Regulation of uncoupling protein-2 mRNA in L6 myotubules: I: Thiazolidinediones stimulate uncoupling protein-2 gene expression by a mechanism requiring ongoing protein synthesis and an active mitogen-activated protein kinase.

The mitochondrial uncoupling protein-2 (UCP2) can uncouple phosphorylation to subserve several functions. It has been reported that the insulin sensitizers, thiazolidinediones (TZDs), increase UCP2 mRNA levels and, more recently, that TZDs stimulate UCP2 reporter genes but that the sequences involved do not bind peroxisome proliferator-activated receptor gamma (PPARgamma). We report here that TZDs stimulated UCP2 gene (ucp2) transcription in L6 myotubules involving an indirect mechanism. L6 cells contained comparatively small amounts of PPARgamma mRNA but clearly detectable amounts of PPARgamma2 protein. UCP2 mRNA levels were increased in a time- and concentration-dependent manner by TZDs. UCP2 mRNA had slow turnover (t 1/2 approximately 38 h), and this was not affected by TZDs. Bisphenol A diglycidyl ether, a PPARy antagonist, concentration dependently inhibited the TZD-induced increase in UCP2 mRNA. Blockade of protein synthesis with cycloheximide as well as abrogation of mitogen-activated protein kinase (MAPK) activity with PD98059 or U0126 also prevented the TZD-induced increase in UCP2 mRNA. As with autologous UCP2 mRNA, TZDs stimulated reporter gene expression directed by ucp2 sequences in transiently transfected L6 cells. The effect was enhanced by cotransfection of PPARgamma + retinoid X receptor gamma and prevented by MEK blockade. TZDs, however, did not increase the activation of MAPK, nor did its activation by other means (change of medium, insulin-like growth factor-1, insulin) increase UCP2 mRNA, indicating that phosphorylation is not limiting. These results suggest that TZDs indirectly stimulate ucp2 transcription by inducing-via PPARgamma-limiting amounts of a protein, which must be phosphorylated by MAPK to stimulate the gene.