Cell experiment:

HepG2 cells (5×105 cells) are plated in 6-well culture plate dishes and then are incubated in the serum-free media for 12 h before transfection. One microgram of plasmid is transfected with FuGENE6 Transfection Reagent. After 5 h of transfection, the culture media are removed and then media supplemented with or without AICAR (0.1-1.0 mM) are added to each well. The stimulation media are changed every 24 h[1].

Animal experiment:

Mice[2] Fourteen-week-old lean (Lepob/+ or Lepob/+) and ob/ob (Lepob/Lepob/) male mice are uesd. After the 14-day experimental treatment (24 h after AICAR injection, including a 12-h fast), the plantar flexor complex muscle is cleanly (tendon-to-tendon) excised from an anesthetized mouse breathing 4% isoflurane. The muscle is quickly weighed and then processed for histology or frozen in liquid nitrogen and stored at –80℃. The anesthetized mice are killed by transection of the diaphragm and removal of the entire heart, after blood collection via needle puncture directly into the heart, while breathing 4% isoflurane. AICAR or saline (control) is injected subcutaneously into the lateral distal portion of the back. AICAR is administered at 0.5 mg*g body wt-1*day-1 one time per day for 14 days. Saline (control) is injected in volumes identical to those used for AICAR treatment in a manner identical to that of AICAR treatment. Body weight is measured prior to death. Rats[3] Male 5-week-old ZDF rats are either subcutaneously injected with a single dose of AICAR (0.5 mg/g body wt) or underwent a single bout of treadmill running (60 min, speed of 25 m/min at a 5% incline). Untreated ZDF rats serve as controls (n=5 in each group). One hour after the subcutaneous AICAR injection or immediately after treadmill running, rats are killed by cervical dislocation. To avoid any effect of muscle spasm and hypoxia, red and white gastrocnemius muscles are removed within seconds and immediately freeze clamped for later determination of AMPK activity.

AICAR phosphate （Acadesine）is an activator of AMPK [1].

AICAR phosphate has been reported to induce apoptosis in a dose-dependent fashion and that the EC50 value is 380±60μM on the viability assay of B-CLL cells. In addition, AICAR phosphate has been revealed to induce caspase activation and cytochrome c release from mitochondria in B-CLL cells. Furthermore, AICAR phosphate has shown that uptake and phosphorylation are required to induce apoptosis and activate AMPK. Apart from these, AICAR phosphate (2-4 mM) has shown to only slightly affects the viability of T cells from B-CLL patients, AICAR phosphate (0.5 mM) has been noted to remarkedly reduces viability of B cells but not T cells [1].

References:
[1] Campàs C1, Lopez JM, Santidrián AF, Barragán M, Bellosillo B, Colomer D, Gil J. Acadesine activates AMPK and induces apoptosis in B-cell chronic lymphocytic leukemia cells but not in T lymphocytes. Blood. 2003 May 1;101(9):3674-80. Epub 2003 Jan 9.