AMPK; Autophagy; Mitophagy; Human Endogenous Metabolite

Acadesine (500 μM) increases the ZMP content in extracts of isolated hepatocytes after up to 30-40 min treatment, then remains fairly constant at approximately 4 nmol/g. Acadesine (500 μM) causes a transient 12-fold activation of AMPK at 15 min in rat hepatocytes and 2-3 fold activation of AMPK in adipocytes, without affecting levels of ATP, ADP or AMP. Acadesine (500 μM) causes a dramatic inhibition of both fatty acid and sterol synthesis in rat hepatocytes. Acadesine (500 μM) also causes a dramatic inactivation of HMG-CoA reductase.[1] With an EC50 of 380 M, cadesine induces apoptosis in B-CLL cells in a dose-dependent manner. The cell viability of B-CLL cells from 20 representative patients is reduced by acadesine (0.5 mM) from 68% to 26%. Caspase activation and mitochondrial cytochrome c release are caused by acadesine (0.5 mM). Acadesine (0.5 mM) must be taken up and phosphorylated in order to cause apoptosis and activate AMPK in B-CLL cells. Acadesine (0.5 mM) noticeably lowers the viability of B cells but not T cells, with only a minimal impact on the viability of T cells from B-CLL patients. [2] In K562, LAMA-84, and JURL-MK1 cells, cadesine causes a loss of cell metabolism. It is also effective in killing imatinib-resistant K562 cells and Ba/F3 cells that have the T315I-BCR-ABL mutation. Since both GF109203X and Ro-32-0432, inhibitors of both classical and new PKCs, negate the effect of Accadesine, Accadesine causes the movement and activation of several PKC isoforms in K562 cells. At day 10, acadesine inhibits K562 colony formation in a dose-dependent manner. Its growth inhibitory effect is already apparent at concentrations of 0.25 mM and is maximal at 2.5 mM. [3] Accadesine decreases the expression of CD18 on LPS-stimulated neutrophils in vitro in a concentration-dependent manner. [4] Granulocyte CD11b up-regulation caused by N-formyl-methionyl-leucyl-phenylalanine is significantly (61% on average) inhibited by cadesine (1 mM) in blood. [5]

In a mouse model of K562 cell xenograft, cadesine (50 mg/kg) significantly lowers tumor development. [3] Pigs' hemodynamic stability requires more fluid when cadesine (10 mg/kg) is administered. Pig dead space ventilation, peak inspiratory pressures on constant tidal volume, and LPS-induced protein permeability of pulmonary capillaries are all inhibited by cadesine (10 mg/kg).[4]

In semisolid methyl cellulose medium, K562 cell lines or primary cells (103 CD34+ cells/mL) are given acadesine. Cell lines and primary CD34+ cells, respectively, are cultured with MethoCult H4100 or H4236. After a 10-day culture period, colonies are found by adding 1 mg/mL of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent, and scoring them using Image J quantification software.

HepG2 cells (5×105 cells) are seeded into 6-well culture plate dishes, where they are then cultured for 12 hours in serum-free media before being transfected. FuGENE6 Transfection Reagent is used to transfect one microgram of plasmid. After 5 hours of transfection, the culture media are removed, and media supplemented with or without AICAR (0.1-1.0 mM) are then added to each well. Every 24 hours, the stimulation medium is changed[1].

Mice: 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. The muscle is rapidly weighed, followed by histology processing or freezing in liquid nitrogen and storing at -80°C. Following a direct needle puncture into the heart to collect blood, the anesthetized mice are killed by transection of the diaphragm and removal of the entire heart. Subcutaneous injections of AICAR or saline (control) are made into the lateral distal region of the back. AICAR is given orally once daily for 14 days at a dose of 0.5 mg/g. Injections of saline (control) are performed in a manner and at volumes identical to those used for AICAR treatment. Prior to death, a person's weight is measured.
Rats: Male ZDF rats aged 5 weeks received a single subcutaneous injection of AICAR (0.5 mg/g body weight) or underwent a single bout of treadmill running (60 minutes, speed of 25 m/min at 5% incline). Controls (n=5 in each group) are untreated ZDF rats. Rats are killed by cervical dislocation one hour following subcutaneous AICAR injection or right away following treadmill use. Red and white gastrocnemius muscles are immediately removed to prevent the effects of muscle spasm and hypoxia, and they are then immediately freeze clamped to measure the AMPK activity later.

[1]. Eur J Biochem. 1995 Apr 15;229(2):558-65.

[2]. Blood. 2003 May 1;101(9):3674-80.

[3]. PLoS One. 2009 Nov 18;4(11):e7889.

C9H14N4O5

258.2313

258.09642

C, 41.86; H, 5.46; N, 21.70; O, 30.98

2627-69-2

AICAR phosphate;681006-28-0;AICAR-13C2,15N;1609374-70-0

Solid powder

C1=NC(=C(N1[C@H]2[C@@H]([C@@H]([C@H](O2)CO)O)O)N)C(=O)N

RTRQQBHATOEIAF-UUOKFMHZSA-N

InChI=1S/C9H14N4O5/c10-7-4(8(11)17)12-2-13(7)9-6(16)5(15)3(1-14)18-9/h2-3,5-6,9,14-16H,1,10H2,(H2,11,17)/t3-,5-,6-,9-/m1/s1

5-amino-1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]imidazole-4-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~51 mg/mL (~197.5 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

2% DMSO+40% PEG 300+2% Tween 80+ddH2O: 6mg/mL (Please use freshly prepared in vivo formulations for optimal results.)