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 obese gene [1-3], and is secreted from adipocytes and binds to receptor sites in the choroid plexus [4-5]. Several studies have implicated serum insulin levels in the upregulation of leptin gene expression [6-8]. It is currently not known whether leptin levels are also subject to regulation at the level of secretion. Leptin is normally produced in adipocytes, the secretory pathways of which are not well characterized. Here, we used pituitary AtT-20 cells, which serve as a model system for both regulated and constitutive secretory pathways, to examine the intracellular targeting and secretion of leptin. Confocal immunofluorescence analysis of AtT-20 cells expressing an epitope-tagged human leptin (FLAG-leptin) demonstrated that FLAG-leptin colocalized with endogenous adrenocorticotrophic hormone (ACTH) at the tips of processes extended from these cells, where regulated secretory granules accumulate. FLAG-leptin secretion was increased in the presence of 8-Br-cAMP, which stimulates the secretion of ACTH. For FLAG-leptin, the calculated sorting index, a quantitative measure of the efficiency of protein sorting to the regulated pathway, was similar to those of other regulated secretory proteins. These results demonstrate that FLAG-leptin behaves like a regulated protein in cells with a biosynthetic regulated secretory pathway.
Chavez
R A
RA
571 Life Sciences Addition, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200, USA.
Moore
H P
HP
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000324:Adrenocorticotropic Hormone / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D000818:Animals; D003594:Cytoplasmic Granules / Q000502:physiology*; D017931:DNA Primers; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation; D006801:Humans; D020738:Leptin; D051379:Mice; D009765:Obesity; D009842:Oligopeptides; D010455:Peptides; D010902:Pituitary Gland; D010911:Pituitary Neoplasms; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D020738:Leptin; D009842:Oligopeptides; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000324:Adrenocorticotropic Hormone; C085706:FLAG peptide