瘦素通过α1肾上腺素受体介导CaMKKβ-AMPKα信号通路在GT1-7细胞系中的作用

doi:10.3877/cma.j.issn.1674-0785.2023.05.012

目的

研究瘦素在仓鼠下丘脑GT1-7细胞系中的作用，通过转染α1-肾上腺素受体影响细胞内钙离子浓度而调节CaMKKβ-AMPKα信号通路。

方法

以仓鼠下丘脑GT1-7细胞株进行细胞培养，用脂质体转染α1-肾上腺素作为受体质粒。细胞在无血清培养基中饥饿过夜后呈指数增长，用不同浓度梯度的瘦素刺激细胞以评估细胞内钙离子浓度：GT1-7细胞系在96孔黑底细胞培养板中用DME/F12培养，每隔一天更换一次无血清的DME/F12，37 ℃培养2 h。用不同浓度梯度瘦素刺激细胞，并用fluo-4（Invitrogen）检测细胞内钙离子浓度变化。同时，未转染的GT1-7细胞用作阴性对照。RT-PCR法检测GT1-7-myc-α1-AR细胞CaMKKβ和AMPKα的mRNA水平，Westernblot法检测GT1-7-myc-α1-AR细胞CaMKKβ和AMPKα的蛋白表达。

结果

瘦素通过α1肾上腺素能受体（α1-adrenoceptor，α1-AR）刺激下丘脑GT1-7细胞内钙离子浓度的变化，从而调节CaMKKβ-AMPKα信号的通路活性。

结论

瘦素作用α1肾上腺素受体，介导交感神经系统G蛋白偶联，通过激活Ca²⁺通道，引起GT1-7-α1-AR细胞［Ca²⁺］i内流，作为钙依赖蛋白CaMKKβ在下丘脑细胞中调节细胞的代谢，随细胞内Ca²⁺浓度的增加来激活AMPKα，促进下丘脑促性腺激素释放激素GT1-7细胞的能量代谢。

关键词: 瘦素, α1-肾上腺素受体, CaMKKβ-AMPKα信号通路, GT1-7细胞系

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.

Key words: Leptin, α1-Adrenergic receptor, CaMKK-AMPKα signaling pathway, GT1-7 cells

表1 寡核苷酸引物序列

Sequence	Name	Length（bp）
TATGGAATCCTGTGGCATC	β-actin（m）-F	87
GTGTTGGCATAGAGGTCTT	β-actin（m）-R	87
CCTTTGGCAGTTGCCTACCA	AMPKa（m）-F	500
GATCCCGATCTCTGTGGAGT	AMPKa（m）-R	500
CATGAATGGACGCTGC	CaMKKβ（m）-F	80
TGACAACGCCATAGGAGCC	CaMKKβ（m）-R	80

表1 寡核苷酸引物序列

Sequence	Name	Length（bp）
TATGGAATCCTGTGGCATC	β-actin（m）-F	87
GTGTTGGCATAGAGGTCTT	β-actin（m）-R	87
CCTTTGGCAGTTGCCTACCA	AMPKa（m）-F	500
GATCCCGATCTCTGTGGAGT	AMPKa（m）-R	500
CATGAATGGACGCTGC	CaMKKβ（m）-F	80
TGACAACGCCATAGGAGCC	CaMKKβ（m）-R	80

图1 瘦素（Leptin）在GT1-7-α1-AR细胞中诱导细胞内钙反应

图2 GT1-7-α1-AR细胞中瘦素（Leptin）诱导的CaMKKβ激活

图3 GT1-7-α1-AR细胞中瘦素（Leptin）诱导的AMPKα激活。

图4 GT1-7-α1-AR细胞中CaMKKβ和AMPKα亚基的蛋白表达不同实验分组及处理下的CaMKKβ和AMPKα亚基的蛋白测定；增加Leptin浓度（0.01、0.05、0.25、1.25、6.25 ng/ml）对GT1-7-α1-AR细胞中AMPKα 和CaMKKβ磷酸化水平的影响。

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Leptin modulates α1-adrenergic receptor-mediated CaMKK-AMPKα signaling in GT1-7 cells

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Leptin modulates α1-adrenergic receptor-mediated CaMKK-AMPKα signaling in GT1-7 cells