1). NCoR Regulates Thyroid Hormone Receptor Isoform-dependent Adipogenesis. We previously showed that two thyroid hormone receptor (TR) isoforms - TRalpha1 and TRbeta1- differentially regulate thyroid hormone (T3)-stimulated adipogenesis in vivo. However, the molecular mechanisms by which TR isoforms differentially regulate adipogenesis remain unknown. Therefore the role of the nuclear receptor corepressor, NCoR, in TR isoform-dependent adipogenesis was studied. We found that T3-stimulated adipogenesis of 3T3-L1 cells was accompanied by progressive loss of NCoR protein levels. In 3T3-L1 cells stably expressing a mutated TRalpha1, PV (L1-alpha1PV cells), the T3-stimulated adipogenesis was more strongly inhibited than in 3T3-L1 cells stably expressing an identical mutation of TRbeta1 (L1-beta1PV cells). The stronger inhibition of adipogenesis in L1-alpha1PV cells was associated with a higher NCoR protein level. These results indicate that the degree of loss of NCoR correlates with the extent of adipogenesis. siRNA knock-down of NCoR promoted adipogenesis of control 3T3-L1 cells and reversed the inhibited adipogenesis of L1-alpha1PV and L1-beta1PV cells. An ubiquitin ligase, mSiah2, that targets NCoR for proteasome degradation was up regulated on day 1 before the onset of progressive loss of NCoR. NCoR was found to associate with mSiah2, and with TR, TRalpha1PV, or TRbeta1PV. Importantly, a stronger interaction of NCoR with TRalpha1PV than with TRbeta1PV was detected. Consistently, TRalpha1PV-NCoR complex was more avidly recruited than TRbeta1PV to the promoter more completely to inhibit the expression of the C/ebpalpha gene. These results indicate that differential interaction of NCoR with TR isoforms accounted for the TR isoform-dependent regulation of adipogenesis. For the first time we found that aberrant interaction of NCoR with TR could underlie the pathogenesis of lipid disorders in hypothyroidism. 2).Thyroid Hormone Receptor Beta PV Mutant Mice Have Defective Adaptive Thermogenesis and Reduced Uncoupling Protein 1 Expression. Cold-induced adaptive (or nonshivering) thermogenesis in small mammals is produced primarily in brown adipose tissue (BAT). Heat production requires Sympathetic Nervous System (SNS) stimulation, thyroid hormone and uncoupling protein 1 (UCP1) expression. Previous studies with a TRbeta-selective agonist showed that TRalpha isoform stimulation is necessary for thyroid hormone augmentation of adrenergic action in BAT, but not for UCP1-stimulation. We utilized mice with a dominant-negative TRbeta PV mutation to determine the role of TRbeta in adaptive thermogenesis and UCP1 expression. Wild-type and PV mutant mice were made hypothyroid and replaced with T3 for 10 days, to achieve normal range serum levels of thyroid hormone in the wild-type and mutant mice. The level of uncoupling protein 1 (UCP1), the key thermogenic protein in BAT, was progressively reduced in heterozygous as well as homozygous PV mutant mice. The thermogenic response of interscapular BAT, as determined by heat production during intravenous infusions of norepinephrine (NE), was defective in PV heterozygous and homozygous mutant mice. Brown adipocytes isolated from PV mutant mice had some reduction in cAMP and glycerol production in response to adrenergic stimulation, but less than that seen with TRbeta simulation alone. Defective adaptive thermogenesis in PV mutant mice is due to reduced UCP1 expression and reduced adrenergic responsiveness. Unique TR isoform-specific functions mediate T3 action in BAT. These studies show that a single tissue, BAT, has TRalpha and TRbeta-specific pathways.
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