Potentiation of Glucose Uptake in 3T3-L1 Adipocytes by PPAR
Agonists Is Maintained in Cells Expressing a PPAR
Dominant-Negative Mutant: Evidence for Selectivity in the Downstream Responses to PPAR
Activation
Claire Nugent,
Johannes B. Prins,
Jonathan P. Whitehead,
David Savage,
John M. Wentworth,
V. Krishna Chatterjee and
Stephen ORahilly
Departments of Clinical Biochemistry and Medicine, University of
Cambridge, Addenbrookes Hospital, Cambridge, United Kingdom, CB2
2QR
Address all correspondence and requests for reprints to: Stephen ORahilly, Department of Medicine, University of Cambridge, Addenbrookes Hospital, Cambridge, United Kingdom, CB2 2QR. E-mail:
sorahill{at}hgmp.mrc.ac.uk
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ABSTRACT
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Pharmacological agonists for the nuclear receptor PPAR
enhance
glucose disposal in a variety of insulin-resistant states in humans and
animals. The precise mechanisms whereby activation of PPAR
leads to
increased glucose uptake in metabolically active cells remain to be
determined. Notably, certain novel, synthetic PPAR
ligands appear to
antagonize thiazolidinedione-induced adipogenesis yet stimulate
cellular glucose uptake. We have explored the molecular mechanisms
underlying the enhancement of glucose uptake produced by PPAR
agonists in 3T3-L1 adipocytes. Rosiglitazone treatment for 48 h
significantly increased basal and insulin-stimulated glucose uptake and
markedly increased the cellular expression of GLUT1 but not GLUT4.
Rosiglitazone increased plasma membrane levels of GLUT1, but not GLUT4,
both basally and after insulin stimulation. Surprisingly,
adenoviral expression of a dominant-negative mutant PPAR
, which was
demonstrated to strongly inhibit adipogenesis, completely failed to
inhibit rosiglitazone-stimulated glucose uptake. Similar findings were
obtained with the non-thiazolidinedione PPAR
agonists, GW1929 and
GW7845. The insensitivity of PPAR
agonist-stimulated glucose uptake
to expression of a dominant-negative mutant, compared with the
latters marked inhibitory effects on preadipocyte differentiation,
suggests that, as is the case for other nuclear receptors, the precise
molecular mechanisms linking PPAR
activation to downstream events
may differ depending on the nature of the biological response. The
growing evidence that the effects of PPAR
on adipogenesis and
glucose uptake can be dissociated may have important implications for
the development of improved antidiabetic drug treatments.
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INTRODUCTION
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THE THIAZOLIDINEDIONE (TZD) class of
drugs was developed through empirical compound screening in rodent
models of type 2 diabetes (1). These compounds were shown
to ameliorate insulin resistance and to lower blood glucose levels,
while having no effect on insulin secretion. The mechanism of action of
the TZDs (1) was initially unknown until strong evidence
emerged in the mid-1990s to suggest that PPAR
was the molecular
target for their antidiabetic activity. The TZDs were shown to function
as selective PPAR
ligands (2, 3) whose rank order of
potency in activating PPAR
in vitro correlated closely
with their glucose lowering activity in rodents (4, 5).
More recently, further evidence has emerged to strengthen the link
between PPAR
and systemic insulin action. First, loss of function
mutations in human PPAR
(hPPAR
) have been identified which result
in extreme insulin resistance and type 2 diabetes (6).
Second, a series of non-TZD, tyrosine-derived PPAR
agonists,
optimized for potency on hPPAR
, have been described, the in
vitro activity of which closely matches their ability to reduce
glucose levels in rodent models of type 2 diabetes (7, 8).
In addition to its role in the control of insulin sensitivity,
PPAR
has also been shown to play a crucial role in adipocyte
differentiation. Retroviral expression of PPAR
in fibroblasts in the
presence of weak PPAR
activators resulted in their efficient
differentiation to adipocytes, as measured by lipid accumulation,
changes in cell morphology, and the expression of an adipocyte-specific
pattern of genes (9). Adipogenesis has since been shown to
involve a complex interplay between PPAR
and other families of
transcription factors, notably the CAAT/enhancer binding proteins
(C/EBPs) and adipocyte determination and differentiation factor 1
(ADD1)/sterol regulatory element-binding protein 1 (SREBP1)
(10). Furthermore, recent gene knockout studies
demonstrated conclusively that PPAR
is essential for adipocyte
differentiation in vivo (11, 12).
It is unclear how activation of a transcription factor that is
predominantly expressed in adipose tissue can improve insulin
sensitization and glucose utilization in muscle, the primary site of
glucose disposal. One explanation for this apparent paradox is that
PPAR
agonists may regulate the storage or secretion of
adipocyte-derived signaling factors that influence glucose metabolism
in muscle. Candidate molecules include FFA, TNF
, and leptin
(13). Alternatively, despite low levels of PPAR
expression in muscle, agonists may directly induce expression of genes
involved in glucose homeostasis in this tissue. Several studies have
demonstrated an enhancement of glucose uptake and glucose transporter
expression in adipocytes treated with TZDs, and there is evidence to
suggest that a similar effect may occur in muscle
(14).
Mukherjee et al. recently reported a novel PPAR
-specific
ligand that blocks TZD-induced adipogenesis but stimulates
insulin-mediated glucose uptake in 3T3-L1 adipocytes (15).
This finding raises the possibility that, as with other nuclear
receptors, the biological response to receptor occupation may depend on
the precise nature of the ligand involved (16, 17). Herein
we report further evidence for the dissociation of downstream
biological responses to PPAR
activation, on this occasion from the
use of genetic, rather than solely pharmacological, tools.
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RESULTS
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Effect of Rosiglitazone on Glucose Uptake in 3T3-L1 Adipocytes
Rosiglitazone-treated cells showed a trend toward increased
insulin-stimulated glucose uptake at all time points (148 h), but the
effect was only statistically significant after 48 h
supplementation (Fig. 1
). Rosiglitazone
significantly enhanced basal glucose uptake at both 24 h and
48 h with the effect being maximal at 48 h supplementation.
This enhancement of basal glucose uptake could be attributed to a
prevention of the time-dependent decrease in basal glucose uptake
observed in untreated cells rather than a potentiation of glucose
uptake per se. Nevertheless, at 48 h,
rosiglitazone-treated cells exhibited a 2.6-fold higher level of basal
glucose uptake compared with untreated cells, and insulin-stimulated
glucose uptake was enhanced by 1.7-fold (both P <
0.001). All further experiments were performed with 48 h of
rosiglitazone treatment.

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Figure 1. Effect of Rosiglitazone on Glucose Uptake
3T3-L1 adipocytes (day 79 post differentiation) were cultured
in six-well plates and supplemented with 10-7
M rosiglitazone in serum-free DMEM (1 h, 2 h, 4
h, and 6 h) or in DMEM/FBS followed by 2 h in serum-free DMEM
(24 h and 48 h). 2-Deoxyglucose uptake was measured over 5 min
following stimulation ± insulin for 30 min. Data are mean
uptakes ± SE from four or more independent
experiments performed in triplicate, normalized to vehicle
insulin-stimulated uptake at each time point (mean insulin responses,
16, 800 dpm/well). Open columns, Vehicle; filled
columns, rosiglitazone.
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Effect of Rosiglitazone on the Expression and Cellular Localization
of Glucose Transporters
Rosiglitazone increased total cellular levels of GLUT1 by 1.5-fold
(P < 0.001) but had no discernible effect on levels of
GLUT4 (Fig. 2A
). Plasma membrane lawns
from disrupted cells were probed with anti-GLUT1 or GLUT4 antibodies
followed by immunofluorescence detection (Fig. 2B
). Consistent with
previously published data (18), insulin treatment of
3T3-L1 adipocytes produced a 2-fold increase in GLUT1 and a 4.5-fold
increase in GLUT4 at the plasma membrane. Rosiglitazone increased
levels of GLUT1 at the plasma membrane in the absence and presence of
insulin by 1.8-fold and 1.3-fold, respectively (both P
< 0.001). Rosiglitazone had no effect on levels of GLUT4 at the plasma
membrane.

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Figure 2. Effect of Rosiglitazone on Glucose Transporters
3T3-L1 adipocytes (day 7 post differentiation) were supplemented
with 10-7 M rosiglitazone in DMEM/FBS for
48 h. A, Western blotting. Cells were lysed as described in
Materials and Methods. Forty micrograms were resolved by
SDS-PAGE and immunoblotted with anti-GLUT1 or GLUT4 antibody. A
representative gel is shown for each data set. Numerical data are
percentage means ± SE obtained by quantitation of
gels from eight independent experiments, normalized to transporter
levels in vehicle-supplemented cells. Open columns,
Vehicle; filled columns, rosiglitazone. B, Plasma
membrane lawn assay. Cells were serum starved in DMEM for 2 h and
stimulated with and without insulin for 30 min before preparation of
plasma membrane lawns and assay of glucose transporter translocation.
Representative images from a typical experiment are shown. Data from
each experiment, utilizing 16 fields for each condition, were
quantified as described in Materials and Methods, and
overall results are shown as means ± SE from five
independent experiments. Open columns, Unstimulated;
filled columns, 10 nM insulin.
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Transduction of 3T3-L1 Preadipocytes with a Dominant-Negative
PPAR
Mutant
A compound hPPAR
1 mutant has previously been described in which
the highly conserved hydrophobic and charged residues, Leu
(468) and Glu (471), in helix 12 of the
ligand-binding domain were mutated to alanine (19). This
mutant receptor retains ligand and DNA binding, but exhibits reduced
transactivation due to impaired coactivator recruitment. In addition,
it silences basal transcription, recruits corepressors more avidly than
wild-type PPAR
, and exhibits delayed ligand-dependent corepressor
release. The L468A/E471A hPPAR
1 mutant is also a potent
dominant-negative inhibitor of wild-type PPAR
action, markedly
inhibiting the effects of TZDs on human preadipocyte differentiation
and aP2 expression (19). We first established that
Ad
m [an adenovirus expressing the PPAR
1
mutant receptor and green fluorescent protein (GFP)] could infect the
3T3-L1 cell line, resulting in a similar marked inhibition of
differentiation induced by the standard differentiation cocktail (Fig. 3A
). Infection with Ad-GFP (an adenovirus
expressing GFP alone) had no effect on 3T3-L1 differentiation.
Ad
m infection of the 3T3-L1 cell line resulted
in a marked increase in levels of PPAR
1 expression (Fig. 3B
).
Infection with Ad-GFP had no effect on the endogenous levels of PPAR
(data not shown). Expression of the dominant-negative PPAR
in 3T3-L1
preadipocytes significantly inhibited the induction of the
differentiation marker, aP2, by rosiglitazone (P <
0.05 compared with both Ad-GFP and nil virus controls) (Fig. 3C
).
Furthermore, Ad
m significantly inhibited
adipocyte differentiation in response to the conventional
differentiation cocktail (P < 0.05 compared with both
Ad-GFP and nil virus controls) (Fig. 3D
). Thus, these studies provide
evidence that the human L468A/E471A PPAR
mutant receptor can block
endogenous PPAR
activity in a murine preadipocyte cell line.

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Figure 3. Transduction of 3T3-L1 Preadipocytes with
Dominant-Negative PPAR
Confluent preadipocytes in 24-well plates were infected for 12 h
with 1 x 109 plaque forming units (pfu)/well Ad-GFP
or Ad m. A, Microscopy. Cells were differentiated as
described in Materials and Methods for 7 d, fixed
with 0.5% glutaraldehyde, and stained with Oil Red O. Representative
images from a typical experiment are shown. Left panel,
Macroscopic view of individual wells of 24-well plate; right
panel, microscopic view of same cells. BD, Medium was changed
to DMEM/FBS with differentiation mix or DMEM/NBCS ±
10-7 M rosiglitazone for 48 h. B, Western
blotting. Infected preadipocytes (minus rosiglitazone) and day 2
adipocytes were scraped and lyzed as described in Materials and
Methods. Ten micrograms of total protein were resolved by
SDS-PAGE and immunoblotted with anti-PPAR antibody. C, aP2 induction
by rosiglitazone in preadipocytes. Infected preadipocytes ±
10-7 M rosiglitazone were lyzed and RNA
extracted and reverse transcribed. aP2 gene expression was quantified
using real time quantitative PCR as described in Materials and
Methods. Data are mean percentage rosiglitazone induction of
aP2 ± SE D, aP2 induction during differentiation.
Infected preadipocytes (minus rosiglitazone) and day 2 adipocytes were
lysed and RNA extracted and reverse transcribed. aP2 gene expression
was quantified using real time quantitative PCR. Data are mean
percentage induction of aP2 during differentiation ±
SE. For experiments C and D, data are from four independent
experiments performed in triplicate, normalized to the endogenous
control, glyceraldehyde-3-phosphate dehydrogenase.
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Effect of Dominant-Negative PPAR
on the Potentiation of Glucose
Uptake by Rosiglitazone
To determine whether fully differentiated 3T3-L1 adipocytes could
also be successfully infected, cells were incubated overnight with
Ad-GFP or Ad
m. Infectivity was assessed
48 h later by fluorescence microscopy and Western blotting with
anti-PPAR
antibody (Fig. 4A
).
Comparable infection efficiencies of 7090% were observed for both
viruses, and Ad
m-infected cells exhibited an
increased PPAR
1 protein expression compared with Ad-GFP-infected and
uninfected cells. Again, adenoviral infection had no effect on
endogenous levels of PPAR
. To assess the effect of the
Ad
m on the potentiation of glucose uptake by
rosiglitazone, cells were infected overnight and the 48 h
incubation with rosiglitazone was subsequently commenced. Rosiglitazone
was observed to enhance basal glucose uptake in the Ad-GFP-infected
cells and there was also a trend for it to increase insulin-stimulated
glucose uptake. Contrary to expectations, Ad
m
did not inhibit this rosiglitazone-mediated potentiation of either
basal or insulin-stimulated glucose uptake (Fig. 4B
). It is noteworthy
that there was a trend for Ad
m to reduce basal
and insulin-stimulated glucose uptake in the absence of rosiglitazone,
while maintaining a similar fold insulin stimulation over basal, and to
reduce insulin-stimulated glucose uptake in the presence of
rosiglitazone. Nevertheless, Ad
m-infected
cells treated with rosiglitazone still exhibited an enhanced basal and
insulin-stimulated glucose uptake compared with untreated cells.

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Figure 4. Effect of Dominant-Negative PPAR on the
Potentiation of Glucose Uptake in 3T3-L1 Adipocytes by Rosiglitazone
3T3-L1 adipocytes (day 7 post differentiation) cultured in
24-well plates were infected for 12 h with 1 x
109 pfu/well Ad-GFP or Ad m. A, Microscopy
and Western blotting. Infection efficiency was estimated using
fluorescence microscopy. Images from a typical experiment are shown.
Images represent an infection efficiency of approximately 90% for both
viruses, with individual infected cells demonstrating variable
fluorescence intensities relating to variable levels of GFP expression.
Infected adipocytes were scraped and lysed as described in
Materials and Methods. Ten micrograms of total protein
were resolved by SDS-PAGE and immunoblotted with anti-PPAR antibody.
A representative gel is shown. B, Glucose uptake. Infected adipocytes
were supplemented with 10-7 M rosiglitazone in
DMEM/FBS for 48 h, serum starved in DMEM for 2 h, and
stimulated with and without 10 nM insulin for 30 min.
2-Deoxyglucose uptake was measured over 5 min. Data are mean
2-deoxyglucose uptake ± SE from four independent
experiments performed in triplicate, normalized to nil virus, vehicle
insulin-stimulated uptake (mean insulin responses, 8,200 dpm/well).
Open columns, Vehicle; filled columns,
rosiglitazone.
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Effect of Non-TZD PPAR
Agonists on Glucose Uptake
A novel class of tyrosine-derived, non-TZD, PPAR
ligands has
recently been developed (7). Two such ligands, optimized
for potency on human (h) PPAR
, are GW1929 and GW7845 (8, 20). GW1929 has been shown to be 2 orders of magnitude more
potent than the TZD, troglitazone, in both in
vitro and in vivo assays (8). It both
lowered plasma glucose levels in Zucker diabetic fatty rats and
transactivated hPPAR
and mouse PPAR
in reporter gene assays more
potently than troglitazone. Similarly, GW7845 was shown to
be significantly more potent than either rosiglitazone or
troglitazone when assayed for induction of adipogenesis in
the 3T3-L1 cell line (20). 3T3-L1 adipocytes were
supplemented for 48 h with 10-7
M rosiglitazone, GW1929, or GW7845 and glucose
uptake was measured (Fig. 5
). All three
compounds had comparable effects on glucose uptake, increasing basal
uptake by between 3- and 4-fold (P < 0.001) and
insulin-stimulated uptake by approximately 1.6-fold (P
< 0.01). Having established that the non-TZD PPAR
agonists act to
increase glucose uptake to a similar extent to the TZD rosiglitazone,
we investigated the effect of the dominant-negative PPAR
mutant in
this system. As before, adipocytes were infected with Ad-GFP or
Ad
m overnight and subsequently supplemented
with the GW compounds for 48 h. As with rosiglitazone, the
tendency of the GW compounds to increase basal and insulin-stimulated
glucose uptake was similar in Ad-GFP and
Ad
m-infected cells (Fig. 6
). In contrast to the lack of effect of
Ad
m on ligand-stimulated glucose uptake, the
mutant PPAR
inhibited the effects of these ligands on GLUT1
expression (Fig. 7
).

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Figure 5. Effect of non-TZD PPAR Agonists on Glucose
Uptake
3T3-L1adipocytes (day 7 post differentiation) cultured in six-well
plates were supplemented with 10-7 M agonist
in DMEM/FBS for 48 h and serum-starved in DMEM for 2 h.
2-Deoxyglucose uptake was measured over 5 min following
stimulation ± 10 nM insulin for 30 min. Data are mean
uptakes ± SE from six or more independent experiments
performed in triplicate, normalized to vehicle insulin-stimulated
uptake (mean insulin responses 31, 500 dpm/well). Open
columns, Unstimulated; filled columns, 10
nM insulin.
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Figure 6. Effect of Dominant-Negative PPAR on the
Potentiation Of Glucose Uptake in 3T3-L1 Adipocytes by GW1929 and
GW7845
3T3-L1adipocytes (day 7 post differentiation) cultured in 24-well
plates were infected for 12 h with 1 x 109
pfu/well Ad-GFP or Ad m. Cells were supplemented with
10-7 M agonist in DMEM/FBS for 48 h,
serum-starved in DMEM for 2 h, and stimulated ± 10
nM insulin for 30 min. 2-Deoxyglucose uptake was measured
over 5 min. Data are mean 2-deoxyglucose uptake ±
SE from two independent experiments performed in
triplicate, normalized to nil virus, vehicle insulin-stimulated uptake
(mean insulin responses, 11,360 dpm/well). Open columns,
Vehicle; filled columns, agonist.
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DISCUSSION
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TZDs are now widely used for the treatment of type 2 diabetes.
Despite this, the precise mechanisms whereby TZDs act to improve
insulin sensitivity and glucose disposal in vivo are
unclear. In the studies reported above we demonstrate that 48 h of
rosiglitazone treatment increases basal and insulin-stimulated
glucose uptake and increases cellular expression of GLUT1 in
3T3-L1 adipocytes. Furthermore, we present new information
regarding the effects of this compound on the translocation of glucose
transporters to the plasma membrane. After recent pharmacological data
that the effects of PPAR
activation on adipogenesis and glucose
uptake can be dissociated (15), we now present evidence
that a dominant-negative PPAR
mutant has inhibitory effects on
ligand-stimulated adipogenesis while having no effects on
ligand-stimulated glucose uptake. Thus, a body of data is accumulating
to suggest that, as is well established with other well studied nuclear
receptors (17, 16), selective modulation of the downstream
biological responses to PPAR
activation may be possible.
Several in vivo studies have demonstrated an increase in
adipose tissue glucose transport activity and GLUT4 expression after
treatment with TZDs (14). Studies in the 3T3-L1 and
3T3-F442A cell lines have similarly reported TZD-induced increases in
glucose transport, although effects on glucose transporters appear to
be dependent on the differentiation state (21, 22, 23, 24, 25, 26). Hence,
increases in both GLUT1 and GLUT4 expression have been reported in
cells supplemented with TZDs during the differentiation process
(21, 22, 23), whereas only GLUT1 expression has been shown to
be increased in fully differentiated cells (26). In our
study, differentiated cells were used to eliminate confounding effects
of rosiglitazone on the adipogenic process itself. In agreement with
previous TZD studies (24, 25, 26), we demonstrated an increase
in basal glucose uptake and cellular GLUT1 expression, with no effect
on GLUT4 expression, following rosiglitazone treatment of 3T3-L1
adipocytes. In addition to the effects of rosiglitazone on the total
cellular expression of GLUT1, we show, for the first time, that this
agent increases plasma membrane levels of GLUT1 in both the basal and
insulin-stimulated state while having no effects on GLUT4 cellular
localization. The marked effects of rosiglitazone on glucose uptake,
GLUT1 expression, and GLUT1 translocation occurring in the absence of
insulin suggests that the frequently used term "insulin sensitizer"
may not fully reflect the molecular mechanisms underlying the effects
of this compound on glucose disposal.
The in vivo importance of PPAR
in the regulation of
glucose disposal and insulin sensitivity in the whole organism is
becoming increasingly apparent. In addition to the large body of
evidence derived from the efficacy of TZD and non-TZD PPAR
agonists
in insulin-resistant and diabetic states in rodents, primates, and
humans (1, 7, 8), genetic studies have recently provided
compelling evidence for a direct link between PPAR
and systemic
insulin action. Thus, two independent naturally occurring
dominant-negative mutations in hPPAR
have been reported in subjects
with severe insulin resistance and type 2 diabetes (6).
PPAR
gene knockout studies in mice, however, were unable to
substantiate this result due to embryonic lethality (11, 12). Surprisingly, mice heterozygous for PPAR
deficiency
exhibited an improved insulin sensitivity and were protected from
high-fat diet-induced insulin resistance (27, 28).
While direct effects of TZDs on glucose uptake have been demonstrated
in cell lines, it is still unclear whether their in vivo
effects on glucose disposal can be attributed to direct or indirect
mechanisms. Thus, TZDs have effects on suppression of the
adipocyte-derived signaling molecules, TNF
and leptin, and on FFA
partitioning between adipose and muscle tissues (29, 30),
all of which may secondarily improve whole organism insulin
sensitivity.
To investigate whether the potentiating effects of rosiglitazone on
glucose uptake in 3T3-L1 adipocytes were mediated via PPAR
, the
experiment was repeated in cells expressing a dominant-negative
hPPAR
mutant receptor. This mutant form of hPPAR
has
previously been shown to powerfully inhibit cotransfected wild-type
receptor action and to inhibit the TZD-induced differentiation of human
preadipocytes (19). We demonstrated that the
transduced hPPAR
mutant receptor was highly expressed in murine
3T3-L1 preadipocytes and adipocytes and that it inhibited 3T3-L1
differentiation induced by both the standard differentiation cocktail
and rosiglitazone. However, there was no discernible effect on
rosiglitazone-induced enhancement of glucose uptake, either basal or
insulin-stimulated, in cells transduced with the mutant receptor. There
have been previous suggestions that TZDs, particularly
troglitazone (31), may have
PPAR
-independent metabolic effects. However, the observation that
the potentiating effects of two structurally distinct, non-TZD PPAR
agonists on glucose uptake were also completely unaffected by the
mutant receptor, would imply that a PPAR
-independent effect on
glucose uptake by all three compounds is unlikely. The ability of
Ad
m to block the PPAR
agonist-induced
increases in total cellular levels of GLUT1 implies that the
potentiation of glucose uptake is mediated via a mechanism independent
of the increase in total cellular GLUT1 protein. Such mechanisms might
include increasing the translocation of GLUT1 to the plasma membrane,
as described above, and/or an increase in the intrinsic activity of the
glucose transporters.
There are several potential explanations to account for the
insensitivity of the PPAR
agonist-induced potentiation of glucose
uptake to the presence of the mutant receptor. A failure to express the
mutant adequately is possible, but we demonstrated that the adenoviral
expression system consistently led to more than 70% infectivity in
preadipocytes and adipocytes, while Western blotting indicated a more
than 30-fold increase of PPAR
expression in the transduced cells.
Another possibility is that PPAR
ligands may exert their effects on
glucose uptake via a nongenomic mechanism, something that has been
established to occur with ligands for other nuclear hormone receptors
(32). However, our time course studies indicated that
rosiglitazone had its maximal effects after 48 h of exposure, a
finding much more consistent with a transcriptional mechanism than with
the expected rapid nature of the nongenomic responses to steroids and
other lipophilic ligands (32). While we did not undertake
formal tests of the dependence of the enhancement of glucose uptake on
new protein synthesis, Kreutter et al. (25)
reported previously that the enhancement of basal glucose uptake by CP
68722 (racemic englitazone) in 3T3-L1 adipocytes can be inhibited by
cycloheximide.
Perhaps the most compelling model to explain our data relates to the
fact that the occupancy of nuclear receptors by ligands may lead to the
activation of selective sets of downstream biological responses that
depend on the precise nature of the activating ligand. Thus, tamoxifen
and raloxifene, both highaffinity agonists for the ER, mimic the
natural ligand in preventing postmenopausal osteoporosis but inhibit
the estrogen-dependent proliferation of breast carcinomas
(17). The possibility that such selective receptor
modulation might occur with PPAR
was recently reported by Mukherjee
et al. (15). A synthetic high-affinity PPAR
agonist, LG100641, was shown to block both TZD-induced differentiation
and target gene activation and repression in 3T3-L1 cells, yet it also
enhanced basal and insulin-stimulated glucose uptake in fully mature
cells.
Selective receptor modulation is thought to relate to the fact that the
pattern of receptor interaction with the complex of coactivator and
corepressor proteins is dependent on the precise conformation of the
particular ligand-receptor complex (16). Compared with
other nuclear receptors, PPAR
has a particularly large
ligand-binding pocket, and there is already strong evidence that
different ligands make distinct structural contacts with receptor
(33, 34). Thus the a priori possibility of
selective receptor modulation is particularly strong for PPAR
. The
growing evidence for the dissociability of biological responses
downstream from nuclear hormone receptors has come largely from
pharmacological rather than genetic studies. Our studies suggest that a
mutant form of PPAR
, which acts as powerful dominant-negative
repressor of adipogenesis, may have no effect on the stimulation of
glucose uptake, providing further support for the idea that the
repertoire of coactivators and corepressors involved in the promotion
of adipogenesis and glucose uptake may be distinct. The existing
structural model of PPAR
would predict that mutating both L468 and
E471 would grossly interfere with the interaction of the AF2 helix with
both ligand and coactivator (19, 35). This suggests that
other areas of the PPAR
molecule, e.g. the N-terminal
activation domain, may be more intimately involved with transcriptional
responses relevant to glucose uptake. Indeed, the coactivators PGC1 and
PGC2 are not thought to require the AF2 helix for binding to PPAR
(36, 37).
In summary, we have demonstrated that PPAR
agonists potentiate basal
and insulin-stimulated glucose uptake in 3T3-L1 adipocytes and increase
total cellular and plasma membrane expression of GLUT1. The
potentiation of glucose uptake is maintained in cells expressing a
PPAR
mutant that inhibits adipogenesis, thus providing further
evidence for selectivity in the downstream responses to PPAR
activation. If such selective modulation of receptor function could be
achieved with PPAR
, drugs might be developed that could improve
insulin sensitivity without promoting fat accumulation. In this regard,
a PPAR
agonist, GW0072, which lowers plasma insulin and
triglycerides in insulin-resistant Zucker rats without causing weight
gain, has recently been described (T. M. Willson, personal
communication).
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MATERIALS AND METHODS
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Materials
-2-Deoxy-D-[2,6-3H]glucose
and the enhanced chemiluminescence kit were purchased from
Amersham Pharmacia Biotech (Piscataway, NJ).
Rosiglitazone was provided by Dr. S. Smith (SmithKline Beecham Pharmaceuticals). GW-1929 and GW-7845 were a
gift from Dr. T. Willson (GlaxoSmithKline). Rabbit anti-GLUT4
antibody was a gift from Professor G. Gould (University of Glasgow,
Glasgow, UK), rabbit anti-GLUT1 was a gift from Professor S. Baldwin
(University of Leeds, Leeds, UK), and rabbit anti-PPAR
was a gift
from Dr. M. Lazar (University of Pennsylvania School of Medicine,
Philadelphia, PA). The RNeasy total RNA kit was from
QIAGEN (Valencia, CA). Reverse transcription reagents
were obtained from Promega Corp. (Madison, WI) and TaqMan
reagents were from PE Applied Biosystems (Foster City,
CA). All other reagents were from Sigma (St. Louis,
MO).
Tissue Culture
3T3-L1 fibroblasts (ATCC, Manassas, VA) were
maintained at no higher than 70% confluence in DMEM containing 10%
newborn calf serum (NBCS), 25 mM glucose, 2 mM
glutamine, and antibiotics (DMEM/NBCS). For differentiation they were
grown 2 d post confluence in DMEM/NBCS and then for 2 d in
medium containing FBS (DMEM/FBS) supplemented with 0.83
µM insulin, 0.25 µM dexamethasone, and 0.5
mM isobutylmethylxanthine. The medium was then changed to
DMEM/FBS supplemented only with 0.83 µM insulin for
2 d and then to DMEM/FBS alone for an additional 35 d.
Differentiated cells were only used when at least 95% of the cells
showed an adipocyte phenotype by accumulation of lipid droplets.
PPAR
Agonist Solutions
Solutions (10-4 M) of
rosiglitazone, GW1929, and GW7845 were prepared in dimethylsulfoxide.
These were diluted 1:1,000 into serum-free DMEM for studies requiring
an incubation time
6 h, or into DMEM/FBS for studies with an
incubation time
6 h, to give a final concentration of
10-7 M.
Glucose Uptake
Adipocytes (at day 79 after initiation of differentiation) in
6 (or 24)-well plates were incubated in medium containing
10-7 M PPAR
agonist for the
specified length of time. In the time course experiment, rosiglitazone
incubations were terminated simultaneously on day 9. Cells that had
been supplemented for 24 h and 48 h were serum starved in
DMEM for 2 h before the assay. Glucose uptake assays were
performed as described previously (18).
Western Blotting
Treated cells (adipocytes/preadipocytes) were solubilized by
scraping and passing 10 times through a 25G needle in lysis buffer as
described previously (18). The lysate was clarified by
centrifugation at 13,500 x g for 10 min at 4 C. Crude
cell extracts were resolved by SDS-PAGE before electroblotting to
polyvinylidene difluoride membranes (Millipore Corp.,
Bedford, MA). Membranes were blocked in 1% BSA, and specific proteins
were detected by incubation with appropriate primary and secondary
(horseradish peroxidase-conjugated) antibodies in 150
mM NaCl, 50 mM Tris, 0.1%
Tween 20. Proteins were then visualized using an enhanced
chemiluminescence kit.
Plasma Membrane Lawn Assay
3T3-L1 adipocytes (day 7), grown on collagen-coated glass
coverslips, were treated for 48 h with 10-7
M rosiglitazone in DMEM/FBS. Cells were serum
starved for 2 h and incubated with and without 10 nM
insulin for 30 min, and a modified version of the plasma membrane lawn
assay (18) was performed. Cells were washed twice in
ice-cold buffer A (50 mM HEPES, 10 mM NaCl, pH
7.2), twice in ice-cold buffer B (20 mM HEPES, 10
mM KCl, 2 mM CaCl2, 1
mM MgCl2, pH 7.2), and sonicated
using a probe sonicator (Kontes, Vineland, NJ) to generate a lawn of
plasma membrane fragments attached to the coverslip. The membranes were
washed twice again in ice-cold buffer B and fixed to the coverslips for
15 min using freshly prepared 3% paraformaldehyde. Membranes were then
serially washed: x3 PBS, x3 50 mM
NH4Cl in PBS over 10 min, x3 PBS, x3
PBS-gelatin (PBS containing 0.2% gelatin and 1 µl/ml goat serum)
over 5 min, and finally x3 PBS. Membranes were incubated in either
anti-GLUT4 or anti-GLUT1 antibody (1:100 dilution in PBS-gelatin) for
1 h at room temperature. After washing x3 PBS-gelatin and x3
PBS, the coverslips were incubated with the secondary antibody,
fluorescein isothiocyanate-conjugated donkey antirabbit IgG, for 1
h at room temperature, washed x3 PBS-gelatin and x3 PBS and mounted
on glass slides. Coverslips were viewed using a 60x objective lens on
a Optiphot-2/Biorad MRC-1000 microscope (Nikon, Melville,
NY) operated in laser scanning confocal mode. Samples were illuminated
at 488 nm, and images were collected at 510 nm. Duplicate coverslips
were prepared at each experimental condition, and eight random images
of plasma membrane lawn were collected from each. The images were
quantified using MRC-1000 confocal microscope operating software
[CoMOS, version 6.05.8 (Bio-Rad Laboratories, Inc.,
Hercules, CA)], on an AST premmia SE P/60 personal
computer.
Adenovirus Expression
Recombinant adenoviruses were generated as described previously
(19), expressing GFP (Ad-GFP) or GFP and full-length
L468A/E471A hPPAR
1 (Ad
m). 3T3-L1
preadipocyte (2 d post confluence) or day 7 adipocyte cultures in
24-well plates were infected with recombinant virus by addition of
1 x 109 plaque-forming units/well. Twelve
hours later medium containing free virus was removed, and appropriate
experimental medium was added. Comparable viral infection efficiency
was verified by microscopy using an axiovert 135 inverted fluorescence
microscope (Carl Zeiss, Thornwood, NY). Only cells with
more than 70% infectivity were used in experiments.
Oil Red O Staining
3T3-L1 adipocytes were fixed with 0.5% glutaraldehyde and
stained with Oil Red O for visualization of lipid droplets, according
to conventional methods (38). Differentiation efficiency
was assessed macroscopically and microscopically using a
Nikon Eclipse TE300 inverted microscope.
RNA Extraction/Quantitative RT-PCR
Virally infected preadipocytes/day 2 adipocytes were scraped and
total RNA was extracted using the RNeasy mini kit from
QIAGEN. Adipocyte P2 (aP2) gene expression was then
quantified using real time quantitative RT-PCR. Briefly, cDNA was
prepared from 100 ng of RNA using 200 U Moloney-murine leukaemia virus
reverse transcriptase (Promega Corp.). Real time
quantitative PCR was performed using an ABI-PRISM 7700 Sequence
Detection System instrument and software (PE Applied Biosystems, Inc., Foster City, CA) as described previously
(39). The primers and probes for aP2 were as follows:
Forward, CACCGCAGACGACAGGAAG; Reverse, GCACCTGCACCAGGG; Probe,
TGAAGAGCATCAAACCCTAGATGGCGG (all 5'-3'). Results were normalized to the
endogenous control, glyceraldehyde-3-phosphate dehydrogenase.
Statistical Analysis
Data are presented as mean ± SE. Statistical
significance of treatments was determined using the paired t
test (*, P < 0.05; **, P < 0.01; ***,
P < 0.001).
 |
FOOTNOTES
|
---|
We would like to thank Dr. Tim Wilson (Glaxo SmithKline) for
his kind gift of GW1929 and GW7845.
C.N. was the recipient of a studentship from Diabetes UK. S.O.R.,
J.B.P., J.P.W., J.M.W., D.S., and V.K.K.C. are supported by the
Wellcome Trust.
Abbreviations: Ad-GFP, An adenovirus expressing GFP alone;
Ad
m, adenovirus expressing the PPAR
1 mutant receptor
and GFP; aP2, adipocyte P2; GFP, green fluorescent protein; NBCS,
newborn calf serum; TZD, thiazolidinedione.
Received for publication January 8, 2001.
Accepted for publication June 28, 2001.
 |
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