A-769662 is an effective, reversible AMPK activator(EC50=0.8 μM).

In ob/ob mice, A-769662 (30 mg/kg, b.i.d.) effectively reduces the hepatic expression of PEPCK, G6Pase, and FAS, decreases plasma glucose levels, and lowers triglyceride levels in both plasma and liver, thereby mitigating weight gain.

A-769662 activates AMPK, purified from various tissues and species, in a dose-dependent manner, demonstrated across purified rat liver (EC50=0.8 μM), rat heart (EC50=2.2 mM), rat muscle (EC50=1.9 mM), and human embryonic kidney cells (HEKs) (EC50=1.1 mM). In hepatocytes, A-769662 enhances ACC phosphorylation and inhibits fatty acid synthesis, with IC50 values of 3.2 μM in primary rat hepatocytes and 3.6 μM in mouse hepatocytes. The activation of AMPK by A-769662 involves preventing the dephosphorylation at Thr-172 and structural modulation. Furthermore, A-769662 inhibits the in vitro activity of the purified 26S proteasome, as well as cell proliferation and DNA synthesis.

96-well AMPK assay: AMPK activity is measured by monitoring phosphorylation of the SAMS peptide substrate (20 mM in standard assays and 100 mM in additivity assays) following a previously described protocol (Anderson et al., 2004). To determine whether A-769662-induced AMPK activation occurs in a reversible manner, AMP or A-769662 are preincubated with rat liver AMPK for 10 minutes at 20 times standard assay concentrations prior to dilution and measurement of AMPK activity.

Cell viability of MEF cells treated or not with A-769662 is performed as follows: cells are harvested by trypsinization and incubated with 0.5 mg/mL RNase and 50 μg/mL propidium iodine at room temperature in the dark; cell viability is analyzed by flow cytometry using a FACScanto flow cytometer, using an excitation laser at 488 nm and a propidium iodine fluorescence detection at 600 nm. To determine the proportion of cells in each phase of the cell cycle, cells are harvested by trypsinization, collected by centrifugation, washed in PBS and fixed overnight in 80% ethanol at -20 °C. Subsequently, these fixed cells are centrifuged to remove the fixative and incubated for 20 minutes in the dark at room temperature in PBS containing 0.5 mg/mL RNase and 50 μg/mL propidium iodine. Flow cytometry analysis is performed as above. The proportion of cells in G1, S, and G2 is determined using the MODFIT program. Cell culture pictures are taken at the indicated times using a camera coupled to an inverted microscope with a 20 × objective. (Only for Reference)