3',5'-CYCLIC ADENOSINE MONOPHOSPHATE-RESPONSE SEQUENCES OF THE UNCOUPLING PROTEIN GENE ARE SEQUENTIALLY RECRUITED DURING DARGLITAZONE-INDUCED BROWN ADIPOCYTE DIFFERENTIATION

Uncoupling protein-1 (UCP) is uniquely expressed in brown adipose tiss ue (BAT) and is essential to the thermogenic function of this tissue. The UCP gene is under the control of norepinephrine (NE) via cAMP. How ever, the precise delineation of the cAMP response sequences and mecha nisms whereby cAMP stimulate the gene have remained elusive. A BAT tum or cell line, HIB-1B, can be differentiated into UCP-expressing brown adipocytes. We report here that when these cells are differentiated wi th a standard differentiation protocol including insulin, T-3, hydroco rtisone, IBMX, and indomethacin (standard differentiation, StD), cAMP stimulation of the rat UCP gene is largely mediated by an upstream 90- bp sequence -2,399/-2,490 (R90) with a lesser contribution of a downst ream sequence -57/+114 (dnCRS). This latter is functional also in non- BAT cells, whereas the cAMP response sequence contained in R90 (upCRS) is BAT-specific. Thiazolidinediones (TZD) are a new group of drugs kn own to increase sensitivity to insulin and, more recently, to induce a dipocyte differentiation (adipogenesis) via PPAR gamma. A TZD, darglit azone (darg), can rapidly induce differentiation of HIB-1B cells, as j udged by the expression of the adipocyte lipid binding protein (aP2), lipoprotein lipase (LPL), uncoupling protein (UCP) and beta(3)-adrener gic receptors. UCP messenger RNA (mRNA) responsive to NE is evidenced as early as one day after exposure to darg. While UCP-CAT vectors (+11 4/-3673 bp of rat UCP gene) are barely responsive to NE in HIB-1B prea dipocytes, both darg and StD markedly enhance NE responsiveness of suc h constructs. However, by 3 days of exposure to darg, the responses we re less vigorous than in StD cells (4- to 10-fold us. 20- to 50-fold), and the deletion of R90 did not affect the response to NE in darg-dif ferentiated cells, whereas this deletion caused a 75% reduction in StD cells. Prolongation of darg exposure to 5-7 days resulted in greater response of UCP mRNA to NE and 50-80% inhibition of the response of UC P-CAT vectors by the deletion of R90. Thus, darg induced differentiati on of HIB-1B cells suggests that the NE-dependent expression of the UC P gene takes place in a step-prise manner: first, the gene is ''enable d,'' as no UCP mRNA is detected in HIB-1B preadipocytes; thereafter an d transiently, the response of the gene to NE is sustained by dnCRS; f inally, as differentiation progresses, a cell-specific and more powerf ul cis-acting sequence, upCRS, is recruited, accounting in the fully d ifferentiated cell for most of the response to NE. These results also suggest that TZDs might increase energy expenditure by inducing termin al differentiation of BAT, and that these drugs may be useful in the d ifferential cloning of the factors involved in the recruitment of the BAT specific cAMP response sequence.