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EXERCISE, MUSCLE GLYCOGEN
AND INSULIN ACTION
Brett Robert Johnstone
B. App. Sci. (Hons)
This thesis is submitted in fulfillment of the degree of Doctor of Philosophy
Supervisor: Prof. Mark Hargreaves, B.Sc., M.A., PhD
Deakin University
September 2004
DEAKIN UNIVERSITY
CANDIDATE DECLARATION
I certify that the thesis entitled:
Exercise, muscle glycogen and insulin action
submitted for the degree of:
Doctor of Philosophy
is the result of my own work and that where reference is made to the work of others,
due acknowledgment is given. I also certify that any material in the thesis which has
been accepted for a degree or diploma by any other university or institution is
identified in the text.
Full Name: Brett Robert Johnstone
Signed .............................................
Date..................................................
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ACKNOWLEGEMENTS
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To Professor Mark Hargreaves for his confidence in allowing me to take on this
project and for his invaluable support and expertise throughout my candidature.
To Doctor Rod Snow for his statistical advice and intellectual input.
To the School of Exercise and Nutrition Sciences (Deakin University) for
providing the opportunity to undertake this research, and for support and resources
throughout the years.
To those who volunteered to be subjects in this series of studies. Without their
dedication and reliability this thesis would not have been possible.
To the Post Graduate students of the School of Health Sciences for their
companionship and support over the course of my candidature.
To Adam Rose and Sean McGee for their assistance with testing and continuing
patience in the face of a bombardment of PKC and p38 analysis questions.
To Glen Wadley, Professor Erik Richter and Doctor Andrew Garnham for their
assistance in setting up and running the hyperinsulinaemic / euglycaemic clamp
technique.
To my friends and family for their ongoing support of my endeavours as a
“professional student”.
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ABBREVIATIONS AICAR 5-amino-4-imidazolecarboxamide riboside
AMP Adenosine monophosphate
AMPK 5'-AMP-activated protein kinase
ANOVA Analysis of Variance
aPKC Atypical PKC
ASIP Atypical PKC isotope-specific interacting protein
ATP Adenosine triphosphate
AUC Area under the curve
cAMP Adenylate cyclase-adenosine 3’, 5’- cyclic monophosphate
CAP c-Cbl associated protein
CP Creatine phosphate
cPKC Conventional PKC
Cr Creatine
DAG Diacylglycerol
EGF Epidermal growth factor
eNOS Endothelial nitric oxide synthase
ERK Extracellular signal-regulated kinase
FFA Free fatty acids
G-6-P Glucose-6-phosphate
GIR Glucose infusion rate
GSK-3 Glycogen synthase kinase-3
IDDM Insulin dependent diabetes mellitus
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IRS-1 Insulin receptor substrate 1
IRS-2 Insulin receptor substrate 2
JNK c-jun NH2-terminal kinase
MAPK Mitogen Activated Protein kinase
MAPKK / MEK Mitogen activated protein kinase kinase
NIDDM Non insulin dependent diabetes mellitus
nNOS Neuronal nitric oxide synthase
NO Nitric oxide
NOS Nitric oxide synthase
nPKC Novel PKC
PDGF Platelet derived growth factor
PDK1 3-phosphoinositide dependent protein kinase
PFK Phosphofructokinase
PI 3-kinase Phospatidylinositiol 3-kinase
PKB Protein Kinase B
PKC Protein kinase C
PLD Phospholipase D
PTB Phosphotyrosine binding construct
RER Respiratory exchange ratio
SAPK Stress activated protein kinases
SNP Sodium nitroprusside
TCr Total creatine
2OV& peak Peak oxygen consumption
2-DG 2-deoxyglucose 6
TABLE OF CONTENTS
CANDIDATE DECLARATION.................................................................................2
ACKNOWLEGEMENTS............................................................................................3
ABBREVIATIONS ......................................................................................................5
ABSTRACT ..................................................................................................................9
CHAPTER 1: INTRODUCTION
1.1 Overview ............................................................................................................11
1.2 Regulation of skeletal muscle glucose uptake....................................................13
1.2.1 Hormonal regulation of glucose uptake ......................................................13
1.2.2 Substrate Availability ..................................................................................15
1.2.3 Glut-4 Translocation....................................................................................18
1.2.3.1 Insulin signalling of muscle glucose uptake.............................................19
1.2.3.2 Contraction signalling of muscle glucose uptake.....................................28
1.3 Mechanisms of enhanced insulin action following exercise ..............................41
1.4 Role of glycogen in the regulation of skeletal muscle glucose uptake ..............46
1.5 Aims of the thesis ...............................................................................................53
CHAPTER 2: GENERAL METHODOLOGY
2.1 Subjects. .............................................................................................................55
2.2 Pre Experimental Procedures. ............................................................................56
2.2.1 Peak Oxygen Consumption. ........................................................................56
2.3 Experimental procedures....................................................................................57
2.3.1 Percutaneous muscle biopsy........................................................................57
2.3.2 Hyperinsulinaemic / euglycaemic clamp ....................................................57
2.3.3 Blood sampling and treatment.....................................................................58
2.4 Data Collection and Analysis .............................................................................59
2.4.1 Collection of Data .......................................................................................59
2.4.2 Analytic Methods ........................................................................................59
2.4.3 Statistical Analysis ......................................................................................63
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CHAPTER 3: INFLUENCE OF MUSCLE GLYCOGEN ON POST-EXERCISE
INSULIN STIMULATED GLUCOSE DISPOSAL AND INSULIN
SIGNALLING IN HUMAN SKELETAL MUSCLE
3.1 Introduction ........................................................................................................65
3.2 Materials and methods .......................................................................................68
3.3 Results ................................................................................................................70
3.4 Discussion ..........................................................................................................82
CHAPTER 4: INFLUENCE OF MUSCLE GLYCOGEN CONCENTRATION
ON WHOLE BODY INSULIN STIMULATED GLUCOSE UPTAKE 24 HRS
AFTER EXERCISE
4.1 Introduction ........................................................................................................95
4.2 Materials and methods .......................................................................................97
4.3 Results ..............................................................................................................101
4.4 Discussion ........................................................................................................116
CHAPTER 5: OVERVIEW AND DISCUSSION
5.1 Overview ..........................................................................................................126
5.2 Conclusions ......................................................................................................133
REFERENCES.........................................................................................................134
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ABSTRACT
The aim of this thesis was to investigate the influence of muscle glycogen
concentration on whole body insulin stimulated glucose uptake in humans and to
examine the potential signalling mechanisms responsible for enhanced insulin action
in the post exercise period. Untrained male subjects were conditioned to achieve a
range of muscle glycogen concentrations via acute exercise or a combination of
exercise and diet. The influence of muscle glycogen content on whole body insulin
stimulated glucose uptake was determined via hyperinsulinaemic / euglycaemic
clamps conducted at rest, 30 min after exercise or 24 hours after exercise. Muscle
glycogen content did not influence insulin mediated glucose disposal either 30 min or
24 hrs after exercise when compared with basal. Conventional insulin signalling to
muscle glucose uptake and signalling through the p38 MAPK cascade was also
largely unaltered by glycogen concentration. Muscle glycogen synthesis was
significantly increased in heavily but not moderately glycogen depleted muscle 30
min after exercise. Enhanced muscle glycogen synthesis occurred in line with a
significant increase in insulin stimulated GSK-3 serine phosphorylation. This finding
suggests that enhanced insulin sensitivity of muscle glycogen synthesis following
glycogen depleting exercise may be mediated via a pathway involving alterations in
insulin stimulated GSK-3 phosphorylation. In summary, whilst glycogen influences
insulin mediated GSK-3 phosphorylation and glycogen synthesis, the findings of the
present series of investigations suggest that the role of muscle glycogen in the process
of insulin stimulated glucose uptake may not be as important as previously theorised.
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CHAPTER 1
❖
INTRODUCTION 1.1 Overview The ability of the hormone insulin to stimulate glucose uptake into skeletal muscle
and adipose tissue is vital in the maintenance of glucose homeostasis in the
postprandial period. Insulin is secreted into the blood stream by pancreatic beta cells
in response to increased blood glucose concentrations, and results in enhanced glucose
entry into muscle cells via the action of the Glut-4 facilitative glucose transporter.
Translocation of Glut-4 from the intracellular membrane compartment to the cell
surface is the major mechanism by which insulin stimulates glucose transport into
skeletal muscle. Whilst the precise signalling mechanisms are unknown, the process
of insulin stimulated Glut-4 translocation is thought to involve activation of
phosphatidylinositiol 3-kinase (PI 3-kinase), protein kinase B (PKB) and protein
kinase C (PKC) at a proximal and possibly phospholipase D (PLD) at a more distal
step (Wojtaszewski, Nielsen et al. 2002). In addition to its role in the process of
glucose uptake into cells, insulin also promotes the storage of glucose as glycogen in
skeletal muscle. These actions, when combined with the ability of insulin to inhibit
glucose production and release from the liver, result in the effective regulation of
blood glucose levels in healthy individuals. Impairment of the metabolic effect of
insulin is associated with the development of non-insulin-dependent diabetes mellitus
(NIDDM) and a wide range of atherosclerotic risk factors including obesity,
11
dislipidaemia and hypertension (Eriksson, Taimela et al. 1997). Accordingly,
strategies aimed at improving insulin action may lead to new therapies for the
treatment and prevention of NIDDM. Physical activity represents one of the most
practical means of improving insulin sensitivity in patients with impaired insulin
action. A single bout of exercise has consistently been shown to improve the
metabolic effect of insulin in the post exercise period, however, the molecular
mechanisms responsible for this adaptation remain elusive. Recent evidence suggests
that post exercise sensitisation to insulin may not be related to activation of the
proximal pathway of insulin signalling intermediates (Wojtaszewski, Hansen et al.
1997; Wojtaszewski, Hansen et al. 2000). These and other findings have led to
substantial research interest in elucidating the mechanisms responsible for enhanced
insulin action following exercise. Several authors have suggested that the mechanism
responsible for post exercise adaptations in insulin action may be dependent upon
muscle glycogen concentrations (Richter, Garetto et al. 1982; Kawanaka, Han et al.
1999; Derave, Hansen et al. 2000). In spite of these findings, the effect of glycogen
on insulin mediated glucose disposal in humans remains equivocal. The molecular
mechanism by which glycogen may influence the action of insulin is unknown,
however, recent investigation suggests that the p38 MAPK cascade and distal insulin
signalling events may be involved in post exercise sensitisation to insulin. The aim of
this thesis was to investigate the effect of muscle glycogen concentration on insulin
stimulated glucose uptake in humans and to examine the potential signalling
mechanisms responsible for enhanced insulin action in the post exercise period.
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1.2 Regulation of skeletal muscle glucose uptake The ability to transport glucose across the plasma membrane is a feature common to
nearly all cell types. In human cells, glucose is required for adenosine triphosphate
(ATP) production via oxidative and non-oxidative metabolism and thus is vital for a
wide range of physiological processes. Impaired glucose uptake is central to the
pathogenesis of non-insulin-dependent diabetes mellitus and is associated with a wide
variety of atherosclerotic risk factors (Eriksson, Taimela et al. 1997). Glucose is also
an important energy source during prolonged exercise and fatigue is often associated
with hypoglycaemia (Coggan and Coyle 1987). Understanding the factors which
regulate glucose uptake and utilisation is therefore important in the prevention of
serious disease and in the maintenance of contractile activity during exercise.
1.2.1 Hormonal regulation of glucose uptake
Insulin and glucagon are the major hormones secreted by the pancreas and have
important but opposing regulatory effects on glucose metabolism. Insulin has a potent
hypoglycaemic effect, and facilitates the entry of glucose into cells. Muscle cells and
adipocytes have been termed the insulin sensitive cell types because they respond to
insulin with a rapid and reversible increase in glucose transport (Mueckler 1994). In
these cells, the effect of insulin is mediated via a cascade of signalling events, which
result in an increase in glucose transport activity at the cell membrane (see 1.2.3).
The importance of insulin in the regulation of glucose uptake is perhaps most
graphically illustrated by the observation that patients who are insulin deficient (type
one diabetes) or insulin resistant (type two diabetes) exhibit severely restricted
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glucose entry into muscle and adipose tissue (Kahn 1996). Whilst insulin is essential
in the regulation of basal glucose metabolism, numerous studies have demonstrated
that muscle contractions stimulate glucose uptake independent of insulin. Despite
these findings, contraction stimulated increases in glucose uptake are greater in the
presence rather than in the absence of insulin (see 1.2.3). In direct opposition to
insulin, glucagon functions to increase blood glucose concentrations via an increase in
liver glycogenolysis and gluconeogenesis. This action is important in maintaining
adequate levels of glucose for vital cell functions in the brain, particularly in instances
of starvation or excessive glucose utilisation during exercise. Under conditions of
severe hypoglycemia, stimulation of the sympathetic nervous system may cause an
increase in epinephrine which further stimulates liver glucose release and inhibits
glucose uptake. In circumstances where hypoglycemia is maintained for many days,
growth hormone and cortisol secretion lead to a decrease in glucose utilisation by
most cells and a concomitant increase in FFA oxidation.
Whilst the effects of insulin and glucagon on glucose metabolism are well established,
the role of adrenaline is less clear. Under resting conditions, an apparent stimulatory
effect of adrenergic mechanisms on Glut-4 expression has been observed. In spite of
this observation, an increase in glucose uptake in the above study was not apparent,
suggesting that the intrinsic activity of the transporter may have been inhibited
(Bonen, Megeney et al. 1992). Recent research suggests that adrenaline can
translocate Glut-4 while at the same time increasing glucose transport when insulin is
absent, or can inhibit glucose transport when insulin is present (Han and Bonen 1998).
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Adrenaline infusion has also been shown to decrease glucose uptake by contracting
forearm (Jansson, Hjemdahl et al. 1986) and leg (Hartling and Trap-Jensen 1982)
muscle. One possibility is that adrenaline induces an increase in adipose tissue
lipolysis which subsequently reduces glucose utilisation. An adrenaline mediated
increase in glycogenolysis may also exert an inhibitory affect on glucose uptake by
increasing G-6-P and subsequently impairing glucose phosphorylation (see 1.2.3).
1.2.2 Substrate Availability
In resting skeletal muscle, glucose utilisation is low with the major contribution to
energy provision occurring via the oxidation of fats. Despite this fact, small changes
in plasma glucose concentration within the physiological range in humans (~3.5 – 7.0
mM) have been shown to result in pronounced increases in resting glucose uptake
(Richter 1996). During exercise, whole body glucose utilisation is increased (Kjaer,
Farrell et al. 1986) largely as a result of increased blood flow (Katz, Broberg et al.
1986) and increased glucose utilisation by the contracting muscles (Kjaer, Kiens et al.
1991). At low to moderate exercise intensities, increased glucose availability results
in enhanced muscle glucose uptake in dogs and in humans (Richter 1996), whilst
reduced glucose availability in the latter stages of prolonged exercise has been shown
to lead to a reduction in glucose uptake (Wolfe, Klein et al. 1988). As exercise
intensity increases, glucose uptake is enhanced (Katz, Broberg et al. 1986), except at
the onset of high intensity exercise where increased glycogenolysis may cause a
transient reversal of the transmembrane glucose gradient.
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Almost four decades ago, the existence of a “glucose free fatty acid cycle” was
proposed whereby increased FFA availability caused enhanced FFA uptake and
oxidation and subsequent citrate mediated inhibition of PFK and glycolysis (Randle,
Garland et al. 1965). Under this proposal, accumulation of G-6-P causes hexokinase
mediated inhibition of glucose phosphorylation and glucose uptake. Whilst research
in this area has produced conflicting results, it is now generally accepted that elevated
plasma FFA concentrations impair resting carbohydrate utilisation, and may under
some conditions decrease muscle glucose uptake and glycogenolysis during exercise.
For a comprehensive examination of this topic the readers attention is directed to
recent reviews (Richter 1996; Zierler 1999). Increased plasma FFA are also
intimately related to peripheral insulin resistance and type 2 diabetes as a result of
their ability to impair insulin mediated glucose uptake and glycogen synthesis (Boden
2003). However, a number of recent observations suggest that operation of the
classical “glucose free fatty acid cycle” may not be responsible for the observed effect
of elevated plasma FFA concentrations on resting and contraction mediated glucose
uptake and glycogen synthesis. During a recent study, increased FFA availability
during high intensity exercise did not influence muscle citrate, G-6-P and FFA
oxidation, leading the authors to suggest direct inhibition of glucose uptake by FFA
levels rather than the operation of the classic “glucose-free fatty acid cycle”
(Hargreaves, Kiens et al. 1991). Furthermore, during hyperinsulinaemic / euglycaemic
clamp conditions at rest, elevation of FFA levels results in a 50% reduction in muscle
glycogen synthesis and whole body glucose oxidation in concert with a reduction in
G-6-P concentration (Roden, Price et al. 1996). This finding is inconsistent with
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impairment of insulin action via the Randle hypothesis which proposes increased G-6-
P levels resulting from the inhibitory effects of FFA on PFK. These findings have
been interpreted as suggesting that elevated plasma FFA concentrations induce insulin
resistance by inhibiting glucose transport or phosphorylation activity, and provide
evidence against the operation of the “glucose-free fatty acid cycle” (Shulman 2000).
A number of studies have investigated whether augmented FFA concentrations
directly influence the Glut-4 transporter, or alter signalling mechanisms involved in
Glut-4 translocation. Insulin stimulated IRS-1 associated PI 3-kinase activity was
recently shown to be impaired when plasma FFA concentrations were increased via
lipid infusion (Dresner, Laurent et al. 1999). It has been further suggested that the
mechanism via which elevated FFA levels cause insulin resistance in humans and rats
may be secondary to impairment of PI 3-kinase activity and associated with
accumulation of diacylglycerol and activation of protein kinase C (PKC) in skeletal
muscle (Griffin, Marcucci et al. 1999; Itani, Ruderman et al. 2002). In support of this
possibility, PKC isoforms have also been shown to be altered when insulin resistance
was induced via by high fat feeding in rats (Schmitz-Peiffer, Browne et al. 1997). In
summary, elevation of FFA concentrations impair carbohydrate utilisation in healthy
humans, are central to the pathogenesis of type two diabetes and probably play a role
in glucose uptake and glycogen metabolism during exercise. Whilst the mechanisms
responsible for these effects are yet to be fully elucidated, recent evidence suggests
that impaired signalling to Glut-4 rather than operation of the “glucose-free fatty acid
cycle” may be involved.
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1.2.3 Glut-4 Translocation Glucose transport is generally considered the rate-limiting factor in muscle and fat cell
glucose utilization. This conclusion has recently been challenged by the observation
that non physiological insulin doses may shift the rate limiting step from glucose
transport to glucose phosphorylation (Kashiwaya, Sato et al. 1994; Manchester, Kong
et al. 1994). Increased glycogenolysis during high intensity exercise may also shift the
rate limiting step to phosphorylation (Katz, Broberg et al. 1986) by increasing G-6-P
levels which in turn inhibit hexokinase and impair glucose phosphorylation. Despite
these findings, a significant body of research suggests that under most physiological
conditions glucose transport remains the rate limiting step for glucose metabolism
(Yki-Jarvinen, Young et al. 1987; Ziel, Venkatesan et al. 1988; Ren, Marshall et al.
1993). Translocation of Glut-4 transporters from the intracellular membrane
compartment to the cell surface is the major mechanism by which insulin and muscle
contractions stimulate glucose transport into muscle and adipose tissue (James, Brown
et al. 1988; Hirshman, Goodyear et al. 1990; Holman, Kozka et al. 1990; Piper, Hess
et al. 1991; Rodnick, Slot et al. 1992). Whilst both insulin and contractions stimulate
glucose uptake, several lines of evidence suggest that there are likely to be differences
between these stimuli. For example it has been established that the effects of insulin
and contraction are additive with regard to glucose transport (Henriksen, Bourey et al.
1990; Ploug, Stallknecht et al. 1990; Brozinick, Etgen et al. 1994) and Glut-4
translocation to the plasma membrane (Gao, Ren et al. 1994). Further evidence in
support of divergent signalling pathways for insulin and contraction stimulated
glucose uptake stems from the observation that contractions stimulate glucose
18
transport in various situations when the ability of insulin to do so is impaired. For
example, inhibition of PI 3-kinase by the fungal product wortmannin completely
inhibits insulin stimulated glucose transport without affecting the stimulation of
transport by contractions (Yeh, Gulve et al. 1995). Furthermore, contractions result in
normal stimulation of glucose transport activity in severely insulin resistant obese
Zucker rats (Dolan, Tapscott et al. 1993). Despite these findings, contraction induced
adaptations in glucose uptake are more pronounced in the presence rather than in the
absence of insulin (Wasserman, Mohr et al. 1992).
1.2.3.1 Insulin signalling of muscle glucose uptake
Insulin binding to its receptor generates a cascade of reactions which ultimately result
in the translocation of Glut 4 transporters from the intracellular compartment to the
cell membrane (Guma, Zierath et al. 1995; Kahn 1996). The proximal step in this
series of reactions has been identified as tyrosine phosphorylation of the insulin
receptor (Kasuga, Karlsson et al. 1982; Cortright and Dohm 1997). The finding that
tyrosine phosphorylation of the insulin receptor ultimately leads to activation of PI 3-
kinase in phosphotyrosine imunoprecipitates has led to the implication of PI 3-kinase
in the insulin signalling pathway (Endemann, Yonezawa et al. 1990; Ruderman,
Kapeller et al. 1990). PI 3-kinase activation has been established as a prerequisite for
insulin stimulated Glut 4 translocation, with the over expression of tyrosine kinase-
deficient insulin receptor in transfected rat adipose cells failing to mediate
translocation of Glut 4 (Quon, Guerre-Millo et al. 1994). The role of PI 3-kinase
activation has been further examined by the inhibition of this enzyme. The fungal
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product wortmannin blocks PI 3-kinase activity and has been shown to inhibit the
stimulation of glucose transport by insulin in adipocytes at 100nmol (Okada, Kawano
et al. 1994) and 500nmol (Lee, Hansen et al. 1995) concentrations. Despite this fact,
inhibition of PI 3-kinase does not affect contraction stimulated glucose uptake,
suggesting that PI 3-kinase activation is exclusive to the insulin signalling pathway
(Yeh, Gulve et al. 1995).
Phosphorylation of insulin receptor substrate 1 (IRS-1) and subsequent association
with the 85-kDa regulatory subunit of PI 3-kinase has been suggested as the
intermediate step in the activation of the catalytic subunit of PI 3-kinase (Cortright
and Dohm 1997). Thus IRS-1 has been postulated as a prerequisite for insulin
stimulated Glut 4 translocation (Cheatham and Kahn 1995). This suggestion has been
challenged by the finding that microinjection of anti IRS-1 antibodies and
phosphotyrosine binding (PTB) domain constructs which reduce interaction of IRS-1
with PI 3-kinase do not affect insulin induced translocation of Glut 4 (Holman and
Kasuga 1997). Additional studies in 3T3-L1 adipocytes and rat 1 fibroblasts have
found that disruption of the insulin receptor / IRS-1 interaction does not affect Glut 4
translocation, but does inhibit membrane ruffling and mitogenesis (Morris, Martin et
al. 1996). In apparent contrast to these findings, maximal stimulation of glucose
transport in IRS-1 null mice is reduced in comparison to control specimens (Araki,
Lipes et al. 1994; Tamemoto, Kadowaki et al. 1994). Interpretation of the above
finding is complicated by the fact that IRS-1 null mice retain normal insulin receptor
substrate 2 (IRS-2) levels. IRS-2 is also phosphorylated in response to tyrosine
20
phosphorylation by the insulin receptor and can bind to the 85-kDa regulatory subunit
of PI 3-kinase. As such IRS-2 may replace the missing signalling intermediate to PI
3-kinase activation in IRS-1 null mice (Holman and Kasuga 1997). In spite of these
findings, a large number of recent papers suggest that IRS-1 plays an important role
in mediating both the metabolic and mitogenic effects of insulin in peripheral tissues
such as muscle and adipose tissue (Quon, Guerre-Millo et al. 1994; Cortright and
Dohm 1997; Holman and Kasuga 1997; Hribal, Federici et al. 2000; Pederson and
Rondinone 2000).
The downstream effectors of PI 3-kinase which mediate the biological effect of
insulin on Glut-4 translocation are poorly understood. Recent research in LT3-L1
adipocytes has implicated Protein Kinase B (PKB), also known as Akt in the insulin
signalling pathway (Kohn, Summers et al. 1996; Hemmings 1997). Further studies in
L6 rat skeletal myoblasts suggest an important role for PKBα /Akt1 in the stimulation
of Glut-4 translocation in cultured muscle cells (Hajduch, Alessi et al. 1998; Wang,
Somwar et al. 1999). The use of dominant negative forms of PKB has produced
conflicting results concerning the role of PKB in insulin stimulated glucose uptake.
Over-expression of PKB with mutations targeted to phosphorylation sites has been
shown to inhibit protein synthesis and P70S6-kinase activity without affecting insulin
stimulated glucose uptake. Recent findings have indicated that the primary
mechanism of PKB activation may be via protein phosphorylation (Kohn, Summers et
al. 1996). It has also been suggested that activation of PKB by insulin may involve
the 3-phosphoinositide dependent protein kinase 1 (PDK1) (Alessi, James et al. 1997).
21
In spite of these findings, increased PKB activity induced by over-expression of
PDK1 is not sufficient for insulin stimulated glucose transport in 3T3-L1 adipocytes
(Yamada, Katagiri et al. 2002). The authors of the above study concluded that PDK1
may be implicated in the process of insulin stimulated glucose uptake via a pathway
which does not involve PKB, however, the existence of an unidentified pathway
downstream of PI 3-kinase and independent of PDK1 could not be excluded (Yamada,
Katagiri et al. 2002). It has recently been suggested that Glut-4 containing vesicles
may be formed as a result of phospholipase D (PLD) activation. In vitro stimulation
of PLD with ammonium sulphate leads to the release of Glut-4 containing vesicles
from donor Glut-4 membranes, a process which may involve binding of PKBβ to Glut-
4 (Kristiansen, Nielsen et al. 2001).
Conventional, novel and atypical isoforms of protein kinase C (PKC) are also thought
to be activated by insulin, however, evidence as to which isoforms are involved
remains equivocal (Schmitz-Peiffer 2000). Recent findings indicate that atypical PKC
isoforms (ζ, λ, ι) are activated by insulin in the in vivo state in humans (Vollenweider,
Menard et al. 2002; Beeson, Sajan et al. 2003; Kim, Kotani et al. 2003). Furthermore,
a number of investigations suggest that aPKC isoforms may play a role in the process
of insulin stimulated glucose uptake in a variety of cell types. For example, over-
expression of constitutively active PKC λ enhances basal glucose uptake to a similar
extent to that observed with insulin stimulation, whilst expression of a kinase deficient
PKC λ completely blocks insulin mediated glucose uptake in skeletal muscle L6 cells
(Bandyopadhyay, Kanoh et al. 2000). It has been suggested that PKC ζ specifically
22
associates with Glut-4 compartments in response to insulin in primary cultures of rat
skeletal muscle, resulting in translocation of PKC ζ and Glut-4 to the plasma
membrane as a complex (Braiman, Alt et al. 2001). The involvement of atypical
PKC’s in insulin stimulated Glut-4 translocation is further supported by studies which
have employed over-expression of atypical PKC isotope-specific interacting protein
(ASIP) to inhibit PKC. Over-expression of ASIP has been shown to inhibit insulin-
induced glucose transport in 3T3-L1 adipocytes by specifically interfering with
signals transmitted through PKC λ (Kotani, Ogawa et al. 2000). A reduction in
insulin stimulated glucose uptake has also been observed when aPKC isoforms were
down-regulated by other chemical inhibitors in L6 myotubes (Bandyopadhyay,
Standaert et al. 1997). Whereas the role of aPKC’s in the process of insulin mediated
glucose uptake is well supported, the significance of the conventional (α, β) and novel
(ε, η, θ) isoforms is less clear. For example, in vivo stimulation with insulin in
humans does not result in translocation of conventional or novel PKC isoforms (Itani,
Ruderman et al. 2002). Unlike aPKC’s, the conventional (cPKC) and novel (nPKC)
isoforms require diacylglycerol (DAG) for their full translocation and activation
(Newton 1995). Interestingly, chronic treatment of L6 skeletal muscle myotubes with
phorbel esters causes downregulation of DAG-sensitive PKC’s without affecting
insulin stimulated glucose uptake (Bandyopadhyay, Standaert et al. 1997). On the
other hand, a number of studies suggest that DAG-sensitive PKC isoforms are
involved in the process of glucose uptake in response to insulin (Chalfant, Ohno et al.
1996; Braiman, Alt et al. 1999). In summary, whilst the above studies suggest an
important role for aPKC in insulin mediated glucose uptake, the exact significance of
23
DAG-sensitive PKC isoforms in this process remains equivocal. In spite of this fact,
the observation that activation of aPKC’s by insulin is defective in skeletal muscle of
type-two diabetic humans, monkeys and rodents (Farese 2002) warrants continued
investigation in this field.
Whilst activation of some isoforms of PKC has been implicated in the enhancement of
post receptor insulin signalling, there is evidence to suggest that PKC may also be
causally related to insulin resistance in a variety of models. Because impaired insulin
action is often associated with increased lipid availability, it is possible that
hyperlipidaemia may activate pathways (including PKC’s) which result in attenuation
of insulin signalling (Schmitz-Peiffer 2000). Indeed, aberrant activation of PKC has
been linked to diminished insulin sensitivity, especially, when the availability of lipid
is increased in target tissues. In the high fat fed model of insulin resistance in rats,
membrane localised PKC ε, θ and δ are increased in red skeletal muscle (Schmitz-
Peiffer, Oakes et al. 1997). Insulin resistance caused by elevation of plasma FFA
during lipid infusion also leads to increased membrane localisation (a marker of
activation) of PKC θ in rat skeletal muscle (Griffin, Marcucci et al. 1999). In humans,
in vitro insulin stimulation increases membrane associated PKC activity in skeletal
muscle of obese but not lean individuals (Itani, Zhou et al. 2000). Protein content of
PKCβ has also been shown to be elevated in skeletal muscle membrane fractions from
obese individuals in the absence of insulin stimulation (Itani, Zhou et al. 2000).
Infusion of lipid during hyperinsulinaemic / euglycaemic clamp conditions also causes
insulin resistance in humans, an effect which may be modulated by increased
24
abundance of PKC-βІІ and -δ isoforms (Itani, Ruderman et al. 2002). Thus a
substantial pool of evidence suggests that activation of PKC’s (particularly the novel
and atypical isoforms) may be related to insulin resistance.
The mitogen-activated protein kinase (MAPK) signalling pathway consists of at least
three parallel cascades known as the extracellular signal-regulated kinase (ERK), the
c-jun NH2 - terminal kinase (JNK) and the p38 kinase. Activation of MAPK
signalling cascades has been shown to occur in response to a wide variety of
mitogenic and stress inducing stimuli and plays an important role in transducing
extracellular signals into cellular responses. Whether MAPK pathways play a role in
the metabolic effect of insulin is yet to be conclusively established. Recent
investigation suggests that p38 MAPK may be involved in insulin stimulation of
glucose uptake in cultured cells. For example, inhibition of the p38 MAPK by the
inhibitor SB203580 has been shown to reduce insulin stimulated glucose transport in
3T3-L1 adipocytes and L6 muscle cells. In spite of this finding, Glut-4 exposure at
the cell surface was not affected by SB203580, suggesting that inhibition of p38 may
affect the intrinsic activity of the glucose transporter (Sweeney, Somwar et al. 1999).
Whilst the above findings suggest a role for p38 MAPK in the insulin signalling
cascade, activation of p38 has not always been shown to be essential for insulin
induced increases in glucose uptake. For instance, suppression of p38 MAPK
activation by over-expression of a dominant negative p38 MAPK mutant does not
reduce insulin stimulated glucose uptake in 3T3-L1 adipocytes (Fujishiro, Gotoh et al.
2001). To the contrary, p38 MAPK activation in the above study resulted in a marked
25
reduction in insulin stimulated glucose uptake via the Glut-4 transporter. Whether
p38 MAPK is activated by insulin in the in vivo state is yet to be conclusively
established. In a recent investigation, intraperitoneal injection of insulin did not
increase p38 activity in rat skeletal muscle (Goodyear, Chang et al. 1996). In contrast,
pharmacological inhibition of p38 has been shown to result in a significant reduction
in insulin stimulated 2DG uptake in isolated rat muscle preparations (Somwar,
Perreault et al. 2000). Whilst the above findings suggest a potential role for p38
MAPK in the metabolic affect of insulin, very little research has examined the role of
p38 in human skeletal muscle. In a recent investigation, insulin infusion resulted in a
modest sustained increase in p38 MAPK phosphorylation during hyperinsulinaemic /
euglycaemic clamp conditions (Thong, Derave et al. 2003). Interestingly, p38
phosphorylation in response to insulin was marginally higher in previously exercised
compared with rested muscle. This finding raises the possibility that p38 may play a
role in the well documented stimulatory effect of acute exercise on insulin stimulated
glucose uptake in the post exercise period (see 1.3).
Recent research in 3T3-L1 adipocytes suggests that activation of the PI 3-kinase and
MAPK signalling cascades may not be sufficient to explain the metabolic effect of
insulin on glucose uptake and glycogen synthesis. For example, inhibition of mitogen
activated protein kinase kinase (MAPKK or MEK) by the specific MEK inhibitor
PD98059 does not block the metabolic effect of insulin (Lazar, Wiese et al. 1995).
Furthermore, activation of the MAPK signalling pathway by thrombin and epidermal
growth factor (EGF) is not sufficient to induce glucose uptake, lipogenesis or
26
glycogen synthesis in 3T3-L1 adipocytes (van den Berghe, Ouwens et al. 1994). In
another study, treatment of 3T3-L1 cells with platelet derived growth factor (PDGF)
or epidermal growth factor (EGF) increased MAPK and PI 3-kinase activity
respectively but did not affect glucose utilisation. These findings raise the possibility
of an alternative signalling pathway that may be uniquely responsive to insulin and
independent of MAPK and PI 3-kinase. The protein product of the c-Cbl proto-
oncogene has emerged as a potential candidate in the PI 3-kinase independent insulin
signalling pathway. Phosphorylation of c-Cbl by insulin requires the c-Cbl associated
protein (CAP), an adaptor molecule that recruits c-Cbl to the insulin receptor (Ribon,
Printen et al. 1998). Translocation of phosphorylated c-Cbl to a Triton-insoluble lipid
raft microdomain in 3T3-L1 adipocytes results in binding of CAP to the caveolar
protein flotillin. Thus CAP functions as an adaptor linking Cbl to the plasma
membrane lipid raft microdomains through the interaction of CAP with flotillin.
Expression of a CAP mutant (CAP∆SH3) interferes with the formation of a CAP-
flotillin-Cbl complex and inhibits insulin stimulated glucose uptake, resulting in
secondary inhibition of glycogen synthase activation and glycogen synthesis in 3T3-
L1 adipocytes (Baumann and Saltiel 2001). Whether c-Cbl plays a role in the
metabolic effect of insulin in other cell types is yet to be conclusively established. In a
recent investigation, tyrosine phosphorylation of Cbl in response to insulin stimulation
was not evident in the skeletal muscle of lean or obese Zucker rats, suggesting that
Cbl may not be involved in the insulin signalling pathway in rodent muscle (Wadley,
Bruce et al. 2004). Whether c-Cbl is activated by insulin in human skeletal muscle is
yet to be thoroughly investigated. Preliminary investigation suggests that protein
27
expression and tyrosine phosphorylation of c-Cbl is unchanged during two hours of
insulin infusion in humans (Wadley et al, 2004, personal communication).
1.2.3.2 Contraction signalling of muscle glucose uptake Several mechanisms have been suggested as mediating contraction induced increases
in glucose transport associated with acute and repeated exercise. Whilst the
intracellular signalling events through which muscle contractions elicit glucose
transport and Glut-4 translocation have not been fully elucidated, an increase in
cytosolic calcium (Ca2+) as a result of membrane depolarisation has been observed for
many years (Holloszy and Narahara 1967). The precise influence of increased Ca2+
concentrations was initially difficult to decipher due to the fact that Ca2+ also resulted
in muscle contraction and subsequent reduction in high energy phosphates. More
recent research has established that an increase in intracellular Ca2+ concentration of
insufficient magnitude to cause muscular contraction results in augmented muscle
glucose transport activity, independent of muscle contraction or a decrease in high
energy phosphate concentrations (Youn, Gulve et al. 1991). These findings have been
interpreted as evidence supporting the hypothesis that Ca2+ mediates the effect of
exercise on muscle glucose uptake (Youn, Gulve et al. 1991). In line with this
hypothesis, increases in cytosolic Ca2+ concentrations have been shown to facilitate
the activation of intracellular signalling proteins that result in the immediate and
prolonged effects of contraction on glucose uptake. In spite of this fact, additional
candidates have recently emerged as potential regulators of contraction induced
28
glucose uptake. Evidence for the involvement of calcium dependent and alternative
signalling pathways in the process of glucose uptake during exercise are presented
below.
Early investigation into the mechanisms responsible for contraction mediated glucose
uptake suggested that various isoforms of PKC may be implicated in the contraction
stimulated signalling pathway. For example, decreased PKC activity in rat skeletal
muscle induced by incubation with phorbol esters, or by using the inhibitor polymyxin
B, was shown to impair contraction induced glucose transport (Henriksen, Sleeper et
al. 1989). Interpretation of these findings is made difficult by the fact that polymyxin
B is not a specific inhibitor of PKC and by the observation that polymyxin B and
phorbol ester treatment can both result in decreased contractility of skeletal muscle.
More specific PKC inhibitors have recently been developed and have been used to
demonstrate the probable role of PKC in the contraction stimulated signalling pathway
(Wojtaszewski, Laustsen et al. 1998; Ihlemann, Galbo et al. 1999). The precise
significance of the different PKC isoforms in the stimulation of glucose transport is
yet to be established, however, recent research in rats suggests that the Ca2+ sensitive
conventional PKCβ2 may be involved (Khayat, Tsakiridis et al. 1998; Kawano,
Rincon et al. 1999). A reduction in Ca2+ sensitive PKC activity induced by chronic
phorbel ester treatment has additionally been shown to result in almost complete
blunting of contraction induced glucose transport (Cleland, Abel et al. 1990).
Calcium insensitive atypical PKC’s have also been shown to be activated by exercise
in skeletal muscle of mice and may play a role in exercise effects on glucose transport
29
(Chen, Bandyopadhyay et al. 2002). During exercise in humans, aPKC activity is
elevated and may be related to enhanced insulin action of glucose transport and
glycogen synthesis (Beeson, Sajan et al. 2003; Nielsen and Richter 2003). In spite of
these findings, it would appear that novel isoforms including PKC δ and PKC ε are
not activated during exercise (Heled, Shapiro et al. 2003; Perrini, Henriksson et al.
2004). Thus whilst PKC may play a role in the process of contraction mediated
glucose uptake, further investigation is warranted in order to determine the exact
significance of the various PKC isoforms.
The observation that adaptations in glucose transport occur only in muscles which are
extensively activated suggests that a signal inherent to increased muscle contractile
activity is at work (Holloszy 1976; Etgen, Farrar et al. 1993). During contractile
activity and hypoxia an inverse relationship between intramuscular creatine phosphate
and glucose uptake has been observed. A reduction in the high-energy phosphate
state of the muscle has hence been proposed as one of the contraction induced
intracellular signals responsible for adaptations in glucose uptake and Glut-4. This
possibility has recently been investigated in rat skeletal muscle using chronic
electrical stimulation and β-guanidinoproprionic acid (β-GPA) diet (Yaspelkis, Castle
et al. 1998). β-GPA is a structural analogue of creatine that competes with creatine
for transport into the muscle, and reduces skeletal muscle creatine phosphate and ATP
levels. In the absence of electrical stimulation, rodents administered a 2% β-GPA diet
displayed a significant reduction in cellular energy supply and a 60% elevation in
30
Glut-4 protein. It was concluded that a reduction in cellular energy supply initiates
the contraction-induced increase in skeletal muscle Glut-4, but that a rise in cAMP
may potentiate this effect (Yaspelkis, Castle et al. 1998).
A reduction in creatine phosphate and increased concentrations of creatine and AMP
in skeletal muscle following acute exercise results in activation of the AMPK
signalling cascade. Recent research suggests that AMPK may be a key regulatory
protein in exercise signalling of insulin independent muscle glucose transport
(Hayashi, Hirshman et al. 2000). In support of this possibility, activation of AMPK
by the 5-amino-4-imidazolecarboxamide riboside (AICAR) has been shown to result
in increased glucose uptake (Merrill, Kurth et al. 1997) and Glut-4 protein (Ojuka,
Nolte et al. 2000) in rats. The mechanism via which AICAR stimulates glucose
uptake would appear to mimic that of contractions given that the effects of AICAR on
glucose uptake are wortmannin insensitive and additive to the effects of insulin but
not contractions (Winder 2001). Whilst the above observations indicate a role for
AMPK in contraction signalling to glucose transport, not all authors share this view.
In a recent investigation, contraction induced stimulation of glucose transport in
glycogen loaded rat slow twitch muscle was shown to occur in the absence of AMPK
activation (Wojtaszewski, Jorgensen et al. 2002). Furthermore, whilst
hyperosmolarity increases AMPK activity and glucose uptake in skeletal muscle L6
cells, inhibition of glucose transport via calcium chelation and PKC inhibition does
not affect AMPK activity (Patel, Khayat et al. 2001). Clarification of the exact
significance of AMPK in the contraction signalling pathway is complicated by the fact
31
that there are no commercially available inhibitors of contraction stimulated AMPK
activity. In spite of this fact, significant insight has been gained via the use of AMPK
deficient animals. Over expression of a dominant negative mutant of AMPK has been
shown to completely block hypoxia stimulated glucose transport whilst only partially
blocking contraction stimulated glucose transport in mice (Mu, Brozinick et al. 2001).
In a further investigation, abolition of the α1 and α2 isoforms of AMPK did not
markedly decrease contraction stimulated glucose uptake in incubated muscle from
AMPK knockout mice (Jorgensen, Viollet et al. 2004). Thus whilst the above
evidence suggests that AMPK activation is likely to be partially involved in the
process of contraction stimulated glucose transport, the existence of one or more
AMPK independent mechanisms cannot be discounted.
Recent findings suggest that both Ca2+ and AMPK may be involved in the stimulation
of glucose transport by muscle contractions (Wright, Hucker et al. 2004). In the
above study, incubation of rat epitroclearis muscles with caffeine (which raises
cytosolic calcium) or AICAR (which mimics contractions) resulted in a threefold
increase in glucose transport. The increase in glucose transport induced by AICAR
and caffeine was additive, and the combined effect was similar to that induced by
contractions (Wright, Hucker et al. 2004). Increased phosphorylation of calmodulin-
dependent protein kinase (CAMK)-II was also observed with caffeine stimulation,
suggesting that CAMK’s may mediate the effect of Ca2+ on glucose transport in rats
(Wright, Hucker et al. 2004). Furthermore, CAMK-II has recently been shown to be
activated by exercise in humans suggesting a role in skeletal muscle function during
32
exercise (Rose 2004). Given that both Ca2+ and AMPK appear to be involved in
contraction signalling, it is possible that Ca2+ may stimulate glucose transport during
moderate exercise which causes minimal decreases in high energy phosphates. Under
this scenario, AMPK may become more important as exercise intensity increases and
the concentration of high-energy phosphates is reduced. Indeed, it has recently been
suggested that AMPK activation may be restricted to intense contractions resulting in
some degree of hypoxia and a reduction in the CrP/Cr and / or ATP/AMP ratios
(Richter, Derave et al. 2001). A number of authors have investigated the effects of
exercise intensity on AMPK activation in contracting human skeletal muscle. In a
recent series of studies, one hour of bicycle exercise at 50% peak had no effect
on AMPK activity, whilst the same duration of exercise at 75% peak resulted in
activation of the α-2 but not the α-1 isoform of AMPK (Wojtaszewski, Nielsen et al.
2000). Activation of both α-1 and α-2 isoforms of AMPK has been shown to occur
following 30 seconds of supramaximal cycling exercise (Chen, McConell et al. 2000).
Thus the notion of progressive activation of AMPK with higher exercise intensities is
supported in humans. Previous studies have shown that glucose uptake is also
progressively activated with exercise intensity (Wahren, Felig et al. 1971; Katz,
Broberg et al. 1986), however, whether there is a causal relationship between AMPK
and glucose uptake in humans is unknown. Determination of such causality has been
difficult to define given that the techniques traditionally used to examine the
relationship between AMPK and glucose uptake in rats (such as specific blockers of
AMPK) are not available in humans. Until this point, research in humans has been
2OV&
2OV&
33
restricted to studies which have investigated whether a correlation exists between
AMPK activation and glucose uptake during exercise. In a recent investigation, no
correlation between AMPK activation and glucose uptake during cycling exercise at
70% peak was observed (Richter, Wojtaszewski et al. 2001), suggesting that
AMPK may not be essential in the regulation of glucose uptake during exercise in
humans.
2OV&
The mechanism by which AMPK increases glucose uptake in skeletal muscle may
involve autocrine / paracrine mechanisms including nitric oxide synthase (NOS).
Activation of NOS has been shown to activate nitric oxide (NO) production in rat
skeletal muscle (Balon and Nadler 1994). Recent investigation suggests that AMPK
phosphorylates and activates endothelial NOS (eNOS) in rat heart muscle (Chen,
Mitchelhill et al. 1999). Furthermore, phosphorylation of neuronal NOS (nNOS) has
been shown to occur when AMPK is activated during exercise in humans (Chen,
McConell et al. 2000). It has been suggested that inhibition of NOS may result in a
decrease in contraction stimulated glucose uptake. For example, inhibition of calcium
dependant NOS has been shown to result in decreased basal (Balon and Nadler 1994)
and contraction (Balon and Nadler 1997) stimulated glucose transport. In contrast,
nitric oxide inhibition had no effect on insulin stimulated glucose uptake, suggesting
that this mechanism is exclusive to the contraction stimulated signalling pathway
(Balon and Nadler 1997). More recent research also suggests that inhibition of NOS
reduces glucose uptake during exercise in control subjects and patients with type two
diabetes (Kingwell, Formosa et al. 2002). In the above study, diabetic subjects
34
displayed a greater reliance on NO for contraction stimulated glucose uptake when
compared with control subjects suggesting a potential link with impaired insulin
action. Studies employing inhibition of NO have been criticised due to the non-
specificity of the inhibitors used. Consequently, some studies have also employed NO
donors such as sodium nitroprusside (SNP) in their investigations of glucose uptake.
SNP has been shown to increase 2-deoxyglucose (2-DG) uptake in a dose dependent
manner, except at concentrations in excess of 2×10-2 M. Beyond this point 2-DG
transport declined to a rate below control levels (Balon and Nadler 1997). The
apparent stimulatory role of NO in contraction stimulated glucose uptake has been
contradicted by some authors (Etgen, Fryburg et al. 1997), suggesting that further
research is required in order to clarify the exact significance of this molecule. A
recent study which employed various combinations of nitric oxide donors (SNP),
nitric oxide inhibitors (L-NMMA & L-NAME), insulin, contractions and wortmannin
concluded that the mechanism by which nitric oxide stimulates glucose uptake is
distinct from that of either insulin or contraction (Higaki, Hirshman et al. 2001).
Should NO prove to be involved in the contraction stimulated signalling pathway, it's
action may be potentiated via the kallikrein molecule. Kallikrein catalyses the
production of bradykinin, and has thus been suggested as a potential stimulator of
nitric oxide synthase and glucose transport (Kishi, Muromoto et al. 1998), however,
not all authors share this view (Constable, Favier et al. 1986).
35
Muscle contraction and exercise induced activation of MAPK signalling has been
shown to occur in skeletal muscle of both rats and humans. Since MAPK signalling
pathways have been implicated in the regulation of gene transcription in a variety of
cell types, it is possible that MAPK activation may play a role in modulating the
cellular processes that occur in skeletal muscle in response to exercise. Investigation
of exercise induced signal transduction in the parallel MAPK pathways may provide
further insight into the mechanisms governing contraction stimulated glucose uptake
in skeletal muscle.
ERK 1/2 MAPK and exercise.
ERK 1/2 signalling has been shown to be influenced by exercise in skeletal muscle of
both rats and humans. In rats, 10-60 min of treadmill running increases ERK 1/2
expression and results in a 1.8 fold increase in the down stream kinase RSK2
(Goodyear, Chang et al. 1996). A 6-14 fold increase in ERK 1/2 phosphorylation has
also been observed following sciatic nerve stimulation in rats (Aronson, Dufresne et
al. 1997; Sherwood, Dufresne et al. 1999). Phosphorylation of ERK 1/2 has
additionally been shown to increase by 1.6 fold and two fold in response to isometric
contraction and static stretch respectively (Wretman, Widegren et al. 2000). Exercise
induced increases in ERK1/2 phosphorylation in humans have been shown to occur
rapidly in response to exercise, with greatest effects observed after 30 minutes
(Widegren, Jiang et al. 1998). In contrast, some authors have reported that
measurable increases in ERK 1/2 phosphorylation are not apparent until after 60 min
36
of exercise (Osman, Pendergrass et al. 2000). Upon cessation of exercise, MAPK
phosphorylation is rapidly decreased and has been shown to return to resting values
within 60 min of the completion of exercise (Widegren, Jiang et al. 1998). The
impact of exercise on MAPK signalling appears to be exercise intensity dependent,
given that ERK 1/2 phosphorylation is greater following high vs. low intensity
exercise (Widegren, Wretman et al. 2000). Furthermore, ERK 1/2 phosphorylation in
response to exercise only occurs in muscles which are extensively activated,
suggesting the influence of local rather than systemic factors (Aronson, Violan et al.
1997). The molecular mechanisms responsible for exercise induced activation of
ERK 1/2 signalling in human skeletal muscle are not completely understood. It has
been suggested that downstream regulation of ERK 1/2 in response to exercise in
humans may involve the Raf-1 / MEK1 signalling pathway (Aronson, Violan et al.
1997; Widegren, Wretman et al. 2000). Investigation of the upstream events in the
ERK 1/2 signalling pathway has been largely restricted to rat skeletal muscle. Recent
research suggests that contraction induced ERK 1/2 phosphorylation in rats does not
involve activation of receptor tyrosine kinases (Sherwood, Dufresne et al. 1999) or
IRS and Shc tyrosine phosphorylation (Sherwood, Dufresne et al. 1999). These
findings suggest that activation of MAPK signalling may not be mediated via classical
receptor tyrosine kinase pathways. It is generally accepted that exercise induced
increases in phosphorylation provide a good indicator of ERK1/2 activation, however,
it is important to note that phosphorylation may not necessarily directly parallel
enzyme activity (Widegren, Ryder et al. 2001). Furthermore, whilst exercise induced
activation of the ERK 1/2 MAPK signalling pathways in skeletal muscle is well
37
documented, recent evidence suggests that ERK signalling is not necessary for
contraction stimulated glucose uptake in rats (Wojtaszewski, Lynge et al. 1999).
JNK MAPK and exercise
Activation of the JNK signalling pathway in response to contraction has been shown
to occur in a number of tissue types. In rat skeletal muscle, sciatic nerve stimulation
results in a two fold increase in phosphorylation and a five-fold increase in activation
of JNK after 30 minutes of contractile activity (Aronson, Dufresne et al. 1997). A
two-fold increase in JNK activity has also been observed following 10-60 minutes of
treadmill running in rats (Goodyear, Chang et al. 1996). In human skeletal muscle,
prolonged cycling exercise has been shown to elicit a 6-fold increase in JNK activity
(Aronson, Boppart et al. 1998). Consistent with the classification of JNK as a
member of the SAPK family, exercise induced increases in JNK activity are
dependent upon the extent of muscle injury sustained. Thus, whilst JNK activity is
significantly increased following 30-60 min of cycling exercise (Osman, Pendergrass
et al. 2000), activation is greatest in response to eccentric exercise (Boppart, Aronson
et al. 1999). An increase in inflammatory cytokines has been suggested as one of the
mechanisms via which injury inducing exercise may activates SAPK cascades
(Fielding, Manfredi et al. 1993). In spite of the fact that exercise has been shown to
be a potent activator of JNK in rat and human skeletal muscle, the JNK signalling
cascade has to this point not been implicated in the well documented stimulatory
effect of exercise on muscle glucose uptake.
38
p38 MAPK and exercise
The second member in the family of SAPK signalling pathways is the p38 MAPK.
Four isoforms of p38 have so far been characterised in mammalian tissue, with these
including p38α, β, γ and δ. Kinase activity and phosphorylation status of the various
isoforms of p38 has been shown to increase in response to treadmill running and
isolated contraction in rat skeletal muscle (Goodyear, Chang et al. 1996; Ryder,
Fahlman et al. 2000; Wretman, Widegren et al. 2000). Recent investigation also
suggests that p38 MAPK and its upstream regulators MEK 4 and MEK 6 are
transiently increased after prolonged strenuous exercise in humans (Boppart, Asp et
al. 2000). Thus it has been suggested that the p38 signalling pathway may play a role
in skeletal muscle adaptation to strenuous exercise. This effect is likely to be isoform
specific, given that marathon running increases p38 γ phosphorylation four-fold
without affecting p38 α phosphorylation or activity. In contrast to ERK 1/2
signalling, p38 phosphorylation has been shown to occur in muscle which is not
extensively activated during exercise, suggesting the influence of both local and
systemic factors. The precise significance of p38 signalling in the vast array of
skeletal muscle adaptations to exercise is unknown. Recent interest has focused on
the potential regulatory role of p38 MAPK in the process of contraction stimulated
glucose uptake. Electrically-induced contraction of isolated extensor digitorum
longus (EDL) muscle has been shown to significantly increase kinase activity of p38
MAPK in rats (Somwar, Perreault et al. 2000). Inhibition of p38 by in vitro
incubation with SB203580 in the above study also resulted in a diminished effect of
contraction on muscle glucose uptake. AICAR treatment has also been shown to
39
activate both the α and β isoforms of p38 with an identical time course to that of
AMPK and glucose transport (Lemieux, Konrad et al. 2003). The authors of the
above investigation suggested that AICAR may increase muscle glucose uptake via
mechanisms which involves recruitment of Glut-4 to the plasma membrane and
activation of p38 MAPK α and β (Lemieux, Konrad et al. 2003). In humans, p38
phosphorylation has recently been shown to be 50% higher in skeletal muscle biopsies
obtained from exercised when compared to rested muscle (Thong, Derave et al. 2003).
The stimulatory effect of exercise on p38 MAPK phosphorylation in the above study
was still apparent 3 hrs after exercise and persisted during subsequent insulin
stimulation. These findings suggest that p38 MAPK signalling may play an important
role in the process of contraction mediated glucose uptake, and may additionally
provide a candidate for investigation of the mechanisms responsible for enhanced
insulin action following an acute bout of exercise. In spite of this possibility, further
research would be required in order to fully elucidate the influence of p38 on
contraction mediated glucose uptake in humans. The potential role of p38 MAPK in
post exercise insulin sensitivity is discussed in section 1.3.
40
1.3 Mechanisms of enhanced insulin action following exercise A single bout of exercise improves the metabolic effect of insulin on glucose uptake
and glycogen synthesis in the post exercise period. This effect has been shown to
occur in both the in vitro state and when whole body glucose uptake is examined
under hyperinsulinaemic / euglycaemic clamp conditions. Enhanced insulin action in
the post exercise period occurs rapidly (3-4 hours) in both healthy individuals
(Bogardus, Thuillez et al. 1983) and patients with impaired insulin action (Devlin,
Hirshman et al. 1987; Perseghin, Price et al. 1996) and may persist for as long as 24
hours after cessation of the exercise bout (Mikines, Sonne et al. 1988). A number of
mechanisms have been suggested to explain post exercise enhancement of insulin
action. One possibility is that the improved metabolic effect of insulin following
exercise is caused by enhanced insulin-mediated capillary perfusion. Under this
proposal, increased capillary recruitment may lead to improved delivery of insulin and
glucose and subsequent enhanced insulin action on skeletal muscle glucose uptake
(Wojtaszewski, Hansen et al. 1997; Wojtaszewski, Hansen et al. 2000). In argument
against such a scenario, contraction induced adaptations in insulin action have been
shown to occur in the absence of changes in microvascular blood volume. For
example, the metabolic effect of insulin on glucose uptake and transport is increased
following muscle contraction in perfused rat muscle (Richter, Garetto et al. 1982) and
in isolated and incubated muscle preparations (Gao, Gulve et al. 1994). Whilst the
role of acute exercise in enhanced insulin mediated capillary perfusion remains
controversial, recent advances in methodology indicate that a major hemodynamic
effect of insulin is to increase nutritive capillary blood flow in muscle. For extensive
41
discussion on this topic, the readers’ attention is directed to a recent review (Clark,
Wallis et al. 2003). Whilst exercise induced adaptations in insulin mediated capillary
perfusion cannot be discounted, the mechanism of enhanced insulin action following
exercise may also involve cellular mechanisms within the exercised muscle. For
example, improved insulin action has been shown to occur only in muscles which are
extensively activated during exercise, suggesting the involvement of local rather than
systemic factors. Insulin stimulated glucose uptake in the period following a single
leg exercise regimen is twofold higher in the exercised compared with the rested leg
(Richter, Mikines et al. 1989). In spite of intense research interest, the molecular
mechanisms responsible for enhanced insulin action following a single bout of
exercise remain elusive. Whilst it is tempting to speculate that exercise induced
adaptations in insulin action may be linked with improved sensitivity of the insulin
signalling pathway, this has not always been shown to be the case. In a recent
investigation, activation or phosphorylation of the insulin receptor, IRS-1, IRS-1-
associated PI 3-kinase, PKB and GSK3 was unchanged in muscle lysates obtained
from exercised compared to resting muscle (Wojtaszewski, Hansen et al. 1997;
Wojtaszewski, Hansen et al. 2000). Furthermore, impaired insulin action in relatives
to patients with NIDDM (Thorell, Hirshman et al. 1999) or in healthy individuals
following caffeine ingestion (Thong, Derave et al. 2002) does not lead to down
regulation of early steps in the insulin signalling cascade. A number of explanations
have been proposed to explain this phenomenon. The most literal interpretation is that
post exercise adaptations in insulin action may not necessarily be causally linked to
changes in insulin signalling. On the other hand, it is possible that the assay
42
procedures which are currently used to measure insulin signalling are not sensitive
enough to detect small but functionally significant changes in signalling.
Alternatively, the spatial arrangement of insulin signalling intermediates may be an
important factor contributing to enhanced insulin action in the post exercise period.
Because insulin signalling is measured in muscle lysates, homogenisation of muscle
samples may not allow the detection of potentially important changes in subcellular
localisation of signalling intermediates. Finally, exercise induced enhancement of
insulin sensitivity may be related to changes in signalling mechanisms which occur
further downstream in the insulin cascade, or may involve an as yet unknown
signalling factor that is upregulated with exercise.
Recent research suggests that improved insulin sensitivity of skeletal muscle glucose
transport after a single bout of exercise may be mediated by enhanced translocation of
glucose transporters to the cell surface (Hansen, Nolte et al. 1998). In the above
study, prior exercise resulted in greater cell surface Glut-4 labeling and glucose
transport activity in response to insulin treatment in exercised compared to sedentary
rats. Insulin receptor substrate-1 tyrosine phosphorylation was unchanged after
exercise, suggesting that the effect of contractions on insulin mediated glucose uptake
was not due to potentiation of insulin-stimulated tyrosine phosphorylation (Hansen,
Nolte et al. 1998). The existence of one or more PI 3-kinase independent signalling
pathways which may act in the stimulation of Glut-4 translocation have been
proposed. Recent evidence suggests that the protein product of the c-Cbl proto-
oncogene may be involved in the process of insulin stimulated glucose uptake in rat
43
adipocytes (see 1.2.3.1). In spite of these findings, c-Cbl tyrosine phosphorylation has
been shown to be reduced in response to insulin stimulation in rat skeletal muscle
(Wadley, Bruce et al. 2004). Furthermore, preliminary investigation in human
skeletal muscle suggests that tyrosine phosphorylation of c-Cbl is unchanged with
insulin stimulation 24 hours after acute exercise (Wadley, 2004 personal
communication). It has been suggested that signals emanating from the p38 MAPK
may comprise a second PI 3-kinase independent insulin signalling pathway (see
1.2.3.1). Whether p38 is implicated in the well documented stimulatory effect of acute
exercise in the immediate post exercise period is yet to be thoroughly investigated. In
a recent investigation, infusion of insulin during a 100 min hyperinsulinaemic /
euglycaemic clamp resulted in a 1.2 and 1.3 fold increase in p38 MAPK
phosphorylation in rested and exercised legs respectively. Interestingly, p38
phosphorylation was ~50% higher before and during insulin infusion in the leg which
had previously performed one hour of knee extensor exercise (Thong, Derave et al.
2003). This finding raises the possibility that p38 MAPK may be located at a
convergence point between exercise and insulin stimulation of glucose uptake. If this
were the case, p38 may prove to be an interesting candidate for investigation of the
mechanisms responsible for improved glucose utilization following acute exercise.
Regulation of carbohydrate metabolism during exercise is governed by a complex
interplay of factors which ensure that muscle glycogenolysis and glucose uptake are
enhanced in line with the increased metabolic demand of the working muscle. These
regulatory factors have recently been reviewed (Roach 2002) and include exercise
44
dependent changes in muscle calcium, inorganic phosphate and metabolites, hormonal
control and substrate availability. The degree of muscle glycogen degradation during
exercise would appear to be important, since exercise induced adaptations in insulin
sensitivity have consistently been linked to muscle glycogen concentrations. For
example, post exercise activation of glycogen synthase is tightly coupled to muscle
glycogen depletion and is only apparent until glycogen stores are replenished. Insulin
stimulated glucose uptake has also been shown to be enhanced in glycogen depleted
skeletal muscle of previously exercised rats (Kawanaka, Han et al. 1999; Derave,
Hansen et al. 2000; Kawanaka, Nolte et al. 2000), although the mechanisms
responsible for this effect are currently unknown. The role of muscle glycogen in the
process of skeletal muscle glucose uptake is further examined in section 1.4.
45
1.4 Role of glycogen in the regulation of skeletal muscle glucose uptake For more than a decade, research has focused on the potential regulatory role of
muscle glycogen on insulin and contraction stimulated glucose uptake in skeletal
muscle. Initial investigations in isolated rat skeletal muscle identified an inverse
relationship between pre contraction muscle glycogen concentration and glucose
uptake during contractions (Richter and Galbo 1986; Hespel and Richter 1990).
Subsequent studies linked the apparent stimulatory effect of lowered muscle glycogen
on glucose uptake to increased contraction induced translocation of Glut-4 to the
plasma membrane (Hespel and Richter 1990; Derave, Lund et al. 1999). A regulatory
effect of muscle glycogen concentration on insulin stimulated glucose uptake in rats
has also been identified (Nolte, Gulve et al. 1994; Kawanaka, Nolte et al. 2000), and
may similarly be modulated by variations in Glut-4 translocation (Kawanaka, Han et
al. 1999; Derave, Hansen et al. 2000). Early investigations in humans have identified
an inverse relationship between muscle glycogen concentration and glucose uptake
during 40 minutes of cycling exercise (Hargreaves, Meredith et al. 1992) and during
dynamic knee extension exercise (Saltin 1986). A potential criticism of such
correlational studies is that the temporal relationship between glycogen concentration
and glucose uptake may reflect the independent effects of exercise on glycogenolysis
and glucose uptake rather than a causal effect. A number of studies have also
investigated the effect of pre contraction muscle glycogen concentration on glucose
uptake during exercise. Glucose uptake in a leg which commenced exercise with low
muscle glycogen has been shown to be greater than that in the contralateral leg which
commenced exercise with higher glycogen levels (Gollnick 1981). More recently, leg
46
glucose clearance during one hour of cycle ergometer exercise at 70% 2OV& peak was
shown to be twice as high in the glycogen depleted state compared to when muscles
were glycogen supercompensated (Wojtaszewski, MacDonald et al. 2003). Whilst
these findings suggest a regulatory effect of glycogen concentration on contraction
stimulated glucose uptake in humans, this has not always been shown to be the case.
A recent study which employed a combination of exercise and diet to manipulate
glycogen found no effect of glycogen concentration on tracer determined glucose
uptake rate during exercise (Hargreaves, McConell et al. 1995). It is possible that a
decrease in plasma insulin and glucose concentrations and an increase in plasma free
fatty acids (FFA) following low carbohydrate feeding in this study may have reduced
glucose uptake during exercise and negated the potential regulatory effect of glycogen
depletion on glucose uptake. In support of this possibility, glucose uptake during
exercise in humans has recently been shown to be higher in the glycogen depleted
state only when the delivery of substrates and hormones remain constant (Steensberg,
Van Hall et al. 2002). At present, very little is known about the influence of muscle
glycogen concentration on insulin stimulated glucose uptake in humans. In a recent
investigation, insulin stimulated glucose disposal was significantly blunted in patients
with McArdle's glycogen storage disease who had chronically elevated muscle
glycogen concentrations (Nielsen, Vissing et al. 2002). Although an inverse
relationship between glycogen concentration and glucose utilization was demonstrated
in the above study, the authors were unable to conclude that blunted glucose uptake in
McArdle’s patients was as a result of glycogen concentration per se, rather than some
47
other regulatory factor linked to the pathogenesis of the disease. Experimental
evidence supporting a regulatory role of muscle glycogen on glucose uptake in non
patient populations is lacking, although some authors have suggested such links
(Richter, Derave et al. 2001). Thus insulin stimulated leg glucose uptake in healthy
men was shown to be positively correlated with the degree of glycogen depletion
during previous exercise when data from two separate studies was combined (Fig 1.1).
The emergence of muscle glycogen as a potential regulator of insulin stimulated
glucose uptake in humans represents an interesting area for further research into the
factors responsible for the regulation of skeletal muscle metabolism. The molecular
mechanisms by which glycogen may influence contraction and insulin stimulated
glucose uptake are currently unknown. Increased muscle glycogen availability results
in an increase in glycogen degradation during submaximal exercise as a result of
enhanced glycogen phosphorylase activity (Hespel and Richter 1992). It has been
suggested that ongoing glycogenolysis during exercise may increase G-6-P and thus
inhibit glucose phosphorylation and decrease or reverse the gradient for glucose
transport. Increased glycogenolysis during exercise also results in higher lactate
levels which may influence glucose transport by reducing muscle pH. Increases and
decreases in muscle pH have been shown to decrease Glut-4 intrinsic activity in rat
skeletal muscle plasma membrane (Kristiansen, Wojtaszewski et al. 1994).
48
Fig. 1.1. Correlation between the degree of muscle glycogen depletion during prior
exercise and insulin stimulated leg glucose uptake during a 120 min
hyperinsulinaemic / euglycaemic clamp initiated 3-4 hrs after exercise in healthy men.
n = 14. Figure reproduced with permission from Richter, Derave et. al., 2001.
49
A direct effect of glycogen on glucose transport has also been proposed. If Glut-4 is
structurally bound to glycogen, ongoing glycogenolysis during exercise may result in
the release of glucose transporters and increased availability for Glut-4 translocation
to the plasma membrane (Hargreaves 1995). Indeed, the increase in cell surface Glut-
4 induced by contractions has been shown to be augmented by 65% in glycogen
depleted when compared to glycogen supercompensated rat muscle (Kawanaka, Nolte
et al. 2000). The residual effects of Glut-4 release during glycogen depleting exercise
may also account for enhanced insulin action of muscle glucose uptake in the post
exercise period. For example, impaired insulin responsiveness of glucose uptake in
glycogen supercompensated rat muscle is mediated by translocation of fewer Glut-4
vesicles to the cell surface when compared to glycogen depleted exercised muscle
(Kawanaka, Han et al. 1999). Whilst the above findings suggest a link between the
glucose transporter and enhanced insulin action in glycogen depleted muscle, a
structural link between glycogen and Glut-4 awaits direct experimental evidence.
Furthermore, inhibition of insulin stimulated glucose uptake by high muscle glycogen
concentration has not always been shown to involve impaired Glut-4 translocation. In
a recent investigation, rats which were exercise trained for a period of two days before
receiving a normal diet (high glycogen) demonstrated no change in insulin stimulated
glucose uptake in spite of an approximately twofold increase in Glut-4 (Host, Hansen
et al. 1998). Thus whilst variations in Glut-4 are likely to be involved in the
mechanism by which glycogen affects insulin stimulated glucose uptake, the existence
of alternative mechanisms cannot be excluded.
50
Recent research has revealed that the apparent stimulatory affect of glycogen
concentration on glucose uptake and Glut-4 content in rat skeletal muscle is specific to
fast twitch muscle fibre types. Thus inhibition of glucose uptake by high glycogen
concentration was not apparent in the slow twitch soleus muscle (Derave, Lund et al.
1999). These findings have led to speculation that inhibition of glucose uptake may
only occur in muscles in which glycogen concentration exceeds 100 µmol/g wet
weight. The limited capacity of the slow twitch soleus muscle for glycogen storage
may therefore negate the possible regulatory effect of glycogen concentration on
glucose uptake in this tissue. An alternative explanation is that the apparent
regulatory role of glycogen on glucose uptake is truly fibre type specific and thus
restricted to glycolytic fibres. This possibility has led to speculation that it may not be
glycogen itself which regulates glucose uptake, but some other indicator of cellular
energy status which changes in concert with glycogen concentration (Derave, Lund et
al. 1999). Indeed a return to basal glucose transport has been demonstrated despite
the maintenance of low glycogen concentrations in the post exercise period,
suggesting an alternative mechanism in the regulation of glucose uptake. Any such
mechanism would need to be more active in fast twitch rather than slow twitch muscle
due to inherent differences between the muscle fibre types or simply because of
different glycogen concentrations (Derave, Lund et al. 1999). Recent research in this
area has indicated a role for 5'-AMP-activated protein kinase (AMPK) in contraction
stimulated glucose uptake (Ojuka, Nolte et al. 2000). AMPK is influenced by cellular
fuel status and thus variations in this signal may help to explain differential regulation
of glucose uptake with high and low glycogen concentrations. In support of this
51
possibility, higher basal levels of AMPK activity have been observed in rodent and
human muscle with lowered muscle glycogen content (Kawanaka, Nolte et al. 2000;
Richter 2001; Wojtaszewski, Jorgensen et al. 2002). Furthermore, pharmacological
activation of AMPK with AICAR results in enhanced insulin sensitivity of glucose
transport in incubated rodent muscle (Fisher, Gao et al. 2002). Recent findings also
suggest that the mechanism by which muscle glycogen affects insulin stimulated
glucose uptake and Glut-4 translocation may involve changes in insulin signalling.
For example, a reduction in muscle glycogen content in rodents has been shown to
result in increased activation of Akt by insulin (Derave, Hansen et al. 2000;
Kawanaka, Nolte et al. 2000). Whilst these findings suggest that glycogen effects on
insulin action may be mediated at least partially by AMPK / Akt, it is interesting to
note that activation of AMPK with AICAR results in enhanced insulin sensitivity of
glucose transport without measurable changes in glycogen content. Furthermore,
whilst Akt activity is increased in glycogen depleted rat muscle, post exercise
enhancement of insulin action does not appear to involve increases in Akt activity or
phosphorylation (Richter, Derave et al. 2001). Thus the mechanism via which muscle
glycogen concentration affects insulin stimulated glucose uptake in the post exercise
period remains equivocal.
52
In summary, a regulatory effect of muscle glycogen on insulin and contraction
stimulated glucose uptake in rats and contraction stimulated glucose uptake in humans
has been observed for many years. In spite of ongoing investigation, the effect of
muscle glycogen content on insulin stimulated glucose uptake in humans is yet to be
conclusively established. Furthermore, the molecular mechanisms via which
glycogen may influence insulin stimulated glucose uptake remain equivocal.
1.5 Aims of the thesis The aim of this thesis was to investigate the influence of muscle glycogen
concentration on insulin stimulated glucose uptake in humans and to examine the
potential signalling mechanisms responsible for enhanced insulin action in the post
exercise period.
The specific aims were;
To investigate the influence of glycogen concentration on insulin stimulated
glucose uptake 30 minutes after high or low intensity exercise.
To examine the influence of muscle glycogen on insulin stimulated glucose uptake
following glycogen depleting exercise and 24 hour dietary manipulation.
To investigate the effect of glycogen and insulin on proposed signalling
mechanisms responsible for enhanced glucose uptake and glycogen synthesis in
skeletal muscle.
53
54
CHAPTER 2
❖
GENERAL METHODOLOGY
The purpose of this thesis was to investigate the influence of muscle glycogen
concentration on insulin stimulated glucose uptake in humans and to examine the
potential signalling mechanisms responsible for enhanced insulin action in the post
exercise period. This chapter presents the general methodology and analytical
techniques employed in the series of studies conducted on the Melbourne campus of
Deakin University. All experimental procedures were approved by the Deakin
University Human Research Ethics Committee prior to the commencement of testing.
2.1 Subjects. Participants in all studies were non endurance trained volunteers recruited via
advertisements on university notice boards and in a local newspaper. Subjects read a
plain language statement detailing the anticipated benefits and potential risks
associated with participation before providing written consent. Each subject
completed a medical questionnaire, and where appropriate, passed a physical
examination before participation in any of the studies. None of the subjects had any
family history of impaired insulin action / diabetes. Subject particulars are presented
in the relevant chapters for each study.
55
2.2 Pre Experimental Procedures.
2.2.1 Peak Oxygen Consumption.
Peak oxygen consumption ( peak) was determined on a separate day at least
seven days prior to testing using a graded exercise test to volitional exhaustion on an
electronically braked cycle ergometer (Lode 1000, Lode Instruments, Groningen, The
Netherlands). Upon entering the laboratory, each subject’s weight and height were
recorded. A heart rate monitor was fitted, and ergometer seat and handle positions
were adjusted to individual specifications and maintained for all subsequent exercise
trials. Subjects rested quietly on the cycle ergometer for three min prior to the
initiation of exercise to allow resting heart rate and oxygen consumption to be
determined. Exercise was then initiated at a workload of 50 watts for a period of three
min, where after resistance was increased by 50 watts for every three-min period to
nine min. Where exercise exceeded nine min, workloads were increased by 25 watts
per min until fatigue. The criteria for the attainment of peak were a plateau in
(<100 ml.min
2OV&
2OV&
2OV& -1) with increasing work rate and a respiratory exchange ratio
(RER) > 1.15. Subjects were required to maintain a self selected pedal frequency,
which did not fall below 70 rpm throughout the peak test and subsequent
exercise trials. Peak was determined by averaging oxygen consumption values
from the final min of the test. Steady state values for oxygen consumption at each
workload were used to determine workloads for subsequent exercise trials based on a
linear regression between power output and oxygen uptake.
2OV&
2OV&
56
2.3 Experimental procedures.
2.3.1 Percutaneous muscle biopsy Where muscle biopsies were obtained in the resting state, subjects reported to the
laboratory after an overnight fast (10-12 hrs), and having abstained from alcohol,
tobacco, caffeine and exercise for a period of 24 hrs. Post exercise muscle biopsies
were obtained 30 min after exercise and immediately prior to the hyperinsulinaemic /
euglycaemic clamp. All samples were obtained under local anaesthesia from the
vastus lateralis using the percutaneous needle biopsy technique (Bergstrom 1962)
modified to include suction (Evans, Phinney et al. 1982). Biopsy needles containing
excised muscle tissue were immediately immersed in liquid nitrogen (< 10 sec).
Frozen muscle samples were then removed from biopsy needles and stored in liquid
nitrogen for later analysis.
2.3.2 Hyperinsulinaemic / euglycaemic clamp Upon entering the laboratory, subjects voided and a 30 mmol KCl tablet was
administered orally in order to prevent a decrease in plasma potassium concentration
during the clamp. Subjects rested in the supine position for a period of fifteen minutes
during which time one hand was wrapped in an electric heating pad to allow
arterialisation of venous blood subsequent to sampling. A 22G Jelco catheter for the
purpose of blood sampling was inserted into a dorsal hand vein on the heated hand
and kept patent with periodic injection of 0.9% saline. A resting blood sample was
then obtained and analysed to establish basal blood glucose concentration. Indwelling
Teflon catheters were inserted into two antecubital veins for the purpose of glucose
57
and insulin infusion. Insulin infusate was prepared in isotonic saline to which 2.5 ml
of the subjects’ blood per 50 ml of infusate was added in order to prevent absorption
of insulin to glassware and plastic surfaces. A single step hyperinsulinaemic /
euglycaemic clamp was then initiated via a one minute priming bolus of insulin
(6mU/kg) followed by a 119 minute continuous insulin infusion
(1.5 - 1.8mU.min-1.kg) designed to maintain plasma insulin concentration at ∼700
pmol·l-1. Insulin infusion was conducted using a syringe pump (IVAC P3000, IVAC
Medical systems, Hampshire, UK). Glucose infusion rate (GIR) was varied at five
minute intervals according to actual plasma glucose levels using a servocorrection
formula as reported elsewhere (DeFronzo, Tobin et al. 1979). Target glucose
concentration for the duration of all clamps was 5 mmol·l-1. Glucose was infused
using a peristaltic pump (IMED PC-1, IMED Corporation, San Diego, USA).
2.3.3 Blood sampling and treatment Venous blood samples taken at five minute intervals throughout the
hyperinsulinaemic / euglycaemic clamps were collected in heparinised syringes (1ml)
and whole blood was immediately analysed for glucose and lactate concentrations. At
10 min intervals, blood samples (0.5 ml) were collected in heparinised syringes,
transferred to labeled eppendorf tubes and kept on ice for later insulin analysis. Blood
samples (1.5 ml) collected at 30 min intervals were transferred to labeled eppendorf
tubes containing 30 µl of EGTA and reduced glutathione and kept on ice for
subsequent FFA determination. All blood samples were spun by centrifuge and
plasma was separated and stored at -80o C for later analysis.
58
2.4 Data Collection and Analysis
2.4.1 Collection of Data
Expired gases during the peak test and subsequent exercise trials were collected
and analysed using an Applied Electrochemistry Instruments (AEI) Metabolic Cart
calibrated to manufacturer specifications with commercial gases of known
composition before each trial. Briefly, O
2OV&
2 and CO2 gas analysers were manually
calibrated against alpha rated gases, accurate to ± 0.01% for CO2 and O2.
2.4.2 Analytic Methods Blood analysis. Plasma glucose was quantified using an automated glucose oxidase
method (EML 105, Radiometer, Copenhagen, DK) while plasma insulin analysis was
conducted via radioimmunoassay (Phadeseph®, Karbi & Pharmacia, Sweden / Human
Insulin Specific RIA Kit, Linco, St Charles, MO). Plasma FFA concentration was
quantified by an enzymatic colorimetric procedure using a commercially available kit
(NEFA C® WAKO, Richmond, VA). Blood lactate concentrations were determined
using an automated analyser (EML 105, Radiometer, Copenhagen, DK).
Muscle analysis. Wet muscle (12-15 mg) was freeze dried for approximately 24
hours, powdered, and divided into aliquots of 1mg and 2 mg for muscle glycogen and
metabolite analysis respectively. Muscle ATP, Cr and PCr was determined via a
flourometric technique (Harris, Hultman et al. 1974). Muscle glycogen concentration
was measured as glucose residues after acid hydrolysis using an enzymatic
flourometric technique (Passonneau and Lauderdale 1974). Glycogen concentration
59
was corrected to the highest total creatine (TCr) content for each subject. Protein
abundance / phosphorylation of p38 MAPK, Akt, IRS-1, GSK-3 and PKC isoforms α,
δ, ζ and θ was examined via immunoblotting of fractionated and whole tissue
preparations as detailed below.
Analysis of p38 MAPK and PKC isoforms. For study one, p38 MAPK protein
abundance / phosphorylation and protein abundance of PKC isoforms α, δ, θ and ζ/ι
were determined via immunoblotting of fractionated tissue preparations. Briefly, wet
muscle (20mg) was homogenised in 8X (Wt/Vol) ice-cold lysis buffer A [50mM Tris-
HCl, pH7.5, 1mM EDTA, 1mM EGTA, 1mM DTT, 50mM NaF, 5mM Na
Pyrophosphate, 2mM Na Orthovanadate, 10% glycerol, 5µL/mL protease inhibitor
cocktail (PIC), 1mM PMSF] in pre cooled ultra centrifuge tubes. Balanced tubes were
spun at 350,000xg for thirty minutes and the resultant supernatant (comprising the
cytosolic fraction) was decanted. The remaining pellet was physically disrupted and
resuspended in 100 – 200 µL ice cold lysis buffer B [buffer A containing 1% (v/v)
NP-40] before being placed on ice for 30 min. Tubes containing the pellet fraction
were then spun by centrifuge at 100,000xg for 60 min and the supernatant containing
solubilised protein (particulate fraction) was removed. Total protein concentration of
particulate and cytosolic fractions was determined using a detergent tolerant protein
assay kit (Pierce BCA) using a microtiter plate protocol. Muscle protein (42 µg) from
cytosolic and particulate fractions was loaded onto 8% separating gels and subjected
to electrophoresis (150 V, 90 min). Protein was transferred to nitrocellulose
membranes using a semi-dry technique (50 mA, 120 min). Membranes were placed in
60
blocking buffer [5% skim milk powder in Tris-buffered saline and 0.25% Tween
(TBST)] for 60 minutes, washed (3 × 10 min in TBST) and incubated over night (4oC)
in primary antibodies (PKC’s: Santa Cruz Biotechnology, Santa Cruz, CA; p38: Cell
Signalling Technology, Beverly, MA). For PKC analysis, membranes were placed in
anti-PKC primary antibodies diluted 1:500 in phosphate buffered saline (PBS). For
p38 protein analysis membranes were placed in anti-p38 antibody diluted 1:1000 in
5% BSA in TBST. The following day, membranes were washed (3 × 10 min) in
TBST and incubated (60 min) in horseradish peroxidase-conjugated secondary
antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:10,000 in blocking
buffer (PKC’s) or 5% BSA in TBST (p38 MAPK). Membranes were washed and
placed in chemiluminescent substrate (Pierce SuperSignal Chemiluminescent) for 60
sec before being exposed (Kodak Image Station). Band density was analysed using
Kodak 1D software. After quantification of p38 MAPK protein abundance, p38
membranes were placed in stripping buffer for 60 min. Membranes were then washed
(3 × 2 min in 1 × TBST) and placed in chemiluminescent substrate for 60 sec before
being re-exposed. Upon confirmation of successful stripping, membranes where
washed in 1 × TBST (5 min) and re-probed overnight (4oC) in anti-phosphotyrosine
p38 antibody. Phosphorylation of p38 was quantified the following day with
membranes treated in an identical manner to that described for measurement of p38
protein.
61
Analysis of p38 / IRS-1 / Akt and GSK 3. Wet muscle (approx. 15 mg) was
homogenised 1:10 (wt:vol) in ice-cold lysis buffer [20 mmol/l HEPES, 2 mmol/l
EGTA, 50 mmol/l β-glycerolphosphate, 1mmol/l DTT, 50mmol/l NaF, 5 mmol/l
Na4P2O7, 1mmol/l Na3VO4, 1% Triton X-100, 10% glycerol, 3mmol/l benzamidine,
10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mmol/l phenylmethonylsulphyl fluoride
(PMSF)] in pre cooled ultra centrifuge tubes. Homogenates were spun by centrifuge
(14,000g) for 15 minutes after which the supernatants were collected and stored at -
80o C for later analysis. Total protein concentration was determined using a detergent
tolerant protein assay kit (Pierce BCA) using a microtiter plate protocol. Muscle
lysate (60 µg) was loaded onto 8% separating gels and subjected to electrophoresis
(150 V, 60-90 min). Protein was transferred to nitrocellulose membranes using a wet
transfer technique (p38 & IRS-1: 100V-100 min / Akt & GSK3: 100V-60 min).
Membranes were placed in blocking buffer containing 5% skim milk in 1 × TBST
(p38 & IRS-1) or 5% BSA in 1 × TBST (Akt & GSK-3) for 60 minutes. After
removal from the blocking buffer, membranes were washed in 1 × TBST (3 × 10
min). For analysis of p38 MAPK protein abundance and IRS-1 phosphorylation,
membranes were cut at a molecular weight of 80 kDa making it possible to probe for
p38 / IRS on separate sections of the original membrane. Incubation of membranes
occurred over night (4oC) in either anti-p38 or anti-IRS pTyr612 antibodies (p38: Cell
Signalling Technology, Beverly, MA; IRS-1: Biosource, Camarillo, CA) diluted
1:1000 in 5% BSA in 1 × TBST. For analysis of Akt / GSK-3, membranes were
incubated over night (4oC) in either anti-Akt or anti-GSK-3 antibodies (Cell
62
Signalling Technology, Beverly, MA) diluted 1:1000 in 5% BSA in 1 × TBST with
the addition 0.01% sodium azide (NaN3). On the following day, membranes were
washed (4 × 5 min) in TBST and incubated (60 min) at room temperature in
horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology,
Santa Cruz, CA) diluted 1:10,000 in 5% BSA in 1 × TBST. Membranes were washed
(total 45 min) and placed in chemiluminescent substrate (Pierce SuperSignal
Chemiluminescent) for 60 sec before being exposed (Kodak Image Station). Band
density was analysed using Kodak 1D software. After quantification of p38 MAPK
protein abundance, p38 membranes were stripped and re-probed for p38
phosphorylation as detailed for study one.
2.4.3 Statistical Analysis Statistical analysis was conducted via a two way analysis of variance (ANOVA) with
repeated measures. Specific differences were identified via Newman-Keuls post hoc
analysis where appropriate. All statistical analysis was conducted using the
Biomedical Data Processing (BMDP) statistical software package and the Statistical
Package for the Social Sciences for Windows (SPSS version 11.0) on an IBM
compatible computer. The level of probability to reject the null hypothesis was P<
0.05. Values throughout this publication are reported as mean ± SE, where SE
(standard error) represents standard deviation of the distribution of sample means.
63
64
CHAPTER 3
❖
INFLUENCE OF MUSCLE GLYCOGEN ON POST-EXERCISE
INSULIN STIMULATED GLUCOSE DISPOSAL AND INSULIN
SIGNALLING IN HUMAN SKELETAL MUSCLE
3.1 Introduction A single bout of exercise has generally been shown to enhance the metabolic effect of
insulin on glucose uptake in the post exercise period. Whilst this phenomenon has
been observed for many years, the molecular mechanisms responsible for enhanced
insulin sensitivity following acute exercise remain equivocal. Several authors have
suggested that post exercise adaptations in insulin action may be dependent upon
prevailing glycogen concentrations. In agreement with this hypothesis, a regulatory
effect of glycogen on insulin stimulated glucose uptake in rat skeletal muscle is well
documented (Host, Hansen et al. 1998; Kawanaka, Han et al. 1999; Derave, Hansen et
al. 2000; Kawanaka, Nolte et al. 2000). Whether muscle glycogen also influences
glucose uptake in response to insulin in humans remains a topic of continuing debate.
Insulin stimulated glucose disposal has recently been shown to be significantly
blunted in patients with McArdle's glycogen storage disease who have chronically
65
elevated muscle glycogen concentrations. The authors of the above study were,
however, unable to conclude that impaired glucose disposal occurred as a result of
elevated muscle glycogen concentrations, rather than some other regulatory factor
linked to the pathogenesis of the disease. (Nielsen, Vissing et al. 2002). Experimental
evidence supporting a regulatory role of muscle glycogen on glucose uptake in non
patient populations is lacking, although some authors have suggested such links
(Richter, Derave et al. 2001). Thus insulin stimulated leg glucose uptake was shown
to be positively correlated with muscle glycogen degradation during prior exercise
when data derived from two separate studies were combined (Richter, Derave et al.
2001).
The mechanism via which muscle glycogen concentration may influence insulin
mediated glucose uptake is poorly understood, although recent observations suggest
that changes in insulin signalling may be involved. For example, insulin induced
activation of Akt in rat skeletal muscle is reduced in the glycogen supercompensated
and increased in the glycogen depleted state (Derave, Hansen et al. 2000; Kawanaka,
Nolte et al. 2000). These findings are perhaps difficult to reconcile with the
observation that enhanced insulin action 3-4 hours after exercise in humans is
apparently not related to adaptations in insulin signalling (Wojtaszewski, Hansen et al.
1997; Wojtaszewski, Hansen et al. 2000; Wojtaszewski, Nielsen et al. 2001). In spite
of this fact, no studies have sought to directly investigate the influence of muscle
glycogen on conventional insulin signalling in the immediate post exercise period. It
has recently been suggested that the p38 MAPK signalling pathway may be involved
66
in insulin stimulation of glucose uptake (Sweeney, Somwar et al. 1999; Somwar,
Perreault et al. 2000). Furthermore, prior exercise has been shown to increase basal
and insulin stimulated p38 phosphorylation in humans (Thong, Derave et al. 2003).
Whether the mechanism responsible for post exercise adaptations in p38 MAPK is
influenced by muscle glycogen concentration is unknown. The purpose of the current
investigation was to investigate the influence of muscle glycogen concentration on
whole body insulin stimulated glucose uptake and insulin signalling in human skeletal
muscle.
67
3.2 Materials and methods
Subjects. Participants in this study were eight untrained male volunteers (age 23.6 ±
4.6 (mean ± SE) yr, weight 72.8 ± 10.0 kg) recruited via advertisements on university
notice boards and in a local newspaper. Physical characteristics of the subjects are
detailed in table 3.1.
Experimental procedures. 2OV& peak was determined prior to the study via an
incremental exercise test to volitional exhaustion on an electromagnetically braked
cycle ergometer. At least seven days after the determination of peak, subjects
underwent a basal (BA) hyperinsulinaemic / euglycaemic clamp in order to determine
the resting rate of whole body insulin stimulated glucose uptake. Muscle biopsies
were obtained prior to initiation of the basal clamp and after 30 and 120 min of insulin
stimulation. Details of the peak and hyperinsulinaemic / euglycaemic clamp
procedures are contained in the General Methodology section of this thesis. At least
three days after the basal clamp, subjects were randomly allocated to either a high
(HI) or a low (LO) intensity cycle ergometer exercise condition. Subjects in HI
completed 45 min of continuous cycle ergometer exercise at approximately 74%
2OV&
2OV&
2OV&
peak, whilst subjects in LO completed an average of 110 min of cycle ergometer
exercise at approximately 38% peak. The total amount of external work
performed under HI and LO exercise conditions was identical. Immediately upon
cessation of exercise, subjects were transferred to an adjacent room where indwelling
catheters for the purpose of blood sampling and insulin and glucose infusion were
inserted into dorsal hand and antecubital veins. Basal blood glucose concentration
2OV&
68
was determined via a resting blood sample before subjects were subjected to a single
step hyperinsulinaemic / euglycaemic clamp as described previously. Muscle biopsies
were obtained immediately prior to the hyperinsulinaemic / euglycaemic clamps (30
min post exercise) and after 30 and 120 min of insulin stimulation. Four subjects
completed HI followed by LO conditions separated by a minimum of seven days with
the order of treatments reversed for the remaining four subjects in a cross over design.
Analytic techniques. Blood samples obtained throughout the clamp procedures were
analysed for plasma glucose, lactate, insulin and FFA. Muscle samples were analysed
for metabolites and muscle glycogen. Protein abundance / phosphorylation of p38
MAPK, IRS-1, Akt and GSK3 were determined via immunoblotting of whole tissue
preparations. The analytical techniques and statistical procedures employed for the
above analyses are detailed in the General Methodology section of this thesis.
69
3.3 Results Prior exercise was successful in producing significant differences in pre clamp muscle
glycogen concentrations between conditions (BA: 416 ± 37; HI 110 ± 27; LO 249 ±
26 mmol⋅kg-1 dw; P<0.05, Fig 3.1). The change in muscle glycogen concentration
across time during the hyperinsulinaemic / euglycaemic clamp was not statistically
significant in the BA and LO treatments. Muscle glycogen in HI was significantly
elevated following 120 min of insulin and glucose infusion when compared to rest (0).
The changes in muscle glycogen values within each condition during the
hyperinsulinaemic / euglycaemic clamp are presented in table 3.2. Plasma insulin was
not significantly different between conditions at rest or at any time point during the
study (Fig 3.2A). Primed continuous infusion of insulin resulted in a significant
(P<0.05) increase in plasma insulin concentration culminating in a steady state plateau
of hyperinsulinaemia (90-120 min) which averaged 727.4 ± 39.4, 658.1 ± 39.7 and
734.7 ± 43.5 pmol⋅l-1 in BA, HI and LO conditions respectively (Fig 3.2A). A
significant time by treatment interaction was observed for plasma lactate in the current
investigation (P<0.05). Post hoc analysis revealed that lactate in HI was significantly
(P<0.05) elevated at time 0 when compared to BA and LO (Fig 3.2B). No significant
differences were observed between HI and LO conditions for the remainder of the
clamp, however, plasma lactate in BA was significantly higher (P<0.05) at 70, 90 100,
110 and 120 minutes when compared to the other treatments (Fig 3.2B). The effect of
insulin infusion on plasma lactate over the duration of the clamp was not different
between treatment conditions. Two way analysis of variance revealed a significant
time by treatment interaction for plasma FFA (P<0.05). Further examination
70
determined that plasma FFA immediately prior to the hyperinsulinaemic /
euglycaemic clamp were significantly lower (P<0.05) in the BA condition (0.45 ±
0.08 mmol⋅l-1) when compared with HI (0.95 ± 0.18 mmol⋅l-1) and LO (0.85 ± 0.13
mmol⋅l-1). Plasma FFA concentrations decreased significantly (P<0.05) over the
duration of the clamp in all treatment conditions (Fig 3.2C). Plasma glucose over the
final 90 minutes of the hyperinsulinaemic / euglycaemic clamp was significantly
(P<0.05) higher in the HI condition when compared with BA and LO. Whole body
insulin stimulated glucose disposal was estimated from the glucose infusion rate in the
final 30 min of the hyperinsulinaemic / euglycaemic clamp and was not significantly
different between conditions (BA 9.11 ± 0.34, HI 8.86 ± 0.33 and LO 8.29 ± 0.29
mg⋅kg-1⋅min-1). Plasma levels of glucose, insulin, lactate and FFA and glucose
infusion rate are summarised in table 3.3.
No significant differences in p38 MAPK protein abundance or phosphorylation were
observed within or between conditions in the current investigation (Fig 3.3). Analysis
of variance on IRS-1 and Akt phosphorylation data identified a significant (P<0.05)
main effect for time indicating that insulin increased both IRS-1 and Akt
phosphorylation when the three conditions were combined (Fig 3.4). Phosphorylation
of IRS-1 and Akt were not significantly different at any time point between conditions
(Fig 3.4). When pre clamp (0) data was examined in isolation, a significant (P<0.05)
reduction in IRS-1 phosphorylation was apparent in HI when compared with BA but
not LO (Fig 3.5). GSK-3 phosphorylation in the LO condition was significantly
(P<0.05) higher at 0 when compared to BA and HI (Fig 3.6). Phosphorylation of
71
GSK-3 following 30 minutes of insulin stimulation was significantly (P<0.05)
elevated in HI when compared to BA and LO (Fig 3.6). Insulin infusion increased
GSK-3 phosphorylation (P<0.05) at 30 and 120 min when compared to 0 in the BA
and HI conditions (Fig 3.6). No effect of insulin on GSK-3 phosphorylation was
observed in the LO condition.
72
Table 3.1. Subject Characteristics
Age, yr 23.6 ± 4.6
Height, cm 177.6 ± 7.4
Weight, kg 72.8 ± 10.0
2OV& peak, l.min-1 3.66 ± 0.8
Values are means ± SE; n = 8. 2OV& peak, peak pulmonary oxygen consumption.
Table 3.2. Change in muscle glycogen concentrations during 120 min of steady state
insulin infusion.
BA
(0-120 min)
HI
(0-120 min)
LO
(0-120 min)
∆ glycogen
(mmol⋅kg dw-1)
-0.96 ± 12.62 53.36 ± 4.90* 1.98 ± 19.59
Values are means ± SE. BA, basal; HI, 30 min post high intensity exercise; LO, 30
min post low intensity exercise. *P<0.05 when compared with BA and LO.
73
Table 3.3. Plasma concentrations of glucose, insulin, lactate and FFA and glucose
infusion rate during steady state insulin infusion
BA
(90-120 min)
HI
(90-120 min)
LO
(90-120 min)
Glucose, mmol⋅l-1 5.0 ± 0.1 5.2 ± 0.1* 5.1 ± 0.0
Insulin, pmol⋅l-1 727.4 ± 39.4 658.1 ± 39.7 734.7 ± 43.5
Lactate, mmol⋅l-1 1.71 ± 0.06 1.31 ± 0.02 1.32 ± 0.03
FFA, mmol⋅l-1 0.06 ± 0.01 0.06 ± 0.02 0.07 ± 0.01
GIR, mg⋅kg-1⋅min-1 9.11 ± 0.34 8.86 ± 0.33 8.29 ± 0.29
Values are means ± SE; n = 8. GIR, glucose infusion rate; BA, basal; HI, 30 min post
high intensity exercise; LO, 30 min post low intensity exercise; Clamp,
hyperinsulinaemic / euglycaemic clamp. *P<0.05 compared with BA and LO.
74
Table 3.4. Muscle concentrations of ATP, PCr and Cr during 0, 30 or 120 min of
steady state insulin infusion
BA HI LO
0
min
30
min
120
min
0 min 30
min
120
min
0 min 30
min
120
min
ATP,
mmol.kg.dw-
1
22.36
±
2.01
21.74
±
1.90
22.22
±
1.73
25.60
± 4.06
25.65
± 4.73
22.96
±
1.49
24.93
± 5.24
27.46
± 7.92
24.07
±
3.01
PCr,
mmol.kg.dw-
1
84.87
±
4.30
81.71
±
5.76
81.84
±
7.79
103.57
±
18.41
101.03
±
17.24
97.66
±
20.14
107.78
±
37.37
102.17
±
38.14
98.65
±
29.81
Cr,
mmol.kg.dw-
1
48.41
±
6.10
51.58
±
5.24
51.45
±
6.26
49.48
±
11.59
52.02
± 8.07
55.39
±
6.74
55.49
±
19.40
61.10
±
17.91
64.63
±
27.00
Values are means ± SE; n = 8. BA, basal; HI, 30 min post high intensity exercise;
LO, 30 min post low intensity exercise.
75
BA HI LO0
100
200
300
400
500
*
**
Mus
cle
Gly
coge
n(m
mol
⋅ kg⋅ dw
-1)
Fig. 3.1. Pre clamp muscle glycogen concentrations in vastus lateralis muscles at
basal (BA), 30 min after high intensity exercise (HI) and 30 min after low intensity
exercise (LO). Data are presented as means ± SE; n = 8. dw, Dry wt. *P<0.05 when
compared to BA and HI. **P<0.05 when compared to BA and LO.
76
0
250
500
750
1000A
Plas
ma
Insu
lin (p
mol
⋅ l-1)
0
1
2
3
4
BAHILO
B
*****
#
Plas
ma
Lact
ate
(mm
ol⋅ l-1
)
0 30 60 90 120
0.0
0.5
1.0
1.5C
*
Plas
ma
FFA
(mm
ol⋅ l-1
)
Fig. 3.2. Plasma insulin (A), plasma lactate (B) and plasma free fatty acids (FFA:C)
during a hyperinsulinaemic / euglycaemic clamp (120 min) at basal (BA), 30 min after
high intensity exercise (HI) and 30 min after low intensity exercise (LO). Data are
means ± SE; n = 8. * P<0.05 compared with HI and LO; # P<0.05 compared with BA
and LO.
77
0.0
0.5
1.0
1.5A
p38
MAP
K ph
osph
oryl
atio
n(a
rbita
ry u
nits
)
BA HI LO
0.0
0.5
1.0
1.5
030120B
p38
MAP
K pr
otei
n(a
rbita
ry u
nits
)
Fig. 3.3. p38 MAPK phosphorylation (A) and protein abundance (B) following 0, 30
or 120 minutes of insulin stimulation in subjects at basal (BA), 30 min after high
intensity exercise (HI) and 30 min after low intensity exercise (LO). Data are means
± SE; n = 8.
78
0.0
0.5
1.0
1.5
2.0
2.5A
IRS-
1 pT
yr61
2 (arb
itary
uni
ts)
*
BA HI LO
0.0
0.5
1.0
1.5
030120B
Akt S
er47
3 (arb
itary
uni
ts)
*
Fig. 3.4. Phosphorylation of Akt (A) and IRS-1 (B) following 0, 30 or 120 minutes of
insulin stimulation in subjects at basal (BA), 30 min after high intensity exercise (HI)
and 30 min after low intensity exercise (LO). Data are means ± SE; n = 8. *P<0.05
main effect for time.
79
BA HI LO
0.0
0.5
1.0
1.5
*
IRS-
1 pT
yr61
2 (arb
itary
uni
ts)
Fig. 3.5. Pre clamp phosphorylation of IRS-1 in the basal condition (BA), 30 min
after high intensity exercise (HI) and 30 min after low intensity exercise (LO). Data
are presented as means ± SE; n = 8. dw, Dry wt. *P<0.05 when compared to BA.
80
Basal High Low
0
1
2
3030120*
**
*
+
#
GSK
-3α
/β S
er21
/9
(arb
itary
uni
ts)
Fig. 3.6. GSK-3 Phosphorylation following 0, 30 or 120 min of insulin stimulation in
basal (BA), 30 min post high intensity exercise (HI) and 30 min post low intensity
exercise (LO) conditions. Data are means ± SE; n = 8. *P<0.05 compared with 0;
+P<0.05 compared with basal and low; #P<0.05 compared with basal.
81
3.4 Discussion In a recent review, insulin stimulated leg glucose uptake 3-4 hrs after exercise was
shown to be positively correlated with muscle glycogen degradation during the prior
exercise bout (Richter, Derave et al. 2001). Whilst the mechanism responsible for this
effect could not be explained, changes in conventional insulin signalling were
apparently not involved. The current investigation sought to examine the influence of
muscle glycogen on insulin mediated glucose uptake and insulin signalling in the
immediate post exercise period. In contrast to previous findings, the results of the
present study suggest that muscle glycogen concentration does not affect insulin
stimulated glucose disposal. One interpretation of this finding is that the potential
regulatory influence of muscle glycogen on insulin mediated glucose uptake is time
dependent and thus present 3-4 hours but not 30 min after exercise. Indeed if the
mechanism via which glycogen affects glucose uptake is indirect, one might expect a
considerable lag time before effects on insulin action are observed. Such a
mechanism is unlikely to involve alterations in conventional signalling, given that no
changes in insulin signalling intermediates were previously observed 3-4 hours after
glycogen depleting exercise (Richter, Derave et al. 2001). Similarly, delayed effects
of glycogen on insulin mediated glucose uptake do not support a structural link
between glycogen and Glut-4 vesicles as such a mechanism would presumably allow
translocation of Glut-4 immediately upon glycogen breakdown. On the other hand, if
the mechanism via which muscle glycogen influences insulin action involves
increased synthesis of Glut-4 in glycogen depleted muscle, it is possible that effects
on insulin sensitivity would not be immediately apparent.
82
Whilst the contrasting results of the current and previous investigations may suggest a
time course effect of glycogen on insulin action, an alternative possibility is that the
effect of muscle glycogen on insulin stimulated glucose uptake is only apparent in the
previously active muscle. Thus a regulatory influence of muscle glycogen on post
exercise insulin action 3-4 hours after exercise is apparent using the leg glucose
uptake method, but is not discernable 30 min after exercise at a whole body level (see
chapter 5). On the other hand, evidence suggesting an inverse relationship between
muscle glycogen and insulin mediated glucose uptake 3-4 hours after exercise is
correlational and based on data from two separate studies (Wojtaszewski, Hansen et
al. 1997; Wojtaszewski, Hansen et al. 2000). Thus direct experimental evidence
suggesting a role for muscle glycogen in the process of insulin stimulated glucose
uptake in humans is still lacking, and such a relationship is not supported by the
findings of the current investigation. In light of these findings, it is possible that a
regulatory influence of muscle glycogen on insulin mediated glucose uptake may not
exist in humans. Indeed, several observations suggest that enhanced insulin mediated
glucose uptake is not always dependent upon a reduction in muscle glycogen (see
chapter 5). For example, recent findings in GSL3-transgenic mice suggest that muscle
glycogen may not influence insulin stimulated glucose uptake (Fogt, Pan et al. 2004).
Should muscle glycogen prove to be involved in regulating glucose uptake in response
to insulin, it is likely that the mechanism responsible for this effect will be
complicated to elucidate. This is partially due to the fact that it is difficult to isolate
the independent effects of glycogen from those of previous glycogen depleting
exercise. For example, acute exercise results in alterations in plasma hormone and
83
catecholamine levels as well as influencing the energy status of the cell. Plasma FFA
concentrations in the current investigation were significantly elevated following high
and low intensity exercise when compared to basal (Fig 3.2C). Elevated FFA
concentrations have previously been associated with impaired insulin action in a
variety of models (Schmitz-Peiffer 2000). Thus exercise induced alterations in FFA
levels may provide an additional explanation as to why the well documented
stimulatory effect of acute exercise on insulin mediated glucose uptake was not
observed in the present investigation. On the other hand, FFA concentrations were
rapidly normalised (30 min) and were not different between conditions at the time
when rates of whole body insulin mediated glucose disposal were determined (90-120
min). Thus it could be argued that FFA would not have influenced whole body
insulin action at this time point. Whilst catecholamines were not measured in the
present study, glycogen depleting exercise has previously been shown to result in
elevated concentrations of both adrenaline and nor-adrenaline (Wojtaszewski,
MacDonald et al. 2003). It is thus possible that alterations in catecholamine levels in
the immediate post exercise period may influence post exercise insulin action. In a
recent study, increased catecholamine concentrations could not be excluded as a
potential explanation for elevated AMPK activity in glycogen depleted human muscle.
Furthermore, in vivo insulin action / signalling has previously been shown to be
impaired immediately after strenuous exercise, possibly due to elevated catecholamine
and FFA levels (Tuominen, Ebeling et al. 1996; Del Aguila, Krishnan et al. 2000).
Thus exercise induced changes in catecholamine and hormone levels can clearly
influence substrate utilisation and signalling processes in skeletal muscle. Although
84
catecholamines were not measured in the current investigation, FFA concentrations
were not significantly higher in HI when compared to LO. Thus whilst FFA
concentrations are unlikely to be responsible for reduced IRS-1 phosphorylation
following high intensity exercise, an effect mediated by elevated catecholamine levels
can not be discounted. In spite of the mechanism involved, the post exercise
reduction in IRS-1 phosphorylation following HI in the current investigation was
rapidly normalised following 30 min of insulin stimulation. As such, alterations in
IRS-1 signalling probably did not impact upon GIR which was measured in the final
30 min of the hyperinsulinaemic / euglycaemic clamp. It was not possible to
determine whether the reduction in pre-clamp IRS-1 phosphorylation had any
functional significance on GIR during the first 30 min of insulin stimulation. This
was due to the fact that plasma glucose levels were quite variable between conditions
during this period, and steady state hyperinsulinaemia had not been achieved.
An exercise induced transient impairment of insulin action may provide a possible
explanation as to why muscle glycogen depletion and / or acute exercise did not
enhance insulin stimulated glucose disposal in the current investigation. It has
previously been suggested that contraction induced increases in intracellular G-6-P
may impair insulin action by impacting upon hexokinase activity. Under this proposal,
ongoing glycogenolysis during high intensity exercise increases G-6-P and impairs
hexokinase activity, thus inhibiting glucose phosphorylation and decreasing or
reversing the gradient for glucose transport (Katz, Broberg et al. 1986; Shulman
2000). Whether the mechanism responsible for this effect involves defects in the
85
insulin signalling pathway is yet to be conclusively established. Of interest in this
respect is the observation that pre-clamp IRS-1 phosphorylation was significantly
reduced in HI when compared with the BA and LO conditions. This finding suggests
that proximal insulin signalling may have been negatively influenced by high intensity
exercise. Such a suggestion is not unprecedented given that in vivo insulin action /
signalling has also been shown to be impaired immediately after strenuous exercise,
possibly due to elevated catecholamine and FFA levels (Tuominen, Ebeling et al.
1996; Del Aguila, Krishnan et al. 2000). It is also worth considering that exercise
which causes significant muscle damage (i.e. eccentric contractions) can lead to
impaired insulin action for as long as 24 hours after exercise in line with a significant
reduction in IRS-1 tyrosine phosphorylation (Del Aguila, Krishnan et al. 2000). Thus
it is possible that factors relating to the acute exercise bout in the current investigation
prevented the potential regulatory influence of muscle glycogen on insulin mediated
glucose uptake from being observed.
Plasma glucose concentrations during the final 30 minutes of the hyperinsulinaemic /
euglycaemic clamp in the current investigation were significantly higher during HI
when compared to BA and LO conditions. This finding was unexpected given that the
purpose of the variable rate glucose infusion was to maintain plasma glucose at the
same level for all conditions (≈ 5 mmol.l-1). The explanation as to why plasma
glucose was elevated during the HI condition most likely relates to technical
difficulties encountered when using the glucose analyser. These technical difficulties
resulted in slightly higher error in maintaining the required plasma glucose level
86
during two clamps in the HI condition. Whilst the increased level of plasma glucose
in the HI condition was statistically significant, the physiological significance of the
disparity between conditions is questionable given that plasma glucose was only 0.17
- 0.20 mmol.l-1 higher in HI when compared with the BA and LO conditions. In
actual fact, the marginally elevated glucose levels in the HI condition may serve to
strengthen the findings of this chapter given that GIR was not greater during the HI
condition (which had the lowest glycogen concentration) even when plasma glucose
was maintained at a slightly higher concentration. In interesting question posed by the
current investigation relates to the eventual fate of the glucose infused during the
hyperinsulinaemic / euglycaemic clamp. Whilst the amount of glucose infused over
the 120 min clamp duration was not significantly different between the three
conditions, this glucose appears only to have been stored as glycogen in HI. The end
point of the infused glucose in the BA and LO conditions is uncertain due to the fact
that whole body insulin stimulated glucose uptake, as measured in the present studies,
provides a measure of glucose uptake into active and non active muscle groups as well
as other tissues. It is interesting to note, however, that previous investigations which
have employed the hyperinsulinaemic / euglycaemic clamp technique have not
observed a significant increase in muscle glycogen concentration during clamp
conditions, even after exercise (Wojtaszewski, Dufresne et al. 1999). One possibility
is that the increase in muscle glycogen which occurred in the HI condition was related
to the independent effects of high intensity exercise on glycogen synthesis 30 min
after exercise, as discussed later in this chapter.
87
At a time when it has been suggested that many acute effects of exercise are no longer
present, no changes in insulin signalling in glycogen depleted muscle have been
observed. Thus insulin induced activation or phosphorylation of the insulin receptor,
IRS-1, IRS-1-associated PI 3-kinase, Akt and GSK3 were unchanged in previously
glycogen depleted muscle 3-4 hours after exercise (Wojtaszewski, Hansen et al. 1997;
Wojtaszewski, Hansen et al. 2000; Wojtaszewski, Nielsen et al. 2001). Even when
the effect of muscle glycogen was examined much earlier after exercise (30 min), the
current investigation also found proximal insulin signalling to be largely unchanged.
For example, insulin induced IRS-1 and Akt phosphorylation were not significantly
different in skeletal muscle expressing a range of glycogen concentrations. This
finding is in contrast to recent investigations which suggest that the mechanism
responsible for enhanced insulin sensitivity of muscle glucose uptake in glycogen
depleted rat skeletal muscle may be linked to alterations in Akt activity (Derave,
Hansen et al. 2000; Kawanaka, Nolte et al. 2000). Thus whilst Akt may be involved
in the mechanism of improved insulin sensitivity in glycogen depleted rats, such a
mechanism is not supported in humans. On the other hand, some signalling pathways
which may be involved in the potential regulatory influence of glycogen on insulin
mediated glucose uptake are less studied. It has recently been suggested that glycogen
dependent alterations in insulin action may be mediated by AMPK (Wojtaszewski,
Nielsen et al. 2002). For example, basal AMPK activity has recently been shown to
be elevated in the glycogen depleted when compared to the glycogen super-
compensated state, although the existence of glycogen independent mechanism in the
regulation of AMPK could not be excluded (Wojtaszewski, MacDonald et al. 2003).
88
The influence of AMPK activity on post exercise insulin mediated glucose uptake was
not examined in the above study and, to our knowledge, remains equivocal. Thus
AMPK may provide a candidate for further investigation into the potential regulatory
influence of muscle glycogen on insulin action in the post exercise period.
In light of the observation that proximal insulin signalling during hyperinsulinaemic /
euglycaemic clamp conditions is largely unaffected by glycogen, it is possible that
muscle glycogen may exert a regulatory influence on insulin action via an alternative
signalling pathway. It has recently been suggested that the p38 MAPK signalling
pathway may be involved in insulin stimulation of glucose uptake (Sweeney, Somwar
et al. 1999; Somwar, Perreault et al. 2000; Somwar, Koterski et al. 2002). In spite of
these observations, the influence of muscle glycogen on p38 signalling was previously
unexplored. The findings of the current investigation suggest that protein abundance /
phosphorylation of p38 MAPK is not altered in human skeletal muscle expressing
different glycogen concentrations. In contrast, we found no evidence to suggest that
either acute exercise and / or insulin stimulation alters p38 MAPK signalling. This
finding was somewhat unexpected given that acute exercise and insulin stimulation
have recently been shown to increase p38 phosphorylation in human skeletal muscle
(Thong, Derave et al. 2003). Interestingly, the insulin induced increase in p38
phosphorylation demonstrated in the above study was quite modest (1.2-1.3 fold).
Thus it is possible that the small sample size employed in the current investigation did
not allow us to detect a stimulatory effect of insulin on p38 signalling. Alternatively,
the disparity between the present and previous investigations may reflect the different
89
time courses employed. Thus an effect of exercise and / or insulin on p38
phosphorylation may be apparent 3 hrs but not 30 min after exercise. As previously
discussed, residual effects of the previous exercise bout have been shown to influence
signalling processes in skeletal muscle. FFA concentrations in the present
investigation were significantly higher following high and low intensity exercise when
compared to basal. It is thus possible that increased p38 signalling previously
observed 3-4 hrs after exercise (Thong, Derave et al. 2003) was masked by elevated
FFA levels 30 min after exercise in the current study. On the other hand, p38
phosphorylation has previously been shown to be elevated 15 minutes after a similar
duration and intensity of exercise as employed in the current study (Widegren, Jiang
et al. 1998). Presumably this effect occurred in spite of an exercise induced increase
in FFA levels, but unfortunately, FFA concentrations in the above study were not
reported. Whilst it could also be argued that elevated FFA levels may have impaired
the potential stimulatory effect of insulin on p38 phosphorylation, this does not
explain the absence of an effect of insulin in the basal condition. Thus our findings
are in contrast to previous investigations suggesting that p38 phosphorylation is
elevated by physiological insulin concentrations in humans (Thong, Derave et al.
2003). To our knowledge, the study conducted by Thong et. al. provided the first in
vivo data indicating physiological hyperinsulinaemia stimulates p38 MAPK signalling
in humans. It is worth considering that the potential stimulatory effect of insulin on
p38 MAPK and its relation to glucose transport in rats are still debated (Goodyear,
Chang et al. 1996; Fujishiro, Gotoh et al. 2001; Kawano, Ryder et al. 2001). Due to
the contrasting results of the present and previous investigations, we suggest that
90
further investigation is warranted in order to elucidate the effect of insulin on p38
MAPK signalling in humans. In spite of this fact, the findings of the current study
suggest that muscle glycogen concentration does not influence basal or insulin
stimulated p38 MAPK signalling in human skeletal muscle.
Insulin stimulated glycogen synthesis in human skeletal muscle has recently been
shown to be inversely correlated with muscle glycogen content, suggesting a potential
role for glycogen in the regulation of it’s own synthesis (Laurent, Hundal et al. 2000).
This mechanism most likely involves alterations in glycogen synthase (GS), since GS
activity is clearly also dependent on glycogen levels (Nielsen, Derave et al. 2001). It
has recently been hypothesised that glycogen may modulate insulin induced GS
activity via de-activation of GSK-3 (Nielsen, Derave et al. 2001). Via this hypothesis
increased GSK-3 serine phosphorylation would result in a reduction in GSK-3 activity
in glycogen depleted muscle, further leading to decreased phosphorylation and
subsequent activation of glycogen synthase. Whilst this is an attractive hypothesis,
changes in insulin mediated phosphorylation of GSK-3 have previously not been
observed 3-4 hours after exercise in glycogen depleted muscle. In contrast to previous
research, we found that GSK-3 phosphorylation was significantly elevated following
30 min of insulin stimulation in heavily (HI) but not moderately (LO) glycogen
depleted muscle. Thus we suggest that increased insulin mediated glycogen synthesis
in the immediate post exercise period may be related to elevated GSK-3
phosphorylation in glycogen depleted muscle. Whilst increased GSK-3 serine
phosphorylation would be expected to exert a positive regulatory influence on GS
91
activity, an obvious criticism of the current investigation is that GS activity was not
directly measured. Furthermore, the mechanism via which muscle glycogen may
influence insulin stimulated GSK-3 phosphorylation is unknown, since changes in Akt
signalling which have been suggested to regulate GSK-3 in glycogen depleted rat
muscle were not observed (see Chapter 5). An interesting finding of the current
investigation was that GSK-3 serine phosphorylation was significantly elevated 30
min after low intensity exercise when compared to basal. Intuitively, one might
expect that an exercise induced increase in GSK-3 serine phosphorylation would
decrease GSK-3 activity and ultimately result in increased glycogen synthase activity.
Indeed, a reduction in GSK-3 activity has recently been observed in previously
exercised rat skeletal muscle (Markuns, Wojtaszewski et al. 1999). Whilst exercise
was shown to reduce GSK-3 activity in the above study, this effect was not due to
increased serine phosphorylation, leading the authors to suggest an alternative
mechanism in the regulation of GSK-3 activation (Markuns, Wojtaszewski et al.
1999). The observation that serine phosphorylation of GSK-3 was increased
following low intensity exercise in the current study could suggest different
mechanisms for exercise induced reductions in GSK-3 activity between rat and human
models. Such an interpretation is highly unlikely given that GSK-3 serine
phosphorylation in the present investigation was not significantly increased following
high intensity exercise which resulted in substantially greater glycogen depletion.
Furthermore, whilst exercise has been shown to reduce GSK-3 activity in rat skeletal
muscle, recent findings suggest that neither high nor low intensity bicycle exercise de-
activates GSK-3 in humans (Wojtaszewski, Nielsen et al. 2001). To the contrary, a
92
small but significant increase in GSK-3α activity was observed following exercise in
the above study. It has also recently been suggested that GSK-3α activity in humans
is not affected by marked differences in muscle glycogen concentrations
(Wojtaszewski, Nielsen et al. 2002). In light of the above observations, it is likely that
the observed influence of low intensity exercise on GSK-3 phosphorylation reflects
random variability rather than a legitimate exercise effect.
In summary, the findings of the current investigation suggest that muscle glycogen
concentration does not influence insulin stimulated glucose uptake in the immediate
post exercise period in humans. Our results further indicate that conventional insulin
signalling to glucose uptake as well as signalling through the p38 MAPK cascade is
not influenced by muscle glycogen content. A potential explanation for this finding is
that high intensity exercise associated with the acute exercise bout resulted in transient
insulin resistance which prevented the potential regulatory influence of muscle
glycogen on insulin mediated glucose uptake and insulin signalling from being
observed. In support of this possibility, IRS-1 tyrosine phosphorylation in the current
investigation was significantly reduced 30 min after high intensity exercise. Finally,
although not consistent with observations made 3-4 hours after exercise, we suggest
that muscle glycogen influences insulin stimulated GSK-3 serine phosphorylation in
the immediate post exercise period. This finding suggests that enhanced insulin
sensitivity of muscle glycogen synthesis following glycogen depleting exercise may
be mediated via a pathway involving alterations in insulin stimulated GSK-3
93
phosphorylation. Due to the disparity between the findings of the current and
previous studies, further investigation is warranted regarding the molecular
mechanisms responsible for enhanced glycogen synthesis in glycogen depleted
muscle.
94
CHAPTER 4
❖
INFLUENCE OF MUSCLE GLYCOGEN CONCENTRATION ON
WHOLE BODY INSULIN STIMULATED GLUCOSE UPTAKE 24
HOURS AFTER EXERCISE
4.1 Introduction
For more than two decades, research has focused on the potential regulatory effect of
muscle glycogen on insulin stimulated glucose disposal in humans. In spite of this
fact, the role of glycogen in the process of insulin mediated glucose uptake remains
controversial. At the center of this controversy is the fact that it is difficult to
distinguish between the isolated effect of glycogen and that of previous glycogen
depleting exercise. Indeed, a potential criticism of study one in the current series of
investigations is that the influence of muscle glycogen on insulin mediated glucose
uptake was investigated in the immediate post exercise period. Recent findings in
patients with McArdle’s disease lend weight to the argument that muscle glycogen
influences insulin stimulated glucose uptake independent of prior exercise. In spite of
this finding, the authors of the above study were unable to conclude that impaired
glucose disposal occurred as a result of elevated muscle glycogen concentrations per
se, rather than some other regulatory factor linked to the pathogenesis of the disease.
(Nielsen, Vissing et al. 2002). Previous investigations in non-patient populations have
95
attempted to elucidate the influence of muscle glycogen on insulin stimulated glucose
uptake whilst minimising the confounding effects of prior exercise. The potential
regulatory effect of glycogen on insulin stimulated glucose uptake in these studies was
evaluated under hyperinsulinaemic / euglycaemic clamp conditions either 24 hrs
(Bogardus, Thuillez et al. 1983) or four days (Laurent, Hundal et al. 2000) after
exercise. Insulin mediated whole body glucose uptake in both investigations was
shown to be independent of the prevailing glycogen concentration. In the above
studies, glucose disposal in subjects with basal glycogen concentrations was compared
to that in the glycogen super-compensated (Laurent, Hundal et al. 2000) or glycogen
depleted (Bogardus, Thuillez et al. 1983) state. Thus neither study made a direct
comparison between whole body insulin stimulated glucose uptake in glycogen
depleted versus glycogen super-compensated muscle. Interestingly, insulin stimulated
glucose uptake in rats with low glycogen (∼90 mmol⋅kg-1 dw) has recently been
shown to be significantly greater than that in rats with high glycogen (∼ 435 mmol⋅kg-
1 dw), but not significantly different when compared to control rats with normal (∼
220 mmol⋅kg-1 dw) glycogen concentrations (Derave, Hansen et al. 2000). Whether
whole body insulin stimulated glucose disposal 24 hrs after exercise is differentially
regulated in glycogen depleted versus glycogen super-compensated muscle remains
equivocal. The aim of the current investigation was to examine the influence of
muscle glycogen concentration on whole body insulin stimulated glucose uptake 24
hours after exercise in humans.
96
4.2 Materials and methods
Subjects. Seven untrained males (age 23.0 ± 2.3 (mean ± SE) yr, weight 73.0 ± 2.8
kg) with no family history of impaired insulin action volunteered to participate in this
study. Physical characteristics of the subjects are detailed in table 4.1.
Experimental procedures. At least seven days prior to the study, 2OV& peak was
determined via an incremental exercise test to volitional exhaustion on an
electromagnetically braked cycle ergometer (Lode 1000, Lode Instruments,
Groningen, The Netherlands). Muscle glycogen concentrations were manipulated
before each experimental protocol by a combination of glycogen lowering exercise
and 24 hour dietary intervention. Subjects completed ∼90 min of continuous cycle
ergometer exercise at 70% 2OV& peak followed by thirty minutes of arm cranking
exercise at 25 watts. Arm cranking exercise was designed to minimize glycogen re-
synthesis in the legs by providing an alternative site for glucose disposal following
cycling exercise. Immediately upon cessation of arm cranking, subjects completed a
maximal number of one minute cycling bouts (7.0 ± 2.3) at 100% 2OV& peak
interspersed with 2 min rest periods. Fatigue was defined as the point where subjects
could no longer maintain a cadence of 70 rpm for one minute. In the ensuing 24 hrs,
participants consumed either a low carbohydrate (LCHO) or a high carbohydrate
(HCHO) diet designed to respectively decrease or increase muscle glycogen
concentrations. Both diets provided 100% of Recommended Daily Energy Intake
(RDEI) based on each subject’s body weight. For a 75kg male subject on the LCHO
diet, energy intake totaled 11,475 kJ with the relative contribution from energy
97
sources being 5% from CHO, 66% from fat and 29% from protein. For a subject of
the same weight on the HCHO diet, energy intake totaled 11,546 kJ with the relative
contribution from each energy source being 87% from CHO, 3% from fat and 10%
from protein. Diets were adjusted accordingly based on each individual’s body
weight in consultation with a qualified nutritionist. On the morning of each trial,
subjects reported to the laboratory after an overnight fast (10-12 hrs), and having
abstained from alcohol, tobacco, caffeine and exercise for a period of 24 hrs. A
resting muscle sample was obtained and indwelling catheters for the purpose of blood
sampling and insulin and glucose infusion were inserted into dorsal hand and
antecubital veins. Basal blood glucose concentration was determined via a resting
blood sample before subjects were subjected to a single step hyperinsulinaemic /
euglycaemic clamp as described previously. A second muscle biopsy was obtained on
completion of the hyperinsulinaemic / euglycaemic clamp.
Analysis of PKC isoforms. Activation of various PKC isoforms, most notably PKC θ,
α and ε has been associated with lipid induced insulin resistance in a variety of rodent
models (Schmitz-Peiffer 2000). Infusion of lipid during hyperinsulinaemic /
euglycaemic clamp conditions also causes insulin resistance in humans, an effect
which may be modulated by increased activation of PKC-βІІ and -δ isoforms (Itani,
Ruderman et al. 2002). An additional aim of the present investigation was to
determine whether overnight feeding of a high fat diet resulted in translocation of
PKC isoforms θ, α and δ from cytosolic to particulate fractions, which is considered
an indicator of PKC activation (Farese 2000). Somewhat paradoxically, some
98
isoforms of PKC have been implicated in the stimulatory effect of insulin on Glut-4
translocation. It has recently been suggested that PKC ζ specifically associates with
Glut-4 compartments in response to insulin in rats, resulting in translocation of PKC ζ
and Glut-4 to the plasma membrane as a complex (Braiman, Alt et al. 2001). In
contrast, insulin stimulation in humans apparently does not result in translocation of
PKC ζ to the plasma membrane (Itani, Ruderman et al. 2002). The current
investigation sought to further examine the potential for translocation of PKC
isoforms from cytosolic to particulate fractions with insulin stimulation in human
skeletal muscle. The effect of muscle glycogen concentration on PKC localisation
within skeletal muscle in humans was also examined.
Analysis of p38 MAPK. Several authors have suggested that enhanced insulin
sensitivity of skeletal muscle after exercise in humans may be linked to adaptations in
the p38 MAPK signalling pathway. For example, insulin induced augmentation of
p38 MAPK phosphorylation three hours after exercise is higher in the previously
exercised when compared to the inactive limb (Thong, Derave et al. 2003). Recent
findings also suggest that inhibition of p38 MAPK signalling may reduce insulin
stimulated glucose uptake by impairing the intrinsic activity of the Glut-4 transporter
(Sweeney, Somwar et al. 1999; Somwar, Koterski et al. 2002). Since muscle
glycogen may also mediate post exercise insulin action, the current study investigated
the effect of glycogen on total p38 MAPK protein content and phosphorylation 24 hrs
after exercise and during subsequent insulin stimulation. Examination of the available
fractionated muscle preparations also allowed us to determine whether changes in
99
subcellular localisation of p38 may occur in response to glycogen depleting exercise
and / or insulin.
Analytic techniques. Blood samples obtained throughout the clamp procedure were
analysed for plasma glucose, lactate, insulin and FFA. Basal and post clamp muscle
samples were analysed for metabolites and muscle glycogen. Protein abundance /
phosphorylation of p38 MAPK and activity of PKC isoforms α, δ, θ and ζ/ι was
examined via immunoblotting of fractionated tissue preparations. Due to insufficient
sample size for one subject, analysis of PKC and p38 MAPK was performed on six of
the seven subjects who initially participated in the study. Total p38 MAPK protein
abundance and phosphorylation was calculated based on the amount of protein present
in particulate and cytosolic fractions when expressed as a percentage of total protein
in each muscle extract. Analytical techniques and statistical procedures employed in
this study are detailed in Chapter 2.
100
4.3 Results The combination of glycogen depleting exercise and dietary intervention was
successful in producing significant differences in pre clamp muscle glycogen
concentrations between LG and HG (LG: 171 ± 13 vs. HG: 293 ± 33 mmol⋅kg-1 dw;
P<.05, Fig 4.1). Infusion of insulin and glucose resulted in an increase in muscle
glycogen concentration over the duration of the clamp which measured 22% in LG
and 18% in HG. The increase in glycogen concentrations from pre to post clamp in
both conditions was not statistically significant. Delta glycogen values during the
hyperinsulinaemic / euglycaemic clamp for each condition are presented in table 4.2.
Plasma insulin, plasma glucose, plasma FFA and blood lactate were not significantly
different between conditions at rest or at any time point during the study (Fig 4.2).
Primed continuous infusion of insulin resulted in a significant (P<.05) increase in
plasma insulin concentration culminating in a steady state plateau of
hyperinsulinaemia (90-120 min) which averaged 341.90 ± 10.79 pmol⋅l-1 in LG and
321.37 ± 13.53 pmol⋅l-1 in HG. Plasma lactate increased significantly over the
duration of the clamp by 26% in HG and by 30% in LG (P<.05). Plasma
concentrations of FFA decreased significantly (P<.05) from 0.58 ± 0.09 to 0.08 ± 0.02
and 0.75 ± 0.11 to 0.10 ± 0.02 mmol⋅l-1 over the duration of the clamp in the HG and
LG conditions respectively. Whole body insulin stimulated glucose uptake as
determined by glucose infusion rate (GIR) in the final 30 min of the
hyperinsulinaemic / euglycaemic clamp was not significantly different between LG
101
and HG conditions (LG: 5.95 ± 0.50 vs. HG 6.25 ± 1.32 mg⋅kg-1⋅min-1). Data for
plasma insulin, plasma glucose, plasma FFA, blood lactate and GIR are summarised
in table 4.3.
Protein abundance of PKC isoforms in particulate and cytosolic fractions and PKC
density ratios are presented in figures 4.3, 4.4, 4.5 and 4.6. PKC density ratio was
calculated as particulate protein abundance divided by cytosolic protein abundance
and provides a measure of PKC fractional activity (Farese 2000). Density ratio of
PKC isoforms α, δ, θ and ζ/ι was not significantly different in the LG and HG
conditions prior to initiation of the hyperinsulinaemic / euglycaemic clamp. Prime
continuous infusion of insulin (120 min) did not significantly alter density ratio of
PKC isoforms α, δ, θ and ζ/ι in either condition. No significant differences in
particulate or cytosolic protein abundance of PKC isoforms α, δ, or θ where observed
prior to or following insulin stimulation. Cytosolic protein abundance of PKC ζ/ι was
not significantly different between treatments before or after insulin stimulation (Fig
4.4B). Protein abundance of PKC ζ/ι in the particulate fraction was significantly
lower following insulin stimulation when compared to basal in the HG condition (Fig
4.4A).
No significant differences in total p38 MAPK protein abundance were observed
within or between conditions in the current investigation (Fig 4.9). Furthermore,
protein abundance of p38 MAPK in particulate and cytosolic fractions and p38 protein
density ratio was not different between conditions before or after insulin stimulation
102
(Fig 4.8). A significant time by treatment interaction was observed for total p38
phosphorylation in the present investigation (4.9A). Post hoc analysis revealed no
significant difference between treatments at basal, however, p38 phosphorylation was
significantly higher following insulin stimulation in LG (Fig 4.9A). In spite of this
finding, insulin stimulation did not significantly alter total p38 phosphorylation in
either condition.
Phosphorylation of p38 in the particulate fraction (Fig 4.8A) and p38 phosphorylation
density ratio (Fig 4.8C) were not significantly different between conditions prior to or
following the hyperinsulinaemic / euglycaemic clamp. A significant time by
treatment interaction (P<.05) was observed for p38 phosphorylation in the cytosolic
fraction (Fig 4.8B). In spite of this finding, post hoc analysis did not reveal any
significant differences in p38 phosphorylation within or between conditions.
103
Table 4.1. Subject Characteristics
Age, yr 23.0 ± 2.3
Height, cm 178.0 ± 2.4
Weight, kg 73.0 ± 2.8
2OV& peak, l.min-1 3.3 ± 0.2
Values are means ± SE; n = 7. 2OV& peak, peak pulmonary oxygen consumption.
Table 4.2. Change in muscle glycogen concentrations during 120 min of steady state
insulin infusion.
LG Clamp (0-120 min)
HG Clamp (0-120 min)
∆ glycogen (mmol⋅kg dw-1) 38.42 ± 20.75 52.60 ± 43.07
Values are means ± SE n = 7. HG, high glycogen; LG, low glycogen. Clamp,
hyperinsulinaemic / euglycaemic clamp.
104
Table 4.3. Plasma concentrations of glucose, insulin, lactate and FFA during steady
state insulin infusion
LG Clamp
(90-120 min)
HG Clamp
(90-120 min)
Glucose, mmol⋅l-1 4.98 ± 0.03 4.83 ± 0.03
Insulin, pmol⋅l-1 341.90 ± 10.79 321.37 ± 13.53
Lactate, mmol⋅l-1 1.08 ± 0.03 1.46 ± 0.07
FFA, mmol⋅l-1 0.10 ± 0.01 0.10 ± 0.01
GIR, mg⋅kg-1⋅min-1 5.95 ± 0.50 6.25 ± 1.32
Values are means ± SE; n = 7. GIR, glucose infusion rate; LG, low glycogen; HG,
high glycogen; Clamp, hyperinsulinaemic / euglycaemic clamp.
105
Table 4.4. Muscle concentrations of ATP, PCr and Cr before (0 min) and after
(120 min) a hyperinsulinaemic / euglycaemic clamp.
LG Clamp HG Clamp
0 min 120 min 0 min 120 min
ATP
mmol.kg.dw-1
20.43 ± 2.70 20.83 ± 3.38 19.2 ± 3.1 19.28 ± 3.10
PCr
mmol.kg.dw-1
86.92 ± 13.38 87.04 ± 10.35 78.80 ± 10.37 81.29 ± 7.65
Cr
mmol.kg.dw-1
43.06 ± 6.76 42.37 ± 10.56 40.64 ± 7.06 38.15 ± 9.87
Values are means ± SE; n = 7. LG, low glycogen; HG, high glycogen; Clamp,
hyperinsulinaemic / euglycaemic clamp.
106
HG LG
0
100
200
300
400
Mus
cle
Gly
coge
n(m
mol
⋅ kg⋅ dw
-1)
*
Fig. 4.1. Pre clamp muscle glycogen concentrations in vastus lateralis muscles
conditioned to obtain high glycogen (HG) and low glycogen (LG). Data are presented
as means ± SE; n = 7. dw, Dry wt. *P<0.05, significant difference compared with
HG.
107
0.0
0.5
1.0
1.5
2.0
2.5A
Lact
ate
(mm
ol ⋅
l-1)
0.00
0.25
0.50
0.75
1.00
HGLG
B
Plas
ma
FFA
(mm
ol⋅ l-1
)
0 20 40 60 80 100 120
0
100
200
300
400C
Time (min)
Plas
ma
Insu
lin (
pmol
⋅ l)
Fig. 4.2. Plasma lactate (A), plasma free fatty acids (FFA;B) and plasma insulin (C)
during a hyperinsulinaemic / euglycaemic clamp (120 min) in subjects pre-
conditioned to obtain high glycogen (HG) and low glycogen (LG). Data are means ±
SE; n = 7.
108
0
1
2
3
4 A
Parti
cula
te P
KCδ
abun
danc
e (A
U)
0
1
2
3
4
BasalInsulin
B
Cyt
osol
ic P
KCδ
abun
danc
e (A
U)
HG LG
0
1
2
3
4 C
PKC
δ de
nsity
ratio
(AU
)
Fig. 4.3. Protein abundance of PKCδ in particulate (A) and cytosolic (B) fractions
and particulate / cytosolic density ratio (C) in subjects conditioned to obtain high
glycogen (HG) and low glycogen (LG) before (Basal) and after (Insulin) a
hyperinsulinaemic / euglycaemic clamp (120 min). Data are presented as means ±
SE; n = 6.
109
0.0
2.5
5.0A
*
Parti
cula
te P
KCζ /
ιab
unda
nce
(AU
)
0.0
2.5
5.0B
BasalInsulin
Cyt
osol
ic P
KCζ /
ιab
unda
nce
(AU
)
HG LG
0
1
2C
PKC
ζ /ι d
ensi
ty ra
tio (A
U)
Fig. 4.4. Protein abundance of PKCζ/ι in particulate (A) and cytosolic (B) fractions
and particulate / cytosolic density ratio (C) in subjects conditioned to obtain high
glycogen (HG) and low glycogen (LG) before (Basal) and after (Insulin) a
hyperinsulinaemic / euglycaemic clamp (120 min). Data are presented as means ±
SE; n = 6. * P<.05 compared with basal.
110
0.0
0.5
1.0
1.5A
Parti
cula
te P
KCα
abun
danc
e (A
U)
0
1
2
3
4B
BasalInsulin
Cyt
osol
ic P
KCα
abun
danc
e (A
U)
HG LG
0.00
0.25
0.50
0.75
1.00 C
PKC
α d
ensi
ty ra
tio (A
U)
Fig. 4.5. Protein abundance of PKCα in particulate (A) and cytosolic (B) fractions
and particulate / cytosolic density ratio (C) in subjects conditioned to obtain high
glycogen (HG) and low glycogen (LG) before (Basal) and after (Insulin) a
hyperinsulinaemic / euglycaemic clamp (120 min). Data are presented as means ±
SE; n = 6.
111
0.0
2.5
5.0
7.5A
Parti
cula
te P
KCθ
abun
danc
e (A
U)
0
1
2
3
BasalInsulin
Cyt
osol
ic P
KCθ
abun
danc
e (A
U) B
HG LG
0
5
10C
PKC
θ de
nsity
ratio
(AU
)
Fig. 4.6. Protein abundance of PKCθ in particulate (A) and cytosolic (B) fractions
and particulate / cytosolic density ratio (C) in subjects conditioned to obtain high
glycogen (HG) and low glycogen (LG) before (Basal) and after (Insulin) a
hyperinsulinaemic / euglycaemic clamp (120 min). Data are presented as means ±
SE; n = 6.
112
0
1
2
3
4A
Parti
cula
te p
38 p
rote
inab
unda
nce
(AU
)
0
5
10
BasalInsulin
B
Cyt
osol
ic p
38 p
rote
inab
unda
nce
(AU
)
HG LG
0.0
0.1
0.2
0.3
0.4C
P38
prot
ein
dens
ity ra
tio (A
U)
Fig. 4.7. Protein abundance of p38 MAPK in particulate (A) and cytosolic (B)
fractions and particulate / cytosolic density ratio (C) in subjects conditioned to obtain
high glycogen (HG) and low glycogen (LG) before (Basal) and after (Insulin) a
hyperinsulinaemic / euglycaemic clamp (120 min). Data are means ± SE; n = 6.
113
0
1
2
3
4Pa
rticu
late
p38
phos
phor
ylat
ion
(AU
) A
0.0
2.5
5.0
7.5
BasalInsulin
Cyt
osol
ic p
38ph
osph
oryl
atio
n (A
U) B
HG LG
0.00
0.25
0.50
0.75
1.00C
P38
phos
phor
ylat
ion
dens
ity ra
tio (A
U)
Fig. 4.8. Phosphorylation of p38 MAPK in particulate (A) and cytosolic (B) fractions
and particulate / cytosolic density ratio (C) in subjects pre-conditioned to obtain high
glycogen (HG) and low glycogen (LG) before (Basal) and after (Insulin) a
hyperinsulinaemic / euglycaemic clamp (120 min). Data are means ± SE; n = 6.
114
0.0
2.5
5.0
7.5
p38
MAP
Kph
osph
oryl
atio
n (A
U) A
*
0.0
2.5
5.0
7.5
BasalInsulin
B
p38
MAP
K pr
otei
nab
unda
nce
(AU
)
HG LG
0.0
0.5
1.0
1.5C
p38
phos
phor
ylat
ion
dens
ity ra
tio (A
U)
Fig. 4.9. Total p38 MAPK phosphorylation (A), protein abundance (B) and
phosphorylation / protein ratio in subjects pre-conditioned to obtain high glycogen
(HG) and low glycogen (LG) before (Basal) and after (Insulin) a hyperinsulinaemic /
euglycaemic clamp (120 min). Data are means ± SE; n = 6. * P<.05 compared to HG.
115
4.4 Discussion The findings of the current investigation suggest that muscle glycogen concentration
in the range seen in this study does not affect glucose uptake in response to
physiological hyperinsulinaemia 24 hrs after exercise. These findings are in agreement
with previous studies which found that insulin stimulated whole body glucose uptake
in humans is not affected by glycogen concentration 24 hrs (Bogardus, Thuillez et al.
1983) or four days after exercise (Laurent, Hundal et al. 2000). The results of the
present and previous investigations are in contrast to a number of studies which have
identified a regulatory effect of glycogen concentration on insulin mediated glucose
uptake in rats (Nolte, Gulve et al. 1994; Host, Hansen et al. 1998; Kawanaka, Han et
al. 1999; Derave, Hansen et al. 2000; Kawanaka, Nolte et al. 2000). Based on these
findings, it would appear that the potential regulatory effect of glycogen concentration
on insulin mediated glucose uptake is blunted in humans when compared with isolated
rat muscle preparations. It is possible that methodological differences when
comparing rat and human studies may be at least partially responsible for this effect.
For example, previous studies which have examined the effect of muscle glycogen on
insulin mediated glucose uptake in rats have employed super-physiological insulin
concentrations (Host, Hansen et al. 1998; Kawanaka, Han et al. 1999). In contrast,
insulin concentrations in the current investigation were within the lower physiological
range (≈ 350 pmol⋅l-1) and were in fact lower than intended for this study. It is
therefore possible that the potential regulatory influence of muscle glycogen on
insulin stimulated glucose uptake may only be apparent in response to non-
physiological insulin concentrations or at least hyperinsulinaemia in the higher
116
physiological range. This hypothesis is further discussed in Chapter 5. An alternative
possibility is that muscle glycogen concentration in the HG condition in our
investigation was not high enough to inhibit glucose uptake in comparison with the
LG trial. Previous studies which have employed a combination of glycogen depleting
exercise and high carbohydrate diet in active but untrained subjects have recorded
glycogen loaded values in the vicinity of 400 mmol⋅kg-1 dw (Hargreaves, McConell et
al. 1995; Steensberg, Van Hall et al. 2002). Furthermore, recent investigations in
which glycogen concentration was shown to influence insulin stimulated glucose
uptake in rats have demonstrated glycogen super-compensation in the range of ∼350
to 450 mmol⋅kg-1 dw (Host, Hansen et al. 1998; Derave, Hansen et al. 2000;
Kawanaka, Nolte et al. 2000). Muscle glycogen concentration in the HG condition in
our study measured 293 ± 33 mmol⋅kg-1 dw and as such may not have been high
enough to inhibit glucose uptake. Partial support for this possibility is provided by a
recent investigation in which rats were conditioned to achieve low, normal and high
glycogen concentrations. In this study, insulin stimulated glucose uptake in rats with
low glycogen (∼90 mmol⋅kg-1 dw) was shown to be significantly greater than that in
rats with high glycogen (∼ 435 mmol⋅kg-1 dw), but not significantly different when
compared to control rats with normal (∼ 220 mmol⋅kg-1 dw) glycogen concentrations
(Derave, Hansen et al. 2000). These findings raise the possibility of a threshold level
of glycogen, which must be present in skeletal muscle before glucose uptake is
inhibited. Whilst this is an attractive hypothesis, further research would be required in
order to confirm the existence of such a mechanism.
117
In contrast to study one in the current series, muscle glycogen concentration did not
influence insulin stimulated glycogen synthesis during the hyperinsulinaemic /
euglycaemic clamp procedure. This raises the possibility that glycogen re-synthesis
after strenuous exercise is intimately related to the prior exercise bout and may not be
directly regulated by glycogen concentration. On the other hand, we previously
suggested that the magnitude of glycogen depletion / super-compensation may be an
important factor in determining the effect of muscle glycogen on insulin action. It is
thus possible that the level of muscle glycogen depletion in the LG condition was not
sufficient to upregulate glycogen synthesis to a greater extent than that observed in
HG. The increased glycogen synthesis observed in study one occurred in muscle with
a substantially lower glycogen content (110 ± 27 mmol⋅kg-1 dw) than that observed in
the current investigation (170 ± 17 mmol⋅kg-1 dw). Thus whilst glycogen depletion
may enhance insulin mediated glycogen synthesis, the magnitude of reduction in
glycogen concentration is seemingly important. Thus the findings of the present
investigation are consistent with the existence of a threshold effect of muscle
glycogen on insulin action in human skeletal muscle.
The aim of the present investigation was to manipulate muscle glycogen in such a
fashion as to create different glycogen levels between conditions prior to the
hyperinsulinaemic / euglycaemic clamp. In spite of this intention, it could be argued
that the absolute difference in glycogen concentrations between conditions was not
sufficient for an effect on insulin action to be observed. At present it is unclear why
118
the experimental protocol employed in the current investigation did not lead to higher
muscle glycogen concentrations in the HG trial. In a previous study employing a
virtually identical protocol, muscle glycogen concentration in HG was ∼40% higher
than in the current investigation (Hargreaves, McConell et al. 1995). One possibility
would be to extend the 24 hour HCHO diet employed in HG to 48 hours in order to
allow additional time for glycogen synthesis. We have previously observed muscle
glycogen concentration to be ∼30% higher when glycogen depleting exercise was
followed by a 48 rather than a 24 hour HCHO intervention (Hargreaves, McConell et
al. 1995). It is important to note, however, that increasing the length of the dietary
intervention to 48 hours for both HG and LG conditions may lead to the undesired
result of higher glycogen concentrations in LG. Whilst the magnitude of muscle
glycogen depletion in the current investigation was not as great as that demonstrated
in study one, the obvious difference was that subjects in this study were allowed an
overnight feeding period before the first biopsy was obtained. Even though this
period was associated with carbohydrate deprivation, some degree of glycogen re-
synthesis was inevitable. Thus it is difficult to conceive a method via which muscle
glycogen could have been further lowered, especially since the exercise protocol
completed in the current investigation was substantially more demanding than that
employed in study one.
119
Overall, our results suggest that muscle glycogen concentration does not influence
insulin stimulated glucose uptake or glycogen synthesis 24 hrs after exercise in
humans. It is possible that the difference in glycogen concentrations between
conditions in the current investigation was not sufficiently pronounced to allow the
potential regulatory effect of muscle glycogen on insulin action to be observed.
Furthermore, any effect of muscle glycogen concentration on insulin mediated glucose
disposal may be apparent only as a localised effect which is not discernable at a whole
body level. This hypothesis will be further discussed in Chapter 5.
Analysis of PKC’s and p38 MAPK.
In a recent study, lipid infusion during hyperinsulinaemic / euglycaemic clamp
conditions caused insulin resistance concomitant with activation of PKC-βІІ and -δ
isoforms in humans (Itani, Ruderman et al. 2002). We sought to investigate whether
the absence of an effect of glycogen depletion on insulin mediated glucose disposal in
the current investigation may have been related to changes in PKC activity induced by
overnight high fat feeding. High fat feeding prior to the hyperinsulinaemic /
euglycaemic clamp in LG did not significantly increase basal activity of PKC
isoforms α, δ, θ and ζ/ι when compared with the HG trial. These findings suggest that
simply elevating plasma FFA is not sufficient to influence PKC activity. Whilst this
finding is in contrast to previous research (Itani, Ruderman et al. 2002), the difference
in basal FFA concentration between conditions in the current investigation was
minimal and did not reach the level of statistical significance. It is thus conceivable
that FFA’s would need to be further elevated before any effect on basal PKC activity
120
could be observed. The current investigation also sought to examine the hypothesis
that changes in subcellular localisation of PKC isoforms may occur in response to
glycogen concentration and / or insulin stimulation. PKC density ratio for all
isoforms studied was not different between conditions suggesting that isoform
localisation was not affected by glycogen or insulin. In spite of this finding, protein
abundance of PKC ζ/ι in the particulate fraction was significantly reduced with insulin
when compared to basal in the HG condition. This was somewhat unexpected given
that PKC ζ has been suggested to translocate to the plasma membrane in response to
insulin (Braiman, Alt et al. 2001). It is also difficult to conceive that insulin would
cause a reduction in PKC ζ/ι in the particulate fraction without influencing cytosolic
protein abundance or density ratio. Thus we suggest that the reduction in PKC ζ/ι
with insulin stimulation in the HG condition may represent random variation possibly
related to the small sample size employed in the present study. In summary the
findings of the present investigation indicate that overnight feeding of a high fat diet
does not influence PKC activity in humans. We further suggest that PKC isoforms are
not translocated from the cytosolic to the particulate fraction in response to insulin
stimulation in human skeletal muscle expressing different glycogen concentrations.
In a previous investigation, p38 MAPK phosphorylation three hours after exercise was
shown to be higher before and after insulin stimulation in the previously exercised
when compared to the inactive limb (Thong, Derave et al. 2003). The authors of the
above study thus postulated that p38 may be involved in the increased insulin
sensitivity of skeletal muscle seen after exercise. The p38 MAPK signalling pathway
121
has also recently been linked to increased intrinsic activity of the Glut-4 transporter
(Sweeney, Somwar et al. 1999; Somwar, Kim et al. 2001; Somwar, Koterski et al.
2002). Given that muscle glycogen concentration has also been suggested as an
important mediator of post exercise insulin action, we investigated the effect of
glycogen concentration on p38 MAPK protein / phosphorylation at rest and following
insulin stimulation in the available fractionated muscle preparations. Consistent with
the findings of study one, no significant differences in p38 protein were observed
between or within conditions in the present study. In contrast, the current
investigation identified a significant time by treatment interaction for p38
phosphorylation. Post hoc analysis revealed that phosphorylation of p38 was not
significantly different between treatments at rest and did not change significantly
within conditions following insulin stimulation. Whilst the effect of insulin on p38
phosphorylation was not significant in either condition, the net effect of a reduction in
phosphorylation in HG and increased phosphorylation in LG meant that a significant
difference was observed between conditions following insulin stimulation (Fig 4.9A).
From a physiological point of view, it is difficult to explain how insulin would cause
p38 phosphorylation to be reduced in one condition and increased in the other. We
therefore suggest that this observation may reflect an artifact of the small sample size
employed, rather than a legitimate directional effect of insulin on p38 phosphorylation
with differing glycogen concentrations. An interesting philosophical argument is
whether p38 phosphorylation is best expressed as total units or as a ratio indicating
phosphorylation per unit of protein. From our point of view it is interesting to note
that the difference in post clamp p38 phosphorylation between conditions is abolished
122
when phosphorylation is expressed in relation to p38 protein abundance as a density
ratio (Fig 4.9C). The findings of both studies in the current series of investigations are
in contrast to recent research suggesting that insulin increases p38 phosphorylation in
rested and exercised human muscle. The effect of insulin on p38 phosphorylation in
the above study was, however, quite modest (1.2-1.3 fold increase). It is therefore
possible that the small sample size and analytical techniques employed in the current
investigations did not allow us to detect a potentially small stimulatory effect of
insulin on p38 MAPK phosphorylation. Examination of the available fractionated
muscle preparations also allowed us to examine whether changes in subcellular
localisation of p38 occurred in response to glycogen / insulin stimulation. No
significant changes in p38 protein abundance or phosphorylation were observed in
cytosolic or particulate fractions within or between conditions. Furthermore, p38
protein / phosphorylation density ratios were not significantly altered by glycogen or
insulin. These results suggest that muscle glycogen does not influence total p38
MAPK protein abundance / phosphorylation or p38 localisation in human skeletal
muscle.
123
In summary, the findings of the present investigation suggest that;
1. Muscle glycogen content in the range demonstrated in this study does not
influence insulin stimulated glucose uptake or glycogen synthesis 24 hrs after
exercise.
2. Overnight feeding of a high fat diet does not increase activity of PKC isoforms in
human skeletal muscle.
3. Insulin does not alter fractional activity of PKC isoforms in human skeletal
muscle with differing glycogen levels.
4. Muscle glycogen concentration does not influence total p38 MAPK protein
abundance / phosphorylation or p38 localisation in human skeletal muscle at rest
or following insulin stimulation.
124
125
CHAPTER 5
❖
OVERVIEW AND CONCLUSIONS
5.1 Overview
The findings of the current series of investigations suggest that muscle glycogen
concentration in the range demonstrated in the present studies does not affect insulin
stimulated glucose uptake in the immediate post exercise period or 24 hrs after
exercise. These findings are in contrast to a number of previous studies which have
suggested a regulatory influence of muscle glycogen on insulin mediated glucose
uptake in rats (Host, Hansen et al. 1998; Kawanaka, Han et al. 1999; Derave, Hansen
et al. 2000; Kawanaka, Nolte et al. 2000). It is possible that methodological
differences between rat and human studies are at least partially responsible for the
disparate findings between models. For example, recent studies which have identified
a regulatory effect of muscle glycogen on insulin stimulated glucose uptake in rats
have employed insulin concentrations well above those which exist in the
physiological state in humans (Host, Hansen et al. 1998; Kawanaka, Han et al. 1999).
In contrast, plasma insulin concentrations in the current series of investigations were
in the range of 350 to 750 pmol⋅l-1. Thus one interpretation is that a regulatory
influence of muscle glycogen on insulin mediated glucose uptake may only be
126
apparent when glucose uptake is stimulated by super-physiological insulin
concentrations. In argument against such a scenario, a regulatory effect of muscle
glycogen concentration on glucose uptake in response to physiological
hyperinsulinaemia (≈ 700 pmol⋅l-1) in perfused rat (Derave, Hansen et al. 2000) and
human (Richter, Derave et al. 2001) muscle has recently been suggested. A potential
criticism of the current series of investigations is that the degree of hyperinsulinaemia
induced via insulin infusion was not identical in both studies. This occurred as a
result of underestimation of the amount of insulin that should be added to the infusate
in the initial study based on the computer algorithm employed. It is possible that the
algorithm did not allow for the potential adherence of insulin to the plastic syringe
utilised for insulin infusion. In support of this possibility, insulin concentrations were
substantially higher during the alternate study when the amount of insulin added to the
infusate was increased by 20 percent. In spite of this fact, no effect of muscle
glycogen on whole body glucose uptake in this study was observed in response to
hyperinsulinaemia in a range (≈ 700 pmol⋅l-1) where muscle glycogen has previously
been shown to exert an influence.
Interestingly, the observed effect of glycogen concentration on glucose uptake in
response to physiological hyperinsulinaemia in rats has been shown to be much less
apparent in the mixed fibre type red gastrocnemius than in the fast twitch white
gastrocnemius (Derave, Hansen et al. 2000). This raises the possibility that the effect
of muscle glycogen concentration on insulin mediated glucose uptake may be muscle
fibre type specific. Whilst this is an interesting possibility, the degree of experimental
127
control required to investigate fibre type specific effects of glycogen concentration on
glucose uptake would be extremely difficult to achieve in human studies. Whole body
insulin stimulated glucose uptake, as measured in the present studies, provides a
measure of glucose uptake into active and non active muscle groups as well as other
tissues. It is thus conceivable that localised or fibre type specific effects on muscle
glucose uptake using this methodology may be masked by changes at a whole body
level. One possibility would be to examine the effect of glycogen on insulin
stimulated glucose uptake in a more localised setting by employing a leg glucose
uptake design. Indeed, indirect evidence suggests that insulin stimulated leg glucose
uptake 3-4 hours after exercise is higher in the glycogen depleted when compared
with the inactive leg (Richter, Derave et al. 2001). Whilst the discrepancy between
the findings of the current investigations and those mentioned above may be explained
by differences in the methodology employed, it is also possible that the timing of the
hyperinsulinaemic / euglycaemic clamp may have played a role. Thus a regulatory
influence of muscle glycogen on insulin stimulated glucose uptake was observed 3-4
hours (Richter, Derave et al. 2001) but not 30 min or 24 hrs after glycogen depleting
exercise. Based on these findings, it is possible that muscle glycogen may have
influenced glucose uptake in study one if the period of the hyperinsulinaemic /
euglycaemic clamp was prolonged or if the clamp was initiated at a later time point
after exercise. This is particularly the case given the possibility that insulin action
may have in fact been transiently impaired as a result of the acute exercise bout in
study one. This interpretation does not explain the inability of muscle glycogen to
influence insulin stimulated glucose uptake 24 hours after exercise, however, as
128
previously discussed, it is possible that this observation was due to insufficient
differences in muscle glycogen between conditions or the lower plasma insulin
concentrations seen in this study. Alternatively, the mechanism responsible for the
observed effect of muscle glycogen on insulin mediated glucose uptake 3-4 hrs after
exercise may not be present after 24 hours in spite of the maintenance of low muscle
glycogen. Irrespective of the mechanism involved, our findings suggest that muscle
glycogen does not affect insulin stimulated glucose uptake 30 min or 24 hours after
exercise.
It may be argued that the overall absence of changes in insulin signalling in the
current series of investigations simply reflects the fact that differences in whole body
insulin action were not observed. On the other hand, we previously postulated that the
methodology employed in the current investigation may not have been sufficiently
sensitive to detect localised changes in insulin sensitivity. In either scenario, it is
conceivable that if glycogen influences insulin action, changes in insulin signalling
which occur at a localised level may be apparent regardless of the effect at a whole
body level. In a recent investigation, conventional insulin signalling was unchanged
even when enhanced insulin sensitivity in glycogen depleted muscle was observed
(Richter, Derave et al. 2001). In support of this finding, we also suggest that proximal
signalling to muscle glucose uptake is not influenced by muscle glycogen
concentration. Furthermore, no changes in signalling or subcellular localisation of
p38 MAPK were observed in the current series of investigations. Thus should muscle
glycogen prove to be involved in the regulation of insulin stimulated glucose uptake,
129
it is likely that the mechanism responsible for this effect will require further
examination. It has recently been suggested that enhanced insulin sensitivity of
muscle glucose uptake in rats may be linked to changes in AMPK (Fisher, Gao et al.
2002). Preliminary investigation suggests that AMPK activity is elevated in rodent
muscle with lowered muscle glycogen content (Derave, Ai et al. 2000; Derave,
Hansen et al. 2000; Kawanaka, Nolte et al. 2000). AMPK α1 and α2 isoforms have
also been shown to be significantly higher in glycogen depleted human skeletal
muscle at rest (Wojtaszewski, MacDonald et al. 2003). Pharmacological activation of
AMPK with AICAR enhances insulin stimulated glucose transport in incubated rodent
muscle (Fisher, Gao et al. 2002). It is thus possible that glycogen dependent insulin
action is mediated by AMPK. In the absence of changes in other signalling
intermediates, AMPK may provide an interesting candidate for further investigation of
the signalling events occurring in glycogen depleted skeletal muscle.
Muscle glycogen synthesis in the current investigation was significantly higher in
heavily but not moderately glycogen depleted skeletal muscle. This finding was in
keeping with previous investigations and supports the notion of a potential regulatory
role for muscle glycogen in the regulation of its own synthesis. In contrast to previous
investigations we suggest that increased insulin mediated glycogen synthesis may be
related to elevated GSK-3 phosphorylation in glycogen depleted muscle. Whilst
increased GSK-3 serine phosphorylation would be expected to exert a positive
regulatory influence on GS activity, an obvious criticism of the current investigation is
that GS activity was not directly measured. Furthermore, the mechanism via which
130
muscle glycogen may influence GSK-3 phosphorylation is unknown, since changes in
Akt signalling which have been suggested to regulate GSK-3 were not observed. On
the other hand, potential factors involved in regulating GSK-3 have recently been
reviewed (Doble and Woodgett 2003) and may involve (1) inactivation of GSK-3 via
serine phosphorylation; (2) activation of GSK-3 through tyrosine phosphorylation; (3)
inactivation of GSK-3 through tyrosine de-phosphorylation; (4) covalent modification
of substrates through priming phosphorylation; (5) inhibition or facilitation of GSK-3-
mediated substrate phosphorylation through interaction of GSK-3 with binding or
scaffolding proteins; (6) targeting of GSK-3 to different subcellular locations; (7)
differential usage of isoforms or splice variants to alter subcellular localisation or
substrate specificity; and (8) integration of parallel signals conveyed by a single
stimulus. Clearly regulation of GSK-3 is a complex process and as such changes in
GSK-3 phosphorylation with differing glycogen concentrations in the present study
are not easily explained. Irrespective of this fact, the observation that GSK-3 serine
phosphorylation was increased in our study suggests that adaptations in conventional
insulin signalling can not be excluded when attempting to explain the regulatory
influence of muscle glycogen on post exercise insulin action. Furthermore, GSK-3
has recently been linked to the insulin resistant state in rats and humans and may play
a role in the modulation of insulin stimulated glucose disposal in both of these models
(Nikoulina, Ciaraldi et al. 2000; Henriksen, Kinnick et al. 2003). Thus further
investigation regarding the role of GSK-3 in the regulation of post exercise insulin
action is warranted.
131
Whilst it is possible that the methodology employed in the current series of
investigations did not allow us to detect a stimulatory effect of muscle glycogen on
insulin mediated glucose uptake, an obvious alternative is that such a relationship does
not exist. Indeed, some evidence suggests that muscle glycogen depletion is not a
necessary component of enhanced insulin action in muscle. For example, treatment of
incubated muscle strips with AICAR results in enhanced insulin sensitivity of glucose
transport without measurable changes in glycogen content (Fisher, Gao et al. 2002).
Furthermore, post exercise improvements of insulin sensitivity have been shown to
persist beyond the point of glycogen normalisation in both rats (Richter, Garetto et al.
1982) and humans (Bergstrom 1966). Recent findings in GSL3-transgenic mice also
suggest that muscle glycogen may not influence insulin stimulated glucose uptake
(Fogt, Pan et al. 2004). GSL-transgenic mice over-express a constitutively active form
of glycogen synthase, which results in abundant muscle glycogen levels. In spite of
markedly elevated glycogen concentrations, insulin stimulated glucose uptake before
and after muscle contraction was not significantly different in GSL-transgenic mice
when compared to their wild type litter mates (Fogt, Pan et al. 2004). These findings
suggest that the inverse correlation between muscle glycogen and insulin stimulated
glucose uptake may be an association rather than a cause and effect relationship. Thus
it is possible that the role of muscle glycogen in insulin stimulated glucose uptake
may not be as important as previously theorised.
132
5.2 Conclusions
In summary, the results of the present series of investigations suggest that muscle
glycogen concentration does not influence insulin stimulated glucose uptake in human
skeletal muscle 30 min or 24 hrs after exercise. We further suggest that conventional
insulin signalling to muscle glucose uptake and signalling through the p38 MAPK
cascade is largely unaltered by muscle glycogen concentration. It is, however,
possible that the potential regulatory influence of muscle glycogen on insulin
mediated glucose uptake in the present series of investigations was not observed due
to the transient effects of acute exercise in study one, and due to insufficient
differences in muscle glycogen concentrations in study two. In contrast to previous
investigations the current series of studies suggest that muscle glycogen influences
insulin stimulated GSK-3 serine phosphorylation. This finding suggests that enhanced
insulin sensitivity of muscle glycogen synthesis following glycogen depleting exercise
may be mediated via a pathway involving alterations in insulin stimulated GSK-3
phosphorylation. Due to the disparity between the findings of the current and
previous studies, further investigation is warranted regarding the influence of muscle
glycogen on insulin action in humans.
133
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