Identification of high-affinity binding sites for the insulin sensitizer rosiglitazone (BRL-49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferator-activated receptor gamma.

A radioiodinated ligand, [125I]SB-236636 [( S )-(−)3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]3-[125I]iodophenyl]2-ethoxy propanoic acid], which is specific for the γ isoform of the peroxisomal proliferator activated receptor (PPARγ), was developed. [125I]SB-236636 binds with high affinity to full-length human recombinant PPARγ1 and to a GST (glutathione S -transferase) fusion protein containing the ligand binding domain of human PPARγ1( K D = 70 nM). Using this ligand, we characterized binding sites in adipose-derived cells from rat, mouse and humans. In competition experiments, rosiglitazone (BRL-49653), a potent antihyperglycemic agent, binds with high affinity to sites in intact adipocytes (IC50 = 12, 4 and 9 nM for rat, 3T3-L1 and human adipocytes, respectively). Binding affinities (IC50) of other thiazolidinediones for the ligand binding domain of PPARγ1 were comparable with those determined in adipocytes and reflected the rank order of potencies of these agents as stimulants of glucose transport in 3T3-L1 adipocytes and antihyperglycemic agents in vivo : rosiglitazone > pioglitazone > troglitazone. Competition of [125I]SB-236636 binding was stereoselective in that the IC50 value of SB-219994, the ( S )-enantiomer of an α-trifluoroethoxy propanoic acid insulin sensitizer, was 770-fold lower than that of SB-219993 [( R )-enantiomer] at recombinant human PPARγ1. The higher binding affinity of SB-219994 also was evident in intact adipocytes and reflected its 100-fold greater potency as an antidiabetic agent. The results strongly suggest that the high-affinity binding site for [125I]SB-236636 in intact adipocytes is PPARγ and that the pharmacology of insulin-sensitizer binding in rodent and human adipocytes is very similar and, moreover, predictive of antihyperglycemic activity in vivo .