LKB1, an upstream AMPK kinase, regulates glucose and lipid metabolism in cultured liver and muscle cells
LKB1, an upstream AMPK kinase, regulates glucose and lipid metabolism in cultured liver and muscle cells
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DOI：
10.1016/j.bbrc.2006.10.056
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发表时间：
2006-12-22
影响因子：
3.1
通讯作者：
Katayama, Shigehiro
中科院分区：
文献类型：
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作者：
Imai, Kenta;Inukai, Kouichi;Katayama, Shigehiro
LKBI 是一种 50 kDa 丝氨酸/苏氨酸激酶，可磷酸化并激活 AMPK 在其 T 环残基 Thr 172 处的催化亚基。我们制备了表达 LKB1 组成型活性（野生型）形式 (CA) 或显性失活（激酶失活，D194A 突变体）形式 (DN) 的腺病毒，并在培养的肌管（C2C12 细胞）和大鼠中过表达这些蛋白肝癌细胞（FAO 细胞）。当用抗Thr172磷酸化AMPK的抗体进行免疫印迹分析时，与表达Lac-Z的对照细胞相比，表达CA和DN LKBI的细胞中AMPK的磷酸化分别增加（2.5倍）和减少（0.4倍）。使用同种型特异性抗体进行的免疫沉淀实验揭示了 AMPK 磷酸化的这些改变可归因于 AMPK α 2 磷酸化的改变，而不是 α 1 催化亚基的磷酸化改变，这强烈表明 α 2 催化亚基是哺乳动物细胞中 LKB1 的主要底物。此外，脂联素或 AICAR 刺激的 AMPK 磷酸化受到 DN LKB1 过表达的抑制，而苯乙双胍刺激的磷酸化不受影响。这些结果可以解释AMP和苯乙双胍之间AMPK激活机制的差异，并且还表明LKB1的AMPK磷酸化参与了AMP刺激的AMPK激活。作为 AMPK 的下游靶标，AICAR 诱导的葡萄糖摄取和 ACCP 磷酸化在表达 DN LKB1 的 C2Cl2 细胞中显着降低。糖异生关键酶、葡萄糖-6-磷酸酶和磷酸烯醇丙酮酸羧激酶的表达也依赖于FAO细胞中LKB1的活性。这些结果表明，LKB1 是肌肉和肝细胞中 AMPK 激活的关键调节因子，因此，LKBI 活性对于我们理解葡萄糖和脂质代谢可能具有重要意义。 (c) 2006 Elsevier Inc. 保留所有权利。
LKBI is a 50 kDa serine/threonine kinase that phosphorylates and activates the catalytic subunit of AMPK at its T-loop residue Thr 172. We prepared adenoviruses expressing the constitutive active (wild-type) form (CA) or dominant negative (kinase inactive, D194A mutant) form (DN) of LKB1 and overexpressed these proteins in cultured myotubes (C2C12 cells) and rat hepatoma cells (FAO cells). When analyzed by immunoblotting with the antibody against Thr172-phosphorylated AMPK, the phosphorylation of AMPK was increased (2.5-fold) and decreased (0.4-fold) in cells expressing CA and DN LKBI, respectively, as compared with Lac-Z expressing control cells. Immunoprecipitation experiments, using isoform-specific antibody, revealed these alterations of AMPK phosphorylation to be attributable to altered phosphorylation of AMPK alpha 2, but not alpha 1 catalytic subunits, strongly suggesting the alpha 2 catalytic subunit to be the major substrate for LKB1 in maminalian cells. In addition, adiponectin or AICAR-stimulated AMPK phosphorylation was inhibited by overexpression of DN LKB1, while phenformin-stimulated phosphorylation was unaffected. These results may explain the difference in AMPK activation mechanisms between AMP and phenformin, and also indicate that AMPK phosphorylation by LKB1 is involved in AMP-stimulated AMPK activation. As a downstream target for AMPK, AICAR-induced glucose uptake and ACCP phosphorylation were found to be significantly reduced in DN LKB1 expressing C2Cl2 cells. The expression of key enzymes for gluconeogenesis, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, was also dependent on LKB1 activities in FAO cells. These results demonstrate that LKB1 is a crucial regulator of AMPK activation in muscle and liver cells and, therefore, that LKBI activity is potentially of importance to our understanding of glucose and lipid metabolism. (c) 2006 Elsevier Inc. All rights reserved.