5-Aminoimidazole-4-Carboxamide Ribonucleoside. A Specific Method for Activating AMP-Activated Protein Kinase in Intact Cells?

Abstract

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‐aminoimidaz‐ole‐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.

Mild caloric restriction reduces blood pressure and activates endothelial AMPK-PI3K-Akt-eNOS pathway in obese Zucker rats

“…This nucleoside is taken up by the cell and accumulates in the cytoplasm as the monophosphorylated derivative, 5′-aminoimidazole-4-carboxamide-ribonucleoside, an AMP analogue that activates AMPK without disturbing cellular adenine nucleotide ratio [32]. In some experiments, the PI3K inhibitor wortmannin (10 −7 M), the Akt inhibitor triciribine (10 −5 M), or the NOS inhibitor N G -nitro-L-arginine methylester (L-NAME, 10 −4 M), were added 25 min before pre-contraction with Phe (10 −7 M).…”

The GID ubiquitin ligase complex is a regulator of AMPK activity and organismal lifespan

“…These data show that the depletion of different GIDsubunits results in a significant elongation of primary cilia. To test whether this is dependent on AMPK activity, we mimicked cellular starvation by treating cells with Torin1 (MTOR signaling inhibitor) or vice versa mimicked a high energy status with Compound C (AMPK inhibitor) to subsequently measure primary cilia length [23,24]. Inhibition of MTOR signaling in WT cells led to a significant elongation of primary cilia ( Figure 5C, compare lanes 1 and 2).…”

5′‐aminoimidazole‐4‐carboxyamide‐ribonucleoside‐activated Glucose Transport Is Not Prevented by Nitric Oxide Synthase Inhibition in Rat Isolated Skeletal Muscle

“…5 Ј -Aminoimidazole-4-carboxyamide-ribonucleoside is taken up by the muscle cell and is converted to 5 Ј -aminoimidazole-4-carboxyamideribonucleotide (ZMP), which mimics the effects of AMP on AMPK. 24 This results in activation of the AMPK signalling pathway, with a dose of 2 mmol/L being shown to be maximal. 25,26 N G -Monomethyl-L -arginine and/ or AICAR was present in the incubation medium for the entire 30 min.…”