(Received for publication, August 10, 1995; and in revised form, October 5, 1995)
From the
Insulin activates glucose transport by recruiting Glut4 glucose
transporters from an intracellular pool to plasma membrane (PM). To
localize intracellular translocating Glut4, we studied the effects of
brefeldin A (BFA), which disassembles Golgi and prevents trans-Golgi
vesicular budding, on the glucose transport system. Isolated rat
adipocytes were treated with and without both BFA (10 µg/ml) and
insulin. BFA did not affect maximal rates of either 2-deoxyglucose or
3-O-methylglucose transport or the insulin:glucose transport
dose-response curve but did increase basal transport by 2-fold (p < 0.05). We also measured Glut4 in PM, low (LDM) and
high density microsome subfractions. In basal cells, BFA increased PM
Glut4 by 58% concomitant with a 18% decrease in LDM (p <
0.05). Insulin alone increased PM Glut4 by 3-fold concomitant with a
56% decrease in LDM. BFA did not affect insulin-induced changes in
Glut4 levels in PM or LDM. Most intracellular Glut4 was localized to
sub-PM vesicles by immunoelectron microscopy in basal cells, and BFA
did not affect insulin-mediated recruitment of immunogold-labeled Glut4
to PM. In summary, 1) in basal cells, BFA led to a small increase in
glucose transport activity and redistribution of a limited number of
transporters from LDM to PM; 2) BFA did not affect insulin's
ability to stimulate glucose transport or recruit normal numbers of LDM
Glut4 to PM; and 3) insulin action is predominantly mediated by a
BFA-insensitive pool of intracellular Glut4, which localizes to sub-PM
vesicles. Thus, the major translocating pool of Glut4 in rat adipocytes
does not involve trans-Golgi.
Insulin stimulates glucose transport by rapidly inducing the
translocation of intracellular glucose transporters to the cell surface (1, 2) . The Glut4 transporter isoform predominates in
insulin target tissues (fat and muscle) and mediates the bulk of
insulin-stimulatable glucose transport activity. The lack of specific
biochemical markers has made it difficult to ascertain the exact
intracellular locus of Glut4 in membrane subfractionation
experiments(3) . Morphological studies employing immunoelectron
microscopy (IEM) ()have localized the major portion of
insulin-responsive Glut4 to
trans-Golgi(4, 5, 6) . However, Smith et
al.(7) have shown in rat adipocytes that most
intracellular Glut4 undergoing insulin-mediated translocation derive
from vesicles adjacent to the endofacial surface of plasma membrane
(PM) or PM invaginations. Also, Lange and Brandt (8) found that
intracellular translocating Glut4 are concentrated in a cell
surface-derived membrane fraction in 3T3-L1 adipocytes by employing
subcellular fractionation method together with a hydrodynamic shearing
technique.
In the current study, we used brefeldin A (BFA) to test whether the intracellular translocating pool of Glut4 is functionally dependent upon an intact Golgi in rat adipocytes. BFA is a fungal macrolide antibiotic, which disrupts the organization of Golgi complex(9) , prevents vesicular budding from Golgi and trans-Golgi(10) , and leads to retrograde movement of Golgi proteins back to the endoplasmic reticulum. If insulin-responsive Glut4 derive from the Golgi network, BFA should inhibit insulin-stimulated glucose transport and Glut4 translocation.
Figure 1: Effect of BFA on 2-deoxyglucose transport. Equal numbers of isolated rat adipocytes were treated without and with the indicated concentrations of BFA for 30 min followed by incubation in the absence and presence of a maximal insulin concentration (100 ng/ml) for 30 min. At the end of incubation, initial rates of 2-DOG uptake were measured. The data are mean ± S.E. of three experiments.
Figure 2: Effect of BFA on dose-response curve for insulin stimulation of glucose transport. Equal numbers of isolated rat adipocytes were treated without and with BFA (10 µg/ml) for 30 min followed by incubation in the absence and presence of indicated insulin concentration for 30 min. At the end of incubation, initial rates of 2-DOG were measured. The data are mean of six experiments.
Figure 3: Effect of BFA on 3-O-methylglucose transport. Equal numbers of isolated rat adipocytes were treated without and with BFA (10 µg/ml) for 30 min followed by incubation in the absence and presence of a maximal insulin concentration (100 ng/ml) for 30 min. At the end of incubation, initial rates of 3-OMG were measured. The data are mean ± S.E. of four experiments.
Figure 4: Reversibility of BFA effect. Isolated rat adipocytes were treated without and with BFA (10 µg/ml) for 30 min followed by incubation in the absence and presence of maximal insulin concentration (100 ng/ml) for 30 min. At the end of incubation, the cells were washed three to four times and resuspended in the same but BFA-free buffer for another 30 min before 2-DOG assay was performed. The data are mean ± S.E. of three experiments.
Figure 5:
Effect of BFA on cellular distribution of
Glut4 in membrane subfractions. Isolated rat adipocytes were treated
without and with BFA (10 µg/ml) for 1 h followed by incubation in
the absence and presence of maximal insulin concentration (100 ng/ml)
for 30 min. The cells were then homogenized, and PM, LDM, and HDM
subfractions were prepared using differential ultracentrifugation.
Membrane proteins (35 µg/ml) were resolved by SDS-polyacrylamide
gel electrophoresis and reacted with Glut4-specific antibody followed
by I-protein A. A, a representative
autoradiograph. B, ± S.E. data from four experiments.
Relative amounts of Glut4 were quantitated by densitometric analysis of
autoradiograms, and the level in basal cells was assigned a value of
1.
From the data in Fig. 5B, it is clear that even maximal insulin was unable to recruit all intracellular Glut4 in LDM to the PM; 40-45% of Glut4 remains associated with LDM as has been consistently reported by multiple investigators. Although BFA caused an 18% decrease in LDM Glut4 in both basal and insulin-stimulated cells, the absolute decrement in LDM Glut4 as a consequence of insulin stimulation was similar in cells treated with and without BFA. Therefore, the BFA-sensitive pool of LDM Glut4 appears to be distinct from insulin-responsive translocating Glut4.
Figure 6: Immunogold labeling of Glut4 in isolated rat adipocytes. Isolated rat adipocytes were treated without and with BFA (10 µg/ml) for 1 h followed by incubation in the absence and presence of maximal insulin concentration (100 ng/ml) for 30 min. At the end of incubation, the cells were prepared for IEM and immunostained with MC2A, the monoclonal antibody to the carboxyl-terminal peptide of Glut4. Antibody binding sites were detected with gold-labeled protein A. A, basal cells; B, insulin-treated cells; C, cells treated by both BFA and insulin. pm, plasma membrane; L, central lipid droplet; arrow heads, sub-PM invaginations or vesicles.
Studies designed to localize the intracellular,
insulin-sensitive, translocating pool of Glut4 have been controversial.
Our experiments, using BFA, support the conclusion that the Golgi
apparatus and trans-Golgi are not the major functional sources of Glut4
undergoing insulin-mediated translocation to PM in rat adipocytes. BFA
is a fungal antibiotic that has been extensively used in studies of
intracellular membrane trafficking. BFA causes a morphological
disassembly of Golgi apparatus. It blocks transport of proteins into
post-Golgi compartments in the cell and redistributes Golgi-resident
proteins back into endoplasmic reticulum(17) . BFA blocks
constitutive secretion by preventing the formation of
non-clathrin-coated vesicles required for vesicular transport through
Golgi cisternae (10) as well as clathrin-coated vesicles that
bud from trans-Golgi network(18) . Molecular mechanisms of BFA
toxicity are not known; however, -cop, ADP-ribosylation factor
(components of non-clathrin-coated vesicles), as well as
-adaptin
(a component of clathrin-coated vesicles) are unable to bind to Golgi
membranes and are released into cytosol. Blockage of vesicle assembly
prevents anterograde transport of proteins from endoplasmic reticulum
through Golgi to the cell surface. Thus, if intracellular translocating
Glut4 derives from trans-Golgi, we would expect to see that
insulin-stimulated glucose transport and Glut4 translocation are
inhibited by BFA. However, in this study, we demonstrated that BFA did
not affect insulin's ability to stimulate glucose transport or
translocate normal numbers of intracellular Glut4 to PM.
Immunoelectron microscopic studies confirm our biochemical data. As previously demonstrated(7) , the major pool of Glut4 undergoing insulin-mediated recruitment to the cell surface is morphologically localized to sub-plasma membrane vesicles or invaginations. The current data confirm this observation and show that BFA did not affect translocation of this pool of Glut4 to PM.
We also found that BFA
led to a small (2-fold) increment in basal glucose transport
activity, which corresponds with redistribution of a limited number of
Glut4 from LDM to PM. The data in Fig. 5define two functional
pools of intracellular Glut4. BFA-sensitive Glut4 constitute a small
pool of intracellular Glut4, which is recruited from LDM to PM
subfractions upon treatment with doses of BFA known to disrupt Golgi.
However, BFA did not affect insulin's ability to maximally
stimulate glucose transport activity or translocate normal numbers of
LDM Glut4 to PM. Thus, the BFA-sensitive pool is distinct from the
absolute net decrement in LDM Glut4 observed as a consequence of
maximal insulin stimulation. This latter insulin-responsive component
comprises the largest pool of intracellular Glut4. The combined
biochemical and immunocytochemical data indicate that the
insulin-responsive pool is not located in trans-Golgi and more likely
resides in PM invaginations, cell surface-connected vesicles, or sub-PM
endosomal compartments(7) , which could sediment or
cofractionate in the LDM subfraction. These ideas are consistent with
data obtained using a hydrodynamic shearing technique applied to
3T
cells before homogenization(8) . This procedure
yielded a low density surface-derived vesicle fraction, which would
sediment in LDM if shearing was not applied. It was shown that the low
density surface-derived vesicle fraction contained nearly 60% of the
cellular glucose transporters and the total insulin-sensitive
transporter pool.
Other authors have recently examined effects of
BFA on Glut4 translocation. Chakrabarti et al.(19) studied effects of BFA on glucose transport and
transporter translocation in 3T-L
cells.
Similar to our data in rat adipocytes, these authors found that BFA
increased PM Glut4 by approximately 2-fold and did not impair
insulin's ability to increase cell surface Glut4 concentration.
However, BFA was found to inhibit both basal and insulin-stimulated
glucose transport activity by up to
60% with a half-maximal effect
being observed at 10 µg/ml. They explained that BFA may directly
inhibit the intrinsic activity of glucose transporters. We found no
evidence in rat adipocytes that 10 µg/ml BFA inhibited intrinsic
activity of glucose transporters. The reason for the difference in BFA
effects between 3T
-L
cells and rat adipocytes
is not clear but could be explained by utilization of different cell
systems or more generalized cellular toxicity at high BFA
concentrations. Lachaal et al.(20) have also studied
the effects of BFA on glucose transport and transporter translocation
in rat adipocytes. In their studies, BFA at 1 µg/ml inhibited
insulin-stimulated glucose transport as well as redistribution of Glut4
from microsomes to PM. These data are not confirmed in the current
study. There were some differences in experimental design. Lachaal et al.(20) used 3-OMG equilibrium exchange to measure
glucose transport activity and did not subfractionate total microsomes
into LDM and HDM while studying Glut4 subcellular redistribution.
However, it is not clear whether these differences can fully explain
the discrepancies between the results. In the current study, we have
performed morphological in addition to biochemical experiments, and
both lines of investigation are consistent with the conclusion that BFA
does not interfere with insulin's ability to stimulate glucose
transport activity ot Glut4 translocation.
In summary, we have shown that 1) in basal cells, BFA led to a small increase on glucose transport activity and to the redistribution of a limited number of Glut4 from LDM to the PM subfraction; 2) BFA did not affect insulin's ability to stimulate transport or recruit normal numbers of Glut4 to PM; 3) there are two functional pools of intracellular Glut4 in rat adipocytes, a large insulin-responsive pool, which is unaffected by BFA, and a smaller BFA-sensitive pool; and 4) BFA-insensitive Glut4, which translocate in response to insulin, are localized by IEM in sub-PM vesicles. In several human diseases characterized by insulin resistance such as obesity and Non-insulin-dependent Diabetes Mellitus, cellular depletion of Glut4 is a major mechanism of insulin resistance in adipocytes(21) . However, defects in translocation of Glut4 may be responsible for insulin resistance in skeletal muscle(22, 23) . In addition, we have shown that abnormalities in Glut4 trafficking may contribute to insulin resistance in adipocytes from women with gestational diabetes(24) . Precise localization of the intracellular Glut4 pool and a better understanding of Glut4 trafficking will permit elucidation of translocation defects causing insulin resistance in human diseases.