Abstract

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 (glutathioneS-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.