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Department of Medicine, University of California, San Diego, La Jolla, California 92093; Veterans Administration Research Service (J.M.O.), San Diego, California 92161; and INSERM U-145, Faculté de Médecine (M.C., Y.L.M.-B.), 06107 Nice, France
Address all correspondence and requests for reprints to: Jerrold M. Olefsky, M.D., Department of Medicine (0673), University of California, 9500 Gilman Drive, La Jolla, California 92093-0673.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast, microinjection of an antibody directed against the highly conserved region of Rab3 proteins had no effect on insulin-induced GLUT4. These results point to a direct role of Rab4 in insulin-induced GLUT4 translocation, and that this effect is dependent on nucleotide binding to the protein. We also studied the effect of microinjection of the same proteins on insulin-induced actin filament rearrangement (membrane ruffling) in the same cell line. Microinjection of Rab4 N121I and Rab4 antibodies inhibited insulin-induced membrane ruffling by 40%, whereas WT Rab4 or a Rab3 antibody injection had no effect on cytoskeletal rearrangement. In summary, 1) Rab4 is a necessary component of the insulin/GLUT4 translocation signaling pathway; 2) the function of Rab4 in this pathway requires GTP binding; 3) Rab4 also participates in the process of insulin-induced membrane ruffling; and 4) Rab3 proteins do not seem to be involved in these processes.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Rab protein family of Ras-related small GTP-binding proteins has been implicated in the regulation of intracellular vesicular traffic (3, 4, 8, 9). These proteins, with a molecular mass between 20–30 kDa, are highly homologous to the yeast YPT1 and SEC4 proteins, which play a crucial role in endocytosis and exocytosis. More than 30 members have been identified in mammalian cells, and individual Rab proteins are localized to distinct compartments of both the endocytotic and exocytotic pathways. All members contain highly conserved domains required for guanine nucleotide binding, GTP/GDP exchange, and GTP hydrolysis (10). Nucleotide binding and subsequent hydrolysis are essential for proper targeting and function of the Rab molecules (11). For example, when Rab5 mutants that are unable to bind GTP are overexpressed, they act as dominant negative mutants for endocytosis in vitro and in vivo (12, 13).
The small GTP-binding protein Rab4 is expressed in rat adipocytes (14) and 3T3-L1 adipocytes (15) and is closely associated with GLUT4-containing vesicles (14, 16, 17). Insulin stimulation induces a redistribution of Rab4 from the GLUT4-containing vesicles to the cytoplasm (14). This effect is reversible after insulin withdrawal. It is, therefore, possible, that Rab4 is involved in insulin-induced GLUT4 translocation. In this regard, it has been recently shown that electroporation of a peptide corresponding to the hypervariable carboxy-terminal domain of Rab4 inhibits insulin-stimulated glucose uptake in rat adipocytes (18), but the effector molecules of such peptides are unknown.
Other members of the Rab protein family are highly expressed in insulin-sensitive tissues. In particular two isoforms of the Rab3 proteins, Rab3A and Rab3D, have been shown to be expressed in rat adipocytes and 3T3-L1 adipocytes (19, 20). Interestingly, messenger RNA levels and expression of Rab3A and Rab3D increase significantly during differentiation of 3T3-L1 fibroblasts into mature adipocytes (19, 20). It has therefore been proposed that these proteins could be involved in exocytotic or endocytotic processes induced by insulin. In contrast to Rab4, Rab3D does not associate with GLUT4-containing vesicles (21) and is not redistributed after insulin stimulation (21). By sucrose density gradient centrifugation in 3T3-L1 adipocytes, Rab3A localizes in a different fraction than GLUT4-containing vesicles (20), but potential functions of theses two proteins in insulin-induced vesicular trafficking remain to be determined.
Besides their role in endocytotic and exocytotic pathways, other potential functions seem to be played by the small GTP-binding proteins of the Rab family. For example, Rab8 has been implicated in actin filament rearrangement in fibroblasts (22). As insulin induces cytoskeletal rearrangement (membrane ruffling) in 3T3-L1 cells (23), we wondered whether Rab4 could also play a role in insulin-induced actin filament rearrangement.
Therefore, to further assess whether Rab4 functions in insulin-induced GLUT4 translocation and actin filament rearrangement, we used single cell microinjection of 3T3-L1 adipocytes coupled with immunofluorescence microscopic detection of GLUT4 or actin filament localization. Here we show that injection of a glutathione S-transferase (GST)-Rab4 protein with a point mutation in the GTP binding site (Rab4 N121I) as well as a Rab4 antibody directed against the C-terminal portion of the protein inhibit both insulin-induced GLUT4 translocation and actin filament rearrangement in 3T3-L1 adipocytes. Microinjection of an antibody directed against a highly conserved region of the Rab3 isoforms had no effect on insulin-induced GLUT4 translocation and actin filament rearrangement. This suggests a direct role for Rab4 in insulin-induced GLUT4 translocation and cytoskeletal rearrangement.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DNA vector construction and GST fusion protein purification
The complementary DNA coding for wild-type (WT) Rab4 was
subcloned in the expression vector pGEX-2T. Mutations of WT Rab4 into
pGEX-2T were performed by site-directed mutagenesis. The residue N-121
was changed into I with the following nucleotide
C-CTT-TGT-GGA-ATC-AAG-AAG-GAC-CTG-G as previously described
(26). For the Q67L mutant, residue Q 67 was changed into L with the
following nucleotide: G-GAT-ACA-GCA-GGA-CTA-GAA-CGA-TTC-AG
(26).
Bacteria transformed with the pGEX-2T Rab4 constructs of interest were cultured until the A600 reached 0.6. Production of the GST-Rab4 was then induced by the addition of 50 µM isopropyl-ß-D-thiogalactoside. Proteins were then purified as described previously (27).
Electrophoresis and immunoblotting
Differentiated 3T3-L1 adipocytes were lysed in a buffer
containing 50 mM HEPES, 10 mM EDTA, 150
mM NaCl, 1% Triton X-100, 2 mM
phenylmethylsulfonylfluoride, 10% glycerol, 4 mM
Na3VO4, 400 mM sodium fluoride, and
20 mM sodium pyrophosphate, pH 7.4, at 4 C. Fifty
micrograms of whole cell lysates were separated by SDS-PAGE (12%
polyacrylamide), and transferred to polyvinylidene difluoride (PVDF)
membranes (Immobilon-P, Millipore Corp., Bedford, MA) in a
Tris-glycine-methanol buffer. After transfer, the membranes were
blocked with Tris-buffered saline-5% non fat milk (wt/vol) or PBS-5%
BSA (wt/vol) overnight at 4 C, then incubated with anti-Rab4 (1:1000)
and anti-Rab3 (1:2000) antibodies. Bound antibodies were visualized
using an enhanced chemiluminescence detection kit (Pierce Chemical Co.,
Rockford, IL).
Cell culture and microinjection
Preparation of antibodies.
For microinjection, antibodies
were purchased without sodium azide or BSA and then concentrated by
centrifugation through 30-kDa cut-off microconcentrators (Amicon Corp.,
Beverly, MA) with at least three washes with a buffer containing 5
mM sodium phosphate (pH 7.2) and 100 mM KCl
(microinjection buffer).
GLUT4 translocation and membrane ruffling.
3T3-L1 cells were
maintained and differentiated into adipocytes, then reseeded onto glass
coverslips as previously described (28). Cells were serum starved
2 h before microinjection for GLUT4 detection and overnight for
actin fiber staining. All reagents for microinjection were dissolved in
microinjection buffer. All reagents, except Rab4 and Rab3 antibodies,
were coinjected with preimmune sheep IgG (10 mg/ml) to allow the
detection of injected cells. After a recovery period of 1 h, the
cells were stimulated with 10 ng/ml insulin (1.7 nM) for 20
min unless otherwise specified. The cells were then fixed for
staining.
Immunostaining and fluorescence microscopy
GLUT4 protein staining.
Immunostaining of GLUT4 was
performed essentially as previously described (28). The cells were
fixed in 3.7% formaldehyde in PBS for 10 min at room temperature.
After washing, the cells were permeabilized and blocked with 0.1%
Triton X-100 and 2% FCS in PBS for 5–10 min. Cells were then
incubated overnight at 4 C with F349 (1 µg/ml, final concentration)
or 1F8 (4 µg/ml), which were diluted in PBS with 2% FCS. After
washing with PBS for 10 min, cells were incubated with
fluorescein-conjugated donkey antirabbit (1:100) or antimouse antibody
(1:100) as appropriate, and with AMCA-conjugated antisheep, or
antirabbit antibody (1:100) to detect injected cells.
Actin staining.
Cells were washed and permeabilized as
described above and then incubated in PBS with rhodamine-phalloidin (1
µg/ml) to visualize the location of polymerized actin (membrane
ruffles) and AMCA-conjugated donkey antisheep or antirabbit antibody to
detect injected cells.
Cell quantification.
Slides were analyzed on a Zeiss
Axiophot immunofluorescence microscope (Zeiss, New York, NY). The
AMCA-positive microinjected cells on each coverslip were evaluated for
the presence of plasma membrane-associated GLUT4 staining or actin
membrane ruffles. The observer was blinded to the experimental
conditions. The percentage of injected cells displaying each phenotype
is represented as GLUT4 translocation (percent positive cells) or the
ruffling index, respectively.
Imaging.
Images were captured using a CCD camera from
Photometrics (Tucson, AZ) and were saved using Isee software from
Inovision (Durham, NC).
Statistics
Statistical significance was assessed by Student’s t
test for paired data.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A and B). Injected cells were
identified by AMCA-labeled anti-IgG antibodies directed against IgG
injected for this purpose (Fig. 1B). As previously shown,
microinjection of IgG does not impair insulin’s effects on 3T3-L1
adipocytes (28). Unstimulated cells display GLUT4 staining mostly
localized around the nucleus, with some staining distributed around the
cytoplasm (Fig. 1A, basal). After insulin stimulation, GLUT4 staining
was seen at the plasma membrane level with a concomitant decrease in
the cytoplasm (Fig. 1A, + insulin). The cells displaying staining
predominantly at the plasma membrane were counted as positive, and
typically, after insulin stimulation, about 50% of the cells were
scored as positive.
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and 2).
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).
Effect of microinjection of a guanosine triphosphatase
(GTPase)-defective mutant (Rab4 Q67L) on insulin-induced GLUT4
translocation
Microinjection of Rab4 Q67L protein at a concentration of 15 mg/ml
in 3T3-L1 adipocytes had no effect on GLUT4 localization on either
basal or insulin-stimulated cells. After insulin stimulation, the
percentage of positive cells after Rab4 Q67L was comparable to that of
control injected cells (50% vs. 52%; P =
0.42; Fig. 3).
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). After insulin stimulation (10
ng/ml for 20 min), GLUT4 translocation was inhibited by 55% compared
with that in control IgG-injected cells (P = 0.0037;
Fig. 3). As Rab3A and Rab3D expression increased in 3T3-L1 fibroblasts
during differentiation into adipocytes, we wondered whether these
proteins could also be involved in insulin-induced GLUT4 translocation.
To assess this question, we used a mouse monoclonal antibody raised
against amino acids 60–220 of rat Rab3A in microinjection studies.
This is a highly conserved region between the different Rab3 isotypes
(>85% identity between Rab3A and Rab3D) (19). Microinjection of this
antibody (6 mg/ml) had no effect on either basal or insulin-induced
GLUT4 translocation in 3T3-L1 adipocytes (54% vs. 53% in
control injected cells; P = 0.57; Fig. 4).
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).
Western blotting of Rab4 and Rab3 proteins in 3T3-L1 adipocytes
After separation by SDS-PAGE and transfer to PVDF membranes, whole
cell lysates were probed with Rab4 and Rab3 antibodies. Figure 5 shows that the Rab4 and Rab3 antibodies
recognize a single band of the expected size in 3T3-L1 adipocyte whole
cell lysates.
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). Rab4 antibody and Rab4 N121I
inhibited insulin-induced membrane ruffling in 3T3-L1 adipocytes by
42% and 39%, respectively, compared with that in control injected
cells (P < 0.0004 and P < 0.05; Fig. 7). WT Rab4 or Rab3 antibody
microinjection had no effect on insulin-induced membrane ruffling
(P = 0.41 and P = 0.8, respectively).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inhibition of insulin-induced GLUT4 translocation
Rab4 is part of the Rab family of Ras-related small GTP-binding
proteins that are essential components of exocytotic and endocytotic
pathways (29). Originally, Rab4 had been shown to be associated with
early endosomes in Chinese hamster ovary cells or HepG2 cells. In
several cell lines, Rab4 controls recycling of transferrin receptors
from early endosomes to the cell surface (30). More recently, Cormont
et al. have shown that Rab4 is present on GLUT4-containing
vesicles in rat adipocytes (14), and that insulin stimulation results
in translocation of Rab4 from the vesicles to the cytosol. Importantly,
similar results have been reported in rat skeletal muscle, another
insulin-sensitive tissue (16, 17).
Rab proteins are small GTPases, and therefore, nucleotide binding as well as GTP/GDP cycling control shuttling and targeting of the protein as well as its function. Almost all Ras-related proteins have a highly conserved GTP-binding pocket (10), and mutations in this GTP/GDP binding motif can result in defective guanine nucleotide binding. We used a mutated protein with a substitution of N for I at position 121. Similar mutations of both YPT1 and SEC4 result in proteins that do not bind GTP and produce dominant lethal phenotypes and secretory defects when expressed in yeast (31, 32). Overexpression of a Rab5 N133I mutant, similar to Rab4 N121I, in BHK-21 cells leads to a complete inhibition of endocytosis and endosome fusion in vitro and in vivo (12). With this as a background, one could ask how could a GTP-binding defective Rab4 protein exerts an inhibitory effect on insulin-induced GLUT4 translocation? One possibility is that the microinjected Rab4 N121I sequesters certain factors required for endogenous Rab4 function. Rab proteins have to interact with a set of proteins, such as GTPase-activationg protein, guanine nucleotide dissociation stimulator proteins, and Rab effector proteins, to be activated and carry out their biological functions. Rab4 N121I could interact with such factors, fail to be activated by GTP binding, and prevent the interaction of endogenous Rab with those proteins.
Interestingly, it has recently been shown, that insulin stimulates GTP binding to Rab4 in rat adipocytes (33). This dose-dependent effect could be blocked by phosphatidylinositol 3-kinase inhibitors that also inhibit insulin-induced GLUT4 translocation (33). We show here that neither wild-type Rab4 nor Rab4 Q67L (a GTPase-defective mutant), which both bind GTP (26), had an effect on insulin-induced GLUT4 translocation. Taken together, our data indicate that GTP binding to Rab4 (rather than GTP hydrolysis) is required for its function in insulin signaling. We speculate that the various Rab4 proteins studied can engage with endogenous effector molecules, but without GTP binding they are not functional; therefore, Rab4 N121I is inhibitory. Furthermore, the fact that WT Rab4 or Rab4 Q67L microinjection had no stimulatory effect on basal or insulin-induced GLUT4 distribution suggests that Rab4 proteins are required, but not sufficient, for GLUT4 translocation.
Vesicular trafficking in the cell requires a precise targeting and docking mechanism. It is now generally agreed that important partners in this process are multisubunit particles, known as soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex (SNARE). The SNARE hypothesis proposes that this is accomplished by the partnering of specific markers, carried by transport vesicles, termed v-SNARES, with their cognate t-SNARES associated with the intended target membrane (34, 35). The SNARE complex assembly is essential for vesicle docking and fusion. Vesicle-associated membrane protein proteins (vesicle-associated membrane protein-2 and cellubrevin), which are part of the SNARE complex, have been localized to GLUT4 vesicles (36, 37), and it has been shown that Rab proteins are required for the assembly of SNARE complexes in the docking of transport vesicles (38, 39). Proteins of the SNARE complex could be effectors of Rab4. The potential role of Rab4 in insulin-induced GLUT4 translocation is further supported by the inhibitory effect observed after Rab4 antibody microinjection. This effect is specific, as premixing the antibody with WT Rab4 almost completely abolishes its inhibitory effect on insulin-induced GLUT4 translocation.
Baldini et al. have shown that two isoforms of the Rab3 proteins, Rab3A and Rab3D, are expressed in rat adipocytes as well as in 3T3-L1 adipocytes (19, 20). Furthermore, they showed that the expression levels of these isoforms of Rab3 increases severalfold during differentiation of 3T3-L1 fibroblasts into 3T3-L1 adipocytes (19, 20). It was speculated that Rab3D or Rab3A could be involved in insulin-regulated secretory pathways, but their function has not yet been characterized. Rab3D does not colocalize with GLUT4 vesicles and, unlike GLUT4, is not redistributed in response to insulin (21). In sucrose gradient fractionation experiments with 3T3-L1 adipocytes, Rab3A does not localize in the same pool as GLUT4-containing vesicles. Rab3A is expressed in neuronal and neuroendocrine cells, where it is associated with synaptic and synaptic-like vesicles. To investigate a potential role for Rab3 proteins in insulin-induced GLUT4 translocation and assess the specificity of the effect observed with the Rab4 antibody, we microinjected a Rab3 antibody, which had no effect on insulin-induced GLUT4 translocation. The Rab3 antibody is a monoclonal antibody against amino acids 60–220 of rat Rab3A, and this region is highly homologous between Rab3A and Rab3D (>85% amino acid identity) (19). This suggests that the antibody cross-reacts with the different isoforms of Rab3. Our results would indicate that Rab3 isoforms are probably not involved in insulin-induced GLUT4 translocation.
Using a different approach, Shibata et al. reported that electroporation into adipocytes of a 20-amino acid peptide corresponding to the hypervariable C-terminal region of the Rab4 protein can inhibit insulin-induced GLUT4 translocation and glucose transport by about 40–50% (18). As the carboxy-terminal domain of Rab proteins contains structural elements necessary for the association with specific target membranes, it was postulated that the peptide could compete with endogenous Rab at target sites. In contrast, electroporation of peptides corresponding to the Rab3C and Rab3D hypervariable region had no effect on insulin-induced glucose transport. This latter result supports our own data showing a lack of effect of Rab3 antibody on insulin-induced GLUT4 translocation. However, effector molecules of such peptides have yet to be characterized.
By cotransfecting freshly isolated rat adipocytes with Rab4 proteins and a GLUT4 transporter tagged with a myc-epitope (GLUT4-myc), Cormont et al. recently showed that Rab4 could play a role in GLUT4 subcellular localization (26). Overexpression of WT Rab4 resulted in a decrease in GLUT4-myc at the cell surface with normal or even increased responses to insulin-induced tagged transporter translocation. No effect on basal or insulin-induced membrane levels of GLUT4-myc was detected by cotransfecting adipocytes with Rab4 N121I, although they did find that expression of a C-terminal-deleted Rab4 inhibited insulin-induced GLUT4 translocation. It is possible that the observed differences in results from those of the current studies are related to the level of overexpression of the Rab4 proteins. Clearly, the responses vary depending on the amount of DNA transfected into the adipocytes. In our system, we measure localization of endogenous GLUT4 molecules present in 3T3-L1 adipocytes rather than transfected overexpressed GLUT4. This might explain why we did not observe an effect of WT Rab4 microinjection on basal GLUT4 localization. Another difference lies in the fact that we microinject GST fusion proteins, rather than expression vectors. Nevertheless, we believe that our results provide further evidence that Rab4 is involved in insulin-induced GLUT4 translocation in 3T3-L1 adipocytes and suggest the importance of nucleotide binding for the function of Rab4.
Actin filament rearrangement
We also studied the effect of the microinjected Rab4 proteins and
Rab4 antibody on insulin-induced actin filament rearrangement (membrane
ruffling) in the same cell line. Our results showed that the
GTP-binding defective Rab4 mutant (Rab4 N121I) as well as the Rab4
antibody inhibit insulin-induced actin filament rearrangement by about
40% when injected into 3T3-L1 adipocytes. WT Rab4 as well as Rab3
antibody microinjection had no effect on membrane ruffling induced by
insulin. To our knowledge, this is the first time that a potential role
for Rab4 in insulin-induced actin filament rearrangement has been
observed. Rac and Rho, which are also small GTP-binding proteins, are
known to function in this pathway (40, 41). Interestingly, it has
recently been shown that a member of the Rab protein family, Rab8,
could be involved in cytoskeletal rearrangement. Transient as well as
stable expression of WT Rab8 and an activated form of Rab8 (Rab8Q67L)
in BHK cells induced reorganization of their actin filaments and
formation of membrane ruffles (22). It was postulated that Rab8 could
play a role in membrane delivery to membrane ruffles and possibly
cross-talk with the small GTP-binding proteins Rho and Rac, leading to
cytoskeletal rearrangement. Our results suggest that Rab4 is involved
in insulin-induced actin filament rearrangement in 3T3-L1 cells and
that GTP binding is important for that function. Rab4 protein could be
a common signaling element in insulin-induced GLUT4 translocation and
membrane ruffling in 3T3-L1 adipocytes.
In summary, our results demonstrate that microinjection of a GTP-binding defective Rab4 protein (Rab4 N121I) as well as Rab4 antibody can inhibit insulin-induced GLUT4 translocation, providing evidence that Rab4 is involved in this metabolic action. We further show that this effect is dependent on nucleotide binding, as WT Rab4 microinjection had no effect on insulin-induced GLUT4 translocation. In addition, we observed an inhibition of insulin-induced membrane ruffling after microinjection of Rab4 N121I and Rab4 antibody, suggesting, for the first time, that Rab4 could play a role in cytoskeletal rearrangement in 3T3-L1 cells.
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Acknowledgments |
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Footnotes |
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Received March 25, 1997.
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References |
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