Leptin mediates the CaMKKβ-AMPKα signaling pathway through α1 adrenergic receptors in the GT1-7 cell line

Abstract:
Objective: To study the effect of leptin on intracellular calcium concentration via transfecting alpha1-adrenoceptors in the Chinese hamster hypothalamus GT1-7 cell line. Materials and Methods: GT1-7 cells were transfected with alpha1-adrenoceptor plasmid using lipofectamine; GT1-7 cells transfected with an NC empty vector were used as a negative control. Cells were then exponentially grown after starvation in a serum-free medium overnight and then stimulated with different concentration gradients ((0.01, 0.05, 0.25, 1.25, 6.25 ng/mL) of leptin to assess intracellular calcium concentrations. Fluo-4 was used to assess intracellular calcium ion concentration. RT-PCR was used to detect the mRNA levels of Ca 2+/Calmodulin-dependent protein kinase (CaMKKbeta) and AMP-activated protein kinase α (AMPKalpha), while Western blot was used to detect the protein expressions of CaMKKbeta and AMPKalpha in GT1-7-myc-alpha1-AR cells. Results: Leptin stimulated an [Ca2+]i response in a dose-dependent manner. Moreover, increasing concentrations of leptin treatment increased CaMKKbeta mRNA and AMPKalpha mRNA levels in GT1-7-alpha1-AR cells. Conclusion: Leptin stimulated changes in intracellular calcium ion concentration in hypothalamic GT1-7 cells through alpha1-adrenoceptor (alpha1-AR), thereby regulating the activity of the CaMKKbeta-AMPKalpha signaling pathway.