Ciliary and Non-Ciliary Protein Kinase A (PKA) Differentially Coordinate 3T3-L1 Adipogenesis

Abstract

The primary cilium (PC) is a compact organelle that regulates cellular processes in virtually all cell types. In this context, research attests that adipocyte progenitor cell (APC) adipogenesis is regulated by PC-specific signaling systems (i.e. cAMP signaling). Recent work identified a role for ciliary expression of free fatty acid receptor 4 (FFAR4), and its ability to increase ciliary cAMP levels, in potentiating pro-adipogenic actions of low concentrations of insulin and dexamethasone in APCs, including those isolated from mouse or human adipose tissues (ATs) and the preadipocyte cell line (i.e. 3T3-L1 cells). We propose that promoting APC hyperplasia, via this PC-specific cAMP system, could reduce AT hypoxia and inflammation in obesity or type 2 diabetes (T2D), therefore, alleviating associated vascular dysfunctions. While earlier work suggested that FFAR4 promoted adipogenesis by stimulating extra-ciliary exchange protein activated by cAMP (EPAC) activity, in contrast, our work hypothesizes that FFAR4-regulated ciliary cAMP promotes adipogenesis by selectively activating ciliary protein kinase A (PKA). Thus, we investigated the role of ciliary PKA activity in promoting FFAR4-dependent adipogenesis in 3T3-L1 preadipocytes, a cell model of APC adipogenesis. Specifically, we determined the impact of expressing a ciliary targeted construct [NPHP3(1-203)-YFP-PKI] that allowed ciliary expression of PKA inhibitory peptide (PKI) on PC, and non-PC-dependent, 3T3-L1 adipogenesis. As a control, a construct showcasing the inactive version of PKI [NPHP3-YFP-PKI(4A)] was expressed in these cells. Adipogenic responses were determined through PPARγ nuclear staining. Our findings are consistent with the idea that specific inhibition of ciliary PKA (i.e. PKI) significantly (p ≤ 0.0001) antagonized FFAR4-mediated pro-adipogenic responses in 3T3-L1 cells (i.e. PPARγ staining was notably absent). In marked contrast, expression of the inactive PKI variant did not alter the pro-adipogenic effects promoted by the FFAR4 agonist TUG-891 in these cells (i.e. PPARγ staining was not altered). Speaking to the specificity of these effects on ciliary FFAR4/PKA signaling, experiments in which adipogenesis was induced using a non-PC-dependent angiogenic cocktail (i.e., high concentrations of insulin, dexamethasone and isobutyl methyl-xanthine (IBMX)) was insensitive to ciliary PKI expression. We believe our work will help identify PC-specific PKA as a pharmacological target in regulating APC adipogenesis.