Thyroid hormone (T3) has profound effects on the metabolism of glucose and lipids in the liver. Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the key initiating step in gluconeogenesis and is regulated by hormones, including T3 primarily at the transcriptional level. The PEPCK gene provides an excellent model for examining T3 action because T3 modulates PEPCK gene expression through at least two mechanisms. First, T3 directly stimulates PEPCK transcription through the binding of the T3 receptor (TR) induction by glucagon (cAMP), which is the major inducer of PEPCK gene expression. The goals of this study are to analyze these mechanisms of transcriptional regulation by T3> The first aim is to characterize the binding of the TR to the TRE in the promoter of the PEPCK gene. The key nucleotides in the PEPCK-TRE required for the binding of the TR and the stimulation of transcription will be identified. The binding of the TR to the PEPCK-TRE will be evaluated with methylation interference assays and gel mobility assays. It will be determined if the TR binds to the PEPCK-TRE as a heterodimer with a liver nuclear factor. The second aim is to identify the putative nuclear protein that heterodimerizes with the TR to bind to the PEPCK- TRE. The retinoic X receptor (RXRalpha) which will form heterodimerizes with the TR will be evaluated for its ability to heterodimerize with the TR and stimulate transcription of the PEPCK gene. The binding properties of RXRalpha will be compared with those of the liver nuclear factor. The third aim is to characterize the interactions between the TR and a liver- specific transcription factor bound to a second site in the PEPCK promoter. These experiments will test for liver interactions between the TR and CCAAT enhancer binding protein (C/EBP). C/EBP contributes to the liver-specific expression and cAMP responsiveness of the PEPCK gene. PEPCK-CAT vectors will be introduced into HepG2 cells along with mammalian expression vectors encoding either the TR and C/EBP. The CAT gene provides a marker to indicate the effects of these proteins on transcription originating from the PEPCK promoter. These studies will characterize the cross-talk between proteins involved in the T3 and cAMP induction of PEPck transcription. The final aim is to determine if the thyroid status of the animal affects the binding of transcription factors to the PEPck promoter. Nuclear proteins will be isolated from hypothyroid and hyperthyroid rats, and binding to the PEPCK promoter will be evaluated with Dnase I footprinting and gel mobility assays. This proposal focuses on the molecular mechanisms by which hormones regulate the transcription of a key enzyme in hepatic glucose production. Elucidation of these mechanisms will promote understanding of the transcriptional regulation of other enzymes affected by T3 and cAMP. Such understanding could lead to clinical benefits for the common endocrine disorders of hypo-and hyperthyroidism and diabetes.