©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Insulin Regulates Transcription of the CCAAT/Enhancer Binding Protein (C/EBP) , , and Genes in Fully-differentiated 3T3-L1 Adipocytes (*)

(Received for publication, August 19, 1994; and in revised form, October 17, 1994)

Ormond A. MacDougald (§) Peter Cornelius(§)(¶) Raymond Liu (**) M. Daniel Lane (§§)

From the Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The effect of insulin on expression of CCAAT/enhancer binding protein (C/EBP) alpha, beta, and was investigated in fully-differentiated 3T3-L1 adipocytes. Treatment of adipocytes with insulin stimulated rapid dephosphorylation of C/EBPalpha, and repressed the expression of C/EBPalpha within 2-4 h, with >90% suppression occurring at 24 h. While insulin induced expression of C/EBPbeta and C/EBP within 1 h and caused a >20-fold increase by 4 h, expression returned to nearly pretreatment levels by 24 h. The insulin concentration dependence of these effects was consistent with involvement of the insulin receptor. Gel shift analysis revealed that 6 h of insulin treatment decreased the binding of nuclear C/EBPalpha while increasing binding of nuclear C/EBPbeta and C/EBP. The reciprocal effects of insulin on the steady-state levels of C/EBP transcription factors can be accounted for kinetically and quantitatively by changes in their mRNA levels, which can be accounted for by effects on gene transcription. The effects of insulin on adipocyte gene transcription (e.g. GLUT4) may be mediated, at least in part, by down-regulation of C/EBPalpha and/or its dephosphorylation.


INTRODUCTION

A large body of evidence has shown that differentiation of 3T3 preadipocytes into adipocytes in cell culture serves as a faithful model of the differentiation process in vivo (reviewed in (1) ). Two cell lines, i.e. the 3T3-L1 and 3T3-F442A, have been most extensively characterized and are now widely used for the study of preadipocyte differentiation(2, 3, 4, 5, 6, 7) . When subjected to an appropriate differentiation protocol, 3T3 preadipocytes lose their fibroblastic features, round-up, and acquire the morphological and biochemical phenotype of adipocytes. Concomitant with the accumulation of cytoplasmic triacylglycerol is the coordinate expression of virtually every enzyme of the pathways of de novo fatty acid and triacylglycerol biosynthesis. In addition, differentiating preadipocytes acquire the complement of proteins for lipolysis of triacylglycerol, uptake, and intracellular translocation of fatty acids, as well as responsiveness to lipogenic and lipolytic hormones (1) . It has been established that these coordinate changes in the cellular levels of proteins that give rise to the adipocyte phenotype are almost entirely due to changes in the transcription rates of the corresponding genes(8, 9) .

Although the sequence of events which prompt preadipocyte differentiation is not fully understood, compelling evidence indicates that C/EBPalpha (^1)plays an essential role in this process(10, 11, 12, 13, 14, 15) . C/EBPalpha appears to function both by inhibiting the clonal expansion that precedes terminal differentiation (16) and by activating the coordinate expression of a group of adipocyte genes whose promoters possess C/EBP-binding sites(10, 11, 17, 18) . Unequivocal proof that C/EBPalpha is essential for differentiation was obtained using the antisense RNA approach(13, 19) . Expression of a truncated C/EBPalpha antisense RNA in 3T3-L1 preadipocytes blocked expression of C/EBPalpha, transcription of several adipocyte genes (i.e. 422/aP2, SCD1, and GLUT4), and accumulation of cytoplasmic triacylglycerol(13) . More recently it was shown that expression of C/EBPalpha is not only necessary, but is sufficient, to induce preadipocyte differentiation(14, 15) . Thus, isopropyl-1-thio-beta-D-galactopyranoside-induced expression of C/EBPalpha by 3T3-L1 preadipocytes harboring a LacSwitch C/EBPalpha expression vector system caused expression of adipocyte markers and acquisition of the adipocyte phenotype(14) . In addition, ectopic expression of C/EBPalpha using a retroviral expression vector was shown to induce adipogenesis in a variety of cell lines(15) .

C/EBPalpha mRNA has been shown to give rise to two major alternative translation products, p42 and p30(18, 20) , both of which are expressed by 3T3-L1 adipocytes, liver, and white adipose tissue. The two C/EBP isoforms possess some similar and some dissimilar functional properties. While both isoforms transactivate the promoters of certain adipocyte genes (18) , only p42 is antimitotic and capable of terminating clonal expansion. Moreover, the relative levels of expression of the two isoforms differ during hepatic development and during the differentiation of 3T3-L1 preadipocytes raising the possibility that they play different roles during differentiation of these cell types. Although expression of p30 precedes expression of p42 during development and differentiation, both isoforms are expressed by terminally differentiated adipocytes and hepatocytes.

The C/EBP family of transcription factors share amino acid sequence similarity within their C-terminal basic region/leucine zipper domain, which confers the capacity to dimerize and bind DNA (reviewed by McKnight(21) ). Members of the C/EBP family can form homo- and heterodimers, all of which can bind to the same cis-regulatory elements within the promoters/enhancers of genes regulated by the C/EBP's. The temporal expression of C/EBPbeta and C/EBP during differentiation of 3T3-L1 preadipocytes, and the presence of a C/EBP-binding site within the C/EBPalpha gene promoter, has led to the hypothesis that C/EBPbeta and/or C/EBP may be responsible for the activation of expression of the C/EBPalpha gene(12) . Further work will be required to clarify the roles of C/EBPbeta and C/EBP in preadipocyte differentiation.

While members of the C/EBP family have been implicated in the differentiation of 3T3-L1 preadipocytes, the role(s) of these transcription factors in the mature, fully-differentiated adipocyte has not been extensively investigated. Recently, we reported that glucocorticoids exert rapid reciprocal effects on the expression of C/EBPalpha, and , largely by altering transcription of the corresponding genes(22) . In view of the established roles of insulin and glucocorticoids on carbohydrate and lipid metabolism in the adipocyte (23, 24, 25, 26) and the fact that a number of genes which function in these processes are regulated by C/EBPalpha, we examined the effect of insulin on the expression of C/EBPalpha, beta and in terminally-differentiated 3T3-L1 adipocytes. Our results suggest that insulin regulates C/EBPalpha through at least three mechanisms: post-translational modification, transcription, and through induction of a dominant negative transcription factor (LIP).


EXPERIMENTAL PROCEDURES

Cell Culture

3T3-L1 preadipocytes were maintained and induced to differentiate into adipocytes as described previously(27) , except that insulin was withdrawn from the medium on day 4. Using this protocol, >95% of the cells begin to acquire the adipocyte phenotype 3-4 days after initiating differentiation. Fully-differentiated 3T3-L1 adipocytes, 11-14 days after induction of differentiation, were switched to fresh media (Dulbecco's modified Eagle's media, 10% fetal bovine serum) 16-24 h before subjecting cells to insulin or IGF-1 treatment. Insulin was dissolved in 0.1 M HCl and IGF-1 was dissolved in 10 mM acetic acid. Okadaic acid (Boehringer Mannheim) was dissolved in dimethyl sulfoxide.

Analysis of RNA

Cellular RNA was isolated from 3T3-L1 cells using guanidine thiocyanate followed by ultracentrifugation through CsCl(28) . The amount of C/EBPalpha and C/EBP mRNA was assessed by Northern blot analysis(28) . Total RNA (20 µg) was separated by electrophoresis in horizontal 1.2% agarose gels containing 6.5% formaldehyde. RNA was transferred to nylon membranes (Hybond-N; Amersham Corp.) overnight and covalently bonded to the membrane by exposure to ultraviolet light (UV Stratalinker 1800; Stratagene). Blots were prehybridized for at least 6 h (42 °C) in a solution containing 50% formamide, 4 times SSC, 5 times Denhardt's solution, 50 mM phosphate buffer, pH 7.0, 100 µg/ml yeast tRNA, 0.5 mg/ml sodium pyrophosphate, and 1% sodium dodecyl sulfate (SDS). Hybridization was carried out for at least 16 h at 42 °C in an identical solution containing 4 times 10^6 dpm of labeled probe per ml. In general, hybridized blots were washed three times in a solution containing 0.5 times SSC, 0.1% SDS at 60 °C. Autoradiography was at -80 °C with Kodak X-Omat AR film (Eastman Kodak Co.) and an intensifying screen for the times indicated in the figure legends. Results were quantified using a laser densitometer (LKB Ultroscan XL).

The DNA fragment used as a probe for C/EBPalpha mRNA was an 900-base pair SacI/HindIII fragment complementary to the 3` end of the C/EBPalpha coding region, as well as part of the 3`-untranslated region (+1175 to +2078 nucleotides relative to transcriptional start site). The cDNA fragment for C/EBPbeta is full length and was cloned from a 3T3-L1 adipocyte library as reported previously(29) . The cDNA fragment used as a probe for C/EBP mRNA was as described(12) . Isolated C/EBPalpha, C/EBPbeta, or C/EBP DNA probes were labeled to high specific activity (1 times 10^9 dpm/µg) by random hexamer priming(30) .

Cell Lysates and Immunoblotting

3T3-L1 adipocyte monolayers (10 cm) were washed once with 10 ml of phosphate-buffered saline and scraped in 1 ml of a lysis buffer containing 1% SDS, 60 mM Tris-Cl, pH 6.8. Lysates were boiled for 3 min, vortexed, then boiled for an additional 7 min prior to storing at -35 °C. Western analysis was performed as described previously(13) . Results were quantified using laser densitometry. When presented graphically for C/EBPalpha, the results represent the sum of p30 and p42, and when presented for C/EBPbeta, the results represent the sum of LAP and LIP.

Preparation of Antibodies

Peptides corresponding to amino acids 278-295 (LRNLFKQLPEPLLASAGH) of C/EBPbeta and 115-130 (ARGPLKREPDWGDGDA) of C/EBP were synthesized (Protein Peptide Facility; Department of Biological Chemistry, Johns Hopkins University School of Medicine) with an additional N-terminal cysteine residue and then cross-linked through the N-terminal-SH to keyhole limpet hemocyanin. Immunization of rabbits and collection of preimmune and immune sera were performed by HRP (Denver, PA). Antiserum used for Western blots or supershifting was specific for either C/EBPbeta or C/EBP and interactions with other C/EBP isoforms were not observed.

Immune serum against a synthetic peptide corresponding to an internal amino acid sequence of C/EBPalpha (present in both p42 and p30) was prepared as described previously(18) . In some experiments, immune sera to C/EBPbeta and C/EBP were generously provided by Dr. Steve McKnight(12) .

Preparation of Nuclear Extracts

Nuclei were purified from 3T3-L1 cells by a modification (31) of the procedure of Dignam et al.(32) . Nuclear extracts were prepared as described by Lavery and Schibler (33) using a 1 times NUN solution (0.3 M NaCl, 1 M urea, 1% Nonidet P-40, 25 mM HEPES, pH 7.9, and 1 mM dithiothreitol). Protein concentration was determined using the BCA Protein Assay Reagent (Pierce Chemical Co.) and ranged from 8.5 to 10 mg/ml.

Gel Shift Analysis

A double stranded oligonucleotide corresponding to the C/EBP-binding site in the 422(aP2) promoter (10) was labeled using [alpha-P]ATP and DNA polymerase (Klenow fragment; New England Biolabs). The gel shift mixture (40 µl) contained 0.33 M urea, 0.1 M NaCl, 0.33% Nonidet P-40, 25 mM HEPES, pH 7.9, 10 mM dithiothreitol, 10% glycerol, 5 µg of acetylated bovine serum albumin, 3 µg of poly[d(I-C)], and 3.0 times 10^5 dpm of P-labeled oligonucleotide. This mixture (including 6 µl of antiserum and/or preimmune sera as indicated) was incubated on ice for 30 min, then at room temperature for 90 min prior to electrophoresis on 6% polyacrylamide gels.

Nuclear Run-on Transcription Analysis

After the indicated treatments of 3T3-L1 adipocytes (day 12), nuclei were isolated from transcriptional run-on assays as described by Cornelius et al.(34) . Equal numbers of nuclei were incubated for 25 min at 28 °C in the presence of [alpha-P]UTP (6000 Ci/mmol; DuPont). Reactions were terminated by addition of RNase-free DNase, and then P-labeled RNA was isolated by guanidine thiocyanate extraction and CsCl density gradient centrifugation. After partial hydrolysis in 0.2 M NaOH, P-labeled RNA was extracted once with chloropane (35) and recovered by ethanol precipitation. Labeled transcripts (20 times 10^6 dpm/ml) were hybridized for 72 h with cDNAs covalently linked to nylon membrane (Hybond-N; Amersham). Blots were washed to high stringency, and hybridized RNA was visualized by autoradiography.


RESULTS

Effects of Insulin on the Cellular Levels of C/EBPalpha, C/EBPbeta, and C/EBP

In view of the well documented effects of insulin on adipose tissue metabolism and of the activating effect of C/EBPalpha on adipocyte gene transcription during preadipocyte differentiation, it was of interest to assess the effect of insulin on the expression of C/EBP family members in fully-differentiated 3T3-L1 adipocytes. As shown in Fig. 1, A and B, treatment of adipocytes with insulin suppressed expression of both the 42- and 30-kDa C/EBPalpha isoforms. A decrease was first observed between 2 and 4 h after the addition of insulin, with repression of C/EBPalpha continuing for at least 24 h. In contrast, insulin caused an increase in C/EBPbeta (LAP and LIP; (36) and (37) ) that was observed at 2 h and was maximal at 4 h. Expression of C/EBP was also induced by insulin within 2 h and was sustained for 10 h. Induction of both C/EBPbeta and C/EBP by insulin was transient, with expression decreasing toward pretreatment levels by 24 h. These results show that insulin reciprocally regulates expression of at least 3 members of the C/EBP family of transcription factors in fully differentiated 3T3-L1 adipocytes.


Figure 1: Effect of insulin on the expression of C/EBP. A, 3T3-L1 adipocytes in monolayer culture were treated with 167 nM insulin (INS) for the indicated times. Whole cell lysates containing equal cell equivalents (200 µg of protein) were subjected to SDS-PAGE, and immunoblotted using antisera against C/EBPalpha, C/EBPbeta, and C/EBP. These results are representative of at least six independent time course experiments. alpha42 and alpha30 refer to p42 and p30 isoforms, respectively. LAP and LIP refer to the liver activator protein and liver inhibitory protein of C/EBPbeta, respectively; and refers to C/EBP. B, results in A were quantified by laser densitometry and the results are shown graphically relative to the maximal level of expression of each C/EBP.



Axel Kahn and colleagues (38) have found that insulin can alter hepatic gene expression by glucose-dependent or glucose-independent mechanisms. To ascertain whether glucose is required for the regulation of C/EBPalpha by insulin, cells were incubated overnight in glucose-free media (with or without pyruvate) prior to insulin treatment. Western blot analysis showed that while overall expression of C/EBPalpha was repressed by incubation in glucose-free medium, regulation of C/EBPalpha by insulin was identical to that shown in Fig. 1. This includes the rapid effect of insulin on post-translational modification and the subsequent suppression of C/EBPalpha protein. Therefore, insulin regulates C/EBPalpha through glucose-independent mechanisms.

Inspection of Fig. 1A reveals that p30 consists of two bands, and that the top band is absent after 2 h of insulin treatment. A more extensive time course of insulin effects on expression of C/EBPalpha in 3T3-L1 adipocytes reveals that the top band of p30 is about 50% depleted by 15 min, and is completely absent at 30 min (Fig. 2). The corresponding increase in the bottom band suggests that these mobilities might reflect structural differences within p30, perhaps due to post-translational modification. A similar, although less obvious, shift in mobility of p42 occurs with similar kinetics (Fig. 2). Evidence described in a later section suggests that insulin treatment alters the phosphorylation of the two C/EBPalpha isoforms.


Figure 2: Rapid effect of insulin on C/EBPalpha. 3T3-L1 adipocytes in monolayer culture were treated with insulin (167 nM) for the indicated times. Whole cell lysates containing equal cell equivalents 200 µg of protein) were subjected to SDS-PAGE, and immunoblotted using antisera against C/EBPalpha.



Effect of Insulin on the Binding of C/EBP Isoforms to a C/EBP Binding-site Oligonucleotide

Gel shift experiments were performed to verify that the insulin-induced changes in C/EBP isoform levels described above correlate with changes in their DNA binding capacity. Thus, nuclear extracts, prepared from 3T3-L1 adipocytes that had been treated or not with insulin for 6 h, were subjected to gel shift analysis using an oligonucleotide probe corresponding to the C/EBP-binding site in the 422(aP2) promoter(10) . All three C/EBP isoforms (alpha, beta, and ) bind at this site. Although the gel shift banding patterns were complex (Fig. 3, lanes 2 and 3), use of isoform-specific antibodies and changes in isoform level due to insulin treatment made it possible to identify DNA-protein complexes containing each of the C/EBP isoforms. While addition of preimmune sera stabilized the binding of C/EBP isoforms to this probe, the relative intensities of the bands were similar, and no ``new'' bands were observed (Fig. 3, lanes 4 and 5). Supershift experiments with antisera against C/EBPbeta and C/EBP gave rise to DNA-protein complexes with low mobility. This facilitated detection of DNA-protein complexes containing C/EBPalpha and revealed that 6 h of insulin treatment suppressed the relative amount of C/EBPalpha available for binding to the C/EBP-binding site (Fig. 3, compare lanes 6 and 7). In a similar fashion, supershifting C/EBPalpha and C/EBP revealed that insulin increased the relative amount of C/EBPbeta-containing DNA-protein complexes (Fig. 3, compare lanes 8 and 9), and supershifting C/EBPalpha and C/EBPbeta revealed that insulin increased the relative amount of C/EBP-containing DNA-protein complex (Fig. 3, compare lanes 10 and 11). Supershifting with antiserum specific to the N-terminal region of C/EBPbeta (recognizes only LAP) revealed that the band of highest mobility contains LIP/LIP homodimers, (^2)and it is presumed that the intermediate C/EBPbeta-containing band is composed of LAP/LIP heterodimers. Therefore, LIP appears to predominate over LAP (and C/EBP) in both the basal and insulin-stimulated states (see ``Discussion''). At 24 h, suppression of C/EBPalpha and induction of C/EBP could still be observed, however, binding of C/EBPbeta was the same as in control adipocyte extracts (data not shown). It should be noted that all of the oligonucleotide-protein complexes detected by gel shift analysis with nuclear extracts from control and insulin-treated adipocytes were supershifted by a combination of antibodies to all three C/EBP isoforms (Fig. 3, lanes 12 and 13). Identical gel shift patterns were observed with the C/EBP-binding site from the C/EBPalpha promoter (data not shown). Taken together, these findings are consistent with the results of Western blot analysis, and show that insulin induces changes in the relative cellular levels of C/EBP transcription factors.


Figure 3: Gel shift analysis of C/EBP isoforms from untreated 3T3-L1 adipocytes (ADIP) and those treated with insulin. Nuclei were isolated and nuclear extracts were prepared from untreated control adipocytes 11 days after initiating differentiation or adipocytes treated with 167 nM insulin for 6 h (INS). Gel shift analysis was performed using 3.0 times 10^5 dpm of a P-labeled oligonucleotide corresponding to the C/EBP-binding site from the 422/aP2 gene promoter, and 8 µg of nuclear protein. Oligonucleotide-protein complexes were separated on a 6% polyacrylamide gel at 9 V/cm for 4 h. Free-labeled oligonucleotide was run off the gel. Autoradiography was performed at -80 °C for 16 h. Supershifting was performed using the indicated combinations of antiserum (2 µl each) with preimmune (PI) serum added to bring the total sera volume to 6 µl. alpha refers to antiserum to C/EBPalpha; beta to antiserum to C/EBPbeta; and to antiserum to C/EBP.



Concentration Dependence and Specificity of the Insulin Response

To ascertain whether the insulin-induced changes in cellular levels of C/EBPalpha, C/EBPbeta, and C/EBP are mediated by the insulin receptor or by the insulin-like growth factor-1 (IGF-1) receptor, the dependence of these changes on concentration of insulin or IGF-1 was assessed. As illustrated in Fig. 4, the concentration of insulin for half-maximal effects on the expression of C/EBPalpha, C/EBPbeta, and C/EBP occurred around 3-10 nM insulin. This is in good agreement with the reported K(d) for insulin with the insulin receptor of intact 3T3-L1 adipocytes(39) . While IGF-1 did not influence the expression of C/EBPalpha or C/EBPbeta in these cells, IGF-1 induced expression of C/EBP with a concentration dependence consistent with an effect mediated by the IGF-1 receptor. These findings support the view that insulin regulates expression of these C/EBP family members through the insulin receptor, and that IGF-1 also induces C/EBP through the IGF-1 receptor.


Figure 4: Effect of insulin or IGF-1 concentration on expression of C/EBPalpha (A), C/EBPbeta (B), and C/EBP (C). The indicated concentrations of insulin or IGF-1 were added to 3T3-L1 adipocytes for 24 h (C/EBPalpha) or 2 h (C/EBPbeta and C/EBP) on day 11 after initiating differentiation. After lysis and Western analysis, results were quantified by laser densitometry and are presented relative to the maximal level of expression for each C/EBP. Results from the insulin concentration dependence are representative of three independent experiments while the IGF-1 experiment was performed once. The half-maximal insulin effect on the expression of C/EBPalpha, C/EBPbeta, and C/EBP was observed at 3-10 nM insulin. While IGF-1 induced expression of C/EBP with a half-maximal effect at 10 nM, IGF-1 did not influence the expression of either C/EBPalpha or C/EBPbeta.



Effect of Insulin on mRNA Levels of C/EBPalpha, beta, and

To determine whether changes in the expression of the C/EBP proteins were due to changes in steady-state levels of mRNA, Northern analysis was performed on total RNA isolated from fully-differentiated 3T3-L1 adipocytes treated with insulin for different periods of time. Analysis of C/EBPalpha, C/EBPbeta, and C/EBP mRNA levels revealed that the effects of insulin on these C/EBP isoforms could be accounted for by changes in the cellular levels of their respective mRNAs (Fig. 5, A and B). Insulin caused a decrease in the expression of C/EBPalpha mRNA, the greatest decrease occurring between 1 and 2 h of treatment, with low levels of expression persisting until at least 24 h. In contrast, insulin transiently induced the level of C/EBPbeta and C/EBP mRNAs, which reached a maximum at 2 and 1 h, respectively (Fig. 5B). It is evident, therefore, that insulin regulates expression of C/EBPalpha, C/EBPbeta, and C/EBP primarily by regulating the steady-state levels of their respective mRNAs.


Figure 5: Comparison of the effect of insulin on the kinetics of expression of C/EBPalpha, C/EBPbeta, and C/EBP mRNAs. A, insulin (167 nM) was added to 3T3-L1 adipocytes on day 12 after initiation of differentiation, and total RNA was prepared from two independent cell monolayers after 0, 1, 2, 4, 6, 10, or 24 h. Equal amounts of RNA (20 µg) were electrophoresed, and analyzed by Northern blotting using DNA fragments complementary to C/EBPalpha, C/EBPbeta, or C/EBP mRNAs. Results are representative of three independent experiments. Autoradiography was for 48 h at -80 °C for C/EBPalpha and C/EBP; 24 h for C/EBPbeta. B, results in A were quantified by laser densitometry and the mean result ± range are shown graphically relative to the maximal level of expression.



Effect of Insulin on Transcription of the C/EBPalpha, C/EBPbeta, and C/EBP Genes

To ascertain whether the rapid changes in the mRNA levels for C/EBPalpha, C/EBPbeta, and C/EBP were due to changes in gene transcription, the effect of insulin on nuclear run-on transcription was evaluated. As shown in Fig. 6, insulin markedly (65%) repressed C/EBPalpha gene transcription within 1 h and this suppressed rate of transcription was maintained for at least 4 h. In contrast, insulin induced (3-fold) transcription of both the C/EBPbeta and C/EBP genes by 1 h (Fig. 6). Other experiments showed induction levels of up to 8-fold. These changes in transcription precede and are comparable in magnitude to effects of insulin on the cellular levels of C/EBPalpha, C/EBPbeta, and C/EBP mRNAs (see Fig. 5). Therefore, insulin rapidly regulates the expression of C/EBPalpha, C/EBPbeta, and C/EBP mRNAs, in large part, by altering rates of transcription.


Figure 6: A) Effect of insulin on nuclear run-on transcription of the C/EBPalpha, C/EBPbeta, and C/EBP genes. Nuclei were prepared from fully-differentiated 3T3-L1 adipocytes that were untreated, or treated with 167 nM insulin for 1 or 4 h. After incubation of nuclei with [P]UTP and isolation of RNA, 18 times 10^6 dpm of P-labeled RNA were used for hybridization of blots containing 1 mg of DNA complementary for C/EBPalpha, C/EBPbeta, C/EBP, pBluescript, or genomic DNA. Filters were washed to high stringency, and exposed to film for 7 days at -80 °C. Results are representative of two independent experiments. B, results in A were quantified by laser densitometry, normalized to the genomic signal, and the results for each C/EBP homologue expressed in arbitrary units.



Effect of Insulin on the Phosphorylation State of C/EBPalpha

As shown above (Fig. 2) insulin rapidly (within 15 min) alters the mobility of p30 and p42 by SDS-PAGE. Following exposure to insulin the initial double-band pattern of each isoform reverts to a single, higher-mobility band, i.e. corresponding to 41 and 29 kDa, reaching 50 and 100% completion within 15 and 30 min, respectively. The rapidity of the response to insulin and the fact that blocking protein synthesis with cycloheximide did not prevent the response (results not shown) suggested that the changes in mobility of the C/EBPalpha isoforms were the result of post-translational modification, such as phosphorylation.

To ascertain whether the mobility differences were due to phosphorylation of the C/EBPalpha isoforms, the effect of okadaic acid, a potent inhibitor of serine/threonine protein phosphatases 1 and 2A(40, 41) , was tested in the absence and presence of insulin. Insulin treatment generated the higher mobility forms of the two C/EBPalphas (most evident with p30), whereas treatment with 1.5 µM okadaic acid for 45 min, either in the absence or presence of insulin, gave rise to the lower mobility forms (Fig. 7A).


Figure 7: Effect of insulin and okadaic acid on the post-translational modification of C/EBPalpha. A, 3T3-L1 adipocytes were treated for 45 min with Me(2)SO (vehicle for okadaic acid; CONT), 167 nM insulin (INS), 1.5 µM okadaic acid (OA), insulin after a 5-min pretreatment with okadaic acid (O+A), or okadaic acid after a 5-min pretreatment with insulin (I+O). Whole cell lysates containing equal cell equivalents (200 µg of protein) were subjected to SDS-PAGE, and immunoblotted using antisera generated against C/EBPalpha. B, 3T3-L1 adipocytes were incubated under serum-free conditions overnight. After a wash in phosphate-free, serum-free media, adipocytes were incubated with [P]orthophosphate for 2 h, then insulin or not for another hour. C/EBPalpha was immunoprecipitated with antiserum against C/EBPalpha using protein A-Sepharose, separated by SDS-PAGE, and visualized with autoradiography at -80 °C. C, nuclear extracts were prepared from adipocytes treated with 1.5 µM okadaic acid for 45 min. After precipitation with 12.5% trichloroacetic acid, the pellet was rinsed with cold acetone, and dissolved in 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl(2), 1 mM dithiothreitol, and 0.1% SDS at 37 °C for 8 h. After addition of Triton X-100 to 1%, 50 units of calf intestinal phosphatase were added to half the sample prior to an overnight incubation at 37 °C. C/EBPalpha was analyzed following SDS-PAGE by Western blot analysis. All results above are representative of at least two independent experiments.



Since inhibition of a phosphatase (by okadaic acid) might be expected to have the opposite effect of insulin, it follows that insulin may function to activate a type 1 or 2A protein phosphatase. Activation of protein phosphatase type 1 by insulin is well documented in several cell types(42, 43) , including 3T3-L1 adipocytes(44) . Moreover, protein phosphatase type 1 has been shown to be regulated during the cell cycle(45) , and this is correlated with the predominance of the higher mobility band in elutriation experiments. (^3)Whether this phosphatase acts to directly dephosphorylate C/EBPalpha or acts through a second messenger pathway which leads to dephosphorylation remains unclear.

Ex vivo labeling of 3T3-L1 adipocytes with [P]orthophosphate followed by immunoprecipitation and SDS-PAGE revealed that both the 42- and 30-kDa forms of C/EBPalpha are highly phosphorylated. Nevertheless, the gross level of phosphorylation of both isoforms did not appear to be affected by insulin treatment (Fig. 7B). It is possible that an insulin (and okadaic acid-)-sensitive phosphatase targets only one of multiple phosphorylation sites on C/EBPalpha and would, therefore, only lead to small fractional changes which would make the dephosphorylation event difficult to detect. Phosphoamino acid analysis of p30 from 293 cells transiently transfected with a p30 expression vector showed that this isoform is phosphorylated on serine and threonine (in an 1:1 ratio), but not on tyrosine.^3 As expected, inhibition of tyrosine kinase activity with genestein (Calbiochem) inhibited the rapid effects of insulin on C/EBPalpha post-translational modification (results not shown). Further evidence that post-translational modification of C/EBPalpha is due to phosphorylation is provided in Fig. 7C. Nuclear extracts from 3T3-L1 adipocytes treated with okadaic acid, and prepared in the presence of phosphatase inhibitors (30 mM beta-glycerol phosphate and 1 mM orthovanadate) gave rise to the low mobility band of both C/EBPalpha isoforms. When the extracts were treated with calf intestinal phosphatase, the low mobility band was lost and the high mobility band was observed (Fig. 7C). These experiments strongly suggest that insulin, in addition to suppressing the expression of C/EBPalpha within several hours, also acutely stimulates dephosphorylation of both C/EBPalpha isoforms.

Temporal Relationship of Down-regulation of C/EBPalpha and GLUT4 mRNA Caused by Insulin

In view of the findings that both C/EBPalpha (Fig. 5) and GLUT4 mRNA(46) ; Fig. 8) are down-regulated by insulin in terminally differentiated 3T3-L1 adipocytes, it was of interest to ascertain whether the kinetics of these processes were compatible with a causal relationship. It should be noted that during differentiation of 3T3-L1 preadipocytes, C/EBPalpha serves as a transactivator of the GLUT4 gene whose promoter possesses a C/EBP-binding site(13, 17) . As shown in Fig. 8, following the insulin-induced drop in the C/EBPalpha message, the decline of C/EBPalpha protein is virtually coincident with the decline of GLUT4 message in 3T3-L1 adipocytes. Although this result is consistent with trans-activation of the GLUT4 gene by C/EBPalpha in the adipocyte, the possibility that dephosphorylation of C/EBPalpha is involved cannot be ruled out (see ``Discussion'').


Figure 8: Kinetic analysis of the effects of insulin on expression of C/EBPalpha mRNA and protein, and GLUT4 mRNAs. 3T3-L1 adipocytes were treated with 167 nM insulin for various times prior to preparation of whole cell lysates or total RNA. The amount of C/EBPalpha protein and C/EBPalpha or GLUT4 mRNAs were analyzed by immunoblot and Northern analyses, respectively. Protein and mRNA levels were quantified using laser densitometry and are represented graphically relative to their maximal levels of expression.



To ascertain whether long-term exposure of 3T3-L1 adipocytes to insulin affects expression of the C/EBP isoforms and the adipocyte-marker GLUT4, preadipocytes were subjected to the standard differentiation protocol, which includes insulin, after which the differentiated adipocytes were maintained for 4 days in medium with or without insulin. Northern analysis revealed that insulin treatment beyond day 4 of adipocyte differentiation markedly suppressed the expression of C/EBPalpha and GLUT4 mRNAs (Fig. 9A) without affecting the expression of C/EBPbeta or C/EBP (Fig. 9B). Some cells were treated with or without insulin for an additional 4 days, i.e. to day 12, at which time cell monolayers were stained for triacylglycerol with oil red-O. Cell monolayers chronically treated with insulin had fewer adipocytes with large unilocular triacylglycerol vacuoles, and had a higher proportion of adipocytes with a multilocular adipocyte phenotype. These findings are consistent with a causal relationship between insulin-induced suppression of C/EBPalpha, and the reduced levels of GLUT4 mRNA and other mRNAs (e.g. SCD1 mRNA; results not shown), which give rise to the adipocyte phenotype.


Figure 9: Effect of chronic exposure to insulin on the expression of C/EBPalpha, C/EBPbeta, C/EBP, and GLUT4. 3T3-L1 preadipocytes were differentiated by the standard protocol until day 4. At this time, half of the cells continued to receive insulin every 2 days with feeding. Total RNA was harvested on day 0, and then daily starting at day 2. Northern analysis was used to evaluate the expression of: A, C/EBPalpha and GLUT4 mRNA levels, as well as, B, C/EBPbeta and C/EBP. Data were quantified using laser densitometry and are represented graphically in arbitrary units.




DISCUSSION

This study shows that insulin reciprocally regulates the gene encoding C/EBPalpha, and those encoding C/EBPbeta and C/EBP in fully-differentiated 3T3-L1 adipocytes. While insulin represses the expression of C/EBPalpha for at least 24 h, insulin rapidly and transiently induces the expression of C/EBPbeta and C/EBP (Fig. 1). These changes are due largely to changes in steady-state levels of their respective mRNAs (Fig. 5), which are correlated with changes in the rates of transcription of the corresponding C/EBP genes (Fig. 6). In addition to regulating C/EBPalpha by repressing its expression, insulin may also regulate the activity of C/EBPalpha itself by controlling its state of phosphorylation. Indeed, it has been reported (47) that C/EBPalpha can be phosphorylated in vitro on serine 299 by protein kinase C, and that phosphorylation attenuated its binding to DNA. Evidence presented in this paper indicates that C/EBPalpha exists in a phosphorylated state in 3T3-L1 adipocytes (Fig. 7B), and that insulin promotes its apparent dephosphorylation by an okadaic acid-sensitive phosphatase (Fig. 7A), presumably protein phosphatase 1 or 2A. Although insulin-activated dephosphorylation of C/EBPalpha has the potential to regulate transcription of adipocyte genes (e.g. GLUT4), the findings presented in this paper do not specifically address this issue. It should be noted, however, that the time frame within which apparent dephosphorylation of C/EBPalpha occurs is consistent with the rate at which GLUT 4 transcription and mRNA fall(46) . Thus, apparent dephosphorylation of C/EBPalpha is complete within 30 min (Fig. 2) and the rate of GLUT4 gene transcription reaches a minimum in less than 2 h (46) . In this connection, it has been reported that insulin transiently activates type 1 protein phosphatase in several cell types including 3T3-L1 adipocytes(42, 43, 44) . Insulin has also recently been shown to promote dephosphorylation and suppression of CREB (cAMP response element-binding protein) activity by a type 1 protein phosphatase(45) . Further work will be necessary to determine whether insulin-promoted dephosphorylation of C/EBPalpha, repressed transcription of the C/EBPalpha gene, or other factors are responsible for the physiological effects of insulin on the expression of GLUT4 and other adipocyte genes.

In addition to regulating the post-translational modification and transcription of C/EBPalpha, insulin transiently induces the expression of C/EBP, as well as both forms of C/EBPbeta (LAP and LIP). Western and gel-shift analyses suggest that the level of LIP predominates over that of LAP or C/EBP in both the basal and insulin-stimulated states ( Fig. 1and Fig. 3). Since LIP can act as a dominant negative inhibitor of C/EBP-regulated gene transcription by forming inactive heterodimers (37) , the induction of LIP by insulin would most likely offset (or at least dampen) the increases in LAP or C/EBP, and would further accentuate the loss of C/EBPalpha.

Reciprocal regulation of the C/EBP transcription factors is a recurring theme. For example, during the acute-phase response of liver or hepatocytes, cytokines suppress the expression of C/EBPalpha while markedly increasing the expression of C/EBPbeta and C/EBP(48) . This results in the induction of a number of acute-phase response proteins such as serum amyloid A(49, 50) , alpha(1)-acid glycoprotein (51, 52, 53) , and complement component C3(54) , whose gene promoters contain critical C/EBP-binding sites.

The C/EBP transcription factors are also reciprocally regulated in fully-differentiated adipocytes. For example, treatment of 3T3-L1 adipocytes with monocyte-conditioned medium (containing tumor necrosis factor alpha) causes an induction of C/EBPbeta while suppressing the expression of C/EBPalpha(55, 56, 57) . A variation of this reciprocity is observed with 3T3-L1 adipocytes treated with glucocorticoids(22) . While expression of C/EBPalpha is transiently decreased and C/EBP is transiently increased, no change is observed in the expression of C/EBPbeta. The current report demonstrates that in mature adipocytes, insulin also reciprocally regulates the C/EBPs. In this case, C/EBPalpha is persistently suppressed, and both C/EBPbeta and C/EBP are transiently induced. The fact that different hormones/cytokines give rise to similar (i.e. reciprocal) patterns of expression of the C/EBP isoforms both during (12) and after terminal cell differentiation (see above) suggests that the C/EBPs play a central role in controlling gene transcription in a variety of metabolic situations. Presumably, the unique, but overlapping, sets of genes that are transcriptionally activated or repressed in each metabolic state would depend on the specific combination of active trans-acting factors (e.g. the glucocorticoid receptor), the complement of C/EBP homo- and heterodimers, and the cis-elements in each of the gene promoters affected. This concept extends the hypothesis (58) that C/EBPalpha serves as a central regulator of energy metabolism.


FOOTNOTES

*
This work was supported in part by a research grant from National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a National Research Service award from the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health.

Supported by a National Research Service award from the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. Present address: The Procter & Gamble Co., Miami Valley Laboratories, 11810 E. Miami River Rd., Ross, OH 45061.

**
Supported by a summer student grant from the Juvenile Diabetes Foundation.

§§
To whom all correspondence should be addressed: Dept. of Biological Chemistry, The Johns Hopkins University, School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-3554; Fax: 410-955-0903.

(^1)
The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein; GLUT4, insulin-responsive glucose transporter; IGF-1, insulin-like growth factor-1; PAGE, polyacrylamide gel electrophoresis; LIP, liver inhibitory protein; LAP, liver activator protein.

(^2)
P. Cornelius and M. D. Lane, unpublished data.

(^3)
F.-T. Lin and M. D. Lane, unpublished results.


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