CARDIOVASCULAR JOURNAL OF AFRICA • Vol 21, No 2, March/April 2010
AFRICA
77
translocation and incorporation into the membrane, while no
associated glucose uptake was detected.
57
In addition, Funaki and
co-workers created a cell-permeable phosphoinositide-binding
peptide that could induce GLUT4 translocation to the plasma
membrane in adipocytes without increasing glucose uptake.
58
We used an antibody directed against the exofacial loop of
the GLUT4 protein, coupled to a flow cytometric method to
determine the amount of GLUT4 exposed on the outer surface
of cardiocytes after stimulation with either insulin or AICAR.
This method clearly demonstrated enhanced exposure with insu-
lin but attenuated exposure with AICAR, similar to the profile
of glucose uptake (Fig. 4). As this was in contrast to the results
obtained by Davey
et al
.
59
showing that ischaemia causes a
marked translocation of GLUT 4 to the sarcolemmal membrane
in whole beating hearts, we speculated on the possible role of
NO in this process.
AMPK phosphorylates endothelial nitric oxide synthase
(eNOS) on Ser
1177
.
60
According to Li
et al
.,
61
this activation modu-
lates glucose uptake and GLUT4 translocation in heart muscle.
Although cardiomyocytes also contain NOS, isolated cardiocytes
produce very little NO in comparison to cardiac endothelial
cells.
44
We therefore argued that the lack of NO formation and
subsequent activation of the cGMP pathway may be responsi-
ble for the lack of glucose uptake in our cells. To test this, we
supplemented the cells with NO with the addition of SNP. This
demonstrated that indeed, simultaneous stimulation with SNP
and AICAR resulted in significantly more GLUT4 protein now
detectable with the antibody against the exofacial loop of the
protein. However, contrary to our expectations, glucose uptake
was not affected, underscoring the concept that there still has to
be activation of the GLUT4 transporter after insertion into the
membrane. These results then reinforce the finding of Li
et al
.
61
that more signals than NO in addition to AMPK are necessary to
induce glucose uptake.
The results obtained in this study therefore argue that other,
hitherto unidentified factors besides AMPK activation and
GLUT4 translocation are necessary to induce glucose transport
via this pathway. We furthermore concluded that these signals
were not activated in the isolated cardiomyocytes via activation
of AMPK, although they were active in beating cardiac muscle.
Both AICAR and ZMP have proved valuable tools in this study.
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