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Chapter 7
GENERAL DISCUSSION7.1 Overview
Changes in lifestyle throughout the last 30 years has resulted in a huge rise in obesity and
concomitant increases in the incidence of the obesity-associated metabolic diseases, insulin
resistance and type 2 diabetes (WHO 2006). This provides a challenge to researchers, as the
development of successful therapeutic interventions requires a thorough understanding of the
underlying mechanisms. Due to its role in disposing the majority of circulating glucose
following the ingestion of a meal (Ferrannini et al 1985), skeletal muscle is vitally important
in maintaining glucose homeostasis. Lipid accumulation in insulin sensitive is associated with
reductions in glucose tolerance, while acute exercise and life-style interventions involving a
reduced calorie and fat intake and an increase in physical activity improve glucose tolerance
(chapter 1). In both cases the underlying mechanisms are only partially understood.
Therefore, the aims of this thesis were i) to compare resistance and endurance exercise as
means to improve glucose tolerance, ii) to investigate whether ingestion of a single high fat
meal reduces glucose tolerance and whether it can be restored with one bout of resistance
exercise, iii) investigate whether near infrared spectroscopy (NIRS) can detect meal-induced
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increases in muscle capillary blood volume and iv) to develop new immunofluorescence
microscopy methods applied to human muscle fibres that can identify defects in lipid
metabolism which potentially contribute to the mechanism leading to insulin resistance.
7.2 The effects of exercise and nutritional interventions on glucose tolerance
Previous studies have demonstrated that acute bouts of endurance and resistance exercise lead
to enhancements in insulin-mediated glucose uptake up to 24-48 h after exercise (chapter 1).
Chapter 2 demonstrated that glucose tolerance measured with an oral glucose tolerance test,
can be improved 6 h after resistance exercise when a carbohydrate/protein-hydrolysate drink
is ingested after exercise to resynthesize at least part of the muscle glycogen oxidised during
the exercise. On the other hand these beneficial effects on glucose tolerance were not seen 6 h
after endurance exercise when the same glycogen repletion protocol was used. These results
were explained (chapter 2) by the fact that resistance exercise not only leads to glycogen
depletion but also to activation of early intermediates in the insulin signalling cascade
(Hernandez et al 2000, Creer et al 2005), while endurance exercise only causes glycogen
depletion and no activation of the insulin signalling cascade (Richter et al 2001;
Wojtaszewski et al 2000). Low concentrations of glycogen following exercise have been
shown to stimulate insulin stimulated glycogen synthase activity and muscle glucose uptake
(chapter 1). The interpretation of the observations made in chapter 2 is based on the
assumption that the muscle glycogen stores were largely or fully restored before the start of
the OGTT. Although this assumption seems to be reasonable as the carbohydrate-protein
beverage consumed in chapter 2 has previously been shown to lead to high rates of muscle
glycogen synthesis (van Loon et al 2000), measurements of the muscle glycogen content and
insulin signalling events are needed to confirm these conclusions.
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At the time that we performed the study described in chapter 2 the available literature
reported that an acute bout of endurance exercise did not activate early events in the insulin
signalling cascade, including insulin-induced insulin receptor kinase activity, IRS-1 tyrosine
phosphorylation, PI 3-kinase activity, and serine phosphorylation of Akt and of glycogen
synthase kinase-3 (chapter 1). However, it has recently been reported that phosphorylation of
Akt substrate of 160 kDa (AS160) occurs in rat skeletal muscle immediately and 4 h after
endurance exercise (Arias et al 2007) and this is mediated by increases in AMPK (Treebak et
al 2006). AS160 is also phosphorylated upon insulin stimulation involving the IRS-1/PI 3kinase/Akt signalling pathway (chapter 1). This indicates that phosphorylation of AS160 is a
cross-point where insulin- and contraction-related stimuli converge to stimulate GLUT4
translocation. This newly discovered mechanism has revealed a potential route by which
GLUT4 translocation to the plasma membrane could occur without increases in proximal
insulin signalling steps in the post-exercise period. Further work is required to investigate
how long AS160 remains phosphorylated after endurance exercise, if the activation remains
when carbohydrates are ingested immediately after exercise (leading to acute inactivation of
AMPK), if AS160 phosphorylation depends on muscle glycogen content, if resistance
exercise also leads to phosphorylation of AS160, and finally whether exercise-induced AS160
phosphorylation also occurs in insulin resistant states.
The results from chapter 2 could have potential implications to type 2 diabetics who want to
reduce the period they are exposed to elevated blood glucose concentrations. Delaying
feeding will lead to more prolonged enhancements in glycogen synthase activity and glucose
transport and a reduction in blood glucose concentrations. On the other hand results from
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chapter 2 suggest that the benefit of exercise remains when a carbohydrate/protein drink is
ingested following resistance exercise. Resistance exercise, therefore, may be the preferential
mode of exercise when the aim is to improve glucose control/homeostasis. Resistance
exercise may also have additional benefits. Koopman et al have shown that ingesting
carbohydrate and protein after resistance exercise improves net protein balance (Koopman et
al 2005b) and enhances downstream insulin signalling events involved in protein synthesis
(Koopman et al 2007).
In chapter 3, the glucose response to an OGTT 6 h after ingestion of a single high fat meal
was 36% higher than that observed after ingestion of a low fat meal, but the difference was
not significant. However, following ingestion of the high fat meal, the glucose concentration
failed to return to the pre-OGTT concentration at 2 h, potentially indicating a small reduction
in the ability of insulin to stimulate glucose uptake in line with the set hypothesis. More
prolonged periods of high fat feeding (3 days) in lean healthy subjects have been shown to
impair insulin-mediated glucose uptake and glucose tolerance (Bachmann et al 2001;
Pehleman et al 2005). The 36% increase in glucose response after ingestion of a high fat meal
found in chapter 3, although not significant, may be relevant to some individuals, as six of
the eight subjects demonstrated an elevated glucose response to the OGTT. This difference in
subject response may be due to differences in individual ability to handle a lipid load. A
recent study demonstrated that rats bred for high endurance running capacity had a higher
basal skeletal muscle oxidative capacity and were resistant to high fat diet-induced obesity
and insulin resistance (Noland et al 2007). In addition, the adipose tissue in obese and obese
insulin resistant groups has a poor lipid buffering capacity leading to higher postprandrial
plasma concentrations of triglycerides and free fatty acids (Bays et al 2004; Frayn 2000).
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Therefore the effect of a single high fat meal may have greater negative effects on glucose
control in populations at risk of lipid-induced metabolic impairments and in those already
insulin resistant.
Chapter 3 also confirmed previous (eg. chapter 2, Koopman et al 2005a, Fluckey et al 1994)
and also more recent studies (Praet et al 2006) showing that a single bout of resistance
exercise can improve insulin-mediated glucose control. The novel finding in chapter 3 was
that a resistance exercise bout performed in the postprandial period following ingestion of a
high fat meal significantly improves glucose tolerance. This study indicates that it is
beneficial to perform resistance exercise following the ingestion of an energy dense meal to
prevent short-term impairments in glucose control. The possible mechanisms for
improvements in glucose tolerance may be related to activation of early insulin signalling
intermediates (PI 3-kinase and Akt) seen in previous studies (Hernandez et al 2000, Creer et
al 2005).
A recent study also demonstrated that the protein concentration of enzymes involved in lipid
synthesis such as diacylglycerol acyltransferase (DGAT) and mitochondrial glycerol-3phosphate acyltransferase (mGPAT) were increased the day after a single bout of endurance
exercise and lead to increases in IMCL content and reduced concentrations of intracellular
DAG and ceramides (Schenk and Horowitz 2007). Interestingly the single bout of endurance
exercise also prevented the development of insulin resistance during a lipid/heparin infusion
leading to high FFA concentrations (Schenk and Horowitz 2007). The effect of resistance
exercise upon the protein expression and activity of such enzymes is not known but resistance
exercise is known to lower IMCL content with resynthesis occuring during the first few hours
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of recovery (Koopman et al 2006). Upregulation of DGAT and/or mGPAT and subsequent
increases in IMCL synthesis may partially explain why an increased glucose tolerance was
seen when resistance exercise was performed in a period of enhanced lipid availability. Fatty
acids originating from chylomicron-TG hydrolysis by muscle lipoprotein lipase in the
postprandrial period would be taken up by the muscle and used for synthesis of IMCL
preventing any accumulation of intracellular FA metabolites. Therefore a bout of resistance
exercise appears to be beneficial to glucose control after the ingestion of a high fat meal. This
may be particularly advisable to those who are already insulin resistant and experience large
daily elevations in plasma glucose concentrations.
7.3 New methods to investigate the mechanisms of insulin resistance
7.3.1 Increases in microvascular blood volume
Over the last 5-10 years it has become clear that insulin-mediated increases in capillary
perfusion is a vital step in the regulation of glucose homeostasis. These findings are mostly
derived from novel techniques utilising contrast-enhanced ultrasound (CEU) applied to
humans to measure the volume of blood in microvascular compartments. In chapter 4, we
investigated whether it was possible to measure increases in microvascular blood volume noninvasively using near-infrared spectroscopy (NIRS), where changes in total haemoglobin
(HbT) concentrations were considered to represent changes in blood volume in the measured
compartment. However NIRS was only able to detect small changes in HbT following muscle
contraction and no change in HbT following ingestion of a beverage known to elicit large
increases in plasma insulin concentrations. It was concluded that the NIR-light is absorbed by
red blood cells in a variety of vessels (arterioles, venules, capillaries) in different tissues (skin,
subcutaneous adipose tissue, muscle, connective tissue) and, therefore, NIRS can not be used
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to gather specific information on the volume of blood present in muscle fibre capillaries.
Therefore, investigations measuring the expansion of muscle capillary blood volume continue
to depend upon the detection of albumin-coated microbubbles with contrast enhanced
ultrasound (Vincent et al 2006) or measurement of the capillary permeability surface area
using invasive techniques (Gudsbjornsdottir et al 2003).
7.3.2 Fluorescence microscopy, skeletal muscle lipid metabolism and insulin resistance
Accumulation of FA metabolites (LCFACoA, DAG and ceramides) in muscle leads to
activation of PKC and downregulation of insulin signalling and is assumed to be the primary
mechanism responsible for reductions in insulin-mediated glucose uptake in obese subjects
with insulin resistance and during intralipid/heparin infusions leading to high plasma FFA
concentrations. Abnormalities in the transport and oxidation of FFA and in the synthesis and
mobilisation of IMCL could all contribute to the increase in intracellular FA metabolites in
insulin resistant states and explain the paradox that athletes combine a high IMCL content
with high insulin sensitivity. The abnormalities in IMCL synthesis and mobilisation have only
partially been identified and much of our current knowledge of the enzymes involved in
IMCL synthesis and mobilisation depends on extrapolation of data obtained in cultured
adipocytes. For a summary of the processes believed to be involved in IMCL synthesis and
metabolism and how they may relate to insulin resistance see figure 7.1. Chapters 5 and 6 in
this thesis developed new immunohistochemical methods that can be used to study lipid
metabolism in skeletal muscle.
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Chapter 5 used double-(immuno)staining protocols to simultaneously identify type I muscle
fibres and intramyocellular lipid droplets, a method previously developed by Koopman et al
(2001) in Maastricht. Quantitative analysis demonstrated that the lipid content is
approximately 2-fold greater in oxidative type 1 fibres than glycolytic type II fibres
confrming the earlier data published by this group (van Loon et al 2003 and 2004).
Additionally identification of the PAT protein adipophilin was combined with a lipid droplet
stain to show that ~60% of lipid droplets colocalise with clusters of adipophilin. Adipophilin
has been
Figure 7.1. The regulation of triglyceride synthesis and lipolysis in skeletal muscle. Both
processes help to maintain low concentrations of intracellular fatty acid metabolites by
directing fatty acids to TG synthesis or transport across the mitochondrial membrane and
oxidation. If defects occur in the synthesis, hydrolysis or transport an accumulation of fatty
acid metabolites activate PKC leading to insulin resistance. ATGL, adipose triglyceride
lipase. DAG, diacylglycerol. DGAT diacylglycerol acyltransferase. FA CoA, fatty acylCoA.
FAT, fatty acid translocase. GPAT, glycerol-3-phosphate acyl transferase. HSL, hormone
sensitive lipase. lysoPAT, lysophosphatidate acyltransferase. M, mitochondria. MG,
monoacylglycerol. MGAT, monoacylglycerol acyltransferase. MGL, monoacylglycerol
lipase. PA phosphatase, phosphatidic acid acyltransferase. PKC, protein kinase C. TG,
triglyceride.
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Fatty acid
Plasma
Skeletal muscle
FAT
Glycerol-3-phosphate
GPAT
Fatty acid
1-acyl-G-3-P
Fattylipase
acyl CoA
synthetase
suggested to potentially mediate access of hormone sensitive
(HSL)
to IMCL (Prats et
lysoPAT
al 2006). We also showed that adipophilin does not colocalise with mitochondria and
Phosphatidic acid
FA CoA
therefore is unlikely to channel fatty acids from the lipid droplets to the mitochondria upon
PA phosphatase
LD hydrolysis as suggested earlier (Chanderbhan et al 1982, Nakamura and Fujimoto 2003).
INSULIN
MG
PKC
DAG
As our studies were
performed
in the resting state this conclusion
is restricted
to this
RESISTANCE
MGAT
condition, but is not necessarily true during exercise.
DGAT
M
Fatty
acid
FAT/CPT
The results from chapter 6 illustrate that the mitochondrial network has a higher density in
type I than in type II fibres and the greatest
region
DAG content is present
MG in the subsarcolemmal
Glycerol
TG
ATGL
HSL
MGL
?
and progressively declines from
plasma
membrane
to
the
interior
of the fibre. The relative
HSL
distribution of LDs is similar to the mitochondria network with the highest content between 0Adrenaline
Contraction
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5 μm from the plasma membrane. Chapter 6 showed that the LDs are situated in the spaces
between neighbouring mitochondria in the network and in close contact to at least one and
often more mitochondria in lean young individuals. Presumably this aids efficient oxidation
of IMCL observed in trained individuals during exercise (van Loon et al 2003). Obese
subjects have been reported to have low subsarcolemmal mitochondria content (Ritov et al
2005) and increased lipid droplets in the interior of muscle fibres (Malenfant et al 2001).
Disturbances in the spatial distribution of LDs and mitochondria in skeletal muscle may
impair the oxidation of LDs during exercise and provide an explanation as to why insulin
resistant subjects are unable to oxidise IMCL during exercise.
7.4 Future Research
Immunofluorescence staining techniques such as the novel methods developed in this thesis
provide a powerful tool to investigate the underlying mechanisms of the relationship between
skeletal muscle lipid metabolism and insulin resistance. Standard biochemical assays applied
to homogenates and extracts have the disadvantage that they do not give information on fibre
type specificity. The use of immunohistochemical techniques has revealed that the
intramyocellular lipid pool significantly contributes to total fat oxidation in type I fibres of
lean active individuals, while no significant IMCL utilisation has been observed in type 2
fibres (van Loon et al 2003). Similarly measurement of mitochondrial content and enzyme
activity is commonly performed on homogenates containing mixed fibre types (Bizeau et al
1998, Holloway et al 2007). With the new immunohistochemical stainings developed in this
thesis it is now possible to investigate the spatial distribution of mitochondria in both
subsarcolemmal and intermyofibrillar regions in a fibre type specific manner in different
populations.
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Great advances have been made in recent years concerning the understanding of the
regulation of the activity of transporters and enzymes involved in glucose and fatty acid
metabolism in skeletal muscle. We know that the activity of transporters and enzymes is not
only modulated by means of phosphorylation/dephosphorylation, but also by translocation
from one subcellular compartment to another upon insulin-stimulation or exercise. For
example, we have known for many years that one of the key steps in glucose uptake is the
translocation of GLUT4 from intracellular vesicles to the plasma membrane (Cushman and
Wardzala 1980). We also know that translocation processes are involved in the transport of
free fatty acids (FFA) into muscle fibres and into the mitochondria via translocation of fatty
acid translocase (FAT) (Campbell et al 2004, Holloway et al 2006). We also know that
colocalisation of enzymes with a substrate or with coactivator proteins can be the end point of
these translocation processes. For example Prats et al (2006) demonstrated that hormone
sensitive lipase (HSL) translocates from intracellular stores upon muscle contraction of intact
rat muscle fibres to colocalise with LDs and two of the PAT proteins in muscle. Although
traditional biochemical techniques combining differential centrifugation and Western blots
can be used to quantify the content of proteins in membrane and cytosolic fractions they do
not give information on the spatial distribution of proteins, on final colocalisation or on fibre
type specificity. Techniques that can visualise fibre-type specific translocation and/or
colocalisation steps in human muscle biopsies with a high resolution have a huge advantage
over many biochemical techniques and will be a new avenue to explore muscle lipid
metabolism in the next few years. With the rise in obesity and obesity-related metabolic
disorders, the effort to develop these new techniques should be substantially increased to fully
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understand the mechanisms by which the current life-style leads to the massive increase in the
frequency of obesity, metabolic syndrome, type 2 diabetes and cardiovascular disease.
Immunofluorescence techniques similar to the ones developed in this thesis provide an
opportunity to further investigate the mechanisms of lipid synthesis, hydrolysis and oxidation
in skeletal muscle. The technique set up in Chapter 5 makes it possible to investigate the
interaction of ADRP and LDs in lean, obese and insulin resistant groups both at rest and
during exercise. We can test now whether a low adipophilin protein content in insulin
resistant muscles plays a role in the mechanisms leading to elevated fatty acid metabolites.
Following on from cross-sectional studies of different populations, acute exercise studies are
also warranted to further investigate IMCL oxidation during exercise. Biopsies pre and postendurance exercise would make it possible to investigate whether the degree of LD and
adipophilin colocalisation is related to the capacity of IMCL oxidation and insulin sensitivity.
Similar methods should also be developed to investigate other determinants of IMCL
synthesis and oxidation. The finding that HSL exerts its effect not only through increased
activity but through direct translocation and interaction with the LD (Prats et al 2006)
explains the large increases in lipolysis with only small increases in enzyme activity. So far
the fluorescent techniques used to illustrate this have been performed on intact rat muscle
fibres (Prats et al 2006). They also showed that HSL translocates to the PAT proteins, which
appear to surround the majority of lipid droplets. It is important to reproduce these results in
human muscle fibres and immunofluorescence on longitudinal muscle sections taken before
and after exercise would be an ideal way to do this. This could also demonstrate if it is only
the LDs that colocalise with adipophilin that can be subjected to HSL-stimulated lipolysis and
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thus oxidation. Additionally defects in the translocation of HSL possibly associated with
adipophilin content could potentially explain the inability of insulin resistant muscle to
oxidise IMCL during exercise. This work should be extended to also define the role of other
PAT proteins in the regulation of LD lipolysis in response to exercise and adrenaline.
Although work has been done on some of these other enzymes/protein in rats (Prats et al
2006), no studies are available in humans and on differences between physically active,
sedentary and insulin resistant populations. Furthermore, adipose triglyceride lipase (ATGL)
has recently been discovered (Zimmerman et al 2004) as the lipase responsible for the first
step in TG breakdown to diacylglycerol (DAG), while HSL primarily acts on DAG (figure
7.1). The role and importance of ATGL in skeletal muscle lipolysis is not known.
The technique set up in chapter 6 is an ideal method to identify the network of mitochondria
and LDs in muscle as both can be observed simultaneously on the same muscle fibres. On the
basis of the regular positioning of the mitochondria in type I fibres we were able to create a
matrix showing the theoretical maximum density of mitochondria and LDs (Figure 7.2A). In
the filled matrix each LD is attached to two neighbouring mitochondria. In the lean subjects
investigated in chapter 6 many of the mitochondrial positions were occupied in type I fibres
while the density of the lipid droplets was lower, meaning that all LDs neighboured at least
one mitochondrion (Figure 7.2B). Previous studies have shown that sedentary, obese and
insulin resistant individuals have a much lower mitochondrial density and only a marginally
lower density of LDs (Figure 7.2C). This could imply that a number of lipid droplets have no
neighbouring mitochondria. This may explain why LDs are not oxidised efficiently during
exercise and why there is an accumulation of fatty acid metabolites. In future studies we will
investigate this theoretical model presented in figure 7.2 in different subject groups. The
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images should preferentially be made in 3 dimensions, which is possible with the software
available in our laboratory. To obtain optimal information on relative occupancy of the
mitochondria and LD networks in different populations, fatty acid diffusion distances and
their potential impact on concentrations of fatty acid metabolites, we will develop
mathematical models in collaboration with Eindhoven University of Technology. These
models also will include comparisons of the network images obtained before and after
exercise, to investigate how fatty acid diffusion distances and mitochondrial density gradients
between subsarcolemmal and deeper layers determine LD oxidation during exercise.
Another interesting protein for future investigations is fatty acid translocase (FAT/CD36).
FAT translocates during exercise and in response to meal induced increases in insulin from
intracellular stores to the plasma membrane (Kiens 2006). Recently it has also been shown
that during exercise FAT translocates to the inner mitochondrial membrane taking a position
next to CPTI, reducing malonylCoA inhibition and thus increasing fatty acid oxidation during
exercise (Bezaire 2006, Holloway et al 2006, Schenk and Horowitz 2006). Suggestions have
been made that an increased FAT content in the plasma membrane leads to increased fatty
acid uptake in animal models of type 2 diabetes (Bonen et al 2004, and 2006). A high FAT
content in the plasma membrane in the presence of a low FAT content in the mitochondria
would lead to an imbalance between fatty acid uptake and oxidation and could lead to
accumulation of fatty acid metabolites and lead to insulin resistance. The development of
immunofluorescence microscopy methods that allow us to simultaneously measure FAT
content of plasma membrane and mitochondrial membranes in resting and exercise biopsies
of various populations with and without insulin resistance therefore should have high priority.
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Mitochondrial (mGPAT) and microsomal (GPAT3) glycerol-3-phophate acyltansferase and
diacylglycerol acyltransferase (DGAT) are key enzymes in the synthesis of triglycerides and
lipid droplets in muscle. Recent studies (Liu et al 2007, Schenk & Horowitz 2007) suggest
that an increased protein content of DGAT and mGPAT leads to increased insulin sensitivity
and protection against acute insulin resistance induced by an intralipid/heparin infusion.
Furthermore one bout of endurance exercise is sufficient to increase the content of DGAT and
mGPAT the next day and offer protection against lipid/heparin induced insulin resistance
(Schenk & Horrowitz 2007). The proposed mechanism behind this observation is that fatty
acids taken up by the muscle will be more efficiently channelled into the IMCL stores leading
to lower concentrations of diacylglycerol, long-chain fatty acylCoAs and potentially
ceramides, the fatty acid intermediates that lead to insulin resistance via PKC activation
(chapter 1). Again immunohistochemical assays able to quantify DGAT, mGPAT and the
recently discovered microsomal GPAT (Cao et al 2006) in muscle again should have a high
priority. Such assays could be used to evaluate the effect of training interventions on the
capacity to oxidise IMCL during exercise and synthesize IMCL during recovery in various
populations with and without insulin resistance.
As explained in chapter 1 and chapter 4, insulin action on the endothelium to increase
muscle capillary perfusion is of great importance in determining glucose uptake in muscle and
glucose tolerance (Rattigan et al 2006). Impairments in the endothelium of the terminal
arterioles in muscle and in feeding and resistance arteries have been suggested to contribute to
the development of insulin resistance, type 2 diabetes and cardiovascular disease
(Wagenmakers et al 2006). Metabolism and mechanisms that operate in the endothelium of
the microvasculature are particularly difficult to study due to the inaccessibility of terminal
arterioles and capillaries in human muscle. Much of what we assume to know today is based
on extrapolation from studies in larger vessels, cultured endothelial cells and animal models
of insulin resistance. Immunofluorescent labelling of basement and plasma membranes with
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anti-laminin shows that capillaries, arterioles and venules are visible on sections of human
muscle biopsies (see figure 7.3, unpublished data of Shaw and Wagenmakers). Therefore
immunolabelling of terminal arterioles with antibodies against insulin signalling intermediates
(eg IRS-1 total, tyrosine and serine phosphorylated IRS-1), against eNOS (total protein and
serine phosphorylated) as an indicator of basal and insulin stimulated NO production could
potentially identify defects in the insulin resistance of terminal arterioles. Furthermore
NADPH oxidase expression was recently shown to be increased in venous vascular
endothelial cells harvested from obese subjects (Silver et al 2007). NADPH oxidase produces
superoxide anions that can take away functional NO in a reaction leading to peroxynitrite
production (Chapter 1.4.5). Future studies should, therefore, explore whether the enzyme
systems and signalling pathways that control endothelial NO production in response to insulin
and exercise can be visualised and quantified with newly developed immuonohistochemical
staining methods of arterioles and capillaries applied to longitudinal sections of human
muscle.