Corton J M, Gillespie J G, Hawley S A, Hardie D G
Department of Biochemistry, University, Dundee, Scotland.
Eur J Biochem. 1995 Apr 15;229(2):558-65. doi: 10.1111/j.1432-1033.1995.tb20498.x.
The AMP-activated protein kinase (AMPK) is believed to protect cells against environmental stress (e.g. heat shock) by switching off biosynthetic pathways, the key signal being elevation of AMP. Identification of novel targets for the kinase cascade would be facilitated by development of a specific agent for activating the kinase in intact cells. Incubation of rat hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) results in accumulation of the monophosphorylated derivative (5-aminoimidazole-4-carboxamide ribonucleoside; ZMP) within the cell. ZMP mimics both activating effects of AMP on AMPK, i.e. direct allosteric activation and promotion of phosphorylation by AMPK kinase. Unlike existing methods for activating AMPK in intact cells (e.g. fructose, heat shock), AICAR does not perturb the cellular contents of ATP, ADP or AMP. Incubation of hepatocytes with AICAR activates AMPK due to increased phosphorylation, causes phosphorylation and inactivation of a known target for AMPK (3-hydroxy-3-methylglutaryl-CoA reductase), and almost total cessation of two of the known target pathways, i.e. fatty acid and sterol synthesis. Incubation of isolated adipocytes with AICAR antagonizes isoprenaline-induced lipolysis. This provides direct evidence that the inhibition by AMPK of activation of hormone-sensitive lipase by cyclic-AMP-dependent protein kinase, previously demonstrated in cell-free assays, also operates in intact cells. AICAR should be a useful tool for identifying new target pathways and processes regulated by the protein kinase cascade.
AMP 激活的蛋白激酶（AMPK）被认为可通过关闭生物合成途径来保护细胞免受环境应激（如热休克），关键信号是 AMP 水平升高。开发一种在完整细胞中激活该激酶的特异性试剂将有助于鉴定激酶级联反应的新靶点。用 5-氨基咪唑-4-甲酰胺核苷（AICAR）孵育大鼠肝细胞会导致细胞内单磷酸化衍生物（5-氨基咪唑-4-甲酰胺核苷；ZMP）积累。ZMP 模拟了 AMP 对 AMPK 的两种激活作用，即直接变构激活和促进 AMPK 激酶介导的磷酸化。与现有在完整细胞中激活 AMPK 的方法（如果糖、热休克）不同，AICAR 不会干扰细胞内 ATP、ADP 或 AMP 的含量。用 AICAR 孵育肝细胞会由于磷酸化增加而激活 AMPK，导致 AMPK 的一个已知靶点（3-羟基-3-甲基戊二酰辅酶 A 还原酶）磷酸化并失活，以及两条已知靶点途径（即脂肪酸和固醇合成途径）几乎完全停止。用 AICAR 孵育分离的脂肪细胞可拮抗异丙肾上腺素诱导的脂解作用。这提供了直接证据，表明此前在无细胞试验中证明的 AMPK 对环磷酸腺苷依赖性蛋白激酶激活激素敏感性脂肪酶的抑制作用在完整细胞中也起作用。AICAR 应该是鉴定由蛋白激酶级联反应调节的新靶点途径和过程的有用工具。
