CARDIOVASCULAR JOURNAL OF AFRICA • Vol 21, No 2, March/April 2010
Cardiovascular Topics
72
AFRICA
AMP kinase activation and glut4 translocation in isolated
cardiomyocytes
INGRID WEBSTER, SVEN O FRIEDRICH, AMANDA LOCHNER, BARBARA HUISAMEN
Summary
Activation of AMP-activated protein kinase (AMPK) results
in glucose transporter 4 (GLUT4) translocation from the
cytosol to the cell membrane, and glucose uptake in the
skeletal muscles. This increased activation of AMPK can be
stimulated by a pharmacological agent,AICAR (5
′
-aminoim-
idazole-4-carboxamide ribonucleoside), which is converted
intracellularly into ZMP (5
′
-aminoimidazole-4-carboxamide-
ribonucleosidephosphate), an AMP analogue. We utilised
AICAR and ZMP to study GLUT4 translocation and glucose
uptake in isolated cardiomyocytes.
Adult ventricular cardiomyocytes were treated with
AICAR or ZMP, and glucose uptake was measured via
[
3
H]-2-deoxyglucose accumulation. PKB/Akt, AMPK and
acetyl-CoA-carboxylase phosphorylation and GLUT4 trans-
location were detected by Western blotting or flow cytom-
etry.
AICAR and ZMP promoted AMPK phosphorylation.
Neither drug increased glucose uptake but on the contrary,
inhibited basal glucose uptake, although GLUT4 transloca-
tion from the cytosol to the membrane occurred. Using flow
cytometry to detect the exofacial loop of the GLUT4 protein,
we showed ineffective insertion in the membrane under these
conditions. Supplementing with nitric oxide improved inser-
tion in the membrane but not glucose uptake.
We concluded that activation of AMPK via AICAR or
ZMP was not sufficient to induce GLUT4-mediated glucose
uptake in isolated cardiomyocytes. Nitric oxide plays a role
in proper insertion of the protein in the membrane but not
in glucose uptake.
Keywords:
AMPK, AICAR, GLUT4, GLUT4 exofacial loop,
cardiomyocytes
Submitted 9/3/09, accepted 19/6/09
Cardiovasc J Afr
2010;
21
: 72–78
AMP-activated protein kinase (AMPK) plays a key role in
maintaining energy homeostasis in the cell
1
by recognising ATP
depletion,
2
initiating changes to restore cellular ATP levels and
inhibiting ATP-utilising anabolic pathways.
3
In heart and skeletal
muscle, AMPK plays an important role in accelerating fatty acid
oxidation, FAT/CD36 translocation and fatty acid uptake, as well
as glut4 translocation, glucose uptake and glycolysis.
4,5
AMPK
is activated by cellular stress, which alters the AMP:ATP ratio
– this can be nutrient stress, lack of oxygen or exercise-induced
stress.
4,6
The AMPK signalling pathway provides an alternative to the
insulin-dependant glucose uptake pathway in muscle. Insulin-
stimulated activation of glut4 translocation via activation of
phosphatidyl-inositol-3 kinase (PI3-K) and PKB/Akt has been
extensively researched. Resistance of muscle to the effects of
insulin is a hallmark of pre-diabetes. This reduction in insulin
sensitivity is reflected in decreased insulin-stimulated glucose
uptake.
7
Since AMPK activates glucose uptake via a distinctly
different signalling pathway than insulin, it may present a
mechanism to manipulate pharmacologically to treat this disease.
Indeed, in insulin-resistant humans and rodents, regular exer-
cise, known to activate AMPK, enhances insulin sensitivity.
8-12
Repeated activation of AMPK may therefore be a mechanism to
improve insulin sensitivity.
13
Furthermore, it was demonstrated that activation of AMPK is
responsible for glucose uptake by hearts subjected to ischaemia,
underscoring the importance of this kinase in the pathophysiol-
ogy of the heart.
14
AICAR (5
′
-aminoimidazole-4-carboxamide
ribonucleoside) is an adenosine analogue which is taken up into
the cells and converted to the monophosphorylated nucleotide,
ZMP (5
′
-aminoimidazole-4-carboxamide-ribonucleosidephos-
phate), by adenosine kinase.
6,15
It was described as a specific acti-
vator of AMPK
16
in intact cells. In has also been found that ZMP,
an analogue of AMP, mimics the effects of AMP on allosteric
activation of AMPK,
17
and the promotion of phosphorylation
and activation of AMPK by the AMPK kinase, LKB1
18
without
changing the ATP:ADP or ATP:AMP ratio in the cell.
16
There are numerous studies showing that AICAR increases
AMPK phosphorylation as well as glucose uptake in skeletal
muscle, the latter mediated via GLUT4 translocation.
19-21
There
are, however, only a few studies to date showing effects of
AICAR on glucose uptake in the heart, some utilising papillary
muscle,
14
whereas Jing and Holman used oligomycin to activate
AMPK in cardiomyocytes.
22
GLUT4 protein in the heart is largely confined to an intracel-
lular vesicle storage site in the basal, non-stimulated state.
23,24
It
becomes recruited to the cell surface under the influence of insu-
Department of Biomedical Sciences, Division of Medical
Physiology, Faculty of Health Sciences, University of
Stellenbosch, Tygerberg, South Africa
INGRID WEBSTER, MSc
SVEN O FRIEDRICH, PhD
AMANDA LOCHNER, PhD, DSc
BARBARA HUISAMEN, PhD,
MRC Cape Heart Centre, Tygerberg, South Africa
AMANDA LOCHNER, PhD, DSc
BARBARA HUISAMEN, PhD
