Leptin modulates α1-adrenergic receptor-mediated CaMKK-AMPKα signaling in GT1-7 cells
Objective:To investigate the leptin regulated CaMKKβ-AMPKα signaling pathway by transfection of α1-adrenergic receptor into hamster hypothalamus GT1-7 cells.Methods:Hamster hypothalamus cell line GT1-7 was cultured, and then α1-adrenaline receptor plasmid (myc-α1-AR) was transfected into the cells with liposomes. The cells grew exponentially after being starved overnight in serum-free medium and then stimulated with leptin at different concentration gradients to assess intracellular calcium concentration as follows: GT1-7 cells were cultured with DME/F12 in a 96-well black-bottom cell culture plate, and serum-free DME/F12 was replaced every other day at 37 ℃ for 2 hours. The cells were stimulated with different concentrations of leptin and the intracellular calcium concentration was measured with Fluo-4. Untransfected GT1-7 cells were used as a negative control. The mRNA levels of CaMKKβ and AMPKα in GT1-7-myc-α1-AR cells were detected by RT-PCR and the protein expression of CaMKKβ and AMPKα was detected by Western blot.Results:Leptin stimulated the change of calcium concentration in GT1-7 cells through α1-adrenoceptor, thus regulating the activity of the CaMKKβ-AMPKα signaling pathway.Conclusion:Leptin acts on α1-adrenergic receptor, mediates G protein coupling in the sympathetic nervous system, and causes [Ca2+]i influx in GT1-7-α1-AR cells by activating Ca2+ channels. As a calcium-dependent protein, CaMKKβ regulates cell metabolism in hypothalamic cells, activates AMPKα with the increase of intracellular Ca2+ concentration, and promotes energy metabolism mediated by hypothalamic gonadotropin-releasing hormone in GT1-7 cells.
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