In accordance with previous studies (for review, see references 25
), we observed that an increase in the cellular level of cAMP by the addition of IBMX or forskolin accelerated adipocyte differentiation of 3T3-L1 cells treated with Dex and Ins. A similar stimulation of adipogenesis was observed in response to the cell-permeable cAMP analog 8-CPT-cAMP, which activates both Epac and PKA (14
). In contrast, cAMP analogs 6-MB-cAMP and 6-Bnz-cAMP, which selectively activate PKA (14
), were inefficient, whether judged by oil red O staining or adipocyte marker gene expression (Fig. ). To demonstrate that the presumed PKA activators efficiently activated PKA in 3T3-L1 cells, we directly determined the PKA activity in lysates obtained from cells treated for 15 min with vehicle IBMX, forskolin, 6-MB-cAMP, or 6-Bnz-cAMP. Both 6-MB-cAMP and 6-Bnz-cAMP led to significant activation of PKA to a degree comparable to that observed when the cells were treated with forskolin or IBMX. These findings suggested that activation of PKA alone was insufficient to promote adipogenesis, challenging the prevailing notion that cAMP stimulates adipogenesis entirely via activation of PKA leading to enhanced phosphorylation and activation of CREB (56
FIG. 1. Elevation of cAMP, but not selective activation of PKA, accelerates differentiation of 3T3-L1 preadipocytes. (A) Two-day-postconfluent 3T3-L1 cells were induced to differentiate by treatment with 1 μM Dex and 1 μg/ml Ins in the presence (more ...)
We studied therefore whether cAMP-mediated activation of Epac1 or Epac2 might be required for the adipogenic effect of cAMP. To determine if Epac1 and/or Epac 2 was expressed in the 3T3-L1 cells, specific primer sets were used for real-time qPCR. While Epac2 mRNA was undetectable, Epac1 mRNA was expressed in 2-day-postconfluent preadipocytes (designated day 0 in the differentiation process) at a level about threefold higher than that in mouse liver (Fig. ). Upon induction of differentiation, the level of Epac1 mRNA declined rapidly during the first 24 h to stabilize at a level approximately 45% of that in day 0 cells. In contrast, Epac2 mRNA was below the detection limit in undifferentiated as well as differentiated 3T3-L1 cells. The Epac2 primer set was validated using liver mRNA as a positive control (Fig. ). Analysis of mRNA isolated from the stromal vascular fraction (SVF) of epididymal white adipose tissue (eWAT) and interscapular brown adipose tissue (iBAT) similarly revealed that Epac1 was more highly expressed in the SVF than in the adipocyte fraction, whereas Epac2 was undetectable (Fig. ).
FIG. 2. Expression of Epac in 3T3-L1 cells and mouse adipose tissues. (A) RNA was isolated on days 0, 1, 2, 3, 4, 6, and 10 from 3T3-L1 cells induced to differentiate by Dex, Ins, and IBMX and from mouse liver. (B) RNA was extracted from SVF or mature adipocytes (more ...)
Having established that Epac1 is expressed in 3T3-L1 cells, we tested whether selective Epac-activating cAMP analogs like 8-pCPT-2′-O
) could mimic the effect of an increase in the endogenous level of cAMP in 3T3-L1 cells treated with Dex and Ins. This was not the case, as the majority of the cells remained fibroblast-like after treatment with the Epac-activating cAMP analogs. However, when the Epac activator 8-pCPT-2′-O
-Me-cAMP was combined with the PKA activator 6-MB-cAMP in the presence of Dex and Ins more than 90% of the cells rounded up and differentiated into mature adipocytes. The degree of differentiation obtained by the combined action of the selective Epac and PKA activators was comparable to that induced by the phosphodiesterase inhibitor IBMX, as determined by oil red O staining (Fig. ). To analyze the effect on gene expression, 3T3-L1 preadipocytes were induced with Dex and Ins together with the Epac-activating analog 8-pCPT-2′-O
-Me-cAMP and the PKA-activating analog 6-MB-cAMP. As shown in Fig. , no significant induction of adipocyte marker genes was observed in cells treated with either analog alone, whereas strong induction occurred with the combined administration of both cAMP analogs.
FIG. 3. Activation of Epac and PKA synergistically induces differentiation of 3T3-L1 cells and MEFs into adipocytes. Two-day-postconfluent 3T3-L1 cells (A) or MEFs (C) were induced to differentiate by treatment with Dex and Ins in the presence of combinations (more ...)
In order to determine if the synergy between PKA and Epac activators was peculiar for the 3T3-L1 cell line, MEFs were tested. As evidenced by oil red O staining of lipids, activation of either Epac or PKA alone was insufficient to achieve adipose conversion. As for the 3T3-L1 cells, only combined activation of Epac and PKA stimulated adipose conversion (Fig. ). Similarly, strong induction of adipocyte marker gene expression required the simultaneous activation of Epac and PKA (Fig. ). Thus, in both models activators of Epac and PKA synergistically promoted adipogenesis.
To support the results obtained by pharmacological activation of Epac1 and PKA, 3T3-L1 cells were transduced with a retroviral vector expressing a dominant-negative form of Epac1 or the empty vector and tested for their ability to undergo cAMP-stimulated adipose conversion. Control cells transduced with the empty vector differentiated when both Epac and PKA were activated by 8-pCPT-2′-O-Me-cAMP and 6-MB-cAMP in combination. In contrast, in cells expressing the dominant-negative form of Epac1, the majority of the cells remained fibroblast-like and were not stained by oil red O (Fig. ). Furthermore, the induction of PPARγ and adipocyte lipid-binding protein (aP2) was severely blunted in the cells transduced with dnEpac1 (Fig. ). Knockdown of Epac1 expression by lentivirus-mediated expression of anti-Epac1 short hairpin RNA similarly blunted differentiation of 3T3-L1 preadipocytes (data not shown).
FIG. 4. Activation of Epac is required for differentiation of 3T3-L1 cells into adipocytes. 3T3-L1 cells were retrovirally transduced with an empty vector, a vector expressing dnEpac1 (A and B), a vector expressing a dominant-negative form of Rap1A (Rap1N17) (more ...)
In order to test the Epac selectivity of the cAMP analogs used and the ability of dnEpac1 to block endogenous Epac action, we determined the level of active (GTP-associated) Rap1 in the 3T3-L1 cells in response to treatment with various cAMP analogs. We found no activation of Rap1 in response to the PKA activator 6-MB-cAMP, while 8-pCPT-2′-O-Me-cAMP activated Rap1 both in the absence and presence of PKA activator. It should be noted that no activation of PKA was observed in cells treated with 8-pCPT-2′-O-Me-cAMP (see Fig. ). Importantly, activation of Rap1 by 8-pCPT-2′-O-Me-cAMP was abolished in cells with forced expression of dnEpac1 (Fig. ).
FIG. 6. Induction of CREB phosphorylation during initiation of adipocyte differentiation is dependent on ERK1/2 activity. (A) 3T3-L1 cells were treated for 15 min in Dex-Ins medium with various combinations of 200 μM 8-pCPT-2′-O-Me-cAMP, 200 μM (more ...)
To determine if the Epac-mediated activation of Rap was a bystander effect or was important for adipogenesis, we tested whether forced expression of the dominant-negative Rap1N17 would inhibit adipogenesis. We noted a robust adipocyte differentiation in 3T3-L1 cells transduced with vector alone, whereas adipocyte differentiation of cells expressing Rap1N17 was suppressed, whether determined by oil red O staining (Fig. ) or adipocyte marker gene expression (Fig. ). Furthermore, retroviral expression of RapGAP, which facilitates conversion of the active Rap-GTP into its inactive GDP-bound form, significantly reduced expression of the adipogenic marker genes PPARγ2 and aP2 (Fig. ). Collectively, these findings indicate that cAMP-dependent activation of the Epac1/Rap1 pathway is required for adipocyte differentiation.
The data in the preceding paragraphs suggested that active PKA was necessary, but not sufficient, for cAMP-stimulation of adipogenesis. The role of PKA was ascertained by the demonstration that the PKA-specific inhibitory cAMP analogs of the equatorial diastereoisomer of adenosine-3′,5′-cyclic monophosphorothioate (R
) counteracted 3T3-L1 cell differentiation (Fig. A) and that differentiation was blocked by forced expression of dominant-negative RIα (Fig. ), which, like the R
p-cAMPS analogs, significantly decreased the PKA activity in 3T3-L1 cell extracts (data not shown). In contrast, the widely used, but nonspecific, PKA inhibitor H89 failed to abrogate cAMP-stimulated adipogenesis (Fig. ). This suggested that PKA could act by inhibiting an additional kinase targeted by H89. In this way H89 would make PKA superfluous for differentiation. H89 is a potent inhibitor of Rho-kinases (42
), which therefore would be a prime candidate for the putative kinase inactivated downstream of PKA; the more so as inhibition of Rho-kinase constitutes a crucial step for initiation of adipocyte differentiation (51
). Since the Rho-kinase is stimulated by GTP-Rho, an obvious mechanism of PKA-induced inhibition of Rho-kinase would be to convert Rho-GTP to the inactive Rho-GDP form. In fact, activation of PKA alone or in combination with activation of Epac reduced the level of Rho-GTP to undetectable levels, whereas activation of Epac alone had no effect, and furthermore, activation of PKA by 6-MB-cAMP decreased phosphorylation of the Rho-kinase substrate MLC (Fig. ). To further substantiate the notion that PKA activation stimulated adipogenesis via downregulation of a Rho/Rho-kinase-dependent pathway, we examined whether pharmacological inhibition of Rho-kinase was sufficient to restore adipogenesis of 3T3-L1 cells expressing the dominant-negative form of the RIα subunit. As predicted, addition of the Rho-kinase inhibitor sc-3536 restored the differentiation of cells expressing the dominant-negative RIα subunit as determined by oil red O staining (Fig. ) and expression of the adipocyte marker proteins PPARγ and aP2 (Fig. ). Based on these findings, we conclude that activation of PKA is dispensable for efficient adipocyte differentiation in the presence of Dex, high levels of Ins, and IBMX, provided that the Rho-kinase is inhibited.
FIG. 5. Activation of PKA is required for differentiation of 3T3-L1 cells, but dispensable when Rho-kinase is inhibited. (A) 3T3-L1 cells were grown to 2 days postconfluence and induced to differentiate by a standard differentiation cocktail consisting of Dex, (more ...)
The finding that the stimulatory effect of PKA activation on adipocyte differentiation could be mimicked by Rho-kinase inhibition and the fact that the dual PKA and Rho-kinase inhibitor enhanced rather than prevented adipocyte differentiation questioned the importance of PKA-mediated phosphorylation of CREB in cAMP-stimulated adipocyte differentiation. To investigate this, we first determined the effects of 6-MB-cAMP, 8-pCPT-2′-O-Me-cAMP, forskolin, IBMX, and H89 on the PKA activity in 3T3-L1 cells. The cells were treated for 15 min, as indicated in Fig. , and PKA activity in the lysates was determined. As expected, the addition of 8-pCPT-2′-O-Me-cAMP did not increase the PKA activity, whereas 6-MB-cAMP, forskolin, and IBMX robustly increased PKA activity. The combination of 6-MB-cAMP with 8-pCPT-2′-O-Me-cAMP increased PKA activity to a level comparable to that observed with 6-MB-cAMP alone. Finally, addition of H89 completely prevented the IBMX-dependent increase in PKA activity (Fig. ). Next, to determine to what extent activation of PKA affected phosphorylation of CREB on Ser-133, 3T3-L1 cells were grown to 2 days postconfluence and then induced with DMEM containing 10% FBS in the absence or presence of cAMP analogs. Cells were harvested after 5, 15, and 30 min. Figure demonstrates the time course of CREB and mitogen-activated protein kinase (MAPK) phosphorylation. Phosphorylation of both CREB and ERK1/2 was stimulated as early as 5 min after induction in the absence of cAMP analogs or IBMX. Maximal phosphorylation of CREB was observed after 15 min, whereas ERK1/2 phosphorylation still increased by 30 min. Interestingly, ERK1/2 phosphorylation increased rapidly in the cells treated with IBMX. Furthermore, the level of phosphorylated ERK1/2 after 15 and 30 min tended to be higher 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 (see below). No significant difference in CREB phosphorylation was 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. ). These findings indicated that robust CREB phosphorylation could be induced without specific activation of PKA, but might depend on ERK1/2 activation. To know if this could be the case, 3T3-L1 cells were transduced with either an empty vector or a vector expressing the dominant-negative form of RIα and induced for 15 min with Dex, Ins, and IBMX, in the absence or presence of the selective PKA inhibitor Rp-cAMPS or the MEK inhibitor U0126. Cell extracts were analyzed for CREB and MAPK phosphorylation by Western blotting. Figure demonstrates that phosphorylation of CREB and ERK1/2 was induced also when the PKA inhibitor was present and in cells expressing the dominant-negative form of RIα. In contrast, the MEK inhibitor almost completely prevented the increased phosphorylation of not only ERK1/2, but also CREB. In vitro kinase assays demonstrated that the MEK kinase inhibitor (and the Rho-kinase inhibitor) did not inhibit PKA, whereas H89 potently inhibited PKA (Fig. ). Finally, we demonstrated that expression of a canonical CREB-responsive gene was induced by Dex, Ins, and IBMX in the presence of the PKA inhibitor Rp-cAMPS, but not in the presence of the MEK inhibitor (Fig. ).
CREB has been shown to play a pivotal role during initiation of adipocyte differentiation, at least in part by regulating the expression of C/EBPβ (72
), although alternative routes for induction of C/EBPβ also seem to exist (26
). It is well established that phosphorylation of Ser-133 is necessary, but not sufficient, for CREB-mediated transactivation and that additional PKA-dependent processes may be needed for transcriptional activation (8
). However, taken together all experiments described above argue against PKA being directly involved in CREB phosphorylation and activation. Rather, the activation of CREB and the ensuing induction of C/EBPβ expression appear to depend on Ins/IGF-1 signaling. Therefore, we propose that Rho-kinase and not CREB is a central target for PKA activation during the onset of the adipocyte differentiation program.
Ins/IGF-1 signaling is crucial for adipogenesis, so it is puzzling that inhibition of Rho-kinase is essential for induction of adipocyte differentiation since inhibition of Rho-kinase also impairs Ins signaling in adipocytes (27
). We reasoned that activation of Epac might overcome the negative effect of Rho-kinase inhibition on Ins/IGF-1 signaling. In fact, Epac activation has been shown to potentiate Ins signaling in muscle cells (7
). The supraphysiological concentration of Ins used in the standard Dex-Ins-IBMX differentiation protocols mimics IGF-1, the main adipogenic inducer, by interacting with the IGF-1 receptor (59
). To demonstrate that inhibition of Rho-kinase impaired IGF-1/Ins signaling in 3T3-L1 preadipocytes, the effect of the Rho-kinase-inhibitor sc-3536 was studied in cells stimulated with increasing concentrations of IGF-1 in the presence or absence of the Epac activator 8-pCPT-2′-O
-Me-cAMP. The fetal calf serum used in the present experiments contained 10 nM IGF-1 (data not shown). Hence, the final concentration of IGF-1 in the medium containing 10% serum was 1 nM. Phosphorylation of PKB was determined as a marker of IGF-1 signaling. We found that the Rho-kinase inhibitor decreased IGF-1-dependent PKB phosphorylation. Importantly, activation of Epac enhanced PKB phosphorylation and restored the IGF-1-dependent PKB phosphorylation in the presence of the Rho-kinase inhibitor (Fig. ). Thus, Epac activation stimulated IGF-1 signaling whether the Rho-kinase was inhibited or not.
FIG. 7. Activation of Epac enhances Ins/IGF-1 signaling in 3T3-L1 cells (A) 3T3-L1 cells were grown to 2 days postconfluence and then treated with Dex and increasing concentrations of IGF-1 in the absence or presence of 10 μM sc-3536 and 200 μM (more ...)
Based on the results above, we predicted that if the Rho-kinase was inhibited the activation of Epac could promote adipogenesis without PKA activation or addition of Ins/IGF-1 on top of the 1 nM basal level of IGF-1 in the medium. To test this prediction, we first treated 3T3-L1 preadipocytes with 8-pCPT-2′-O-Me-cAMP and the Rho-kinase inhibitor sc-3536 in the absence of added Ins/IGF-1 and IBMX. Pharmacological inhibition of the Rho-kinase by sc-3536 had no effect on adipogenesis, whether determined by oil red O staining or adipocyte 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, inhibition of Rho-kinase dramatically enhanced differentiation when combined with the Epac activator 8-pCPT-2′-O-Me-cAMP (Fig. ). Expression of the dnEpac prevented, as expected, the 8-pCPT-2′-O-Me-cAMP-induced enhancement of adipocyte differentiation (Fig. ). To corroborate the results obtained by pharmacological inhibition of the Rho-kinase, we performed parallel experiments in which 3T3-L1 cells were transduced with either an empty retroviral vector or a retroviral vector expressing a dominant-negative version of RhoA (RhoA-N19). In agreement with the results obtained with the chemical Rho-kinase inhibitor sc-3536, activation of Epac induced adipocyte differentiation, as determined by oil red O staining and adipocyte marker gene expression in cells expressing the dominant-negative version of RhoA, but not in cells transduced with the empty vector (Fig. ). We conclude that Epac activation is essential for adipogenesis in cells exposed to physiological levels of Ins/IGF-1 when the Rho-kinase is inhibited.
FIG. 8. Activation of Epac is sufficient to induce differentiation of 3T3-L1 cells when Rho-kinase is inhibited. 3T3-L1 cells were grown to 2 days postconfluence and induced to differentiate by Dex in the absence or presence of 10 μM sc-3536. Additionally, (more ...)