Thesis defended in public on Friday 29th February 2008
by
Johan WE Jocken
SUMMARY
Obesity is characterized by increased fat storage, as triacylglycerol (TAG), mainly in
adipose tissue. This, increased adipose tissue mass results in lipid overflow into the
circulation. Inappropriately elevated fatty acid (FA) levels have many adverse
metabolic effects and are associated with an increased risk for the development of
insulin resistance, type 2 diabetes and cardiovascular diseases. Furthermore, lipid
overflow may lead to an increased storage of fat in non-adipose tissues (e.g. skeletal
muscle), which is associated with insulin resistance. An impaired lipolysis in adipose
tissue and skeletal muscle might contribute to the development or maintenance of
increased fat stores and obesity. Unraveling the underlying mechanism of a blunted
lipolysis might increase pharmacological and lifestyle strategies to prevent or treat
obesity, type 2 diabetes and cardiovascular diseases.
This thesis is focused on the molecular and physiological regulation of human adipose
tissue and skeletal muscle lipolysis in obesity. Therefore, this thesis describes a
variety of human in vitro and in vivo studies specially designed to investigate lipolytic
regulation in both tissues, comparing obese and lean subjects
In human adipose tissue and skeletal muscle the regulation of lipolysis depends on the
balance between lipolytic and anti-lipolytic hormones, as extensively reviewed in
chapter 1. Catecholamines are the major lipolytic hormones that regulate lipolysis
through beta-adrenoceptors. There are indications, from in vitro and in situ
microdialysis studies, for the existence of a blunted catecholamine-induced lipolysis
in obesity. However, real in vivo evidence is scarce. Therefore, in chapter 2
abdominal subcutaneous adipose tissue lipolysis was examined in vivo using [2H5]glycerol tracer methodology in combination with the measurement of arteriovenous
concentration differences. We demonstrated that in vivo, after an overnight fast, the
lipolytic rate related to fat mass is decreased in obesity. Furthermore, we showed a
blunted isoprenaline (non-selective beta-agonist)-induced increase in net FA and
glycerol release across abdominal subcutaneous adipose tissue of obese men. This
suggests a down-regulation of lipolysis per unit adipose tissue in obese men, which
might be attributed to hyperinsulinemia in obesity. Alternatively, a blunted lipolysis
per unit fat mass might be an early, even primary factor in the development of
increased adipose tissue stores and obesity.
Post-receptor signaling and activation of lipases (e.g. hormone-sensitive lipase; HSL)
and lipid droplet associated proteins (e.g. perilipin) results in increased TAG
hydrolysis and the release of glycerol and FA in the circulation. Recently the lipolytic
pathway has been revisited by the identification of a new lipase: adipose triglyceride
lipase (ATGL). In chapter 3 we used RNA interference (RNAi) to unravel the
physiological relevance of ATGL and HSL for human adipose tissue lipolysis. A
superior role of HSL in promoting catecholamine-induced lipolysis was clearly
observed in human adipocytes, contrasting previous findings in murine adipocytes.
However, we cannot preclude the possibility that ATGL may have roles in adipose
tissue lipolysis, beside promoting basal lipolytic rate, which are revealed until more in
known regarding its regulation.
Several molecular receptor and post-receptor defects in the lipolytic pathway might
contribute to the observed catecholamine resistance in obesity and provide new
therapeutic targets. In chapter 3 we demonstrated that a decreased HSL expression is
associated with a blunted in vitro catecholamine-induced lipolysis in human
adipocytes. In contrast, we showed for the first time that adipose tissue ATGL
expression is not reduced in abdominal subcutaneous adipocytes derived from obese
subjects. Given that HSL is of greater importance than ATGL in promoting
catecholamine-induced lipolysis, (chapter 3) it seems unlikely that that ATGL
contributes to the catecholamine resistance observed in adipose tissue of obese
subjects. However, in chapter 4 we showed that when the obese state has already
developed, adipose tissue ATGL and HSL mRNA and protein expression is decreased
as a consequence of hyperinsulinemia and the insulin resistant state. This decreased
lipase expression might reduce FA outflow from the adipose tissue and subsequently
protect against worsening of the insulin resistant state. Alternatively, there is
substantial evidence that a reduced lipase expression is and early defect in obesity.
Indeed, we showed that genetic variability in different steps of the lipolytic patway,
like the beta-2 adrenoceptor (chapter 7) and HSL (chapter 8) contribute to a blunted
in vivo catecholamine induced lipolysis and fat oxidation in obesity. It should be
metioned, however, that both primary disturbances and secondary adaptational
responses might coexist in obesity. This then leaves the question whether we have to
stimulate or inhibit lipases as potential treatment of obese insulin resistant conditions.
As the lipolytic process critically affects the concentration of circulating FA,
inhibiting lipases to decrease FA release is considered a potential target for the
treatment of insulin resistance in type 2 diabetes. Alternatively, lipase activators may
have potential benefits for the treatment of obesity, by reducing fat mass. Thereby,
FA oxidation should be increased to clear the released FA from the circulation.
As mentioned earlier, lipid overflow in obesity may lead to increased storage of fat in
skeletal muscle, which is associated with insulin resistance. Besides an impaired FA
handling, intrinsic disturbances in skeletal muscle lipolysis might contribute to this
increased fat storage. We therefore examined skeletal muscle lipolysis in vivo using
[2H5]-glycerol tracer methodology in combination with the forearm balance model. In
chapter 5, we demonstrated significantly lower glycerol release across the forearm of
obese compared with lean subjects after an overnight fast. Interestingly, this blunted
fasting lipolysis was accompanied by a reduced total HSL expression and HSL serine
phosphorylation. Furthermore, we were the first to demonstrate ATGL protein
expression in human skeletal muscle, being exclusively expressed in type 1 oxidative
fibres (chapter 6). This suggests an important role for ATGL in muscle FA handling
and lipolyis. Since in particular lipid metabolites (e.g. diacylglycerol (DAG), and
ceramides) interfere with insulin signaling, it is tempting to speculate that a
dysbalance between ATGL and HSL might increase the storage of these lipid
metabolites in muscle of obese insulin resistant subjects. Future research is necessary
to examine whether ATGL expression and activity is impaired in skeletal muscle of
obese subjects.
In summary, from the series of studies described in this thesis the main conclusions
are that:

in vivo and in vitro catecholamine-induced lipolysis is blunted in abdominal
subcutaneous adipose tissue of obese subjects. This catecholamine resistance
might be an important factor contributing to the development or maintenance of
increased adipose tissue fat stores and obesity.

a reduced HSL expression in abdominal subcutaneous adipocytes is one of the
best characterized defects that is associated with this blunted lipolytic response in
obese subjects. In contrast to HSL, adipose tissue ATGL protein expression is not
altered by obesity per se.

HSL and not ATGL is the predominant lipase for stimulated lipolysis in human
adipose tissue, suggesting ATGL might not play an important role in the
catecholamine resistance of lipolysis observed in abdominal subcutaneous adipose
tissue of obese subjects. However, this does not exclude the possibility that ATGL
may play an important role in human adipose tissue lipolysis, which is not
revealed until more is known regarding the regulation of this lipase.

when the obese state has already developed, adipose tissue ATGL and HSL
mRNA and protein expression is decreased as a consequense of hyperinsulinemia
and the insulin resistant state. Alternatively, genetic variability in different steps
of the lipolytic pathway, like the beta-2 adrenoceptor, and HSL contribute to the
blunted in vivo catecholamine-induced lipolysis and fat oxidation in obesity. This
suggests that early genetic defects in the lipolytic pathway are present in obesity.
It should be mentioned, however, that both primary disturbances and secondary
adaptational responses might coexist in obesity.

obese subjects have a blunted fasting forearm muscle lipolysis, which is
accompanied by a lower total HSL protein expression and HSL serine
phosphorylation, most probably reducing HSL activity. These data highlight that
beside an impaired FA handling also intrinsic disturbances in muscle lipolysis
may contribute to the increased lipid and lipid metabolites storage in skeletal
muscle of obese subjects.

ATGL protein is expressed in a fibre type specific way in human skeletal muscle.
This indicates that beside HSL also ATGL might play an important role in skeletal
muscle lipolysis, FA handling, and could have contributed to the observed blunted
fasting skeletal muscle lipolysis in obesity.