Volume 24 October 1, 2013
MBoC | ARTICLE
Insulin responsiveness of glucose transporter 4 in
3T3-L1 cells depends on the presence of sortilin
Guanrong Huang*,†, Dana Buckler-Pena*, Tessa Nauta‡, Maneet Singh, Agnes Asmar, Jun Shi§,
Ju Youn Kim¶, and Konstantin V. Kandror
Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118
ABSTRACT Insulin-dependent translocation of glucose transporter 4 (Glut4) to the plasma
membrane of fat and skeletal muscle cells plays the key role in postprandial clearance of
blood glucose. Glut4 represents the major cell-specific component of the insulin-responsive
vesicles (IRVs). It is not clear, however, whether the presence of Glut4 in the IRVs is essential
for their ability to respond to insulin stimulation. We prepared two lines of 3T3-L1 cells with
low and high expression of myc7-Glut4 and studied its translocation to the plasma membrane
upon insulin stimulation, using fluorescence-assisted cell sorting and cell surface biotinylation.
In undifferentiated 3T3-L1 preadipocytes, translocation of myc7-Glut4 was low regardless of
its expression levels. Coexpression of sortilin increased targeting of myc7-Glut4 to the IRVs,
and its insulin responsiveness rose to the maximal levels observed in fully differentiated adi-
pocytes. Sortilin ectopically expressed in undifferentiated cells was translocated to the plas-
ma membrane regardless of the presence or absence of myc7-Glut4. AS160/TBC1D4 is ex-
pressed at low levels in preadipocytes but is induced in differentiation and provides an
additional mechanism for the intracellular retention and insulin-stimulated release of Glut4.
Adipocytes, skeletal muscle cells, and some neurons respond to
insulin stimulation by translocating intracellular glucose transporter
4 (Glut4) to the plasma membrane. In all these cells, the insulin-
responsive pool of Glut4 is localized in small membrane vesicles,
the insulin-responsive vesicles (IRVs; Kandror and Pilch, 2011;
Bogan, 2012). The protein composition of these vesicles has been
largely characterized (Kandror and Pilch, 2011; Bogan, 2012). The
IRVs consist predominantly of Glut4, insulin-responsive aminopep-
tidase (IRAP), sortilin, low-density-lipoprotein receptor–related pro-
tein 1 (LRP1), SCAMPs, and VAMP2. Glut4, IRAP, and sortilin physi-
cally interact with each other, which might be important for the
biogenesis of the IRVs (Shi and Kandror, 2007; Shi et al., 2008). In
addition, the IRVs compartmentalize recycling receptors, such as
the transferrin receptor and the IGF2/mannose 6-phosphate recep-
tor, although it is not clear whether these receptors represent oblig-
atory vesicular components or their presence in the IRVs is explained
by mass action (Pilch, 2008), inefficient sorting, or other reasons.
Deciphering of the protein composition of the IRVs is important
because it is likely to explain their unique functional property: trans-
location to the plasma membrane in response to insulin stimulation.
Even if we presume that IRV trafficking is controlled by loosely asso-
ciated peripheral membrane proteins, the latter should still some-
how recognize the core vesicular components that create the “bio-
chemical individuality” of this compartment. In spite of our knowledge
of the IRV protein composition, however, the identity of the protein(s)
that confer insulin sensitivity to these vesicles is unknown.
Insulin responsiveness of the IRVs was associated with either
IRAP or Glut4. Thus it was shown that Glut4 interacted with the in-
tracellular anchor TUG (Bogan et al., 2003, 2012), whereas IRAP
Thomas F. J. Martin
University of Wisconsin
Received: Oct 24, 2012
Revised: Jul 29, 2013
Accepted: Aug 2, 2013
This article was published online ahead of print in MBoC in Press (http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E12-10-0765) on August 21, 2013.
*These authors contributed equally to this study.
Present addresses: †Biogen Idec, Cambridge, MA 02142; ‡VU University Medical
Center, 1081BT Amsterdam, Netherlands; §Novartis Institutes for Biomedical
Research, Cambridge, MA 02139; ¶University of California School of Medicine,
San Diego, La Jolla, CA 92093.
Address correspondence to: K. V. Kandror (firstname.lastname@example.org).
Abbreviations used: ACAP1, ArfGAP with coiled-coil, ankyrin repeat, and PH do-
mains 1; Ad, adipocyte; AS160, Akt substrate of 160 kDa; BS, bovine serum; BSA,
bovine serum albumin; EV, empty vector; FACS, fluorescence-activated cell
sorting; Fb, fibroblast; FBS, fetal bovine serum; GFP, green fluorescent protein;
GGA, Golgi-localizing gamma-adaptin ear homology ARF-binding protein;
HRP, horseradish peroxidase; IRAP, insulin-responsive aminopeptidase; IRVs,
insulin responsive vesicles; LRP1, low-density-lipoprotein receptor–related
protein 1; SCAMP, secretory carrier membrane protein; TBC1D4, TBC1 family
member D4; TGN, trans-Golgi network; TUG, tether containing UBX domain for
Glut4; VAMP, vesicle-associated membrane protein; Vps10p, vacuolar protein
sorting 10 protein.
© 2013 Huang et al. This article is distributed by The American Society for Cell
Biology under license from the author(s). Two months after publication it is avail-
able to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
“ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of
the Cell®” are registered trademarks of The American Society of Cell Biology.
3116 | G. Huang et al. Molecular Biology of the Cell
Ectopically expressed sortilin shows insulin responsiveness
in 3T3-L1 preadipocytes
In differentiating 3T3-L1 adipocytes, sortilin and Glut4 are induced
on days 3 and 4, respectively (Shi and Kandror, 2005). Immediately
upon induction, Glut4 is incorporated in the preexisting IRVs (ElJack
et al., 1999; Shi and Kandror, 2005). Of importance, the IRVs do not
exist in undifferentiated preadipocytes but are formed on day 3 of
differentiation simultaneously with induction of sortilin (Shi and
Kandror, 2005). On the basis of these results, we hypothesized that
sortilin played the key role in the formation of insulin-responsive
vesicles in differentiating adipocytes (Shi and Kandror, 2005). To fur-
ther clarify the role of this protein, we stably expressed sortilin
tagged with myc/histidine (His) epitopes at the C-terminus in 3T3-L1
cells, which are named S cells. Note that expression of sortilin-myc/
His in undifferentiated S preadipocytes is equivalent to expression
of endogenous sortilin in differentiated adipocytes (Figure 1A).
Using sucrose gradient centrifugation, we showed that, in undiffer-
entiated preadipocytes, ectopically expressed sortilin is localized in
the IRV-like vesicles (Figure 1B; see also Shi and Kandror, 2008).
We showed that cell surface biotinylation of sortilin-myc/His in S
preadipocytes is increased by insulin (2.00 ± 0.23)-fold, suggesting
that this protein is translocated to the plasma membrane (Figure 1C).
associated with other proteins implemented in the regulation of
Glut4 translocation, such as AS160 (Larance et al., 2005; Peck et al.,
2006), p115 (Hosaka et al., 2005), tankyrase (Yeh et al., 2007), and
several others (reviewed in Bogan, 2012). Results of these studies, or
at least their interpretations, are not necessarily consistent with each
other, as the existence of multiple independent anchors for the IRVs
is, although possible, unlikely.
Ablation of the individual IRV proteins has also led to controver-
sial data. Thus knockout of IRAP decreases total protein levels of
Glut4 but does not affect its translocation in the mouse model
(Keller et al., 2002). On the contrary, knockdown of IRAP in 3T3-L1
adipocytes has a strong inhibitory effect on translocation of Glut4
(Yeh et al., 2007). In yet another study, knockdown of IRAP in 3T3-L1
adipocytes did not affect insulin-stimulated translocation of Glut4
but increased its plasma membrane content under basal conditions
(Jordens et al., 2010). By the same token, total or partial ablation of
Glut4 had various effects on expression levels, intracellular localiza-
tion, and translocation of IRAP (Jiang et al., 2001; Abel et al., 2004;
Carvalho et al., 2004; Gross et al., 2004; Yeh et al., 2007). Knock-
down of either sortilin or LRP1 decreased protein levels of Glut4 (Shi
and Kandror, 2005; Jedrychowski et al., 2010).
One model that might explain these complicated and somewhat
inconsistent results is that depletion of either major integral protein of
the IRVs disrupts the network of interactions between vesicular pro-
teins and thus decreases the efficiency of protein sorting into the IRVs
(Kandror and Pilch, 2011). Correspondingly, the remaining IRV com-
ponents that cannot be faithfully compartmentalized in the vesicles
are either degraded (Jiang et al., 2001; Keller et al., 2002; Abel et al.,
2004; Carvalho et al., 2004; Shi and Kandror, 2005; Yeh et al., 2007;
Jedrychowski et al., 2010) or mistargeted (Jiang et al., 2001; Jordens
et al., 2010), depending on experimental conditions and types of
cells used in these studies. In other words, knockdown of any major
IRV component may decrease vesicle formation along with insulin
responsiveness. Thus, in spite of a large body of literature, the iden-
tity of protein(s) that confer insulin responsiveness to the IRVs is
Here we used a gain-of-function approach to address this ques-
tion. Specifically, we attempted to “build” functional IRVs in undif-
ferentiated 3T3-L1 preadipocytes by forced expression of the
relevant proteins. Undifferentiated preadipocytes do not express
Glut4 or sortilin and lack IRVs (ElJack et al., 1999; Shi and Kandror,
2005; Shi et al., 2008). Correspondingly, IRAP, which is expressed
in these cells, shows low insulin response (Ross et al., 1998; Shi
et al., 2008). We found that ectopic expression of increasing
amounts of Glut4 in undifferentiated preadipocytes does not lead
to its marked translocation to the plasma membrane upon insulin
stimulation. On the contrary, sortilin expressed in undifferentiated
preadipocytes was localized in the IRVs and was translocated to
the plasma membrane in response to insulin stimulation. More-
over, upon coexpression with Glut4, sortilin dramatically increased
its insulin responsiveness to the levels observed in fully differenti-
ated adipocytes. Thus sortilin may represent the key component of
the IRVs, which is responsible not only for the formation of vesicles
(Shi and Kandror, 2005; Ariga et al., 2008; Hatakeyama and Kan-
zaki, 2011), but also for their insulin responsiveness. It is worth not-
ing that sortilin levels are significantly decreased in obese and dia-
betic humans and mice (Kaddai et al., 2009). We thus suggest that
sortilin may be a novel and important target in the fight against
insulin resistance and diabetes.
Our experiments also demonstrate that undifferentiated preadi-
pocytes lack a mechanism for the full intracellular retention of Glut4
that can be achieved by ectopic expression of AS160/TBC1D4.
FIGURE 1: Sortilin ectopically expressed in undifferentiated 3T3-L1
preadipocytes is localized in the IRV-like vesicles and is translocated
to the cell surface in response to insulin stimulation.
(A) Undifferentiated and differentiated 3T3-L1 cells were
homogenized, and total cell lysates (40 μg) were analyzed by Western
blotting with antibodies against sortilin. Cyclophilin A was used as a
loading control. Dotted line indicates that irrelevant lanes have been
spliced out. Representative result of three independent experiments.
(B) Undifferentiated S preadipocytes and differentiated wild-type
3T3-L1 adipocytes were homogenized and centrifuged at 27,000 × g
for 35 min, and supernatants (1 mg of total protein) were separated
by continuous sucrose gradient centrifugation. The arrow indicates
the direction of sedimentation. Representative result of at least 10
independent experiments. (C) Undifferentiated S preadipocytes were
biotinylated with sulfo-NHS-S-S-biotin, and biotinylated proteins were
isolated from 100–140 μg of total cell lysates using streptavidin-
agarose and analyzed by Western blotting along with total lysates and
unbound material (40 μg each). Representative result of four
independent experiments. (D) Undifferentiated empty vector
(EV)–infected and S preadipocytes were incubated with 7 nM [3H]
neurotensin for the indicated periods of time, washed as described in
Materials and Methods, and counted in the scintillation counter.
Normalized mean values ± SEM of three experiments.
Volume 24 October 1, 2013 Sortilin controls Glut4 translocation | 3117
undifferentiated GS preadipocytes and differentiated high-G adipo-
cytes that express sortilin endogenously (Figure 1A). Figure 3, C, D,
and H, shows that insulin response of myc7-Glut4 in GS preadipo-
cytes is even higher than in high-G adipocytes. The explanation for
this “overshoot” is not known. It might be attributed to the fact that,
in differentiated cells, myc7-Glut4 is “diluted” by endogenously ex-
pressed Glut4. At the same time, undifferentiated cells have signifi-
cantly more myc7-Glut4 at the plasma membrane under the basal
conditions than differentiated cells. Thus GS preadipocytes may
have the same amount of the IRVs as differentiated adipocytes but,
in agreement with results in Figure 3, B, C, and G, do not have a
mechanism for the efficient intracellular sequestration and/or basal
retention of Glut4. Apparently the latter mechanism is independent
of sortilin-driven biogenesis of the IRVs (see next section).
In the next experiment, we directly demonstrated that intracel-
lular sequestration of myc7-Glut4 is achieved upon differentiation of
preadipocytes. Indeed, Figure 3, C, E, and I, shows that basal GS
preadipocytes have much more myc7-Glut4 at the plasma mem-
brane than differentiated cells in spite of the fact that the latter ex-
press significantly more myc7-Glut4 (Supplemental Figure S1). The
latter observation suggests that activity of lentiviral promoters that
drive the expression of myc7-Glut4 and sortilin-myc/His is markedly
stimulated upon adipocyte differentiation.
We decided to support this result by an independent approach based
on the uptake of radioactive neurotensin. It was reported that sortilin
binds neurotensin and, in fact, may represent neurotensin receptor in
neurons (Mazella et al., 1998). We used this property of sortilin to
study its response to insulin stimulation. As is shown in Figure 1D,
internalization of radioactive neurotensin is increased by insulin in S
cells but not in control 3T3-L1 preadipocytes, suggesting that sortilin
is translocated to the plasma membrane in response to insulin.
Sortilin confers insulin responsiveness to Glut4
Insulin responsiveness of Glut4 ectopically expressed in undifferen-
tiated cells is a controversial issue. Earlier studies (Haney et al., 1991;
Hudson et al., 1992), as well as our subsequent experiments (Shi
and Kandror, 2005), showed that Glut4 expressed in undifferenti-
ated fibroblasts is not insulin responsive. McGraw’s group, however,
reported that Glut4 can undergo insulin-dependent translocation to
the cell surface even in undifferentiated cells (Kanai et al., 1993;
Lampson et al., 2000; Sadacca et al., 2013). These studies raise a
possibility that translocation of Glut4 in undifferentiated cells has
not been observed due to technical reasons, such as low levels of its
expression and/or low stability of the transporter (Shi and Kandror,
2005; Liu et al., 2007). Given that sortilin is known to stabilize Glut4
in preadipocytes (Shi and Kandror, 2005; Hatakeyama and Kanzaki,
2011), this effect alone might account for the effect of sortilin on
translocation of Glut4 in response to insulin.
To test this possibility, we used the retroviral expression system to
prepare two lines of stably transfected 3T3-L1 preadipocytes, one
with low (low G) and one with high (high G) content of Glut4 tagged
with seven consecutive myc epitopes in the first luminal loop (myc7-
Glut4). Then we stably expressed sortilin-myc/His in high-G cells with
the help of the lentivirus carrying a different selection marker. This cell
line was named GS. Figure 2A shows expression of various proteins in
all three cell lines. Note that, in agreement with our earlier report (Shi
and Kandror, 2005), expression of sortilin in high-G cells causes a small
but significant increase in the total myc7-Glut4 content (Figure 2B).
In the next experiment, we separated the total lysate of low-G,
high-G, and GS preadipocytes by centrifugation into two fractions:
a vesicular fraction (27,000 × g supernatant) and a heavy membrane
fraction (27,000 × g pellet). As shown in Figure 2C, expression of
sortilin-myc/His increases myc7-Glut4 content specifically in the ve-
sicular fraction. Then we analyzed the intracellular compartmental-
ization of myc7-Glut4 in the vesicular fraction with the help of su-
crose gradient centrifugation. Figure 2D demonstrates that the
presence of myc7-Glut4 in small vesicles is increased in GS preadi-
pocytes in comparison to high G cells. Further analysis shows that
the sedimentational properties of Glut4 vesicles formed in undiffer-
entiated cells by ectopic expression of myc7-Glut4 and sortilin-myc/
His are close to those of “classic” IRVs from fat and skeletal muscle
cells (compare Figures 2D and 1B).
Using fluorescence-assisted cell sorting, we found that myc7-
Glut4 is translocated to the cell surface in both low-G and high-G
cells, but only to a small degree (Figure 3, A, B, and F). Note that a
fourfold difference in the total content of myc7-Glut4 between these
two cell lines does not lead to corresponding changes in transloca-
tion of the transporter in these cells. Then we compared insulin re-
sponsiveness of myc7-Glut4 in high-G and GS preadipocytes that
express close amounts of the transporter. We found that the mean
insulin responsiveness of myc7-Glut4 in GS preadipocytes is mark-
edly higher than in G cells (Figure 3 B, C, and G).
To compare the maximal insulin response of GS preadipocytes
with that of differentiated adipocytes, we performed fluorescence-
activated cell sorting (FACS) analysis of myc7-Glut4 translocation in
FIGURE 2: Compartmentalization of ectopically expressed myc7-Glut4
in undifferentiated preadipocytes. (A) Undifferentiated and
differentiated cells expressing low levels of myc7-Glut4 (LG), high
levels of myc7-Glut4 (HG), or both myc7-Glut4 and sortilin-myc/His
(GS) were homogenized, and total cell lysates were analyzed by
Western blotting. Cellugyrin and glyceraldehyde 3-phosphate
dehydrogenase were used as a loading control. (B) Quantification of
myc7-Glut4 signal shown in A, based on three independent
experiments. *p < 0.05, **p < 0.01. (C) Undifferentiated LG, HG, and
GS preadipocytes were homogenized and centrifuged at 27,000 × g
for 35 min. Supernatants were pelleted by centrifugation at 200,000 ×
g for 90 min and analyzed by Western blotting along with aliquots of
27,000 × g pellets. Representative result of three independent
experiments. (D) Undifferentiated HG and GS preadipocytes were
homogenized and centrifuged at 27,000 × g for 35 min. Supernatants
were analyzed by continuous sucrose gradient centrifugation for
55 min in a SW55 rotor at 48,000 rpm. The arrow indicates direction of
sedimentation. Fractions with myc7-Glut4 from HG and GS
preadipocytes were transferred to the same membrane and processed
simultaneously and therefore are directly comparable to each other.
Representative result of at least four independent experiments.
3118 | G. Huang et al. Molecular Biology of the Cell
We supported data shown in Figure 3 by
manual analysis of cells using ImageJ soft-
ware, with essentially the same results
(Supplemental Figure S2). Note that be-
cause of differentiation-induced changes in
the shape of the cell, preadipocytes and
adipocytes have to be analyzed separately.
We decided to confirm the effect of
sortilin on translocation of Glut4 by an inde-
pendent approach, unrelated to immuno-
fluorescence staining, that is, by cell surface
biotinylation. Although wild-type Glut4 has
but one lysine in its extracellular regions and
thus cannot be efficiently biotinylated, every
myc epitope has a lysine residue, so that
myc7-Glut4 represents a good target for bi-
otinylation. As is shown in Figure 4A, cell
surface biotinylation of myc7-Glut4 in high-
G cells is not increased by insulin (1.01 ±
0.22). We believe that the sensitivity of this
approach is not sufficient to detect a rela-
tively small change in the plasma membrane
myc7-Glut4 caused by insulin administration
in high-G cells. On the contrary, biotinyla-
tion of myc7-Glut4 and sortilin-myc/His is
clearly increased by insulin in GS preadipo-
cytes (2.44 ± 0.26 and 1.95 ± 0.38, respec-
tively; Figure 4B), suggesting that both pro-
teins are translocated to the cell surface in
response to insulin stimulation.
Of importance, insulin-stimulated in-
crease in sortilin biotinylation is similar in GS
and S preadipocytes (Figure 4B). Thus Glut4
does not contribute much in terms of insulin
responsiveness of corresponding vesicles,
and the protein that renders this compart-
ment insulin sensitive could be sortilin.
Intracellular sequestration of the IRVs
during differentiation depends on the
expression of AS160/TBC1D4
As shown in Figure 3 and Supplemental
Figure S2, differentiation of high-G and GS
preadipocytes is associated with efficient
intracellular sequestration of myc7-Glut4.
Two mechanisms for such sequestration
have been described. One depends on the
expression of TUG (Bogan et al., 2003) and
another on AS160/TBC1D4 together with
its target Rab10 (Sano et al., 2003, 2007,
2008; Eguez et al., 2005; Chen et al., 2012).
As is shown in Figure 5A, all of these pro-
teins are expressed in both undifferentiated
and differentiated cells; however, AS160/
TBC1D4 is strongly induced upon differen-
tiation (Figure 5A and Supplemental Figure
S3). Low expression of AS160/TBC1D4 in
preadipocytes may explain our observation
that the intracellular retention of myc7-
Glut4 in these cells is not complete. Indeed,
a substantial amount of myc7-Glut4 in
FIGURE 3: Translocation of ectopically expressed myc7-Glut4 in undifferentiated and
differentiated 3T3-L1 cells. Undifferentiated (Fb) and differentiated for 5–7 d (Ad) cells were
treated (+) or not treated (–) with insulin for 15 min. (A–E) Immunofluorescence staining of
representative cells with anti-myc antibody. (F–I) Translocation of myc7-Glut4 analyzed by
fluorescence-assisted cell sorting as described in Materials and Methods. Each is a
representative result of at least two independent experiments.
Volume 24 October 1, 2013 Sortilin controls Glut4 translocation | 3119
endosomes and the TGN (Nielsen et al., 2001) and is abundant in
endosome-to-TGN transport vesicles (Mari et al., 2008).
To characterize the mechanism of sortilin action, we prepared
and compared four stably transfected lines of 3T3-L1 cells: with low
Glut4 expression (low-G cells), high Glut4 expression (high-G cells),
preadipocytes is localized at the plasma membrane and is seques-
tered inside the cell upon differentiation (Figure 3). To determine
whether the latter effect is associated with the induction of AS160/
TBC1D4, we overexpressed this protein in undifferentiated GS
preadipocytes. cDNA for green fluorescent protein (GFP) was used
to visualize transfected cells. Immunofluorescence staining demon-
strates that expression of AS160/TBC1D4 leads to an efficient in-
tracellular sequestration of myc7-Glut4 in preadipocytes (Figure 5,
B and C). Of importance, treatment of transfected preadipocytes
with insulin leads to phosphorylation of ectopically expressed
AS160/TBC1D4 (Figure 5C) and reconstitutes PM localization of
myc7-Glut4 (Figure 5B). In agreement with our results, Sadacca
et al. (2013) recently found that AS160/TBC1D4 increases intracel-
lular retention and insulin responsiveness of Glut4 ectopically ex-
pressed in CHO cells.
In the next experiment, we transfected preadipocytes with the
cDNA for the unphosphorylated 6-P mutant of AS160/TBC1D4,
which also provides efficient intracellular retention for myc7-Glut4 in
basal cells (Figure 5D). This protein, however, is not phosphorylated
in response to insulin stimulation (Figure 5C) and blocks the effect of
insulin on translocation of myc7-Glut4 (Figure 5D).
Sortilin and other members of the sortilin family represent multili-
gand protein receptors in mammalian cells with various functions in
protein sorting and signaling (Hermey, 2009). We (Shi and Kandror,
2005; Kim and Kandror, 2012) and others (Ariga et al., 2008;
Hatakeyama and Kanzaki, 2011) found that sortilin is essential for
the acquisition of insulin-stimulated glucose uptake in cells, al-
though the mechanism of sortilin action is uncertain. According to
one model, sortilin is directly involved in the formation of the IRVs
on the perinuclear “donor” membranes that represent a specialized
domain of the trans-Golgi network (TGN; Shewan et al., 2003), recy-
cling endosomes (Karylowski et al., 2004), or both. It is also feasible
that sortilin is involved in stabilization of Glut4 (Shi and Kandror,
2005) by, for example, facilitating its retrieval from early endosomes
to the TGN (Hatakeyama and Kanzaki, 2011). In agreement with
the latter hypothesis, it was shown that sortilin cycles between
FIGURE 4: Translocation of ectopically expressed myc7-Glut4 and
sortilin-myc/His in undifferentiated preadipocytes by cell surface
biotinylation. Undifferentiated preadipocytes were biotinylated with
sulfo-NHS-biotin, and myc-tagged proteins were isolated by
immunoprecipitation and analyzed by Western blotting.
Representative result of three independent experiments.
FIGURE 5: AS160/TBC1D4 is induced upon differentiation of 3T3-L1
cells and is required for intracellular sequestration of myc7-Glut4.
(A) Expression of proteins in undifferentiated and differentiated 3T3-L1
cells. Representative results of two independent experiments. (B) GS
preadipocytes were electroporated with either GFP cDNA alone or a
mixture of cDNAs for GFP and AS160/TBC1D4 as indicated. After
48 h, cells were treated or not treated with insulin for 15 min and
immunostained with antibody against the myc epitope without
permeabilization. Left, representative cells. Right, percentage of
myc-positive cells among randomly chosen GFP-positive cells
(35–56 cells for each field). Representative results of three experiments.
(C) GS preadipocytes were transfected with cDNA for AS160/TBC1D4
and 6-P mutant using Lipofectamine 2000. After 48 h, cells were
treated or not treated with insulin for 15 min and harvested, and total
cell lysates (40 μg) were analyzed by Western blotting. Representative
result of three independent experiments. (D) GS preadipocytes were
transfected with either GFP cDNA alone or with the mixture of cDNAs
for GFP and 6-P mutant as indicated using Lipofectamine 2000. After
48 h, cells were treated or not treated with insulin for 15 min and
immunostained with antibody against the myc epitope without
permeabilization. Left, repre sentative cells. Right, percentage of
myc-positive cells among randomly chosen GFP-positive cells.
Representative results of three experiments, with 286–299 insulin-
treated and untreated cells counted. *p < 0.05, **p < 0.01.
3120 | G. Huang et al. Molecular Biology of the Cell
Once AS160/TBC1D4 is expressed, however, it provides an addi-
tional mechanism for the intracellular retention and insulin-regulated
release of Glut4 (Figure 5B).
MATERIALS AND METHODS
Reagents and antibodies
Insulin, bovine serum albumin, and other chemicals were obtained
from Sigma-Aldrich (St. Louis, MO). Bovine serum and fetal bovine
serum (FBS) were from Atlanta Biologicals (Lawrenceville, GA).
DMEM, Opti-MEM, D-PBS, Slowfade antifade solution, and Lipo-
fectamine 2000 were purchased from Invitrogen (Carlsbad, CA).
Alexa 488–conjugated transferrin was from Molecular Probes (Carls-
bad, CA). Sulfo-NHS-biotin, sulfo-NHS-S-S-biotin, streptavidin-aga-
rose, and horseradish peroxidase–conjugated streptavidin were
from Thermo Scientific (Rockford, IL). [3,11-tyrosyl-3,5-3H(N)]-Neu-
rotensin and [3H]2-deoxyglucose were purchased from PerkinElmer
(Waltham, MA). Monoclonal and polyclonal antibodies against myc
epitope and rabbit polyclonal antibody against the phosphorylated
form of AS160 (Thr-642) were from Cell Signaling Technology (Dan-
vers, MA). Mouse monoclonal antibody against sortilin was from BD
Bioscience PharMingen (San Diego, CA). Rabbit monoclonal anti-
body against LRP1 was from Abcam (Cambridge, MA). Rabbit poly-
clonal antibody against cellugyrin (Ac-CQNVETTEGYQPPPVY-OH)
was raised and affinity purified by BioSource International (Cama-
rillo, CA; Xu and Kandror, 2002). Cy3-conjugated anti-mouse im-
munoglobulin G (IgG) was obtained from Jackson ImmunoResearch
(West Grove, PA). Rabbit polyclonal antibody against TUG was a
kind gift of Jonathan Bogan (Yale Medical School, New Haven, CT).
Chicken polyclonal antibody against the C-terminus of AS160/
TBC1D4 (HPTNDKAKAGNKP), generated by Quality Controlled
Biochemicals (Hopkinton, MA), was a kind gift of Michael Czech
(University of Massachusetts Medical School, Worcester, MA). cDNA
for Flag-tagged AS160/TBC1D4 and Flag-tagged unphosphory-
lated 6-P mutant (Sano et al., 2003) with Ser-318, Ser-341, Ser-570,
Ser-588, Thr-642, and Thr-751 replaced for Ala was a kind gift of
Gustav Lienhard (Dartmouth Medical School, Hanover, NH) and
Takahiro Nagase (Kazusa DNA Research Institute, Chiba, Japan).
Stable cell lines
Preparation, culturing, and differentiation of 3T3-L1 cells stably
transfected with mLNCX2 (EV cells), mLNCX2-sortilin-myc/His
(S cells), and pBabe-myc7-Glut4 (G cells), as well as 3T3-L1 double
transfected with pBabe-myc7-Glut4 and mLNCX2-sortilin-myc/His
(GS cells), were described previously (Shi and Kandror, 2005). Cells
were grown in DMEM containing 10% calf bovine serum. Two days
after confluence, cells were transferred to the differentiation me-
dium (DMEM with 10% FBS, 1.67 μM insulin, 1 μM dexamethasone,
and 0.5 mM 3-isobutyl-1-methylxanthine). After 48 h, differentiation
medium was replaced with DMEM containing 10% FBS.
Transient transfection of undifferentiated 3T3-L1
3T3-L1 cells were trypsinized, washed with D-PBS twice, and resus-
pended in 500 μl of electroporation buffer with 60 μg of cDNA in a
Gene Pulser cuvette with 0.4-cm electrode gap (Bio-Rad, Hercules,
CA). Electroporation was performed with a Gene Pulser MXcell
Electroporation System at 950 μF, 0.16 kV. After electroporation,
1 ml of DMEM containing 10% FBS was added to the cuvette, and
cells were left to recover for 10 min at room temperature and
replated on collagen IV–coated cover slips (Fisher Scientific,
Pittsburgh, PA). Alternatively, 3T3-L1 cells were transfected with
Lipofectamine 2000 according to the manufacturer’s instructions
cells expressing both Glut4 and sortilin (GS cells), and cells express-
ing only sortilin (S cells). We found that Glut4 has a small degree of
insulin responsiveness in undifferentiated cells (Figure 3F and Sup-
plemental Figure S3). Increase in levels of Glut4 protein per se, how-
ever, is not sufficient to confer full insulin responsiveness to the
transporter in undifferentiated preadipocytes. Indeed, a fourfold up-
regulation of myc7-Glut4 expression in high-G cells in comparison to
low-G cells results in only a minute increase in translocation of the
transporter (Figure 3, A, B, and F, and Supplemental Figure S2). At
the same time, expression of sortilin results in a relatively small in-
crease in total myc7-Glut4, whereas its insulin responsiveness is in-
creased to the maximal level observed in differentiated adipocytes
(Figure 3, B–D, G, and H). This result suggests that sortilin not only
stabilizes myc7-Glut4 but also directly facilitates formation of the
IRVs, a notion supported by the sucrose gradient analysis of Glut4-
containing vesicles in preadipocytes (Figure 2, C and D).
According to our model of IRV biogenesis, the cytoplasmic tails
target Glut4, IRAP, and sortilin to the perinuclear compartment
where the IRVs are formed. In the lumen of this compartment, the
Vps10p domain of sortilin interacts with the first luminal loop of
Glut4 and the luminal domains of IRAP and, likely, LRP1. The het-
eromeric complex consisting of the major IRV proteins is then dis-
tributed from the donor membranes to the IRV as a single entity
with the help of GGA adaptors, which bind to the cytoplasmic tail
of sortilin, and ACAP1, which interacts with the central loop of
Glut4 (reviewed in Kandror and Pilch, 2011).
On ectopic expression in undifferentiated preadipocytes that do
not express sortilin, targeting of Glut4 (this study) and IRAP (Shi
et al., 2008) to small vesicles is inefficient, and plasma membrane
translocation of these proteins is low. On the contrary, sortilin ec-
topically expressed in these cells is recovered in small, IRV-like vesi-
cles (Figure 1B), shows considerable insulin responsiveness (Figures
1, C and D, and 4B), and facilitates recruitment of both Glut4 and
IRAP into the IRVs (Figure 2D; Shi et al., 2008). We suggest, there-
fore, that sortilin not only plays the key role in vesicle biogenesis
but may also represent the long-sought IRV component responsible
for their insulin sensitivity. The mechanism of this effect is unknown.
We were unable to detect phosphorylation of sortilin in response to
insulin stimulation (G.H. and K.V.K., results not shown). We believe
that the search for sortilin-binding proteins in adipocytes may help
to find an answer to this question.
Of importance, acquisition of insulin-stimulated glucose uptake
in differentiating adipocytes requires not only formation of the IRVs,
but also efficient sequestration of glucose transporters from the
plasma membrane under basal conditions. Previous studies showed
that in the process of differentiation, 3T3-L1 cells acquire a mecha-
nism for the efficient sequestration of Glut1 (Yang et al., 1992) and
IRAP (Ross et al., 1998). Here we show that the same is true for
Glut4, as preadipocytes have significantly more myc7-Glut4 at the
plasma membrane under basal conditions than differentiated adi-
pocytes (Figure 3). We also show that complete sequestration of
myc7-Glut4 is associated with an increase in expression of AS160/
TBC1D4 (Figure 5).
Still, undifferentiated GS cells possess a significant pool of intra-
cellular IRVs (Figure 2). Thus low endogenous levels of either AS160/
TBC1D4 or TUG, which is abundant in preadipocytes, may be re-
sponsible for intracellular retention and insulin-stimulated release of
the IRVs. Of note, massive translocation of myc7-Glut4 in GS preadi-
pocytes that express low levels of AS160/TBC1D4 (Figure 3, G–I)
implies that the latter protein may not regulate the principal insulin-
sensitive event in Glut4 exocytosis, which is consistent with previous
studies (Eguez et al., 2005; Bai et al., 2007; Brewer et al., 2011).
Volume 24 October 1, 2013 Sortilin controls Glut4 translocation | 3121
quenching buffer with 1% Triton X-100, and cell lysates were incu-
bated with 15–30 μl of monoclonal anti-myc antibody or nonspecific
mouse IgG-conjugated agarose beads overnight at 4°C. Alterna-
tively, biotinylated proteins were isolated using streptavidin-aga-
rose. The beads were washed three times with 1% Triton X-100 in
quenching buffer, and elution was carried out with Laemmli sample
buffer at room temperature for 30 min.
EV or S cells were grown in 35-mm dishes. Cells were starved in se-
rum-free DMEM for 2 h and treated with 100 nM insulin or carrier
along with 7 nM [3,11-tyrosyl-3,5-3H(N)]-neurotensin for indicated
times at 37°C. Cells were then rinsed once with ice-cold PBS and
washed with ice-cold stripping buffer (0.15 M NaCl, 50 mM [2-(N-
morpholino)] ethanesulfonate, pH 5.0) for 2 min, followed by two
washes with PBS. Cells were then lysed with 400 μl of 1% SDS in
KRH buffer (Krebs Ringer HEPES buffer; 121 mM NaCl, 12 mM
HEPES, 4.9 mM KCl, 1.2 ml MgSO4, 0.33 mM CaCl2) without glu-
cose, and 300-μl aliquots were used for determination of radioactiv-
ity by liquid scintillation counter (LKB-Wallac, Bromma, Sweden).
Protein concentration was determined using the bicinchoninic acid
protein assay kit and was used to normalize counts.
Subcellular fractionation of 3T3-L1 cells
3T3-Ll adipocytes or preadipocytes were washed three times with
serum-free DMEM warmed to 37°C and starved in the same media
for 2 h. Cells were treated with 100 nM insulin or carrier (5 mM HCl
at 1000× dilution) in DMEM for 15 min at 37°C. Cells were then
washed three times with cold HES buffer (250 mM sucrose, 20 mM
HEPES, 1 mM EDTA, pH 7.4, 1 μM aprotinin, 2 μM leupeptin, 1 μM
pepstatin, 5 mM benzamidine, and 1 mM PMSF) and harvested in
the same buffer (0.3–1 ml/10-cm dish). Homogenization was
performed in a ball-bearing homogenizer (Isobiotec, Heidelberg,
Germany) with a 12-μm clearance by 10 strokes for adipocytes and
15 strokes for preadipocytes. Homogenates were centrifuged at
27,000 × g for 35 min. In some experiments, membrane vesicles in
supernatants were concentrated by pelleting at 200,000 × g for
90 min. Supernatants (or thoroughly resuspended 200,000 × g pel-
lets) were loaded onto a 4.6-ml linear 10–30% (wt/vol) sucrose gra-
dient in HES buffer without sucrose and centrifuged for 55 min in a
SW55 rotor (Beckman Coulter, Fullerton, CA) at 48,000 rpm. Each
gradient was separated into 22–26 fractions starting from the bot-
tom of the tube. The fractions were further analyzed by SDS–PAGE
and Western blotting.
Gel electrophoresis and Western blotting
Proteins were separated in SDS–polyacrylamide gels and trans-
ferred to Immobilon-P membranes (Millipore, Bedford, MA) in
25 mM Tris and 192 mM glycine. After transfer, the membrane was
blocked with 10% BSA in PBS with 0.5% Tween 20 for 1 h. Blots
were probed overnight with specific primary antibodies at 4°C, fol-
lowed by 1-h incubation at room temperature with horseradish
peroxidase–conjugated secondary antibodies (Sigma-Aldrich).
Protein bands were detected with the enhanced chemilumines-
cence substrate kit (PerkinElmer Life Sciences, Boston, MA) using a
Kodak Image Station 440CF (Eastman Kodak, Rochester, NY).
using 0.2 μg of cDNA/well of a 24-well plate or 2 μg/60-mm dish.
After 48 h, cells were analyzed by immunofluorescence and Western
Undifferentiated and differentiated 3T3-L1 cells were grown on cov-
erslips coated with collagen IV (Sigma Aldrich). Serum-starved cells
were treated with either insulin (100 nM) or carrier (5 mM HCl) for
15 min and fixed with 4% paraformaldehyde in phosphate-buffered
saline (PBS; pH 7.4) for 20 min. Fixed cells were stained overnight at
4ºC with mouse monoclonal anti-myc antibody or nonspecific
mouse IgG, followed by incubation with Cy3-conjugated anti-mouse
IgG for 1 h at room temperature. Antifade solution was used for
mounting cells on slides. Slides were examined with an Axio Ob-
server Z1 fluorescence microscope equipped with C10600/ORCA-
R2 digital camera (Hamamatsu, Hamamatsu, Japan) and AxioVision
4.8.1 (Carl Zeiss, Thornwood, NY). Quantitative analysis of immu-
nostaining was carried out with ImageJ software (National Institutes
of Health, Bethesda, MD).
Fluorescence-assisted cell sorting
Fluorescence-activated cell sorter analysis was performed as de-
scribed previously (Shi and Kandror, 2008) with minor modifications.
In brief, 3T3-L1 cells were grown in six-well plates. Before the ex-
periment, cells were incubated in serum-free DMEM for 2 h, and
insulin (100 nM) was administered for 15 min. Cells were then cooled
to 4°C and washed with cold PBS containing 0.9 mM CaCl2 and
0.5 mM MgCl2 (PBS++). All subsequent steps were carried out at
4°C. Cells were incubated with anti-myc (1:1000) antibody or non-
specific mouse IgG in PBS++ containing 5% BSA and 5% donkey
serum (1 ml/well) for 1 h and washed twice with PBS++ for 5 min each
time. Cells were then incubated with 5 μg/ml phycoerythrin-conju-
gated donkey F(ab)2 anti-mouse IgG (Jackson ImmunoResearch) for
1 h. At the end of the incubation, cells were rinsed twice with PBS++
and additionally washed three times with PBS++ for 10 min each
wash. Cells were incubated with 1 ml of 0.25% trypsin and 0.5 mg/ml
collagenase in PBS without calcium and magnesium (to decrease
cell adhesion) at 37°C for 3–5 min until most cells detached from the
plate. Detached cells were washed once by centrifugation at 300 ×
g for 4 min and resuspended in 1 ml of PBS. Immediately before
sorting, cells were passed through a 40-μm cell strainer (BD Biosci-
ences, San Jose, CA). Data were acquired using a FACScan (BD
Biosciences) maintained by the Boston University Medical Campus
Flow Cytometry Core Facility. At least 20,000 cells were counted in
each sample. Specific phycoerythrin fluorescence signal was deter-
mined by subtracting the signal from transfected cells treated under
the same conditions as the experimental group and stained with
nonspecific IgG and phycoerythrin-conjugated secondary antibody.
Mean fluorescence intensities were used for quantification.
Cell surface biotinylation
Serum-starved cells in KRP buffer (12.5 mM 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid [HEPES], 120 mM NaCl, 6 mM KCl,
1.2 mM MgSO4, 1.0 mM CaCl2, 0.6 mM Na2HPO4, 0.4 mM
NaH2PO4, 2.5 mM d-glucose, pH 7.4) were pretreated with 100 nM
insulin or carrier for 2 min at 37°C, and sulfo-NHS-biotin or sulfo-
NHS-S-S-biotin was added to cells to final concentration 0.5 mg/ml
for 15 min. The reaction was stopped by adding quenching buffer
(50 mM Tris, 10 mM EDTA, 150 mM NaCl, 1 μM aprotinin, 2 μM leu-
peptin, 1 μM pepstatin, 5 mM benzamidine, and 1 mM phenylmeth-
ylsulfonyl fluoride [PMSF], pH 7.4) for 15 min at 4°C, followed by two
washes with the same buffer. Cells were then lysed in 400 μl of
This work was supported by National Institutes of Health Research
Grants DK52057 and AG039612, Research Award 7-11-BS-76 from
the American Diabetes Association, and a Research Award from the
Allen Foundation to K.V.K.
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