ACY1 Mouse

What is ACY1 and what is its primary function in mice?

ACY1 (Aminoacylase-1) is a metalloenzyme that catalyzes the deacetylation of N-acetylated amino acids into free amino acids. In mice, it plays critical roles in:

• Amino acid metabolism and recycling

• Myocardial fibrosis regulation through the TGF-β1/Smad3 pathway

• Insulin secretion and glucose homeostasis

The enzyme is functionally significant as inhibition of ACY1 in mice exacerbates cardiac dysfunction and fibrosis, while administration of ACY1 alters amino acid metabolism and increases insulin secretion .

What experimental models are available for studying ACY1 in mice?

The most effective approach involves combining protein expression analysis with functional enzymatic assessment:

• Protein quantification methods:

  • Western blotting for protein levels in tissue or plasma

  • Immunohistochemistry for tissue localization

• Functional activity assessment:

  • Quantify N-acetylated amino acid/free amino acid ratios using mass spectrometry

  • Key substrate-product pairs include:

    • N-acetyl-methionine/methionine

    • N-acetyl-glutamine/glutamine

    • N-acetyl-isoleucine/isoleucine

When analyzing data, decreased ratios indicate increased ACY1 activity. In AAV8-ACY1 treated mice, ratios decreased by 68.5-80.2% compared to controls, confirming functional enzymatic activity .

What is the relationship between ACY1 and the TGF-β1/Smad3 pathway in myocardial fibrosis?

ACY1 functions as a negative regulator of the TGF-β1/Smad3 signaling pathway in cardiac fibrosis models. The relationship is characterized by:

• Inhibitory effect: ACY1 overexpression suppresses TGF-β1/Smad3 signaling in mouse cardiac fibroblasts (MCFs) treated with Angiotensin II, reducing collagen deposition (COL3A1, COL1A) .

• Loss-of-function consequences: ACY1 inhibition with MTBM significantly:

  • Increases TGF-β1 expression and Smad3 phosphorylation (p-Smad3/Smad3 ratio)

  • Exacerbates myocardial fibrosis and collagen deposition

  • Worsens cardiac function in heart failure models

• Cellular validation: In MCFs, ACY1 silencing via siRNA promotes collagen expression and activates TGF-β1/Smad3 pathway both with and without Angiotensin II stimulation .

This regulatory axis presents a potential therapeutic target for addressing myocardial fibrosis in heart failure.

How does ACY1 affect insulin secretion and glucose metabolism in mice?

ACY1 demonstrates significant metabolic effects through specific mechanisms:

• Insulin secretion stimulation:

  • Acute administration of purified ACY1 (100 mg/kg i.p.) increases fasting insulin levels 1.6-fold in mice

  • Similar effects observed with AAV8-mediated ACY1 expression (1.43 ± 0.09 vs 0.91 ± 0.06 ng/mL in controls)

• Proposed mechanism:

  • ACY1 produces free amino acids through deacetylation

  • These free amino acids (particularly methionine, glutamine, and isoleucine) act as potent insulin secretagogues

  • Increased insulin promotes glucose uptake and utilization

• Genetic evidence:

  • A loss-of-function variant (rs121912698, 1057C>T, Arg353Cys) is associated with decreased fasting blood glucose levels in humans

These findings establish ACY1 as a potential therapeutic target for metabolic disorders involving insulin secretion deficiencies.

What are the divergent roles of ACY1 in cardiac versus metabolic disease models?

ACY1 exhibits context-dependent functions across different disease states:

For rigorous experimental design, consider these validated approaches:

• Pharmacological intervention:

  • Inhibition: MTBM administration via intraperitoneal injection

  • Supplementation: Purified ACY1 protein (100 mg/kg, i.p.)

  • Assessment timeframe: Effects observed within 6 hours

• Genetic approaches:

  • Viral vectors: AAV8-ACY1 (2×10¹¹ genome copies) for sustained expression

  • Expression validation: Western blot showing approximately 3.3-fold increase in plasma ACY1 levels

  • Functional validation: Measure substrate/product ratios by mass spectrometry

• Experimental controls:

  • AAV8-GFP for viral delivery control

  • Saline injection for protein administration control

  • Control-plasmid/siRNA for in vitro manipulations

Each approach requires appropriate validation by measuring both protein levels and enzymatic activity to confirm successful manipulation.

What is the role of N-acetylglutamine in ACY1 research and disease biomarkers?

N-acetylglutamine has emerged as a key metabolite in ACY1 research with significant biomarker potential:

• Biomarker significance:

  • Significantly elevated in both human heart failure patients and CAL-induced heart failure mice

  • Inversely correlated with ACY1 expression in heart failure

• Mechanistic implications:

  • N-acetylglutamine is a specific substrate for ACY1

  • Its accumulation directly reflects decreased ACY1 activity

  • May contribute to disease pathophysiology through unknown mechanisms

• Clinical applications:

  • Potential diagnostic marker for heart failure

  • May indicate severity of myocardial fibrosis

  • Metabolomic analysis using HPLC-QqQ-MS provides targeted quantification

The elevated N-acetylglutamine levels associated with heart failure suggest that this metabolite could serve as a surrogate marker for ACY1 dysfunction in cardiovascular disease.

How do I optimize AAV8-mediated ACY1 expression for long-term studies?

For successful long-term ACY1 expression studies using AAV8 vectors:

• Vector design considerations:

  • Use tissue-specific promoters for targeted expression

  • Optimize codon usage for mouse expression

  • Include histidine tag for detection if antibody availability is limited

• Delivery parameters:

  • Optimal dose: 2×10¹¹ genome copies showed effective expression

  • Route: Intravenous delivery for systemic effects

  • Expected expression: 3-4 fold increase over endogenous levels

• Validation approaches:

  • Western blot analysis of plasma samples

  • Functional validation through N-acetylated amino acid/free amino acid ratios

  • Physiological readouts (insulin levels, glucose tolerance)

This approach provides stable, long-term ACY1 overexpression suitable for chronic disease models and metabolic studies.

What gene variants of ACY1 have functional significance in mice?

Current research highlights several ACY1 variants with functional consequences:

• Key variant with established impact:

  • 1057C>T, Arg353Cys missense substitution (human variant rs121912698)

  • Disrupts the catalytic subunit of ACY1

  • Associated with decreased circulating ACY1 levels (β = –1.8) and reduced fasting glucose

• Experimental considerations for variant studies:

  • CRISPR/Cas9-mediated introduction of specific variants

  • Functional validation through enzymatic activity assays

  • Metabolic phenotyping (glucose tolerance, insulin secretion)

  • Cardiac phenotyping (fibrosis susceptibility, TGF-β1/Smad3 activation)

Understanding these functional variants provides insight into structure-function relationships and potential therapeutic targeting strategies.

What emerging technologies might advance ACY1 mouse research?

Several cutting-edge approaches show promise for advancing ACY1 research:

• Single-cell metabolomics:

  • Cell-type specific ACY1 activity in heterogeneous tissues

  • Identifying cellular sources of N-acetylated amino acids

• Tissue-specific conditional knockouts:

  • CRISPR-based tissue-specific manipulation

  • Temporal control of ACY1 expression

• Metabolic tracing:

  • Isotope-labeled N-acetylated amino acids to track metabolism

  • Flux analysis for enzymatic activity in vivo

• AI-integrated analysis:

  • As demonstrated in recent research, large language models can facilitate genetic discovery and hypothesis generation in complex systems like ACY1 regulation

These emerging approaches will help resolve remaining questions about tissue-specific functions and therapeutic potential of ACY1 in multiple disease contexts.

How can ACY1 mouse research translate to human therapeutic applications?

Translational research on ACY1 shows promise in several therapeutic areas:

• Cardiovascular applications:

  • ACY1 enhancement as a cardioprotective strategy against fibrosis

  • N-acetylglutamine as a biomarker for heart failure monitoring

  • TGF-β1/Smad3 pathway modulation through ACY1-based interventions

• Metabolic disease applications:

  • ACY1 supplementation to enhance insulin secretion

  • Targeted amino acid metabolism in diabetes management

  • Personalized approaches based on ACY1 genetic variants

• Translational considerations:

  • Species differences in ACY1 substrate specificity

  • Tissue-specific delivery methods for therapeutic applications

  • Potential off-target effects requiring careful safety assessment

The dual role of ACY1 in both cardiac and metabolic pathways makes it a particularly interesting therapeutic target with potential applications across multiple disease domains.

What are the established protocols for measuring N-acetylated amino acids in mouse plasma?

For accurate quantification of N-acetylated amino acids:

• Sample preparation:

  • Collect plasma 6 hours after intervention (ACY1 administration)

  • Protein precipitation with organic solvent

  • Centrifugation to remove precipitated proteins

• Analytical methods:

  • HPLC-QqQ-MS (High-Performance Liquid Chromatography coupled to Triple Quadrupole Mass Spectrometry)

  • Targeted metabolomic approach for specific N-acetylated amino acids

  • Internal standards for quantification

• Data analysis:

  • Calculate substrate/product ratios (e.g., N-acetyl-methionine/methionine)

  • Compare treated vs. control groups

  • Assess correlations with physiological parameters