TARGETING OF LEPTIN TO THE REGULATED SECRETORY PATHWAY IN PITUITARY ATT-20 CELLS

Leptin, a key regulator of fat homeostasis, is the product of the obes e gene [1-3], and is secreted from adipocytes and binds to receptor si tes in the choroid plexus [4,5]. Several studies have implicated serum insulin levels in the upregulation of leptin gene expression [6-8]. I t is currently not known whether leptin levels are also subject to reg ulation at the level of secretion. Leptin is normally produced in adip ocytes, the secretory pathways of which are not well characterized. He re, we used pituitary AtT-20 cells, which serve as a model system for both regulated and constitutive secretary pathways, to examine the int racellular targeting and secretion of leptin. Confocal immunofluoresce nce analysis of AtT-20 cells expressing an epitope-tagged human leptin (FLAG-leptin) demonstrated that FLAG-leptin colocalized with endogeno us adrenocorticotrophic hormone (ACTH) at the tips of processes extend ed from these cells, where regulated secretary granules accumulate. FL AG-leptin secretion was increased in the presence of 8-Br-cAMP, which stimulates the secretion of ACTH. For FLAG-leptin, the calculated sort ing index, a quantitative measure of the efficiency of protein sorting to the regulated pathway, was similar to those of other regulated sec retary proteins. These results demonstrate that FLAG-leptin behaves li ke a regulated protein in cells with a biosynthetic regulated secretar y pathway.