Regulation of Insulin Signalling by Exercise
in Skeletal Muscle
Glenn Wadley
Bachelor of Education (Secondary)
Bachelor of Applied Science (Honours)
Master of Applied Science (Human Movement)
Submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
Deakin University
July 2003
i
DEAKIN UNIVERSITY
CANDIDATE DECLARATION
I am the author of the thesis
entitled
Regulation of insulin signalling by exercise in skeletal muscle
Submitted for the degree
of
Doctor of Philosophy
Is the result of my own research, except where otherwise acknowledged, and that this
thesis in whole or in part has not been submitted for an award, including a higher
degree, to any other university or institution.
Full Name: Glenn Wadley
Signed: __________________________________ Date: ________________
ii
ACKNOWLEDGEMENTS
The author wishes to acknowledge and thank the following people for their help
and contribution to the current project:
Dr. David Cameron-Smith and Professor Mark Hargreaves for their guidance and
supervision.
Dr. Lance Macaulay, Dr. Nicky Konstantopuolos and Peter Hoyne (CSIRO) for their
help with the protein analysis (Chapters 4 – 6).
Dr. Andrew Garnham for his excellent medical assistance.
Rebecca Tunstall for her major contribution with data collection, including RNA
extraction from skeletal muscle, in the endurance training and gene expression study
(Chapter 2). Kate Mehan for all the data collection, including the RNA extraction from
skeletal muscle, in the resistance training study (Chapter 3). Clinton Bruce, Dr. Jong
Sam and Dr. John Hawley (RMIT University) for their major contributions to data
collection and manuscript preparation in the rat study (Chapter 6). Dr. Kirsten Howlett
for her much appreciated advice on analysis and with the revisions. Adam Rose and
Brett Johnstone for their invaluable and much appreciated assistance with the testing
and analysis (Chapter 4 and 5). Dr. Rod Snow for all his statistical advice. Janelle
Mollica and Andrew Howarth for all of their help in the lab. Andrew Sanigorski and Dr.
Robyn Murphy for teaching me real-time PCR. To all the other postgraduate students
who helped me in some shape or form along the way.
To the subjects who participated and made the study worthwhile.
To my Mum for her support and understanding over the years.
To Kathleen for her love and support.
iii
PUBLICATIONS ARISING FROM THIS THESIS
Wadley, G.D., R.J. Tunstall, A. Sanigorski, G.R. Collier, M. Hargreaves, and D.
Cameron-Smith. Differential effects of exercise on insulin-signaling gene expression in
human skeletal muscle. Journal of Applied Physiology, 90(2): 436-440, 2001.
Wadley, G.D., C.R. Bruce, N. Konstantopoulos, S.L. Macaulay, K.F. Howlett,
J.A. Hawley and D. Cameron-Smith. The effect of insulin and exercise on c-cbl protein
abundance and phosphorylation in insulin resistant skeletal muscle. Submitted to
Diabetologia, May 2003.
Wadley, G.D., N. Konstantopoulos, S.L. Macaulay, K. Howlett, A. Garnham, M.
Hargreaves and D. Cameron-Smith. The effect of an acute bout of exercise and exercise
training on insulin signalling in human skeletal muscle. Manuscript in preparation
iv
DEFINITION OF TERMS
The symbols and definition of terms used in this thesis are:
BMI body mass index (kg.m-2)
BSA bovine serum albumin
CAP c-Cbl associating protein
CHO carbohydrate
DMEM Dulbecco's modified Eagle’s medium
EGF extracellular growth factor
ELISA enzyme-linked immunoabsorbent assay
g relative centrifugal force
GIR glucose infusion rate
GLUT4 glucose transporter isoform 4
HOMA homeostatic model of assessment
HRP horse radish peroxidase
IDDM insulin dependent diabetes mellitus
IgG immunoglobulin G
IR Insulin receptor
IRS Insulin receptor substrate
LAR leukocyte antigen-related PTPase
LRP leukocyte common antigen-related PTPase
MAPK mitogen activated protein kinase
min minute
mRNA messenger ribonucleic acid
v
DEFINITION OF TERMS CONTINUED
NIDDM non-insulin dependent diabetes mellitus
NS not significant
p level of probability
PBS phosphate buffered saline
PCR polymerase chain reaction
PI 3-kinase phosphatidylinositol 3-kinase
PTEN tensin homolog deleted on chromosome ten
PTP1B protein tyrosine phosphatase 1B
PTPase protein tyrosine phosphatase
pY phosphotyrosine
SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis
SHIP2 Src Homology 2 domain containing inositol 5-phosphatase 2
SHPTP2 Src Homology (SH) PTPase 2
TBS tris buffered saline
U units
VO2 rate of oxygen uptake
VO2 peak peak rate of oxygen uptake per minute
VCO2 rate of carbon dioxide production
vi
TABLE OF CONTENTS Page No.
CANDIDATE DECLARATION i ACKNOWLEDGEMENTS ii PUBLICATIONS ARISING FROM THIS THESIS iii DEFINITION OF TERMS v TABLE OF CONTENTS vii LIST OF TABLES viii LIST OF FIGURES xi ABSTRACT
CHAPTER ONE. REVIEW OF LITERATURE 1.1 Introduction 1 1.2 Physical activity, insulin resistance and type 2 diabetes 2 1.3 Pathway of insulin signalling 6 1.4 A second pathway for insulin signalling 12 1.5 Exercise and glucose transport 13 1.6 Defective insulin signalling in insulin resistant states 14 1.7 Interactions between exercise and the insulin signalling pathway 15 1.8 Protein tyrosine phosphatases as regulators of insulin signalling 25 1.9 Summary 32 1.10 Aims 33 CHAPTER TWO. DIFFERENTIAL EFFECTS OF EXERCISE ON INSULIN SIGNALLING GENE EXPRESSION IN HUMAN SKELETAL MUSCLE
2.1 Introduction 35 2.2 Materials and Methods 36 2.3 Results 40 2.4 Discussion 43 2.5 Conclusion 46 CHAPTER THREE. IRS-2 GENE EXPRESSION IS UPREGULATED BY RESISTANCE EXERCISE IN HUMAN SKELETAL MUSCLE
3.1 Introduction 47 3.2 Materials and Methods 48 3.3 Results 51 3.4 Discussion 54 3.5 Conclusion 57
vii
TABLE OF CONTENTS CONTINUED Page No. CHAPTER FOUR. THE EFFECT OF EXERCISE ON POTENTIAL MEDIATORS OF INSULIN SIGNALLING IN HUMAN SKELETAL MUSCLE 4.1 Introduction 584.2 Materials and Methods 614.3 Results 704.4 Discussion 834.5 Conclusion 92 CHAPTER FIVE. EFFECTS OF EXERCISE ON PROTEIN ABUNDANCE OF IRS-2 AND THE P85α SUB UNIT PI 3-KINASE 5.1 Introduction 945.2 Materials and Methods 955.3 Results 965.4 Discussion 995.5 Conclusion 102 CHAPTER SIX. THE EFFECT OF INSULIN AND EXERCISE ON C-CBL PROTEIN ABUNDANCE AND PHOSPHORYLATION IN INSULIN RESISTANT SKELETAL MUSCLE 6.1 Introduction 1036.2 Materials and Methods 1066.3 Results 1116.4 Discussion 1246.5 Conclusion 128 CHAPTER SEVEN. CONCLUSION 7.1 Conclusions 1307.2 Future Directions 1357.3 Concluding Statement 140 REFERENCES 141
viii
LIST OF TABLES
Table No.
Page No.
1.1 Classification of major protein tyrosine phosphatases (PTPases) implicated in the regulation of insulin signalling in skeletal muscle.
28
2.1 Gene primer sequences
39
3.1 Gene primer sequences
50
3.2 Average torque during each repetition of isokinetic thigh exercise.
52
4.1 Summary of the testing sessions. 64
4.2 2OV& peak prior to the study and exercise characteristics during
the exercise-training period (n = 8 subjects). 72
4.3 Fasting plasma characteristics prior to the 3 euglycemic, hyperinsulinemic clamps.
73
6.1 Plasma glucose and insulin concentrations measured in lean and obese Zucker rats.
113
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LIST OF FIGURES Figure No.
Page No.
1.1 Pathway of insulin signalling.
9
1.2 Potential regulation of the insulin-signalling pathway.
28
2.1 Effect of a single bout of exercise and short-term endurance training on the gene expression of β-actin.
41
2.2 Effect of a single bout of exercise and short-term endurance training on the gene expression of A: the insulin receptor (IR), B: insulin receptor substrate-1 (IRS-1), C: insulin receptor substrate-2 (IRS-2) and D: p85α subunit of phosphatidylinositol 3-kinase (p85α PI 3-kinase).
42
3.1 Effect of a single bout of resistance training on the gene expression of β-actin.
52
3.2 Effect of a single bout of resistance exercise on the mRNA concentration of A: insulin receptor substrate-1 (IRS-1), B: insulin receptor substrate-2 (IRS-2). And C: p85α subunit of PI 3-kinase.
53
4.1 Average plasma insulin concentration for 90-120min during the euglycemic, hyperinsulinemic clamps.
72
4.2 Glucose infusion rate during the euglycemic, hyperinsulinemic clamps.
73
4.3 Insulin receptor protein expression during the euglycemic, hyperinsulinemic clamps as measured by immunoprecipitation.
76
4.4 Insulin receptor tyrosine phosphorylation during the euglycemic, hyperinsulinemic clamps as measured by immunoprecipitation.
76
4.5 Insulin receptor number during the euglycemic,
hyperinsulinemic clamps as measured by ELISA.
77
4.6 Insulin receptor tyrosine phosphorylation relative to receptor number during the euglycemic, hyperinsulinemic clamps as measured by ELISA.
77
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LIST OF FIGURES CONTINUED Figure No.
Page No.
4.7 IRS-1 protein abundance during the euglycemic,
hyperinsulinemic clamps.
79
4.8 IRS-1 tyrosine phosphorylation relative to protein abundance during the euglycemic, hyperinsulinemic clamps.
79
4.9 Akt protein abundance during the euglycemic, hyperinsulinemic clamps.
80
4.10 pAkt (Ser473) during the euglycemic, hyperinsulinemic clamps.
80
4.11 SHPTP2 protein abundance in the cytosolic fraction during
the euglycemic, hyperinsulinemic clamps.
81
4.12 SHPTP2 protein abundance in the particulate fraction during the euglycemic, hyperinsulinemic clamps.
81
4.13 PTP1B protein abundance in the cytosolic fraction during the euglycemic, hyperinsulinemic clamps.
82
4.14 PTP1B protein abundance in the particulate fraction during the euglycemic, hyperinsulinemic clamps.
82
5.1 p85α PI 3-kinase protein abundance during the euglycemic, hyperinsulinemic clamps.
98
5.2 IRS-2 protein abundance during the euglycemic,
hyperinsulinemic clamps. 98
6.1 Effects of acute exercise and insulin stimulation on insulin
receptor (IR) phosphorylation in lean (open bar) and obese (closed bar) Zucker rats.
115
6.2 Effects of exercise and insulin on IRβ protein abundance in
lean (open bar) and obese (closed bar) Zucker rats. 115
6.3 Effects of exercise and insulin on Akt protein abundance in
lean and obese Zucker rats.
117
xi
LIST OF FIGURES CONTINUED Figure No.
Page No.
6.4 Effects of exercise and insulin on Akt protein abundance in
lean (open bar) and obese (closed bar) Zucker rats. 118
6.5 Insulin treatment but not exercise increases pAkt (Ser473) in
lean (open bar) and obese (closed bar) Zucker rats.
118
6.6 No difference in c-Cbl protein abundance between lean (open bar) and obese (closed bar) Zucker rats.
120
6.7 Insulin treatment but not exercise increases c-Cbl protein abundance in Zucker rats.
120
6.8 c- Cbl is not tyrosine phosphorylated by insulin treatment in rat skeletal muscle.
121
6.9 CAP protein expression appears more abundant in 3T3L1 adipocytes compared to rat skeletal muscle.
123
xii
ABSTRACT
Regulation of insulin signalling by exercise in skeletal muscle
Regular physical activity improves insulin action and is an effective therapy for the treatment
and prevention of type 2 diabetes. However, little is known of the mechanisms by which
exercise improves insulin action in muscle. These studies investigate the actions of a single bout
of exercise and short-term endurance training on insulin signalling. Twenty-four hours
following the completion of a single bout of endurance exercise insulin action improved,
although greater enhancement of insulin action was demonstrated following the completion of
endurance training, implying that cumulative bouts of exercise substantially increase insulin
action above that seen from the residual effects of an acute bout of prior exercise. No alteration
in the abundance and phosphorylation of proximal members of the insulin-signalling cascade in
skeletal muscle, including the insulin receptor and IRS-1 were found. A major finding however,
was the significant increase in the serine phosphorylation of a known downstream signalling
protein, Akt (1.5 fold, p<0.05) following an acute bout of exercise and exercise training. This
was matched by the observed increase in protein abundance of SHPTP2 (1.6 fold, p<0.05) a
protein tyrosine phosphatase, in the cytosolic fraction of skeletal muscle following endurance
exercise. These data suggest a small positive role for SHPTP2 on insulin stimulated glucose
transport consistent with transgenic mice models. Further studies were aimed at examining the
gene expression following a single bout of either resistance or endurance exercise. There were
significant transient increases in IRS-2 mRNA concentration in the few hours following a single
bout of both endurance and resistance exercise. IRS-2 protein abundance was also observed to
significantly increase 24-hours following a single bout of endurance exercise indicating
transcriptional regulation of IRS-2 following muscular contraction. One final component of this
PhD project was to examine a second novel insulin-signalling pathway via c-Cbl tyrosine
phosphorylation that has recently been shown to be essential for insulin stimulated glucose
uptake in adipocytes. No evidence was found for the tyrosine phosphorylation of c-Cbl in the
skeletal muscle of Zucker rats despite demonstrating significant phosphorylation of the insulin
receptor and Akt by insulin treatment and successfully immunoprecipitating c-Cbl protein.
Surprisingly, there was a small but significant increase in c-Cbl protein expression following
insulin-stimulation, however c-Cbl tyrosine phosphorylation does not appear to be associated
with insulin or exercise-mediated glucose transport in skeletal muscle.
1
CHAPTER ONE
REVIEW OF LITERATURE
1.1 Introduction
Currently around 1 million Australians and 124 million people worldwide have diabetes
mellitus, and this figure is expected to increase to approximately 300 million worldwide
by the year 2025 (Amos et al., 1997; Dunstan et al., 2001). Diabetes mellitus is a
collection of disorders characterised by high glucose levels in the blood and body
tissues. The two major forms of diabetes mellitus are insulin dependent (IDDM or type
1 diabetes) and non insulin dependent diabetes mellitus (NIDDM or type 2 diabetes).
Type 1 diabetes is caused by the destruction of the insulin secretory capacity of the
pancreatic beta cells by an autoimmune response and affects approximately 15% of all
cases worldwide (Alberti et al., 1997). Type 2 diabetes is by far the more prevalent form
of the disease, accounting for up to 90% of cases in Western countries (Alberti et al.,
1997) and is characterised by defects in both insulin secretion and insulin resistance of
peripheral target tissues such as skeletal muscle (DeFronzo et al., 1985). The
burgeoning epidemic of type 2 diabetes and obesity will be among the most significant
public health issues facing industrialised nations within the next decade. The
development of insulin resistance is a particularly crucial and an early event in the
aetiology of syndrome X, a cluster of overlapping conditions, which include obesity,
atherosclerosis, type 2 diabetes and hypertension (Reaven, 1994; Weyer et al., 1999).
The most important site of insulin resistance in type 2 diabetics has been found to be
skeletal muscle; which can account for approximately 80 – 90% of insulin stimulated
2
glucose uptake (DeFronzo et al., 1985). Therefore, the intracellular defects that
contribute to insulin resistance in skeletal muscle will be a focus of this discussion.
There is considerable evidence suggesting physical activity is an effective therapy for
the treatment and prevention of type 2 diabetes, however little is known of the
intracellular mechanisms by which physical activity exerts such a positive influence on
skeletal muscle insulin action. Considerable recent gains have been made in
determining the cellular basis of the actions of insulin and intracellular sites of insulin
resistance, providing new clues in the hunt to identify the mechanisms by which regular
physical activity is able to exert a positive benefit on insulin sensitivity in humans. This
review will provide an overview of the current literature that has examined how insulin
and exercise regulate glucose uptake in skeletal muscle, identifying what is known and
hypothesised regarding the interactions between these pathways. There are significant
benefits in determining the intracellular pathways through which physical activity
improves insulin action as this knowledge may provide a basis for the development of
more efficacious therapies in the treatment and prevention of type 2 diabetes.
1.2 Physical activity, insulin resistance and type 2 diabetes
Physical inactivity is believed to be causally associated with the development of chronic
diseases such as obesity, atherosclerosis, type 2 diabetes and hypertension. Indeed,
regular physical activity is inversely correlated with type 2 diabetes (Helmrich et al.,
1991; Manson et al., 1992; Manson et al., 1991) coronary heart disease (Manson et al.,
1999), and importantly total mortality (Powell & Blair, 1994), a relationship which has
been shown to be independent of body fat mass (Lee et al., 1999). A large proportion of
3
Australians do not currently undertake sufficient physical activity to obtain some health
benefits. Approximately half of all Australians over 25 years of age accumulate less
than the recommended minimum of 150 minutes of physical activity per week
(including walking, moderate and vigorous activity), which also coincides with 24% of
Australians over the age of 25 having some form of impaired glucose metabolism
(Dunstan et al., 2001). Vigorous exercise at least once per week has been found to
significantly reduce the risk of developing type 2 diabetes (Manson et al., 1991) whilst
increased frequency of vigorous physical activity per week progressively decreases the
risk of developing type 2 diabetes (Manson et al., 1992). It is not only vigorous
exercise that has such a profound therapeutic benefit, since at least 180 minutes of brisk
walking per week is able to reduce the risk of type 2 diabetes and coronary heart disease
to a similar extent as regular vigorous exercise (Hu et al., 1999; Manson et al., 1999).
Furthermore, long-term interventions (3-6 years), which involve physical activity and
diet modification have been shown to reduce the progression to type 2 diabetes in
subjects already with impaired glucose tolerance (Eriksson & Lindgarde, 1991;
Knowler et al., 2002; Tuomilehto et al., 2001). A landmark study (Knowler et al., 2002)
recently demonstrated that lifestyle interventions involving both diet and physical
activity modification are more effective at preventing type 2 diabetes than treatment
with common antihyperglycemic drugs. Over 3000 obese non-diabetics with impaired
glucose tolerance underwent long-term treatment involving standard lifestyle
recommendations plus metformin, a placebo or an intensive lifestyle intervention
program involving 150 minutes of brisk walking per week, individualised dietary
education promoting weight reduction and a healthy low calorie, low fat diet. After 3
years the incidence of diabetes was 58% lower in the lifestyle intervention group and
only 31% lower in the metformin group when compared to the placebo group. These
4
studies demonstrate the powerful effect that a healthy lifestyle has on the prevention of
metabolic disorders. However they do not address whether physical activity
independently of diet modification provides protection against type 2 diabetes.
There does however appear to be a distinct therapeutic benefit of physical activity alone
in the prevention of type 2 diabetes. A study into a cohort of the Amish population of
America revealed that despite similar rates of obesity when compared to the general
Caucasian population in America, there was a significantly lower prevalence of type 2
diabetes (Hsueh et al., 2000). The Amish population of America are a cultural group
that forgo many of the labour saving devices of the typical American population and are
therefore thought to be significantly more physically active (Hsueh et al., 2000), which
supports the independent benefits of physical activity in diabetes prevention. More
direct evidence also demonstrates the powerful effect of physical activity on diabetes
prevention. In one of the most definitive studies so far, almost 600 Chinese citizens with
impaired glucose tolerance underwent a randomised clinical control trial over a six-year
period. Regular physical activity was found to be significantly more effective in
preventing type 2 diabetes than diet modification with no further benefit when diet and
exercise were combined (Pan et al., 1997). Therefore, the reasons why physical activity
is effective in reducing the prevalence and development of type 2 diabetes are probably
twofold.
Firstly, regular physical activity is protective against excessive weight gain
(Paffenbarger et al., 1986), with adiposity strongly associated with insulin resistance
and diabetes development obesity (Goodyear et al., 1995a; Kelley et al., 1999). High fat
feeding in rats induces insulin resistance (Kim et al., 2000a), while weight loss in
5
humans via a low calorie diet improves insulin sensitivity (Franssila Kallunki et al.,
1992) demonstrating the clear link between body fatness and insulin action.
Secondly, independent of body fatness physical activity improves skeletal muscle
insulin sensitivity. The sensitivity of tissues such as muscle and fat to exogenous insulin
is commonly measured in research by a euglycemic, hyperinsulinemic clamp. A primed,
constant infusion of insulin is used to raise and maintain plasma insulin levels. The
negative feedback principle is used to hold plasma glucose concentration at basal levels
via a variable glucose infusion (DeFronzo et al., 1979). Under steady-state euglycemia,
the glucose infusion rate is equal to the glucose uptake by all insulin sensitive tissues in
the body, primarily skeletal muscle and fat (DeFronzo et al., 1985). The euglycemic,
hyperinsulinemic clamp is the recognised gold standard for measuring whole-body
insulin sensitivity (DeFronzo et al., 1979). In cross-sectional studies, those who are
physically active have greater insulin sensitivity than sedentary individuals (Ebeling et
al., 1993; Kirwan et al., 2000; Takala et al., 1999). Indeed, a single bout of exercise
provides sufficient stimuli to improve insulin sensitivity in humans (Mikines et al.,
1988; Thorell et al., 1999; Wojtaszewski et al., 1997) and rodents (Hansen et al., 1998).
Whilst endurance training in humans (1-12 weeks, 30-60 min/ day) has also been shown
in many studies to improve insulin sensitivity (DeFronzo et al., 1987; Dela et al., 1994;
Houmard et al., 1999; Hughes et al., 1993) with even one week of endurance training
being sufficient to improve glucose tolerance in humans with mild type 2 diabetes
(Rogers et al., 1988). Recently, a 6-month walking program involving moderate or
vigorous walking for 30 minutes per day, 3-7 days per week in older, mildly overweight
non-diabetics was sufficient to improve insulin sensitivity despite no changes in
bodyweight (Duncan et al., 2003). Thus, there is considerable evidence that physical
6
training and more specifically, endurance training has a powerful impact on insulin
action in human skeletal muscle. Yet it remains to be fully elucidated how regular
muscle contraction is able to elicit such a profound effect on the intracellular actions of
insulin. An obvious starting point when searching for the underlying mechanisms of
insulin resistance is to discover where differences lie along the pathway of insulin
signalling to glucose transport and glycogen synthesis in type 2 diabetics compared with
subjects displaying normal glucose tolerance. Downstream of insulin signalling, defects
in the skeletal muscle of type 2 diabetics are apparent, including reduced hexokinase II
and glycogen synthase activity (Cusi et al., 2000; Kruszynska et al., 1998). Type 2
diabetics also have a downregulation of a number of steps involved in insulin-signalling
and glucose transport (Cusi et al., 2000). To understand this further it is important to
examine firstly the pathway of insulin action, how this pathway is dysregulated in
insulin resistant states and then identify how exercise might interact with the insulin-
signalling cascade to enhance insulin action.
1.3 Pathway of insulin signalling
The binding of insulin to its receptor initiates a complex cascade of signalling events,
which ultimately leads to the translocation of the GLUT4 glucose transporter to the
plasma membrane. The key proteins involved in insulin signalling and their interaction,
resulting in GLUT4 translocation, is not fully understood. However, several steps have
been well elucidated and are known to be crucial in this pathway (Figure 1.1).
The insulin receptor is a transmembrane protein comprising two alpha subunits, each
linked to a beta subunit and each other by disulfide bonds. The two alpha subunits are
7
extracellular and contain the insulin binding sites, whilst the two beta subunits are
transmembraneous with the intracellular portion containing the insulin regulated
tyrosine protein kinase (for reviews, see (Kellerer et al., 1999; White & Kahn, 1994).
Binding of insulin to the alpha subunits activates the tyrosine kinase of the beta
subunits, enabling autophosphorylation of the insulin receptor (IR) via tyrosine
phosphorylation. This autophosphorylation of the IR in turn enables phosphorylation of
a family of multi-site docking proteins called insulin receptor substrates (IRS), each
containing numerous tyrosine and serine phosphorylation sites including at least 8
tyrosine sites on IRS-1 alone that are phosphorylated by the activated IR. (Lavan et al.,
1997a; Lavan et al., 1997b; Sun et al., 1993; Sun et al., 1995). The 4 known members of
the IRS family are IRS-1, IRS-2, IRS-3 and IRS-4 (Lavan et al., 1997a; Lavan et al.,
1997b; Sun et al., 1993; Sun et al., 1995). Of the 4 IRS proteins, various combinations
of genetic knockout mice models have demonstrated a major role for IRS-1 and IRS-2
in insulin signalling, with IRS-1 being a major regulator in muscle and IRS-2 being
prominent in liver (Kido et al., 2000). Recent findings also point to a partial insulin-
signalling role for IRS-2 in skeletal muscle immediately after exercise (Howlett et al.,
2002). The tyrosine phosphorylation of IRS-1 binds a number of proteins containing the
Src-homology 2 and 3 domains (SH2 and SH3). These include Src homology protein
tyrosine phosphatase 2 (SHPTP2, also called SHP2); a protein tyrosine phosphatase,
GRB2; an adaptor molecule that links the RAS pathway and is involved in cell growth
and metabolism, Nck; an adaptor protein involved in cell proliferation and the lipid
cleavage enzyme phosphatidylinositol 3-kinase (PI 3-kinase) that is a key enzyme
required for insulin stimulated glucose transport (Figure 1.1) (Lee et al., 1993; Myers et
al., 1992; Skolnik et al., 1993; Sun et al., 1993). In addition to phosphorylating PI 3-
kinase the IRS proteins traffic insulin signalling via the mitogenic pathways (for
8
reviews see (Kellerer et al., 1999; Virkamaki et al., 1999; White & Kahn, 1994). Recent
evidence also points to a role in GLUT4 activation for the p38 subgroup of the mitogen
activated protein kinase (MAPK) pathways (Somwar et al., 2001; Thong et al., 2003)
independently of IRS’s and PI 3-kinase, although the mitogenic pathways are not the
focus of this review.
To add further to this complexity, PI 3-kinase is a heterodimeric enzyme composed of a
p85 regulatory subunit and a p110 catalytic subunit. The p85 regulatory subunit resides
predominantly in the cytosol in the basal state. Following insulin stimulation, the p85
subunit translocates from the cytosol to the intracellular membrane whereby it
associates the activated IRS proteins and is activated (Clark et al., 1998; Inoue et al.,
1998). Currently, five isoforms of the regulatory subunits have been identified, two 85
kDa proteins (p85α, p85β), two 55 kDa proteins (p55α, p55γ) and a 50 kDa protein
(p50α) (Inukai et al., 1997; Shepherd et al., 1997). The isoforms have tissue-specific
distribution and exhibits different degrees of activation in response to stimulation by
insulin (Inukai et al. 1997). Of these isoforms, p85α is ubiquitous and is expressed most
abundantly in muscle, fat and liver (Inukai et al., 1997; Shepherd et al., 1997). Chemical
inhibition, and more recently genetic manipulation to generate dominant negative
mutant p85 cells have demonstrated the key role of PI 3-kinase in the stimulation of
GLUT4 translocation by insulin (Cheatham et al., 1994; Lund et al., 1995;
Wojtaszewski et al., 1996).
9
Figure 1.1 Pathway of insulin signalling. Upon insulin binding to its receptor it undergoes
autophosphorylation, whereby it catalyses phosphorylation of a number of proteins, including
the IRS family that is involved in glucose transport. Insulin receptor autophosphorylation also
initiates numerous cellular processes involved in glucose metabolism, glycogen synthesis, cell
growth and differentiation, protein synthesis and gene expression. Adapted from (Saltiel &
Kahn, 2001). Note: SHP2 is also called SHPTP2 and is known to associate with IRS-1 upon
insulin-stimulation and is thought to be involved in glucose transport (Kuhne et al., 1993;
Maegawa et al., 1999).
10
Much of the complexity of this pathway resides beyond PI 3-kinase activation, with
many of the steps not yet identified. Little is known of the proteins downstream of PI 3-
kinase that are involved in the regulation of GLUT4 translocation. Akt, also known as
protein kinase B, is potentially a key protein involved in insulin stimulated GLUT4
translocation (Kim et al., 1999b; Thorell et al., 1999; Wojtaszewski et al., 1999) whilst
some atypical isoforms of protein kinase C (aPKC) such as zeta (ζ) and lambda (λ) may
also have a role (Standaert et al., 1999).
Akt is a downstream target of PI 3-kinase, that is activated by insulin and has a potential
role in glucose transport since overexpression of constitutively active Akt increases
GLUT4 translocation and glucose transport in 3T3L1 adipocytes and L6 muscle cells
(Hajduch et al., 1998; Kohn et al., 1996; Tanti et al., 1997; Ueki et al., 1998). Recently,
some isoforms of Akt (Akt-2 and Akt-3) have shown defective activity in obese, insulin
resistant humans compared with lean controls (Brozinick et al., 2003). The precise role
of Akt in glucose transport is somewhat unclear since overexpression of a dominant
negative mutant Akt in chinese hamster ovary (CHO) cells does not effect insulin
stimulated glucose transport (Kitamura et al., 1998) suggesting that it may not play a
major role in glucose transport. However, inhibitory mutant models are problematic
with chronic mutant overexpression potentially allowing the adaptation of alternate
pathways (Hill et al., 1999). Recently, depletion of Akt-1 and/or Akt-2 isoforms in
3T3L1 adipocytes by RNA directed gene silencing suggest a primary role for Akt-2 and
a lesser role for Akt-1 in insulin-stimulated glucose transport (Jiang et al., 2003).
Insulin stimulation of Akt has been shown to increase glycogen synthesis in L6
myotubes by inhibiting glycogen synthase kinase-3 (GSK-3), a known inhibitor of
glycogen synthase (Cross et al., 1995; Cross et al., 1997; Ueki et al., 1998) whilst in cell
11
lines lacking GSK-3 such as 3T3L1 adipocytes, overexpression of Akt has no effect of
glycogen synthesis (Ueki et al., 1998). Activation of Akt also has been shown to
increase protein synthesis in both L6 myotubes and adipocytes (Hajduch et al., 1998;
Ueki et al., 1998). The pathway for insulin stimulated protein synthesis via the
activation of Akt appears to involve the downstream activation of p70 S6 kinase, since
inhibition of Akt reduces activation of p70 S6 kinase in CHO cells and 3T3L1
adipocytes (Kitamura et al., 1998). These findings suggest that not only is Akt a crucial
insulin signalling protein that resides downstream of PI 3-kinase, but that it has multiple
signalling roles in response to insulin stimulation that appear to involve glucose
transport as well as glycogen and protein synthesis.
Other components of the insulin signalling pathway downstream of PI 3-kinase that
have been shown to have a role in insulin stimulated glucose transport is the activation
of atypical PKC isoforms ζ and λ via activation of 3-phosphoinositide-dependent
protein kinase 1 (PDK1) (Grillo et al., 1999; Standaert et al., 1999). The role of these
downstream kinases in the insulin stimulated translocation of GLUT4 has still to be
fully elucidated and will not be a major focus of this review although they are a
promising area of future investigation since atypical PKC activity is associated with
impaired insulin-stimulated GLUT4 translocation brought about by high fat feeding and
fatty acid infusion in rodents (Kim et al., 2002; Tremblay et al., 2001).
This leaves the perplexing situation in which it has yet to be adequately described how
insulin stimulates glucose transport. Clearly such research is a focus of major research
endeavours.
12
1.4 A second pathway for insulin signalling
Recently, a second novel insulin-signalling pathway has been described (Baumann et
al., 2000) that has been shown to be essential for insulin stimulated glucose uptake in
adipocytes. This pathway involves the recruitment of the protein c-Cbl to the IR by the
c-Cbl associating protein (CAP) (Figure 1.1). c-Cbl is a 120-kDa cellular homologue of
the transforming v-Cbl oncogene. In 3T3L1 adipocytes c-Cbl is rapidly tyrosine
phosphorylated by insulin (Ribon & Saltiel, 1997) and is recruited to the IR by CAP
(Ribon et al., 1998). Tyrosine phosphorylation via the IR initiates the dissociation of the
CAP-Cbl complex enabling c-Cbl interaction with the caveolar protein flotillin. Flotillin
forms a ternary complex with both CAP and c-Cbl directing its localisation to lipid rich
regions of the plasma membrane known as lipid rafts. Expression of a non-functional
CAP mutant in 3T3-L1 adipocytes prevents insulin stimulated GLUT4 translocation and
glucose transport, demonstrating the importance of this pathway (Baumann et al., 2000).
A number of other stimuli are able to tyrosine phosphorylate c-Cbl, including
extracellular growth factor (EGF) and interleuken-3 (Anderson et al., 1997; Fukazawa
et al., 1996; Marcilla et al., 1995; Meisner et al., 1995; Ribon & Saltiel, 1997). Shear
stress also tyrosine phosphorylates c-Cbl in endothelial cells (Miao et al., 2002).
However, studies are yet to be undertaken to determine the role of this pathway in the
primary target for glucose disposal, skeletal muscle. There is evidence that c-Cbl’s role
in insulin signalling may be tissue specific. Cell lines such as 3T3L1 fibroblasts do not
contain the c-Cbl associating protein, CAP and do not show the same insulin stimulated
tyrosine phosphorylation of c-Cbl as 3T3L1 adipocytes (Ribon et al., 1998). In cell lines
expressing high levels of functional insulin receptors, such as chinese hamster ovary
(CHO) cells overexpressing the human insulin receptor, rat 1 fibroblasts (HIRC) and
KRC-7 cells insulin treatment does not induce tyrosine phosphorylation of c-Cbl (Ribon
13
& Saltiel, 1997). Unfortunately, there has been no investigation into the role of c-Cbl in
insulin action in the primary site of glucose disposal, skeletal muscle to confirm if the
pathway is a central component of insulin signalling or if it is an ancillary pathway used
for insulin signalling in cells lacking sufficient abundance of CAP or insulin receptors.
1.5 Exercise and glucose transport
Exercise alone enables GLUT4 translocation to the cell surface and increased GLUT4
glucose transport (Richter et al., 1998; Thorell et al., 1999). The mechanisms for this
contraction-mediated activation of GLUT4 remain unknown, although it is now well
established that the pathway is independent from the proximal steps in the insulin-
signalling cascade. Type 2 diabetics also have normal contraction-stimulated GLUT4
translocation and glucose uptake (Kennedy et al., 1999; Kingwell et al., 2002) so it is
unlikely that the contraction-stimulated pathway for GLUT4 translocation is directly
involved in the increased insulin sensitivity observed following exercise cessation. The
independent effects of exercise on GLUT4 translocation and subsequent glucose
transport will therefore not be covered in great detail in this review. For recent insights
detailing the independent mechanisms by which muscle contraction alone stimulates
glucose transport see reviews (Cortright & Dohm, 1997; Hayashi et al., 1997; Richter et
al., 2001; Ryder et al., 2001). Although contraction and insulin seem to stimulate
GLUT4 translocation and glucose uptake via independent pathways, it has been
mentioned earlier that following exercise cessation there is a substantial increase in
insulin action above that exhibited by contraction or insulin alone and can last up to 48
hours following a single exercise bout (Mikines et al., 1988). Acute exercise and
exercise training increases hexokinase II and glycogen synthase activity along with
insulin sensitivity in normal glucose tolerant rodents (Kim et al., 2000a; O'Doherty et
14
al., 1993) and also increases glycogen synthase (GS) activity in type 2 diabetic humans
(Cusi et al., 2000). Similar improvements have also been found at a number of steps
involved in glucose transport (Cusi et al., 2000; Houmard et al., 1999). However
nuclear magnetic resonance (NMR) combined with microdialysis measurements of
skeletal muscle found no association between reduced glycogen synthesis and
intracellular glucose accumulation in type 2 diabetics (Cline et al., 1999). This would
suggest that glucose transport is a major rate-limiting step for insulin-stimulated glucose
metabolism in type 2 diabetic muscle. Recent exercise training and high fat diet studies
in rodents would support this view (Kim et al., 2000a).
1.6 Defective insulin signalling in insulin resistant states
Critical to the impairment in insulin action observed in human diseases of insulin
resistance, including obesity and type 2 diabetes, is attenuated activation of the tyrosine
phosphorylation of the key insulin-signalling proteins. Reduced insulin stimulated
tyrosine phosphorylation of IR, IRS-1 and IRS-1 associated PI 3-kinase activity is
evident in human obesity (Cusi et al., 2000), demonstrating reduced activation of the
initial steps of insulin action. In addition, morbid obesity (BMI > 40 kg/m2) is also
accompanied by a reduction in the protein content of IR, IRS-1 and the p85 subunit of
PI 3-kinase in their muscles (Goodyear et al., 1995a). Similar defects are observed in
obese rats (Anai et al., 1998; Folli et al., 1993; Kerouz et al., 1997). Type 2 diabetics
demonstrate comparable suppression of insulin stimulated IRS-1 tyrosine
phosphorylation and lower activation of PI 3-kinase activity by either IRS-1 or IRS-2
(Bjornholm et al., 1997; Cusi et al., 2000; Kim et al., 1999b; Krook et al., 2000).
15
Downstream of PI 3-kinase, there is some controversy regarding the defective activation
of Akt by insulin in muscle that is insulin resistant. Some studies have shown that obese
subjects and type 2 diabetics have normal activation of Akt isoforms (Kim et al.,
1999b), whilst recently Brozinick et al. (Brozinick et al., 2003) has shown defective
activity of Akt-2 and Akt-3 isoforms but not Akt-1 in morbidly obese subjects.
Interestingly, there was an impairment of Akt-1 serine (473) phosphorylation in obese
subjects despite normal activation of Akt-1 kinase activity suggesting other sites of
regulation for Akt-1 apart from phosphorylation on serine site 473 such as the threonine
308 site (Brozinick et al., 2003). The discrepancies between studies could be due to a
number of factors such as different subject populations or muscle analysis although the
differences are probably due to antibody specificity. Kim et al. (Kim et al., 1999b)
found no defective activation of Akt immunoprecipitated with an antibody that
recognises both Akt-1 and Akt-2 isoforms. If Akt-1 were normal in type 2 diabetics then
this would mask any defective regulation of Akt-2 using an antibody that recognises
both isoforms. Clearly, more investigation into the functional regulation of proteins
distal to PI 3-kinase in insulin resistant states is required. However the pattern emerges
that in insulin resistant states, a major defect in insulin signalling resides in an impaired
capacity to generate a tyrosine phosphorylation signal from the IR through to PI 3-
kinase. Further downstream in the insulin signalling pathway it is unlikely that GLUT4
protein abundance is primarily involved in the development of insulin resistance since
type 2 diabetics have normal levels of GLUT4 protein (Andersen et al., 1993; Kennedy
et al., 1999).
16
1.7 Interactions between exercise and the insulin signalling
pathway
It was mentioned earlier that a remarkable effect of exercise is that enhanced insulin
sensitivity is observed for up to 48 hours following the cessation of a single exercise
bout (Mikines et al., 1988). There is some evidence that during the later stages of this
period enhanced insulin sensitivity may be due to an upregulation of the insulin-
signalling cascade. In the hours following a single bout of endurance exercise, improved
insulin sensitivity is not due to an upregulation of the proximal steps in the insulin-
signalling cascade. Indeed there is no increase in insulin binding and activation of the
IR despite blood flow potentially increasing the amount of available insulin to the
contracted muscle (Bonen et al., 1985; Treadway et al., 1989). There is no increase in
the insulin-stimulated tyrosine phosphorylation of IRS-1, or its associated activation of
PI 3-kinase despite significant increases in insulin sensitivity (Goodyear et al., 1995b;
Wojtaszewski et al., 1997). Prior exercise alone also reduces basal IR tyrosine
phosphorylation without significantly activating IRS-1 or its association with the p85
subunit of PI 3-kinase (Koval et al., 1999). However it appears that following exercise
and insulin stimulation, IR/ IRS-1 independent pathways can activate PI 3-kinase.
Studies in rodents have demonstrated an insulin-stimulated increase in phosphotyrosine
associated PI 3-kinase activity following acute exercise (Zhou & Dohm, 1997) with part
of the increase in phosphotyrosine associated PI 3-kinase activity attributed to IRS-2
associated PI 3-kinase activity (Howlett et al., 2002). Also, recent work using IR
knockout mice suggest that an as yet unidentified tyrosine phosphoprotein is binding
and activating PI 3-kinase following exercise (Wojtaszewski et al., 1999).
17
Also, there is evidence that in the few hours following exercise, a serum factor, most
likely a protein, may be responsible for enhanced insulin sensitivity although little
research has followed up on this theory (Gao et al., 1994). Alternative mechanisms
regulating tyrosine phosphoproteins may also be involved in the enhancement of insulin
signalling and these will be discussed at a later stage.
There is also some evidence that some of the downstream insulin signalling proteins
may be activated by contraction. Although the activation of Akt by insulin is well
known, its activation by contraction is somewhat controversial in skeletal muscle
(Brozinick Jr. & Birnbaum, 1998; Sherwood et al., 1999; Turinsky & Damrau-Abney,
1999; Whitehead et al., 2000). Total Akt kinase activity does not increase in response to
contraction (Brozinick Jr. & Birnbaum, 1998; Sherwood et al., 1999) although a small
increase in Akt-1 kinase activity and serine (473) phosphorylation has been observed
following electrical stimulation in rat muscle (Turinsky & Damrau-Abney, 1999;
Whitehead et al., 2000). The serine (473) and threonine (308) phosphorylation of all
three isoforms (Akt-1 > Akt-2 > Akt-3) by contraction has recently been confirmed
(Sakamoto et al., 2002) however this effect is relatively minor and transient compared
to the insulin stimulated activation of Akt (Sakamoto et al., 2002; Turinsky & Damrau-
Abney, 1999; Whitehead et al., 2000) since maximal activation occurs by 3 minutes and
then returns to basal levels by 15 minutes of contraction. In humans, pAkt (Ser473) is
increased immediately following exercise (Thorell et al., 1999) although pAkt (Ser473)
is not elevated in humans 4-hours after the cessation of exercise (Wojtaszewski et al.,
2000). However, it may play a role in enhanced insulin-stimulated glucose uptake in the
longer term (24–48 hour period) following exercise cessation. Rodent studies point
towards a role for an additive effect of exercise on insulin-stimulated Akt
18
phosphorylation since it has been shown to be increased in the 16-48 hour period
following 5 days and 6 weeks of exercise training in lean, non-diabetic rats (Chibalin et
al., 2000; Luciano et al., 2002) with a trend for increased pAkt (Ser473) following one
day of exercise (Chibalin et al., 2000). Clearly, more work needs to be done to
investigate the potential effect of muscular contraction on Akt activation although it is a
potential candidate for the regulation of insulin-stimulated glucose uptake following
exercise.
A single bout of exercise and exercise training also enhances GLUT4 gene expression,
protein concentration and activation (Cox et al., 1999; Goodyear et al., 1990; Kraniou et
al., 2000). Furthermore, overexpression of GLUT4 in transgenic mice improves basal
and insulin-stimulated glucose transport (Brozinick et al., 2001; Brozinick et al., 1997)
so it is possible that increases in GLUT4 content following exercise could be at least be
partly responsible for improvements in insulin sensitivity.
Exactly, how exercise is then able to increase insulin action remains unclear. Based on
available data 2 mechanisms can be proposed.
• An acute effect, which lasts for a few hours following exercise cessation and
appears to act independently of the early steps in the insulin-signalling cascade.
• And a second chronic response that can last for up to 48 hours following a single
exercise bout (Mikines et al., 1988) and may be related to improvements in
insulin-signalling transduction or increased protein abundance that may account
for greater activation (Chibalin et al., 2000; Cusi et al., 2000). It is this longer
19
lasting effect of a single bout of exercise or endurance training on insulin action
and insulin signalling transduction and the potential regulatory mechanisms that
will be a major focus of this thesis. Therefore, unless otherwise stated, the
chronic response of insulin action and/or insulin signalling to either a single
(acute) bout of exercise or endurance training in the period > 15 hours post
exercise will form the basis of the proceeding discussion.
1.7.1 Insulin signalling and acute exercise
One of the remarkable effects of a single exercise bout at a moderate intensity is its
ability to exert improvements on insulin sensitivity for up to 48 hours following
exercise (Mikines et al., 1988). Investigations into the mechanisms of how insulin
sensitivity is increased 24-48 hours after a single exercise bout is an area of
considerable interest. Along with the improvements in insulin sensitivity there are also
improvements in a number of the early signalling events following insulin stimulation
including IRS-1 and IRS-2 associated PI 3-kinase activity in lean, non-diabetic rodents
examined 16 hours following exercise (Chibalin et al., 2000). There are few
comparative results in humans, particularly in subjects with normal glucose tolerance.
In obese non-diabetics, insulin sensitivity was improved 24 hours following a single
exercise bout and this was associated with improvements in IR tyrosine phosphorylation
and the association of the p85 subunit of PI 3-kinase with IRS-1 (Cusi et al., 2000).
When the same experiment was performed in type 2 diabetics, there was no change in
insulin sensitivity despite improvements in IR and IRS-1 tyrosine phosphorylation.
There was also no improvement in the p85 association with IRS-1 (Cusi et al., 2000) in
type 2 diabetics, suggesting that this may be a key rate-limiting step in insulin action.
Clearly, further work needs to clarify the differences in insulin action and the signalling
20
cascade between normal and insulin resistant subjects. Exercise however, provides a
powerful intervention in which to understand the mechanisms behind the intracellular
defects in those individuals with impaired glucose tolerance.
It is possible that acute exercise may be exerting changes in the protein abundance of
some of the proteins involved in insulin signalling. Most of the primary signalling
proteins such as IR and IRS-1 do not change in response to a single bout of exercise
although IRS-2 protein abundance and tyrosine phosphorylation significantly increase
in the 24 hours following acute exercise (Chibalin et al., 2000). IRS-2 may exert a
major role in mediating insulin action in liver and in the development and maintenance
of pancreatic β-cell mass, yet its precise role in skeletal muscle is uncertain (Kido et al.,
2000; Withers et al., 1999). In isolated muscle and fat cells, IRS-2 has been shown to
have a similar role to IRS-1 in insulin-stimulated glucose transport (Miele et al., 1999;
Zhou & Dohm, 1997) and may act as an alternative pathway of insulin action. Although
IRS-2 has been shown not to play a major role in exercise or insulin stimulated glucose
transport (Higaki et al., 1999), insulin stimulated IRS-2 tyrosine phosphorylation and
associated PI 3-kinase activity are elevated immediately post exercise and can account
for some of the improvements in insulin stimulated glucose uptake (Howlett et al.,
2002) immediately post exercise, however much less is known of its role in the 12-24
hours period after the cessation of acute exercise. Even less is known of the
transcriptional regulation of IRS-2 following exercise. If the protein abundance of IRS-
2 is altered 24 hours following acute exercise, increases in IRS-2 gene expression may
be initiated via contraction, thus upregulating protein expression, although this has not
been investigated. It is also unknown if similar transient increases in IRS-2 protein
expression are observed in humans following acute exercise.
21
1.7.2 Insulin signalling and exercise training
Exercise training is typically used to describe bouts of exercise performed on a regular
basis. Previous studies examining the effect of exercise training on insulin action have
typically used bouts of moderate endurance exercise on most days of the week for a
minimum of 5 days up to several weeks (Chibalin et al., 2000; Houmard et al., 1999;
Luciano et al., 2002; Tanner et al., 2002). Recent human studies have found significant
improvements in whole-body insulin sensitivity following short-term endurance training
involving several consecutive days of cycling for 60 minutes per day at an intensity of
around 75% peak (Houmard et al., 1999; Tanner et al., 2002; Youngren et al.,
2001). However, the mechanisms which insulin enhances signalling following exercise
training remain unclear. Principally, the insulin-signalling pathway could be regulated
by several means. Exercise training could be mediating improvements in the abundance
of key proteins such as IR, IRS-1, and IRS-2 or increased protein abundance of protein
kinases such as Akt or specific components in the PI 3-kinase complex. Although
skeletal muscle IR protein content in rats is significantly elevated following both a
single exercise bout and exercise training (Chibalin et al., 2000) a similar effect is not
apparent in humans (Cusi et al., 2000; Youngren et al., 2001). Indeed, IRS-1 protein
abundance has been shown to be decreased following short term training in rats, whilst
IRS-2 protein content increases after one bout of exercise and returns to sedentary
levels following training (Chibalin et al., 2000). Longer-term training (6-7 weeks) in
rodents shows no change in protein abundance of any of the major signalling proteins
(Christ et al., 2002; Luciano et al., 2002). Therefore transcriptional control of these
insulin-signalling proteins does not appear to be a central component of the adaptive
response to exercise training. This does not imply that the gene expression of these
2OV&
22
proteins cannot change in response to exercise training. The mRNAs of IR, IRS-1 and
PI 3-kinase increase in response to 9 weeks of endurance training in rats (Kim et al.,
1995; Kim et al., 1999a) however these changes probably occur subsequent to exercise
induced improvements in insulin sensitivity. The findings discussed above would
therefore suggest that changes in the protein abundance of the various insulin signalling
proteins do not appear to be the main mechanisms by which endurance training exerts
its influence on insulin sensitivity.
In humans and rats it appears that insulin signal transduction is a major factor, although
probably not the only factor, contributing to increased insulin sensitivity following
training. In the insulin resistant state, exercise training induced improvements in insulin
sensitivity may not be mediated by changes in the proximal components of the insulin
signalling pathway such as the IR, IRS-1, PI 3-kinase or Akt (Christ et al., 2002). In
obese rats, several weeks of endurance training improves insulin sensitivity despite no
changes in IR, IRS-1 tyrosine or Akt serine phosphorylation (Christ et al., 2002).
Moderately obese middle-aged men were observed following short term training to have
improved insulin sensitivity without any improvement in PI 3-kinase, also suggesting
that enhanced insulin action may occur downstream of PI 3-kinase (Tanner et al., 2002).
In support of this finding is the recent investigation that 5 months of exercise training in
patients with chronic heart failure did not improve insulin signalling at the level of IRS-
1, PI 3-kinase or Akt despite improvements in insulin sensitivity (Kemppainen et al.,
2003). This would imply that in the insulin resistant state, exercise-induced
improvements in insulin sensitivity are either occurring at more distal and as yet
unknown steps in the insulin-signalling cascade or via a mechanism independent from
insulin signalling.
23
In the healthy state with normal glucose tolerance, the changes in insulin signal
transduction following training are not clearly defined or understood even for the
proximal components of the cascade. Endurance training for six weeks in lean, healthy
rodents upregulates numerous steps in the insulin-signalling cascade (Luciano et al.,
2002) and IRS-1 associated PI 3-kinase activity is significantly higher in trained
subjects compared with sedentary subjects (Kirwan et al., 2000). However with such a
long time course, most of these changes could be occurring secondary to improvements
in insulin sensitivity. Short-term endurance training of between 5-7 days does however
significantly improve insulin stimulated IR function and PI 3-kinase activity in both
humans and rodents (Chibalin et al., 2000; Houmard et al., 1999; Youngren et al.,
2001). However in humans, IR autophosphorylation was measured in vitro (Youngren
et al., 2001) in relatively high insulin concentrations and it is unknown if it is improved
in vivo at normal physiological levels of insulin. It is also unknown what intracellular
mechanisms are regulating this improvement in insulin signalling and this will be
covered in some detail at a later stage. Even though in the insulin resistant or diabetic
state exercise training may be mediating improvements in the insulin-signalling cascade
distal to PI 3-kinase, it is still important to understand how exercise training in the
healthy, glucose-tolerant state regulates the insulin-signalling pathway. Not only does
this further our understanding of the overall regulation of the insulin signalling cascade,
but as discussed above, physically active people not only have enhanced insulin
sensitivity compared to sedentary subjects with normal glucose tolerance, they also
have an upregulation of numerous steps in the insulin signalling cascade, that may
ultimately provide protection from metabolic diseases such as type 2 diabetes.
24
1.7.3 Insulin sensitivity, insulin signalling and resistance training
Recently, insulin action has shown to be improved by resistance exercise (Dunstan et
al., 2002; Ishii et al., 1998; Miller et al., 1994; Poehlman et al., 2000). The mechanisms
to account for improvements in insulin action via resistance exercise remain unknown.
Some cross sectional and longitudinal studies suggest resistance training mediates
improvements in insulin action via increases in lean muscle mass alone without
improving insulin sensitivity per unit of skeletal muscle mass (Poehlman et al., 2000;
Takala et al., 1999). Other resistance training studies have shown insulin action to be
improved independently of changes in lean muscle mass or body composition (Dunstan
et al., 2002; Ishii et al., 1998). Muscle overload in rodents has been shown to increase
glucose uptake, protein synthesis and IRS-1 and p85 associated PI 3-kinase activity
independently of insulin (Carlson et al., 2001; Hernandez et al., 2000) suggesting
multiple and divergent roles for these proteins. However the effects of resistance
exercise on any aspect of the insulin-signalling pathway have yet to be studied in
humans.
1.7.4 Unresolved issues regarding improved insulin signalling and sensitivity via
exercise
Even though short-term endurance training in humans has also been shown in many
studies to improve insulin sensitivity (DeFronzo et al., 1987; Dela et al., 1994;
Houmard et al., 1999; Youngren et al., 2001) one constant criticism is that the effect is
primarily due to the residual effects of the last bout of exercise and not the result of
cumulative bouts of exercise. Indeed, early studies using an oral glucose tolerance test
(OGTT) to measure insulin action have found that a large portion of the improvement in
insulin sensitivity following exercise could be attributed to the last bout (Heath et al.,
25
1983). A single bout of exercise is sufficient to improve insulin sensitivity in humans
(Mikines et al., 1988; Thorell et al., 1999; Wojtaszewski et al., 1997) and rodents
(Hansen et al., 1998) for up to 48 hours (Mikines et al., 1988) and may therefore be
solely responsible for the improvements in insulin action. There is some data in rodents
and insulin resistant humans to suggest that this is not the case and that there is likely an
additive effect of short-term training on insulin sensitivity. In humans with impaired
glucose tolerance, a single bout of exercise does not increase glucose tolerance as
measured by an OGTT although following 7 days of exercise-training glucose tolerance
is increased by approximately 30% (Rogers et al., 1988). In rats measured 16 hours
following cessation of exercise, a single bout of exercise increased insulin-stimulated
glucose transport by 30%, while 5 days of exercise training increased glucose transport
by 50% (Chibalin et al., 2000) suggesting that cumulative bouts of exercise have an
additive effect above that of a single exercise bout, however this effect has not been
fully investigated in humans, particularly those with normal glucose tolerance.
Importantly, the development of type 2 diabetes, from impaired glucose tolerance, is
associated with decreased insulin signalling transduction. The findings that endurance
training increases insulin stimulated IR function and PI 3-kinase activity (Chibalin et
al., 2000; Houmard et al., 1999; Youngren et al., 2001) provides the possibility that the
major beneficial action of exercise training is to exert a positive influence on insulin
signalling via improvements in signal transduction that may ultimately prevent skeletal
muscle from becoming insulin resistant.
Further, it remains unresolved if acute endurance exercise has an impact on the
transcriptional regulation and abundance of proteins such as IRS-2, particularly in
26
humans. Also, the impact of acute endurance exercise and exercise training on the
insulin-stimulated activation of key proteins has yet to be fully resolved, particularly in
humans while even less in known regarding the impact of resistance exercise on insulin
signalling. Additionally, little is known of the molecular mechanisms that may be
mediating the improvements in the activation of these signalling proteins.
1.8 Protein tyrosine phosphatases as regulators of insulin
signalling
Significant interest is currently being focussed on the regulation of the insulin-
signalling cascade by protein tyrosine phosphatases (PTPases). This diverse family of
enzymes have a common conserved catalytic domain made up of around 250 amino
acids with a cysteine residue that is essential for catalysing the hydrolysis of
phosphotyrosine residues (for reviews see (Goldstein et al., 1998; Ostman & Bohmer,
2001) whilst the rest of the protein differs greatly among this group of enzymes.
PTPases act to either dampen or amplify the insulin signal by removing the activated
phosphate group from tyrosine amino acids (Elchebly et al., 1999) and may therefore be
key regulators of the insulin-signalling cascade. The subcellular localisation of PTPases
may also be important since readily abundant PTPases in skeletal muscle and
adipocytes each display characteristic subcellular distribution between the cytosol and
particulate fractions (Table 1.1) suggesting preferential sites of action towards the
insulin receptor and insulin receptor substrates depending upon subcellular localisation
(Figure 1.2).
Immunoblot analysis has confirmed the expression of 4 major PTPases in skeletal
muscle, leukocyte antigen-related (LAR), leukocyte common antigen related (LRP),
27
protein tyrosine phosphatase 1B (PTP1B) and Src Homology (SH) SHPTP2 (Ahmad et
al., 1997a; Ahmad & Goldstein, 1995) although LRP expression appears to be
expressed at relatively lower levels than the other three PTPases (Ahmad & Goldstein,
1995).
The transmembrane enzyme, LAR has been implicated as a negative regulator of insulin
signalling. Reduction of LAR protein abundance via antisense RNA expression in rat
hepatoma cells increased IR signalling (Kulas et al., 1995). Also, elevated LAR
expression accounts for enhanced PTPase activity in the adipose tissue of obese humans
(Ahmad et al., 1995a) suggesting a role for this PTPase in the regulation of insulin
signalling. The regulation of insulin signalling by LAR is thought to be primarily via its
association with the insulin receptor (Ahmad & Goldstein, 1997). However, conflicting
findings have cast some doubt onto the role of LAR in the regulation of insulin
signalling. Unexpectedly, LAR knockout mice display significant insulin resistance
compared to their wild type littermates and post insulin receptor defects involving PI 3-
kinase (Ren et al., 1998) in the liver. In transgenic mice that specifically overexpress
LAR in skeletal muscle, insulin stimulated glucose uptake is suppressed as expected but
the primary sites of action for LAR, namely the IR and IRS-1 were unaltered
(Zabolotny et al., 2001). Rather, the effects of LAR overexpression appear to be
directed towards downstream targets such as IRS-2 and PI 3-kinase (Zabolotny et al.,
2001). The exact role of LAR on insulin signalling remains unclear however it may not
reside at the proximal sites of the insulin-signalling cascade as previously thought.
28
Table 1.1. Classification of Major Protein Tyrosine Phosphatases (PTPases)
implicated in the regulation of insulin signalling in skeletal muscle.
Name Other Names Cellular Distribution
LAR Particulate fraction
LRP RPTP-α Particulate fraction
PTP1B Cytosol & Particulate
SHPTP2 Syp, SHP-2, PTP1D, SHPTP3 & PTP2C Cytosol & Particulate
(Ahmad & Goldstein, 1995; Calera et al., 2000; Maegawa et al., 1999)
Figure 1.2. Potential regulation of the insulin-signalling pathway.
LAR LRP
Insulin Receptor
?
?
GLUT4
Translocation IRS-1
?
aPKC
Akt
PTP1B
SHPTP2
+
PI 3-kinase
+
?
29
PTP1B was the first PTPase to be isolated in homogenous form in 1988 from human
placenta (Tonks et al., 1988). PTP1B is distributed between the cytosol and particulate
fractions in skeletal muscle (Ahmad & Goldstein, 1995) and has been shown in vitro to
negatively regulate insulin signalling via dephosphorylation of the IR and IRS-1
(Ahmad et al., 1995b; Calera et al., 2000; Goldstein et al., 2000; Kenner et al., 1996).
Perhaps the strongest evidence for the role of PTPases in insulin signalling comes from
mice lacking a PTPase gene. Elchebly et al. (1999) generated PTP1B null mice by
targeted disruption of exons 5 and 6 (PTP1B Ex5/6-/-). These mice demonstrated
enhanced insulin sensitivity and increased phosphorylation of IR and IRS-1 in muscle
and liver following insulin infusion. Interestingly, these mice were resistant to weight
gain when fed a high fat diet. Klaman et al. (2000) has also generated PTP1B deficient
mice however they targeted exon 1 (PTP1B Ex1-/-) and also found increased insulin
sensitivity. PTP1B Ex1-/- mice were also shown to have low body fat stores, which is
largely due to an increase in energy expenditure via increases in basal metabolic rate
(Klaman et al., 2000). The relationship between enhanced insulin sensitivity and
increased energy expenditure in PTP1B deficient mice is unclear, however these
findings do point towards an important role for PTP1B in the regulation of insulin
sensitivity. These effects also appear to be tissue specific since overexpression of
PTP1B in adipose cells results in no change in insulin stimulated glucose transport
despite significant reductions in signal transduction through IR, IRS-1 and PI 3-kinase
(Venable et al., 2000). Inhibition of PTP1B activity or disruption of PTP1B protein
abundance in vivo also has a profound effect on insulin signalling. Treatment of obese
Zucker rats with a known inhibitor of PTP1B activity, Bis(maltolato)oxovanadium(IV),
not only reduced PTP1B activity in vivo but reduced plasma insulin levels (Mohammad
et al., 2002). Reduction of PTP1B protein abundance in liver and fat by antisense
30
oligonucleotide treatment in mice has not only been shown to improve insulin
sensitivity but improve insulin stimulated phosphorylation of the IR, IRS-1, IRS-2 and
Akt-1 (Gum et al., 2003; Zinker et al., 2002). Not surprisingly, research is now
underway into specific inhibitors of PTP1B (Iversen et al., 2000) due to its therapeutic
potential in patients with insulin resistance.
Following insulin stimulation, SHPTP2 associates with IRS-1 (Kuhne et al., 1993) and
unlike PTP1B, the major site of SHPTP2 activity appears to reside in the cytosolic
fraction (Ahmad & Goldstein, 1995), implicating SHPTP2 as a potential candidate to
dephosphorylate IRS-1. However, unlike most other PTPases in skeletal muscle,
SHPTP2 has been implicated to have a small role as a positive regulator of insulin
signalling. In rat fibroblasts there is an increase in insulin stimulated binding of IRS-1
with SHPTP2, IRS-1 tyrosine phosphorylation and PI 3-kinase activity when SHPTP2
is overexpressed, with the opposite observed when SHPTP2 is inhibited (Ugi et al.,
1996). Inhibiting SHPTP2 by transfection of a dominant negative mutant in rat
adipocytes also reduces insulin-stimulated translocation of GLUT4 (Chen et al., 1997).
In vivo studies also implicate SHPTP2 as a positive regulator of insulin signalling.
Transgenic mice have been generated to express a dominant negative mutant that lacks
a PTPase domain thereby inhibiting SHPTP2 (Maegawa et al., 1999). These transgenic
mice exhibit impaired insulin stimulated glucose uptake along with impaired activation
of IRS-1, PI 3-kinase and Akt-1 in skeletal muscle. It is unclear how SHPTP2 may
positively influence insulin signalling. Ugi et al., (Ugi et al., 1996) postulated that
SHPTP2 may be increasing tyrosine phosphorylation of IRS-1 by either inhibiting an as
yet unknown PTPase(s) or by activating other tyrosine kinases. SHPTP2 is clearly a
31
candidate involved in the regulation of insulin signalling but as yet it’s precise role and
relative contribution to insulin signalling is yet to be determined.
There is some evidence to suggest that PTPases are negative regulators of insulin
signalling in humans. PTPase activity is significantly increased in skeletal muscle and
adipose tissue of obese subjects compared to lean subjects (Ahmad et al., 1997a; Ahmad
et al., 1995a). Furthermore, enhanced insulin sensitivity following weight loss in obese
subjects correlates with a reduction in PTPase abundance (Ahmad et al., 1997b).
However, PTPase activity is lower in obese type 2 diabetics suggesting that different
regulatory mechanisms may be operating in the diseased state (Ahmad et al., 1997a).
Unfortunately, no studies to date have examined the impact of exercise on PTPase
abundance or subcellular distribution let alone activity. If PTPase abundance,
subcellular distribution or activity were decreased in response to exercise then it is
tempting to speculate that insulin sensitivity might increase due to the amplified
tyrosine phosphorylation on a number of key proteins, including PI 3-kinase.
There are other potential regulators of the insulin-signalling cascade that will not be the
focus of this review but are nevertheless worthy of consideration. Briefly, these include
the lipid phosphatases Src homology 2 domain containing inositol 5-phosphatase 2
(SHIP2) and tensin homolog deleted on chromosome ten (PTEN) as potential
modulators of the proximal and distal actions of PI 3-kinase (for reviews see (Jiang &
Zhang, 2002). SHIP2 is thought to be a negative regulator of insulin signalling via its
ability to dephosphorylate PI(3,4,5)P3 which is a product of PI 3-kinase and a known
activator of Akt (Jiang & Zhang, 2002). Mice deficient in the SHIP2 gene have
increased glucose tolerance and insulin sensitivity along with increased glycogen
32
synthesis (Clement et al., 2001). PTEN is also a potential regulator of insulin signalling
since it dephosphorylates PI(3,4,5)P3 and PI(3,4)P2 and may also be involved in the
modulation of Akt activity by PI 3-kinase (Jiang & Zhang, 2002). Inhibition of PTEN
via an antisense oligonucleotide decreases PTEN protein expression in liver and fat of
mice, thereby increasing insulin sensitivity and insulin-stimulated Akt activity (Butler et
al., 2002). These findings clearly implicate SHIP2 and PTEN as potential regulators of
the insulin-signalling pathway and therapeutic targets for the treatment of insulin
resistance although there is currently no data linking them to the exercise-induced
improvements in insulin sensitivity.
1.9 Summary
Based on the literature reviewed there is a clear need for research to examine the
physiological regulation of the insulin-signalling cascade by exercise in human subjects.
An effective intervention that would provide valuable insights into the regulation of the
insulin-signalling pathway involves both acute exercise and exercise training. There is
currently little known of the cellular adaptations to endurance exercise, which enables
greater insulin sensitivity. Indeed, it is currently unknown if insulin sensitivity is
unregulated solely by the residual effects of acute exercise or if exercise training further
improves insulin sensitivity. The regulation of the proximal aspects of the insulin
signalling cascade including tyrosine phosphorylation of the IR and IRS-1in response to
a physiological in vivo insulin stimulus following both acute exercise and exercise
training still requires clarification even in healthy, glucose tolerant humans. Less is
known of the impact of exercise on more distal components of the insulin-signalling
cascade, such as Akt. Few studies have examined the transcriptional regulation of key
33
insulin signalling proteins such as IRS-2 in skeletal muscle by exercise. There is also
now some evidence in humans and rodents suggesting that resistance exercise can also
improve the intrinsic capacity of skeletal muscle to respond to insulin independent of
changes in lean muscle mass, although even less is known of the molecular mechanisms
that may be regulating this when compared to endurance exercise. The emergence of
PTPases as regulators of insulin signalling provides potential mechanisms through
which muscle contraction may improve insulin-signalling transduction. Furthermore, in
vitro studies have provided an alternative or adjunct pathway by which insulin may
stimulate GLUT4 translocation and glucose uptake. No studies to date have examined if
this pathway is activated by insulin or contraction in vivo, let alone in skeletal muscle,
which is the primary site for glucose uptake in mammals. Understanding the
physiological significance of these pathways in vivo and responsiveness to exercise will
have a profound impact upon the delivery of diabetic care and management as well as
providing insights into the development of safer therapies for the treatment and
prevention of this and other metabolic diseases.
1.10 Aims
The aims of this thesis are:
1. To determine the impact of acute endurance exercise and endurance training on
the mRNA levels of the key insulin signalling proteins IR, IRS-1, IRS-2 and the
p85α subunit of PI 3-kinase in human skeletal muscle.
2. To determine the impact of acute resistance exercise on the mRNA levels of key
insulin signalling proteins in human skeletal muscle.
34
3. To determine if short-term endurance training results in significantly higher
levels of whole body insulin sensitivity compared to a single bout of endurance
exercise in sedentary males.
4. To determine if a single bout of acute exercise and short-term endurance training
results in increased protein abundance and/or insulin-stimulated phosphorylation
of IR, IRS-1 and Akt in sedentary humans.
5. To examine in detail whether acute endurance exercise or short-term endurance
training alters the abundance or subcellular distribution of PTPases such as
SHPTP2 and PTP1B in human skeletal muscle and to see if this coincides with
improvements in tyrosine phosphorylation of IR, IRS-1 or serine
phosphorylation of Akt.
6. To determine if acute endurance exercise or training alters IRS-2 protein
expression in human muscle.
7. To determine if insulin stimulation or acute exercise activates the novel insulin
signalling pathway involving tyrosine phosphorylation of c-Cbl in skeletal
muscle of rodents.
8. To determine if there are any differences in c-Cbl protein expression or tyrosine
phosphorylation in the muscle of lean and insulin resistant rodents.
35
CHAPTER TWO
DIFFERENTIAL EFFECTS OF EXERCISE ON INSULIN
SIGNALLING GENE EXPRESSION IN HUMAN
SKELETAL MUSCLE
2.1 Introduction
Despite the epidemiological evidence correlating regular physical activity with
improvements in insulin action (Helmrich et al., 1991; Manson et al., 1992; Manson et
al., 1991) there is a paucity of consistent data examining the cellular mechanisms of
action, particularly in humans. In recent years, it has become apparent that
transcriptional regulation of gene expression is an integral component of skeletal
muscle adaptation to exercise (Goldspink, 1998; Neufer et al., 1998; Puntschart et al.,
1998). Although the mechanisms by which insulin signalling is enhanced following
exercise remain unclear, the gene expression of key proteins involved in the insulin-
signalling pathway could be mediated by endurance training and may account for some
of the improvements in insulin signalling following chronic exercise.
The mRNAs of insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and
phosphatidylinositol 3-kinase (PI 3-kinase) increase in response to endurance training in
rats (Kim et al., 1995; Kim et al., 1999a), while skeletal muscle IR protein content is
significantly elevated following both a single exercise bout and exercise training in rats
(Chibalin et al., 2000). In humans, PI 3-kinase activity during insulin infusion is
increased following endurance training (Houmard et al., 1999), although mRNA
abundance was not reported. In addition to IRS-1, skeletal muscle contains an
36
alternative substrate of the IR, insulin receptor substrate-2 (IRS-2). The response of
IRS-2 appears to differ from IRS-1, with IRS-2 protein content increasing after a single
bout of exercise, although this increase is abolished following several bouts of
endurance exercise (Chibalin et al., 2000). The regulation of IRS-2 gene expression in
human skeletal muscle by exercise has yet to be described.
In this study, it was hypothesised that a mechanism contributing to the enhancement of
insulin action following endurance training in human subjects is upregulated gene
expression of key members of the insulin-signalling cascade. To examine this
hypothesis, the impact of a single bout of exercise and short term endurance training on
the mRNA levels of IR, IRS-1, IRS-2 and the p85α subunit PI 3-kinase in untrained
human subjects was determined.
2.2 Materials and Methods
2.2.1 Experimental protocol
Seven healthy subjects volunteered to be involved in the study. Four were female and
three were male. The age, peak and BMI (mean ± SE) of the subjects prior to the
study were 28.9 ± 3.1 years, 37.1 ± 2.7 ml.kg
2OV&
-1.min-1 and 22.6 ± 1.4 kg.m-2,
respectively. Body mass did not change with the 9 days of exercise training (67.7 ± 5.4
vs. 67.2 ± 5.3 kg). The study was approved by the Deakin University Human Research
Ethics Committee and subjects gave their written consent to participate in this study
after all procedures and the possible risks of participation were explained. Prior to
testing, all subjects performed an incremental cycling test to exhaustion for the
37
determination of peak pulmonary oxygen uptake ( peak). All cycling tests were
performed on a cycle ergometer (Quinton Excalibur, Groningen, The Netherlands).
was measured using indirect calorimetry (Gould metabolic systems, Ohio, USA).
2OV&
2OV&
All subjects reported to the laboratory in the morning after an overnight fast (10-12
hours). For the 24 hours preceding all test sessions, subjects abstained from alcohol,
tobacco, caffeine and exercise. Muscle samples were obtained prior to, immediately
after and 3 hr after exercise (from the vastus lateralis) on the first and last days of a 9-
day training program using the percutaneous needle biopsy technique. Excised muscle
tissue from the biopsy was immediately frozen in liquid nitrogen for subsequent
analysis. Exercise was 60 min on a cycle ergometer at 63 ± 2 % peak, which was
repeated for nine consecutive days.
2OV&
2.2.2 Analytical methods
Total RNA was isolated using FastRNA™ Kit-Green (BIO 101, Vista CA) (Dana et al.,
1995). RNA was reverse transcribed to synthesise first strand cDNA using AMV
Reverse Transcriptase (Promega, Madison, WI). Briefly, the RNA was added to a
mixture containing a final concentration of 5 mM MgCl, 10 mM Tris-HCl (pH 8.8), 50
mM KCl, 1% Triton® X-100, 1 mM of each dNTP, 20 U Recombinant Rnasin
Ribonuclease Inhibitor (40 U/µl) and 0.5 µg Oligo (dT)15. Primers were designed using
the Primer Express™ software package version 1.0 (Perkin–Elmer, Norwalk, USA)
from gene sequences obtained from GeneBank (β-actin: X00351, IR: M10051, IRS-1:
NM_005544, IRS-2: AF073310, PI 3-kinase p85α subunit: M61906). The primer
sequences were validated using BLAST (Altschul et al., 1990) to ensure each primer
38
was homologous with the desired mRNA of human skeletal muscle. The primer
sequences are shown in Table 2.1. Real time PCR was used to quantify mRNA
expression and has been described in detail previously (Gerber et al., 1997). This
technique has been modified to include SYBR® Green chemistry rather than the
oligonucleotide probe used elsewhere (Gerber et al., 1997; Xin et al., 1999). Direct
detection of PCR product was monitored by measuring the increase in fluorescence
caused by the binding of SYBR® Green to double stranded DNA (Perkin-Elmer,
Norwalk, USA). mRNA levels were quantitated using the threshold cycle (CT) value,
which is the cycle at which the fluorescence emission increases above a threshold level
(10 times the standard deviation of the background). Therefore, CT values are calculated
at the initiation of the logarithmic phase of PCR amplification and provide accurate
measurement of starting cDNA concentrations (Gerber et al., 1997). Real-time PCR was
performed in triplicate using the ABI PRISM® 5700 sequence detection system (Perkin-
Elmer, Norwalk, USA). A real-time PCR mix of 2X SYBR® Green Universal PCR
Master Mix (Perkin-Elmer, Norwalk, USA); forward and reverse primer (2 µM) and
cDNA was run for 40 cycles of PCR in a volume of 25 µl. To compensate for variations
in input RNA amounts, and efficiency of reverse transcription, β-actin mRNA was also
quantified, and results were normalised to these values. β-actin mRNA levels have been
reported not to change in response to 3, 6 and 12 weeks of training in rat skeletal muscle
(Murakami et al., 1994) and therefore considered adequate as an internal control for this
study.
Samples were analysed using two-way ANOVA with repeated measures. Post Hoc
analysis was performed to determine differences between groups using Newman-Keuls
test, where appropriate.
39
Table 2.1: Gene primer sequences
Gene Sense Primer (5’-3’) Antisense Primer (5’-3’)
β-actin GAC AGG ATG CAG AAG GAG ATT ACT TGA TCC ACA TCT GCT GGA AGG T
IR GCC ACC AAT ACG TCA TTC ACA A GTT GCT GGA ATT CAT CGT GTA C
IRS-1 CCA CTC GGA AAA CTT CTT CTT CAT AGA GTC ATC CAC CTG CAT CCA
IRS-2 ACG CCA GCA TTG ACT TCT TGT TGA CAT GTG ACA TCC TGG TGA
TAA
p85α GGA AGC AGC AAC CGA AAC AA TTC GCC GTC CAC CAC TAC A
IR: insulin receptor; IRS-1: insulin receptor substrate-1; IRS-2: insulin receptor
substrate-2; p85α: p85α subunit of phosphatidylinositol 3-kinase.
40
2.3 Results
The impact of acute exercise and exercise training was determined relative to the
abundance of β-actin, a commonly used housekeeping gene. To confirm that the
expression of β-actin is not modified in human muscle following exercise training,
absolute gene expression of β-actin is shown in Figure 2.1. Fluorescent detection by
real-time PCR demonstrated no significant training effect on the expression of β-actin.
A small reduction in CT was evident immediately following the completion of the first
exercise bout, suggesting increased mRNA recovery or cDNA synthesis (reverse
transcriptase) efficiency at this time-point. Subsequent analysis of gene expression is
normalised against β-actin (housekeeping gene), to account for the variability in cDNA
abundance between samples.
The gene expression of IR (Figure 2.2A) and IRS-1 (Figure 2.2B) were not significantly
altered either immediately post-exercise or 3 hours following a single bout of exercise.
Furthermore, there was no significant change in the mRNA expression of these genes
following 9 days of endurance training. The gene expression of IRS-2 was found to be
11 ± 4 fold higher (p< 0.01) 3 hours after exercise compared with basal levels, although
this effect was reduced to 4 ± 1 fold (p< 0.05) after exercise training (Figure 2.2C).
Similarly, the mRNA levels of PI 3-kinase were also higher (p<0.05) 3 hours following
exercise in the untrained state but no changes in expression were found after exercise in
the trained state (Figure 2.2D).
41
Pre P10
15
20
25
β -actin
Crit
ical
Thr
esho
ld (C
T)(c
ycle
num
ber)
Figure 2.1. Effect of a single bou
the gene expression of β-actin.
Closed bars: untrained; Hatch
immediately after, and 3hr Post:
Threshold (CT) represents the PC
increases above a threshold level.
mRNA concentration. , * p< 0.05
*
ost 3hr Post Pre Post 3hr Post
t of exercise and short-term endurance training on
Bars on graph represent means ± SE of 7 subjects.
ed bars: trained; Pre: immediately before, Post:
3 hours after a single exercise bout. Note that Critical
R cycle number at which the fluorescence emission
Therefore, a lower CT value represents a higher initial
vs. trained values.
42
Pre Post 3hr Post Pre Post 3hr Post0.00
0.01
0.02
0.03
0.04
A IR
Arbi
trar
y U
nits
Pre Post 3hr Post Pre Post 3hr Post0.00
0.01
0.02
0.03
0.04
0.05
B IRS-1
Arbi
trar
y U
nits
Pre Post 3hr Post Pre Post 3hr Post0.0000
0.0025
0.0050
0.0075 #
*
C IRS-2
Arbi
trar
y U
nits
Pre Post 3hr Post Pre Post 3hr Post0.000
0.025
0.050
0.075
*+
D p85 PI 3-kinase
Arbi
trar
y U
nits
Figure. 2.2. Effect of a single bout of exercise and short-term endurance training
on the gene expression of A: the insulin receptor (IR), B: insulin receptor
substrate-1 (IRS-1), C: insulin receptor substrate-2 (IRS-2) and D: p85α subunit of
phosphatidylinositol 3-kinase (p85α PI 3-kinase). Bars on graph represent means ±
SE of 7 subjects. Closed bars: untrained; Hatched bars: trained; Pre: immediately
before, Post: immediately after, and 3hr Post: 3 hours after a single exercise bout. # p<
0.01 vs. all other values, * p< 0.05 vs. trained values, + p< 0.05 vs. Post untrained
values.
43
2.4 Discussion
The increased sensitivity of insulin-mediated glucose uptake following exercise persists
for up to 48 hours, during which time the acute effects of glucose on GLUT4
translocation have been reversed and muscle glycogen concentrations are largely
restored (Goodyear et al., 1990; Mikines et al., 1988). The results from this study and
others have shown that a single bout of exercise is capable of eliciting increased gene
expression of key components of the glucose disposal pathway, including GLUT4 and
hexokinase II (Kraniou et al., 2000; Neufer & Dohm, 1993). Recent data have also
shown increased levels of GLUT4 protein 22 hours following a single bout in humans
(Greiwe et al., 2000). In addition to the upregulated glucose transport capacity,
enhanced transduction of the insulin signal cascade is evident in humans and rodents
(Chibalin et al., 2000; Houmard et al., 1999), however the impact of exercise on the
gene transcription of critical components of this pathway is unknown. The present study
examined whether a single bout of exercise and endurance training alters the gene
expression of the key insulin-signalling proteins in human subjects. The results of this
study demonstrated that gene expression of IRS-2, and to a lesser extent, PI 3-kinase, is
increased by a single bout of exercise in untrained individuals. However, other key
insulin-signalling intermediates, including IR and IRS-1 were not regulated either by a
single exercise bout or by short-term training in humans.
The present study found significantly increased IRS-2 gene expression in the hours
following a single bout of exercise, but not immediately following exercise in humans.
This finding is in agreement with an increase in IRS-2 protein levels and insulin
stimulated IRS-2 associated PI 3-kinase activity measured 16 hours following a single
44
exercise bout (Chibalin et al., 2000) in rat skeletal muscle. IRS-2 may exert a major role
in mediating insulin action in liver and in the development and maintenance of
pancreatic β-cell mass, yet its precise role in skeletal muscle is uncertain (Kido et al.,
2000; Withers et al., 1999). In isolated muscle and fat cells, IRS-2 has been shown to
have a similar role to IRS-1 in insulin-stimulated glucose transport (Miele et al., 1999;
Zhou et al., 1997) and may act as an alternative pathway of insulin action. However,
IRS-2 is not involved in glucose transport immediately following either a single
exercise bout or short-term training (Chibalin et al., 2000; Higaki et al., 1999). A
complex interplay between IRS-1 and IRS-2 is evident in the regulation of insulin
action in skeletal muscle, with alterations in the balance of these proteins impacting on
insulin action (Withers et al., 1999). Therefore, it is tempting to speculate of an early
adaptive role of IRS-2 in the exercise-induced increases in insulin signalling in the
hours following exercise. This role for IRS-2 might then become redundant following
the more slowly initiated adaptive responses of other members of the insulin signalling
intermediates, such as IRS-1. In the current study no changes in IRS-1 gene expression,
either acutely or following training were observed. However, insulin-stimulated
function of IRS-1 has been observed to increase by 5 days of training (Chibalin et al.,
2000) in rats. As tempting as it is to speculate on the role of IRS-2 to insulin signalling
in the hours following exercise, further evidence is required to establish its contribution.
It was somewhat surprising that there were no observable changes in IR and IRS-1
following endurance training given the enhanced gene expression found previously in
rodents following endurance training (Kim et al., 1995; Kim et al., 1999a). This may
reflect species differences, the composition of fiber-types analysed and/ or differences
in training protocol. The present study used a relatively short (9 days and 60 min per
45
day) training period, whereas the rodents were trained for 90 min per day over a 9-week
period. A further limitation of the present study was that subjects were relatively young
and lean, without a family history of type 2 diabetes. Thus, any potential changes in
insulin action may be small, such that large changes in the gene expression of IR and
IRS-1 might not be expected. However, similar studies have demonstrated enhanced
insulin-sensitivity following only a single bout of exercise (Wojtaszewski et al., 1997)
and in training programs utilizing relatively short-term programs such as the one used in
the present study (Houmard et al., 1999). Thus, it can be concluded that it is unlikely
that enhanced gene expression of IR and IRS-1 are central components of the adaptive
response to exercise. Exercise-induced improvements in translational control such as
RNA stability, protein processing transport and protein stability, as identified
previously following exercise training (Welle et al., 1999) may exert a greater influence
on IR and IRS-1 protein abundance and activity.
In the present study it is also possible that any significant changes in gene expression
occurred outside or between the sampling periods. There are few data on induction of
increased gene expression following an exercise stimulus, or the half-lives of many
mRNA species in humans (Neufer, 1999). Therefore, in the current study, timing of the
biopsy was aimed to maximize the likelihood of identifying altered gene expression in
the target genes. A spectrum of responses in human skeletal muscle has been reported
previously, with increased mRNA abundance demonstrated within minutes of exercise
initiation (Puntschart et al., 1998), immediately following the completion (Gustafsson et
al., 1999; Kraniou et al., 2000) and for up to eight hours post-exercise (Seip et al.,
1997). Indeed significantly increased gene expression of IR, IRS-1 and PI 3-kinase have
been shown in rodents 48 hours following the termination of a 9-week training program
46
(Kim et al., 1995; Kim et al., 1999a). In the present study, there were no changes in
mRNA levels for IR and IRS-1 either immediately post-exercise or when measured
again 3 hours post-exercise. Furthermore there was no evidence of a training effect on
the gene expression of IR and IRS-1 as the second pre-exercise biopsy sample was
obtained 24 hours following the last training session. These results would therefore
suggest that significantly altered gene expression to moderate exercise of the IR and
IRS-1 genes is unlikely.
2.5 Conclusion
In summary, skeletal muscle IRS-2 gene expression is significantly increased in the few
hours following a single bout of exercise, but following training this effect is
diminished in humans. Similarly, the mRNA levels of PI 3-kinase appear to be
increased in the hours following a single bout of exercise, however this effect was not
observed again following short-term training. A single bout of exercise or short-term
endurance training does not increase the gene expression of either the IR and IRS-1
genes. Therefore, in untrained human subjects undertaking moderate exercise training
there is little evidence of sustained and substantial alterations in the gene expression of
key members of the insulin-signalling pathway. Further studies are required to elucidate
the mechanisms regulating the increased activity of the insulin-signalling cascade
following exercise.
47
CHAPTER THREE
IRS-2 GENE EXPRESSION IS UPREGULATED BY
RESISTANCE EXERCISE IN HUMAN SKELETAL
MUSCLE
3.1 Introduction
As discussed in Chapter 1, insulin action is improved by resistance exercise (Dunstan et
al., 2002; Ishii et al., 1998; Miller et al., 1994; Poehlman et al., 2000). There is a small
amount of evidence in rodents to suggest that resistance exercise is able to increase
some of the components of the insulin signalling pathway such as IRS-1 and p85
associated PI 3-kinase activity independently of insulin (Carlson et al., 2001; Hernandez
et al., 2000) along with increased glucose uptake and protein synthesis. However, the
mechanisms to account for improvements in insulin action via resistance exercise
remain unknown. The results obtained in Chapter 2 of this thesis show that endurance
exercise has a marked yet temporary impact on the transcriptional activation of the
genes encoding major insulin-signalling proteins including IRS-2 and PI 3-kinase whilst
the gene expression of other key proteins in this pathway remain unaffected. The
effects of resistance exercise on any aspect of the insulin-signalling pathway have yet to
be studied in humans. Therefore, this study investigated the impact of a single bout of
resistance exercise on the mRNA concentration of key insulin signalling proteins that
are differentially regulated following endurance exercise.
48
3.2 Materials and Methods
3.2.1 Experimental protocol
Ten healthy but untrained male subjects volunteered to be involved in the study. The
age, weight, height and BMI (mean ± SE) of the subjects prior to the study were 27.2 ±
1.7 years, 78.4 ± 2.7 kg, 179.5 ± 1.6 cm and 24.2 ± 0.9 kg.m-2, respectively. The Deakin
University Human Research Ethics Committee approved the study and subjects gave
their written consent to participate in this study after all procedures and the possible
risks of participation were explained.
One week prior to the experiment, subjects underwent a familiarisation trial on the
Cybex dynamometer (Cybex International Inc. UK). During this session, seat and leg
positions were recorded and subjects performed 3 sets of 5 maximal isokinetic
concentric and eccentric muscle contractions at an angular velocity of 60 deg.sec-1.
For the 24 hours preceding the trial, subjects abstained from alcohol, tobacco, caffeine
and exercise and consumed a standardised diet consisting of 66% CHO, 20% fat and
14% protein. Subjects were permitted to consume water ad libitum. On the day of the
trial, subjects reported to the laboratory in the morning after an overnight fast (10-12
hours). Muscle samples were obtained from the vastus lateralis using the percutaneous
needle biopsy technique prior to, 30 min, 4 hours and 24 hours after the conclusion of
the exercise bout. After the muscle biopsy at 30 min, subjects were given a small
breakfast consisting of 2 slices of toasted white bread with a small amount of butter,
jam and 250ml of orange juice. Excised muscle tissue from the biopsy was immediately
frozen in liquid nitrogen for subsequent analysis. The exercise bout consisted of 3 sets
49
of 12 repetitions of maximal isokinetic thigh exercise on the Cybex Dynamometer at an
angular velocity of 60 deg.sec-1 with each set being separated by 2 min. This involved
both concentric and eccentric muscle contractions of the knee extensors. Concentric and
eccentric muscle contractions combined have been shown to produce the largest
alterations in myofibrillar disruption (Dudley et al., 1991; Gibala et al., 2000). During
each trial, peak and average torque were measured and recorded.
3.2.2 Analytical methods
Total RNA was isolated and reverse transcribed as described in Chapter 2. Primers were
identical to those used in Chapter 2 and the primer sequences are shown in Table 3.1.
Real time PCR was used to quantify mRNA expression and has been described in detail
previously (Chapter 2) and cDNA was run for 40 cycles of PCR in a volume of 20 µl.
To compensate for variations in input RNA amounts, and efficiency of reverse
transcription, β-actin mRNA was also quantified, and results were normalised to these
values. The previous study (Chapter 2) in this thesis found that β-actin mRNA levels
did not change in response to endurance exercise in humans and was therefore
considered as an adequate internal control for this study.
Samples were analysed using repeated measures ANOVA. Post Hoc analysis was
performed to determine differences between groups using Newman-Keuls test, where
appropriate.
50
Table 3.1: Gene primer sequences
Gene Sense Primer (5’-3’) Antisense Primer (5’-3’)
β-actin GAC AGG ATG CAG AAG GAG ATT ACT TGA TCC ACA TCT GCT GGA AGG T
IRS-1 CCA CTC GGA AAA CTT CTT CTT CAT AGA GTC ATC CAC CTG CAT CCA
IRS-2 ACG CCA GCA TTG ACT TCT TGT TGA CAT GTG ACA TCC TGG TGA TAA
p85α GGA AGC AGC AAC CGA AAC AA TTC GCC GTC CAC CAC TAC A
IRS-1: insulin receptor substrate-1; IRS-2: insulin receptor substrate-2; p85α: p85α
subunit of phosphatidylinositol 3-kinase.
51
3.3 Results
The peak torque (mean ± SE) recorded for each subject during the trials was 229.6 ±
10.0 and 273.4 ± 9.5 N.m-1 for the concentric and eccentric contractions, respectively.
The resistance exercise was performed in 3 sets, and the work intensity increased as
each set progressed and as the subjects became more familiar with the protocol (Table
3.2). It was also observed that the subjects fatigued rapidly towards the latter stages of
the last exercise set.
The impact of resistance exercise was determined relative to the abundance of β-actin, a
commonly used housekeeping gene. To confirm that the expression of β-actin is not
modified in human muscle following resistance exercise, absolute gene expression of β-
actin is shown in Figure 3.1. Fluorescent detection by real-time PCR demonstrated no
significant exercise effect on the expression of β-actin. Subsequent analysis of gene
expression is normalised against β-actin (housekeeping gene), to account for the
variability in cDNA abundance between samples.
Thirty minutes following resistance exercise mRNA concentration of IRS-1 was
decreased approximately 2.3 ± 0.6 fold (Figure 3.2A, not significant) and was then
increased 3.4 ± 1.1 fold (Figure 3.2A, p<0.05) 3.5 hours later. IRS-2 gene expression
increased 3.4 ± 1.2 fold (Figure 3.2B, p<0.05) 4 hours following resistance exercise and
was restored to basal levels by 24 hours (Figure 3.2B, p<0.05). There was no change in
the mRNA concentration of the p85α subunit of PI 3-kinase (Figure 3.2C).
52
Table 3.2. Average torque during each repetition of isokinetic thigh exercise.
Average Torque (N.m-1)
Concentric
Average Torque (N.m-1)
Eccentric
Set 1 153.7 ± 9.9 147.3 ± 17.9
Set 2 188.8 ± 11.5 205.5 ± 18.6
Set 3 192.7 ± 12.1 215.1 ± 17.5
Values are means ± SE; n = 10 for all sets
β-actin
0 30min 4hrs 24hrs22
24
26
28
Cri
tical
Thr
esho
ld (C
T)(c
ycle
num
ber)
Figure 3.1. Effect of a single bout of resistance training on the gene expression of β-actin.
Bars on graph represent means ± SE of 8 subjects. 0: immediately before exercise, 30min, 4hrs
and 24hrs: 30 min, 4 hours and 24 hours following a single bout of resistance exercise,
respectively. Note that Critical Threshold (CT) represents the PCR cycle number at which the
fluorescence emission increases above a threshold level. Therefore, a lower CT value represents
a higher initial mRNA concentration.
53
IRS1
0 30min 4hrs 24hrs0.000
0.005
0.010
0.015
0.020
0.025
+
A
Arbi
trar
y un
its
IRS2
0 30min 4hrs 24hrs0.00
0.01
0.02B
*
Arbi
trar
y un
its
p85 PI 3-kinase
0 30min 4hrs 24hrs0.0
0.2
0.4
0.6
C
Arbi
trar
y un
its
Figure 3.2. Effect of a single bout of resistance exercise on the mRNA concentration of A:
insulin receptor substrate-1 (IRS-1), B: insulin receptor substrate-2 (IRS-2). And C: p85α
subunit of PI 3-kinase. Bars on graph represent means ± SE of 8 subjects. 0, 30min, 4hrs and
24hrs: immediately before, 30 min, 4 hours and 24 hours following resistance exercise,
respectively. +p<0.05 vs. 4hrs, * p< 0.05 vs. 0hrs.
54
3.4 Discussion
There is a considerable amount of data now on the impact of endurance exercise on
insulin sensitivity and the molecular components of the insulin-signalling pathway.
Resistance exercise has also been shown to improve insulin action although conflicting
findings have yet to establish if this improvement is due to an enhanced ability of
skeletal muscle to respond to insulin or if this is simply due to increases in lean muscle
mass (Dunstan et al., 2002; Ishii et al., 1998; Poehlman et al., 2000; Takala et al., 1999).
Furthermore, there has been no investigation into the impact of resistance exercise on
any molecular aspect of the insulin-signalling pathway in humans.
This study has for the first time shown that a single bout of resistance exercise is
sufficient to significantly alter the gene expression of IRS-1 and IRS-2 in the skeletal
muscle of humans. The overall response of IRS-1 suggests that the mRNA
concentration tends to decrease 30 min following resistance exercise whereupon it is
restored to pre exercise levels over the following 3.5 hours. IRS-2 on the other hand,
significantly increases in the few hours following resistance exercise and is restored to
basal levels within 24 hours.
Only one study has investigated mRNA concentration of select genes following
resistance exercise in humans (Willoughby & Nelson, 2002). They found a small but
significant upregulation of the gene expression for myosin heavy chain (MHC) isoform
Type IIx, and myogenic regulatory factors (MRF) Myo-D and myogenin immediately
after heavy resistance exercise. This effect was also found to be significantly greater 6
hours post exercise with MHC isoforms Type I and IIa also elevated. An upregulation
55
of genes involved in muscle phenotype and the enlargement or repair of the myofibre is
therefore not surprising following heavy resistance exercise.
The major finding of this study was that resistance exercise significantly increases IRS-
2 gene expression in the few hours post exercise in humans. A similar response has
been found for this gene in the few hours following endurance exercise (Chapter 2)
although it appears endurance training resulted in a much more marked response (11 ± 4
fold increase, p<0.01) (Chapter 2) compared to the 3 ± 1 fold increase following
resistance exercise in the present study. It is however difficult to compare the magnitude
of the two results since although the two studies used healthy untrained volunteers, the
endurance study involved both men and women and the time course for the sampling of
muscle differed. What is clear however is that intense muscular contraction using very
different exercise modalities is sufficient to upregulate IRS-2 mRNA concentration for
several hours following exercise. This may point towards a role for IRS-2 in muscle
fibre growth in addition to a small role on insulin action. The physiological role/s for
IRS-2 remain unclear although gene knockout studies have shown it to be important in
insulin stimulated PI 3-kinase activity (Howlett et al., 2002) in skeletal muscle and may
act as an alterative pathway for insulin action when IRS-1 function is compromised
(Kido et al., 2000; Rondinone et al., 1997; Withers et al., 1999). IRS-2 has also been
shown to be particularly important in the liver where it plays a major role in insulin
action and is critical to the maintenance of β-cell mass in the pancreas (Kubota et al.,
2000; Withers et al., 1999).
The present study is limited in its findings as subjects were fed a small meal
approximately 35 minutes after exercise, which may have had an effect on the gene
56
expression of IRS-2. This confounding factor cannot be discounted, but it is unlikely to
be a major factor since similar transient increases were observed following endurance
exercise despite no food being consumed. There were also no observable changes in the
gene expression of IRS-1 or the p85α sub unit of PI 3-kinase compared to basal levels
during this period. Also, IRS-2 gene expression was not increased above basal levels in
the longer term suggesting that the increase in IRS-2 gene expression in the few hours
following exercise is largely attributable to the preceding muscular contraction.
Tyrosine phosphorylation of IRS-1 is known to play a major role in insulin stimulated
mitogenesis and glucose uptake (for reviews see (Kellerer et al., 1999; Virkamaki et al.,
1999; White & Kahn, 1994). Also, IRS-1 and p85 associated PI 3-kinase activity have
been shown to be significantly elevated for 6 – 24 hours in rats who underwent muscle
overload by either short term resistance training or surgical ablation, which coincided
with enhanced glucose uptake and protein synthesis (Carlson et al., 2001; Hernandez et
al., 2000). Unfortunately, IRS-2 tyrosine phosphorylation and any associated activation
of PI 3-kinase following such types of intense muscular contraction have yet to be
investigated. A constitutively active form of the Ras proto-oncogene that selectively
stimulates the PI 3-kinase pathway was found to induce muscle fibre growth in
regenerating denervated mammalian muscle (Murgia et al., 2000). The activation of PI
3-kinase by insulin receptor substrates following muscular contraction may therefore
play a role in mediating protein synthesis independently from insulin mediated glucose
uptake.
Endurance exercise was observed in the previous chapter not to have any effect on the
mRNA concentration of IRS-1 in untrained humans. It is therefore surprising to observe
57
a trend for IRS-1 gene expression to decrease in the short period following resistance
exercise. A physiological explanation for the tendency of IRS-1 mRNA concentration to
decrease post exercise could be postulated that since resistance exercise results in a
large degree of cellular disruption and damage, perhaps there is little need or ability to
resynthesise any proteins that are already in abundance at that particular time, although
there is no evidence to support this.
3.5 Conclusion
In summary, this is the first study to demonstrate altered mRNA concentration of key
insulin signalling proteins in skeletal muscle of humans following high intensity
resistance exercise. There was a tendency for IRS-1 gene expression to decrease in the
short period following resistance exercise, whereas IRS-2 gene expression markedly
increased several hours after exercise. The mRNA expression of the p85α subunit of PI
3-kinase was not however altered by resistance exercise. The observation that IRS-2
gene expression is increased following intense muscular contractions following either
endurance and resistance exercise points towards roles for the protein not only in insulin
action and glucose transport but perhaps in myofibre growth in skeletal muscle
independent from insulin.
58
CHAPTER FOUR
THE EFFECT OF EXERCISE ON POTENTIAL
MEDIATORS OF INSULIN SIGNALLING IN HUMAN
SKELETAL MUSCLE
4.1 Introduction
As discussed in Chapter 1, exercise improves insulin sensitivity although the effects of
exercise on insulin signalling appear time dependent. In the 12-24 hour period
following the cessation of exercise the observed improvements in insulin sensitivity
may be related to an upregulation of the insulin-signalling cascade. It is this delayed
period following exercise cessation that will be the focus of this study rather than the
initial few hours following exercise cessation. Understanding the intracellular pathways
and mechanisms that are upregulated during sustained periods of enhanced insulin
sensitivity provides valuable insights into the development of safer therapies for the
prevention and treatment of metabolic diseases such as diabetes.
Insulin receptor (IR) and IRS-1 tyrosine phosphorylation appear to be upregulated in
response to short-term exercise training, although it is unclear if these effects are
apparent under physiological insulin levels in vivo. It is also yet to be resolved if any
improvements in insulin signalling during this period are due to increased protein
abundance of key insulin signalling proteins. Furthermore, there is little data on the
upregulation of distal parts of the insulin-signalling cascade such as Akt in the 12-24
hours following exercise. Even though short-term endurance training in humans has
been shown in many studies to improve insulin sensitivity (DeFronzo et al., 1987; Dela
59
et al., 1994; Houmard et al., 1999; Youngren et al., 2001) one constant criticism is that
the effect is primarily due to the residual effects of the last bout of exercise and not the
result of cumulative bouts of exercise. There are some data in rodents and insulin
resistant humans (Chibalin et al., 2000; Rogers et al., 1988) to suggest that cumulative
bouts of exercise further increase insulin sensitivity when compared to an acute bout of
exercise although this effect has not been fully investigated in humans, particularly
those with normal glucose tolerance. The primary aims of this study were to determine
if a single bout of acute exercise and short-term endurance training resulted in increased
protein abundance and/or insulin-stimulated phosphorylation of IR, IRS-1 and Akt in
sedentary humans. Another aim of this study was to determine if short-term endurance
training resulted in significantly higher levels of whole-body insulin sensitivity
compared to a single bout of exercise.
It was also discussed in Chapter 1 that significant interest is currently being focussed on
the regulation of the insulin-signalling cascade by protein tyrosine phosphatases
(PTPases). This diverse family of PTPases are novel mediators of the insulin-signalling
cascade although little is known of the effects of exercise on PTPase abundance,
particularly in human muscle. The subcellular localisation of PTPases could be
important since readily abundant PTPases in skeletal muscle and adipocytes each
display characteristic subcellular distribution between the cytosol and particulate
fractions (Ahmad & Goldstein, 1995; Calera et al., 2000). Therefore it is likely there are
preferential sites of action between the IR and insulin receptor substrates (IRS)
depending upon subcellular localisation.
60
PTP1B is an abundant PTPase in skeletal muscle and PTP1B knockout mice display
markedly enhanced insulin sensitivity along with increased phosphorylation of IR and
IRS-1 in muscle and liver following insulin infusion (Elchebly et al., 1999). Recently,
reduction of PTP1B protein in liver and fat have been associated with improved insulin
action along with increased tyrosine phosphorylation of the IR, IRS-1, IRS-2 and serine
phosphorylation of pAkt (Ser473) (Gum et al., 2003; Mohammad et al., 2002; Zinker et
al., 2002).
Following insulin stimulation, SHPTP2 associates with IRS-1 (Kuhne et al., 1993) and
unlike PTP1B, the major site of SHPTP2 activity appears to reside in the cytosolic
fraction (Ahmad & Goldstein, 1995), implicating IRS-1 as a potential candidate for
dephosphorylation. However, unlike most other PTPases in skeletal muscle, SHPTP2
has actually been implicated as a positive regulator of insulin signalling. As mentioned
in Chapter 1, overexpression of SHPTP2 in various cell lines upregulates insulin
signalling at multiple sites including IRS-1 tyrosine phosphorylation and PI 3-kinase
activity, with the opposite observed when SHPTP2 is inhibited (Chen et al., 1997; Ugi
et al., 1996). In vivo studies also implicate SHPTP2 as a positive regulator of insulin
signalling since transgenic mice with impaired SHPTP2 have decreased insulin action
and impaired activation of IRS-1, PI 3-kinase and Akt in skeletal muscle (Maegawa et
al., 1999). SHPTP2 is clearly a candidate involved in the regulation of insulin signalling
although a precise role and relative contribution to insulin signalling is yet to be
determined. Furthermore, no studies have sought to investigate the actions of exercise
training on SHPTP or PTP1B.
61
Crucial to the analysis of SHPTP2 and PTP1B activity is examination of their
subcellular localisation. If abundance of particular PTPases were altered in response to
exercise then insulin sensitivity might increase due to the amplified phosphorylation of
key signalling proteins. It could be hypothesised that decreases in PTP1B abundance in
the particulate and cytosolic fractions by exercise could increase both IR and IRS-1
tyrosine phosphorylation, with concomitant enhanced phosphorylation of distal
signalling proteins such as Akt. Furthermore, if SHPTP2 protein abundance were
enhanced in the cytosolic fraction following exercise, then increased IRS-1 tyrosine
phosphorylation, along with enhanced Akt activation/phosphorylation may be expected.
The secondary aim of this study was therefore to determine if exercise altered the
abundance or subcellular distribution of PTP1B or SHPTP2 in humans and if this
coincided with exercise-induced increases in the tyrosine phosphorylation of IR and
IRS-1 or serine phosphorylation of Akt.
4.2 Materials And Methods
4.2.1 Materials
PTP1B mouse monoclonal IgG (BD, Cat. # 610140) and SHPTP2 mouse monoclonal
IgG (BD, Cat. # 610621) were from BD Biosciences (San Diego, USA). IRS-1 rabbit
polyclonal IgG (UBI, Cat. # 06-248) was purchased from Upstate Biotechnology (New
York, USA). Phosphotyrosine (PY99) mouse monoclonal IgG (SC, Cat. # 7020) and
IRS-1 (A-19) rabbit polyclonal IgG (SC, Cat. #560) were from Santa Cruz
Biotechnology (California, USA). 83.7 IRβ mouse monoclonal IgG was a gift from
Prof. Ken Siddle (University of Cambridge). Phospho-Akt (pAkt) Ser473 rabbit
polyclonal IgG (Cat. # 9271) and Akt rabbit polyclonal IgG (Cat. # 9272) were from
62
Cell Signaling Technology (New England BioLabs, Hartsfordshire, England). Affinity
purified peroxidase labelled anti-mouse IgG and anti-rabbit IgG were purchased from
Silenus (Victoria, Australia). All other reagents were analytical grade (Sigma, NSW,
Australia).
4.2.2 Subjects
A group of 8 untrained, but healthy males volunteered to be involved in this study. The
experimental protocol and consent form was approved by the Deakin University Human
Research Ethics Committee prior to the commencement of the study. All subjects gave
their written consent to participate in this study after all procedures and the possible
risks of participation were explained. Subject characteristics were (means ± SE) age, 24
± 1yrs; height, 180 ± 3cm; mass, 82.3 ± 4.3kg; and body mass index, 25.6 ± 1.5kg·m-2.
4.2.3 Experimental protocol
Subjects visited the Exercise Physiology Laboratories at Deakin University for
determination of peak and then on ten further occasions. Eight subjects underwent
3 euglycemic-hyperinsulinemic clamps on separate days, each following an overnight
fast (Table 4.1). Subjects were requested to refrain from any moderate or strenuous
activity or exercise for at least 48 hours prior to the first euglycemic clamp (day 1).
Several days later, each subject cycled at 75% peak for 60 min (day 7). Twenty-
four hours after finishing the acute bout of exercise (day 8) each subject underwent a
second euglycemic clamp procedure. Each subject then cycled for 60 min each day at
75% peak for the following 7 days. During each exercise session, was
2OV&
2OV&
2OV& 2OV&
63
monitored and the workload was increased accordingly to maintain a relative exercise
intensity of 75% peak. Twenty-four hours following the last exercise bout, each
subject underwent a third euglycemic clamp procedure (Table 4.1).
2OV&
In the 24-hour period prior to each euglycemic clamp subjects avoided the consumption
of tobacco, alcohol and caffeine and were told to avoid any physical activity other than
that prescribed by the study. Furthermore, the day prior to each euglycemic clamp, each
subject ate a standardised diet consisting of approximately 14,500KJ with 76% of
energy derived from carbohydrate, 12% from protein and 12% from fat.
All cycling tests were performed on a Quinton Excalibur cycle ergometer (Groningen,
The Netherlands). The seat height and handlebar position on the cycle ergometer were
recorded on the first cycle session and replicated on subsequent visits. During each
cycling test, a large domestic fan circulated air onto the subject’s head and chest in
order to aid body temperature regulation. was measured on line and calculated
every 30 seconds using Vista/Turbofit software, version 4.045 (Vacumetrics Inc.
California, USA). Ventilation was measured by a turbine flow transducer (KL
Engineering Co. California, USA), which was calibrated using a 3.0 L syringe. Expired
CO
2OV&
2 and O2 were measured with infrared carbon dioxide and zirconia cell oxygen
analysers, respectively (AEI Technologies Inc. Pittsburgh, USA) which were calibrated
to 0.01% using alpha rated standard gases (Linde Gases Pty Ltd, Villawood, NSW,
Australia). Heart rates were measured continuously using a Polar heart rate monitor
(Polar Electro, Finland).
64
Table 4.1. Summary of the testing sessions.
Day 1 Euglycemic, hyperinsulinemic clamp 1 Day 7 Exercise, 75% VO2 peak Day 8 Euglycemic, hyperinsulinemic clamp 2 Days 8-14 Exercise, 75% VO2 peak Day 15 Euglycemic, hyperinsulinemic clamp 3 Time (hrs) 0 1 2 Biopsies ↑ ↑
65
Each subject performed an incremental cycling test until volitional exhaustion and the
results were used to later estimate submaximal exercise workloads from the linear
relationship between oxygen uptake and power output. peak was deemed as the
highest (L⋅min
2OV&
2OV& -1) value during a 60 second period.
4.2.4 Euglycemic hyperinsulinemic clamp
Following an overnight fast, a dose of 1.2 g KCl (Novartis Pharmaceticals, North Ryde,
NSW, Australia) was administered orally to prevent a decrease in plasma potassium
concentration during the clamp. A 22 gauge polyurethane catheter was inserted into an
antecubital vein in the left arm for infusion of glucose and into the right arm for
infusion of insulin. A third polyurethane catheter was inserted retrograde into a dorsal
vein of the right hand and the hand was wrapped in an electric heating pad and warmed
(approximately 50 0C) for sampling of arterialised blood. Five millilitres of the subject’s
blood and 95 ml of 0.9% (w/v) saline were then mixed with 20U of human insulin
(Actrapid, Novo Nordisk, Denmark) to create the insulin infusate. A one-step
euglycemic-hyperinsulinemic clamp was initiated by an intravenous bolus injection
given over 1-min (9mU.kg-1) followed by a 120-min constant infusion of insulin (40
mU.m-2.min-1). Arterialised venous blood samples obtained immediately before and at 5
min intervals during the infusion were analysed for glucose concentration using a
glucose analyser (EML105, Radiometer Pacific, Melbourne, Australia). Blood glucose
levels were clamped at 4.83 ± 0.02 mM for the final 30 min of hyperinsulinemia in each
clamp by the use of a variable infusion of 25% glucose. Arterialised venous blood
samples were also obtained immediately before and at 10 min intervals during the
infusion for the determination of plasma insulin concentrations. These blood samples
66
were spun in a centrifuge at 13,000 rpm for 3 min and the plasma was frozen at -200C
for later analysis. Glucose infusion rate (GIR) was calculated as the average from the
final 30 min of the clamp.
In addition, homeostasis model assessment (HOMA) measures of insulin action were
calculated from fasting glucose and insulin values obtained prior to the initiation of
insulin infusion. HOMA = fasting plasma insulin (µU/ml) × fasting plasma glucose
(mmol.l-1) ÷ 22.5 (Matthews et al., 1985). HOMA values provide a determination of the
effectiveness of fasting insulin levels to regulate blood glucose levels (Matthews et al.,
1985). HOMA has been used in numerous studies as a measure of insulin action and has
been found to correlate highly with whole-body insulin sensitivity as measured by the
euglycemic, hyperinsulinemic clamp (Katsuki et al., 2001; Matthews et al., 1985).
4.2.5 Blood biochemistry
Plasma insulin concentrations were determined in duplicate by radioimmunoassay using
a commercially available kit (Phadaseph, insulin RIA, Pharmacia and Upjohn
Diagnostics, Uppsala, Sweden).
4.2.6 Muscle biopsy procedure
Percutaneous muscle samples (137 ± 6 mg) were obtained under local anaesthesia
(Xylocaine, 1% plain) from the vastus lateralis of each subject by using the needle
biopsy technique (Bergström, 1962) modified to include suction. Biopsies were taken 5
min prior to and 120 min following the initiation of the insulin infusion on the same leg
with the second biopsy being at least 5 cm proximal to the first. Muscle samples were
67
frozen in the needles within 5 seconds in liquid nitrogen and stored in vented cryotubes.
These samples were stored in liquid nitrogen for later analysis.
4.2.7 Preparation of whole muscle lysates
Frozen muscle (10µl of buffer per mg of muscle) was homogenised using a polytron at
maximum speed for 30-seconds in freshly prepared ice-cold Buffer A, (50mM HEPES
at pH 7.6 containing 150mM NaCl, 20mM Na4P2O7, 20mM β-glycerophosphate, 10mM
NaF, 2mM EDTA, 1% v/v Nonidet P-40, 10% v/v glycerol, 1mM MgCl2, 1mM CaCl2,
2mM Na3VO4, 2mM PMSF and 5µl.ml-1 Protease Inhibitor Cocktail (P8340, Sigma)).
Tissue lysates were incubated on ice for 20 min and then spun at 10,000 × g for 20 min
at 40C. Protein concentration was determined using the Bio-Rad protein assay (Bio-Rad,
NSW, Australia) with BSA as the standard. The supernatants were stored at –800C until
analysis.
4.2.8 Preparation of fractionated muscle lysates
Frozen muscle (10µl of buffer per mg of muscle) was homogenised using a polytron at
maximum speed for 30-seconds in freshly prepared ice-cold Buffer B, (50mM HEPES
at pH 7.4 containing 1mM DTT, 4mM EDTA, 2.5 mM Benzamidine, 2mM PMSF,
5µl.ml-1 Protease Inhibitor Cocktail (P8340, Sigma)). The crude homogenate was spun
at 350,000 × g for 30 min at 40C and the resulting supernatant was taken as the cytosol
fraction. Proteins in the particulate fraction were then solublised by treating the pellet
with ice cold Buffer B containing 1% v/v Triton X-100 and 0.2 M NaCl, incubating for
45min at 40C, then spinning at 150,000 × g for 60 min at 40C. The resulting supernatant
was taken as the particulate fraction. Protein concentration was determined using a
68
bicinchoninic (BCA) protein assay (Pierce, Il, USA) with BSA as the standard. The
supernatants were stored at –800C until analysis.
4.2.9 Immunoprecipitations and immunoblotting
For immunoprecipitation, 2µg of anti-IRβ monoclonal antibody (Upstate
Biotechnology) and 3µg of anti- IRS-1 (A-19) polyclonal antibody (Santa Cruz) was
coupled to 30 µl (50% w/v) Protein A-sepharose beads (Zymed Laboratories, CA, USA)
for 20 min at room temperature and then washed once in PBS. Tissue lysates (350µg
protein) were incubated with the antibody coupled beads overnight at 40C with rotation.
Immunoprecipitated proteins were then washed once in PBS containing 0.5M NaCl and
0.2% v/v Triton X-100 and twice in PBS containing 0.2% v/v Triton X-100.
Immunoprecipitated proteins or equal amounts of protein for determination of pAkt
(Ser473) (75µg), PTP1B (10µg), SHPTP2 (10µg), IRS-1 (45µg), and Akt protein
abundance (75µg) were suspended in Laemmli sample buffer. Bound proteins were
separated by SDS-PAGE and electrotransfer of proteins from the gel to nitrocellulose
membranes in towbin transfer buffer (25mM Tris, pH 8.3, 192mM glycine, and 20% v/v
methanol) was performed for 100min at 100V (constant). Blots were probed with anti-
pAkt (Ser473) rabbit polyclonal (1:1000), anti-PTP1B monoclonal mouse (1:1000),
anti-IRS-1 rabbit polyclonal (UBI, 1:1000), anti-Akt rabbit polyclonal (1:1000) or anti-
phosphotyrosine PY99 monoclonal mouse (1:1000) antibodies. IRβ immunoprecipitated
phosphotyrosine and PTP1B blots were stripped using RestoreTM (Pierce, IL, USA), and
reprobed with either anti-IRβ monoclonal mouse antibody (1:1000) or anti-SHPTP2
monoclonal mouse (1:1000), respectively, to determine IRβ and SHPTP2 protein
abundance. Binding was viewed by enhanced chemiluminescence (Pierce, IL, USA) and
69
quantified with Kodak 1D version 3.5 densitometry software (Eastman Kodak Co., CT,
USA).
4.2.10 IR content and autophosphorylation by ELISA
The IR content and the level of autophosphorylation in skeletal muscle extracts was also
quantified by an IR antibody “trap” activation assay using time resolved fluorescence
and Europium conjugated reagents. Microtiter 96-well plates were coated with 50µl of
83.7 anti-insulin receptor antibody (a gift from Prof. Ken Siddle, University of
Cambridge) (215 µg/ml in 50mM NaHCO3 buffer, pH 9.6) for 16 hours at 4oC.
Antibody was removed from the plate wells and the plate was blocked with 75µl of
0.5% ovalbumin in 1×TBS for 2 hours at room temperature. After removal of blocking
solution the plates were washed three times with 1×TBST. Aqueous samples (50ul) of
skeletal muscle extracts containing 100µg of protein were applied to wells in triplicate
and allowed to bind overnight at 4oC. After washing three times (1×TBST), 50 µl of
either Europium-labelled human recombinant insulin (1:4000, a gift from Peter Hoyne,
CSIRO, Health Sciences and Nutrition, Melbourne) or anti-phosphotyrosine (1:10000,
pY100 Perkin Elmer) antibody in binding buffer consisting of 100mM Hepes at pH 8.0
containing 100mM NaCl, 0.05%Tween 20 and 2µM DTPA was added to each well and
allowed to incubate overnight at 4oC. After washing three times (1×TBST), 100µl of
europium enhancement solution was added and the wells were counted by a Wallac
Victor fluorometer (Perkin Elmer).
70
4.2.11 Statistical analysis
Standards were included in all immunoblots and interassay variation was accounted for
by normalising data to control samples. Data are presented as mean ± SEM.
Differences were determined using either one or two-way analysis of variance
(ANOVA) with Newman-Keuls post-hoc analysis, where appropriate. Significance was
accepted when p<0.05.
4.3 Results
4.3.1 Subjects
Body mass did not change with exercise training (82.3 ± 4.3 vs. 82.4 ± 4.4 kg). The
cardiovascular fitness (as measured by peak) prior to the study and the exercise
characteristics for the duration of the study are presented in Table 4.2.
2OV&
There was a measurable increase in cardiovascular fitness following the short-term
training program. Exercise heart rate during the final training session was significantly
lower (159 ± 3 beats⋅min-1) when compared to the first session (166 ± 3 beats⋅min-1; p <
0.05) despite exercising remaining the same (2.67 ± 0.17 L⋅min2OV& -1 vs. 2.62 ± 0.16
L⋅min-1; NS) between the first and final sessions, respectively.
4.3.2 Euglycemic Hyperinsulinemic clamp
The euglycemic, hyperinsulinemic clamp is the recognised gold standard for measuring
whole-body insulin sensitivity (DeFronzo et al., 1979). This study performed a 2 hour
71
clamp procedure on 8 sedentary subjects as outlined in Table 4.1 This procedure allows
the present study to measure changes to whole body insulin-sensitivity brought about 24
hours following a single bout of endurance exercise and short-term endurance training.
Percutaneous muscle biopsies taken before and during the clamp procedures provided
this study with skeletal muscle that was stimulated with or without high physiological
doses of insulin. The muscle samples obtained from this study were then measured to
examine if changes in whole-body insulin sensitivity via exercise are also matched by
alterations in protein abundance and phosphorylation of key insulin-signalling proteins
and the abundance of putative mediators of insulin signalling such as PTPases.
Plasma glucose concentration was not significantly different at any time point after 30
min of insulin infusion and was clamped at 4.83 ± 0.02 mM for the final 30 min of each
clamp. There was also no significant (NS) difference in plasma glucose concentration
during the final 30 min of each clamp between the three clamp conditions (sedentary vs.
acute exercise vs. endurance training; NS). Plasma insulin concentration reached a
steady state of 421.5 ± 15.2 pmol/l by 20 min of infusion in the sedentary condition.
Although insulin infusion rates remained the same during each of the clamps, there was
an average 9% decrease (p<0.05, Figure 4.1) in plasma insulin concentration from 90-
120 min following 8 days of endurance training, possibly reflecting an increase in
insulin clearance.
72
Table 4.2. peak prior to the study and exercise characteristics during the
exercise-training period (n = 8 subjects).
2OV&
Variable Means ± SE
2OV& peak (mL⋅kg-1⋅min-1) 42.2 ± 1.6
Training (%peak) 2OV& 76 ± 1
Training heart rate (beats⋅min-1) 161 ± 3
Training heart rate (%max) 83 ± 2
Training ; average for the eight days of endurance training, expressed as a
percentage of peak.
2OV& 2OV&
2OV&
sedentary acute training0
100
200
300
400
500*
[pla
sma
insu
lin] (
pmol
/l)
Figure 4.1. Average plasma insulin concentration for 90-120 min during the
euglycemic, hyperinsulinemic clamps. Sedentary: no prior exercise, Acute: 24 hours
following 60 min of exercise, Training: 24 hours following 8 days of endurance
training. Bars on graph represent means ± SE of 8 subjects. *p<0.05 vs. other variables.
73
Table 4.3 Fasting plasma characteristics prior to the 3 euglycemic,
hyperinsulinemic clamps
Sedentary Acute Training
Fasting plasma glucose
(pmol/l)
4.86 ± 0.23 4.75 ± 0.12 4.84 ± 0.13
Fasting plasma insulin
(mmol/l)
79.4 ± 8.5 59.2 ± 8.2** 49.1 ± 6.0+
HOMA 2.75 ± 0.41 2.08 ± 0.29* 1.77 ± 0.23**
HOMA, homeostasis model assessment; Sedentary: no prior exercise, Acute: 24 hours
following 60 min of exercise, Training: 24 hours following 8 days of endurance
training. Bars on graph represent means ± SE of 8 subjects. *p<0.05 vs. sedentary;
**p<0.01 vs. sedentary; +p<0.001 vs. sedentary.
sedentary acute training0
2
4
6
8
10*
Glu
cose
Infu
sion
Rat
e(m
g.kg
-1.m
in-1
)
Figure 4.2. Glucose infusion rate during the euglycemic, hyperinsulinemic clamps.
Sedentary: no prior exercise, Acute: 24 hours following 60 min of exercise, Training:
24 hours following 8 days of endurance training. Bars on graph represent means ± SE of
8 subjects. *p<0.05 vs. other variables.
74
Fasting plasma glucose was not altered by a single bout of exercise or endurance
training (Table 4.3; NS). However insulin action was significantly improved following
an acute bout of endurance exercise and was further improved following short-term
training. Firstly, fasting plasma insulin following a single bout of exercise significantly
decreased 21% (p<0.01, Table 4.3) and was reduced 34% (p<0.001, Table 4.3)
following endurance training. Secondly, as assessed via HOMA values at rest, HOMA
values were significantly reduced 24% (p<0.05, Table 4.3) following acute exercise and
36% following training (p<0.01, Table 4.3). GIR during the final 30 min of the clamp
was not significantly increased following an acute bout of exercise, however there was a
significant 26% (p<0.05, Figure 4.2) increase following 8 days of endurance training
(5.8 ± 1.1 vs. 6.2 ± 1.0 vs. 7.3 ± 1.0 mg.kg-1.min-1, respectively, Figure 4.2), indicating
an improvement in insulin sensitivity that is in agreement with other short term training
studies (Houmard et al., 1999; Tanner et al., 2002; Youngren et al., 2001).
4.3.3 Insulin receptor measured by immunoprecipitation
Figure 4.3 and 4.4 shows IR protein expression and tyrosine phosphorylation for the 3-
euglycemic, hyperinsulinemic clamps performed for each of the subjects, respectively.
There was no significant difference in IR protein abundance or tyrosine phosphorylation
following endurance training. Insulin induced a 2.9 ± 0.7 fold increase in IR tyrosine
phosphorylation in the sedentary state, a 2.2 ± 0.4 fold increase following acute exercise
and a 2.2 ± 0.9 fold increase following endurance training (NS).
75
4.3.5 IR content and autophosphorylation by ELISA
There was also no significant difference in IR protein abundance following insulin
infusion, acute exercise or endurance training for the 3-euglycemic, hyperinsulinemic
clamps performed for each of the subjects (Figure 4.5) measured by ELISA. Insulin
significantly increased IR tyrosine phosphorylation by approximately 2.1 fold
(p<0.0001, Figure 4.6) similar to the immunoprecipitation approach. Also, acute
exercise and endurance training were not found to alter IR tyrosine phosphorylation
similar to the immunoprecipitation approach. It is evident from the standard errors
observed in Figure 4.5 and 4.6 compared to the size of the means that IR protein
abundance and tyrosine phosphorylation as measured by the antibody “trap” assay were
more quantitative and less variable than the method of immunoprecipitation followed by
Western blotting. Nevertheless, both methods provided essentially the same finding.
Insulin-stimulation induced an approximately 2-fold increase in IR tyrosine
phosphorylation, but neither IR protein abundance or tyrosine phosphorylation is altered
by acute exercise or training.
76
0
25000
50000
75000
sedentary acute training
IR p
rote
in a
bund
ance
(arb
itrar
y un
its)
Figure 4.3. Insulin receptor protein expression during the euglycemic, hyperinsulinemic
clamps as measured by immunoprecipitation. Western blots are representative from one
subject. Sedentary: no prior exercise, Acute: 24 hours following 60 min of exercise, Training:
24 hours following 8 days of endurance training. Open bars: basal values, closed bars: insulin
stimulated values. Bars on graph represent means ± SE of 8 subjects.
0
2500
5000
7500
10000
12500
sedentary acute training
* * *
IR ty
rosi
ne p
hosp
hory
latio
n(a
rbitr
ary
units
)
Figure 4.4. Insulin receptor tyrosine phosphorylation during the euglycemic,
hyperinsulinemic clamps as measured by immunoprecipitation. Western blots are
representative from one subject. Sedentary: no prior exercise, Acute: 24 hours following 60 min
of exercise, Training: 24 hours following 8 days of endurance training. Open bars: basal values,
77
closed bars: insulin stimulated values. Bars on graph represent means ± SE of 8 subjects.
*p<0.05 vs. basal in the sedentary condition.
0
2500
5000
7500
sedentary acute training
IR n
umbe
r(a
rbitr
ary
units
)
Figure 4.5. Insulin receptor number during the euglycemic, hyperinsulinemic clamps as
measured by ELISA. Sedentary: no prior exercise, Acute: 24 hours following 60 min of
exercise, Training: 24 hours following 8 days of endurance training. Open bars: basal values,
closed bars: insulin stimulated values. Bars on graph represent means ± SE of 8 subjects.
0.0
0.5
1.0
1.5
sedentary acute training
* * *
IR ty
rosi
neph
osph
oryl
atio
n(a
rbitr
ary
units
)
Figure 4.6. Insulin receptor tyrosine phosphorylation relative to receptor number during
the euglycemic, hyperinsulinemic clamps as measured by ELISA. Sedentary: no prior
exercise, Acute: 24 hours following 60 min of exercise, Training: 24 hours following 8 days of
endurance training. Open bars: basal values, closed bars: insulin stimulated values. Bars on
graph represent means ± SE of 8 subjects. *p<0.0001 basal vs. insulin-stimulated values.
78
4.3.5 IRS-1
IRS-1 protein expression was not altered by insulin infusion, acute exercise or
endurance training (Figure 4.7). Insulin increased IRS-1 tyrosine phosphorylation
approximately 2.9 fold (p<0.05, Figure 4.8). Due to technical difficulties, only 7
subjects were analysed for IRS-1 tyrosine phosphorylation. However neither acute
exercise nor endurance training was found to alter the insulin stimulated tyrosine
phosphorylation of IRS-1.
4.3.6 Akt
There was no significant effect of insulin or exercise on total Akt protein abundance
(Figure 4.9). Insulin induced an approximate 3.8 fold increase in pAkt (Ser473)
(p<0.0001, Figure 4.10). Acute exercise and exercise training were found to increase
overall pAkt (Ser473) by approximately 1.5 fold (p<0.05, Figure 4.10). However
ANOVA showed no additive effect of exercise on insulin-stimulated pAkt (Ser473), nor
were basal levels of pAkt (Ser473) found to be significantly increased following
exercise.
4.3.7 SHPTP2 and PTP1B
SHPTP2 protein abundance in the cytosolic fraction was increased approximately 1.6
fold following acute exercise and endurance training (p<0.05, Figure 4.11) when
compared to the sedentary condition. SHPTP2 protein abundance in the particulate
fraction was not altered by exercise (Figure 4.12). There were no significant changes in
PTP1B protein abundance in either the cytosol or particulate fractions (Figures 4.13 and
4.14, respectively).
79
0
10000
20000
30000
sedentary acute training
IRS-
1 pr
otei
n ab
unda
nce
(arb
itrar
y un
its)
Figure 4.7. IRS-1 protein abundance during the euglycemic, hyperinsulinemic clamps.
Protein abundance is determined following western blotting of skeletal muscle extracts with
anti-IRS-1 antibody. Western blots are representative from one subject. Sedentary: no prior
exercise, Acute: 24 hours following 60 min of exercise, Training: 24 hours following 8 days of
endurance training. Open bars: basal values, closed bars: insulin stimulated values. Bars on
graph represent means ± SE of 8 subjects.
0.0
0.4
0.8
1.2
1.6
2.0
sedentary acute training
* * *
IRS-
1 ty
rosi
ne p
hosp
hory
latio
n(a
rbitr
ary
units
)
Figure 4.8. IRS-1 tyrosine phosphorylation relative to protein abundance during the
euglycemic, hyperinsulinemic clamps. Skeletal muscle extracts were immunoprecipitated with
anti-IRS-1 antibody and blots were probed for anti-phosphotyrosine (pY99) antibody. Western
blots are representative from one subject. Sedentary: no prior exercise, Acute: 24 hours
following 60 min of exercise, Training: 24 hours following 8 days of endurance training. Open
80
bars: basal values, closed bars: insulin stimulated values. Bars on graph represent means ± SE of
7 subjects. *p<0.05 basal vs. insulin-stimulated values.
0
5000
10000
15000
sedentary acute training
Akt p
rote
in a
bund
ance
(arb
itrar
y un
its)
Figure 4.9. Akt protein abundance during the euglycemic, hyperinsulinemic clamps.
Protein abundance is determined following western blotting of skeletal muscle extracts with
anti-Akt antibody. Western blots are representative from one subject. Sedentary: no prior
exercise, Acute: 24 hours following 60 min of exercise, Training: 24 hours following 8 days of
endurance training. Open bars: basal values, closed bars: insulin stimulated values. Bars on
graph represent means ± SE of 8 subjects.
0
4000
8000
12000
16000
sedentary acute training
* * *# #
pAkt
(Ser
473)
(arb
itrar
y un
its)
Figure 4.10. pAkt (Ser473) during the euglycemic, hyperinsulinemic clamps. Protein
abundance is determined following western blotting of skeletal muscle extracts with anti-pAkt
(Ser473) antibody. Western blots are representative from one subject. Sedentary: no prior
exercise, Acute: 24 hours following 60 min of exercise, Training: 24 hours following 8 days of
endurance training. Open bars: basal values, closed bars: insulin stimulated values. Bars on
81
graph represent means ± SE of 8 subjects. *p<0.0001 basal vs. insulin stimulation, #p<0.05 vs.
sedentary condition.
0
10000
20000
sedentary acute training
# #
SHPT
P2 p
rote
in a
bund
ance
Cyt
osol
frac
tion
(arb
itrar
y un
its)
Figure 4.11. SHPTP2 protein abundance in the cytosolic fractions during the euglycemic,
hyperinsulinemic clamps. Protein abundance is determined following western blotting of
fractionated skeletal muscle extracts with anti-SHPTP2 antibody. Western blots are
representative from one subject. Sedentary: no prior exercise, Acute: 24 hours following 60 min
of exercise, Training: 24 hours following 8 days of endurance training. Open bars: basal values,
closed bars: insulin stimulated values. Bars on graph represent means ± SE of 8 subjects.
#p<0.05 vs. sedentary condition.
0
1000
2000
3000
4000
5000
6000
7000
sedentary acute training
SHPT
P2 p
rote
in a
bund
ance
Part
icul
ate
frac
tion
(arb
itrar
y un
its)
Figure 4.12. SHPTP2 protein abundance in the particulate fraction during the euglycemic,
hyperinsulinemic clamps. Protein abundance is determined following western blotting of
fractionated skeletal muscle extracts with anti-SHPTP2 antibody. Western blots are
representative from one subject. Sedentary: no prior exercise, Acute: 24 hours following 60 min
82
of exercise, Training: 24 hours following 8 days of endurance training. Open bars: basal values,
closed bars: insulin stimulated values. Bars on graph represent means ± SE of 8 subjects.
0
2000
4000
6000
8000
sedentary acute training
PTP1
B p
rote
in a
bund
ance
cyto
solic
frac
tion
(arb
itrar
y un
its)
Figure 4.13. PTP1B protein abundance in the cytosolic fraction during the euglycemic,
hyperinsulinemic clamps. Protein abundance is determined following western blotting of
fractionated skeletal muscle extracts with anti-PTP1B antibody. Western blots are representative
from one subject. Sedentary: no prior exercise, Acute: 24 hours following 60 min of exercise,
Training: 24 hours following 8 days of endurance training. Open bars: basal values, closed bars:
insulin stimulated values. Bars on graph represent Means ± SE of 8 subjects.
0
10000
20000
30000
sedentary acute training
PTP1
B p
rote
in a
bund
ance
part
icul
ate
frac
tion
(arb
itrar
y un
its)
Figure 4.14. PTP1B protein abundance in the particulate fraction during the euglycemic,
hyperinsulinemic clamps. Protein abundance is determined following western blotting of
fractionated skeletal muscle extracts with anti-SHPTP2 antibody. Western blots are
representative from one subject. Sedentary: no prior exercise, Acute: 24 hours following 60 min
83
of exercise, Training: 24 hours following 8 days of endurance training. Open bars: basal values,
closed bars: insulin stimulated values. Bars on graph represent Means ± SE of 8 subjects.
84
4.4 Discussion
The present study observed significant increases in the insulin action of sedentary
individuals following an acute bout of exercise and exercise training. Although the
volunteers in the present study were healthy non-diabetics, it is still important to
understand how exercise training in the healthy, glucose-tolerant state regulates the
insulin-signalling pathway. As discussed in Chapter 1, physically active people have
enhanced insulin sensitivity compared to sedentary subjects with normal glucose
tolerance (Duncan et al., 2003; Kirwan et al., 2000). Furthermore, physically active
people have an upregulation of steps in the insulin-signalling cascade that may
ultimately provide protection from metabolic diseases such as type 2 diabetes (Kirwan
et al., 2000). In the present study, fasting plasma insulin and HOMA values, used as
measures of whole-body insulin action, significantly decreased following acute exercise
and further decreased following short-term training. Whilst the GIR measured during a
euglycemic, hyperinsulinemic clamp is the gold standard for measuring insulin
sensitivity; HOMA is a useful measure of insulin action since it has been used in a
number of exercise studies as an index to measure improvements in insulin sensitivity
(Katsuki et al., 2001; Kinoshita et al., 2002; Youngren et al., 2001) and correlates well
with insulin sensitivity (Katsuki et al., 2001; Matthews et al., 1985). GIR during
hyperinsulinemic, euglycemic clamps did not increase after an acute bout of exercise,
however a significant 26% increase was observed following 8 days of endurance
training. These changes occurred despite no significant change in body mass and
therefore any improvement in insulin action could largely be attributed to endurance
exercise rather than any associated alterations in body composition. These
improvements in insulin action are consistent with similar studies using short-term
85
endurance exercise (Houmard et al., 1999; Tanner et al., 2002; Youngren et al., 2001).
Rodents show further increases in insulin action following 5 days of training compared
to a single day (Chibalin et al., 2000) and there is a similar improvement in glucose
intolerant humans using an oral glucose tolerance test (Rogers et al., 1988). However
this is the first study to show in humans with normal glucose tolerance that insulin
action is further enhanced following cumulative bouts of exercise compared to a single
bout of exercise. Also, the significant increase in GIR following short-term training
could potentially be higher since there was an observed decrease in plasma insulin
levels during the euglycemic clamp following short-term training, despite infusion rates
remaining the same for each subject.
The lack of statistical significance for GIR following acute exercise when compared to
previous studies could be explained by differences in methodology. Mikines et al.,
(Mikines et al., 1988) used a 4-step euglycemic, hyperinsulinemic clamp following an
acute bout of exercise which allowed them to measure insulin sensitivity and insulin
responsiveness quite accurately compared to the one-step clamp procedure used in the
present study. Other studies to show improvements in insulin sensitivity following a
single bout of exercise have employed the one-step clamp procedure within a few hours
of the cessation of exercise (Thorell et al., 1999; Wojtaszewski et al., 1997) or have
used subjects with impaired glucose tolerance (Cusi et al., 2000).
The alterations in insulin sensitivity detected by HOMA are largely due to reduced
circulating insulin concentrations prior to the euglycemic clamp. Furthermore, it was
also observed in the present study that plasma insulin concentrations during the clamp
were significantly reduced following training despite insulin infusion rates remaining
86
constant. This could be put down to an increase in insulin clearance with exercise. The
internalisation of the IR to the endosomal compartments of the cell following insulin
binding and the subsequent dissociation of insulin from the IR and recycling back to the
plasma membrane is an important mechanism for insulin clearance (Authier et al., 1994;
Burgess et al., 1992; Khan et al., 1989) although the proportional contribution of the
liver and muscle to insulin clearance are unknown. Endurance training has been shown
to increase hepatic insulin clearance in rodents (Wirth et al., 1982) although relatively
little is known of the effects of exercise on insulin clearance, particularly in humans. A
single bout of exercise has been observed to increase whole body insulin clearance
immediately or up to 18 hours following the cessation of exercise when plasma insulin
concentrations are within the range observed in the present study (Brambrink et al.,
1997; Mikines et al., 1989; Thorell et al., 1999). When supraphysiological levels of
insulin are infused the exercise induced improvement in insulin clearance are not
observed (Mikines et al., 1988; Mikines et al., 1989) suggesting that the maximum
clearing capacity of insulin is not altered by exercise. None of the recent studies to
examine the effects of short-term training on insulin sensitivity and signalling have
observed a similar reduction in plasma insulin following training although much higher
levels of insulin stimulation (2-3 fold higher) (Houmard et al., 1999; Tanner et al.,
2002; Youngren et al., 2001) were utilised compared to the present study, and may
therefore account for the lack of exercise effect on insulin clearance.
The results presented in this chapter are consistent with previous studies that
demonstrate insulin induces a significant increase in the tyrosine phosphorylation of the
IR and IRS-1. However, interestingly exercise did not alter the insulin-stimulated
increase in either IR or IRS-1 tyrosine phosphorylation or protein expression despite
87
improvements in whole-body insulin action. A significant increase in in vitro IR
autophosphorylation has previously been reported in human muscle following a similar
short-term endurance-training program (Youngren et al., 2001) when compared to the
present study. However the increases observed by Youngren et al., (Youngren et al.,
2001) were only observed when in vitro insulin concentrations were at least 960 pmol/l;
approximately 2.5 fold higher than the in vivo levels observed in the present study,
suggesting that IR function may only be increased following training at
supraphysiological insulin levels. The present study has shown using two different
approaches to measure IR tyrosine phosphorylation that acute exercise and endurance
training do not increase insulin-stimulated IR tyrosine phosphorylation, despite
observed improvements in insulin action. Few short-term training studies in humans
have measured IRS-1 tyrosine phosphorylation. IRS-1 associated PI 3-kinase activity
has been shown to be elevated in trained vs. untrained males (Kirwan et al., 2000) and
has also been elevated following 7 days of training in young males (Houmard et al.,
1999) although these studies also used supraphysiological doses of insulin that resulted
in plasma insulin levels approximately 3 times higher than those used in the present
study (Houmard et al., 1999; Tanner et al., 2002) making comparisons difficult. These
findings suggest that proximal steps in the insulin-signalling cascade are not the primary
mechanisms for exercise-induced improvements in insulin signalling in human skeletal
muscle. The effects of exercise on glucose transport are more likely mediated on more
distal and perhaps as yet unknown signalling proteins.
The major finding of this study was that 24-hours following acute exercise and
endurance training; overall pAkt (Ser473) was significantly increased. This is a
surprising finding since prior exercise alone has not yet been reported to have any long-
88
term (12-24 hours) effect on pAkt (Ser473) in humans. Previous studies have shown
that muscular contraction is associated with increased serine (473) and threonine (308)
phosphorylation of Akt (Sakamoto et al., 2002; Sherwood et al., 1999; Turinsky &
Damrau-Abney, 1999) with maximal activation of Akt by 3 min of contraction, then
returning to basal levels by 15 min (Sakamoto et al., 2002). In humans, pAkt (Ser473) is
increased immediately following exercise (Thorell et al., 1999) although a separate
study showed pAkt (Ser473) is not elevated in humans 4-hours after the cessation of
exercise (Wojtaszewski et al., 2000) so it is unusual that pAkt (Ser473) was upregulated
24 hours following the cessation of exercise in the present study. A short-term training
study in middle-aged men found no change in insulin-stimulated Akt activity when
measured 17-hours following the last bout of exercise, although only 4 subjects were
analysed so the results must be viewed with caution (Tanner et al., 2002). Insulin-
stimulated pAkt (Ser473) has been shown to be increased following 5 days and 6 weeks
of training in lean, non-diabetic rats (Chibalin et al., 2000; Luciano et al., 2002) with a
trend for increased pAkt (Ser473) following one day of exercise (Chibalin et al., 2000).
These findings of increased insulin-stimulated pAkt (Ser473) in rodents 16-48 hours
after the last bout of exercise training are slightly different to the observations in the
present study where overall pAkt (Ser473) was found to be increased following exercise
without an additive effect of exercise on insulin-stimulated pAkt (Ser473). However,
the findings from these rodent studies and the present study suggest that in the 16-48
hour period following the cessation of exercise there is a significant effect of exercise
on pAkt (Ser473). There are currently few clues as to the precise mechanism/s whereby
exercise may be acting upon Akt. Growth hormone, intracellular calcium and cAMP all
activate Akt in vitro (Sable et al., 1997; Sakaue et al., 1997; Yano et al., 1998) and may
account for the increased pAkt (Ser473) observed following exercise in the present
89
study. However, cAMP is generally thought to be a short-lived second messenger, so it
may not be stimulating Akt 24 hours following exercise. Indeed, cAMP levels are not
significantly elevated in rodent muscle several hours after a single bout of exercise, or
exercise training (Sheldon et al., 1993; Yaspelkis et al., 1999). Little is known of the
role of intracellular calcium on the activation of Akt, particularly in the skeletal muscle
of humans following exercise and will therefore not be the focus of this study. There is
some evidence to suggest growth hormone may be elevated in the 24 hour period
following an acute bout of endurance exercise in humans, although the response
following training may be blunted, for review see (Godfrey et al., 2003). Attenuated
growth hormone levels following training would not be consistent with increased pAkt
(Ser473), suggesting it may not be a major regulator of Akt activity following exercise.
Nevertheless there is sufficient evidence to warrant further investigation into the
exercise-mediated increase in pAkt (Ser473) by growth hormone.
Despite exercise and insulin increasing pAkt (Ser473), there was no interaction between
insulin and exercise, suggesting that the effects are not additive. This would imply that
exercise might be increasing signalling through Akt for purposes other than, or in
addition to insulin-stimulated glucose uptake. Akt seems to have multiple signalling
roles, including insulin-stimulated glucose transport, glycogen synthesis and mitogenic
signalling although its exact contributions are still controversial (Kitamura et al., 1998).
Overexpression of Akt in L6 myotubes increases glucose uptake, glycogen and protein
synthesis (Ueki et al., 1998). Also, PI 3-kinase inhibitors not only block insulin-
stimulated glucose uptake but Akt activity in isolated rodent muscle (Brozinick Jr. &
Birnbaum, 1998; Whitehead et al., 2000) demonstrating a key role for Akt on glucose
uptake. Insulin stimulation of Akt has been shown to increase glycogen synthesis in L6
90
myotubes by inhibiting glycogen synthase kinase-3 (GSK-3), a known inhibitor of
glycogen synthase (Cross et al., 1995; Cross et al., 1997; Ueki et al., 1998) whilst in cell
lines lacking GSK-3 such as 3T3L1 adipocytes, overexpression of Akt has no effect of
glycogen synthesis (Ueki et al., 1998). The pathway for insulin stimulated protein
synthesis via the activation of Akt also appears to involve the downstream activation of
p70 S6 kinase, since inhibition of Akt reduces activation of p70 S6 kinase in CHO cells
and 3T3L1 adipocytes (Kitamura et al., 1998). Also, in isolated rodent muscle, PI 3-
kinase inhibitors not only impair insulin-stimulated Akt kinase activity but also p70 S6
kinase activity (Brozinick Jr. & Birnbaum, 1998) further supporting the involvement of
Akt in insulin-stimulated protein synthesis. There is some evidence to suggest that the
different isoforms of Akt have preferential roles towards the regulation of glucose
uptake, glycogen synthesis and mitogenesis. Akt-1 knockout mice have normal insulin
sensitivity but are much smaller than their wild type littermates (Cho et al., 2001b),
whilst Akt-2 deficient mice have normal growth development but are insulin resistant
(Cho et al., 2001a). Akt-2 inhibition in 3T3L1 adipocytes also inhibits insulin-
stimulated GLUT4 translocation (Hill et al., 1999). Recently, depletion of Akt-1 and/or
Akt-2 isoforms in 3T3L1 adipocytes by RNA directed gene silencing suggest a primary
role for Akt-2 and a lesser role for Akt-1 in insulin-stimulated glucose transport (Jiang
et al., 2003). Similar preferential roles for Akt-1 and Akt-2 were found for glycogen
synthesis (Jiang et al., 2003). Whilst further work is required to elucidate the exact
roles of the different Akt isoforms, it appears that Akt is a key regulatory protein
involved in insulin-stimulated glucose transport, glycogen synthesis and mitogenesis.
However, without downstream measures of the mitogenic or glycogen synthesis
pathways such as phosphorylated MAP kinase, p70 S6 kinase or glycogen synthase
activity there is no direct evidence for this in the present study. Due to a lack of
91
available sample, time and financial constraints these variables were not measured in
the present study. Nevertheless, this study has successfully shown for the first time that
there is a small increase in key component of insulin signalling in response to acute
exercise and short-term training that is distal to PI 3-kinase in human skeletal muscle.
Another major finding of this study was that exercise selectively increases the protein
expression of SHPTP2 in human skeletal muscle. The finding that SHPTP2 protein
abundance was selectively increased in the cytosolic fraction suggests this to be a major
site for its intracellular action. In support of this SHPTP2 has been implicated in rat
skeletal muscle to account for a large majority of cytosolic PTPase activity (Ahmad &
Goldstein, 1995). However, increased cytosolic levels of SHPTP2 are not always
associated with states of improved insulin sensitivity. Both SHPTP2 and PTP1B are
increased in obese subjects and decreased in obese type 2 diabetics compared to lean
controls. These findings suggest a dysregulation of PTPases in the insulin resistant and
diabetic states (Ahmad et al., 1997a). Nevertheless, SHPTP2 has been implicated to
play a role in the positive regulation of insulin-stimulated glucose transport, glycogen
synthesis and mitogenic signalling (Chapter 1). Over expression of SHPTP2 in rat
fibroblasts increases the insulin stimulated binding of IRS-1 with SHPTP2, IRS-1
tyrosine phosphorylation, and the associated PI 3-kinase activity whilst inhibition of
SHPTP2 has the opposite effect (Ugi et al., 1996). Transgenic mice with impaired
SHPTP2 not only display reduced insulin-stimulated glucose transport, but glycogen
synthase activity and MAP kinase phosphorylation are also impaired, suggesting
multiple regulatory roles for SHPTP2 in skeletal muscle (Maegawa et al., 1999). Both
overexpression and inhibition of SHPTP2 in rat fibroblasts also supports the multiple
actions of SHPTP2 on insulin signalling (Ugi et al., 1996).
92
How exactly a PTPase such as SHPTP2 is able to positively influence insulin signalling
is unclear. It has been postulated that SHPTP2 amplifies tyrosine phosphorylation of
IRS-1 by either inhibiting an as yet undefined PTPase(s) (Ugi et al., 1996) or by the
activation of tyrosine kinases. As mentioned in Chapter 1, one of the main regulatory
mechanisms for SHPTP2 on insulin signalling involves increased IRS-1 tyrosine
phosphorylation (Maegawa et al., 1999; Ugi et al., 1996). However, the present study
observed no changes in IRS-1 tyrosine phosphorylation following acute exercise and
exercise training. Although it appears that increased SHPTP2 protein abundance is not
mediating IRS-1 tyrosine phosphorylation in the present study it is possible that
enhanced cytosolic SHPTP2 protein expression indirectly increases pAkt (Ser473). An
indirect link between increased SHPTP2 abundance and increased Akt activity has been
demonstrated in the past. Overexpression of SHPTP2 is known to increase the activity
of the tyrosine kinase, Src (Walter et al., 1999) via the binding to its SH3 domain. The
association of Src through its SH3 domain with the C-terminal regulatory region of Akt
has been shown to be necessary for the tyrosine phosphorylation and activation of Akt
(Jiang & Qiu, 2003). Src association with Akt is also required for its extracellular
growth factor (EGF) induced activation (Jiang & Qiu, 2003), although the association of
SHPTP2 with Src on the SH3 domain and the subsequent activation of Akt following
insulin stimulation are unknown. Although highly speculative, the observation in the
current study that SHPTP2 cytosolic protein abundance is associated with increased
pAkt (Ser473) following both acute exercise and short-term training, combined with the
previous findings of others, indirectly linking SHPTP2 and Akt via the tyrosine kinase,
Src, is sufficient to warrant further investigation into this poorly described aspect of
insulin signalling.
93
The other significant finding of the present study was that PTP1B protein abundance or
subcellular distribution is unaltered by exercise. There is evidence for the inactivation
of PTP1B by insulin and reactive oxygen species such as hydrogen peroxide (Mahadev
et al., 2001; Tao et al., 2001) without alteration in protein expression. It is however
unlikely that alterations in PTP1B activity may be regulating any exercise-induced
improvements in insulin sensitivity since the IR and IRS-1, which are substrates of
PTP1B, were not altered by exercise. The vast majority of studies investigating the role
of PTPases in the regulation of insulin signalling have focussed on PTP1B. This is not
surprising since PTP1B null mice display enhanced insulin sensitivity and improved
insulin stimulated IR and IRS-1 tyrosine phosphorylation (Elchebly et al., 1999).
Similar results have been found when PTP1B is disrupted by either antisense
oligonucleotide or specific inhibitor treatment (Mohammad et al., 2002; Zinker et al.,
2002). Weight loss in humans is also associated with reduction of PTP1B in adipose
tissue (Ahmad et al., 1997b). Clearly, PTP1B has an important role to play in insulin
signalling and therapeutic benefits may be gained for type 2 diabetics via the
development of specific PTP1B inhibitors (Iversen et al., 2000; Mohammad et al.,
2002). However, it would appear based on the findings of this study that exercise-
induced improvements in insulin action are not mediated by changes in PTP1B protein
abundance or subcellular localisation.
4.5 Conclusions
In summary, this study has found that 24 hours following a single bout of exercise there
was improved whole-body insulin action in sedentary humans. Larger improvements in
94
insulin action were found 24 hours following the completion of endurance training,
which implies that increased insulin action is not only due to the residual effects of the
prior bout of exercise, but that cumulative bouts of exercise substantially increase this
response. This study also found that there was no effect of exercise on the upstream
components of the insulin-signalling pathway such as the IR or IRS-1 protein
expression or tyrosine phosphorylation, suggesting that increased insulin sensitivity
following exercise is not mediated by changes in the proximal section of the insulin
signalling pathway. A major finding of this study was that there was an observed
increase in the serine phosphorylation of a known downstream signalling protein, Akt,
in human skeletal muscle following an acute bout of exercise and exercise training.
Finally, the present study also found increased abundance of SHPTP2 protein in the
cytosolic fraction of skeletal muscle following both acute exercise and short-term
training. Previous studies have shown a possible link between SHPTP2 and Akt, so it is
possible, although highly speculative, that the upregulation of both SHPTP2 and pAkt
(Ser473) following exercise in the present study is directly related, although further
research is required to establish this link. It is also possible that the increase in pAkt
(Ser473) and/or cytosolic SHPTP2 by exercise could be responsible for increased
mitogenic signalling and/or glycogen synthesis and future research should examine
these pathways. Also, PTP1B protein expression and cellular distribution was unaltered
by exercise, as were its targets for protein tyrosine dephosphorylation, IR and IRS-1,
suggesting that PTP1B is not involved in any improvements in insulin stimulated
glucose uptake by exercise.
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CHAPTER FIVE
EFFECTS OF EXERCISE ON PROTEIN ABUNDANCE OF
IRS-2 AND THE p85α SUB UNIT PI 3-KINASE
5.1 Introduction
In Chapter 2 it was observed that a single bout of endurance exercise is sufficient to
increase the mRNA concentration of insulin signalling proteins such as IRS-2 and the
p85α sub unit of PI 3-kinase in humans, whilst mRNA concentration of other key
insulin signalling proteins such as IR and IRS-1 are unchanged by acute exercise. IRS-2
mRNA concentration is also upregulated following a single bout of resistance exercise,
demonstrating the acute transcriptional activation of IRS-2 by intense muscle
contraction (Chapter 3). Improvement in IRS-2 protein abundance and insulin
stimulated PI 3-kinase activity in rodents has further been shown following an acute
bout of exercise pointing to a specialised role for IRS-2 in insulin stimulated glucose
transport and/or mitogenesis (Chibalin et al., 2000; Howlett et al., 2002). It remains
unclear if this transient, transcriptional activation of IRS-2 and the p85α sub unit of PI
3-kinase gene expression following a single bout of endurance exercise in humans
results in an increase in protein abundance. It was therefore decided that subsequent to
the results obtained from Chapter 4 further analysis was required to determine if
exercise increased the protein abundance of these insulin-signalling components.
Therefore, the aim of this study was to determine if acute exercise is sufficient to
increase IRS-2 and p85α PI 3-kinase protein expression in humans skeletal muscle.
96
5.2 Materials And Methods
5.2.1 Materials
PI 3-kinase p85α rabbit polyclonal IgG (UBI, Cat. # 06-195) was purchased from
Upstate Biotechnology (New York, USA). IRS-2 (A-19) goat polyclonal (SC, Cat.
#1556) was from Santa Cruz Biotechnology (California, USA). Affinity purified
peroxidase labelled anti-mouse IgG and anti-rabbit IgG were purchased from Silenus
(Victoria, Australia). All other reagents were analytical grade (Sigma, NSW, Australia).
5.2.2 Subjects
This study forms part of the human intervention that was covered in the previous
chapter. The subjects recruited, and the methods for exercise training and the
euglycemic hyperinsulinemic clamp procedure are the same as per the previous chapter.
Briefly, A group of 8 untrained, but healthy males volunteered to be involved in this
study. Subject characteristics were as described in the previous chapter.
5.2.3 Experimental protocol
The subjects underwent 3 euglycemic-hyperinsulinemic clamps on separate days, each
following an overnight fast and performed acute exercise and endurance training as per
the previous chapter.
5.2.4 Immunoprecipitations and immunoblotting
Equal amounts of protein for determination of IRS-2 (75µg) and PI 3-kinase p85 (60µg)
were solubilised in Laemmli sample buffer. Bound proteins were separated by SDS-
97
PAGE and electrotransfer of proteins from the gel to nitrocellulose membranes (25mM
Tris, pH 8.3, 192mM glycine, and 20% v/v methanol) was performed for 100 min at
100V (constant). Blots were probed with anti-IRS-2 goat polyclonal (1:200) or anti-p85
PI 3-kinase rabbit polyclonal (1:1000) antibodies. Binding was viewed by enhanced
chemiluminescence (Pierce, IL, USA) and quantified with Kodak 1D version 3.5
software (Eastman Kodak Co., CT, USA).
5.2.5 Statistical analysis
Standards were included in all immunoblots and interassay variation was accounted for
by normalising data to control samples. Data are presented as mean ± SEM.
Differences were determined using 2-way analysis of variance (ANOVA) with
Newman-Keuls post-hoc analysis, where appropriate. Significance was accepted when
p<0.05.
5.3 Results
The subject and exercise characteristics are presented in the previous chapter (Table
4.2). Also, refer to the previous chapter for the results of the euglycemic,
hyperinsulinemic clamps (Table 4.3 and Figure 4.1 and 4.2).
5.3.1 IRS-2 and p85α PI 3-kinase
The protein expression of the p85α catalytic subunit of PI 3-kinase was not altered by
exercise (Figure 5.1). IRS-2 protein expression increased approximately 1.5 fold
98
following insulin stimulation in the sedentary condition (p<0.05, Figure 5.2). There was
also a 1.9 fold increase in IRS-2 protein abundance following acute exercise (p<0.05,
Figure 5.2), which decreased back to sedentary levels following endurance training
(p<0.05, Figure 5.2).
99
0
6000
12000
18000
24000
30000
sedentary acute training
p85α
pro
tein
abu
ndan
ce(a
rbitr
ary
units
)
Figure 5.1. p85α PI 3-kinase protein abundance during the euglycemic, hyperinsulinemic
clamps. Protein abundance is determined following western blotting of skeletal muscle extracts
with anti-p85α PI 3-kinase antibody. Western blots are representative from one subject.
Sedentary: no prior exercise, Acute: 24 hours following 60 min of exercise, Training: 24 hours
following 8 days of endurance training. Open bars: basal values, closed bars: insulin stimulated
values. Bars on graph represent means ± SE of 8 subjects.
0
20000
40000
60000
80000
sedentary acute training
**
IRS-
2 pr
otei
n ab
unda
nce
(arb
itrar
y un
its)
Figure 5.2. IRS-2 protein abundance during the euglycemic, hyperinsulinemic clamps.
Protein abundance is determined following western blotting of skeletal muscle extracts with
anti-IRS-2 antibody. Western blots are representative from one subject. Sedentary: no prior
exercise, Acute: 24 hours following 60 min of exercise, Training: 24 hours following 8 days of
endurance training. Open bars: basal values, closed bars: insulin stimulated values. Bars on
graph represent means ± SE of 8 subjects. *p<0.05 vs. basal value in the sedentary state.
100
5.4 Discussion
It was discussed in the previous chapter that the present study observed significant
increases in the insulin action of sedentary individuals following an acute bout of
exercise and further improvements following short-term endurance training that could
largely be attributed to exercise rather than any associated alterations in body
composition.
We found a small but significant increase in IRS-2 protein expression following
prolonged insulin stimulation in the sedentary state. This result was surprising since
little is known regarding increases in protein abundance following acute insulin
stimulation. Even more surprising was that it was only increased by insulin in the
sedentary state and not following acute exercise or endurance training. No studies to our
knowledge have investigated the effects of insulin stimulation on IRS-2 protein
abundance. Increased IRS-2 protein abundance can account for a significant amount of
increased IRS-2 associated PI 3-kinase activity following an acute bout of exercise
(Chibalin et al., 2000) although the effects of prolonged insulin stimulation on IRS-2
protein abundance have not been previously measured. It could be postulated that unlike
the sedentary condition, no change in insulin-stimulated IRS-2 protein expression was
observed following acute exercise since protein abundance was already significantly
elevated by the exercise bout, reducing the stimulus for additional protein synthesis. For
some as yet unknown reason cumulative bouts of exercise attenuate the transcriptional
regulation of IRS-2 compared to acute exercise (Chapter 2) and this may explain the
lack of effect for insulin stimulation or endurance training on IRS-2 protein abundance
observed in the present study.
101
The protein expression of IRS-2 also significantly increased by 1.9 fold in the 24 hours
following a single bout of endurance exercise and returned to sedentary levels after 8
days of endurance training. Chapter 2 of this thesis reported a significant increase in
IRS-2 gene expression several hours following an acute bout of exercise, which would
now appear to coincide with increased protein abundance approximately 20 hours later.
Furthermore, short-term endurance training diminished the effect of a single bout of
exercise on IRS-2 gene expression (Chapter 2), which also coincides with the findings
of the present study that IRS-2 protein abundance returned to sedentary levels following
training. It is often difficult to correlate mRNA with protein abundance due to the
transient nature of mRNA and the longer synthesis and half lives of the proteins.
However, it would appear that for IRS-2 at least, large changes in gene expression in
the few hours following exercise are associated with significant increases in protein
expression up to 20 hours later. It would also suggest that alterations in IRS-2 protein
abundance by exercise are mediated by transcriptional factors rather than via translation
control. The protein abundance of IRS-2 in rodents (Chibalin et al., 2000) 16 hours
following a single bout of exercise also significantly increases with a similar pattern to
return to sedentary levels following short term training when compared to the findings
of the present study. Chibalin et al. (Chibalin et al., 2000) also found increases in IRS-2
associated PI 3-kinase activity in both the basal and insulin stimulated state, which
could largely be attributed to increases in protein abundance. Unfortunately, the
measurement of IRS-2 tyrosine phosphorylation requires relatively large volumes of
sample and for this reason IRS-2 tyrosine phosphorylation not measured in the present
study. Nevertheless, the finding in the present study of increased IRS-2 protein
abundance following acute exercise, combined with previous findings in rodents
(Chibalin et al., 2000) would suggest an early adaptive role for IRS-2 that is different to
102
the other insulin receptor substrates with regards to glucose transport and/or
mitogenesis. Although IRS-2 has been shown not to play a major role in exercise or
insulin stimulated glucose transport (Higaki et al., 1999), insulin stimulated IRS-2
tyrosine phosphorylation and associated PI 3-kinase activity are elevated immediately
post exercise and can account for some of the improvements in insulin stimulated
glucose uptake (Howlett et al., 2002) immediately post exercise, however much less is
known of its role in the 12-24 hours period after the cessation of acute exercise. In the
present study, there was only a small improvement in insulin action following acute
exercise when compared with exercise training and in light of this it would appear that
the observed increases in IRS-2 protein abundance if they are related to any
improvements in insulin sensitivity are likely to be small. One other potential role for
IRS-2 is via the mitogenic pathway. IRS-2 has a major role in the maintenance of β-cell
mass in the pancreas (Withers et al., 1999). Little is known of the role for IRS-2 in the
mitogenic pathway, particularly in skeletal muscle although as discussed earlier there is
a sustained activation of IRS-2 associated PI 3-kinase activity for up to 16 hours
following an acute bout of endurance exercise in rodents independently of insulin that
could largely be attributed to increased IRS-2 protein abundance (Chibalin et al., 2000).
Selective stimulation of PI 3-kinase independently from insulin has been shown in
regenerating rodent muscle to be important for myofiber growth (Murgia et al., 2000).
The activation of PI 3-kinase via association with its p85α catalytic subunit and by IRS-
1 independent from insulin has been shown following muscle overload in rodents to
coincide with increased glucose uptake and protein synthesis (Carlson et al., 2001;
Hernandez et al., 2000). It would therefore appear that following intense muscular
contractions IRS-2 protein abundance increases, which may activate PI 3-kinase via
concomitant increases in the tyrosine phosphorylation of IRS-2. There is some evidence
103
to suggest that this pathway may play a role in protein synthesis and perhaps following
insulin stimulation IRS-2 may also play a minor role in glucose uptake following an
acute bout of exercise. This pathway may have an early transitory role in both
mitogenesis and insulin stimulated glucose uptake since IRS-2 protein expression and
function appear to return to sedentary levels with repeated bouts of intense muscular
contractions.
The finding that protein abundance of the p85α sub unit of PI 3-kinase was not
increased 24 hours following acute exercise or exercise training suggests that even
though acute exercise can stimulate transcriptional activation of the p85α gene, this
does not appear to result in improved protein abundance.
5.5 Conclusions
In summary, prolonged insulin stimulation in the sedentary state was found to increase
IRS-2 protein abundance and that IRS-2 protein abundance, like its gene expression
found previously in Chapter 2, is augmented following acute exercise, but attenuated
following cumulative bouts of exercise. In light of these novel findings, future studies
investigating if any functional changes occur in IRS-2 tyrosine phosphorylation or
related PI 3-kinase activity following a single bout of exercise and exercise training are
therefore warranted. This would further support a transient role for IRS-2 in insulin-
stimulated glucose uptake and mitogenic signalling in human skeletal muscle.
104
CHAPTER SIX
THE EFFECT OF INSULIN AND EXERCISE ON c-Cbl
PROTEIN ABUNDANCE AND PHOSPHORYLATION IN
INSULIN RESISTANT SKELETAL MUSCLE
6.1 Introduction
While the activation of the PI 3-kinase pathway is pivotal in the stimulation of glucose
transport, its activation alone is not sufficient to mediate glucose transport. Activation
of IRS-1 and/or PI 3-kinase with ligands such as platelet derived growth factor (PDGF),
insulin in cell lines with impaired IR function or via integrin engagement on the cell
membrane do not result in increased glucose transport (Baumann et al., 2000;
Guilherme & Czech, 1998; Krook et al., 1997; Wiese et al., 1995). These studies
provide some evidence for another insulin signalling pathway that is required for
insulin-stimulated glucose transport, but is independent of PI 3-kinase activation.
It was discussed in Chapter 1 that there has been recent identification of an alternate
insulin-signalling pathway for glucose transport involving the recruitment of the protein
c-Cbl to the IR by the adapter protein CAP (Baumann et al., 2000). c-Cbl is heavily
tyrosine phosphorylated by a variety of kinase signalling pathways (Anderson et al.,
1997; Fukazawa et al., 1996; Marcilla et al., 1995; Meisner et al., 1995) and in 3T3-L1
adipocytes c-Cbl is markedly and rapidly tyrosine phosphorylated in response to insulin.
(Ribon et al., 1998).
105
Tyrosine phosphorylation of c-Cbl and subsequent localization to the lipid rafts in
conjunction with the CAP and flotillin complex may be a necessary insulin-dependent
pathway for glucose transport in adipocytes. However, involvement of this pathway in
skeletal muscle, the primary tissue of insulin-mediated glucose uptake has yet to be
demonstrated. Unlike adipocytes which are responsible for only 10% of insulin
stimulated glucose disposal, skeletal muscle accounts for as much as 90% of whole-
body glucose uptake (DeFronzo et al., 1985). Furthermore, in insulin resistant states the
activation of signalling proteins such as the insulin receptor, IRS-1, PI 3-kinase and
some isoforms of Akt are dysregulated (Brozinick et al., 2003; Christ et al., 2002; Cusi
et al., 2000; Krook et al., 2000). Therefore, investigation of c-Cbl activation in normal
healthy and insulin resistant skeletal muscle is important in order to elucidate the
potential role of this novel insulin-signalling cascade.
Exercise is a potent stimuli for the translocation of GLUT4 to the cell surface and the
subsequent increase in glucose transport into muscle (Richter et al., 1998; Thorell et al.,
1999). The signalling pathways mediating translocation of GLUT4 in response to
exercise remain unknown, although it is now well established that the pathway is
independent from the insulin-mediated activation of PI 3-kinase (Wojtaszewski et al.,
2002). c-Cbl is tyrosine phosphorylated by a wide variety of cell surface receptors
including growth factors, cytokines, and lymphocyte antigens (Galisteo et al., 1995;
Ribon & Saltiel, 1997; Taher et al., 2002). Additionally, shear stress in endothelial cells,
initiates rapid and sustained tyrosine phosphorylation of c-Cbl (Miao et al., 2002).
Collectively the results of these studies suggest a possible role for skeletal muscle
contractile activity to result in tyrosine phosphorylation of c-Cbl and hence downstream
106
activation of the flotillin related complex involved in GLUT4 translocation to the cell
membrane lipid rafts.
Furthermore, the expression of CAP in skeletal muscle is important since failure of
insulin to tyrosine phosphorylate c-Cbl in 3T3L1 fibroblasts has been shown to be
associated with a lack of CAP expression in 3T3L1 fibroblasts (Ribon et al., 1998).
Skeletal muscle of mice has been previously shown to express CAP mRNA, implying
expression of functional CAP protein in skeletal muscle (Ribon et al., 1998). However
the expression of CAP protein in skeletal muscle and its abundance relative to other
tissues such as 3T3L1 adipocytes is unknown. Therefore, the measurement of CAP
expression in skeletal muscle will further elucidate the role of this novel pathway
In the present study the impact of insulin-stimulation and an acute bout of exhaustive
swimming exercise on c-Cbl tyrosine phosphorylation in the skeletal muscle of lean and
insulin-resistant obese Zucker rats was investigated. Soleus muscle from these animals
was harvested for analysis of c-Cbl, IR and Akt phosphorylation and protein abundance
following rest, acute exercise or insulin stimulation. A further aim of this study was to
briefly determine if CAP protein abundance is much higher in 3T3L1 adipocytes
compared to rat skeletal muscle.
107
6.2 Materials and Methods
6.2.1 Materials
c-Cbl (7G10) mouse monoclonal IgG (UBI, Cat. # 05-440), CAP rabbit polyclonal IgG
(UBI, Cat. # 06-994), insulin-receptor (IRβ) rabbit polyclonal IgG (UBI, Cat. # 06-492)
and phosphotyrosine (4G10) mouse monoclonal IgG (UBI, Cat. # 05-321) were
purchased from Upstate Biotechnology (NY, USA). CT1 IRβ mouse monoclonal IgG
was a gift from Prof. Ken Siddle (University of Cambridge). Phospho-Akt (pAkt)
Ser473 rabbit polyclonal IgG (Cat. # 9271) and Akt rabbit polyclonal IgG (Cat. # 9272)
were from Cell Signaling Technology (New England BioLabs, Hartsfordshire,
England). Affinity purified peroxidase labelled anti-mouse IgG and anti-rabbit IgG
were purchased from Silenus (Victoria, Australia). All other reagents were analytical
grade (Sigma, NSW, Australia).
6.2.2 Animal care and dietary treatment
Female lean (fa/?; n = 18) and obese (fa/fa; n = 18) Zucker rats aged 10-11 wk and
weighing ~ 176 and ~296 g, respectively, were obtained from Monash University
Animal Services, Victoria, Australia. Animals were housed two per cage in an
environmentally controlled laboratory (temperature 22 ± 1oC, relative humidity 50 ±
2%) with a 12:12-h light-dark cycle (light 0700-1900). Animals were fed standard
rodent chow (67.5% carbohydrate, 11.7% fat, 20.8% protein; Barastock, Victoria,
Australia), given ad libitum access to water; and familiarized to laboratory conditions
for 1 wk prior to experimentation. The Animal Experimentation Ethics Committee of
RMIT University approved all experimental procedures.
108
Animals were assigned to one of three subgroups on the basis of 1) whether they
remained sedentary control (Con), 2) were exercised (Ex), or 3) were insulin stimulated
(Insulin).
6.2.3 Sedentary controls
At 1700 h on the day before the experiment, lean (ZL) animals were restricted to 10 g,
and obese (ZO) animals to 12 g, of chow (this amount being ~60% of the animals
average daily food consumption from the previous 7 d). ZL rats were assigned to one of
three experimental groups: sedentary control (ZL-Con; n = 6), exercised (ZL-Ex; n = 6),
and insulin treated (ZL-Insulin; n = 6). ZO rats were also assigned to one of three
experimental groups: sedentary control (ZO-Con; n = 6), exercised (ZO-Ex; n = 6), and
insulin treated (ZO-Insulin; n = 6).
6.2.4 Exercise
Two groups of rats (ZL-Ex and ZO-Ex) performed a standard exercise regimen in order
to deplete their skeletal muscle glycogen stores. Three rats swam together in a steel
barrel measuring 60 cm in diameter and filled to a depth of ~60 cm. Water temperature
was maintained at 35˚C. Prior to the commencement of an experiment, all animals had
been familiarized to swimming for 10 min/d for 3 d. The swimming protocol was a
modification of the procedure used extensively in previous exercise studies with rats
(Bruce et al., 2001; Cartee et al., 1989; Gulve et al., 1990). Rats swam for eight 30 min
bouts separated by 5 min rest periods. In the case of the obese animals, a weight equal
to ~2.5% of body mass (BM) was attached to the base of the tail after the first 30 min
exercise bout in an attempt to compensate for their increased buoyancy. The obese
109
animals swam with the weight attached for the remaining seven exercise bouts. Weights
were chosen so that during the swimming protocol the body angles relative to the
surface of the water were similar for both the obese and lean rats (Walberg et al., 1982).
Rats were sacrificed immediately after the exercise.
6.2.5 Insulin treatment
Two groups of non-exercised rats (ZL-Insulin and ZO-Insulin) were injected
intraperitoneally with insulin (0.15 U.g-1 body weight, Human, Actrapid, Novo Nordisk,
Denmark) (Howlett et al., 2002). After 5 min the animals were anaesthetized with an
intraperitoneal injection of pentobarbital sodium (60 mg.kg-1 BM) and sacrificed.
6.2.6 Preparation of rat tissue and blood samples
Approximately 5 min before the due time of death, rats were anaesthetized with an
intraperitoneal injection of pentobarbital sodium (60 mg.kg-1 BM) and soleus muscle
was rapidly excised and frozen in liquid N2. The soleus (84% type I, slow-twitch,
oxidative fibers) was deliberately chosen to sample because previous investigations
have reported there to be no differences in GLUT4 content between ZL and ZO animals
(Brozinick et al., 1992). The exercise protocol used in the present study has been shown
in previous studies to significantly reduce muscle glycogen in the soleus, indicating
active recruitment of this muscle group during the exercise protocol (Bruce et al., 2001).
Furthermore, type I fibers are more insulin sensitive (Kern et al., 1990) and have a
higher maximal glucose transport rate than type II fibers (Richter et al., 1988). A blood
sample (~1 mL) was obtained via cardiac puncture.
110
Frozen rat soleus were homogenised using a polytron at maximum speed for 30 sec on
ice in 600µl of freshly prepared ice-cold buffer consisting of 50mmol/l HEPES at pH
7.6 containing 150 mmol/l NaCl, 20 mmol/l Na4P2O7, 20 mmol/l β-glycerophosphate,
10 mmol/l NaF, 20 mmol/l EDTA, 1% v/v Nonidet P-40, 10% v/v glycerol, 1 mmol/l
MgCl2, 1 mmol/l CaCl2, 2 mmol/l Na3VO4, 2 mmol/l PMSF, 5µl/ml Protease Inhibitor
Cocktail (P8340, Sigma). Tissue lysates were incubated on ice for 20 min and then
centrifuged at 10,000 × g for 20 min at 4 0C. Protein concentration was determined
using the Bio-Rad protein assay (Bio-Rad, NSW, Australia). The supernatants were
stored at –80 0C until subsequent analysis.
6.2.7 Cells and culture conditions
Differentiated 3T3-L1 adipocytes were a gift from Dr. Lance Macaulay (CSIRO).
Briefly, 3T3L1 fibroblasts were grown and passaged in Dulbecco's modified Eagle’s
medium (DMEM) supplemented with 10% (v/v) newborn calf serum at 37oC.
Fibroblasts were differentiated 1 to 2 days post-confluence. The differentiation medium
contained 10% (v/v) fetal calf serum (FCS), 250nM dexamethasone, 500nM isobutyl
methylxanthine and 500nM insulin. After 3 days, the differentiation medium was
replaced with post-differentiation medium containing 10% (v/v) FCS and 500nM
insulin for a further 2 days. Cells were then fed for another 2 days post-differentiation
in DMEM supplemented with 5% (v/v) FCS. Prior to cell lysis, adipocytes were serum
starved overnight in DMEM containing 0.5% (v/v) FCS. Adipocytes were then washed
twice with ice-cold PBS and incubated for 15 min on ice in lysis buffer containing
50mmol/l Tris-HCl at pH 7.4 containing 150 mmol/l NaCl, 1 mmol/l Na4P2O7, 0.25%
sodium deoxycholate (w/v), 1 mmol/l NaF, 1 mmol/l EGTA, 1% Nonidet P-40, 1
111
mmol/l Na3VO4, 1 mmol/l PMSF, 1µg/ml aprotinin, 1µg/ml leupeptin and 1µg/ml
pepstatin. Tissue lysates were then spun at 10,000 × g for 15 min at 4 0C. Protein
concentration was determined using the Bio-Rad protein assay (Bio-Rad, NSW,
Australia). The supernatants were stored at –80 0C until analysis. The lysis analysed
was from 2 separate 10 cm-diameter dishes.
6.2.8 Immunoprecipitations and immunoblotting
For immunoprecipitation, 1µg of anti-c-Cbl or 2µg of anti-IRβ monoclonal antibody
(Upstate Biotechnology) was coupled to 30 µl protein A-sepharose beads (Zymed
Laboratories, CA, USA) for 20 min at room temperature and then washed once in PBS.
Tissue lysates (375µg protein) were incubated with the antibody coupled beads
overnight at 4 0C with rotation. Immunoprecipitated proteins were then washed once in
PBS containing 0.5 mol/l NaCl and 0.2% v/v Triton X-100 and twice in PBS containing
0.2% v/v Triton X-100. Immunoprecipitated proteins or total lysates for determination
of pAkt (Ser473) (75µg), IRβ (45µg), Akt protein abundance (75µg) in rat skeletal
muscle and CAP protein abundance in 3T3L1 adipocytes (20µg of total protein per
lane) and rat skeletal muscle (75µg of total protein per lane) and were solubilised in
Laemmli sample buffer. Bound proteins were separated by SDS-PAGE and
electrotransfer of proteins from the gel to nitrocellulose membranes (25 mmol/l Tris, pH
8.3, 192 mmol/l glycine, and 20% v/v methanol) was performed for 100 min at 100V
(constant). Blots were probed with anti-c-Cbl monoclonal mouse, anti-CAP polyclonal
rabbit, anti-phosphotyrosine 4G10 mouse monoclonal, anti-IRβ CT-1 mouse
monoclonal, anti-Akt rabbit polyclonal or anti-pAkt (Ser473) rabbit polyclonal
antibodies. Binding was detected with HRP coupled secondary antibodies and by
112
enhanced chemiluminescence (Pierce, IL, USA). Blots were quantified with Kodak 1D
version 3.5 software (Eastman Kodak Co., CT, USA).
6.2.9 Blood biochemistry
Whole blood (~1 mL) was transferred to an EDTA administered tube and was spun in a
centrifuge at 12,000 rpm for 3 min. The plasma was analysed for plasma glucose
concentration using an automated analyser (Yellow Springs Instruments 2300 Stat Plus
Glucose Analyser, Yellow Springs, OH, USA). The remaining plasma was stored at –
80˚C and was subsequently analysed for plasma insulin concentration by
radioimmunoassay using a commercially available kit (Phadeseph, Insulin RIA,
Pharmacia & Upjohn Diagnostics AB, Uppsala, Sweden).
6.2.10 Statistical analyses
Standards were included in all immunoblots and interassay variation was accounted for
by normalising data to control samples. Data are presented as mean ± SEM. Analysis of
differences between two treatments within a genotype and differences between lean and
obese animals was performed using an unpaired t test. All other differences were
determined using a two-way analysis of variance with Newman-Keuls post-hoc
analysis, where appropriate. Significance was accepted when p<0.05.
113
6.3 Results
6.3.1 Characteristics of lean and obese Zucker rats
As expected, ZO animals were significantly heavier than ZL (296 ± 7g versus 176 ± 3g,
p<0.0001). Table 6.1 shows the concentrations of plasma glucose and plasma insulin at
rest and following the two treatment interventions. Resting plasma glucose
concentrations were similar between ZO and ZL. However, as expected, resting plasma
insulin concentrations were 3 to 4-fold higher in ZO compared with ZL rats (p<0.05,
Table 1). In ZL rats, exercise was associated with a 1.6 and 2.6 fold reduction in plasma
glucose and plasma insulin concentrations, respectively (p<0.05, Table 6.1). Insulin
treatment resulted in supra-physiological plasma insulin levels. Insulin treated rats were
2000-fold and 800-fold higher in ZL and ZO rats, respectively when compared to the
control groups (p<0.05, Table 6.1). Following insulin treatment, plasma glucose
concentrations decreased 2.0 and 1.6 fold in ZL and ZO rats, respectively (p<0.05,
Table 6.1).
114
Table 6.1 Plasma glucose and insulin concentrations measured in lean and obese
Zucker rats.
Glucose, mmol/l Insulin, pmol/l
Lean Obese Lean Obese
Control 8.7 ± 0.1 9.4 ± 0.4 80 ± 3 293 ± 44+
Exercise 5.5 ± 0.9* 10.4 ± 0.9+ 31 ± 6* 212 ± 30+
Insulin 4.4 ± 0.6* 7.7 ± 0.5*+ 193 ± 22 (× 103)* 245 ± 21 (× 103)*
Values are means ± SE for 6 rats in each group. *p<0.05 vs. control group, +p<0.05 vs.
lean group in identical conditions.
115
6.3.2 Insulin receptor protein expression and tyrosine phosphorylation in muscle
of Zucker rats
Lean and obese Zucker rats either undertook a single bout of exhaustive swimming
exercise, or were treated with a supra-physiological dose of insulin.
Immunoprecipitation and immunoblotting were performed on the extracts prepared
from skeletal muscle and analysed for protein abundance and tyrosine phosphorylation
of the IR and c-Cbl. pAkt (Ser473) and protein abundance of Akt, a kinase
phosphorylated following the insulin-stimulated activation of PI 3-kinase was also
determined. The results clearly show that insulin treatment markedly increased tyrosine
phosphorylation of the IR. ZL-insulin treated rats had a 12-fold higher tyrosine
phosphorylation of the IR compared to the ZL-control rats (p<0.0001, Figure 6.1). This
effect was blunted in obese Zucker rats with IR tyrosine phosphorylation only 3-fold
higher for the ZO-insulin treated compared to the ZO-control rats (p<0.001, Figure 6.1).
This observation may partly be explained by the 40% lower abundance of IR protein in
ZO compared to ZL animals (p<0.0001, Figure 6.2). IR protein abundance was not
altered by either insulin treatment or acute exercise (not significant, Figure 6.2),
although there was a trend (p=0.06) for ZO animals to have higher IR tyrosine
phosphorylation levels than ZL in the control group (Figure 6.1). This finding can
probably be explained by the significantly higher basal plasma insulin concentrations of
the ZO animals in the two control groups (Table 6.1). Exercise did not increase IR
phosphorylation in either lean or obese Zucker rats (Figure 6.1). Indeed, IR tyrosine
phosphorylation was unable to be detected following exercise in the ZL rats.
Accordingly, a nominal value was assigned to this group relative to the fold changes
seen in the ZO animals between the control and exercised groups.
116
0
50
100
150
200
250
300* *
Control Exercise Insulin
#IR
pY
(arb
itrar
y un
its)
Figure 6.1. Effects of acute exercise and insulin stimulation on insulin receptor (IR)
phosphorylation in lean (open bar) and obese (closed bar) Zucker rats. Skeletal muscle
extracts were immunoprecipitated with anti-IRβ antibody and blots were probed for anti-
phosphotyrosine (4G10) antibody. Western blots are representative of one rat in each group.
Bars on graph represent means ± SEM of 6 rats per group. *p<0.0001 vs. control, #p<0.0001 vs.
ZL-insulin.
0
5000
10000
15000
20000
25000
30000
35000
Control Exercise Insulin
* * *
IRβ p
rote
in(a
rbitr
ary
units
)
Figure 6.2. Effects of exercise and insulin on IRβ protein abundance in lean (open bar) and
obese (closed bar) Zucker rats. Protein abundance is determined following western blotting of
skeletal muscle extracts with anti-IRβ antibody. Western blots are representative of one rat in
each group. Bars on graph represent means ± SEM of 6 rats per group. *p<0.05 vs. ZL
117
6.3.3 Akt protein expression and serine (473) phosphorylation in muscle of
Zucker rats
ANOVA revealed a main effect for treatment (p<0.0001, Figure 6.3), suggesting that
exercise increased Akt protein abundance when the genotypes are analysed together.
This is probably explained by the 1.7 fold higher protein abundance in the ZL rats
following exercise (Figure 6.4) although the lack of interaction between genotype and
treatment does not allow this interpretation to be made in statistical terms. Akt protein
expression was similar between obese and lean animals (not significant, Figure 6.4).
Figure 6.5 shows that insulin treatment significantly increased pAkt (Ser473) in ZL-
insulin and ZO-insulin skeletal muscle by 9 and 14 fold respectively, compared to the
control groups (p<0.0001, Figure 6.5). pAkt (Ser473) was not significantly altered by
exercise nor was it different in the control groups between genotypes. However pAkt
(Ser473) following insulin treatment was reduced by ~30% in ZO rats compared with
lean littermates (p<0.0001, Figure 6.5), an effect that cannot be explained by differences
in Akt protein expression.
118
control exercise insulin0
2500
5000
7500 *
Akt
pro
tein
abu
ndan
ce(a
rbitr
ary
Uni
ts)
Figure 6.3. Effects of exercise and insulin on Akt protein abundance in lean and obese
Zucker rats. Protein abundance is determined following western blotting of skeletal muscle
extracts with anti-Akt antibody. Bars on graph represent means ± SEM of 12 rats per
intervention (lean and obese rats were combined for each intervention group). *p<0.0001 vs.
control.
119
0
3000
6000
9000
Control Exercise Insulin
Akt
pro
tein
abu
ndan
ce(a
rbitr
ary
units
)
Figure 6.4. Effects of exercise and insulin on Akt protein abundance in lean (open bar) and
obese (closed bar) Zucker rats. Protein abundance is determined following western blotting of
skeletal muscle extracts with anti-Akt antibody. Western blots are representative of one rat in
each group. Bars on graph represent means ± SEM of 6 rats per group.
0
10
20
30
40
Control Exercise Insulin
* *#
pAkt
(Ser
473)
(arb
itrar
y un
its)
Figure 6.5. Insulin treatment but not exercise increases pAkt (Ser473) in lean (open bar)
and obese (closed bar) Zucker rats. Protein abundance is determined following western
blotting of skeletal muscle extracts with anti-pAkt (Ser473) antibody. Western blots are
representative of one rat in each group. Bars on graph represent means ± SEM of 6 rats per
group. *p<0.0001 vs. control, #p<0.0001 vs. ZL-insulin.
120
6.3.4 c-Cbl protein expression and tyrosine phosphorylation in muscle of Zucker
rats
Figure 6.6 shows that c-Cbl is an abundant protein in skeletal muscle. However there
was no significance difference in c-Cbl protein abundance between genotypes. ANOVA
detected a significant treatment effect (p<0.05, Figure 6.7) suggesting that c-Cbl protein
abundance increases 1.5-fold in skeletal muscle in response to supraphysiological
insulin treatment. However tyrosine phosphorylation of c-Cbl was undetectable
(immunoprecipitating Cbl and immunoblotting for tyrosine phosphorylation) between
any of the interventions, despite detecting both immunoprecipitated c-Cbl protein and
tyrosine phosphorylation of the IR in rodent skeletal muscle (Figure 6.8).
121
0
5
10
15
20
25
Control Exercise Insulin
c-C
bl p
rote
inab
unda
nce
(arb
itrar
y un
its)
Figure 6.6. No difference in c-Cbl protein abundance between lean (open bar) and obese
(closed bar) Zucker rats. Skeletal muscle extracts were immunoprecipitated with anti-c-Cbl
antibody and blots were probed for anti-c-Cbl antibody. Western blots are representative of one
rat in each group. Bars on graph represent means ± SEM of 6 rats per group.
Control Exercise Insulin0
5
10
15
20
25*
Cbl
pro
tein
abu
ndan
ce(a
rbitr
ary
units
)
Figure 6.7. Insulin treatment but not exercise increases c-Cbl protein abundance in
Zucker rats. Skeletal muscle extracts were immunoprecipitated with anti-c-Cbl antibody and
blots were probed for anti-c-Cbl antibody. Bars on graph represent means ± SEM of 12 rats per
intervention (lean and obese rats were combined for each intervention group). *p<0.05 vs.
control.
122
IP
lot
Insulin + + + + + + + +
75kD
100kD
150kD
75kD
100kD
150kD
Anti-pY (4G10) Anti-c-Cbl B
IR c-Cbl IR c-Cbl IP
Figure 6.8. c- Cbl is not tyrosine phosphorylated by insulin treatment in rat skeletal
muscle. The skeletal muscle extracts from 2 lean Zucker rats (insulin treated) were prepared and
immunoprecipitated with anti-insulin receptor (IR) or anti-c-Cbl antibodies. The resulting
immunoprecipitates were then subjected to immunoblotting with anti-c-Cbl or anti-
phosphotyrosine (anti-pY 4G10 blot) antibodies. The positions of molecular mass markers (in
kilo Daltons) are indicated on the left. IP: immunoprecipitation. Insulin +: insulin treated.
123
6.3.5 CAP protein expression in 3T3L1 adipocytes and skeletal muscle of
Zucker rats
For determination of CAP protein expression between rat skeletal muscle and 3T3L1
adipocytes, only one experiment was performed due to time and financial constraints.
This provides a qualitative comparison of CAP protein expression between 3T3L1
adipocytes and skeletal muscle of Zucker rats. Figure 6.9 shows the expression of CAP
protein from whole cell lysates prepared from 2 separate dishes of 3T3L1 adipocytes
and skeletal muscle from 3 lean Zucker rats. The 99kD and 125kD isoforms appear
more abundant in 3T3L1 adipocytes compared to rat skeletal muscle.
124
111kD
79kD
61kD
47kD
36kD
3T3L1 20 µ ell
Rat le7 l g/w
skeletal musc5 µg/wel
171kD
Figure 6.9. CAP protein expression appears more abundant in 3T3L1 adipocytes
compared to rat skeletal muscle. Cell lysates (20µg of 3T3L1 adipocytes and 75µg of
rat skeletal muscle total protein per well) were solubilised in Laemmli sample buffer
and subjected to immunoblotting with anti-CAP antibody. The positions of molecular
mass markers (in kilo Daltons) are indicated on the left. The arrows indicate the 2 major
isoforms of CAP (125kD and 99kD) in 3T3L1 adipocytes. Two separate dishes of
3T3L1 adipocytes and skeletal muscle from 3 lean Zucker rats.
125
6.4 Discussion
To determine the role and responsiveness of the c-Cbl pathway, skeletal muscle from
normal and obese insulin resistant Zucker rats was examined under resting conditions,
after insulin-stimulation and immediately following a single bout of exhaustive
swimming exercise. There was no evidence of tyrosine phosphorylation of c-Cbl in the
muscle samples analysed under any of the experimental conditions, despite clearly
demonstrating insulin-stimulation of its receptor and downstream activation of insulin
signalling via Akt phosphorylation. These data suggest that tyrosine phosphorylation of
c-Cbl may not be integral to GLUT4 translocation and activation of glucose transport in
healthy and insulin resistant skeletal muscle. There was however a very small increase
in c-Cbl protein abundance following insulin treatment. Given that the time course of
insulin stimulation was relatively short, these results suggest that insulin may cause the
translocation of c-Cbl out of a cellular compartment not solubilized by the extraction
protocol.
Analysis of the responsiveness of the proximal members of the classical insulin
signalling cascade demonstrated that basal tyrosine phosphorylation of the IR was
elevated in the obese animals under resting conditions. Insulin stimulation resulted in a
significant increase in tyrosine phosphorylation of the IR, and pAkt (Ser473) and these
effects were blunted in obese Zucker rats compared with lean littermates. These results
are in agreement with previous findings in this animal model (Christ et al., 2002; Kim et
al., 2000b; Zhou et al., 1999). A novel finding from the present study was that
intraperitoneal insulin administration failed to induce tyrosine phosphorylation of c-Cbl.
There is evidence that c-Cbl’s role in insulin signalling may be tissue specific. Cell lines
126
such as 3T3L1 fibroblasts do not contain the c-Cbl associating protein, CAP and do not
show the same insulin stimulated tyrosine phosphorylation of c-Cbl as 3T3L1
adipocytes (Ribon et al., 1998) although CAP abundance in skeletal muscle has not
been previously documented. In cell lines expressing high levels of functional insulin
receptors, such as chinese hamster ovary (CHO) cells overexpressing the human IR and
KRC-7 cells insulin treatment does not induce tyrosine phosphorylation of c-Cbl (Ribon
& Saltiel, 1997). This would suggest that in cell lines abundant in functional IR or
lacking CAP, the c-Cbl pathway is not a central component for insulin signalling but
rather an ancillary pathway. It is however difficult to explain the small, significant
increase in c-Cbl protein abundance following a supraphysiological dose of insulin. It is
unlikely that 5 min of insulin treatment in the current study was sufficient time for
increases to occur via protein synthesis. It is possible that the increase in protein
resulted from another storage site within the cell that was not extracted upon cell lysis.
The measurement of c-Cbl abundance and tyrosine phosphorylation from the detergent-
soluble fraction of the cell was chosen for analysis since it has been shown in 3T3L1
adipocytes that c-Cbl is predominantly in the detergent-soluble fraction under basal
conditions and is a major site for its tyrosine phosphorylation following insulin
stimulation (Mastick & Saltiel, 1997; Ribon & Saltiel, 1997). Upon insulin stimulation
c-Cbl is tyrosine phosphorylated in the detergent-soluble fraction as early as 1 min and
for at least 30 min following stimulation (Ribon & Saltiel, 1997). This time course also
coincides with partial translocation of c-Cbl into the detergent-insoluble fraction
(Baumann et al., 2000; Mastick & Saltiel, 1997). The whole cell lysis method employed
in the present study would not extract c-Cbl from the detergent-insoluble fraction of the
cell assuming there was any c-Cbl present in this fraction. Small amounts of c-Cbl have
been reported in the detergent-insoluble fraction of 3T3L1 adipocytes under basal
127
conditions (Mastick & Saltiel, 1997) although the subcellular distribution of c-Cbl has
not been reported in skeletal muscle. It is possible in skeletal muscle that the small
increase measured in c-Cbl protein following insulin treatment could result from release
of c-Cbl from the detergent-insoluble fraction following insulin stimulation. Rodent
studies examining c-Cbl abundance in the various cellular fractions of adipose tissue
and skeletal muscle following insulin treatment may confirm this conjecture.
c-Cbl undergoes activation and tyrosine phosphorylation by a diverse range of cellular
stimuli including shear stress (Miao et al., 2002) and a wide array of cytokines and
growth factors, including hepatocyte growth factor and epidermal growth factor
(Galisteo et al., 1995; Ribon & Saltiel, 1997; Taher et al., 2002). Exercise stimulates the
translocation of GLUT4 to the cell surface, increasing glucose transport via a pathway
that is independent from the insulin-mediated activation of PI 3-kinase (Richter et al.,
1998; Thorell et al., 1999), suggesting a possible role of the alternative c-Cbl pathway.
Therefore the present study also examined whether c-Cbl would undergo tyrosine
phosphorylation in response to an intense bout of prolonged exercise. The exercise
stimuli chosen has been previously shown to result in pronounced reductions in muscle
glycogen concentrations (Bruce et al., 2001), demonstrating the energetic demands of
this exercise mode. However, c-Cbl tyrosine phosphorylation was unaffected by a
single bout of swimming exercise.
An additional finding of the current investigation was that c-Cbl is a readily abundant
protein in rodent skeletal muscle. Given the apparent abundance of this protein and the
lack of subsequent tyrosine phosphorylation to either supra-maximal insulin-
administration or acute, exhaustive exercise the physiological action of c-Cbl in skeletal
128
muscle remains to be elucidated. Considerable interest is focused on the possible
actions of c-Cbl as a negative regulator of growth hormone mediated gene transcription
(Goh et al., 2002) or as a negative regulator of non-receptor tyrosine kinases such by
enhancement of their ubiquitin-dependent degradation (Ota & Samelson, 1997).
Interestingly, despite the many perturbations in cellular homeostasis in the insulin
resistant Zucker rat, c-Cbl protein expression was not altered between genotypes.
There was also a significant increase in Akt protein abundance following exercise,
which could largely be attributed to a 1.7-fold increase in Akt protein in the lean group.
This finding is unusual since there is no published data to support this observation. It is
known that pAkt (Ser473 and Thr308) increases transiently during electrical stimulation
of short duration in rodent muscle and decreases back to basal levels by 15 min of
stimulation (Sakamoto et al., 2002) without any increases in Akt protein abundance.
Further research is required to investigate the physiological significance of this finding.
This study also sought to compare the protein abundance of CAP in rodent skeletal
muscle with its expression in 3T3L1 adipocytes, since CAP expression is involved in
the insulin-stimulated tyrosine phosphorylation of c-Cbl in 3T3L1 adipocytes
(Baumann et al., 2000). The major isoforms of CAP in 3T3L1 adipocytes occurs at
99kD and 125kD, whilst other isoforms or spliced variants occur at 75, 53 and 45kD
(Ribon et al., 1998). It would appear from this preliminary investigation that the 99 and
125kD isoforms of CAP are much more abundant in 3T3L1 adipocytes compared to rat
skeletal muscle, despite approximately 375% more total protein from the skeletal
muscle lysates being loaded per well. This particular analysis has obvious limitations
since due to time and financial constraints; the results are from one experiment. This
129
experiment would need to be repeated several times to allow for a thorough statistical
examination of the data compared to the qualitative findings presented here.
Furthermore, the lysis of the 3T3L1 adipocytes was performed with a slightly different
buffer, which may have altered the efficiency of CAP extraction between the two cell
types although the total protein assay would have partially controlled for this.
Nevertheless, this is the first reported measurement of CAP protein expression in
skeletal muscle, although CAP protein has previously been reported to be abundant in
adipose tissue of the same animals (Ribon et al., 1998). Further investigation comparing
CAP protein abundance between adipose tissue and skeletal muscle would help to
explain the role of the c-Cbl pathway in insulin responsive tissue. It has been shown
previously that a lack of CAP protein expression in 3T3L1 fibroblasts is associated with
the lack of c-Cbl tyrosine phosphorylation in response to insulin stimulation (Ribon et
al., 1998). However the qualitative findings of the present study suggest that CAP may
not be an abundant protein in skeletal muscle and this may explain why there is no
tyrosine phosphorylation of c-Cbl in this tissue in response to insulin stimulation.
6.5 Conclusion
In summary, this is the first study to report that c-Cbl is a readily abundant protein in
both healthy and insulin resistant rodent skeletal muscle. The tyrosine phosphorylation
of c-Cbl in response to exercise or insulin treatment was undetectable using the
protocols employed in the present study despite detecting both immunoprecipitated c-
Cbl protein and the tyrosine phosphorylation of the IR in rodent skeletal muscle.
Therefore, unlike 3T3L1 adipocytes, 5 minutes of insulin stimulation in rodent skeletal
130
muscle does not appear to tyrosine phosphorylate c-Cbl. Furthermore, exercise does not
seem to have an effect on either c-Cbl protein abundance or tyrosine phosphorylation
but there was however a small increase in protein abundance following
supraphysiological insulin treatment. Based on the limited findings of the present study,
it appears as if CAP protein is much more readily expressed in 3T3L1 adipocytes
compared to skeletal muscle. Insufficient abundance of CAP protein in skeletal muscle
may explain why insulin stimulation does not tyrosine phosphorylate c-Cbl in this tissue
as it has been shown previously by others that CAP protein expression is necessary for
the insulin stimulated tyrosine phosphorylation of c-Cbl. Finally, insulin resistant states
such as obesity do not appear to result in differential expression of c-Cbl protein in the
Zucker rat, which may suggest that it is not a defect associated with insulin resistance in
skeletal muscle.
131
CHAPTER SEVEN
CONCLUSION
7.1 Conclusions
Regular physical activity is an effective therapy for the treatment and prevention of type
2 diabetes. Importantly, physical activity improves insulin sensitivity although there is a
paucity of data examining the intracellular mechanisms that account for this improved
insulin action that proceed greater muscular contractile activity. Understanding how
exercise improves insulin action will not only improve diabetic care and management,
but also lead to greater insight into future effective therapies for the prevention and
treatment of this and other metabolic diseases. This thesis investigated several aspects
involved in the regulation of the insulin-signalling cascade by exercise and sought to
clarify the benefits of exercise training on improved insulin action. The thesis also
sought to investigate if a novel insulin signalling pathway that has been reported in
adipocytes was also involved in the insulin signalling of skeletal muscle.
The residual effect of the last bout of exercise alone has often been attributed to the
observed improvements in insulin action following endurance training. A finding of this
thesis was that 24 hours following a single bout of endurance exercise insulin action
was improved although larger improvements in insulin action were found following the
completion of endurance training. This would imply that increased insulin action is not
only due to the residual effects of the prior bout of exercise, but that cumulative bouts
of exercise provide further enhancement of this response.
132
Little was previously known of the transcriptional regulation of key insulin signalling
proteins by exercise. This thesis found a significant transient increase in IRS-2 mRNA
concentration in the few hours following a single bout of both endurance and resistance
exercise. While endurance and resistance exercise is not directly comparable, the data
from this thesis demonstrates that resistance exercise acts to elicit some alterations in
some of the insulin signalling genes. The recent findings of increased insulin action
following resistance training (Dunstan et al., 2002) suggest the involvement of as yet
unknown mechanisms. The gene expression of the p85α subunit of PI 3-kinase also
increased significantly in the few hours following a single bout of endurance but not
resistance exercise. This effect was attenuated following several days of endurance
training. A single bout of exercise or short-term endurance training also failed to alter
the gene expression of either the IR or IRS-1 genes providing little evidence of
sustained and substantial alterations in the gene expression of most of the key proteins
of the insulin-signalling pathway. Surprisingly, IRS-2 protein abundance was however
observed to significantly increase 24 hrs following a single bout of endurance exercise
which then decreased back to sedentary levels following short term endurance training
indicating transcriptional regulation of this protein via intense muscular contraction.
IRS-2 protein abundance was also observed to increase significantly in the sedentary
state following prolonged insulin stimulation. The transient nature of this regulation
following prolonged insulin stimulation or a single bout of exercise but not cumulative
bouts of exercise indicates an early adaptive role for IRS-2 in both insulin stimulated
glucose uptake and mitogenesis. Recent work has focussed on the unique actions and
roles for IRS-2 in skeletal muscle (Chibalin et al., 2000; Howlett et al., 2002) although
the exact physiological function is still to be determined.
133
This thesis found that there was no effect of a single bout of exercise or short-term
endurance training on proximal components of the insulin-signalling cascade such as IR
and IRS-1 protein expression or tyrosine phosphorylation. However a major finding of
this thesis was that there was an observed increase in the serine phosphorylation of a
known downstream signalling protein, Akt, following both an acute bout of exercise
and with exercise training. This finding suggests a role for Akt in the upregulation of
glucose transport following exercise. However, Akt has been shown by other to have
multiple signalling roles, including glycogen synthesis (Cross et al., 1995; Cross et al.,
1997; Ueki et al., 1998) and mitogenic signalling (Hajduch et al., 1998; Ueki et al.,
1998) and the relative contribution of Akt to all these pathways following exercise
cannot be determined from the present study.
Protein tyrosine phosphatases (PTPases) are a diverse family of enzymes that have
generated considerable interest as novel targets for the treatment of type 2 diabetes and
are currently regarded as key regulators of the insulin-signalling cascade. Readily
abundant PTPases in skeletal muscle and adipocytes each display characteristic
subcellular distribution between the cytosol and particulate fractions (Ahmad &
Goldstein, 1995; Calera et al., 2000) and are therefore likely to have preferential sites of
action throughout the insulin-signalling cascade. The major finding of this thesis was
that exercise selectively increases the protein expression of SHPTP2 in human skeletal
muscle. This is the first study to report of an increase in a positive regulator of insulin
signalling in vivo following exercise in humans. The increase in SHPTP2 protein
abundance at a major site of PTPase activity, the cytosolic fraction, in skeletal muscle
following exercise was associated with an increase in pAkt (Ser473); an enzyme that is
activated downstream of PI 3-kinase by insulin. This finding has important
134
implications in the search for alternative therapies to improve insulin sensitivity in
patients with insulin resistance. Inhibiting SHPTP2 in rat adipocytes and transgenic
mice also reduces insulin-stimulated glucose uptake, translocation of GLUT4 and
impairs activation of IRS-1, PI 3-kinase and Akt-1 (Chen et al., 1997; Maegawa et al.,
1999). If increased SHPTP2 protein abundance is directly involved in insulin-stimulated
glucose transport then it provides another target for the pharmacological treatment of
type 2 diabetes. And even if SHPTP2 is not directly responsible for improved insulin-
stimulated glucose transport following exercise then it at least highlights a poorly
described section of the insulin signalling cascade potentially involved in the
upregulation of insulin sensitivity by exercise. There was however, no observed
improvement in IRS-1 tyrosine phosphorylation following exercise, which is reported to
be a major target for the action of SHPTP2 suggesting that it may also be involved in
other actions such as mitogenesis and glycogen synthesis (Maegawa et al., 1999; Ugi et
al., 1996) although there is no direct evidence for this in the present study. It is possible
that SHPTP2 is mediating its effects on insulin-stimulated glucose transport
independently of IRS-1. It was discussed in chapter 4 that SHPTP2 could be signalling
to downstream components of the cascade, such as Akt, via other tyrosine kinases like
Src, although the evidence for this is far from conclusive. The protein expression of
another prominent PTPase, PTP1B, was not altered by acute exercise or exercise
training in either the particulate or cytosolic fractions, nor was the insulin-stimulated
tyrosine phosphorylation of the IR, which is one of its main targets for
dephosphorylation. This would suggest that any improvements in insulin action via
exercise are not mediated by PTP1B and do not involve alterations in the abundance
and tyrosine phosphorylation of proximal members of the insulin signalling such as IR
and IRS-1.
135
A novel insulin-signalling pathway involving c-Cbl tyrosine phosphorylation is
essential for insulin stimulated glucose transport in adipocytes although the role of this
pathway in skeletal muscle, the primary site for glucose disposal, is unknown. This
thesis found no evidence of tyrosine phosphorylation of c-Cbl in the skeletal muscle of
Zucker rats by insulin treatment or exercise despite demonstrating significant
phosphorylation of the insulin receptor and Akt by insulin treatment and successful
immunoprecipitation of c-Cbl protein. There was also no differential expression of c-
Cbl protein between lean and obese Zucker rats, suggesting that c-Cbl tyrosine
phosphorylation is not associated with insulin or exercise-mediated stimulation of
glucose transport in skeletal muscle nor does it participate in insulin resistance in
skeletal muscle. Surprisingly, there was a small but significant increase in c-Cbl protein
expression following insulin-stimulation. It is possible in skeletal muscle that the small
increase in c-Cbl protein observed following insulin treatment could result from release
of c-Cbl from the detergent-insoluble fraction following insulin stimulation, as the
whole-cell lysis method employed in the present study would not extract c-Cbl from the
detergent-insoluble fraction of the cell.
In summary, this thesis evaluated several key aspects of insulin signalling in human
skeletal muscle. This work provided new insights into the insulin signalling cascade and
response to exercise. Cumulative bouts of exercise were found to increase insulin action
above that of a single bout, debunking the commonly held belief that insulin sensitivity
is primarily due to the residual effects of a single bout of prior exercise. The thesis
found that there is a transient upregulation of IRS-2 gene expression and protein
136
abundance following acute exercise indicating a role for this protein in insulin-
stimulated glucose transport and mitogenesis. There was no observed increase in the
protein expression or tyrosine phosphorylation of IR and IRS-1 following acute exercise
or short-term training. This would suggest that proximal members of the insulin-
signalling cascade such as IR and IRS-1 are not involved in the enhanced insulin-
stimulated glucose uptake observed following exercise in human skeletal muscle. A
major finding of this thesis was the observed increase in serine phosphorylation of a
known downstream signalling protein, Akt, in human skeletal muscle following an
acute bout of exercise and exercise training. It is unclear just how exercise is able to
increase pAkt (Ser473) however it does suggest that downstream components of the
insulin signalling cascade may be involved in enhanced insulin-stimulated glucose
uptake. The thesis found that a poorly described section of the insulin signalling cascade
involving SHPTP2 in the cytosolic fraction of the cell is increased following exercise. It
is unclear if the increased cytosolic SHPTP2 protein abundance is directly related to
observed increase in pAkt (Ser473) following exercise although the findings from this
thesis warrant further investigation into this area. This thesis also found that a novel
insulin-signalling pathway involving c-Cbl tyrosine phosphorylation is not involved
with insulin or exercise mediated stimulation of glucose transport in rodent skeletal
muscle, although insulin treatment may be involved in c-Cbl translocation.
7.2 Future Directions
There are many unresolved issues surrounding the actions of acute and repeated
exercise on insulin action. Emerging insights into the control, regulation and
downstream signalling components of the insulin-signalling cascade will make this an
interesting area of on-going research. The results obtained from the present study
137
indicate that many unresolved issues remain with respect to some of the aspects of the
insulin-signalling cascade and the impact of exercise on insulin signalling.
1. The present investigation observed an increase in SHPTP2 protein abundance
and pAkt (Ser473) following acute exercise and exercise training. It is unclear
based on the present findings if these changes are directly related. As mentioned
earlier, transgenic mouse models involving SHPTP2 inhibition result in
decreased Akt activity and insulin-stimulated glucose transport. However a
direct link between SHPTP2 and Akt has yet to be established, although as
mentioned in Chapter 4 there is some indirect evidence suggesting SHPTP2
activates Akt via the tyrosine kinase Src following EGF stimulation. It is
unknown if Src is activated following muscular contraction or if there is an
increased association of Src with SHPTP2 following exercise. It is also
unknown if this increased association then increases the binding of Src with the
regulatory region of Akt, which subsequently increases Akt activation or
phosphorylation. Further work would also be required to show the interaction of
this pathway following insulin stimulation.
2. The finding that SHPTP2 protein abundance and pAkt (Ser473) are increased by
a single bout of endurance exercise and endurance training with concomitant
improvements in whole body insulin action is not sufficient to conclude that
these intracellular changes are directly involved in improved insulin-stimulated
glucose transport. Both SHPTP2 and Akt have been implicated in insulin
stimulated glucose transport, glycogen synthesis and mitogenic signalling
(Brozinick Jr. & Birnbaum, 1998; Cross et al., 1995; Cross et al., 1997;
138
Maegawa et al., 1999; Ueki et al., 1998; Ugi et al., 1996). It is possible that
upregulation of SHPTP2 protein abundance and pAKT (Ser473) are involved in
a combination of these pathways in response to exercise. Further research needs
to measure changes in insulin-stimulated glycogen synthase, MAP kinase and
p70 S6 kinase activity following acute exercise and short-term training. This
would help to establish if an upregulation of these pathways are mediated by
increased cytosolic SHPTP2 protein abundance and/or Akt activity/
phosphorylation.
3. Two of the predominant PTPases in skeletal muscle were investigated for their
role in the regulation of insulin signalling by exercise. There are a number of
other PTPases thought to be involved in the regulation of insulin signalling that
were not investigated due to time and financial constraints. The PTPase LAR is
a highly expressed in skeletal muscle and thought to play a major role in the
regulation of the insulin receptor. It is unlikely that LAR is involved in the
regulation of insulin signalling by exercise since no changes were observed in
the insulin-stimulated tyrosine phosphorylation of the insulin receptor following
acute exercise or exercise training. Furthermore, lipid phosphatases such as
PTEN and SHIP2 that were briefly mentioned in Chapter 1 are thought to act
downstream of IRS-1 and may potentially be involved in the regulation of
insulin signalling by exercise.
4. Although the insulin-signalling pathway proximal to PI 3-kinase is well
described, the hunt is on worldwide to discover the downstream components of
the insulin-signalling cascade that ultimately lead to GLUT4 translocation and
139
glucose uptake. It has been discussed briefly in Chapter 1 that PDK-1, Akt and
the atypical PKC’s (PKCλ and PKCζ) are all thought to be involved in insulin-
stimulated GLUT4 translocation. The upregulation of insulin stimulated glucose
uptake by exercise could very well occur in the insulin signalling pathway distal
to PI 3-kinase as this thesis found in support of the majority of previously
published work no evidence for improvements in the proximal section of the
pathway involving the insulin receptor or IRS-1. There is good evidence as
discussed in Chapter 1, that Akt and atypical PKC activation is downregulated
in insulin resistant muscle brought about by fatty acid infusion or high fat
feeding (Kim et al., 2002; Tremblay et al., 2001). Future research should
examine if exercise induced improvements in insulin stimulated glucose uptake
occur via upregulation of these atypical PKC’s. There is also some recent
evidence that insulin-stimulated p38 MAPK is upregulated in the few hours
following endurance exercise in humans (Thong et al., 2003), suggesting a role
for this pathway in the exercise induced improvement of insulin sensitivity.
5. It has been found that in the few hours following resistance and endurance
exercise, there is a transient increase in IRS-2 mRNA concentration. The
resistance exercise study is limited in its findings as subjects were fed a small
meal immediately after exercise, which may have had an effect on the gene
expression of IRS-2. Controlling for food intake on IRS-2 gene expression after
exercise therefore warrants further investigation. However gene expression of
IRS-1 and the p85α subunit of PI 3-kinase did not change during this period and
there were no changes in IRS-2 gene expression in the longer term. Also, similar
transient increases were observed following endurance exercise despite no food
140
being consumed. Therefore the results of the present study do justify increasing
the scope of the investigation to determine if resistance exercise is able to
increase IRS-2 protein content in the 24 hour period post exercise, and if IRS-2
associated PI 3-kinase activity is also improved during this period, which may in
part explain small increases in insulin sensitivity observed following resistance
exercise. As discussed earlier, IRS-2 potentially plays a role in insulin-
stimulated glucose uptake and muscle fibre growth in skeletal muscle (Howlett
et al., 2002; Kido et al., 2000; Kubota et al., 2000; Rondinone et al., 1997;
Withers et al., 1999) although its exact role in glucose uptake and mitogenesis
following acute exercise is still unclear. The finding that IRS-2 gene expression
is upregulated following a single bout of endurance and resistance exercise may
point towards an early adaptive role for IRS-2 in the combined actions of
glucose uptake and mitogenesis following very different types of muscle
contraction. Further study investigating IRS-2 protein abundance and basal and
insulin-stimulated tyrosine phosphorylation following acute resistance and
endurance exercise will help to resolve the physiological role/s of IRS-2
following intense muscular contraction.
6. Although it appears that insulin or exercise do not tyrosine phosphorylate c-Cbl
in skeletal muscle there does appear to be tissue specific role for c-Cbl in insulin
stimulated glucose transport. A study examining if insulin stimulates tyrosine
phosphorylation of c-Cbl in adipose tissue but not skeletal muscle of rodents is
required to confirm this. CAP expression appears to be crucial in the tyrosine
phosphorylation and subsequent recruitment of c-Cbl to the insulin receptor and
translocation to lipid rafts. Preliminary evidence was presented demonstrating
141
that CAP is expressed more abundantly in 3T3L1 adipocytes compared to
skeletal muscle. Therefore the relative abundance of CAP in both skeletal
muscle and adipose tissue of rodents would help to explain any tissue specific
differences that may exist for this pathway.
7.3 Concluding statement
Many grey and unknown areas exist in the hunt to determine the cellular basis for the
beneficial actions of exercise on insulin action. This research area will undoubtedly
provide many key insights into the malfunction in insulin resistant states. However, the
immutable fact remains that even in young and relatively fit male subjects, as
investigated in the current study, exercise is a necessary and central component
involved in the optimal regulation of insulin signalling transduction.
142
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