Abstract
Lipid is stored as triacylglycerol (TG) in lipid droplets and is in skeletal muscle stored as intra
muscular triacylglycerol (IMTG). IMTG is considered an energy pool that is utilized by lipolysis
during situations with low cellular energy availability, such as exercise. Lipolysis is in skeletal
muscle regulated by the IMTG lipase Adipose triglyceride lipase (ATGL) releasing FA and DAG,
by the DAG lipase hormone sensitive lipase (HSL) releasing monoacylglycerol (MAG) and finally
the MAG lipase (MAGL) releasing FA and glycerol. Lipolysis is regulated by different factors and
stimuli and especially the energy sensor 5' adenosine monophosphate-activated protein kinase
(AMPK), which is activated during exercise, has received increased attention. However, whether
AMPK is an activator or inhibitor of lipolysis in skeletal muscle is not clear.
Therefore, we in study I aimed to identify the role of AMPK in regulation of lipid handling and
lipolysis in the basal non-contracting state and during muscle contractions in skeletal muscle. To
evaluate the role of AMPK, we measured protein expression and phosphorylation as well as gene
expression of proteins important for regulation of lipid handling and lipolysis in skeletal muscle
from wildtype mice and mice overexpressing a kinase dead AMPKα2 construct (AMPKα2 KD) in
the basal non-contracting state and during in situ stimulated muscle contractions. We found, that
IMTG levels were ~50% lower in AMPKα2 KD in the basal resting state, explained by a lower
protein and gene expression of 1) fatty acid translocase cluster of differentiation 36 (FAT/CD36)
reducing the intracellular FA availability and re-esterification of FA into IMTG and 2) the lipid
droplet-associated protein PLIN3, making the lipid droplet more accessible for lipolytic lipases and
lipolysis. IMTG was in wildtype mice reduced with ~50% after muscle contractions with no effect
of contractions in AMPKα2 KD mice. Concomitantly, ATGL was phosphorylated at ser406 and HSL
on ser565 with muscle contractions in an AMPK dependent manner, suggesting that these sites
actives lipolysis during muscle contraction. Accordingly, AMPK is suggested to be an important
regulator of basal IMTG and lipid handling in skeletal muscle as well as an important activator of
lipolysis by phosphorylation of ATGL and HSL during muscle contractions.
Oversupply of energy and dietary fat leads to obesity and accumulation of IMTG and toxic lipid
intermediates in skeletal muscle, which is associated with insulin resistance and type 2 diabetes.
Especially the lipid intimidate diacylglycerol (DAG) is considered a key molecule in the
mechanism behind lipid-induced insulin resistance, however not all studies can detect this
association between DAG accumulation and impaired insulin sensitivity. However, DAG appears in
different stereoisomers depending on their origin and cellular localization and has different
reactivity in vitro Thus, the role of DAG in lipid-induced insulin resistance in skeletal muscle may
be dependent on the stereo chemical structure and cellular localization of DAG.
Therefore, we in study II aimed to identify the role of dysregulation of lipolysis on insulin
sensitivity in skeletal muscle and if the role of DAG in insulin resistance in skeletal muscle is
dependent on the cellular localization and stereo chemical structure of DAG. We took advantage of
the fact that insulin sensitivity is increased after exercise, and that mice knocked out (KO) of HSL
accumulate DAG after exercise, and measured insulin stimulated glucose uptake after treadmill
running in skeletal muscle from HSL KO mice and wildtype control mice. We found that insulin
stimulated glucose uptake is significantly higher in skeletal muscle from HSL KO mice compared
to wildtype control mice after treadmill running, concomitantly with an accumulation of total
intramuscular DAG after exercise in HSL KO mice. Further analysis of the chemical structure of
DAG revealed that the overall level of sn-1,3 DAG was significantly higher than sn-1,2 DAG and
that HSL KO mice accumulated sn-1,3 DAG after exercise compared to wildtype and not sn-1,2
DAG. Accordingly, these data clearly shows that sn-1,3 DAG accumulation, originating from
hydrolysis of IMTG, is not an important signaling molecule in the mechanism behind insulin
resistance and type 2 diabetes
The findings of this PhD thesis are presented in one manuscript and in one published paper. In
addition, the thesis comprises unpublished work.
muscular triacylglycerol (IMTG). IMTG is considered an energy pool that is utilized by lipolysis
during situations with low cellular energy availability, such as exercise. Lipolysis is in skeletal
muscle regulated by the IMTG lipase Adipose triglyceride lipase (ATGL) releasing FA and DAG,
by the DAG lipase hormone sensitive lipase (HSL) releasing monoacylglycerol (MAG) and finally
the MAG lipase (MAGL) releasing FA and glycerol. Lipolysis is regulated by different factors and
stimuli and especially the energy sensor 5' adenosine monophosphate-activated protein kinase
(AMPK), which is activated during exercise, has received increased attention. However, whether
AMPK is an activator or inhibitor of lipolysis in skeletal muscle is not clear.
Therefore, we in study I aimed to identify the role of AMPK in regulation of lipid handling and
lipolysis in the basal non-contracting state and during muscle contractions in skeletal muscle. To
evaluate the role of AMPK, we measured protein expression and phosphorylation as well as gene
expression of proteins important for regulation of lipid handling and lipolysis in skeletal muscle
from wildtype mice and mice overexpressing a kinase dead AMPKα2 construct (AMPKα2 KD) in
the basal non-contracting state and during in situ stimulated muscle contractions. We found, that
IMTG levels were ~50% lower in AMPKα2 KD in the basal resting state, explained by a lower
protein and gene expression of 1) fatty acid translocase cluster of differentiation 36 (FAT/CD36)
reducing the intracellular FA availability and re-esterification of FA into IMTG and 2) the lipid
droplet-associated protein PLIN3, making the lipid droplet more accessible for lipolytic lipases and
lipolysis. IMTG was in wildtype mice reduced with ~50% after muscle contractions with no effect
of contractions in AMPKα2 KD mice. Concomitantly, ATGL was phosphorylated at ser406 and HSL
on ser565 with muscle contractions in an AMPK dependent manner, suggesting that these sites
actives lipolysis during muscle contraction. Accordingly, AMPK is suggested to be an important
regulator of basal IMTG and lipid handling in skeletal muscle as well as an important activator of
lipolysis by phosphorylation of ATGL and HSL during muscle contractions.
Oversupply of energy and dietary fat leads to obesity and accumulation of IMTG and toxic lipid
intermediates in skeletal muscle, which is associated with insulin resistance and type 2 diabetes.
Especially the lipid intimidate diacylglycerol (DAG) is considered a key molecule in the
mechanism behind lipid-induced insulin resistance, however not all studies can detect this
association between DAG accumulation and impaired insulin sensitivity. However, DAG appears in
different stereoisomers depending on their origin and cellular localization and has different
reactivity in vitro Thus, the role of DAG in lipid-induced insulin resistance in skeletal muscle may
be dependent on the stereo chemical structure and cellular localization of DAG.
Therefore, we in study II aimed to identify the role of dysregulation of lipolysis on insulin
sensitivity in skeletal muscle and if the role of DAG in insulin resistance in skeletal muscle is
dependent on the cellular localization and stereo chemical structure of DAG. We took advantage of
the fact that insulin sensitivity is increased after exercise, and that mice knocked out (KO) of HSL
accumulate DAG after exercise, and measured insulin stimulated glucose uptake after treadmill
running in skeletal muscle from HSL KO mice and wildtype control mice. We found that insulin
stimulated glucose uptake is significantly higher in skeletal muscle from HSL KO mice compared
to wildtype control mice after treadmill running, concomitantly with an accumulation of total
intramuscular DAG after exercise in HSL KO mice. Further analysis of the chemical structure of
DAG revealed that the overall level of sn-1,3 DAG was significantly higher than sn-1,2 DAG and
that HSL KO mice accumulated sn-1,3 DAG after exercise compared to wildtype and not sn-1,2
DAG. Accordingly, these data clearly shows that sn-1,3 DAG accumulation, originating from
hydrolysis of IMTG, is not an important signaling molecule in the mechanism behind insulin
resistance and type 2 diabetes
The findings of this PhD thesis are presented in one manuscript and in one published paper. In
addition, the thesis comprises unpublished work.
| Originalsprog | Engelsk |
|---|
| Forlag | Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen |
|---|---|
| Antal sider | 189 |
| Status | Udgivet - 2017 |
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