AMPK; Autophagy; Mitophagy; Human Endogenous Metabolite
AICAR phosphate targets AMP-activated protein kinase (AMPK), a key regulator of cellular energy homeostasis. By activating AMPK, it promotes glucose uptake, fatty acid oxidation, and metabolic adaptations. The compound also inhibits autophagy, YAP (Yes-associated protein), and mitophagy. Additionally, AICAR phosphate regulates glucose and lipid metabolism and inhibits the production of pro-inflammatory cytokines and iNOS. Its effects on multiple cellular processes, including metabolism, inflammation, and cell survival, make it a valuable tool for studying AMPK signaling and its role in various physiological and pathological conditions. The compound's ability to induce apoptosis in a dose-dependent fashion further suggests its potential in cancer research.

HepG2 cells were treated with varied doses of AICAR (0.1-1.0 mM) for 12, 24, and 48 hours, respectively. The expression levels of IR-β were considerably reduced to 50%, 53% and 46% of the control at 48 hours with 0.25, 0.5 and 1.0 mM AICAR, respectively [1].
In vitro studies have shown that AICAR phosphate is a potent activator of AMPK. In HepG2 cells treated with varied doses of AICAR (0.1-1.0 mM) for 12, 24, and 48 hours, the expression levels of IR-β were considerably reduced to 50%, 53%, and 46% of the control at 48 hours with 0.25, 0.5, and 1.0 mM AICAR, respectively. The compound has been reported to induce apoptosis in a dose-dependent fashion, with an EC₅₀ value of 380±60 μM on the viability assay of B-CLL cells. AICAR phosphate regulates glucose and lipid metabolism and inhibits the production of pro-inflammatory cytokines and iNOS in various cell types. Its ability to modulate multiple cellular processes makes it a valuable tool for studying AMPK signaling and its role in metabolism, inflammation, and cell survival.

For 14 days, 0.5 mg of the AMP-activated kinase (AMPK) activator AICAR (A) *g body weight wt-1*day-1 or saline control (C) was injected into 14-week-old male lean (L; 31.3 g body weight) wild-type and ob/ob (O; 59.6 g body weight) mice. The gastrocnemius, soleus, and plantaris muscles of the plantarflexor complex were removed for analysis twenty-four hours following the last injection, which included a 12-hour fast. All animals were then euthanized. OC mice had a reduced muscle mass (159±12 mg) compared to LC, LA, and OA mice (176±10, 178±9, and 166±16 mg, respectively), regardless of body weight variations [3]. Compared to the exercise group and the AICAR (0.5 mg/g body weight) group, the kidney weight of the untreated group was considerably higher. The exercise group had a higher heart weight than the other groups, but the AICAR-treated group's liver weight was considerably larger than that of the exercise group and the untreated group [4].
In vivo, AICAR phosphate has been studied in various animal models. In a study using 14-week-old male lean wild-type and ob/ob mice, 0.5 mg of AICAR per gram body weight per day was injected for 14 days. The results showed that OC mice had reduced muscle mass compared to other groups, regardless of body weight variations. Compared to the exercise group and the AICAR-treated group, the kidney weight of the untreated group was considerably higher. The exercise group had a higher heart weight than the other groups, but the AICAR-treated group's liver weight was considerably larger than that of the exercise group and the untreated group. These findings suggest that AICAR phosphate has significant effects on organ weights and metabolism in vivo. The compound's ability to regulate glucose and lipid metabolism and inhibit pro-inflammatory cytokine production supports its potential in treating metabolic disorders and inflammatory conditions.

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.
In vitro non-cell enzyme assays for AICAR phosphate typically involve measuring AMPK activation using purified AMPK enzyme and a peptide substrate. The compound is incubated with AMPK, ATP, and a fluorogenic or radiolabeled peptide substrate, and kinase activity is measured by fluorescence or scintillation counting. IC₅₀ or EC₅₀ values are calculated from dose-response curves. The compound's effects on iNOS activity can be measured using a cell-free assay with purified iNOS enzyme and a substrate such as L-arginine, with nitrite production measured by the Griess reaction. These assays provide quantitative data on the compound's direct effects on enzyme activity.

epG2 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.
In vitro cell-based assays for AICAR phosphate use various cell lines, including HepG2 hepatoma cells and B-CLL cells. Cells are cultured in appropriate media and treated with varying concentrations of AICAR phosphate. Parameters such as cell viability (MTT or CCK-8 assays), apoptosis (Annexin V/PI staining), and AMPK activation (Western blotting for phospho-AMPK) are assessed. The expression of IR-β and other metabolic markers is measured by Western blotting or qPCR. The production of pro-inflammatory cytokines and iNOS is measured by ELISA or qPCR. Autophagy and mitophagy are assessed by Western blotting for LC3-II/LC3-I ratio, p62 levels, and mitochondrial markers.

Lifexstyle interventions including exercise programs are cornerstones in the prevention of obesity-related diabetes. The AMP-activated protein kinase (AMPK) has been proposed to be responsible for many of the beneficial effects of exercise on glucose and lipid metabolism. The effects of long-term exercise training or 5-aminoimidazole-4-carboxamide-1-beta-d-riboruranoside (AICAR) treatment, both known AMPK activators, on the development of diabetes in male Zucker diabetic fatty (ZDF) rats were examined. Five-week-old, pre-diabetic ZDF rats underwent daily treadmill running or AICAR treatment over an 8-week period and were compared with an untreated group. In contrast to the untreated, both the exercised and AICAR-treated rats did not develop hyperglycemia during the intervention period. Whole-body insulin sensitivity, as assessed by a hyperinsulinemic-euglycemic clamp at the end of the intervention period, was markedly increased in the exercised and AICAR-treated animals compared with the untreated ZDF rats (P < 0.01). In addition, pancreatic beta-cell morphology was almost normal in the exercised and AICAR-treated animals, indicating that chronic AMPK activation in vivo might preserve beta-cell function. Our results suggest that activation of AMPK may represent a therapeutic approach to improve insulin action and prevent a decrease in beta-cell function associated with type 2 diabetes.[4]
In vivo animal studies for AICAR phosphate employ mouse models, including lean wild-type and ob/ob mice. The compound is administered via intraperitoneal injection at doses such as 0.5 mg/g body weight per day for 14 days. Parameters assessed include body weight, muscle mass, organ weights (kidney, heart, liver), and metabolic markers. Blood glucose and lipid levels are measured to evaluate the compound's effects on metabolism. Inflammatory markers are assessed in serum and tissues. Histological examination of tissues is performed to evaluate organ-specific effects. Pharmacokinetic studies in these models provide information about the compound's absorption, distribution, metabolism, and excretion.

AICAR phosphate has a molecular weight of approximately 338.21 g/mol for the phosphate salt and a molecular formula of C₉H₁₄N₄O₅·xH₃O₄P. It is soluble in water and DMSO at concentrations up to 71 mg/mL. The compound should be stored as a powder at -20°C for long-term stability. For in vivo studies, a homogeneous suspension can be prepared in CMC-Na solution at concentrations up to 5 mg/mL. Detailed pharmacokinetic parameters such as half-life, volume of distribution, and clearance have been characterized in preclinical studies. As an adenosine analog, AICAR phosphate is expected to be taken up by cells via nucleoside transporters and phosphorylated to its active form, ZMP, which activates AMPK.

The toxicity profile of AICAR phosphate has been evaluated in preclinical studies. In animal studies at doses of 0.5 mg/g body weight per day for 14 days, the compound caused significant changes in organ weights, including increased liver weight. No significant acute toxicity was reported at these doses. The compound's ability to induce apoptosis in B-CLL cells suggests potential for therapeutic applications in cancer, though it may also have effects on normal cells. As a research compound, AICAR phosphate is not intended for human therapeutic use without appropriate regulatory approval. Standard laboratory safety precautions should be followed when handling the compound, including the use of appropriate personal protective equipment and working in a well-ventilated area.

[1]. AICAR, an activator of AMP-activated protein kinase, down-regulates the IR expression in HepG2 cells. Biochem Biophys Res Commun. 2005 Mar 11;328(2):449-54.

[2]. 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside inhibits proinflammatory response in glial cells: a possible role of AMP-activated protein kinase. J Neurosci. 2004 Jan 14;24(2):479-87.

[3]. AICAR treatment for 14 days normalizes obesity-induced dysregulation of TORC1 signaling and translational capacity in fasted skeletal muscle. Am J Physiol Regul Integr Comp Physiol. 2010 Dec;299(6):R1546-54.

[4]. Long-term AICAR administration and exercise prevents diabetes in ZDF rats.Diabetes. 2005 Apr;54(4):928-34.

[5]. A combat with the YAP/TAZ-TEAD oncoproteins for cancer therapy. Theranostics. 2020 Feb 18;10(8):3622-3635.

The liver is one of the main target organs of insulin, with abundant expression of the insulin receptor. We analyzed the effect of the AMPK activator AICAR on insulin receptor expression in the human hepatocellular carcinoma line HepG2. After 48 hours of AICAR treatment, insulin receptor protein expression was significantly reduced in a dose-dependent manner, but this effect was not observed in 3T3-L1 adipocytes or CHO cells. Insulin receptor mRNA expression was also reduced after AICAR treatment. Furthermore, luciferase reporter gene assays revealed that AICAR treatment downregulated the transcriptional activity of the insulin receptor gene promoter. The adenosine transporter inhibitor dipyridamole and the adenosine kinase inhibitor 5'-amino-5'-deoxyadenosine both blocked the downregulation of insulin receptor protein, mRNA, and promoter activity by AICAR. Our results demonstrate for the first time that AMPK activation may reduce insulin receptor expression, at least partially, by downregulating transcriptional levels, and that this effect may be liver-specific. [1]

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This study aimed to investigate the effects of 14-day treatment with 5-aminoimidazole-4-carboxamide-1β-4-furanoside (AICAR) on the mammalian target of rapamycin (mTOR) signaling pathway and its regulatory processes (e.g., translation initiation) in the skeletal muscle of obese mice. We hypothesized that daily AICAR treatment (14 days) could restore obesity-induced alterations in the skeletal muscle mTOR signaling pathway and its regulatory processes to normal levels and have a positive effect on muscle mass. Fourteen-week-old male lean (L; 31.3 g) wild-type mice and ob/ob (O; 59.6 g) mice were injected with the AMP-activated protein kinase (AMPK) activator AICAR (A) at a dose of 0.5 mg·g body weight⁻¹·day⁻¹, or with saline control (C), for 14 consecutive days. Twenty-four hours after the last injection (including a 12-hour fast), all mice were sacrificed, and plantar flexor muscles (gastrocnemius, soleus, and plantar muscles) were excised for analysis. The results showed that, regardless of body weight change, the muscle mass of mice in the OC group (159 ± 12 mg) was lower than that in the LC, LA, and OA groups (176 ± 10 mg, 178 ± 9 mg, and 166 ± 16 mg, respectively). In obese mice, the reduction in muscle mass corresponded to increased staining intensity of lipid and glycogen cross-sections, elevated blood glucose and insulin levels, and decreased nuclear enrichment of peroxisome proliferator-activated receptor gamma coactivator-1α protein expression; AICAR treatment restored these indicators to normal. Obese mice showed decreased phosphorylation levels of AMPK and acetyl-CoA carboxylase, while AICAR treatment increased these levels in osteoarthritis mice. Conversely, compared to mildly obese mice, obese mice exhibited higher activation levels of mTOR downstream targets (S6 kinase-1 and ribosomal protein S6) and lower levels of raptor-related mTOR; these indicators showed the opposite changes after 14 days of AICAR treatment. By measuring the content of ribosomes, total RNA, and ribosome-associated RNA, the formation of eukaryotic initiation factor 4F complex, and the phosphorylation level of eukaryotic initiation factor 4G using sucrose density gradient centrifugation, it was found that the translational dysfunction in OA mice was improved. These data suggest that short-term (14 days) AMPK agonist treatment can enhance the regulatory process of skeletal muscle in atrophic obese mice by restoring the mTOR signaling pathway and mRNA translation to normal levels, thereby bringing them closer to the level of lean mice. [3]
AICAR phosphate is an adenosine analog and a potent activator of AMP-activated protein kinase (AMPK). It is also known as Acadesine phosphate and AICA Riboside phosphate. The compound regulates glucose and lipid metabolism and inhibits the production of pro-inflammatory cytokines and iNOS. It is also an inhibitor of autophagy, YAP, and mitophagy. AICAR phosphate promotes glucose uptake, fatty acid oxidation, and metabolic adaptations, making it valuable in research on metabolic disorders, diabetes, and exercise physiology. It has been reported to induce apoptosis in a dose-dependent fashion, with an EC₅₀ of 380±60 μM on B-CLL cells. Not approved for clinical use; intended for research purposes only.

C9H17N4O9P

356.2264

356.073

681006-28-0

AICAR;2627-69-2

67675098

White to off-white solid powder

8

11

3

23

380

4

P(=O)(O[H])(O[H])O[H].O1[C@]([H])(C([H])([H])O[H])[C@]([H])([C@]([H])([C@]1([H])N1C([H])=NC(C(N([H])[H])=O)=C1N([H])[H])O[H])O[H]

BPVGMEHURDEDAZ-GWTDSMLYSA-N

InChI=1S/C9H14N4O5.H3O4P/c10-7-4(8(11)17)12-2-13(7)9-6(16)5(15)3(1-14)18-9;1-5(2,3)4/h2-3,5-6,9,14-16H,1,10H2,(H2,11,17);(H3,1,2,3,4)/t3-,5-,6-,9-;/m1./s1

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

AICAR (phosphate); AICAR phosphate; 681006-28-0; 5-amino-1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]imidazole-4-carboxamide;phosphoric acid; SCHEMBL8722270;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

H2O : ~100 mg/mL (~280.72 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.84 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 20.8 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: ≥ 2.08 mg/mL (5.84 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 20.8 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: ≥ 2.08 mg/mL (5.84 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 33.33 mg/mL (93.56 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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