The small molecule phenamil is a modulator of adipocyte differentiation and PPARγ expression

We previously described the use of a cell-based screening approach to identify small molecules that regulate adipocyte differentiation. Here we identify the amiloride derivative phenamil as an adipogenic compound. Phenamil acutely induces expression of the key transcription factor of adipogenesis, peroxisome proliferator-activated receptor γ (PPARγ) and, consequently, promotes the differentiation of multiple preadipocyte cell lines, including 3T3-L1 and F442A. Interestingly, the adipogenic action of phenamil is distinct from and additive with both PPARγ ligands and the previously identified adipogenic small molecule harmine. To identify signaling pathways mediating phenamil's effects, we performed transcriptional profiling of 3T3-F442A preadipocytes. ETS variant 4 (ETV4) was identified as a gene rapidly induced by phenamil but not by other adipogenic small molecules or PPARγ agonists. Transient expression of ETV4 in preadipocytes enhances the expression of PPARγ. Stable overexpression of ETV4 promotes expression of PPARγ and its downstream target genes and enhances morphological differentiation. Finally, knockdown of PPARγ expression by shRNA blocks the effects of phenamil on adipocyte differentiation and gene expression, but it does not block phenamil induction of ETV4, which suggests that ETV4 acts upstream of PPARγ in differentiation processes. These results identify a phenamil as new small molecule tool for the probing of adipocyte differentiation that acts, at least in part, through induction of ETV4 expression.