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(Received for publication, October 11,
1995; and in revised form, January 17, 1996) From the
Adipocytes of transgenic mice overexpressing Glut4 selectively
in adipose tissue (Shepherd, P. R., Gnudi, L., Tozzo, E., Yang, H.,
Leach, F., and Kahn, B. B.(1993) J. Biol. Chem. 268,
22243-22246) have 15-20-fold more Glut4 than normal
adipocytes. To study compartmentalization of intracellular Glut4 in
these cells, we fractionated light microsomes prepared from transgenic
and normal adipocytes in velocity and density sucrose gradients.
Glut4-containing intracellular membranes from both cell types have a
specific and narrow distribution in these gradients, i.e. behave as homogeneous vesicles with identical sedimentation
coefficients and different buoyant densities. Immunoadsorption of
Glut4-containing vesicles with covalently immobilized monoclonal
anti-transporter antibody demonstrated that the total polypeptide
composition of these vesicles from transgenic and normal cells was
identical, with the exception of Glut4 itself, which was much more
abundant in the transgenic cells. Both preparations also had comparable
levels of secretory carrier membrane proteins and of aminopeptidase
activity (gp160). Glut4-containing vesicles from both normal and
transgenic adipocytes excluded Glut1, which in both cell types formed a
different vesicle population. Thus, even under conditions of high level
overexpression, Glut4 is still specifically targeted to the same unique
type of structurally defined insulin-sensitive vesicles as in normal
cells. Insulin-sensitive adipose and skeletal muscle tissues express a
specific isoform of glucose transporter protein, Glut4, which under
basal conditions, i.e. in the absence of insulin, resides in
intracellular microsomal vesicles. After insulin administration,
Glut4-containing vesicles fuse with the plasma membrane and deliver the
transporter to the cell surface (for recent reviews, see Birnbaum
(1992), Bell et al.(1993), Mueckler(1994), James and Piper
(1994), James et al.(1994), Holman and Cushman(1994), and
Stephens and Pilch(1995)). It is likely that this process largely
accounts for insulin's effect on blood glucose update. Therefore,
transporter translocation has enormous physiological significance, and
its molecular mechanism has been studied in many laboratories.
Important questions in this regard are how Glut4 is retained inside a
cell and how its movement to the cell surface is regulated by insulin,
while other members of the glucose transporter family stay permanently
at the plasma membrane and are not insulin-regulatable. A general
agreement has been reached that the C terminus of Glut4 defines its
intracellular localization (Czech et al., 1993; Verhey et
al., 1993; Marshall et al., 1993; Haney et al.,
1995), although this is not a unanimous conclusion (Piper et
al., 1992, 1993; Asano et al., 1992). The nature of the
Glut4-containing intracellular compartment(s) and the mechanism of its
insulin-sensitive translocation to the cell surface remain unknown. To define the nature of this compartment, it will be important to
understand its relation to various other intracellular membrane
structures and, in particular, to transport vesicles that shuttle
between different subcellular membrane structures such as the
endoplasmic reticulum, Golgi apparatus, and plasma membrane. We need to
determine whether Glut4-containing structures represent unique and
specialized subcellular particles (organelles?) or a subpopulation of
Golgi-to-plasma membrane transport vesicles, a component of the normal
endocytotic pathway, or something else. Immunocytochemical studies of
Glut4 in fat and muscle cells (Smith et al., 1991; Slot et
al., 1991a, 1991b; Rodnick et al., 1992) revealed some of
the morphological features characteristic of Glut4-containing
structures, but still left this question open. Biochemical studies
demonstrated that Glut4-containing vesicles from adipose and muscle
tissues are very homogeneous in size, sedimentation coefficient,
buoyant density, and total polypeptide composition (Kandror et
al., 1995a); have at least one marker protein besides Glut4,
namely the aminopeptidase gp160 (Kandror and Pilch, 1994b; Kandror et al., 1994); and thus may represent a unique specialized
compartment. The development of transgenic mice overexpressing Glut4
selectively in fat tissue (Shepherd et al., 1993) provided an
excellent physiological model for studies on compartmentalization and
targeting of the transporter. Transgenic mice give the advantages of
transfection experiments and still allow for studies with native
adipocytes instead of less differentiated or undifferentiated cell
lines. The central question addressed in this study was whether
overexpressed Glut4 was compartmentalized in the specific vesicles, as
in normal adipocytes, or whether it ``spilled'' out into
other cellular membranes. The data that we present here are consistent
with the interpretation that overexpressed Glut4 is retained in a
specific vesicle population that is biochemically homogeneous to
intracellular Glut4 vesicles from nontransgenic adipocytes.
The fraction of intracellular LM that is enriched in Golgi
apparatus protein markers (Simpson et al., 1983) was isolated
from nontransgenic and transgenic adipocytes as described under
``Materials and Methods.'' Fig. 1(A and B) demonstrates by Coomassie Blue staining and Western
blotting that transgenic mice have at least 15-20 times more
Glut4 in this fraction in comparison with normal control mice, whereas
the total polypeptide composition of their LM is not changed. This
demonstrates that overexpression of Glut4 is highly specific and does
not cause major perturbations in the protein composition of microsomal
membranes. Our further work was directed toward comparative
characterization of Glut4-containing compartments from adipocytes of
transgenic and normal mice.
Figure 1:
Coomassie Blue staining of (A)
and Glut4 content in (B) light microsomes from transgenic and
nontransgenic adipocytes. 15 µg of light microsomes from transgenic (tg) and nontransgenic (ntg) adipocytes were
electrophoresed in a 7.5% polyacrylamide gel. A, Coomassie
Blue staining of the gel, with molecular masses of the protein
standards shown on the right; B, Western blotting of the same
preparation with anti-Glut4 monoclonal antibody 1F8 after transfer to
polyvinylidene difluoride membrane. Results are representative of five
different experiments with 10-15 mice per
experiment.
We have previously demonstrated that
Glut4-containing vesicles from rat adipose and muscle tissues behave as
individual particles with a sedimentation coefficient of
Figure 2:
Fractionation of light microsomes from
transgenic and nontransgenic adipocytes in sucrose velocity gradients.
Light microsomes (0.8 mg of protein) from nontransgenic (open
circles) and transgenic (closed circles) adipocytes were
centrifuged in a 4.6-ml 1%-30% sucrose gradient as described under
``Materials and Methods.'' A, protein distribution; B, quantitation of the Glut4 distribution shown in C; C, Western blotting of odd gradient fractions with anti-Glut4
antibody 1F8. Results are representative of two different experiments
with 10-15 mice per experiment.
In the next experiment,
we ran equilibrium density sucrose gradients to determine the buoyant
density of Glut4-containing membrane structures. Fig. 3(B and C) demonstrates that, as in the previous experiment,
much more Glut4 is recovered when LM from transgenic mice were loaded
on the gradient in comparison with the nontransgenic control mice.
Moreover, Glut4-containing structures from transgenic animals have a
higher buoyant density than Glut4-containing vesicles from normal mice,
which is consistent with the possibility of more Glut4 molecules per
vesicle. However, paradoxically, the distribution of the total protein
was also shifted in these gradients to the high density zone (Fig. 3A). To clarify these data, we assessed the
density distribution of Glut1-containing vesicles, which are known to
represent a different vesicle population than those containing Glut4
(Zorzano et al., 1989; Kandror et al., 1995a; this
paper), and found out that the buoyant density of Glut1 vesicles was
also increased (Fig. 3D), although there is no
overexpression of Glut1 in the transgenic mice. Several other proteins
abundant in LM, like SCAMPs and Rab4, also demonstrate a shift to the
high density zone (data not shown). However, caveolin-containing
vesicles retain the same buoyant density both in normal and transgenic
adipocytes (Fig. 3E). This further proves that caveolin
is not associated with Glut4 in the same vesicles (Kandror et
al., 1995b) and suggests that the nature of caveolin-containing
structures is different from that of Glut1- and Glut4-containing
vesicles.
Figure 3:
Fractionation of light microsome fractions
from transgenic and nontransgenic adipocytes in sucrose density
gradients. Light microsomes (1.1 mg of protein) from nontransgenic (open squares) and transgenic (closed squares)
adipocytes were centrifuged in a 4.6-ml 10-50% sucrose gradient
as described under ``Materials and Methods.'' A,
protein distribution; B, quantitation of the Glut4
distribution shown in C; C, Western blotting of odd
gradient fractions with anti-Glut4 antibody 1F8; D,
quantitation of the Glut1 distribution; E, quantitation of the
caveolin distribution. Original Western blots are not shown for D and E. Results are representative of two different
experiments with 10-15 mice per experiment. ntg,
nontransgenic; tg, transgenic.
The direct experiment to compare Glut4-containing
compartments from adipocytes of transgenic and normal mice is the
comparison of their polypeptide composition. To isolate these
structures, we applied the same experimental protocol that we use for
immunoadsorption of Glut4-containing vesicles from rat adipocytes
(Kandror and Pilch, 1994a), and this proved to be very efficient for
mouse-derived vesicles as well. We were able to immunoadsorb
Figure 4:
Isolation of Glut4-containing vesicles
from nontransgenic and transgenic mice. Light microsomes (0.4 mg of
protein) isolated from nontransgenic (ntg) and transgenic (tg) adipocytes were immunoadsorbed with 70 µl
(nontransgenic) or 300 µl (transgenic) of 1F8 beads or nonspecific
IgG beads as described under ``Materials and Methods.''
Eluted material was electrophoresed, transferred, and blotted for Glut4 (A) and Glut1 (B). Results are representative of two
different experiments with 10-15 mice per experiment. sup, supernatant.
By silver staining of the
immunoadsorbed material (Fig. 5A), we have shown that
Glut4-containing vesicles from transgenic and normal mice are composed
of precisely the same constituent proteins. The only protein that
appears different is Glut4 itself, which is the expected result.
Western blotting with anti-SCAMP antibody (Thoidis et al.,
1993) does not reveal any differences in the amount or electrophoretic
mobility of these vesicle proteins (Fig. 5B).
Unfortunately, our antibody against another marker protein for
Glut4-containing vesicles, the aminopeptidase gp160, appeared to be
rat-specific, and although we could recognize a major protein of 160
kDa on a silver-stained gel, we could not confirm its identity by
Western blot analysis. Instead, we measured the enzymatic activity of
gp160 in Triton X-100 eluates from Immunobeads and showed in three
independent experiments that its activity was elevated only
1.3-1.5-fold in transgenic animals, which is still far from the
increase in Glut4 content.
Figure 5:
Polypeptide composition of
Glut4-containing vesicles from nontransgenic and transgenic mice. Light
microsomes (0.9 mg) from nontransgenic (ntg) and transgenic (tg) adipocytes were immunoadsorbed with 150 µl
(nontransgenic) or 400 µl (transgenic) of 1F8 beads or nonspecific
IgG beads as described under ``Materials and Methods.''
Eluted material was electrophoresed in a 10% polyacrylamide gel. A, silver staining of the gel, with molecular masses of the
protein standards shown on the right; B, Western blotting of
the same preparation with anti-SCAMP antibody after transfer to
polyvinylidene difluoride membrane. Results are representative of two
different experiments with 10-15 mice per
experiment.
In this paper, we studied compartmentalization of Glut4 in
adipocytes from transgenic mice overexpressing Glut4 at high levels
selectively in adipose tissue (Shepherd et al., 1993). Under
these conditions, Glut4 is found in the same type of vesicles as in
normal cells according to protein composition and sedimentation
analysis. We propose that in adipocytes from transgenic animals, there
may be more Glut4 molecules packed into one vesicle than in control
cells. An alternative explanation for our result is formation of a
higher number of vesicles with normal Glut4 content. Since no drastic
increase in any other vesicle protein component was detected, we think
that the first explanation is more likely to be true. Of course, the
most direct way to prove this hypothesis would be to compare the
buoyant density of Glut4-containing vesicles from normal and transgenic
animals. This analysis was complicated by the unexpected result that
the buoyant density of the total LM fraction is much higher in
transgenic mice than in control mice. We do not have an explanation for
this phenomenon yet. Since the total protein composition of the LM
fraction does not seem to change (Fig. 1), it may be the
phospholipid portion of the membrane that accounts for this very
significant effect. We plan to perform a high pressure liquid
chromatography-based analysis of the LM lipids to determine whether
there are major alterations in the amount and/or composition of the
lipids. In general, transgenic adipocytes are quite similar to
control cells and have very close physiological parameters such as
their size (0.3 mg of lipid/cell for both nontransgenic and transgenic
adipocytes used in this study). Elevated basal and insulin-stimulated
glucose transport in transgenic adipocytes results in increased glucose
metabolism at both rate-limiting and physiological glucose
concentrations with preferential effects on regulation of de novo fatty acid synthesis (Tozzo et al., 1995). A question may
arise as to how the results described in this paper can be interpreted
in light of the previous observation that basal glucose transport is
elevated in transgenic adipocytes. In fact, these findings are
consistent. It has been shown that under basal conditions, i.e. in the absence of insulin, Glut4 constantly recycles between its
intracellular vesicular compartment and the plasma membrane (Jhun et al., 1992; Yang et al., 1992; Czech and Buxton,
1993; Satoh et al., 1993; Yang and Holman, 1993) in such a way
that Naturally, in transgenic animals
expressing 15-20 times more Glut4, a dramatic increase in plasma
membrane transporter would be expected in unstimulated adipocytes,
which in turn would lead to a considerable elevation of basal glucose
transport. Along with this, we cannot entirely rule out the possibility
that some Glut4 may be also present in other structures that are more
likely to target to the plasma membrane. To check this hypothesis, we
tried to determine a potential presence of extra synthesized Glut4 in
Glut1- and caveolin-containing vesicles. We considered that since there
is more Glut1 and, especially, caveolin on the cell surface than inside
the cell, the equilibrium between these intracellular vesicles and the
plasma membrane must be shifted toward the latter compartment. However,
in full agreement with previously published data (Zorzano et
al., 1989; Kandror et al., 1995a, 1995b), we found no
significant colocalization of these proteins with Glut4, neither by
immunoadsorption (Glut1; Fig. 4) nor by sedimentation analysis
(caveolin; Fig. 3). As has been shown earlier (Shepherd et al., 1993), insulin-stimulated glucose transport is also
increased severalfold in transgenic adipocytes in comparison with
control normal cells. This is consistent with the results from our
present study that overexpressed Glut4 is compartmentalized in specific
vesicles that must be capable of delivering it to the cell surface in
an insulin-dependent fashion. This suggests the following. 1)
Adipocytes have a powerful intracellular machinery of precise targeting
of Glut4 to specific vesicles. 2) These vesicles appear to be different
from other intracellular microsomal structures since only they are
selected as a target for Glut4 compartmentalization. 3) These vesicles
appear to have a high reserve capacity to accept (and to translocate to
the cell surface) much more Glut4 than under normal conditions. Another
important conclusion is that overexpression of Glut4 in
insulin-sensitive tissues, which appears to enhance both basal and
insulin-stimulated glucose uptake, may lead to development of a genetic
treatment for diabetes.
Volume 271,
Number 18,
Issue of May 3, 1996 pp. 10490-10494
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Antibodies
In this study, we used the monoclonal
anti-Glut4 antibody 1F8 (James et al., 1988), polyclonal
anti-Glut1 antibody (a gift from Dr. Bernard Thorens), monoclonal
anti-GTV3/SCAMP (
)antibody (Thoidis et al., 1993),
and monoclonal anti-caveolin antibody (Transduction Laboratories)Transgenic Animals
Transgenic mice were engineered
by injection of the following DNA construct into the pronucleus of
fertilized zygotes from FVB mice and transfer to pseudopregnant
females. The adipose-specific Glut4 transgene was constructed
as described previously using the 5.4-kilobase adipose-specific
promoter/enhancer (a gift of Drs. B. M. Spiegelman and R. Graves) from
the fatty acid-binding protein gene aP2 ligated to a
6.3-kilobase BamHI-PvuII genomic DNA fragment
corresponding to bases 2061-8396 of the human GLUT4 gene
(a gift of Drs. J. Buse and G. I. Bell). The sequence contains all
exons and introns and a consensus polyadenylation signal of Glut4. To
identify transgenic mice, DNA was extracted from tail clippings and
subjected to Southern blotting or polymerase chain reaction using
primers 5`-TAT-CAT-CTC-TCA-GTG-GCT-TGG-AAG-3` and
5`-CTC-GAA-GAT-GCT-GGT-CGA-ATA-ATA-G-3`. Mice were housed at 21 °C
with a 12-h light/dark cycle and were fed standard Purina mouse chow
5008 ad libitum. All studies were carried out in heterozygous
mice at 12-14 weeks of age. Results were confirmed in both male
and female mice.Preparation of Light Microsomes from Adipose
Cells
Subcellular fractionation of adipocytes was performed as
described by Simpson et al.(1983). Briefly, adipocytes were
isolated from gonadal fat pads of 10-15 mice per genotype for
each experiment by collagenase digestion (1 mg/ml). Fat pads were
incubated at 37 °C with constant shaking in Krebs-Ringer phosphate
buffer (2 ml/g of fat) with 20 mM Hepes, 2.5% bovine serum
albumin (fraction V), and 200 nM adenosine (pH 7.4). Cells
were washed four times with buffer and twice with TES to reduce the
bovine serum albumin content and homogenized, and light microsomes (LM)
were prepared.Isolation of Glut4-containing Vesicles
This was
carried out by immunoadsorption of light microsomes on monoclonal
anti-Glut4 antibody 1F8 immobilized on polyacrylamide beads.
Nonspecific adsorption of microsomes was monitored by passage of
membranes over IgG immobilized on the same beads at the same protein
concentration (Kandror and Pilch, 1994a, 1994b). After overnight
incubation with light microsomes, beads were thoroughly washed with PBS
and eluted either with 1% Triton X-100 in PBS or with Laemmli sample
buffer.Subfractionation of Intracellular Microsomes on Sucrose
Gradients
LM from mouse adipocytes were suspended in PBS and
loaded onto a 4.6-ml continuous sucrose gradient (10-30% sucrose
for velocity centrifugation and 10-50% sucrose for density
gradient centrifugation) and centrifuged for 50 min and 18 h,
respectively, in an SW 50.1 rotor at 48,000 rpm. Each gradient was
collected into 26 fractions starting from the bottom of the tube.Aminopeptidase Activity
This was measured by the
fluorometric method described by Little et al.(1976). Briefly,
1% Triton X-100 eluates from 1F8 or IgG beads were mixed with 0.5 ml of
3 mML-alanyl-
-naphthylamide in a total volume
of 1.5 ml of PBS with 0.5% Triton X-100 to a final substrate
concentration of 1 mM. The mixture was incubated for 30 min at
37 °C, and fluorescence in fractions was measured at 410 nm
relative to the -naphthylamine standard (0.1 mM in the
assay mixture). The excitation wavelength was 340 nm.Gel Electrophoresis and Immunoblotting
Proteins
were separated in 10% SDS-polyacrylamide gels according to
Laemmli(1970) and transferred to Immobilon-P membrane (Millipore Corp.)
in 25 mM Tris, 192 mM glycine. Following transfer,
the membrane was blocked with 10% non-fat dry milk in PBS for 2 h at 37
°C for SCAMPs and with 5% non-fat dry milk in Tris-buffered saline,
0.1% Tween for 1 h at room temperature for Glut1 and Glut4 blotting.
Proteins were visualized with a chemiluminescent substrate kit (DuPont
NEN).Protein Content
This was determined with a BCA kit
(Pierce) according to the manufacturer's instructions.
100-120 S (Kandror and Pilch, 1994b; Kandror et
al., 1995a). So, in the next experiment (Fig. 2, A and B), we compared the sedimentation behavior of
Glut4-containing structures from normal and transgenic mice. As
expected, the total protein distribution was similar in both gradients (Fig. 2A), whereas the amount of Glut4 was much higher
in the vesicles from transgenic mice (Fig. 2, B and C). The striking result of this experiment was that the
sedimentation coefficients of Glut4-containing structures from
transgenic and control animals were similar (and the same as
Glut4-containing vesicles from rat adipocytes; data not shown) and that
these structures in both cases were very efficiently separated from the
bulk of total protein in the LM fraction. This strongly suggests that
in transgenic animals, extra synthesized Glut4 does not spill into
intracellular microsomes of a different size, but rather is
compartmentalized in specific particles similar or identical to those
from fat cells obtained from normal animals.
90%
of Glut4 from mouse LM with very low nonspecific adsorption (Fig. 4A). In agreement with previously published data,
we immunoadsorbed only 2-3% of the total protein, which is
consistent with the data illustrated in Fig. 2and proves that
Glut4-containing structures represent a specific minor population of
total LM membranes even from adipocytes of transgenic animals.
Interestingly enough, there was no Glut1 found in immunoadsorbed
material (Fig. 4B), which once again demonstrates the
existence of distinct vesicle populations for each of the transporters.
This fact suggests that adipocytes have a high fidelity sorting
mechanism that does not mix Glut1 and Glut4 even under conditions of
considerable overexpression of Glut4. More important, the intracellular
sorting of Glut4 to this distinct vesicle population is not saturated
even at 15-20-fold overexpression.
2-5% of the transporter is present on the cell surface
at any given moment of time.
)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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