CARDIOVASCULAR JOURNAL OF AFRICA • Vol 21, No 2, March/April 2010
AFRICA
73
lin
25-27
or stimuli such as contraction or hypoxia. GLUT4 vesicles
respond to insulin in a marked and dramatic way, increasing
GLUT4 levels in the membrane up to nine times that of basal
levels.
28
The protein is recycled via endocytosis in clathrin-
coated vesicles.
29
It has, however, been recognised that translocation of GLUT4
to the membrane, and glucose uptake by the GLUT4 transport-
ers are not always reconcilable. There is a marked discrepancy
between glucose uptake and translocation of GLUT4.
30
Although
our understanding of the regulation of GLUT4 translocation
from the intracellular to the membrane compartments has
expanded rapidly over the past years,
31
the exact mechanisms
governing these events, especially at the site of fusion with
the cell membrane, is not fully understood. Events elicited by
stimulation with insulin have been the focus of intense research,
whereas less is known of those elicited by AMPK activation.
However, stimulation with activators of AMPK share some of the
downstream signalling events of the insulin pathway.
32,33
We used isolated, adult ventricular myocytes stimulated with
AICAR and ZMP to correlate glucose uptake and GLUT4 trans-
location after AMPK activation. The results demonstrated that in
cardiomyocytes, activation of AMPK was not sufficient to affect
GLUT4 insertion into the cell membrane and therefore glucose
uptake.
Methods
Materials
AICAR, ZMP, insulin and Triton X-100 were obtained from
Sigma, type 2 collagenase was from Worthington, bovine
serum albumin (BSA) fraction V, fatty acid-free from Roche
Diagnostics and 2-deoxy-D-[
3
H]glucose from New England
Nuclear. The ECL Western blotting detection reagents, anti-
rabbit Ig, horseradish peroxidase-linked whole secondary anti-
body were from Amersham Biosciences, UK Ltd, GLUT4
(H-61): sc-7938 rabbit polyclonal antibody was from Santa
Cruz Biotechnology Inc., phospho-PKB/Akt (Ser
473
), phospho-
AMPK-
α
(Thr
172
) and Phospho-ACC antibodies were from Cell
Signalling technology. The anti-GLUT4 (exofacial loop) was
purchased from Chemicon International (Temecula, California,
USA), and Zenon Alexa Fluor 488 Rabbit IgG Labeling Kit
was acquired from Molecular Probes (Eugene, Oregon, USA).
Paraformaldehyde was purchased from Merck (Cape Town,
RSA) and Dulbecco’s phosphate-buffered saline (PBS) from
Gibco (Grand Island, NewYork, USA).
Animals
Male Wistar rats weighing between 250 and 300 g were used in
all experiments. Animals were bred and kept in the AAALAC-
accredited facility of this institution. The project was approved
by the Ethics committee of the Faculty of Health Sciences,
University of Stellenbosch and conformed to the
Guide for the
Care and Use of Laboratory Animals
of the NIH (Publication
No. 85-23, revised 1996). Animals were anaesthetised with
sodium pentobarbital (160 mg/kg) before experimentation.
Preparation of cardiomyocytes
Rod-shaped ventricular cardiomyocytes were obtained by colla-
genase perfusion, essentially as described previously.
34
Isolated
cells were filtered through a nylon mesh (200
×
200
µ
m) and
gently spun down (3 min, 100 rpm). The resulting pellet was
resuspended in HEPES buffer (10 mM HEPES pH 7.4, 6 mM
KCl, 1 mM Na
2
HPO
4
, 0.2 mM NaH
2
PO
4
, 1.4 mM MgSO
4
, 128
mM NaCl, 5.5 mM glucose, 2 mM pyruvate, containing 1.25
mM CaCl
2
, 2% BSA (fraction V, fatty acid free) and the cells
were allowed to recover for 60 min on a slowly rotating plat-
form. Osmotic fragility coupled to trypan blue exclusion (TBE)
and cell morphology were used as indices for assessment of cell
viability.
35
Viable cells varied between 70 and 80% and all cell
isolates of less than 70% viability were discarded. At the end
of experimentation, the viability of cell preparations was deter-
mined with propidium iodide.
Propidium iodide staining
Propidium iodide (PI) staining was used to assess cell membrane
permeability. Nuclear staining by PI was assessed with flow
cytometric (FACS) analysis. Cardiac myocytes were incubated
with 10
μ
M PI for 15 min before analysis. Data are expressed
as mean fluorescence intensity as a percentage of control. FACS
analysis was done with a FACSCalibur using Cellquest 3.3 soft-
ware (Becton Dickinson, San Jose, California, USA).
Detection of glucose uptake
Myocyte uptake of 2-deoxy-D-[
3
H] glucose (2DG) was meas-
ured as described previously.
34
Cardiomyocytes (approximately
0.5 mg protein) were placed in a total volume of 750
µ
l assay
medium containing (in mmol/l): KCl 6, Na
2
HPO4 1, NaH
2
PO4
0.2, MgSO
4
1.4, NaCl 128, HEPES 10, CaCl
2
1.25 plus 2%
BSA (fraction V, fatty acid free) pH 7.4, 37°C, equilibrated with
oxygen. Cells were equilibrated for 15 min in a shaking water
bath (180 strokes/min) with or without phloretin (400
µ
M) for
measurement of non-carrier-mediated glucose uptake. They were
stimulated with or without insulin (15 min
×
100 nM), AICAR
(30 min
×
1 mM) or ZMP (30 min
×
1 mM) as indicated. Glucose
uptake was initiated by the addition of 2-deoxy-D-[
3
H] glucose
(1.5
µ
Ci/ml; final 2-deoxy-D-glucose concentration 1.8
µ
M) and
allowed to progress for 30 min before the reaction was stopped
with phloretin (final concentration 400
µ
M). The cells were then
centrifuged and the pellet was washed twice with HEPES buffer
and dissolved in 1 ml 0.5 N NaOH. An aliquot of this solution
was then used to assay protein concentration by the method of
Lowry,
36
and the rest was counted for radioactivity.
Preparation of lysates for Western blotting
Cardiomyocytes were removed after stimulation with insulin,
AICAR or ZMP as described above and placed on ice. The cells
were centrifuged and washed three times with ice-cold HEPES
buffer without albumin and lysed in 100
µ
l of lysis buffer (25
mM HEPES, 50 mM
β
-glycerophosphate, 1 mM EGTA, 1%
Triton X-100, 10 mM p-nitrophenyl phosphate, 1 mM Na
3
VO
4
,
2.5 mM MgCl
2
, 10
µ
g/ml aprotonin, 10
µ
g/ml leupeptin, 1 mM
phenylmethylsulphonyl fluoride and 1 mM 1-4-dithiothreitol,
pH 7.4). The lysates were centrifuged at 15 000
×
g for 10 min
and the supernatants diluted with Laemmli sample buffer for
SDS-PAGE. An aliquot of the supernatant was used for protein
determination.
37
