Cyclic AMP (cAMP)-Mediated Stimulation of Adipocyte Differentiation Requires the Synergistic Action of Epac- and cAMP-Dependent Protein Kinase-Dependent Processes

MOLECULAR AND CELLULAR BIOLOGY, June 2008, p. 3804–3816 Vol. 28, No. 110270-7306/08/$08.00�0 doi:10.1128/MCB.00709-07Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cyclic AMP (cAMP)-Mediated Stimulation of AdipocyteDifferentiation Requires the Synergistic Action of

Epac- and cAMP-Dependent ProteinKinase-Dependent Processes�

Rasmus Koefoed Petersen,2† Lise Madsen,1,3,4† Lone Møller Pedersen,1 Philip Hallenborg,1Hanne Hagland,3 Kristin Viste,3 Stein Ove Døskeland,3 and Karsten Kristiansen1*

Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark1;BioLigands, International Science Park Odense, DK-5230 Odense M, Denmark2; Department of Biomedicine,

University of Bergen, N-5009 Bergen, Norway3; and National Institute ofNutrition and Seafood Research, 5817 Bergen, Norway4

Received 23 April 2007/Returned for modification 23 July 2007/Accepted 17 March 2008

Cyclic AMP (cAMP)-dependent processes are pivotal during the early stages of adipocyte differentiation. Weshow that exchange protein directly activated by cAMP (Epac), which functions as a guanine nucleotideexchange factor for the Ras-like GTPases Rap1 and Rap2, was required for cAMP-dependent stimulation ofadipocyte differentiation. Epac, working via Rap, acted synergistically with cAMP-dependent protein kinase(protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate Rho andRho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho-kinase impairedproadipogenic insulin/insulin-like growth factor 1 signaling, which was restored by activation of Epac. Thisinterplay between PKA and Epac-mediated processes not only provides novel insight into the initiation andtuning of adipocyte differentiation, but also demonstrates a new mechanism of cAMP signaling whereby cAMPuses both PKA and Epac to achieve an appropriate cellular response.

Adipocytes are derived from multipotent mesenchymal stemcells in a process involving commitment to the adipocyte lin-eage followed by terminal differentiation of the committedpreadipocytes. The process is regulated via complex interac-tion of external and internal clues, where cell shape and cy-toskeletal tension converging on regulation of Rho and Rho-kinase activity have been demonstrated to play pivotal roles(48, 63). Whereas our understanding of the early steps oflineage determination still is limited, regulatory cascades con-trolling terminal adipocyte differentiation have been eluci-dated in great detail, particularly the sequential action of dif-ferent transcription factors culminating in the expression ofadipocyte-specific genes (25, 30, 58). Much information onterminal adipocyte differentiation has been obtained usingmodel cell lines such as 3T3-L1 and 3T3-F442A or mouseembryo fibroblasts (MEFs). In both MEFs and 3T3-L1 prea-dipocytes, terminal differentiation is initiated upon treatmentwith fetal calf serum, glucocorticoids, and high levels of insulinor physiological concentrations of insulin-like growth factor 1(IGF-1). Factors that increase cellular cyclic AMP (cAMP),such as isobutylmethylxanthine (IBMX) or forskolin, stronglyaccelerate the initiation of the differentiation program (forreview, see references 25 and 45).

Elevation of cellular cAMP concentration has been associ-

ated with crucial events in the early program of differentiation,such as suppression of Wnt10b (5) and Sp1 (64) and inductionof CCAAT/enhancer-binding protein � (C/EBP�) (10, 29, 70).Moreover, the transcriptional activity of peroxisome prolifera-tor-activated receptor � (PPAR�) is regulated synergisticallyby ligands and cAMP (32). In addition, cAMP has been impli-cated in the production of endogenous PPAR� ligand(s) oc-curring during the initial stages of differentiation (46, 67). ThecAMP-responsive element-binding protein (CREB) is a cen-tral transcriptional activator of the adipocyte differentiationprogram. Activated CREB induces expression of C/EBP�, trig-gering expression of a number of transcription factors, includ-ing C/EBP� and PPAR� (16, 64–66, 70, 72). Indeed, forcedexpression of constitutively active CREB can induce adipogen-esis, whereas expression of a dominant-negative form of CREBblocks differentiation (56). The importance of CREB is under-scored by the finding that adipocyte differentiation of CREB-deficient mouse embryo fibroblast is impaired (72) and thatsmall interfering RNA-mediated depletion of CREB and theclosely related activating transcription factor 1 (ATF1) blocksadipocyte differentiation (26). CREB was initially character-ized as a cAMP target whose transcriptional activity was stim-ulated by cAMP-dependent protein kinase (protein kinase A[PKA])-catalyzed phosphorylation on serine 133 (28), but in-sulin (Ins) signaling may also activate CREB in 3T3-L1 cellsthrough Ser-133 phosphorylation via the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway (40).

While cAMP signaling via PKA has been investigated fordecades, the complexity of cAMP signaling via interplay be-tween PKA and the exchange proteins directly activated bycAMP (Epac1 and Epac2) is only beginning to be understood.

* Corresponding author. Mailing address: Department of Biochem-istry and Molecular Biology, University of Southern Denmark, Cam-pusvej 55, DK-5230 Odense M, Denmark. Phone: 45 6550 2408. Fax:45 6550 2467. E-mail: [email protected].

† R.K.P. and L.M. contributed equally to this work.� Published ahead of print on 7 April 2008.

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Epac1 and Epac2 function as guanine nucleotide exchangefactors (GEFs) for the Ras-like small GTPases Rap1 and Rap2(6), and possibly Rit (60), and several cAMP-dependent pro-cesses are now believed to be modulated by Epac. Epac maymediate cAMP-dependent exocytosis (36, 37, 52) and integrin-dependent cell adhesion (17, 24, 54). Whereas Epac and PKAcan exert opposing effects in regulating downstream targetssuch as protein kinase B (PKB) (49), they act synergistically topromote PC-12 cell differentiation, as judged by neurite exten-sion (15).

The present work was undertaken to determine if Epac hadany role in cAMP-stimulated adipocyte differentiation of3T3-L1 preadipocytes and, if so, to dissect the contributions ofEpac and PKA. We demonstrate that cAMP stimulated adi-pocyte differentiation through the concerted action of PKAand Epac/Rap. A similar finding was made for cAMP-stimu-lated adipocyte differentiation of MEFs. While stimulation ofPKA activity was not required for the increased phosphoryla-tion of CREB during the initiation of adipocyte differentiation,it was important for the suppression of Rho/Rho-kinase activ-ity. Inhibition of Rho-kinase activity in 3T3-L1 preadipocytesdecreased Ins/IGF-1 signaling, but concomitant activation ofEpac restored Ins/IGF-1 sensitivity. Accordingly, adipocytedifferentiation was still Epac dependent when Rho-kinase wasinhibited, whereas PKA activity was dispensable under suchconditions. This interplay between PKA-, Epac-, and Rho-kinase-mediated processes provides novel insight into regula-tory circuits controlling the initiation of adipocyte differentia-tion and provides a new example of how cAMP can use bothPKA and Epac to achieve an appropriate cellular response.

MATERIALS AND METHODS

Plasmids. The retroviral expression plasmid encoding dominant-negativeRap1A (pBABE-Rap1-N17) was constructed by inserting the BamHI/XhoI frag-ment of pcDNA3-HA-Rap1A-N17 (kindly provided by Eva Pålsson-McDermott)into the BamHI/SalI sites of a polylinker-modified plasmid, pBABE-puro (kindlyprovided by Ormond MacDougald). The plasmid encoding dominant-negativeRhoA for retroviral expression (pLXSN-RhoA-N19) was constructed by insert-ing the HindIII/NotI fragment from pcDNA3-RhoA-N19 (kindly provided by R.Regazzi) into the HindIII/NotI sites of pLXSN (kindly provided by OrmondMacDougald). The vector encoding dominant-negative Epac1 (dnEpac1;Gly269-to-Glu mutation) for retroviral expression (pLXSN-dnEpac1) was con-structed by insertion of the EcoRI (blunt ended)/NotI fragment of pGEX-dnEpac1 (kindly provided by Johannes Bos) into the HpaI/NotI sites of pLXSN.pMT2-HA-RapGAP (55) was kindly provided by Johannes L. Bos. pBABE-RapGAP was generated by subcloning full-length Rap-GTPase-activating(RapGAP; BglII/XbaI blunt) fragment from pMT2-HA-RapGAP into BamHI/HpaI-digested pBABE-Puro.IpJim-RI�DN, and pJim was a kind gift fromReidun Kopperud.

Cell culture and differentiation. 3T3-L1 cells were cultured to confluence inDulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% calfserum. Two-days-postconfluent (designated day 0) cells were induced to differ-entiate with DMEM supplemented with 10% fetal bovine serum (FBS) and 1�M dexamethasone (Dex) (Sigma). One microgram per milliliter Ins (Sigma) orIGF-1 (Sigma), 0.5 mM IBMX (Sigma), 100 �M 8-para-chloro-phenylthio-cAMP (8-CPT-cAMP), 200 �M 8-(4-chlorophenylthio)-2�-O-methyl-cAMP (8-pCPT-2�-O-Me-cAMP) (Biolog), 100 �M N6-monobutyryl-cAMP (6-MB-cAMP) (Biolog), and/or 100 �M N6-benzoyl-cAMP (6-Bnz-cAMP) was includedas indicated in the figure legends. After 48 h, the media were replaced withDMEM supplemented with 10% FBS and 1 �g/ml Ins or IGF-1 as indicated inthe figure legends. The cells were subsequently refed every 48 h with DMEMsupplemented with 10% fetal bovine serum. When included, H-89 (10 �M)(Biomol), sc-3536 (10 �M), (Santa Cruz), and Rp-8-Br-cAMPS/Rp-cAMPS (100�M) (Biolog) were present from day 0 to day 2. The preparation of MEFs hasbeen described previously (44). MEFs were grown in AmnioMax basal medium

(Life Technologies, Inc.) supplemented with 7.5% FBS, 7.5% AmnioMax-C100supplement, and 2 mM glutamine and were induced to differentiate as 3T3-L1cells. Staining of lipid by oil red O was performed as described previously (32).

Retroviral transduction. Phoenix-Eco cells were plated at 30 to 40% conflu-ence in DMEM supplemented with 10% FBS. Next day, the cells were trans-fected using a standard calcium phosphate method by adding 10 �g retroviralexpression vector (pJim-RI�DN, pLXSN-dnEpac1, pLXSN-RhoA-N19,pBABE-Rap1N17, or the empty retroviral vectors) and 15 �g pBSK (Stratagene)to a total of 25 �g DNA per 9-cm dish. Two days posttransfection, the virus-containing media were collected by centrifugation and immediately used to infect30 to 40% confluent 3T3-L1 cells by mixing viral supernatant 1:1 with DMEMsupplemented with 10% calf serum. Polybrene (Sigma) was added to a finalconcentration. of 7 �g/ml. After 24 h, the transduced cells were split and sub-jected to selection (400 �g/ml hygromycin B [Calbiochem] or 3 �g/ml puromy-cin). After approximately 4 days, the selected clones were pooled and replatedfor differentiation.

Real-time RT-PCR. Total RNA was purified from cells using Trizol, andcDNA was synthesized as described earlier (46) and quantified by real-timequantitative PCR (qPCR) using the ABI PRISM 7700 sequence detection system(Applied Biosystems). Each PCR mixture contained, in a final volume of 25 �l,1 �l of first-strand cDNA, 12.5 �l of 2� Sybr green PCR master mix, and 5 pmolof each primer. All reactions were performed using the following cycling condi-tions: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 sand 60°C for 1 min. PCR was carried out in 96-well plates and in duplicate.Primers for real-time reverse transcription-PCR (RT-PCR) were designed usingPrimer Express 2.0 (Applied Biosystems). Target gene mRNA expression wasnormalized to transcription factor IIB (TFIIB) or TATA-binding protein (TBP)mRNA expression, and the relative amounts of all mRNAs were calculated. Thefollowing primers were used (upstream and downstream, respectively): Epac1,5�-GGGACTCCGCTGGACACC and 5�-CGGCCAGAGCAGCAATGCCG;Epac2, 5�-CAATCGGATTCTGAGGGACG and 5�-CATTTAAAACCGAATCTG; C/EBP�, 5�-CAAGAACAGCAACGAGTACCG and 5�-GTCACTGGTCAACTCCAGCAC; aP2, 5�-CTGGGCGTGGAATTCGAT and 5�-GCTCTTCACCTTCCTGTCGTCT; PPAR�2, 5�-ACAGCAAATCTCTGTTTTATGC and5�-TGCTGGAGAAATCAACTGTGG; LXR�, 5�-GAGTTGTGGAAGACAGAACCTCAA and 5�-GGGCATCCTGGCTTCCTC; and TBP, 5�-ACCCTTCACCAATGACTCCTATG and 5�-ATGATGACGGCAGCAAATCGC.

Western blotting. Obtaining whole-cell extracts, electrophoresis, blotting, vi-sualization, and stripping of membranes were performed as described previously(31). The primary antibodies used were obtained from the following sources: CellSignaling Technology, mouse anti phospho-ERK1/2 (Thr-202/Tyr-204), rabbitanti-ERK1/2, rabbit anti-phospho-PKB (Ser-473), rabbit anti-PKB, mouse anti-CREB, mouse anti-phospho-myosin light chain (MLC [Ser-19]), and rabbit anti-MLC; Upstate, mouse anti-phospho-CREB (Ser-133); Cayman Chemical, rabbitanti FABP4/aP2; and Santa Cruz, rabbit anti-PPAR� and rabbit anti-TFIIB. Thesecondary antibodies were horseradish peroxidase-conjugated antimouse or an-tirabbit antibodies obtained from DAKO.

Determination of PKA activity in cell lysates. Cells were incubated for 15 minwith various agents supposed to modulate their cAMP level or PKA activity,washed in ice-cold phosphate-buffered saline, and lysed in 0.5 ml of 50 mMpotassium phosphate buffer (pH 7.0) with 1 mM EGTA, 0.3 mM EDTA, 2 mM1,4-dithioerythriol (DTE), 0.15% Triton X-100, and Complete protease inhibitorcocktail (Roche). Lysates were snap-frozen in liquid nitrogen and thawed im-mediately before assay of kinase activity, which was performed essentially asdescribed by Ekanger et al. (22). Briefly, incubations were for 5 to 10 min at 30°Cin 15 mM HEPES-NaOH, 50 mM potassium phosphate, 10 mM magnesiumacetate, 0.5 mM EGTA, 100 �M [�-32P]ATP, and 70 �M kemptide (LRRASLG)substrate. Some incubations contained cAMP, cAMP analogs, or kinase inhibi-tors (see the relevant figure legend for details). The blank value was determinedby incubating in the presence of 100 nM of the inhibitor peptide from theheat-stable PKA inhibitor.

Other assays. Rap1 and Rho activity were measured using the Rap1 activationassay kit (no. 17-321; Upstate) and the Rho activity kit (no. 17-294; Upstate).

Statistical analysis. Statistical evaluation of the data was performed usingStudent t test. A P value of 0.05 was considered to be significant. n denotes thenumber of independent assays or experiments.

RESULTS

In accordance with previous studies (for review, see refer-ences 25 and 45), we observed that an increase in the cellularlevel of cAMP by the addition of IBMX or forskolin acceler-

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ated adipocyte differentiation of 3T3-L1 cells treated with Dexand Ins. A similar stimulation of adipogenesis was observed inresponse to the cell-permeable cAMP analog 8-CPT-cAMP,which activates both Epac and PKA (14). In contrast, cAMPanalogs 6-MB-cAMP and 6-Bnz-cAMP, which selectively acti-vate PKA (14, 15), were inefficient, whether judged by oil redO staining or adipocyte marker gene expression (Fig. 1A andB). To demonstrate that the presumed PKA activators effi-ciently activated PKA in 3T3-L1 cells, we directly determinedthe PKA activity in lysates obtained from cells treated for 15min with vehicle IBMX, forskolin, 6-MB-cAMP, or 6-Bnz-cAMP. Both 6-MB-cAMP and 6-Bnz-cAMP led to significantactivation of PKA to a degree comparable to that observedwhen the cells were treated with forskolin or IBMX. Thesefindings suggested that activation of PKA alone was insufficientto promote adipogenesis, challenging the prevailing notion thatcAMP stimulates adipogenesis entirely via activation of PKAleading to enhanced phosphorylation and activation of CREB(56, 57, 72).

We studied therefore whether cAMP-mediated activation ofEpac1 or Epac2 might be required for the adipogenic effect ofcAMP. To determine if Epac1 and/or Epac 2 was expressed inthe 3T3-L1 cells, specific primer sets were used for real-timeqPCR. While Epac2 mRNA was undetectable, Epac1 mRNA

was expressed in 2-day-postconfluent preadipocytes (desig-nated day 0 in the differentiation process) at a level aboutthreefold higher than that in mouse liver (Fig. 2A). Uponinduction of differentiation, the level of Epac1 mRNA declinedrapidly during the first 24 h to stabilize at a level approximately45% of that in day 0 cells. In contrast, Epac2 mRNA was belowthe detection limit in undifferentiated as well as differentiated3T3-L1 cells. The Epac2 primer set was validated using livermRNA as a positive control (Fig. 2A and B). Analysis ofmRNA isolated from the stromal vascular fraction (SVF) ofepididymal white adipose tissue (eWAT) and interscapularbrown adipose tissue (iBAT) similarly revealed that Epac1 wasmore highly expressed in the SVF than in the adipocyte frac-tion, whereas Epac2 was undetectable (Fig. 2B).

Having established that Epac1 is expressed in 3T3-L1 cells,we tested whether selective Epac-activating cAMP analogs like8-pCPT-2�-O-Me-cAMP (15, 23) could mimic the effect of anincrease in the endogenous level of cAMP in 3T3-L1 cellstreated with Dex and Ins. This was not the case, as the majorityof the cells remained fibroblast-like after treatment with theEpac-activating cAMP analogs. However, when the Epac ac-tivator 8-pCPT-2�-O-Me-cAMP was combined with the PKAactivator 6-MB-cAMP in the presence of Dex and Ins morethan 90% of the cells rounded up and differentiated into ma-

FIG. 1. Elevation of cAMP, but not selective activation of PKA, accelerates differentiation of 3T3-L1 preadipocytes. (A) Two-day-postcon-fluent 3T3-L1 cells were induced to differentiate by treatment with 1 �M Dex and 1 �g/ml Ins in the presence of 0.5 mM IBMX, 10 �M forskolin,100 �M 8-CPT-cAMP, 100 �M 6-MB-cAMP, or 100 �M 6-Bnz-cAMP, as described in Materials and Methods. The cells were stained with oil redO and photographed on day 8. (B) RNA was isolated on day 8, and expression of PPAR�2, LXR�, C/EBP�, and aP2 was determined by RT-qPCR.The error bars represent standard deviations (n 3). (C) Relative PKA activity in lysates of 3T3-L1 cells pretreated for 15 min in mediumcontaining Dex plus Ins with vehicle, 0.5 mM IBMX, 50 �M forskolin, or 100 �M 6MB-cAMP. The error bars represent standard errors of themeans (n 3 to 5).

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ture adipocytes. The degree of differentiation obtained by thecombined action of the selective Epac and PKA activators wascomparable to that induced by the phosphodiesterase inhibitorIBMX, as determined by oil red O staining (Fig. 3A). Toanalyze the effect on gene expression, 3T3-L1 preadipocyteswere induced with Dex and Ins together with the Epac-acti-vating analog 8-pCPT-2�-O-Me-cAMP and the PKA-activatinganalog 6-MB-cAMP. As shown in Fig. 3B, no significant in-duction of adipocyte marker genes was observed in cellstreated with either analog alone, whereas strong inductionoccurred with the combined administration of both cAMPanalogs.

In order to determine if the synergy between PKA and Epacactivators was peculiar for the 3T3-L1 cell line, MEFs were

tested. As evidenced by oil red O staining of lipids, activationof either Epac or PKA alone was insufficient to achieve adi-pose conversion. As for the 3T3-L1 cells, only combined acti-vation of Epac and PKA stimulated adipose conversion (Fig.3C). Similarly, strong induction of adipocyte marker gene ex-pression required the simultaneous activation of Epac andPKA (Fig. 3D). Thus, in both models activators of Epac andPKA synergistically promoted adipogenesis.

To support the results obtained by pharmacological activa-tion of Epac1 and PKA, 3T3-L1 cells were transduced with aretroviral vector expressing a dominant-negative form ofEpac1 or the empty vector and tested for their ability to un-dergo cAMP-stimulated adipose conversion. Control cellstransduced with the empty vector differentiated when bothEpac and PKA were activated by 8-pCPT-2�-O-Me-cAMP and6-MB-cAMP in combination. In contrast, in cells expressingthe dominant-negative form of Epac1, the majority of the cellsremained fibroblast-like and were not stained by oil red O (Fig.4A). Furthermore, the induction of PPAR� and adipocytelipid-binding protein (aP2) was severely blunted in the cellstransduced with dnEpac1 (Fig. 4B). Knockdown of Epac1 ex-pression by lentivirus-mediated expression of anti-Epac1 shorthairpin RNA similarly blunted differentiation of 3T3-L1 pre-adipocytes (data not shown).

In order to test the Epac selectivity of the cAMP analogsused and the ability of dnEpac1 to block endogenous Epacaction, we determined the level of active (GTP-associated)Rap1 in the 3T3-L1 cells in response to treatment with variouscAMP analogs. We found no activation of Rap1 in response tothe PKA activator 6-MB-cAMP, while 8-pCPT-2�-O-Me-cAMP activated Rap1 both in the absence and presence ofPKA activator. It should be noted that no activation of PKAwas observed in cells treated with 8-pCPT-2�-O-Me-cAMP(see Fig. 6A). Importantly, activation of Rap1 by 8-pCPT-2�-O-Me-cAMP was abolished in cells with forced expression ofdnEpac1 (Fig. 4B).

To determine if the Epac-mediated activation of Rap was abystander effect or was important for adipogenesis, we testedwhether forced expression of the dominant-negative Rap1N17would inhibit adipogenesis. We noted a robust adipocyte dif-ferentiation in 3T3-L1 cells transduced with vector alone,whereas adipocyte differentiation of cells expressing Rap1N17was suppressed, whether determined by oil red O staining (Fig.4C) or adipocyte marker gene expression (Fig. 4D). Further-more, retroviral expression of RapGAP, which facilitates con-version of the active Rap-GTP into its inactive GDP-boundform, significantly reduced expression of the adipogenicmarker genes PPAR�2 and aP2 (Fig. 4E). Collectively, thesefindings indicate that cAMP-dependent activation of theEpac1/Rap1 pathway is required for adipocyte differentiation.

The data in the preceding paragraphs suggested that activePKA was necessary, but not sufficient, for cAMP-stimulation ofadipogenesis. The role of PKA was ascertained by the demon-stration that the PKA-specific inhibitory cAMP analogs of theequatorial diastereoisomer of adenosine-3�,5�-cyclic mono-phosphorothioate (Rp-8-Br-cAMPS/Rp-cAMPS) (15) counter-acted 3T3-L1 cell differentiation (Fig. 5 A) and that differen-tiation was blocked by forced expression of dominant-negativeRI� (Fig. 5C), which, like the Rp-cAMPS analogs, significantlydecreased the PKA activity in 3T3-L1 cell extracts (data not

FIG. 2. Expression of Epac in 3T3-L1 cells and mouse adiposetissues. (A) RNA was isolated on days 0, 1, 2, 3, 4, 6, and 10 from3T3-L1 cells induced to differentiate by Dex, Ins, and IBMX and frommouse liver. (B) RNA was extracted from SVF or mature adipocytes(Ads) isolated from mouse eWAT, iBAT, and liver as described inMaterials and Methods. The expression of Epac1 and Epac2 wasdetermined using RT-qPCR and normalized to TBP expression. Theerror bars represent standard deviations (n 3).



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shown). In contrast, the widely used, but nonspecific, PKAinhibitor H89 failed to abrogate cAMP-stimulated adipogene-sis (Fig. 5A). This suggested that PKA could act by inhibitingan additional kinase targeted by H89. In this way H89 wouldmake PKA superfluous for differentiation. H89 is a potentinhibitor of Rho-kinases (42), which therefore would be aprime candidate for the putative kinase inactivated down-stream of PKA; the more so as inhibition of Rho-kinase con-stitutes a crucial step for initiation of adipocyte differentiation(51). Since the Rho-kinase is stimulated by GTP-Rho, an ob-vious mechanism of PKA-induced inhibition of Rho-kinasewould be to convert Rho-GTP to the inactive Rho-GDP form.In fact, activation of PKA alone or in combination with acti-vation of Epac reduced the level of Rho-GTP to undetectablelevels, whereas activation of Epac alone had no effect, andfurthermore, activation of PKA by 6-MB-cAMP decreasedphosphorylation of the Rho-kinase substrate MLC (Fig. 5B).To further substantiate the notion that PKA activation stimu-lated adipogenesis via downregulation of a Rho/Rho-kinase-dependent pathway, we examined whether pharmacologicalinhibition of Rho-kinase was sufficient to restore adipogenesisof 3T3-L1 cells expressing the dominant-negative form of theRI� subunit. As predicted, addition of the Rho-kinase inhibi-tor sc-3536 restored the differentiation of cells expressing thedominant-negative RI� subunit as determined by oil red Ostaining (Fig. 5C) and expression of the adipocyte markerproteins PPAR� and aP2 (Fig. 5D). Based on these findings,we conclude that activation of PKA is dispensable for efficient

adipocyte differentiation in the presence of Dex, high levels ofIns, and IBMX, provided that the Rho-kinase is inhibited.

The finding that the stimulatory effect of PKA activation onadipocyte differentiation could be mimicked by Rho-kinaseinhibition and the fact that the dual PKA and Rho-kinaseinhibitor enhanced rather than prevented adipocyte differenti-ation questioned the importance of PKA-mediated phosphor-ylation of CREB in cAMP-stimulated adipocyte differentia-tion. To investigate this, we first determined the effects of6-MB-cAMP, 8-pCPT-2�-O-Me-cAMP, forskolin, IBMX, andH89 on the PKA activity in 3T3-L1 cells. The cells were treatedfor 15 min, as indicated in Fig. 6A, and PKA activity in thelysates was determined. As expected, the addition of 8-pCPT-2�-O-Me-cAMP did not increase the PKA activity, whereas6-MB-cAMP, forskolin, and IBMX robustly increased PKAactivity. The combination of 6-MB-cAMP with 8-pCPT-2�-O-Me-cAMP increased PKA activity to a level comparable to thatobserved with 6-MB-cAMP alone. Finally, addition of H89completely prevented the IBMX-dependent increase in PKAactivity (Fig. 6A). Next, to determine to what extent activationof PKA affected phosphorylation of CREB on Ser-133, 3T3-L1cells were grown to 2 days postconfluence and then inducedwith DMEM containing 10% FBS in the absence or presenceof cAMP analogs. Cells were harvested after 5, 15, and 30 min.Figure 6A demonstrates the time course of CREB and mito-gen-activated protein kinase (MAPK) phosphorylation. Phos-phorylation of both CREB and ERK1/2 was stimulated as earlyas 5 min after induction in the absence of cAMP analogs or

FIG. 3. Activation of Epac and PKA synergistically induces differentiation of 3T3-L1 cells and MEFs into adipocytes. Two-day-postconfluent3T3-L1 cells (A) or MEFs (C) were induced to differentiate by treatment with Dex and Ins in the presence of combinations of 200 �M8-pCPT-2�-O-Me-cAMP and 100 �M 6-MB-cAMP or 0.5 mM IBMX as indicated. The cells were stained with oil red O on day 8. RNA was isolatedon day 8, and expression of PPAR�2, LXR�, C/EBP�, and aP2 in 3T3-L1 cells (B) and MEFs (D) was determined by RT-qPCR. The error barsrepresent standard deviations (n 3).



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IBMX. Maximal phosphorylation of CREB was observed after15 min, whereas ERK1/2 phosphorylation still increased by 30min. Interestingly, ERK1/2 phosphorylation increased rapidlyin the cells treated with IBMX. Furthermore, the level ofphosphorylated ERK1/2 after 15 and 30 min tended to behigher in cells receiving IBMX or the Epac activator 8-pCPT-2�-O-Me-cAMP alone or in combination with 6-MB-cAMP,possibly reflecting an enhanced Ins-dependent signaling (seebelow). No significant difference in CREB phosphorylationwas observed in cells treated with IBMX, 6-MB-cAMP,8-pCPT-2�-O-Me-cAMP, or 6-MB-cAMP plus 8-pCPT-2�-O-Me-cAMP (Fig. 6B). These findings indicated that robustCREB phosphorylation could be induced without specific ac-tivation of PKA, but might depend on ERK1/2 activation. Toknow if this could be the case, 3T3-L1 cells were transducedwith either an empty vector or a vector expressing the domi-nant-negative form of RI� and induced for 15 min with Dex,Ins, and IBMX, in the absence or presence of the selectivePKA inhibitor Rp-cAMPS or the MEK inhibitor U0126. Cellextracts were analyzed for CREB and MAPK phosphorylationby Western blotting. Figure 6C demonstrates that phosphory-lation of CREB and ERK1/2 was induced also when the PKAinhibitor was present and in cells expressing the dominant-negative form of RI�. In contrast, the MEK inhibitor almostcompletely prevented the increased phosphorylation of not

only ERK1/2, but also CREB. In vitro kinase assays demon-strated that the MEK kinase inhibitor (and the Rho-kinaseinhibitor) did not inhibit PKA, whereas H89 potently inhibitedPKA (Fig. 6D). Finally, we demonstrated that expression of acanonical CREB-responsive gene was induced by Dex, Ins, andIBMX in the presence of the PKA inhibitor Rp-cAMPS, butnot in the presence of the MEK inhibitor (Fig. 6E).

CREB has been shown to play a pivotal role during initiationof adipocyte differentiation, at least in part by regulating theexpression of C/EBP� (72), although alternative routes forinduction of C/EBP� also seem to exist (26). It is well estab-lished that phosphorylation of Ser-133 is necessary, but notsufficient, for CREB-mediated transactivation and that addi-tional PKA-dependent processes may be needed for transcrip-tional activation (8). However, taken together all experimentsdescribed above argue against PKA being directly involved inCREB phosphorylation and activation. Rather, the activationof CREB and the ensuing induction of C/EBP� expressionappear to depend on Ins/IGF-1 signaling. Therefore, we pro-pose that Rho-kinase and not CREB is a central target forPKA activation during the onset of the adipocyte differentia-tion program.

Ins/IGF-1 signaling is crucial for adipogenesis, so it is puz-zling that inhibition of Rho-kinase is essential for induction ofadipocyte differentiation since inhibition of Rho-kinase also

FIG. 4. Activation of Epac is required for differentiation of 3T3-L1 cells into adipocytes. 3T3-L1 cells were retrovirally transduced with anempty vector, a vector expressing dnEpac1 (A and B), a vector expressing a dominant-negative form of Rap1A (Rap1N17) (C and D), or a vectorexpressing Rap GTPase activating protein (RapGAP) (E). The cells were grown to confluence, and at 2 days postconfluence they were inducedto differentiate by Dex and Ins in the presence of combinations of 200 �M 8-pCPT-2�-O-Me-cAMP and 100 �M 6-MB-cAMP as indicated in thefigure. (A and C) The cells were stained with oil red O and photographed on day 8. (B) GTP-bound Rap1 was measured by a Rap1 activationpull-down assay as described in Materials and Methods. Expression of aP2 and PPAR� was determined by Western blotting on day 8. TFIIB wasused for control of equal protein loading on the gel. One representative experiment out of three independent experiments is shown. (D and E)RNA was isolated on day 8, and expression of LXR�, C/EBP� (D), PPAR�2, and aP2 (D and E) was determined by RT-qPCR. The error barsrepresent standard deviations (n 3).



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impairs Ins signaling in adipocytes (27). We reasoned thatactivation of Epac might overcome the negative effect of Rho-kinase inhibition on Ins/IGF-1 signaling. In fact, Epac activa-tion has been shown to potentiate Ins signaling in muscle cells(7). The supraphysiological concentration of Ins used in thestandard Dex-Ins-IBMX differentiation protocols mimicsIGF-1, the main adipogenic inducer, by interacting with theIGF-1 receptor (59, 61). To demonstrate that inhibition ofRho-kinase impaired IGF-1/Ins signaling in 3T3-L1 preadipo-cytes, the effect of the Rho-kinase-inhibitor sc-3536 was stud-ied in cells stimulated with increasing concentrations of IGF-1in the presence or absence of the Epac activator 8-pCPT-2�-O-Me-cAMP. The fetal calf serum used in the present exper-iments contained 10 nM IGF-1 (data not shown). Hence, thefinal concentration of IGF-1 in the medium containing 10%serum was 1 nM. Phosphorylation of PKB was determined asa marker of IGF-1 signaling. We found that the Rho-kinaseinhibitor decreased IGF-1-dependent PKB phosphorylation.Importantly, activation of Epac enhanced PKB phosphoryla-tion and restored the IGF-1-dependent PKB phosphorylationin the presence of the Rho-kinase inhibitor (Fig. 7). Thus,

Epac activation stimulated IGF-1 signaling whether the Rho-kinase was inhibited or not.

Based on the results above, we predicted that if the Rho-kinase was inhibited the activation of Epac could promoteadipogenesis without PKA activation or addition of Ins/IGF-1on top of the 1 nM basal level of IGF-1 in the medium. To testthis prediction, we first treated 3T3-L1 preadipocytes with8-pCPT-2�-O-Me-cAMP and the Rho-kinase inhibitor sc-3536in the absence of added Ins/IGF-1 and IBMX. Pharmacolog-ical inhibition of the Rho-kinase by sc-3536 had no effect onadipogenesis, whether determined by oil red O staining oradipocyte marker gene expression when administered alone.Similarly, addition of the Epac activator 8-pCPT-2�-O-Me-cAMP alone had no effect on adipogenesis. However, inhibi-tion of Rho-kinase dramatically enhanced differentiation whencombined with the Epac activator 8-pCPT-2�-O-Me-cAMP(Fig. 8A and B). Expression of the dnEpac prevented, as ex-pected, the 8-pCPT-2�-O-Me-cAMP-induced enhancement ofadipocyte differentiation (Fig. 8C). To corroborate the resultsobtained by pharmacological inhibition of the Rho-kinase, weperformed parallel experiments in which 3T3-L1 cells were

FIG. 5. Activation of PKA is required for differentiation of 3T3-L1 cells, but dispensable when Rho-kinase is inhibited. (A) 3T3-L1 cells weregrown to 2 days postconfluence and induced to differentiate by a standard differentiation cocktail consisting of Dex, Ins, and IBMX. Additionally,100 �M Rp-8-Br-cAMPS/Rp-cAMPS or 10 �M H89 was present from day 0 to day 2. On day 8, the cells were stained with oil red O andphotographed. (B) GTP-bound Rho was measured by a Rho activation pull-down assay after 15 min of treatment with 200 �M 8-pCPT-2�-O-Me-cAMP and 100 �M 6-MB-cAMP alone or in combination. Phosphorylated MLC and MLC were determined by Western blotting after 15 minof treatment with 100 �M 6-MB-cAMP or vehicle. One representative experiment out of three independent experiments is shown. (C) 3T3-L1 cellswere retrovirally transduced with an empty vector or a vector expressing a dominant-negative form of the RI� PKA subunit (RI�DN). At 2 dayspostconfluence, the cells were induced to differentiate by Dex, IBMX, and Ins in the absence and presence of 10 �M sc-3536. On day 8, the cellswere stained with oil red O and photographed or whole-cell extracts were prepared and the expression of PPAR� and aP2 was determinedby Western blotting using TFIIB as a control for equal protein loading. One representative experiment out of three independent experimentsis shown (D).



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FIG. 6. Induction of CREB phosphorylation during initiation of adipocyte differentiation is dependent on ERK1/2 activity. (A) 3T3-L1 cellswere treated for 15 min in Dex-Ins medium with various combinations of 200 �M 8-pCPT-2�-O-Me-cAMP, 200 �M 6MB-cAMP, 0.5 mM IBMX,and 10 �M H89 as indicated. Subsequently the PKA activity in lysates of the cells was determined. The error bars represent standard errors of themeans (n 3). (B) 3T3-L1 preadipocytes at 2 days postconfluence were treated with Dex and Ins with combinations of 200 �M 8-pCPT-2�-O-Me-cAMP, 200 �M 6MB-cAMP, and 0.5 mM IBMX. Whole-cell extracts were prepared after 5, 15, and 30 min and analyzed for phosphorylationof CREB and ERK1/2 by Western blotting. One representative experiment out of three independent experiments is shown. (C) 3T3-L1preadipocytes at 2 days postconfluence were treated with Dex, Ins, and IBMX with or without 100 �M Rp-8-Br-cAMPS/Rp-cAMPS or 10 �MU0126 as indicated. Whole-cell extracts were prepared after 15 min and analyzed for phosphorylation of CREB and ERK1/2 by Western blotting.One representative experiment out of three independent experiments is shown. (D) Effects of protein kinase inhibitors on PKA activity in 3T3-L1lysates. The lysates were incubated with 10 �M H89, 10 �M sc-3536, or 10 �M U0126 in the presence of a maximally PKA-stimulatingconcentration of cAMP (1 �M). The error bars represent standard errors of the means (n 3). (E) Two-day-postconfluent 3T3-L1 cells wereinduced to differentiate with Dex, Ins, and IBMX with or without 100 �M Rp-8-Br-cAMPS/Rp-cAMPS or 10 �M U0126. RNA was isolated after6 h, and expression of CREB was determined by RT-qPCR. d0, day 0. The error bars represent standard deviations (n 3).



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transduced with either an empty retroviral vector or a retrovi-ral vector expressing a dominant-negative version of RhoA(RhoA-N19). In agreement with the results obtained with thechemical Rho-kinase inhibitor sc-3536, activation of Epac in-duced adipocyte differentiation, as determined by oil red Ostaining and adipocyte marker gene expression in cells express-ing the dominant-negative version of RhoA, but not in cellstransduced with the empty vector (Fig. 8D and E). We con-clude that Epac activation is essential for adipogenesis in cellsexposed to physiological levels of Ins/IGF-1 when the Rho-kinase is inhibited.

DISCUSSION

The differentiation of fibroblast-like cells into adipocytesinvolves dramatic changes of cell shape and function, whichcan be studied in vitro. In spite of a large number of studiespointing to the importance of elevated cAMP for initiation ofadipocyte differentiation, the contribution of the two cAMPreceptor families PKA and Epac has not been addressed. Nei-ther has the interplay between cAMP and Rho and Rho-ki-nase. The present study shows that the effect of elevated cAMPlevels depends on the concerted activation of both PKA andEpac1 to promote adipogenesis of both 3T3-L1 preadipocytes

and mouse embryo fibroblasts. A model of such activation isshown in Fig. 9. The stimulation via Epac1 involves activationof Rap and is counteracted by dominant-negative Rap1, whilethe action of PKA involves inhibition of Rho and can besubstituted for by dominant-negative Rho or inhibitors of Rho-kinase. These new findings require a reassessment of the roleof cAMP in adipogenesis.

In the context of adipocyte differentiation, PKA has previ-ously been considered as the sole target of cAMP, whosedifferentiation-promoting effect has been ascribed to the acti-vating phosphorylation of Ser-133 of CREB, an activator of theC/EBP� promoter and pivotal regulator of adipocyte differen-tiation (26, 56, 57, 72). While our results do not question theimportance of CREB-dependent activation of the adipogenicprogram, our results argue against PKA-mediated phosphory-lation of CREB being indispensable for CREB activation dur-ing the initiation of adipocyte differentiation of 3T3-L1 pre-adipocytes using the standard Dex, Ins, and IBMX protocol.The selective PKA inhibitors Rp-8-Br-cAMPS/Rp-cAMPS didnot diminish MDI-induced CREB phosphorylation. In con-trast, the MEK inhibitor U0126 completely prevented CREBphosphorylation, underscoring the importance of signalingthrough the Ins/IGF-1-stimulated Ras/Raf/MEK/ERK path-way for phosphorylation of CREB (40). ERK1/2-dependentphosphorylation and activation of CREB and induction ofC/EBP� expression were also reported for leukemia inhibitoryfactor-induced adipocyte differentiation of Ob1771 and 3T3-F442A preadipocytes (4). It has been demonstrated that non-PKA-dependent phosphorylation of CREB may require addi-tional cAMP-dependent signaling to activate CREB-mediatedtranscription (8, 12, 47). Our finding that adipocyte differen-tiation takes place and even is enhanced in the presence of thedual PKA and Rho kinase inhibitor H89 (see below for furtherdiscussion) indicates that if such cAMP-dependent activity isrequired, PKA is not directly involved. It remains to be estab-lished whether activation of the Epac-Rap branch of cAMP-dependent signaling may enhance CREB-mediated gene ex-pression.

The first clue to the importance of Rho-kinase inhibition forthe adipogenic action of PKA came through the observationthat the PKA/Rho-kinase inhibitor H89 could substitute forPKA in supporting differentiation (Fig. 5A) (38, 51, 53). In thepresent report, we show in addition that a more specific Rho-kinase inhibitor as well as dominant-negative RhoA also couldsubstitute for PKA and that PKA activation decreased activeRho-GTP and abolished the phosphorylation of the Rho-ki-nase target MLC. We propose therefore that the major role ofPKA in promoting adipocyte differentiation may be throughinhibition of Rho/Rho-kinase.

Mesenchymal determination and terminal differentiationalong the adipocyte, myocyte, and osteocyte lineages are con-trolled by cell shape and cytoskeletal tension (34, 48, 50, 62),converging on regulation of Rho and Rho-kinase activity (48,51, 63). We propose that PKA can decrease cell tension byconverting RhoA to the inactive GTP-free form, which leads torelaxation of microfilaments, in part via inhibition of Rho-kinase-catalyzed phosphorylation of MLC. This relaxation ofcell tension will lead to further inhibition of Rho. There areseveral known mechanisms for PKA-mediated inhibition ofRho. One is inactivating phosphorylation of RhoA by PKA

FIG. 7. Activation of Epac enhances Ins/IGF-1 signaling in 3T3-L1cells (A) 3T3-L1 cells were grown to 2 days postconfluence and thentreated with Dex and increasing concentrations of IGF-1 in the ab-sence or presence of 10 �M sc-3536 and 200 �M 8-pCPT-2�-O-Me-cAMP, as indicated in the figure. After 15 min, whole-cell extractswere prepared and the levels of phosphorylated PKB and total PKBwere determined by Western blotting. Shown is one representativeexperiment out of three independent experiments. (B) Quantificationof the relative levels of PKB phosphorylation. Autoradiographs wereanalyzed by densitometric scanning, and the levels of phosphorylatedPKB relative to total PKB were determined. The error bars representstandard deviations (n 3).



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(21, 41), a second is AKAP-Lbc phosphorylation by anchoredPKA followed by recruitment of 14-3-3 and decreased Rho-GEF activity (20), and a third is PKA-catalyzed phosphoryla-tion of G�13 with consequent inhibition of Rho activation (1).Results presented here and previously published data showthat repression of Rho activity is a point of convergence foradipogenesis-promoting signals. Sordella and coworkers havedemonstrated that IGF-1-dependent phosphorylation of p190-BRhoGAP induces a translocation of p190-B RhoGAP to lipidrafts, leading to a down-regulation of Rho activity (63) Con-versely, forced expression of the selective Rho-GEF, GEFT,prevents 3T3-L1 preadipocyte differentiation (9). Recently, itwas furthermore shown that inhibition or genetic ablation ofRho-kinase (ROCK-II/ROK�) but not ROCK-I/ROK� pro-moted adipocyte differentiation (51).

Epac was discovered as a cAMP-stimulated Rap activator(19), but has also been reported to act independently of Rap tostimulate the Jun-kinase (18) or activate R-ras (33, 39). In the3T3-L1 model of adipocyte differentiation, we found that ac-tivation of Epac1 increases Rap1-GTP and that its stimulationof adipocyte differentiation was counteracted by dnRap1. Thissuggests that Epac1 acted to promote adipogenesis via activa-tion of Rap1. A further clue to what role Epac1 might have inadipogenesis was provided by our observation that Epac1 ac-tivation could overcome the paradoxical negative effect of in-hibition of Rho-kinase on IGF-1/Ins signaling. This may berelated to the observations that inhibition of Rho-kinase activ-ity impairs Ins signaling in cultured adipocytes and myocytesand causes Ins resistance with impaired glucose uptake inskeletal muscle of mice (27). Rho-kinase-dependent stimula-tion of Ins signaling was found to be associated with phosphor-ylation of serine residues 632 and 635 of Ins receptor substrate1 (IRS-1) (27). On the other hand, Rho-kinase can inhibit Inssignaling through phosphorylation of serine 612 of IRS-1 (2, 3,63). Thus, to achieve optimal insulin/IGF-1 signaling the ac-tivity of the Rho-kinase must be regulated within a very narrowwindow, a situation reminiscent of the p120 catenin-dependentregulation of adhesion junctions, where both inhibition of Rho

FIG. 8. Activation of Epac is sufficient to induce differentiation of3T3-L1 cells when Rho-kinase is inhibited. 3T3-L1 cells were grown to2 days postconfluence and induced to differentiate by Dex in theabsence or presence of 10 �M sc-3536. Additionally, 200 �M 8-pCPT-2�-O-Me-cAMP was present from day 0 to day 2 as indicated. On day8, the cells were stained with oil red O and photographed (A), totalRNA was isolated, and the expression of PPAR�, LXR�, and aP2 wasdetermined by RT-qPCR. The error bars represent standard devia-tions (n 3) (B). 3T3-L1 cells were retrovirally transduced with anempty vector or a vector expressing a dominant negative form of Epac1(dnEpac1). At 2-days post confluence they were induced to differen-tiate by Dex in the absence or presence of 10 �M sc-3536. Additionally,200 �M 8-pCPT-2�-O-Me-cAMP was present from day 0 to day 2 asindicated. On day 8, the cells were stained with oil red O and photo-graphed (C). 3T3-L1 cells were retrovirally transduced with an emptyvector or a vector expressing a dominant-negative form of RhoA(RhoA-N19). At 2 days postconfluence, they were induced to differ-entiate by Dex in the absence or presence of 200 �M 8-pCPT-2�-O-Me-cAMP. On day 8, the cells were stained with oil red O and pho-tographed. (D) Total RNA was isolated, and the expression of PPAR�,LXR�, and aP2 was determined by RT-qPCR. The error bars repre-sent standard deviations (n 3) (E).



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activity and strong activation of Rho can block the formationof junctions (69). Thus, under conditions with elevated levelsof cAMP resulting in PKA-dependent inhibition of Rho activ-ity, effective Ins/IGF-1 signaling might dependent on a com-pensatory sensitizing effect by the Epac branch of the cAMPeffector machinery.

Using phosphorylation of PKB as a measure of IGF-1 sig-naling, we demonstrate that Rho-kinase inhibition impairedand Epac1 activation enhanced IGF-1-dependent activation ofPKB and, furthermore, we show that activation of Epac1 fullyrestored or even enhanced PKB activation in the presence of aRho-kinase inhibitor. Similarly, activation of Epac1 potenti-ated Ins-dependent activation of PKB in skeletal muscle (7).

We speculate that activation of Rap may be one commonfeature of Epac and IGF-1 signaling, explaining at least in partthe ability of Epac activation to compensate for limiting levelsof Ins/IGF-1. Thus, the adaptor protein c-Crk, which recruitsC3G, a GEF for Rap1, has been shown to be required as anearly signaling mediator of IGF-1-induced adipocyte differen-tiation of 3T3-L1 preadipocytes (35). C3G is able to activateRap1 in NIH 3T3 cells (43), and hence, it is conceivable thatthe same is true for 3T3-L1 adipocytes. How Epac-Rap1 acti-vation sensitizes Ins/IGF-1 signaling in preadipocytes remainsto be elucidated. In muscle, it was shown that activation ofEpac promoted Ins-stimulated recruitment of phosphatidyl-inositol 3-kinase (PI3-K) to signaling complexes. However, nointeraction between the p85 subunit of the PI3-K and Rap1analogous to the interaction between cdc42 and p85 (68) could

be detected (7), and the molecular basis for the increasedPI3-K recruitment remains to be established.

The synergistic action of Ins/IGF-1 and cAMP signalingduring initiation of adipocyte differentiation is remarkable andcontrasts the normal interplay between insulin and cAMP sig-naling in liver, muscle, and mature adipocytes. In mature adi-pocytes, stimulation of �-adrenergic receptors increases cAMPlevels, leading to PKA activation and stimulation of lipolysis byPKA-mediated phosphorylation of hormone-sensitive lipaseand perilipin. In this setting, Ins disrupts the coupling betweenthe �2-adrenergic receptor and PKA presumably throughmodulation of AKAP scaffolding proteins (71). Similarly, thechanneling of cAMP signaling via Epac also seems to varybetween preadipocytes and adipocytes. In preadipocytes, ourresults show that activation of Epac increases Ins/IGF-1 sig-naling and PKB activation, whereas activation of Epac by8-pCPT-2�-O-Me-cAMP in primary rat adipocytes decreasesIns-dependent activation of PKB (73). Thus, cAMP-dependentsignals appear to follow different routes in preadipocytes andmature adipocytes. In addition, the marked differences in thenumber of receptors for Ins and IGF-1, respectively, in pre-adipocytes and mature adipocytes (61) coupled with the in-creased expression of small G-proteins such as TC10�, which isrecruited to CAP-Cbl-C3G complexes in lipid rafts in responseto Ins and required for Ins non-PI3-K-dependent translocationof Glut4 (11, 13), may well in a competitive manner affectEpac-independent Rap activation. Further deciphering of thecross talk between cAMP-dependent signaling and Ins/IGF-1

FIG. 9. Model for the role of cAMP-stimulated adipogenesis via PKA- end Epac1-dependent processes. Increased levels of cAMP activate bothPKA- and Epac-dependent pathways. Activation of PKA leads to repression of Rho-kinase activity by targeting either the Rho-kinase or theupstream regulator Rho. High levels of Rho kinase activity inhibit Ins/IGF-1-dependent signaling, and attenuation of Rho-kinase activity is crucialfor adipogenesis. However, low levels of Rho-kinase activity also enhance Ins/IGF-1-dependent signaling, and PKA-mediated inhibition of theRho-kinase impairs Ins/IGF-1-dependent signaling. This is counteracted by the simultaneous activation of an Epac1/Rap1-dependent pathway,which also induces important changes in cytoskeletal organization, adhesion, and extracellular matrix. Activation of CREB is not dependent onPKA activity, but rather requires ERK activity during the initial stages of adipogenesis.



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signaling promises novel avenues for insight into the complexsignaling governing the transition from preadipocytes and ad-ipocytes as well as metabolic regulation in mature adipocytes.

ACKNOWLEDGMENTS

We thank Johannes Bos, Ormond MacDougald, Eva Pålsson-Mc-Dermott, R Regazzi, and Reidun Kopperud for valuable plasmids.

This work was carried out as a part of the research program of theDanish Obesity Research Centre (DanORC). DanORC is supportedby The Danish Council for Strategic Research (grant no. 2101-06-0005). This work was in addition supported by the Danish NaturalScience Research Council, the Norwegian Research Council, and theNOVO Foundation.

L.M. and K.K. are founders and members of the board of BioLi-gands ApS, and R.K.P. is an employee of BioLigands ApS.

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Cyclic AMP (cAMP)-Mediated Stimulation of Adipocyte Differentiation Requires the Synergistic Action of Epac- and cAMP-Dependent Protein Kinase-Dependent Processes