<P><B>1) Distinct dysregulation of lipid metabolism by mutant TR isoforms</B></P><P>We showed that in contrast to the TRalpha1PV mouse, TRbetaPV mice exhibited no significant reduction in white adipose tissue, but had significant increases in serum free fatty acids and total triglycerides. Moreover, the liver of TRbetaPV mice was markedly increased (34%) with excess lipid accumulation, but the liver mass of the TRalpha1PV mouse was decreased (19%) with a paucity of lipids. These results indicated that TRbeta and TRalpha1 mutants exerted distinct abnormalities in lipid metabolism. Further biochemical analyses indicated that increased lipogenic enzyme expression, activated peroxisome proliferators-activated receptor gamma (PPARgamma) signaling, and decreased fatty acid beta-oxidation activity contributed to the adipogenic steatosis and lipid accumulation in the liver of TRbetaPV mice. In contrast, the expression of lipogenic enzymes and PPARgamma was decreased in the liver of TRalpha1PV mice. These results suggested that the regulation of genes critical for lipid metabolism by TRs in the liver is isoform-dependent. These results indicated that TRbeta and TRalpha1 mutants had different effects on lipid metabolism and that TR isoforms differentially contribute to the pathogenesis of lipid metabolism in hypothyroidism and lipid disorders.</P><P><B>2) Differential impairment of adipogenesis by mutant TR isoforms</B></P><P>To dissect the molecular mechanism underlying the distinct phenotypic manifestation in white adipose tissue between TRalpha1PV and TRbetaPV mice, we used the 3T3-L1 cell line. This system has long been used by investigators as a model cell line to study adipogenesis. To establish the cause-effect relationship, we stably expressed TRalpha1PV or TRbetaPV in 3T3-L1 cells and evaluated the alterations in adipogenesis as compared with parental cells that contained only the vector backbone (control cells). Clones with an equal abundance of TRalpha1PV (denoted as L1-alpha1PV cells) or TRbetaPV (L1-betaPV cells) were used to elucidate the molecular basis of the distinct phenotypic expression in adipogenesis of white adipose tissue observed in TRalpha1 and TRbeta mice. We found that the T3-induced adipogenesis was more severely impaired in L1-alpha1PV cells than in L1-betaPV cells. The expression of two master regulatorsPPARgamma and CCAAT enhancer binding protein alpha (C/EBPalpha)was found to be more repressed in L1-alpha1PV cells than in L1-betaPV cells. Consistent with the gene expression patterns, the expression of several key lipogenic enzymes was more severely inhibited by TRalpha1PV than TRbetaPV. Further analysis indicated that the preferential repression of the C/EBPalpha gene by TRalpha1PV was mediated by more avid recruitment of TRalpha1PV to the promoter complex of the C/EBPalpha gene. That TR-mediated adipogenesis is isoform-dependent raised the possibility that isoform-specific TR ligands could be considered for therapeutic intervention aimed at lipid abnormalities.</P>
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