Epac, not PKA catalytic subunit, is required for 3T3-L1 preadipocyte differentiation

Abstract

Cyclic AMP plays a critical role in adipocyte differentiation and maturation. However, it is not clear which of the two intracellular cAMP receptors, exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor or protein kinase A/cAMP-dependent protein kinase, is essential for cAMP-mediated adipocyte differentiation. In this study, we utilized a well-defined adipose differentiation model system, the murine preadipocyte line 3T3-L1, to address this issue. We showed that knocking down Epac expression in 3T3-L1 cells using lentiviral based small hairpin RNAs down-regulated peroxisome proliferator-activated receptor gamma expression and dramatically inhibited adipogenic conversion of 3T3-L1 cells while inhibiting PKA catalytic subunit activity by two mechanistically distinct inhibitors, heat stable protein kinase inhibitor and H89, had no effect on 3T3-L1 adipocyte differentiation. Moreover, cAMP analog selectively activating Epac was not able to stimulate adipogenic conversion. Our study demonstrated that while PKA catalytic activity is dispensable, activation of Epac is necessary but not sufficient for adipogenic conversion of 3T3-L1 cells.

Figure 1

PKA catalytic activity is not required for the differentiation of 3T3-L1 cells. 3T3-L1 cells, two days post-confluence, were treated with various reagents as described below and induced to differentiate. Effects of treatments were documented either by cell imaging using a digital camera under a phase-contrast microscope (A) or by monitoring the cellular glycerophosphate dehydrogenase (B). Treatments include: 1, vehicles only; 2, 1 μM insulin and 1 μM Dex; 3, 1 μM insulin, 1 μM Dex, and 0.5 mM IBMX; 4, 1 μM insulin, 1 μM Dex, 0.5 mM IBMX and 5 μM H-89; and 5, 1 μM insulin, 1 μM Dex, 0.5 mM IBMX and 50 μM mPKI.

Figure 2

Epac1 expression is required for 3T3-L1 adipogenic differentiation. (A) mRNA expression levels of Epac1 after lentiviral shRNA silencing as measured by RT-PCR. (B) Protein levels of Epac1 after lentiviral shRNA silencing as measured by Western blot. (C) Effects of Epac1 gene silencing by lentiviral shRNAs on adipocyte differentiation efficiency of 3T3-L1 cells as determined by Oil Red O staining. NTC: non-targeting control shRNA.

Figure 3

Silencing of Epac1 expression suppresses PPAR_γ expression in 3T3-L1 cells. Confluent 3T3-L1 cells treated with or without Epac1-specific or non-targeting control lentiviral shRNAs were incubated with 1 μM insulin, 1 μM Dex, and 0.5 mM IBMX (induced) or vehicle (uninduced) for two days. Total RNAs were then isolated and the levels of PPAR_γ expression were measured by real time PCR.

Figure 4

Activation of Epac is not sufficient for cAMP-stimulated differentiation of 3T3-L1 cells. 3T3-L1 cells, two days post-confluent, were treated with various reagents as described below and induced to differentiate. Effects of treatments were documented either by cell imaging using a digital camera under a phase-contrast microscope (A) or by monitoring the cellular glycerophosphate dehydrogenase (B). Treatments include: 1, vehicles only; 2, 1 μM insulin and 1 μM Dex; 3, 1 μM insulin, 1 μM Dex, and 0.5 mM IBMX; and 4 and 5, 1 μM insulin, 1 μM Dex, and 100 or 200 μM 8-pCPT-2’-O-Me-cAMP, respectively.