Reconstitution of Insulin-sensitive Glucose Transport in Fibroblasts Requires Expression of Both PPARgamma and C/EBPalpha *

Amr K. El-JackDagger , Jonathan K. HammDagger , Paul F. Pilch, and Stephen R. Farmer§

From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
REFERENCES

Adipocyte differentiation is regulated by at least two major transcription factors, CCAAT/enhancer-binding protein alpha  (C/EBPalpha ) and peroxisome proliferator-activated receptor gamma  (PPARgamma ). Expression of PPARgamma in fibroblasts converts them to fat-laden cells with an adipocyte-like morphology. Here, we investigate the ability of PPARgamma to confer insulin-sensitive glucose transport to a variety of murine fibroblast cell lines. When cultured in the presence of a PPARgamma ligand, Swiss-3T3 and BALB/c-3T3 cells ectopically expressing PPARgamma accumulate lipid droplets, express C/EBPalpha , aP2, insulin-responsive aminopeptidase, and glucose transporter isoform 4 (GLUT4), and exhibit highly insulin-responsive 2-deoxyglucose uptake. In contrast, PPARgamma -expressing NIH-3T3 cells, despite similar lipid accumulation, adipocyte morphology, and aP2 expression, do not express C/EBPalpha or GLUT4 and fail to acquire insulin sensitivity. In cells ectopically expressing PPARgamma , the development of insulin-responsive glucose uptake correlates with C/EBPalpha expression. Furthermore, ectopic expression of C/EBPalpha 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 PPARgamma is sufficient to trigger the adipogenic program, C/EBPalpha is required for establishment of insulin-sensitive glucose transport.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

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).

PPARgamma , a member of the PPAR subfamily of nuclear hormone receptors, is the most fat-specific of its family (15-17). Ectopic expression of PPARgamma activates adipogenesis in several fibroblast cell lines (10, 12, 15, 18). PPARgamma 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). PPARgamma ligands and activators can induce adipocyte differentiation in preadipocytes, myoblasts, and multipotential stem cells (20, 21).

Of the C/EBP family members, C/EBPalpha 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 PPARgamma , forced expression of C/EBPalpha is adipogenic in several fibroblastic cell lines (5) and in 3T3-L1 preadipocytes (6). Expression of antisense C/EBPalpha 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/EBPalpha -null mice fail to develop white adipose tissue (24).

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 PPARgamma by the ectopic expression of C/EBPbeta and C/EBPdelta 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/EBPalpha , a transcription factor with a well established role in terminal adipocyte differentiation (8, 22). These results prompted us to determine the effect of PPARgamma on C/EBPalpha expression and on the program leading to insulin-sensitive glucose transport. We report here that despite similar ability of PPARgamma -expressing fibroblasts to exhibit a morphologically defined adipocyte phenotype, only those cells that permit expression of C/EBPalpha are insulin-sensitive with regard to glucose uptake. In addition, ectopic expression of C/EBPalpha in NIH-3T3 cells (normally deficient in C/EBPalpha ) results in expression of PPARgamma and conversion to the adipocyte phenotype, and confers insulin-responsive glucose uptake.

    EXPERIMENTAL PROCEDURES

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.; [alpha -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.

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/EBPalpha (Santa Cruz Biotechnology, Santa Cruz, CA), a monoclonal anti-caveolin-1 antibody (Transduction Laboratories, Lexington, KY), and anti-insulin-responsive aminopeptidase (IRAP) serum (27).

Plasmids and Stable Cell Lines-- The BOSC 23 packaging cells (28), pBabe-Puro, pBabe-PPARgamma -Puro and pBabe-C/EBPalpha -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.

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 PPARgamma or control vector and NIH-3T3 cells expressing retroviral C/EBPalpha 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.

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 -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).

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 M guanidinium isothiocyanate. Lysates were extracted with acid phenol/chloroform and RNA was isopropanol-precipitated overnight at -20 °C. Northern blot analysis was performed on 20 µg of each sample RNA as described (10). cDNA probes for aP2, PPARgamma , and C/EBPalpha (10) were labeled using Klenow fragment of DNA polymerase I and [alpha -32P]dCTP by random priming method.

    RESULTS

To understand the mechanisms by which PPARgamma regulates development of the adipocyte phenotype, we introduced PPARgamma 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-Pgamma , BALB/c-Pgamma , and NIH-Pgamma 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 PPARgamma 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 PPARgamma -dependent. Also, whereas NIH-Pgamma and Swiss-Pgamma cells required troglitazone for adipocytic conversion, the differentiation of BALB/c-Pgamma 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 PPARgamma expression. Furthermore, this effect was dependent on the presence of troglitazone in the culture medium except for BALB/c-Pgamma 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 PPARgamma ligand and/or PPARgamma is reflected in the low but obvious adipose conversion (Fig. 1A), and the expression of aP2 in absence of troglitazone or PPARgamma (Fig. 1B).


View larger version (69K):
[in this window]
[in a new window]
 
Fig. 1.   Ectopic expression of PPARgamma in various fibroblast cell lines activates the adipogenic program. 3T3-L1 preadipocytes and NIH-3T3, Swiss-3T3, and BALB/c-3T3 fibroblasts expressing retroviral PPARgamma 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 PPARgamma expression in these fibroblasts results in C/EBPalpha expression during their differentiation into adipocytes and compared that with differentiating 3T3-L1 preadipocytes (Fig. 2A). Consistent with previous reports (10), NIH-Pgamma cells do not express significant levels of C/EBPalpha (Fig. 2B). In contrast, C/EBPalpha protein expression is induced during the differentiation of Swiss-Pgamma , BALB/c-Pgamma , and 3T3-L1 cells. The relative abundance of C/EBPalpha protein in the different cell lines is shown in Fig. 2C. Swiss-Pgamma and BALB/c-Pgamma cells express C/EBPalpha at levels comparable with 3T3-L1 adipocytes, whereas NIH-Pgamma cells do not express appreciable C/EBPalpha . In BALB/c-Pgamma cells, C/EBPalpha levels are approximately doubled by the inclusion of troglitazone in the culture medium (Fig. 2C).


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 2.   Time course of C/EBPalpha expression during adipose conversion of 3T3-L1 preadipocytes and PPARgamma -expressing fibroblasts. On the indicated days following the induction of differentiation, whole cell extracts were prepared from the indicated cell lines. Equal amounts (100 µg) of protein were electrophoresed and Western blotted for C/EBPalpha . A, time course of C/EBPalpha expression during 3T3-L1 preadipocyte differentiation. B, time course of C/EBPalpha expression during adipose conversion of Swiss-Pgamma , BALB/c-Pgamma , and NIH-Pgamma cells. C, comparison of C/EBPalpha expression by the indicated cell lines on day 8 of differentiation (500 µg of protein/lane). These data are representative of two independent experiments.

To determine the effect of PPARgamma expression and resultant endogenous C/EBPalpha 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 PPARgamma -expressing fatty fibroblasts express PI3-kinase, IRAP, GLUT1, and caveolin-1. An important difference, however, is in the expression of GLUT4. Whereas Swiss-Pgamma (Fig. 3B), BALB/c-Pgamma (Fig. 3C), and 3T3-L1 cells (Fig. 3A) express GLUT4 in the latter stages of the differentiation program, GLUT4 is undetectable in NIH-Pgamma cells at all times (Fig. 3D).


View larger version (53K):
[in this window]
[in a new window]
 
Fig. 3.   A comparison of protein expression during adipose conversion of 3T3-L1 preadipocytes versus PPARgamma - expressing fibroblasts. On the indicated days post-induction, whole cell extracts were prepared from differentiating 3T3-L1 (A), Swiss-Pgamma (B), BALB/c-Pgamma (C), and NIH-Pgamma (D) cells. Equal amounts of protein (100 µg; 500 µg for GLUT4 and caveolin) were electrophoresed and Western blotted for PI3-kinase, IRAP, GLUT4, GLUT1, and caveolin-1. These data are representative of two experiments.

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-Pgamma 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-Pgamma and BALB/c-Pgamma 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-Pgamma cells, whose adipose conversion is PPARgamma 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-Pgamma cells do not differentiate (see Fig. 1) and do not become insulin-sensitive (Fig. 4B).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 4.   Ectopic expression of PPARgamma confers insulin-responsive 2-deoxyglucose transport to some but not all fibroblast cell lines. Differentiating 3T3-L1 preadipocytes (A) and Swiss-3T3 (B), BALB/c-3T3 (C), and NIH-3T3 (D) fibroblasts expressing PPARgamma or vector were serum-starved for 2 h before treatment with (black bars) or without (striped bars) 100 nM insulin for 15 min at 37 °C. 3H-2-deoxyglucose uptake was then determined. Each bar and error bar represent the average and difference between duplicate determinations, respectively. These data are representative of two independent experiments. Trog, troglitazone.

To examine the effect of expression of C/EBPalpha and PPARgamma together in NIH-3T3 fibroblasts, we introduced C/EBPalpha by retroviral infection into NIH-3T3 cells and monitored their adipose conversion. As shown in Fig. 5A, these cells (deemed NIH-C/Ealpha cells) round up and accumulate lipid droplets to a significant extent (80-90%) when cultured under standard conditions, independent of PPARgamma ligand. In addition, the expression of C/EBPalpha in NIH-C/Ealpha cells results in the troglitazone-independent expression of PPARgamma mRNA at levels similar to those in 3T3-L1 adipocyte controls (Fig. 5B). Like the Swiss-Pgamma and BALB/c-Pgamma cells, NIH-C/Ealpha 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/Ealpha cells were measured at day 7 of differentiation as described (Fig. 5D). As shown, NIH-C/Ealpha cells, unlike their PPARgamma -transfected counterparts (Fig. 4D), exhibit a significant insulin-responsive glucose uptake (2-3-fold) when differentiated in the presence or absence of troglitazone.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 5.   Ectopic expression of C/EBPalpha in NIH-3T3 fibroblasts converts them to the adipocyte phenotype, induces PPARgamma , and confers insulin-sensitive glucose uptake. NIH 3T3 cells expressing retroviral C/EBPalpha were differentiated in the presence or absence of 5 µM troglitazone (Trog). Seven days later (day 7), cells were used for experimentation. A, cells were fixed, stained with Oil Red O, and photographed as described; B, Northern blot analysis of PPARgamma and C/EBPalpha mRNAs in NIH-C/Ealpha cells; C, Western blot analysis of GLUT4, GLUT1, and IRAP protein levels in NIH-C/Ealpha cells; and D, insulin-responsive 2-deoxyglucose uptake assay of NIH-C/Ealpha cells performed as described for Fig. 4. These data are representative of two experiments. L1, 3T3-L1 (day 7) adipocyte control.


    DISCUSSION

In this study, we have examined the ability of PPARgamma to activate the program leading to insulin-dependent glucose transport in various murine fibroblast cell lines. We report here that although ectopic expression of PPARgamma activates the adipogenic program as evidenced by the accumulation of cytoplasmic fat droplets and the expression of adipocyte markers, "fatty" PPARgamma -expressing fibroblasts do not all acquire the capacity for insulin-stimulated glucose transport (Fig. 4). Indeed, whereas ectopic expression of PPARgamma 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/EBPalpha (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 PPARgamma , in addition to becoming fatty (Fig. 1A) and turning on aP2 (Fig. 1B), express C/EBPalpha (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/EBPalpha transactivates the GLUT4 gene (40). Consistent with this report, the induction of GLUT4 expression in differentiating 3T3-L1, Swiss-Pgamma , and BALB/c-Pgamma cells (Fig. 3) immediately follows the onset of C/EBPalpha expression (Fig. 2).

The acquisition of insulin-sensitive glucose transport by the Swiss-Pgamma and BALB-Pgamma 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-Pgamma 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/EBPalpha normally nor as a result of ectopic PPARgamma expression, do convert to "adipocyte-like" cells when infected with retroviral PPARgamma . However, these NIH-Pgamma cells fail to develop insulin-regulated glucose transport. Introduction of retroviral C/EBPalpha into NIH-3T3 fibroblasts converts them to an adipose phenotype (cell rounding and lipid accumulation, Fig. 5A), leads to expression of PPARgamma 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-Pgamma , and BALB/c-Pgamma 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 PPARgamma and GLUT4 genes are activated by ectopic expression of C/EBPbeta and C/EBPdelta (beta delta 39 cells), are only mildly responsive to insulin (10).

Obviously, expression of PPARgamma alone is sufficient to activate adipocyte differentiation, including the generation of cytoplasmic fat droplets and expression of aP2 (Fig. 1). However, because only those PPARgamma -expressing fibroblasts that permit expression of endogenous C/EBPalpha or NIH-3T3 cells ectopically expressing C/EBPalpha acquire the capacity for insulin-regulated glucose uptake (Figs. 2, 4, and 5), we propose that C/EBPalpha plays an important role in driving the development of insulin sensitivity with regard to glucose transport.

Although ectopic expression of either C/EBPalpha or PPARgamma 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 PPARgamma and C/EBPalpha 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/EBPalpha in adipocyte differentiation is well documented (8, 22). In light of the fact that expression of antisense C/EBPalpha RNA in 3T3-L1 preadipocytes inhibits their differentiation (23), it is somewhat surprising that NIH-3T3 fibroblasts ectopically expressing C/EBPbeta , C/EBPbeta and C/EBPdelta together, or PPARgamma alone are capable of differentiating into adipocyte-like cells, even though they do not express C/EBPalpha (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/EBPalpha by binding to C/EBP motifs in target genes. This may explain the modest capacity for insulin-regulated glucose uptake exhibited by beta delta 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 PPARgamma can phenotypically differentiate fat cells, but only those cell lines that also express C/EBPalpha 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
REFERENCES
  1. Shulman, R. G. (1996) Mol. Med. 2, 533-540[Medline] [Order article via Infotrieve]
  2. Kandror, K. V., and Pilch, P. F. (1996) Am. J. Physiol. Endocrinol. Metab. 271, E1-E14[Abstract/Free Full Text]
  3. Rea, S., and James, D. E. (1997) Diabetes 46, 1667-1677[Abstract]
  4. Cornelius, P., MacDougald, O. A., and Lane, M. D. (1994) Annu. Rev. Nutr. 14, 99-129[CrossRef][Medline] [Order article via Infotrieve]
  5. Freytag, S. O., Paielli, D. L., and Gilbert, J. D. (1994) Genes Dev. 8, 1654-1663[Abstract]
  6. Lin, F.-T., and Lane, M. D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8757-8761[Abstract]
  7. Wu, Z., Xie, Y., Bucher, N. L., and Farmer, S. R. (1995) Genes Dev. 9, 2350-2363[Abstract]
  8. Yeh, W. C., Cao, Z., Classon, M., and McKnight, S. L. (1995) Genes Dev. 9, 168-181[Abstract]
  9. Schwarz, E. J., Reginato, M. J., Shao, D., Krakow, S. L., and Lazar, M. A. (1997) Mol. Cell. Biol. 17, 1552-1561[Abstract]
  10. Wu, Z. D., Xie, Y. H., Morrison, R. F., Bucher, N. L. R., and Farmer, S. R. (1998) J. Clin. Invest. 101, 22-32[Abstract/Free Full Text]
  11. Darlington, G. J., Ross, S. E., and MacDougald, O. A. (1998) J. Biol. Chem. 273, 30057-30060[Free Full Text]
  12. Tontonoz, P., Hu, E., and Spiegelman, B. M. (1994) Cell 79, 1147-1156[Medline] [Order article via Infotrieve]
  13. Tontonoz, P., Hu, E., Graves, R. A., Budavari, A. I., and Spiegelman, B. M. (1994) Genes Dev. 8, 1224-1234[Abstract]
  14. Tontonoz, P., Hu, E., Devine, J., Beale, E. G., and Spiegelman, B. M. (1995) Mol. Cell. Biol. 15, 351-357[Abstract]
  15. Brun, R. P., Tontonoz, P., Forman, B. M., Ellis, R., Chen, J., Evans, R. M., and Spiegelman, B. M. (1996) Genes Dev. 10, 974-984[Abstract]
  16. Braissant, O., Foufelle, F., Scotto, C., Dauca, M., and Wahli, W. (1996) Endocrinology 137, 354-366[Abstract]
  17. Schoonjans, K., Staels, B., and Auwerx, J. (1996) Biochim. Biophys. Acta 1302, 93-109[Medline] [Order article via Infotrieve]
  18. Forman, B. M., Tontonoz, P., Chen, J., Brun, R. P., Spiegelman, B. M., and Evans, R. M. (1995) Cell 83, 803-812[Medline] [Order article via Infotrieve]
  19. Lehmann, J. M., Moore, L. B., Smith-Oliver, T. A., Wilkison, W. O., Wilson, T. M., and Kliewer, S. A. (1995) J. Biol. Chem. 270, 12953-12956[Abstract/Free Full Text]
  20. Hu, E. D., Tontonoz, P., and Spiegelman, B. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9856-9860[Abstract]
  21. Teboul, L., Gaillard, D., Staccini, L., Inadera, H., Amri, E.-Z., and Grimaldi, P. A. (1995) J. Biol. Chem. 270, 28183-28187[Abstract/Free Full Text]
  22. Mandrup, S., and Lane, M. D. (1997) J. Biol. Chem. 272, 5367-5370[Free Full Text]
  23. Lin, F.-T., and Lane, M. D. (1992) Genes Dev. 6, 533-544[Abstract]
  24. Wang, N.-D., Finegold, M. J., Bradley, A., Ou, C. N., Abdelsayed, S. V., Wilde, M. D., Taylor, L. R., Wilson, D. R., and Darlington, G. J. (1995) Science 269, 1108-1112[Medline] [Order article via Infotrieve]
  25. James, D. E., Brown, R., Navarro, J., and Pilch, P. F. (1988) Nature 333, 183-185[CrossRef][Medline] [Order article via Infotrieve]
  26. Kandror, K. V., Coderre, L., Pushkin, A. V., and Pilch, P. F. (1995) Biochem. J. 307, 383-390[Medline] [Order article via Infotrieve]
  27. Kandror, K. V., and Pilch, P. F. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8017-8021[Abstract]
  28. Pear, W. S., Nolan, G. P., Scott, M. L., and Baltimore, D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8392-8396[Abstract/Free Full Text]
  29. Stephens, J. M., Lee, J., and Pilch, P. F. (1997) J. Biol. Chem. 272, 971-976[Abstract/Free Full Text]
  30. Green, H., and Kehinde, O. (1974) Cell 1, 113-116
  31. Cornelius, P., Marlowe, M., Lee, M. D., and Pekala, P. H. (1990) J. Biol. Chem. 265, 20506-20516[Abstract/Free Full Text]
  32. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  33. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[CrossRef][Medline] [Order article via Infotrieve]
  34. Ogawa, W., Matozaki, T., and Kasuga, M. (1998) Mol. Cell. Biochem. 182, 13-22[CrossRef][Medline] [Order article via Infotrieve]
  35. Keller, S. R., Scott, H. M., Mastick, C. C., Aebersold, R., and Lienhard, G. E. (1995) J. Biol. Chem. 270, 23612-23618[Abstract/Free Full Text]
  36. Ross, S. A., Scott, H. M., Morris, N. J., Leung, W. Y., Mao, F., Lienhard, G. E., and Keller, S. R. (1996) J. Biol. Chem. 271, 3328-3332[Abstract/Free Full Text]
  37. Scherer, P. E., Lisanti, M. P., Baldini, G., Sargiacomo, M., Mastick, C. C., and Lodish, H. F. (1994) J. Cell Biol. 127, 1233-1243[Abstract]
  38. Kandror, K. V., Stephens, J. M., and Pilch, P. F. (1995) J. Cell Biol. 129, 999-1006[Abstract]
  39. Garcia de Herreros, A., and Birnbaum, M. J. (1989) J. Biol. Chem. 264, 19994-19999[Abstract/Free Full Text]
  40. Kaestner, K. H., Christy, R. J., and Lane, M. D. (1991) Proc. Natl. Acad. Sci. U. S. A. 87, 251-255[Abstract]
  41. Haney, P. M., Slot, J. W., Piper, R. C., James, D. E., and Mueckler, M. (1991) J. Cell Biol. 114, 689-699[Abstract]
  42. Hudson, A. W., Ruiz, M., and Birnbaum, M. J. (1992) J. Cell Biol. 116, 785-797[Abstract]
  43. Kotliar, N., and Pilch, P. F. (1992) Mol. Endocrinol. 6, 337-345[Abstract]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.