Numerous studies have focused on the regulation of leptin signalling and the functions of leptin in energy homoeostasis; however, little is known about how leptin secretion is regulated. In the present study we studied leptin storage and secretion regulation in 3T3-L1 and primary adipocytes. Leptin is stored in membrane-bound vesicles that are localized predominantly in the ER (endoplasmic reticulum) and close to the plasma membrane of both 3T3-L1 and primary adipocytes. Insulin increases leptin secretion as early as 15 min without affecting the leptin mRNA level. Interestingly, treatment with the protein synthesis inhibitor cycloheximide and the ER–Golgi trafficking blocker Brefeldin A inhibit both basal and ISLS (insulin-stimulated leptin secretion), suggesting that insulin stimulates leptin secretion by up-regulating leptin synthesis and that leptin-containing vesicles go through the ER–Golgi route. The PI3K (phosphoinositide 3-kinase)/Akt, but not MAPK (mitogen-activated protein kinase), pathway is involved in ISLS in vitro and in vivo. Although Ca2+ triggers synaptic vesicle and secretory granule exocytosis, Ca2+ influx alone is not sufficient to induce leptin secretion. Remarkably, Ca2+ is required for ISLS possibly due to its involvement in insulin-stimulated Akt phosphorylation. We conclude that insulin stimulates leptin release through the PI3K/Akt pathway and that Ca2+ is required for robust Akt phosphorylation and leptin secretion.

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