[Frontiers in Bioscience E2, 392-398, January 1, 2010]

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

Zhenyu Ji, Fang C. Mei, Xiaodong Cheng

Department of Pharmacology and Toxicology, Sealy Center for Cancer Cell Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Brach, Galveston, Texas 77555-0616, USA

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Reagents
3.2. Cell culture and adipocyte differentiation in vitro
3.3. GPDH activity assay
3.4. Oil Red O staining
3.5. Stable shRNA transfection of 3T3-L1 cells by lentiviral infection
3.6 .Reverse transcriptase polymerase chain reaction (RT-PCR) analysis
3.7. Immunoblotting analysis
4. Results
4.1. Activation of PKA catalytic subunit is not necessary for 3T3-L1 preadipocyte differentiation
4.2. Epac1 is required for 3T3-L1 differentiation
4.3. Epac1 knockdown suppresses PPARγ
4.4. Activation of Epac is not sufficient for cAMP-mediated 3T3-L1 differentiation
5. Discussion
6. Acknowledgement
7. References

1. 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.