Aicar

Research Overview

AICAR serves as a fundamental research tool for investigating AMPK-dependent cellular metabolism and energy sensing mechanisms in laboratory settings. This nucleoside analog has become indispensable in metabolic research due to its ability to pharmacologically activate AMPK, enabling researchers to dissect the role of this critical energy sensor in various biological processes. Research applications span cellular metabolism, exercise physiology, mitochondrial function, metabolic disease modeling, and therapeutic target investigation.

The compound’s designation as an AMPK activator reflects its mechanism of cellular uptake and intracellular phosphorylation. Upon entering cells through equilibrative nucleoside transporters, AICAR is phosphorylated by adenosine kinase to form ZMP (AICAR monophosphate). ZMP mimics 5′-AMP, the natural activator of AMPK, binding to the gamma regulatory subunit and causing conformational changes that activate the kinase. This activation occurs without depleting cellular ATP levels, providing a clean experimental system for studying AMPK-dependent effects independent of energy stress.

AICAR research has significantly advanced understanding of metabolic regulation, particularly in contexts of exercise, metabolic disease, and cellular adaptation to energy challenges. Laboratory studies demonstrate AICAR’s ability to stimulate glucose uptake, enhance fatty acid oxidation, promote mitochondrial biogenesis, and modulate numerous metabolic pathways. These effects have earned AICAR the designation exercise mimetic in research literature, as it can activate many of the same metabolic adaptations observed during physical exercise.

Molecular Characteristics

Complete Specifications:

• CAS Registry Number: 2627-69-2
• Molecular Weight: 338.3 Da
• Molecular Formula: C₉H₁₄N₄O₅
• Chemical Name: 5-Aminoimidazole-4-carboxamide ribonucleoside
• Alternate Names: AICA riboside, Acadesine, ZMP precursor
• PubChem CID: 17513
• Classification: Nucleoside analog, AMPK activator
• Appearance: White to off-white crystalline powder
• Solubility: Water, DMSO, buffered aqueous solutions

The molecular structure of AICAR consists of a ribose sugar moiety attached to an aminoimidazole carboxamide base. This structure closely resembles adenosine, allowing AICAR to be recognized by nucleoside transporters and adenosine kinase. The key structural difference – the substitution of the purine base with an aminoimidazole carboxamide – confers unique properties that make AICAR an AMPK activator rather than simply an adenosine analog.

Upon intracellular phosphorylation, AICAR-monophosphate (ZMP) structurally mimics AMP sufficiently to bind AMPK’s regulatory gamma subunit at the same sites that recognize AMP. This binding prevents ATP from binding to the same site, maintaining AMPK in its active conformation. The structural similarity to AMP extends to other AMP-binding proteins, meaning researchers must consider potential off-target effects on other AMP-sensitive proteins when designing experiments.

Mechanism of Action and AMPK Activation

AMPK Activation Pathway:

AICAR’s mechanism provides a sophisticated tool for metabolic research. The activation cascade proceeds through several steps:

1. Cellular Uptake: AICAR enters cells via equilibrative nucleoside transporters (ENTs), particularly ENT1 and ENT2, which normally transport adenosine and other nucleosides.

2. Intracellular Phosphorylation: Adenosine kinase phosphorylates AICAR to form AICAR monophosphate (ZMP), the active metabolite that mimics endogenous AMP.

3. AMPK Activation: ZMP binds to the gamma regulatory subunit of AMPK, specifically to the CBS (cystathionine beta-synthase) domains that normally bind AMP. This binding:

• Promotes phosphorylation of AMPK at Thr172 by upstream kinases (primarily LKB1)
• Protects phospho-Thr172 from dephosphorylation
• Causes allosteric activation of AMPK enzymatic activity

4. Metabolic Reprogramming: Activated AMPK phosphorylates numerous downstream targets, switching cellular metabolism from anabolic to catabolic pathways.

Selectivity and Specificity:

While AICAR serves as an excellent research tool, investigators should be aware of its selectivity profile. ZMP can interact with other AMP-binding proteins beyond AMPK, including:

• Fructose-1,6-bisphosphatase (FBPase)
• Glycogen phosphorylase
• Certain AMP-binding enzymes in nucleotide metabolism

This broader binding profile means that some AICAR effects may occur through AMPK-independent mechanisms. Research protocols should include appropriate controls and complementary approaches (genetic AMPK manipulation, alternative AMPK activators) to definitively attribute effects to AMPK activation.

Pharmacokinetic Profile in Research Models

AICAR pharmacokinetic characterization in preclinical research reveals important properties for experimental design:

Absorption and Distribution:

• Cellular uptake mediated by nucleoside transporters (ENTs)
• Rapid cellular entry in most tissue types
• Intracellular accumulation as ZMP (phosphorylated form)
• ZMP cannot exit cells readily, providing sustained intracellular AMPK activation
• Distribution to multiple tissue types following systemic administration

Metabolism and Intracellular Fate:

• Phosphorylation by adenosine kinase to form ZMP (AICAR-monophosphate)
• Further phosphorylation to AICAR-diphosphate and AICAR-triphosphate is limited
• ZMP accumulation reaches steady state within 30-60 minutes in many cell types
• Slow clearance of intracellular ZMP (hours after AICAR removal)
• Potential incorporation into nucleotide pools at very high concentrations

Effective Concentrations:

• In vitro cell culture: Typically 0.25-2.0 mM for AMPK activation
• Lower concentrations (50-250 μM) sometimes sufficient depending on cell type
• In vivo rodent models: 50-500 mg/kg dosing range reported in literature
• Duration of AMPK activation depends on ZMP accumulation and clearance kinetics

Experimental Design Considerations:
The pharmacokinetic profile informs several aspects of research protocol design:

• Pre-treatment time: Allow 30-60 minutes for ZMP accumulation and AMPK activation
• Duration of effects: ZMP persists for hours after AICAR removal from media
• Concentration selection: Higher concentrations increase ZMP levels but may have off-target effects
• Route considerations: For in vivo studies, IP and IV routes provide systemic exposure

Research Applications

Metabolic Regulation Studies

AICAR serves as a primary research tool for investigating AMPK’s role in metabolic regulation:

• Glucose Metabolism Research: Investigation of AMPK-mediated glucose uptake, GLUT4 translocation, glycolysis activation, and insulin-independent glucose disposal mechanisms
• Fatty Acid Metabolism Studies: Analysis of fatty acid oxidation, acetyl-CoA carboxylase (ACC) phosphorylation, carnitine palmitoyltransferase 1 (CPT1) activity, and lipid oxidation pathways
• Lipid Metabolism Research: Examination of lipogenesis inhibition, malonyl-CoA reduction, fatty acid synthesis suppression, and triglyceride metabolism
• Hepatic Metabolism Studies: Investigation of hepatic glucose production suppression, gluconeogenesis inhibition, and liver metabolic regulation
• Mitochondrial Function Research: Studies on mitochondrial biogenesis, PGC-1α activation, oxidative phosphorylation, and cellular energetics

Research protocols employ metabolic assays including glucose uptake measurements, fatty acid oxidation assays, oxygen consumption analysis, and metabolic flux studies to characterize AICAR’s effects on cellular metabolism.

Exercise Physiology and Muscle Metabolism Research

AICAR has become known as an exercise mimetic in research contexts, based on its ability to activate metabolic pathways typically associated with physical exercise:

• Exercise Adaptation Research: Investigation of endurance capacity enhancement, metabolic adaptations, and exercise-independent activation of exercise-responsive genes
• Muscle Fiber Type Studies: Analysis of slow-twitch (oxidative) fiber type promotion, fast-to-slow fiber type conversion, and myosin heavy chain expression changes
• Mitochondrial Biogenesis Research: Examination of mitochondrial content increases, PGC-1α pathway activation, and oxidative capacity enhancement
• Metabolic Switching Studies: Investigation of fuel preference shifts toward fatty acid oxidation and enhanced oxidative metabolism
• Endurance and Fatigue Research: Studies on exercise endurance, fatigue resistance, and metabolic determinants of performance

Laboratory studies in rodent models demonstrate that AICAR treatment can increase running endurance, enhance oxidative capacity, and induce gene expression changes similar to those produced by endurance training. These findings have made AICAR valuable for studying the molecular basis of exercise adaptations.

Metabolic Disease Research

AICAR provides a research tool for investigating AMPK’s therapeutic potential in metabolic disease models:

• Diabetes Research: Investigation of glucose homeostasis, insulin sensitivity enhancement, glucose uptake mechanisms, and glycemic control
• Obesity Studies: Analysis of energy expenditure, fat oxidation, adiposity reduction, and metabolic rate modulation
• Metabolic Syndrome Research: Examination of multiple metabolic parameters, dyslipidemia, insulin resistance, and cardiovascular risk factors
• Fatty Liver Disease Studies: Investigation of hepatic steatosis reduction, lipid accumulation mechanisms, and liver metabolic dysfunction
• Cardiovascular Metabolism Research: Studies on cardiac metabolism, myocardial energy substrate utilization, and cardioprotection mechanisms

Research models include diet-induced obesity models, genetic diabetes models, metabolic syndrome models, and tissue-specific metabolic assessments to characterize AICAR’s effects on disease-relevant metabolic parameters.

Autophagy and Cellular Stress Response Research

AMPK activation by AICAR influences cellular quality control and stress response pathways:

• Autophagy Induction Studies: Investigation of AMPK-mediated autophagy activation, ULK1 phosphorylation, autophagic flux, and cellular degradation pathways
• mTOR Pathway Research: Analysis of mTOR suppression, mTORC1 inhibition, and mTOR-AMPK signaling crosstalk
• Cellular Stress Research: Examination of stress response activation, cellular adaptation mechanisms, and metabolic stress resilience
• Protein Quality Control Studies: Investigation of proteostasis, protein aggregation, and cellular clearance mechanisms
• Cellular Longevity Research: Studies on AMPK’s role in lifespan extension, cellular aging, and longevity pathways

Laboratory protocols employ autophagy markers (LC3 processing, p62 degradation), mTOR activity assays, and stress response measurements to characterize AICAR’s effects on these cellular processes.

Cardiovascular Research Applications

Research applications extend to cardiovascular system investigation:

• Cardiac Metabolism Studies: Examination of myocardial substrate utilization, cardiac energy metabolism, and metabolic flexibility
• Cardioprotection Research: Investigation of ischemia protection, reperfusion injury reduction, and cardiac stress resistance
• Vascular Function Studies: Analysis of endothelial function, nitric oxide production, vascular reactivity, and blood flow regulation
• Hypertrophy and Remodeling Research: Studies on pathological cardiac hypertrophy, maladaptive remodeling, and AMPK’s protective effects
• Atherosclerosis Research: Investigation of endothelial dysfunction, lipid metabolism, inflammatory pathways, and vascular disease mechanisms

Research models include cardiac ischemia-reperfusion models, pressure overload models, endothelial cell studies, and vascular function assessments.

Cancer Metabolism Research

AICAR serves as a tool for investigating metabolic aspects of cancer biology:

• Cancer Cell Metabolism Studies: Investigation of cancer cell energy metabolism, metabolic dependencies, and metabolic vulnerabilities
• Proliferation Research: Analysis of AMPK’s effects on cell proliferation, growth inhibition, and cell cycle regulation
• Metabolic Reprogramming Research: Examination of cancer metabolic phenotypes, Warburg effect modulation, and metabolic adaptation
• Autophagy in Cancer Studies: Investigation of autophagy’s dual roles in cancer, cell survival vs. death, and therapeutic implications
• Combination Therapy Research: Studies on AICAR combined with other therapeutic agents, metabolic targeting strategies, and synthetic lethality approaches

Research protocols examine cancer cell line responses, tumor metabolism, proliferation assays, and mechanistic studies of AMPK’s complex roles in cancer biology.

Anti-inflammatory and Immunometabolism Research

AMPK activation influences immune cell function and inflammatory responses:

• Inflammation Research: Investigation of anti-inflammatory effects, cytokine production modulation, and inflammatory signaling pathways
• Immune Cell Metabolism Studies: Analysis of immune cell metabolic reprogramming, T cell metabolism, macrophage polarization, and metabolic immune regulation
• NF-κB Pathway Research: Examination of NF-κB inhibition, inflammatory transcription factor modulation, and anti-inflammatory mechanisms
• Macrophage Polarization Studies: Investigation of M1/M2 polarization, metabolic influences on macrophage phenotype, and inflammation resolution
• Neuroinflammation Research: Studies on microglial activation, neuroinflammatory pathways, and AMPK’s neuroprotective effects

Laboratory approaches include immune cell culture studies, inflammatory marker measurements, metabolic profiling, and inflammatory disease models.

Laboratory Handling and Storage Protocols

Powder Storage:

• Store at -20°C to -80°C in original sealed container
• Protect from light exposure and moisture
• Desiccated storage environment required
• Stability data available for 12+ months at -20°C
• Allow powder to reach room temperature before opening to prevent moisture condensation

Solution Preparation:

• AICAR is soluble in water at concentrations up to 100 mM
• For aqueous solutions: Dissolve in sterile water or appropriate buffer
• For cell culture: Prepare stock solutions in sterile water or buffer, filter sterilize (0.22 μm)
• For in vivo studies: Dissolve in sterile saline or appropriate vehicle
• DMSO can be used as alternative solvent (solubility >100 mM)
• Final pH should be 7.0-7.4 for biological applications

Stock Solution Storage:

• Aqueous solutions: Store at -20°C in aliquots
• Stable for several months when frozen
• Avoid repeated freeze-thaw cycles (maximum 2-3 cycles)
• Single-use aliquots recommended to maintain compound integrity
• DMSO stocks: Can be stored at -20°C for extended periods

Working Solution Preparation:

• Prepare fresh working dilutions from frozen stock on day of experiment
• For cell culture: Add appropriate volume to culture medium
• Typical final concentrations: 0.25-2.0 mM for cell culture studies
• Filter sterilization recommended for sterile applications

Stability Considerations:

• AICAR is relatively stable in aqueous solution at neutral pH
• Avoid extreme pH conditions which may cause degradation
• Light protection recommended but not as critical as for some compounds
• Aqueous solutions at 4°C remain stable for several days

Quality Assurance and Analytical Testing

Each AICAR batch undergoes comprehensive analytical characterization:

Purity Analysis:

• High-Performance Liquid Chromatography (HPLC): ≥98% purity
• Analytical method: Reversed-phase HPLC with UV detection at 260nm
• Multiple peak integration to ensure accurate purity determination
• Impurity profiling to identify and quantify related substances

Structural Verification:

• Mass Spectrometry (ESI-MS): Confirms molecular weight 338.3 Da
• Nuclear Magnetic Resonance (NMR): Structural confirmation by ¹H-NMR and ¹³C-NMR
• Infrared Spectroscopy (IR): Functional group verification
• Melting point determination: Quality control parameter

Contaminant Testing:

• Bacterial endotoxin: <5 EU/mg (LAL method)
• Heavy metals: Below detection limits per USP standards
• Residual solvents: Analysis by gas chromatography
• Water content: Karl Fischer titration (<5% for powder)

Documentation:

• Certificate of Analysis (COA) provided with each batch
• Third-party analytical verification available upon request
• Stability data documented for recommended storage conditions
• Batch-specific QC results traceable by lot number
• Comprehensive analytical data package available

Research Considerations

Experimental Design Factors:

Researchers should consider several factors when designing AICAR experiments:

1. Concentration Selection: Published research reports broad concentration ranges (0.1-2.0 mM in vitro). Optimal concentration depends on cell type, treatment duration, and experimental objectives. Higher concentrations may have AMPK-independent effects.

2. Temporal Considerations: AICAR requires cellular uptake and phosphorylation to ZMP for activity. Pre-treatment time of 30-60 minutes typically needed before maximal AMPK activation. Duration of treatment influences the scope of metabolic changes.

3. Cell Type Variations: Different cell types express varying levels of nucleoside transporters and adenosine kinase, affecting AICAR sensitivity. Preliminary concentration-response studies recommended.

4. AMPK Dependency Verification: Use complementary approaches to confirm AMPK-dependence of observed effects:

• Genetic approaches: AMPK knockout cells or dominant-negative AMPK
• Pharmacological approaches: Alternative AMPK activators (A-769662, 991)
• Biochemical confirmation: Measure AMPK phosphorylation and substrate phosphorylation

5. Control Groups: Include appropriate vehicle controls, time-matched controls, and relevant positive/negative controls for the specific research question.

6. Off-Target Effects: At higher concentrations, AICAR may affect AMP-binding proteins beyond AMPK. Complementary approaches help distinguish AMPK-dependent from AMPK-independent effects.

Mechanism Investigation:

AICAR’s mechanism of action involves multiple steps, providing opportunities for mechanistic research:

• Transport Studies: Investigation of ENT-mediated uptake, transporter expression, and cellular entry mechanisms
• Metabolism Studies: Analysis of AICAR phosphorylation by adenosine kinase, ZMP accumulation kinetics, and nucleotide pool effects
• AMPK Activation Studies: Direct measurement of AMPK phosphorylation (Thr172), AMPK activity assays, and downstream substrate phosphorylation
• Downstream Signaling Research: Examination of metabolic pathway changes, gene expression alterations, and phenotypic outcomes

Comparison with Other AMPK Activators:

AICAR should be considered within the context of alternative AMPK activation strategies:

• A-769662: Direct AMPK activator that binds AMPK’s beta subunit, does not require metabolism
• 991 (Ex229): Another direct AMPK activator with different binding properties
• Metformin: Complex I inhibitor that activates AMPK secondarily through ATP depletion
• Genetic activation: Constitutively active AMPK mutants, AMPK overexpression
• Energy stress: Glucose deprivation, hypoxia, other metabolic stresses

Each approach has distinct advantages and limitations. AICAR’s advantage is that it activates AMPK without depleting cellular ATP, while its limitation is potential off-target effects on other AMP-binding proteins.

Compliance and Safety Information

Regulatory Status:
AICAR is provided as a research chemical for in-vitro laboratory studies and preclinical research only. This product has not been approved by the FDA for human therapeutic use, dietary supplementation, or medical applications. AICAR has been investigated in clinical trials for other indications but is not approved for general therapeutic use.

Sports Regulatory Status:
Researchers should be aware that AICAR is prohibited by the World Anti-Doping Agency (WADA) due to its exercise mimetic properties. This prohibition is relevant for sports science research contexts and compliance requirements.

Intended Use:

• In-vitro cell culture studies
• In-vivo preclinical research in approved animal models
• Laboratory investigation of AMPK biology and metabolism
• Academic and institutional research applications
• Mechanistic studies of metabolic regulation

NOT Intended For:

• Human consumption or administration
• Therapeutic treatment or diagnosis
• Dietary supplementation or performance enhancement
• Veterinary therapeutic applications without appropriate oversight
• Any applications outside controlled research settings

Safety Protocols:
Researchers should follow standard laboratory safety practices when handling AICAR:

• Use appropriate personal protective equipment (lab coat, gloves, safety glasses)
• Handle in well-ventilated areas
• Follow institutional chemical safety guidelines
• Dispose of waste according to local regulations for chemical waste
• Consult safety data sheet (SDS) for additional safety information
• Be aware of potential biological activity when handling

—