A. Kessler et al., Rab11 is associated with GLUT4-containing vesicles and redistributes in response to insulin, DIABETOLOG, 43(12), 2000, pp. 1518-1527
Aims/hypothesis. To identify a GTPase of 24 000 M-r which we recently found
to co-localize with GLUT4 in cardiac muscle.
Methods. A 24 000 M-r-GTP-binding fraction was purified from pig heart by a
three-step chromatographic procedure, followed by two-dimensional electrop
horesis and electrospray ionization-mass spectrometry, Subcellular distribu
tion of the GTPase was assessed by western blotting. Go-localization with G
LUT4 was assessed by continuous sucrose density gradient fractionation and
immunoadsorption of GLUT4-containing vesicles.
Results. The Rab11 protein was identified as a major component of the GTP-b
inding fraction and its expression in rat cardiac muscle was confirmed. In
vivo insulin treatment resulted in the recruitment of Rab11 from the micros
omal fraction to the plasma membrane. Subcellular fractionation indicated t
wo immunoreactive GLUT4 pools. Most of the intracellular pool of Rab11 over
lapped with the high-density GLUT4 pool and most of the transferrin recepto
r pool. The Rab11 protein also co-sedimented with the low-density, non-endo
somal GLUT4 pool and substantially increased in this fraction after insulin
treatment. It was specifically present in GLUT4-containing vesicles and in
sulin increased its abundance in these vesicles 2.2-fold relative to the am
ount of GLUT4. These vesicles also containend Rab4 and Akt-2, the latter be
ing only associated after insulin stimulation. Insulin was unable to alter
the cellular localization of Rab11 in insulin-resistant obese Zucker rats.
Conclusion/interpretation. These results support the hypothesis that at lea
st two GTPases of the Rab family participate in GLUT4-vesicle trafficking.
We suggest that Rab11 is involved in the endosomal recycling, sorting and e
xocytotic movement of the glucose transporter.