AMPK α1β1γ1 (Kd = 0.06 μM); AMPK α2β1γ1 (Kd = 0.06 μM); AMPK α1β2γ1 (Kd = 0.51 μM)
AMP-activated protein kinase (AMPK) [1]
AMPK [2]

EX229 activates AMPK in incubated rat epitrochlearis skeletal muscle and incubation of rat skeletal muscle with ex229 increases glucose uptake. Ex229 stimulates skeletal muscle glucose uptake, but this stimulation is AMPK-dependent and PI3K/PKB-independent. In L6 myotubes, ex229 boosts fatty acid oxidation and glucose uptake[1].

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EX-229 treatment (100 μM for 90 min) in isolated rat epitrochlearis muscle increased AMPK Thr172 phosphorylation dose-dependently, becoming significant at 50 μM, and ACC Ser218 phosphorylation became significant at 10 μM. [1]
Incubation with 100 μM EX-229 increased AMPK α1-, α2-, β1-, and β2-containing complex activities in rat muscle, with α1 and β2 activities reaching levels not significantly different from those induced by contraction. [1]
In rat muscle, EX-229 (100 μM) increased glucose uptake ~2-fold, and increased TBC1D1 Ser237 phosphorylation dose-dependently (significant at 50 μM). [1]
In L6 myotubes, EX-229 (0.5-1 μM) dose-dependently increased fatty acid oxidation (significant at 0.5 μM) and glucose uptake (~1.5-fold at 0.5-1 μM). [1]
In mouse EDL and soleus muscles, EX-229 (100 μM) increased AMPK activity more than 2-fold and glucose uptake ~2-fold. [1]
In AMPK α1/α2 double-knockout myotubes, the increase in glucose uptake and ACC phosphorylation induced by EX-229 was completely abolished, indicating AMPK-dependent effects. [1]
In muscles from starved (24 h) rats with reduced glycogen content, EX-229 (100 μM) induced higher AMPK activity and greater glucose uptake compared to fed rats. [1]
The increase in glucose uptake by EX-229 was not inhibited by the PI3K inhibitor wortmannin (1 μM). [1]
EX-229 (100 μM) did not change purine nucleotide levels (ATP, ADP, AMP) in resting rat muscle. [1]
EX-229 (10-100 μM) reduced the rise in AMP, adenosine, inosine, and AMP/ATP and ADP/ATP ratios in electrically stimulated rat muscle. [1]
EX-229 (25-100 μM) dose-dependently increased raptor Ser792 phosphorylation and decreased 4E-BP1 Thr37/Thr46 phosphorylation. [1]
EX-229 (100 μM) caused a small (<20%) but significant decrease in protein synthesis rate in rat muscle. [1]
In HEK293T cells, EX-229 (compound 991) at 50 μM for 60 min increased phosphorylation of Myc-Rabep1 at Ser407 as detected by AMPK substrate motif antibody. [2]
In primary mouse hepatocytes, EX-229 (991) at 10, 25, and 50 μM for 60 min induced dose-dependent mobility shifts in endogenous STIM1 and STIM2, paralleling AMPK-dependent phosphorylation of Raptor and ACC; these effects were abolished in AMPK DKO hepatocytes. [2]
In mouse embryonic fibroblasts (MEFs), EX-229 (991) at 50 μM for 1 h induced STIM1 Ser521 phosphorylation, which was lost in Ser521Ala mutants or in AMPK DKO MEFs. [2]

Rat epitrochlearis muscles incubated with EX-229 (100 μM for 90 min) showed increased AMPK activity and glucose uptake. Muscles from fed or 24-h-starved rats were used. Glucose uptake was measured by adding 2-deoxy-D-[1-3H]glucose and D-[1-14C]mannitol during the last 30 min of incubation. [1]
Mouse EDL and soleus muscles incubated with EX-229 (100 μM for 60 min) showed increased AMPK activity and glucose uptake (measured over an additional 20 min). [1]
In L6 myotubes, fatty acid oxidation was measured with [9,10-3H]palmitic acid over 4 h incubation with EX-229 (0.05-1 μM). Glucose uptake was measured with 2-deoxy-D-[1-3H]glucose over 10 min after 2 h pre-incubation with EX-229 (0.05-1 μM). [1]
In AMPK α1/α2 double-knockout myotubes, EX-229 (0.5-1 μM for 2 h) failed to increase glucose uptake, confirming AMPK dependence. [1]
In HEK293T cells, EX-229 (991) at 50 μM for 60 min was used to treat cells transfected with Myc-Rabep1 WT or S407A; Myc immunoprecipitations were performed and probed with anti-AMPK pMOTIF antibody. [2]
In primary mouse hepatocytes from AMPK WT and DKO mice, EX-229 (991) at 0, 10, 25, 50 μM for 60 min was used; cell lysates were immunoblotted for p-AMPK, p-ACC, and STIM1/STIM2 mobility shifts. [2]
In MEFs (WT or AMPK DKO) stably expressing empty vector, flag-STIM1-WT or flag-STIM1-Ser521Ala, cells were treated with vehicle, 50 μM EX-229 (MK-991) or 2 mM phenformin for 1 h; flag immunoprecipitates were blotted with anti-p-STIM1 Ser521 antibody. [2]

Vehicle or 50 μM 991 were applied to cells transfected with Myc-Rabep1 WT or Ser407Ala for 60 min.

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For isolated muscle incubation: Rat epitrochlearis muscles (110-130 g male Wistar rats) were incubated in Krebs-Henseleit bicarbonate buffer with 0.1-0.2% DMSO (vehicle) or EX-229 (0.5-100 μM) for 90 min at 30°C. For contraction, electrical stimulation (200 ms trains every 2 s at 100 Hz for 30 min) was applied. For glucose uptake, 2-deoxy-D-[1-3H]glucose (0.25 μCi/ml) and D-[1-14C]mannitol (0.1 μCi/ml) were added. Muscles were blotted and frozen in liquid nitrogen. [1]
For mouse muscle incubation: Mouse EDL and soleus muscles (3-4-month-old C57/B16 mice) were incubated with 0.1% DMSO, 100 μM EX-229, 300 μM A769662, or 2 mM AICAR for 60 min. Glucose uptake was measured over an additional 20 min. [1]
For L6 myotube fatty acid oxidation: Differentiated L6 myotubes were serum-starved for 2 h in low glucose DMEM, then incubated with EX-229 or AICAR in glucose-free DMEM with 2 mM pyruvate and 0.1 mM [9,10-3H]palmitic acid (2 μCi/well) for 4 h. ³H₂O was collected. [1]
For L6 myotube glucose uptake: Overnight serum-starved L6 myotubes were incubated with EX-229 (0.05-1 μM), 2 mM AICAR, or 1 μM insulin for 2 h, then 2-deoxy-D-[1-3H]glucose (0.5 μCi/well) was added for 10 min at room temperature. [1]
For AMPK DKO myotubes: Control or AMPK α1/α2 double-knockout myotubes were serum-starved for 4 h, then incubated with EX-229 (0.5-1 μM), 2 mM AICAR, or 1 μM insulin for 2 h. Glucose uptake measured as above. [1]
For HEK293T cell treatment: Cells were transfected with Myc-Rabep1 WT or S407A, treated with vehicle or 50 μM EX-229 (991) for 60 min. Myc immunoprecipitations were performed. [2]
For primary hepatocyte treatment: Primary hepatocytes from AMPK WT or DKO mice were treated with a dose-response curve of EX-229 (991) at 0, 10, 25, 50 μM for 60 min, or 2 mM phenformin. [2]
For MEF treatment: MEFs (WT or AMPK DKO) were treated with vehicle, 50 μM EX-229 (991), or 2 mM phenformin for 1 h. Flag immunoprecipitations were performed. [2]

For rat muscle: Male Wistar rats (110-130 g) were obtained. For starvation, rats were fasted for 24 h to reduce muscle glycogen content. Muscles (epitrochlearis) were dissected and incubated ex vivo as described in the Cell Assay and Animal Protocol sections. No in vivo administration of EX-229 to live animals is reported in this study. [1]
For mouse muscle: Male C57/B16 mice (3-4 months old) were used. EDL and soleus muscles were dissected and incubated ex vivo with EX-229 as described. No in vivo administration of EX-229 to live animals is reported. [1]
For primary hepatocytes: AMPKα1fl/flα2fl/fl mice ± Albumin-CreERT2 were treated with tamoxifen (1 mg every other day, 3 injections) to generate AMPK DKO. Primary hepatocytes were isolated about 2 weeks later. Cells were treated with EX-229 (991) ex vivo. No in vivo administration of EX-229 to live animals is reported in this study. [2]
For MEFs: Mouse embryonic fibroblasts from wild-type or AMPKα1/α2 DKO mice were used. No animal dosing with EX-229 is reported. [2]

[1]. Biochem J. 2014 Jun 15;460(3):363-75.

[2]. Cell Rep. 2019 Dec 3;29(10):3331-3348.e7.

AMP-activated protein kinase activators

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EX-229 is a cyclic benzimidazole derivative with the chemical name 5-({6-chloro-5-(1-methylindol-5-yl)-1H-benzimidazol-2-yl}oxy)-2-methyl-benzoic acid. It is a direct AMPK activator that binds at the interface between the N-terminal lobe of the kinase domain and the glycogen-binding module of the β-subunit. It activates AMPK both allosterically and by protecting against dephosphorylation of Thr172 on the α-subunit. EX-229 has been shown to be 5-10-fold more potent than A769662 in activating purified AMPK. [1]
EX-229 (also called compound 991) was used as a tool compound to validate AMPK-dependent phosphorylation of STIM1/STIM2 and Rabep1. It directly activates AMPK without causing changes to cellular ATP/AMP levels. [2]

C24H18CLN3O3

431.8710

431.103

C, 66.75; H, 4.20; Cl, 8.21; N, 9.73; O, 11.11

1219739-36-2

45256693

Off-white to pink solid powder

5.6

2

4

4

31

666

0

CC1=C(C=C(C=C1)OC2=NC3=C(N2)C=C(C(=C3)C4=CC5=C(C=C4)N(C=C5)C)Cl)C(=O)O

FHWSAZXFPUMKFL-UHFFFAOYSA-N

InChI=1S/C24H18ClN3O3/c1-13-3-5-16(10-17(13)23(29)30)31-24-26-20-11-18(19(25)12-21(20)27-24)14-4-6-22-15(9-14)7-8-28(22)2/h3-12H,1-2H3,(H,26,27)(H,29,30)

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)

Solubility in Formulation 1: ≥ 1.3 mg/mL (3.01 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 13.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.3 mg/mL (3.01 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 13.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1.3 mg/mL (3.01 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 13.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)