| Obesity has become a major health problem in Western society. Discovery of the ob gene product, leptin, was a breakthrough in understanding body-weight regulation. Leptin is produced mainly by adipocytes and regulates food intake and whole-body energy balance. In agreement with its role, leptin levels in serum increase with feeding and decrease upon food deprivation. The mechanism that couples leptin biosynthesis and secretion to the nutritional status of the body remains unknown. Free nutrients potentiate the actions of insulin on protein metabolism by stimulating signaling pathways such as the TOR (target of rapamycin) pathway. Mammalian TOR is a Ser/Thr kinase which is activated by free amino acids, especially leucine as well as by insulin via an unknown mechanism. Upon activation, mTOR phosphorylates the translation initiation repressor protein, PHAS which then dissociates from and unmasks the rate-limiting cap-binding initiation factor eIF-4E for translation initiation. To determine if PHAS inhibits translation of leptin mRNA, we performed immunoprecipitation experiments using antibodies against PHAS. We discovered that this antibody specifically immunoprecipitates leptin mRNA from adipocyte extracts. Addition of leucine to rat adipocytes caused phosphorylation of PHAS and stimulated leptin secretion in a rapamycin-sensitive/actinomycin D-resistant manner. Thus, leptin expression in adipocytes is regulated at the translational level via the mTOR pathway and provides a direct link between dietary leucine and leptin production. We also found that adipocytes compartmentalize a major pool of pre-synthesized leptin in specialized secretory vesicles. These vesicles were isolated and characterized by means of sucrose and iodixanol gradient centrifugation and also studied using immunoadsorption and confocal microscopy. These results indicate that exocytosis of leptin-containing secretory vesicles is stimulated by insulin and serum. Thus, in addition to the well characterized hormonal regulation of leptin expression at the transcriptional level, we found acute regulation of leptin release from a specialized storage compartment and regulation of leptin biosynthesis at the translational level. This latter step provides a long sought connection between food intake and blood leptin levels. This multilevel regulation appears to allow the body to accurately adjust leptin levels to suit its nutritional needs. |
