LOCALIZATION OF A PIOGLITAZONE RESPONSE ELEMENT IN THE ADIPOCYTE FATTY-ACID-BINDING PROTEIN GENE

The thiazolidinediones are a class of antidiabetic compounds that incr ease the sensitivity of target tissues to insulin. An earlier study ha s shown that these compounds enhance the insulin-stimulated differenti ation of 3T3-L1 cells and up-regulate expression of differentiation-de pendent genes. We have observed that the mRNA encoding the adipocyte f atty acid-binding protein (aFABP) increases shortly after incubation o f cells with pioglitazone, a thiazolidinedione analogue. The drug was found to enhance, in a time- and dose-dependent fashion, the expressio n of a chimeric gene that was constructed by fusing the aFABP promoter upstream of the chloramphenicol acetyltransferase (CAT) gene. To loca lize the sequence within the promoter that is responsive to pioglitazo ne, a series of chimeric genes containing sections of the aFABP promot er fused to the CAT gene were analyzed after transfection of 3T3-L1 ce lls. A section of DNA located at approximately 5.2 kilobases and known to encompass a tissue-specific and differentiation-dependent enhancer element was found to confer responsiveness to the drug. Analysis of s equences in this region of the aFABP promoter by DNA gel retardation a ssays revealed the presence of a protein in nuclear extracts from drug -treated cells that bound to a specific sequence (ARE-6). The presence of the protein could be demonstrated in differentiated adipocytes, bu t the protein was present at only low levels in preadipocytes. Treatme nt of preadipocytes with pioglitazone resulted in the precocious appea rance of this protein in nuclear extracts. Multiple copies of the ARE- 6 sequence inserted upstream of a heterologous promoter linked to the CAT gene conferred pioglitazone responsiveness. The experiments report ed in this study establish that the insulin-sensitizing agent pioglita zone up-regulates expression of the aFABP gene through an element loca ted within a region of DNA responsible for tissue-specific and differe ntiation-dependent expression of the gene.