose uptake. Insulin prornotes glycolysis and glycogen storage and suppresses glycogenolysis and gluconeogenesis in the liver. In type 2 diabetes, defects at the level of skeletaî muscle and adipose tissue (insulin resistance, lack of response to circulating insulin), the pancreas (dysregulated insulin secretion) and the liver (inappropriate hepatic glucose output) are evident.

BlOLOGlCAL ACTIONS OF INSULIN

Insulin is the predominant hormone responsible for the maintenance of glucose

homeostasis, through iü regulation of metabolic activities in skeletai muscle, liver and adipose

tissue. Insulin is released from the pcells of the pancreas in response to an elevation in plasma

glucose and amino acid concentrations. The rapid action of this hormone results in increased

glucose uptake into penpheral tissues, specifically skeletal muscle and adipose tissue. Conversely,

in the liver, the hormone decreases giuconeogenesis, thereby reducing hepatic glucose output

(Figure B. 1). Additionally. insulin stimulates anabolic processes including the promotion of

glycogen, lipid and protein synthesis. Collectively. these effects are a consequence of both the

rapid and long-terni metabolic actions of the hormone. Insulin can also function as a growth factor

in a variety of cell types in culture influencing cellular prolifention and growth. In addition, insulin

is responsible for the modification of expression and activity levels of a variety of metabolic

enzymes and transport systems. Intensive research has yielded an explosion of valuable insight

into the molecular mechanisms that are responsible for the diverse actions of insulin. Complex

signalling cascades, initiated by the binding of insulin to its cellular receptor, involve the

participation of tyrosine, lipid, and serine/threonine kinases and phosphatases. These participants

convey the insulin signal to the final biological effectors of the hormone. The cascade of events

involved in the stimulation of glucose transport will be described in detail below.

Glucose Transport And Trans~orters

Glucose is the principal source of carbon and energy, essential to cellular homeostasis and

metabolism. Accordingly, the transport of glucose across the plasma membrane of mammalian

cells represents one of the most important cellular nutrient transport events (339). The transport is

rnediated by a family of highiy related transporters, designated GLUTl to GLUï7 based on their

chronological order of discovery. The glucose transporters (GLUTs) are the products of distinct

genes and are expressed in a tissue-specific fashion, each likely to play a distinct role in whole

body glucose homeostasis (25,92.93). This family of transporters, of the facilitative difision

5

type, provide a transport system for D-glucose across the plasma membrane, down a concentration

gradient (25, 114).

The sizes of the mammalian facilitative glucose transporters Vary between 492 and 524

amino acid (92). There is 39-65% identity and 50-76% similarity between the arnino acid

sequences of the different isoforms (25.282) and a high degree of sequence conservation is

retained across different species.

The common structural features revealed by sequence alignment and anaiysis of al1 the

transporters predicts the protein to fold into 12 arnphipathic helices arranged so that both the N-

and C-termini are cytoplasmic. There are large loops between helices 1 and 2 and between helices 6

and 7, the latter divides the structure into two halves, the N and C-terminal domains. The loops

between the remainder of the helices at the cytoplasmic surface are relatively short and represent a

conserved feature of the entire farnily (93). The N- and C-termini and the large exofacial loops

between helices 1 and 2 and between 6 and 7, represent unique regions of each glucose transporter

isofonn, diffenng in both amino acid sequence and size (93). It is predicted that the clustering of

helicies 7,8 and 1 1 form a hydrophilic channel for hexose transport (19,93). Cytochalasin B, a

ce11 permeable metabolite which specifically inhibits D-glucose transport, binds to a trytophan

residue (388) on the cytosolic side of helix 10 (85). The last 12 amino acids of each glucose

transporter are unique, hence isoform-specific antibodies can be readily raised against these regions

(134).

Structure. Function and Tissue-S~ecific Ex~ression of Glucose Trans~orter Isoforms.

A summary of the different glucose transporter isoforms including tissue-specific

expression patterns, kinetic properties and sugar specificity's is discussed in Table B. 1. However,

the two glucose transporters that respond to insulin in 3T3-Ll adipocytes in culture are discussed

in greater detail below.

CLUTI

The glucose transporter GLUTl was isolated from human red blood ce11 membranes (148)

and its identity was later confirmed by the cloning of this glucose transporter cDNA from the

libraries of the Hep G2 hepatoma cell line (224) and rat brain (29). GLUT 1 is most highly

expressed in the brain (glia) and in cells of the blood-braidnerve banier. It is also enriched in the

placenta, retina. adipose tissue and skeletal muscle (93). Expressed in virtually al1 tissues, the level

of expression of this transporter is also markedly elevated in trmsformed ce11 lines in culture (77,

93).

The repoaed Km of GLUTl for D-glucose ranges between 1-10 mM (3 14). A role for

GLUTl in mediating basal glucose uptake has been suggested as the ubiquitous expression of this

transporter coupled to its relatively low Km value indicate that GLUTl would be saturated at the

normal circulating levels of glucose.

GLUT4

The GLUT4 glucose transporter isoform, also known as the insulin-responsive glucose

transporter, is predominantly expressed in peripheral insulin responsive tissues, specifically

cardiac and skeletal muscle (28,42,84) and in adipose tissue (135). GLUT4 expression is also

evident in the insulin-responsive 3T3-LI adipocytes and L6 skeletal muscle ce11 lines in culture

(216,275). The Km value of GLUT4 for D-glucose is 2-5 mM (25.94, 155). The Km of GLUT4

indicates that this transporter is only half sanirated at nomoglycemia (4-6 mM) and would becorne

fully saturated upon a rise in blood glucose concentration typical of the fed state (93). The most

distinguishing property of GLUT4 from the other glucose transporters is its propensity to remain

localized in intracellular storage compamnents in the absence of insulin. Under conditions where

7

glucose transport is rate-limiting for metabolism. such as in the postprandial state, insulin recniits

this transporter to the plasma membrane. The increased flux of glucose across the plasma

membrane mediated by GLüT4 ensures that the transport of glucose is not rate limiting for glucose

metabolism (93).

8

Table Bala Mammalian Facilitative Glucose Transporters: Major Sites of

Expression and Physiological Functions.

isoform

GLUTl

GLUT2

GLUT3

r

GLUT4

L

GLUTS

GLUT6

GLUT7

Major Sites of

Expression

Erythrocytes, Blood-Brain

Barrier, tissue culture cells,

most cells at low levels

Liver, kidney, pancreatic B

cells, small intestine

- -- -- - -

neurons, fetal muscle,

placenta, testis

Skeletal and cardiac muscle,

brown and white fat

Small intestine

Liver

Physiological Function

Basal glucose uptake in most

cells (excluding neurons)

Glucose-sensor in the

pancreas, bi-directional

transport of glucose in liver,

High capacity, low affinity

glucose transporter,

Major function in mediating

neuronal glucose uptake

Insulin-responsive transporter

Fructose transporter

Pseudogene

"Cloning artifact" - does not

mode a rat Iiver endoplasmic

reticulum GLUT

Acute Rqplation of Glucose Trans~ort bv Insulin

One of the fundamental actions of insulin is to stimulate the transport of glucose across the

plasma membrane into muscle and adipose cells. These tissues are the main sites for postprandial

glucose utilization. Transport of glucose across the plasma membrane of these tissues represents

the rate limiting step in glucose utilization (165). in an atternpt to define the mechanism underlying

the ability of insulin to stimulate glucose transport, it was demonstrated that in unstimulated rat

adipose cells, glucose transporters (now known to be GLUT4) were predominantly associated

with an intracellular light density microsornal fraction, enriched in intemal membranes (61,309).

In response to insulin, these transporters were found to be recniited or translocated to the plasma

membrane thereby becoming available to take up extracellular glucose into cells. This phenomenon

was also described analyzing muscle membranes (163). When GLUT4 was cloned and antibodies

to its C-terminus were raised, studies confirmed that in both fat and muscle cells the recruited

transporter was GLUT4. This phenomenon of intracellular sequestration and insulin-induced

translocation of glucose transporters was demonstrated in other insulin-responsive tissues

including brown adipose tissue (287), heart (286), diaphragm (344), skeletal muscle (1 19, 164,

166), and in cultured ce11 lines including L6 skeletal muscle cells (2 17) and 3T3-LI adipocytes (39,

50).

The insulin-stimuiated increase in cell-surface abundance of GLUT4 is the result of an

increase in the exocytosis of this transporter to the membrane and a reduction in the rate of

endocytosis (62,362, 363). The acute stimulation of glucose transport by insulin is rapid in cells

in culture, reaching a stable maximum by twenty to thirty minutes. This initial phase of the

stimulation of transport is independent of new transporter biosynthesis (338). In addition to

GLUT4, other glucose transporter isoforms translocate to the plasma membrane in response to

insulin. GLUTI has been shown to uanslocate in response to an acute insulin challenge in rat

adipocytes, 3T3-L1 adipocytes (39), L6 skeletal muscle cells (217) and in Chinese hamster ovary

cells (104). The GLüT3 isoform, although largely present on the ce11 surface, also experiences a

small degree of further translocation to the plasma membrane of L6 skeletal muscle cells in

response to acute insulin treatment (27).

SlGNALLlNG MECHANISMS REGULATING INSULIN-STIMULATED GLUCOSE TRANSPORT

During the past two decades there has been an explosion in the understanding of the

molecular mechanisms undedying normal insulin action and glucose disposal. It was discovered

that insulin binding to its ceIl surface receptor activates the receptor's intrinsic tyrosine kinase

leading to phosphorylation of cellular substrates (149, 15 1). This finding, coupled to the discovery

that insulin stimulation lead to an increase in the recruitment of presynthesized glucose transporters

(GLUT4) to the plasma membrane (61.309) revealed the beginning and end of a cascade of

reactions which linked the interaction of insulin with its receptor to the stimulation of glucose

uptake in marnrnalian cells. Emerging details conceming the beginning and end of the cascade, in

addition to the cornplex intermediate signalling events and glucose transporter recruitment, are

being elucidated (122). A schematic representation of the signals necessary for insulin-stimulated

GLUT4 translocation is illustrated in Figure 8.2. As stated earlier. a molecular undentanding of

the cellular mechanism of insulin action is required for our undentanding of the defects that

underlie type 2 diabetes, and should ultimately lead to the design of successful therapeutic

interventions. A detailed summary of the cascade of events leading the stimulation of glucose

uptdce will be discussed in the following sections.

Figure B.2. The Insulin Signalliag Cascade.

lnsulin Receptor

Class IA PI 3-Kinase

Translocation to Plasma Membrane 1

1 GLUCOSE TRANSPORT 1

The Insulin Rece~tor

Following its release by the P cells of the pancreas, the first step in insulin action at the

cellular level is binding of the hormone to its transmembrane receptor which is a tetramenc pmtein

composed of two a-subunits (molecular weight - 135 kDa) and two P subunits (molecular weight -

95 m a ) (150,209). The two a-subunits are linked to each other and each to a P subunit through

disulfide bonds. The a-subunits are located entirely outside the ce11 and contain the insulin binding

site(s), whereas the intracellular portion of the P subunit, which spans the membrane, contains

several functional regions and includes the insulin-regulated tyrosine kinase (348). A schematic

diagram of the insulin receptor demonstrating its different functional domains is illustnted in

Figure 8.3. The functional regions of the B subunit are: the juxtamembrane region, essential for

signal transmission as it mediates (downstream) substrate selection; the ATP binding domain,

essential for the kinase activity; the regulatory domain. containing three tyrosine residues essential

for insulin-stimulated kinase activity; the tyrosine kinase domain; and the C-terminal tail, essential

for regulation of insulin signals (348). Insulin binding activates the tyrosine kinase. leading to

autophosphorylation of tyrosine residues in several regions of the intracellular P subunit including

T y r 9 ~ in the juxtamembrane region; Tyr1 146, Tyr1 150, and Tyr1 15 1 in the regulatory loop; and

Tyr 13 16 and Tyr 1322 in the C-terminus (204,2 12.350). Thus, autophosphorylation activates the

kinase activity of the receptor towards other substrate proteins.

Figure B.3. The Insulin Receptor

r-953 f- Juxatmembrane NPXY-Motif n m -1 1 -AT? Binding Site u ml

Tyr-1 146 Tyr-1 150 , Regulatory Loop 1 1 ITyr-11511

Important structural and functional features of the insulin receptor. The insulin receptor is a

disulfide bonded heterotetramer composed of two a- and f3-subunits. The a-subunits are

extracellular and contain the insulin binding domains. The p-subunits contain a short extracellular

domain, a trammembrane spanning region, and intracellular hinctional regions including the

juxtamembrane region, regulatory (lunase) domain, and the C-terminal tail. Adapted fiom: White,

M.F. (1997). The Insulin Signalling System and the IRS Proteins. Diabetologia 40, S2-Sl7.

14

Considerable evidence has been collected indicating that the tyrosine kinase activity of the

insulin receptor is essential for insulin signalling (146), as follows: Site-directed point mutations in

the ATP binding domain destroy ATP binding and result in abolished kinase activity of the receptor

and abrogation of insulin signalling (47.2 13). Naturally occumng mutations in the insulin

receptor, which result in an inhibition of kinase activity, are accompanied by severe insulin

resistance (218,236). Mutation at one, two or three tyrosine residues in the regulatory domain

progressively reduce insulin-stimulated kinase activity in parallel with a loss in biological activity

(333,353). Further evidence also suggests that activation of the insulin receptor tyrosine kinase is

required for the stimulation of glucose transport. Mutation of the putative ATP-binding region of

the insulin receptor abolished insulin-stimulsted glucose transport (7 1) and overexpression of

tyrosine kinase-deficient insulin receptors in rat adipose cells failed to mediate an increase in

GLUT4 translocation (256). Accordingly, the activation of the insulin receptor tyrosine kinase and

the subsequent phosphorylation of cellular substrates predominates as an important mechanism of

insulin signal transduction (348). Thus, failure to activate the tyrosine kinase of the insulin receptor

has been demonstrated to be accompanied by a loss in the ability of the receptor to transmit signals

to metabolic and mitogenic endpoints.

In addition to tyrosine phosphorylation, the insulin receptor has been shown to be regulated

by serinelthreonine phosphorylation. An increase in the level of serinelthreonine phosphorylation

of the insulin receptor is associated with a decrease or inhibition of the insulin receptor tyrosine

kinase activity (68,201,3 10). As a result, the insulin receptor and its ability to transduce

necessary signals is directly influenced by ligand binding, tyrosine autophosphorylation, and

serinehreonine phosphorylation.

Recently, specific protein tyrosine phosphatases (PTPase) have gained considerable

attention in the regulation of insulin signalling. A central role for reveeible tyrosine

phosphorylation in the regulation of the steady state baiance of insulin receptor has also been

established. In particular, the receptor-type, transmembrane PTPase LAR Ueukocyte comrnon

antigen-plated) has emerged as a candidate insulin receptor PTPase. LAR is expressed in insulin- -

sensitive tissues and it is localized to the celi membrane fraction of the ceîi where insulin receptor

dephosphorylation occurs rapidly in situ (108,368). In addition, a physical interaction between

15

LAR and the insulin receptor has k e n demonstrated and overexpression of LAR lead to an

attenuation of insulin receptor autophosphorylation (2, 189,202). The trammembrane protein

tyrosine phosphatase alpha (PTPa) has also k e n shown to dephosphorylate the insulin receptor in

intact cells (193). thus functioning as a negative regulator of the insulin receptor tyrosine kinase.

This carries over to impact on downstream endpoints of insulin action as. overexpression of m a

has been shown to inhibit insulin-stimulated GLUT4 translocation in rat adipocytes (54). Yet

another PTPase, protein tyrosine phosphatase 1 B (PTP 1 B) has been demonsated to directly

interact with and dephosphorylate the activated insulin receptor (3. 108). Again, overexpression of

wildtype FTPIB resulted in a reduction in the Ievel of GLUT4 translocation in response to insulin

(45). Recently, a more defïnitive role for PTPlB in insulin signalling was established. Disruption

of the mouse homologue of the gene encoding PrPl B yielded mice (WPIB-1-) which displayed

enhanced insulin sensitivity, increased phosphorylation of the insulin receptor and insulin receptor

substrate (1RS)-1, and resistance to weight gain (73). Taken together, these results indicate that

PTPases play an integral role in the regulation of the insulin receptor and mediating downstream

insulin signalling.

Insulin Rece~tor Su bstrate Proteins

In contrast to other receptor tyrosine kinases, such as the epidemal growth factor (EGF)

receptor and the platelet derived growth factor (PDGF) receptor, the insulin receptor does not

directly engage or phosphorylate Src homology 2 (SH2) domain-containing proteins (discussed in

following sections). Instead, insulin receptor autophosphorylation causes activation of its substrate

kinase activity, which in tum binds and phosphorylates "docking"1adaptor proteins of the insulin

receptor substrate (IRS) farnily (350). These "docking" proteins then serve to recruit and link the

activated tyrosine kinases to other SH2 domain proteins involved in signal transduction. Mernbers

of this family of insulin receptor-docking proteins include Shc (Src homology-çollagen-like

protein), Gab-1. p62dok. and the insulin receptor substrate (RS) proteins, of which four rnernbers

have been identified (IRS 1-4) (121,308,359). The following section will highlight the

importance of the IRS-proteins in mediating insulin signals by binding and activating various

16

enzymes or adaptor molecules. In particular, I will focus on the role of the LRS proteins in the

stimulation of glucose transport.

Insulin Receator Substrate-1 (IR$-1)

IRS- 1 was the first insulin receptor substrate to be purified and cloned (15, 156. 157, 264,

5 1 ) and functions as an insulin receptor-docking protein capable of engaging multiple

downstream signalling molecules during insulin signalling (349). IRS- 1 is a cytoplasrnic protein

with an apparent molecular weight of 185 kDa on sodium dodecyl sulfate (SDS) gels that

undergoes rapid tyrosine phosphorylation in response to insulin (226). IRS-1 is widely expressed

is tissues and cells in culture and is highly conserved arnong species (146). Several common

structural features are characteristic of IRS proteins including an N-terminal PH (gleckstrin

homology) andor FTB (phospho~rosine-binding) domain, multiple C-terminal tyrosine residues, - praline rich residues and serine/threonine-nch regions. A schematic diagram of the stmcture of

IRS-1 is illustrated in Figure 8.4.

17

Figure B.4. Structural Features of Insulin Receptor Substrate-1 (IRS-1).

A schematic diagram of the structure of IRS-1 (rat). The pleckstrin homology (PH) domain and

phosphotyrosine binding (PTB) domain are thought to mediate interactions with the insulin

receptor. Putative tyrosine phosphorylation sites are indicated in the C-terminal tail; these tyrosine

phosphorylation motifs facilitate interactions with downstream SHZcontaining proteins, including

PI 3-kinase (PI 3-K), Grb-2, and SHP-2.

18

The interaction between the insulin receptor and IRS-1 is facilitated by the PH and PTB

protein interacting domains in the highly conserved N-terminal region of IRS- 1. The PH domain is

a poorly conserved region of approximately 120 amino acids that was first identified as intemal

repeat sequences in pleckstrin (major substrate of protein kinase C in platelets) and is present in a

variety of signalling moiecules (1 10,S 1 1,225). It has been dernonstrated that deletion of the PH

domain results in a reduction in the level of tyrosine phosphorylation of IRS-1 (365). This is

suggestive that the PH dornain contributes to the interaction of IRS-1 with the insulin receptor and

provides the most sensitive coupling of IRS- 1 to the insulin receptor (365). Located irnmediately

downstream of the PH domain is the PTB domain. This protein module is cornposed of

approximately 150 arnino acids which binds to phosphotyrosine (154, 327). This domain binds

specifically, but weakly, to the phosphorylated ~ ~ ~ ~ g m - m o t i f located in the juxtamembrane

region of the insulin receptor (97,235,308, 341). Taken together, these protein interaction

domains may provide the specific mechanisms for coupling of the tyrosine phosphorylated insulin

receptor and IRS- 1.

A unique structural feature of IRS- 1 is the presence of multiple tyrosine phosphorylation

sites in the C-terminus. These sites account for the ability of IRS-1 to become tyrosine

phosphorylated in response to insulin and io participate in insulin signalling. (146). IRS- 1 contains

21 putative tyrosine phosphorylation sites including 6 in YMXM motifs, three in YXXM motifs,

and twelve in other hydrophobic motifs (350). At least 8 of these tyrosine residues, some of

which are in the YMXM motif, undergo phosphorylation by the activated insulin receptor (303,

349). As a result of the ability of IRS-I to bind several intracellular signalling molecules, IRS-1 is

viewed as a "docking" protein which provides a site for the assembly of subsequent downstrearn

signalling molecules. The binding of IRS- 1 to downstream intracellular proteins is mediated by the

tyrosine phosphorylation motifs in IRS-1 to specific domains on the target proteins termed SH2

a r c homology 2) domains for their homology to a viral oncogene product src (146, 170). SH2

domains are protein modules of approximately 100 arnino acids that recognize shon

phosphopeptide motifs composed of a phosphotyrosine followed by three to five carboxyl-terminal

residues, for example pYMXM or pYXXM motifs (249). Several SH2 containing proteins have

been identified which associate with iRS-1 including phosphatidylinositol3-kinase (PI 3-kinase),

19

SHP2, Fyn, Grb-2, nck and Crk (24, 187, 199,227,305). As a result, IRS-1 serves as a docking

protein for several intracellular enzymes and a&ptor molecules which further propagate the signals

emanating from the insulin receptor.

In addition to regulation by tyrosine phosphorylation, IRS-1 contains over 30 potential

senneheonine phosphorylation sites in motifs that are recognized by various kinases (348). In

the basal state, IRS-1 is strongly senne phosphorylated (307). During insulin stimulation it appears

that an elevation in the level of serine and threonine phosphorylation of IRS-1 inhibits the tyrosine

phosphorylation of IRS- 1. This increase in the level of serinelthreonine phosphory!ation is

associated with a reduction in the ability of IRS- 1 to interact with downstream SH2-containing

signalling proteins (222). Importantly, insulin resistance is also associated with elevated

sennelthreonine phosphorylation levels of IRS- 1. Furtherrnore, elevated levels of the circulating

cytokine nimor necrosis factor - alpha (TNF-a), a mediator of insulin resistance in obesity,

diminishes insulin-induced tyrosine phosphory lation of IRS- 1 while it induces serine/threonine

phosphorylation of IRS- 1 ( 124). These effects of TNF-a are presumably mediated through

inhibition of serine phosphatases or activation of sennelthreonine kinases (147,250). Casein

kinase-2, MAP kinase, protein kinase C, and PI 3-kinase have been proposed as the kinases which

may be responsible for increasing the level of sennelthreonine phosphorylation of IRS- 1 (304,

3 12). Consequently, elevated levels of senneheonine phosphorylation of IRS- 1 negatively

modulate insulin action and are implicated in the downregulation of hormone signalling.

PTPases interact with IRS-1, thereby regulating downstream insulin signalling. SHP-2 is a

novel nontrammembrane PTPase which contains two SH2 domains necessary for mediating the

interaction with phosphotyrosine motifs on LRS-1 (302,324). It has been demonstrated that SHP-

2 participates as a positive mediator of the mitogenic actions of insulin and other growth factors

(1 12,233). However, conflicting evidence of the involvement of SHP-2 in the regulation of the

metabolic responses of insulin have k e n reported. Microinjection of the SH2 domains of SHP-2

or anti-SHP-2 antibodies failed to inhibit GLUT4 translocation in 3T3-LI adipocytes (1 12). In

conaast, a mutant IRS-1 molecule that does not bind SHP-2 became more highly phosphorylated

in response to insulin and lead to a stronger activation of phosphatidylinositol3-kinase (PI 3-

kinase) (228). Thus, although SHP-2 has been demonstrated to regulate multiple levels of insulin

20

action, its association with RS- 1 appears to attenuate certain insulin signals important for

metabolic responses. including GLüT4 translocation. These results aiso highlight the possibility

bat, in addition to negative regulation, SHP-2 may potentiate insulin action. although the

mechanism for a potentiating effect remains unknown.

Insulin Receptor Substrate-2 (IRS-2)

Shonly after the cloning of iRS- 1 another high molecular weight tyrosyl phosphoprotein

was identified with several structural and functionai features similar to IRS-1 (215,308) . This

finding, coupled to the observation that mice made IRS- 1 deficient became only rnildly insulin

resistant and continued to exhibit some insulin-stimulated glucose disposal and PI 3-kinase (see

below) activation (14.3 11) suggested that a second member of the IRS-protein family could also

be responsible for mediating insulin signals.

A cornparison of the amino acid sequences of IRS- 1 and IRS-2 frorn mouse, rat and human

sources revealed a highly conserved amino terminus containing the PH and Fil3 domains between

these two proteins (348). The PH domains of IRS- 1 and IRS-2 are 69% identical, and the I T B

domains share 75% identity. In contrast, the C-terminal portions of IRS-1 and IRS-2 are poorly

conserved (35% identity ), but contain multiple tyrosine phosphorylation sites in relatively similar

positions (349). In addition to the ability of the PH and PTB domains to confer binding specificity

of IRS proteins to the insulin receptor, IRS-2 contains a unique region cornprising residues 591-

786 that interact specifically with the phosphorylated regulatory loop of the insulin receptor (97).

Hence, this unique domain of IRS-2 reveals an important difference between IRS- 1 and IRS-2 and

may serve to determine fùnctional specificity between these two proteins.

In parallel to IRS-1, insulin stimulation leads to a rapid increase in tyrosine

phosphorylation of IRS-2 leading to the binding of IRS-2 to SH2-containing proteins (248,308).

However, it has k e n demonstrated that in muscle cells in culture IRS-2 is more rapidly

dephosphorylated than IRS-1 and leads to a more transient activation of PI 3-kinase in response to

insulin (238). The more transient activation of the IRS-2-mediated insulin signais has aiso been

demonstrated in 3T3-LI adipocytes in culture (126). In addition, distinct cellular distribution

patterns of IRS-1 and IRS-2 have been observed with IRS-2 predorninating in murine

21

hematopoietic cells and by IRS- 1 predominating in adipocytes and differentiated 3T3-L1

adipocytes (306). Interestingly, in IRS- 1 deficient mice the phosphorylation of IRS-2 and its

association with the downstream signalling effector PI 3-kinase was markedly enhanced, perhaps

in an attempt to compensate for the deficiency (248). Thus, signalling specificity through the iRS

proteins is accomplished through their specific expression patterns, distinct phosphorylation

pattems and fûnctional differences allowing for the regulation of insulin responses in an IRS-

specific rnanner (306)

Insulin Rece~tor Substrate-3 (IRS-3)

Initially, a 60 kDa pmtein was identified in adipocytes and hepatocytes which becarne

tyrosine phosphorylated in response to insulin (197,2 14). This insulin receptor substrate,

originally referred to as pp60, was purified and cloned from rat adipocytes (196) and from a mouse

expression sequence tag library (276). It was designated IRS-3, a new member of the insulin

receptor substrate family (196). Despite the fact that IRS-3 is 700-800 amino acids smaller than

IRS- 1 and IRS-2, the overall structure of IRS-3 is well conserved, especially the N-terminal

domain in which the PH and PTB domains share approximately 50% sequence identity with the

corresponding domains of IRS- 1 and IRS-2 (196). The COOH-tail contains multiple tyrosine

phosphorylation sites which occur in motifs recognized to bind PI 3-kinase, SHP2 and Grb-2

(196,262). In addition, it was dernonstrated in adipocytes that IRS-3 bound more rapidly to p85,

the regulatory subunit of PI 3-kinase, suggesting that IRS-3 is a principal regulator of PI 3-kinase

(288). Munne IRS-3 messenger RNA (mRNA) is expressed in numerous tissues, including the

liver and lung (276). Mouse IRS-3 is also expressed dunng early embryonic life, when IRS-1 is

barely detectable (276). The differences in tissue distribution and in structural and functional

capacities of IRS-3 may contribute to the diversity of the cellular responses mediated by the

different IRS proteins.

Insulin Receotor Substrate-4 (IRS-4)

It was demonstrated that a 160 kDa protein in human embryonic kidney (HEK) 293 cells

was rapidly tyrosine phosphorylated in response to insulin (188). This protein, originally

designated as PY 160, was found to be immunologically unreiated to IRS-1. Subsequent cloning of

22

this protein from insulin-treated HEK 293 cells revealed that PY 160 was a new rnember of the IRS

family (IRS-4) based on its predicted amino acid sequence (194). IRS-4 contains an N-terminal

PH dornain, a R B dornain, and 12 potential tyrosine phosphorylation sites in its C-terminus. The

PH and PTB domains of IRS-4 share at least 40% identity with IRS-1, IRS-2 and IRS-3 (194).

IRS-4 was also found to be associated with the SH2 domain-containing proteins, PI 3-kinase and

Grb2 in insulin-stimulated HEK 293 cells (75). Initial characterization of the properties of IRS-4

has revealed that this protein is located in cellular membranes of HEK 293 cells, with the majority

of IRS-4 concentrated at the cytoplasrnic surface of the plasma membrane (75).

Role of Insulin Receator Substrate Proteins in Insulin-Stimulated Glucose Trans~ort

IRS- 1 was the first insulin receptor substrate protein to be characterized and rnay play an

important role in insulin signalling (349). However, conflicting evidence has been presented for

the role of IRS-1 in mediating insulin signals necessary for the stimulation of glucose transport. In

support of the involvement of IRS-1 in glucose transport, a decrease in the level of endogenous

IRS-I with an antisense ribozyme in rat adipose cells lead to a rightward shift in the insulin dose-

response curve, whereas no change in maximal responsiveness occurred (255). Altematively.

experiments in 3T3-L 1 adipocytes which involved the inhibition of insulin receptor/IRS- l

interactions, revealed the possibility that insulin may activate novel signalling pathways that are

independent of IRS- 1 phosphorylation (22 1,277,278,294). For example, overexpression of the

W B domain of IRS-1 in 3T3-L1 adipocytes resulted in a decrease in the insulin-stimulated

tyrosine phosphorylation of IRS-1 and its association with PI 3-kinase without any effect on

insulin-stirnulated Akt activation or glucose transport (277). Furthemore, platelet-derived growth

factor (PDGF) treatment of 3T3-L1 adipocytes lead to an increase in the level of serine/threonine

phosphorylation of IRS- 1 and this was accornpanied by a reduction in the level of insulin-

stimulated tyrosine phosphorylation of IRS-1 and IRS-1-associated PI 3-kinase activity. Despite

the reduction in IRS-1 phosphorylation, insulin-stimulated Akt activation and glucose transport

were unaffected by PDGF treatment (294). Further evidence in support of an IRS- l-independent

pathway leading to the stimulation of glucose transport was provided through the use of the IRS-1

deficient rnice. These mice displayed growth retardaiion, but were only rnildly insulin resistant and

23

were not diabetic (14,3 1 1). In addition, fat cells derived h m IRS- 1 knockout mice displayed an

approximately 50% decrease in glucose transport, despite the complete lack of IRS-1 (14,360).

These studies demonstrate that IRS- 1 is not entirely sufficient to mediate glucose transport,

suggesting the participation of an alternative insulin receptor substrate protein.

In contrast to IRS-1 knockout rnice, mice deficient in IRS-2 display severe insulin

resistance and develop overt diabetes (354). This indicates that IRS-2 rnay play a more crucial role

in the mechanisms regulating fuel homeostasis. Structural similarities between IRS-1 and IRS-2

are supportive of the ability of IRS-2 to compensate or even predominate as the rnediator of

insulin-stimulated glucose transport. Overexpression of IRS-2 in rat adipocytes (369) lead to an

increase in the amount of G L U 4 at the surface of unstimulated (basal state) cells, supportive of

the notion that IRS-2 is capable of mediating the metabolic responses of insulin action.

Additionally, it has also k e n suggested that IRS-3 may mediate insulin action on glucose transport

and GLUT4 translocation in adipocytes from IRS-1 deficient mice (140). In the absence of IRS- 1

and marginally detectable levels of tyrosine phosphorylated IRS-2, IRS-3 became the major

tyrosine-phosphorylated protein that associated with PI 3-kinase, and was suggested to support the

level of glucose transport and GLUT4 translocation (52 and 68% vs. wild-type, respectively)

remaining in adipocytes from the IRS- 1 deficient rnice (140).

However, it has also been suggested thai an 1RS-independent, but PI 3-kinase dependent,

pathway leading to the stimulation of glucose transport may exist (129, 140, 184,294). For

example, a study in which GLUT4 and the interleukinl receptor were overexpressed in L6

myoblasts, it was shown that stimulation with interleukin-4 had no effect on glucose transport,

despite the fact that interleukin-4 strongly stimulated tyrosine phosphorylation of IRS-1 and its

association with PI 3-kinase (129). Similarly, Krook et al. have shown that the expression of two

insulin receptor mutants expressed in CHO cells could still mediate IRS-1 phosphorylation but

failed to stimulate glycogen synthesis (186). Taken together, both of these studies demonstrate that

IRS- 1 phosphorylation, with PI 3-kinase activation, is not sufficient to initiate metabolic

signalling. Thus, M e r studies are necessary to determine if the stimulation of glucose transport

is mediated by parallel pathways and to define the necessary signalling cornponents of these

pathways involved in rnediating the ability of insuiin to stimulate glucose transport.

Phos~hatidvlinositol 3-Kinase (PI 3-kinase)

A large body of evidence has accumulated to suggest that the activity of the enzyme

phosphatidylinositol3-kinase (PI 3-kinase) is necessary for insulin regulation of glucose

metabolism. PI 3-kinases exhibit inainsic lipid and senne kinase activities and have k e n

implicated in mitogenic signalling, ceIl survival, cytoskeleton remodeling, metabolic control and

vesicular trac (358). PI 3-kinases are a family of enzymes which cataiyze the phosphorylation of

the D-3 position of the inositol head group of phosphoinositides (297). In vitro, PI 3-kinases

convert phosphoinositol (PtdIns), PtdIns(4)P and PtdIns(4,5)P2 to PtdIns(3)P, PtdIns(3,4)P2 and

PtdIns(3,4,5)P3, respectively (330).

The first marnrnalian PI 3-kinase charactenzed was an 85 kDa phosphoprotein (56).

Subsequent cloning and characterization of this enzyme revealed that the enzyme was a heterodimer

composed of an 85 kDa regulatory adaptor subunit (p85) and a catalytic subunit of 110 kDa (p 1 10)

(83,280). The catalytic subunits of PI 3-kinase can be divided into three main classes (Table B.2)

on the basis of their in vitro lipid substrate specificity, structure and likely mode of regulation

(330). It is widely accepted that class IA PI 3-kinases are regulated by insulin and play an

important role in the stimulation of glucose transport (described in detail below). For this reason,

this thesis will focus on the structure, function, regulation and role of class IA PI 3-kinases in the


25

Table B.2. A Classification of Phosphatidylinositol (PI) 3-Kinase Family

Members.

Class

1 A

II3

II

III

ln vitro Lipid Substrates

PtdIns, PtdIns(4)P, PtdIns(4,5)P2

PtdIns,

PtdIns(4)P

-- -

PtdIns

Catalytic Subunits

PI3 K-C2a (Cpk-dp l7O), PI3 K-C2P

PI 3-kinase

( P m (Vps34p, yeast)

Adaptor Subunits

p 150 (Vps 15p)

Phosphotyrosine residues and Ras

G protein By subunits and Ras

Constitutive ?

Adapted frorn: Vanhaesebroek. B., S.J. Leevers, G. Panayotou, and M.D. Waterfield.

Phosphoinositide 3-kinases: a conserved family of signal transducers. Trends Biochem. Sci. 22:

267-272, 1997.

26

Three highly homologous isoforms of the catalytic subunits of class IA PI 3-kinases have

been cloned and have been designated as pl lOa (1 18). pl 10p (125) and pl 106 (329). The a and

isoforms are most likely to participate in insulin signalling as they are most widely expressed,

whereas the 6 isoform is restricted to hematopoetic cells (280).These three isoforms share similar

stmctures including the kinase domain at the C-terminus, a PI-kinase domain (PIK; of unknown

function), binding domains for p85 and Ras association located at the N-terminus, and a domain

that binds to the inter-SH2 (iSH2) region of the adaptor subunit (260,280).

The class IA PI 3-kinases adaptor/regulatory subunits are encoded by at least three genes

which generate highly homologous products (280). The adaptorlregulatory subunits contain two

SH2 domains which have a high selectivity for binding phosphorylated YXXM and YMXM

sequences (290), providing a link to upstream signalling events. The SH2 domains are linked by

the iSH2 region, which is both necessary and sufficient for binding the adaptodregulatory subunits

to the catalytic subunits of PI 3-kinase. p85a and p85P also contain an SH3 domain. a Bcr/Rac

GTP-ase-activating protein (GAP) homology (BH) domain, and two proiine rich regions which

flank either side of the BH domain (280). Three spliced variants of p85a have been reponed

including one form arising from the addition of eight amino acids in the iSH2 domain, p85ai, and

two truncated versions ds ing from alternative splicing, p55a and p50a. The p55a isoform is

highly expressed in brain and muscle (13, 128) and p50u is highly expressed in brain, liver,

muscle, and kidney (128). A third PI 3-kinase adaptor subunit gene has also been characterized

termed p55P1K/p55y (127,254) which encodes for a protein that is highly homologous to p55a

and whose expression is restricted to neural tissues (254). It has also been demonstrated that the

adaptor subunits differentiaily modulate IRS-associated PI 3-kinase activity. This difference,

roupled to distinctive tissue expression patterns, provide a basis for the regulation of PI fkinase-

mediated glucose metabolism and transport according to tissue specific needs (5).

27

Two relatively specific and ce!l-permeable inhibitors of PI 3-kinase have facilitated

investigation into the role of PI 3-kinases in cellular processes. The fungal metabolite wortmannin

acts as potent inhibitor of the lipid and protein kinase (192) activities of PI 3-kinase. Worunannin

covalently modifies Lys802 within the conserved core catalytic domain a residue that is essential in

the phosphate transfer reaction (357). This cell-permeant inhibitor inhibits class I A PI 3-kinase

with an ICs0 of approxirnately 3 nM in vitro and ai 10-30 nM in intact cells (325). At

concentrations greater than 100 nM the effecu of wortmannin have been demonstrated to be less

specific as some isoforms of PI Clcinase (230) and phospholipase A2 (58) become wortmannin-

sensitive.

The synthetic compound 2-(4-morpholiny1)-8-phenyl-4H- I -benzopyran-4-one (LY 294OO2)

is another specific inhibitor of class 1~ PI 3-kinases (332). LY294002 binds competitively to the

ATP binding site of the catalytic subunit of PI 3-kinase and at micromolar concentrations this

inhibitor cause a dose-dependent inhibition of PI 3-kinase (43).

The Role of PI 3-kinase in lnsulin-Reeulated Glucose Trans~ort.

Class IA heterodimeric PI-3 kinases are involved in many insulin-regulated responses

including the stimulation of glucose uptake. It was initially demonstrated that insulin increased PI

3-kinase activity in anti-phosphotyrosine immunoprecipitates (159,266) with a similar time frame

and with a similar dose-dependence to its stimulation of glucose transport. It has since been

demonstrated that insulin stimulates PI 3-kinase activity in skeletal muscle, liver, isolated rat

adipocytes and in 3T3-L1 adipocytes and L6 skeletal muscle cells in culture. Increased tyrosine

phosphoiylation of YMXM motifs in iRS-proteins with subsequent binding and activation of PI 3-

kinase in response to insulin provides a means of coupling the insulin receptor tyrosine kinase

activity with the activation of intracellular PI 3-kinase (18, 195,366).

Recently, it has aiso been demonstrated that class II PI 3-kinases can be regulated by

insulin (280), but they are wortmannin insensitive (69,33 1). Class II PI 3-kinases cannot utilize

PtdIns(4,5)P2 as a substrate and so do not generate PtdIns(3,4,5)P3 (69, 331). For these reasons,

it is believed that class II PI 3-kinases do not participate in insulin signalling responsible for the


28

Several subsequent expenrnents have dernonstrated the role of class IA PI 3-kinase in

mediating insulin action. Overexpression of a mutant p85 lacking the binding site for the catalytic

pl 10 subunit resulted in attenuation of the insulin-induced increase in anti-phosphotyrosine

associated PI 3-kinase activity and glucose transport (67, 104). These expenments indicated bat

the anti-p85 coupled PI 3-kinase activity is necessary for the insulin-dependent increase in glucose

transport. Furthemore, it has been shown that microinjection of the mutant p85 inhibits the

translocation of GLUT4 in 3T3-L1 adipocytes (181). GLUT4 translocation is also inhibited by

microinjection of the SH2 domains of p85, expressed as a glutathione S-transferase fusion protein

(106,278). Moreover, it has been demonstrated that an overexpression of the pl 10 catalytic

subunit of PI3 kinase in rat adipocytes (3 13) and in 3T3-L1 adipocytes (152) leads to an increase

in basal levels of GLüTl and GLUT4 at the ce11 surface. These results further support the

importance of PI 3-kinase activation in insulin-stimulated glucose transporter u;uislocation and

glucose transport.

The altemate use of inhibitors of the catalytic activity of PI 3-kinase also implicates PI 3-

kinase as an important signalling intermediate required for insulin-stimulated glucose transport. It

has been demonstrated that wortmannin inhibits insulin action on glucose transport and the

translocation of GLUTl and GLUT4 in rat adipocytes (239), 3T3-L1 adipocytes (50), L6 skeletal

muscle cells (3 19), and skeletal muscle (364).

Although necessary, activation of PI 3-kinase alone may not be sufficient to stimulate

glucose transport as several growth factors activate PI 3-kinase to a sirnilar extent as insulin yet fail

to activate glucose transport in muscle or fat cells in culture (129,23 1,352). This suggests that

mechanisms additional to the activation of PI 3-kinase maybe required for insulin-stimulated

glucose transport.

Distinct spatial distribution patterns of signalling events induced by growth factors may

provide a mechanism to explain the specificity of insulin action on glucose transport (82). For

example, PDGF treatment potently stimulates PI 3-kinase activity but only produces a small

stimulation of glucose transport in 3T3-L1 adipocytes (23 1,352). PDGF stimulates an increase in

PI 3-kinase activity in the plasma membrane whereas insulin stimulates an increase in the level of

29

PI 3-kinase activity associated with intracellular membrane compartments (279). These intracellular

membrane cornpartments have also been demonstmted to be enriched with IRS proteins, tyrosine

phosphorylated IRS proteins and GLUT4-containing vesicles (49, 126,208). Furthermore, it has

k e n shown that in response to insulin. PI 3-kinase activity is elevated in intracellular

compartments containing GLUT4 (1 15) and this event requires the participation of intact actin

filaments (343). Thus. the unique ability of insulin to direct the localization of PI 3-kinase to

intracellular membrane fractions may account for the specificity of insulin action on glucose

transport.

The lack of correlation between PI 3-kinase activation and GLUT4 translocation may also

be reflective of additionalpathways required for insulin-stimulated glucose tmsport. This is

supported by a recent finding whereby an inhibition of the insulin-mediated IRS protein tyrosine

phosphorylation and recruitrnent of PI 3-kinase was mediated by an increase in the level of

serinelthreonine phosphorylation in response to PDGF treatment, which failed to diminish insulin-

stimulated glucose transport (294). Furthermore, treatment of 3T3-LI adipocytes with a ceIl

penneable analog of the Pi 3-kinase product PtdIns(3,4,5)P3 alone did not increase glucose

uptake, but partially rescued the inhibition of insulin-stimulated glucose transport by wortmannin

(136). These results suggest that indeed insulin rnay induce PI 3-kinase-independent and

-dependent signalling events. In addition to these events, the ability of insulin to spatially regulate

signalling molecules rnay also contribute to the mediation of signals necessary for the stimulation

of glucose transport.

a n a l s Downstream of PI 3-kinase: Involvernent of Serine and Threonine Kinases

Several lines of evidence suggest that the lipid products of PI 3-kinase are involved in

regulating signal transduction cascades. These lipid products, specificall y PtdIns(3,4)P2 and

PtdIns(3,4,5)P3, act as both membrane anchors and allostenc regulaton which serve to localize

and activate downstream enzymes and their protein substrates (280). The lipid products of PI 3-

kinase function in insulin signalling by binding to the PH domains of downstream kinases

including the phosphoinositide-dependent protein kinase (PDK) -1, Akt [also referred to as protein

kinase B (PKB)], and atypical protein kinase Cs (aPKCs) (see following sections). Thus. the lipid

products of PI 3-kinase serve to connect PI 3-kinase as the molecular switch which is capable of

regulating the activity of serindthreonine-specific kinase cascades implicated in mediating insulin

action (280).

Akt -

The serindthreonine kinase Aktlprotein kinase B (PKB) was identified independently by

different groups as a result of its homology to protein kinase A (PKA) and protein kinase C

(PKC) giving nse to the names PKB and RAC (related to the A and C kinases) (5 1, 138).

SimuItaneously, the kinase was identified as the product of the oncogene v-akz of the acutely

transforming retrovirus AKT8, found in a rodent T-ce11 lymphorna (26). To date, Akt has been

implicated in the regulation of physiological processes including cellular growth and metabolism.

This 50-60 kDa protein has been demonstrated to participate in the regulation of glycogen

synthesis, cardiac muscle glycolysis, glucose transport (see following section), activation of

p70S6K, and prevention from apoptosis.

Akt is a PH domain-containing serindthreonine kinase that is activated acutely by a range

of growth factors including epidermal growth factor (EGF), PDGF, basic fibroblast growth factor,

insulin-like growth factor (IGF)- 1 and insulin (6,33,57,81). The N-terminal PH dornain is

followed by a central catalytic domain (1 10,2 10). Three mammalian isoforms have k e n

identified: Aktl (PKBa) (26,51), Akt2 (PKBB) (46) and Akt3 (PKBy) (177) which have >80%

sequence identity. Aktl and Akt2 are sirnilar in size whereas Akt3 is smaller, lacking 23 amino

acids at the C-terminus (177). It has also been demonstrated that the isoforms of Akt are

3 1

differentially regulated by insulin in a tissue-specific manner (337). Aktl is the major isoform

activated by insulin in liver and skeled muscle, whereas Akt2 is the major insulin-responsive

isoform in rat adipocytes. Akt3 is the major isoform activated by insulin in L6 skeletal muscle cells

(337).

Activation of PI 3-kinase in intact cells is both necessary and sufficient for the activation of

al1 Akt isoforrns by growth factors. This is supported by the findings that: growth factor-induced

Akt activation is sensitive to wortmannin, an inhibitor of PI 3-kinase (1 1,33,81); expression of

dominant negative foms of PI 3-kinase prevents activation of Akt (1 1.33); mutants of the PDGF

receptor that cannot interact with PI 3-kinase are incapable of Akt activation (33.81); constitutively

active foms of PI 3-kinase are able to activate Akt in intact cells (169). The magnitude and timing

of activation of Akt is also closely comlated with the magnitude and timing of increases in the

levels of the PI 3-kinase lipid products, PtdIns(3,4,5)P3 and PtdIns(3,4)P2 (57, 328).

It was initially suggested that a direct mechanism for PI 3-kinase-dependent activation of

Akt was a result of the ability of Akt, via its PH domain, to bind to PtdIns(3,4,5)P3 and

PtdIns(3,4)P2 in vitro leading to its activation (80, 81). However, it was subsequently

demonstrated that the binding of inositol phospholipids to Akt did not lead to activation (8, 168,

298). Several studies supported this latter finding that Akt could not be activated soleiy through the

interaction of phosphoinositol lipids with its PH domain. For example, deletion of the entire PH

domain failed to affect signalling through growth factor receptors (172, 174). Additiondly, it has

ken recently demonstrated that deletion of the PH domain of Akt does not impair the kinase

activity; in contrast, this deletion lead to an increase in the basal activity in cornparison to wild-type

Akt (271). These results imply that the PH domain of Akt may exen an inhibitory effect which is

relieved upon removal of the domain or by binding to phosphoinositol lipids which can then allow

for phosphorylation and thereby activation of Akt.

Thus, it is now believed that the pnmary mechanism for the activation of Aktl by insulin

results from the phosphorylation of two residues, 'I'hr308 and se873 (Thr309 and se874 in Akt2

and ~h r305 of Akt3), by distinct enzymes (6). It has been shown that phosphorylation of the two

sites occurs independently and causes strong activation of the kinase activity of Akt which can be

reversed by phosphatase treatment (12,33). Following stimulation of growth factor receptors, PI

32

3-kinase is activated, resulting in the production of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 at the

plasma membrane (330). This is followed by the interaction of Akt, via its PH domain, with the

phosphoinositides leading to its recruitment from the cytosol to the membrane (1 1). This

interaction with membrane phosphoinositol lipids results in conformational changes in Akt such

that Thr and Ser becomes accessible to phosphorylation. Subsequentiy, Akt is released fiom the

membrane enabling it to phosphorylate its specific targets. The recently cloned 3-phosphoinositide-

dependent protein kinase-1 (PDK-1) is responsible for the phosphorylation of and the

kinase responsible for the phosphorylation of se873 has been designated as PDK-2. (6. 8,298).

PDK-1 was initially purified from rabbit skeletal muscle (7) and this 67 kDa protein kinase

is cornprised of an N-terminal kinase domain that is distantly related to Akt and a C-terminal PH

domain capable of binding PtdIns(3,4,5)P3. This enzyme has been demonstrated to specifically

phosphorylate Thr308 in the presence of PtdIns(3,4,5)P3 and Ptdlns(3,4)P2 (7, 8,298) resulting

in the rapid activation of Akt in vitro (60). Interestingly, the kinase activity of PDK- 1 is not

influenced by binding to PldIns(3,4,5)P3 in vitro (9, 296); it is not stimulated by insulin; and it is

not inhibited by pharmacological inhibitors of PI 3-kinase (9,70). The high afïinity of PDK-1 for

PtdIns(3,4,5)P3 and PtdIns(3,4)P2 ensures that under basal conditions, the kinase can still

associnte with the plasma membrane (60). For these reasons, it is currently believed thai PDK- 1 is

a constitutively active, membrane-associated kinase. However, this does not preclude regulation of

the kinase at the level of substnte availability.

The insulidIGF1-induced phosphorylation of ~ e P 7 3 , similar to the phosphorylation of

~hr308, is prevented by inhibition of PI Ikinase (6), suggesting that phosphorylation of this

residue may occur by an analogous mechanism. A recent report suggests that the se873 kinase,

designated as PDK-2, may be the integrin-linked kinase (ILK) (65). It was reported that insulin

induced a transient activation of ILK in a PI 3-kinase-dependent manner, likely through the

interaction of PtdIns(3,4,5)P3 with the PH-like domain of ILK. Furthemore, it was demonstrated

that ILK could directly phosphorylate Akt on se473 in vitro, whereas a kinase-deficient fonn of

LLK severely inhibited Akt s ~ P ~ ~ phosphorylation in vivo (65). This fvst report suggests that

ILK may be PDK2, an important mediator of PI 3-kinase signals responsible for the regulation of

the kinase activity of Akt.

33

In summary, a current mode1 of signal transduction immediately downstrem of

PtdIns(3,4,5)P3 leading to activation of Akt (60) suggests that PtdIns(3,4,5)P3 andor

PtdIns(3,4)P2 mediate the localization of PDKl to the plasma membrane under basal conditions.

Upon stimulation with insulin or growth factors, Akt is recruited to the plasma membrane. where it

colocalizes with PDKl and most likely PDK-2. This localization, coupled with a PtdIns(3,4,5)P3-

mediated enhancement in the ability of Akt to becorne a substrate for PDK-1 (9,298), leads to the

phosphorylation and activation of Akt. A schematic mode1 for the activation of AktlPKB by PI 3-

kinase-dependent mechanisms is illustrated in Figure B.5.

34

Figure B.S. Activation of AktRKB by PI 3-Kinase-Dependent Mechanisms.

Basal State: Membrane

ctivated Stam

PH Domain

(Y-!3

L c=e>-L ornai -

Growth Factors

embrane

The mechanism of activation of Akt. Stimulation of cells with growth factors leads to an increase in

the concentration of PtdIns (3,4,5) P j and PtdIns(3,4)P2 in the plasma membrane. The binding of

the PH domain of Akt to the phosphoinositides recruits Akt to the plasma membrane and alters the

conformation of Akt such that Thr308 and Ser473 become accessible for phosphorylation by

PDK 1 and PDK2, respectively. The localization/regulation of PDKl , and perhaps PD=, may

also be influenced by the lipid products of PI 3-kinase. Adapted Frorn: Alessi, D.R., and P.

Cohen. Mechanism of activation and function of protein kinase B. Curr Opin Gen Devel8: 55-62,

1998.

35

Several recent reports have suggested that a PI 3-kinase-independent mechanism leading to

the activation of Akt also exists. Agents which increase intracellular CAMP levels such as forskolin

and prostaglandin El produce Akt activation in a wortmannin-insensitive manner (272).

Additionally, isoproterenol, heat-shock, hyperosmolarity and sodium arsenite-mediated activation

of Akt are also insensitive to inhibition by wortmannin (178,223). Thus. although it appears that

activation of PI 3-kinase is the major factor regulating the activation of Akt, several alternative

mechanisms leading to the activation of Akt, which have yet to be determined, may be employed

by various agents.

Akt has emerged as a downstream element of PI 3-kinase involved in the regulation of

GLUT4 translocation and glucose uptake. It bas been demonstrated that overexpression of wild-

type or constitutively active Akt 1 in rat adipocytes (53), skeletal muscle cells (99) and 3T3-L1

adipocytes (17 1) results in an upregulation of glucose transport and redistribution of GLUT4 to the

plasma membrane. These results indicate that activation of Akt 1 may be sufficient to stimulate

glucose transport to a sirnilar extent as insulin. In addition, it has been shown that insulin increases

the association of Akt2 with GLUT4-containing vesicles (19 1). However, a more definitive role

for Akt in the regulation of GLUT4 translocation remains to be established. In the absence of

specific pharmacological inhibitors to Akt, only Akt 1 mutants that act as dominant negative

inhibiton of endogenous Aktl have been used to establish a more definitive role for Akt. These

studies have yielded controversial results that may reflect the different Akt mutants and ce11 systems

used. Expression of a kinase-inactive mutant of Akt with a point mutation in the ATP-binding

domain, resulted in only a 20% reduction in insulin-stimulated GLUT4 translocation in rat

adipocytes and a rightward shift of the insulin dose-response curve (53). Expression of a second

mutant Aktl (PKBa-AA) in which the phosphorylation sites (Thr308 and Ser473) were replaced

by alanine, failed to affect insulin-stimulated glucose uptake in 3 n - L l adipocytes (162).

However, overexpression of tliis dominant negative mutant did not lead to a complete inhibition of

endogenous Aktl. In contrast, a transiently expressed kinase-dead and unphosphorylatable Aktl

(Akt 1 -AAA) successfully prevented, in full, the insulin-stimulated Akt l activation in L6 muscle

36

cells (342). Transient expression of this construct, which contains three mutations (K179A,

T308A and S473A), was also able to prevent to a large extent the insulin-induced GLUT4

translocation (342). Taken together, these results suggest that Aktl functions as an important

physiological effector downstream of PI 3-kinase in the stimulation of glucose transport, perhaps

in a cell-specific manner.

Atv~ical PKC's: Emer~ing Role in Insulin-Stimulated Glucose Trans~ort

The protein kinase C (PKC) family comprises distinct sennehhreonine protein kinases

which are ubiquitously expressed. They are currently believed to participate in many biological

functions such as ce11 growth and protein synthesis (232). PKCs have been subdivided into three

subfamilies according to their lipid-activation profiles: conventional PKCs (a, BI, PII, y) are

activated by both diacylglycerol (DAG) and calcium; novel PKCs (6, E, q/L, 0, and p) do not

respond to calcium, but require DAG; and atypical PKCs (5 and Ni) are not activated by either

DAG or calcium (4,48, 229, 240).

Atypical PKCs (aPKCs) have received considenble attention recently as they are now

believed to mediate some of the PI 3-kinase dependent signals. Evidence in favour of aPKCs

functioning as downstream targets of PI 3-kinase include: aPKCs have been shown to bind to, and

become activated by, the lipid products of PI 3-kinase in vitro (48,229,293); PDKl

phosphorylates and activates PKCC in a PtdIns-3,4.5-P3-dependent manner (48, 198); insulin

increases the activity of imrnunoprecipitable PKCh from 3T3-Li adipocytes (182) and PKCG from

3T3-LI adipocytes (20), rat adipocytes(293), and L6 skeletal muscle cells (22) in a wortrnannin-

sensitive manner (22,293). However, at present, it remains unclear which isoform, PKCC or h

are regulated by PI 3-kinase in vivo. It has been shown that the PKCC antibodies that were used in

the original studies also recognize PKCh (182). Using PCR techniques, PKCG mRNA was not

detected in 3T3-L 1 adipocytes (1 82). although it had been show that insulin could evoke a rapid

increase in the activity of immunoprecipitable PKCG from 3n-LI adipocytes (20). Furthemore,

studies in our labontory have failed to reproduce the ability of insulin to increase endogenous or

transfected PKCC activity in L6 skeletal myoblasts (342).

37

Nonetheless, it is currently believed that aPKCs participate in the PI 3-kinase dependent

stimulation of glucose transpon. For example, expression of either wild-type PKCG or PKCh

increased GLUT4 translocation and elevated basal and insulin-stimulated glucose transport (20,

2 1, 182,293). Consistent with this observation. overexpression of either a dominant negative

PKCC or PKCh mutant lead to a partial inhibition of GLUT4 recruitment to the plasma membrane

and a decrease in glucose uptake (20-22, 182,293). These results, coupled to a recent study

conducted in rat adipocytes, suggest that the aPKCs appear to be interchangeably required for the

insulin effect on the stimulation of glucose transport and GLUT4 translocation (21). Additionally,

it is conceivable that both Akt and aPKCs may function as physiological effectors of PI 3-kinase.

The results presented above indicate that both Akt and aPKCs may be required to different degrees

for the stimulation of glucose transport by insulin in a cell-specific manner. A schematic diagram of

the signals currently believed to be necessary for insulin-stirnulated glucose transport is illustrated

in Figure B.6.

38

Figure B.6. The Insulin Signalling Cascade: Proposed Signals Necessary for

Insulin-Stimulated Glucose Transport.

lnsulin

I Activating Signal

Inhibitory Slgnal -1 Containing ~esicles \ Our current hypothesis of the signals necessary for the stimulation of glucose transport.

GLUCOSE TRANSPORTERS IN TYPE 2 DIABETES

The effect of insulin to rapidly stimulate glucose uptake into muscle and adipose tissue

represents an essentiai component of normal glucose homeostasis. The first detectable step in the

pathogenesis of some patients with type 2 diabetes is resistance to insulin-stimulated glucose

uptake in peripheral tissues, including adipose tissue and skeletal muscle, the latter king

responsible for 70-908 of the glucose disposai following a carbohydrate load (63,203,207). For

this reason, many studies have focused on the involvement of glucose transporten and the role of

inherited defects in insulin signalling molecules in the pathogenesis of type 2 diabetes. It follows

that the diminished response to insulin of glucose uptake into muscle and adipose tissue could

result from any of the following possibilities: defects in the production, amplitude, tirne course,

localization of insulin signals; defects in the detection or transduction of the signal; a reduction in

the total amount of glucose transporters; andor inability of the transporters to properly dock with,

and incorponte into the plasma membrane. Of these, defects in insulin signalling and glucose

transporter levels have been identified, and emerging evidence indicates defects in glucose

transporter translocation as well. No studies have examined the mechanism of glucose transporter

interaction with the plasma membrane in either hurnans or animals with diabetes. Finally, because

the mechanism whereby the intracellular organelle detects the insulin signal is largely unknown, the

possibility that this step is defective remains unexplored. A bnef account of the status of glucose

transporter levels, GLUT4 translocation, and defects in the insulin signaling pathway in diabetes is

provided below. In addition, reference to recent studies which utilize genetic techniques to disrupt

genes of proteins in the insulin-signalling cascade in mice will also be discussed. These "knock

out" studies aim to provide models of type 2 diabetes in which investigation into the defects and/or

the source of insulin resistance can be successfully accomplished.

GLUT4 Expression.

The levels of expression of the GLUT4 glucose transporter have been analyzed in a variety

of animal models of type 2 diabetes as well as in tissue from individuals with type 2 diabetes (167,

3 18,373). It has been found that GLUT4 expression in adipocytes decreases as diabetes develops

40

in older Zucker rats (167, 252, 318). and adipose cells taken from humans with type 2 diabetes

also display a reduction in GLUT4 content (87,261,284). However, this change in GLUT4 levels

is restricted to adipose tissue and is not seen in skeletal muscle of these animal models of type 2

diabetes as normal expression of GLUT4 is observed in muscle of db/db mice and Zucker rats

(144, 145, 180). Muscle biopsies taken from individuals with type 2 diabetes also show normal

skeletal muscle GLUT4 content (IO, 88, 101,25 1). These studies highlight the tissue-specific

regulation of GLUT4 and may suggest that the net synthesis of the transporter is a key factor in

adipose tissue, whereas sorting/translocation of the transporter is more pertinent in muscle.

In an attempt to address the question of whether the changes in GLUT4 expression could

cause the diabetic state, GLUT4 was ablated from brown adipose tissue in transgenic mice (10) .

The ablation of GLUT4 resulted in a decrease in the total GLUT4 protein in adipocytes and lead to

the development of diabetes. Heterozygous GLüT4 (+/-) deficient male rnice which displayed a

generalized deficiency of GLUT4 exhibited a reduction in skeletal niuscle glucose transport. These

mice also exhibited hyperinsulinemia and were overtly diabetic (295). Further, this phenotype was

reversed through the crossing of these mice with transgenic mice expressing GLUT4 from a

muscle-specific promoter (MLC-GLUT4) (32 1). The revend of the diabetic phenotype by the

cornplementation of GLUT4 revealed that skeletal muscle GLUT4 was a major regulator of skeletal

muscle and whole body glucose metabolism. Mice that lacked GLUT4 (GLUT4 null) surpnsingly

did not become diabetic, yet exhibited abnormalities in glucose and lipid metabolism (153).

Complementation of GLUT4 nul1 mice wih the muscle-specific MLC-GLUT4 transgene generated

a population of MLC-GLüT4 null mice with restored glucose metabolism (322). However, lipid

abnormalities generated in the GLUT4 nul1 mouse were not reversed by the complementation of

muscle-specific GLUT4. In combination, these results suggest that GLUT4 rnay be the major

regulator of whole body glucose metabolism and that the defects in glucose and lipid metabolism in

the GLUT4 nul1 mice may arise separately. Moreover, GLUT4 overexpression in adipose tissue

improved glucose tolerance in insulin-resistant mice (3 16). This finding indicated that the increased

glucose flux into various tissues was capable of improving glucose homeostasis in insuiin-resistant

States. Additionally, overexpression of GLUT4 in skeletai muscle resulted in increased glucose

uptake and rnetabolism and provided protection against the development of insulin resistance in

41

transgenic mice (90, 103,200,3? 1). Collectively. these studies underscore the importance of

GLUT4 and have furthered the understanding of the role of GLUT4 in glucose metabolism.

GLUT4 Translocation

In skeletal muscle of individuals with type 2 diabetes, the reduced ability of insulin to

stimulate glucose transport is associated with a reduction in the insulin-induced translocation of

glucose transporters to the membrane. It has k e n demonstrated that the membranes of these

muscles display a diminished gain in glucose transporters in response to an insulin challenge

(372). A similar observation was also made in two animal models of diabetes (90, 161) and

defective GLUT4 translocation is also evident in fat cells from humans with type 2 diabetes (261).

Thus, it has been proposed that human insulin resistance may result from a defect in GLUT4 trafic

and targeting. In support of this, a recent study demonstrated that GLUT4 was found to

accumulate in a dense membrane compartment in hurnan skeletai muscle from which insulin was

unable to recruit GLUT4 to the ce11 surface (89). In addition, several other explanations have been

put forward to account for this reduced translocation of GLUT4 to the ceIl surface in skeletal

muscle and adipocytes. These include impaired translocation rnachinery and an inability of the

transporters to functionally incorporate into the plasma membrane, in addition to the next topic to

be discussed, an alteration in the signalling emanating from the insulin receptor.

Insuiin Si~nal l in~.

In animal models of type 2 diabetes and in individuals with type 2 diabetes, there is

considerable evidence that defects in the early stages of insulin action are associated with altered

whole body glucose homeostasis (78, 117, 146,270). There is an approximately 50% decrease in

insulin receptor phosphorylation and an 80% decrease in IRS-1 phosphorylation in liver and

skeletal muscle of ob/ob mice (78). This was associated with a more than 90% decrease in insulin-

stimulated PI 3-kinase activity associated with IRS-1 and no detectable stimulation of total PI 3-

kinase activity. Skeletal muscle isolated ftom obese subjects and individuals with type 2 diabetes

also show defects at the level of the insulin receptor tyrosine kinase activity, IRS-1 expression and

phosphorylation, and IRS- 1 -associated PI 3-kinase activity (30,9 1). A reduction in IRS- 1

expression (by 70 1) and RS-1 associated PI 3-kinase activity has also been reported in adipose

42

cells isolated from individuals with type 2 diabetes (261). In addition, insulin-stimulated Akt

kinase activity in skeletal muscle of the lean diabetic Goto-Kakizaki (GK) rat was reduced by 68%

(1 83). It has been recently demonstrated that the insulin-stimulated Akt 1 kinase activity was

reduced in skeletal muscle from individuals with type 2 diabetes, despite an increased level of Akt 1

protein expression (185). Thus, in type 2 diabetes, there are defects at four early steps of insulin

action. Whether there are also defects distal to the initial signaling events that contribute to irnpaired

translocation of GLUT4 remains to be deterrnined.

In an attempt to further understand the role of insulin signalling molecules in glucose

metabolism, disruption of the genes of proteins in the insulin signdling cascade in mice have been

investigated. Mice which lack the insulin receptor (IR null) die within 72 h after birth from severe

ketoacidosis (1, 139). In contrast, as described in an earlier section, rnice genetically manipulated

to lack IRS- 1 do not develop diabetes, despite irnpaired glucose tolerance, since pancreatic P ceIl

hyperplasia compensates for the increased insulin demand (14). Double heterozygous IMRS-1

(+/-) mice display marked insulin resistance, and 50% develop diabetes (32). Mice lacking IRS-2

develop OveR diabetes due to defects in both insulin action in peripheral tissues and in the growth

of the pancreatic p cells (354). However, in each of these models the specific role of insulin

resistance of individual tissues in the pathogenesis of diabetes is difficult to evaluate.

For this reason, the development of techniques to inactivate specific genes in specific

tissues has arisen. Very recently, a muscle-specific insulin receptor knockout mouse (MIRKO)

was generated in an attempt to determine the contribution of muscle insulin resistance to the

metabolic phenotype of diabetes (3 1). Surprisingly, the MIRKO mouse exhibited normal glucose

metabolism yet abnormaiities in lipid metabolism were evident. The lack of disruption of glucose

metabolisrn in the MIRKO mice suggested that insulin signailing in tissues other than skeletal

muscle may be more involved in insulin-regulated glucose disposal than previously recognized and

may encornpass a pnmary defect in the pathogenesis of type 2 diabetes.

In an anempt to define the pnniary site of insulin resistance which underlies the

pathophysiology of type 2 diabetes funher studies in which expression of the insulin receptor had

k e n specificaily diminished from specific tissues has recently been published. Mice lacking the

insulin receptor in the p ce11 (~IRKO) exhibited a selective loss of insulin secretion in response to

glucose, a reduced f3 cet1 mass and a progressive impairment of glucose tolerance (190). The use

of the BIRKO mice has also further established an important functional role for the insulin receptor

in glucose sensing andor survival of the P cell. Evidence from the MIRKO and PIRKO mouse

models support a novel hypothesis in which insulin resistance rt the ce11 could result in the

genesis of insulin resistance. Once established, the other tissues could acquire secondary insulin

resistance.

In addition to alteraiions in the level of expression or activation of the signaling molecules

in type 2 diabetes, the isoform selectiuity of signalling also changes in the diseased state. In

adipose cells isolated from humans with type 2 diabetes IRS-2 becomes the main docking protein

for PI 3-kinase in response to insulin (261). This is not surprising as expression of IRS-2 was

shown to increase, thereby predorninating as the main insulin receptor substrate in mice lacking

IRS-1 (14, 311).

In surnrnary, it is apparent that changes in the levels of glucose transporter expression,

defects in the insulin signaling pathway and aiterations in pattern of signalling molecules may al1

contribute to the insulin resistance associated with type 2 diabetes. Targeted gene deletion has

provided further insight into the role of specific molecules in glucose metabolism however, these

studies fail to define the actual defect(s) responsible for primary or successive insulin resistance.

Despite these obstacles in defining the primas, defect(s), the centrality of insulin resistance in type

2 diabetes invites for future research towards understanding this phenomenon in order to create

successful therapeutic strategies.

Factors That Mav Tri-

It is largely acknowledged that insulin resistance, genetically determined or acquired, is a

primary factor responsible for the manifestation of type 2 diabetes. Clinical manifestations of

insulin resistance include glucose intolerance, hyperglycemia and hypennsulinemia which arise, at

least in part, as a consequence of the inability of insulin to stimulate glucose transport into muscle

and fat. As previously discussed, it is conceivable that an impairment in the signals that emanate

from the insulin receptor andor the translocation or function of GLUT4 may represent a primary

defect responsible for the progression into an insulin resistant state. However, it has been strongîy

44

suggested that both genetic and environmental components contribute to the manifestation of

insulin resistance in the pre-diabetic state (143). Envuonmental factors, particularly those

contnbuting to obesity, may funher enhance the propensity for diabetes by accentuating the insulin

resistance.

The etiology of insulin resistance could be influenced by circulating and metabolic factors.

It was demonstrated that a state of insulin resistance could be induced in vitro upon exposure of

cells in culture to circulating factors which are thought to inhibit insulin action in vivo. These

include the cytokine tumor necrosis factor (TNF)-a (123, 124,326), and high levels of free fatty

acids (FFA) ( 158,205,28 1). Both of these are believed to act as mediators of obesity-related

insulin resistance. In addition, it has been observed that an increased flux of intracellular glucose

through the glucosamine biosynthetic pathway may represent the mechanisrn by which prolonged

exposure to high levels of glucose and insulin levels induce insulin resistance through the

downregulation of the glucose transport system (86, 113, 206, 234). This is supported by the

observation that insulin resistance of in viim muscle prepantions c m be reversed by incubation in

solutions of normal insulin and glucose levels (37 1). Thus, circulating factors and intracellular

metabolites are capable of inducing an insulin resistant state in vitro and may contribute to the

development of insulin resistance in vivo.

In contrast to the ample evidence suggesting that circulating factors and intracellular

metabolites may be involved in the induction of insulin resistance, less is known about the role of

oxidative stress in the ongin of insulin resistance. Oxidative stress is defined as the oxidative

darnage inflicted by an excess of reactive oxygen species on a ce11 or organ. The darnage reflects an

increase in free radical concentration andor a decrease in the antioxidant capacity - or oxidant

scavenging - ability of the ce11 (52,245). Several studies outline the coexistence of oxidative stress

and diabetes. For example, increased generation of oxygen free radicals and abnormally high levels

of oxidatively modified proteins have been found in diabetic BB rats (237,317); alterations in the

antioxidant capacity of serum of individuals with type 2 diabetes (34,41,244) have also been

noted. Suspected causative agents of the increased level of oxidative stress associated with type 2

diabetes include hyperglycemia, hyperinsulinernia, and altered senun antioxidant capacity. These

factors are believed to contribute to oxidative stress by prducing free radicals, oxidizing proteins,

45

and by depleting inuacellular reducing or antioxidant stores (64,355,367). The fint prospective

study undertaken to address the role of oxidative stress and antioxidants in relation to the incidence

of diabetes revealed that low serum vitamin E concentrations was associated with an increased risk

of developing diabetes four years later (273). In addition, exposure of 3T3-L1 adipocytes and L6

skeletal muscle cells in culture to reactive oxygen species lead to an impairment in insulin-

stimulated glucose transport (268) supporting an intemlationship between oxidative stress and

insulin resistance in vitro. Taken together, an interrelationship between oxidative stress and

diabetes is supported by recent in vivo studies and the use of in vitro systems may facilitate the

understanding of how oxidative stress could impair insulin action and contribute to the insulin

resistance associated with the diabetic state. Additionally, a recent study demonstrated that the

antioxidant, a-lipoic acid, provided protection against oxidative stress-induced impairment of

insulin-stimulated GLUT4 translocation and Akt activation in 3T3-L1 adipocytes (269). The ability

of antioxidants to protect against oxidative stress highlight the therapeutic potential of antioxidant

therapy in the prevention of insulin resistance and type 2 diabetes.

Anti-Diabetic Drps: Potential nierapies for Insulin Resistance and Type 2 Diabetes

Anti-diabetic dmgs represent a class of compounds with blood glucose lowenng

properties. These agents aim to norrnalize glycemic values and improve glucose tolerance, yet their

mechanisms of action and target tissues vary. A brief sumrnary of the most commonly used dmgs

as well as 'expenmental' dmgs currently under investigation is presented in Table B.3. A sumrnary

of the potential anti-diabetic properties of a-lipoic acid, an 'experimental drug', is described in

greater detail below.

Table B.3. Anti-Diabetic Drugs: Mechanisms

DRUG

Sulfonylureas

Biguanides

~hiazolidinedion;

. - -- - - -

Vanadium Derivatives ('experimental')

Primary Mechanism of Action

Hypoglycernic effects: Potentiate insulin secretion from the pancreas

Antihyperglycemic effects: Reduce hepatic glucose output

Promote adipocyte

differentiation: Ac tivate the nuclear PPARy

receptor

and Sites of Action

Secondary Effects

Extra-pancreatic effects:

Stimulate glucose transport in L6 muscle cells, 3T3-L 1 adipocytes (chronic treatmen t). Enhance transporter translocation in adipose tissue and skeletal muscle.

Stimulate glucose transport in skeletal

and cardiac muscle cells via transporter translocation (chronic treatment).

"Insulin-sensitizer", reduce FFA and

circulating triglyceride levels. Enhance insulin-stimulatd GLUT4 translocation in adipocytes and skeletal muscle after prolonged treatment only.

Insulin-mimetic properties: Enhance insulin-mediated glucose disposal in models of insulin resistance. S tirnulate transporter translocation in adipocytes and Li5 muscle cells - independent of PI 3-kinase.

a-Li~oic Acid

a-Lipoic acid (1.2-dithiolane-3-pentanoic acid, thioctic acid) is a naturally occuming

cofactor of mitochondrial enzymes involved in oxidative metabolism. It is found as lipoarnide

covalently bound to a lysyl residue in mitochondrial dehydrogenase complexes including the

pyruvate dehydrogenase complex (PDC), a-ketoglutarate and branched chah a-ketoacid

dehydrogenases (241). A natural antioxidant, exogenous administration of a-lipoic acid has been

utilized for the treatment of diabetic neuropathy (370), ischemia-repemision injury (243) and has

been shown to improve glucose metabolism (13 1). For this reason, the beneficial effects of this

compound on glucose utilization and its potential as an anti-diabetic agent is under current

investigation.

In vitro and in vivo studies have demonstrated that exogenously applied a-lipoic acid is

taken up and reduced to dihydrolipoic acid (DHLA) by NADH- or NADPH-dependent enzymes in

a variety of cells and tissues (102, 105). The chernical structures of a-lipoic acid and dihydrolipoic

acid are illustrated in Figure B.7A. More specifically, a-lipoic acid is reduced to dihydrolipoic acid

(DHLA) in the mitochondna by dihydrolipoamide dehydrogenase, the E3 component of the

pyruvate dehydrogenase complex. Dihydrolipoamide dehydrogenase is specific for the R (+)

enantiomer of a-lipoic acid and is dependent on NADH (105). Therefore, in this way a-lipoic acid

has the ability to decrease cellular NADH/NAD+ ratios elevated under such conditions as

hyperglycernia by utilizing NADH as a cofactor for its own reduction process (265). ln

erythrocytes however, glutathione reductase is more important for the reduction of Q-lipoic acid.

Glutathione reductase is localized rnainly in the cytosol, is specific for the S (-) enantiomer of a-

lipoic acid, and is dependent on NADPH (105). A schematic representation of the reduction of a-

lipoic acid to dihydrolipoic acid and its ability to recycle the intracellular antioxidant glutathione is

ilhstrated in Figure B.7B.

48

Figure B.7. Schematic Represeotation of a-Lipoic Acid, Dihydrolipoic Acid and

the Reduction Process.

a-Lipoic Acid (LA)

Dihydrolipoic Acid (DHLA)

NADH, NADPH GSH

Dihydrolipoamide Dehydrogenase, Glutathione Reductase

NAD+, NADP+ GSSG

The chernical structures of a-lipoic acid (LA) and its reduced form. dihydrolipoic acid (DHLA),

are depicted in A. (B) a-Lipoic acid utilizes NADWNADPH in its reduction process. DHLA is

then capable of regenerating other intracelluiar antioxidants including glutathione [GSH, from its

oxidized f o m glutathione disulfide (GSSG)]. Note: dihydrolipoamide dehydrogenase is specific

for the R (+) enantiomer of a-lipoic acid and is dependent on NADH, whereas glutathione

reductase is specific for the S (-) enantiomer of a-lipoic acid and is dependent on NADPH.

The important antioxidant properties attributed to a-lipoic acid include its ability to directly

scavenge reactive oxygen species and to recycle other intracellular antioxidants. The redox

muction-~idation) potential of the DHLAh-lipoic acid couple is -0.32 V (137) thus, this strong

reductant is capable of the regeneration of glutathione, vitamin C a d vitamin E (14 1). It can also

prevent lipid peroxidation, probably through its ability to scavenge free radicals such as hydroxyl,

peroxyl. and superoxide (242). The ability of a-lipoic acid to protect against oxidative stress is an

important feature that rnight be applied to counteract diabetic complications induced by oxidative

stress. However, it is not known whether these potent antioxidant properties of a-lipoic acid or its

action as a cofactor of key mitochondnal enzymes involved in the regulation of glucose metabolism

contribute to its ability to improve glucose utilization. A sumrnary of the effects of a-lipoic acid on

glucose metabolism in various animal models of insulin resistance, individuals with type 2 diabetes

and in cells in culture is presented below.

a-Lipoic acid has been shown in vitro to enhance glucose utilization in isolated rat

diaphragms (1 1 1) and glucose uptake in rat heart (283). A marked improvement in insulin-

stirnulated glucose metabolism following chronic treatment with a-lipoic acid (ncemic mixture)

was observed in insulin-resistant skeletal muscle of obese Zucker rats (133). a-Lipoic acid

(racemic mixture) treatment also stimulated glucose transport activity and enhanced insulin-

stimulated glucose uptake in skeletal muscle isolated from both lem and obese Zucker rats (1 16).

Furthermore, chronic treatment with the R (+) enantiomer of a-lipoic acid was shown to be more

effective than the S (-) enantiomer in enhancing insulin-stirnulated glucose uptake and non-

oxidative and oxidative glucose rnetabolism in skeletal muscle from obese Zucker rats (299).

Chronic R (+) a-lipoic acid treatment also significantly reduced plasma insulin and free fatty acid

levels relative to the untreated obese Zucker rats (299). In streptozotocin-induced diabetic rats,

chronic a-lipoic acid (racemic mixture) treatment reduced blood glucose concentrations with no

effect on plasma insulin levels (160). The reduction in blood glucose concen~ations was

accornpanied by an enhancement of muscle GLUT4 protein content and increased muscle glucose

utilization, as determined by increased insulin-stimulated glucose uptake (160).

The relevance of these important actions of a-lipoic acid on glucose metabolism were

further evaluated in individuals with type 2 àiabetes. Acute and repeated parenteral administration

of a-lipoic acid (racemic mixture) improved insulin-stimulated glucose disposal in individuals with

type 2 diabetes by 50 and 3096. respectively (13 1, 132). A recent study evaluated the effect of

chronic treaunent of a-lipoic acid (racemic mixture) on glucose utilization in lean and obese

patients with type 2 diabetes. Four weeks of a-lipoic acid treatment lead to a reduction in fasting

lactate and pyruvate concentrations in both diabetic groups and prevented hyperglycemia-induced

increments in serurn pyruvate and lactate levels (179). In iean diabetic patients. a-lipoic acid

treatrnent was associated with significantly reduced fasting glucose concentrations and improved

insulin sensitivity and glucose effectiveness (179). Despite the emerging evidence supponing the

beneficial effects of this compound on glucose utilization and its potential anti-diabetic properties.

the exact cellular mechanism of action of a-lipoic acid remains to be elucidated.

In order to investigate the exact cellular mode of action, studies in our labontory have

established the effect of a-lipoic acid on glucose uptake in ce11 culture systems. We have shown

that a-lipoic acid stimulated glucose uptake into the insulin-responsive L6 skeletal muscle cells in

culture (74). The naturally occumng R (+) isoform of lipoic acid had a significantly greater effect

on the stimulation of glucose uptake in L6 cells in cornparison with the S (-) isoform or the racemic

mixture (74). In addition, R (+) lipoic acid had a positive effect on both basal and insulin-

stimulated glucose uptake in L6 muscle cells, but it did not improve the sensitivity of glucose

uptake to submaximal concentrations of insulin (74). It was suggested that the increase in glucose

uptake was not attributed to the antioxidant abilities of this agent alone. Subsequently, it was

demonstrated that the increase in glucose uptake in L6 myotubes was mediated by a rapid

translocation of the GLUTl and GLUT4 glucose transporter isoforms From the intemal membrane

fraction to the plasma membrane (74).

The results presented in the following section, Chapter 1, will attempt to define a cellular

mechanism of action of R (+) a-lipoic acid in the insulin responsive 37'3-LI adipocytes in culture.

Elucidation of a-lipoic acid's mechanism of action will funher Our understanding of the

physiological role of this compound. Furthemore, a more detailed understanding of the action of

51

a-lipoic acid may exploit novel therapeutic strategies and strengthen the therapeutic potential of a-

lipoic acid as an anti-diabetic agent for the treatment of insulin resistance in type 2 diabetes.

CHAPTER ONE

UNIQUE ACTION OF AN ANTI-DIABETIC AGENT:


STIMULATION OF GLUCOSE TRANSPORT BY a-LIPOIC ACID IN

3T3-Ll ADIPOCYTES.

RATIONALE AND HYPOTHESIS

In the previous background section, 1 discussed that insulin acutely regulates glucose

uptake though the recruitment of GLUT4, and to a lesser extent GLUTl, from an intracellular

membrane stonge pool to the plasma membrane (61, 166,309). This metabolic action of insulin is

elicited by a senes of intracellular signals initiated by the binding of insulin to its receptor. The

activated receptor phosphorylates memben of the insulin receptor substrate (IRS) family, thereby

facilitating the recruitment and activation of type IA phosphatidylinositol (PI) 3-kinase (280,349).

The subsequent activation of downstream effectors of PI 3-kinase, including Akt, are also

necessary for insulin-mediated GLUT4 translocation (55,99, 173,342).

There is considerable evidence that a defect in glucose transport, such as an altention in

GLUT4 expression or translocation andor defects in the insulin signalling pathway, may be

responsible for the acquired insulin resistance of glucose utilization observed in diabetes (142).

Currently used anti-diabetic agents do not directly target glucose uptake in muscle and fat cells.

Instead, they function by regulating glycemia though the promotion of insulin release

(sulfonylureas), reduction in hepatic glucose output (biguanides) or increased gene expression

(thiazolidinediones) in fat cells.

In search of agents with anti-diabetic properties which rnay directly function at the level of

muscle and fat ceils, Our attention has focused on a-lipoic acid, a potent biological antioxidant and

a natural cofactor of oxidative metabolism (241,242). Administration of a-lipoic acid has k e n

shown to enhance insulin-stimulated glucose metabolism in various animal rnodels of insulin-

resistance (1 16, 133, 160) and to improve insulin-stimulated glucose disposal in individuals with

type 2 diabetes (13 1, 132, 179). Studies in our laboratory have shown that a-lipoic acid rapidly

stimulates glucose aansport into the insulin-responsive L6 skeletal muscle cells in culture via the

rapid redistribution of GLUTl and GLUT4 (74). The stimulation of glucose uptake by lipoic acid

in L6 skeletal muscle cells was wortmannin sensitive, which revealed the possibility that Lipoic acid

utilized PI 3-kinase in its mechanism of action. in contrast to conditions of contractile activity and

hypoxia (364), or to agents such as dinitrophenol(320) or vanadium cornpounds (246) which

increase glucose transport independentiy of PI 3-kinase, a-lipoic acid similar to insulin, utilizes

54

this enzyme. Given the possible involvement of PI 3-kinase, we predicted that a-lipoic acid may

engage elements of the insulin signai transduction cascade necessary for the stimulation of glucose

uptake. Thus. the aim of this thesis was to investigate the involvernent of lipid, tyrosine and

sennekhreonine kinases which participate in insulin signalling in R (+) a-lipoic acid' s mechanism

of action, in 3T3-LI adipocytes.

EXPERIMENTAL PROCEDURES

Materials

Dulbecco's Modified Eiagles Medium (DMEM), calf semm (CS), fetal bovine serurn (FBS)

and other tissue culture reagents were purchased from GIBCO/BRL (Burlington, ON, Canada). R

(+) a-Lipoic acid was obtained from ASTA Medica (Frankfurt, Germany). For simplicity, R (+)

a-lipoic acid will be referred to as a-lipoic acid. Human insulin (Humulin R) was obtained from

Eli Lilly Canada Inc. (Toronto, ON, Canada). Protein A-Sepharose and protein G-Sepharose were

from Phamacia (Upssala, Sweden). The antibody to the a subunit of the insulin receptor (ARS-2)

was a kind gift from Dr. C. Yip (Department of Physiology, University of Toronto). Polyclonal

anti-Akt 1 (C-20), monoclonal anti-phosphotyrosine (PY99) antibody, and monoclonal anti-insulin

receptor p (29(34) antibody were purchased from Santa Cruz Biotechnology (Santa Cruz. CA,

USA). Polyclonal anti-IRS- 1 antibody, monoclonal anti-phosphotyrosine antibody and Akt

substrate peptide (Crosstide) were from Upstate Biotechnology (Lake Placid, NY, USA).

Polyclonal anti-GLUTl and anti-GLUT4 glucose transporter antisera were from East Acres

Laboratones (Southbridge, MA, USA). Purified L-a-phosphatidylinositol (PI) was purchased

from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Oxalate-treated TLC Silica gel H plates (250

microns) were from Anaitech (Newark, DE, USA). Wortmannin was from Sigma (Si. Louis, MO,

USA). Microcystin, erbstatin and okadaic acid were from BioMol (Plymouth Meeting, PA, USA).

Enhanced Cherniluminescence (ECL) reagents and [ Y ~ ~ P I - A T P (6000 Ci/mmol) were purchased

from Amersham (Oakville, ON, Canada). Al1 electrophoresis and irnrnunoblotting reagents were

purchased from BioRad (Mississauga, ON, Canada). AI1 other reagents were of the highest

analyticai grade.

Methods

Cell Culture and Incubations

Mouse 3T3-L1 fibroblasts received from ATCC (Arnerican Type Culture Collection) were

cultured in DMEM (containing 25 mM glucose) supplemented with 20% (voVvol) CS and 1%

(voVvol) antibiotic/antimycotic solution (10 000 unitslml penicillin, 10 mdml streptomycin). Cells

were passaged and maintained in an atmosphere of 5% C02-95% air at 370C. For expenments.

cells were trypsinized with 0.05% trypsin and seeded into 12 well dishes (glucose transport

studies), 6 well dishes (imrnunoprecipitation andor kinase assays), or into 10 cm dishes

(subcellular fractionation experiments). Cells were rnaintained in DMEM/CS, which was changed

every 48 h, until2 days post confluence. At this time, differentiation into adipocytes was induced

by the addition of 0.25 pM dexamethazone, 0.5 mM 1-methyl-34sobutylxanthine and 10 pg/ml

porcine insulin in DMEM containing 10% FBS (voVvol) (301). After four days the medium was

changed to DMEM/FBS containing 10 pg/ml insulin for an additional two days. Differentiated

adipocytes were rnaintained in DMEM/10% FBS which was changed every 48 h. Cells were used

12- 13 days post initiation of differentiation. Prior to al1 experimentai manipulations, 3T3-Ll

adipocytes were deprived of serurn for at least 3 houn.

2 - ~ e o x v - 3 ~ - D - ~ l u c o s e Ugtake

3T3-L1 Adipocytes were cultured in 12-well plates and subjected to the appropriate

treatments as follows: 2.5 rnM a-lipoic acid (1 h), LOO nM insulin (unless otherwise specified, 20

min), 100 nM wortmannin (30 min pretreatment), 10 ~g/d erbstatin (20 min pretreatment). Ce11

monolayers were then nnsed two times with glucose-free HEPES-buffered saline solution (HBS,

140 m . NaCl, 2.4 m M MgS04,5 m . KI, 1 m M CaC12,20 mM Na-HEPES pH 7.4) and any

remaining liquid was aspirated. Cells were then incubated for 5 min in HBS containing 10 pA4

unlabelled 2-deoxy-D-glucose and 10 p.M 2-deoxy-~~-~-glucose (1 pCi/ml) in the absence of

insulin. The reaction was terminated by washing three times with ice-cold 0.9% NaCl. Non-

specific uptake was determined in the presence of 10 ph4 cytochalasin B and was subtracted from

total uptake. Cell-associated radioactivity was determined by lysing the cells with 0.05 N NaOH

and the cellular radioactivity of a 0.8 ml aliquot of the cell lysate was quantitated by liquid

57

scintillation counting. Each upiake expriment was perforrned in triplicate, and the results of

specific uptake are expressed as mean I SEM in picornoles per milligram total cellular protein per

minute.

bcellular Fractionation of 3T3-L1 Adi~ocvtes

Subcellular fractionation of 3T3-LI adipocytes was carried out as descnbed (263) to obtain

plasma membranes (PM), high density rnicrosomes (DM) and low density microsornes (LDM).

Briefly, cells grown in 10 cm dishes were treated with 2.5 mM a-lipoic acid for 1 h or with 100

nM insulin for 20 min and were nnsed twice with cold homogenizatinn buffer (250 rnM sucrose,

20 mM HEPES, pH 7.4.5 m M NaN3,2 mM ethylenediamine-tetraacetic acid (EDTA), 1 mM

Na3V04.200 j&l phenylmethylsulfonyl fluoride (PMSF), 1 leupeptin, and 1 pM pepstatin) . Cell monolayers were scraped into cold homogenization buffer and homogenized with 10 strokes

though a Cell Cracker (clearance 0.0016 in). The homogenate was centrifuged at 19,000 g for 20

min, the pellet was resuspended in 5 ml homogenization buffer, layered ont0 Buffer 2 (1.12 M

sucrose, 1 mM EDTA and 20 rnM HEPES, pH 7.4) and centrifuged at 100,000 g for 60 min. The

membranes recovered on top of Buffer 2 were resuspended in homogenization buffer and pelleted

ai 40,000 g for 20 min. The pelleted membranes were designated PM based on enzyme marker

composition. The supernatant frorn the 19,000 g spin was sedimented at 40,000 g for 20 min to

yield HDM as the pellet and this supernatant was centrifûged at 195,000 g for 75 min to pellet the

LDM.

Irnmuno~recioitation and Assav of Phosohatidvlinositol 3-Kinase Activitv.

3T3-L1 adipocytes grown in 6 cm dishes were treated with 2.5 mM a-lipoic acid or 100 nM

insulin for the indicated time pends as described in the figure legend and were washed twice with

ice-cold phosphate-buffered saline (PBS) and lysed in 1 ml Buffer A (20 mM Tris, pH 7.5, 137

mM NaCI, 1 mM MgCI2, 1 mM CaC12, 1% NP-40 (voVvol), 10% glycerol (voVvol), 10 rnM

sodium pyrophosphate, 100 mM NaF and 1 mM Na3V04 containing a mixture of protease

inhibitors (1 p M leupeptin, 1 pM pepstatin A, and 200 ph4 PMSF). After 15 min of slow agitation

and centrifugation (15, M)O g for 15 min), 500 pg of total ceilular protein was subjected to

immunoprecipitation using 2 pg anti-phosphotyrosine (PY99) antibody for 2-3 h under constant

58

rotation (40C). Irnmunoprecipitates were complexed to a combination of 20 pl each of protein A-

and protein G- Sepharose beads (100 rnghl) for 1-2 h under constant rotation (40C). The

immunocomplexes were washed three times with Buffer 3 (PBS containing 1% NP40 (voVvol)

and 100 ph4 Na3V04), three times with Buffer 4 (100 mM Tris, pH 7 5 5 0 0 m M LiCl and 100

pM Na3V04) and twice with Buffer 5 (10 m M Tris, pH 7.5, LOO rnM NaCl, 1 m . EDTA and

100 Na3V04). The pellets were resuspended in 55 pl of 10 mM Tris, pH 7.5, 100 m . NaCI,

1 rnM EDTA, 100 pM Na3V04, 11 pl of 100 mM MgCl2 and 10 pl phosphatidylinositol(2

rnghl) in 10 m M Tris, pH 7.5 and 1 rnM EGTA. The reaction was initiated by the addition of 5 pl

of 440 pM ATP containing 10 pCi [Y~*P]ATP. After 15 min at 30°C, the reaction was terminated

by the addition of 20 11 of 8 M HCl and 160 pl of CHCl~:methanol(l: 1, vovvol). The samples

were centrifuged for 5 min at maximum speed in a microcentrifuge, and 40 pl of the lower organic

phase was removed and applied to a potassium oxalate (1 96) pretreated Silica gel 60 TLC plate

which was treated with trans- 1 ,2-diaminocyclohexane-N,N,N',N1,N1-tetra-acetic acid and prebaked

for 10 min at 1000C. The PtdIns3P and PtdIns4P (phosphatidylinositoI3- and Cphosphate,

produced by PI 3-kinase and PI Clcinase, respectively) were separated in the presence of boric acid

(340).The detection and quantitation of [ 3 2 ~ ] ~ ~ 3 ~ on TLC plates were done using a Molecular

Dynamics PhosphorIrnager System (Sunnyvale, CA, USA).

Irnmuno~reci~iiation and Assav of Akt l Protein Kinase Activitv.

3T3-Li adipocytes grown in 6 cm dishes were treated with 2.5 rnM a-lipoic acid or 100 nM

insulin for the indicated time periods as described in the figure legend. Cells were lysed with lysis

buffer containing 50 rnM HEPES, pH 7.6, 150 rnM NaCl, 10% glycerol (voVvol), 1% Triton X-

100 (volhol), 30 rnM sodium pyrophosphate, 10 rnM NaF, 1 mM EDTA, 1 mM PMSF, 1 pM

leupeptin, 1 ph4 pepstatin A, 1 mM benzamidine, 1 mM Na3 VOq, 1 mM dithiothreitol (Dm) and

100 nM okadaic acid. Anti-Aktl antibody (C-20) was pre-coupled to a mixture of protein A- and

protein G-Sepharose beads by incubating 2 ~g of antibody per condition with 20 pl each of the

protein A-and protein G-Sephamse beads (100 mglrnl) for a minimum of 2 h. These anti-Aktl

coupled beads were washed twice with ice-cold PBS and once with ice-cold lysis buffer. Aktl was

immunoprecipitated by incubating 200 pg of total cellular protein with the anti-Akt l bead complex

59

for 2-3 houn under constant rotation (40C). Akt 1 imrnunocornplexes were isolated and washed 4

times with 1 ml wash buffer (25 m M HEPES, pH 7.8, 10% glycerol (voUvol), 1% Triton X-100

(voVvol), 0.1% bovine serum albumin (voVvol), 1 M NaCl, 1 mM DïT, 1 mM PMSF, 1 pM

rnicrocystin and 100 nM okadaic acid) and twice with 1 ml kinase buffer (50 rnM TrisMCl, pH

7.5, 10 m M MgCl2 and 1 mM Dm). This was then incubated under constant agitation for 30 min

at 30oC with 30 pl of reaction mixture (kinase buffer containing 5 pM ATP, 2 pCi [~~*P]ATP and

LOO pM Crosstide). Following the reaction, 30 pl of the supernatant was transferred ont0

Whatman p81 filter paper and washed 4 times for 10 min with 3 ml of 175 mM phosphonc acid

and once with distilled water for 5 min. Filters were air-dried and then subjected to liquid

scintillation counting.

Detection of Insulin Rece~tor Substrate-1 Phosahorvlation.

3T3-L1 adipocytes grown in 6 cm dishes were treated with 2.5 rnM a-lipoic acid or with

100 nM insulin as indicated in the figure legend, rinsed twice with ice-cold phosphate-buffered

saline containing 100 pM Na3V04 and lysed with 0.5 ml of Buffer A 120 mM Tris, pH 7.5, 137

mM NaCI, 1 mM MgCI2, 1 rnM CaC12, 1% NP-40 (voVvol), 10% glycerol (voYvol), 10 rnM

sodium pyrophosphate, 100 mM NaF and 1 m M Na3V04 containing a mixture of protease

inhibitors (1 pM leupeptin, 1 FM pepstatin A, and 200 pM PMSF)]. Lysates were passed five

times though a 25-gauge syringe and then incubated for 15 minutes at 4QC under constant rotation.

Debris and unbroken cells were removeci by centrifugation. 500 kg of total cellular protein was

incubated with 2 pg anti-IRS- 1 antibody for 2-3 hours under constant rotation (4QC) followed by a

1 hour incubation with 30 pl protein A Sepharose beads (100 mglml). Immunocomplexes were

washed 4 times witb phosphate-buffered sidine containing 100 pM sodium orthovanadate and

0.1 % NP-40. Pellets were resuspended in 30 pl 2X Laemmli sarnple buffer (125 mM Tris-HCl pH

6.8,4% SDS, 10% 2-mercaptoethanol, 20% glycerol, and 0.01% biomophenol blue) and boiled

for 5 minutes. Roteins were resolved by 7.5% sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) and then electrotransferred ont0 polyvinylidene difluoride (PVDF)

membranes. To detect tyrosine-phosphorylated [RS-1, the blots were probed with anti-

phosphotyrosine antibody ( 1 5000 dilution) and protein detected by the enhanced

60

chemiluminescence method using sheep anti-mouse immunoglobulin conjugated to horseradish

peroxidase (HRP, 1 :2000 dilution) as the secondary antibody. Autoradiograms of X-ny films

exposed to produce bands within the linear range for quantitation were scanned in a Microtek

ScanMaker IIHR and quantitated using the computer software NIH Image.

Detection of Insulin Rece~tor Phos~horvlation.

3T3-L1 adipocytes grown in 6 cm dishes were treated with 2.5 mM a-lipoic acid or with

100 nM insulin as indicated in the figure legend, rinsed twice with ice-cold PBS containing 100

pM Na3V04 and lysed with 0.5 ml of lysis buffer [150 mM NaCI, 50 m M Tris, pH 7.2,0.25%

(voVvol) deoxycholate, 1% (voUvol) NP-40, 10 m M sodium pyrophosphate, 100 m M NaF, 2 rnM

EDTA, 1 rnM Na3V04 containing a mixture of protease inhibitors (1 leupeptin, 1 pM

pepstatin A, and 200 pM PMSF)]. Lysates were passed five times though a 25-gauge syringe and

then incubated for 15 minutes at 40C under constant rotation. Debris and unbroken cells were

removed by centrifugation. One rnilligram of total cellular protein was incubated with 0.5 mglm1

anti-ARS-2 antibody for 2-3 hours at 40C under constant rotation, followed by a 1 h incubation

with 30 pl protein A-Sepharose beads (LOO mglml). Immunocornplexes were washed 5 tirnes with

phosphate-buffered saline containing 100 FM Na3V04 and 0.1% NP-40 (voVvol). Pellets were

resuspended in 30 pl 2X Laemmli sample buffer and boiled for 5 minutes. Proteins were resolved

by 7.5% SDS-PAGE and then electrotransferred onto PVDF membranes. To detect tyrosine-

phosphorylated IR, the blot was probed with anti-phosphotyrosine antibody (l:5000 dilution), and

protein was detected by the enhanced chemiluminescence method using sheep anti-mouse

imrnunoglobulin conjugated to horseradish peroxidase (HRP) (1:2000 dilution) as the secondary

an ti body.

Unstirnulated 3T3-L1 adipocytes grown in 6 cm dishes were rinsed twice with ice-cold PBS

containing 100 pM Na3V04 and lysed with 0.8 ml of lysis buffer [150 mM NaCl, 50 m M Tris,

pH 7.2,0.25% (voVvol) deoxycholate, 1% (voVvol) NP-40, 10 mM sodium pyrophosphate, LOO

mM NaF, 2 m M EDTA, 1 m M Na3V04) containing a mixture of protease inhibitors (1 pM

leupeptin, 1 pM pepstatin A, and 200 plbl PMSF)]. Lysates were passed five times though a 25-

61

gauge syringe and then incubated for 15 min at 40C under constant rotation. Debris and unbroken

cells were removed by centrifugation. One miIligram of total cellular protein was incubated with 2

pg of anti-insulin receptor (2964) antibody for 2-3 h at 40C under constant rotation, followed by a

1 h incubation with 20 pl each of protein A- and G-Sepharose beads (100 mgfml).

Irnrnunocomplexes were isolated and washed 2 times with 1 ml wash buffer (50 mM HEPES. pH

7.5, O. 1% Triton X- 100 (voVvol), 150 mM NaCl. 1 m M Na3V04, 1 pM leupeptin. 1

pepstatin A, and 200 pM PMSF) and twice with 1 ml kinase buffer (50 rnM HEPES, pH 7.4, 150

m M NaCI, 12 rnM MgC12,2 mM MnC12,0.2% Triton X-100 (voVvol) and 1 mM Na3V04). The

irnmunocomplexes were then treated in vitro with 1.0 ml kinase buffer containing either 2.5 mM

a-lipoic acid or 100 nM insulin as indicated in the figure legend. This was then incubated under

constant agitation for 10 min at 300C with 65 pl of kinase buffer containing 25 pM ATP and 2 1Ci

[ y 3 2 ~ ] ~ ~ ~ . The reaction was terminated with the addition of 30 pl of 2X Laemmli sample buffer

and irnmunocornplexes were boiled for 5 minutes. Proteins were resolved by 7.5% SDS-PAGE.

The gel was dried and the detection and quantitation of 3 2 ~ incorporated into the insulin receptor

were accomplished though the use of a Molecular Dynarnics PhosphorIrnager System (Sunnyvale.

CA, USA).

Statistical Analvsis.

Autoradiograrns of X-ray films exposed to produce bands within the linear range for

quantitation were scanned in a Microtek ScanMAker IMR and quantitated using the cornputer

software NIH Image. Statistical analysis was performed using the analysis of variance test

(ANOVA, Fisher's multiple cornparisons test) or the paired two-tailed Z test as indicated in figure

legends.

RESULTS

a-Li-pic Acid Stimulates Glucose U~take in 3T3-L1 Adiwcvtes.

We have previously demonstnted that acute treatment of L6 skeletal muscle cells in culture

with a maximally effective dose of R (+) a-lipoic acid (2.5 mM) stimulated glucose transport (74).

Here we dernonstrate that treatment of 3T3-LI adipocytes for 1 h with 2.5 rnM R (+) a-lipoic acid

rapidly stimulated glucose uptake as shown in Figure 1.1. For simplicity, R (+) a-lipoic acid will

be referred to as a-lipoic acid in ail subsequent experirnents. The rate of glucose transport

increased Cfold in response to a-lipoic acid (basal: 4.2 pmol. min-l.mg-1 protein, a-lipoic acid:

17.1 pmol.rnin-l.mg-l protein, pcO.05). In cornparison, treatment of 3T3-LI adipocytes with the

maximally effective dose of 100 nM insulin elicited a 6.7-fold increase in glucose uptake (basal:

4.2 pmol.min-l.mg-l protein . insulin: 28.0 pmol.min-l.rng-l protein. p<0.005).

63

Figure 1.1. a-Lipoic Acid Stimulates Glucose Uptake in 3T3-Ll Adipocytes.

Figure 1.1.3T3-L1 Adipocytes grown in 12-well dishes were deprived of semm for 3 h, then

treated with 2.5 m M a-lipoic acid (LA) for 1 h or with LOO nM insulin (1) for 20 min. 2-Deoxy-

~ H - D - ~ I U C O S ~ uptake was subsequentiy determined over a 5 min period. Results represent the

mean i SEM of 4-7 experiments, with al1 conditions assayed in triplicate. * Significantly different

from control (C), ~~0.05 (ANOVA, Fisher's multiple cornparisons test).

The Effect of a-Li~oic acid on Insulin-Stimulated Glucose U~take in 3T3-LI Adi~ocvtes.

The rapid stimulatory effect of a-lipoic acid on glucose uptake is shared with the action of

insulin, but not of other currently used anti-diabetic agents. In order to determine the effect, if any,

of this compound on insulin sensitivity and responsiveness we examined the effect of a-lipoic

acid on insulin-stimulated glucose transport in 3T3-L1 adipocytes. For these expenments, the cells

were incubated with a-lipoic acid (2.5 mM) for 60 min, and insulin was added during the last 20

min at the indicated concentrations (concentrations ranging from 1 to 1000 nM). Figure 1.2

demonstrates that the combination of a-lipoic acid and insulin treatment was not additive on the

stimulation of glucose uptake. Insulin was able to stimulate glucose uptake in a dose-dependent

manner and upon the addition of a-lipoic acid a leftward shift in the insulin dose-response cume

was observed. These results demonstrated that a-lipoic acid had a small, positive effect on insulin

sensitivity, but did not affect insulin responsiveness. However, the ability of a-lipoic acid to

potentiate the effect of insulin on the stimulation of glucose transport failed to reach statisticd

significance at any of the concentrations tested.

65

Figure 1.2. The Effect of a-Lipoic Acid on Insulin-Stimulated Glucose Uptake in

3T3-L1 Adipocytes.

- 1 10 100 1000

Log insulin (nmolll)

Figure 1.2. 3T3-L1 Adipocytes grown in 12 well dishes were treated with 2.5 mM a-lipoic acid

for 60 min and with insulin at the indicated concentrations during the last 20 min. ~ - D ~ O X ~ - ~ H - D -

glucose uptake was subsequently deterrnined over a 5 min period. Results represent the mean f

SEM of 3-6 experiments that were performed in tripiicates. (C): Insulin alone. (LA): a-lipoic acid

+ insulin.

a-Limic Acid Stimulates the Translocation of GLUTl and GLUT4 to the Plasma Membrane.

To determine the mechanism underlying the ability of a-iipoic acid to stimulate glucose

transport in 3T3-Ll adipocytes, we examined the effect of a-lipoic acid on the subcellular

distribution of the glucose transporters GLUTl and GLUT4. Equal arnounts of protein from each

fraction was resolved by 10% SDS-PAGE and immunoblotied for GLUTl or GLUT4. The results

of four independent experiments were averaged and representative irnmunoblots of GLUTl and

GLUT4 redistribution are depicted in Figure 1.3. Figure 1.3A illustrates that treatrnent of 3T3-LI

adipocytes with a-lipoic acid for 60 min augmented the level of GLUTl in the plasma membrane

fraction (PM) by 2.1 1: 0.4 fold (p<0.05), relative to levels in the PM of untreated cells (control,

C). Insulin caused a similar gain in GLUTl in the plasma membrane (2.1 I 0.3 fold, pc0.05). a-

Lipoic acid and insulin reduced GLUTl content in the low density microsomal fiaction (LDM) of

3T3-LI adipocytes by 26% and 61 %, respectively.

The effect of GLUT4 translocation in response to a-lipoic acid and insulin is illustrated in

Figure 1.38. a-Lipoic acid caused a 2.9 f 0.9 fold (pc0.05) increase in GLUT4 content in the

PM, relative to PM of control cells. Insulin raised the level of GLUT4 at the plasma membrane by

4.8 I 1.6 fold (peO.05) above control. There was no statistically significant difference beiween the

mount of GLUT.1 in the PM of insulin- and a-lipoic acid-treated cells. The concomitant reduction

in the level of GLUT4 in the LDM in response to lipoic acid and insulin was reflected by a

reduction in the level of this transporter by 17 % and 378, respectively. These results indicate that

in 3T3-L1 adipocytes, a-lipoic acid is able to cause a rapid translocation of GLUTl and GLUT4 to

the plasma membrane which was associated with a reduction in the level of these transporters in the

low density microsomal fraction.

67

Figure 1.3. a-Lipoic Acid Stimulates the Translocation of GLUTl and GLUT4 to

the Plasma Membrane.

PM LDM

CLUT1 - C I L A C I L A

PM LDM

PM LDM

GLUT4 - LDM

68

Figure 1.3. Effect of a-lipoic acid on the distribution of glucose transporters in 3T3-L1

adipocytes. Plasma membranes (PM) and low density microsornes (LDM) were isolated by

subcellular fractionation from 3T3-Ll adipocytes that were treated for 1 h with 2.5 mM a-lipoic

acid (LA) or for 20 min with 100 nM insulin (I). Fifteen micrograms of protein from the indicated

fractions were resolved by 10% SDS-PAGE and immunoblotted for GLUTl (A) or GLUT4 (B).

Immunoreactive bands were scanned within the linear range and the protein was quantitated using

the computer software NIH Image. A representative immunoblot of 4 independent experiments is

shown. Results are the means it SEM of 4 independent experiments and are expressed in relative

units, with the average reading of control (C) PM assigned a value of 1. *Significantly different

from control PM, pe0.05 (Z-test, two tailed). #significantly different from control LDM, pc0.05

(ANOVA. Fisher's multiple cornparisons test).

Womnannin Revents the Stimulation of Glucose Transmrt bv a-Liwic Acid in 3T3-LI

To elucidate the mechanism underlying the stimulation of glucose bansport by a-lipoic

acid. we examined the possible engagement of molecules that are known to mediate insulin action.

We first examined the effect of wortmannin, a relatively specific inhibitor of class 1 PI 3-kinases,

on the a-lipoic acid-stimulated glucose transport. a-Lipoic acid stimulated glucose vansport in

3T3-L1 adipocytes by Cfold (basal: 4.2 pmol.min-l.mg-l, a-lipoic acid: 17.1 pmol.min-lmg-1

protein. pcO.05). Figure 1.4 shows that pretreatment of 3T3-L1 adipocytes with 100 nM

wortmannin for 30 min abrogated the ability of a-lipoic acid to stimulate glucose transport (control:

3.7 prnol.min-l.mg- l protein. a-lipoic acid + wortmannin: 2.7 pmol.min-l.mg-l protein,

p<0.05). Similarly, wortmannin abolished insulin-siimulated glucose transport (insulin + wortmannin: 3.4 pmol.min- l .mg- 1 protein, pc0.005). The reduction in the ability of a-lipoic acid

to stimulate glucose transport in the presence of wortmannin suggests that PI 3-kinase is a

necessary component in the signalling pathway utilized by a-lipoic acid.

70

Figure 1.4. Wortmannin Prevents the Stimulation of Glucose Transport by a-

Lipoic Acid in 3T3-L1 Adipocytes

Figure 1.4.3T3-L1 adipocytes grown in 12-well dishes were treated for 60 min with 2.5 rnM a-

lipoic acid (LA) or with 100 nM insulin (1) for 20 min, in the absence or presence of 100 nM

wortmannin (W, 30 min preneatment). 2 - ~ e o x ~ - 3 ~ - ~ - ~ l u c o s e uptake was subsequently

determined over a 5 min period. Results represent the mean f SEM of 3-7 experiments in which * each condition was assayed in triplkate. Significantly different from conaol, p<0.05 (ANOVA,

Fisher's multiple comparisons test). #significantiy different from corresponding controls, p<0.05

(ANOVA, Fisher's multiple comparisons test).

Activation of PI 3-Kinase b~ a-Lipic Acid in 3T3-LI Adiwcvtes.

The results obtained above with wortmannin suggested that a-lipoic acid may engage PI 3-

kinase. To further explore this possibility. we determined the effect of a-Iipoic acid on PI 3-kinase

activity associated with anti-phosphotyrosine immunoprecipitaies, using an in vitro kinase assay

(Figure 1.5). Treatment of 3T3-LI adipocytes with a-lipoic acid raised the phosphotyrosine-

associated PI 3-kinase activity to 4.0 i 1.3 fold (pc0.05) at 5 min, relative to control, and to 4.4 k

0.9 fold (pc0.05) at 10 min, relative to control. Although elevated, PI 3-kinase activity stimulated

by a-lipoic acid ai 20 and 30 min were not significantly different from basal (3.4 f 0.2 and 2.2 k

0.3, respectively). Insulin treatment (5 min) induced a greater increase in the level of

phosphotyrosine-associûied PI 3-kinase activity (13.2 f 1.3 fold, pc0.0001). In vitro treatment

with wortmannin (100 nM) abolished the insulin and a-lipoic acid stimulation of PI 3-kinase

activity (data not shown).

Figure 1.5. Activation of PI 3-Kinase by a-Lipoic Acid in 3T3-L1 Adipocytes.

Figure 1 S. 3T3-L 1 adipocytes were treated with 2.5 m M a-lipoic acid (LA) or with 100 nM

insulin (1) for the indicated time periods. PI 3-kinase activity associated wiih anti-phosphotyrosine

irnmunoprecipitates was then determined using an in vitro kinase assay as described in

METHODS. Resuits are expressed in relative units and represent the mean i SEM of 4

independent experiments. Kinase activity in the absence of any treatment was assigned a value of

1 .O. *significantlY different from control, p<0.05 (ANOVA, Fisher's multiple cornparisons test).

Effect of cx-lipoic Acid on Aktl Activihr in 3T3-L1 Adiwcvtes.

Aktl is one of the downstream effectors of PI 3-kinase activated by insulin. There is strong

evidence to support a role for Aktl in insulin-stimulated glucose transport, yet the regulation of

Akt 1 by insulinomirnetic agents has been inadequately described. Given the ability of a-lipoic acid

to increase PI 3-kinase activity, we next examined whether Aktl is aiso activated by a-lipoic acid.

Figure 1.6 shows that a-lipoic acid increased the activity of Akt 1 by 2.4 I 0.5 fold (pc0.05) in 5

min and by 2.2 f 1.0 fold ( ~ ~ 0 . 0 5 ) in 10 min. Activation of Aktl by a-lipoic acid subsided in

tirne, so that at 20 and 30 min it did not reach statistical significance (1.6 + 0.2 and 1.7 f 0.2,

respectively). Hence, the rise in a-lipoic acid-stimulated Akt 1 activation followed a similar

temporal pattern to the stimulation of PI 3-kinase activity. By cornparison, Aktl activity was

elevated 4.1 5 0.8 fold after 5 min of exposure to insuiin (peO.0001).

Prewatment of the cells with 100 n . wortmannin prevented the activation of Aktl by a-

lipoic acid similar to the inhibition of insulin-stimulated Aktl activity. This suggests that the

activation of Aktl by a-lipoic acid is indeed downstream of PI 3-kinase as inhibition of PI 3-

kinase with wortmannin abolished the nse in Akt 1 activity in response to a-lipoic acid.

Figure 1.6. Effect of a-Lipoic Acid on Aktl Activity in 3T3-L1 Adipocytes.

Figure 1.6. Total ce11 lysates were prepared frorn 3T3-L1 adipocytes that had been treated for the

indicated times with 2.5 mM a-lipoic acid (LA) or with 100 nM insulin (1) in the presence or

absence of 100 nM wortrnannin (W, 30 min pretreatment). Aktl was immunoprecipitated and

kinase activity subsequently determined using an in vitro kinase assay. Results are expressed in

relative uni& and represent the mean f SEM of 3-5 independent experiments. Kinase activity in the

absence of any treatment was assigned a value of 1.0. *significantly different fiom control,

pc0.05 (ANOVA, Fisher's multiple comparisons test). *significantly different from insulin (1,5

min), pc0.0001 (ANOVA, Fisher's multiple comparisons test). A~ignificantly different from a-

lipoic acid (LA, 5 min), pc0.05 (ANOVA, Fisher's multiple comparisons tests).

Effect of Erbstatin on a-Lipoic Acid-Stimulated Glucose U take in 3T3-L1 Adi-tes.

It was demonstrated in Figure 1.5 that a-lipoic acid induced the activation of PI 3-kinase

bound to phosphotyrosine-containing proteins. To further examine the role of phosphotyrosyl

proteins in the ability of a-lipoic acid to stimulate glucose transport we utilized a tyrosine kinase

inhibitor. erbstatin, and studied its effect on a-lipoic acid-stimulated glucose uptake. Figure 1.7

demonstrates that pretreatment of 3T3-L1 adipocytes with erbstatin (10 pg/ml) for 20 min partially

reduced the ability of a-lipoic acid to stimulate glucose uptake (a-lipoic acid: 30.1 pmolmin-

l.mg-l protein, a-lipoic acid + erbstatin: 21.4 pmolmin-l.mg-1 protein). However, this

reduction failed to reach statisticd significance (p= 0.07 ANOVA, Fisher's multiple comparisons

tests). Similarly, treatrnent of 3T3-LI adipocytes with erbstatin partially reduced the ability of

insulin to stimulate glucose uptake [insulin: 36.8 pmolmin-l.mg-1 protein, insulin + erbstatin:

27.4 pmol.min-l.mg-l protein, p= 0.059 (ANOVA, Fisher's multiple comparisons tests)].

Preliminary studies in L6 skeletal muscle cells in culture demonstrated that increasing

concentrations of erbstatin prevented the stimulation of glucose uptake by both a-lipoic acid and

insulin in a dose-dependent manner. with maximal inhibition of glucose uptake evident at 40 pghi

erbstatin (data not shown). Thus, the reduction in the ability of a-lipoic acid to stimulate glucose

uptake in the presence of a tyrosine kinase inhibitor suggests the involvement of tyrosine

phosphorylation in a-lipoic acid's mechanism of action.

76

Figure 1.7. Effect of Erbstatin on a-Lipoic Acid Stimulated Glucose Uptake in

3T3-L1 Adipocytes.

Figure 1.7. 3T3-L1 adipocytes grown in 12-well dishes were treated for 60 min with 2.5 mM a-

lipoic acid (LA) or with 100 nM insulin (I) for 20 min, in the absence or presence of 10 pghl

erbstatin (E, 20 min pretreatment). 2 - ~ e o x ~ - 3 ~ - ~ - ~ l u c o s e uptake was subsequently determined

over a 5 min period. Results represent the mean f SEM of 5 experiments in which each condition

was assayed in triplicate.

Effect of a-Lipoic Acid on IRS- 1 Phosphoqlation in 3 n - L 1 Adiwcvtes.

To specifically address the involvement of phosphotyrosyl proteins in a-lipoic acid's

mechanism of action we explored whether the cornpound affected the level of tyrosine

phosphorylation of IRS- 1. iRS-1 is the predominant IRS protein in differentiated 3T3-LI

adipocytes and it may be a major mediator of the metabolic actions of insulin in these cells (306,

362,369). Immunoprecipitates of IRS- 1 from 3T3-L 1 adipocytes that were treated with a-lipoic

acid or insulin for the indicated time periods were resolved by 7.5% SDS-PAGE and

immunoblotted with anti-phosphotyrosine antibody. Figure 1.8 illustrates that a-lipoic acid

treatment for 2 or 5 min stimulated tyrosine phosphorylation of IRS-I by 3.1 f 1.1 fold (p~0.05)

or 3.3 f 1.1 fold (pc0.05), relative to control. By cornparison, tyrosine phosphorylation of IRS-I

was stimulated 4.7 I 1.3 fold (pc0.05) by a 2 min insulin challenge. These results indicate that a-

lipoic acid increases the level of tyrosine phosphorylation of IRS-l, and suggests that a-lipoic

acid rnay activate an upstrearn tyrosine kinase in order to facilitate this increase in IRS-I tyrosine

phosphorylation.

78

Figure 1.8. Effect of a-Lipoic Acid on IRS-1 Phosphorylation in 3T3-LI

Adipocytes.

C 2 2 5 min - I LA

Figure 1.8. Total cell lysates were prepared from 3T3-L1 adipocytes that had been treated with 2.5

rnM a-Iipoic acid (LA) or with 100 nM insulin (1) for the indicated time periods. IRS-1 was

irnmunoprecipitated from total ce11 lysates, imrnunoprecipitates were resolved by 7.5% SDS-

PAGE, and immunoblotted with anti-phosphotyrosine antibody. A typical irnmunoblot is

illustrated. Immunoblots fiom 4 independent expenments were scanned within the linear range and

tyrosine phosphorylated ES- 1 quantitated using the cornputer software NIH Image. The results

(means f SEM) are expressed in relative units, with the average reading of the controls (C)

assigned a value of 1 .O. *significantly different from control, p< 0.05 (2 test, two tailed).

Induction of Tvrosine Phos~horvlation of the Insulin Rece~tor bv a-Limic Acid in Intact

Cells.

Insulin activates the inbinsic tyrosine kinase activity of its receptor, leading to

autophosphorylation of tyrosine residues in several regions of the intracellular P subunit of the

receptor (348). The activation of the insulin receptor tyrosine kinase activity is necessary for the

interaction and subsequent activation of downstrearn signalling molecules, including IRS-1. As

previously demonstnted in Figure 1.8, the increase in the level of tyrosine phosphorylation of

IRS-1 suggests that upstream tyrosine kinases may also be activated by a-lipoic acid. In order to

define the involvement of tyrosine kinases in the action of a-lipoic acid, we explored the

possibility that in vivo treatment of 3T3-L1 adipocytes with a-lipoic acid might activate the insulin

receptor itself. Figure 1.9 illustrates that a-lipoic acid treatment of intact cells increased the level of

tyrosine phosphorylation of the insulin receptor. Despite the noticeable increase in the level of

tyrosine phosphorylation, relative to control, of the insulin receptor following 5 and 10 min of a-

lipoic acid treatment (3.9 f l .2 fold, p<0.05 and 4.5 f 1.3 fold, pc0.05, respectively) these levels

were lower than thnt induced by insulin treatment at 5 min (15.5 1: 4.4 fold, relative to control,

pcO.005).

80

Figure 1.9. Induction of Tyrosine Phosphorylation of the Insulin Receptor by a-

Lipoic acid in Intact Cells.

2 - 5 10 min I LA

Figure 1.9. Total ce11 lysates were prepared from 3T3-LI adipocytes that were treated in vivo

with 2.5 rnM lipoic acid (LA) or with 100 nM insulin (1) for the indicaied time periods. The

insulin receptor (R) was immunoprecipitated from total ce11 lysates, immunoprecipitates were

resolved by 7.5% SDS-PAGE, and immunoblotted with anti-phosphotyrosine antibody.

Immunoblots were scanned within the linear range and insulin receptor protein was quantitated

using the computer software NIH Image. A representative immunoblot of 5 independent

experiments is illustrated. The results (means f SEM) are expressed in relative units, with the

average reading of the controls (C) assigned a value of 1.0. *significantly different from control,

pc 0.05 (2 test, two tailed). %ignificantly different fiom control, p< 0.005 (Z test, two tailed)

Effect of a-lipoic Acid on Phosphorylation of the Insulin Receptor In Vitro.

To explore the possibility that or-lipoic acid might directly activatc the insulin receptor, we

measured the effect of a-lipoic acid on insulin receptor phosphory lation in vitro. Insulin receptor

immunoprecipitates from untreated 3T3-L1 adipocytes were exposed in vitro to 32~-ATP in the

presence or absence of insulin or a-lipoic acid. 32~-1ncor~oration into the P subunit was assessed

by SDS-PAGE and autoradiography. As demonstrated in Figure 1.10, in vitro treatment with a-

lipoic acid failed to increase the level of phosphorylation of immunopurified insulin receptors (1 .O

f 0.3 and 1.0 i 0.2 at 5 and 10 min treatment, respectively, relative to the control which was

assigned a value of 1.0). This was markedly different from the ability of insulin treatment to

increase the level of insulin receptor phosphorylation in vitro (3.3 t 1 .O, relative to control,

p<0.05). These results may identify a point of divergence in the mechanisms of action of a-lipoic

acid and insulin to stimulate components of the insulin signal transduction patliway necessary for

the stimulation of glucose transport. However, these results may be reflective of the difference in

the sensitivity of the in vivo and in vitro assays utilized to measure the effect of a-lipoic acid on

the insulin receptor. In vivo, insulin increased tyrosine phosphorylation of the insulin receptor to a

much larger extent than a-Iipoic acid, 15.5 fold vs. 3.9 fold, respectively. However, the insulin-

mediated increase in the level of insulin receptor phosphorylation in vitro was 3.3 fold relative to

control. In this in vitro assay, a-lipoic acid failed to stimulate insulin receptor phosphorylation

above control. Thus, it remains to be determined whether the in vitro assay utilized would even

have the capacity to detect any small increase in insulin receptor phosphorylation in response to a-

lipoic acid treatment.

82

Figure 1.10. Effect of a-Lipoic Acid on Phosphorylation of the Insulin Receptor

In vitro.

Figure 1.10. Total ce11 lysates were prepared from untreated 3T3-L1 adipocytes. An mtibody

raised against the P-subunit was used to immunopurify the insulin receptor (IR). The

irnmunocomplex was treated in vitro for the indicated time periods with 2.5 rnM a-lipoic acid

(LA) or 100 nM insulin (1). The levels of insulin receptor autophosphorylation were determined

using an in vitro kinase assay. Proteins were resolved by 7.5% SDS-PAGE, the gel was dried and

exposed to a Phosphorimager cassette. Quantitation of insulin receptor autophosphorylation was

accomplished using a MolecuIar Dynamics PhosphorIrnager System. Results are the means f SEM

of 4-5 independent experiments and are expressed in relative units, with the average reading of

controls (C) assigned a value of 1.W. *significantly different from control, p<0.05 (ANOVA,

Fisher's multiple cornparisons tests). #significantly different from a-lipoic acid (5 and 10 min),

p<0.05 (ANOVA, Fisher's multiple comp~sons tests).

DISCUSSION

a) Action of a-Lipoic Acid on Glucose Transporters and Glucose Transport

The ability of insulin to npidly stimulate glucose upiake into muscle and adipose tissue

represents a critical component of normal glucose homeostasis. Upon binding to its receptor,

insulin initiates a senes of intracellular signals involving tyrosine, lipid and senne/threonine

kinases. These signals result in the mobilization of glucose transporten to the plasma membrane

and the subsequent uptake of glucose. Currently, the cascade of signalling molecules believed to be

necessary for insulin-stimulated glucose uptake is IR--> IRS--> PI 3-kinase--> Akt I aPKCs-->

GLUT4 translocation.

There is considerable evidence that a defect in glucose transport may be responsible for the

acquired insulin resistance of glucose utilization observed in diabetes (142, 143). Conceivably, this

metabolic impairment could be explained by a variety of defects in GLUT4 regulation including

alterations in GLUT4 expression and translocation, defects in the insulin signalling pathway and

alterations in the temporal and spatial pattern of signalling molecules. Cumnt anti-diabetic

therapeutic strategies to reduce glycemia involve phmacological agents which promote insulin

release (sulfonylureas), curb hepatic glucose output (biguanides). or increase gene expression in

fat cells (thiazolidinediones) (120, 130,274). These compounds also have secondary peripheral

effects, such as the stimulation of glucose transport into insulin-responsive tissues. In contrast to

available anti-diabetic strategies, a successful therapeutic approach could directly target steps of the

insulin signalling cascade involved in the stimulation of glucose uptake into insulin-responsive

tissues. In this manner, an agent capable of primarily activating key components of the insulin

signalling pathway, which may be downregulated in the diseased state, could effectively rescue or

enhance the ability of insulin to increase glucose uptake, thereby regulating glycemia.

In search of physiologically relevant compounds that may be useful in the control of

glycemia our attention has focused on the potent biological antioxidant and natural cofactor of

oxidative metabolism, a-lipoic acid (242). As previously mentioned, administration of a-lipoic

acid enhanced insulin-stimulated glucose metabolism in various animal mcdels of insulin-resistance

84

(1 16, 133, 160) and improved insulin-stimulated glucose disposal in individuals with type 2

diabetes (13 1. 132. 179). We have also shown that a-lipoic acid rapidly stimulates glucose

transport into L6 sketetal muscle cells in culture (74).

In this thesis, 1 have shown that a-lipoic acid rapidly stimulates glucose uptake into the

insulin responsive 3T3-L1 adipocytes in culture. The stimulation of glucose transport by a-lipoic

acid warranted investigation into its mechanism of action. Thus, in an attempt to understand the

mechanism underlying the stimulation of glucose vansport by a-lipoic acid in 3T3-L1 adipocytes,

we examined the effect of this compound on GLUTl and GLUT4 redistribution. Subcellular

fractionation of 3T3-LI adipocytes indicated that a-lipoic acid induced a rapid translocation of

GLUTl and GLUT4 to the plasma membrane, concomitantiy with a reduction in the levels of these

transporters in the low density microsornal membrane fraction. This action of a-lipoic acid

qualitatively resembled the ability of insulin to redistribute GLUTl and GLUT4. However, the

quantitative differences in the action of a-lipoic acid and insulin may be due to the quantitative

differences in signalling described below. The rapid redistribution of glucose transporten is likely

to be the mechanism by which a-lipoic acid can acutely stimulate glucose transport into 3T3-LI

adipocytes. More importantly, to our knowledge, a-lipoic acid is the only known agent with anti-

diabetic properties that c m directly induce a rapid redistribution of glucose transporters in its target

cells, thereby allowing for the subsequent increase in the uptake of glucose.

b) Effect of a-Lipoic Acid on Lipid and Serinemhreonine Kinases

To analyze the signals involved in the stimulation of glucose transport and transporter

translocation by a-lipoic acid, we examined several elements of the insulin signaliing cascade.

Studies involving the use of selective inhibitors of PI 3-kinase (43,50,239,364), dominant

negative mutants (104) and overexpression of constinitively active forms of PI 3-kinase (152,313)

have demonstrated the necessity of this lipid kinase in the stimulation of glucose transport by

insulin. Here we demonstrate that wortmannin, a relatively specific inhibitor of PI 3-kinase,

prevents the stimulation of glucose aanspon by a-lipoic acid in 3T3-L1 adipocytes. This finding

indicated that a-lipoic acid engaged elements of the insulin signalling pathway necessary for the

stimulation of glucose uptake. With this in mind, we exarnined the effect of this compound on

85

insulin sensitivity and responsiveness. a-lipoic acid did not further increase the maximal insulin-

stimulated glucose transport in 3T3-Ll adipocytes, indicating that it may not utilize an alternative

pathway towards the stimulation of glucose transport. Although not statistically significant, a-

lipoic acid had a small, positive effect on the sensitivity of insulin-stimulated glucose uptake in

3T3-Ll adipocytes. This lends further support of the ability of a-lipoic acid to target insulin-

sensitive signalling pathways that mediate the stimulation of glucose transport.

We further demonstrated that a-lipoic acid could directly increase the level of PI 3-kinase

activity associated with anti-phosphotyrosine immunoprecipitates. In contrast to conditions of

contractile activity and hypoxia (364) or to agents such as dinitrophenol(320) or vanadium

compounds (246) which increase glucose transport independently of PI 3-kinase, a-Iipoic acid

has the ability, sirnilar to insulin, to utilize this enzyme. Thus, this is the Ant demonstration of an

agent with anti-diabetic propenies to increase glucose uptake in a PI 3-kinase dependent manner.

In suppon of the ability of a-lipoic acid to engage cornponents of the insulin-sensitive

pathway, we demonstrated that this compound activated the serine/threonine kinase Akt 1, a

downstrearn effector of PI 3-kinase in the stimulation of glucose transport. Surprisingly, there is

little evidence for any effect of agents capable of stimulating glucose transport on the activation of

Aktl. A recent report demonsüated that vanadium compounds activated Akt l and this activation

was inhibited by wortmannin. However, this is inconsequential for glucose transport since the

stimulation of glucose transport by vanadate is wortmannin-insensitive (323). In contrast, both the

stimulation of glucose transport and activation of Aktl by a-lipoic acid in 3T3-L1 adipocytes were

sensitive to inhibition of PI 3-kinase with wortmannin. The engagement by a-lipoic acid of lipid

and sennefthreonine kinases implicated in the stimulation of glucose transport by insulin, lends

further support of the notion that a-lipoic acid targets the insulin-sensitive pathway leading to the


c) Effect of a-Lipoic Acid on Tyrosine Kinases

Tyrosine kinases acting upstream of PI 3-kinase also represent critical components of the

insulin signalling pathway involved in the stimulation of glucose transport. Given that a-lipoic

acid induced the activation of PI 3-kinase bound to phosphotyrosine-containing proteins, we

86

investigated whether this compound could effect the level of tyrosine phosphorylation of 3T3-LI

adipocytes. Erbstatin. a tyrosine kinase inhibitor, partially reduced the ability of a-lipoic acid to

stimulate glucose uptake, suggesting the involvement of tyrosine phosphorylation in a-lipoic

acid's mechanism of action. Wilh this in mind, we exarnined the effect of a-lipoic acid on tyrosine

phosphorylated proteins implicated to be involved in the stimulation of glucose uptake by insulin.

The level of IRS- 1 tyrosine phosphory Iation in 3T3-L1 adipocytes was increased in

response to a-lipoic acid which suggested that a-lipoic acid may activate an upstrearn tyrosine

kinase. Surprisingly, we found that treatment of 3T3-L1 adipocytes with a-lipoic acid also

increased the level of tyrosine phosphorylation of the insulin receptor, although to a lesser extent

than insulin. To undentand the actual involvement of the insulin receptor in response to a-lipoic

acid, we tested the effect of a-lipoic acid added in vitro to immunopurified insulin receptors. This

experimental paradigm enabled us to dissociate the involvernent of any other cytosolic factors that

may be responsible for the increase in receptor phosphorylation in intact cells. Addition of a-lipoic

acid to immunopunfied receptors did not increase the level of receptor phosphorylation, in contrast

to the ability of in vitro treatment of insulin to increase receptor phosphorylation. A schematic

summary of the signals engaged by a-lipoic acid in its ability to stimulate glucose transport is

illustrated in Figure 1.1 1.

Figure 1.11. The Signalling Pathway of a-Lipoic Acid.

lnsulin Receptor 1 a-Lipyc Acid 1

J Translocation to Plasma Membrane

The proposed signalling pathway of a-Iipoic acid in 3T3-L1 adipocytes. a-Lipoic acid induces a

rapid redistribution of GLUT1 and GLUT4, leading to stimulation of glucose uptake. The signals

engaged by a-lipoic acid in its ability to stimuiate glucose transport include: Nt vivo tyrosine

phosphorylation of the insulin receptor; tyrosine phosphorylation of IRS- 1; antiphosphotyrosine-

associated PI 3-kinase activation; and activation of Akt 1.

88

The finding that a-lipoic acid could not induce an increase in the level of insulin receptor

autophosphorylation implies that a-lipoic acid may engage an additional factor in its ability to

stimulate components of the insulin signailing pathway. It is possible that an additional kinase is

activated in response to a-lipoic acid in intact cells, causing phosphorylation of the insulin

receptor. In support of this hypothesis, it has been demonstrated that a nonhydrolyzable GTP

analog [guanosine 5'-0-(3-thiotriphosphate) (GTPyS)] stimulates GLUT4 translocation

independently of the insulin receptor or RS-112 phosphorylation in 3T3-L1 adipocytes (107).

Furthemore, GTPyS induced tyrosine phosphorylation of several proteins (one at 70-80 kDa,

another at 120- 130 kDa) which may be involved in GLUT4 translocation as microinjection of

antiphosphotyrosine antibodies inhibited GTPyS-stimulated GLUT4 translocation (107). This

study indicated that the effect of GTPyS is dependent on tyrosine-phosphorylated proteins and this

effect could be mediated by an alternative pathway parallel to the insulin receptor and IRS-1/2

leading to GLUT4 translocation. Moreover, the insulin-like effects of vanadate have been shown to

be independent of the insulin receptor and IRS-1 phosphorylation (95,300). It was demonstrated

that vanadate treatment of rat adipocytes activated a membrane-associated nonreceptor protein

tyrosine kinase (55-60 kDa) suggested to be involved in glucose uptake (72). This finding also

highlighted a possible alternative pathway involving tyrosine kinases independent of the insulin

receptor in the stimulation of glucose uptake. In an analogous fashion, phosphorylation of

additional proteins through tyrosine kinases may be necessary to mediate the increase in glucose

uptake by a-lipoic acid.

d) Possible Effects of a-Lipoic Acid on Protein Tyrosine Phosphatases

Alternatively, the ability of a-lipoic acid to increase glucose uptake may also be a result of

its ability to modulate the activity of protein tyrosine phosphatases (PTPases) in the cell. PrPases

play an essential role in the regulation of signalhg pathways and in the regulation of reversible

tyrosine phosphorylation of cellular proteins. Such an inhibition could result in elevated levels of

tyrosine phosphorylation of the receptor, if the unoccupied insulin receptor has a degree of

autoactivity. Hence, the receptor may be under dual kinase and phosphatase regulation. Several

89

PTPases have been implicated in mediating insulin signalling. LAR, PTPlB and W a have been

suggested to act as negative regulators of insulin signalling by acting as physiological regulators of

the insulin receptor (2,45,54,73,368). Overexpression of PTPa and PTPlB have also been

shown to inhibit insulin-stimulated GLüT4 translocation in rat adipocytes (45, 54). SHP2 has

been shown to interact with IRS-1, and may exert negative regulation of the rnetabolic responses of

insulin though this interaction (228). On the other hand, the action of certain PrPases as positive

regulators of insulin action has also been reported (1 12). These data suggest that PTPases may

function as both positive and negative regulators. Although a definitive role for PTPases in the

direct regulation of glucose transport has not been firmly established, these recent findings indicate

that PTPases play an integral role in the regulation of the insulin receptor and mediating

downstream insulin signalling.

In relation to a-lipoic acid, it is feasible that a-lipoic acid may modulate the activity level

or interaction of PTPases with key signalling molecules involved in the stimulation of glucose

transport. KPases are susceptible to oxidative inactivation and are potential targets of redox

(duction-aidation) regulation (59,66,220). Hypothetically, a-lipoic acid could potentially

oxidize a PTPase, thereby rendenng the PTPase inactive. Thus, the redox modulating potential of

a-lipoic acid may mediate its ability to regulate PTPases involved in the stimulation of glucose

transport. To this end. a recent report has demonstrated the reversible inactivation of a low

molecular weight PTPase (LMW-FTP) though redox rnechanisms. Oxidation of the LMW-ITP at

two cysteine residues in the active site by hydrogen peroxide, at concentrations reflective of

conditions of oxidative stress or during signalling processes initiated by a variety of ligands

including growth factors, rendered the enzyme inactive (40). That study demonstrated that a redox

mechanism was capable of regulating the function of a PTPase. Furthemore, that study tends

support to the notion that a-lipoic acid, a strong redox modulator, could modulate the statu of

FTPases potentially involved in glucose transport.

Future studies are needed to address the precise role of an upstream tyrosine kinase or

phosphatase engaged in the a-lipoic acid-stimulated signals leading to glucose transport. The

ability of redox agents to directly modify PTPases and signalling molecules coupled to the

90

existence of alternative pathways capable of stimulating glucose transport may provide the

theoretical means for the ability of a-lipoic acid to initiate or complement the engagement of

components of the insulin signalling pathway necessary for the stimulation of glucose transport.

e) Does a-Lipoic Acid Function as an Antioxidant?

The ability of a-lipoic acid to enhance glucose uptake may also be reflective of the potent

antioxidant capacity of this compound. a-Lipoic acid has the ability to directly scavenge reactive

oxygen species and to recycle intracellular antioxidants including glutathione. vitamin E and

vitarnin C (141,242). The reduced form of a-lipoic acid, DHLA, is a strong reductant and is

capable of functioning as a redox regulator of intracellular proteins (14 1). For exarnple, the ability

of DHLA to reduce cellular sulfhydryl groups is essential for the regeneration of the intracellular

antioxidant glutathione and is essential for its ability to provide protective celiular defenses against

oxidative stress (242). The exposure of 3T3-L1 adipocytes to micro molar concentrations of Hz02

resulted in the generation of an insulin resistant state in vitro (267,268). This in vitro system of

oxidative stress was associated with a decrease in reduced glutathione content in addition to a

reduction in the level of insulin-stimulated glucose transport, GLUT4 translocation, and insulin-

stimulated Akt senne 473 phosphorylation (269). However. pretreatment wiih a-lipoic acid

(racemic) provided a significant protection against the decrease in cellular reduced glutathione

content and preserved the insulin-stimulated increase in glucose transport, GLUT4 translocation

and Akt serine 473 phosphorylation (269). This study demonstrates the unique ability of a-lipoic

acid to provide protection against impaired insulin action induced by oxidative stress. It was

suggested that the protective effects of a-lipoic acid are mediated by its capacity to sustain levels of

reduced glutathione essential for the maintenance of a balanced intracellular redox state.

Importantiy, the ability of a-lipoic acid to protect against oxidative stress, by maintaining

intracellular antioxidant levels, represents a critical feature that might be applied to counteract

oxidative stress-induced insulin resistance at the level of insulin-stimulated glucose transport.

f) Does a-Lipoic Acid Affect Glucose Uptake Through a Metabolic Action on

the Pyruvate Dehydrogenase Cornplex?

91

Furthemore, the ability of a-lipoic acid to increase glucose uptake may be reflective of iü

intimate involvement as a cofactor with the pymvate dehydrogenase complex. The pymvate

dehydrogenase complex (PDC) plays an essential role in controlling the oxidative consumption of

body carbohydrate stores by converting pyruvate into acetyl CoA, thereby facilitating its entry in

the tricarboxcylic acid (TCA) cycle. A schematic mode1 of the relation of the PDC and glucose

metabolism is illusûated in Figure 1.12. The PDC is composed of ihree main catalytic enzymes: the

El pyruvate decarboxylase; the E2 pyruvate transacetylase component (which contains two a-

lipoic acid domains covalently attached though a lysine residue); and the E3 dihydrolipoamide

dehydrogenase. The PDC is controlled by an inûicately regulated cycle whereby phosphorylation

by a specitic pynivate dehydrogenase kinase inactivates the complex, and dephosphorylation by a

pyruvate dehydrogenase phosphatase leads to activation of the PDC (258,259,361). Various

studies have described a reduction in activity of the PDC in models of type 2 diabetes (2 19,257,

336,346). Therefore, a potential molecular target for exogenously administered a-lipoic acid could

be to activate the PDC, likely downregulated in the diabetic stûte. We propose that activation of the

PDC would generate a 'pull' effect on glucose rnetabolism resulting in increased glucose transport.

92

Figure 1.12. Relationship of the Pyruvate Dehydrogenase Complex Reaction and

Glucose Metabolism

Glucose

J. Glycolysis

Pyruvate

Fatty Acids Amino Acids

1 Pyruvate 1 Dehydrogenase

Corn plex

4 io] A Acetyl CoA

NADH - FADH2

Electron Transfer and Oxidative Phosphorylation

ATP

The mamrnalian pyruvate dehydrogenase complex (PDC) has a strategic role in controlling the

oxidative consumption of glucose. The PDC is dso controlled by regulatory inputs; increased

levels of the products of this enzyme complex provide a negative feedback in hiel selection.

93

Moreover, the PDC is aiso controlled by regulatory inputs including changes in the levels

of acetyl CoA and NADH. Pymvate dehydrogenase activity is decreased upon an increase in the

ratios of NADH:NAD+ and acetyl CoA:CoA (please refer to Figure 1.12). Thus, increased levels

of the products of this enzyme complex provide a negative feedback in fuel selection (247). It is

possible that oc-lipoic acid may influence the activity of the PDC by influencing int~acellular NADH

levels. Since a-lipoic acid is reduced to dihydrolipoic acid by dihydrolipoamide dehydrogenase,

the E3 component of the PDC, the compound has the capacity to decrease cellular NADH/NAD+

ratios by utilizing NADH as a cofactor for its own reduction process (265). A reduction in the

intracellular levels of NADH rnay also provide a mechanism whereby a-lipoic acid could accelerate

pyruvate rnetabolism again resulting in a 'pull' effect on glucose metabolism and a subsequent

increase in glucose transport.

It has k e n demonstrated that the PDC plays an important role in regulating intracellular

glucose metabolisrn. Activation of the PDC via pharmacological doses of dichloroacetate (DCA)

was shown to decrease semm pyruvate and lactate concentrations and improve overall glucose

homeostasis (292). However, due to the toxic side effects of DCA its use has been discontinued

(291). Individuals with type 2 diabetes display elevated fasting levels of pyruvate and lactate (17,

179). A downregulation of the activity of the PDC in the diabetic state could explain the increase in

the levels of pyruvate. A reduction in PDC activity would contribute to increased conversion of

pyruvate to lactate, resulting in the elevated levels of lactate also observed in individuals with type

2 diabetes (219). A recent study demonstrated that chronic treatment with a-lipoic acid reduced the

elevated fasting lactate and pyruvate concentrations in individuals with type 2 diabetes and

prevented the increase in pymvate and lactate concentrations after a glucose load. (179). It was

proposed that the reduction in pyruvate levels in individuals with type 2 diabetes treated with a-

lipoic acid reflected the ability of a-lipoic acid to stimulate the PDC. hportantly, this proposal was

designed on conelative evidence only. Further in vivo snidies are needed to address a direct

relationship between a-lipoic acid administration and changes in PDC activity. Regardless, the

improvement of intracellular glucose utilization with a-lipoic acid treatment, possibly though the

stimulation of the PDC, highlights the effectiveness of this agent in controiling metabolic

parameters associated with type 2 diabetes.

g) Concluding Remarks

In conclusion. the results presented in this thesis highlight the ability of a physiologically

relevant compound, a-lipoic acid, to increase glucose uptake into the insulin-responsive 3T3-LI

adipocytes in culture. More specifically, we have demonstrated that a-lipoic acid induces a rapid

redistribution of GLUTl and GLUT4, leading to stimulation of glucose uptake. a-Lipoic acid

increased the level of antiphosphotyrosine-associated PI 3-kinase activity and activity of Aktl.

Tyrosine phosphorylation of IRS-1 and the insulin receptor were induced by a-lipoic acid

treatment. However, a-lipoic acid did not seem to cause insulin receptor autophosphorylation in

vitro, indicating that in intact cells, a-lipoic acid may indirectly induce tyrosine phosphorylation of

the insulin receptor. This is the first demonstration that a-lipoic acid utilizes tyrosine, lipid, and

serinehhreonine kinases in its mechanism of action. The results presented here suggest that a-

lipoic acid may directly target some elements of the insulin-sensitive signalling pathway, in contrast

to the actions of al1 other agents known CO stimulate glucose transport. Interestingly, signals

engaged in the action of a-lipoic acid are not due to direct effects of the compound on the insulin

receptor. We have proposed that the antioxidant and redox-rnodulating capacities of this compound

and/or its association with the pyruvate dehydrogenase complex may provide the mechanisms

whereby a-lipoic acid can improve glucose metabolism though an enhancement in glucose

transport (See Figure 1.13). These unique metabolic actions of a-lipoic acid, including the rapid

stimulation of glucose transport via components of an insulin sensitive pathway, support the

potential therapeutic importance of this agent in the treatment of diabetes.

Figure 1.13. a-Lipoic Acid: Potential Mechanisms of Action.

1 cc-Lipoic Acid 1 lnsulin Receptor II

I - Kinase ' 3

-- I

Class IA PI 3-Kinase

GLUCOSE TRANSPORT

Schematic representation of the proposed intracellular targets/mechanisms of action of a-lipoic

acid. The activation of a tyrosine kinase or inhibition of a protein tyrosine phosphatase (PTPase, a

possible result of the redox-modulating capacities of this compound) coupled to its association with

the pyruvate dehydrogenase complex (PDC) may provide the mechanismsrintracellular targets

which facilitate the ability of a-lipoic acid to improve glucose metabolism though an enhancement

in glucose transport.

FUTURE DIRECTIONS

In this thesis 1 have attempted to descnbe the mechanism by which a-lipoic acid stimulates

glucose uptake in 3T3-L1 adipocytes. Specifically, 1 showed that a-lipoic acid stimulated glucose

uptake via the npid translocation of GLUTl and GLUT4. Furthemore, this is most likely

accomplished by engaging some of the components of the insulin signalling cascade. necessary for

the stimulation of glucose uptake. Unlike any other anti-diabetic drug, a-lipoic acid engaged PI 3-

kinase in i ts mechanism of action and was able to alter the phosphorylation state of IRS- 1 and the

insulin receptor.

The discrepancy between the ability of a-lipoic acid to increase the level of tyrosine

phosphorylation of the insulin receptor in vivo, but not in vitro, raised the possibility that a-lipoic

acid rnay utilize an alternative mechanism(s) ta recruit molecules of the insulin signalling cascade

necessary for the stimulation of glucose uptake. It is possible that a-lipoic acid may initiate

signalling by activating a membrane-bound or cytosolic tyrosine kinase which would then

phosphorylate the insulin receptor. To examine this scenario, inhibiton which prevent the

activation of tyrosine kinases could be employed. This approach will reveal if the in vivo

phosphorylation of the insulin receptor that is observed with a-lipoic acid treatment is, indeed, due

to the activation of an additional kinase.

Altematively, a-lipoic acid rnay elevate the level of insulin receptor tyrosine

phosphorylation by inhibiting a protein tyrosine phosphatase. Increased interest in the role of

PTPases in insulin action have yielded insight into PTPases implicated in modulating signals

necessary for glucose uptake. To establish a role of FTPases in a-lipoic acid's mechanisrn of

action, we must first examine the level of expression of the different FTPases in 3T3-L1

adipocytes. Assays to measure specific PTPase activity would further clarify the ability of a-lipoic

acid to modulate the activity of PrPases in 3T3-LI adipocytes. Recently, a specific inhibitor of

PTP 1B has been utilized to demonstrate a role for P?'PlB in insulin-stimulated GLUT4

translocation in rat adipocytes (44). Use of this inhibitor may provide an important cornpaison to

understand the action of a-lipoic acid on this PTPase and its relation to GLUT4 translocation.

Failing to establish a definitive role of a-lipoic acid on PTPases by the above stnitegies,

97

overexpression of a specific PTPase in 3T3-LI adipocytes may provide the system necessary to

study the action of a-lipoic acid on PTPases involved in mediating glucose uptake.

In surnmary, 1 have demonstrated that a-lipoic acid is effective at stimulating glucose

transport in cells in culture. Studies described hem add credence to the suitability of a-lipoic acid

as a potential agent io control glycemia in diabetic individuals. The benefits of a-lipoic acid as a

therapeutic agent are two-fold: the ability of this agent to improve glucose metabolism. and its

beneficial effects on the conuol of the devastating complications of type 2 diabetes including

diabetic neuropathy.

INVOLVEMENT OF HRS-2 IN INSULIN REGULATED VESICLE

TRAFFIC.

RATIONALE/HY POTHESIS

Translocation of glucose transporter-containing vesicles in response to insulin and the

subsequent docking and fusion with the target membrane mediates the ability of insulin to increase

glucose uptake into insulin responsive tissues. The latter step bears some resernblance to the

regulated docking and fusion of vesicles in neuroendocrine cells which involves the interaction of

the core complex of SNARE (soluble wM sensitive factor-attachment protein ~ceptor) proteins

(37,289). The original SNARE hypothesis was put forth to describe a framework mode1 for the

role of SNAREs and soluble partnen dunng vesicle docking and fusion. The SNARE hypothesis

proposed that synaptic vesicles dock and subsequently fuse with the presynaptic membranes via

interactions among the vesicular, integral membrane proteins VAMPs (vesicle-associated

membrane protein) and the target membrane proteins SNAP25 (synaptosome-associated protein of

25 D a ) and syntaxin- 1. Non-neuronal celis, including insulin-responsive tissues and cells in

culture, also express isoforms of the SNARE proteins, namely VAMP-2, SNAP23, and syntaxin-

4 (35, 334, 335, 356).

It is also known that SNAREs cm bind additional proteins which regulate complex

formation. Modulators of neuronal v- and t-SNARE interactions include: Munc 18 binding to

syntarin-1 (253); synaptophysin interaction with VAMP-2 (38); and VAMP-associated protein of

33 kDa (VAP-33) association with VAMP-2 (285). Homologues of sorne of these neuronal

proteins have been detected in insulin-responsive tissues. Munc- 18c, a Munc- 18a isofom, has

been shown to bind syntaxin-4, thereby inhibiting interactions between syntaxin-4 and VAMP-2

necessary for the docking/fÙsion of GLUT4-containing vesicles with the plasma membrane of

3T3-L1 adipocytes (3 15). A recent study demonstrated that VAP-33 has a broad tissue

distribution, which suggests chat this protein may also regulate vesicle in non-neuronal

tissues (347). Pantophysin, a homologue of synaptophysin, is present in non-neuronal cells (98)

and may potentially bind VAMPs. Hence, the universality of the docking and fusion phenornenon

involving SNARE proteins may extend to the existence of functional isoforms of the regulatory

proteins ( 109, 285).

100

Recently, Hrs-2 (hepatocyte growth factor ggulated tyrosine kinase ~ubstrate-2), a novel

ATPase implicated in calcium-regulated secretion, was shown to interact with SNAP-25 (23). In

addition, homologues of this protein, mouse and human Hrs, were identified as substrates of the

hepatocyte growth factor receptor tyrosine kinase and of cytokine teceptors, respectively (Figure

A. 1, Table A. 1) (16, 175, 176). Collectively, these recent findings rnay implicate a potential role

of Hrs-2 in the regulation of a SNARE complex protein in response to growth factors. This leads

to the hypothesis that Hrs-2, or a homologue thereof, may be present in insulin-responsive tissues

and may be involved in the regulation of SNAP-23 (Figure A.2). In order to address this

hypothesis, we exarnined the expression of Hrs-2 in the insulin responsive L6 skeletal muscle cells

and 3T3-L1 adipocytes in culture. We also determined the ability of Hn-2 to interact with SNAP23

and studied any changes in the level of tyrosine phosphorylation of Hrs-2 in response to insulin.

101

Figure A.1. Schematic Structures of Mouse Hrs, Human Hrs, and Rat Hrs-2.

100 200 300 400 500 600 700 800 900 amino acids I m I 8 8 I I I

zinc Pro coi1 ProIGln

mouse Hrs

Based on sequence predictions, mouse Hrs, human Hn and rat Hrs-2 contain many readily

identifiable regions; zinc-finger motifs (zinc), proline-nch regions (Pro), proline and glutamine-

rich regions (ProIGln), putative coiled-coi1 sequences (coil), and three or four putative nucleotide-

binding sites (Gl, G2, G3, and G4). Note: Hrs-2 has an additional 150 amino acids at its C-

terminal tail.

human Hrs , - I

, , , :' -1

102

Table A.1. Functional Characteristics of Rat Hrs-2, Mouse Hrs, and Human Hrs.

Homoiogue

Hrs-2 (rat)

r

Hrs (mouse)

Hrs (human)

CharacteristicdFunction Direct and saturable binding of

Hrs-2 to SNAP25.

Regulates secretory apparatus; recombinant Hrs-2 inhibited c&-triggered noradrenaline

release from PC- 12 cells.

ATP-preferring nucleotidase. Localized to cytoplasmic surface

of early endosomes.

Shares homology to yeast protein essential for protein traffic.

Interacts with STAM (signal- mnsduction daptor molecule) which is involved in cytokine- mediated signal transduction.

Regulation/Phosphorylation Hn-2 binding to SNAP25

reduced in presence of ~ a 2 + . ~ n * +

Phosphorylated by hepatocyte growth factor, epidermal growth

factor, PDGF.

Phosphorylated by the cytokines, IL-2 and GM-CSF (granulocyte- macrophage olony-ztimulating -

factor). -

Suppress IL-2- and GM-CSF- stirnulated DNA synthesis.

103

Figure A.2. Schernatic Model of the Proposed Involvement of Hrs-2 in Insulin-

Regulated Vesicle Traffic.

The presence of Hrs-2, or a homologue thereof, in insulin responsive tissues implicates a possible

role of Hrs-2 in the regulation of SNARE complex proteins, specifically SNAP23. Insulin-

mediated signals may lead to the phosphorylation of Hrs-2, thereby regulating its interaction with

SNAP23 and its role in insulin-regulated vesicle

METHODS Çell Culture

A ctonal line of L6 rat skeletai muscle cells selected for high fusion potential was grown in

Minimum Essentiai Medium (a-MEM) containing 2% FBS; these cells were allowed to fuse and

differentiate as previously described (2 17). Cultures were studied after more than 90% of the ceIls

had fused into fully differentiated myotubes. Prior to al1 experimental manipulations, L6 muscle

cells were depnved of serurn for at least 5 hours.

Culture of mouse 3T3-LI adipocytes was as described in Chapter 1.

Total Membrane Pre~aration and Immunoblottin~.

Total membranes from 3T3-LI fibroblasts and adipocytes as well as L6 fibroblasts and

myotubes were prepared as follows: monolayers were rinsed twice with ice-cold homogenization

biiffer (250 mM sucrose, 20 m M HEPES, pH 7.4,s m M NaN3,2 rnM EDTA), scraped into

homogenization buffer containing 1 mM Na3V04,200 pM PMSF, 1 pM leupeptin, and 1 pM

pepstatin and homogenized with 20 strokes in a Dounce type A homogenizer. The homogenate was

centrifuged at 1,000 x g for 5 min at 40C to pellet the nuclei and large cellular debris. The

supernatant was centrifuged at 190,000 x g for 90 min to sediment the total membranes. The

membrane samples (20 pg) were resolved by 7.5% SDS-PAGE and immunoblotted with anti-

Hn-2 antibody (polyclonal, 1 :2500 dilution). Hn-2 protein was detected by the enhanced

cherniluminescence method using protein A conjugated to horseradish peroxidase (HRP, 1:SOûû

dilution) as the secondary antibody. The polyclonal H m 2 antibody was raised against a GST

fusion protein encoding the entire Hrs-2 protein.

Recombinant Fusion Proteins.

GST fusion proteins containing the full-length SNAP23 and SNAP25 were prepared as previously

described (36,79,356).The full-length Hrs-2 was expressed as N-terminal six-His fusion protein

using the pRSET expression vector in BSJ72-comptent Eschenchia coli. Cnide bactenal lysate of

His-tagged Hrs-2 was prepared as previously described (76) with the following modifications. The

N-terminal six-His fusion protein was not purified from the bacterial lysate, instead foilowing

bacterial lysis the concentration of the cmde bactenal lysate was determined and subsequently

stored at -80oC.

In vitro Bindin~ Assavs.

Ten micrograms of GST. recombinant GST-SNAP23 or GST-SNAP25 fusion protein

bound to glutathione-agarose beads were incubated with 6 mg of crude bacterial lysate containing

Hrs-2 for 3 h at 40C. The bead complexes were washed four tirnes in lysis buffer (20 rnM

HEPES, 100 m M KCI, 1% Triton X-100, 1 mM D?T. 2 mM EDTA, 0.5 m M PMSF, 1 pM

pepstatin A. I pM Ieupeptin, pH 7.4), bound materiai was eluted with 30 pl of 2X concentrated

Laemmli sarnple buffer containing 8 M urea and boiled for 5 min. Proteins were resolved by 7.5%

SDS-PAGE and irnmunoblotted with anti-Hrs-2 antibody (polyclonal, 1 :2500).

Lvsate Pre~aration and Anti-Phos~hotvrosine Immuno~reci~itation.

L6 Myotubes grown in 6 cm dishes were treated with 100 nM insulin for 20 min, rinsed

twice with ice-cold PBS and lysed in 0.5 ml of lysis buffer [20 m M Tris. pH 7.5, 137 mM NaCl,

1 rnM MgCI2, 1 rnM CaC12, 1% NP-40 (voVvol), 10% glycerol (voUvol), 10 rnM sodium

pyrophosphate, 1 ûû rnM NaF and 1 m M Na3V04 containing a mixture of protease inhibitors (1

pM leupeptin. 1 pli4 pepstatin A. and 200 p.M PMSF)]. Lysates were passed five times through a

25-gauge syringe and then incubated for 15 minutes at 40C under constant rotation. Debris and

unbroken ceils were removed by centrifugation. One miIligram of total cellular protein was

incubated with 2 pg anti-phosphotyrosine antibody for 2-3 houn under constant rotation (40C)

followed by a 1 hour incubation with 30 pl protein A Sepharose beads (100 mghl).

Immunocomplexes were washed 4 times with PBS containing 100 PM sodium orthovanadate and

0.1% NP-40. Pellets were resuspended in 30 pl 2X Laemmli sample buffer and boiled for 5

minutes. Proteins were resolved by 7.5% SDS-PAGE and irnrnunoblotted with anti-Hrs-2

an tibody (polyclonal, 1 :2500).

Exoression and Subcellular Distribution of Hrs-2 in L6 Muscle Cells and 3T3-LI Adi~ocvtes.

To begin, we examined the expression of Hrs-2 in the insulin responsive L6 skeletal

muscle cells and 3T3-L1 adipocytes in culture. The level of expression of H m 2 in both the total

membrane and cytosolic fractions increased upon differentiation of L6 myoblasts to myotubes as

illustrated in Figure A.3. The majority of Hrs-2 was found in the cytosolic fraction of both L6

rnyoblasts and myotubes, although a detectable level of Hrs-2 was also seen in the total membrane

fraction of both L6 rnyoblasts and myotubes. Furthemore, subcellular fractionation of L6

myotubes revealed that Hrs-2 exclusively associated with the plasma membrane fhction (data not

shown). There was no significant change in the level of Hn-2 in the plasma membrane fraction of

L6 myotubes upon insulin stimulation. Figure A.4 illustrates that the level of expression of Hrs-2

is also elevated upon differentiation of 3T3-LI fibroblasts to adipocytes in both total membrane and

cytosolic fractions although to a lesser degree than in L6 skeletal muscle cells. Similar to L6 muscle

cells, the majority of Hrs-2 disuibuted to the cytosolic fraction of 3T3-L1 fibroblasts and

adipocytes. However, a substantial amount Hrs-2 was also found associated with total membrane

fractions of both 3T3-L 1 fibroblasts and adipocytes.

108

Figure A.3. Expression and Subcellular Distribution of Hrs-2 in L6 Muscle Cells.

c O Mb Mt Mb Mt

TM CYT

Total membranes (TM) and cytosolic fractions (CYT) were isolated from L6 myoblasts (Mb) and

myotubes (Mt). Twenty micrograms of protein were resolved by 7.5% SDS-PAGE and

irnrnunoblotted with anti-Hrs-2 antibody (polyclonal, 1 :2500). Immunoreactive bands were

scanned within the linear range and the protein was digitally quantitated using NIH image (NIH,

Bethesda. MD). Results are the mean f SEM of three independent experirnents. * Significantly

di fferent from Mb TM, pc0.0005 ( ANOVA, Fisher's multiple cornparisons test). # Significantly

different fiom Mt TM, pc0.0001 (ANOVA, Fisher's multiple cornparison's test). Note: Values

represent arbitrary units brised on total protein per fraction.

109

Figure A.4. Expression and Subcellular Distribution of Hrs-2 in 3T3-LI

Adipocytes.

Total membranes (TM) and cytosolic fractions (CYT) were isolated from 3T3-L1 fibroblasts (Fb)

and adipocytes (Ad). Twenty rnicrograms of protein from the isolated fractions were resolved by

7.5% SDS-PAGE and immunoblotted with anti-Hrs-2 antibody (polyclonal, 1 :2500).

Immunoreactive bands were scanned within the linear range and the protein was quantitated using

the cornputer software NIH Image. Results are the rnean f SEM of three independent experiments.

In Vitro Binding of Recombinant SNAP25 and SNAP23 to Hrs-2

Hrs-2 has been shown to bind SNAP25 both in vitro and in vivo (23). In an attempt to

define a similar interaction between Hrs-2 and SNAP23, we examined the ability of Hrs-2 to bind

recombinant SNAP23 bound to glutathione-agarose beads. Figure A S illustrates that recombinant

Hn-2, from a crude bacterial lysate, was able to bind to recombinant SNAP25. In contrast,

recombinant SNAP23 did not bind Hrs-2. We further demonstrated that any 'binding' between

Hn-2 and SNAP23 evident after prolonged exposure of the irnmunoblot (Figure A S B) was

nonspecific (data not shown) as Hrs-2 interacted with GST bound to glutathione-agarose beads

alone. In addition, we were unable to demonstrate any in vivo interaction between SNAP23 and

Hrs-2 as immunoprecipitates of SNAP23 from 3T3-LI adipocytes did not contain endogenous

Hrs-S (data not shown).

111

Figure A S . In Vitro Binding of Recombinant SNAP25 and SNAP23 to Hrs-2.

Ten micrograrns of recombinant SNAP25 and SNAP23 bound to glutathione beads were cornbined

with six milligrms of cmde bacterial lysate containing His-tagged Hrs-2. Bound material was

eluted with 2X concentrated Laemrnli sample buffer containing 8 M urea, boiled for 5 min and

resolved by 7.5% SDS-PAGE. Bound Hrs-2 was detected by immunoblotting with Hrs-2

antibody (polyclonal, 1:2500). (A) Represents a lighter exposure of the sarne immunoblot as

illustrated in (B).

Phosphorylation of Hrs-2 in L6 M~otubes in Reswnse to Insulin.

The finding that a homologue of Hrs-2, mouse Hrs, was a substnte of the hepatocyte

growth factor receptor tyrosine kinase and was tyrosine phosphorylated in response to hepatocyte

growth factor, epidemal growth factor and PDGF suggests that Hrs-2 may also be regulated by

growth factors. The presence of Hrs-2 in insulin-responsive cells in culture reveals the possibility

that Hrs-2 rnay also become tyrosine phosphorylated in response to insulin. To address this

possibility , phosphotyrosy 1 proteins were immunoprecipitated from L6 rnyotubes treated wi th 100

nM insulin for 20 min, proteins were resolved by 7.5% SDS-PAGE and irnmunoblotted with anti-

Hrs-2 antibody. Immunoprecipitates of phosphotyrosyl proteins from lysates of L6 myotubes were

devoid of Hrs-2, in the presence or absence of insulin treatment (Figure A.6). Instead, Hrs-2

remained in the supernatant fraction following anti-phosphotyrosine immunoprecipitation. This

indicated that the majority of Hrs-2 remained unphosphorylated in response to insulin.

113

Figure A.6. Phosphorylation of Hrs-2 in L6 Myotubes in Response to Insulin.

C I IgG L6 Sol

L6 Myotubes were treated with LOO nM insulin (1) for 20 min, lysed and 1 mg of total cellular

lysaies were immunoprecipitated with 2 pg of anti-phosphotyrosine antibody . The

immunocomplexes were resolved by 7.5% SDS-PAGE and immunoblotted with anti-Hrs-2

antibody (polyclonal, 1 :2500). Fifty rnicrograrns of a L6 myotube soluble fraction (L6 sol) were

run as a positive control for the presence of Hrs-2.

Concluding Remarks

In conclusion, we have demonstrated that Hrs-2 is predominantiy expressed in the

cytosolic fractions of 3T3-Ll fibroblasts and adipocytes as well as in L6 skeletal muscle myoblasts

and myotubes. However, in vitro binding studies reveaied that Hrs-2 from a crude bacterial lysate

did not form a detectable complex with recombinant SNAP-23 in a similar manner to the binding of

recombinant SNAP-25 to Hrs-2. Immunoprecipitation of SNAP23 from 3T3-L 1 adipocytes failed

to reveal any in vivo association with endogenous Hrs-2. In addition. Hrs-2 did not become

tyrosine phosphoiylated in response to 100 nM insulin treatrnent in L6 myotubes. This finding

suggests that Hrs-2 is not a substrate of the insulin receptor, unlike the mouse Hrs which has been

identified as substrate of the hepatocyte growth factor receptor.

The presence of Hrs-2 in insulin responsive cells revealed the possibility that Hrs-2 may

play an important functional role in insulin-stimulated vesicle traffic. However, in the context of

cells and systems employed in this snidy, Hrs-2 did not become phosphorylated in response to

insulin and did not bind SNAP-23. Thus, these results imply that Hn-2 may not be involved in the

docking and fusion of glucose transporter-containing vesicles with the target membrane in insulin-

responsive cells.

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