From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118
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ABSTRACT |
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Adipocyte differentiation is regulated by at
least two major transcription factors, CCAAT/enhancer-binding protein
The ability of metabolically active cells to respond to insulin is
central to the proper regulation of lipid and glucose metabolism in the
body. Compromised insulin sensitivity or insulin resistance results in
impairment of glucose tolerance, and in some cases, to type 2 diabetes
mellitus (1). In cardiac muscle, skeletal muscle, and adipose tissue,
insulin stimulates glucose transport by inducing the rapid
relocalization of glucose transporter proteins from intracellular pools
to the cell surface (2, 3).
In adipose tissue, the ability of cells to respond to insulin is
acquired during their differentiation into mature adipocytes (4). Fully
differentiated adipocytes express proteins involved in fatty acid
binding, lipogenesis and lipolysis, hormone action and signaling, and
insulin-sensitive glucose uptake (4). Transcription of the genes for
these proteins is regulated in a coordinate manner by at least two
families of transcription factors, CCAAT/enhancer-binding proteins
(C/EBPs)1 (5-11) and
peroxisome proliferator-activated receptors (PPARs) (12-15).
PPAR Of the C/EBP family members, C/EBP Recent studies have measured adipose differentiation by Oil Red O
staining of intracellular triglyceride, changes in morphology, and the
expression of fat-specific or fat-enriched proteins, such as aP2 and
adipsin (22), whereas the development of insulin-responsive glucose
uptake, a characteristic of the mature adipocyte, has not been
extensively monitored. Previously, we have demonstrated that activation
of endogenous PPAR Materials--
Materials were obtained from the following
sources: dexamethasone, 3-isobutyl-1-methylxanthine, insulin,
puromycin, benzamidine, Oil Red O, and digitonin from Sigma; aprotinin,
leupeptin, pepstatin A, and phenylmethylsulfonyl fluoride from American
Bioanalytical (Natick, MA); Dulbecco's modified Eagle's medium (DMEM)
and fetal bovine serum from Life Technologies, Inc.;
[ Antibodies--
Monoclonal anti-GLUT4 antibody 1F8 (25),
polyclonal antibodies against GLUT1 (a kind gift from Dr. C. Carter-Su)
(26), PI3-kinase (Upstate Biotechnology, Lake Placid, NY), C/EBP Plasmids and Stable Cell Lines--
The BOSC 23 packaging cells
(28), pBabe-Puro, pBabe-PPAR Cell Culture--
Murine 3T3-L1 preadipocytes were cultured,
maintained, and differentiated as described previously (29). Briefly,
cells were plated and grown for 2 days post-confluence in DMEM
supplemented with 10% calf serum. Differentiation was then induced
(day 0) by changing the medium to DMEM containing 10% fetal bovine
serum, 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM dexamethasone, and 1.7 µM insulin. After
48 h, cells were maintained in DMEM containing 10% fetal bovine
serum. NIH-3T3, Swiss-3T3, and BALB/c-3T3 cells expressing either
retroviral PPAR Oil Red O Staining--
Oil Red O staining was performed
following the procedure described by Green and Kehinde (30) with minor
modifications. The cells were then photographed using Hoffman
optics microscopy.
3H-2-Deoxyglucose Uptake--
This assay was
performed in 3.5-cm dishes as described previously (31) with minor
modifications (29). Measurements were made in duplicate and corrected
for specific activity and nonspecific diffusion (as determined in the
presence of 5 µM cytochalasin B), which was less than
10% of the total uptake. The protein concentration was determined
using the Bio-Rad protein assay kit and was used to normalize counts.
Preparation of Whole Cell Extracts--
At the indicated
times, cultured cells grown in 10-cm dishes were rinsed with
phosphate-buffered saline and then harvested in ice-cold buffer
containing 50 mM Tris (pH 7.4), 100 mM NaCl, 1% sodium deoxycholate, 4% Nonidet P-40, 0.4% SDS, 1 mM
phenylmethylsulfonyl fluoride, 5 µM pepstatin, 5 µM aprotinin, and 50 µM leupeptin. Lysates
were vortexed and stored at Gel Electrophoresis and Immunoblotting--
Proteins were
separated in SDS-polyacrylamide (acrylamide from National Diagnostics)
gels as described by Laemmli (32) and transferred to polyvinylidene
difluoride membrane (Bio-Rad) in 25 mM Tris, 192 mM glycine. After transfer, the membrane was blocked with
10% nonfat dry milk in phosphate-buffered saline for 1 h at room
temperature. After incubation with the primary antibodies specified
above, horseradish peroxidase-conjugated secondary antibodies (Sigma)
and an enhanced chemiluminescent substrate kit (Amersham Pharmacia
Biotech) were used for detection.
RNA Analysis--
Total RNA was harvested according to the
procedure of Chomczynski and Sacchi (33). Cells were lysed in buffer
containing 4 M guanidinium isothiocyanate. Lysates were
extracted with acid phenol/chloroform and RNA was
isopropanol-precipitated overnight at To understand the mechanisms by which PPAR (C/EBP
) and peroxisome proliferator-activated receptor
(PPAR
). Expression of PPAR
in fibroblasts converts them to
fat-laden cells with an adipocyte-like morphology. Here, we investigate
the ability of PPAR
to confer insulin-sensitive glucose transport to
a variety of murine fibroblast cell lines. When cultured in the
presence of a PPAR
ligand, Swiss-3T3 and BALB/c-3T3 cells
ectopically expressing PPAR
accumulate lipid droplets, express
C/EBP
, aP2, insulin-responsive aminopeptidase, and glucose
transporter isoform 4 (GLUT4), and exhibit highly insulin-responsive
2-deoxyglucose uptake. In contrast, PPAR
-expressing NIH-3T3 cells,
despite similar lipid accumulation, adipocyte morphology, and aP2
expression, do not express C/EBP
or GLUT4 and fail to acquire
insulin sensitivity. In cells ectopically expressing PPAR
, the
development of insulin-responsive glucose uptake correlates with
C/EBP
expression. Furthermore, ectopic expression of C/EBP
in
NIH-3T3 cells converts them to the adipocyte phenotype and restores
insulin-sensitive glucose uptake. We propose that the pathway(s)
leading to fat accumulation and morphological changes are distinct from
that leading to insulin-dependent glucose transport. Our
results suggest that although PPAR
is sufficient to trigger the
adipogenic program, C/EBP
is required for establishment of
insulin-sensitive glucose transport.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
, a member of the PPAR subfamily of nuclear hormone receptors,
is the most fat-specific of its family (15-17). Ectopic expression of
PPAR
activates adipogenesis in several fibroblast cell lines (10,
12, 15, 18). PPAR
can be activated in a ligand-related manner by
15d-PGJ2, a natural prostaglandin (18), and by members of the
insulin-sensitizing thiazolidinedione drug family (19). PPAR
ligands
and activators can induce adipocyte differentiation in preadipocytes,
myoblasts, and multipotential stem cells (20, 21).
is most highly expressed in
adipose tissue and in liver, and has been implicated in maintenance of
the terminally differentiated adipocyte phenotype (8, 22). As with
PPAR
, forced expression of C/EBP
is adipogenic in several fibroblastic cell lines (5) and in 3T3-L1 preadipocytes (6). Expression
of antisense C/EBP
prevents its own expression and that of fatty
acid-binding protein (aP2) and of the muscle/fat-specific glucose
transporter isoform, GLUT4, and it blocks triglyceride accumulation
(23). In addition, C/EBP
-null mice fail to develop white adipose
tissue (24).
by the ectopic expression of C/EBP
and
C/EBP
in NIH-3T3 fibroblasts results in expression of the adipocyte
phenotype (10). However, compared with mature 3T3-L1 adipocytes, these
fibroblasts have a minimal capacity for insulin-responsive glucose
uptake and fail to express C/EBP
, a transcription factor with a well
established role in terminal adipocyte differentiation (8, 22). These
results prompted us to determine the effect of PPAR
on C/EBP
expression and on the program leading to insulin-sensitive glucose
transport. We report here that despite similar ability of
PPAR
-expressing fibroblasts to exhibit a morphologically defined
adipocyte phenotype, only those cells that permit expression of
C/EBP
are insulin-sensitive with regard to glucose uptake. In
addition, ectopic expression of C/EBP
in NIH-3T3 cells (normally
deficient in C/EBP
) results in expression of PPAR
and conversion
to the adipocyte phenotype, and confers insulin-responsive glucose uptake.
EXPERIMENTAL PROCEDURES
-32P]dCTP and 3H-2-deoxyglucose from NEN
Life Science Products, Inc.; Klenow fragment of DNA polymerase I from
Promega Corp. (Madison, WI). Troglitazone was a kind gift from Dr. John
Johnson, (Parke-Davis/Warner Lambert, Ann Arbor, MI). BALB/c-3T3 cells
and Swiss-3T3 cells were kind gifts from Drs. Matthew Nugent and
Douglas Faller (Boston University School of Medicine), respectively.
(Santa Cruz Biotechnology, Santa Cruz, CA), a monoclonal
anti-caveolin-1 antibody (Transduction Laboratories, Lexington, KY),
and anti-insulin-responsive aminopeptidase (IRAP) serum (27).
-Puro and pBabe-C/EBP
-Puro retroviral
expression vectors (12) were kind gifts of Dr. Bruce Spiegelman (Dana
Farber Cancer Institute, Harvard Medical School). Transfection of BOSC
23 packaging cells and subsequent infection of target cells was
performed as described (12, 28). Infected target cells were selected
for 6-10 days in medium containing 2.0 µg/ml puromycin.
or control vector and NIH-3T3 cells expressing
retroviral C/EBP
were differentiated by the same protocol for 3T3-L1
cells, except growth medium was DMEM containing 10% fetal bovine serum
and 2.0 µg puromycin, and the cells were differentiated and
maintained in the presence or absence of 5 µM troglitazone.
80 °C. When ready to be analyzed, samples were thawed, vortexed, and spun for 15 min at full speed (13,000 rpm) in a microcentrifuge. The protein content of the supernatants was determined using the bicinchoninic acid kit (Pierce).
20 °C. Northern blot
analysis was performed on 20 µg of each sample RNA as described (10).
cDNA probes for aP2, PPAR
, and C/EBP
(10) were labeled using
Klenow fragment of DNA polymerase I and [
-32P]dCTP by
random priming method.
RESULTS
regulates
development of the adipocyte phenotype, we introduced PPAR
by
retroviral infection into three well characterized murine fibroblast
cell lines: Swiss-3T3, BALB/c-3T3, and NIH-3T3. The adipose conversion of the resulting cell lines (deemed Swiss-P
, BALB/c-P
, and
NIH-P
cells, see Fig. 2) was monitored by Oil Red O staining.
Stained cells were then photographed using Hoffman optics microscopy
(Fig. 1A). As shown, the
majority (75-95%) of each of these lines exhibited an adipocyte
phenotype (cell rounding and accumulation of cytoplasmic lipid
droplets) when cultured in medium containing the PPAR
ligand, troglitazone. Control cells infected with virus harboring vector DNA
alone, and cultured under the same conditions did not differentiate to
any considerable extent, indicating that adipose conversion of these
engineered cells was PPAR
-dependent. Also, whereas
NIH-P
and Swiss-P
cells required troglitazone for adipocytic
conversion, the differentiation of BALB/c-P
cells was
troglitazone-independent. The mRNA level of the fatty acid-binding
protein, aP2, was also determined in these cells by Northern blot
analysis (Fig. 1B). In all instances, the level of aP2
mRNA was markedly induced by retroviral PPAR
expression.
Furthermore, this effect was dependent on the presence of troglitazone
in the culture medium except for BALB/c-P
cells, which expressed
similarly elevated levels of aP2 independent of ligand. Thus, aP2
expression in all the cell lines correlated closely with lipid droplet
accumulation. Interestingly, the "adipogenic tendency" of the Swiss
and BALB/c cell lines to convert to fat in the absence of PPAR
ligand and/or PPAR
is reflected in the low but obvious adipose
conversion (Fig. 1A), and the expression of aP2 in absence
of troglitazone or PPAR
(Fig. 1B).
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Fig. 1.
Ectopic expression of
PPAR in various fibroblast cell lines
activates the adipogenic program. 3T3-L1 preadipocytes and
NIH-3T3, Swiss-3T3, and BALB/c-3T3 fibroblasts expressing
retroviral PPAR
or vector DNA alone were differentiated in the
presence or absence of 5 µM troglitazone. Eight days
later, the cells were used for experimentation. A, cells
were fixed and then stained with Oil Red O. Shown are micrographs taken
using Hoffman optics microscopy. B, total RNA was extracted,
and 20 µg of each sample was subjected to Northern blot
analysis for aP2. These data are representative of three
experiments.
Next, we examined whether ectopic PPAR expression in these
fibroblasts results in C/EBP
expression during their differentiation into adipocytes and compared that with differentiating 3T3-L1 preadipocytes (Fig. 2A).
Consistent with previous reports (10), NIH-P
cells do not express
significant levels of C/EBP
(Fig. 2B). In contrast,
C/EBP
protein expression is induced during the differentiation of
Swiss-P
, BALB/c-P
, and 3T3-L1 cells. The relative abundance of
C/EBP
protein in the different cell lines is shown in Fig.
2C. Swiss-P
and BALB/c-P
cells express C/EBP
at
levels comparable with 3T3-L1 adipocytes, whereas NIH-P
cells do not
express appreciable C/EBP
. In BALB/c-P
cells, C/EBP
levels are
approximately doubled by the inclusion of troglitazone in the culture
medium (Fig. 2C).
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To determine the effect of PPAR expression and resultant endogenous
C/EBP
expression (or lack thereof) upon adipocyte differentiation, the level of several proteins related to adipocyte function was measured by Western blot analysis. All three engineered cell lines were
differentiated in the presence of troglitazone, whereas the 3T3-L1
cells were differentiated according to the standard protocol. On the
indicated days after the induction of differentiation, cell lysates
were prepared and subjected to Western blot analysis for
phosphatidylinositol 3-kinase (PI3-kinase), IRAP, the
muscle/fat-specific glucose transporter GLUT4 isoform, the ubiquitously
expressed glucose transporter isoform GLUT1, and caveolin-1, the
structural protein of caveolae. As shown in Fig.
3, the expression of most of these
proteins during adipose conversion of the engineered cell lines is
similar to 3T3-L1 cells. The level of PI3-kinase, whose activation is
required for insulin-stimulated glucose transport (for review see Ref.
34), is relatively constant during differentiation. IRAP, which
traffics identically to and is completely colocalized with GLUT4 (26,
27, 35, 36), increases during the differentiation of all the cell lines
as does caveolin-1 (37, 38), which is unlikely to be directly involved
in GLUT4 trafficking (38). GLUT1 is expressed at all times in all of
the cell lines (Fig. 3). This result is consistent with previous
studies that have shown GLUT1 levels to either decrease (10) or remain
relatively constant (39) during 3T3-L1 differentiation. Thus, just like 3T3-L1 adipocytes, all of the PPAR
-expressing fatty fibroblasts express PI3-kinase, IRAP, GLUT1, and caveolin-1. An important difference, however, is in the expression of GLUT4. Whereas Swiss-P
(Fig. 3B), BALB/c-P
(Fig. 3C), and 3T3-L1
cells (Fig. 3A) express GLUT4 in the latter stages of the
differentiation program, GLUT4 is undetectable in NIH-P
cells at all
times (Fig. 3D).
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Finally, the ability of the various engineered fibroblasts to
take up glucose in response to insulin was measured by assaying 2-deoxyglucose uptake. The cells were differentiated in the presence or
absence of troglitazone as described under "Experimental
Procedures." The rates of basal and insulin-stimulated glucose
transport were then measured on days 0, 4, and 8 of differentiation. As
shown in Fig. 4D, NIH-P
cells do not become insulin-sensitive, even under conditions (+ troglitazone) in which greater than 75% of the cells exhibit an
adipocyte phenotype (see Fig. 1A) and aP2 is highly
expressed (Fig. 1B). None of the control cells infected with
vector DNA alone (Swiss-Vector, BALB-Vector, and NIH-Vector) showed any
significant levels of insulin-regulated 2-deoxyglucose uptake even when
cultured in the presence of troglitazone, in correlation with their
lack of adipose conversion. In contrast, Swiss-P
and BALB/c-P
cells cultured in the presence of troglitazone become significantly
insulin-responsive (3-4-fold stimulation on day 8) (Fig. 4,
B and C). Interestingly, under these conditions, the absolute rate of insulin-stimulated 2-deoxyglucose uptake on days 4 and 8 is very similar to that in differentiating 3T3-L1 cells (Fig.
4A). In the absence of troglitazone, BALB/c-P
cells, whose adipose conversion is PPAR
ligand-independent (see Fig. 1),
still acquire insulin-responsiveness (>4-fold stimulation) but the
absolute rate of transport is much smaller. Under these conditions (no troglitazone), Swiss-P
cells do not differentiate (see Fig. 1) and do not become insulin-sensitive (Fig.
4B).
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To examine the effect of expression of C/EBP and PPAR
together in NIH-3T3 fibroblasts, we introduced C/EBP
by retroviral infection into NIH-3T3 cells and monitored their adipose conversion. As
shown in Fig. 5A, these cells
(deemed NIH-C/E
cells) round up and accumulate lipid droplets to a
significant extent (80-90%) when cultured under standard conditions,
independent of PPAR
ligand. In addition, the expression of C/EBP
in NIH-C/E
cells results in the troglitazone-independent expression
of PPAR
mRNA at levels similar to those in 3T3-L1 adipocyte
controls (Fig. 5B). Like the Swiss-P
and BALB/c-P
cells, NIH-C/E
cells express GLUT4, GLUT1, and IRAP proteins
at levels similar to 3T3-L1 adipocytes (Fig. 5C). Finally,
basal and insulin-stimulated levels of 2-deoxyglucose uptake in
NIH-C/E
cells were measured at day 7 of differentiation as described
(Fig. 5D). As shown, NIH-C/E
cells, unlike their PPAR
-transfected counterparts (Fig. 4D), exhibit a
significant insulin-responsive glucose uptake (2-3-fold) when
differentiated in the presence or absence of troglitazone.
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DISCUSSION |
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In this study, we have examined the ability of PPAR to activate
the program leading to insulin-dependent glucose transport in various murine fibroblast cell lines. We report here that although ectopic expression of PPAR
activates the adipogenic program as evidenced by the accumulation of cytoplasmic fat droplets and the
expression of adipocyte markers, "fatty" PPAR
-expressing fibroblasts do not all acquire the capacity for insulin-stimulated glucose transport (Fig. 4). Indeed, whereas ectopic expression of
PPAR
in NIH-3T3 fibroblasts causes the accumulation of lipid droplets (Fig. 1A) and the expression of aP2 at levels
comparable with 3T3-L1 adipocytes (Fig. 1B), these cells do
not express C/EBP
(Fig. 2) or GLUT4 (Fig. 3), and they are
completely insensitive to insulin with respect to glucose transport
(Fig. 4). In contrast, the Swiss-3T3 and BALB/c-3T3 fibroblasts
ectopically expressing PPAR
, in addition to becoming fatty (Fig.
1A) and turning on aP2 (Fig. 1B), express
C/EBP
(Fig. 2), GLUT4 (Fig. 3), and substantially increase
(3-4-fold) their rate of glucose transport in response to insulin
(Fig. 4). Previously it has been shown that C/EBP
transactivates the
GLUT4 gene (40). Consistent with this report, the induction of GLUT4
expression in differentiating 3T3-L1, Swiss-P
, and BALB/c-P
cells
(Fig. 3) immediately follows the onset of C/EBP
expression (Fig.
2).
The acquisition of insulin-sensitive glucose transport by the
Swiss-P and BALB-P
cells, as assessed by 2-deoxyglucose uptake, bears a strong resemblance to this process in 3T3-L1 preadipocytes (Fig. 4). In particular, the onset of insulin sensitivity in all three
cell lines is, primarily if not completely, because of a decrease in
the basal rate of transport. In 3T3-L1 cells, we showed that this was
the result of the sequestration of glucose transporters into a newly
forming hormone-responsive vesicular
pool.2 Our more recent
results showing that insulin stimulates the translocation of GLUT4,
GLUT1, IRAP, and transferrin receptor in differentiated BALB/c-P
cells (data not shown) indicate that we have, at least partially,
recapitulated insulin-regulated vesicular trafficking in an engineered
fibroblast. It will be interesting to study the development of the
insulin-sensitive compartment in these transfected cells and to see if
it occurs in a short time frame and before the expression of GLUT4, as
it does in 3T3-L1 preadipocytes.2 Based upon the similar
timing of these events in the different cell lines and the acquisition
of insulin sensitivity before GLUT4 expression (Figs. 3 and 4), we
expect this to be the case.
Our results show that NIH-3T3 fibroblasts, which do not express
C/EBP normally nor as a result of ectopic PPAR
expression, do
convert to "adipocyte-like" cells when infected with retroviral PPAR
. However, these NIH-P
cells fail to develop
insulin-regulated glucose transport. Introduction of retroviral
C/EBP
into NIH-3T3 fibroblasts converts them to an adipose phenotype
(cell rounding and lipid accumulation, Fig. 5A), leads to
expression of PPAR
mRNA (Fig. 5B) and several
adipocyte-related proteins, including GLUT4 (Fig. 5C), and
most importantly, results in acquisition of insulin-sensitive glucose
uptake (Fig. 5D).
Although only the cell lines that express GLUT4 become insulin
responsive, it is unlikely that the expression of GLUT4 confers insulin
sensitivity for the following reasons. First, ectopic expression of
GLUT4 alone does not confer insulin-sensitive glucose transport to
cells (41-43). Second, on day 4 of differentiation, 3T3-L1,
Swiss-P, and BALB/c-P
cells are all insulin responsive (see Fig.
4), and at that time, they express little to no GLUT4 (see Fig. 3).
Third, NIH-3T3 cells, whose endogenous PPAR
and GLUT4 genes are
activated by ectopic expression of C/EBP
and C/EBP
(
39
cells), are only mildly responsive to insulin (10).
Obviously, expression of PPAR alone is sufficient to activate
adipocyte differentiation, including the generation of cytoplasmic fat
droplets and expression of aP2 (Fig. 1). However, because only those
PPAR
-expressing fibroblasts that permit expression of endogenous
C/EBP
or NIH-3T3 cells ectopically expressing C/EBP
acquire the
capacity for insulin-regulated glucose uptake (Figs. 2, 4, and 5), we
propose that C/EBP
plays an important role in driving the
development of insulin sensitivity with regard to glucose transport.
Although ectopic expression of either C/EBP or PPAR
alone in
fibroblasts can stimulate the adipogenic program, there are numerous
lines of evidence to suggest that they act synergistically with one
another (12, 15, 20). For example, ectopic expression of either of
these transcription factors alone in NIH-3T3 fibroblasts appears to
cause commitment to the adipocyte lineage such that these cells undergo
differentiation upon exposure to the hormonal inducers (5, 12).
However, co-expression of both PPAR
and C/EBP
activates the
terminal differentiation program in NIH-3T3 fibroblasts in the absence
of any external agents (12). Thus, it appears that although the targets
of these two transcription factors overlap somewhat, both are required
to activate the complete repertoire of adipocyte gene expression.
The essential role of C/EBP in adipocyte differentiation is
well documented (8, 22). In light of the fact that expression of
antisense C/EBP
RNA in 3T3-L1 preadipocytes inhibits their differentiation (23), it is somewhat surprising that NIH-3T3 fibroblasts ectopically expressing C/EBP
, C/EBP
and C/EBP
together, or PPAR
alone are capable of differentiating into
adipocyte-like cells, even though they do not express C/EBP
(7, 8,
10). One possible reason for this is that the overexpression of the other C/EBP isoforms may compensate to some degree for C/EBP
by
binding to C/EBP motifs in target genes. This may explain the modest
capacity for insulin-regulated glucose uptake exhibited by
cells
in our previous study (10).
It has long been recognized that the onset of insulin-stimulated
glucose transport is a late event in the process of fat cell differentiation in cell culture. The marked increase in
insulin-stimulated glucose transport occurs from days 6 to 8 of the
differentiation program, whereas marked lipid accumulation and cell
rounding are evident by days 4 to 5 of this process. Thus,
insulin-sensitive glucose transport (and possibly other
insulin-regulated metabolic processes) are the late and definitive
markers of a mature fat cell. This conclusion is further supported by
the fact that even fully differentiated 3T3-L1 adipocytes express more
GLUT1 and less GLUT4 than primary isolates of rat fat cells. In other
words, like most or all tissue culture cells, they are not quite as
mature as the same cell type as acutely isolated from the animal. We propose that there are subprograms involved in adipocyte
differentiation under the regulation of several transcription factors,
insulin sensitivity being an ultimate program. Our data support this
notion by demonstrating that ectopic expression of PPAR can
phenotypically differentiate fat cells, but only those cell lines that
also express C/EBP
show an insulin response. As previously noted,
both these transcription factors are known to be critical for fat cell
differentiation and to influence one another. We are in the process of
determining their relative roles and whether there are additional
transcription factors whose expression regulates the insulin response.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Ron F. Morrison for valuable discussions and insight. We thank Dr. Zhidan Wu for helpful advice on the retroviral system. We also thank Sean Coughlin for help with some of the Western blot analyses and 2-deoxyglucose uptake assays. We acknowledge Dr. Bruce Spiegelman for the gift of the BOSC cells and retroviral vectors.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK30425, DK36424 (to P. F. P.), DK51586 (to S. R. F.), a Medical Student Research Fellowship from the American Heart Association (to A. K. E.-J.), and Public Health Service Training Grant HL07035 (to J. K. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors have contributed equally to this work.
§ To whom correspondence should be addressed: Dept. of Biochemistry, Boston University School of Medicine, 715 Albany St., Boston, MA 02118. Tel.: 617-638-4186; Fax: 617-638-5339; E-mail: farmer{at}med-biochem.bu.edu.
2 A. K. El-Jack, K. V. Kandror, and P. F. Pilch, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein; PPAR, peroxisome proliferator-activated receptor; IRAP, insulin-responsive aminopeptidase; GLUT, glucose transporter isoform; aP2, adipose protein 2/fatty acid-binding protein; DMEM, Dulbecco's modified Eagle's medium; PI3-kinase, phosphatidylinositol 3-kinase.
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REFERENCES |
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