(Received for publication, August 28, 1995; and in revised form, January 30, 1996)
From the
The present study was conducted to examine the involvement of
Rab4, a low molecular weight GTP-binding protein, in the action of
insulin on glucose transport. A synthetic peptide corresponding to the
Rab4 hypervariable carboxyl-terminal domain, Rab4-(191-210), was
successfully transferred into rat adipocytes by electroporation and
inhibited insulin-stimulated glucose transport by about 50% without
affecting the basal transport activity. In contrast, synthetic peptides
corresponding to the Rab3C and Rab3D carboxyl-terminal hypervariable
domain had little effect on insulin action on glucose transport. The
Rab4-(191-210) peptide also reduced insulin-induced GLUT4
translocation from the intracellular pool to the plasma membrane.
Furthermore, the Rab4-(191-210) peptide reduced both
insulin-induced glucose transport and GLUT4 translocation in the
presence of a major histocompatibility complex class I antigen-derived
peptide, D-(62-85), which is a potent inhibitor of
GLUT4 internalization, suggesting that the peptide inhibited exocytotic
recruitment of GLUT4-containing vesicles. The Rab4-(191-210)
peptide also inhibited GTP
S-stimulated glucose transport. In
addition, insulin-stimulated glucose transport was inhibited by the
addition of anti-Rab4 antibody. These results suggest that Rab4 protein
plays a crucial role in insulin action on GLUT4 translocation,
especially in exocytotic recruitment by the hormone of the glucose
transporter to the plasma membrane from the intracellular retention
pool.
Insulin stimulates glucose uptake in skeletal/cardiac muscles
and adipocytes by promotion of translocation of glucose transporter
isoform, GLUT4, from the intracellular pool to the plasma membrane (1, 2, 3) . Although the precise mechanism(s)
of insulin-induced GLUT4 redistribution is still unclear, several lines
of evidence indicate that GTP-binding protein(s) is involved in the
insulin action. Thus, nonhydrolyzable GTP analogues induce GLUT4
translocation (4, 5) and stimulate glucose transport (6, 7) in permeabilized adipocytes. Furthermore, by
dissecting the recycling pathway of GLUT4 into exocytosis and
endocytosis, we have demonstrated recently that GTPS (
)stimulates exocytotic fusion of GLUT4-containing vesicles,
whereas the nucleotide inhibits endocytosis of the glucose
transporter(8) . Our previous study also indicated that the
number of GLUT4 molecules in recycling pool is not large in the basal
state and the rate-limiting step of insulin-induced redistribution of
GLUT4 is the exocytotic recruitment from the intracellular retention
pool to the plasma membrane(8) .
The Rab family proteins of
Ras-related small GTP-binding proteins have been implicated in
regulation of intracellular vesicular traffic(9) . Given that
exocytotic movement of vesicles is regulated by the Rab family
protein(s), GTPS may promote exocytosis of GLUT4-containing
vesicles by stimulation of the Rab protein(s) associated with the
vesicles, although other candidates (e.g. trimeric GTP-binding
proteins) cannot be ruled out. In this regard, Baldini et al.(10) reported that Rab3D is predominantly expressed in
adipocytes and is induced during differentiation of 3T3-L1 cells into
adipocytes. However, it remains to be demonstrated whether Rab3D is
associated with the GLUT4-containing vesicles(11) . On the
other hand, Cormont et al.(12) recently revealed that
Rab4 is associated with GLUT4-containing vesicles in rat adipocytes and
insulin stimulation resulted in redistribution of the protein from the
vesicle to the cytosol. In contrast, Uphues et al.(13) reported that Rab4A was barely detectable in
GLUT4-containing vesicles in rat cardiac muscle, although insulin
induced an extensive shift of Rab4A from the cytosol and the microsomal
fraction to the plasma membrane.
In order to investigate whether Rab4 plays an essential role in insulin-induced GLUT4 translocation, we incorporated a synthetic peptide corresponding to the Rab4 hypervariable carboxyl-terminal domain into rat adipocytes and examined its effect on the insulin action. The carboxyl-terminal domain of Rab family proteins is thought to be responsible for targetting the proteins to the appropriate subcellular membranes (14) and to interact with the guanosine diphosphate dissociation inhibitor(15) . Perez et al.(16) reported that synthetic peptides corresponding to the carboxyl-terminal domains of Rab3B and Rab3C blocked calcium-triggered prolactin release from anterior pituitary cells. Our study shows that the insulin-induced GLUT4 translocation was markedly inhibited by incorporation of the Rab4 carboxyl-terminal domain peptide, suggesting that Rab4 protein plays a crucial role in the insulin-stimulated glucose transport in rat adipocytes.
In the present study, we first examined whether a sufficient amount of the Rab4-(191-210) peptide could be transferred into rat adipocytes by electroporation and whether transferred peptides had any effect on insulin action on glucose transport. As shown in Fig. 1, neither the basal nor the insulin-stimulated glucose transport activity was affected by the Rab4-(191-210) peptides in intact cells, whereas the insulin effect was reduced by about 50% in cells electroporated in the presence of the peptide. The extent of inhibition of insulin-stimulated glucose transport by the peptide varied from 30 to 90% (data not shown) probably because the efficiency of transfer of the peptide by electroporation was not consistent among the batches of cells. These results indicate that the Rab4-(191-210) peptides were successfully transferred into the cells by electroporation and inhibited insulin action on glucose transport.
Figure 1:
Effects of Rab4-(191-210)
peptide on the glucose transport activities in intact and
electroporated adipocytes. Adipocytesin Buffer X were incubated for 30
min at 37 °C to stabilize the basal transport activity. Left
panel, the cells were then incubated for 15 min with () or
without (
) 100 nM insulin in the absence or the presence
of 0.4 mM of the Rab4-(191-210) peptide and subjected to
the glucose transport assay under ``Experimental
Procedures.'' Right panel, the cells were electroporated
as described under ``Experimental Procedures'' in the absence
or the presence of 0.4 mM of the Rab4-(191-210) peptide,
then incubated with (
) or without (
) 100 nM insulin
for 15 min, and subjected to the glucose transport assay. The results
are the means ± S.E. (n =
3-6).
As presented in Fig. 2, the Rab4-(191-210) peptide inhibited the insulin-stimulated glucose transport in a dose-dependent manner without affecting the basal transport activity. In contrast, a synthetic peptide of the Rab3C carboxyl-terminal domain, which was shown to inhibit prolactin release in anterior pituitary cells(16) , had little effect on the insulin-induced glucose transport. Similarly, a synthetic peptide of the Rab3D carboxyl-terminal domain did not affect the insulin-induced glucose transport (data not shown). These results suggest that the insulin action was specifically inhibited by the Rab4 carboxyl-terminal domain peptide.
Figure 2:
Effects of Rab4-(191-210) and
Rab3C-(196-215) peptides on the insulin-stimulated glucose
transport activities. Adipocytes in Buffer X were incubated for 30 min
at 37 °C and then electroporated in the presence of the indicated
concentrations of the Rab4-(191-210) (,
) or
Rab3C-(196-215) (
,
) peptides as described under
``Experimental Procedures.'' The cells were then incubated
for 15 min without (open symbols) or with (closed
symbols) 100 nM insulin and subjected to the glucose
transport assay. The results are the means ± S.E. (n = 3-6).
We also investigated the effect of the Rab4-(191-210) peptide on GLUT4 translocation induced by insulin. The results of immunoblot analysis (Fig. 3) showed that insulin-stimulated translocation of GLUT4 was prevented by about 40% in the peptide-incorporated cells, indicating that the Rab4-(191-210) peptide inhibited the insulin action on glucose transport by attenuating GLUT4 translocation from the intracellular pool to the plasma membrane. These results also provide evidence that Rab4 protein plays an essential role in insulin-induced GLUT4 translocation.
Figure 3: Inhibition by Rab4-(191-210) peptide of insulin-induced GLUT4 translocation. Adipocytes in Buffer X were incubated for 30 min and then electroporated in the absence or the presence of the Rab4-(191-210) peptide. The cells were then incubated for 20 min with or without 100 nM insulin. At the end of the incubation, the cells were washed with STE buffer, homogenized, and then subjected to subcellular fractionation and immunoblotting as described under ``Experimental Procedures.'' A, representative immunoblot data. PM, plasma membrane fractions; SS, slowly sedimenting fractions. B, relative amounts of GLUT4. The relative intensities of GLUT4 bands were quantified by using FUJIX BAS2000 bio-imaging analyzer. The results are the means ± S.D. of three determinations. *, p < 0.05.
In
the next set of experiments, we studied the effect of the
Rab4-(191-210) peptide on insulin-stimulated glucose transport in
the presence of the major histocompatibility complex class I
antigen-derived peptide, D-(62-85), which is a potent
inhibitor of endocytosis of GLUT4(24) . As we reported
previously(8) , under these conditions, exclusively exocytotic
accumulation of GLUT4 on the plasma membrane could be measured. As
illustrated in Fig. 4, Rab4-(191-210) peptide inhibited
insulin-induced glucose transport by about 40% in the presence of 50
µMD
-(62-85) without affecting the basal
transport activity. Likewise, GLUT4 translocation by insulin in the
presence of D
-(62-85) was significantly inhibited by
the Rab4-(191-210) peptide (Fig. 5). These results
indicate that the Rab4-(191-210) peptide inhibited exocytotic
recruitment by insulin of GLUT4 to the plasma membrane from the
intracellular pool.
Figure 4:
Inhibition by Rab4-(191-210) peptide
of the insulin-stimulated glucose transport in the presence of
D-(62-85) peptide. Adipocytes in Buffer X were
incubated for 60 min at 37 °C to stabilize the basal transport
activity and then electroporated in the absence or the presence of 0.4
mM Rab4-(191-210) peptide. The cells were then incubated
for 30 min in the absence (right panel) or the presence (left panel) of 50 µMD
-(62-85)
without (
) or with (
) 100 nM insulin, and the
glucose transport activity was assayed. The results are the means
± S.E. (n = 3).
Figure 5:
Inhibition by Rab4-(191-210) peptide
of the insulin-stimulated GLUT4 translocation in the presence of
D-(62-85) peptide. Adipocytes in Buffer X were
incubated for 60 min at 37 °C to stabilize the basal transport
activity and then electroporated in the absence or the presence of 0.4
mM Rab4-(191-210) peptide. The cells were then incubated
for 30 min in the presence of 50
µMD
-(62-85) with or without 100 nM insulin. At the end of the incubation, the cells were washed,
homogenized, and subjected to subcellular fractionation and
immunoblotting as described under ``Experimental
Procedures.'' A, representative immunoblot data. PM, plasma membrane fractions; SS, slowly sedimenting
fractions. B, relative amounts of GLUT4. The relative
intensities of GLUT4 bands were quantified by using FUJIX BAS2000
bio-imaging analyzer. The results are the means ± S.D. of three
determinations. *, p < 0.05.
Studies from many laboratories including ours (4, 5, 6, 7) showed that
nonhydrolyzable GTP analogues stimulate GLUT4 translocation.
Furthermore, by dissecting the recycling pathway of GLUT4 into
exocytosis and endocytosis, we have demonstrated that GTPS
promotes exocytotic fusion of the GLUT4-containing
vesicles(8) . These observations led us to investigate whether
the Rab4-(191-210) peptide inhibits the GTP
S-stimulated
glucose transport. As presented in Fig. 6, the glucose transport
activity stimulated by 1 mMGTP
S was reduced by about 40%
in the cells electroporated with the Rab4-(191-210) peptide but
not with the Rab3C-(196-215) peptides.
Figure 6:
Effects of Rab4-(191-210) and
Rab3C-(196-215) peptides on the GTPS-stimulated glucose
transport activities. Adipocytes in Buffer X were incubated for 30 min
at 37 °C and then electroporated in the presence of the indicated
concentrations of Rab4-(191-210) (
,
) or
Rab3C-(196-215) (
,
) peptides as described under
``Experimental Procedures.'' The cells were then incubated
for 15 min without (open symbols) or with (closed
symbols) 1 mM GTP
S and subjected to the glucose
transport assay. The results are the means ± S.E. (n = 3-6).
In the final set of experiments, we examined the effect of anti-Rab4 antibodies on insulin-induced glucose transport activity. A number of studies have demonstrated that electroporation can be employed to introduce antibodies into mammalian cells without affecting the physiological integrity of the cells(25, 26, 27, 28) . As shown in Fig. 7, in adipocytes electroporated in the presence of anti-Rab4 IgG, insulin effect on glucose transport was markedly attenuated. In contrast, nonimmune IgG did not show any inhibitory effect on insulin-induced glucose transport. The anti-Rab4 IgG was ineffective on the insulin action in intact cells (data not shown). These results provide further evidence for the forementioned notion that Rab4 protein plays an essential role in the insulin action on glucose transport.
Figure 7:
Effects of anti-Rab4 IgG and nonimmune IgG
on the insulin-stimulated glucose transport activities. Adipocytes in
Buffer X were incubated for 30 min at 37 °C and then electroporated
in the presence of the indicated concentrations of anti-Rab4 IgG
(,
) or nonimmune IgG (
,
). The cells were then
incubated for 15 min with (closed symbols) or without (open symbols) 100 nM insulin and subjected to the
glucose transport assay as described under ``Experimental
Procedures.'' The results are the means ± S.E. (n = 3-6).
The Rab4 is a member of the Rab family of Ras-related small GTP-binding proteins and has been found to be associated with early endosomes in Chinese hamster ovary cells (29) or HepG2 cells(30) . Functionally, Rab4 has been shown to control recycling of transferrin receptor from the early endosome to the cell surface(31) . Cormont et al.(12) have recently revealed that Rab4 is present in the GLUT4-containing vesicles in rat adipocytes, and insulin stimulation resulted in redistribution of the protein from the vesicles to the cytosol. They also reported that Rab4 is phosphorylated by the insulin-stimulated MAP kinase (32) and argued that the insulin-induced redistribution of Rab4 from the GLUT4-containing vesicles to the cytosol could result from Rab4 phosphorylation by the kinase. Although the significance of Rab4 phosphorylation and redistribution in the insulin-stimulated GLUT4 translocation is yet to be clarified, the presence of Rab4 in the GLUT4-containing vesicles implies the endosomal origin of the vesicles and raises the possibility that Rab4 may play an essential role in the insulin action.
In the present study, to investigate the physiological significance of Rab4 protein in the insulin action on glucose transport, we incorporated a synthetic peptide corresponding to the hypervariable carboxyl-terminal domain of Rab4 into rat adipocytes. The carboxyl-terminal domain of Rab family proteins is highly divergent (33) and contains structural elements necessary for the association of the proteins with specific target membranes(14) . Synthetic peptides corresponding to the carboxyl-terminal domain of Rab3B and Rab3C have been shown to block calcium-triggered prolactin release from pituitary cells(16) . As illustrated in Fig. 1, the Rab4-(191-210) peptide was successfully transferred into rat adipocytes by electroporation and inhibited insulin action on glucose transport. Immunoblot analysis indicated that the peptide significantly attenuated the insulin-induced GLUT4 translocation from the intracellular pool to the plasma membrane (Fig. 3). The inhibitory effects were specific to Rab4 because synthetic peptides of the Rab3C and Rab3D carboxyl-terminal domain were without effect.
Importantly, the inhibition by the peptide was also
observed in the presence of a major histocompatibility complex class I
antigen-derived peptide, D-(62-85), a potent
inhibitor of GLUT4 endocytosis (24) ( Fig. 4and Fig. 5). As we reported previously(8) , in the presence
of a sufficient concentration of D
-(62-85), the
endocytosis of GLUT4 is almost completely inhibited; consequently
recycling of the glucose transporter from the early endosomes to the
plasma membrane does not take place, and exclusively exocytotic
recruitment of GLUT4 onto the plasma membrane from the intracellular
retention pool, which may be a rate-limiting step for GLUT4
translocation by insulin, can be measured. Although we cannot totally
rule out the possibility that Rab4 may be involved in the
internalization step of GLUT4 molecules, the present results indicate
that one of the steps affected by the Rab4-(191-210) peptide is
the insulin-induced exocytotic recruitment from the intracellular
retention pool to the plasma membrane and that Rab4 is involved in this
crucial step for the hormonal action. However, it is also possible that
insulin may activate a step(s) other than the Rab4-regulated one and
Rab4 protein may play a more passive role, just like VAMP (38) or cellubrevin(39) , in exocytosis of the
GLUT4-containing vesicles. Recent kinetic and morphological studies
have proposed a three-compartment model for the subcellular trafficking
of GLUT4(3, 40, 41) . In the model, GLUT4
molecules are associated with at least two intracellular compartments.
One is the early endosome in which GLUT4 molecules seem to appear only
in the presence of insulin, and the second one is a more specialized
compartment where GLUT4 molecules are sequestrated in the basal state.
It is possible that GLUT4 moves to the plasma membrane from either
compartment. The results of present study that the extent of inhibiton
by the peptide of the insulin effect is somewhat modest in the range of
40-50% may indicate that the Rab4-(191-210) peptide may be
involved in only one pathway. In either way, our study provides
evidence that Rab4 protein is indispensable for the insulin-induced
GLUT4 translocation.
At present, the mechanism of the inhibition by the Rab4-(191-210) peptide is not clear. Because the carboxyl-terminal domain of Rab proteins contains structural elements necessary for the association with specific target membranes(14) , the Rab4-(191-210) peptide probably competes with endogenous Rab4 at the target site(s). In agreement with the study by Perez et al.(16) , the absence in Rab4-(191-210) peptide of two cysteine residues necessary for geranylgeranylation (34) seems to exclude Rab-guanosine diphosphate dissociation inhibitor as one of the target molecule because Rab-guanosine diphosphate dissociation inhibitor only associates with geranylgeranylated Rab protein(35) . Further studies are clearly needed to identify the target molecules of the Rab4-(191-210) peptide.
Rab4 has been shown to control
recycling of transferrin receptor from the early endosome to the cell
surface(31) . In Chinese hamster ovary cells overexpressing a
mutant Rab4, transferrin receptor remains intracellular(31) .
These observations raise a possibility that the inhibition by the
Rab4-(191-210) peptide of the insulin action may result from the
reduction of the insulin receptor number on the cell surface. However,
reduction of the receptor number may not account for the inhibition of
glucose transport by the peptide, because the Rab4-(191-210)
peptide also inhibited the glucose transport stimulated by GTPS (Fig. 6). The effect of GTP
S on exocytosis of
GLUT4-containing vesicles was not blocked by wortmannin, (
)a
potent and specific inhibitor of phosphatidylinositol
3-kinase(36) , an observation indicating that GTP
S acts at
a step(s) distal to phosphatidylinositol 3-kinase presumably by a
direct interaction with vesicle trafficking machinery. Furthermore, as
shown in Fig. 4and Fig. 5, the inhibitory effect of the
Rab4-(191-210) peptide on the insulin action was also observed in
the presence of D
-(62-85). Because the
D
-(62-85)peptide is a potent inhibitor of the
internalization of the insulin receptor (37) as well as GLUT4,
the insulin receptor remains on the cell surface in the presence of the
peptide.
The notion that Rab4 is involved in the insulin action on glucose transport is further supported by the experiment using antibodies against the carboxyl-terminal domain of Rab4. As demonstrated in Fig. 7, the anti-Rab4 antibody markedly reduced the insulin-stimulated glucose transport without affecting the basal transport activity. Hence, in the present study, two independent methods to modify the function of the Rab4 carboxyl-terminal domain provided essentially similar results.
In summary, the present results demonstrate that a synthetic peptide corresponding to the Rab4 hypervariable domain specifically inhibits the insulin-stimulated glucose transport as well as GLUT4 translocation, providing evidence that Rab4 is involved in the insulin action, especially in exocytotic recruitment by the hormone of the glucose transporter to the plasma membrane from the intracellular pool. In the light of the observation that insulin stimulates exocytosis of the receptors for transferrin(42) , it would be very intriguing to examine whether the Rab4 function is directly activated by the hormone or not.