Characterization of leptin intracellular trafficking

• Published in: European journal of histochemistry Vol. 44; no. 4; pp. 325 - 334
• Main Authors: Wilcke, M, Walum, E
• Format: Journal Article
• Language: English
• Published: Italy PAGEPress Publications 01.01.2000
• Subjects: Anti-Bacterial Agents - metabolism; Anti-Bacterial Agents - pharmacology; Cell Line; Endosomes - metabolism; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Humans; Intracellular Fluid - metabolism; Ionophores - metabolism; Ionophores - pharmacology; Leptin - metabolism; Macrolides; Monensin - metabolism; Monensin - pharmacology; Proton-Translocating ATPases - antagonists & inhibitors
• ISSN: 1121-760X; 2038-8306

Leptin is produced by adipose tissue, and its concentration in plasma is related to the amount of fat in the body. The leptin receptor (OBR) is a member of the class I cytokine receptor family and several different isoforms, produced by alternative mRNA splicing are found in many tissues, including the hypothalamus. The two predominant isoforms includes a long form (OBR) with an intracellular domain of 303 amino acids and a shorter form (OBR) with an intracellular domain of 34 amino acids. Since OBR, is mainly expressed in the hypotalamus, it has been suggested to be the main signalling form. The peripheral production of leptin by adipocyte tissue and its effects as a signal of satiety in the central nervous system imply that leptin gains access to regions of the brain regulating in energy balance by crossing the blood-brain barrier. In an attempt to characterize the intracellular transport of leptin, we have followed binding internalization and degradation of leptin in HEK293 cells. We have also monitored the intracellular transport pathway of fluorescent conjugated leptin in HEK293 cells. Phenylarsine oxide, a general inhibitor of endocytosis, as well as incubation at mild hypertonic conditions, prevented the uptake of leptin, confirming a receptor-mediated internalization process. When internalized, 125I-leptin was rapidly accumulated inside the cells and reached a maximum after 10 min. After 70 minutes about 40-50% of total counts in each time point were found in the medium as TCA-soluble material. Leptin sorting, at the level of early endosomes, did not seem to involve recycling endosomes, since FITC-leptin was sorted from Cy3-transferrin containing compartments at 37 degrees C. At 45 minutes of continuos internalization, FITC-leptin appeared mainly accumulated in late endocytic structures colocalizing with internalized rhodamine coupled epidermial growth factor (EGF) and the lysosomal marker protein lamp-1. The transport of leptin was also shown to engage a monensin and bafilomycin sensitive degradation process in lysosomes. Together, our results provide novel data concerning the uptake, intracellular localization and transport of leptin.