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From PHYSIOLOGY AND PHARMACOLOGY
Karolinska Institutet, Stockholm, Sweden
REGULATORS OF GLUCOSE
AND LIPID METABOLISM IN
SKELETAL MUSCLE AND
SERUM
IMPLICATIONS FOR OBESITY AND
TYPE 2 DIABETES
Fredirick Mashili
Stockholm 2012
All previously published papers were reproduced with permission from the publisher.
Published by Karolinska Institutet. Printed by Karolinska University Service US-AB.
© Fredirick Mashili, 2012
ISBN 978-91-7457-952-9
Happiness lies in the joy of achievement and the thrill of creative effort.
Franklin D. Roosevelt.
To my Wife
ABSTRACT
Type 2 diabetes mellitus (T2DM) has become a growing worldwide problem of public
health importance. Insulin resistance is commonly associated with obesity and a key
factor mediating the progression to T2DM. The failure of insulin-sensitive peripheral
tissues to respond to insulin results in an increase in serum glucose levels that leads to
an impaired homeostatic state. Skeletal muscle plays a crucial role in maintaining
glucose metabolism. Impairments in both glucose and lipid metabolism arising from a
dysregulation of hormones, free fatty acids, or other factors contribute significantly to
the pathogenesis of T2DM.
The roles of several circulating metabolites in the development of insulin resistance
have been described. However the molecular mechanisms involved in skeletal muscle
insulin resistance remain poorly defined. Furthermore, the biological interactions
between skeletal muscle, novel circulating factors, and lifestyle factors such as exercise
in the regulation of glucose and lipid metabolism need to be investigated. This thesis
aims at examining the role of novel regulators of glucose and lipid metabolism,
uncovering the molecular targets involved in the development of skeletal muscle
insulin resistance, and describing their clinical implications in obesity and T2DM.
Physical exercise has beneficial effects on glucose and lipid metabolism and hence
improves cardiovascular risk factors. In Study I, we report differential effects of Nordic
walking (low-moderate intensity exercise) on cardiovascular risk factors in normal and
impaired glucose tolerant individuals. We provide evidence to support the
recommendation of a more intense and supervised exercise modality for significant
improvements in cardiovascular risk factors.
Fibroblast growth factor (FGF)-21 is a member of the FGF family that plays a role in a
variety of endocrine functions, including the regulation of glucose and lipid
metabolism. Observations from animal models have suggested a potential therapeutic
role of this growth factor in T2DM. In Study II, we provide evidence for direct effects
of FGF-21 in skeletal muscle glucose uptake. Using cell-surface photolabeling of
human myotubes, we report enhanced glucose transporter-1 abundance at the cell
membrane, coincident with increased basal and insulin-stimulated glucose uptake. We
further confirm a paradoxical increase in serum FGF-21 in T2DM in humans, and
identify BMI as the strongest independent predictor of FGF-21 serum levels. The
mechanisms controlling the metabolic actions of FGF-21 are currently being resolved.
Signal transducer and activator of transcription factor 3 (STAT3) is involved in
cytokine- and nutrient-induced insulin resistance. The role of STAT3 in the
development of skeletal muscle insulin resistance and T2DM pathogenesis is
incompletely defined. In Study III, we report an increased STAT3 phosphorylation in
T2DM. Using palmitate and STAT3 specific siRNA treatment of myotubes in vitro, we
provide evidence for the role of STAT3 in the development of lipid-induced skeletal
muscle insulin resistance.
Collectively, the work presented in this thesis contributes to the understanding of
various regulators of glucose and lipid metabolism from the whole body physiology
context to molecular mechanisms in skeletal muscle. Metabolic alterations result from
the interplay between biological processes within the cells, tissues and organs. These
alterations may translate into ill health such as T2DM. Information from Translational
studies like the ones presented in this thesis will help to identify molecules with both
clinical significance and therapeutic potential.
LIST OF PUBLICATIONS
I. Fritz T, Caidahl K, Krook A, Lundström P, Mashili F, Osler M, Szekeres F,
Östenson CG, Wändell P, Zierath JR. Effects of Nordic walking on cardiovascular
risk factors in overweight individuals with type 2 diabetes, impaired or normal
glucose tolerance. Diabetes Metab Res Rev. Aug 8 2012. doi: 10.1002/dmrr.2321.
[Epub ahead of print].
II. Mashili FL, Austin RL, Deshmukh AS, Fritz T, Caidahl K, Bergdahl K, Zierath JR,
Chibalin AV, Moller DE, Kharitonenkov A, Krook A. Direct effects of FGF-21 in
human skeletal muscle: implications for type 2 diabetes and obesity. Diabetes Metab
Res Rev 27:286-97, 2011.
III. Mashili FL, Chibalin AV, Krook A, Zierath JR. Constitutive STAT3
phosphoryation in skeletal muscle contributes to skeletal muscle insulin resistance in
type 2 diabetes. In press Diabetes Oct 5 2012 [Epub ahead of print].
CONTENTS
List of publications .............................................................................................. 7
1 Introduction .................................................................................................. 1
1.1 Epidemiology ..................................................................................... 1
1.2 Obesity and T2DM ............................................................................. 1
1.3 Pathophysiology and natural history of T2DM ................................. 2
1.3.1 Interfering with the natural history of T2DM ....................... 4
1.4 Metabolism and T2DM ...................................................................... 6
1.4.1 Regulation of glucose metabolism ........................................ 6
1.4.2 Glucose uptake ....................................................................... 6
1.4.3 Glucose uptake as a target for intervention ........................... 7
1.5 Lipid metabolism and glucose Homeostasis ..................................... 8
1.6 Insulin resistance .............................................................................. 10
1.6.1 Insulin resistance in different target organs......................... 10
1.6.2 Obesity, insulin resistance and metabolic cross-talk........... 10
1.7 Novel circulating factors, glucose and lipid metabolism. ............... 11
1.7.1 Clinical importance of FGF-21 in human disease .............. 11
1.7.2 Insulin signaling in skeletal muscle ..................................... 12
2 AIMS .......................................................................................................... 16
3 Materials and methods ............................................................................... 17
3.1 Subjects ............................................................................................. 17
3.2 Clinical characterisation of the study subjects................................. 17
Exercise study protocol .................................................................... 17
3.2.1 General clinical characteristics ............................................ 18
3.2.2 Exercise testing .................................................................... 19
3.2.3 Muscle biopsy procedure ..................................................... 19
3.2.4 Physical activity estimation ................................................. 19
3.3 SERUM ANALYSIS ....................................................................... 20
3.3.1 FGF-21 serum analysis ........................................................ 20
3.3.2 Plasma FFA analysis ............................................................ 20
3.4 Materials ........................................................................................... 20
3.5 Cell culture procedures ..................................................................... 20
3.5.1 Primary human skeletal muscle cell culture ........................ 20
3.5.2 L6 cell culture ....................................................................... 21
3.6 Metabolic studies in cell culture ...................................................... 21
3.6.1 Glycogen synthesis .............................................................. 21
3.6.2 Glucose uptake ..................................................................... 21
3.6.3 Media lactate determination ................................................. 21
3.6.4 siRNA transfection and fatty acid treatment ....................... 21
3.6.5 Cell surface GLUT1 and GLUT4 determination ................ 22
3.7 Animal Studies ................................................................................. 22
3.7.1 Animal models ..................................................................... 22
3.7.2 Isolated skeletal muscle procedures .................................... 22
3.8 experimental assays and analysis ..................................................... 23
3.8.1 Protein concentration assay and western blot analysis ....... 23
3.8.2 Gene expression studies ....................................................... 23
3.9 Statistical analyses ............................................................................ 24
4 RESULTS AND DISCUSSION ................................................................ 25
4.1 Clinical implications for exercise-induced benefits in T2DM ........ 25
4.1.1 Pronounced beneficial effects of Nordic walking in normal
as compared to impaired glucose metabolism. ................................ 25
4.2 Systemic regulators of glucose and lipid metabolism ..................... 28
4.2.1 FGF-21 as a metabolic regulator of glucose metabolism in
skeletal muscle ……………………………………………………28
4.2.2 FGF-21 as a potential biomarker ......................................... 30
4.3 Molecular regulators of glucose and lipid metabolism in skeletal
muscle. ........................................................................................................ 33
4.3.1 STAT3 is constitutively phosphorylated in skeletal muscle
from T2DM patients ......................................................................... 33
4.3.2 STAT3 phosphorylation and skeletal muscle insulin
resistance ………………………………………………………...34
4.3.3 STAT3 as a potential therapeutic target .............................. 35
4.4 SUMMARY OF FINDINGS ........................................................... 37
5 CONCLUSIONS AND FUTURE PERSPECTIVES ............................... 39
6 Acknowledgements .................................................................................... 41
7 References ................................................................................................... 43
LIST OF ABBREVIATIONS
2-DOG
AMP
AMPK
AS160
ATP
BMI
caMKK
C-NMR
EDL
FFA
FGF
GAPDH
GLUT
HOMA-IR
IDF
IGT
IRS
JAK
mTOR
NGT
OGTT
PBS
PI3K
PKB
PKC
siRNA
SOCS
STAT
T2DM
WHO
2-Deoxyglucose
Adenosine monophosphate
Activated protein kinase
Akt substrate of 160 kDa
Adenosine triphosphate
Body mass index
Ca2+/calmodulin-dependent protein kinase kinase
Carbon-nuclear magnetic resonance
Extensor digitorum longus
Free fatty acids
Fibroblast growth factor
Glyceraldehyde phosphate dehydrogenase
Glucose transporter
Homeostatic model of assessment for insulin resistance
International diabetes federation
Impaired glucose tolerance
Insulin receptor substrate proteins
Janus activated kinase
Mammalian target of rapamycin
Normal glucose tolerance
Oral glucose tolerance test
Phosphate buffered saline
Phosphatidylinositol 3 kinase
Protein kinase B (or Akt)
Protein kinase C
Small interfering ribonucleic acid
Suppressor of cytokine signaling
Signal transducer and activator of transcription factor
Type 2 diabetes mellitus
World health organization
1 INTRODUCTION
1.1 EPIDEMIOLOGY
The prevalence of diabetes has increased rapidly all over the world. According to
the International Diabetes Federation (IDF), the number of people with diabetes in the
world is estimated to be 346 million. This number is projected to grow to 552 million
by the year 2030 (Whiting et al., 2011). Type 2 diabetes (T2DM) accounts for 90% of
all diabetes cases. Therefore, the continuously growing epidemic reflects an increased
incidence and prevalence of T2DM. Epidemiologically, T2DM is well-documented in
populations in the United States and Europe, notably among Native Americans, Pacific
Islanders, people of Asian Indian origin, Hispanics and African Americans. The
prevalence of T2DM grew substantially in these populations and ethnic groups during
the 20th century (Acton et al., 2003; Harris et al., 1998). Increased intake of high-
caloric foods and physical inactivity has contributed to the development and
progression of T2DM (Berlin and Colditz, 1990; Powell et al., 1987).
Increased incidence of obesity is closely linked to the rising prevalence of T2DM.
Between 70 to 90 percent of T2DM is attributable to obesity. Approximately 197
million people have impaired glucose tolerance globally, mostly due to obesity and the
associated metabolic syndrome. Obesity and T2DM are rare in communities where a
traditional lifestyle has been preserved (Fall, 2001; King and Rewers, 1993; Swai et al.,
1993a; Swai et al., 1993b). By contrast, urbanization along with westernization has
become a major driving force for these co-morbidities, mostly in developing countries
where the rate of growth of T2DM is rapid (Fall, 2001; Haslam and James, 2005; Wild
et al., 2004). Prevention of obesity is therefore an early strategy to slow the rapidly
growing prevalence of T2DM and its associated cardiovascular complications, which
exert a great socio-economic toll on affected countries.
The serious cardiovascular complications of obesity and diabetes are
overwhelming. In developing countries, this creates a double burden of disease since
these countries are already straining under the burden of communicable diseases
(Ramaiya, 2005). The risk of cardiovascular complications is higher among obese
people (Lee, 2003; Wannamethee et al., 2011c; Wild et al., 2004), and the effect of
diabetes on cardiovascular complications is more severe among people of most ethnic
minorities in western countries and in the populations of the developing countries,
where an increased waist-to-hip ratio strongly predicts ischemic heart disease and
stroke (Lee, 2003; Wannamethee et al., 2011b, c). Approximately 3.6% deaths per year
are attributable to diabetes complications (Barcelo et al., 2003). Around 2.5% to 25%
of the annual health care budgets are directed to direct health care costs for diabetes and
according to estimates derived from a number of Latin America countries, the indirect
costs might be as much as five times the direct costs (Barcelo et al., 2003).
1.2 OBESITY AND T2DM
Generally overweight and obesity are defined as abnormal or excessive fat
accumulation that can cause health problems. According to the World Health
Organization (WHO), individuals with a body mass index (BMI) greater or equal to 30
kg/m2 are considered obese, while those with BMI greater or equal to 25 kg/m2 are
categorized as overweight (Alberti et al., 1998; Klein et al., 2007; Pi-Sunyer et al.,
1998). Waist circumference, a good surrogate marker for visceral fat, may reflect
obesity. However due to different standards on how to measure it, the use of waist
circumference in the definition of obesity is debatable (Klein et al., 2007). Different
criteria are used in the diagnosis of T2DM, but in general, the oral glucose tolerance
1
test (OGTT) has been widely employed in the clinical diagnosis of T2DM. The choice
of detection method is of particular relevance in T2DM diagnosis since, in the earlier
stages of disease progression, individuals with “pre-diabetes” can present normal
fasting glucose values. With an OGTT, impaired glucose tolerance is easily apparent
from an abnormal response following a glucose challenge. Furthermore, an OGTT
provides both the fasting and postprandial glucose levels, which reflect liver and
skeletal muscle phenotypes, respectively. WHO defines diabetes as fasting plasma
glucose ≥ 7.0 mmol/l (126 mg/dl) or plasma glucose ≥ 11.1 mmol/l (200 mg/dl) 2 hours
after an oral glucose load.
1.3 PATHOPHYSIOLOGY AND NATURAL HISTORY OF T2DM
T2DM is a chronic, progressive disease characterized by hyperglycemia, insulin
resistance, and importantly, decreased β-cell number and secretory function (Harris et
al., 1998; Haslam and James, 2005; Wild et al., 2004). It progresses from an early ‘pre-
diabetes’ stage, which is often asymptomatic, with insulin resistance, to a relatively
mild postprandial hyperglycemia, before ultimately developing into overt diabetes,
requiring pharmacological intervention. A clear understanding of the pathogenesis of
T2DM is a cornerstone to its management. Effective treatment regimens should be
directed at the pathological characteristics within the different stages of the disease.
The metabolic defects underlying T2DM are insulin resistance, β-cell dysfunction
and impaired hepatic glucose production (Wild et al., 2004). Insulin resistance is both a
primary defect and an early feature in the development of T2DM, and tends to manifest
in liver as well as in peripheral tissues such as skeletal muscle and adipocytes (Lee,
2003; Wild et al., 2004). While insulin resistance in liver is characterized by the
inability of insulin to suppress glucose production, insulin resistance in peripheral
tissues is characterized by the inability of tissues to take up glucose in response to
insulin. Skeletal muscle is a key player in glucose metabolism and accounts for 75% of
the whole body insulin stimulated glucose uptake (Hjeltnes et al., 1998; Wannamethee
et al., 2011b; Wannamethee et al., 2011d; Zierath et al., 1998). Defects resulting in
skeletal muscle insulin resistance originate from both genetic and environmental
etiology (Jones et al., 2011; Wild et al., 2004).
Insulin resistance is a primary defect in T2DM. It is early trigger for the
progression of normal glucose tolerance (NGT) into impaired glucose tolerance (IGT)
and finally to frank T2DM. At an early stage, β-cells compensate for nominal insulin
resistance by secreting more insulin. Elevated levels of secreted insulin counteract the
effect of hyperglycemia that result from decreased glucose disposal at the periphery.
Thus, in “pre-diabetes”, normal glycemia is maintained by hyperinsulinemia (Reiber et
al., 1993; Wannamethee et al., 2011e). This compensation is able to maintain normal
glucose levels for several years. Continuous assault from harmful metabolites such as
free fatty acids (FFA) worsens the insulin resistance, demanding for even higher levels
of insulin to maintain physiological fasting glucose levels (Fall, 2001; Kitange et al.,
1993). Although the fasting glucose levels are within physiological limits at this stage,
response to a glucose challenge is impaired, resulting in a state of impaired glucose
tolerance (Figure 1). In addition to the deleterious effects of insulin resistance, Current
evidence point to the genetic defect in β-cells as an important trigger in the
pathogenesis of T2DM (Griffen et al., 2001; Moran et al., 2012). Genome wide
association studies (GWAS), have provided a new insight on the role of β-cell function
in the pathogenesis of T2DM, reviewed in (McCarthy, 2009; McCarthy and Zeggini,
2009), further highlighting the important role of the β-cell in T2DM pathogenetis.
Initially, IGT is characterized by mild postprandial hyperglycemia, but as the
degree of insulin resistance worsens, more global derangements in insulin production
2
occur that result in progressive hyperglycemia (Wannamethee et al., 2011e). Clinically,
IGT represents an essentially asymptomatic but potentially pathologic stage in a
continuum between normal glucose metabolism and the development of overt T2DM.
IGT is a suitable predictor for both T2DM and cardiovascular diseases. In fact, several
cardiovascular complications such as macroangiopathies may already be present at this
stage indicating that macroangiopathies might not be secondary to diabetes but rather
contribute to development of the diabetes phenotype (Wannamethee et al., 2011a).
Figure 1: Progression of IGT to clinical T2DM in skeletal muscle. Insulin resistance in peripheral
tissues including skeletal muscle is an important trigger for the progression of IGT into T2DM. Initially,
β cells compensate by producing more insulin that normalizes the glycemia. At this stage, however, the
body displays an impaired response to a glucose challenge (IGT). Worsening insulin resistance exhausts
β cell function, resulting in impaired insulin production. Insufficient insulin production fails to counteract
the increasing insulin resistance and, hence, a state of hyperglycemia that triggers clinical T2DM ensues.
Progression of IGT to T2DM is marked by diminished β-cell function and concomitant
reduction in insulin secretion (Wannamethee et al., 2011e). At this point, the quantity of
secreted insulin becomes insufficient to normalize the progressively increasing
hyperglycemia. Although T2DM might be asymptomatic as the case of IGT but severe
hyperglycemia is sufficient to trigger the development of micro vascular complications
(Lee, 2003). Early intervention is therefore crucial to counteract the development of
T2DM and to prevent cardiovascular complications. At this stage lifestyle intervention
involving physical activity could play a crucial role in slowing disease progression.
3
Investigating the effects of glucose tolerance on the cardiovascular benefits of physical
activity is a key area covered in this thesis.
1.3.1 Interfering with the natural history of T2DM
1.3.1.1 Physical activity, glucose and lipid metabolism
Physical activity has a beneficial role in glucose and lipid metabolism. Insulin-
independent effects on glucose transport in isolated skeletal muscle have been reviewed
elsewhere (Fontana et al., 2007; Holloszy, 2005; Krook et al., 2003). This key finding
provides evidence for the existence of functional exercise/contraction-mediated glucose
uptake pathways, despite the impaired insulin-mediated pathway in T2DM (Chibalin et
al., 2000). Direct evidence for exercise-specific effects on glucose and lipid metabolism
is available; however, additional studies are still needed for a comprehensive
understanding of the effects of exercise on human health. Clinical studies are therefore
necessary to translate basic/experimental research observations to public health.
Physical exercise offers both preventive and curative benefits on T2DM, making
it an important component of an early control strategy. Physical activity, coupled with a
reduction of caloric intake, may drastically slow or even prevent the development of
T2DM in people with IGT (Corpeleijn et al., 2009;). Reduced mortality has been
reported in physically active T2DM patients compared to those leading a more
sedentary lifestyle (Leibiger et al., 2001). Furthermore, regular physical exercise has
beneficial effects on cardiovascular risk factors in people with IGT and T2DM
(Tsuruzoe et al., 2001). However, disease progression is associated with problems such
as musculoskeletal complications and low motivation, which may affect compliance to
a training program (Saltiel and Kahn, 2001). This suggests that a more effective
exercise intervention should be initiated during the early stages of T2DM.
1.3.1.2 Physical activity and weight loss
Obesity and physical inactivity are both independent risk factors for T2DM.
Elevated levels of FFA, resulting from dysregulated metabolism in adipose tissue, leads
to impaired insulin sensitivity in liver and skeletal muscle, as well as adipose tissue,
which triggers the pathogenesis of T2DM in obesity (Feldstein et al., 2008; Henquin,
2009; Henquin et al., 2009; Longo et al., 2008). Physical activity may counteract the
diabetogenic effect of obesity by reducing fat mass, or through other biological
pathways. Weight loss, as little as 5% to 10%, can reduce hyperglycemia and improve
other cardiovascular risk factors in T2DM (Andres and Zierler, 1956; Obici et al.,
2002b; Ravier et al., 2009). Improvements in insulin sensitivity in obese individuals
have also been reported following a similar reduction in body weight (Andres and
Zierler, 1956). Indeed, weight loss can reduce the fatty acid supply and thereby reduce
the amount of lipid contained within liver (Campbell et al., 1994b) and skeletal muscle
(Jensen, 1998b; Levine et al., 1998b). Together, these observations underscore the
importance of a negative energy balance to prevent insulin resistance and
hyperglycemia, as well as other cardiovascular risk factors in T2DM. However whether
a similar exercise modality (low-moderate intensity exercise) will have comparable
effects on weight loss and other cardiovascular risk factors across different stages
(NGT, IGT, and T2DM) of T2DM pathogenesis is unclear.
1.3.1.3 Physical activity and insulin sensitivity
Positive effects of physical activity on insulin sensitivity in both normal and
insulin resistant states are well documented. As early as the 5th century, physical
activity was already advocated in the treatment of diseases, reviewed by Levine et al
4
(Levine et al., 1998a). Today, the robust effect of exercise on insulin sensitivity has
become evident. Cross sectional studies have reported increased insulin sensitivity in
trained compared to non-trained subjects (Abel et al., 2001; Arcaro et al., 1999;
Shankar et al., 2004; Shankar and Steinberg, 2005). Likewise, regular endurance
training can prevent age-induced insulin resistance in the elderly (Paradisi et al., 2001).
Interestingly, older but active individuals were found to be more insulin sensitive
compared to young sedentary subjects (Paradisi et al., 2001). Additionally, a growing
body of evidence from epidemiological studies has linked physical inactivity with the
increasing prevalence of T2DM (Jensen, 1998a; Jensen and Levine, 1998; Jensen et al.,
1998). Physical activity is therefore an effective way to improve insulin sensitivity
or/and prevent the development of insulin resistance.
Over the last two decades, the physiological and molecular mechanisms through
which physical activity improves skeletal muscle glucose uptake have been extensively
investigated (Outlined in Figure 2). The positive effects of exercise in skeletal muscle,
together with the effects in other organs, help to maintain glucose homeostasis, even in
states of insulin resistance. Aerobic exercise has long been considered the most
effective mode of physical activity for improving insulin sensitivity. However, greater
consideration of a minimal and more realistic exercise intervention that is practically
applicable in a normal primary care setting is warranted. Investigating the effect of a
minimal unsupervised exercise intervention on insulin sensitivity (Study 3) will
therefore provide scientific evidence for therapeutic effects of this exercise modality in
obesity and T2DM.
Figure 2: Exercise-dependent glucose uptake in skeletal muscle. In insulin resistance, insulin-
dependent pathways for glucose uptake are impaired while contraction-dependent pathways remain
intact. Exercise facilitates the translocation of GLUT4 to the plasma membrane through molecular
mechanisms other than the insulin receptor, subsequently inducing glucose uptake into skeletal muscle.
5
1.3.1.4 Exercise capacity and T2DM
Impairments in cardiopulmonary fitness have been observed in obesity and
T2DM (Gupta et al., 1998; Steinberg et al., 1997). In most clinical exercise studies,
cardiopulmonary fitness is often estimated by measuring the maximum oxygen uptake
during an exercise test, hence expressed as maximum oxygen uptake (VO2 max)(Ong
and Ong, 2000; Pi-Sunyer et al., 1998; Seyoum et al., 2006).The existence of T2DM
confers an additional reduction in cardiopulmonary fitness beyond that seen with
obesity (Kristen J. Nadeau et al., 2009). This suggests that progression from obesity to
overt T2DM is accompanied by worsening cardiopulmonary fitness, emphasizing the
importance of early lifestyle interventions to prevent T2DM. Cardiopulmonary fitness
is a good predictor of cardiovascular events both in obese and T2DM subjects. Thus,
improvement in fitness offers a protective advantage against cardiovascular risk factors
in T2DM (Seyoum et al., 2006; Wei et al., 2000). Physical exercise is the main
treatment modality with a positive impact on cardiopulmonary fitness. Evidence from
both epidemiological and clinical studies suggests that regular exercise improves
cardiopulmonary fitness in normal, obese and T2DM subjects, and lowers
cardiovascular events and mortality in subjects with T2DM (Gupta et al., 1998; Kristen
J. Nadeau et al., 2009; Seyoum et al., 2006; Steinberg et al., 1997; Wei et al., 2000).
1.4 METABOLISM AND T2DM
1.4.1 Regulation of glucose metabolism
Glucose is the endpoint breakdown product of carbohydrate digestion that is
used by all living organisms as an important energy substrate and metabolic
intermediate in many pathways. Uptake and metabolism of glucose is crucial for
cellular functioning and is tightly regulated by the hormone insulin (Corpeleijn et al.,
2009; Longo et al., 2008; Saltiel and Kahn, 2001). In a normal physiological state,
pancreatic ß-cells secrete insulin in response to a meal (Feldstein et al., 2008;
Henquin, 2009). The postprandial presence of insulin in the circulation diminishes
hepatic glucose production and facilitates glucose uptake into peripheral tissues.
Skeletal muscle accounts for ~75% of whole body insulin-stimulated glucose uptake,
rendering it a critical tissue for glucose metabolism (Wannamethee et al., 2011b;
Wannamethee et al., 2011d). Moreover, resting skeletal muscle has a low rate of
glucose utilization in overnight fasted humans (Andres et al., 1956), reflecting a
conservation of fuel for tissues with a compulsory glucose requirement (e.g. brain).
1.4.2 Glucose uptake
Glucose uptake in skeletal muscle occurs by facilitated diffusion, a process that is
stimulated by insulin and mediated by glucose transporters. GLUT4 is the primary
glucose transporter in skeletal muscle, but GLUT1 may also facilitate glucose uptake to
a lesser extent (Gerrits et al., 1993; Klip and Paquet, 1990; Olson and Pessin, 1996).
Once glucose enters the cell, it can either be transported back outside or, in the presence
of hexokinase, irreversibly phosphorylated to glucose-6-phosphate, which can be
channeled into different metabolic pathways including glycogen synthesis, as
summarized in Figure 3.
6
Figure 3: Fate of cellular glucose. Glucose enters skeletal muscle cells and is phosphorylated by
Heokinase into glucose-6-phosphate. After phosphorylation, glucose can either be used for energy
production or stored in the form of glycogen. A small percentage of glucose can either be channeled to
hexose biosynthesis or pentose phosphate pathways for a variety of metabolic functions.
1.4.3 Glucose uptake as a target for intervention
A primary defect in T2DM is insulin resistance, which result in perturbations in
glucose and lipid metabolism. Hyperglycemia, as a result of impaired glucose uptake
in skeletal muscle and unregulated hepatic glucose production, is the main trigger for
clinical diabetes. Interventions directed towards improving skeletal muscle glucose
uptake are, therefore, important in the ongoing battle against the rise of T2DM. A
number of novel pharmaceutical agents for treatment of T2DM have been proposed
and widely investigated. This include among others, sulfonylureas, biguanides, and
thiazolidinediones. Most of these agents have shown a positive effect on glucose
uptake. However, low efficacy and mechanism-based side effects favor the continued
development of new avenues of safe and potentially effective agents.
7
1.5 LIPID METABOLISM AND GLUCOSE HOMEOSTASIS
Free fatty acids are elevated in obesity (Boden, 2002; Boden and Shulman,
2002), providing evidence that availability of excess fat in the form of FFA may lead to
impairments in muscle glucose metabolism and storage, and consequently to glucose
intolerance and T2DM (Boden and Shulman, 2002). Defects such as increased hepatic
gluconeogenesis and decreased glucose oxidation in skeletal muscle have been linked
to excess availability of FFA. In obesity, the excess FFAs are readily available for
oxidation in skeletal muscle at the expense of glucose (Randle et al., 1963).
Furthermore elevated serum FFA can interfere with glucose utilization both in vitro and
in vivo (Boden and Jadali, 1991; Boden et al., 1991; Randle et al., 1963). Together this
evidence implicate disturbed lipid metabolism in the pathogenesis of T2DM.
Quantitatively, insulin-stimulated glucose disposal in skeletal muscle is of major
importance compared to that of adipose tissue. However, insulin is a potent inhibitor of
lipolysis in adipose tissue, a physiological process with major implications for glucose
homeostasis. Elevated levels and increased oxidation of FFAs contribute to the
development of insulin resistance in skeletal muscle (Boden, 1997; Randle et al.,
1994a; Randle et al., 1994b). FFAs blunt the effect of insulin on glucose metabolism,
resulting in increased hepatic glucose production, both by stimulating key enzymes and
providing energy for gluconeogenesis (Foley et al., 1992; Toft et al., 1992). Moreover,
uncontrolled lipolysis as a result of insulin resistance increases the production of
glycerol that acts as a substrate for gluconeogenesis (Campbell et al., 1992; Nurjhan et
al., 1992a; Nurjhan et al., 1992b; Toft et al., 1992). Consequently, increased production
of FFAs and glycerol, resulting from blunted insulin effects on lipolysis, may result in
deleterious effects on glucose homeostasis. Dysregulated metabolic cross-talk between
skeletal muscle and adipose tissue that occurs in obesity plays a role in the development
of skeletal muscle insulin resistance as illustrated in Figure 4. According to Randle, a
competition for oxidation between glucose and FFA occurs in muscles (Randle et al.,
1963), such that high flux of FFA favors its oxidation at the expense of glucose,
consequently inhibiting muscle glucose uptake. Generally, increased plasma FFA levels
could lead to impaired skeletal muscle insulin sensitivity and as a result affect whole
body glucose metabolism.
Recent observations have however challenged the Randle hypothesis (Shulman,
2004; Wolfe, 1998). Contrary to what Randle et al. observed in rat heart and
hemidiaphragm, the decrease in glucose uptake was not due to increased fatty acid
oxidation, rather a primary defect in glucose uptake resulted in secondary defects in
glucose oxidation (Wolfe, 1998). In addition, intracellular glucose-6-phosphate
decreased in response to increased FFA availability (Roden, 2004; Shulman, 2004).
Using the euglycemic/hyperinsulinemic clamp technique together with C-NMR, a
substantial decrease in intracellular glucose-6-phosphate in obese and T2DM patients
was observed, rather than an increase as predicted by Randle (Shulman, 2004).
Moreover, a defect in glycogen synthesis without concomitant increase in glucose-6-
phosphate in lipid-infused humans has also been reported (Roden, 2004; Shulman,
2004). This has led to the proposal of alternative mechanisms involving intrinsic
defects in skeletal muscle insulin signaling (Griffin et al., 1999; Roden et al., 1996;
Shulman, 2004; Yu et al., 2002). Mechanistic studies have uncovered potential
mechanisms through which different lipid derivatives including FFAs can cause insulin
resistance in skeletal muscle. A number of pathways related to these mechanisms have
also been proposed. Targeting FFA-related pathways in skeletal muscle to reverse
insulin resistance may have clinical implications in T2DM treatment.
8
Figure 4: Role of obesity and dysregulated lipid metabolism in the development of skeletal muscle
insulin resistance. Dysregulated metabolism in adipose tissue as a result of obesity causes an increase in
the levels of pro-inflammatory cytokines and free fatty acid in the circulation. These metabolites impact
skeletal muscle and cause insulin resistance. Other factors related to obesity can directly target skeletal
muscle also causing insulin resistance.
9
1.6 INSULIN RESISTANCE
1.6.1 Insulin resistance in different target organs
Insulin resistance is an insufficiency in insulin action, which results in an
increase in endogenous hepatic glucose production, and impaired stimulatory effects of
insulin on peripheral organs. In skeletal muscle, failure of insulin action is manifested
as impaired glucose uptake and glycogen synthesis. In adipose tissue, insulin resistance
is characterized by increased lipolysis as a result of impaired insulin action. Increased
lipolysis is the cause of elevated levels of free fatty acids in the circulation. The
suppressive effect of insulin on free fatty acids is indeed impaired in obesity (Campbell
et al., 1994a; Levine et al., 1998a) and T2DM (Groop et al., 1991). Furthermore,
impaired glucose uptake into adipose tissue contributes to both hepatic and skeletal
muscle insulin resistance (Abel et al., 2001), providing evidence for metabolic cross-
talk in the pathogenesis of insulin resistance. This metabolic communication between
adipose tissue and skeletal muscle suggests that excessive metabolites such as free fatty
acids target skeletal muscle and could subsequently trigger or worsen the already
existing insulin resistance in skeletal muscle (see Figure 4 above). The quantitative
importance of skeletal muscle in insulin-stimulated glucose disposal, and its role as a
target for many metabolically active molecules in the course of insulin resistance,
brings skeletal muscle into the forefront of this thesis.
Insulin-dependent signaling pathways are present in virtually all tissues,
including for example the vascular endothelium (Arcaro et al., 1999; Steinberg et al.,
1996) . Since the metabolic cross-talk between tissues coordinates the whole-body
response to insulin in obesity and T2DM, a brief understanding of insulin resistance in
the brain and blood vessels, is necessary to understand pathogenesis from a systemic
perspective. In obesity, an impaired insulin-mediated vasodilatation, which is a
recognized precursor of atherosclerosis, is noted (Arcaro et al., 1999; Steinberg et al.,
1996). Whether glucose metabolism in skeletal muscle is affected by insulin-mediated
vasodilatation of blood vessels supplying skeletal muscle remains disputed (Clark,
2008; Steinberg and Baron, 1999; Yki-Jarvinen and Utriainen, 1998) . However, it has
been shown that access of insulin to target tissues is inhibited by a high fat diet,
potentially by inhibiting capillary recruitment (Ellmerer et al., 2006).
Central inhibition of insulin action such as resistance in the central appetite-
suppressing and metabolic action of insulin plays an important role in the development
of skeletal muscle insulin resistance. Recently it has become clear that physiological
glucose homeostasis in the brain requires insulin action (Obici et al., 2002a; Obici et
al., 2002b; Okamoto et al., 2004), contrary to the traditional understanding of the brain
as an insulin-independent organ (Seaquist et al., 2001). Although basal levels of insulin
can stimulate brain glucose uptake (Bingham et al., 2002), this effect is significantly
reduced in insulin resistance (Anthony et al., 2006). Furthermore neurons in the
hypothalamus express the insulin-responsive insulin transporter GLUT4 (Leloup et al.,
1996). Impairments in insulin receptors in the hypothalamus consequently cause
hyperphagia that lead to diet-induced obesity (Leloup et al., 1996).
1.6.2 Obesity, insulin resistance and metabolic cross-talk
The following section will review the available evidence related to the interaction
between different tissues and organs (metabolic cross-talk) in the development of
skeletal muscle insulin resistance in obesity. Emphasis will be placed on the adipose
10
tissue/skeletal muscle cross-talk, especially the role of free fatty acids and circulating
adipokines in the development of insulin resistance. Novel tissuekines, produced by
liver and adipocytes that hold potential therapeutic effects in skeletal muscle, will also
be discussed. Finally, evidence for potential drug targets to modulate the negative
effects of excessive metabolites on insulin signaling in skeletal muscle will be reviewed
and discussed. Perturbations in FFA metabolism play a crucial role in the pathogenesis
of insulin resistance in obesity. Plasma FFA turnover is related to whole-body lipolysis.
The increased lipolysis occurring in obesity leads to a chronic elevation of FFA in
skeletal muscle and other tissues. Circulating FFA mediate the metabolic cross-talk
between adipose tissue and skeletal muscle.
1.7 NOVEL CIRCULATING FACTORS, GLUCOSE AND LIPID
METABOLISM.
Accumulating evidence suggests that secreted factors from adipocytes and
skeletal muscle participate in the physiological regulation of glucose and lipid
metabolism in energy homeostasis (Guilherme et al., 2008; Pedersen and Febbraio,
2008). The identification of adipocyte- and skeletal muscle-derived molecules, which
interact with insulin-sensitive tissues, has expanded the understanding of glucose
metabolism. There is a growing appreciation that Fibroblast Growth Factor (FGF)-21, a
novel member of the FGF family, participates in a number of endocrine functions
including the regulation of glucose and lipid metabolism (Coskun et al., 2008;
Kharitonenkov et al., 2005; Ryden, 2009; Wente et al., 2006). FGF-21 is a potent
regulator of insulin-dependent glucose uptake in both murine 3T3-L1 adipocytes and
primary human adipocytes (Kharitonenkov et al., 2005). Transgenic overexpression of
FGF-21 improves insulin sensitivity and lowers blood glucose and triglycerides levels
in animal models of obesity (Kharitonenkov et al., 2005). Together, these observations
emphasize the role of FGF-21 in modulating glucose and lipid metabolism.
1.7.1 Clinical importance of FGF-21 in human disease
Current therapeutic options for treatment of T2DM are suboptimal, since the
majority of patients on oral agents fail to achieve the targeted clinical outcomes (1995;
Scheen, 2003). Limited efficacy, tolerability and reported side effects are common to
all the available therapies (e.g. insulin, metformin, peroxisome proliferator-activated
receptor-gamma agonists, alpha glucosidase inhibitors), necessitating a intensive search
for new agents. Furthermore, a continuous search for a potential biomarker that will aid
in early prediction or/and detection of T2DM is crucial. Fibroblast Growth Factor 21
(FGF-21) is an emerging novel circulating tissuekine with possible diagnostic and
therapeutic potentials.
1.7.1.1 FGF-21 as a potential drug target
Evidence arising primarily from in vitro and in vivo studies in animals suggests
that FGF-21 exerts beneficial metabolic effects on both carbohydrate and lipid
metabolism (Coskun et al., 2008; Kharitonenkov et al., 2005; Ryden, 2009; Wente et
al., 2006). In diabetic animal models, FGF-21 treatment improved glucose and lipid
homeostasis and preserves β-cell function (Coskun et al., 2008; Hotta et al., 2009;
Kharitonenkov et al., 2005; Kharitonenkov et al., 2007; Kralisch and Fasshauer, 2011;
Sarruf et al., 2010; Wente et al., 2006). Systemic administration of recombinant FGF-
21 decreases plasma triglycerides, FFA, and cholesterol in genetically-modified obese
and diabetic rodents (Kharitonenkov et al., 2005; Xu et al., 2009). Moreover, long-term
11
FGF-21 therapy in diabetic rhesus monkeys improves lipid profiles (Kharitonenkov et
al., 2007). Interestingly, unlike classic FGFs, proliferation or mitosis is unaffected by
FGF-21 (Huang et al., 2006; Kharitonenkov et al., 2005; Wente et al., 2006), offering a
promising and potentially safe treatment option for T2DM. Whether FGF-21 has direct
effects on glucose metabolism in skeletal muscle, is unknown. Furthermore the
relationship between FGF-21 serum levels and other metabolic parameters of clinical
importance needs further clarification.
Several adipokines (tissuekines secreted from adipose tissue) have been identified
and shown to influence insulin action in skeletal muscle (Pittas et al., 2004). These
include TNFα, IL-6, and adiponectin that, together with other possibly unknown
factors, might constitute the missing link(s) between adipose tissue and skeletal muscle
insulin resistance (Greenberg and McDaniel, 2002). The involvement of the cytokine-
responsive JAK/STAT pathway in the cross-talk between adipocyte/liver and skeletal
muscle has been reported (Emanuelli et al., 2001; Rui et al., 2002; Ueki et al., 2004).
However, knowledge regarding the role of STAT3 in the development of skeletal
muscle insulin resistance in humans is sparse and inconclusive. Further, detailed
investigations on the role of STAT3 in the development of skeletal muscle insulin
resistance may identify targets of therapeutic potential for the treatment of T2DM.
1.7.2 Insulin signaling in skeletal muscle
Insulin stimulates glucose uptake in skeletal muscle by increasing the abundance
of glucose transport proteins, mainly GLUT4 (Czech and Corvera, 1999), at the plasma
membrane. This process is initiated upon binding of insulin to insulin receptors at the
cell surface. Insulin binding triggers a cascade of intracellular signaling events,
including the consecutive phosphorylation of several cytosolic proteins, such as the
insulin receptor substrate molecules (IRS), phosphatidyl inositol 3 kinase (PI3K), and
protein kinase B (PKB/Akt) (Czech and Corvera, 1999). Akt/PKB and members of the
protein kinase C (PKC) family are key molecules in the canonical insulin signaling
cascade that ultimately lead to increased intracellular glucose transport. Total GLUT4
protein content is unaltered in skeletal muscle from T2DM patients (Handberg et al.,
1990; Pedersen et al., 1990; Shepherd and Kahn, 1999). Thus, impaired glucose uptake
in insulin-resistant skeletal muscle cannot be explained by a decrease in the
biosynthesis of GLUT 4. Rather, impairments in insulin signaling or GLUT4
trafficking are likely to play a role (Cusi et al., 2000; Krook et al., 2000; Krook et al.,
1998) in T2DM-related defects in glucose metabolism.
Both in vitro and in vivo studies in humans and laboratory animals provide
evidence to support a role for a selective insulin signaling defect in skeletal muscle
(Kim et al., 2003; Krook et al., 2000; Leng et al., 2004). In T2DM patients, insulin-
mediated glucose uptake in skeletal muscle is reduced by approximately 50%. A
molecular explanation for the development of insulin resistance in skeletal muscle
points to specific alterations in the insulin signaling pathways (Bjornholm et al., 1997;
Bouzakri et al., 2003; Cusi et al., 2000; Krook et al., 1998). Alterations in the
expression or translocation of GLUT4 to plasma membrane have also been implicated
as a potential cause of skeletal muscle insulin resistance.
Furthermore, the impairment in glucose uptake that causes skeletal muscle
insulin resistance arises from an aberrant insulin response, but also involves influence
of growth factors and locally acting hormones (sometimes referred to as tissuekines)
secreted by different organs. In humans and animal models of T2DM, factors secreted
from liver, adipose tissues and other organs, play a crucial role in the development of
skeletal muscle insulin resistance. This provides a mechanism for metabolic crosstalk
between different tissues and organs.
12
1.7.2.1 The JAK-STAT pathway
Various biological processes, such as the cellular response to cytokines and
growth factors, are mediated by the evolutionary conserved Janus kinase/signal
transducers and activators of transcription (JAK/STAT) signaling pathways (Figure 5).
Depending on the signal, tissue, and cellular milieu, activation of this pathway results
in a wide range of responses. These responses include apoptosis, cell survival,
differentiation, proliferation and migration, highlighting the essential role for
JAK/STAT signaling in homeostatic processes like glucose and lipid dynamics (Manea
et al., 2010; Marrero et al., 2006).
The JAK family contains four tyrosine kinase cytosolic proteins known as JAK1,
JAK2, JAK3 and TYK 2, all of which are coupled to different receptors including those
of cytokines (Darnell et al., 1994; Persico et al., 1995). In response to ligand binding to
cytokine receptors, JAKs tyrosine-phosphorylate and activates these receptors.
Activated JAKs may also tyrosine phoshorylate and activate other signaling molecules
including the signal transducer and activators of transcription (STAT) factor family
(Darnell et al., 1994; Persico et al., 1995). Seven STAT isoforms are known, i.e.
STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6, and are
differentially expressed in a tissue-specific manner (Aaronson and Horvath, 2002; Levy
and Darnell, 2002; Pellegrini and Dusanter-Fourt, 1997).
The JAK/STAT pathway is therefore a link between cell surface receptor
activation, nuclear transcriptional events, and various physiological outcomes. A
growing body of evidence implicates this pathway in the adipokine-mediated cross-talk
between adipocytes and liver or skeletal muscle (Emanuelli et al., 2001; Rui et al.,
2002; Ueki et al., 2004). However, the specific role of STAT3 in the development of
skeletal muscle insulin resistance in humans is inconclusive (Carey et al., 2006; Kim et
al., 2011; Weigert et al., 2004). Further investigations to uncover STAT3-dependent
mechanisms contributing to the development of skeletal muscle insulin resistance is
therefore warranted.
13
Figure 5: The JAK/STAT pathway. Various factors such as nutrients and cytokines signal through the
JAK/STAT pathway. Activation of this pathway results in a number of biological processes mainly by
transcriptional regulation. Activation of STAT3, a key member of the STAT family, has been linked to
insulin resistance in liver and adipose tissue.
1.7.2.2 STAT3 and insulin sensitivity
STAT3 is a transcription factor expressed in multiple metabolic tissues that is
activated through phosphorylation of Tyr705 and Tyr727 in response to cytokines, growth
factors and nutrients. Targeted disruption of STAT3 leads to embryonic lethality in
mice (Takeda et al., 1997), an effect not observed following disruption of other STAT
family members. Moreover, accumulating evidence suggests that STAT3 responds to a
wide array of physiological stimuli, implying a fundamental and novel biological
importance of STAT3 versus other related family members. For that reason, the role of
STAT3 in the development of insulin resistance in T2DM was explored. Studies
involving STAT3 are therefore covered in this thesis (Study 3).
Signaling pathways involving STAT3 play a role in regulating both the hepatic
and peripheral insulin sensitivity. Activation of STAT3 may have a dual effect on
insulin signaling depending on the stimulus, physiological context and/or duration of
activation (Carey et al., 2006; Kim et al., 2011; Weigert et al., 2004). In liver
hepatocarcinoma cell lines, STAT3 knockdown prevents amino acid-induced insulin
resistance (Kim et al., 2009a). Activation of STAT3 in adipocytes is linked to growth
hormone-induced insulin resistance in rats chronically treated with arginine (de Castro
Barbosa et al., 2009). In contrast, acute IL-6-induced activation of STAT3 in rat liver
inhibits hepatic glucose production (Inoue et al., 2006). These findings underscore the
14
importance of understanding the varying effects of acute and chronic activation of this
signaling molecule on insulin signaling.
1.7.2.3 Circulating factors and STAT3 activation
Various circulating metabolites such as cytokines, hormones and FFA activate
STAT3 (Kim et al., 2008; Kim et al., 2011; Oberbach et al., 2010). In human smooth
muscle cells, short-term palmitate exposure up-regulates STAT3 phosphorylation (p-
STAT3) whereas long-term exposure down-regulates p-STAT3 and concomitantly
increases SOCS3 protein abundance, implying negative feedback regulation of this
signaling cascade (Oberbach et al., 2010). In mouse primary hepatocytes chronically
treated with IL-6, mTOR upregulated phosphorylation of STAT3 and incresed SOCS3
expression, causing an impairment of insulin signaling (Kim et al., 2008). Furthermore,
IL-6 induced insulin resistance in cultured myotubes derived from people with IGT
(Kim et al., 2011). Collectively, these studies provide evidence to suggest circulating
factors and hormones indirectly signal through STAT3 and differentially regulate
insulin signaling in a variety of tissues. Thus, excessive STAT3 signaling may impose
negative feedback regulation on canonical insulin signaling pathways controlling
metabolic action in T2DM.
Given the complex regulation of whole body metabolism, several questions
regarding the role of circulatory factors including glucose, lipids and cytokines are
worthy of further penetration. Molecular mechanisms involved in skeletal muscle
glucose and lipid metabolism are not completely resolved. Furthermore the collective
role of physical activity in the clinical management of obesity and T2DM needs further
evaluation. Investigation of the regulation of glucose and lipid metabolism integrated
with description of the corresponding clinical implication, from a whole body
physiology context to cellular mechanisms is therefore important.
15
2 AIMS
The overall aim of this thesis is to investigate the regulation of glucose and lipid
metabolism in states of normal and impaired insulin sensitivity. The studies are
designed to investigate the molecular mechanisms involved in the development of
skeletal muscle insulin resistance. An overall evaluation of study findings further
relates to the clinical significance in obesity and T2DM.
Specifically the following questions were posed:
1. Does low moderate-intensity exercise have similar effects on cardiovascular risk
factors in overweight individuals with varying degrees of insulin sensitivity?
2. Is FGF-21 differentially regulated in normal and impaired glucose homeostasis, and
does FGF-21 exert direct effects on glucose metabolism in human skeletal muscle?
3. Is STAT3 differentially regulated in normal and T2DM, and does STAT3 plays a role
in the development of lipid-induced skeletal muscle insulin resistance?
16
3 MATERIALS AND METHODS
3.1 SUBJECTS
The subjects examined in Study I, II and III were recruited from Gustavsberg, a
suburban area proximal to Stockholm, Sweden. Recruitment was achieved through
newspaper advertisement and letters of invitation to former participants in the
Stockholm Diabetes Prevention Program (SDPP) who lived within the catchment area
(Eriksson et al., 2008). Clinical evaluations of the participants were performed at the
Gustavsberg Vårdcentral primary health care center and at the Department of Clinical
Physiology, Karolinska University Hospital, Stockholm.
Individuals aged 45 to 69 years, with BMI >25 kg/m2, were included in the study.
Study participants were qualified as T2DM if HbA1c values were between 7.4 and
9.3% National Glycohemoglobin Standardization Program standard (57 to 78
mmol/mol International Federation of Clinical Chemistry (IFCC) standards). Exclusion
criteria were as follows: physical impairments, symptoms of angina pectoris, atrial
fibrillation as determined by electrocardiogram, systolic or diastolic blood pressure of
>160 or >100 mmHg respectively, and insulin treatment. Insulin treatment was an
exclusion criterion since it would interfere with the calculation of HOMA-IR.
Upon inclusion into the study, the participants were classified into T2DM, IGT, or
NGT by an oral glucose tolerance test (OGTT). The duration of diabetes was 5.1±3.7
years for people with T2DM (mean ±SD). Participants were stratified based on glucose
tolerance state (NGT, IGT, or T2DM). In Study I, a total of 213 subjects [NGT
(n=128), IGT (n=35), or T2D (n=50)] were analyzed before and after the study
timeframe of 4 months. A single-blind randomization procedure was used to assign
individuals to either a control or intervention group. For Study II and III, only T2DM
patients (40 in Study II and 20 in Study III) and BMI- and age-matched NGT subjects
were analyzed. A skeletal muscle biopsy was obtained from T2DM (10 in Study II and
20 in Study III) patients as well as an equivalent number of BMI- and age-matched
NGT subjects. The clinical characteristics of the study subjects are presented in the
respective articles. Primary human muscle cells used in Study II were obtained from a
separate cohort of subjects who were scheduled for abdominal surgery at the
Karolinska University Hospital, Huddinge, Sweden. These subjects were free from
metabolic disorders and presented normal fasting glycemia.
3.2 CLINICAL CHARACTERISATION OF THE STUDY SUBJECTS
Exercise study protocol
For Study I, the participants in each category (NGT, IGT or T2DM) were
randomized to the exercise or control group (only 10 T2DM patients on exercise
intervention were analyzed in Study II). Subjects were asked to maintain their usual
dietary habits. The participants in the exercise group were instructed to increase their
weekly level of physical activity by 5 hours of walking with poles (Nordic walking) for
4 months. Instructions for Nordic walking were given by an exercise physiologist.
Walking intensity was prescribed as a pace that caused slight shortness of breath and
perspiration. Written informed consent was obtained from all participants. The study
was approved by the Ethics Committee of Karolinska Institutet, Stockholm.
17
3.2.1 General clinical characteristics
Body weight was measured with a calibrated electronic scale (Hugin, Mustelia,
EB5011). Systolic (SBP) and diastolic (DBP) blood pressures were determined to the
nearest 5 mm Hg, in the seated position (Speidell & Keller Tonometer). At the time of
inclusion, an OGTT was performed. A mean fasting plasma glucose was determined
prior to and 2 hours after the ingestion of 75g of glucose solution. Glucose was
assessed with a HemoCue B-Glucose analyzer. Fasting venous blood samples were
analyzed for total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, and
HbA1c at the Laboratory of Clinical Chemistry at Karolinska University Hospital,
Stockholm. Fasting serum insulin was determined using an adipokine (HADK2-61K-
B09) assay kit from Linco Research (Electra-Box Diagnostica AB, Tyresö, Sweden),
according to the manufacturer’s instructions. Plasma samples from each subject were
extracted from whole blood and analyzed in duplicate. Results were quantified using
the Luminex Bio-Plex 200 system (Bio-Rad, Stockholm, Sweden). Insulin resistance
was calculated using the HOMA IR model (15), as fasting insulin (μU/ml) x fasting
glucose (mM)/22.5. These measurements were repeated after four months of
intervention (Figure 6). Tables with clinical characteristics of the study subjects are
presented in respective articles.
Figure 6: The overall human study protocol. Clinical material included in the three different studies.
Clinical evaluation, blood samples and muscle biopsies from individuals with NGT, IGT and T2DM
before and after intervention in Study I, II and III.
18
3.2.2 Exercise testing
Bicycle exercise tests were performed using a Rodby ergometer RE 820/830. A steady
baseline was obtained prior to the initiation of the test. The initial work load was set at
50 W and continuously increased by 10 W/min. A Sensor Medics ergo spirometer
Vmax Encore (Sensor Medics Corporation; Yorba Linda, USA) was used to measure
gas exchange. The gas analyzers were calibrated with two calibrated gases containing
16.0% O2, 4.0% CO2 and 26.0% O2, 0.0% CO2. Oxygen uptake (VO2) was calculated
breath by breath, and the mean value for the latest 30 seconds was used. The VO2 at
peak exercise (peak VO2) was determined at the point of subjective exhaustion.
3.2.3 Muscle biopsy procedure
Muscle biopsies in Study II and III were obtained from individuals with NGT (10 in
Study II and 20 in Study III) and T2DM patients (10 in Study II and 20 in Study III). A
local anesthesia (Lidocaine hydrochloride 5mg/ml) was injected at the site of incision.
An incision (5 mm long/10 mm deep) was made to expose the quadriceps femoris
muscle. A biopsy (20-100 mg) was taken from the vastus lateralis portion of the
quadriceps femoris using a Weil-Blakesley contochome. Biopsies were immediately
frozen in liquid nitrogen and stored at -80°C to await further analysis.
3.2.4 Physical activity estimation
3.2.4.1 Self-reported physical activity
In Study I, self-reported exercise was obtained from both the control and intervention
participants at baseline and after four months. Participants were asked to complete a
questionnaire to report their physical activity and indicate their average weekly amount
of low, medium and high intensity exercise. The frequency and duration of activities
were also recorded. Exercise diaries were used by participants in the exercise group to
record details such as date and duration of each Nordic walking bout.
3.2.4.2 Accelerometer
To validate the self-reported physical activity assessment method, several individuals
also used personal accelerometers for seven days shortly after randomization (Haffner
et al., 1997), in order to generate a second, objective assessment of activity. Of the 214
individuals in entire cohort, 25 participants (n=11 from the control group and n=14
from the intervention group) wore a belt accelerometer during operative hours
(ActiGraph model GT1M; ActiGraph, Pensacola, Florida, USA). Physical activity was
recorded as total activity counts per minute and minutes per day of inactivity, low,
moderate, or vigorous activity.
19
3.3 SERUM ANALYSIS
3.3.1 FGF-21 serum analysis
In Study II, circulating FGF21 was measured in serum using a commercially available
enzyme-linked immunosorbent assay kit (Human FGF21, Bio vendor, Czech
Republic), following the manufacturer’s instructions. Serum samples were diluted 1:2
with the dilution buffer prior to assay. Positive and negative controls were included. All
samples were measured in duplicate. All values were within the standard curve range.
3.3.2 Plasma FFA analysis
In Study III, circulating FFA was measured in plasma using the plasma Human Free
Fatty Acids detection kit (Zenbio), according to the manufacturer’s instructions.
Samples were diluted 10 times with the dilution buffer before dispersal into a 96-well
plate in duplicate. A standard curve using standards of known concentration was used
to calculate the concentration of FFA within the samples. All measurements fell within
the acceptable range of the standard curve. Plasma concentration of IL-6 and TNF-a
was measured using a Novex multiplex Luminex assay for quantitation and detection of
Cytokines (Life Technologies, Carlsbad, CA).
3.4 MATERIALS
Dulbecco's modified Eagle's medium, fetal bovine serum, penicillin, streptomycin, and
Fungizone were obtained from Invitrogen (Stockholm, Sweden). Recombinant human
FGF21 used in Study II was provided by Lilly Research Laboratories (Lilly Corporate
Center, Indianapolis, IN) or purchased from ProSpec-Tany TechnoGene Ltd (Rehovot,
Israel). General laboratory reagents, including palmitic acid for Study II, were obtained
from Sigma (St. Louis, MO). Radioactive reagents were purchased from Amersham
(Uppsala, Sweden). Oligonucleotide primers used in Study II and III were purchased
from Oregene (Oregene) and SYBR Green probes were from Invitrogen (Invitrogen,
Carlsbad, CA). Total and phospho-specific protein antibodies were from Cell Signaling
Technology (Danvers, MA, USA).
3.5 CELL CULTURE PROCEDURES
3.5.1 Primary human skeletal muscle cell culture
Rectus abdominus biopsies were obtained from four males and four females who
underwent abdominal surgery. The mean age and BMI of the subjects were 54±6 years
and 25.6±1.5 kg/m2, respectively. The subjects had no known metabolic disorders and
they presented with normal plasma glucose. Trypsin digestion was used to separate
muscle satellite cells from the muscle biopsies. Isolated myoblasts were propagated in
growth media [DMEM (1 g/L glucose) with 20% FBS, 1% Penicillin/streptomycin, 1%
fungizone] and grown to >80% confluence prior to exposure to differentiation media
(DMEM with 2% FBS, 1% Penicillin/streptomycin, 1% fungizone), which stimulates
differentiate and formation of myotubes (Al-Khalili et al., 2004a) . Differentiated
myotubes were exposed to dimethyl sulfoxide (DMSO, vehicle) or FGF21 (1 μg/mL)
for 2, 6, and 24 h. Cells were serum-starved with or without FGF21, as appropriate, for
4 hours prior to a specific metabolic experiment (glycogen synthesis or glucose
uptake).
20
3.5.2 L6 cell culture
Rat L6 muscle cells (received as a gift from Professor Amira Klip, Hospital for Sick
Children, Toronto, ON, Canada) were grown in MEM-α media (10% FBS, 1%
penicillin/streptomycin, and 1% Fungizone) until confluent (>80% confluence) and
then cultured with differentiating media (MEM-a with 2% FBS, 1%
penicillin/streptomycin, and 1% Fungizone) for 4 days.
3.6 METABOLIC STUDIES IN CELL CULTURE
3.6.1 Glycogen synthesis
Differentiated skeletal muscle myotubes were serum-starved for 4-18 hours prior to the
experiment. Myotubes were then incubated in the absence or presence of 60 or 120nM
insulin for 30 min followed by a 90 min incubation with 5mM of glucose containing
insulin and D-glucose [U-14C] (1 µCi/mL; Amersham, Uppsala, Sweden). Myotubes
were washed in ice-cold PBS and then lysed in 0.03% of sodium dodecyl sulphate
(SDS). Carrier glycogen was added to the lysate, heated at 95°C and incubated for 30
min. Subsequently, 95% ethanol was added and the samples were incubated overnight
in -20°C to precipitate glycogen. Following centrifugation, the precipitating glycogen
pellets were washed with 70% ethanol and resuspended in distilled water. Liquid
scintillation counting was used to measure radioactivity (WinSpectral 1414 liquid
scintillation counter; Wallac/PerkinElmer, Waltham, MA, USA). Protein concentration
was estimated using the lysate from the same experiment.
3.6.2 Glucose uptake
Following approximately 18 hours of serum starvation, differentiated myotubes were
incubated in glucose-free DMEM in the absence or presence of 120 nM insulin at 37°C
for 30 min. Myotubes were subsequently incubated for 10 min in 5 mM 2-[G-H3]
deoxy-D-glucose. After washing cells 3 times in ice-cold PBS, cells were lysed in 0.5
M NaOH. Scintillation fluid was added to 500 µl of lysate and radioactivity was
measured by liquid scintillation counting (WinSpectral 1414 liquid scintillation
counter; Wallac/PerkinElmer, Waltham, MA, USA).
3.6.3 Media lactate determination
Lactate concentration in the media was determined using a lactate kit from Biomedical
Research Service Centre, University at Buffalo (Buffalo, NY). Media samples were
diluted 1:20 and then 20 μL of the dilution was added to a 96-well microplate. The
enzymatic reaction was initiated with the addition of 50 μL Lactate Assay Solution to
each well. The solutions were mixed by gentle agitation and the microplate was
covered and incubated at 37°C for 1 h. The reaction was terminated by adding 50 μL of
3% (0.5 M) acetic acid per well and the samples were mixed by brief agitation.
Absorbance was measured at 492 nM.
3.6.4 siRNA transfection and fatty acid treatment
siRNA oligos for STAT3, SOCS3, or scrambled sequences (OnTargetplus) were
purchased from Dharmacon (Chicago, IL). On day 3 of differentiation at ~70%
confluence, myotubes were cultured in antibiotic-free MEM-α media and transfected
with the specific siRNA (1 mg/mL) by calcium phosphate precipitation (Cell Phect
Transfection kit, Amersham Pharmacia). On day 6 of differentiation, transfected
21
myotubes were exposed to BSA (control) or BSA-conjugated fatty acid (0.25 mmol/L
palmitate) for 24 hour. During the last 4 h of the incubation procedure, cells were
cultured in serum-free media in the presence or absence of palmitate. Thereafter,
myotubes were incubated in the presence or absence of 60 or 120 nmol/L insulin for
determination of glucose incorporation into glycogen and protein phosphorylation. For
time-course experiments, differentiated L6 myotubes were exposed to BSA (control) or
BSA-conjugated fatty acids (0.25 mmol/L palmitate) or mouse recombinant IL-6 (20
ng/mL) for 0, 2, 6, 12, 24, or 36 h. Cells were then harvested and lysates were prepared
for Western blot analysis.
3.6.5 Cell surface GLUT1 and GLUT4 determination
In order to determine the abundance of GLUT1 and GLUT4 at the cell surface,
myotubes were treated with either FGF-21 or vehicle (DMSO). The incubation protocol
previously described for glucose uptake in myotubes was used, followed by an
additional incubation for 5 min at 18°C. Myotubes were then washed and biotinlyated
with Krebs- Henseleit bircabonate buffer (KHB) supplemented with 5 mM HEPES and
0.1 % BSA with 100 µM Bio-LC-ATB-BGPA {4, 4 - O - [2 - [2 - [2 - [2 - [2 - [6 -
(biotinylamino) hexanoyl] amino ] ethoxy]ethoxy]ethoxy] -4 - (1-azi - 2, 2, 2, rifluoro-
ethyl) benzoyl] amino-1, 3-propanedyl bis-D-glucose} for 8 min, followed by 3 min
irradiation. Myotubes were then washed with PBS before being solubilized and scraped
into 1 ml PBS with 2 % thesit (C12E9) and protease inhibitors. Cell extracts were rotated
for 60 min in microtubes at 4°C, followed by 10 min centrifugation at 20,000 g. The
supernatant was removed for protein measurement. Equal amounts of protein were
mixed with 50 µl of PBS-washed streptavidin agarose beads (50% slurry; Pierce, Inc.,
Rockford, IL, USA). The streptavidin-biotin complex was incubated > 16 hours at 4°C
with end-to-end rotation. The streptavidin agarose beads were then washed several
times in PBS with varying concentration of thesit. Photolabeled glucose transporters
were eluted from the streptavidin agarose beads by heating in 4 x Laemmli buffer for
20 min at 56°C. Proteins were separated by electrophoresis on pre-cast gels (biorad)
and Western immunoblot analysis was performed to determine GLUT1 and GLUT4
cell surface abundance.
3.7 ANIMAL STUDIES
3.7.1 Animal models
For Study II, male wild-type C57BL/6 mice were maintained on a 12-h light-dark cycle
and allowed free access to standard rodent chow. Mice were fasted 4 h prior to study.
Mice were anaesthetized via an intraperitoneal injection of 2.5% avertin (0.02 mL/g of
body weight), and the extensor digitorum longus (EDL) and soleus muscles were
rapidly removed for in vitro studies, as described below. Mice were euthanized by
cervical dislocation immediately after muscle dissection. The Ethics Committee on
Animal Research in Northern Stockholm approved all experimental procedures.
3.7.2 Isolated skeletal muscle procedures
Following dissection, EDL and soleus muscles were incubated in
oxygenated Krebs-Henseleit prebuffer at 30°C under a constant gas phase (95% O2/5%
CO2) in separate vials. Muscles were exposed to either DMSO (vehicle control) or
FGF21 (1 μg/mL) for 6 h. The final concentration of DMSO in all vials was adjusted to
2.25 mM. Media was refreshed every hour. During the last hour of the incubation, the
muscles were also incubated in the absence or presence of insulin (0.36 nM Actrapid;
22
Novo Nordisk, Bagsværd, Denmark). Thus, the muscles have been subjected to four
different conditions: basal (DMSO vehicle control), insulin, FGF21, or FGF21 and
insulin. Finally, for measurement of glucose uptake, the muscles were incubated under
the same conditions as described earlier. Thereafter, the muscles were incubated in a
glucose-free pre-buffer for 10 min. They were then transferred to new vials containing
pre-oxygenized Krebs–Henseleit buffer supplemented with 1 mM 2-deoxy-
[1,2,3H]glucose (2.5 μCi/mL) and 19 mM mannitol and incubated for 20 min. After the
incubation period, the muscles were washed in ice-cold Krebs-Henseleit buffer, blotted
on filter paper, and quickly frozen with aluminium tongs pre-cooled in liquid nitrogen
and stored at -80°C. Scintillation fluid containing 2-deoxyglucose was used to assess
glucose uptake for 20 min at 30°C. The muscles were pulverized in microcentrifuge
tubes over liquid nitrogen. Powdered muscle was homogenized in 0.4 mL of ice-cold
lysis buffer [20 mM Tris (pH 8.0), 137 mM NaCl, 2.7 mM KCl, 10 mM NaF, 1 mM
MgCl,1 mMNa3VO4, 0.2 mM PMSF, 10% glycerol, 1% Triton X-100, 1 μg/mL
aprotinin, 1 μg/mL leupeptin, and 1 μg/mL pepstatin A]. Homogenates were rotated for
60 min at 4°C and subjected to centrifugation (20 000 g for 10 min at 4°C), and the
supernatant was transferred to a second microcentrifuge tube for protein determination.
Protein concentration was assessed using the Pierce method. Glucose transport activity
is expressed as nM 2-deoxyglucose × mg/(protein*20 min).
3.8 EXPERIMENTAL ASSAYS AND ANALYSIS
3.8.1 Protein concentration assay and western blot analysis
Protein concentration for Western blot and other analyses was measured in homogenate
prepared from skeletal muscle cells or biopsies by the Braford (Bio-rad, Richmond CA,
USA) or Pierce (Rockford, IL, USA) methods. The assays were performed according to
the respective manufacturer’s instructions.
Aliquots of cell lysates were mixed with 4x Laemmli buffer and heated at 56°C
for 30 min. Proteins were resolved by SDS-PAGE using pre-casted gels, and
transferred to nitrocellulose membranes. Following protein transfer, membranes were
blocked in 7.5% low fat milk in TBST at room temperature for 1 hour in order to
prevent non-specific binding. Membranes were washed to remove milk solution and
then incubated overnight at 4°C with antigen-specific antibodies. Membranes were
washed 5 times with TBST and incubated with appropriate secondary antibodies
(horseradish peroxidase-conjugated) for 1 hour at room temperature. Proteins were
visualized by enhanced chemiluminescence and quantified by densitometry. Results
were normalized to a respective total protein or GAPDH, a housekeeping protein.
3.8.2 Gene expression studies
Gene expression studies using real time polymerase chain reaction (RT-PCR) were
used in Study II and III. mRNA expression was measured in vastus lateralis skeletal
muscle, human and L6 myotubes using quantitative RT-PCR (ABIPRISM 7000
Sequence Detection System, Applied Biosystems). Total RNA was purified from
skeletal muscle specimens using Trizol reagent (Invitrogen, Carlsbad, CA) and from
human and L6 myotubes using RNeasy Mini Kit (Invitrogen). Purified RNA was
treated with DNase using a DNA-free kit (Ambion), and cDNA synthesis was
performed with SuperScript First Strand Synthesis system (Invitrogen). A SYBR green-
based gene expression assay was used to assess mRNA (Origene). All samples were
assayed in duplicate and values were compared against the housekeeping genes β-actin
and 18S as internal controls. Standard curve and relative expression methods were used
to quantify mRNA expression (Applied Biosystems).
23
3.9 STATISTICAL ANALYSES
In the Studies included in this thesis, data is presented as Mean ± SEM. Paired
and unpaired Student t-tests were used to compare differences within and between
groups, respectively. Data was examined for normality before performing any further
statistical analysis and appropriate statistical tests were assigned for each set of data.
Log transformation was performed on data sets that did not display normal distribution.
Pearson’s correlation analysis was used to establish relationships between two
variables. Further details of statistical analysis are explained specifically in each
separate study.
24
4 RESULTS AND DISCUSSION
The biological processes regulating glucose and lipid metabolism are critical to the
maintenance of whole-body energy homeostasis. Appropriate intracellular glucose and
lipid breakdown satisfies the intrinsic fuel requirement and thereby translates into
systemic physiological health. Conversely, perturbations in this delicate substrate
balance leads to impaired fuel utilization, which can ultimately manifest as ill health.
Obesity and T2DM represent a culmination of imbalances in energy homeostasis that
persist over time as a result of impaired glucose and lipid metabolism. Understanding
both the cellular and whole-body regulation of substrate homeostasis in normal and
insulin resistant states is key to the management of T2DM. A combination of
experimental and clinical approaches to directly translate basic mechanistic discoveries
to the treatment and prevention of T2DM is therefore warranted. To this end, the
studies presented in this thesis were conducted in humans, animal models and in vitro
cell systems to ensure a proper translation of basic scientific enquiries to clinical
application.
4.1 CLINICAL IMPLICATIONS FOR EXERCISE-INDUCED BENEFITS IN
T2DM
The exercise-induced benefits on glucose and lipid metabolism are of clinical
relevance in the prevention and treatment of T2DM (Chibalin et al., 2000).
Additionally, accumulating evidence stresses the importance of regular exercise as
effective intervention against cardiovascular risk factors (Tsuruzoe et al., 2001), obesity
(Feldstein et al., 2008; Henquin, 2009; Henquin et al., 2009; Longo et al., 2008), and
impaired insulin metabolism in T2DM (Corpeleijn et al., 2009;). Aerobic exercise is
regarded as a highly effective method of physical activity to improve insulin sensitivity.
However, greater consideration of a minimal and more realistic exercise intervention
that is practically applicable in a normal primary care setting is warranted. In Study I,
the effects of a moderate, unsupervised exercise intervention (Nordic walking) has been
investigated on cardiovascular risk factors in overweight individuals with NGT, IGT
and T2DM.
4.1.1 Pronounced beneficial effects of Nordic walking in normal as compared
to impaired glucose metabolism.
The vast majority of exercise intervention studies involve relatively intense,
expensive, and closely supervised exercise programs that demand a large number of
personnel and considerable resources. Often, these exercise programs do not reflect
lifestyle interventions that are achievable in a normal primary care setting. Studies
aimed at defining a physical activity level that is more realistic in terms of time and
effort for older sedentary individuals, yet still confers vital health benefits, are therefore
warranted. Nordic walking is a validated moderate exercise modality, which is easy to
perform. Importantly, Nordic walking is associated with improved adherence (Figard-
Fabre et al., 2011) and is associated with low risk for injury or other complications. The
focus of Study I was to determine the effects of a low-moderate, unsupervised exercise
intervention on cardiovascular risk factors and metabolic control in a traditional clinical
setting of a local primary health care center. The hypothesis that Nordic walking
(Church et al., 2002), will reduce cardiovascular risk and improve metabolic control
was tested in overweight people with NGT, IGT and T2DM.
25
4.1.1.1 Clinical characteristics of the study volunteers
A total of 213 subjects were included and classified on the basis of an oral
glucose tolerance test (OGTT) into either NGT (n=128), IGT (n=35) or T2DM (n=50).
The participants in each category (NGT, IGT and T2DM) were randomized to the
exercise or control group and asked to maintain their usual eating habits. Baseline
clinical characteristics for the study participants are presented in Table 1 of article
number 1 at the articles section. Self-reported high-intensity physical activity was more
frequent in the NGT control group than in the NGT intervention group. Subjects in the
three cohorts were age and BMI-matched, but some slight differences across
intervention groups in several clinical parameters were present. The total cholesterol
level was lower and lipid-lowering statin medication was more frequent in the NGT
intervention group, compared with the NGT control group. The triglyceride level was
lower in the T2DM control group, but statin medication did not differ in comparison
with the T2DM intervention group. There were no other significant differences
between the control and intervention groups.
4.1.1.2 Effect of regular low-moderate intensity exercise (Nordic walking).
The aim of the study was to achieve cardio-protective benefits and better
metabolic control in a cohort of sedentary subjects by implementing a relatively
moderate-intensity exercise modality in a normal primary health care setting. A
previous study examining a cohort of individuals with similar clinical characteristics to
the present cohort provided evidence that four months of Nordic walking intervention,
performed at intervals of 45-60 min x 3 times per week, was insufficient to improve
cardiovascular risk factors in T2DM (Fritz et al., 2006). Therefore, increasing the
frequency and intensity of exercise may be necessary to induce health benefits in
T2DM. Compared to the previous study (Fritz et al., 2006), the current study
participants increased not only the exercise intensity, but also number of individual
exercise sessions.
Self-reported physical activity and anthropometric parameters (BMI and waist
circumference) were improved after 4 months of regular walking in the NGT-exercise
group compared to NGT-control group. Exercise capacity was improved in the IGT-
exercise group compared to the IGT-control group. Four months of Nordic walking did
not result in any significant difference between the T2DM-exercise and T2DM-control
group. However, improvements in HbA1c, 2 hour glucose and exercise power output
was noted in NGT, IGT and T2DM individuals who reported ≥80% of the prescribed
recommended amount of Nordic walking in their exercise diaries. Together, these
findings provide evidence for a varying response to a similar exercise modality in NGT,
IGT and T2DM individuals, and stress the importance of adherence to achieving better
cardio-protective benefits.
Overall, the clinical exercise intervention study (Paper II) highlights that a 4-
month low-moderate intensity Nordic walking program improves body weight, BMI,
and waist circumference in overweight people with NGT. Improvements in weight,
BMI and waist circumference constitute cardio-protective benefits. Moreover, in
participants who reported ≥80% of the prescribed exercise, HbA1c, 2 h glucose, and
exercise capacity improved, underscoring the importance of adherence to achieving
favorable metabolic control (Saltiel and Kahn, 2001).
26
4.1.1.3 Clinical implications
Visceral fat is a predictor of mortality and waist circumference may be one
simple measure of cardiovascular risk to monitor improvement following a period of
lifestyle intervention. The findings presented here suggest that exercise intervention has
a more pronounced effect on the anthropometric risk factors in people who do not have
derangements in glucose metabolism.
Although Nordic walking exercise improves cardiovascular risk-factors in
overweight T2DM participants, participants with NGT achieved greater improvements
(paper II). A three month Nordic walking program reduces fat mass and blood pressure
in obese people with NGT (Figard-Fabre et al., 2011). Moreover, previous study has
reported improvements in fat mass, but not HbA1c in T2DM patients after completing
a four month Nordic walking program (Gram et al., 2010). In paper II improvements in
metabolic control were reported in obese participants after four months of Nordic
walking in exercise responders but not in non-responders. Although varying frequency,
intensity and adherence to exercise protocols may explain the inconsistencies in
exercise effects, intrinsic factors governing exercise-response/non-response need
further investigation.
The results presented here may indicate that the ability to respond to lifestyle
intervention, such as exercise, is more effective during the early stages of the T2DM
pathogenesis. Moreover low motivation and musculoskeletal complications that may
occur in T2DM result in lack of adherence to exercise programs (Saltiel and Kahn,
2001). Together these observations highlight the importance of early exercise
intervention in the prevention and treatment of T2DM.
4.1.1.4 Study limitation
A low-cost moderate intensity exercise was investigated in the current study.
Higher intensity exercise protocols such as aerobics and/or resistance training can result
in a more beneficial outcome (Roumen et al., 2008; Sigal et al., 2006; Snowling and
Hopkins, 2006); however positive effects on cardiovascular risk factors could still be
achieved with this relatively low level of physical activity. Even though more
pronounced effects were noted in the NGT group. However, IGT and T2DM
participants who reported good compliance achieved a better metabolic control in a
number of clinical parameters. Of clinical relevance, this study could be achieved
without occurrence of musculoskeletal complications which is more prevalent in
T2DM (Arkkila and Gautier, 2003). Thus, Nordic walking if performed regularly,
might offer a safe mode of introductory exercise, even in T2DM.
While the current study highlights Nordic walking improves cardiovascular risk
factors in people with varying degrees of glucose tolerance, the following study
limitations need to be considered when interpreting the data. First individual
participants reported a considerably varying physical activity levels at baseline. This
might have affected the outcome hence explaining part of the variation in Nordic
walking effects. Furthermore this study involved unsupervised, self-reported exercise
and whether the participants in the intervention group added Nordic walking in their
pre-existing daily activity or replaced their daily activities with Nordic walking is not
known.
Secondly, the relatively small number of study participants might have affected
the statistical power. A greater number of participants would have strengthened the
statistical power, hence increasing the chances of attaining significance in many tested
parameters. Lastly while food intake can affect the study outcomes, individual food
intake was not addressed in this study, despite the possibility of increased food intake
27
with increased physical activity levels. Of note, this study aimed at investigating
physical activity per se, and not lifestyle modification.
4.2 SYSTEMIC REGULATORS OF GLUCOSE AND LIPID METABOLISM
Various hormones, cytokines, and growth factors circulating in serum play a role
in the regulation of glucose and lipid metabolism (Pittas et al., 2004). These factors
exert their effects in an autocrine, paracrine and/or endocrine manner; hence, their
effects are typically systemic. Fibroblast growth factor 21 (FGF-21) is an endocrine
metabolic regulator with systemic effects on both glucose and lipid metabolism.
Reports from studies conducted in animal models and in vitro systems have provided
evidence for the role of FGF-21 in the regulation of glucose and lipid metabolism in
liver, adipose tissue and pancreas (Coskun et al., 2008; Hotta et al., 2009;
Kharitonenkov et al., 2005; Kharitonenkov et al., 2007; Kralisch and Fasshauer, 2011;
Sarruf et al., 2010; Wente et al., 2006). Whether FGF-21 has a direct effect on glucose
metabolism in skeletal muscle is still unknown. The focus of Study II was therefore to
investigate the direct effects of FGF-21 on glucose metabolism in skeletal muscle.
Furthermore, using serum from NGT and T2DM subjects, the aim was to establish the
relationship between circulating FGF-21 levels and clinical parameters related to
glucose and lipid metabolism. The findings in this study provide further support for the
clinical significance of this novel tissuekine in the diagnosis and treatment of obesity
and T2DM.
4.2.1 FGF-21 as a metabolic regulator of glucose metabolism in skeletal muscle
4.2.1.1 FGF-21 has a direct effect on glucose uptake in human skeletal muscle
Owing to its endocrine properties, FGF-21 circulates in the serum and exerts its
effects in a variety of tissues and organs such as liver, pancreas, and skeletal muscle.
The beneficial effects of this endocrine regulator on glucose metabolism in liver and
adipose tissue are well established (Coskun et al., 2008; Hotta et al., 2009;
Kharitonenkov et al., 2005; Kharitonenkov et al., 2007; Kralisch and Fasshauer, 2011;
Sarruf et al., 2010; Wente et al., 2006). Skeletal muscle is quantitatively an important
tissue in glucose metabolism (Wannamethee et al., 2011d). However, evidence related
to the the metabolic effects of FGF-21 on glucose uptake in skeletal muscle is lacking.
Cultured human skeletal muscle and isolated rodent muscle was used to
determine whether FGF-21 could directly regulate glucose uptake. Differentiated
myotubes were exposed to 1 μg/ml of recombinant FGF-21 and glucose uptake was
measured after 6 and 24 hours. FGF-21 increased basal glucose uptake in a time-course
dependent manner. Glucose uptake tended to increase after 6 hours and was increased
after 24 hours of exposure with FGF-21. In cultured murine adipocytes, FGF-21
enhances glucose uptake as a result of an increased GLUT1 mRNA and protein
expression (Kharitonenkov et al., 2005). To determine whether a similar effect occurs
in skeletal muscle cells, GLUT1 mRNA was measured in cultured myotubes and cell
surface photolabelling was assessed to determine the relative abundance of GLUT1 and
GLUT4 proteins at the plasma membrane. GLUT1 mRNA expression was increased
following 24 hours exposure to FGF-21. Moreover, exposure to FGF-21 increased the
relative abundance of GLUT1 protein on the cell surface. To determine whether FGF-
21 has an additive effect with insulin on glucose uptake, myotubes were exposed to
FGF-21 or DMSO as control for 6 or 24 hours and measured glucose uptake under
basal and insulin-stimulated states. Indeed, both 6 and 24 hour FGF-21 exposure had an
28
additive effect with insulin on glucose uptake. These results provide evidence for a
synergistic action between insulin and FGF-21 on glucose uptake.
Based on the observations from human cell culture system, these findings were
validated in whole skeletal muscle. Using an in vitro muscle glucose uptake assay,
isolated mouse EDL muscle was pre-exposed to FGF-21 for 6 hours, followed by 20
min incubation with insulin. Consistent with the findings in cultured human myotubes,
insulin-stimulated glucose uptake was increased in EDL muscle pre-exposed with FGF-
21. Conversely, FGF-21 was without effect on the basal glucose uptake in EDL muscle.
Taken together, FGF-21 increased glucose uptake in both the absence and presence
of insulin in primary human myotubes,. However, in isolated mouse skeletal muscle,
FGF-21 only increased glucose uptake under insulin-stimulated conditions. These
disparities may be related to the FGF-21 exposure time or intrinsic differences between
cultured and whole skeletal muscle. In cultured human muscle cells, FGF-21 promotes
increased protein abundance of cell surface GLUT1, but not GLUT4 protein. As
cultured muscle has a higher GLUT1: GLUT4 ratio as compared to whole muscle, this
may explain the enhanced sensitivity to FGF-21 on glucose uptake in cultured muscle.
Furthermore, in cultured human myotubes, the most pronounced effect of FGF-21 on
basal glucose uptake was noted after 24 hour, a time point that is challenging to
establish in in vitro muscle incubation experiments. Alternatively, primary muscle
cultures are derived from muscle satellite cells, which do not fully recapitulate whole
muscle (Al-Khalili et al., 2004b); consequently, this could also explain the
discrepancies between the model systems.
4.2.1.2 Therapeutic implications in treatment of T2DM.
The results presented in Study II are consistent with the reported metabolic
effects of FGF-21 in adipose tissue (Kharitonenkov et al., 2005). FGF-21 increases
glucose uptake in mouse 3T3-L1 cells, and in primary human adipocytes
(Kharitonenkov et al., 2005). Here, these findings are extended to skeletal muscle, a
quantitatively important tissue in glucose homeostasis. Previous studies reported
improvements in metabolic profiles in obese animal models following treatment with
FGF-21. Enhanced β cell function in the pancreas and regulation of lipid and glucose
metabolism in liver are among other reported beneficial effects of FGF-21(Coskun et
al., 2008; Hotta et al., 2009; Kharitonenkov et al., 2005; Kharitonenkov et al., 2007;
Kralisch and Fasshauer, 2011; Sarruf et al., 2010; Wente et al., 2006). Collectively,
these findings suggest that FGF-21 could act as a potential therapeutic option for the
treatment of T2DM.
29
Figure 7: Effects of FGF-21 on glucose metabolism. Systemic FGF-21 circulation promotes enhanced
glucose uptake in skeletal muscle and adipose tissue, improved β cell function in pancreas, and greater
regulation of glucose and lipid metabolism in liver.
4.2.2 FGF-21 as a potential biomarker
4.2.2.1 FGF-21 serum levels are increased in T2DM.
Liver and adipose tissue are the main source of FGF-21, but skeletal muscle and
thymus (Hojman et al., 2009; Mraz et al., 2009; Nishimura et al., 2000) have also been
reported to secrete FGF-21. The relative tissue-specific contribution to serum FGF-21
levels is unknown. A number of clinical and epidemiological studies have analyzed
circulating FGF-21 and described the relationship to diabetes and other pathologies
related to glucose homeostasis. Serum levels of FGF-21 are higher in states of
30
abnormal glucose metabolism as compared to normal states (Hojman et al., 2009; Mraz
et al., 2009; Semba et al., 2012). In Study II, levels of FGF-21 were found in T2DM
patients compared to age and BMI-matched normal glucose tolerant subjects,
confirming the previously observed paradox. Higher FGF-21 levels have also been
found in insulin resistant adults (Semba et al., 2012). Furthermore, a positive
association between FGF-21 serum levels and markers of diabetes complications has
been reported in clinical and epidemiological studies (An et al., 2012; Jian et al., 2012).
FGF-21 is an independent marker for the presence of the metabolic syndrome in
obesity in adults (Tyynismaa et al., 2011; Zhang et al., 2010; Zhang et al., 2008). In the
cohort examined in Study II, serum FGF-21 levels were significantly greater in T2DM
patients in the tertile of subjects presenting the highest fasting insulin and BMI.
Moreover, BMI was identified as an independent predictor of serum FGF-21 levels.
Recent studies reported an increase in levels of FGF-21 in serum and a positive
correlation with intra-hepatic lipid content in NAFLD, reflecting the ability of FGF-21
to independently predict liver steatosis (Dushay et al., 2010; Li et al., 2010; Yan et al.,
2011; Yilmaz et al., 2010). Collectively these findings suggest that FGF-21 could act as
a potential biomarker for metabolic diseases.
4.2.2.2 Clinical implications in obesity and T2DM.
Based on the positive effects of FGF-21 on glucose and lipid metabolism, the
paradoxical higher levels of FGF-21 in serum from people with disturbed glucose
homeostasis may reflect a compensatory mechanism as a physiological defense against
dysregulated state. A state of FGF-21 resistance may also account for its higher levels
in obesity and T2DM. Obesity is an “FGF-21-resistant” state (Fisher et al., 2010). In
Study II, parallel reduction in BMI and FGF-21 serum levels was noted following a
four month lifestyle intervention that involved regular walking in T2DM patients
(figure 8). This was accompanied by concomitant improvements in insulin sensitivity.
Changes in FGF-21 serum levels following lifestyle and pharmacological interventions
have been reported elsewhere (Cuevas-Ramos et al., 2012; Dutchak et al., 2012;
Fletcher et al., 2012; Wei et al., 2012). Thus, because FGF-21 levels are dynamically
influenced by various intervention modalities, it could be used as a marker to monitor
clinical progress following an intervention. Furthermore, the continuously increasing
levels of FGF-21 with increase in the severity of obesity and insulin resistance
observed in Study II and other published reports suggests that FGF-21 may be used to
monitor the progression of obesity and T2DM. However the validity of this notion
requires further investigations in prospective follow-up studies.
31
Figure 8: Serum FGF21 concentrations before and after four months of participation in an adult
fitness program (low-moderate intensity exercise). The T2DM subjects were divided into two groups
based on whether or not they achieved an improvement in BMI of at least one unit (n=10 in each group).
Solid and empty bars represent before and after four months of exercise intervention respectively. Results
are presented as mean ± SEM *p-value <0.05, ** <0.01, *** <0.001.
4.2.2.3 Study limitation
The cross-sectional nature of Study II, including the correlation analysis used
to establish associations between different parameters might not explain causality.
Furthermore the relatively small sample size used to analyze FGF-21 serum levels
might limit the translation of this result to situations beyond the scope of this study.
However, the findings in this study and others provide important information on the
metabolic effects of FGF-21, including its clinical significance. This information will
aid in the design and execution of more comprehensive prospective follow-up studies
in order to provide a better understanding of the novel metabolic regulator, FGF-21.
32
4.3 MOLECULAR REGULATORS OF GLUCOSE AND LIPID
METABOLISM IN SKELETAL MUSCLE.
High levels of circulating metabolites such as FFAs and cytokines significantly
contribute to the development of insulin resistance in liver and skeletal muscle. These
metabolites signal through STAT3 and regulate a variety of biological processes. The
involvement of STAT3 in the development of insulin resistance in liver and adipose
tissue has previously been reported (Inoue et al., 2004; Kim et al., 2007). Whether this
transcription factor plays a role in the development of skeletal muscle insulin resistance
and T2DM is incompletely understood.
4.3.1 STAT3 is constitutively phosphorylated in skeletal muscle from T2DM
patients
4.3.1.1 Clinical characteristics of the study subjects
In Study III, 20 overweight, but otherwise healthy participants with normal glucose
tolerance (NGT) and 20 T2DM patients were selected from a primary health care
clinic. The NGT and T2DM participants were matched for age and BMI. Individuals on
insulin or with symptomatic coronary heart disease were excluded. Venous blood was
collected for standard clinical chemistry analysis and vastus lateralis skeletal muscle
biopsies was obtained from the participants following an overnight fast, as described
earlier (Al-Khalili et al., 2003).
4.3.1.2 Increased STAT3 phosphorylation in T2DM
Protein abundance and phosphorylation of STAT3 was measured in skeletal muscle
biopsies from NGT and T2DM subjects. Phosphorylated STAT3 was increased in
skeletal muscle biopsy from T2DM patients, compared to age- and BMI-matched NGT
subjects (Figure 9A). Protein phosphorylation of JAK2, an upstream regulator of
STAT3, was also increased in skeletal muscle from T2DM patients (Fig. 9B). To
further investigate the JAK/STAT pathway downstream of STAT3, SOCS3 mRNA and
protein abundance was measured in muscle from similar cohort. Increased SOCS3
mRNA and protein abundance was observed in biopsies from T2DM patients.
Interestingly, p-STAT3 protein positively correlated with SOCS3 protein and mRNA
expression in individuals with NGT and T2DM. Since STAT3 is involved in
adipogenesis (Zhang et al., 2011), obesity may directly influence STAT3 signaling.
However, these findings are consistent with an observed increase in skeletal muscle p-
STAT3 abundance in non-obese people with IGT (Kim et al., 2011). Furthermore, to
avoid misinterpretation of the effect of obesity, the subjects were matched for BMI.
Thus, aberrant skeletal muscle STAT3 signaling appears to be an early marker of
insulin resistance that precedes clinical diagnosis of T2DM.
33
Fig 9: Protein phosphorylation in skeletal muscle from people with normal glucose tolerance or
T2DM. Phosphorylation of (A) STAT3 (B) JAK2. Normal glucose tolerance (Open Bar) and T2DM
(Closed Bar), n = 20 subjects. Results are mean ± SEM. ***P < 0.001, **P< 0.01 and *P < 0.05 vs.
NGT, respectively.
4.3.1.3 A link between increased JAK/STAT signaling and insulin resistance
SOCS3, a key player linking the JAK/STAT pathway to insulin signaling, is
implicated in the development of insulin resistance in obesity and T2DM (Howard and
Flier, 2006). SOCS3 protein and mRNA are increased in skeletal muscle from severely
obese or T2DM patients, compared to lean people with normal glucose tolerance
(Rieusset et al., 2004). Consistently, an upregulation of SOCS3 protein abundance and
mRNA expression was observed in skeletal muscle from T2DM patients, supporting a
link between aberrant signal transduction and reduced insulin sensitivity. A positive
association between p-STAT3 and SOCS3 protein and mRNA levels in normal glucose
tolerance and T2DM was also observed, suggesting a physiological link between
phosphorylation of STAT3 and SOCS3 induction. In liver, STAT3 phosphorylation
upregulates SOCS3 protein and subsequently causes insulin resistance (Kim et al.,
2009a; Kim et al., 2008). In Study III, we extend these findings to skeletal muscle and
to states of T2DM.
4.3.2 STAT3 phosphorylation and skeletal muscle insulin resistance
4.3.2.1 Association between circulating FFA and STAT3 phosphorylation in skeletal
muscle.
Elevated FFA serum levels are associated with insulin resistance and T2DM.
Several other molecules circulating in serum such as Tumor necrotic factor alpha
(TNFα), negatively regulate insulin signaling in skeletal muscle and cause insulin
resistance. To investigate the possible cause of the increased STAT3 phosphorylation in
T2DM at the whole body level, various clinical parameters were measured and
correlation analysis was performed. Plasma FFA level was positively correlated with
skeletal muscle p-STAT3 abundance, and inversely correlated with measures of insulin
sensitivity in normal glucose tolerance individuals. However, despite the finding of
elevated FFA levels and insulin resistance in T2DM, the correlation between FFA and
p-STAT3 observed in NGT subjects was lost with T2DM. Plasma FFA level accounted
34
for greatest variation in skeletal muscle p-STAT3 abundance, highlighting a
relationship between circulating FFAs, STAT3 phosphorylation, and measures of
insulin sensitivity. The positive association between circulating FFA levels and p-
STAT3 might indicate that FFAs are indeed the cause of increased STAT3
phosphorylation in T2DM. Several nutrients and circulating metabolites have been
linked to STAT3 activation in different tissues (He et al., 2006; Kim et al., 2008;
Rieusset et al., 2004; Senn et al., 2003). Given the clinical evidence that elevated FFA
levels are a biomarker for the conversion from IGT to T2D (Charles et al., 1997;
Paolisso et al., 1995), interventions that lower FFA may prevent excessive p-STAT3
and maintain appropriate insulin signaling responses in skeletal muscle to control
glucose and lipid metabolism.
4.3.2.2 Palmitate induces insulin resistance via STAT3 phosphorylation
Phosphorylation and subsequent activation of STAT3 has been reported in
cultured myotubes exposed to either FFAs (Weigert et al., 2004) or IL-6 (Kim et al.,
2011; Weigert et al., 2004). To determine whether these systemic factors cause skeletal
muscle insulin resistance via a STAT3-mediated mechanism,the direct effect on L6
cultured myotubes was studied. Exposure of cultured myotubes to palmitate resulted in
a slow, but persistent phosphorylation of STAT3 and reduced insulin-stimulated Akt
phosphorylation. However, IL-6 exposure resulted in a rapid, but transient
phosphorylation of STAT3, without altering insulin action on p-Akt abundance. This
time-related difference in STAT3 phosphorylation between IL-6 and palmitate
exposure may explain the divergent effects between these two stimuli on insulin
signaling. Interestingly, the slow but persistent phosphorylation of STAT3 resulted into
impairments in insulin signaling as opposed to the rapid, acute STAT3
phosphorylation. This finding may suggest that chronic but not acute STAT3
phosphorylation is a culprit in the pathogenesis of insulin resistance in skeletal muscle.
4.3.3 STAT3 as a potential therapeutic target
Studies performed in tissue-specific knockout mice reveal that STAT3 plays a
role in the development of insulin resistance in liver (Inoue et al., 2004). Using siRNA,
the direct role of STAT3 on lipid-induced insulin resistance in skeletal muscle was
determined. A parallel down-regulation of SOCS3 protein abundance was observed
following STAT3 silencing. Palmitate exposure triggered STAT3 phosphorylation,
consequently causing a reduction in insulin-stimulated Akt phosphorylation and
glucose incorporation into glycogen. Importantly, STAT3 silencing prevented the
palmitate-induced increase in SOCS3 protein abundance, as well as the lipid-induced
reduction in insulin-stimulated Akt phosphorylation and glucose incorporation into
glycogen. This finding that STAT3 silencing improves insulin sensitivity is consistent
with earlier parallel findings in liver hepatocarcinoma cells and human myotubes
following treatment with amino acids or IL-6, respectively (Kim et al., 2009a; Kim et
al., 2008; Kim et al., 2009b; Kim et al., 2011). (Kim et al., 2009a; Kim et al., 2008;
Kim et al., 2009b; Kim et al., 2011). Collectively these findings suggest that STAT3
could act as a potential therapeutic target for T2DM. However, tissue-specific effects of
STAT3 silencing require further clarifications.
STAT3 is constitutively phosphorylated in skeletal muscle from T2DM
patients. Chronic elevation in circulating metabolites like FFAs is likely to cause a
persistent phosphorylation of STAT3 and negatively impact skeletal muscle insulin
signaling and glucose uptake. siRNA-mediated silencing of STAT3 protein prevents
the development of lipid-induced insulin resistance in skeletal muscle. Therefore,
interventions targeting STAT3 directly or focused towards normalizing elevated
35
circulating metabolites might prevent the development of skeletal muscle insulin
resistance early in the pathogenesis of T2DM.
Fig 10: Lipid-induced skeletal muscle insulin resistance. Elevated levels of FFA cause constitutive
phosphorylation of STAT3, resulting in the induction of SOCS3 protein and consequently causing insulin
resistance through negative regulation of insulin signaling. Interventions targeting STAT3 or lowering
FFAs levels in serum may have significant effects in T2DM prevention and treatment.
36
4.4 SUMMARY OF FINDINGS
Obesity and T2DM are metabolic disorders characterized by impaired glucose and lipid
homeostasis. Impairments in the normal physiological regulation of glucose and lipid
metabolism, resulting from both genetic and environmental factors, cause
hyperglycemia which triggers clinical diabetes. Studies presented in this thesis, which
are aimed at investigating the regulation of glucose and lipid metabolism in skeletal
muscle and serum, describe molecular interactions in a whole-body physiology context
and evaluate the corresponding clinical implications in obesity and T2DM.
• In Study I, the effects of a moderate-intensity exercise on cardiovascular risk
factors in overweight individuals with T2DM are reported. The beneficial
effects of moderate physical activity on cardiovascular risk factors were more
pronounced in normal glucose tolerant individuals as compared to people with
impaired glucose tolerance. This highlights the importance of early lifestyle
intervention (exercise) in the treatment of T2DM.
• In Study II, a paradoxically higher level of FGF-21 in obesity and T2DM was
reported. Evidence for the role of this novel endocrine regulator on glucose
metabolism in skeletal muscle was provided. The findings in Study II extend
the metabolic effects of FGF-21 to skeletal muscle, a qualitatively important
tissue in glucose homeostasis.
• In Study III, a differential regulation of STAT3 in normal and T2DM states was
reported. Study III provides evidence that constitutive STAT3 phosphorylation
plays a role in the development of lipid-induced insulin resistance in skeletal
muscle. Indeed, silencing of STAT3 in L6 myotubes prevents palmitate-
induced insulin resistance, as measured by glycogen synthesis and p-Akt.
Targeting STAT3 in skeletal muscle could therefore present therapeutic benefits
in T2DM.
Collectively, substrate (glucose and lipid) regulation and its clinical significance
in obesity and T2DM has been described. Metabolic characteristics from the
whole body physiology perspective into specific systemic modulators in serum
has been analyzed. The studies presented in this thesis dissect basic molecular
mechanisms that fuel the pathogenesis of T2DM in skeletal muscle, as
summarized in Figure 11.
37
Figure 11. Regulators of glucose and lipid metabolism investigated in this thesis. Physical activity
has positive effects on glucose and lipid metabolism. These effects are noted at the systemic level.
Circulating factors like FGF-21 target different organs and tissues and also regulate glucose and lipid
metabolism. Skeletal muscle is an important consumer of both glucose and lipids, and thus crucial for the
regulation of these two key substrates.
38
5 CONCLUSIONS AND FUTURE PERSPECTIVES
The overall objective of work presented in this thesis was to investigate the
regulators of glucose and lipid metabolism in skeletal muscle and serum, describe their
interactions and finally, evaluate their clinical implications in obesity and T2DM. The
approach taken involved investigating the interaction between physical exercise and
glucose metabolism at the whole-body level. The studies were advanced to undertake a
more specific investigation of a novel regulator in serum, and finally to dissect the
molecular mechanisms involved in the development of insulin resistance and T2DM in
skeletal muscle.
Studies from this thesis provide evidence for differential effects of low-
moderate exercise (Nordic walking), in subjects with normal and impaired glucose
tolerance. Anthropometric variables related to cardiovascular risk factors improved in
NGT individuals, but not in those with impaired glucose tolerance. This underscores
the importance of early lifestyle intervention in the prevention of cardiovascular
complications in overweight individuals. Nordic walking offers a safe mode of exercise
that can easily be tolerated with T2DM patients. The findings reported in Study I show
that, with high compliance, individuals with T2DM can also achieve significant
metabolic improvements with Nordic walking. Indeed, in Study II, T2DM patients who
underwent exercise intervention and responded by lowering their BMI, were able to
improve metabolic control, as well as trigger a decrease in serum FGF-21 levels. While
differences in compliance could explain the varying effects of exercise in different
individuals, other extrinsic and intrinsic factors could play a paramount role in this
biological phenomenon. Future research should therefore focus on identifying
exogenous and endogenous regulators of exercise response and non-response.
Treatment of T2DM is relatively challenging. The available pharmacological
agents have limited efficacy and mechanism-based side effects. An urgent need for safe
and more effective agents has stimulated research in the field, and a number of novel
molecules with therapeutic potential are continuously being identified. Current
evidence points to FGF-21 as a novel metabolic regulator with therapeutic potential in
the treatment of T2DM. Earlier studies investigating FGF-21 support its role in glucose
and lipid metabolism in liver, adipose tissue and pancreas. Study II extends the findings
to skeletal muscle. Mechanisms governing FGF-21-dependent glucose uptake
previously described in adipose tissue were shown to also occur in skeletal muscle.
Results from the analysis of FGF-21 in serum confirmed the earlier reported paradox of
higher FGF-21 levels in obesity and T2DM. This phenomenon is hypothesized to arise
from FGF-21 resistance that occurs in obesity and T2DM. Indeed moderate intensity
exercise, which resulted in a minimal weight loss, lowered the levels of FGF-21 in
serum of T2DM patients who participated in Study II. Whether the decreased FGF-21
serum levels was a result of a decrease in its production due to decreased fat mass, or
improvements in FGF-21 resistance per se, is a question for further research.
Future research on the metabolic regulator FGF-21 should address the reported
paradox on its serum levels in obesity and T2DM. Even though the available evidence
implicates FGF-21 resistance, the possibility of increased FGF-21 serum levels as a
compensatory mechanism against impaired metabolism, should not be overlooked.
However, in both cases, serum FGF-21 levels could reflect a state of impaired glucose
and lipid metabolism, a phenomenon that can be harnessed as a biomarker.
Investigating the role of FGF-21 as a potential biomarker should constitute future
research opportunities.
The mechanisms involved in the pathogenesis of skeletal muscle insulin
resistance in T2DM remain incompletely resolved. A wide array of nutrients and
39
hormones interact with insulin signaling via complex pathways and cause insulin
resistance in skeletal muscle. Until recently, the involvement of STAT3 in the
development of insulin resistance was known to involve only the liver and adipose
tissue. A recent study showed that STAT3 is involved in the development of cytokine-
induced insulin resistance in skeletal muscle. Indeed, the findings in Study II confirmed
the involvement of STAT3 in the development of skeletal muscle insulin resistance.
This finding was further extended to T2DM pathogenesis. Constitutive STAT3
phosphorylation appears to be involved in lipid-induced skeletal muscle insulin
resistance since silencing STAT3 in cultured rat myotubes could prevent palmitate-
induced insulin resistance. STAT3 could therefore present a potential drug target for
treatment of T2DM. Furthermore, these findings provide evidence that early
intervention aimed at normalizing FFA levels in serum could prevent the development
of insulin resistance.
Collectively, the work presented in this thesis emphasizes the importance of
understanding various regulators of glucose and lipid metabolism from the whole body
physiology context to molecular mechanisms in skeletal muscle. Metabolic alterations
result from the interplay between biological processes within the cells, tissues and
organs. These alterations may translate into ill health such as T2DM. Translational
studies involving both molecular and clinical studies will help to identify molecules
with both clinical significance and therapeutic potential. Identification of these
molecules is crucial for the fight against obesity and T2DM.
40
6 ACKNOWLEDGEMENTS
The whole process towards the production of this thesis required
dedication, team work, supervision, financial support and, above all, the right
motivation. This would not have been possible without the support of different people
from within and outside the scientific community. I wish to express my immense
gratitude and thanks to people whose helping hands were always there for me.
I wish to convey my deepest gratitude to Professor Anna Krook, my main
supervisor, for constant motivation, advice and endless support that were of great help
for my projects. Thank you, Anna, for being kind and always positive towards my
work. Your positivity gave me a reason to keep going, and your kindness fueled my
motivation. You have shared so much knowledge on science and communication that
helped me to significantly boost my scientific standard. This will always guide my
scientific journey. You are such a wonderful supervisor.
My sincere gratitude and thanks to my co-supervisor Professor Juleen Zierath.
Thank you, Juleen, for trusting me and giving me the opportunity to join Integrative
Physiology, and above all, for giving me valuable advice on how to communicate
science. Your advice, both scientific and general, has been a cornerstone to my carrier
growth. You have been a wonderful leader and a great source of inspiration.
Special thanks to my external mentor, Professor Jan Lindsten, for always being
there. Your wisdom has always helped me to face and overcome challenges. Our
constant meetings translated into energy and motivation that helped me to move
forward. Thank you, Jan, for making my scientific journey fine-tuned .You have been a
true and wonderful mentor.
I would also like to acknowledge Docent Alexander Chibalin, for helping me
with technical and core scientific methods. Your broad scientific and general
knowledge has really been instrumental towards accomplishing this work. I really
enjoyed working with you.
I would like to thank Dr. Megan Osler for introducing me to the world of
scientific research and organization. Thank you, Megan, for guiding me through
material organization and processing. I learned a lot from your high organizing skills,
including the proper use of excel spreadsheet that helped a lot with data handling and
analysis. Special thanks to Dr. Boubacar Benziane for teaching me the science and
art of Western blotting, Dr. Marie Björnholm for guiding me through journal clubs
and literature discussion in general, and Docent Lubna Al-Khalili for your profound
expertise in cell culture. My work wouldn’t have been complete without your support.
My humble appreciation to Dr. Tomas Fritz for sharing his expertise in muscle
biopsy and exercise test techniques. Thank you, Tomas, for giving me the opportunity
to activate and extend my clinical knowledge and skills. Many thanks to Professor
Kenneth Caidahl, Department of Clinical Physiology, for allowing me to run the
ergometer cycle exercise test under your supervision.
I would also like to express my immerse gratitude and thanks to our previous
administrator at Integrative physiology, Mrs. Margareta Svedlund. Thank you so
much, Margareta, for always helping me with the visa, in addition to so many other
administrative works. You have really helped me getting along with the Swedish
administrative system. Special thanks to Arja Kantz, our current administrator, for so
much help, especially towards the end of my studies. Your support means a lot to this
work. Special thanks to Docent Dana Galuska for your help with ethical clearance,
especially so for helping me with translation and processing the ethical clearance for
my Tanzania study. Thank you, Professor Marc Gilbert, for sharing your in depth
knowledge in biochemistry. My deepest gratitude to Dr. Stefan Nobel for organizing
41
wonderful SRP-diabetes seminars that helped to improve my knowledge in diabetes
research.
Special thanks to my present colleagues in the lab, Dr. Håkan Karlsson, Dr.
Mutsumi Katayama, Dr. Ulrika Widegren, Rasmus Sjögren, Dr. Thais De Castro
Barbosa, Leonidas Lundell, Dr. Jonathan Mudry, Dr. Qunfeng Jiang, Eva Palmer,
Torbjörn Morein, Katrin Bergdahl, Dr. Carolina Nylen, Ann-Marie Pettersson,
Milena Schönke, Dr. Laurene Vetterli and my previous colleagues, Dr. Ferenc
Szekeres and Dr. Jie Yang for your kind support. Thank you, Dr. Julie Massart, for
words of encouragement when I was writing this thesis.
My deepest gratitude to my office mates at integrative physiology, David
Lassiter, Isabelle Riedel, Maria Holmström, Dr. Emmani Nascimento, Dr.
Hanneke Boon, Dr. Henriette Kirchner, and Dr. Louise Mannerås Holm. Thank
you for valuable discussions and your energy that made our office a better place. You
guys are wonderful.
I wish to extend my deep gratitude to my friends, Dr. Sameer Kulkarni, Dr.
Reginald Austin, Dr. Sydney Carter, Stephen Ochaya and Robby Tom, for the
valuable discussions that we shared. Thank you very much for your friendship.
I am deeply grateful to our family friends Mr. and Mrs. Ubena John, and their
daughter Janelle for their friendly support. All my Tanzanian friends in Stockholm, for
bringing the Tanzanian taste in Sweden. My friend Ngesa Ezekiel, for your deep
scientific insight and critical thinking. My life in Stockholm wouldn’t have been
complete without you.
My special thanks to colleagues at the department of physiology at MUHAS
in Tanzania, Dr. Benjamin Mtinangi, Dr. Josiah Ntogwisangu, Dr. Emmanuel
Ballandya, Dr. Omary Chillo, Dr. Davis Ngarashi and Dr. Mwanamkuu
Maghembe for your kind support during my absence.
Thank you so much Professor Kisali Pallangyo for inspiring me into
academia and giving me the necessary support whenever I needed. Special thanks to
my local supervisor in Tanzania, Professor Janeth Lutale for your professional
support. Sincere gratitude to Dr. Marina Njelekela, for your kind support. Thank you
Dr. Julie Makani for ever ending motivation. Special thanks to my colleagues and
close friends, Drs. Abel Makubi and Francis Dida, for acting on my behalf during my
absence at MUHAS. Special thanks to my friends, Dr. Joel Msafiri and Dr. Goodluck
Tesha for your assistance during my absence in Tanzania.
My deepest appreciation to my dear wife, Juliana Masaulwa, and my family.
Thank you Juliana for taking care of the kids and always making sure I am healthy and
happy. Many thanks to Ethan and Kenedy. I wish to thanks my mother Mrs. Pulkeria
Mashili for her everlasting love and support and my father Dr. Lazaro Mashili for
inspiring me into the medical field. To my sister, Joyce Mashili and my brothers,
Jerry and Jamal, I say Thank you so much.
My sincere gratitude to my beloved neighbors in Dar es Salaam, Mr. and Mrs.
Gudluck Mosha. Your kindness and wisdom guided my family during my absence.
Thank you very much for your true friendship and wisdom.
Finally I would like to thank the Novo Nordisk Foundation for financial support,
without which, anything towards accomplishing this work would have been impossible.
42
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