Abstract
Adipogenesis is a complex process modulated by several factors, including cAMP signaling. The main cAMP target is protein kinase A (PKA), a tetrameric enzyme with four regulatory subunits showing tissue-specific expression and function: PRKAR2B is the main regulatory subunit in adipose tissue in mice and in adult humans. This study aimed to evaluate the expression of PKA regulatory subunits in human adipose tissue during fetal development and to investigate their role in the differentiation of 3T3-L1 and primary human preadipocytes. The expression of PKA regulatory subunits was evaluated in fetal adipose tissue (immunohistochemistry) and in cultured 3T3-L1 and primary human preadipocytes (western blot analysis). Cultured cells were transiently transfected with siRNA against PRKAR2B and induced to differentiate. Differentiation was evaluated by intracellular triglyceride staining (Oil Red O) and expression of molecular markers of adipocyte differentiation. In this study, we found that PRKAR2B is the main regulatory subunit in human adipose tissue during fetal development, from 12 weeks of gestation to the end of gestation, as well as in 3T3-L1 and primary human preadipocytes. The expression of PRKAR2B increases progressively during in vitro differentiation. The silencing of PRKAR2B abolishes the increase in the expression of peroxisome proliferator-activated receptor gamma (PPARg (PPARG)), fatty acid synthase, aP2 (FABP4), and lipoprotein lipase, as well as intracellular triglyceride accumulation, resulting in impaired adipocyte differentiation in both mouse and human cell systems. In conclusion, PRKAR2B is the key PKA regulatory subunit involved in mouse and human adipose tissue development. The physiological increase in the expression of PRKAR2B is an essential event in adipogenesis in both mice and humans, and it might represent a possible target for future strategies for obesity treatment.
Highlights
White adipose tissue (WAT) is mainly involved in energy storage and mobilization in the form of triglycerides, a paracrine and endocrine function of white adipocytes has been recognized
In order to investigate the expression of protein kinase A (PKA) regulatory subunits in human adipose tissue during fetal development, IHC was carried out on tissue sections of adipose tissue obtained from fetuses of different gestational ages
In line with previous reports on mice and adult humans [11, 12, 13], the present study provides data indicating that PRKAR2B is the key PKA regulatory subunit in human adipose tissue during fetal development
Summary
White adipose tissue (WAT) is mainly involved in energy storage and mobilization in the form of triglycerides, a paracrine and endocrine function of white adipocytes has been recognized. CAMP acts as an intracellular second messenger in the regulation of a number of different cellular processes, such as cell growth and differentiation. 3-isobutyl-1-methylxanthine (IBMX), one of the inducers of adipogenesis, acts through the increase in intracellular cAMP levels. The activation of cAMP-responsive elementbinding protein (CREB) induces the expression of several transcription factors that promote adipogenesis, mainly CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor gamma (PPARg (PPARG)) [3, 4, 5, 6]. A role for exchange protein directly activated by cAMP (EPAC (RAPGEF3)) in cAMP-mediated adipocyte differentiation has been suggested [8], evidence indicates that the activation of CREB is mainly modulated by cAMPdependent protein kinase A (PKA) [9]
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