This project aims to determine the regulation of ob gene expression in immortalized human brown adipocytes. The ob gene product, leptin, is a putative hormone that acts on hypothalamic centres, recently shown to induce weight loss in obese mice. Leptin functions as a central component in the body weight regulatory system, including insulin, B3-adrenergic receptor and hypothalamic centres. Since obesity constitutes the greatest health problem in Western society, the elucidation of leptin regulation is of considerable scientific and social relevance. A unique cellular system of immortalized human brown adipocytes forms the basis of this work Northern blot, PCR and nuclear run-on analyses will examine the dependence of ob gene expression on insulin and various B3-adrenergic ligands. The ob gene and its regulatory regions will be examined to define enhancer and transcription elements. Cell lines with a mutant B3-adrenergic receptor will be generated and selective inhibitors/activators of signal transduction pathway components will be employed to define intracellular processes in ob gene expression.